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Regulation of insulin effector systems in the brain

Material Information

Title:
Regulation of insulin effector systems in the brain
Creator:
Mudd, Laura Mary, 1958-
Publication Date:
Language:
English
Physical Description:
v, 137 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Brain ( jstor )
Cells ( jstor )
Cultured cells ( jstor )
Insulin ( jstor )
Neuroglia ( jstor )
Neurons ( jstor )
Phorbol esters ( jstor )
Rats ( jstor )
Receptors ( jstor )
Brain -- enzymology ( mesh )
Dissertations, Academic -- Physiology -- UF ( mesh )
Physiology thesis Ph.D ( mesh )
Protein Kinase C ( mesh )
Receptor, Insulin ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1989.
Bibliography:
Bibliography: leaves 123-136.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Laura Mary Mudd.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
026374546 ( ALEPH )
25072955 ( OCLC )
AFK3052 ( NOTIS )

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Full Text










REGULATION OF INSULIN EFFECTOR SYSTEMS IN THE BRAIN


LAURA MARY MUDD












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1989








TABLE OF CONTENTS


PAGE

A B S T R A C T ................................................ ... ...........................................................iv

CHAPTER

I. INTRODUCTION........................................................ ............................. 1

Insulin and Insulin Receptors in the Brain........................................ 2
Insulin in the Brain: Potential Functions......................... ............ 4
Protein Kinase C (PKC): Regulation of Insulin and IGF I
R eceptors....................................................... .......................... 6
Ligand-Receptor Interactions.......................................................8
Phosphorylation of Receptors.................... .......... .............. 8
Receptor-Induced Effects......................... .........................10
Effects ofGrowth Factors on PKC.................................... ..11
PKC in the Brain: Distribution........................................ ........12
PKC Regulation of Glucose Uptake and Insulin Receptors
in the B rain................................................. ................. ...............13
Cultured Brain Cells: A Model for the Study of Neurotrophic/
Neuroactive Substances.......................... ..............................14

II. METHODS.................................. ..........................................................20

Preparation of Primary Neuronal Cultures from Rat Brains......20
Preparation of Primary Astrocytic Glial Cultures from Rat
Brains.....................................................................................................20
Immunocytochemistry of Neuron-Specific Enolase/Glial
Fibrillary Acidic Protein.......................... ........................ 22
Neuronal Depolarization..........................................................23
Insulin Radioimmunoassay.......................... .......................................... 23
Characterization of Immunoprecipitable Insulin by HPLC..........24
Labelling of Immunoreactive Insulin in Neuronal
Cultures...............................................................................................25
Characterization of the Regulation of Neuronal Insulin Release
by Glucose................................................................................. 25
2-Deoxy-D-glucose (2-dGlc) Uptake...............................................26
lodination of Insulin................................................ ........................26
Insulin Binding........................................................ ..... .................. 28
Protein Determinations...................................... ............................29








PKC Im m unocytochemistry.................................. ............................... 31
Statistical Analysis........................... ...... ..........................31

III. INSULIN SYNTHESIS AND RELEASE FROM NEURONAL CULTURES.37

Introduction.................................... .................................................... 37
Results......................................................................... ............. ........... 39
D iscussion...................................... ...................................................... 40

IV. CHARACTERIZATION OF PKC IN NEURONAL AND GLIAL CELLS
IN PRIMARY CULTURE......................... .................................51

Introduction........................................... ................ ........................... 51
R esults....................................................................... ................. .......... 52
Discussion....................................... ..................................................... 53

V. THE REGULATION OF SUGAR TRANSPORT IN PRIMARY
NEURONAL AND GLIAL CELL CULTURES BY PHORBOL
E S T E R S ........................................................... ...... ......................71

Introduction.................................... ..................................................... 71
Results.............................. ...............................................72
Discussion....................................... ..................................................... 72

VI. THE REGULATION OF INSULIN RECEPTORS IN NEURONAL AND
GLIAL PRIMARY CULTURES BY PHORBOL ESTERS..................76

Introduction.................................... .................................................... 76
R esults........................................... ....................................................... 77
D iscussion............................ ................................... ................................ 79

VII. THE EFFECTS OF INSUUN AND DEXAMETHASONE ON NEURONAL
AND GLIAL PKC........................................................... 97

Introduction............................................................. ........................... 97
R esults............................................................................ ............................. 98
Discussion...................................... ..................................................... 99

VIII. DISCUSSION AND SUMMARY...............................................................113

R EFERENC ES ....................................................................... ................................. 123

BIOGRAPHICAL SKETCH........................ ...... ..........................137










Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

REGULATION OF INSULIN EFFECTOR SYSTEMS IN THE BRAIN

By

Laura Mary Mudd

May, 1989

Chairman: Mohan K. Raizada
Major Department: Physiology


In spite of insulin's effects on glucose uptake in the periphery and the fact
that the brain is a glucose-obligate organ, the brain was long believed to be
insulin-independent because insulin does not cross the blood-brain barrier.
Many reports over the course of the past ten years have localized both insulin
and insulin receptors in the brain, although the source and function of the
peptide in the brain and the mechanisms by which receptors are regulated have
remained the subjects of some mystery.
It was found that an immunoreactive insulin was released from cultured
neurons under depolarizing conditions, that labelled leucine could be
incorporated into the released peptide and that the peptide coeluted with a rat
insulin standard when the two were applied to a reverse-phase high pressure
liquid chromatography column. Thus, the peptide resembles insulin
chromatographically as well as immunologically, is synthesized in neuronal
cultures and is released under depolarizing conditions. In the brain, insulin
may act as a neurotransmitter.








Protein kinase C (PKC) occurs in many tissues, but it occurs at highest
concentration in the brain. It has been shown to regulate glucose uptake and
the insulin receptor in many peripheral tissues. PKC stimulates both glucose
uptake and insulin binding in glial cells without affecting either in neurons. The
stimulation of insulin receptor binding in glial cells is not accompanied by an
increase in activity; thus it appears to inactivate the receptors. Activation of PKC
by phorbol esters, as evidenced by translocation from the cytosol to the
membrane, occurs in both neurons and glial cells, although the fraction of
cytosolic, and thus, translocatable, PKC is much smaller in the former. In glial
cells, Translocation of PKC precedes other effects of PKC, and thus, may be
involved in the mechanism. Insulin stimulates the level of immunoreactive PKC
in glial cells, but not in neurons; thus, in glial cells, this appears to be a
feedback loop whereby insulin regulates its own receptor. In neurons, both
arms of the circuit, that is, regulation of the PKC concentration by insulin and
regulation of the insulin receptor by PKC, are missing. That neuronal and glial
insulin receptors are differentially regulated is not surprising as the receptors
differ from one another with respect to both structure and function.

















CHAPTER I
INTRODUCTION


Historically, insulin has been used in the treatment of diabetes
mellitus and psychosis. Pancreatic insulin was first isolated in
1922 at which time it was used clinically in the treatment of Type I
diabetes (1). In classical Type I diabetes the insulin-secreting 8-
cells of the pancreas are destroyed and the patients become
dependent on exogenous insulin to control their blood sugar. Several
years after Banting and Best began using insulin to treat diabetes,
Schmidt (2) and Sakel (3) separately reported successful treatment
of psychotic patients with insulin in the presence and absence of
carbohydrate, respectively, that is, treatment with and without
hypoglycemic shock. Insulin-induced hypoglycemic shock was used
successfully in the treatment of schitzophrenia and depression as
well as psychosis until the development of electroshock therapy and
more sophisticated psychoactive drugs (4).
The mechanisms of pancreatic insulin synthesis and release, as
well as physiological effects on liver, muscle and adipose tissue,
have been well characterized. Pancreatic insulin is synthesized as
preproinsulin, cleaved to proinsulin almost immediately and








packaged into secretary granules where the C-peptide is cleaved
prior to secretion (5). Basal beta-cell secretion of insulin is about
0.5 U/hour. This can be increased 10-30 times with acute
stimulation. Glucose is the most potent stimulator of pancreatic
insulin release in man (6) although amino acids (especially arg, lys,
leu and val), glucagon, B-adrenergic and vagal stimulation of the
pancreas stimulate secretion as well (7,8). Somatostatin,
serotonin, dopamine, prostaglandin E and splanchnic stimulation are
inhibitory to insulin release (8,9). Chronic glucose stimulation
induces a biphasic release of insulin (10); this is thought to be due
to the release of short- and long-term insulin stores (8).
The first step in the action of insulin on the peripheral target
tissues is its interaction with specific receptors on the cell
surface. This interaction of insulin with its receptor stimulates
autophosphorylation of a tyrosine residue (11) as well as receptor
tyrosine kinase activity (12). Binding of insulin induces an increase
in glucose and amino acid uptake by liver, muscle and adipose
tissues and consequently an increase in the synthesis of glycogen
and protein with a corresponding decrease in lipolysis and
gluconeogenesis (13).


Insulin and Insulin Receptors in the Brain
In spite of insulin's effects on glucose uptake in the periphery
and the fact that the brain is a glucose-obligate organ, the brain was
long believed to be insulin-independent because insulin does not
cross the blood-brain barrier. Havrankova et al., established the
presence of insulin in the brain at concentrations that averaged 25








times that of plasma insulin (14). Since then many reports have
appeared in the literature confirming this finding although the
actual amount of insulin in the brain is the subject of some
controversy (15,16). Insulin is found in cultures from mouse brain
and neuronal cultures from rat brain (17,18). The presence of this
insulin-like peptide in the brain raises two major questions; what
is the source of this peptide and what is its function?
The following observations suggest that insulin may be
synthesized in the brain: (a) central insulin concentrations appear to
remain constant in pathological situations in which peripheral
insulin concentrations vary widely (19); (b) insulin-like activity
has been demonstrated in cultured brain cells which are removed
from pancreatic insulin for weeks (18); (c) radioactive amino acids
can be incorporated into an insulin-like peptide in cultured neurons
by a cycloheximide-sensitive process (18) and (d) there is an mRNA
in the brain which hybridizes with insulin cDNA (20-4). In addition,
our experiments have shown that insulin is synthesized and released
by neurons (25). Furthermore, when neuronal cultures were labelled
with leucine and depolarized, a labelled peptide was released which
could be precipatated by an insulin antibody. Cycloheximide
decreased insulin synthesis by 80% (18).
In recent years specific insulin receptors in the brain have been
demonstrated conclusively (26). These receptor sites, which are
distributed non-uniformly throughout the brain (27), are evenly
distributed between neurons and glial cells (28-30). Studies have
shown that neuronal and glial insulin receptors are structurally and
physiologically distinct in several respects. The brain insulin








receptor is generally similar in subunit structure to the more
familiar peripheral insulin receptors although the alpha-subunit is
somewhat smaller in the brain (31) due to decreased glycosylation
of the receptor in the brain. In particular, the neuronal alpha- and
beta-subunits are smaller, while the glial subunits resemble those
from the liver (29,32) in size. That the glial receptor is
structurally similar to the peripheral receptor is not surprising,
since insulin appears to have traditional metabolic effects in glia
and neuromodulatory effects in neurons. Insulin stimulates glucose
uptake by glia, but not by neurons (28) and inhibits norepinephrine
uptake by neurons, but not by glia (30).


Insulin in the Brain: Potential Functions
Insulin in the central nervous system (CNS) has been implicated
in the control of brain growth and development (28,33-5),
catecholamine release (36-9), and diabetes (40) in in vitro studies
and satiety (41) in in vivo studies. In addition, insulin may itself
act as a neurotransmitter in the CNS. In vitro studies have been
essential in developing an understanding of insulin's actions in the
CNS at the cellular level. With regard to insulin's role in growth and
development, insulin stimulates DNA, RNA and protein synthesis in
mixed brain cell cultures and astrocyte glial cultures. It also
stimulates 2-deoxy-D-glucose uptake in astrocyte glial cultures
(28,33). These are all events which are associated with cell growth
and proliferation. In addition, insulin has differentiative effects on
developing rat brain. When immature rat retinal cultures were
exposed to insulin, precocious synaptic release of acetylcholine was








evoked within one hour while control and glutamate treatment of the
cells elicited no such response (34). Insulin induces ornithine
decarboxylase activity and neurite outgrowth in cultured embryonic
mouse brain cells and rat brain cultures (35). Thus, insulin induces
maturation of brain cells in culture as well as growth.
Insulin alters the content of serotonin and catecholamines in the
brain by increasing the rate of uptake of their precursors (36-8). It
also stimulates the release of dopamine, epinephrine and
norepinephrine from hypothalamic slices (39) and changes the firing
rates of neurons in the striatum (42) and hippocampus (43). Insulin,
then, definitely acts as a neuromodulator in the CNS and it may act
as a neurotransmitter itself. Recently, the neuromodulatory effects
of insulin have been suggested to be mediated by insulin receptors
present on the neurons (44). Insulin has, then, satisfied several
criteria by which putative neurotransmitters are classified as such:
it is synthesized in neurons, released under depolarizing conditions,
bound with specificity and high affinity to a receptor, degraded and
modulates neuronal activity.
CNS insulin may be involved in the pathophysiology of obesity and
Type II, or insulin-independent, diabetes. In vivo studies in baboons
indicate that insulin has a direct effect on satiety (41). When
insulin was infused into the lateral cerebral ventricles, a
significant, dose-related decrease in food intake and body weight
was observed. This may occur by interaction with neurons in the
hypothalamus. Injection of insulin in low doses causes electrical
activity to increase in hypothalamic neurons (45). In addition to its
association with obesity, central insulin may play a role in Type II








diabetes. Lesions of the ventromedial hypothalamus cause
hypersecretion of hypothalamic insulin (46).
In studies involving cultures of brain cells from diabetic mice
the ratio of externalized insulin receptors to total insulin receptors
was substantially decreased in cultures from diabetic mice versus
control cultures (40). As obesity is a major risk factor for Type II
diabetes there may be a relationship between these two effects of
central insulin. This hypothesis is supported by the observation that
insulin receptor number is significantly decreased in the olfactory
bulb of obese rats (47).


Protein Kinase C (PKCI: Regulation of Insulin and IGF I Receotors
PKC is a serine/threonine kinase which is present in many
tissues but occurs at highest concentrations in brain (48). PKC is
calcium-dependent and 1,2-diacylglycerol (DAG) a product of
membrane phospholipid metabolism, increases the affinity of the
enzyme for calcium (49-50). Reports of the molecular weight of
PKC vary. The different values may reflect the method by which the
relative molecular weight (Mr) is determined, as evidenced by a
study in which values of 77,000 and 82,000 daltons were obtained
from sucrose density gradient and polyamide gel electrophoresis,
respectively (51). Differences may also be attributable to subunit
aggregation (52) or to the existence of different isozymes of PKC
(53-4). Currently, seven highly-homologous isozymes of PKC have
been isolated and characterized. Four are single polypeptide chains
with four constant and five variable regions, while three subspecies
differ slightly. The isozyme distributions differ with respect to one








another (55-6) and with respect to the development of the organism
(53,57), however, the kinetic properties of the isozymes are very
similar. For a review see Nishizuka (54).
Tumor-promoting phorbol esters, such as 12-O-tetradecanoyl-
phorbol-13-acetate (TPA), act as exogenous stimulators of PKC (58).
Upon activation by TPA or calcium, there is a rapid decrease in
cytoplasmic PKC and a corresponding increase in membrane PKC (59-
60, Figure 1-1). Following this translocation, the membrane-bound
PKC catalyzes the phosphorylation of specific proteins (61-2).
Translocation appears to mediate other activities of PKC such as
neuronal potentiation (63) as well. Prevention of PKC
redistribution, as with concanavalin A, has been shown to block
activation of PKC (64). Once activated, PKC acts in many types of
cells to block hormone-stimulated phosphoinositide hydrolysis, and
thus exerts negative feedback over its own activation (65-8).
PKC has different effects in different tissues (48). In the brain
PKC is involved in the regulation of neuronal ion channels (69-71),
synaptic plasticity (72-3), neurite outgrowth (74) and
neurotransmitter release (75-7), as well as the differentiation of
astrocytes and oligodendrocytes (78-9) and changes in membrane
conductance in astrocytes (80). Many of the varied effects of PKC
appear to result from its interactions with growth factor receptors
with tyrosine kinase activities. PKC-induced receptor
phosphorylations alter the affinities, activities and effects of some
of these receptors. These growth factors, in turn, have been shown
to regulate either content or activity of PKC in certain cells.








Ligand-Receptor Interactions
PKC regulates the specific binding of insulin and insulin-like
growth factor I (IGF I). Phorbol esters regulate the insulin receptor
in lymphocytes, adipocytes and monocytes (81-3). In each of these
types of cells, phorbol esters inhibit the binding of insulin by
increasing the Km of the high affinity receptor. The endogenous
analogues of TPA, the DAGs, also reversibly inhibit the binding of
insulin to its receptor (84) by altering the affinity of the receptor.
The calcium ionophore A23187 potentiates the effect of TPA on
insulin binding in monocytes (85). TPA does not, however, decrease
insulin binding in all cell types. TPA has no effect on insulin binding
in either 3T3 cells or in hepatoma cells (86-7) although the
response to insulin is impaired in the latter. Thus, PKC-induced
decreases in insulin binding may result from either decreased
affinity of the receptors or increased internalization of the insulin-
receptor complex, depending on the type of tissue or cell.
The case for regulation of the IGF I receptor by PKC is similar to
that for the insulin receptor. DAGs inhibit IGF I binding to the IGF I
receptor in monocytes. TPA also inhibits IGF I binding in
lymphocytes, monocytes and adipocytes by altering the high-affinity
binding site without altering the number of receptors (84). This
differs from insulin or IGF I down-regulation of the IGF I receptor,
which results from a decrease in the number of receptors (88).


Phosohorvlation of Receotors
The mechanism for PKC-stimulated alterations in the insulin and
IGF I receptors appears to involve serine/threonine phosphorylation








of those receptors. In 1983, TPA was first shown to stimulate
phosphorylation of both insulin and IGF I receptors in IM-9 cells that
had been preincubated with H332p04 (61). Insulin- and TPA-
stimulated phosphorylation appeared to be additive, suggesting that
there was no interaction between the sites. In 1984, TPA was
shown to enhance serine/threonine phosphorylation of the insulin
receptor in rat hepatoma cells at nine sites (87). Insulin was shown
to stimulate phosphorylation of tyrosine and serine residues at six
sites, three of which were similar to the TPA-phosphorylated sites.
In addition, the phorbol ester decreased insulin-stimulated
phosphorylation, suggesting that there was, in fact, an interaction
between the sites of action of the two agents. In later studies on
IM-9 and HepG2 cells, TPA was found to phosphorylate four major
serine residues, which were not phosphorylated in untreated cells
and to increase the phosphorylation of one threonine residue on the
insulin receptor. These serine residues were not phosphorylated by
insulin, which, however, did phosphorylate three tyrosine residues
(89). PKC acts directly on the insulin receptor as it phosphorylates
the insulin receptor in vitro (90). Similar results were seen with
the IGF I receptor. These very different profiles of phosphorylation
induced by insulin and phorbol esters give strong evidence that
insulin and IGF I were not acting through PKC. In 1988, TPA was
found to enhance predominantly the phosphorylation of one serine
residue on the insulin receptor in hepatoma cells (91). TPA-
treatment of cells inhibits insulin-stimulated receptor
phosphorylation of exogenous substrates by 50 percent. These
changes in the receptor are maintained when the receptors are








isolated and are reversed by incubation with alkaline phosphatase,
suggesting that PKC decreases the tyrosine kinase activity of the
insulin receptor and that this decrease is due to the phosphorylative
changes induced in the receptor. Studies on rat adipocytes have also
shown that TPA increases the Km of the insulin receptor for ATP,
thus suggesting a mechanism for insulin resistance in adipocytes
(82).


Receptor-Induced Effects
The tyrosine kinase activity of the insulin receptor is necessary
for normal receptor function and down-regulation (92-3). This has
been demonstrated by studies in which kinase-defective mutant
insulin receptors were used to transfect cells. The mutant
receptors demonstrate normal binding of insulin but do not possess
tyrosine kinase activity, are not internalized and do not possess
biological activity. Treatment of endogenous, biologically-active
insulin receptors with monoclonal antibodies against the receptor
kinase inhibits insulin-stimulated effects as well (94). Inhibition
of receptor tyrosine kinase activity by TPA leads to the same types
of defects. Treatment with TPA is associated with inhibition of
insulin-mediated DNA synthesis (95), phosphorylation of metabolic
enzymes (87), glycogen synthesis (96) and glucose uptake (97),
among others. Thus, an impaired PKC pathway can have dire
consequences for the cell or organism. This is demonstrated by
genetically obese (fa/fa) rats, in whose hearts and hepatocytes both
the basal distribution and the translocation of PKC are abnormal.
The resultant insulin-insensitivity can be duplicated by treating








lean rats with TPA to down-regulate PKC (98). The same is not true
for all tissues, however, as phorbol esters have only minimal
effects on insulin sensitivity in rat skeletal muscle (99). The
interaction of PKC and the insulin receptor is somewhat complicated
by reports of synergism in mitotic stimulation (100). In addition,
there are proteins which have phosphorylation sites for both PKC and
receptor kinases (101) and are stimulated by both types of mitogens
(102). This last effect would account for the paradoxical way in
which PKC both inhibits the insulin receptor and mimics many of
insulin's effects within the cell.


Effects of Growth Factors on PKC
While the majority of studies on PKC/growth factor interactions
in the literature focus on regulation of growth factor receptors by
PKC, those receptors frequently regulate PKC as well. Treatment
with insulin in the presence of glucose has been shown to increase
both the binding capacity of PKC and enzymatic activity in
adipocytes (103). As the insulin effect is eliminated in the presence
of high glucose, the effect may be secondary to increased glucose
uptake in the insulin-treated cells. Insulin also increases the level
of cytosolic calcium ion in adipocytes (104), which could account
for the increased PKC activity. Studies also demonstrate
enhancement of PKC activity in myocytes and mammary tumor cells
by insulin (105-6). In the former the increase occurs in both the
cytosolic and membrane fractions and is not inhibited by
cycloheximide. This increase in the activity of PKC is reportedly
mediated via increased DAG generated by phospholipid hydrolysis and








phospholipid synthesis. There is some controversy on this point,
with other work suggesting that insulin does not increase the
activity of PKC in myocytes (107). It is suggested that the
increases in phosphorylation observed after the administration of
insulin are mediated by S6 kinase which is activated by both insulin
and TPA. Growth factors, then, may increase PKC indirectly by
increasing either cytosolic free calcium ion or DAG or by acting at a
point in the pathway beyond the PKC molecule itself. A direct effect
of insulin on PKC must necessarily be demonstrated on the isolated
enzyme.


PKC in the Brain : Distribution
Although PKC is present in many tissues, it occurs at highest
concentration in the brain. The seven subspecies of PKC have
different distributions in the brain (54) and these change with brain
development. The gamma subspecies, which occurs only in the
central nervous system in the rat and monkey, has a
developmentally-regulated distribution, with expression increasing
from birth until it reaches a maximum at about three weeks of age.
Total PKC is also developmentally regulated in brain in studies in
cultured neurons and in vivo (57). Interestingly, insulin and IGF 1
receptors are developmentally regulated in the rat brain with
increases occurring in the first weeks of life, followed by a decline
(108-9). Immunohistochemical analyses show different antibody
staining patterns. There appear to be subspecies which are present
almost exclusively in neurons (55,110), in astrocyte-glial cells and
in oligodendrocytes (55,111). The enzymatic activity is also








unevenly distributed, with the left cerebral hemisphere expressing
more than the right in the rat (112). Binding studies using
radiolabeled phorbol esters show two to three times more PKC in
neurons than in glial cells cultured from the same rat brains (113).
The subcellular distribution of PKC has been the subject of many
investigations. It is localized in dendrites, axons, perikarya and
nuclei (110) of neurons with particularly high concentrations in
presynaptic terminals (111) and in growth cones (114). This is not
unexpected as PKC mediates both neurotransmission and neurite
outgrowth (115). Fractionation of glial cells demonstrated that the
majority of the PKC was cytoplasmic (116). The same study showed
that the majority of PKC in whole brain tissue is associated with
the membrane, suggesting that the majority of neuronal PKC is
membrane-bound. Seventy-five percent of the glial cell cytoplasmic
PKC can be translocated to the membrane within 30-60 minutes of
TPA-treatment. This is similar to the situation seen in peripheral
tissues.


PKC Regulation of Glucose Uptake and Insulin Receptors in the Brain
PKC has been shown to regulate glucose uptake in many tissues.
Phorbol ester-induced decreases in the binding of insulin to its
receptor are not associated with decreased glucose uptake as, in
fact, phorbol esters stimulate glucose uptake in adipocytes,
myocytes, fibroblasts and astrocytes (97,117-9).
As discussed, in the brain there are distinctions between neurons
and glia with regard to PKC: PKC is present in higher concentrations
in neurons than in glia from the same brains as demonstrated by the








binding of phorbol esters (113); different isotypes of PKC are
present in neurons and glia (55,110,111) and phorbol esters
stimulate glucose uptake in glia but not in neurons (119).
The joint observations that neurons and glia differ with respect
to the physiological activities of the insulin receptor and PKC and
that phorbol esters regulate the insulin receptor and glucose uptake
peripherally, led us to investigate the role of PKC in the regulation
of insulin receptors in neurons and glia from the central nervous
system. Because activation of PKC by TPA has been shown to involve
translocation of PKC from the cytosolic to the membranous fraction
in many types of cells, we chose to examine TPA's effects on
neurons and glia to determine the relative concentrations of
immunoactive PKC in neurons and glia, whether TPA stimulates the
translocation of PKC in both neurons and glia, and whether the
translocation in glia, if it occurs, precedes TPA's effects on glucose
uptake and the insulin receptor and, thus, might be involved in a
mechanism. Differences in the ability of TPA to induce
translocation or the time-course of the translocation might
presumably explain TPA's differential effects on neurons and glia.
Lastly, in order to determine whether insulin regulates its own
receptor by this pathway, we chose to study insulin's effects on PKC
in neurons and glia from the brain.

Cultured Brain Cells: A Model for the Study of Neurotrophic/
Neuroactive Substances
The importance of neurons in brain function is unquestioned.
Proper neuronal growth, development and maintenance are essential








for every aspect of normal brain function. The importance of growth
factors, such as nerve growth factor, insulin and the insulin-like
growth factors, in neuronal development and activity has been well
documented although the mechanisms by which these agents act are
still objects of intense study (115,120).
In contrast, although glial cells are the predominant cell type in
the mature nervous system, their involvement in the growth,
development, differentiation and function of the brain has only
recently become a subject of investigation. Glial cells have recently
been implicated in processes involving the growth, development and
function of the nervous system. Glia are not only responsive to
trophic factors but may produce them as well. They facilitate
neuronal migration in fetal life but induce scarring to inhibit
regeneration in the mature nervous system. In addition, they alter
the levels of neurotransmitters available at the synapse, thus
altering neuronal excitability and they may even be excitable
themselves.
Glia respond to trophic factors such as insulin and insulin-like
growth factor I (IGF I) with an increase in glucose uptake in contrast
to neurons (119,121). This is of interest developmentally as both
insulin and IGF I receptors in brain increase to a maximal level
during brain development and then show a gradual decline (108-9).
Glia also exhibit an increase in macromolecular synthesis in
response to both insulin (33) and IGF I (121). Epidermal growth
factor receptors in the brain appear predominantly in glia, as well
(122).








In addition to being responsive to trophic factors, glial cells may
produce them as well. A substance which is immunologically
identical to nerve growth factor is present in glia (123) and glia,
which develop at the same time as neurons, provide other
extracellular molecules which enhance neuronal migration (124).
This glial stimulus to neuronal growth is lost with age as astrocytic
glial cells block axonal regeneration (125) and synapse formation
(126) in the mature nervous system. Inflammation of the mature
nervous system causes a reactive gliosis which prevents neuronal
repair as in multiple sclerosis (124).
Finally, glia contribute to the regulation of nervous system
excitability. Glia take up, and thus inactivate, glutamate, GABA,
aspartate and serotonin (127) at the synapse. Insulin decreases
levels of alpha2-adrenergic receptors in glia; this would tend to
regulate the amount of norepinephrine in the synaptic cleft (128).
Glia do not only take up neurotransmitters but also act on several
via specific enzymes such as glutamine synthetase and GABA
transaminase. In addition, they have specific receptors for alpha-
and beta-adrenergic agonists, dopamine, prostaglandin El, secretin,
somatostatin and vasoactive intestinal peptide (127), among others.
Lastly, glia have themselves been shown to possess some voltage-
gated channels and, thus, may act as excitable cells (129).
Because both neurons and glia are important for appropriate
development and function of the nervous system and because of the
afore-mentioned differences in neurons and glia with regard to both
the insulin receptor and PKC, we chose to study the interaction of





17


insulin and PKC in differential cultures of neurons and glia from the
same rat brains.






















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CHAPTER II
METHODS


Preparation of Primary Neuronal Cultures from Rat Brains
Brains from 8-10 one-day-old rats were removed in a sterile
manner from the cranium at the level of the medulla oblongata and
placed in an isotonic buffer solution containing 100 units penicillin,
100ltg streptomycin and 2.5jg Fungizone/ml. All pia mater and
blood vessels were removed and the brains were minced into 1-
2mm3 pieces. Brain cells were dissociated by trypsin and
deoxyribose treatment as described previously (18). Dissociated
cells were suspended in 50 ml of Dulbecco's modified Eagle's
medium (DMEM) containing 10 percent plasma-derived horse serum
(PDHS) and sedimented at 1000 x g for 10 minutes at 240C. The cell
pellet was suspended in the same medium at a concentration of 1.5 x
106 cells/ml. Fifteen million cells were placed in each Falcon
tissue culture dish pretreated with poly-L-lysine and the cells were
placed in a humidified incubator at 370C with 6 percent C02/94
percent air. The cells began to attach immediately and 90-96
percent were attached to culture dishes within 30-60 min. After
three days in culture, the cells were treated with 10.M cytosine








arabinoside in DMEM containing 10 percent PDHS. This treatment
resulted in the death of rapidly dividing cells. After 48 hours
cytosine arabinoside was removed and the cells were refed with 10
percent PDHS in DMEM and the cells were grown in culture for
another 7-10 days prior to use.
These cultures contain 80-85 percent neurons as demonstrated
by light microscopy and immunocytochemical markers (18, Figure 2-
1). Since insulin and insulin receptors were widely distributed
throughout the brain, in contrast with the localized distribution of
other neuropeptides and their receptors, cultures from the whole
brain were used. We considered growing the cells in a chemically
defined medium. However, since one of the essential components of
this defined medium is insulin in relatively high concentration, this
possibility was abandoned. It was felt that the presence of insulin
in the medium would complicate studies related to the expression
and action of insulin receptors. Thus, we proposed to continue to
culture cells in DMEM containing either PDHS or fetal bovine serum
(FBS). PDHS and FBS were purchased from Hyclone and Gibco
Laboratories, respectively, with an insulin concentration of 1-4
ng/ml. At this concentration, insulin did not cause either down-
regulation or other biological effects on insulin receptors of either
neurons or glia (130).


Preparation of Primary Astrocytic Glial Cultures from Rat Brains
The procedure for removing the brains and dissociating the cells
was the same as that described above. After the cells were
centrifuged, they were suspended in medium containing 10 percent








FBS and plated onto 100mm culture dishes at a density of 10 million
cells/dish. After three days the cells were refed with 10 percent
FBS in DMEM. After an additional three days, cultures were rinsed
once with an isotonic buffer and dissociated by treatment with
trypsin. The cells were centrifuged at 1000 x g for 10 min at room
temperature and the pellet was resuspended in DMEM containing 10
percent FBS. Five hundred thousand cells were placed on each
100mm culture dish and the cells were returned to the incubator
until they were confluent, at which time they were used. Phase
contrast microscopic examination revealed a confluent monolayer of
large flat cells by day 6 or 7 after transfer. These cells have
previously been demonstrated to be of glial origin (131). Greater
than 98 percent of these cells have been identified as astrocytic
glial cells. Neuronal cells did not survive the transfer (Figure 2-2).

Immunocvtochemistry of Neuron-Specific Enolase/Glial Fibrillarv
Acidic Protein
Either cultured neurons or glia were washed with PBS and the
cells were fixed in a solution of 4 percent paraformaldehyde and 10
percent picric acid in PBS (pH 7.4) for 30 minutes at 40C. Cultures
were again washed 3 times with PBS and permeabilized in 0.1
percent Triton-X 100, 5 percent low-fat, dry milk in PBS for 30
minutes at room temperature. After being washed again with PBS,
the cells were exposed to a polyclonal antibody (either 1:100
neuron-specific enolase or 1:20 glial fibrillary acidic protein)
diluted in 0.1 percent NaN3/5 percent non-fat dry milk in PBS for 24
hours at 4C. PBS was used to wash the excess primary antibodies








from the cultured cells and a rhodamine-linked, goat anti-rabbit Ig
was diluted 1:100 in 5 percent non-fat dry milk in PBS and applied
to the cells for 30 minutes at room temperature. Excess secondary
antibody was washed from the cultured cells with PBS and the cells
were photographed under fluorescent light at 400x magnification on
a Zeiss D-7082 Axiophot photomicroscope.




Neuronal Deoolarization
Medium was aspirated from 14 day old neuronal cultures and
replaced by 8 ml of various solutions: the control solution contained
140 mM NaCI and 1.2 mM MgSO4; the depolarizing solution contained
78 mM NaCI, 60 mM KCI, 2 mM CaCl2 and 1.2 mM MgSO4; a potassium
solution contained 80 mM NaCI, 60 mM KCI and 1.2 mM MgSO4; a
calcium solution contained 138 mM NaCI, 2 mM CaCl2 and 1.2 mM
MgSO4, and a veratridine solution contained 10 uM veratridine, 138
mM NaCI, 2 mM CaCl2 and 1.2 mM MgSO4. Cultures were incubated
for 30 minutes at 37C. The solutions were aspirated, lyophilized,
reconstituted in distilled water and subjected to radioimmunoassay
for insulin.


Insulin Radioimmunoassav
A 100 ul aliquot of a sample of human insulin standard (0-300
uU/ml) was mixed with 5000 cpm of [1251]-insulin. One hundred ul
of guinea pig anti-human insulin (Serono) was added and the samples
and standards were vortexed and incubated at room temperature for
2 hours. A secondary antibody (sheep anti-guinea pig Ig, Serono) was








added and the sample and standard tubes were vortexed and
incubated for a further 30 minutes at room temperature. Following
the incubation, all tubes were centrifuged at 2500 g for 30 minutes
at 4C. Supernatants were discarded. All tubes were swabbed dry of
supernatant and the radioactivity in the pellets was counted.
Samples were compared to a standard curve and the cpms were
expressed as international units of insulin/ml.


Characterization of Immunoorecipitable Insulin by HPLC
Neuronal cultures grown in 100mm culture dishes were washed
twice with a solution of 138mM NaCI, 1.2mM MgSO4 and 2mM CaC12
(pH 7.4) at room temperature to remove the growth medium. Eight
ml of a depolarizing solution (78mM NaCI, 1.2mM MgSO4, 2mM CaCl2
and 60mM KCI, adjusted to pH 7.4) were placed on each culture dish
and the dishes were incubated at 370C for 30 min. in a 94 percent
air/6 percent C02 incubator. After incubation the solution was
aspirated from the plates.
High-pressure liquid chromatography (HPLC) was used to
determine whether the released peptide had the same
chromatographic properties as commercial rat insulin (Eli Lilly).
Two buffers were prepared. Buffer A consisted of 0.1 percent
trifluoroacetic acid (TFA) and 0.1 percent triethylamine (TEA) in
water. Buffer B consisted of 0.1 percent TFA and 0.1 percent TEA in
acetonitrile. The sample was dissolved in Buffer A and injected
onto a BioRad HiPore C4 column. A buffer system of 1:9, A:B was
graduated to 5:5, A:B over the course of 60 min. The column was run
at room temperature with a flow rate of 2 ml/min. Fractions were








collected every 0.2 min. and absorbance was monitored at 210 and
280 nm. Fractions were subjected to radioimmunoassay (Serono) to
determine whether the fraction which bound the insulin antibody
corresponded to insulin chromatographically.


Labelling of Immunoreactive Insulin in Neuronal Cultures
Neuronal cultures grown in 100mm culture dishes were incubated
in leucine-free DMEM, 10 percent dialyzed PDHS and 50p.Ci [3H]-
leucine (146.5 Ci/mmole) for 24 hours at 370C. Medium was
aspirated from the culture dishes; cultures were washed 4 times
with PBS and used for experiments as described above. A 100 pl
aliquot of each solution was exposed to a guinea pig anti-human
insulin antibody (Serono) for 2 hours at room temperature. Sheep
anti guinea pig Ig was added to each sample for 30 minutes to
precipitate the primary antibody and all samples were centrifuged
at 2500g to pellet the antibody-antigen complexes. Supernatants
were decanted and the radioactivity in each pellet was counted
(Liquiscint, National Diagnostics, LKB1217 Rack Beta Counter).

Characterization of the Regulation of Neuronal Insulin Release by
Glucose
Neuronal cultures prepared in 100mm culture dishes were washed
twice with a control buffer [25mM NaHCO3, 1.2mM NaH2PO4, 122mM
NaCI, 1.2mM MgSO4 and 2.5mM CaCl2 (pH 7.4)] at room temperature
to remove media. Glucose buffers were prepared such that they
were isotonic to the control buffer (eg. 25mM NaHCO3, 5.5mM
glucose, 119mM NaCI, 2.5mM CaCl2 and 1.2mM MgSO4). Eight ml of








either the control or a glucose buffer were placed on each culture
dish. Cells were incubated at 370C for 30 min. in 6 percent CO2/94
percent air with 90 percent relative humidity. After the incubation
the buffers were aspirated from the dishes, lyophilized, and insulin
in the buffers was quantitated by radioimmunoassay.


2-Deoxv-D-Glucose (2-dGlc) Uptake
Medium was aspirated from the culture dishes and the cells were
washed three times with phosphate-buffered saline (PBS) at pH 7.4.
The cells were then incubated in PBS containing 1mM CaCl2, 0.5 mM
MgSO4 and 0.5 mM 2-dGIc (1 pCi/plate). After a five min. incubation
at 37C, the cells were washed three times with ice-cold PBS to
remove excess radioactivity. Cells were dissolved in 0.2 N NaOH;
they were then scraped from the culture dishes and radioactivity
was counted (Ecoscint, National Diagnostics, LKB 1217 Rack Beta
scintillation counter). The cpm were converted to dpm by the
counter (efficiency about 30 percent) and normalized for protein
values. The specific activity was then used to convert results to
nmoles of 2-dGlc/mg protein.


lodination of Insulin
One hundred ml of a pH 6.7 phosphate buffer was prepared from
55 ml of 0.3M KH2PO4 and 45 ml of 0.3M Na2HPO4. Chloramine T was
prepared as follows: the surface was scraped and chloramine T was
weighed and placed in a foil-covered tube; the salt was diluted to 4
mg/ml in the phosphate buffer and again diluted 1:100 (i.e. 25 l in
2.5 ml) in phosphate buffer just before use; all dilutions were in








foil-covered tubes as the compound is light-sensitive. One mg of
porcine insulin was weighed and dissolved in 2 ml of 0.01 N HCI.
Sodium metabisulfite was weighed and diluted similarly to the
chloramine T except that it was made to 8 mg/ml in the first
dilution such that the concentration after 1:100 dilution was 80
pg/ml. The decay chart for 1251 was checked to determine what
volume of 1251 contained 1 mCi. Fifty ml of a solution of 1 mg/ml
of insulin-free BSA (eg Sigma A-7030) in phosphate buffer was
prepared and about 20 ml was used to wash a sephadex-G25 column
(PD 10 column from Pharmacia). The following were added in
sequence to a disposable plastic test tube in a fume hood: 1) 25 pI
phosphate buffer, 2) 5 Ig insulin (10 I1), 3) 1 mCi 1251 and 4) 10 pl
chloramine T. The tube was capped, vortexed and contents were
incubated at room temperature for 5 min. Ten ul of sodium
metabisulfite was added to stop the reaction and the tube was
capped and vortexed once again. Two hundred Il of phosphate buffer
was added to increase the volume and the solution was added to the
top of the column. After the reaction mixture was absorbed by the
column, the column was washed with the BSA/phosphate buffer and
fractions were collected. [1251]-insulin usually eluted in about 3-5
ml. Ten pl of each fraction was removed and placed in a test tube,
capped and counted on a scintillation counter. To calculate percent
incorporation, 50 p. of 3 percent BSA and 1 ml of ice-cold 10
percent trichloroacetic acid were added to the test tube containing
the highest counts. Then the sample was incubated at 4C for 5 min.
and centrifuged at 1000 x g for 1 min. Radioactivity in the pellet
represented incorporated insulin. This should be greater than 90








percent. To calculate specific activity the number of cpm per ml
was determined. The counting efficiency was used to convert to
dpm/ml (75 percent for the LKB Rack Beta). Two million two
hundred thousand dpm/pCi was used to convert to .Curies. The
number of ig/ml was divided by the number of piCi/ml. Insulin
specific activity was generally about 40 I.Ci/g. Labelled insulin
was aliquoted into microfuge tubes, capped and stored at -700C until
use. The labelled material was tested for bioactivity by
displacement with a high concentration of unlabelled insulin (100
g.M) in a binding assay.


Insulin Binding
Medium was aspirated from culture dishes and the cells were
washed three times with PBS. Total binding was determined by
incubating triplicate dishes with a binding buffer (100mM Hepes,
30mM NaCI, 10mM glucose, 1mM CaCl2, 0.5 mM MgSO4, 0.1 percent
bovine serum albumin (BSA) at pH 7.4) containing [1251]-insulin
(100,000 cpm/plate). Non-specific binding was determined with a
similar buffer containing, in addition, 100p.M unlabelled insulin.
After a 1 hour incubation (2 hours for competition experiments) at
room temperature, cells were washed three times with ice-cold PBS
to remove excess, unbound insulin. The cells were then dissolved in
0.2 N NaOH and scraped from the culture dishes. Radioactivity was
counted on a Beckman Gamma 5500 counter. All values were
normalized for protein content and specific binding was determined
by subtracting non-specific binding from total binding.








Protein Determinations
One hundred to two hundred ul of a solution of protein in 0.1 N
NaOH was used for protein determinations. Bovine serum albumin
standards (10-100 ug of protein) were prepared in the same volume
of 0.1 N. NaOH. Samples and standards were made to 500 ul with
deionized, distilled water and protein determinations were made by
the method of Lowry (132).


Western Blot
Treated or untreated cultures of neurons and glia were washed 3
times with PBS, scraped from the culture dishes and centrifuged at
1000 x g for 5 min. to pellet cells. The supernatant was poured off
and the cells were resuspended in a homogenizing buffer consisting
of 20mM Tris HCI, 2mM EDTA, 0.5mM EGTA, 0.1mM PMSF and 1
percent 28-mercaptoethanol at pH 7.5. The suspension was
homogenized with 15 strokes of a glass homogenizer and centrifuged
at 1500 x g at 40C for 8 min. to remove nuclei and large particles.
The supernatant was recentrifuged at 100,000 x g at 2-40C for 30
min. to isolate cytosolic and membranous fractions. The membrane
was resuspended in homogenizing buffer containing 0.1 percent
Triton X-100. Samples of homogenates from whole cell were used
after the homogenization step. The protein content of samples was
determined and samples were made to a final concentration of 10mM
Tris base, 2 percent sodium dodecyl sulfate (SDS), 15 percent
sucrose, 0.002 percent bromophenol blue and 10 percent 28-
mercaptoethanol at pH 8.3. Samples were boiled for one min. and
stored at 40C before proteins were separated on discontinuous







polyacrylamide gels (5 percent stacking gel, 7.5 percent separating
gel, 30mA for 6 hours) and transferred to a nitrocellulose membrane
in a buffer consisting of 25mM Tris base, 150mM glycine and 20%
v/v methanol at pH 8.3 for three hours at 150mA. Total transfer
was demonstrated by transfer of prestained standards and by the
lack of a band as demonstrated by Coommassie staining of the gel
after the transfer. The nitrocellulose membrane was stored in 3
percent BSA in PBS overnight to decrease non-specific binding of the
antibody.
The nitrocellulose membrane was incubated with a monoclonal
antibody against protein kinase C (Amersham, diluted 1:100 in PBS
with 0.1 percent BSA) for 4 hours at room temperature. This
antibody recognized the alpha and beta subtypes (Types II and III) of
PKC, which are the majority of PKC in the brain (159). It was then
washed 4 times for 5 min. with 0.1% Tween-20 in PBS (pH 7.4) and
incubated with horseradish-peroxidase-linked anti-mouse Ig diluted
1:100 with 0.1 percent BSA/0.1 percent Tween-20 in PBS for 30 min.
at room temperature. The membrane was washed as before and
incubated in 0.03 percent hydrogen peroxide/0.5 mg/ml 3,3'-
diaminobenzidine (DAB) in PBS prepared immediately prior to use,
until bands appeared. It was then washed and allowed to air dry.
Bands were quantitated via densitometry. Immunological PKC will
be referred to as iPKC throughout this dissertation, while PKC
activity will be designated aPKC.








PKC Immunocvtochemistrv
Cells were grown on sterile glass coverslips in culture dishes.
Prior to staining, they were washed three times with PBS and fixed
in 3.5 percent paraformaldehyde/0.25 percent glutaraldehyde in PBS
(pH 7.4) on ice for 30 min. The cultured cells were then
permeabilized with 0.1 percent Triton X-100 in PBS for 30 min. at
room temperature. Following the fixing and permeabilizing steps,
the cells were rinsed three times with PBS and a 1:10 dilution of a
monoclonal anti-PKC antibody in 1 percent BSA, 0.1 percent sodium
azide in PBS was applied. After a 24 hour exposure to this antibody
at 40C and they were again rinsed with PBS. Control cells were
treated with the same solution without the primary antibody. A
1:100 dilution of an anti-mouse Ig-peroxidase conjugate in 0.1
percent BSA in PBS was applied for 30 min. at room temperature and
then the excess was removed by washing with PBS. Finally, the
cells were incubated in a solution of 0.5 mg/ml DAB/0.03 percent
hydrogen peroxide in PBS prepared immediately prior to use. After
10 min. they were washed and a drop of 9:1 glycerol:PBS was applied
to the coverslip. The coverslips were inverted, placed on a glass
microscope slide and the edges were sealed with nail polish.
Photographs were taken at 400 and 1000 x magnification with a
Zeiss D-7082 Axiophot photomicroscope.


Statistical Analysis
Statistical analysis was by analysis of variance (ANOVA)
followed by Duncan's post hoc test when the means of several groups
were to be compared or Dunnett's post hoc test when the means of





32


several groups were to be compared to that of one control group.
Significance was determined for p<0.05. Experiments whose results
were expressed as a percent of control were converted to arcsin
prior to ANOVA if all values were equal to or less than 100 percent
or to the log if any of the values exceeded 100 percent. The
particular test used was specified in the legend of each figure. The
one exception to these rules was Figure 3-1, for which a two-tailed
Wilcoxan Rank Sum nonparametric test was employed because of one
outlying value in the depolarized group.

























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CHAPTER III
INSULIN SYNTHESIS AND RELEASE FROM NEURONAL CULTURES

Introduction
Insulin alters the content of several neurotransmitters in the
brain (36-8), stimulates the release of others (39) and changes
neuronal electrical activity in specific regions of the brain (42-3).
Thus, it definitely acts as a neuromodulator. As insulin is suggested
to act via specific insulin receptors (44), it may act as a
neurotransmitter as well. In order to be classified as a
neurotransmitter, however, synthesis and release of insulin under
depolarizing conditions must be demonstrated in neurons.
Two separate bodies of evidence suggest that the insulin present
in the brain is also synthesized there: 1) preproinsulin mRNA is
present in brain (20-23) and 2) the level of insulin in the brain is
independent of the level of insulin in the periphery (19,23). Several
investigators have identified an mRNA in brain tissue that
hybridizes to a cDNA for insulin. Insulin mRNA is found in neurons,
but not in glial cells in cultures from the brains of both rats and
rabbits (22-3). Only 3-5 percent of cultured neurons from rabbits
contain the mRNA. This is in agreement with an earlier study








localizing insulin immunoreactivity to 3-5 percent of cultured
neurons from rat brain (18). The mRNA species is larger than that
observed in the human pancreas when the two are compared by
Northern blotting techniques (24). In situ hybridization studies
localize neurons containing the insulin mRNA to the periventricular
hypothalamus and cerebral cortex in rat brain (21) and rat, mouse
and hamster anterior pituitary cells (20). Other areas of the brain,
including the olfactory bulb and choroid plexus do not contain the an
mRNA for insulin (21). In the anterior pituitary, only 5-10 percent
of the cells are positive for the mRNA; those cells are epithelial
and the immunoreactive insulin that they contain is localized in
secretary granules (20).
The second body of evidence relates to the independent regulation
of brain and peripheral insulin levels. Many investigators have
reported brain insulin concentrations that are higher than those
observed in plasma (14,23). It is unlikely that this represents
sequestration and concentration of plasma insulin for two reasons.
First, brain insulin concentrations are not altered by disease states
which raise or lower plasma insulin concentrations, although the
concentration in CSF is lowered in response to lowered plasma
insulin. Secondly, the capillaries of the blood-brain barrier do not
transport active insulin into the brain.
As discussed previously, insulin acts in the brain to alter
neuronal electrical activity. This, in combination with the evidence
that insulin may be synthesized in the brain, led us to investigate
whether pulse-labelled immunoreactive insulin could be synthesized
in the brain and whether this immunoreactive insulin could be








released under depolarizing conditions. The chromatographic
properties of the released immunoreactive insulin were then
compared to those of an insulin purified from rat pancreas by HPLC.





RESULTS
In this study, primary neuronal cultures from rat brains were
treated with depolarizing solutions containing a high concentration
of potassium (60mM) with or without calcium (2mM). After 30 min.
the solutions were aspirated and lyophilized and insulin was
quantitated by radioimmunoassay. Depolarized neuronal cultures
released more than three times as much insulin as saline controls
(103.21U/ml vs 31.7pU/ml) in the presence of calcium. Potassium-
stimulated release was calcium-dependent as in the absence of
calcium, insulin release was negligible (34.0iU/ml, Figure 3-1).
The sodium ionophore, veratridine, similarly stimulated the release
of insulin from neurons by 379 percent. In contrast, release of
insulin from glial cultures was not stimulated by depolarization.
This, in combination with the evidence that no mRNA for insulin has
been demonstrated in glial cells and that glial cells are not
immunoreactive for insulin (18), suggested that the insulin released
from neuronal cultures was not due to glial contamination of those
cultures. The very low level of insulin released by glial cells may,
in fact, have represented insulin taken up from the medium. The
immunoprecipitable insulin that was released from neurons under
depolarizing conditions coeluted with an insulin purified from rat








pancreas (supplied by Eli Lilly) when subjected to reverse phase
HPLC. Both the released material and the standard showed peaks for
both rat 1 insulin and rat 2 insulin (Figure 3-2).
When primary neuronal cultures were treated with D-glucose
(0.1-0.6 percent) in the presence of 2mM calcium, a dose-dependent
stimulation of insulin release was observed (Figure 3-3). Neuronal
cultures were exposed to [3H]-leucine for 24 hours prior to a timed-
release experiment. These cultures were washed and incubated at
37C with control and depolarizing solutions which were aspirated
from the cultures at time intervals from 1 to 60 min. A biphasic
pattern of insulin release was observed (Figure 3-4); this pattern of
release was similar to that observed with stimulation of pancreatic
insulin release by glucose. The fact that exogenous [3H]-leucine was
incorporated into the immunoreactive insulin suggested that this
insulin was synthesized within the neurons.


Discussion
There is some controversy as to the origin of insulin in the brain.
Some investigators have shown that the concentration of insulin in
the brain is a fraction of that found in the plasma when quantitated
by different antibodies than the one first used by Havrankova, et al.
(16, 14). These same investigators have also found that the
concentration of insulin in the CSF is dependent on the plasma
concentration. They have suggested that these two pieces of
information, in conjunction with the lack of a demonstrable
proinsulin in the brain and the inability of one investigator to
demonstrate an mRNA for insulin in brain prove that insulin is not








synthesized in the brain (16). They propose that insulin passes from
the plasma into the CSF and is retained in brain to the extent that it
binds to local insulin receptors. They further propose that this
accounts for insulin immunocytochemistry in the brain.
Both an mRNA for insulin and concentrations of insulin which are
independent of those in the plasma and CSF have been demonstrated
in the brain in several other studies, as described previously. The
results of this study suggest that insulin is synthesized in neurons
from the brain and released under depolarizing conditions. The
insulin in the brain has immunological and chromatographic
properties that resemble those of pancreatic insulin. While this
evidence suggests that the material is, in fact, insulin, it is not
definitive proof. Rat proinsulin is not commercially available and
would be likely to have similar properties. Sequencing with an
amino acid analyzer would be the most appropriate method of
identifying the peptide, but it requires a larger sample of the
material than is available.
Depolarized release was measured using the solutions described
in methods in order to duplicate the methods used by Yalow to study
cholecystokinin release from synaptosomes. These unbuffered
solutions had an acidic pH after 30 min. This low pH may have
altered the release and/or the viability of the cells, although the
cells were still attached to the culture dish and appeared normal
under the microscope. Acidity was observed in all groups, but
onlythe depolarized groups showed increased release of insulin,
suggesting that it was the depolarization, and not the acidity that
induced release. In any case, glucose-induced release was measured








in a Krebs buffered solution, in order to eliminate this problem.
Insulin degradation in the release solutions was probably not
significant as degradation of insulin in binding studies, in which the
insulin is exposed to the cells for an hour or more, is generally less
than 10 percent.
It is likely that at least some of the insulin released from
neuronal cultures is also synthesized there: The cultured cells have
been removed from any peripheral insulin for at least 10 days prior
to use; the serum in which the cells are grown is plasma-derived
horse serum, which contains only one insulin, not the two observed
in the rat, and exogenous leucine is incorporated into the
immunoprecipitable insulin. The inability of some researchers to
demonstrate synthesis of insulin in the brain may be related either
to the small percentage of cells producing insulin or to their very
specific localization. In addition, insulin acts to promote survival
of brain cells in culture (115) and, thus, insulin-producing neurons
may survive preferentially in culture and may represent a larger
percentage of neurons than are present in whole brain. That
immunocytochemical evidence localizing insulin to neurons is the
result of insulin bound to surface receptors, is unlikely as glial
cells have specific, high-affinity insulin receptors as well (28) The
evidence presented here that insulin is synthesized in neurons from
the brain and is released under depolarizing conditions, in addition
to the evidence that insulin in the brain binds to specific, high-
affinity receptors and has electrical and physiological effects in the
brain suggest that insulin is a neurotransmitter in the brain.





















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CHAPTER IV
CHARACTERIZATION OF PKC IN NEURONAL AND GLIAL CELLS IN
PRIMARY CULTURES


Introduction
PKC is present in both neurons and glial cells from the brain
(55,110-11). It is present in dendrites, axons, perikarya and nuclei
(110) of neurons with particularly high concentrations in
presynaptic terminals (111) and in growth cones (114). The last two
are predictable, as PKC mediates both neurotransmission and neurite
outgrowth (115). In glial cells, PKC is involved in the regulation of
differentiation (78) and membrane conductance (80). Thus, PKC is
also physiologically active in both types of cells, although its
activity takes different forms in neurons and glial cells.
Because of these differences and the existence of different
isotypes of PKC we elected to study neurons and glia in culture to
determine the relative quantities of PKC, the relative molecular
weight (Mr) of PKC and the distribution of PKC within the cells. As
TPA-activation of PKC involves translocation of the enzyme, we
chose to observe the time-course of the translocation to determine
whether this differed in the different types of cells.








Results
Immunoreactive PKC was present in both neurons and glia.
Immunocytochemistry showed neurons staining darker than glial
cells in the same cultures with the stain distributed throughout both
types of cells. In glial cells the staining was unevenly distributed
with particularly dark staining in nuclear and perinuclear areas. In
neurons, both perikarya and processes stained. No further
differences in staining could be seen in neurons at this
magnification. No unstained cells were observed in either the
neuronal or glial cultures (Figure 4-1,2). Control cells, which
received the same treatment except for the primary antibody, had no
staining whatsoever. The concentration of iPKC was 4.60.5 (mean
standard error) times higher in neuronal than in glial cultures from
the same brains when measured by Western blot and densitometry
(Figure 4-3) of whole cell homogenates. Immunoactive PKC in both
neurons and glia had a relative molecular weight of about 80 kD as
determined by polyacrylamide gel electrophoresis followed by
Western blotting and comparison with commercial molecular weight
standards.
The cytosol contained 63 9 percent of the iPKC in glial cells
(Figure 4-4). In contrast, the neuronal iPKC resided primarily in the
membrane with only 12 2.3 percent in the cytosolic fraction when
quantitated by Western blot and densitometry (Figure 4-5).
Treatment of both neurons and glia with 100 nM TPA induced a
time-dependent translocation of iPKC from the cytosolic fraction.
In glial cells treatment with TPA decreased the cytosolic level of
iPKC to 33 7 percent of the basal level within 5 min. Within 15








min. the cytosolic iPKC was barely detectable by Western blot (6 4
percent of the control level). At 24 hours after treatment with
TPA, the concentration of iPKC in the cytosol remained low. In
contrast, the iPKC in glial membranes was increased to 150 39
percent of the control within 5 min. of the administration of TPA,
after which it declined. Within 24 hours the membrane-bound iPKC
had downregulated such that it was barely detectable as well
(Figures 4-4,6).
Treatment with 100 nM TPA induced a downregulation of iPKC in
neuronal cells. The cytosolic iPKC decreased to 59 percent of the
control concentration within 1 hour, to 16 percent within 2 hours
and did not increase again over the course of a 24 hour treatment
with TPA. The level of iPKC bound to the membrane did not change
within 15 min. after treatment with TPA but decreased to 54
percent within 1 hour and continued to decline such that the iPKC
was only 17 percent of the control iPKC concentration after 24
hours (Figures 4-5,7).


Discussion
An iPKC of 80 kD is present in both neurons and glia, although
neurons express several times the cconcentration of iPKC as that
seen in glia. This is seen with both the immunocytochemistry and
the Western blot experiments. This is in agreement with studies
showing that the binding of phorbol esters is 2-3 times higher in
cultured neurons than in glial cells from the same rat brains (113).
No differences with regard to immunostaining of different regions
of the neurons were observed. As the vast majority of the neuronal








enzyme is associated with the membranous fraction the membrane
staining is very dark and differences within the cell cannot be seen.
In contrast, in glia, the majority of the iPKC is cytoplasmic. This
was not unexpected, as an earlier study reported that whereas glial
PKC was predominantly cytosolic, PKC from homogenized whole
brain tissue was predominantly membrane-bound (116). The nuclei
in glial cells appear to stain darkly for iPKC, but nuclear PKC would
not contribute to either the cytosolic or membranous fractions as
the nuclei were removed in a centrifugation step prior to the
separation of membrane and cytosol. The aPKC in the membrane-
bound fraction is latent in the liver (52,133). The concentration of
PKC associated with neuronal cytosol was, in fact, low enough to be
the result of glial cell contamination. Neuronal cultures contain 15-
20 percent glial cells, as described in methods.
Complementary studies on neuronal and glial aPKC demonstrate
that glial cells have far greater aPKC than neurons (134). The
different results obtained by immunological and bioassay suggest
that either our antibody does not recognize a large percentage of
glial aPKC or that a large percentage of the neuronal iPKC is
inactive. The first could result from the presence of different
isozymes of PKC in different types of cells, one or several of which
are not recognized by an antibody directed against the alpha and beta
(beta-1 and -2) forms of the enzyme, although these Type II and III
subtypes of PKC do represent the majority of PKC in the brain, there
is another subtype, Type I, which is found only in the brain, and
which is not recognized by our antibody (159). The existence of this
subtype, which is not immunoactive could lead to a false negative








result in studies in which iPKC was measured. In the brain,
different subspecies of PKC have been identified at different stages
of development, as discussed previously (54,57). This cannot
explain the disparity between the immunoreactive levels and enzyme
activities in neurons and glia, as experiments were done on cultures
of the same age. The increase in neuronal aPKC when stimulated
was much smaller than the increase in glial activity under the same
circumstances. This suggests that a subset of the neuronal PKC
enzymes may be physiologically inactive or less active than their
glial counterparts. The second situation could result from inactivity
of the neuronal, predominantly membrane-bound iPKC. As discussed
previously, there is evidence to support either of these hypotheses;
that is, neurons and glial cells express different isozymes of PKC
and the membrane-bound enzyme may be latent. TPA stimulates
translocation of the glial aPKC over a similar time-course and to a
similar extent as that observed immunologically. This is similar to
the situation in the periphery, in which PKC is primarily localized in
the soluble fraction and is translocated to the membrane in a time-
dependent fashion when stimulated (48). It is unlikely that the
enzyme is translocated during the process by which the cells are
prepared for electrophoresis as they are washed extensively to
remove TPA and the procedure is carried out in the presence of
calcium chelators, which prevent endogenous stimulation of
translocation as well as inhibiting proteases.
Other investigators have found that only 25-35% of the PKC in
whole brain is in the soluble fraction (51,116). It is likely that this
is due to the presence of the predominantly membrane-bound enzyme





56


in neurons. Our failure to observe a consistent increase in the
membranous fraction at 5-15 min. after TPA stimulation may be due
to the very small percentage of cytosolic iPKC available to be
translocated. It may be that the change in the concentration of iPKC
in the membrane is too small to be observed consistently.























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CHAPTER V
THE REGULATION OF SUGAR TRANSPORT IN PRIMARY NEURONAL AND
GUAL CELL CULTURES BY PHORBOL ESTERS


Introduction
Insulin has been shown to stimulate sugar uptake in astrocytic
glial cells from rat brain. This effect was time- and dose-
dependent with a maximal stimulation observed at 18 nM and a half-
maximal effect at 0.1 nM insulin (28). The latter value has been
shown to be well within the concentrations found in the brain (15)
indicating that the endogenous insulin is sufficient to induce this
stimulatory effect. This effect was unique to glial cells as neurons
prepared from the same brains failed to express similar properties.
Because of these and other observations it has been proposed that
insulin's lack of effect on sugar uptake in neurons may be due to the
absence of an intracellular pool of glucose transporters and/or an
inability of insulin to translocate intracellular transporters or to
activate membrane-bound transporters, or to a combination of all
three of these.
In this study we utilized TPA to determine its effects on
neuronal and glial glucose uptake as TPA has been shown to
stimulate glucose uptake in other tissues (97, 117-9). The








activation of PKC will provide us with an additional parameter to
study the differences in the regulation of neuronal and glial glucose
uptake.


Results
TPA stimulated [3H]2-dGlc uptake in glial cells in a time-
dependent manner. Glucose uptake was increased as early as 20 min.
after the administration of TPA with maximal increases occurring
after 4 hours of treatment with 100 nM TPA. The maximal level of
increase was 204.5 12.5 percent (Figure 5-1). In contrast, TPA
failed to influence 2-dGlc uptake in neuronal cultures under similar
conditions. The stimulatory effect of TPA on glial glucose uptake
was selective and was due to an increase in the number of glucose
transporters rather than a change in the Km of the transporter (119).


Discussion
The differences in PKC-stimulated glucose uptake between
neurons and glial cells may be due to the different isozymes of PKC
present in the two types of cells, or to the presence of a smaller
pool of PKC available to be translocated in neurons. In other related
work in this area, our group found that phorbol ester-induced
stimulation of glucose uptake in glial cells is also concentration-
dependent with a maximal effect at 100 nM TPA. It is likely that
TPA is acting through PKC as the potency of five phorbol esters
paralleled their abilities to bind and activate PKC (119). However,
PKC has many effects on cells within the brain; even if these
effects occur by way of activastion of PKC, they may be indirect





73


effects that occur as a result of PKC's other actions in the brain,
such as neurotransmitter release (75-77), or alterations in
neuronal ion channels (69-710 or membrane conductance in
astrocytes (80)




















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CHAPTER VI
THE REGULATION OF INSULIN RECEPTORS IN NEURONAL AND GLIAL
PRIMARY CULTURES BY PHORBOL ESTERS


Introduction
Neuronal and glial insulin receptors are structurally and
physiologically distinct in several respects: neurons have a receptor
of lower molecular weight (29,32); insulin stimulates the uptake of
glucose in glia but not in neurons (28), and insulin inhibits the
uptake of norepinephrine in neurons, but not in glia (30). In addition,
there are distinctions between neurons and glia with regard to PKC:
PKC is present in higher concentration in neurons than in glia from
the same brains as demonstrated immunologically and by binding
studies with phorbol esters (113) although the activity is higher in
glial cells; different isotypes of PKC are present in neurons and glia
(55,110,111), and phorbol esters stimulate glucose uptake in glia,
but not in neurons (119). As neurons and glia differ with respect to
the physiological activities of both insulin receptors and PKC and
because PKC regulates the binding, autophosphorylation, tyrosine
kinase activity and some cellular responses of the insulin receptor
peripherally (81-3,87,91,95-7), we chose to investigate the role of
PKC in the regulation of the insulin receptor in neurons and glia from








the central nervous system to determine whether PKC might
differentially regulate these two receptors.


Results
TPA induced a dose-dependent increase in the binding of insulin
in glial cells with no effect on binding in neurons over the same
range of concentrations (Figure 6-1). TPA treatment of glial cells
for two hours did not alter the binding of insulin at a concentration
of 1 nM TPA, began to increase binding at 10 nM TPA and caused a
maximal increase at a dose of 50 nM TPA. The ED50 was 15 nM and
50 nM caused a maximal increase of 77 percent. The effect of TPA
on the binding of insulin was time-dependent as well (Figure 6-2).
Treatment with 100 nM TPA induced an increase in the binding of
insulin in glial cells within 30 min. with a maximal increase at two
hours, followed by a decline. The amount of bound insulin stabilized
at four hours after treatment with TPA. Translocation of PKC in
glial cells from the cytosol to the membrane began within 5 min. and
was virtually complete within 15 min. The effects of TPA on insulin
binding in glial cells and 2-dGlc uptake followed TPA-stimulated
translocation of PKC and, thus, PKC may be involved in TPA's effect
on insulin binding (Figure 6-3). Treatment of neurons with 10 and
100 nM doses of TPA had no effect on the binding of insulin (Figure
6-1).
Glial cells were treated with 100 nM TPA for 2 hours. Following
this treatment, competitive inhibition experiments for untreated
and TPA-treated glial cultures were conducted by adding a constant
amount of [1251]insulin (100,000 cpm/dish) and increasing amounts








of unlabelled insulin (0.167 nM-133 nM) to binding buffers. An IC50
of 40 nM insulin was observed for both curves (Figure 6-4). The data
were used for Scatchard analysis. The Kds for the high and low
affinity insulin receptors were 18.5 and 131.6 nM, respectively, in
control cells. Treatment with TPA induced a 184 percent increase in
the number of high affinity binding sites on glial cells without
altering their affinity. TPA increased the number of low affinity
sites by 74 percent while decreasing the affinity of those sites only
minimally (Table 6-1). Glial cells were exposed to three different
phorbol esters for 2 hours to evaluate the specificity of the
stimulation of insulin binding by TPA. TPA stimulated the binding of
insulin by 109 percent at a concentration of 50 nM. The same dose
which caused maximal stimulation by TPA had no effect when cells
were treated with 48-phorbol 128,13a-didecanoate (B-PDD). B-PDD
exhibited an increase of 82 percent only at a concentration of 500
nM while 4a-phorbol 12B,13a-didecanoate (a-PDD) had no effect at
any concentration (Figure 6-5). Thus, the potencies of these drugs
corresponded to their abilities to bind and activate PKC, that is, TPA
> B-PDD > the inactive analog, a-PDD (147). Pretreatment of glial
cultures for two hours with 100 nM TPA followed by its removal
resulted in a time-dependent recovery of insulin binding. The
binding recovered by 59 percent within 2 hours. Recovery was
complete within 6 hours (Figure 6-6).
The next question concerned the TPA-stimulated increase in the
number of glial insulin receptors and whether it corresponded to an
increase in responsiveness to insulin. Insulin and/or TPA was added
directly to the glial medium and the dishes were returned to the








incubator for the appropriate preincubation time prior to
quantitation of 2-dGlc uptake. Insulin induced a 34 percent increase
in glial 2-dGlc uptake when cells were treated with a dose of 167
nM for 15 min. Treatment of cells for 2 hours with 100 nM TPA
stimulated 2-dGlc uptake by 112 percent. However, TPA eliminated
the response to insulin. Administration of insulin in combination
with TPA resulted in a stimulation of 144 percent (Figure 6-7).
Thus, the binding of insulin increased, but the response to insulin did
not when glial cells were treated with TPA.


Discussion
TPA stimulates the binding of insulin in glial cells from the brain
without altering neuronal binding of insulin. This stimulation in glia
occurs without a parallel increase in the responsiveness to insulin,
as demonstrated by the lack of effect of TPA on insulin stimulated
2-dGlc uptake. Thus, it represents an increase in the number of
binding sites, not necessarily an increase in the number of
receptors. The potency of the phorbol esters in effecting a
stimulation of glial insulin binding corresponds to their respective
abilities to activate PKC (147), suggesting that TPA is acting
through PKC. As with TPA stimulation of glial glucose uptake,
however, caution must be used in translating an effect of phorbol
esters to an effect of PKC. Again also, the effect may well be an
indirect effect as PKC has other effects in the brain, as described in
the introduction. TPA's effects on glial insulin binding occur more
slowly than PKC translocation, but over the same range of doses.
Thus, translocation of PKC may be involved in TPA's stimulation of








insulin binding. This is to be expected as translocation precedes and
is necessary for other PKC-induced effects, both centrally and
peripherally (61-4). This increase in the binding of insulin could
also be interpreted as an increase in ionternalization of insulin.
Further studies using an acid wash to separate bound versus
internalized insulin will be necessary to resolve this question.
As was demonstrated, treatment with TPA for 24 hours
downregulates PKC in the brain. As the binding of insulin in glial
cells recovered to the level of the control, but not below, 24 hours
after TPA administration and removal, it appears that the binding of
insulin in glial cells is not under chronic control by PKC.
PKC-induced inactivation of receptors has generally been
associated with a decrease in binding of the appropriate ligand in
studies in peripheral tissues. This example of differential
regulation of brain and peripheral insulin receptors as well as
distinct regulation of insulin receptors in neuronal and glial cells is
by no means unprecedented. Insulin, which generally downregulates
its receptor peripherally, downregulates its receptor in glia but
upregulates the receptor in neurons (148). Glucocorticoids, which
increase the binding of insulin in hepatocytes (149) and
lymphocytes(150), have no effect in adipocytes (151) and decrease
binding in an astrocytic cell line (152). As insulin has different
actions in different types of cells, it is to be expected that
regulation of insulin receptors might vary among cell and tissue
types.
TPA induces insensitivity to insulin insofar as glial 2-dGlc
uptake is concerned. This is true even at doses of TPA which elicit a








less than maximal increase in binding. This suggests that these
glial insulin receptors, like their peripheral counterparts, may be
inactivated by PKC. Inactivation of tyrosine kinase prevents
internalization of insulin receptors in Chinese hamster ovary cells
(93). A similar proposal could be made for the glial insulin receptor.
The most intriguing aspect of this study concerned TPA's lack of
effect on neuronal insulin binding, although this does not necessarily
indicate that PKC does not inactivate the receptors. Both neurons
and glia contain a PKC which is capable of binding phorbol esters,
and neurons and glia both respond physiologically when stimulated
by phorbol esters (69-80). PKC is certainly involved in neuronal
receptor regulation as phorbol esters induce an increase in the
binding of angiotensin II by a calcium-dependent mechanism (153).
Thus, TPA's failure to alter the binding of insulin in neurons can
neither be attributed to a lack of PKC, nor to a non-functional PKC.
As PKC's effects on the insulin receptor are direct effects (90), the
different effects of TPA on the binding of insulin must necessarily
be due to differences in neuronal and glial PKC or to structural
differences within the insulin receptors themselves.


























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Control


High Affinity
Receptor number
Ka


Low Affinity
Receptor number
Ka


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Full Text
135
144. Kayano, T., Fukomoto, H.( Eddy, R., Fan, Y., Byers, M., Shows, T. and
Bell,G. (1988) Evidence for a family of glucose transporter-like [
proteins: Sequence and gene localization of a protein expressed in fetal
skeletal muscle and other tissues. J. Biol. Chem. 263:15245.
145. James, D., Brown, R,, Navarro, J. and Pilch, P. (1988) Insulin-
regulatabie tissues express a unique insulin-sensitive glucose transport
protein. Nature 333:183.
146. Oka, Y., Asano, T., Shibasaki, Y., Kasuga, M., Kanazawa, Y. and Tanaku,
F. (1988) Studies with anti-peptide antibody suggest the presence of at
least two types of glucose transporter in rat brain and adipocyte, J. Biol.
Chem. 263:13432.
147. Leach, K., James, M. and Blumberg, P. (1983) Characterization of a
specific phorbot ester aporeceptor in mouse brain cytosol. Proc. Natl.
Acad. Sci. USA 80:4208.
148. Raizada, M., Boyd, F., Clarke, D. and LeRoith, D. (1987) Physiologically
unique insulin receptors on neuronal cells. In: Insulin. Insulin-like Growth
Factors and Their Receptors in the Central Nervous System. Raizada,
M., Phillips, M. and LeRoith, D. Plenum Press, New York.
149. Salhanick, A., Krupp, M. and Amatruda, J. (1983) Dexamethasone
stimulates insulin receptor synthesis and release in cultured rat
hepatocytes. J. Biol. Chem. 258:14130.
150. Fantus, I., Saviolakis, G., Hedo, J. and Gorden, P. (1982) Mechanism of
glucocorticoid-induced increase in insulin receptors in cultured human
lymphocytes. J. Biol. Chem. 257:8277.
151. Olefsky, J. Johnson, J., Liu, F., Jen, P. and Reaven, G. (1975) The effects
of acute and chronic dexamethasone administration on insulin binding to
isolated rat hepatocytes and adipocytes. Metab. 24:517.
152. Montiel, F. Ortiz-Caro, J., Villa, A., Pascual, A. and Aranda, A. (1987)
Glucocorticoid regulation of insulin binding in a rat glial cell line. Endocr.
121:258.
153. Sumners, C., Rueth, S., Myers, L, Kalberg, C., Crews, F. and Raizada, M.
(1988) Phorboi ester-induced upregulation of angiotensin II receptors in
neuronal cultures is potentiated by a calcium ionophore. J. Neurochem.
51:1.
154. Nishizuka, Y. (1984) Turnover of inositol phospholipids and signal
transduction. Science 225:1365.
155. Berridge, M. (1981) Phosphotidylinositol hydrolysis: a multifunctional
transducing mechanism. Melar Cell. Endocr. 24:115.


Figure 7-5: The effect of dexamethasone on glial iPKC.
Dexamethasone (2.55 uM) was added to the medium of
confluent glial cultures and the cells were returned to
the incubator for the times specified. The cells were
then Western blotted for PKC as described in methods.
Densitometric readings are (from left to right) 6007,
6167, 6219, 6195, 8286 and 6536. The experiment was
repeated once.


lean rats with TPA to down-regulate PKC (98). The same is not true
for all tissues, however, as phorbol esters have only minimal
effects on insulin sensitivity in rat skeletal muscle (99). The
interaction of PKC and the insulin receptor is somewhat complicated
by reports of synergism in mitotic stimulation (100). In addition,
there are proteins which have phosphorylation sites for both PKC and
receptor kinases (101) and are stimulated by both types of mitogens
(102). This last effect would account for the paradoxical way in
which PKC both inhibits the insulin receptor and mimics many of
insulins effects within the cell.
Effects of Growth Factors on PKC
While the majority of studies on PKC/growth factor interactions
in the literature focus on regulation of growth factor receptors by
PKC, those receptors frequently regulate PKC as well. Treatment
with insulin in the presence of glucose has been shown to increase
both the binding capacity of PKC and enzymatic activity in
adipocytes (103). As the insulin effect is eliminated in the presence
of high glucose, the effect may be secondary to increased glucose
uptake in the insulin-treated cells. Insulin also increases the level
of cytosolic calcium ion in adipocytes (104), which could account
for the increased PKC activity. Studies also demonstrate
enhancement of PKC activity in myocytes and mammary tumor cells
by insulin (105-6). In the former the increase occurs in both the
cytosolic and membrane fractions and is not inhibited by
cycloheximide. This increase in the activity of PKC is reportedly
mediated via increased DAG generated by phospholipid hydrolysis and


34


4
receptor is generally similar in subunit structure to the more
familiar peripheral insulin receptors although the alpha-subunit is
somewhat smaller in the brain (31) due to decreased glycosylation
of the receptor in the brain. In particular, the neuronal alpha- and
beta-subunits are smaller, while the glial subunits resemble those
from the liver (29,32) in size. That the glial receptor is
structurally similar to the peripheral receptor is not surprising,
since insulin appears to have traditional metabolic effects in glia
and neuromoduiatory effects in neurons. Insulin stimulates glucose
uptake by glia, but not by neurons (28) and inhibits norepinephrine
uptake by neurons, but not by glia (30).
Insulin .in the Brain: Potential Function?
Insulin in the central nervous system (CNS) has been implicated
in the control of brain growth and development (28,33-5),
catecholamine release (36-9), and diabetes (40) in in vitro studies
and satiety (41) in in vivo studies. In addition, insulin may itself
act as a neurotransmitter in the CNS. In vitro studies have been
essential in developing an understanding of insulins actions in the
CNS at the cellular level. With regard to insulin's role in growth and
development, insulin stimulates DNA, RNA and protein synthesis in
mixed brain cell cultures and astrocyte glial cultures. It also
stimulates 2-deoxy-D-giucose uptake in astrocyte glial cultures
(28,33). These are all events which are associated with cell growth
and proliferation. In addition, insulin has differentiative effects on
developing rat brain. When immature rat retinal cultures were
exposed to insulin, precocious synaptic release of acetylcholine was


55
result in studies in which iPKC was measured. In the brain,
different subspecies of PKC have been identified at different stages
of development, as discussed previously (54,57). This cannot
explain the disparity between the immunoreactive levels and enzyme
activities in neurons and glia, as experiments were done on cultures
of the same age. The increase in neuronal aPKC when stimulated
was much smaller than the increase in glial activity under the same
circumstances. This suggests that a subset of the neuronal PKC
enzymes may be physiologically inactive or less active than their
glial counterparts. The second situation could result from inactivity
of the neuronal, predominantly membrane-bound iPKC. As discussed
previously, there is evidence to support either of these hypotheses;
that is, neurons and glial cells express different isozymes of PKC
and the membrane-bound enzyme may be latent. TPA stimulates
translocation of the glial aPKC over a similar time-course and to a
similar extent as that observed immunologically. This is similar to
the situation in the periphery, in which PKC is primarily localized in
the soluble fraction and is translocated to the membrane in a time-
dependent fashion when stimulated (48). It is unlikely that the
enzyme is translocated during the process by which the cells are
prepared for electrophoresis as they are washed extensively to
remove TPA and the procedure is carried out in the presence of
calcium chelators, which prevent endogenous stimulation of
translocation as well as inhibiting proteases.
Other investigators have found that only 25-35% of the PKC in
whole brain is in the soluble fraction (51,116). It is likely that this
is due to the presence of the predominantly membrane-bound enzyme


80kD
Control Insulin
106


Insulin
r
T

1
log nM


Figure 4-7: TPA-induced PKC redistribution in neurons.
One hundred nM TPA was added to the neuronal medium
for the times specified and samples were separated into
cytosolic and membrane fractions and subjected to
Western blotting. The densitometric values were divided
by the values for the control samples. Each bar
represents the mean of at least three experiments SEM.
Means of treated groups were compared to that of the
control group by two-way analysis of variance, followed
by by Dunnett's post hoc test (p<0.05).


28
percent. To calculate specific activity the number of cpm per ml
was determined. The counting efficiency was used to convert to
dpm/ml (75 percent for the LKB Rack Beta). Two million two
hundred thousand dpm/jiCi was used to convert to jiCunes. The
number of pg/ml was divided by the number of |iCi/ml. Insulin
specific activity was generally about 40 (iCi/pg. Labelled insulin
was aliquoted into microfuge tubes, capped and stored at -70C until
use. The labelled material was tested for bioactivity by
displacement with a high concentration of unlabelled insulin (100
jiM) in a binding assay.
Insulin Binding
Medium was aspirated from culture dishes and the cells were
washed three times with PBS. Total binding was determined by
incubating triplicate dishes with a binding buffer (100mM Hepes,
30mM NaCI, 10mM glucose, 1mM CaCl2, 0.5 mM MgS04, 0.1 percent
bovine serum albumin (BSA) at pH 7.4) containing [125|]-jnsulin
(100,000 cpm/plate). Non-specific binding was determined with a
similar buffer containing, in addition, 100pM unlabelled insulin.
After a 1 hour incubation (2 hours for competition experiments) at
room temperature, cells were washed three times with ice-cold PBS
to remove excess, unbound insulin. The cells were then dissolved in
0.2 N NaOH and scraped from the culture dishes. Radioactivity was
counted on a Beckman Gamma 5500 counter. All values were
normalized for protein content and specific binding was determined
by subtracting non-specific binding from total binding.


14
binding of phorbol esters (113); different isotypes of PKC are
present in neurons and glia (55,110,111) and phorbol esters
stimulate glucose uptake in glia but not in neurons (119).
The joint observations that neurons and glia differ with respect
to the physiological activities of the insulin receptor and PKC and
that phorbol esters regulate the insulin receptor and glucose uptake
peripherally, led us to investigate the role of PKC in the regulation
of insulin receptors in neurons and glia from the central nervous
system. Because activation of PKC by TPA has been shown to involve
translocation of PKC from the cytosolic to the membranous fraction
in many types of cells, we chose to examine TPA's effects on
neurons and glia to determine the relative concentrations of
immunoactive PKC in neurons and glia, whether TPA stimulates the
translocation of PKC in both neurons and glia, and whether the
translocation in glia, if it occurs, precedes TPA's effects on glucose
uptake and the insulin receptor and, thus, might be involved in a
mechanism. Differences in the ability of TPA to induce
translocation or the time-course of the translocation might
presumably explain TPA's differential effects on neurons and glia.
Lastly, in order to determine whether insulin regulates its own
receptor by this pathway, we chose to study insulins effects on PKC
in neurons and glia from the brain.
Cultured Brain Cells; a Model for the Study of Neurotrophic/
Neureactiye., Substances
The importance of neurons in brain function is unquestioned.
Proper neuronal growth, development and maintenance are essential


ZOV


6
diabetes. Lesions of the ventromedial hypothalamus cause
hypersecretion of hypothalamic insulin (46).
In studies involving cultures of brain cells from diabetic mice
the ratio of externalized insulin receptors to total insulin receptors
was substantially decreased in cultures from diabetic mice versus
control cultures (40). As obesity is a major risk factor for Type II
diabetes there may be a relationship between these two effects of
central insulin. This hypothesis is supported by the observation that
insulin receptor number is significantly decreased in the olfactory
bulb of obese rats (47).
Protein Kinase C (PKC): Regulation of Insulin and IGF I Receptors
PKC is a serine/threonine kinase which is present in many
tissues but occurs at highest concentrations in brain (48). PKC is
calcium-dependent and 1,2-diacylglycerol (DAG) a product of
membrane phospholipid metabolism, increases the affinity of the
enzyme for calcium (49-50). Reports of the molecular weight of
PKC vary. The different values may reflect the method by which the
relative molecular weight (Mr) is determined, as evidenced by a
study in which values of 77,000 and 82,000 daltons were obtained
from sucrose density gradient and polyamide gel electrophoresis,
respectively (51). Differences may also be attributable to subunit
aggregation (52) or to the existence of different isozymes of PKC
(53-4). Currently, seven highly-homologous isozymes of PKC have
been isolated and characterized. Four are single polypeptide chains
with four constant and five variable regions, while three subspecies
differ slightly. The isozyme distributions differ with respect to one


Figure 3-1: Effect of depolarizing conditions on the
release of insulin from neuronal and glial cells from the
rat brain. Medium was aspirated from 14 day old
neuronal cultures and replaced by 8 ml of various
solutions: C (control) contained 140 mM NaCI, 1.2 mM
MgSC>4; K+Ca contained 78 mM NaCI, 60 mM KCL, 2 mM
CaCl2, 1.2 mM MgS04; K contained 80 mM NaCI, 60 mM
KCI, 1.2 mM MgS04; Ca contained 138 mM NaCI, 2 mM
CaCl2, 1.2 mM MgSC>4; V contained 10 uM veratridine,
138 mM NaCI, 2 mM CaCl2, 1.2 mM MgSC>4. Cultures were
incubated for 30 min at 37C. The solutions were
aspirated, lyophilized, reconstituted in distilled water
and subjected to radioimmunoassay for insulin. Means of
data from five to seven experiments are represented and
statistical significance evaluated by two-tailed
Wiicoxan Rank Sum Test.


CHAPTER Vil
THE EFFECTS OF INSULIN AND DEXAMETHASONE ON NEURONAL AND
GLIAL PKC
Introduction
Insulin increases PKC activity in adipocytes and myocytes
(103,105). Both insulin and PKC appear to have the same effects in
astrocytes as in these peripheral cells. That is, insulin stimulates
glucose uptake and macromolecular synthesis (28,33) and PKC
stimulates glucose uptake (119) and inactivates the insulin receptor
in glial cells, as in the periphery. Each, however, has different
effects in neurons. Insulin appears to have neuromodulator/
neurotransmitter effects (36-44) as opposed to its more traditional
metabolic effects (13) and PKC regulates neuronal differentiation
and function (69-77) as opposed to glucose uptake. We elected to
study insulins effect on PKC to determine whether neuronal and
glial cells responded to insulin with an increase in PKC as peripheral
cells do (103,105,106).
In order to determine whether these effects were specific to
insulin, the effects of dexamethasone on neuronal and glial PKC were
also studied. Dexamethasone is a synthetic glucocorticoid.
Glucocorticoids regulate glucose homeostasis (13) as well as the
97


Figure 6-7: The effect of TPA on [3|-l]2-dGlc uptake
responsiveness to insulin in glial cultures. TPA-treated
cells were treated with 100 nM TPA for two hours.
Insulin-treated cells were treated for 15 minutes with
167 nM porcine-derived insulin. Following treatments,
cells were washed and [3H]2-dGlc uptake was determined
as described in methods. Each point represents the mean
SEM. Means of insulin-treated groups were compared to
those of the appropriate non-insulin-treated groups by
two-way analysis of variance, followed by Newman-
Keuls post hoc test (p<0.05). This experiment was
repeated 5 times


56
in neurons. Our failure to observe a consistent increase in the
membranous fraction at 5-15 min. after TPA stimulation may be due
to the very small percentage of cytosolic iPKC available to be
translocated. It may be that the change in the concentration of iPKC
in the membrane is too small to be observed consistently.


Figure 6-5: Specificity of TPA's effect on glial insulin
binding. The appropriate phorbol ester (50, 100 or 500
nM) was added to the medium of confluent gliai cultures
for 2 hours at 37C. Following treatment, cells were
washed to remove phorbol esters and insulin binding was
measured as described in methods. Each bar represents
the mean SEM. This is 1 representative experiment of
3.


Figure 4-6: TPA-induced PKC translocation in astrocytic
glial cells. One hundred nM TPA was added to the medium
of confluent glial cultures for the times specified and
samples were separated into cytosolic () and membrane
() fractions and subjected to Western blotting. The
densitometric values were divided by the values for the
control samples. Each point represents the mean of at
least three experiments SEM. Means of treated groups
were compared to that of the control group by two-way
analysis of variance, followed by Dunnett's post hoc test
(p<0.05).


Time (Hr)
Immunoactive PKC (% of Control)
ro co
104


TABLE OF CONTENTS
PAGE
ABSTRACT iv
CHAPTER
I. INTRODUCTION 1
Insulin and Insulin Receptors in the Brain 2
Insulin in the Brain: Potential Functions 4
Protein Kinase C (PKC): Regulation of Insulin and IGF I
Receptors 6
Ligand-Receptor Interactions 8
Phosphorylation of Receptors 8
Receptor-Induced Effects 10
Effects ofGrowth Factors on PKC 11
PKC in the Brain: Distribution 12
PKC Regulation of Glucose Uptake and Insulin Receptors
in the Brain 13
Cultured Brain Cells: A Model for the Study of Neurotrophic/
Neuroactive Substances 14
II. METHODS 20
Preparation of Primary Neuronal Cultures from Rat Brains 20
Preparation of Primary Astrocytic Glial Cultures from Rat
Brains ....20
Immunocytochemistry of Neuron-Specific Enolase/Glial
Fibrillary Acidic Protein 22
Neuronal Depolarization 23
Insulin Radioimmunoassay 23
Characterization of Immunoprecipitable Insulin by HPLC 24
Labelling of Immunoreactive Insulin in Neuronal
Cultures 25
Characterization of the Regulation of Neuronal Insulin Release
by Glucose... 25
2-Deoxy-D-glucose (2-dGlc) Uptake 26
lodination of Insulin 26
Insulin Binding 28
Protein Determinations 29
i i


glucose
neuron
Astrocyte
fnsullnergic neuron
insulin
122


25
collected every 0.2 min. and absorbance was monitored at 210 and
280 nm. Fractions were subjected to radioimmunoassay (Serono) to
determine whether the fraction which bound the insulin antibody
corresponded to insulin chromatographically.
Labelling of Immunoreactive Insulin in_.Neuronal Cultures
Neuronal cultures grown in 100mm culture dishes were incubated
in leucine-free DMEM, 10 percent dialyzed PDHS and 50pCi [3H]-
leucine (146.5 Ci/mmole) for 24 hours at 37C. Medium was
aspirated from the culture dishes; cultures were washed 4 times
with PBS and used for experiments as described above, A 100 pi
aliquot of each solution was exposed to a guinea pig anti-human
insulin antibody (Serono) for 2 hours at room temperature. Sheep
anti guinea pig Ig was added to each sample for 30 minutes to
precipitate the primary antibody and all samples were centrifuged
at 2500g to pellet the antibody-antigen complexes. Supernatants
were decanted and the radioactivity in each pellet was counted
(Liquiscint, National Diagnostics, LKB1217 Rack Beta Counter).
Characterization of the Regulation of Neuronal Insulin Release bv
Ohm se
Neuronal cultures prepared in 100mm culture dishes were washed
twice with a control buffer [25mM NaHC03, 1,2mM NaH2P04, 122mM
NaCI, 1.2mM MgS4 and 2.5mM CaCl2 (pH 7.4)] at room temperature
to remove media. Glucose buffers were prepared such that they
were isotonic to the control buffer (eg. 25mM NaHC03, 5.5mM
glucose, 119mM NaCI, 2.5mM CaCl2 and 1.2mM MgS04). Eight ml of


10
isolated and are reversed by incubation with alkaline phosphatase,
suggesting that PKC decreases the tyrosine kinase activity of the
insulin receptor and that this decrease is due to the phosphorylative
changes induced in the receptor. Studies on rat adipocytes have also
shown that TPA increases the Km of the insulin receptor for ATP,
thus suggesting a mechanism for insulin resistance in adipocytes
(82).
aefiSBifiriliiduMci Effects
The tyrosine kinase activity of the insulin receptor is necessary
for normal receptor function and down-regulation (92-3). This has
been demonstrated by studies in which kinase-defective mutant
insulin receptors were used to transfect cells. The mutant
receptors demonstrate normal binding of insulin but do not possess
tyrosine kinase activity, are not internalized and do not possess
biological activity. Treatment of endogenous, biologically-active
insulin receptors with monoclonal antibodies against the receptor
kinase inhibits insulin-stimulated effects as well (94). Inhibition
of receptor tyrosine kinase activity by TPA leads to the same types
of defects. Treatment with TPA is associated with inhibition of
insulin-mediated DNA synthesis (95), phosphorylation of metabolic
enzymes (87), glycogen synthesis (96) and glucose uptake (97),
among others. Thus, an impaired PKC pathway can have dire
consequences for the cell or organism. This is demonstrated by
genetically obese (fa/fa) rats, in whose hearts and hepatocytes both
the basal distribution and the translocation of PKC are abnormal.
The resultant insulin-insensitivity can be duplicated by treating


30
polyacrylamide gels (5 percent stacking gel, 7.5 percent separating
gel, 30mA for 6 hours) and transferred to a nitrocellulose membrane
in a buffer consisting of 25mM Tris base, 150mM glycine and 20%
v/v methanol at pH 8.3 for three hours at 150mA. Total transfer
was demonstrated by transfer of prestained standards and by the
lack of a band as demonstrated by Coommassie staining of the gel
after the transfer. The nitrocellulose membrane was stored in 3
percent BSA in PBS overnight to decrease non-specific binding of the
antibody.
The nitrocellulose membrane was incubated with a monoclonal
antibody against protein kinase C (Amersham, diluted 1:100 in PBS
with 0.1 percent BSA) for 4 hours at room temperature. This
antibody recognized the alpha and beta subtypes (Types II and III) of
PKC, which are the majority of PKC in the brain (159). It was then
washed 4 times for 5 min. with 0.1% Tween-20 in PBS (pH 7.4) and
incubated with horseradish-peroxidase-linked anti-mouse Ig diluted
1:100 with 0.1 percent BSA/0.1 percent Tween-20 in PBS for 30 min.
at room temperature. The membrane was washed as before and
incubated in 0.03 percent hydrogen peroxide/0.5 mg/ml 3,3'-
diaminobenzidine (DAB) in PBS prepared immediately prior to use,
until bands appeared. It was then washed and allowed to air dry.
Bands were quantitated via densitometry. Immunological PKC will
be referred to as iPKC throughout this dissertation, while PKC
activity will be designated aPKC.


58


a b c a b c
Gia Neurons
180 kD
80kD
4-36.5 kD
b c
Brain


41
synthesized in the brain (16). They propose that insulin passes from
the plasma into the CSF and is retained in brain to the extent that it
binds to local insulin receptors. They further propose that this
accounts for insulin immunocytochemistry in the brain.
Both an mRNA for insulin and concentrations of insulin which are
independent of those in the plasma and CSF have been demonstrated
in the brain in several other studies, as described previously. The
results of this study suggest that insulin is synthesized in neurons
from the brain and released under depolarizing conditions. The
insulin in the brain has immunological and chromatographic
properties that resemble those of pancreatic insulin. While this
evidence suggests that the material is, in fact, insulin, it is not
definitive proof. Rat proinsulin is not commercially available and
would be likely to have similar properties. Sequencing with an
amino acid analyzer would be the most appropriate method of
identifying the peptide, but it requires a larger sample of the
material than is available.
Depolarized release was measured using the solutions described
in methods in order to duplicate the methods used by Yalow to study
cholecystokinin release from synaptosomes. These unbuffered
solutions had an acidic pH after 30 min. This low pH may have
altered the release and/or the viability of the cells, although the
cells were still attached to the culture dish and appeared normal
under the microscope. Acidity was observed in all groups, but
onlythe depolarized groups showed increased release of insulin,
suggesting that it was the depolarization, and not the acidity that
induced release. In any case, glucose-induced release was measured


CHAPTER VIII
DISCUSSION AND SUMMARY
Although the brain has long been considered to be an insulin-
independent organ, both insulin and high affinity insulin receptors
have been localized in the brain within the past ten years. Three
major questions have arisen with regard to these findings: what is
the source of this peptide; what is its function, and how are the
receptors regulated in the brain. Of the three questions, the most
controversial is the first. Several investigators have independently
measured concentrations of insulin in the brain that are higher than,
and independent of, plasma concentrations of insulin (14,23). The
latter finding suggests that the high level of insulin in the brain is
not the result of sequestration and concentration of the peptide. In
addition, many investigators have independently identified an mRNA
in the brain that binds with a cDNA for insulin (20-4). These have
been identified in both cultured cells and in tissue slices from the
brain. As insulin acts to promote neurite outgrowth and neuronal
survival in the brain (115), the higher incidence of reports of an
mRNA for insulin in culture may be due to preferential survival of
insulin-producing and/or -responsive cells in culture.
113


36


29
Protein Determinations
One hundred to two hundred ul of a solution of protein in 0.1 N
NaOH was used for protein determinations. Bovine serum albumin
standards (10-100 ug of protein) were prepared in the same volume
of 0.1 N. NaOH. Samples and standards were made to 500 ul with
deionized, distilled water and protein determinations were made by
the method of Lowry (132).
Western Blot
Treated or untreated cultures of neurons and glia were washed 3
times with PBS, scraped from the culture dishes and centrifuged at
1000 x g for 5 min. to pellet cells. The supernatant was poured off
and the cells were resuspended in a homogenizing buffer consisting
of 20mM Tris HCI, 2mM EDTA, 0.5mM EGTA, 0.1 mM PMSF and 1
percent 213-mercaptoethanoi at pH 7.5. The suspension was
homogenized with 15 strokes of a glass homogenizer and centrifuged
at 1500 x g at 4C for 8 min. to remove nuclei and large particles.
The supernatant was recentrifuged at 100,000 x g at 2-4C for 30
min. to isolate cytosolic and membranous fractions. The membrane
was resuspended in homogenizing buffer containing 0.1 percent
Triton X-100. Samples of homogenates from whole cell were used
after the homogenization step. The protein content of samples was
determined and samples were made to a final concentration of 10mM
Tris base, 2 percent sodium dodecyl sulfate (SDS), 15 percent
sucrose, 0.002 percent bromophenol blue and 10 percent 28-
mercaptoethanol at pH 8.3. Samples were boiled for one min. and
stored at 4C before proteins were separated on discontinuous


42
in a Krebs buffered solution, in order to eliminate this problem.
Insulin degradation in the release solutions was probably not
significant as degradation of insulin in binding studies, in which the
insulin is exposed to the cells for an hour or more, is generally less
than 10 percent.
It is likely that at least some of the insulin released from
neuronal cultures is also synthesized there; The cultured cells have
been removed from any peripheral insulin for at least 10 days prior
to use; the serum in which the cells are grown is plasma-derived
horse serum, which contains only one insulin, not the two observed
in the rat, and exogenous leucine is incorporated into the
immunoprecipitable insulin. The inability of some researchers to
demonstrate synthesis of insulin in the brain may be related either
to the small percentage of cells producing insulin or to their very
specific localization. In addition, insulin acts to promote survival
of brain cells in culture (115) and, thus, insulin-producing neurons
may survive preferentially in culture and may represent a larger
percentage of neurons than are present in whole brain. That
immunocytochemical evidence localizing insulin to neurons is the
result of insulin bound to surface receptors, is unlikely as glial
cells have specific, high-affinity insulin receptors as well (28) The
evidence presented here that insulin is synthesized in neurons from
the brain and is released under depolarizing conditions, in addition
to the evidence that insulin in the brain binds to specific, high-
affinity receptors and has electrical and physiological effects in the
brain suggest that insulin is a neurotransmitter in the brain.


134
132. Lowry, O., Rosebrough, N., Farr, A. and Randall, R. (1951) Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193:265.
133. Azhar, S., Butte, J. and Reaven, E. (1988) Identification of isoenzymic
forms of hepatic calcium-activated phospholipid-dependent protein
kinase in various animal models. Bchm. Bphys. Res. Comm. 155:1017.
134. Mudd, l., Azhar, S. and Raizada, M. (1989) unpublished observation.
135. Farese, R., Barnes, D., Davis, J., Standaert, M. and Poliet, R. (1984)
Effects of insulin and protein synthesis inhibition on phospholipid
metabolism, diacylglycerol levels and pyruvate dehydrogenase activity in
BC3H-1 cultured myocytes. J. Biol. Chem. 259:7094.
136. Witters, I., Vater, C. and Lienhard, G. (1985) Phosphorylation of the
glucose transporter in vitro and in vivo by protein kinase C. Nature
315:777.
137. Amir, S., Schechter, Y. (1988) Apparent involvement of protein kinase
C in the central glucoregulatory action of insulin. Brain Res. 450:272.
138. Pierre, M., Toru-Delbauffe, D., Gavaret, J., Pomerance, M. and
Jacquemin, C. (1986) Activation of S6 kinase activity in astrocytes by
insulin, somatomedin C and TPA. FEBS Lett. 206:162.
139. Klip, A., Ramlal, T. (1987) Protein kinase C is not required for insulin
stimulationof hexose uptake in muscle cells in culture. Biochem. J.
242:131.
140. Spach, D., Nemenoff, R., Blackshear, P. (1986) Protein phosphorylation
and protein kinase activities in BC3H-1 myocytes: Differences between
the effects of insulin and phorbol esters. J. Biol. Chem. 261:12750.
141. Pelech, S., Krebs, E. (1987) Mitogen-activated S6 kinase is stimulated
via protein kinase C- dependent and -independent pathways in Swiss
3T3 cells. J. Biol. Chem. 262:11598.
142. Werner, H., Raizada, M., Mudd, L., Foyd, H., Simpson, I., Roberts, C. and
LeRoith, D. Regulation of rat brain/HepG2 glucose transporter gene
expression by insulin and insutin-like growth factor 1 in primary cultures
of neuronal and glial cells. In Press.
143. Hiraki, Y., Rosen, O. and Birnbaum, M. (1988) Growth factors rapidly
induce expression of the glucose transporter gene. J. Biol. Chem.
263:13655.


5
evoked within one hour while control and glutamate treatment of the
cells elicited no such response (34). Insulin induces ornithine
decarboxylase activity and neurite outgrowth in cultured embryonic
mouse brain cells and rat brain cultures (35). Thus, insulin induces
maturation of brain cells in culture as well as growth.
Insulin alters the content of serotonin and catecholamines in the
brain by increasing the rate of uptake of their precursors (36-8). It
also stimulates the release of dopamine, epinephrine and
norepinephrine from hypothalamic slices (39) and changes the firing
rates of neurons in the striatum (42) and hippocampus (43). Insulin,
then, definitely acts as a neuromodulator in the CNS and it may act
as a neurotransmitter itself. Recently, the neuromodulatory effects
of insulin have been suggested to be mediated by insulin receptors
present on the neurons (44). Insulin has, then, satisfied several
criteria by which putative neurotransmitters are classified as such:
it is synthesized in neurons, released under depolarizing conditions,
bound with specificity and high affinity to a receptor, degraded and
modulates neuronal activity.
CNS insulin may be involved in the pathophysiology of obesity and
Type II, or insulin-independent, diabetes. In vivo studies in baboons
indicate that insulin has a direct effect on satiety (41). When
insulin was infused into the lateral cerebral ventricles, a
significant, dose-related decrease in food intake and body weight
was observed. This may occur by interaction with neurons in the
hypothalamus. Injection of insulin in low doses causes electrical
activity to increase in hypothalamic neurons (45). In addition to its
association with obesity, central insulin may play a role in Type II


114
This research addressed the question of the site of insulin
synthesis in the brain as well. Exogenous, radioactively labelled
leucine was applied to neuronal cultures which had been removed
from any peripheral source of insulin for at least ten days. The cells
incorporated the radioactive amino acid into a peptide which was
precipitable with an antibody against insulin. When samples of this
material were compared to insulin chromatographicaily on a
reverse-phase HPLC column, they coeluted with the an insulin
purified from rat pancreas. The medium in which the cells were
grown contained horse serum, in which only one type of insulin was
expressed. In contrast, the rat expresses two different insulin
molecules. The sample isolated from the brain had two peaks which
were precipitated by the antibody against insulin and which were
similar chromatographicaily to the two peaks for rat insulin. This
suggests that insulin may be synthesized in the brain, although the
question cannot be answered definitively until the material is
sequenced. A number of other molecules are similar to insulin and
may both react with the antibody and have chromatographic
properties similar to those of insulin.
The next question addressed by this study concerned the function
of the insulin-like peptide found in the brain. Other investigators
have found that administration of insulin into the brain has specific
effects both in vivo and in vitro (28, 33-41). Insulin appears to act
as a neuromodulator in the brain and may act as a neurotransmitter
as well (36-39, 42-44). It appears to be synthesized in the brain,
and it acts on specific, high-affinity receptors to alter electrical
activity in selected areas of the brain (14, 20-3, 42-44). A fourth


126
37. Cooper, J., Bloom, F. and Roth, R. (1978) The Biochemical Basis of
Neuropharmacology. Oxford University Press, New York.
38. Wurtman, R., Larin, F., Mostafapour, S. and Fernstrom, J. (1974) Brain
catechol synthesis: control by brain tyrosine concentration. Science
185:183.
39. Sauter, A., Goldstein, M., Engel, J. and Ueta, K. (1983) Effect of insulin
on central catecholamines. Brain Res. 260:330.
40. Kadle, R., Raizada, M. and Fellows, R. (1985) Increased turnover of
surface insulin receptor in fibroblastic cultures from genetically diabetic
db/db mice. J. Cell Biochem. 28:59.
41. Woods, S., Letter, E., McKay, L. and Porte, D. (1979) Chronic
intracerebroventricular infusion of insulin reduces food intake and body
weight of baboons. Nature 282:503.
42. Sailer, D. and Chiodo, L. (1980) Glucose supresses basal firing and
galoperidol-induced increases in the firing rate of central dopaminergic
neurons. Science 210:1269.
43. Palovcik, R., Phillips, M., Kappy, M. and Raizada, M. (1984) Insulin
inhibits pyramidal neurons in hippocampal slices. Brain Res. 309:187.
44. Boyd, F., Clarke, D. and Raizada. (1985) Insulin receptors and
modulation of norepinephrine uptake by insulin in neuronal cells in
culture. J. Biol. Chem. 260:15880.
45. Oomura, Y. (1976) Significance of glucose, insulin and free fatty acids
on the hypothalamic feeding and satiety neurons. In: Hunger: Basic
Mechanisms and Clinical Implications. Sacher, E., ed. Raven Press,
New York.
46. Steffans, A., Mogenson, G. and Stevenson, J. (1972) Blood glucose,
insulin and free fatty acids after stimulations and lesions of the
hypothalamus. Am. J. Physiol. 22:1446.
47. Figlewicz, D. Brain insulin binding in obese rats. Endocr. 117:1537.
48. Nishizuka, Y. (1986) Studies and perspectives of protein kinase C.
Science 233:305.
49. Takai, Y., Kishimoto, A., Iwasa, Y. Kawahara, Y., Mori, T. and Nishizuka,
Y. (1979) Calcium-dependent activation of a multifunctional protein
kinase by membrane phospholipid. J. Biol. Chem. 252:7603.


32
several groups were to be compared to that of one control group.
Significance was determined for p<0.05. Experiments whose results
were expressed as a percent of control were converted to arcsin
prior to ANOVA if all values were equal to or less than 100 percent
or to the log if any of the values exceeded 100 percent. The
particular test used was specified in the legend of each figure. The
one exception to these rules was Figure 3-1, for which a two-tailed
Wilcoxan Rank Sum nonparametric test was employed because of one
outlying value in the depolarized group.


Figure 4-1: PKC immunostaining of cultured glial cells.
Cells were cultured on sterile glass coverslips. Prior to
immunostaining with a monoclonal PKC antibody, cells
were fixed and permeabilized. Following staining of the
cells, photographs were taken at 400x magnification.
This experiment was repeated three times.


54
enzyme is associated with the membranous fraction the membrane
staining is very dark and differences within the cell cannot be seen.
In contrast, in glia, the majority of the iPKC is cytoplasmic. This
was not unexpected, as an earlier study reported that whereas glial
PKC was predominantly cytosolic, PKC from homogenized whole
brain tissue was predominantly membrane-bound (116), The nuclei
in glial cells appear to stain darkly for iPKC, but nuclear PKC would
not contribute to either the cytosolic or membranous fractions as
the nuclei were removed in a centrifugation step prior to the
separation of membrane and cytosol. The aPKC in the membrane-
bound fraction is latent in the liver (52,133). The concentration of
PKC associated with neuronal cytosol was, in fact, low enough to be
the result of glial cell contamination. Neuronal cultures contain 15-
20 percent glial cells, as described in methods.
Complementary studies on neuronal and glial aPKC demonstrate
that glial cells have far greater aPKC than neurons (134). The
different results obtained by immunological and bioassay suggest
that either our antibody does not recognize a large percentage of
glial aPKC or that a large percentage of the neuronal iPKC is
inactive. The first could result from the presence of different
isozymes of PKC in different types of cells, one or several of which
are not recognized by an antibody directed against the alpha and beta
(beta-1 and -2) forms of the enzyme, although these Type II and III
subtypes of PKC do represent the majority of PKC in the brain, there
is another subtype, Type I, which is found only in the brain, and
which is not recognized by our antibody (159). The existence of this
subtype, which is not immunoactive could lead to a false negative


80KD-
0 min
15 min
1 hr 2 hr
24 hr


119
to determine whether there were differences which might explain
both of these phenomena. Both neurons and glia express PKC, with
neurons expressing 3-4 times greater iPKC and phorbol ester
binding activity than glial cells (113). Interestingly, similarly
prepared glial cells of the same age express a much higher aPKC.
This suggests that either our antibody does not recognize one or
more major isozymes of PKC in glia or that there is a large fraction
of neuronal iPKC which is inactive. Either of these is entirely
possible. Different isozymes have been identified in neurons and
glia (55,110-11). As these were all identified with different
antibodies, it is not clear whether these subtypes are recognized by
an antibody to the alpha and beta-subtypes. One subtype, the gamma
isozyme, is expressed entirely or predominantly in brain and is not
recognized by this antibody (54). The second possibility is that
some fraction of the neuronal PKC is inactive. Neuronal iPKC is
predominantly localized in the membrane, as opposed to the cytosol.
This is the opposite of the situation seen in glial cells. Membrane-
bound PKC is latent in liver (52,133), and may be so in neurons as
well. PKC has many other effects in neurons, but these may require
a smaller concentration of the enzyme.
Activation of PKC generally involves a translocation of the
enzyme from the cytosol to the membrane (59, 60, 63-4). In this
study we showed that TPA stimulated a translocation of glial iPKC
within 5-15 minutes after the cells were exposed. Within hours, the
cytosolic iPKC was immeasurably low, and the membranous iPKC
was downregulated to very low levels within 24 hours. TPA had the
same effect on translocation of aPKC in studies on glial cells (134).


79
incubator for the appropriate preincubation time prior to
quantitation of 2-dGlc uptake. Insulin induced a 34 percent increase
in glial 2-dGlc uptake when cells were treated with a dose of 167
nM for 15 min. Treatment of cells for 2 hours with 100 nM TPA
stimulated 2-dGlc uptake by 112 percent. However, TPA eliminated
the response to insulin. Administration of insulin in combination
with TPA resulted in a stimulation of 144 percent (Figure 6-7).
Thus, the binding of insulin increased, but the response to insulin did
not when glial cells were treated with TPA.
Discussion
TPA stimulates the binding of insulin in glial cells from the brain
without altering neuronal binding of insulin. This stimulation in glia
occurs without a parallel increase in the responsiveness to insulin,
as demonstrated by the lack of effect of TPA on insulin stimulated
2-dGlc uptake. Thus, it represents an increase in the number of
binding sites, not necessarily an increase in the number of
receptors. The potency of the phorbol esters in effecting a
stimulation of glial insulin binding corresponds to their respective
abilities to activate PKC (147), suggesting that TPA is acting
through PKC. As with TPA stimulation of glial glucose uptake,
however, caution must be used in translating an effect of phorbol
esters to an effect of PKC. Again also, the effect may well be an
indirect effect as PKC has other effects in the brain, as described in
the introduction. TPA's effects on glial insulin binding occur more
slowly than PKC translocation, but over the same range of doses.
Thus, translocation of PKC may be involved in TPA's stimulation of


115
quality must necessarily be present for insulin to be characterized
as neurotransmitter. It must be released under depolarizing
conditions. When cultured neurons were treated with depolarizing
solutions containing potassium and calcium, the amount of insulin
released into the medium was increased dramatically. This
depolarized release was calcium-dependent, as in the absence of
calcium, no such stimulation of release was observed. This further
suggests that insulin may act as a neurotransmitter in the central
nervous system. Release of insulin from insulinergic neurons was
also stimulated by glucose. Insulin acts on glial cells to stimulate
glucose uptake, and thus, acts indirectly to decrease one stimulus
for its own release (Figure 8-1).
The third question addressed by this work concerned the
regulation of insulin receptors in the brain. Earlier work had already
demonstrated that the insulin receptors in different types of cells
from the brain differed from peripheral receptors and from each
other (29, 31-2). These differences were found in structure,
function and regulation. Our particular interest was with regard to
the regulation of insulin receptors in the brain by PKC and
subsequent actions of insulin. This enzyme has many actions which
mimic the effects of insulin, and yet, the enzyme inactivates the
kinase activity of the insulin receptor and effectively turns off the
receptor in most tissues (87, 91, 95-7, 117-9). In our study of
insulin regulation of PKC in the brain, we first asked whether PKC
mimicked one specific action of insulin in the brain, the stimulation
of glial glucose uptake. We then went on to look at the effects of
PKC on the insulin receptors themselves. Lastly, we looked at


Figure 3-3: The effect of glucose on insulin release by
neuronal cultures from the rat brain. Medium was
aspirated from 14 day old neuronal cultures and replaced
by 8 ml of various solutions: the control solution
contained 140 mM NaCI, 1.2 mM MgS04 and 2 mM CaCl2;
Glucose solutions contained 5.5-33 mM glucose, 1.2 mM
MgSC>4 and NaCI to make the solution isotonic with the
control solution. Cultures were incubated for 30 min at
37C. Solutions were aspirated, lyophilized,
reconstituted in distilled water and subjected to
radioimmunoassay for insulin. Each bar represents the
mean SEM for 6-8 experiments.


REGULATION OF INSULIN EFFECTOR SYSTEMS IN THE BRAIN
BY
LAURA MARY MUDD
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989


Figure 7-1: The effect of insulin on glial iPKC. One
hundred sixty-seven nM insulin was added to the medium
of confluent glial cultures for the times specified. The
cells were then washed, prepared for Western blotting as
described in methods, and bands were read on a
densitometer. Each band represents 200 ug protein.
Densitometric readings are (from left to right) 610,
1488, 2549 and 2127. The experiment was repeated
eight times.


23
from the cultured cells and a rhodamine-linked, goat anti-rabbit Ig
was diluted 1:100 in 5 percent non-fat dry milk in PBS and applied
to the cells for 30 minutes at room temperature- Excess secondary
antibody was washed from the cultured cells with PBS and the cells
were photographed under fluorescent light at 400x magnification on
a Zeiss D-7082 Axiophot photomicroscope.
Neuronal Depolarization
Medium was aspirated from 14 day old neuronal cultures and
replaced by 8 ml of various solutions: the control solution contained
140 mM NaCI and 1.2 mM MgS04; the depolarizing solution contained
78 mM NaCI, 60 mM KCI, 2 mM CaCl2 and 1.2 mM MgSC>4; a potassium
solution contained 80 mM NaCI, 60 mM KCI and 1.2 mM MgSC>4; a
calcium solution contained 138 mM NaCI, 2 mM CaCl2 and 1.2 mM
MgS4, and a veratridine solution contained 10 uM veratridine, 138
mM NaCI, 2 mM CaC2 and 1.2 mM MgSC>4. Cultures were incubated
for 30 minutes at 37C. The solutions were aspirated, lyophilized,
reconstituted in distilled water and subjected to radioimmunoassay
for insulin.
Insulin Radioimmunoassay
A 100 ul aliquot of a sample of human insulin standard (0-300
uU/ml) was mixed with 5000 cpm of [125|]-insulin. One hundred ul
of guinea pig anti-human insulin (Serono) was added and the samples
and standards were vortexed and incubated at room temperature for
2 hours. A secondary antibody (sheep anti-guinea pig Ig, Serono) was


Cytosoi Membrane
Immunoactive PKC
(% Control)
Control
5 ml
15
1
2 hr
24
0 L


[125l]-lnsulin Binding
(% of controi)
0 1 10 100
Concentration (nM)


22
FBS and plated onto 100mm culture dishes at a density of 10 million
cells/dish. After three days the cells were refed with 10 percent
FBS in DMEM. After an additional three days, cultures were rinsed
once with an isotonic buffer and dissociated by treatment with
trypsin. The cells were centrifuged at 1000 x g for 10 min at room
temperature and the pellet was resuspended in DMEM containing 10
percent FBS. Five hundred thousand cells were placed on each
100mm culture dish and the cells were returned to the incubator
until they were confluent, at which time they were used. Phase
contrast microscopic examination revealed a confluent monolayer of
large flat cells by day 6 or 7 after transfer. These cells have
previously been demonstrated to be of glial origin (131). Greater
than 98 percent of these cells have been identified as astrocytic
glial cells. Neuronal cells did not survive the transfer (Figure 2-2).
Immunocvtochemistrv of Neuron-Specific Enolase/Glial Fibrillary
Acidic Protein
Either cultured neurons or glia were washed with PBS and the
cells were fixed in a solution of 4 percent paraformaldehyde and 10
percent picric acid in PBS (pH 7.4) for 30 minutes at 4C. Cultures
were again washed 3 times with PBS and permeabilized in 0.1
percent Triton-X 100, 5 percent low-fat, dry milk in PBS for 30
minutes at room temperature. After being washed again with PBS,
the cells were exposed to a polyclonal antibody (either 1:100
neuron-specific enolase or 1:20 glial fibrillary acidic protein)
diluted in 0.1 percent NaN3/5 percent non-fat dry milk in PBS for 24
hours at 4C. PBS was used to wash the excess primary antibodies


Figure 6-3: Time-courses of TPA-stimulated events in
glial cultures. One hundred nM TPA was added to the
medium of confluent glial cultures for the times
specified. Following treatment with TPA, cells were
washed and 2-dGLC uptake, insulin binding and iPKC
translocation were measured as described in methods.
Each point represents the mean SEM for at least three
separate experiments.


UNIVERSITY OF FLORIDA
3 1262 08554 6256


(fmoles/100mm dish)


na
Control
TPA
a-PDD
lAIUQOS
CD
ro
B-PDD


53
min. the cytosolic iPKC was barely detectable by Western blot (6 4
percent of the control level). At 24 hours after treatment with
TPA, the concentration of iPKC in the cytosol remained low. In
contrast, the iPKC in glial membranes was increased to 150 39
percent of the control within 5 min. of the administration of TPA,
after which it declined. Within 24 hours the membrane-bound ¡PKC
had downregulated such that it was barely detectable as well
(Figures 4-4,6).
Treatment with 100 nM TPA induced a downregulation of iPKC in
neuronal cells. The cytosolic iPKC decreased to 59 percent of the
control concentration within 1 hour, to 16 percent within 2 hours
and did not increase again over the course of a 24 hour treatment
with TPA. The level of iPKC bound to the membrane did not change
within 15 min. after treatment with TPA but decreased to 54
percent within 1 hour and continued to decline such that the iPKC
was only 17 percent of the control iPKC concentration after 24
hours (Figures 4-5,7).
Discussion
An iPKC of 80 kD is present in both neurons and glia, although
neurons express several times the cconcentration of iPKC as that
seen in glia. This is seen with both the immunocytochemistry and
the Western blot experiments. This is in agreement with studies
showing that the binding of phorbol esters is 2-3 times higher in
cultured neurons than in glial cells from the same rat brains (113).
No differences with regard to immunostaining of different regions
of the neurons were observed. As the vast majority of the neuronal


CHAPTER I
INTRODUCTION
Historically, insulin has been used in the treatment of diabetes
mellitus and psychosis. Pancreatic insulin was first isolated in
1922 at which time it was used clinically in the treatment of Type I
diabetes (1). In classical Type I diabetes the insulin-secreting (3-
cells of the pancreas are destroyed and the patients become
dependent on exogenous insulin to control their blood sugar. Several
years after Banting and Best began using insulin to treat diabetes,
Schmidt (2) and Sakel (3) separately reported successful treatment
of psychotic patients with insulin in the presence and absence of
carbohydrate, respectively, that is, treatment with and without
hypoglycemic shock. Insulin-induced hypoglycemic shock was used
successfully in the treatment of schitzophrenia and depression as
well as psychosis until the development of electroshock therapy and
more sophisticated psychoactive drugs (4).
The mechanisms of pancreatic insulin synthesis and release, as
well as physiological effects on liver, muscle and adipose tissue,
have been well characterized. Pancreatic insulin is synthesized as
preproinsulin, cleaved to proinsulin almost immediately and
1


24
added and the sample and standard tubes were vortexed and
incubated for a further 30 minutes at room temperature. Following
the incubation, all tubes were centrifuged at 2500 g for 30 minutes
at 4C. Supernatants were discarded. All tubes were swabbed dry of
supernatant and the radioactivity in the pellets was counted.
Samples were compared to a standard curve and the cpms were
expressed as international units of insulin/ml.
Characterization of Immunoorecipitable Insulin bv HPLC
Neuronal cultures grown in 100mm culture dishes were washed
twice with a solution of 138mM NaCI, 1.2mM MgS04 and 2mM CaCl2
(pH 7.4) at room temperature to remove the growth medium. Eight
ml of a depolarizing solution (78mM NaCI, 1.2mM MgS04, 2mM CaCl2
and 60mM KCI, adjusted to pH 7.4) were placed on each culture dish
and the dishes were incubated at 37C for 30 min. in a 94 percent
air/6 percent CO2 incubator. After incubation the solution was
aspirated from the plates.
High-pressure liquid chromatography (HPLC) was used to
determine whether the released peptide had the same
chromatographic properties as commercial rat insulin (Eli Lilly).
Two buffers were prepared. Buffer A consisted of 0.1 percent
trifluoroacetic acid (TFA) and 0.1 percent triethylamine (TEA) in
water. Buffer B consisted of 0.1 percent TFA and 0.1 percent TEA in
acetonitrile. The sample was dissolved in Buffer A and injected
onto a BioRad HiPore C4 column. A buffer system of 1:9, A:B was
graduated to 5:5, A:B over the course of 60 min. The column was run
at room temperature with a flow rate of 2 ml/min. Fractions were


Figure 7-4: The effect of insulin on neuronal iPKC. One
hundred sixty-seven nM insulin was added to the neuronal
medium for the times specified. The cells were then
washed, prepared for Western blotting as described in
methods, and bands were read on a densitometer.
Densitometric readings are (from left to right) 2158,
1320, 1347, 2364, 1724 and 2162. The experiment was
repeated 3 times.


17
insulin and PKC in differential cultures of neurons and glia from the
same rat brains.


12
phospholipid synthesis. There is some controversy on this point,
with other work suggesting that insulin does not increase the
activity of PKC in myocytes (107). It is suggested that the
increases in phosphorylation observed after the administration of
insulin are mediated by S6 kinase which is activated by both insulin
and TPA. Growth factors, then, may increase PKC indirectly by
increasing either cytosolic free calcium ion or DAG or by acting at a
point in the pathway beyond the PKC molecule itself. A direct effect
of insulin on PKC must necessarily be demonstrated on the isolated
enzyme.
PKC in the Brain : Distribution
Although PKC is present in many tissues, it occurs at highest
concentration in the brain. The seven subspecies of PKC have
different distributions in the brain (54) and these change with brain
development. The gamma subspecies, which occurs only in the
central nervous system in the rat and monkey, has a
developmentally-regulated distribution, with expression increasing
from birth until it reaches a maximum at about three weeks of age.
Total PKC is also developmentally regulated in brain in studies in
cultured neurons and in vivo (57). Interestingly, insulin and IGF 1
receptors are developmentally regulated in the rat brain with
increases occurring in the first weeks of life, followed by a decline
(108-9). Immunohistochemical analyses show different antibody
staining patterns. There appear to be subspecies which are present
almost exclusively in neurons (55,110), in astrocyte-glial cells and
in oligodendrocytes (55,111). The enzymatic activity is also


Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
REGULATION OF INSULIN EFFECTOR SYSTEMS IN THE BRAIN
By
Laura Mary Mudd
May, 1989
Chairman: Mohan K. Raizada
Major Department: Physiology
In spite of insulin's effects on glucose uptake in the periphery and the fact
that the brain is a glucose-obligate organ, the brain was long believed to be
insulin-independent because insulin does not cross the blood-brain barrier.
Many reports over the course of the past ten years have localized both insulin
and insulin receptors in the brain, although the source and function of the
peptide in the brain and the mechanisms by which receptors are regulated have
remained the subjects of some mystery.
It was found that an immunoreactive insulin was released from cultured
neurons under depolarizing conditions, that labelled leucine could be
incorporated into the released peptide and that the peptide coeluted with a rat
insulin standard when the two were applied to a reverse-phase high pressure
liquid chromatography column. Thus, the peptide resembles insulin
chromatographically as well as immunologically, is synthesized in neuronal
cultures and is released under depolarizing conditions. In the brain, insulin
may act as a neurotransmitter.
IV


Figure 2-2: Glial fibrillary acidic protein staining of
cultured glial cells. Cultured glial cells were fixed and
permeabilized prior to immunostaining for glial
fibrillary acidic protein. Following staining the glial
cells were photographed at 400x.


C M C M C M
80 kD
0 min 5 min 15 min
Time
C M C M C M
CD
4^


26
either the control or a glucose buffer were placed on each culture
dish. Cells were incubated at 37C for 30 min. in 6 percent C2/94
percent air with 90 percent relative humidity. After the incubation
the buffers were aspirated from the dishes, lyophilized, and insulin
in the buffers was quantitated by radioimmunoassay.
2-Peoxv-D-Glucose (2-dGlc) Uptake
Medium was aspirated from the culture dishes and the cells were
washed three times with phosphate-buffered saline (PBS) at pH 7.4.
The cells were then incubated in PBS containing 1mM CaCl2, 0.5 mM
MgS04 and 0.5 mM 2-dGlc (1 pCi/plate). After a five min. incubation
at 37C, the ceils were washed three times with ice-cold PBS to
remove excess radioactivity. Cells were dissolved in 0.2 N NaOH;
they were then scraped from the culture dishes and radioactivity
was counted (Ecoscint, National Diagnostics, LKB 1217 Rack Beta
scintillation counter). The cpm were converted to dpm by the
counter (efficiency about 30 percent) and normalized for protein
values. The specific activity was then used to convert results to
nmoles of 2-dGlc/mg protein.
lodination of Insulin
One hundred ml of a pH 6.7 phosphate buffer was prepared from
55 ml of 0.3M KH2PO4 and 45 ml of 0.3M Na2HP04. Chloramine T was
prepared as follows: the surface was scraped and chloramine T was
weighed and placed in a foil-covered tube; the salt was diluted to 4
mg/ml in the phosphate buffer and again diluted 1:100 (i.e. 25 pi in
2.5 ml) in phosphate buffer just before use; all dilutions were in


98
insulin receptor peripherally (149-51), and downregulate the insulin
receptor in an astrocyte cell line (152). In addition, there are
interactions between PKC and the glucocorticoids, as there are
between PKC and insulin. Adrenocorticotropic hormone (ACTH) may
be a stimulus for PKC (154). ACTH is reported to increase
diphosphoinositide (DPI) in the adrenal cortex and to modulate DPI
kinase in brain (155). In a conflicting study, no effect of ACTH on
adrenal phosphoinositides was observed although corticotropin
releasing hormone (CRH) did increase phosphoinositide metabolism
in the anterior pituitary (156). PKC, in turn, inhibits the production
of cortisol by the adrenal and phosphorylates mammalian stress
proteins (157-8) when activated. As glucocorticoids act on the
insulin receptor centrally, and glucorticoids, ACTH and CRH were
reported to interact with PKC both centrally and peripherally, we
chose to study dexamethasone's effects on neuronal and glial PKC as
well.
Results
Incubation of glial cells with insulin resulted in a time-
dependent increase in iPKC. Increases were observed within hours,
with a significant increase 224 percent within six hours of
treatment with 167 nM insulin. The iPKC remained elevated for up
to 24 hours after the administration of insulin (Figures 7-1,2).
Preliminary experiments suggested that the increase was primarily
cytoplasmic as after 2 hours of treatment with insulin the
cytoplasmic iPKC was 383 percent of the control concentration,
while membrane-bound iPKC was 113 percent of the basal level


CO


132
107. Spach, D., Nemenoff, R. and Biackshear, P. (1986) Protein
phosphorylation and protein kinase activities in BC3H-1 myocytes. J.
Biol. Chem. 261:12750.
108. Kappy, M., Sellinger, S. and Raizada, M. (1984) Insulin binding in four
regions of the developing rat brain. J. Neurochem. 42:198.
109. Bassas, L, DePablo, F., Lesniak, M. and Roth, J. (1985) Ontogeny of
receptors for insulin-like peptides in chick embryo tissues: Early
dominance of insulin-like growth factor over insulin receptor in brain.
Endocr. 117:2321.
110. Saito, N., Kikkawa, U., Nishizuka, Y. and Tanaka, C. (1988) Distribution
of protein kinase C-like immunoreactive neurons in rat brain. J.
Neurosci. 8:369.
111. Wood, J., Girard, P., Mazzei, G. and Kuo, J. (1986) Immunocytochemical
localization of protein kinase C in identified neuronal components of rat
brain. J. Neurosci. 6:2571.
112. Ginobili de Martinez, M. and Barrantes, F. (1988) Calcium and
phospholipid-dependent protein kinase activity in rat cerebral
hemispheres. Brain Res. 440:386.
113. Raizada, M., Morse, C., Gonzales, R., Crews, F. and Sumners, C. (1988)
Receptors for phorbol esters are primarily localized in neurons:
Comparison of neuronal and glial cultures. Neurochem. Res. 13:51.
114. Girard, P.f Wood, J., Freschi, J. and Kuo, J. (1988) Immunocytochemical
localization of protein kinase C in developing brain tissue and in primary
neuronal brain cultures. Dev. Biol. 126:98.
115. Redo-Pinto, E. and Ishii, D. (1988) Insulin and related growth factors:
Effects on the nervous system and mechanism for neurite growth and
regeneration. Neurochem. Int. 12:397.
116. Neary, J., Norenberg, L. and Norenberg, M. (1988) Protein kinase C in
primary astrocyte cultures: Cytoplasmic localization and translocation by
a phorbol ester. J. Neurochem. 50:1179.
117. Farese, R., Standaert, M., Barnes, D., Davis, J. and Pollet, R. (1985)
Phorbol ester provokes insulin-like effects on glucose transport, amino
acid uptake and pyruvate dehydrogenase activity in BC3H-1 cultured
myocytes. Endocr. 116:2650.
118. Driedger, P., Blumberg, P., (1977) The effect of phorbol esters on chick
embryo fibroblasts. Cancer Res. 37:3257.


2
packaged into secretory granules where the C-peptide is cleaved
prior to secretion (5). Basal beta-cell secretion of insulin is about
0.5 U/hour. This can be increased 10-30 times with acute
stimulation. Glucose is the most potent stimulator of pancreatic
insulin release in man (6) although amino acids (especially arg, lys,
leu and val), glucagon, 6-adrenergic and vagal stimulation of the
pancreas stimulate secretion as well (7,8). Somatostatin,
serotonin, dopamine, prostaglandin E and splanchnic stimulation are
inhibitory to insulin release (8,9). Chronic glucose stimulation
induces a biphasic release of insulin (10); this is thought to be due
to the release of short- and long-term insulin stores (8).
The first step in the action of insulin on the peripheral target
tissues is its interaction with specific receptors on the cell
surface. This interaction of insulin with its receptor stimulates
autophosphorylation of a tyrosine residue (11) as well as receptor
tyrosine kinase activity (12). Binding of insulin induces an increase
in glucose and amino acid uptake by liver, muscle and adipose
tissues and consequently an increase in the synthesis of glycogen
and protein with a corresponding decrease in lipolysis and
gluconeogenesis (13).
Insulin and Ingglin Receptor? in the Brain
In spite of insulins effects on glucose uptake in the periphery
and the fact that the brain is a glucose-obligate organ, the brain was
long believed to be insulin-independent because insulin does not
cross the blood-brain barrier. Havrankova et al., established the
presence of insulin in the brain at concentrations that averaged 25


juU Insulin Released/mg Protein


15
for every aspect of normal brain function. The importance of growth
factors, such as nerve growth factor, insulin and the insulin-like
growth factors, in neuronal development and activity has been well
documented although the mechanisms by which these agents act are
still objects of intense study (115,120).
In contrast, although glial cells are the predominant cell type in
the mature nervous system, their involvement in the growth,
development, differentiation and function of the brain has only
recently become a subject of investigation. Glial cells have recently
been implicated in processes involving the growth, development and
function of the nervous system. Glia are not only responsive to
trophic factors but may produce them as well. They facilitate
neuronal migration in fetal life but induce scarring to inhibit
regeneration in the mature nervous system. In addition, they alter
the levels of neurotransmitters available at the synapse, thus
altering neuronal excitability and they may even be excitable
themselves.
Glia respond to trophic factors such as insulin and insulin-like
growth factor I (IGF I) with an increase in glucose uptake in contrast
to neurons (119,121). This is of interest developmental^ as both
insulin and IGF I receptors in brain increase to a maximal level
during brain development and then show a gradual decline (108-9).
Glia also exhibit an increase in macromolecular synthesis in
response to both insulin (33) and IGF I (121). Epidermal growth
factor receptors in the brain appear predominantly in glia, as well
(122).


52
Bfi suits
Immunoreactive PKC was present in both neurons and glia.
Immunocytochemistry showed neurons staining darker than glial
cells in the same cultures with the stain distributed throughout both
types of cells. In glial cells the staining was unevenly distributed
with particularly dark staining in nuclear and perinuclear areas. In
neurons, both perikarya and processes stained. No further
differences in staining could be seen in neurons at this
magnification. No unstained cells were observed in either the
neuronal or glial cultures (Figure 4-1,2). Control cells, which
received the same treatment except for the primary antibody, had no
staining whatsoever. The concentration of iPKC was 4.6+0.5 (mean
standard error) times higher in neuronal than in glial cultures from
the same brains when measured by Western blot and densitometry
(Figure 4-3) of whole cell homogenates. Immunoactive PKC in both
neurons and glia had a relative molecular weight of about 80 kD as
determined by polyacrylamide gel electrophoresis followed by
Western blotting and comparison with commercial molecular weight
standards.
The cytosol contained 63 9 percent of the iPKC in glial cells
(Figure 4-4). In contrast, the neuronal iPKC resided primarily in the
membrane with only 12 2.3 percent in the cytosolic fraction when
quantitated by Western blot and densitometry (Figure 4-5).
Treatment of both neurons and glia with 100 nM TPA induced a
time-dependent translocation of iPKC from the cytosolic fraction.
In glial cells treatment with TPA decreased the cytosolic level of
iPKC to 33 7 percent of the basal level within 5 min. Within 15


130
85. Rouis, M., Thomopoulos, P., Cherier, C. and Testa, U. (1985) Inhibition
of insulin receptor binding by A23187: Synergy with phorbol esters.
Bchm. Bphys. Res. Comm. 130:9.
86. Shoyab, M., DeLarco, J. and Todaro, G. (1979) Biologically active
phorbol esters specifically alter affinity of epidermal growth factor
membrane receptors. Nature 279:387.
87. Takayama, S,, White, M., Lauris, V. and Kahn, R. (1984) Phorbol esters
modulate insulin receptor phosphorylation and insulin action in cultured
hepatoma cells. Proc. Natl. Acad. Sci. USA 81:7797.
88. Rosenfeld, R. Hintz, R. and Dollar, L. (1982) Insulin-induced loss of an
insulin-like growth factor-l receptor on IM-9 lymphocytes. Diabetes
31:375.
89. Jacobs, S. and Cuatrecasas, P. (1986) Phosphorylation of receptors for
insulin and insulin-like growth factor I: Effects of hormones and phorbol
esters. J. Biol. Chem. 261:934.
90. Bollag, G., Roth, R., Beaudoin, J., Mochly-Rosen, D. and Koshland, D.
(1986) Protein kinase C directly phosphorylates the insulin receptor in
vitro and reduces its protein kinase activity. Proc. Natl. Acad. Sci. USA
83:5822.
91. Takayama, S., White, M. and Kahn, C. (1988) Phorbol ester-induced
serine phosphorylation of the insulin receptor decreases its tyrosine
kinase activity. J. Biol. Chem. 263:3440.
92. Maegawa, H., Olefsky, J., Thies, S., Boyd, D., Ullrich, A. and McClain, D.
(1988) Insulin receptors with defective tyrosine kinase inhibit normal
receptor function at the level of substrate phosphorylation. J. Biol. Chem.
263:12629.
93. Russell, D., Gherzi, R.Johnson, E., Chou, C. and Rosen, O. (1987) The
protein-tyrosine kinase activity of the insulin receptor is necessary for
insulin-mediated receptor down-regulation. J. Biol. Chem. 262:11833.
94. Morgan, D. and Roth, R. (1987) Acute insulin action requires insulin
receptor kinase activity: introduction of an inhibitory monoclonal
antibody into mammalian cells blocks the rapid effects of insulin. Proc.
Natl. Acad. Sci. USA 84:41.
95. Takada, K., Amino, N., Tetsumoto, T. and Miyai, K. (1988) Phorbol esters
have a dual action through protein kinase C in regulation of proliferation
of FRTL-5 cells. FEBS Lett. 234:13.


Figure 6-4: Competitive inhibition of 2^l]-insulin
binding to untreated and TPA-treated glial cultures.
Cultures were incubated without () or with () 100 nM
TPA for 2 hours at 37C. Cells were washed and
incubated with increasing concentrations of insulin (0.8-
133nM) in the presence of 100,000 cpm of [125l]insulin.
The experiment was reproduced four times.


125
25. Clarke, D., Mudd, L, Boyd, F., Fields, M. and Raizada, M. (1986) Insulin
is released from rat brain neuronal cells in culture. J. Neurochem.
47:831.
26. Raizada, M., Phillips, M. and LeRoith, D., eds. (1986) Insulin. Insulin-like
Growth Factors andTheiLBaceptors in.lhe Central Nervous System-
Plenum Press, New York.
27. Hill, J., Lesniak, M., Rojeski, M., Pert, C. and Roth, J. (1986) Receptors for
insulin and insulin-related peptides in the CNS: Studies of localization
in rat brain. In: Insulin. Insulin-like Growth Factors and Their Receptors
in the Central Nervous System. Raizada, M., Phillips, M. and LeRoith, D.,
eds. Plenum Press, New York.
28. Clarke, D., Boyd, F Kappy, M. and Raizada, M. (1984) Insulin binds to
specific receptors and stimulates 2-deoxy-D-giucose uptake in cultured
glial cells from rat brain. J. Biol. Chem. 259:11672.
29. Lowe, W., Boyd, F., Clarke, D., Raizada, M., Hart, C. and LeRoith, D.
(1986) Development of brain insulin receptors: structural and functional
studies of insulin receptors from whole brain and primary cell cultures.
Endocr. 119:25.
30. Boyd, F., Clarke, D., Muther, T. and Raizada, M. (1985) Insulin receptors
and insulin modulation of norepinephrine uptake in neuronal cultures
from rat brain. J. Biol. Chem. 260:15880.
31. Heidenreich, K. (1985) The Mr difference between insulin receptors in
brain and peripheral tissues is due to variation in carbohydrate content.
Diabetes (Suppl.) 34:56A.
32. Masters, B., Shemer, J., Judkins, J., Clarke, D., LeRoith, D. and Raizada,
M. (1987) Insulin receptors and insulin action in dissociated brain cells.
Brain Res. 417:247.
33. Clarke, D., Boyd, F., Kappy, M. and Raizada, M. (1985) Insulin
stimulates macromolecule synthesis in cultured glial cells from rat brain.
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34. Puro, D. and Agardh, E. (1984) Insulin-mediated regulation of neuronal
maturation. Science 225:1170.
35. Bhat, N. (1983) Insulin dependent neurite outgrowth in cultured
embryonic mouse brain cells. Dev. Brain Res. 11:315.
36. Tagliamonte, A. DeMontis, M., Olianas, M., Onali, P. and Gessa, G.
(1975) Possible role of insulin in the transport of tyrosine and tryptophan
from blood to brain. Pharm. Res. Comm. 7:493.


Figure 4-2: PKC immunostaining of cultured neurons.
Cells were cultured on sterile glass cover slips. Prior to
immunostaining with a monoclonal anti-PKC antibody,
cells were fixed and permeabilized. Following staining,
the neurons and glial cells were photographed at 1000x
magnification. This experiment was repeated three
times.


Figure 1-1: Activation and translocation of PKC. As the
receptor is activated, the membrane-bound enzyme,
phospholipase C (PLC), is activated and phosphoinositide
(PI) is hydrolyzed to inositol-phosphate (IP), -
diphosphate (IP2) -triphosphate (IP3) and diacylglycerol
(DAG). IP3 stimulates calcium mobilization. DAG
increases the affinity of the calcium-activated,
phospholipid-dependent PKC for calcium. As PKC is
activated it is translocated to the membrane. Phorbol
esters act in a manner which is analogous to DAG.


129
73. Baranyi, A., Szente, M. and Woody, C. (1988) Activation of protein
kinase C induces long-term changes of postsynaptic currents in
neocortical neurons. Brain Res. 440:341.
74. Hall, F., Fernyhough, P., Ishii, D. and Viulliet, P. (1988) Suppression of
nerve growth factor-directed neurite outgrowth in PC12 cells by
sphingosine, an inhibitor of protein kinase C. J. Biol. Chem. 263:4460.
75. Wang, H. and Friedman, E. (1987) Protein kinase C: regulation of
serotonin release from rat brain cortical slices. Eur. J. Pharm. 141:15.
76. Matthies, H., Palfrey, H., Hirning, L. and Miller, R. (1987) Down-
regulation of protein kinase C in neuronal cells: Effects on
neurotransmitter release. J. Neurosci. 7:1192.
77. Tanaka, C., Taniyama, K. and Kusonoki, M. (1984) A phorbol ester and
A23187 act synergistically to release acetylcholine from the guinea pig
ileum. FEBSLett. 175:165.
78. Honegger, P. (1986) Protein kinase C-activating tumor promoters
enhance the differentiation of astrocytes in aggregating fetal brain cell
cultures. J. Neurochem. 46:1561.
79. Yong, V., Sekiguchi, M. and Kim, S. (1988) Phorbol ester enhances
morpholigical differentiation of oligodendrocytes in culture. J. Neurosci.
Res. 19:187.
80. MacVicar, B., Crichton, S., Burnard, D. and Tse, F. (1987) Membrane
conductance oscillations induced in astrocytes induced by phorbol ester.
Nature 329:242.
81. Grunberger, G. and Gorden, P. (1982) Affinity alteration of insulin
receptor induced by a phorbol ester. Amer. J. Physiol. 243:E319.
82. Haring, H., Kirsch, D., Obermaier, B., Ermel, B. and Machicao, F. (1986)
Tumor-promoting phorbol esters increase the Km of the ATP-binding site
of the insulin receptor kinase from rat adipocytes. J. Biol. Chem.
261:3869.
83. Thomopoulos, P., Testa, U., Gourdin, M., Hervy, C.,Titeux, M. and
Vainchenker, W. (1982) Inhibition of insulin receptor binding by phorbol
esters. Eur. J. Bchm. 129:389.
84. Grunberger, G., lacopetta, B., Carpentier, J. and Gorden, P. (1986)
Diacylglycerol modulation of insulin receptor from cultured human
mononuclear cells. Diabetes 35:1364.


120
This TPA-induced translocation of PKC preceded TPA's other effects
in glial cells and thus, might have been involved in the mechanism.
That those effects continued to occur long after the translocation
even as PKC was downregulated suggested that there were changes
beyond the translocation of PKC. TPA induced a downregulation of
neuronal ¡PKC within hours as well. The increase in membranous
iPKC, which would demonstrate a translocation, was only seen in
some experiments. This might have been because the cytosolic ¡PKC
represented a much smaller fraction in the neurons and its
translocation was too small to be observed. It was equally likely
that the particular isozymes identified by this antibody were not
translocated, although others were. In any case, the smaller
fraction of iPKC available to be translocated may play a role in
PKC's different effects in the different types of cells.
Thus, insulin receptors are differentially regulated by phorbol
esters in neurons and glial cells in the central nervous system.
These differences between the types of cells with regard to the
synthesis and function of insulin and the regulation of its receptors
are appropriate for the cells' requirements. In glial cells, which
continue to grow and multiply throughout the life of the organism,
insulin serves a metabolic function. In neurons, insulin's primary
effects are not metabolic in nature. The receptors are differentially
regulated accordingly.


136
156. Michell, R. (1975) Inositol phospholipid and cell surface receptor
function. Bchm. Bphys. Acta 415:81.
157. McAllister, J. and Hornsby, P. (1987) TPA inhibits the synthesis of
androgens and cortisol and enhances the synthesis of non-17 alpha*
hydroxylated steroids in cultured human adrenocortical cells. Endocr.
121:1908.
158. Welch, W. (1985) Phorbol ester, calcium ionophore or serum added to
quiescent rat embryo fibroblast cells all result in the elevated
phosphorylation of two 28,000-Dalton mammalian stress proteins. J.
Biol. Chem. 260:3058.
159. Yoshida, Y.t Huang, F., Nakabayashi, H. and Huang, K. (1988) Tissue
distribution and developmental expression of protein kinase C isozymes.
J. Biol. Chem. 263:9868.


CHAPTER V
THE REGULATION OF SUGAR TRANSPORT IN PRIMARY NEURONAL AND
GLIAL CELL CULTURES BY PHORBOL ESTERS
Introduction
Insulin has been shown to stimulate sugar uptake in astrocytic
glial cells from rat brain. This effect was time- and dose-
dependent with a maximal stimulation observed at 18 nM and a half-
maximal effect at 0.1 nM insulin (28). The latter value has been
shown to be well within the concentrations found in the brain (15)
indicating that the endogenous insulin is sufficient to induce this
stimulatory effect. This effect was unique to glial cells as neurons
prepared from the same brains failed to express similar properties.
Because of these and other observations it has been proposed that
insulin's lack of effect on sugar uptake in neurons may be due to the
absence of an intracellular pool of glucose transporters and/or an
inability of insulin to translocate intracellular transporters or to
activate membrane-bound transporters, or to a combination of all
three of these.
In this study we utilized TPA to determine its effects on
neuronal and glial glucose uptake as TPA has been shown to
stimulate glucose uptake in other tissues (97, 117-9). The
71


BIOGRAPHICAL SKETCH
Laura Mary Mudd was born to John and Barbara Mudd on September
24, 1958, in Washington, D.C. She is the eldest of five children.
John (attorney/corporate executive) and Barbara
(theologian/teacher) reside in Miami with one sister, Ellen
(insurance/accountant). Philip (political writer), Clare (artist) and
David (attorney) are currently living in Washington. Laura graduated
from LaSalle High School in Miami in 1976. She then attended
Georgetown University, where she studied chemistry and English
literature. Following graduation in 1980, she worked at Gillette
Research Institute in Maryland, where she studied steroid and
enzyme biochemistry. From 1984-89 she attended the University of
Florida, where she received her doctorate in physiology in the spring
of 1989. Her graduate work involved the regulation of growth
factors in cultured brain cells. She hopes to continue her study of
brain development with postdoctoral research following her
graduation. Laura also enjoys literature, sailing, cooking and the
companionship of her two cats, Langston and Electra Mudd.
137


Time (hours)


39
released under depolarizing conditions. The chromatographic
properties of the released immunoreactive insulin were then
compared to those of an insulin purified from rat pancreas by HPLC.
RESULTS
In this study, primary neuronal cultures from rat brains were
treated with depolarizing solutions containing a high concentration
of potassium (60mM) with or without calcium (2mM). After 30 min.
the solutions were aspirated and lyophilized and insulin was
quantitated by radioimmunoassay. Depolarized neuronal cultures
released more than three times as much insulin as saline controls
(103.2pU/ml vs 31.7pU/ml) in the presence of calcium. Potassium-
stimulated release was calcium-dependent as in the absence of
calcium, insulin release was negligible (34.0pU/ml, Figure 3-1).
The sodium ionophore, veratridine, similarly stimulated the release
of insulin from neurons by 379 percent. In contrast, release of
insulin from glial cultures was not stimulated by depolarization.
This, in combination with the evidence that no mRNA for insulin has
been demonstrated in glial cells and that glial cells are not
immunoreactive for insulin (18), suggested that the insulin released
from neuronal cultures was not due to glial contamination of those
cultures. The very low level of insulin released by glial cells may,
in fact, have represented insulin taken up from the medium. The
immunoprecipitable insulin that was released from neurons under
depolarizing conditions coeluted with an insulin purified from rat


[3H]2-dGlc Uptake
(nmol/mg protein/5 min)
96


Figure 7-6: The effect of dexamethasone on neuronal
iPKC. Dexamethasone (2.55 uM) was added to the
neuronal medium for the times specified. The cells were
then washed, prepared for Western blotting as described
in methods, and bands were read on a densitometer.
Densitometric readings are (from left to right) 1142,
1201, 2371, 1771, 2374 and 3661. The experiment was
repeated once.


Protein kinase C (PKC) occurs in many tissues, but it occurs at highest
concentration in the brain. It has been shown to regulate glucose uptake and
the insulin receptor in many peripheral tissues. PKC stimulates both glucose
uptake and insulin binding in glial cells without affecting either in neurons. The
stimulation of insulin receptor binding in glial cells is not accompanied by an
increase in activity; thus it appears to inactivate the receptors. Activation of PKC
by phorbol esters, as evidenced by translocation from the cytosol to the
membrane, occurs in both neurons and glial cells, although the fraction of
cytosolic, and thus, translocatable, PKC is much smaller in the former. In glial
cells, Translocation of PKC precedes other effects of PKC, and thus, may be
involved in the mechanism. Insulin stimulates the level of im mu noreactive PKC
;n glial cells, but not in neurons; thus, in glial cells, this appears to be a
feedback loop whereby insulin regulates its own receptor. In neurons, both
arms of the circuit, that is, regulation of the PKC concentration by insulin and
regulation of the insulin receptor by PKC, are missing. That neuronal and glial
insulin receptors are differentially regulated is not surprising as the receptors
differ from one another with respect to both structure and function.
v


40
pancreas (supplied by Eli Lilly) when subjected to reverse phase
HPLC. Both the released material and the standard showed peaks for
both rat 1 insulin and rat 2 insulin (Figure 3-2).
When primary neuronal cultures were treated with D-glucose
(0.1-0.6 percent) in the presence of 2mM calcium, a dose-dependent
stimulation of insulin release was observed (Figure 3-3). Neuronal
cultures were exposed to [^Hj-leucine for 24 hours prior to a timed-
release experiment. These cultures were washed and incubated at
37C with control and depolarizing solutions which were aspirated
from the cultures at time intervals from 1 to 60 min. A biphasic
pattern of insulin release was observed (Figure 3-4); this pattern of
release was similar to that observed with stimulation of pancreatic
insulin release by glucose. The fact that exogenous [^Hj-leucine was
incorporated into the immunoreactive insulin suggested that this
insulin was synthesized within the neurons.
Discussion
There is some controversy as to the origin of insulin in the brain.
Some investigators have shown that the concentration of insulin in
the brain is a fraction of that found in the plasma when quantitated
by different antibodies than the one first used by Havrankova, et al.
(16, 14). These same investigators have also found that the
concentration of insulin in the CSF is dependent on the plasma
concentration. They have suggested that these two pieces of
information, in conjunction with the lack of a demonstrable
proinsulin in the brain and the inability of one investigator to
demonstrate an mRNA for insulin in brain prove that insulin is not


Figure 8-1: Proposed regulation of insulin effector
systems in the CNS. As the concentration of glucose
rises in the CNS, release of insulin from insulinergic
neurons increases in a dose-dependent manner. Insulin in
the brain binds to specific, high affinity receptors (R) on
both neurons and glial cells. Insulin acts to decrease
norepinephrine uptake in neuronal cells (30). In glial
cells, insulin acts to stimulate glucose uptake (28) by
activating and/or translocating glucose transporters
(GT), thus decreasing the stimulus for its own release by
a form of negative feedback.
Insulin acts in glial cells to increase PKC. PKC feeds
back to inactivate the glial insulin receptor and inhibit
insulin-stimulated glucose uptake, providing another
level of control over the glucose concentration within
the cell and in the interstitial spaces. Both arms of the
circuit, that is, stimulation of iPKC by insulin and
inhibition of the insulin receptor by PKC are missing in
the neurons.


73
effects that occur as a result of PKC's other actions in the brain,
such as neurotransmitter release (75-77), or alterations in
neuronal ion channels (69-710 or membrane conductance in
astrocytes (80)


60


Figure 4-4: TPA-induced PKC translocation in astrocytic
glial cells. One hundred nM TPA was added to the medium
of confluent glial cultures for the times specified,
following which samples were separated into cytosolic
(C) and membrane (M) fractions and subjected to gel
electrophoresis and Western blotting as described in
Methods. Each lane represents 165 ug protein.
Densitometric values for the 80 kD band are (from left to
right), 885, 732, 229, 1688, 29, 952, 9, 540, 0, 471, 0, 0.
This is one representative experiment of three.


80
insulin binding. This is to be expected as translocation precedes and
is necessary for other PKC-induced effects, both centrally and
peripherally (61-4). This increase in the binding of insulin could
also be interpreted as an increase in ionternalization of insulin.
Further studies using an acid wash to separate bound versus
internalized insulin will be necessary to resolve this question.
As was demonstrated, treatment with TPA for 24 hours
downregulates PKC in the brain. As the binding of insulin in glial
cells recovered to the level of the control, but not below, 24 hours
after TPA administration and removal, it appears that the binding of
insulin in glial cells is not under chronic control by PKC.
PKC-induced inactivation of receptors has generally been
associated with a decrease in binding of the appropriate ligand in
studies in peripheral tissues. This example of differential
regulation of brain and peripheral insulin receptors as well as
distinct regulation of insulin receptors in neuronal and glial ceils is
by no means unprecedented. Insulin, which generally downregulates
its receptor peripherally, downregulates its receptor in glia but
upregulates the receptor in neurons (148). Glucocorticoids, which
increase the binding of insulin in hepatocytes (149) and
lymphocytes(150), have no effect in adipocytes (151) and decrease
binding in an astrocytic cell line (152). As insulin has different
actions in different types of cells, it is to be expected that
regulation of insulin receptors might vary among cell and tissue
types.
TPA induces insensitivity to insulin insofar as glial 2-dGlc
uptake is concerned. This is true even at doses of TPA which elicit a


7
another (55-6) and with respect to the development of the organism
(53,57), however, the kinetic properties of the isozymes are very
similar. For a review see Nishizuka (54).
Tumor-promoting phorbol esters, such as 12-O-tetradecanoyl-
phorbol-13-acetate (TPA), act as exogenous stimulators of PKC (58).
Upon activation by TPA or calcium, there is a rapid decrease in
cytoplasmic PKC and a corresponding increase in membrane PKC (59-
60, Figure 1-1). Following this translocation, the membrane-bound
PKC catalyzes the phosphorylation of specific proteins (61-2).
Translocation appears to mediate other activities of PKC such as
neuronal potentiation (63) as well. Prevention of PKC
redistribution, as with concanavalin A, has been shown to block
activation of PKC (64). Once activated, PKC acts in many types of
cells to block hormone-stimulated phosphoinositide hydrolysis, and
thus exerts negative feedback over its own activation (65-8).
PKC has different effects in different tissues (48). In the brain
PKC is involved in the regulation of neuronal ion channels (69-71),
synaptic plasticity (72-3), neurite outgrowth (74) and
neurotransmitter release (75-7), as well as the differentiation of
astrocytes and oligodendrocytes (78-9) and changes in membrane
conductance in astrocytes (80). Many of the varied effects of PKC
appear to result from its interactions with growth factor receptors
with tyrosine kinase activities. PKC-induced receptor
phosphorylations alter the affinities, activities and effects of some
of these receptors. These growth factors, in turn, have been shown
to regulate either content or activity of PKC in certain cells.


46


124
12. Kahn, C. (1985) The molecular mechanism of insulin action. Annu. Rev.
Med. 36:429.
13. Guyton, A. (1976) Textbook of Medical Physiology. (5th ed.) W.B.
Saunders Co., Philadelphia.
14. Havrankova, J., Schmechel, D., Roth, J. and Brownstein, M. (1978)
Identification of insulin in rat brain. Proc. Natl. Acad. Sci. USA. 75:5737.
15. LeRoith, D., Hendricks, S.t Lesniak, M.f Rishi, S., Becker, K.,
Havrankova, J., Rosensweig, J., Brownstein, M. and Roth, J. (1983)
Insulin in brain and other extrapancreatic tissues of vertebrates and
nonvertebrates. In: Advances in Metabolic Disorders. Vol. 10 Szabo, A.,
ed. Academic Press, New York.
16. Yalow, R. and Eng, J. (1983) Insulin in the central nervous system. In:
Advances in Metabolic Disorders. Vol. 10 Szabo, A. ed. Academic Press,
New York.
17. Birch, N., Christie, D. and Renwick, A. (1984) Immunoreactive insulin
from mouse brain cells in culture and whole rat brain. Biochem. J.
218:19.
18. Raizada, M. (1983) Localization of insulin-like immunoreactivity in the
neurons from primary cultures of rat brain. Exp. Cell Res. 143:351.
19. Havrankova, J., Roth, J. and Brownstein, M. (1979) Concentrations of
insulin and of insulin receptors in the brain are independent of peripheral
insulin levels. J. Clin. Invest. 64:636.
20. Budd, G., Pansky, B. and Cordell, B. (1986) Detection of insulin
synthesis in mammalian anterior pituitary cells by immunohistochemistry
and demonstration of insulin-related transcripts by in situ RNA-DNA
hybridization. J. Histochem. Cytochem. 34:673.
21. Young, W. (1986) Periventricular hypothalamic cells in the rat brain
contain insulin mRNA. Neuropep. 8:93.
22. Schechter, R., Holtzclaw, L, Sadiq, F., Kahn, A. and Devaskar S. (1988)
Insulin synthesis by isolated rabbit neurons. Endocr. 123:505.
23. Clarke, D., Poulakos, J., Mudd,.L., Raizada, M. and Cooper, D. (1986)
Evidence for central nervous system insulin synthesis. In: Insulin.
Insulin-like Growth Factors and Their Receptors in the Central Nervous
System. Plenum Press, New York.
24. Villa-Komaroff, L, Gonzalez, A., Song, H., Wentworth, B., Dobnes, P.
(1984) Novel insulin-related sequences in fetal brain. Adv. Exp. Med.
Biol. 181:65.


128
61. Jacobs, S., Sahyoun, N., Saltiel, A. and Cuatrecasas, P. (1983) Phorbol
esters stimulate the phosphorylation of receptors for insulin and
somatomedin C. Proc. Natl. Acad. Sci. USA 80:6211.
62. Cochet, C., Gill, G., Meisenhelder, J., Cooper, J. and Hunter, T. (1984)
C-Kinase phosphorylates the epidermal growth factor receptor and
reduces its epidermal growth factor-stimulated tyrosine protein kinase
activity. J. Biol. Chem. 259:2553.
63. Akers, R.} Lovinger, D., Colley, P., Linden, D. and Routenberg, A. (1986)
Translocation of protein kinase C activity may mediate hippocampal
long-term potentiation. Science 231:587.
64. Patel, J. and Kassis, J. (1987) Concanavalin A prevents phorbol-
mediated redistribution of protein kinase C and beta-adrenergic
receptors in rat glioma C6 cells. Bchm. Bphys. Res. Comm. 144:1265.
65. Orrelano, S., Solski, P. and Heller-Brown, J. (1985) Phorbol ester
inhibits phosphoinositide hydrolysis and calcium mobilization in cultured
astrocytoma cells. J. Biol. Chem. 260:5236.
66. Drummond, A. (1985) Bidirectional control of cytosolic free calcium by
thyrotropin-releasing hormone in pituitary cells. Nature 315:752.
67. Naccache, P., Molski, T., Borgeat, P., White, J. and Shaafi, R. (1985)
Leu-Phe- and leukotriene B4-stimulated calcium mobilization and
enzyme secretion in rabbit neutrophils. J. Biol. Chem. 260:2125.
68. Vicentini, L., Virgilio, F., Ambrosini, A., Pozzan, T. and Mendolesi, J.
(1985) Tumor promoter phorbol 12-myristate, 13-acetate inhibits
phosphoinositide hydrolysis and Ca++ rise induced by the activation of
muscarinic receptors in PC12 cells. Bchm. Bphys. Res. Comm. 127:310.
69. Baraban, J., Snyder, S. and Alger, B. (1985) Protein kinase C-regulated
ionic conductance in hippocampal pyramidal neurons:
Electrophysiological effects of phorbol esters. Proc. Natl. Acad. Sci. USA
82:2538.
70. Kaczmarek, L. (1986) Phorbol esters, protein phosphorylation and the
regulation of neuronal ion channels. J. Exp. Biol. 124:375.
71. Strong, J., Fox, A., Tsien, R. and Kaczmarek, L. (1987) Stimulation of
protein kinase C recruits covert calcium channels in Aplysia bag cell
neurons. Nature 325:714.
Routtenberg, A. (1987) Phospholipid and fatty acid regulation of signal
transduction at synapses: potential role for protein kinase C in
information storage. J. Neur. Transm. (Suppl.)24:239.
72,


81
less than maximal increase in binding. This suggests that these
glial insulin receptors, like their peripheral counterparts, may be
inactivated by PKC. Inactivation of tyrosine kinase prevents
internalization of insulin receptors in Chinese hamster ovary cells
(93). A similar proposal could be made for the glial insulin receptor.
The most intriguing aspect of this study concerned TPAs lack of
effect on neuronal insulin binding, although this does not necessarily
indicate that PKC does not inactivate the receptors. Both neurons
and glia contain a PKC which is capable of binding phorbol esters,
and neurons and glia both respond physiologically when stimulated
by phorbol esters (69-80). PKC is certainly involved in neuronal
receptor regulation as phorbol esters induce an increase in the
binding of angiotensin II by a calcium-dependent mechanism (153).
Thus, TPAs failure to alter the binding of insulin in neurons can
neither be attributed to a lack of PKC, nor to a non-functional PKC.
As PKC's effects on the insulin receptor are direct effects (90), the
different effects of TPA on the binding of insulin must necessarily
be due to differences in neuronal and glial PKC or to structural
differences within the insulin receptors themselves.


CHAPTER II
METHODS
Preparation of Primary Neuronal Cultures from Rat Brains
Brains from 8-10 one-day-old rats were removed in a sterile
manner from the cranium at the level of the medulla oblongata and
placed in an isotonic buffer solution containing 100 units penicillin,
100pg streptomycin and 2.5jig Fungizone/ml. All pia mater and
blood vessels were removed and the brains were minced into 1-
2mm3 pieces. Brain cells were dissociated by trypsin and
deoxyribose treatment as described previously (18). Dissociated
cells were suspended in 50 ml of Dulbecco's modified Eagle's
medium (DMEM) containing 10 percent plasma-derived horse serum
(PDHS) and sedimented at 1000 x g for 10 minutes at 24C. The cell
pellet was suspended in the same medium at a concentration of 1.5 x
10$ cells/ml. Fifteen million cells were placed in each Falcon
tissue culture dish pretreated with poly-L-lysine and the cells were
placed in a humidified incubator at 37C with 6 percent C02/94
percent air. The cells began to attach immediately and 90-96
percent were attached to culture dishes within 30-60 min. After
three days in culture, the cells were treated with 10|iM cytosine
20


133
119. Clarke, D., Ramaswamy, A., Holmes, L, Mudd, L., Poulakos, J. and
Raizada, M. (1987) Phorbol esters stimulate 2-deoxyglucose uptake in
glia but not neurons. Brain Res. 421:358.
120. Levi-Montalcini, R. and Angeletti, R. (1968) Nerve growth factor.
Physiol. Rev. 48:534.
121. Shemer.J., Raizada, M., Masters, B., Ota, A. and LeRoith, D. (1987)
Insulin-like growth factor 1 receptors in neuronal and glial cells:
Characterization and biological effects in primary culture. J. Biol. Chem.
262:7693.
122. Wang, S., Shiverick, K.t Ogilvie, S., Dunn, W. and Raizada, M. (1989)
Characterization of epidermal growth factor receptors in astrocyte glial
and neuronal cells in primary culture. Endocr. 124:240.
123. Tarris, R., Weichsel, M. and Fisher, D. (1986) Synthesis and secretion of
a nerve growth-stimulating factor by neonatal mouse astrocyte cells in
vitro. Pediat. Res. 20:367.
124. Bunge, R., and Waksman, B. (1985) Glial development and interactions.
Trends in Neurosci. 424.
125. Liuzzi, F. and Lasek, R. (1987) Astrocytes block axonal regeneration in
mammals by activating the physiological stop pathway. Science
237:642.
126. Meshul, C., Seil, F. and Herndon, R. (1987) Astrocytes play a role in
synaptic density. Brain Res. 402:139.
127. Kimelberg.H. (ed) (1988) Glial Cell Receptors. Raven Press, New York.
128. Richards, E., Raizada, M. and Sumners, C. (1987) Insulin
downregulates alpha-2 adrenergic receptors in cultured glial cells. In:
Insulin, Insulin-like Growth Factors and Their Receptors in the Central
Nervous System. Raizada, M., Phillips, M. and LeRoith, D. (eds.) Plenum
Press, New York.
129. Walz, W. and MacVicar, B. (1988) Electrophysiologicai properties of
glial cells: Comparison of brain slices with primary cultures. Brain Res.
443:321.
130. Boyd, F. (1985) Characterization and Physiological Significance of
Brain Insulin Receptors. Dissertation, Univ. of Fla.
131. Raizada, M. Yang, J. and Fellows, R. (1980) Binding of 125l-insulin to
specific receptors and stimulation of nucleotide incorporation in cells
cultured from rat brain. Brain Res. 200:389.


CHAPTER IV
CHARACTERIZATION OF PKC IN NEURONAL AND GLIAL CELLS IN
PRIMARY CULTURES
Introduction
PKC is present In both neurons and glial cells from the brain
(55,110-11). It is present in dendrites, axons, perikarya and nuclei
(110) of neurons with particularly high concentrations in
presynaptic terminals (111) and in growth cones (114). The last two
are predictable, as PKC mediates both neurotransmission and neurite
outgrowth (115). In glial cells, PKC is involved in the regulation of
differentiation (78) and membrane conductance (80). Thus, PKC is
also physiologically active in both types of cells, although its
activity takes different forms in neurons and glial cells.
Because of these differences and the existence of different
isotypes of PKC we elected to study neurons and glia in culture to
determine the relative quantities of PKC, the relative molecular
weight (Mr) of PKC and the distribution of PKC within the cells. As
TPA-activation of PKC involves translocation of the enzyme, we
chose to observe the time-course of the translocation to determine
whether this differed in the different types of cells.
51


0 min
15 min
1 hr
2 hr
4 hr
24 hr
80KD
110


100
inappropriate for PKC to feed back negatively on a receptor which
apparently does not regulate it in the first place. Because an
antibody which did not bind all subtypes of PKC was used, however,
there is a possibility that negative results were false negatives;
that is'insulin may induce an increase in the gamma subtype of PKC.
The increase in glial PKC in response to insulin occurs in the
cytoplasmic fraction, and thus, the enzyme is likely to be active.
Although insulin stimulates immunoreactive PKC in glial cells,
insulin's effect on aPKC has yet to be determined. Activity does not
necessarily increase with increased concentration as demonstrated
by the effect of PKC on the insulin receptor.
Dexamethasone has exactly the opposite effect. While
glucocorticoids have peripheral metabolic effects which are in
opposition to those of insulin (13), the relationship between
glucocorticoids and PKC is, on the surface, similar to that between
insulin and PKC. The dexamethasone stimulation of neuronal, but not
glial, immunoreactive PKC indicates that the effect of insulin is not
a general one. The effect of PKC on glucocorticoid receptors in
neuronal and glial cells is not known. It may be that PKC acts
differentially on those as well.


REGULATION OF INSULIN EFFECTOR SYSTEMS IN THE BRAIN
BT
LAURA MARY MUDD
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989

TABLE OF CONTENTS
PAGE
ABSTRACT iv
CHAPTER
I. INTRODUCTION 1
Insulin and Insulin Receptors in the Brain 2
Insulin in the Brain: Potential Functions 4
Protein Kinase C (PKC): Regulation of Insulin and IGF I
Receptors 6
Ligand-Receptor Interactions 8
Phosphorylation of Receptors 8
Receptor-Induced Effects 10
Effects ofGrowth Factors on PKC 11
PKC in the Brain: Distribution 12
PKC Regulation of Glucose Uptake and Insulin Receptors
in the Brain 13
Cultured Brain Cells: A Model for the Study of Neurotrophic/
Neuroactive Substances 14
II. METHODS 20
Preparation of Primary Neuronal Cultures from Rat Brains 20
Preparation of Primary Astrocytic Glial Cultures from Rat
Brains 20
Immunocytochemistry of Neuron-Specific Enolase/Glial
Fibrillary Acidic Protein 22
Neuronal Depolarization 23
Insulin Radioimmunoassay 23
Characterization of Immunoprecipitable Insulin by HPLC 24
Labelling of Immunoreactive Insulin in Neuronal
Cultures 25
Characterization of the Regulation of Neuronal Insulin Release
by Glucose 25
2-Deoxy-D-glucose (2-dGlc) Uptake 26
lodination of Insulin 26
Insulin Binding 28
Protein Determinations 29

PKC Immunocytochemistry 31
Statistical Analysis 31
III. INSULIN SYNTHESIS AND RELEASE FROM NEURONAL CULTURES.37
Introduction 37
Results 39
Discussion 40
IV. CHARACTERIZATION OF PKC IN NEURONAL AND GLIAL CELLS
IN PRIMARY CULTURE 51
Introduction 51
Results 52
Discussion 53
V. THE REGULATION OF SUGAR TRANSPORT IN PRIMARY
NEURONAL AND GLIAL CELL CULTURES BY PHORBOL
ESTERS 71
Introduction 71
Results 72
Discussion 72
VI. THE REGULATION OF INSULIN RECEPTORS IN NEURONAL AND
GLIAL PRIMARY CULTURES BY PHORBOL ESTERS 76
Introduction 76
Results 77
Discussion 79
VII. THE EFFECTS OF INSULIN AND DEXAMETHASONE ON NEURONAL
AND GLIAL PKC 97
Introduction 97
Results 98
Discussion 99
VIII. DISCUSSION AND SUMMARY 113
REFERENCES 123
BIOGRAPHICAL SKETCH
137

Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
REGULATION OF INSULIN EFFECTOR SYSTEMS IN THE BRAIN
By
Laura Mary Mudd
May, 1989
Chairman: Mohan K. Raizada
Major Department: Physiology
In spite of insulin's effects on glucose uptake in the periphery and the fact
that the brain is a glucose-obligate organ, the brain was long believed to be
insulin-independent because insulin does not cross the blood-brain barrier.
Many reports over the course of the past ten years have localized both insulin
and insulin receptors in the brain, although the source and function of the
peptide in the brain and the mechanisms by which receptors are regulated have
remained the subjects of some mystery.
It was found that an immunoreactive insulin was released from cultured
neurons under depolarizing conditions, that labelled leucine could be
incorporated into the released peptide and that the peptide coeluted with a rat
insulin standard when the two were applied to a reverse-phase high pressure
liquid chromatography column. Thus, the peptide resembles insulin
chromatographically as well as immunologically, is synthesized in neuronal
cultures and is released under depolarizing conditions. In the brain, insulin
may act as a neurotransmitter.
IV

Protein kinase C (PKC) occurs in many tissues, but it occurs at highest
concentration in the brain. It has been shown to regulate glucose uptake and
the insulin receptor in many peripheral tissues. PKC stimulates both glucose
uptake and insulin binding in glial cells without affecting either in neurons. The
stimulation of insulin receptor binding in gliai cells is not accompanied by an
increase in activity; thus it appears to inactivate the receptors. Activation of PKC
by phorbol esters, as evidenced by translocation from the cytosol to the
membrane, occurs in both neurons and glial cells, although the fraction of
cytosolic, and thus, translocatable, PKC is much smaller in the former. In glial
cells, Translocation of PKC precedes other effects of PKC, and thus, may be
involved in the mechanism. Insulin stimulates the level of immunoreactive PKC
in glial cells, but not in neurons; thus, in glial cells, this appears to be a
feedback loop whereby insulin regulates its own receptor. In neurons, both
arms of the circuit, that is, regulation of the PKC concentration by insulin and
regulation of the insulin receptor by PKC, are missing. That neuronal and glial
insulin receptors are differentially regulated is not surprising as the receptors
differ from one another with respect to both structure and function.
v

CHAPTER I
INTRODUCTION
Historically, Insulin has been used in the treatment of diabetes
mellitus and psychosis. Pancreatic Insulin was first isolated in
1922 at which time it was used clinically in the treatment of Type I
diabetes (1). In classical Type I diabetes the insulin-secreting 13-
cells of the pancreas are destroyed and the patients become
dependent on exogenous insulin to control their blood sugar. Several
years after Banting and Best began using insulin to treat diabetes,
Schmidt (2) and Sakel (3) separately reported successful treatment
of psychotic patients with insulin in the presence and absence of
carbohydrate, respectively, that is, treatment with and without
hypoglycemic shock. Insulin-induced hypoglycemic shock was used
successfully in the treatment of schitzophrenia and depression as
well as psychosis until the development of electroshock therapy and
more sophisticated psychoactive drugs (4).
The mechanisms of pancreatic insulin synthesis and release, as
well as physiological effects on liver, muscle and adipose tissue,
have been well characterized. Pancreatic insulin is synthesized as
preproinsulin, cleaved to proinsulin almost immediately and
1

2
packaged into secretory granules where the C-peptide is cleaved
prior to secretion (5). Basal beta-cell secretion of insulin is about
0.5 U/hour. This can be increased 10-30 times with acute
stimulation. Glucose is the most potent stimulator of pancreatic
insulin release in man (6) although amino acids (especially arg, lys,
leu and val), glucagon, IB-adrenergic and vagal stimulation of the
pancreas stimulate secretion as well (7,8). Somatostatin,
serotonin, dopamine, prostaglandin E and splanchnic stimulation are
inhibitory to insulin release (8,9). Chronic glucose stimulation
induces a biphasic release of insulin (10); this is thought to be due
to the release of short- and long-term insulin stores (8).
The first step in the action of insulin on the peripheral target
tissues is its interaction with specific receptors on the cell
surface. This interaction of insulin with its receptor stimulates
autophosphorylation of a tyrosine residue (11) as well as receptor
tyrosine kinase activity (12). Binding of insulin induces an increase
in glucose and amino acid uptake by liver, muscle and adipose
tissues and consequently an increase in the synthesis of glycogen
and protein with a corresponding decrease in lipolysis and
gluconeogenesis (13).
Insulin and Insulin Receptors in the Brain
In spite of insulin's effects on glucose uptake in the periphery
and the fact that the brain is a glucose-obligate organ, the brain was
long believed to be insulin-independent because insulin does not
cross the blood-brain barrier. Havrankova et al., established the
presence of insulin in the brain at concentrations that averaged 25

3
times that of plasma insulin (14). Since then many reports have
appeared in the literature confirming this finding although the
actual amount of insulin in the brain is the subject of some
controversy (15,16). Insulin is found in cultures from mouse brain
and neuronal cultures from rat brain (17,18). The presence of this
insulin-like peptide in the brain raises two major questions; what
is the source of this peptide and what is its function?
The following observations suggest that insulin may be
synthesized in the brain: (a) central insulin concentrations appear to
remain constant in pathological situations in which peripheral
insulin concentrations vary widely (19); (b) insulin-like activity
has been demonstrated in cultured brain cells which are removed
from pancreatic insulin for weeks (18); (c) radioactive amino acids
can be incorporated into an insulin-like peptide in cultured neurons
by a cycloheximlde-sensitive process (18) and (d) there is an mRNA
in the brain which hybridizes with insulin cDNA (20-4). In addition,
our experiments have shown that insulin is synthesized and released
by neurons (25). Furthermore, when neuronal cultures were labelled
with leucine and depolarized, a labelled peptide was released which
could be precipatated by an insulin antibody. Cycloheximlde
decreased insulin synthesis by 80% (18).
In recent years specific insulin receptors in the brain have been
demonstrated conclusively (26). These receptor sites, which are
distributed non-uniformly throughout the brain (27), are evenly
distributed between neurons and glial cells (28-30). Studies have
shown that neuronal and glial insulin receptors are structurally and
physiologically distinct in several respects. The brain insulin

4
receptor is generally similar in subunit structure to the more
familiar peripheral insulin receptors although the alpha-subunit Is
somewhat smaller in the brain (31) due to decreased glycosylation
of the receptor in the brain. In particular, the neuronal alpha- and
beta-subunits are smaller, while the glial subunits resemble those
from the liver (29,32) in size. That the glial receptor Is
structurally similar to the peripheral receptor Is not surprising,
since insulin appears to have traditional metabolic effects in glia
and neuromodulatory effects in neurons. Insulin stimulates glucose
uptake by glia, but not by neurons (28) and inhibits norepinephrine
uptake by neurons, but not by glia (30).
Insulin ..in the Brain: Potential. .FiiocliQns
Insulin in the central nervous system (CNS) has been implicated
in the control of brain growth and development (28,33-5),
catecholamine release (36-9), and diabetes (40) in in vitro studies
and satiety (41) in in vivo studies. In addition, insulin may itself
act as a neurotransmitter in the CNS. In vitro studies have been
essential in developing an understanding of insulin's actions in the
CNS at the cellular level. With regard to insulin's role in growth and
development, insulin stimulates DNA, RNA and protein synthesis in
mixed brain cell cultures and astrocyte glial cultures. It also
stimulates 2-deoxy-D-glucose uptake in astrocyte glial cultures
(28,33). These are all events which are associated with cell growth
and proliferation. In addition, insulin has differentiative effects on
developing rat brain. When immature rat retinal cultures were
exposed to insulin, precocious synaptic release of acetylcholine was

5
evoked within one hour while control and glutamate treatment of the
cells elicited no such response (34). Insulin induces ornithine
decarboxylase activity and neurite outgrowth in cultured embryonic
mouse brain cells and rat brain cultures (35). Thus, insulin induces
maturation of brain cells in culture as well as growth.
Insulin alters the content of serotonin and catecholamines in the
brain by increasing the rate of uptake of their precursors (36-8). It
also stimulates the release of dopamine, epinephrine and
norepinephrine from hypothalamic slices (39) and changes the firing
rates of neurons in the striatum (42) and hippocampus (43). Insulin,
then, definitely acts as a neuromodulator in the CNS and it may act
as a neurotransmitter itself. Recently, the neuromodulatory effects
of insulin have been suggested to be mediated by insulin receptors
present on the neurons (44). Insulin has, then, satisfied several
criteria by which putative neurotransmitters are classified as such:
it is synthesized in neurons, released under depolarizing conditions,
bound with specificity and high affinity to a receptor, degraded and
modulates neuronal activity.
CNS insulin may be involved in the pathophysiology of obesity and
Type II, or insulin-independent, diabetes. In vivo studies in baboons
indicate that insulin has a direct effect on satiety (41). When
insulin was infused into the lateral cerebral ventricles, a
significant, dose-related decrease in food intake and body weight
was observed. This may occur by interaction with neurons in the
hypothalamus. Injection of insulin in low doses causes electrical
activity to increase in hypothalamic neurons (45). In addition to its
association with obesity, central insulin may play a role in Type II

6
diabetes. Lesions of the ventromedial hypothalamus cause
hypersecretion of hypothalamic insulin (46).
In studies involving cultures of brain cells from diabetic mice
the ratio of externalized insulin receptors to total insulin receptors
was substantially decreased in cultures from diabetic mice versus
control cultures (40). As obesity is a major risk factor for Type II
diabetes there may be a relationship between these two effects of
central insulin. This hypothesis is supported by the observation that
insulin receptor number is significantly decreased in the olfactory
bulb of obese rats (47).
Protein Kinase C (PKC); Regulation of Insulin and IGF I Receptors
PKC is a serine/threonine kinase which is present in many
tissues but occurs at highest concentrations in brain (48). PKC is
calcium-dependent and 1,2-diacylglycerol (DAG) , a product of
membrane phospholipid metabolism, increases the affinity of the
enzyme for calcium (49-50). Reports of the molecular weight of
PKC vary. The different values may reflect the method by which the
relative molecular weight (Mr) is determined, as evidenced by a
study in which values of 77,000 and 82,000 daltons were obtained
from sucrose density gradient and polyamide gel electrophoresis,
respectively (51). Differences may also be attributable to subunit
aggregation (52) or to the existence of different isozymes of PKC
(53-4). Currently, seven highly-homologous isozymes of PKC have
been isolated and characterized. Four are single polypeptide chains
with four constant and five variable regions, while three subspecies
differ slightly. The isozyme distributions differ with respect to one

7
another (55-6) and with respect to the development of the organism
(53,57), however, the kinetic properties of the isozymes are very
similar. For a review see Nishizuka (54).
Tumor-promoting phorbol esters, such as 12-O-tetradecanoyl-
phorbol-13-acetate (TPA), act as exogenous stimulators of PKC (58).
Upon activation by TPA or calcium, there is a rapid decrease in
cytoplasmic PKC and a corresponding increase in membrane PKC (59-
60, Figure 1-1). Following this translocation, the membrane-bound
PKC catalyzes the phosphorylation of specific proteins (61-2).
Translocation appears to mediate other activities of PKC such as
neuronal potentiation (63) as well. Prevention of PKC
redistribution, as with concanavalin A, has been shown to block
activation of PKC (64). Once activated, PKC acts in many types of
cells to block hormone-stimulated phosphoinositide hydrolysis, and
thus exerts negative feedback over its own activation (65-8).
PKC has different effects in different tissues (48). In the brain
PKC is involved in the regulation of neuronal ion channels (69-71),
synaptic plasticity (72-3), neurite outgrowth (74) and
neurotransmitter release (75-7), as well as the differentiation of
astrocytes and oligodendrocytes (78-9) and changes in membrane
conductance in astrocytes (80). Many of the varied effects of PKC
appear to result from its interactions with growth factor receptors
with tyrosine kinase activities. PKC-induced receptor
phosphorylations alter the affinities, activities and effects of some
of these receptors. These growth factors, in turn, have been shown
to regulate either content or activity of PKC in certain cells.

8
Ligand-Receptor Interactions
PKC regulates the specific binding of insulin and insulin-like
growth factor I (IGF I). Phorbol esters regulate the insulin receptor
in lymphocytes, adipocytes and monocytes (81-3). In each of these
types of cells, phorbol esters inhibit the binding of insulin by
increasing the Km of the high affinity receptor. The endogenous
analogues of TPA, the DAGs, also reversibly inhibit the binding of
insulin to its receptor (84) by altering the affinity of the receptor.
The calcium ionophore A23187 potentiates the effect of TPA on
insulin binding in monocytes (85). TPA does not, however, decrease
insulin binding in all cell types. TPA has no effect on insulin binding
in either 3T3 cells or in hepatoma cells (86-7) although the
response to insulin is impaired in the latter. Thus, PKC-induced
decreases in insulin binding may result from either decreased
affinity of the receptors or increased internalization of the insulin-
receptor complex, depending on the type of tissue or cell.
The case for regulation of the IGF I receptor by PKC is similar to
that for the insulin receptor. DAGs inhibit IGF I binding to the IGF I
receptor in monocytes. TPA also inhibits IGF I binding in
lymphocytes, monocytes and adipocytes by altering the high-affinity
binding site without altering the number of receptors (84). This
differs from insulin or IGF I down-regulation of the IGF I receptor,
which results from a decrease in the number of receptors (88).
Phosphorylation of Receptors
The mechanism for PKC-stimulated alterations in the insulin and
IGF I receptors appears to involve serine/threonine phosphorylation

9
of those receptors. In 1983, TPA was first shown to stimulate
phosphorylation of both insulin and IGF I receptors in IM-9 cells that
had been preincubated with H332PC>4 (61). Insulin- and TPA-
stimulated phosphorylation appeared to be additive, suggesting that
there was no interaction between the sites. In 1984, TPA was
shown to enhance serine/threonine phosphorylation of the insulin
receptor in rat hepatoma cells at nine sites (87). Insulin was shown
to stimulate phosphorylation of tyrosine and serine residues at six
sites, three of which were similar to the TPA-phosphorylated sites.
In addition, the phorbol ester decreased insulin-stimulated
phosphorylation, suggesting that there was, in fact, an interaction
between the sites of action of the two agents. In later studies on
IM-9 and HepG2 cells, TPA was found to phosphorylate four major
serine residues, which were not phosphorylated in untreated cells
and to increase the phosphorylation of one threonine residue on the
insulin receptor. These serine residues were not phosphorylated by
insulin, which, however, did phosphorylate three tyrosine residues
(89). PKC acts directly on the insulin receptor as it phosphorylates
the insulin receptor in vitro (90). Similar results were seen with
the IGF I receptor. These very different profiles of phosphorylation
induced by insulin and phorbol esters give strong evidence that
insulin and IGF I were not acting through PKC. In 1988, TPA was
found to enhance predominantly the phosphorylation of one serine
residue on the insulin receptor in hepatoma cells (91). TPA-
treatment of cells inhibits insulin-stimulated receptor
phosphorylation of exogenous substrates by 50 percent. These
changes in the receptor are maintained when the receptors are

isolated and are reversed by incubation with alkaline phosphatase,
suggesting that PKC decreases the tyrosine kinase activity of the
insulin receptor and that this decrease is due to the phosphorylative
changes induced in the receptor. Studies on rat adipocytes have also
shown that TPA increases the Km of the insulin receptor for ATP,
thus suggesting a mechanism for insulin resistance in adipocytes
(82).
Receotor-lnduced Effects
The tyrosine kinase activity of the insulin receptor is necessary
for normal receptor function and down-regulation (92-3). This has
been demonstrated by studies in which kinase-defective mutant
insulin receptors were used to transfect cells. The mutant
receptors demonstrate normal binding of insulin but do not possess
tyrosine kinase activity, are not internalized and do not possess
biological activity. Treatment of endogenous, biologically-active
insulin receptors with monoclonal antibodies against the receptor
kinase inhibits insulin-stimulated effects as well (94). Inhibition
of receptor tyrosine kinase activity by TPA leads to the same types
of defects. Treatment with TPA is associated with inhibition of
insulin-mediated DNA synthesis (95), phosphorylation of metabolic
enzymes (87), glycogen synthesis (96) and glucose uptake (97),
among others. Thus, an impaired PKC pathway can have dire
consequences for the cell or organism. This is demonstrated by
genetically obese (fa/fa) rats, in whose hearts and hepatocytes both
the basal distribution and the translocation of PKC are abnormal.
The resultant insulin-insensitivity can be duplicated by treating

lean rats with TPA to down-regulate PKC (98). The same is not true
for all tissues, however, as phorbol esters have only minimal
effects on insulin sensitivity in rat skeletal muscle (99). The
interaction of PKC and the Insulin receptor Is somewhat complicated
by reports of synergism In mitotic stimulation (100). In addition,
there are proteins which have phosphorylation sites for both PKC and
receptor kinases (101) and are stimulated by both types of mitogens
(102). This last effect would account for the paradoxical way in
which PKC both Inhibits the insulin receptor and mimics many of
Insulin's effects within the cell.
Effects of Growth Factors on PKC
While the majority of studies on PKC/growth factor interactions
In the literature focus on regulation of growth factor receptors by
PKC, those receptors frequently regulate PKC as well. Treatment
with insulin In the presence of glucose has been shown to increase
both the binding capacity of PKC and enzymatic activity in
adipocytes (103). As the insulin effect is eliminated in the presence
of high glucose, the effect may be secondary to increased glucose
uptake in the insulin-treated cells. Insulin also increases the level
of cytosolic calcium ion in adipocytes (104), which could account
for the increased PKC activity. Studies also demonstrate
enhancement of PKC activity In myocytes and mammary tumor cells
by insulin (105-6). In the former the increase occurs in both the
cytosolic and membrane fractions and is not inhibited by
cycloheximide. This Increase in the activity of PKC is reportedly
mediated via increased DAG generated by phospholipid hydrolysis and

12
phospholipid synthesis. There is some controversy on this point,
with other work suggesting that insulin does not increase the
activity of PKC in myocytes (107). It is suggested that the
increases in phosphorylation observed after the administration of
insulin are mediated by S6 kinase which is activated by both insulin
and TPA. Growth factors, then, may increase PKC indirectly by
increasing either cytosolic free calcium ion or DAG or by acting at a
point in the pathway beyond the PKC molecule itself. A direct effect
of insulin on PKC must necessarily be demonstrated on the isolated
enzyme.
PKC in the Brain : Distribution
Although PKC is present in many tissues, it occurs at highest
concentration in the brain. The seven subspecies of PKC have
different distributions in the brain (54) and these change with brain
development. The gamma subspecies, which occurs only in the
central nervous system in the rat and monkey, has a
developmentally-regulated distribution, with expression increasing
from birth until it reaches a maximum at about three weeks of age.
Total PKC is also developmentally regulated in brain in studies in
cultured neurons and in vivo (57). Interestingly, insulin and IGF 1
receptors are developmentally regulated in the rat brain with
increases occurring in the first weeks of life, followed by a decline
(108-9). Immunohistochemical analyses show different antibody
staining patterns. There appear to be subspecies which are present
almost exclusively in neurons (55,110), in astrocyte-glial cells and
in oligodendrocytes (55,111). The enzymatic activity is also

unevenly distributed, with the left cerebral hemisphere expressing
more than the right in the rat (112). Binding studies using
radiolabeled phorbol esters show two to three times more PKC in
neurons than in glial cells cultured from the same rat brains (113).
The subcellular distribution of PKC has been the subject of many
investigations. It is localized in dendrites, axons, perikarya and
nuclei (110) of neurons with particularly high concentrations in
presynaptic terminals (111) and in growth cones (114). This is not
unexpected as PKC mediates both neurotransmission and neurite
outgrowth (115). Fractionation of glial cells demonstrated that the
majority of the PKC was cytoplasmic (116). The same study showed
that the majority of PKC in whole brain tissue is associated with
the membrane, suggesting that the majority of neuronal PKC is
membrane-bound. Seventy-five percent of the glial cell cytoplasmic
PKC can be translocated to the membrane within 30-60 minutes of
TPA-treatment. This is similar to the situation seen in peripheral
tissues.
PKC Regulation of Glucose Uptake and Insulin Receptors in the Brain
PKC has been shown to regulate glucose uptake in many tissues.
Phorbol ester-induced decreases in the binding of insulin to its
receptor are not associated with decreased glucose uptake as, in
fact, phorbol esters stimulate glucose uptake in adipocytes,
myocytes, fibroblasts and astrocytes (97,117-9).
As discussed, in the brain there are distinctions between neurons
and glia with regard to PKC: PKC is present in higher concentrations
in neurons than in glia from the same brains as demonstrated by the

14
binding of phorboi esters (113); different isotypes of PKC are
present in neurons and glia (55,110,111) and phorboi esters
stimulate glucose uptake in glia but not in neurons (119).
The joint observations that neurons and glia differ with respect
to the physiological activities of the Insulin receptor and PKC and
that phorboi esters regulate the Insulin receptor and glucose uptake
peripherally, led us to investigate the role of PKC in the regulation
of insulin receptors in neurons and glia from the central nervous
system. Because activation of PKC by TPA has been shown to involve
translocation of PKC from the cytosolic to the membranous fraction
in many types of cells, we chose to examine TPA's effects on
neurons and glia to determine the relative concentrations of
immunoactive PKC in neurons and glia, whether TPA stimulates the
translocation of PKC In both neurons and glia, and whether the
translocation in glia, if it occurs, precedes TPA's effects on glucose
uptake and the insulin receptor and, thus, might be involved in a
mechanism. Differences in the ability of TPA to induce
translocation or the time-course of the translocation might
presumably explain TPA's differential effects on neurons and glia.
Lastly, in order to determine whether insulin regulates its own
receptor by this pathway, we chose to study insulin's effects on PKC
in neurons and glia from the brain.
Cultured Brain Cells; A Model for the Study of Neurotrophic/
Neuroactive Substances
The importance of neurons in brain function is unquestioned.
Proper neuronal growth, development and maintenance are essential

1 5
for every aspect of normal brain function. The importance of growth
factors, such as nerve growth factor, insulin and the insulin-like
growth factors, in neuronal development and activity has been well
documented although the mechanisms by which these agents act are
still objects of intense study (115,120).
In contrast, although glial cells are the predominant cell type in
the mature nervous system, their involvement in the growth,
development, differentiation and function of the brain has only
recently become a subject of investigation. Glial cells have recently
been implicated in processes involving the growth, development and
function of the nervous system. Glia are not only responsive to
trophic factors but may produce them as well. They facilitate
neuronal migration in fetal life but induce scarring to inhibit
regeneration in the mature nervous system. In addition, they alter
the levels of neurotransmitters available at the synapse, thus
altering neuronal excitability and they may even be excitable
themselves.
Glia respond to trophic factors such as insulin and insulin-like
growth factor I (IGF I) with an increase in glucose uptake in contrast
to neurons (119,121). This is of interest developmental^ as both
insulin and IGF I receptors in brain increase to a maximal level
during brain development and then show a gradual decline (108-9).
Glia also exhibit an increase in macromolecular synthesis in
response to both insulin (33) and IGF I (121). Epidermal growth
factor receptors in the brain appear predominantly in glia, as well
(122).

16
In addition to being responsive to trophic factors, glial cells may
produce them as well. A substance which is immunologically
identical to nerve growth factor is present in glia (123) and glia,
which develop at the same time as neurons, provide other
extracellular molecules which enhance neuronal migration (124).
This glial stimulus to neuronal growth is lost with age as astrocytic
glial cells block axonal regeneration (125) and synapse formation
(126) in the mature nervous system. Inflammation of the mature
nervous system causes a reactive gliosis which prevents neuronal
repair as in multiple sclerosis (124).
Finally, glia contribute to the regulation of nervous system
excitability. Glia take up, and thus inactivate, glutamate, GABA,
aspartate and serotonin (127) at the synapse. Insulin decreases
levels of alpha2-adrenergic receptors in glia; this would tend to
regulate the amount of norepinephrine in the synaptic cleft (128).
Glia do not only take up neurotransmitters but also act on several
via specific enzymes such as glutamine synthetase and GABA
transaminase. In addition, they have specific receptors for alpha-
and beta-adrenergic agonists, dopamine, prostaglandin Ei, secretin,
somatostatin and vasoactive intestinal peptide (127), among others.
Lastly, glia have themselves been shown to possess some voltage¬
gated channels and, thus, may act as excitable cells (129).
Because both neurons and glia are important for appropriate
development and function of the nervous system and because of the
afore-mentioned differences in neurons and glia with regard to both
the insulin receptor and PKC, we chose to study the interaction of

17
insulin and PKC in differential cultures of neurons and glia from the
same rat brains.

Figure 1-1: Activation and translocation of PKC. As the
receptor is activated, the membrane-bound enzyme,
phospholipase C (PLC), is activated and phosphoinositide
(PI) is hydrolyzed to inositol-phosphate (IP), -
diphosphate (IP2) -triphosphate (IP3) and diacylglycerol
(DAG). IP3 stimulates calcium mobilization. DAG
increases the affinity of the calcium-activated,
phospholipid-dependent PKC for calcium. As PKC is
activated it is translocated to the membrane. Phorbol
esters act in a manner which is analogous to DAG.

TPA

CHAPTER II
METHODS
Preparation of Primary Neuronal Cultures from Rat Brains
Brains from 8-10 one-day-old rats were removed in a sterile
manner from the cranium at the level of the medulla oblongata and
placed In an isotonic buffer solution containing 100 units penicillin,
100pg streptomycin and 2.5pg Fungizone/ml. All pia mater and
blood vessels were removed and the brains were minced into 1-
2mm3 pieces. Brain cells were dissociated by trypsin and
deoxyribose treatment as described previously (18). Dissociated
cells were suspended in 50 ml of Dulbecco's modified Eagle's
medium (DMEM) containing 10 percent plasma-derived horse serum
(PDHS) and sedimented at 1000 x g for 10 minutes at 24°C. The cell
pellet was suspended in the same medium at a concentration of 1.5 x
106 cells/ml. Fifteen million cells were placed in each Falcon
tissue culture dish pretreated with poly-L-lysine and the cells were
placed in a humidified incubator at 37°C with 6 percent C02/94
percent air. The cells began to attach immediately and 90-96
percent were attached to culture dishes within 30-60 min. After
three days in culture, the cells were treated with 10pM cytosine
20

21
arabinoside in DMEM containing 10 percent PDHS. This treatment
resulted in the death of rapidly dividing cells. After 48 hours
cytosine arabinoside was removed and the cells were refed with 10
percent PDHS in DMEM and the cells were grown in culture for
another 7-10 days prior to use.
These cultures contain 80-85 percent neurons as demonstrated
by light microscopy and immunocytochemical markers (18, Figure 2-
1). Since insulin and insulin receptors were widely distributed
throughout the brain, in contrast with the localized distribution of
other neuropeptides and their receptors, cultures from the whole
brain were used. We considered growing the cells in a chemically
defined medium. However, since one of the essential components of
this defined medium is insulin in relatively high concentration, this
possibility was abandoned. It was felt that the presence of insulin
in the medium would complicate studies related to the expression
and action of insulin receptors. Thus, we proposed to continue to
culture cells in DMEM containing either PDHS or fetal bovine serum
(FBS). PDHS and FBS were purchased from Hyclone and Gibco
Laboratories, respectively, with an insulin concentration of 1-4
ng/ml. At this concentration, insulin did not cause either down-
regulation or other biological effects on insulin receptors of either
neurons or glia (130).
Preparation of Primary Astrocytic Glial Cultures from Rat Brains
The procedure for removing the brains and dissociating the cells
was the same as that described above. After the cells were
centrifuged, they were suspended in medium containing 10 percent

22
FBS and plated onto 100mm culture dishes at a density of 10 million
cells/dish. After three days the cells were refed with 10 percent
FBS in DMEM. After an additional three days, cultures were rinsed
once with an isotonic buffer and dissociated by treatment with
trypsin. The cells were centrifuged at 1000 x g for 10 min at room
temperature and the pellet was resuspended in DMEM containing 10
percent FBS. Five hundred thousand cells were placed on each
100mm culture dish and the cells were returned to the incubator
until they were confluent, at which time they were used. Phase
contrast microscopic examination revealed a confluent monolayer of
large flat cells by day 6 or 7 after transfer. These cells have
previously been demonstrated to be of glial origin (131). Greater
than 98 percent of these cells have been identified as astrocytic
glial cells. Neuronal cells did not survive the transfer (Figure 2-2).
Immunocytochemistrv of Neuron-Specific Enolase/Glial Fibrillary
Acidic Protein
Either cultured neurons or glia were washed with PBS and the
cells were fixed in a solution of 4 percent paraformaldehyde and 10
percent picric acid in PBS (pH 7.4) for 30 minutes at 4°C. Cultures
were again washed 3 times with PBS and permeabilized in 0.1
percent Triton-X 100, 5 percent low-fat, dry milk in PBS for 30
minutes at room temperature. After being washed again with PBS,
the cells were exposed to a polyclonal antibody (either 1:100
neuron-specific enolase or 1:20 glial fibrillary acidic protein)
diluted in 0.1 percent NaN3/5 percent non-fat dry milk in PBS for 24
hours at 4°C. PBS was used to wash the excess primary antibodies

23
from the cultured cells and a rhodamlne-linked, goat anti-rabbit Ig
was diluted 1:100 In 5 percent non-fat dry milk in PBS and applied
to the cells for 30 minutes at room temperature. Excess secondary
antibody was washed from the cultured cells with PBS and the cells
were photographed under fluorescent light at 400x magnification on
a Zeiss D-7082 Axiophot photomicroscope.
Neuronal Depolarization
Medium was aspirated from 14 day old neuronal cultures and
replaced by 8 ml of various solutions: the control solution contained
140 mM NaCI and 1.2 mM MgSCH; the depolarizing solution contained
78 mM NaCI, 60 mM KCI, 2 mM CaCl2 and 1.2 mM MgSC>4; a potassium
solution contained 80 mM NaCI, 60 mM KCI and 1.2 mM MgSCH; a
calcium solution contained 138 mM NaCI, 2 mM CaCl2 and 1.2 mM
MgSÜ4, and a veratridine solution contained 10 uM veratrldlne, 138
mM NaCI, 2 mM CaCl2 and 1.2 mM MgSC>4. Cultures were Incubated
for 30 minutes at 37°C. The solutions were aspirated, lyophilized,
reconstituted in distilled water and subjected to radioimmunoassay
for insulin.
Insulin Radioimmunoassay
A 100 ul aliquot of a sample of human insulin standard (0-300
uU/ml) was mixed with 5000 cpm of [125l]-insulin. One hundred ul
of guinea pig anti-human insulin (Serano) was added and the samples
and standards were vortexed and incubated at room temperature for
2 hours. A secondary antibody (sheep anti-guinea pig Ig, Serano) was

24
added and the sample and standard tubes were vortexed and
incubated for a further 30 minutes at room temperature. Following
the incubation, all tubes were centrifuged at 2500 g for 30 minutes
at 4°C. Supernatants were discarded. All tubes were swabbed dry of
supernatant and the radioactivity in the pellets was counted.
Samples were compared to a standard curve and the cpms were
expressed as international units of Insulin/ml.
Characterization of Immunoprecipitable Insulin bv HPLC
Neuronal cultures grown in 100mm culture dishes were washed
twice with a solution of 138mM NaCI, 1.2mM MgSC>4 and 2mM CaCl2
(pH 7.4) at room temperature to remove the growth medium. Eight
ml of a depolarizing solution (78mM NaCI, 1.2mM MgSC>4, 2mM CaCl2
and 60mM KCI, adjusted to pH 7.4) were placed on each culture dish
and the dishes were incubated at 37°C for 30 min. in a 94 percent
air/6 percent CO2 incubator. After incubation the solution was
aspirated from the plates.
High-pressure liquid chromatography (HPLC) was used to
determine whether the released peptide had the same
chromatographic properties as commercial rat insulin (Eli Lilly).
Two buffers were prepared. Buffer A consisted of 0.1 percent
trifluoroacetic acid (TFA) and 0.1 percent triethylamine (TEA) in
water. Buffer B consisted of 0.1 percent TFA and 0.1 percent TEA in
acetonitrile. The sample was dissolved in Buffer A and injected
onto a BioRad HiPore C4 column. A buffer system of 1:9, A:B was
graduated to 5:5, A:B over the course of 60 min. The column was run
at room temperature with a flow rate of 2 ml/mln. Fractions were

25
collected every 0.2 min. and absorbance was monitored at 210 and
280 nm. Fractions were subjected to radioimmunoassay (Serono) to
determine whether the fraction which bound the insulin antibody
corresponded to insulin chromatographically.
Labelling of Immunoreactive Insulin in Neuronal Cultures
Neuronal cultures grown in 100mm culture dishes were incubated
in leucine-free DMEM, 10 percent dialyzed PDHS and 50pCi [3H]-
leucine (146.5 Ci/mmole) for 24 hours at 37°C. Medium was
aspirated from the culture dishes; cultures were washed 4 times
with PBS and used for experiments as described above. A 100 pi
aliquot of each solution was exposed to a guinea pig anti-human
insulin antibody (Serono) for 2 hours at room temperature. Sheep
anti guinea pig Ig was added to each sample for 30 minutes to
precipitate the primary antibody and all samples were centrifuged
at 2500g to pellet the antibody-antigen complexes. Supernatants
were decanted and the radioactivity in each pellet was counted
(Liquiscint, National Diagnostics, LKB1217 Rack Beta Counter).
Characterization .af the Regulation of Neuronal-Insulin .Release by
Glucose
Neuronal cultures prepared in 100mm culture dishes were washed
twice with a control buffer [25mM NaHC03, 1.2mM NaH2PC>4, 122mM
NaCI, 1.2mM MgS04 and 2.5mM CaCl2 (pH 7.4)] at room temperature
to remove media. Glucose buffers were prepared such that they
were isotonic to the control buffer (eg. 25mM NaHC03, 5.5mM
glucose, 119mM NaCI, 2.5mM CaCl2 and 1.2mM MgSC>4). Eight ml of

26
either the control or a glucose buffer were placed on each culture
dish. Cells were incubated at 37°C for 30 min. in 6 percent CÜ2/94
percent air with 90 percent relative humidity. After the Incubation
the buffers were aspirated from the dishes, lyophillzed, and Insulin
In the buffers was quantitated by radioimmunoassay.
2-Deoxv-D-Glucose (2-dGlc) Uptake
Medium was aspirated from the culture dishes and the cells were
washed three times with phosphate-buffered saline (PBS) at pH 7.4.
The cells were then Incubated in PBS containing 1mM CaCl2, 0.5 mM
MgSC>4 and 0.5 mM 2-dGlc (1 pCi/plate). After a five min. Incubation
at 37°C, the cells were washed three times with Ice-cold PBS to
remove excess radioactivity. Cells were dissolved in 0.2 N NaOH;
they were then scraped from the culture dishes and radioactivity
was counted (Ecosclnt, National Diagnostics, LKB 1217 Rack Beta
scintillation counter). The cpm were converted to dpm by the
counter (efficiency about 30 percent) and normalized for protein
values. The specific activity was then used to convert results to
nmoles of 2-dGlc/mg protein.
lodination of Insulin
One hundred ml of a pH 6.7 phosphate buffer was prepared from
55 ml of 0.3M KH2PO4 and 45 ml of 0.3M Na2HP04. Chloramine T was
prepared as follows: the surface was scraped and chloramine T was
weighed and placed In a foil-covered tube; the salt was diluted to 4
mg/ml in the phosphate buffer and again diluted 1:100 (I.e. 25 pi In
2.5 ml) in phosphate buffer just before use; all dilutions were in

27
foil-covered tubes as the compound is light-sensitive. One mg of
porcine insulin was weighed and dissolved in 2 ml of 0.01 N HCI.
Sodium metabisulfite was weighed and diluted similarly to the
chloramine T except that it was made to 8 mg/ml in the first
dilution such that the concentration after 1:100 dilution was 80
pg/ml. The decay chart for 125¡ was checked to determine what
volume of 125| contained 1 mCi. Fifty ml of a solution of 1 mg/ml
of insulin-free BSA (eg Sigma A-7030) in phosphate buffer was
prepared and about 20 ml was used to wash a sephadex-G25 column
(PD 10 column from Pharmacia). The following were added in
sequence to a disposable plastic test tube in a fume hood: 1) 25 pi
phosphate buffer, 2) 5 pg insulin (10 pi), 3) 1 mCi 125l and 4) 10 pi
chloramine T. The tube was capped, vortexed and contents were
incubated at room temperature for 5 min. Ten pi of sodium
metabisulfite was added to stop the reaction and the tube was
capped and vortexed once again. Two hundred pi of phosphate buffer
was added to increase the volume and the solution was added to the
top of the column. After the reaction mixture was absorbed by the
column, the column was washed with the BSA/phosphate buffer and
fractions were collected. [125|]-insulin usually eluted in about 3-5
ml. Ten pi of each fraction was removed and placed in a test tube,
capped and counted on a scintillation counter. To calculate percent
incorporation, 50 pi of 3 percent BSA and 1 ml of ice-cold 10
percent trichloroacetic acid were added to the test tube containing
the highest counts. Then the sample was incubated at 4°C for 5 min.
and centrifuged at 1000 x g for 1 min. Radioactivity in the pellet
represented incorporated insulin. This should be greater than 90

28
percent. To calculate specific activity the number of cpm per ml
was determined. The counting efficiency was used to convert to
dpm/ml (75 percent for the LKB Rack Beta). Two million two
hundred thousand dpm/pCi was used to convert to pCuries. The
number of pg/ml was divided by the number of pCi/ml. Insulin
specific activity was generally about 40 pCi/pg. Labelled insulin
was aliquoted into microfuge tubes, capped and stored at -70°C until
use. The labelled material was tested for bioactivity by
displacement with a high concentration of unlabelled insulin (100
pM) in a binding assay.
Insulin Binding
Medium was aspirated from culture dishes and the cells were
washed three times with PBS. Total binding was determined by
incubating triplicate dishes with a binding buffer (100mM Hepes,
30mM NaCI, 10mM glucose, 1mM CaCl2, 0.5 mM MgSC>4, 0.1 percent
bovine serum albumin (BSA) at pH 7.4) containing [125|]-insulin
(100,000 cpm/plate). Non-specific binding was determined with a
similar buffer containing, in addition, 100pM unlabelled insulin.
After a 1 hour incubation (2 hours for competition experiments) at
room temperature, cells were washed three times with ice-cold PBS
to remove excess, unbound insulin. The cells were then dissolved in
0.2 N NaOH and scraped from the culture dishes. Radioactivity was
counted on a Beckman Gamma 5500 counter. All values were
normalized for protein content and specific binding was determined
by subtracting non-specific binding from total binding.

29
Protein Determinations
One hundred to two hundred ul of a solution of protein in 0.1 N
NaOH was used for protein determinations. Bovine serum albumin
standards (10-100 ug of protein) were prepared in the same volume
of 0.1 N. NaOH. Samples and standards were made to 500 ul with
deionized, distilled water and protein determinations were made by
the method of Lowry (132).
Western Blot
Treated or untreated cultures of neurons and glia were washed 3
times with PBS, scraped from the culture dishes and centrifuged at
1000 x g for 5 min. to pellet cells. The supernatant was poured off
and the cells were resuspended in a homogenizing buffer consisting
of 20mM Tris HCI, 2mM EDTA, 0.5mM EGTA, 0.1 mM PMSF and 1
percent 2B-mercaptoethanol at pH 7.5. The suspension was
homogenized with 15 strokes of a glass homogenizer and centrifuged
at 1500 x g at 4°C for 8 min. to remove nuclei and large particles.
The supernatant was recentrifuged at 100,000 x g at 2-4°C for 30
min. to isolate cytosolic and membranous fractions. The membrane
was resuspended in homogenizing buffer containing 0.1 percent
Triton X-100. Samples of homogenates from whole cell were used
after the homogenization step. The protein content of samples was
determined and samples were made to a final concentration of 10mM
Tris base, 2 percent sodium dodecyl sulfate (SDS), 15 percent
sucrose, 0.002 percent bromophenol blue and 10 percent 28-
mercaptoethanol at pH 8.3. Samples were boiled for one min. and
stored at 4°C before proteins were separated on discontinuous

30
polyacrylamide gels (5 percent stacking gel, 7.5 percent separating
gel, 30mA for 6 hours) and transferred to a nitrocellulose membrane
in a buffer consisting of 25mM Tris base, 150mM glycine and 20%
v/v methanol at pH 8.3 for three hours at 150mA. Total transfer
was demonstrated by transfer of prestained standards and by the
lack of a band as demonstrated by Coommassie staining of the gel
after the transfer. The nitrocellulose membrane was stored in 3
percent BSA in PBS overnight to decrease non-specific binding of the
antibody.
The nitrocellulose membrane was incubated with a monoclonal
antibody against protein kinase C (Amersham, diluted 1:100 in PBS
with 0.1 percent BSA) for 4 hours at room temperature. This
antibody recognized the alpha and beta subtypes (Types II and III) of
PKC, which are the majority of PKC in the brain (159). It was then
washed 4 times for 5 min. with 0.1% Tween-20 in PBS (pH 7.4) and
incubated with horseradish-peroxidase-linked anti-mouse Ig diluted
1:100 with 0.1 percent BSA/0.1 percent Tween-20 in PBS for 30 min.
at room temperature. The membrane was washed as before and
incubated in 0.03 percent hydrogen peroxide/0.5 mg/ml 3,3'-
diaminobenzidine (DAB) in PBS prepared immediately prior to use,
until bands appeared. It was then washed and allowed to air dry.
Bands were quantitated via densitometry. Immunological PKC will
be referred to as iPKC throughout this dissertation, while PKC
activity will be designated aPKC.

31
PKC Immunocvtochemistrv
Cells were grown on sterile glass coversllps in culture dishes.
Prior to staining, they were washed three times with PBS and fixed
in 3.5 percent paraformaldehyde/0.25 percent glutaraldehyde in PBS
(pH 7.4) on ice for 30 min. The cultured cells were then
permeabillzed with 0.1 percent Triton X-100 in PBS for 30 min. at
room temperature. Following the fixing and permeabilizing steps,
the cells were rinsed three times with PBS and a 1:10 dilution of a
monoclonal antl-PKC antibody in 1 percent BSA, 0.1 percent sodium
azide in PBS was applied. After a 24 hour exposure to this antibody
at 4°C and they were again rinsed with PBS. Control cells were
treated with the same solution without the primary antibody. A
1:100 dilution of an anti-mouse lg-peroxidase conjugate in 0.1
percent BSA in PBS was applied for 30 min. at room temperature and
then the excess was removed by washing with PBS. Finally, the
cells were incubated in a solution of 0.5 mg/ml DAB/0.03 percent
hydrogen peroxide in PBS prepared immediately prior to use. After
10 min. they were washed and a drop of 9:1 glycerol:PBS was applied
to the coverslip. The coversllps were inverted, placed on a glass
microscope slide and the edges were sealed with nail polish.
Photographs were taken at 400 and 1000 x magnification with a
Zeiss D-7082 Axiophot photomicroscope.
Statistical Analysis
Statistical analysis was by analysis of variance (ANOVA)
followed by Duncan's post hoc test when the means of several groups
were to be compared or Dunnett's post hoc test when the means of

32
several groups were to be compared to that of one control group.
Significance was determined for p<0.05. Experiments whose results
were expressed as a percent of control were converted to arcsin
prior to ANOVA if all values were equal to or less than 100 percent
or to the log if any of the values exceeded 100 percent. The
particular test used was specified in the legend of each figure. The
one exception to these rules was Figure 3-1, for which a two-tailed
Wilcoxan Rank Sum nonparametrlc test was employed because of one
outlying value in the depolarized group.

Figure 2-1: Neuron-specific enolase staining of cultured
neurons. Cultured neurons on a bed of glial cells were
fixed and permeabilized prior to immunostaining for
neuron-specific enolase. Following staining the neurons
were photographed at 400x.

34

Figure 2-2: Glial fibrillary acidic protein staining of
cultured glial cells. Cultured glial cells were fixed and
permeabilized prior to immunostaining for glial
fibrillary acidic protein. Following staining the glial
cells were photographed at 400x.

36

CHAPTER III
INSULIN SYNTHESIS AND RELEASE FROM NEURONAL CULTURES
Introduction
Insulin alters the content of several neurotransmitters in the
brain (36-8), stimulates the release of others (39) and changes
neuronal electrical activity in specific regions of the brain (42-3).
Thus, it definitely acts as a neuromodulator. As insulin is suggested
to act via specific insulin receptors (44), it may act as a
neurotransmitter as well. In order to be classified as a
neurotransmitter, however, synthesis and release of insulin under
depolarizing conditions must be demonstrated in neurons.
Two separate bodies of evidence suggest that the insulin present
in the brain is also synthesized there: 1) preproinsulin mRNA is
present in brain (20-23) and 2) the level of insulin in the brain is
independent of the level of insulin in the periphery (19,23). Several
investigators have identified an mRNA in brain tissue that
hybridizes to a cDNA for insulin. Insulin mRNA is found in neurons,
but not in glial cells in cultures from the brains of both rats and
rabbits (22-3). Only 3-5 percent of cultured neurons from rabbits
contain the mRNA. This is in agreement with an earlier study
37

38
localizing insulin immunoreactivity to 3-5 percent of cultured
neurons from rat brain (18). The mRNA species is larger than that
observed in the human pancreas when the two are compared by
Northern blotting techniques (24). In situ hybridization studies
localize neurons containing the insulin mRNA to the periventricular
hypothalamus and cerebral cortex in rat brain (21) and rat, mouse
and hamster anterior pituitary cells (20). Other areas of the brain,
including the olfactory bulb and choroid plexus do not contain the an
mRNA for insulin (21). In the anterior pituitary, only 5-10 percent
of the cells are positive for the mRNA; those cells are epithelial
and the immunoreactive insulin that they contain is localized in
secretory granules (20).
The second body of evidence relates to the independent regulation
of brain and peripheral insulin levels. Many investigators have
reported brain insulin concentrations that are higher than those
observed in plasma (14,23). It is unlikely that this represents
sequestration and concentration of plasma insulin for two reasons.
First, brain insulin concentrations are not altered by disease states
which raise or lower plasma insulin concentrations, although the
concentration in CSF is lowered in response to lowered plasma
insulin. Secondly, the capillaries of the blood-brain barrier do not
transport active insulin into the brain.
As discussed previously, insulin acts in the brain to alter
neuronal electrical activity. This, in combination with the evidence
that insulin may be synthesized in the brain, led us to investigate
whether pulse-labelled immunoreactive insulin could be synthesized
In the brain and whether this immunoreactive insulin could be

39
released under depolarizing conditions. The chromatographic
properties of the released immunoreactive insulin were then
compared to those of an insulin purified from rat pancreas by HPLC.
RESULTS
In this study, primary neuronal cultures from rat brains were
treated with depolarizing solutions containing a high concentration
of potassium (60mM) with or without calcium (2mM). After 30 min.
the solutions were aspirated and lyophilized and insulin was
quantitated by radioimmunoassay. Depolarized neuronal cultures
released more than three times as much Insulin as saline controls
(103.2pU/ml vs 31.7pU/ml) in the presence of calcium. Potassium-
stimulated release was calcium-dependent as In the absence of
calcium, insulin release was negligible (34.0pU/ml, Figure 3-1).
The sodium ionophore, veratridine, similarly stimulated the release
of insulin from neurons by 379 percent. In contrast, release of
insulin from glial cultures was not stimulated by depolarization.
This, in combination with the evidence that no mRNA for insulin has
been demonstrated in glial cells and that glial cells are not
Immunoreactive for insulin (18), suggested that the insulin released
from neuronal cultures was not due to glial contamination of those
cultures. The very low level of insulin released by glial cells may,
In fact, have represented insulin taken up from the medium. The
immunoprecipitable insulin that was released from neurons under
depolarizing conditions coeluted with an insulin purified from rat

40
pancreas (supplied by Eli Lilly) when subjected to reverse phase
HPLC. Both the released material and the standard showed peaks for
both rat 1 insulin and rat 2 insulin (Figure 3-2).
When primary neuronal cultures were treated with D-glucose
(0.1-0.6 percent) in the presence of 2mM calcium, a dose-dependent
stimulation of insulin release was observed (Figure 3-3). Neuronal
cultures were exposed to [3H]-leucine for 24 hours prior to a timed-
release experiment. These cultures were washed and incubated at
37°C with control and depolarizing solutions which were aspirated
from the cultures at time intervals from 1 to 60 min. A biphasic
pattern of insulin release was observed (Figure 3-4); this pattern of
release was similar to that observed with stimulation of pancreatic
insulin release by glucose. The fact that exogenous [3H]-leucine was
incorporated into the immunoreactive insulin suggested that this
insulin was synthesized within the neurons.
Discussion
There is some controversy as to the origin of insulin in the brain.
Some investigators have shown that the concentration of insulin in
the brain is a fraction of that found in the plasma when quantitated
by different antibodies than the one first used by Havrankova, et al.
(16, 14). These same investigators have also found that the
concentration of insulin in the CSF is dependent on the plasma
concentration. They have suggested that these two pieces of
information, in conjunction with the lack of a demonstrable
proinsulln In the brain and the inability of one investigator to
demonstrate an mRNA for insulin in brain prove that insulin is not

41
synthesized in the brain (16). They propose that insulin passes from
the plasma into the CSF and is retained in brain to the extent that it
binds to local insulin receptors. They further propose that this
accounts for insulin immunocytochemistry in the brain.
Both an mRNA for insulin and concentrations of insulin which are
independent of those in the plasma and CSF have been demonstrated
in the brain in several other studies, as described previously. The
results of this study suggest that insulin is synthesized in neurons
from the brain and released under depolarizing conditions. The
insulin in the brain has immunological and chromatographic
properties that resemble those of pancreatic insulin. While this
evidence suggests that the material is, in fact, insulin, it is not
definitive proof. Rat proinsulin is not commercially available and
would be likely to have similar properties. Sequencing with an
amino acid analyzer would be the most appropriate method of
identifying the peptide, but it requires a larger sample of the
material than is available.
Depolarized release was measured using the solutions described
in methods in order to duplicate the methods used by Valow to study
cholecystokinin release from synaptosomes. These unbuffered
solutions had an acidic pH after 30 min. This low pH may have
altered the release and/or the viability of the cells, although the
cells were still attached to the culture dish and appeared normal
under the microscope. Acidity was observed in all groups, but
onlythe depolarized groups showed increased release of insulin,
suggesting that it was the depolarization, and not the acidity that
induced release. In any case, glucose-induced release was measured

42
in a Krebs buffered solution, in order to eliminate this problem.
Insulin degradation in the release solutions was probably not
significant as degradation of insulin in binding studies, in which the
insulin is exposed to the cells for an hour or more, is generally less
than 10 percent.
It is likely that at least some of the insulin released from
neuronal cultures is also synthesized there: The cultured cells have
been removed from any peripheral insulin for at least 10 days prior
to use; the serum in which the cells are grown is plasma-derived
horse serum, which contains only one insulin, not the two observed
in the rat, and exogenous leucine is incorporated into the
immunoprecipitable insulin. The inability of some researchers to
demonstrate synthesis of insulin in the brain may be related either
to the small percentage of cells producing insulin or to their very
specific localization. In addition, insulin acts to promote survival
of brain cells in culture (115) and, thus, Insulin-producing neurons
may survive preferentially in culture and may represent a larger
percentage of neurons than are present in whole brain. That
immunocytochemical evidence localizing insulin to neurons is the
result of insulin bound to surface receptors, is unlikely as glial
cells have specific, high-affinity insulin receptors as well (28) The
evidence presented here that insulin is synthesized in neurons from
the brain and is released under depolarizing conditions, in addition
to the evidence that insulin in the brain binds to specific, high-
affinity receptors and has electrical and physiological effects in the
brain suggest that insulin is a neurotransmitter in the brain.

Figure 3-1: Effect of depolarizing conditions on the
release of insulin from neuronal and glial cells from the
rat brain. Medium was aspirated from 14 day old
neuronal cultures and replaced by 8 ml of various
solutions: C (control) contained 140 mM NaCI, 1.2 mM
MgSCH; K+Ca contained 78 mM NaCI, 60 mM KCL, 2 mM
CaCl2, 1.2 mM MgS04; K contained 80 mM NaCI, 60 mM
KCI, 1.2 mM MgSC>4; Ca contained 138 mM NaCI, 2 mM
CaCl2, 1.2 mM MgSC>4; V contained 10 uM veratridine,
138 mM NaCI, 2 mM CaCl2, 1.2 mM MgSCH. Cultures were
incubated for 30 min at 37°C. The solutions were
aspirated, lyophilized, reconstituted in distilled water
and subjected to radioimmunoassay for insulin. Means of
data from five to seven experiments are represented and
statistical significance evaluated by two-tailed
Wilcoxan Rank Sum Test.

A »0
juU Insulin Released/mg Protein

Figure 3-2: Radioimmunoassay of HPLC fractions of
released insulin. Pooled samples of the material
released under depolarizing conditions were
chromatographed on a BioRad-C4 column as described in
Methods. The fractions were assayed by routine
radioimmunoassay for insulin. The top panel represents
the purified insulin from rat pancreas and the bottom
panel represents the radioimmunoassayable insulin of
each fraction of the pooled samples. This representative
experiment was repeated once.

46

Figure 3-3: The effect of glucose on insulin release by
neuronal cultures from the rat brain. Medium was
aspirated from 14 day old neuronal cultures and replaced
by 8 ml of various solutions: the control solution
contained 140 mM NaCI, 1.2 mM MgS04 and 2 mM CaCl2;
Glucose solutions contained 5.5-33 mM glucose, 1.2 mM
MgSC>4 and NaCI to make the solution isotonic with the
control solution. Cultures were incubated for 30 min at
37°C. Solutions were aspirated, lyophilized,
reconstituted in distilled water and subjected to
radioimmunoassay for insulin. Each bar represents the
mean ± SEM for 6-8 experiments.

Glucose (mM)
Insulin Released (% Control)
_* ro to A
o o o o
o o o °

Figure 3-4: Time course of depolarization-induced
release of [3H]-leucine labelled insulin. One hundred uCi
of [3H]-leucine was added to 100 mm culture plates 24
hours in advance of the depolarization-induced release
experiment. Medium was aspirated from 14 day old
neuronal cultures and replaced by 8 ml of a control or a
depolarizing solution. Cultures were incubated at 37°C
for periods ranging from 0-60 minutes. The solutions
were aspirated, lyophilized, reconstituted in distilled
water and subjected to radioimmunoassay. Control
release was subtracted from depolarized release at each
time point and results were converted to femtomoles of
insulin released. This is one representative experiment
of three.

20 30 40
Time (minutes)
cn
o
50

CHAPTER IV
CHARACTERIZATION OF PKC IN NEURONAL AND GLIAL CELLS IN
PRIMARY CULTURES
Introduction
PKC is present in both neurons and glial cells from the brain
(55,110-11). It is present in dendrites, axons, perikarya and nuclei
(110) of neurons with particularly high concentrations in
presynaptic terminals (111) and in growth cones (114). The last two
are predictable, as PKC mediates both neurotransmission and neurite
outgrowth (115). In glial cells, PKC is Involved in the regulation of
differentiation (78) and membrane conductance (80). Thus, PKC is
also physiologically active in both types of cells, although its
activity takes different forms in neurons and glial cells.
Because of these differences and the existence of different
isotypes of PKC we elected to study neurons and glia in culture to
determine the relative quantities of PKC, the relative molecular
weight (Mr) of PKC and the distribution of PKC within the cells. As
TPA-activation of PKC involves translocation of the enzyme, we
chose to observe the time-course of the translocation to determine
whether this differed in the different types of cells.
51

52
Results
Immunoreactive PKC was present in both neurons and glia.
Immunocytochemistry showed neurons staining darker than glial
cells in the same cultures with the stain distributed throughout both
types of cells. In glial cells the staining was unevenly distributed
with particularly dark staining In nuclear and perinuclear areas. In
neurons, both perikarya and processes stained. No further
differences in staining could be seen in neurons at this
magnification. No unstained cells were observed In either the
neuronal or glial cultures (Figure 4-1,2). Control cells, which
received the same treatment except for the primary antibody, had no
staining whatsoever. The concentration of iPKC was 4.6±0.5 (mean ±
standard error) times higher in neuronal than in glial cultures from
the same brains when measured by Western blot and densitometry
(Figure 4-3) of whole cell homogenates. Immunoactive PKC in both
neurons and glia had a relative molecular weight of about 80 kD as
determined by polyacrylamide gel electrophoresis followed by
Western blotting and comparison with commercial molecular weight
standards.
The cytosol contained 63 ± 9 percent of the iPKC in glial cells
(Figure 4-4). In contrast, the neuronal IPKC resided primarily In the
membrane with only 12 ± 2.3 percent in the cytosolic fraction when
quantitated by Western blot and densitometry (Figure 4-5).
Treatment of both neurons and glia with 100 nM TPA induced a
time-dependent translocation of iPKC from the cytosolic fraction.
In glial cells treatment with TPA decreased the cytosolic level of
iPKC to 33 ± 7 percent of the basal level within 5 min. Within 15

53
min. the cytosolic iPKC was barely detectable by Western blot (6 ± 4
percent of the control level). At 24 hours after treatment with
TPA, the concentration of iPKC in the cytosol remained low. In
contrast, the iPKC in glial membranes was increased to 150 ± 39
percent of the control within 5 min. of the administration of TPA,
after which it declined. Within 24 hours the membrane-bound ¡PKC
had downregulated such that it was barely detectable as well
(Figures 4-4,6).
Treatment with 100 nM TPA induced a downregulation of iPKC in
neuronal cells. The cytosolic iPKC decreased to 59 percent of the
control concentration within 1 hour, to 16 percent within 2 hours
and did not increase again over the course of a 24 hour treatment
with TPA. The level of iPKC bound to the membrane did not change
within 15 min. after treatment with TPA but decreased to 54
percent within 1 hour and continued to decline such that the ¡PKC
was only 17 percent of the control iPKC concentration after 24
hours (Figures 4-5,7).
Discussion
An iPKC of 80 kD is present in both neurons and glia, although
neurons express several times the cconcentration of iPKC as that
seen in glia. This is seen with both the immunocytochemistry and
the Western blot experiments. This is in agreement with studies
showing that the binding of phorbol esters is 2-3 times higher in
cultured neurons than in glial cells from the same rat brains (113).
No differences with regard to immunostaining of different regions
of the neurons were observed. As the vast majority of the neuronal

54
enzyme is associated with the membranous fraction the membrane
staining is very dark and differences within the cell cannot be seen.
In contrast, in glia, the majority of the iPKC is cytoplasmic. This
was not unexpected, as an earlier study reported that whereas glial
PKC was predominantly cytosolic, PKC from homogenized whole
brain tissue was predominantly membrane-bound (116). The nuclei
in glial cells appear to stain darkly for iPKC, but nuclear PKC would
not contribute to either the cytosolic or membranous fractions as
the nuclei were removed in a centrifugation step prior to the
separation of membrane and cytosol. The aPKC in the membrane-
bound fraction is latent in the liver (52,133). The concentration of
PKC associated with neuronal cytosol was, in fact, low enough to be
the result of glial cell contamination. Neuronal cultures contain 15-
20 percent glial cells, as described in methods.
Complementary studies on neuronal and glial aPKC demonstrate
that glial cells have far greater aPKC than neurons (134). The
different results obtained by immunological and bioassay suggest
that either our antibody does not recognize a large percentage of
glial aPKC or that a large percentage of the neuronal iPKC is
inactive. The first could result from the presence of different
isozymes of PKC in different types of cells, one or several of which
are not recognized by an antibody directed against the alpha and beta
(beta-1 and -2) forms of the enzyme, although these Type II and III
subtypes of PKC do represent the majority of PKC in the brain, there
is another subtype, Type I, which is found only in the brain, and
which is not recognized by our antibody (159). The existence of this
subtype, which is not immunoactive could lead to a false negative

55
result in studies in which iPKC was measured. In the brain,
different subspecies of PKC have been identified at different stages
of development, as discussed previously (54,57). This cannot
explain the disparity between the immunoreactive levels and enzyme
activities in neurons and glia, as experiments were done on cultures
of the same age. The increase in neuronal aPKC when stimulated
was much smaller than the increase in glial activity under the same
circumstances. This suggests that a subset of the neuronal PKC
enzymes may be physiologically inactive or less active than their
glial counterparts. The second situation could result from inactivity
of the neuronal, predominantly membrane-bound iPKC. As discussed
previously, there is evidence to support either of these hypotheses;
that is, neurons and glial cells express different isozymes of PKC
and the membrane-bound enzyme may be latent. TPA stimulates
translocation of the glial aPKC over a similar time-course and to a
similar extent as that observed immunologically. This is similar to
the situation in the periphery, in which PKC is primarily localized in
the soluble fraction and is translocated to the membrane In a time-
dependent fashion when stimulated (48). It is unlikely that the
enzyme is translocated during the process by which the cells are
prepared for electrophoresis as they are washed extensively to
remove TPA and the procedure is carried out in the presence of
calcium chelators, which prevent endogenous stimulation of
translocation as well as inhibiting proteases.
Other investigators have found that only 25-35% of the PKC in
whole brain is in the soluble fraction (51,116). It is likely that this
is due to the presence of the predominantly membrane-bound enzyme

56
in neurons. Our failure to observe a consistent increase in the
membranous fraction at 5-15 min. after TPA stimulation may be due
to the very small percentage of cytosolic ¡PKC available to be
translocated. It may be that the change in the concentration of iPKC
in the membrane is too small to be observed consistently.

Figure 4-1: PKC immunostaining of cultured glial cells.
Cells were cultured on sterile glass coverslips. Prior to
immunostaining with a monoclonal PKC antibody, cells
were fixed and permeabilized. Following staining of the
cells, photographs were taken at 400x magnification.
This experiment was repeated three times.

58

Figure 4-2: PKC immunostaining of cultured neurons.
Cells were cultured on sterile glass cover slips. Prior to
immunostaining with a monoclonal anti-PKC antibody,
cells were fixed and permeabilized. Following staining,
the neurons and glial cells were photographed at 1000x
magnification. This experiment was repeated three
times.

60

Figure 4-3: Demonstration and quantitation of PKC
protein in neuronal and glial cells. Homogenates of glial,
neuronal and 1-day-old rat brains were prepared for
Western blot as described in Methods. Lanes containing
a) 25 b) 50 and c) 100 ug of protein were run for each
preparation. Densitometric values for the 80 kD band are
(from left to right), 113, 117, 425, 377, 636, 2164, 67,
230 and 1441.

abe abe
Qia Neurons
<-180kD
— — <-80kD
t nf-
-36.5 kD
b c
Brain

Figure 4-4: TPA-induced PKC translocation in astrocytic
glial cells. One hundred nM TPA was added to the medium
of confluent glial cultures for the times specified,
following which samples were separated into cytosolic
(C) and membrane (M) fractions and subjected to gel
electrophoresis and Western blotting as described in
Methods. Each lane represents 165 ug protein.
Densitometric values for the 80 kD band are (from left to
right), 885, 732, 229, 1688, 29, 952, 9, 540, 0, 471, 0, 0.
This is one representative experiment of three.

C M
ti
0 min
C M C M
5 min 15 min
Time
C M
1 hr
80 kD-

Figure 4-5: TPA-induced PKC redistribution in neurons.
One hundred nM TPA was added to the neuronal medium
for the times specified, following which samples were
separated into cytosolic (C) and membrane (M) fractions
and prepared for Western blotting as described in
Methods. Each lane represents 330 ug protein.
Densitometric values for the 80 kD band are (from left to
right), 2122, 10352, 4863, 5156, 1709, 4272, 1325,
6493, 900 and 1581. This is one representative
experiment of four.

80KD -»
C M
C M
C M
C M C M
- ¿há m Mm
—wk
m t- 'Httb
sSRfc*r;,i
. —
r 1-*
0 min
15 min
1 hr
2 hr 24 hr

Figure 4-6: TPA-induced PKC translocation in astrocytic
glial cells. One hundred nM TPA was added to the medium
of confluent glial cultures for the times specified and
samples were separated into cytosolic (â– ) and membrane
(•) fractions and subjected to Western blotting. The
densitometric values were divided by the values for the
control samples. Each point represents the mean of at
least three experiments ± SEM. Means of treated groups
were compared to that of the control group by two-way
analysis of variance, followed by Dunnett's post hoc test
(p<0.05).

Immunoactive PKC (% of

Figure 4-7: TPA-induced PKC redistribution in neurons.
One hundred nM TPA was added to the neuronal medium
for the times specified and samples were separated Into
cytosolic and membrane fractions and subjected to
Western blotting. The densitometric values were divided
by the values for the control samples. Each bar
represents the mean of at least three experiments ± SEM.
Means of treated groups were compared to that of the
control group by two-way analysis of variance, followed
by by Dunnett's post hoc test (p<0.05).

Cytosoi Membrane
Immunoactive PKC
(% Control)
r\3
CD
00
o
o
O
o
0¿
120

CHAPTER V
THE REGULATION OF SUGAR TRANSPORT IN PRIMARY NEURONAL AND
GLIAL CELL CULTURES BY PHORBOL ESTERS
Introduclian
Insulin has been shown to stimulate sugar uptake in astrocytic
glial cells from rat brain. This effect was time- and dose-
dependent with a maximal stimulation observed at 18 nM and a half-
maximal effect at 0.1 nM insulin (28). The latter value has been
shown to be well within the concentrations found in the brain (15)
indicating that the endogenous insulin is sufficient to induce this
stimulatory effect. This effect was unique to glial cells as neurons
prepared from the same brains failed to express similar properties.
Because of these and other observations it has been proposed that
insulin's lack of effect on sugar uptake in neurons may be due to the
absence of an intracellular pool of glucose transporters and/or an
inability of insulin to translocate intracellular transporters or to
activate membrane-bound transporters, or to a combination of all
three of these.
In this study we utilized TPA to determine its effects on
neuronal and glial glucose uptake as TPA has been shown to
stimulate glucose uptake in other tissues (97, 117-9). The
71

72
activation of PKC will provide us with an additional parameter to
study the differences in the regulation of neuronal and glial glucose
uptake.
Results
TPA stimulated [3H]2-dGlc uptake in glial cells in a time-
dependent manner. Glucose uptake was increased as early as 20 min.
after the administration of TPA with maximal increases occurring
after 4 hours of treatment with 100 nM TPA. The maximal level of
increase was 204.5 ± 12.5 percent (Figure 5-1). In contrast, TPA
failed to influence 2-dGlc uptake in neuronal cultures under similar
conditions. The stimulatory effect of TPA on glial glucose uptake
was selective and was due to an increase in the number of glucose
transporters rather than a change in the Km of the transporter (119).
Discussion
The differences in PKC-stimulated glucose uptake between
neurons and glial cells may be due to the different isozymes of PKC
present in the two types of cells, or to the presence of a smaller
pool of PKC available to be translocated in neurons. In other related
work in this area, our group found that phorbol ester-induced
stimulation of glucose uptake in glial cells is also concentration-
dependent with a maximal effect at 100 nM TPA. It is likely that
TPA is acting through PKC as the potency of five phorbol esters
paralleled their abilities to bind and activate PKC (119). However,
PKC has many effects on cells within the brain; even if these
effects occur by way of activastion of PKC, they may be indirect

73
effects that occur as a result of PKC's other actions in the brain,
such as neurotransmitter release (75-77), or alterations in
neuronal ion channels (69-710 or membrane conductance in
astrocytes (80)

Figure 5-1: The effect of TPA on neuronal and glial 2-
dGIc uptake. One hundred nM TPA was added to the
medium of either neuronal (a) or glial (â– ) cultures for the
times specified. Following this incubation, cells were
rinsed twice with phosphate-buffered saline (PBS) and
exposed to a solution containing labelled 2-dGlc for 5
min at 37°C. The cultures were again rinsed with PBS.
Cells were dissolved in 0.2 N NaOH and scraped from the
dishes; radioactivity was counted and normalized for
content of protein. Each point represents the mean ± SEM
of at least three experiments.

[3H]2-dGlc Uptake (% Control)
o
2 3
Time (Hr)
-«4
cn
1
4
5

CHAPTER VI
THE REGULATION OF INSULIN RECEPTORS IN NEURONAL AND GLIAL
PRIMARY CULTURES BY PHORBOL ESTERS
Introduction
Neuronal and glial insulin receptors are structurally and
physiologically distinct in several respects: neurons have a receptor
of lower molecular weight (29,32); insulin stimulates the uptake of
glucose in glia but not in neurons (28), and insulin inhibits the
uptake of norepinephrine in neurons, but not in glia (30). In addition,
there are distinctions between neurons and glia with regard to PKC:
PKC is present in higher concentration in neurons than in glia from
the same brains as demonstrated immunologically and by binding
studies with phorbol esters (113) although the activity is higher in
glial cells; different isotypes of PKC are present in neurons and glia
(55,110,111), and phorbol esters stimulate glucose uptake in glia,
but not in neurons (119). As neurons and glia differ with respect to
the physiological activities of both insulin receptors and PKC and
because PKC regulates the binding, autophosphorylation, tyrosine
kinase activity and some cellular responses of the insulin receptor
peripherally (81-3,87,91,95-7), we chose to investigate the role of
PKC in the regulation of the insulin receptor in neurons and glia from
76

77
the central nervous system to determine whether PKC might
differentially regulate these two receptors.
Results
TPA induced a dose-dependent increase in the binding of insulin
in glial cells with no effect on binding in neurons over the same
range of concentrations (Figure 6-1). TPA treatment of glial cells
for two hours did not alter the binding of insulin at a concentration
of 1 nM TPA, began to increase binding at 10 nM TPA and caused a
maximal increase at a dose of 50 nM TPA. The ED50 was 15 nM and
50 nM caused a maximal increase of 77 percent. The effect of TPA
on the binding of insulin was time-dependent as well (Figure 6-2).
Treatment with 100 nM TPA induced an increase in the binding of
insulin in glial cells within 30 min. with a maximal increase at two
hours, followed by a decline. The amount of bound insulin stabilized
at four hours after treatment with TPA. Translocation of PKC in
glial cells from the cytosol to the membrane began within 5 min. and
was virtually complete within 15 min. The effects of TPA on insulin
binding in glial cells and 2-dGlc uptake followed TPA-stimulated
translocation of PKC and, thus, PKC may be involved in TPA's effect
on insulin binding (Figure 6-3). Treatment of neurons with 10 and
100 nM doses of TPA had no effect on the binding of insulin (Figure
6-1).
Glial cells were treated with 100 nM TPA for 2 hours. Following
this treatment, competitive inhibition experiments for untreated
and TPA-treated glial cultures were conducted by adding a constant
amount of [125l]insuiin (100,000 cpm/dish) and increasing amounts

78
of unlabelled insulin (0.167 nM-133 nM) to binding buffers. An IC50
of 40 nM insulin was observed for both curves (Figure 6-4). The data
were used for Scatchard analysis. The Kds for the high and low
affinity insulin receptors were 18.5 and 131.6 nM, respectively, in
control cells. Treatment with TPA induced a 184 percent increase in
the number of high affinity binding sites on glial cells without
altering their affinity. TPA increased the number of low affinity
sites by 74 percent while decreasing the affinity of those sites only
minimally (Table 6-1). Glial cells were exposed to three different
phorbol esters for 2 hours to evaluate the specificity of the
stimulation of insulin binding by TPA. TPA stimulated the binding of
insulin by 109 percent at a concentration of 50 nM. The same dose
which caused maximal stimulation by TPA had no effect when cells
were treated with 46-phorbol 128,13a-didecanoate (13-PDD). B-PDD
exhibited an increase of 82 percent only at a concentration of 500
nM while 4a-phorbol 12B,13a-didecanoate (a-PDD) had no effect at
any concentration (Figure 6-5). Thus, the potencies of these drugs
corresponded to their abilities to bind and activate PKC, that is, TPA
> B-PDD > the inactive analog, a-PDD (147). Pretreatment of glial
cultures for two hours with 100 nM TPA followed by its removal
resulted in a time-dependent recovery of insulin binding. The
binding recovered by 59 percent within 2 hours. Recovery was
complete within 6 hours (Figure 6-6).
The next question concerned the TPA-stimulated increase in the
number of glial insulin receptors and whether it corresponded to an
increase in responsiveness to insulin. Insulin and/or TPA was added
directly to the glial medium and the dishes were returned to the

79
incubator for the appropriate preincubation time prior to
quantitation of 2-dGlc uptake. Insulin induced a 34 percent increase
in glial 2-dGlc uptake when cells were treated with a dose of 167
nM for 15 min. Treatment of cells for 2 hours with 100 nM TPA
stimulated 2-dGlc uptake by 112 percent. However, TPA eliminated
the response to insulin. Administration of insulin in combination
with TPA resulted in a stimulation of 144 percent (Figure 6-7).
Thus, the binding of insulin increased, but the response to insulin did
not when glial cells were treated with TPA.
.Discussion
TPA stimulates the binding of insulin in glial cells from the brain
without altering neuronal binding of insulin. This stimulation in glia
occurs without a parallel increase in the responsiveness to insulin,
as demonstrated by the lack of effect of TPA on insulin stimulated
2-dGlc uptake. Thus, it represents an increase in the number of
binding sites, not necessarily an increase in the number of
receptors. The potency of the phorbol esters in effecting a
stimulation of glial insulin binding corresponds to their respective
abilities to activate PKC (147), suggesting that TPA is acting
through PKC. As with TPA stimulation of glial glucose uptake,
however, caution must be used in translating an effect of phorbol
esters to an effect of PKC. Again also, the effect may well be an
indirect effect as PKC has other effects in the brain, as described in
the introduction. TPA's effects on glial insulin binding occur more
slowly than PKC translocation, but over the same range of doses.
Thus, translocation of PKC may be involved in TPA's stimulation of

80
insulin binding. This is to be expected as translocation precedes and
is necessary for other PKC-induced effects, both centrally and
peripherally (61-4). This increase in the binding of insulin could
also be interpreted as an increase in ionternalization of insulin.
Further studies using an acid wash to separate bound versus
internalized insulin will be necessary to resolve this question.
As was demonstrated, treatment with TPA for 24 hours
downregulates PKC in the brain. As the binding of insulin in glial
cells recovered to the level of the control, but not below, 24 hours
after TPA administration and removal, it appears that the binding of
insulin in glial cells is not under chronic control by PKC.
PKC-induced inactivation of receptors has generally been
associated with a decrease in binding of the appropriate ligand in
studies in peripheral tissues. This example of differential
regulation of brain and peripheral insulin receptors as well as
distinct regulation of insulin receptors in neuronal and glial cells is
by no means unprecedented. Insulin, which generally downregulates
its receptor peripherally, downregulates its receptor in glia but
upregulates the receptor in neurons (148). Glucocorticoids, which
increase the binding of insulin in hepatocytes (149) and
lymphocytes(150), have no effect in adipocytes (151) and decrease
binding in an astrocytic cell line (152). As insulin has different
actions in different types of cells, it is to be expected that
regulation of insulin receptors might vary among cell and tissue
types.
TPA induces insensitivity to insulin insofar as glial 2-dGlc
uptake is concerned. This is true even at doses of TPA which elicit a

81
less than maximal Increase in binding. This suggests that these
glial insulin receptors, like their peripheral counterparts, may be
inactivated by PKC. Inactivation of tyrosine kinase prevents
internalization of insulin receptors in Chinese hamster ovary cells
(93). A similar proposal could be made for the glial insulin receptor.
The most Intriguing aspect of this study concerned TPA's lack of
effect on neuronal insulin binding, although this does not necessarily
indicate that PKC does not inactivate the receptors. Both neurons
and glia contain a PKC which is capable of binding phorbol esters,
and neurons and glia both respond physiologically when stimulated
by phorbol esters (69-80). PKC is certainly involved in neuronal
receptor regulation as phorbol esters induce an increase in the
binding of angiotensin II by a calcium-dependent mechanism (153).
Thus, TPA's failure to alter the binding of insulin in neurons can
neither be attributed to a lack of PKC, nor to a non-functional PKC.
As PKC's effects on the Insulin receptor are direct effects (90), the
different effects of TPA on the binding of insulin must necessarily
be due to differences in neuronal and glial PKC or to structural
differences within the insulin receptors themselves.

Figure 6-1: The effect of TPA on insulin binding in glial
and neuronal cultures. Cells were incubated with TPA
for two hours at 37°C. TPA was removed by washing and
insulin binding was measured as described in methods.
Each point represents the mean ± SEM of three
experiments.

[125l]-lnsulin Binding
(% of control)

Figure 6-2: Time-course of stimulation of insulin
binding by TPA in glial cultures. One hundred nM TPA was
added to culture medium for the times specified.
Following this incubation, TPA was removed by washing
and insulin binding was measured as described in
methods. Each point represents the mean ± SEM of four
experiments.

[125l]-lnsulm Binding
(% of control)
Time (hours)

Figure 6-3: Time-courses of TPA-stimulated events in
glial cultures. One hundred nM TPA was added to the
medium of confluent glial cultures for the times
specified. Following treatment with TPA, cells were
washed and 2-dGLC uptake, insulin binding and iPKC
translocation were measured as described in methods.
Each point represents the mean ± SEM for at least three
separate experiments.

Time (Hr)
% of Control
PKC Translocation Insulin Binding 2-dGlc uptake
2001

Figure 6-4: Competitive inhibition of [125l]-insulin
binding to untreated and TPA-treated glial cultures.
Cultures were incubated without (•) or with (■) 100 nM
TPA for 2 hours at 37°C. Cells were washed and
incubated with increasing concentrations of insulin (0.8-
133nM) in the presence of 100,000 cpm of [125l]insulin.
The experiment was reproduced four times.

% [125l]lnsulin Bound

90
Table 6-1: Data from the Scatchard plot of TPA effect on glial cell
High Affinity
Receptor number
Ka
Low Affinity
Receptor number
Ka
[125l]insulin binding
Control
1.13+0.13 pmol/mg
0.054±0.008 nM’1
5.02±0.81 pmol/mg
0.0076±0.0005 nM-
TPA
3.21±0.65 pmol/mg
0.043±0.006 nM'1
8.77±1.69 pmol/mg
0.0066±0.002 nM’1

Figure 6-5: Specificity of TPA's effect on glial insulin
binding. The appropriate phorbol ester (50, 100 or 500
nM) was added to the medium of confluent gliai cultures
for 2 hours at 37°C. Following treatment, cells were
washed to remove phorbol esters and insulin binding was
measured as described in methods. Each bar represents
the mean ± SEM. This is 1 representative experiment of
3.

Control
TPA
a-PDD
B-PDD

Figure 6-6: Recovery of insulin binding in glial cultures
after removal of TPA. Cells were treated with 100 nM
TPA for 2 hours at 37°C and washed three times with
DMEM. Both TPA-treated (■) and control (•) cells were
refed with DMEM containing 10% FBS and returned to the
incubator for the specified time. Cells were then washed
and the binding of insulin was measured as described in
methods. Each point represents the mean + SEM. Means
of treated and control groups were compared by two-way
analysis of variance, followed by Duncan's post hoc test
(p<0.05). This is 1 representative experiment of 4.

% P25l]lnsulin Binding
f
_L
2
-t
24
Time (Hr)

Figure 6-7: The effect of TPA on [3p|]2-dGlc uptake
responsiveness to insulin in glial cultures. TPA-treated
cells were treated with 100 nM TPA for two hours.
Insulin-treated cells were treated for 15 minutes with
167 nM porcine-derived insulin. Following treatments,
cells were washed and [3H]2-dGlc uptake was determined
as described in methods. Each point represents the mean
± SEM. Means of insulin-treated groups were compared to
those of the appropriate non-insulin-treated groups by
two-way analysis of variance, followed by Newman-
Keuls post hoc test (p<0.05). This experiment was
repeated 5 times

H]2—dGlc Uptake
(nmol/mg protein/5 min)
-» IO OJ
O O O
96

CHAPTER Vil
THE EFFECTS OF INSULIN AND DEXAMETHASONE ON NEURONAL AND
GLIAL PKC
Introduction
Insulin increases PKC activity in adipocytes and myocytes
(103,105). Both insulin and PKC appear to have the same effects in
astrocytes as in these peripheral cells. That is, insulin stimulates
glucose uptake and macromolecular synthesis (28,33) and PKC
stimulates glucose uptake (119) and inactivates the insulin receptor
in glial cells, as in the periphery. Each, however, has different
effects in neurons. Insulin appears to have neuromodulator/
neurotransmitter effects (36-44) as opposed to its more traditional
metabolic effects (13) and PKC regulates neuronal differentiation
and function (69-77) as opposed to glucose uptake. We elected to
study insulin's effect on PKC to determine whether neuronal and
glial cells responded to insulin with an increase in PKC as peripheral
cells do (103,105,106).
In order to determine whether these effects were specific to
insulin, the effects of dexamethasone on neuronal and glial PKC were
also studied. Dexamethasone is a synthetic glucocorticoid.
Glucocorticoids regulate glucose homeostasis (13) as well as the
97

98
insulin receptor peripherally (149-51), and downregulate the insulin
receptor in an astrocyte cell line (152). In addition, there are
interactions between PKC and the glucocorticoids, as there are
between PKC and insulin. Adrenocorticotropic hormone (ACTH) may
be a stimulus for PKC (154). ACTH Is reported to Increase
dlphosphoinositide (DPI) In the adrenal cortex and to modulate DPI
kinase in brain (155). In a conflicting study, no effect of ACTH on
adrenal phosphoinosltldes was observed although corticotropin
releasing hormone (CRH) did Increase phosphoinosltlde metabolism
In the anterior pituitary (156). PKC, in turn, inhibits the production
of cortisol by the adrenal and phosphorylates mammalian stress
proteins (157-8) when activated. As glucocorticoids act on the
insulin receptor centrally, and glucorticoids, ACTH and CRH were
reported to interact with PKC both centrally and peripherally, we
chose to study dexamethasone's effects on neuronal and glial PKC as
well.
Results
Incubation of glial cells with insulin resulted in a time-
dependent increase in iPKC. Increases were observed within hours,
with a significant increase 224 percent within six hours of
treatment with 167 nM insulin. The ¡PKC remained elevated for up
to 24 hours after the administration of insulin (Figures 7-1,2).
Preliminary experiments suggested that the increase was primarily
cytoplasmic as after 2 hours of treatment with insulin the
cytoplasmic iPKC was 383 percent of the control concentration,
while membrane-bound iPKC was 113 percent of the basal level

99
(Figure 7-3). In contrast, treatment of neurons with insulin under
the same conditions did not elevate iPKC in neurons. This was true
even after 24 hours of treatment (Figures 7-4). When cells were
treated with dexamethasone, a different effect was observed.
Treatment with dexamethasone (2.55 uM) had no effect on glial iPKC
over the course of 24 hours (Figure 7-5) but markedly stimulated the
level of iPKC in neurons in a time-dependent manner. Neuronal iPKC
was elevated by greater than 100 percent within 4 hours and
continued to rise over the course of 24 hours, at which time it was
increased 4.4-fold over the control (Figure 7-6).
Discussion
These experiments show that insulin stimulates iPKC in the same
cells in which PKC regulates the insulin receptor, that is, in
cultured glial cells. It appears that PKC acts as a part of a feedback
loop to regulate the insulin receptor. The increase in iPKC following
treatment with insulin could be due to increased transcription of an
mRNA for PKC, increased translation of existing mRNA or to the
alteration of a non-immunoreactive molecule to one that is
recognized by the antibody. As the level of iPKC increases over the
course of hours, it is likely that synthesis of protein is involved.
Additional experiments with cycloheximide would be necessary to
support this. In contrast, insulin does not elevate neuronal PKC
under the same conditions of time and concentration. This
concentration of insulin has been shown to induce other effects in
the brain (28, 30). As PKC does not alter neuronal binding of insulin,
it appears that both arms of the circuit are missing. It would be

100
inappropriate for PKC to feed back negatively on a receptor which
apparently does not regulate It in the first place. Because an
antibody which did not bind all subtypes of PKC was used, however,
there is a possibility that negative results were false negatives;
that is'insulin may induce an increase in the gamma subtype of PKC.
The increase in glial PKC in response to insulin occurs in the
cytoplasmic fraction, and thus, the enzyme is likely to be active.
Although insulin stimulates Immunoreactive PKC in glial cells,
insulin's effect on aPKC has yet to be determined. Activity does not
necessarily increase with increased concentration as demonstrated
by the effect of PKC on the insulin receptor.
Dexamethasone has exactly the opposite effect. While
glucocorticoids have peripheral metabolic effects which are in
opposition to those of insulin (13), the relationship between
glucocorticoids and PKC is, on the surface, similar to that between
insulin and PKC. The dexamethasone stimulation of neuronal, but not
glial, immunoreactive PKC indicates that the effect of insulin is not
a general one. The effect of PKC on glucocorticoid receptors in
neuronal and glial cells is not known. It may be that PKC acts
differentially on those as well.

Figure 7-1: The effect of insulin on glial iPKC. One
hundred sixty-seven nM insulin was added to the medium
of confluent glial cultures for the times specified. The
cells were then washed, prepared for Western blotting as
described in methods, and bands were read on a
densitometer. Each band represents 200 ug protein.
Densitometric readings are (from left to right) 610,
1488, 2549 and 2127. The experiment was repeated
eight times.

00
o
7s
O
i
20 l

Figure 7-2: The effect of insulin on glial iPKC. One
hundred sixty-seven nM insulin was added to the medium
of confluent glial cultures for the specified period of
time. PKC was quantitated by Western blotting and
densitometry. Each point represents the mean ± SEM for
at least three experiments. Means of insulin-treated
groups were compared to those of controls by a two-way
analysis of variance, followed by Dunnett's post hoc test
(p<0.05)

Time (Hr)
Immunoactive PKC {% of Control)
1 04

Figure 7-3. The effect of insulin on glial iPKC. Insulin
was added to the culture medium for 2 hours, following
which the medium was aspirated from the culture dishes
and the glial cells were separated into cytosolic (C) and
membranous (M) fractions and subjected to gel
electrophoresis and Western blotting as described in
methods. Each lane represents 160 ug protein.
Densitometric values are (from left to right) 944, 788,
3673 and 888. The experiment was repeated twice.

80kD-
Control Insulin
106

Figure 7-4: The effect of insulin on neuronal iPKC. One
hundred sixty-seven nM insulin was added to the neuronal
medium for the times specified. The cells were then
washed, prepared for Western blotting as described in
methods, and bands were read on a densitometer.
Densitometric readings are (from left to right) 2158,
1320, 1347, 2364, 1724 and 2162. The experiment was
repeated 3 times.

00
o
o
i
0 min |
15 min
v 4
1 hr j
2 hr |
4 hr
24 hr
80 i

Figure 7-5: The effect of dexamethasone on glial iPKC.
Dexamethasone (2.55 uM) was added to the medium of
confluent glial cultures and the cells were returned to
the incubator for the times specified. The cells were
then Western blotted for PKC as described in methods.
Densitometric readings are (from left to right) 6007,
6167, 6219, 6195, 8286 and 6536. The experiment was
repeated once.

0 min
15 min
1 hr
2 hr
4 hr
24 hr
o

Figure 7-6: The effect of dexamethasone on neuronal
iPKC. Dexamethasone (2.55 uM) was added to the
neuronal medium for the times specified. The cells were
then washed, prepared for Western blotting as described
in methods, and bands were read on a densitometer.
Densitometric readings are (from left to right) 1142,
1201, 2371, 1771, 2374 and 3661. The experiment was
repeated once.

0 min
15 min
1 hr
2 hr
4 hr
24 hr
i
2U
80KD

CHAPTER VIII
DISCUSSION AND SUMMARY
Although the brain has long been considered to be an insulin-
independent organ, both insulin and high affinity insulin receptors
have been localized in the brain within the past ten years. Three
major questions have arisen with regard to these findings: what is
the source of this peptide; what is its function, and how are the
receptors regulated in the brain. Of the three questions, the most
controversial is the first. Several investigators have independently
measured concentrations of insulin in the brain that are higher than,
and independent of, plasma concentrations of insulin (14,23). The
latter finding suggests that the high level of insulin in the brain is
not the result of sequestration and concentration of the peptide. In
addition, many investigators have independently identified an mRNA
in the brain that binds with a cDNA for insulin (20-4). These have
been identified in both cultured cells and in tissue slices from the
brain. As insulin acts to promote neurite outgrowth and neuronal
survival in the brain (115), the higher incidence of reports of an
mRNA for insulin in culture may be due to preferential survival of
insulin-producing and/or -responsive cells in culture.
113

114
This research addressed the question of the site of insulin
synthesis in the brain as well. Exogenous, radioactively labelled
leucine was applied to neuronal cultures which had been removed
from any peripheral source of insulin for at least ten days. The cells
incorporated the radioactive amino acid into a peptide which was
precipitable with an antibody against insulin. When samples of this
material were compared to insulin chromatographically on a
reverse-phase HPLC column, they coeluted with the an insulin
purified from rat pancreas. The medium in which the cells were
grown contained horse serum, in which only one type of Insulin was
expressed. In contrast, the rat expresses two different insulin
molecules. The sample isolated from the brain had two peaks which
were precipitated by the antibody against insulin and which were
similar chromatographically to the two peaks for rat Insulin. This
suggests that insulin may be synthesized in the brain, although the
question cannot be answered definitively until the material is
sequenced. A number of other molecules are similar to insulin and
may both react with the antibody and have chromatographic
properties similar to those of insulin.
The next question addressed by this study concerned the function
of the insulin-like peptide found in the brain. Other investigators
have found that administration of insulin into the brain has specific
effects both in vivo and in vitro (28, 33-41). Insulin appears to act
as a neuromodulator in the brain and may act as a neurotransmitter
as well (36-39, 42-44). It appears to be synthesized in the brain,
and it acts on specific, high-affinity receptors to alter electrical
activity in selected areas of the brain (14, 20-3, 42-44). A fourth

115
quality must necessarily be present for insulin to be characterized
as neurotransmitter. It must be released under depolarizing
conditions. When cultured neurons were treated with depolarizing
solutions containing potassium and calcium, the amount of insulin
released into the medium was increased dramatically. This
depolarized release was calcium-dependent, as in the absence of
calcium, no such stimulation of release was observed. This further
suggests that insulin may act as a neurotransmitter in the central
nervous system. Release of insulin from insulinergic neurons was
also stimulated by glucose. Insulin acts on glial cells to stimulate
glucose uptake, and thus, acts indirectly to decrease one stimulus
for its own release (Figure 8-1).
The third question addressed by this work concerned the
regulation of insulin receptors in the brain. Earlier work had already
demonstrated that the insulin receptors in different types of cells
from the brain differed from peripheral receptors and from each
other (29, 31-2). These differences were found in structure,
function and regulation. Our particular interest was with regard to
the regulation of insulin receptors in the brain by PKC and
subsequent actions of insulin. This enzyme has many actions which
mimic the effects of insulin, and yet, the enzyme inactivates the
kinase activity of the insulin receptor and effectively turns off the
receptor in most tissues (87, 91, 95-7, 117-9). In our study of
insulin regulation of PKC in the brain, we first asked whether PKC
mimicked one specific action of insulin in the brain, the stimulation
of glial glucose uptake. We then went on to look at the effects of
PKC on the insulin receptors themselves. Lastly, we looked at

116
insulin's effects on PKC in brain cells to determine whether PKC
might be involved in a feedback loop of some kind. Because
glucocorticoids appear to interact with PKC in peripheral tissues as
well (157), we chose to study dexamethasone's effects on PKC, to
see whether insulin's effects were general or specific to insulin.
When PKC was stimulated by TPA, it acted to stimulate glucose
uptake in glial cells but not in neurons from the brain. This
stimulation was both time- and concentration-dependent. TPA
stimulated glucose uptake to a greater extent than did insulin, and
the stimulation did not reach a maximum as quickly as did
stimulation of glial glucose uptake by insulin. Insulin acts within
minutes, presumably by inducing activation and/or translocation of
the glucose transporter to the membrane. Insulin also stimulates
the transcription of an mRNA for the transporter in glial cells (142).
As PKC acted very quickly to stimulate glial glucose uptake it is
likely that It also induced activation and/or translocation of the
transporter to the membrane. As the effect continued to increase
for hours, it is equally likely that PKC was stimulating synthesis of
the transporter by stimulating the production of an mRNA for the
transporter. Other possibilities cannot be excluded. PKC activation
by TPA induces changes in the membranes of many types of cells and
might have been acting less specifically to modulate membrane
conductances and open channels within the membrane by
phosphorylating channels, pumps and/or ion exchange proteins (48).
This study showed that PKC had no effect on neuronal binding of
insulin but dramatically stimulated glial binding of insulin. This is
unusual, as in peripheral tissues, PKC generally decreases the

117
binding of insulin (81-3). The increase in glial cells was both time-
and concentration-dependent. Specificity studies with other phorbol
esters showed that the most active phorbol esters, with regard to
the activation of PKC were also the most active in stimulating glial
insulin binding, suggesting that TPA was acting through PKC. While
TPA stimulated the binding of insulin in glial cells, it prevented
stimulation of glucose uptake by insulin. As this occured at doses of
TPA which did not induce maximal glucose uptake (119), this could
not have been due to a transport maximum for glucose. This
inactivation of the insulin receptor is similar to the situation
observed with growth factor receptors in other tissues. As
inactivation of the tyrosine kinase activity has been shown to block
internalization of the receptor in some tissues (92-4), this may be
the reason for the increase in the binding of insulin. Scatchard
analysis showed that the increase was due to an increase in the
number of receptors, not the affinity.
Insulin has been shown to stimulate PKC activity in several types
of cells (103, 105-6). In this study insulin stimulated the
concentration of iPKC in glial but not neuronal cells from the brain.
This effect of insulin was time-dependent. Thus, the inactivation of
glial insulin receptors by PKC is a form of negative feedback. In
neurons, both arms of the loop appear to be missing; that is, insulin
failed to stimulate the level of iPKC and PKC did not alter neuronal
binding of insulin. The last statement does not preclude an effect of
PKC on the neuronal insulin receptor, though. PKC may well decrease
the tyrosine kinase activity of the receptor or the response to the

118
receptor without altering the binding characteristics of the
receptor.
Although PKC inactivates the insulin receptor, it has effects
which mimic those of insulin in glial cells as in other cells. Both
insulin and PKC stimulate the activity of S6 kinase as well as other
substrates (101-2, 107). This design is highly advantageous for the
cell which is so regulated. The insulin receptor can be turned off by
feedback inhibition without the loss of those receptor-stimulated
effects which are necessary for the survival and function of the cell.
The effect of insulin to increase glial iPKC without altering
neuronal PKC is not a general effect. Glucocorticoids are involved
peripherally in the regulation of glucose, as is insulin (13). ACTH
may stimulate PKC and PKC, in turn, inhibits the synthesis of
cortisol and has effects of its own on proteins involved in the stress
response (154-8). Thus the interactions between glucocorticoids
and PKC are somewhat similar to the interactions between insulin
and PKC. When cultured neurons and glial cells were treated with
dexamethasone, there was a time-dependent increase in the
concentration of neuronal iPKC, but glial ¡PKC was unaltered.
PKC, then, has different effects on glucose uptake and the binding
of insulin in glial cells and neurons. In addition, it has different
effects on differentiation and function of these cells (69-80). The
different effects on glucose uptake may represent differences in the
glucose transporter, and the differences in the effects on the
binding of insulin may result from differences in those receptors.
However, since both effects were missing in neurons, we chose to
study the relative amounts and activation of PKC in neurons and glia

119
to determine whether there were differences which might explain
both of these phenomena. Both neurons and glia express PKC, with
neurons expressing 3-4 times greater iPKC and phorbol ester¬
binding activity than glial cells (113). Interestingly, similarly
prepared glial cells of the same age express a much higher aPKC.
This suggests that either our antibody does not recognize one or
more major isozymes of PKC in glia or that there is a large fraction
of neuronal ¡PKC which is inactive. Either of these is entirely
possible. Different isozymes have been identified in neurons and
glia (55,110-11). As these were all identified with different
antibodies, it is not clear whether these subtypes are recognized by
an antibody to the alpha and beta-subtypes. One subtype, the gamma
isozyme, is expressed entirely or predominantly in brain and is not
recognized by this antibody (54). The second possibility is that
some fraction of the neuronal PKC is inactive. Neuronal iPKC is
predominantly localized in the membrane, as opposed to the cytosol.
This is the opposite of the situation seen in glial cells. Membrane-
bound PKC is latent in liver (52,133), and may be so in neurons as
well. PKC has many other effects in neurons, but these may require
a smaller concentration of the enzyme.
Activation of PKC generally involves a translocation of the
enzyme from the cytosol to the membrane (59, 60, 63-4). In this
study we showed that TPA stimulated a translocation of glial iPKC
within 5-15 minutes after the cells were exposed. Within hours, the
cytosolic iPKC was immeasurably low, and the membranous iPKC
was downregulated to very low levels within 24 hours. TPA had the
same effect on translocation of aPKC in studies on glial cells (134).

120
This TPA-induced translocation of PKC preceded TPA's other effects
in glial cells and thus, might have been involved in the mechanism.
That those effects continued to occur long after the translocation
even as PKC was downregulated suggested that there were changes
beyond the translocation of PKC. TPA induced a downregulation of
neuronal iPKC within hours as well. The increase in membranous
iPKC, which would demonstrate a translocation, was only seen in
some experiments. This might have been because the cytosolic iPKC
represented a much smaller fraction in the neurons and its
translocation was too small to be observed. It was equally likely
that the particular isozymes identified by this antibody were not
translocated, although others were. In any case, the smaller
fraction of iPKC available to be translocated may play a role in
PKC's different effects in the different types of cells.
Thus, insulin receptors are differentially regulated by phorbol
esters in neurons and glial cells in the central nervous system.
These differences between the types of cells with regard to the
synthesis and function of insulin and the regulation of its receptors
are appropriate for the cells' requirements. In glial cells, which
continue to grow and multiply throughout the life of the organism,
insulin serves a metabolic function. In neurons, insulin's primary
effects are not metabolic in nature. The receptors are differentially
regulated accordingly.

Figure 8-1: Proposed regulation of insulin effector
systems in the CNS. As the concentration of glucose
rises in the CNS, release of insulin from insulinergic
neurons increases in a dose-dependent manner. Insulin in
the brain binds to specific, high affinity receptors (R) on
both neurons and glial cells. Insulin acts to decrease
norepinephrine uptake in neuronal cells (30). In glial
cells, Insulin acts to stimulate glucose uptake (28) by
activating and/or translocating glucose transporters
(GT), thus decreasing the stimulus for its own release by
a form of negative feedback.
Insulin acts in glial cells to increase PKC. PKC feeds
back to inactivate the glial insulin receptor and inhibit
insulin-stimulated glucose uptake, providing another
level of control over the glucose concentration within
the cell and in the interstitial spaces. Both arms of the
circuit, that is, stimulation of iPKC by insulin and
inhibition of the insulin receptor by PKC are missing in
the neurons.

Insullnergíc neuron
Astrocyte

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J. Biol. Chem. 263:9868.

BIOGRAPHICAL SKETCH
Laura Mary Mudd was born to John and Barbara Mudd on September
24, 1958, in Washington, D.C. She Is the eldest of five children.
John (attorney/corporate executive) and Barbara
(theologian/teacher) reside in Miami with one sister, Ellen
(insurance/accountant). Philip (political writer), Clare (artist) and
David (attorney) are currently living in Washington. Laura graduated
from LaSalle High School in Miami in 1976. She then attended
Georgetown University, where she studied chemistry and English
literature. Following graduation in 1980, she worked at Gillette
Research Institute in Maryland, where she studied steroid and
enzyme biochemistry. From 1984-89 she attended the University of
Florida, where she received her doctorate in physiology in the spring
of 1989. Her graduate work involved the regulation of growth
factors in cultured brain cells. She hopes to continue her study of
brain development with postdoctoral research following her
graduation. Laura also enjoys literature, sailing, cooking and the
companionship of her two cats, Langston and Electra Mudd.
137

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Mohan K. Raizada, Chairman
Professor of Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy. _ v ^
â– .
Melvin J.IFregly; f\
Graduate "Research Professor
of Physiology
lfc.M \
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Colin Sumners
Associate Professor of
Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Edwin M. Meyer
Associate Professor of
Pharmacology and
Therapeutics

This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate School and was accepted in
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
May, 1989
J. Lee Dockery
Dean, College of Medicine
Madelyn M. Lockhart
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08554 6256



118
receptor without altering the binding characteristics of the
receptor.
Although PKC inactivates the insulin receptor, it has effects
which mimic those of insulin in glial cells as in other cells. Both
insulin and PKC stimulate the activity of S6 kinase as well as other
substrates (101-2, 107). This design is highly advantageous for the
cell which is so regulated. The insulin receptor can be turned off by
feedback inhibition without the loss of those receptor-stimulated
effects which are necessary for the survival and function of the cell.
The effect of insulin to increase glial ¡PKC without altering
neuronal PKC is not a general effect. Glucocorticoids are involved
peripherally in the regulation of glucose, as is insulin (13). ACTH
may stimulate PKC and PKC, in turn, inhibits the synthesis of
cortisol and has effects of its own on proteins involved in the stress
response (154-8). Thus the interactions between glucocorticoids
and PKC are somewhat similar to the interactions between insulin
and PKC. When cultured neurons and glial cells were treated with
dexamethasone, there was a time-dependent increase in the
concentration of neuronal iPKC, but glial iPKC was unaltered.
PKC, then, has different effects on glucose uptake and the binding
of insulin in glial cells and neurons. In addition, it has different
effects on differentiation and function of these cells (69-80). The
different effects on glucose uptake may represent differences in the
glucose transporter, and the differences in the effects on the
binding of insulin may result from differences in those receptors.
However, since both effects were missing in neurons, we chose to
study the relative amounts and activation of PKC in neurons and glia


16
In addition to being responsive to trophic factors, glial cells may
produce them as well. A substance which is immunologically
identical to nerve growth factor is present in glia (123) and glia,
which develop at the same time as neurons, provide other
extracellular molecules which enhance neuronal migration (124).
This glial stimulus to neuronal growth is lost with age as astrocytic
glial cells block axonal regeneration (125) and synapse formation
(126) in the mature nervous system. Inflammation of the mature
nervous system causes a reactive gliosis which prevents neuronal
repair as in multiple sclerosis (124).
Finally, glia contribute to the regulation of nervous system
excitability. Glia take up, and thus inactivate, glutamate, GABA,
aspartate and serotonin (127) at the synapse. Insulin decreases
levels of alpha2-adrenergic receptors in glia; this would tend to
regulate the amount of norepinephrine in the synaptic cleft (128).
Glia do not only take up neurotransmitters but also act on several
via specific enzymes such as glutamine synthetase and GABA
transaminase. In addition, they have specific receptors for alpha-
and beta-adrenergic agonists, dopamine, prostaglandin Ei, secretin,
somatostatin and vasoactive intestinal peptide (127), among others.
Lastly, glia have themselves been shown to possess some voltage
gated channels and, thus, may act as excitable cells (129).
Because both neurons and glia are important for appropriate
development and function of the nervous system and because of the
afore-mentioned differences in neurons and glia with regard to both
the insulin receptor and PKC, we chose to study the interaction of


% [125l]lnsulin Binding
Time (Hr)


117
binding of insulin (81-3). The increase in glial cells was both time-
and concentration-dependent. Specificity studies with other phorbol
esters showed that the most active phorbol esters, with regard to
the activation of PKC were also the most active in stimulating glial
insulin binding, suggesting that TPA was acting through PKC. While
TPA stimulated the binding of insulin in glial cells, it prevented
stimulation of glucose uptake by insulin. As this occured at doses of
TPA which did not induce maximal glucose uptake (119), this could
not have been due to a transport maximum for glucose. This
inactivation of the insulin receptor is similar to the situation
observed with growth factor receptors in other tissues. As
inactivation of the tyrosine kinase activity has been shown to block
internalization of the receptor in some tissues (92-4), this may be
the reason for the increase in the binding of insulin. Scatchard
analysis showed that the increase was due to an increase in the
number of receptors, not the affinity.
Insulin has been shown to stimulate PKC activity in several types
of cells (103, 105-6). In this study insulin stimulated the
concentration of iPKC in glial but not neuronal cells from the brain.
This effect of insulin was time-dependent. Thus, the inactivation of
glial insulin receptors by PKC is a form of negative feedback. In
neurons, both arms of the loop appear to be missing; that is, insulin
failed to stimulate the level of ¡PKC and PKC did not alter neuronal
binding of insulin. The last statement does not preclude an effect of
PKC on the neuronal insulin receptor, though. PKC may well decrease
the tyrosine kinase activity of the receptor or the response to the


Figure 3-4: Time course of depolarization-induced
release of [3H]-leucine labelled insulin. One hundred uCi
of [3H]-leucine was added to 100 mm culture plates 24
hours in advance of the depolarization-induced release
experiment. Medium was aspirated from 14 day old
neuronal cultures and replaced by 8 ml of a control or a
depolarizing solution. Cultures were incubated at 37C
for periods ranging from 0-60 minutes. The solutions
were aspirated, lyophilized, reconstituted in distilled
water and subjected to radioimmunoassay. Control
release was subtracted from depolarized release at each
time point and results were converted to femtomoles of
insulin released. This is one representative experiment
of three.


3H]2-dGlc Uptake (% Control)
250
200
150
100
50
0
2 3
Time (Hr)
1
4
5


CHAPTER VI
THE REGULATION OF INSULIN RECEPTORS IN NEURONAL AND GLIAL
PRIMARY CULTURES BY PHORBOL ESTERS
Intmduclian
Neuronal and glial insulin receptors are structurally and
physiologically distinct in several respects: neurons have a receptor
of lower molecular weight (29,32); insulin stimulates the uptake of
glucose in glia but not in neurons (28), and insulin inhibits the
uptake of norepinephrine in neurons, but not in glia (30). In addition,
there are distinctions between neurons and glia with regard to PKC:
PKC is present in higher concentration in neurons than in glia from
the same brains as demonstrated immunologically and by binding
studies with phorbol esters (113) although the activity is higher in
glial cells; different isotypes of PKC are present in neurons and glia
(55,110,111), and phorbol esters stimulate glucose uptake in glia,
but not in neurons (119). As neurons and glia differ with respect to
the physiological activities of both insulin receptors and PKC and
because PKC regulates the binding, autophosphorylation, tyrosine
kinase activity and some cellular responses of the insulin receptor
peripherally (81-3,87,91,95-7), we chose to investigate the role of
PKC in the regulation of the insulin receptor in neurons and glia from
76


Figure 6-1: The effect of TPA on insulin binding in glial
and neuronal cultures. Cells were incubated with TPA
for two hours at 37C. TPA was removed by washing and
insulin binding was measured as described in methods.
Each point represents the mean SEM of three
experiments.


Figure 6-2: Time-course of stimulation of insulin
binding by TPA in glial cultures. One hundred nM TPA was
added to culture medium for the times specified.
Following this incubation, TPA was removed by washing
and insulin binding was measured as described in
methods. Each point represents the mean SEM of four
experiments.


Figure 6-6: Recovery of insulin binding in glial cultures
after removal of TPA. Cells were treated with 100 nM
TPA for 2 hours at 37C and washed three times with
DMEM. Both TPA-treated () and control () cells were
refed with DMEM containing 10% FBS and returned to the
incubator for the specified time. Cells were then washed
and the binding of insulin was measured as described in
methods. Each point represents the mean SEM. Means
of treated and control groups were compared by two-way
analysis of variance, followed by Duncans post hoc test
(p<0.05). This is 1 representative experiment of 4.


Figure 7-3. The effect of insulin on glial iPKC. Insulin
was added to the culture medium for 2 hours, following
which the medium was aspirated from the culture dishes
and the glial cells were separated into cytosolic (C) and
membranous (M) fractions and subjected to gel
electrophoresis and Western blotting as described in
methods. Each lane represents 160 ug protein.
Densitometric values are (from left to right) 944, 788,
3673 and 888. The experiment was repeated twice.


0 min
15 min
1 hr
2 hr
4 hr
24 hr
2U
80KD


PKC Immunocytochemistry 31
Statistical Analysis 31
III. INSULIN SYNTHESIS AND RELEASE FROM NEURONAL CULTURES.37
Introduction 37
Results 39
Discussion 40
IV. CHARACTERIZATION OF PKC IN NEURONAL AND GLIAL CELLS
IN PRIMARY CULTURE 51
Introduction 51
Results 52
Discussion 53
V. THE REGULATION OF SUGAR TRANSPORT IN PRIMARY
NEURONAL AND GLIAL CELL CULTURES BY PHORBOL
ESTERS 71
Introduction 71
Results 72
Discussion 72
VI. THE REGULATION OF INSULIN RECEPTORS IN NEURONAL AND
GLIAL PRIMARY CULTURES BY PHORBOL ESTERS 76
Introduction 76
Results 77
Discussion 79
VII. THE EFFECTS OF INSULIN AND DEXAMETHASONE ON NEURONAL
AND GLIAL PKC 97
Introduction 97
Results 98
Discussion 99
VIII. DISCUSSION AND SUMMARY 113
REFERENCES 123
BIOGRAPHICAL SKETCH
.137


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
m
Mohan K. Raizada, Chairman
Professor of Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
mi.
>aiy/ /)
Melvin J. Fregly/
Graduate Research Professor
of Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
l7L= Sz
Colin Sumners
Associate Professor of
Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Edwin M. Meyer
Associate Professor of
Pharmacology and
Therapeutics


38
localizing insulin immunoreactivity to 3-5 percent of cultured
neurons from rat brain (18). The mRNA species is larger than that
observed in the human pancreas when the two are compared by
Northern blotting techniques (24). In situ hybridization studies
localize neurons containing the insulin mRNA to the periventricular
hypothalamus and cerebral cortex in rat brain (21) and rat, mouse
and hamster anterior pituitary cells (20). Other areas of the brain,
including the olfactory bulb and choroid plexus do not contain the an
mRNA for insulin (21). In the anterior pituitary, only 5-10 percent
of the cells are positive for the mRNA; those cells are epithelial
and the immunoreactive insulin that they contain is localized in
secretory granules (20).
The second body of evidence relates to the independent regulation
of brain and peripheral insulin levels. Many investigators have
reported brain insulin concentrations that are higher than those
observed in plasma (14,23). It is unlikely that this represents
sequestration and concentration of plasma insulin for two reasons.
First, brain insulin concentrations are not altered by disease states
which raise or lower plasma insulin concentrations, although the
concentration in CSF is lowered in response to lowered plasma
insulin. Secondly, the capillaries of the blood-brain barrier do not
transport active insulin into the brain.
As discussed previously, insulin acts in the brain to alter
neuronal electrical activity. This, in combination with the evidence
that insulin may be synthesized in the brain, led us to investigate
whether pulse-labelled immunoreactive insulin could be synthesized
in the brain and whether this immunoreactive insulin could be


9
of those receptors. In 1983, TPA was first shown to stimulate
phosphorylation of both insulin and IGF I receptors in IM-9 cells that
had been preincubated with H332P04 (61). Insulin- and TPA-
stimulated phosphorylation appeared to be additive, suggesting that
there was no interaction between the sites. In 1984, TPA was
shown to enhance serine/threonine phosphorylation of the insulin
receptor in rat hepatoma cells at nine sites (87). Insulin was shown
to stimulate phosphorylation of tyrosine and serine residues at six
sites, three of which were similar to the TPA-phosphorylated sites.
In addition, the phorbol ester decreased insulin-stimulated
phosphorylation, suggesting that there was, in fact, an interaction
between the sites of action of the two agents. In later studies on
IM-9 and HepG2 cells, TPA was found to phosphorylate four major
serine residues, which were not phosphorylated in untreated cells
and to increase the phosphorylation of one threonine residue on the
insulin receptor. These serine residues were not phosphorylated by
insulin, which, however, did phosphorylate three tyrosine residues
(89). PKC acts directly on the insulin receptor as it phosphorylates
the insulin receptor in vitro (90). Similar results were seen with
the IGF I receptor. These very different profiles of phosphorylation
induced by insulin and phorbol esters give strong evidence that
insulin and IGF I were not acting through PKC. In 1988, TPA was
found to enhance predominantly the phosphorylation of one serine
residue on the insulin receptor in hepatoma cells (91). TPA-
treatment of cells inhibits insulin-stimulated receptor
phosphorylation of exogenous substrates by 50 percent. These
changes in the receptor are maintained when the receptors are


77
the central nervous system to determine whether PKC might
differentially regulate these two receptors.
Results
TPA induced a dose-dependent increase in the binding of insulin
in glial cells with no effect on binding in neurons over the same
range of concentrations (Figure 6-1). TPA treatment of glial cells
for two hours did not alter the binding of insulin at a concentration
of 1 nM TPA, began to increase binding at 10 nM TPA and caused a
maximal increase at a dose of 50 nM TPA. The ED50 was 15 nM and
50 nM caused a maximal increase of 77 percent. The effect of TPA
on the binding of insulin was time-dependent as well (Figure 6-2).
Treatment with 100 nM TPA induced an increase in the binding of
insulin in glia! cells within 30 min. with a maximal increase at two
hours, followed by a decline. The amount of bound insulin stabilized
at four hours after treatment with TPA. Translocation of PKC in
glial cells from the cytosol to the membrane began within 5 min. and
was virtually complete within 15 min. The effects of TPA on insulin
binding in glial cells and 2-dGlc uptake followed TPA-stimulated
translocation of PKC and, thus, PKC may be involved in TPA's effect
on insulin binding (Figure 6-3). Treatment of neurons with 10 and
100 nM doses of TPA had no effect on the binding of insulin (Figure
6-1).
Glial cells were treated with 100 nM TPA for 2 hours. Following
this treatment, competitive inhibition experiments for untreated
and TPA-treated glial cultures were conducted by adding a constant
amount of [125|]jrisulin (100,000 cpm/dish) and increasing amounts


Time (Hr)
% of Control
PKC Translocation
cn o on
G>
Insulin Binding
o
x
200
2-dGlc uptake


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This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate School and was accepted in
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
May, 1989
J. Lee Dockery
Dean, College of Medicine
Madelyn M. Lockhart
Dean, Graduate School


72
activation of PKC will provide us with an additional parameter to
study the differences in the regulation of neuronal and glial glucose
uptake.
Results
TPA stimulated [3H]2-dGlc uptake in glial cells in a time-
dependent manner. Glucose uptake was increased as early as 20 min.
after the administration of TPA with maximal increases occurring
after 4 hours of treatment with 100 nM TPA. The maximal level of
increase was 204.5 12.5 percent (Figure 5-1). In contrast, TPA
failed to influence 2-dGlc uptake in neuronal cultures under similar
conditions. The stimulatory effect of TPA on glial glucose uptake
was selective and was due to an increase in the number of glucose
transporters rather than a change in the Km of the transporter (119).
Piscussion
The differences in PKC-stimulated glucose uptake between
neurons and glial cells may be due to the different isozymes of PKC
present in the two types of cells, or to the presence of a smaller
pool of PKC available to be translocated in neurons. In other related
work in this area, our group found that phorbol ester-induced
stimulation of glucose uptake in glial cells is also concentration-
dependent with a maximal effect at 100 nM TPA. It is likely that
TPA is acting through PKC as the potency of five phorbol esters
paralleled their abilities to bind and activate PKC (119). However,
PKC has many effects on cells within the brain; even if these
effects occur by way of activastion of PKC, they may be indirect


99
(Figure 7-3). In contrast, treatment of neurons with insulin under
the same conditions did not elevate iPKC in neurons. This was true
even after 24 hours of treatment (Figures 7-4). When cells were
treated with dexamethasone, a different effect was observed.
Treatment with dexamethasone (2.55 uM) had no effect on glial iPKC
over the course of 24 hours (Figure 7-5) but markedly stimulated the
level of iPKC in neurons in a time-dependent manner. Neuronal iPKC
was elevated by greater than 100 percent within 4 hours and
continued to rise over the course of 24 hours, at which time it was
increased 4.4-fold over the control (Figure 7-6).
Di.§cusi£n
These experiments show that insulin stimulates iPKC in the same
cells in which PKC regulates the insulin receptor, that is, in
cultured glial cells. It appears that PKC acts as a part of a feedback
loop to regulate the insulin receptor. The increase in iPKC following
treatment with insulin could be due to increased transcription of an
mRNA for PKC, increased translation of existing mRNA or to the
alteration of a non-immunoreactive molecule to one that is
recognized by the antibody. As the level of iPKC increases over the
course of hours, it is likely that synthesis of protein is involved.
Additional experiments with cycloheximide would be necessary to
support this. In contrast, insulin does not elevate neuronal PKC
under the same conditions of time and concentration. This
concentration of insulin has been shown to induce other effects in
the brain (28, 30). As PKC does not alter neuronal binding of insulin,
it appears that both arms of the circuit are missing. It would be


78
of unlabelled insulin (0.167 nM-133 nM) to binding buffers. An IC50
of 40 nM insulin was observed for both curves (Figure 6-4). The data
were used for Scatchard analysis. The Kds for the high and low
affinity insulin receptors were 18.5 and 131.6 nM, respectively, in
control cells. Treatment with TPA induced a 184 percent increase in
the number of high affinity binding sites on glial cells without
altering their affinity. TPA increased the number of low affinity
sites by 74 percent while decreasing the affinity of those sites only
minimally (Table 6-1). Glial cells were exposed to three different
phorbol esters for 2 hours to evaluate the specificity of the
stimulation of insulin binding by TPA. TPA stimulated the binding of
insulin by 109 percent at a concentration of 50 nM. The same dose
which caused maximal stimulation by TPA had no effect when cells
were treated with 43-phorbol 123,13a-didecanoate (3-PDD). (3-PDD
exhibited an increase of 82 percent only at a concentration of 500
nM while 4a-phorbol 1213,13a-didecanoate (a-PDD) had no effect at
any concentration (Figure 6-5). Thus, the potencies of these drugs
corresponded to their abilities to bind and activate PKC, that is, TPA
> 6-PDD > the inactive analog, a-PDD (147). Pretreatment of glial
cultures for two hours with 100 nM TPA followed by its removal
resulted in a time-dependent recovery of insulin binding. The
binding recovered by 59 percent within 2 hours. Recovery was
complete within 6 hours (Figure 6-6).
The next question concerned the TPA-stimulated increase in the
number of glial insulin receptors and whether it corresponded to an
increase in responsiveness to insulin. Insulin and/or TPA was added
directly to the glial medium and the dishes were returned to the


Immunoactive PKC (% of Control)
89


Figure 3-2: Radioimmunoassay of HPLC fractions of
released insulin. Pooled samples of the material
released under depolarizing conditions were
chromatographed on a BioRad-C4 column as described in
Methods. The fractions were assayed by routine
radioimmunoassay for insulin. The top panel represents
the purified insulin from rat pancreas and the bottom
panel represents the radioimmunoassayable insulin of
each fraction of the pooled samples. This representative
experiment was repeated once.


21
arabinoside in DMEM containing 10 percent PDHS. This treatment
resulted in the death of rapidly dividing cells. After 48 hours
cytosine arabinoside was removed and the cells were refed with 10
percent PDHS in DMEM and the cells were grown in culture for
another 7-10 days prior to use.
These cultures contain 80-85 percent neurons as demonstrated
by light microscopy and immunocytochemical markers (18, Figure 2-
1). Since insulin and insulin receptors were widely distributed
throughout the brain, in contrast with the localized distribution of
other neuropeptides and their receptors, cultures from the whole
brain were used. We considered growing the cells in a chemically
defined medium. However, since one of the essential components of
this defined medium is insulin in relatively high concentration, this
possibility was abandoned. It was felt that the presence of insulin
in the medium would complicate studies related to the expression
and action of insulin receptors. Thus, we proposed to continue to
culture cells in DMEM containing either PDHS or fetal bovine serum
(FBS). PDHS and FBS were purchased from Hyclone and Gibco
Laboratories, respectively, with an insulin concentration of 1-4
ng/ml. At this concentration, insulin did not cause either down-
regulation or other biological effects on insulin receptors of either
neurons or glia (130).
Preparation of Primary Astrocytic Glial Cultures from Rat Brains
The procedure for removing the brains and dissociating the cells
was the same as that described above. After the cells were
centrifuged, they were suspended in medium containing 10 percent


8(H
80KD


REFERENCES
1. Humbel, R., Bosshard, H. and Zahn, H. (1972) Chemistry of insulin. In:
Handbook of Physiology. Sect. 7: Endocrinology. VoL l. Endocrine
Pancreas. R. Greep and E. Astwood, Eds., American Physiological Soc.f
Washington, D.C.
2. Schmidt, P. (1928) Uber organtherapie und insulin behandlung bei
endogenen geistesstorungen. Klin. Wochenschr. 7:839.
3. Sakel, M. (1935) Neue behandlungsmethode der schizophrenic.
Verlag Moritz Perles, Vienna.
4. Prange, A., Jr., Loosen, P, (1984) Peptides in depression. In: Frontiers
ip-Biogiiemicai 3Qd, Ph?irrnagoiiqicaLB9ge9rciun,.PgpresiQn. E. Usein,
ed. Raven Press, New York.
5. Steiner, D., Kemmler, W., Clark, J., et al. (1972) The biosynthesis of
insulin. In: Handbook of Physiology. Endocrinology 1. Steiner, D. and
Freinkel, N., eds. Williams and Wilkins, Baltimore.
6. Goodner, C. and Porte, D. (1972) Determinants of basal islet secretion i
n man. Handbook of Physiology. Steiner, D. and Freinkel, N., eds.
Williams and Wilkins, Baltimore.
7. Fajans, S. and Floyd, J. (1972) Stimulation of islet cell secretion by
nutrients and by gastrointestinal hormones released during digestion. In:
Handbook of Physiology. Steiner, D. and Freinkel, N. eds. Williams and
Wilkins, Baltimore.
8. Porte, D. and Halter, J. (1981) The endocrine pancreas and diabetes
mellitus. In: Textbook of Endocrinology. (6th ed.) Williams, R., ed. W.B.
Saunders Co., Philadelphia.
9. Lockhart-Ewart, R., Mok, C. and Martin, J. (1976) Neuroendocrine
control of insulin secretion. Diabetes 25:96.
10. Grodsky, G., Landahl, H. and Curry, D. (1970) In vitro studies suggesting
a two-compartmental model for insulin secretion. In: Structure and
Metabolism of the Pancreatic Islets. Falmer, S, Heilman, B. and Taljedal,
I., eds. Pergamon Press, Oxford.
11. Gammeltoft, S., Kowalski, A., Fehlmann, M. and Obberghen, E. (1984)
Insulin receptors in rat brain: Insulin stimulates phosphorylation of its
receptor beta-subunit. FEBS Lett. 172:87.
123


31
PKC Immunocytochemistry
Cells were grown on sterile glass coverslips in culture dishes.
Prior to staining, they were washed three times with PBS and fixed
in 3.5 percent paraformaldehyde/0.25 percent glutaraldehyde in PBS
(pH 7.4) on ice for 30 min. The cultured cells were then
permeabilized with 0.1 percent Triton X-100 in PBS for 30 min. at
room temperature. Following the fixing and permeabilizing steps,
the cells were rinsed three times with PBS and a 1:10 dilution of a
monoclonal anti-PKC antibody in 1 percent BSA, 0.1 percent sodium
azide in PBS was applied. After a 24 hour exposure to this antibody
at 4C and they were again rinsed with PBS. Control cells were
treated with the same solution without the primary antibody. A
1:100 dilution of an anti-mouse 1g-peroxidase conjugate in 0.1
percent BSA in PBS was applied for 30 min. at room temperature and
then the excess was removed by washing with PBS. Finally, the
cells were incubated in a solution of 0.5 mg/ml DAB/0.03 percent
hydrogen peroxide in PBS prepared immediately prior to use. After
10 min. they were washed and a drop of 9:1 glycerol:PBS was applied
to the coverslip. The coverslips were inverted, placed on a glass
microscope slide and the edges were sealed with nail polish.
Photographs were taken at 400 and 1000 x magnification with a
Zeiss D-7082 Axiophot photomicroscope.
Statistical Analysis
Statistical analysis was by analysis of variance (ANOVA)
followed by Duncan's post hoc test when the means of several groups
were to be compared or Dunnett's post hoc test when the means of


8
Ligand-Receptor Interactions
PKC regulates the specific binding of insulin and insulin-like
growth factor I (IGF I). Phorbol esters regulate the insulin receptor
in lymphocytes, adipocytes and monocytes (81-3). In each of these
types of cells, phorbol esters inhibit the binding of insulin by
increasing the Km of the high affinity receptor. The endogenous
analogues of TPA, the DAGs, also reversibly inhibit the binding of
insulin to its receptor (84) by altering the affinity of the receptor.
The calcium ionophore A23187 potentiates the effect of TPA on
insulin binding in monocytes (85). TPA does not, however, decrease
insulin binding in all cell types. TPA has no effect on insulin binding
in either 3T3 cells or in hepatoma cells (86-7) although the
response to insulin is impaired in the latter. Thus, PKC-induced
decreases in insulin binding may result from either decreased
affinity of the receptors or increased internalization of the insulin-
receptor complex, depending on the type of tissue or cell.
The case for regulation of the IGF I receptor by PKC is similar to
that for the insulin receptor. DAGs inhibit IGF I binding to the IGF I
receptor in monocytes. TPA also inhibits IGF I binding in
lymphocytes, monocytes and adipocytes by altering the high-affinity
binding site without altering the number of receptors (84). This
differs from insulin or IGF I down-regulation of the IGF I receptor,
which results from a decrease in the number of receptors (88).
Phosphorylation of Receptors
The mechanism for PKC-stimulated alterations in the insulin and
IGF I receptors appears to involve serine/threonine phosphorylation


13
unevenly distributed, with the left cerebral hemisphere expressing
more than the right in the rat (112). Binding studies using
radiolabeled phorbol esters show two to three times more PKC in
neurons than in glial cells cultured from the same rat brains (113).
The subcellular distribution of PKC has been the subject of many
investigations. It is localized in dendrites, axons, perikarya and
nuclei (110) of neurons with particularly high concentrations in
presynaptic terminals (111) and in growth cones (114). This is not
unexpected as PKC mediates both neurotransmission and neurite
outgrowth (115). Fractionation of glial cells demonstrated that the
majority of the PKC was cytoplasmic (116). The same study showed
that the majority of PKC in whole brain tissue is associated with
the membrane, suggesting that the majority of neuronal PKC is
membrane-bound. Seventy-five percent of the glial cell cytoplasmic
PKC can be translocated to the membrane within 30-60 minutes of
TPA-treatment. This is similar to the situation seen in peripheral
tissues.
PKC Regulation of Glucose Uptake and Insulin Receptors in the Brain
PKC has been shown to regulate glucose uptake in many tissues.
Phorbol ester-induced decreases in the binding of insulin to its
receptor are not associated with decreased glucose uptake as, in
fact, phorbol esters stimulate glucose uptake in adipocytes,
myocytes, fibroblasts and astrocytes (97,117-9).
As discussed, in the brain there are distinctions between neurons
and glia with regard to PKC: PKC is present in higher concentrations
in neurons than in glia from the same brains as demonstrated by the


27
foil-covered tubes as the compound is light-sensitive. One mg of
porcine insulin was weighed and dissolved in 2 ml of 0.01 N HCl.
Sodium metabisulfite was weighed and diluted similarly to the
chloramine T except that it was made to 8 mg/ml in the first
dilution such that the concentration after 1:100 dilution was 80
pg/ml. The decay chart for 125j Was checked to determine what
volume of 125| contained 1 mCi. Fifty ml of a solution of 1 mg/ml
of insulin-free BSA (eg Sigma A-7030) in phosphate buffer was
prepared and about 20 ml was used to wash a sephadex-G25 column
(PD 10 column from Pharmacia). The following were added in
sequence to a disposable plastic test tube in a fume hood: 1) 25 pi
phosphate buffer, 2) 5 pg insulin (10 pi), 3) 1 mCi and 4) 10 pi
chloramine T. The tube was capped, vortexed and contents were
incubated at room temperature for 5 min. Ten pi of sodium
metabisulfite was added to stop the reaction and the tube was
capped and vortexed once again. Two hundred pi of phosphate buffer
was added to increase the volume and the solution was added to the
top of the column. After the reaction mixture was absorbed by the
column, the column was washed with the BSA/phosphate buffer and
fractions were collected. [125|]-jnsulin usually eluted in about 3-5
ml. Ten pi of each fraction was removed and placed in a test tube,
capped and counted on a scintillation counter. To calculate percent
incorporation, 50 pi of 3 percent BSA and 1 ml of ice-cold 10
percent trichloroacetic acid were added to the test tube containing
the highest counts. Then the sample was incubated at 4C for 5 min.
and centrifuged at 1000 x g for 1 min. Radioactivity in the pellet
represented incorporated insulin. This should be greater than 90


Figure 7-2: The effect of insulin on glial iPKC. One
hundred sixty-seven nM insulin was added to the medium
of confluent glial cultures for the specified period of
time. PKC was quantitated by Western blotting and
densitometry. Each point represents the mean + SEM for
at least three experiments. Means of insulin-treated
groups were compared to those of controls by a two-way
analysis of variance, followed by Dunnett's post hoc test
(p<0.05)


90
Table 6-1: Data from the Scatchard plot of TPA effect on glial cell
High Affinity
Receptor number
Ka
Low Affinity
Receptor number
Ka
[125|]jnsulin binding
Control
1.13+0.13 pmol/mg
0.0540.008 nM"1
5.020.81 pmol/mg
0.0076+0.0005 nM"
TPA
3.21+0.65 pmol/mg
0.043+0.006 nM"1
8.77+1.69 pmol/mg
0.0066+0.002 nM-1


CHAPTER Hi
INSULIN SYNTHESIS AND RELEASE FROM NEURONAL CULTURES
in.iradu.cti, on
Insulin alters the content of several neurotransmitters in the
brain (36-8), stimulates the release of others (39) and changes
neuronal electrical activity in specific regions of the brain (42-3).
Thus, it definitely acts as a neuromodulator. As insulin is suggested
to act via specific insulin receptors (44), it may act as a
neurotransmitter as well. In order to be classified as a
neurotransmitter, however, synthesis and release of insulin under
depolarizing conditions must be demonstrated in neurons.
Two separate bodies of evidence suggest that the insulin present
in the brain is also synthesized there: 1) preproinsulin mRNA is
present in brain (20-23) and 2) the level of insulin in the brain is
independent of the level of insulin in the periphery (19,23). Several
investigators have identified an mRNA in brain tissue that
hybridizes to a cDNA for insulin. Insulin mRNA is found in neurons,
but not in glial cells in cultures from the brains of both rats and
rabbits (22-3). Only 3-5 percent of cultured neurons from rabbits
contain the mRNA. This is in agreement with an earlier study
37


5.5 11 2
Glucose (mM)
Insulin Released (% Control)
4 ro go **
O o o O
o o o
T | 1 r


116
insulin's effects on PKC in brain cells to determine whether PKC
might be involved in a feedback loop of some kind. Because
glucocorticoids appear to interact with PKC in peripheral tissues as
well (157), we chose to study dexamethasone's effects on PKC, to
see whether insulin's effects were general or specific to insulin.
When PKC was stimulated by TPA, it acted to stimulate glucose
uptake in glial cells but not in neurons from the brain. This
stimulation was both time- and concentration-dependent. TPA
stimulated glucose uptake to a greater extent than did insulin, and
the stimulation did not reach a maximum as quickly as did
stimulation of glial glucose uptake by insulin. Insulin acts within
minutes, presumably by inducing activation and/or translocation of
the glucose transporter to the membrane. Insulin also stimulates
the transcription of an mRNA for the transporter in glial cells (142).
As PKC acted very quickly to stimulate glial glucose uptake it is
likely that it also induced activation and/or translocation of the
transporter to the membrane. As the effect continued to increase
for hours, it is equally likely that PKC was stimulating synthesis of
the transporter by stimulating the production of an mRNA for the
transporter. Other possibilities cannot be excluded. PKC activation
by TPA induces changes in the membranes of many types of cells and
might have been acting less specifically to modulate membrane
conductances and open channels within the membrane by
phosphorylating channels, pumps and/or ion exchange proteins (48).
This study showed that PKC had no effect on neuronal binding of
insulin but dramatically stimulated glial binding of insulin. This is
unusual, as in peripheral tissues, PKC generally decreases the


Figure 5-1: The effect of TPA on neuronal and glial 2-
dGIc uptake. One hundred nM TPA was added to the
medium of either neuronal (*) or glial () cultures for the
times specified. Following this incubation, cells were
rinsed twice with phosphate-buffered saline (PBS) and
exposed to a solution containing labelled 2-dGlc for 5
min at 37C. The cultures were again rinsed with PBS.
Cells were dissolved in 0.2 N NaOH and scraped from the
dishes; radioactivity was counted and normalized for
content of protein. Each point represents the mean SEM
of at least three experiments.


Figure 4-5: TPA-induced PKC redistribution in neurons.
One hundred nM TPA was added to the neuronal medium
for the times specified, following which samples were
separated into cytosolic (C) and membrane (M) fractions
and prepared for Western blotting as described in
Methods. Each lane represents 330 ug protein.
Densitometric values for the 80 kD band are (from left to
right), 2122, 10352, 4863, 5156, 1709, 4272, 1325,
6493, 900 and 1581. This is one representative
experiment of four.


131
96. Caron, M., Cherqui, G., Wicek, D., Capeau, J., Bertrand, J. and Picard, J.
(1988) Effect of protein kinase C activation and depletion on insulin
stimulation of glycogen synthesis in cultured hepatoma cells. Exp.
44:34.
97. Kirsch, D., Obermaier, B. and Haring, H. (1985) Phorbol esters enhance
basal D-glucose transport but inhibit insulin stimulation of D-gluccse
transport and insulin binding in isolated rat adipocytes. Bchm. Bphys.
Res. Comm. 128:824.
98. van de Werve, G., Zaninetti, D., Lang, U., Vallotton, M. and Jeanrenaud,
B. (1987) Identification of a major defect in insulin-resistant tissues of
genetically obese (fa/fa) rats: Impaired protein kinase C. Diabetes
36:310.
99. Sowell, M., Tretelaar, M., Burant, C. and Buse, M. (1988) Minimal effects
of phorbol esters on glucose transport and insulin sensitivity in rat
skeletal muscle. Diabetes 37:499.
100. Shimizu, Y.( Fujiki, H., Sugimura, T. and Shimizu, N. (1986) Mouse 3T3-
L1 cell variants unable to respond to mitogenic stimulation of
dihydroteleocidin B: Genetic evidence for the synergism of tumor
promoters with growth factors. Cane. Res. 46:4027.
101. Zick, Y., Sagi-Eisenberg, R., Pines, M., Gierschik, P. and Spiegel, A.
(1986) Multisite phosphorylation of the alpha subunit of transducin by
the insulin receptor kinase and protein kinase C. Proc. Natl. Acad. Sci.
USA 83:9294.
102. Pelech, S. and Krebs, E. (1987) Mitogen-activated S6 kinase is
stimulated via protein kinase C-dependent and independent pathways in
Swiss 3T3 cells. J. Biol. Chem. 262:11598.
103. Draznin, B., Leitner, J., Sussman, K. and Sherman, N. (1988) Insulin
and glucose modulate protein kinase C activity in rat adipocytes. Bchm.
Bphys. Res. Comm. 156:570.
104. Draznin, B., Kao, M. and Sussman, K. (1987) insulin and glyburide
increase cytosolic free calcium concentration in isolated rat adipocytes.
Diabetes 36:174.
105. Cooper, D., Konda, T., Standaert, M., Davis, J., Pollet, R. and Farese, R.
(1987) Insulin increases membrane and cytosolic protein kinase C
activity in BC3H-1 myocytes. J. Biol. Chem. 262:3633.
106. Gomez, M., Medrano, E., Cafferatta, E. and Tellez-lnon, M. (1988)
Protein kinase C is differentially regulated by thrombin, insulin and
epidermal growth factor in human mammary tumor cells. Exp. Cell. Res.
175:74.


Figure 2-1: Neuron-specific enolase staining of cultured
neurons. Cultured neurons on a bed of glial cells were
fixed and permeabilized prior to immunostaining for
neuron-specific enolase. Following staining the neurons
were photographed at 400x.


3
times that of plasma insulin (14). Since then many reports have
appeared in the literature confirming this finding although the
actual amount of insulin in the brain is the subject of some
controversy (15,16). Insulin is found in cultures from mouse brain
and neuronal cultures from rat brain (17,18). The presence of this
insulin-like peptide in the brain raises two major questions; what
is the source of this peptide and what is its function?
The following observations suggest that insulin may be
synthesized in the brain: (a) central insulin concentrations appear to
remain constant in pathological situations in which peripheral
insulin concentrations vary widely (19); (b) insulin-like activity
has been demonstrated in cultured brain cells which are removed
from pancreatic insulin for weeks (18); (c) radioactive amino acids
can be incorporated into an insulin-like peptide in cultured neurons
by a cycloheximide-sensitive process (18) and (d) there is an mRNA
in the brain which hybridizes with insulin cDNA (20-4). In addition,
our experiments have shown that insulin is synthesized and released
by neurons (25). Furthermore, when neuronal cultures were labelled
with leucine and depolarized, a labelled peptide was released which
could be precipatated by an insulin antibody. Cycloheximide
decreased insulin synthesis by 80% (18).
In recent years specific insulin receptors in the brain have been
demonstrated conclusively (26). These receptor sites, which are
distributed non-uniformly throughout the brain (27), are evenly
distributed between neurons and glial cells (28-30). Studies have
shown that neuronal and glial insulin receptors are structurally and
physiologically distinct in several respects. The brain insulin


127
50. Kaibuch, K., Takai, Y. and Nishizuka, Y. (1981) Cooperative roles of
various membrane phospholipids in the activation of calcium-activated,
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51. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S. and Nishizuka, Y.
(1982) Calcium-activated, phospholipid-dependent protein kinase from
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52. Azhar, S., Butte, J. and Reaven, E. (1987) Calcium-activated,
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53. Kikkawa, U., Ogita, K., Ono, Y., Asaoka, Y., Shearman, M., Fujii, T., Ase,
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54. Nishizuka, Y. (1988) The molecular heterogeneity of protein kinase C
and its implications for cellular recognition. Nature 334:661.
55. Mochly-Rosen, D., Basbaum, A. and Koshland, D. (1987) Distinct
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56. Ono, Y. and Kikkawa, U. (1987) Do multiple species of protein kinase C
transduce different signals? Trends Bchm. Sci. 143:421.
57. Burgess, S., Sahyoun, N., Blanchard, S., LeVine, H., Chang, K. and
Cuatrecasas, P. (1986) Phorbol ester receptors and protein kinase C in
primary neuronal cultures: development and stimulation of endogenous
phosphorylation. J. Cell Biol. 102:312.
58. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and
Nishizuka, Y. (1982) Direct activation of calcium-activated,
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esters. J. Biol. Chem. 257:7847.
59. Kraft, A. and Anderson, W. (1983) Phorbol esters increase the amount of
Ca++, phospholipid-dependent protein kinase associated with plasma
membrane. Nature 301:621.
60. Wolf.M., LeVine, H., May, W., Cuatrecasas, P. and Sahyoun, N. (1985) A
model for intracellular translocation of protein kinase C involving
synergism between calcium and phorbol ester. Nature 317:546.


Figure 4-3: Demonstration and quantitation of PKC
protein in neuronal and glial cells. Homogenates of glial,
neuronal and 1-day-old rat brains were prepared for
Western blot as described in Methods. Lanes containing
a) 25 b) 50 and c) 100 ug of protein were run for each
preparation. Densitometric values for the 80 kD band are
(from left to right), 113, 117, 425, 377, 636, 2164, 67,
230 and 1441.