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Regulation of expression of the glucose transporter GLUT1 by glucose in 3T3-L1 adipocytes

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Title:
Regulation of expression of the glucose transporter GLUT1 by glucose in 3T3-L1 adipocytes
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McMahon, Robert Joseph, 1968-
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English
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xii, 165 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Adipocytes ( jstor )
Antibodies ( jstor )
Antiserum ( jstor )
Cell membranes ( jstor )
Cells ( jstor )
CHO cells ( jstor )
Glycogen ( jstor )
Membrane proteins ( jstor )
Molecular weight ( jstor )
Oligosaccharides ( jstor )
Adipocytes -- physiology ( mesh )
Cricetulus ( mesh )
Department of Biochemistry and Molecular Biology thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Biochemistry and Molecular Biology -- UF ( mesh )
Gene Expression Regulation ( mesh )
Glucose -- physiology ( mesh )
Glycosylation ( mesh )
Monosaccharide Transport Proteins -- genetics ( mesh )
Monosaccharide Transport Proteins -- isolation & purification ( mesh )
Monosaccharide Transport Proteins -- metabolism ( mesh )
Research ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1995.
Bibliography:
Bibliography: leaves 158-164.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Joseph McMahon.

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University of Florida
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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.
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REGULATION OF EXPRESSION OF THE GLUCOSE TRANSPORTER GLUT1 BY
GLUCOSE IN 3T3-L1 ADIPOCYTES














By


ROBERT JOSEPH MCMAHON















A THESIS 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

1995




























This work is dedicated to my wife Laura and my daughter

Amelia.













ACKNOWLEDGMENTS


I would like to acknowledge the people without whose

help and encouragement this project could not have been

completed. I would first like to acknowledge the members of

my committee, Dr. Charles Allen, Dr. Christopher West, Dr.

Michael Kilberg, and Dr. John Gander, who graciously agreed

to help partway through the completion of this thesis. A

debt of thanks goes to Dr. Susan Frost, whose ability to

simultaneously allow her students the freedom to investigate

problems in their own fashion and yet maintain their focus

constitute only two of the many admirable qualities I hope to

bring to my own lab.

I would also like to acknowledge the assistance given to

me on numerous occasions by Dr. Ron Laine and Marc Malandro.

My discussions with them have been invaluable in overcoming

obstacles that arise during any thesis. I would also like to

thank Maxine Fisher for always reminding me to cultivate

personal relationships between scientists which are essential

for a successful career.

I would also like to thank my parents, Joseph and

Florence, for the freedom to always pursue my ambitions with

their constant support. Lastly, I would like to thank my

wife Laura for her unending support and encouragement during








this period of immense personal and professional growth. Her

love and involvement have enriched during this experience

beyond measure.














TABLE OF CONTENTS

ACKNOWLEDGMENTS ........................................... iii

LIST OF FIGURES ..........................................viii

ABSTRACT ....................................................x

OVERVIEW ....................................................1

GLUT Proteins and Glucose Transport....................... 1
GLUT Protein Family ....................................1
Glucose Transporter Structure ..........................4
Regulation of Glucose Transport...........................7
Regulation of Glucose Transport Activity by Glucose
Deprivation .........................................8
Regulation of Glucose Transporter Protein ..............8
3T3-L1 Adipocytes ........................................10

MATERIALS AND METHODS ...................................... 16

Materials ................................................16
Methods ..................................... ... ......... 17
Cell Culture ..........................................17
Antibody Production ...................................18
Peptide Purification of Anti-GTl Antibody ............. 21
Membrane Fraction Isolation ...........................23
Markwell Assay for Protein Determination .............. 26
Gel Electrophoresis ...................................27
Electrotransfer and Western Blotting .................. 27
Metabolic Radiolabeling of 3T3-L1 Adipocytes .......... 28
GLUT1 Immunoprecipitation .............................30
Endoglycosidase Digestions .........................32
Glycogen Isolation ....................................33
Glucose Assay .........................................34
Subfractionation of 3T3-L1 Adipocytes .................35
Cell Surface Biotinylation ............................37
Plasma Membrane Fragment Isolation ..................40

CHARACTERIZATION OF GLUT1 GLYCOFORMS .......................43

Introduction .............................................43
Results.................................................44
Characterization of Anti-GTl Antiserum ................ 44
Glucose Deprivation and GLUT1 Protein Expression ......48
Tryptic Digest of GLUT1 Transporter ................... 51
Effect of Tunicamycin Treatment on GLUT1 Protein ......54
Glycosidase Digestion of GLUT1 Glycoforms .............60








Specificity and Efficiency of GLUT1
Immunoprecipitation ...............................64
Conclusions...............................................67

REGULATION OF GLUT1 BIOSYNTHESIS, PROCESSING AND
TURNOVER BY GLUCOSE IN 3T3-L1 ADIPOCYTES..................70

Introduction............................................70
Results.................................................75
Synthesis and Glycosylation of GLUT1 in Control and
Glucose-deprived 3T3-L1 Adipocytes .................75
Regulation of GLUT1 Processing by Glucose ............. 78
Synthesis of GLUT1 Glycoforms During Glucose
Deprivation and Glucose Refeeding .................. 81
GLUT1 Half-Life in Control and Glucose-Deprived
Adipocytes ..........................................87
Conclusions.............................................92

REGULATION OF GLYCOGEN CONTENT AND GLYCOSYLATION OF
GLUT1 GLUCOSE TRANSPORTER ................................ 98

Introduction .............................................98
Results................................................102
Time Course and Concentration Curve of Glucose
Assay ................. ................... ........102
Effect of Glucose-Deprivation on Glycogen Content
in 3T3-L1 Adipocytes .............................. 103
Glycogen Accumulation During Refeeding ............... 105
Effect of Fructose on Glycogen Depletion in
Glucose-Deprived Adipocytes .......................105
Effect of Insulin and 3-O-methylglucose on the
Glycogen Level in Glucose-Deprived 3T3-L1
Adipocytes Incubated with Fructose ................107
Prior Depletion of the Cellular Glycogen Pool
Results in a More Rapid Alteration of
Glycosylation in the Absence of Glucose ........... 111
Glycogen Content in the CHO Cell Line ................ 113
GLUT1 Glycosylation from Glucose-Deprived CHO
Cells ............................................. 118
Conclusions............................................118

GLUT1 TARGETING IN NORMAL AND GLUCOSE-DEPRIVED 3T3-L1
ADIPOCYTES ..............................................126

Introduction ............................................126
Results.................................................131
Subfractionation of Control and Glucose-Deprived
3T3-L1 Adipocytes ................................. 131
Cell Surface Biotinylation of Control and Glucose-
deprived 3T3-L1 Adipocytes. ....................... 138
Conclusions............................................145









CONCLUSIONS AND FUTURE DIRECTIONS .........................147

Conclusions............................................. 147
Future Directions....................................... 154

LIST OF REFERENCES ........................................158

BIOGRAPHICAL SKETCH ....................................... 165





















LIST OF FIGURES


Figure page

1-1 Predicted Secondary Structure of Glucose Transporters. .4

2-1 Elution of GT1-Specific Antibodies. ................... 23

3-1 Characterization of Anti-GTl Antiserum. ...............45

3-2 Glucose Deprivation and GLUT1 Expression ............. 49

3-3 Tryptic Digest of p46 and p37. ........................52

3-4 Effect of Tunicamycin on Expression of GLUT1.......... 55

3-5 GLUT1 Expression Following Tunicamycin Washout. .......58

3-6 Glycosidase Digestion of GLUT1 Glycoforms. ............61

3-7 Immunoprecipitation of GLUT1 from Normal and Glucose-
Deprived 3T3-L1 Adipocytes ............................65

4-1 Biosynthesis of GLUT1 in Normal and Glucose-Deprived
Adipocytes ............................................76

4-2 Processing of GLUT1 in Normal and Glucose-Deprived
Adipocytes ...........................................79

4-3 Time Course of p37 GLUT1 Biosynthesis in Glucose-
Deprived Adipocytes ..................................82

4-4 Biiosynthesis of p37 GLUT1 During Glucose-Refeeding. ..85

4-5 Turnover of GLUT1 in Glucose-Deprived Adipocytes. .....88

4-6 Turnover of GLUT1 in Glucose-Refed Adipocytes. ........90

5-1 Time Course and Concentration Curve of Glucose Assay. 104


viii









5-2 Glycogen Content and Glucose Deprivation. ............ 106

5-3 Glycogen Content and Glucose Refeeding. .............. 108

5-4 Glycogen Content in Fructose-Incubated Adipocytes. ... 109

5-5 Effect of Insulin and 3-O-methylglucose on Glycogen
Content ............................................112

5-6 Glycogen Depletion and GLUT1 Glycosylation. .......... 114

5-7 Glycogen Depletion and GLUT1 Glycosylation. .......... 116

5-8 GLUT1 Glycosylation and Glucose Deprivation in CHO
cells. ............................................... 119

5-9 GLUT1 Glycosylation and Glucose Deprivation in CHO
cells. ..................................... .........121

6-1 Subcellular Fractionation of 3T3-L1 Adipocytes ....... 133

6-2 Subcellular Fractionation of 3T3-L1 Adipocytes ....... 135

6-3 Cell Surface Biotinylation ...........................139

6-4 Plasma Membrane Fragment Isolation ................... 143















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 GLUT1 GLUCOSE TRANSPORTER EXPRESSION BY GLUCOSE
IN 3T3-L1 ADIPOCYTES

By

ROBERT JOSEPH MCMAHON

DECEMBER, 1995




Chairperson: Susan C. Frost
Major Department: Biochemistry and Molecular Biology


The regulation of GLUT1 expression by glucose was

analyzed in the 3T3-L1 adipocyte. Glucose deprivation of

3T3-L1 adipocytes for more than 12 hours resulted in the

accumulation of a second GLUT1 glycoform (p37). 48 hours of

glucose deprivation resulted in a 2-fold increase in total

GLUT1, a reflection of the accumulation of p37.

Metabolically labeled GLUT1 migrated as a wide band of

approximately 46 kDa. In contrast, metabolically labeled

GLUT1 protein in glucose-deprived adipocytes migrated at 37

kDa. Although at least 12 hours of glucose-deprivation were

required to affect GLUT1 glycosylation, glucose-deprived

cells quickly recovered the ability to correctly glycosylate

nascent GLUT1 upon the readdition of glucose.









The regulation of GLUT1 turnover by glucose was analyzed

by metabolic labeling and pulse-chase analysis. GLUT1 in

normal adipocytes exhibited a half-life of 14 hours. In

contrast, the turnover of the transporter in glucose-deprived

adipocytes was greater than 50 hours. The inhibition of

transporter turnover was readily reversed upon the re-

addition of glucose.

The glycogen content of 3T3-L1 adipocytes was assessed

as a potential carbohydrate source for protein glycosylation.

Normal 3T3-L1 adipocytes contained 0.537 0.097 pmol

glucose/106 adipocytes, which decreased in a first order

fashion upon glucose deprivation (0.0.052 pmol glucose/106

cells/hr, tl/2 = 6 hours). Fructose was unable to prevent the

depletion of glycogen in glucose-deprived adipocytes. In

glycogen-depleted cells, alterations in GLUT1 glycosylation

occurred more rapidly upon glucose deprivation than in

adipocytes with normal glycogen levels. Chinese Hamster

Ovary cells (CHO), which contain only 1% of the glycogen of

3T3-L1 adipocytes, exhibited the most rapid alteration in

GLUT1 glycosylation, occurring within 3 hours of glucose

deprivation.

The targeting of the alternatively glycosylated GLUT1

transporter was assessed by plasma membrane fragment

isolation, cell surface biotinylation, and subcellular

fractionation. All three techniques detected both GLUT1

glycoforms (p46 and p37) at the plasma membrane, indicating

that the targeting of the GLUT1 protein was not affected by









either the withdrawal of glucose or the alteration in N-

linked glycosylation.













CHAPTER 1
OVERVIEW


GLUT Proteins and Glucose Transport



GLUT Protein Family


The maintenance of intracellular metabolism relies upon

the ability of the cell to incorporate solutes that cannot

readily diffuse across the plasma membrane. The uptake of

organic solutes, such as sugars and amino acids, is

accomplished by specific integral membrane proteins that

mediate the movement of molecules from the extracellular

environment to the cytosol. The transport of solutes across

the plasma membrane is accomplished by one of three

mechanisms (Voet and Voet, 1990). The first is passive

diffusion across the plasma membrane, a slow process due to

the low solubility of organic molecules in the hydrophobic

membrane interior. The second transport mechanism, termed

facilitated transport, is mediated by specific integral

membrane proteins that undergo conformational changes that

allow the solute access to the cytosol. The direction of

flux in this type of transport is determined solely by the

concentration gradient of the solute. Facilitated transport

describes many transport systems, including many salt, sugar,

and amino acid transporters. In some instances, solutes can








be transported against its concentration gradient if

transport is coupled to that of another solute that has a

concentration gradient of greater magnitude. The third

mechanism of transport is termed active transport. In this

process, the cleavage of high energy phosphoryl bonds

(usually in the form of ATP) is coupled to changes in the

conformation of the transport protein that result in the

translocation of the solute across the plasma membrane.

Since energy is expended in this process, solutes can be

transported against their concentration gradients. Examples

of this include the Na+, K+ ATPase and the Ca2+ ATPase.

Due to a large volume of scientific study, the transport

of glucose has served as a paradigm for the facilitated

transport of small, neutral organic solutes. The most well

understood glucose transport system exists in the mammalian

erythrocyte. The partial purification of a glucose

transporter protein was facilitated by its high concentration

in the erythrocyte plasma membrane. The purified transporter

from erythrocyte ghosts migrated as a wide band with an

average molecular weight of 55 kDa (Kasahara and Hinkle,

1977; Baldwin et al., 1979). Screening a cDNA expression

library with antiserum against the purified transporter

resulted in the isolation of a clone coding for a protein

capable of saturable, D-stereoisomer specific, glucose

transport (Mueckler et al., 1985; Birnbaum et al., 1986).

The study of glucose transport then underwent another

expansion when it was discovered that the transport of








glucose was not accomplished by the same protein in every

cell type. Screening cDNA libraries from other tissues at

low stringency led to the cloning of additional glucose

transporters, which in total are now known as the GLUT

protein family (GLUcose Transporter), a group of structurally

and functionally related integral membrane glycoproteins that

mediate the tissue-specific uptake of glucose (reviewed by

Gould and Bell, 1990). The expression of GLUT1 is virtually

ubiquitous; in most tissues, it is thought to be responsible

for a basal, constitutive transport activity. GLUT2 is

expressed predominantly in liver and is characterized by a

relatively high affinity constant; its role has been ascribed

to equilibrating glucose between the hepatocyte and blood

plasma according to the extracellular glucose concentration.

GLUT3 is expressed primarily in fetal muscle and brain

tissue. GLUT4 is expressed in insulin-sensitive tissues such

as heart, muscle, and adipose. The localization of GLUT4 can

be altered upon the binding of insulin, where the transporter

is translocated from an intracellular compartment to the

plasma membrane. GLUT5 is a recently cloned transporter that

is characterized by a high affinity for fructose; its role in

glucose transport and utilization has not yet been

elucidated. GLUT 6, a recently cloned gene, is a non-

expressed pseudogene. GLUT7, a component of the glucose-6-

phosphatase system, is localized in the endoplasmic reticulum

of the hepatocyte where its role is vital in gluconeogenesis.

The complex arrangement of transporter family, exhibiting








tissue-specific expression, developmental regulation, and

hormonal regulation, lends itself well to being capable of

delivering glucose to different tissues with varying

requirements for carbohydrate.


Glucose Transporter Structure


Although GLUT proteins exhibit a high degree of tissue-

specific expression, analysis of the primary sequences of the

cloned transporters reveals a high degree of homology. The

GLUT family exhibits approximately 68% overall homology; the

highest degree occurs in the membrane spanning regions, and

the lowest conservation occurs in the amino and carboxy

terminal ends and the large intracellular loop between

membrane spanning regions 6 and 7 (Carruthers, 1990). The

predicted topology of the transporter is depicted in Figure

1. The disposition of the transporter in the membrane was

based upon hydrophobicity and hydrophilicity measurements

which predict putative membrane spanning domains (Mueckler et

al., 1985). This model predicts that the GLUT proteins

exhibit 12 membrane spanning regions, and places both the

amino and carboxy terminal ends facing the cytoplasm

(Mueckler et al., 1985). Infrared spectroscopy and circular

dichroism have revealed a high degree of a-helical nature in

the transporter protein, which likely represents the membrane

spanning regions (Chin et al., 1986; Alvarez et al., 1987).

This topological model has received a significant amount of

experimental support (Shanahan et al., 1984; Cairns et al.,






























Plasma
Membrane 1 3 4 5 7 9 10 11s



NH,I1 COOH












Figure 1-1 Predicted Secondary Structure of GLUT1 The
predicted topology of the GLUT1 transporter, based on
hydropathy values (Mueckler et al., 1985). The amino- and
carboxy-terminal ends of the transporter are designated by
[N] and [C], respectively. The intracellular- and
extracellular-facing sides of the transporter are designated
by [IN] and [OUT], respectively. The boxes represent
membrane spanning regions and the branched chain represents
oligosaccharide attached to the extracellular loop between
membrane spanning regions 1 and 2.








1984; Cairns et al., 1987; Davies et al., 1987). Utilization

of peptide-specific antibodies directed against amino or

carboxyl terminal ends of GLUT1, in conjunction with tryptic

digestion, allowed the analysis of the predicted

intracellular and extracellular faces of the transporter.

Using an antiserum that recognized GLUT1 in its native form

and a preparation of erythrocyte membranes of known

orientation, it was determined that the carboxy terminus

resided intracellularly because the transporter was

recognized only in vesicles which were predominantly "inside

out". Furthermore, tryptic digests of the transporter

resulted in two large fragments of only slightly different

molecular weights, supporting the hypothesis that the major

cleavage site in the protein is near the middle of the large

intracellular loop between membrane spanning regions 6 and 7.

Antiserum specific for the amino terminus of the transporter

recognized a large diffuse band, suggesting that this

fragment contained the oligosaccharide, confirming that the

putative N-linked glycosylation site in the first

extracellular domain was utilized. Labeling the erythrocyte

transporter with the competitive ligand cytochalasin B before

proteolysis demonstrated that the binding site for this

ligand resided in the carboxy terminal half of the protein

(Holman and Rees, 1987).

Apart from the amino acid sequence, a second aspect that

deserves review is the glycosylation of the glucose

transporters. All glucose transporters are glycosylated at








an asparagine residue located between the first and second

membrane spanning region (Carruthers, 1990). Although the

complete oligosaccharide sequence is not known, some

characteristics have been elucidated. All members of the

glucose transporter family can be digested with N-glycanase F

or endoglycosidase F to release the oligosaccharide,

indicating N-linked glycosylation (Haspel et al., 1986;

Kitzman et al., 1994). The oligosaccharide is usually a

large structure that accounts for approximately 10 kDa of

migration by SDS-PAGE, or 20% of its total relative molecular

weight. Additionally, GLUT proteins migrate as diffuse bands

when analyzed by SDS-PAGE, but whether this heterogeneity

results from differences in the oligosaccharide structure

(known as microheterogeneity), or from different charges

associated with the terminal sugar chains, is not known.


Regulation of Glucose Transport


The regulation of glucose transport has received much

attention due to the central role the transporter plays in

the utilization of glucose. Glucose transport is regulated by

many factors, including hormonal influence and cellular

stress. The influence of hormones on the transport process

has been extensively reviewed elsewhere (Czech et al., 1992;

Czech, 1995) and will not be discussed in this work. Rather,

I will highlight the role of cellular stress, particularly

nutrient deprivation, on the regulation of glucose

transporter expression.








Regulation of Glucose Transport Activity by Glucose
Deprivation


Glucose deprivation, a specific type of nutrient

deprivation, augments glucose uptake in a variety of cell

types, including glial cells (Walker et al., 1989), avian

fibroblasts (Martineau et al., 1972; Kletzein and Purdue

1975; Pessin et al., 1982; Yamada et al., 1983), mammalian

fibroblasts (Germinario et al., 1982; Haspel et al., 1986,

Ortiz and Haspel, 1993), muscle cells (Walker et al., 1989;

Koivisto et al., 1991; Maher and Harrison, 1991), and

adipocytes (van Putten and Krans, 1985; Reed et al., 1990;

Tordjman et al., 1990). Several characteristics of this

enhanced uptake are common among these cell types. Glucose

uptake enhancement results from an increase in the Vm, of the

transport process with only a small change in the affinity

(Km) (Kletzien and Purdue, 1975; van Putten and Krans, 1975).

Transport activation also contains a protein synthesis-

dependent component, since exposure to cycloheximide

attenuates this response (Tordjman et al., 1990; Kitzman et

al., 1994). Metabolism or metabolites of glucose are not

required since incubation with 3-0-methyl glucose and 2-

deoxyglucose (2-deoxyglucose is only phosphorylated) can

prevent transport activation (Kitzman et al., 1994).


Regulation of Glucose Transporter Protein


Glucose deprivation also regulates glucose transport by

regulating the GLUT1 transporter protein level. Although not







as intensely studied as the regulation of transport activity,

progress has been made in elucidating the regulation of GLUT1

protein by glucose. The regulation of transporter expression

can be categorized into two classes; the first, where the

level of the transporter changes in response to glucose

deprivation, and the second, where a second transporter form

accumulates. Glucose deprivation results in an increase in

GLUT1 protein levels in some cell types, including the

fibroblast cell line 3T3-C2 and the kidney cell line NRK

(Haspel et al., 1986; Haspel et al., 1991). This effect,

however, is not conserved in all cell types; in 3T3-L1

adipocytes, there is no change in the level of normal GLUT1

transporter (Reed et al., 1990; Kitzman et al., 1994). With

extended periods of glucose deprivation, an additional

protein recognized by anti-GLUT1 antiserum accumulates. This

phenomenon is observed in several other cell types, including

the 3T3-L1 fibroblasts, normal rat kidney cells, and a Wilm's

tumor cell line (Haspel et al., 1986, Haspel et al., 1990;

Shawver et al., 1987; Haspel et al., 1986, Haspel et al.,

1990; Kitzman et al., 1994). This second protein migrates as

analyzed by SDS-PAGE approximately 10 kDa lower than the

normal GLUT1 protein. The glycosylation state of this

protein was investigated by treatment with endoglycosidase F

or N-Glycanase F, which remove all N-linked oligosaccharides.

The migration of this second protein can be altered slightly

upon this treatment, indicating that like the normal GLUT1

protein, it is modified with N-linked oligosaccharide (Haspel








et al., 1986; Kitzman et al., 1994). Furthermore, the

migration of the normal GLUT1 protein and the low molecular

weight protein were found to be identical upon digestion with

N-Glycanase F, indicating that resultant core proteins were

of identical molecular weight. Most investigators have now

accepted the assumption that the second protein is indeed a

different form of the GLUT1 transporter (Haspel et al., 1986;

Haspel et al., 1990; Kitzman, et al., 1993).


3T3-L1 Adipocytes


White adipose tissue plays an important role in the

storage of triglyceride resulting from excess blood glucose.

With this important role in the disposal of glucose, the

transport system for glucose is highly regulated. Many

studies involving the transport of glucose utilize adipocytes

isolated from fat pads, but these cells exhibit several

disadvantages, including the propensity to dedifferentiate

once isolated, and the inability to culture these cells for

extended periods of time. For these reasons, the work

described herein utilized the 3T3-L1 adipocyte, an adipocyte

model cell line, to study glucose transporter regulation.

In 1974, Green and Kehinde isolated several subclones of

mouse fibroblasts 3T3 cells that exhibited the propensity to

accumulate large lipid droplets once confluent. This

phenomenon could be prevented or reversed by passing and

reculturing the cells at low density. Extensive subcloning

of these cells resulted in the establishment of the 3T3-L1








(Lipid accumulating subclone 1) cell line in which

essentially all cells accumulate fat droplets (Green and

Meuth, 1974).

After observation of the onset of lipogenesis during the

differentiation of 3T3-L1 cells, it was established that the

3T3-L1 adipocytes express the proteins necessary to carry out

the metabolism characteristic of true adipose tissue.

Lipoprotein lipase (Spooner et al., 1979), fatty acid

synthase (Student et al., 1980; Wise et al., 1980),

acquisition of hormonal sensitivity, including to insulin

(Rubin et al., 1977; Resh et al., 1982), enzymes involved in

phosphatidylcholine and phosphoethanolamine biosynthesis

(Coleman et al., 1978), stearoyl CoA desaturase and fatty

acid binding protein (Cook et al., 1988) are all expressed

specifically during the onset of differentiation of 3T3-L1

cells. The large body of information regarding the

metabolism of the 3T3-L1 adipocyte has established this cell

line as a valid model for both the differentiation and

physiology of true adipose tissue.

3T3-L1 adipocytes express two members of the glucose

transporter family; GLUT1, the basal transporter, and GLUT4,

the insulin-sensitive glucose transporter. It has been

estimated by cell surface labeling techniques in normal and

insulin-treated adipocytes that each cell expresses

approximately 1 x 106 and 0.3 x 106 GLUT1 and GLUT4

transporter molecules, respectively (Calderhead et al.,

1990). The 3T3-L1 cell line has been used extensively in the








study of glucose transport regulation, including regulation

by insulin (reviewed by Czech, 1995), cholera toxin and

dibutyryl cyclic AMP (Clancy and Czech, 1990), cadmium, and
tumor necrosis factor a (Stephens et al., 1992).

In particular, the 3T3-L1 adipocyte has been used to

investigate the effects of glucose deprivation on glucose

transport activity and glucose transporter expression. The

withdrawal of glucose from the culture medium of 3T3-L1

adipocytes results in a 10-fold increase in the rate of

glucose transport, consistent with the responses of other

cell types to glucose deprivation (van Putten and Krans,

1985, Kitzman et al., 1994). Furthermore, the transport

activation results from an increase in the V, of the

transport system, with little change in the affinity of the

transporter for glucose (Km)((van Putten and Krans, 1985).

Other groups have since confirmed this result (Tordjman et

al., 1990; Kitzman et al., 1994). An increase in the maximal

velocity of transport without a change in the Km suggests

that there is either a greater number of transporter

molecules at the cell surface available for transport, or

that the activity of the transporter has been increased,

possibly by allosteric or covalent modification. Transport

activation, beginning at 3 hours post glucose withdrawal and

reaching a maximum at 24 hours, is protein synthesis-

dependent as assessed by cycloheximide sensitivity (Tordjman

et al., 1985, Kitzman et al., 1994). Additionally,

cycloheximide attenuated transport activation at any time








during glucose deprivation (Kitzman et al., 1994). Up-

regulation of glucose transport by glucose deprivation was

concentration-dependent, with a transport activation

threshold of approximately 0.5 mM. Additionally, transport

activation was completely reversible upon the re-addition of

glucose, indicating that down-regulation of transport was

also glucose-dependent (Kitzman et al., 1994). The

dependence of transport activation upon glucose or one of the

metabolites of glucose was tested by analyzing the up-

regulation of transport in the presence of several other

sugars; some of which are not transported by glucose

transporters, some of which are only ligands of the

transporter, and some which are transported but not

metabolized (Haspel et al., 1990; Kitzman et al., 1994).

Incubation of glucose-deprived 3T3-L1 adipocytes with

fructose, galactose, or maltose, none of which are

internalized by the glucose transporter, although maltose (a

disaccharide of glucose) is bound by the transporter, had no

effect on the up-regulation of transport activity. 3-0-

methylglucose, which is transported by the glucose

transporter but not metabolized, and 2-deoxyglucose, which is

only phosphorylated, completely abolish transport activation

(Kitzman et al., 1994). These observations suggest that

neither the binding of the transporter nor the metabolism of

glucose is required for prevention of transport activation;

rather, they suggest that a molecule that is either glucose








itself or a molecule closely resembling glucose is the

regulatory factor controlling transport activity.

Another effect of glucose deprivation in 3T3-L1

adipocytes is the appearance of a second protein recognized

by anti-GLUT1 antiserum that migrates at a relative molecular

weight of 37 kDa, in contrast to the normal GLUT1 transporter

which migrates at a relative molecular weight of 46 kDa (Reed

and Vang, 1990; Kitzman et al., 1994). The migration of

these two proteins, when treated with N-Glycanase F, is

identical (36 kDa) (Haspel et al., 1986; Kitzman et al.,

1994). This led several groups to conclude that the p37

protein is a second GLUT1 transporter which contains a

different oligosaccharide than the normal transporter. Like

transport activation, the appearance of p37 was prevented by

exposure to cycloheximide, even when added 12 hours after the

withdrawal of glucose, indicating that the appearance of p37

required new protein synthesis (Kitzman et al., 1994). The

threshold glucose concentration required to elicit the

accumulation of p37 was 0.005 mM. The difference in glucose

concentration (approximately 100-fold) for the activation of

glucose transport and the appearance of p37 strongly suggests

that p37 is not involved in, or responsible for, the

activation of glucose uptake (Kitzman et al., 1994). In

agreement with this conclusion, incubation of glucose-

deprived cells with fructose prevents the appearance of p37

while allowing the activation of glucose transport (Haspel et

al., 1986, Kitzman et al., 1994). Aside from the








determination of the glycosylation state of p37 and the

sensitivity of its appearance to cycloheximide, little else

is known about the regulation of the glucose transporter

protein by glucose. It should be noted that there is one

report where glucose deprivation resulted in an increase in

the level of the normal transporter protein, but the

discrepancy between this and other reports may lie in the

fact that equal numbers of cell equivalents were not analyzed

(Tordjman et al., 1990). The hypothesis that p37 is a second

GLUT1 protein synthesized in glucose-deprived 3T3-L1

adipocytes evokes several important questions regarding its

genesis, processing, turnover, and targeting. The goal of

this work is to describe how the expression of GLUT1 is

altered in the absence of glucose. This includes how the

transporter is processed to its final form, how it is

degraded, and to determine whether its altered glycosylation

state affects its targeting to the plasma membrane.













CHAPTER 2
MATERIALS AND METHODS


Materials


Dulbecco's Modified Eagle's Medium (D-MEM), Modified

Earle' Medium, a modification (a-MEM), and D-MEM base powder

was obtained from Life Technologies, Inc. Calf serum (Cat.

No. 1100-90) and fetal bovine serum (FBS) (Cat. No. 1020-75)

were obtained from Intergen. Glucose-free FBS was prepared

by dialyzing against phosphate buffered saline, pH 7.4, (PBS)

for 48 hours at 4C, with a molecular weight cutoff of 13,000

(dialysis tubing obtained from SpectraPor). L-methionine, L-

cysteine, L-proline, D-glucose, D-fructose, D-xylose, 3-0-

methylglucose, methylisobutylxanthine, dexamethasone, protein

A Sepharose, Keyhole Limpet Hemocyanin, Sephadex G-100,

Freund's Complete and Incomplete Adjuvant, polyoxyethylene 9

lauryl ether (C12E9), octanoic acid, bovine serum albumin,

poly-L-lysine (M.W. > 150,000), and anti-rabbit IgG

conjugated horseradish peroxidase were obtained from Sigma.

Insulin was a generous gift of Dr. Ronald Chance of Eli Lilly

Corp. Tran35S-Label (1100 Ci/mmol) was obtained from ICN.

Aquacide was obtained from Calbiochem. Streptavidin-agarose,

m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-

MBS), the Sulfolink matrix, and sulfosuccinimidyl-6-

(biotinamido) hexanoate (NHS-LC-Biotin) were obtained from








Pierce. All other chemicals were obtained through either

Fisher or Sigma.


Methods


Cell Culture


Cell culture and differentiation of 3T3-L1 fibroblasts

were performed as previously described (Frost and Lane,

1985). Cells were seeded in plastic tissue culture dishes

and cultured in D-MEM containing 10% calf serum (CS) for

seven days, refeeding every other day until confluent. After

seven days, the cells were incubated in D-MEM containing 10%

fetal bovine serum (FBS), 1 pg/ml insulin, 0.5 mM

methylisobutylxanthine, and 0.25 mM dexamethasone (Kuri-

Haruch and Green, 1978; Rubin et al., 1978; Reed and Lane,

1980). Two days later, the cells were incubated in D-MEM/10%

FBS containing 1 pM insulin only. Thereafter, the cells were

fed with D-MEM/10% FBS alone. Cells were used for

experiments between days 8 and 12 post-differentiation. To

prepare glucose-deprived 3T3-L1 adipocytes, the normal

culture medium was aspirated and replaced with glucose-free

D-MEM containing 10% dialyzed FBS for the indicated periods

of time.

CHO-K1 cells, obtained from American Type Cell Culture,

Rockville, MD, and LEC1 CHO cells, obtained as a kind gift of

Dr. Pamela Stanley, (Albert Einstein College of Medicine, NY,

NY) were seeded onto plastic tissue culture dishes (100 cm2








for most experiments) and grown to confluence in a-MEM medium

containing 10% FBS. Cells were used between days 1 and 5

post-confluence. For glucose deprivation of the CHO-K1 cells

and the LEC1 CHO cells, the a-MEM medium was aspirated and

replaced with glucose-free DMEM containing 10% dialyzed FBS.

This medium was supplemented with 0.1 mM proline in order to

satisfy the auxotrophic requirement of the CHO cell line .
Incubation of all cells with other sugars (i.e., xylose,

fructose, 3-0-methylglucose) was performed by adding the

appropriate amount of crystalline sugar in glucose-free DMEM

containing 10% dialyzed FBS to obtain a final concentration

of 25 mM.


Antibody Production


The detection of GLUT1 transporter protein was

accomplished through the generation of a polyclonal antiserum

against a peptide corresponding to the carboxy terminus of

the amino acid sequence. This region was chosen because of

its charged nature and its low degree of identity compared to

the same region in other members of the GLUT family.

Although use of this crude antiserum is adequate for the

detection of GLUT1 in Western blotting applications, it was

necessary to purify the GLUT1-specific antibodies for use in

immunoprecipitation experiments. The following sections

describe the generation of the anti-GLUT1 antiserum and the

purification of the peptide-specific antibodies.








Some small peptides are poorly immunogenic; therefore,

in order to ensure antigenicity, they are often coupled to

larger proteins, termed immune carriers, (Harlow and Lane,

1988). Covalent conjugation of small proteins or peptides to

these immune carriers results in an antigen large enough to

elicit an immune response. Antiserum prepared in this manner

contain antibodies against the immune carrier and the

antigen. The proteins typcially chosen as immune carriers

are BSA, ovalbumin, or keyhole limpet hemocyanin. Careful

choice of the immune carrier can result in antiserum which

recognizes only the peptide because the immune carrier is

absent from the experimental system. Antisera prepared

against synthetic peptides have several advantages over

antisera generated against whole proteins (Harlow and Lane,

1988). First, the antibody can be generated without knowing

the entire sequence of the protein or purifying the protein.

Second, the antiserum can be generated in such a way that it

will be specific for specific regions of the protein of

interest. Finally, peptide-specific antibodies have a higher

potential of being conformation specific.

For the preparation of an antiserum against GLUT1, a

peptide (CEELFHPLGADSQV) that corresponds to amino acid

residues 480-492 of the GLUT1 sequence was chosen as the

antigenic region. This peptide, designated GT1, was

synthesized by the Protein Chemistry Core facility at the

University of Florida and verified by amino acid analysis.

An additional cysteine was added to the amino terminus of the








peptide in order to facilitate covalent coupling to the

immune carrier. A keyhole limpet hemocyanin-GTl conjugate

was generated by using a thiol-specific cross-linking

reagent, sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide ester

(sulfo-MBS). Keyhole limpet hemocyanin (KLH) (60 mg) was

dissolved in 10 ml 10 mM potassium phosphate, pH 7.3 and

dialyzed at 4C against the same for 48 hours. The

dialysate was concentrated to approximately 4 ml by covering

the dialysis bag in a liberal amount of Aquacide and wrapping

in foil (Calbiochem). The concentrated dialysate was divided

into microcentrifuge tubes and centrifuged in microcentrifuge

tube for 10 minutes to remove any protein aggregates. The

concentration of protein in the supernatant was estimated by

measuring the absorbance at 280 nm using the milligram

extinction coefficient for KLH of 1.6. KLH (6 mg) from a 12

mg/ml solution in 10 mM potassium phosphate, pH 7.3 was

reacted at room temperature with 0.6 mg sulfo-MBS for 45 min.

The KLH-MBS conjugate was separated from free sulfo-MBS by

passing the reaction mixture over a 10 ml G-100 column pre-

equilibrated in 50 mM potassium phosphate, pH 6.0. The KLH-

MBS conjugate is excluded in the void volume, and the free

sulfo-MBS is retarded in the column. Ten fractions (1 ml

each) were collected and measured at 280 nm. Peak fractions,

containing KLH-MBS were pooled and reacted with the GT1

peptide (6 mg) for 3 hours at room temperature with end-over-

end rotation. The pH of the reaction mixture was adjusted

with HC1 in order to produce the maximum amount of








precipitate. The reaction mixture was then allowed to stand

upright overnight at 4C to collect precipitate, which

represents the KLH-peptide conjugate.

A polyclonal antiserum to this conjugate was then

generated by injection of the KLH-GT1 conjugate (200 yg)

emulsified in Freund's Complete Adjuvant into the popliteal

lymph node of a New Zealand white rabbit (Sigel et al.,

1983). The rabbit was boosted by intradermal injection with

an equal amount of the GT1-KLH conjugate emulsified in

Freund's Incomplete Adjuvant 28 days later, and then again

two weeks later. Serum (designated as anti-GT1) was

collected every week by laceration of the medial ear vein.

Additional boost injections were administered approximately

every 6 weeks. This crude antiserum was divided into 1 ml

aliquots and stored at -200C.

Antiserum against a carboxy-terminal peptide of the

insulin-responsive glucose transporter GLUT4 (CSTELEYLGPDEND)

and an amino-terminal peptide of the glucose-regulated

protein GRP78 (EEEDLLEDVGTVC) were generated in the same

manner. This antiserum were used unpurified for Western

blotting.


Peptide Purification of Anti-GT1 Antibody


Although the crude antiserum generated against the KLH-

GT1 conjugate recognized the peptide, a fraction of the whole

serum containing the total IgG was purified using a

modification of a previously described method (Dankert et








al., 1985). The eventual purpose of this technique was to

produce an antibody fraction that could be used for the

purification of peptide specific antibodies.

First, the pH of approximately 15 ml whole rabbit serum

was adjusted to 5 with 3M acetic acid. While vigorously

stirring, 0.75 ml octanoic acid (1 part in 20) was added

dropwise; the solution was then stirred an additional 30 min

at room temperature. The precipitate was collected at 41,000

x g for 30 min at 4C and the pellet discarded. The

supernatant was then removed and its volume determined. An

equal volume of ammonium sulfate (saturated solution in

water) was added and stirred overnight at 4C. The

precipitate was then collected by centrifugation at 41,000 x

g for 30 min at 4C. The pellet, consisting of primarily

immunoglobulin, was dissolved in PBS and the concentration of

this solution was estimated by measuring its absorbance at

280 nm (using a milligram extinction coefficient of 1.4).

The ammonium sulfate was then removed by dialysis against

three changes of PBS over 48 hours at 4C with a molecular

weight cutoff of 13,000 daltons.

The purification of peptide specific antibodies from the

total antibody fraction provides an advantage of reducing

non-specific protein--antibody interactions that could

complicate the interpretation of data gathered by

immunoprecipitation. Peptide-specific antibodies were

purified from the total IgG fraction by affinity

chromatography over a column of GT1 peptide immobilized on an








inert substrate. The GT1 peptide (2 mg) was covalently

coupled to a Sulfolink matrix through the additional cysteine

residue on the GT1 peptide following the manufacturer's

instructions. Sulfolink matrix consists of 6% cross-linked

beaded agarose with a terminal iodoacetyl group that confers

reactivity with sulfhydryl groups under mild (neutral pH)

conditions. An additional advantage of the Sulfolink matrix

is that the linkage between the peptide and the matrix is

irreversible; other matrices utilizing sulfhydryl chemistry

can be reversed under reducing conditions. Anti-GTl

antibodies were purified using the following method. The

total IgG fraction (15 mg each batch) purified from whole

serum was incubated with the peptide column and rotated end-

over-end at room temperature for 2 h. Unbound IgG was washed

from the column with PBS, monitoring the eluate by absorbance

at 280 nm until a baseline was reached. Specifically bound

IgG was then eluted with 0.1 M glycine, pH 3.0, by collecting

1 ml fractions. The eluate was immediately neutralized with

0.1 ml 1M Tris-Base. Peak fractions, as assessed by

absorbance at 280 nm, were pooled and dialyzed against PBS

for 12 h at 4C. The final IgG was stored at -20C as a 0.5

mg/ml solution in PBS.


Membrane Fraction Isolation


To facilitate the detection of integral membrane proteins

such as the glucose transporter, it is often desirable to





















0.5
E
0
00 0.4





0 0.2


S 0.1
0


0.0
1 2 3 4 5 6 7 8 9 10

Fraction Number


Figure 2-1 Elution Profile of Peptide-Purified anti-GT1
Antibody IgG from a total antibody fraction was incubated
with the anti-GT1 column for 2 hours at room temperature.
Unbound antibody was washed from the column with PBS, and
specifically bound antibody was eluted with 0.1 M glycine, pH
3.0. The absorbance of 1 ml fractions collected during the
elution was then measured at 280 nm. This profile is
representative of at least ten elution profiles performed on
this column.








partially purify a cellular fraction which is enriched in

membrane protein. This affords the advantage of preparing

relatively concentrated preparations of the membrane protein

of interest, and furthermore, reduces non-specific

interactions in procedures such as Western blotting and

immunoprecipitation. To this end, GLUT1 was routinely

partially purified by preparing a cell lysate fraction which

was depleted of cytosolic and nuclear proteins. To prepare

this fraction, the culture medium was aspirated and the cell

monolayer rinsed in phosphate-buffered saline (PBS) at 40C.

Cells were then scraped into 5 ml TES buffer [20 mM Tris-HCl,

pH 7.4, 1 mM EDTA, 250 mM sucrose, and protease inhibitors

(20 pg/ml each of aprotonin, leupeptin, pepstatin, TPCK,

TLCK, and 1 mM PMSF)] and homogenized by 20 strokes in a 10

ml Potter-Elvejhem homogenizing flask with a teflon pestle.

The homogenate was centrifuged at 1300 x g for 5 min at 4C

to remove nuclei and unbroken cells. The supernatant was

centrifuged at 212,000 x g for 1 hour at 4C to pellet the

remaining particulate fraction. The supernatant, containing

cytosolic proteins, was discarded and the final membrane

pellet resuspended in TES buffer (usually 0.3 0.5 ml) by

homogenization. The resuspended membrane fraction was

quickly frozen by submersion in liquid nitrogen and then

stored until use at -20oC.








Markwell Assay for Protein Determination


Several assays have been developed for the determination

of protein concentration in biological samples. All protein

determinations, however, suffer the limitation that certain

compounds, such as detergents or urea, can interfere with the

accuracy of the assay. For the determination of protein in

samples containing membranes, the sample must be extracted

with detergent in order to obtain a true reflection of the

protein content. The most popular method, a procedure

developed by Lowry, has come into wide use because of its

ease and sensitivity (Lowry et al., 1951). The Lowry protein

assay, however, is interfered by the presence of detergents,

especially sodium dodecyl sulfate (SDS) (Peterson, 1979).

Protein determinations in this work were most commonly

performed on membrane fractions, necessitating the routine

use of the Markwell modification of the Lowry assay for

protein determination. The Markwell assay is a modification

of the Lowry protein determination which is compatible with

certain detergents, including SDS.

To generate a standard curve, samples of purified bovine

serum albumin (BSA) (0-100 pg) were diluted to a total of 0.1

ml with water. Aliquots of the unknown samples (usually 5 -

25 pl) were also diluted to 0.1 ml water. If the unknown

protein samples were in buffer, an equal volume of that

buffer was also added to the protein standards. Assay

solution (1 ml of [100 parts 2.0% Na2CO3, 0.4% NaOH, 0.16%








Na', K+ tartrate, 1.0% SDS to 1 part 4% CuSO4]), was added,

mixed well, and incubated at room temperature for 10 minutes.

Folin Phenol (0.1 ml, IN) was then added to each sample and

mixed well immediately after addition. The samples were

incubated for 45 min at room temperature for color

development. The absorbance of the samples was then

determined at 650 nm. The protein concentration of the

unknown samples was then determined from the standard curve.

Gel Electrophoresis


Gel electrophoresis was performed essentially as

originally described (Laemmli et al., 1970). Protein samples

were diluted with an equal volume of sample dilution buffer

consisting of 4% SDS, 6M urea, 10% B-mercaptoethanol, 0.15

mg/ml bromophenol blue, 40% glycerol and 20 mM Tris-base, pH

6.6. Gels were run at approximately 45 V overnight at room

temperature.


Electrotransfer and Western Blotting


The detection of proteins by the Western blotting method

requires that the protein sample be transferred to a

substrate, usually nitrocellulose. The transfer buffer and

conditions closely follow the original procedure outlined by

Towbin et al., (1979). The electrophoresis gel was removed

from the gel apparatus and soaked in transfer buffer for 30

min at room temperature prior to transfer. Resolved proteins

were then transferred to nitrocellulose at 250 mA for 2.5








hours at 4'C in 25 mM Tris-base (unadjusted pH), 192 mM

glycine, 20% methanol. The blot was stained briefly in amido

black (0.2% in 40% methanol, 10% acetic acid) and destined

(40% methanol, 10% acetic acid). The blot was blocked in

Tris-buffered saline/0.1% Tween-20 (TBS-T) containing 5% non-

fat dry milk (NFDM) for 30 min at room temperature. The blot

was agitated constantly with an orbital shaker throughout the

procedure. The blot was then incubated in the same solution

containing a 1:500 dilution of the anti-GT1 antiserum for 1

hour at room temperature. After extensively washing the blot

in TBS-T, the blot was incubated in 1:50,000 dilution of goat

anti-rabbit antibody coupled to horseradish peroxidase in

TBS-T/5% NFDM for 1 hour at room temperature. The blot was

then extensively washed in TBS-T before before being

visualized with Amersham's Enhanced Chemiluminescence

detection reagents following manufacturer's directions.

Light emissions from the blot were captured on Hyperfilm MP

(Amersham). Band intensity was quantitated in the linear

range of the film on a Visage Bioscan video densitometer.


Metabolic Radiolabelinq of 3T3-L1 Adipocytes


In order to characterize individual proteins for their

turnover and processing, they need to be metabolically

labeled for a defined period of time. Usually, this is

accomplished with the use of radioactively labeled amino

acids, such as 35S labeled methionine and/or cysteine. In

order to determine the time required for the degradation of








the labeled protein, the label is then "chased" by incubating

the cells in a large excess of unlabeled amino acids in order

to dilute the specific activity of the intracellular amino

acid pools (Harlow and Lane, 1988). Immunoprecipitation and

recovery of the protein of interest during this period allows

the analysis of the radioactivity present in the initially

labeled protein pool and its disappearance due to degradation

as a function of time. The same strategy can be used for

very short labeling periods where the protein is labeled, and

then the processing or glycosylation of the protein can then

be followed by observing shifts in relative molecular weight

as assessed by gel electrophoresis. This section describes a

metabolic labeling procedure optimized for the labeling and

immunoprecipitation of GLUT1 expressed at endogenous levels

in 3T3-L1 adipocytes for subsequent gel electrophoresis and

fluorography.

Cells were incubated in 8 ml methionine- and cysteine-

free D-MEM without added serum for 1 hour to deplete the

intracellular pools of methionine and cysteine. The

depletion medium was aspirated and the cells incubated in 2

ml methionine- and cysteine-free DMEM containing 200 pCi/ml

Tran35S-Label for the specific times indicated. To initiate

the chase periods, the labeling medium was aspirated and

replaced with 8 ml complete DMEM/10% FBS containing 2 mM

methionine and 2 mM cysteine. These conditions were

established to generate an adequate signal for fluorography

while minimizing the time of methionine and cysteine








depletion based on previous reports of possible alterations

in protein turnover with prolonged amino acid depletion

(Haspel et al., 1985).


GLUT1 Immunoprecipitation


Immunoprecipitation is a procedure by which a particular

protein can be isolated from a crude mixture of proteins by

incubation and binding of specific antibodies. Once this

immunocomplex is formed, it is captured on a substrate,

commonly protein A Sepharose, so that it can be removed from

solution. Proteins associated in a non-specific manner can

then be removed by washing the protein A Sepharose-

immunocomplex. Finally, the immunoprecipitated protein can

be released by destroying the interaction between the

antibody and the protein A, usually with strong denaturing

solutions such as urea, low pH, SDS, or a combination of

these conditions (Harlow and Lane, 1988).

GLUT1 was immunoprecipitated from the total membrane

fraction in the following manner. A total membrane fraction

was obtained by first washing the monolayer in cold PBS. The

cells were then scraped into TES buffer and homogenized by 20

strokes in a 10 ml Potter-Elvejhem flask with a teflon

pestle. The homogenate was then centrifuged at 212,000 x g

for 1 hour at 40C to collect a total membrane pellet. The

final membrane pellet was then homogenized with 20 strokes of

a 2 ml Potter-Elvehjem homogenizing flask with a teflon

pestle in 1 ml extraction buffer [PBS containing 1 mM EDTA,








2% C12E9, 0.1% SDS and protease inhibitor cocktail as above].

Any insoluble material was then removed by centrifugation in

a microcentrifuge for 5 min at 4C. The supernatant was then

recovered, frozen in liquid nitrogen, and stored at -20"C.

In order to eliminate non-specific interactions between

membrane proteins and immunoglobulin, thawed extracts

(containing between 1.5 2 mg membrane protein) were

incubated with an unrelated non-immune antiserum (5 pl) and

collected with a 50% suspension of Protein A Sepharose (25

pl) for 1 h at 4C. Samples were centrifuged briefly in a

microcentrifuge to pellet the protein A Sepharose pellet and

the pre-cleared supernatants transferred to new

microcentrifuge tubes. Extracts were adjusted to equal

protein concentration in 1 ml with additional extraction

buffer and incubated with the anti-GT1 antibody (5 pg) for 3

hours at 4C with end-over-end rotation. The 50% Protein A

Sepharose suspension in PBS was then added (25 pl) for 2

hours to collect the immunoprecipitates. The Protein A

Sepharose was then washed 3 times with extraction buffer,

followed by 4 times for 10 min with extraction buffer

containing 1M NaCl. Immunoprecipitates were released by

incubation in 0.1 ml sample dilution buffer containing 6 M

urea and 10% BME for 15 min at 37C. The supernatants were

then loaded onto an 8% SDS-PAGE gel and run overnight at 50

V. For fluorography, the gels were fixed in 10% TCA/40% MeOH

for 30 min, soaked in water for 30 min, and then soaked in 1M

sodium salicylate for 1 h before drying at 60C under vacuum.








Dried gels were exposed to Amersham Hyperfilm typically for 4

days.


Endoqlycosidase Digestions


Endoglycosidases refer to a class of enzymes that

catalyze the cleavage of oligosaccharide chains at specific

sugar residues. These enzymes are often useful for the

characterization of oligosaccharides on glycoproteins. For

the analysis of the oligosaccharide on GLUT1, two specific

endoglycosidases were chosen. The first, N-Glycanase F,

cleaves between the two GlcNAc residues adjacent to the

asparagine residue on the protein. This enzyme can be used

to determine if glycoproteins are post-translationally

modified with an N-linked oligosaccharide. This enzyme will

not cleave oligosaccharides attached to serine or threonine

resides (O-linked). The second endoglycosidase,

endoglycosidsase H, also cleaves between the two GlcNAc

residues but is specific for the high mannose type of N-

linked oligosaccharide. This enzyme is useful for not only

determining if a protein contains an N-linked

oligosaccharide, but can be used to distinguish among high

mannose type and complex type oligosaccharides. Like N-

Glycanase F, this enzyme will not cleave oligosaccharides

attached in an O-linked manner.

Membrane protein (20 pg) from control or glucose-

deprived adipocytes and 10 pg from LEC1 CHO cells were

denatured in 50 mM BME/0.5% SDS in a final volume of 50 Mp








for 20 min at 37C. Then, for N-glycosidase F digestions,

the samples were brought to 150 mM Tris-HC1, pH 8.0, 1% NP-

40, protease inhibitors as above, 1.25 U N-glycosidase F and

then incubated at 37C for 2 hours. For endoglycosidase H

digestions, the denatured protein sample was brought to 75 mM

sodium citrate, pH 5.5, protease inhibitors as above, and 2.5

mU endoglycosidase H and incubated at 37C for 2 hours. An

equal volume of 2X sample dilution buffer was added and

loaded onto an 8% SDS-PAGE gel for separation.


Glycoqen Isolation


Glycogen, a polymerized form of glucose, is stored by

most cells (Voet and Voet, 1990). Glycogen breakdown

provides a source of fuel for energy production by glycolysis

as well as other pathways. The determination of glycogen in

this work is not made directly; rather the glycogen is

hydrolyzed with acid to produce free glucose which can then

be assayed directly by the method described below. The

results of this assay are therefore represented as the amount

of glucose contained in glycogen. The procedure used was

based on a previously described method for the isolation of

glycogen from liver (Pfleiderer 1963). After rinsing in

phosphate-buffered saline, pH 7.2 (PBS) at 40C, cells were

scraped into a 15 ml polypropylene tube in 1 ml PBS. The

cells were sonicated for 10 sec on power 1.5 at 50% duty

cycle on a Branson Sonifier 450. KOH (2.0 ml, 30% w/v) was

added and the mixture mixed vigorously. The sample was then








incubated in a boiling water bath for 15 min. The sample was

removed and cooled briefly before the addition of 3.5 ml 95%

EtOH. After mixing, the samples were heated in a boiling

water bath for 3 min and then cooled to room temp. The

precipitate was collected in a clinical centrifuge at 1300 x

g for 5 min at room temperature. The clear supernatant was

removed by aspiration and the precipitate washed in 1 ml 95%

EtOH by resuspension. The suspension was transferred to a

microcentrifuge tube and recollected at 13,300 x g for 5 min.

The supernatant was aspirated and the precipitate stored at

-200C. When all samples were collected, the precipitate was

dissolved in 0.2 ml 2N H2S04 and incubated in a boiling water

bath for 2 hours. The samples were cooled before the

addition of 0.15 ml 2N NaOH and 0.65 ml H20. The hydrolysates

were used for glucose determination as described below.


Glucose Assay


The assay chosen for the determination of glucose

released from glycogen is based upon the following reaction

mechanism. Glucose is first phosphorylated by hexokinase in

the presence of ATP. Glucose-6-phosphate is then oxidized in

the presence of NAD+ by glucose-6-phosphate dehydrogenase,

forming 6-phosphogluconate. This results in the production

of NADH in direct proportion to the initial concentration of

glucose. To perform the assay, 0.1 ml glycogen hydrolysate

was diluted with 0.4 ml H2SO4 / NaOH/ H20 mixture (2/1.5/6.5).

Samples of this solution (0.1 ml) were assayed in triplicate.








Samples, as well as the assay buffer, were pre-warmed to

37C. Assay reagent (1.0 ml) was mixed with 0.1 ml sample

and incubated at 370C for 30 min. The absorbance of the

samples was then measured at 340 nm.


Subfractionation of 3T3-L1 Adipocytes


Subfractionation is a procedure for separating and

purifying the different compartments of the cell. This

usually involves breaking the cell open while attempting to

maintain the integrity of the intracellular organelles. When

these individual organelles are then subjected to centrifugal

force, these organelles separate from each other on the basis

of both their size and density. In this manner, the

localization of proteins can be determined. One disadvantage

of this technique, is that the difference in density and size

between different cell organelles is relatively small. This

allows incomplete separation of these organelles resulting in

cross-contamination; that is, that a part of the golgi

fraction may be present in the plasma membrane, and so forth.

The following section describes a protocol used to separate

fractions enriched in plasma membrane, high density

microsomes, and low density microsomes. The high density

membrane fraction is usually enriched in proteins contained

in the endoplasmic reticulum and the golgi apparatus. The

low density membrane fraction is enriched in proteins

characteristic of endosomes, lysosomes, and the trans-golgi

network. Although all subfractionation procedures exhibit








some cross-contamination, this procedure will still provide

useful information in conjunction with other procedures to

analyze protein targeting.

Plasma membrane, high density, and low density membrane

fractions were isolated with the following subfractionation

procedure developed by Fisher and Frost (in preparation).

3T3-L1 adipocytes cultured in 10 cm tissue culture plates (5

plates per condition) were washed in PBS at 40C before being

scraped into 20 ml TES buffer. The cells were passed by a

ball bearing in a homogenizing block with a 0.0025 inch

clearance ten times at 40C. The homogenate was centrifuged

at 17,369 x g for 15 min at 4C [A]. The supernatant from

[A] was centrifuged at 48,254 x g for 30 min at 40C [B].

The pellet from [A] was resuspended into 25 ml TES buffer and

layered over 11.5 ml sucrose cushion of TES containing 1.12M

sucrose. This step gradient was centrifuged at 95,133 x g

for 65 min at 4C in a swinging bucket rotor [C]. The

interface from the step gradient, representing the plasma

membrane enriched fraction, was centrifuged at 48,254 x g for

30 min at 40C [D]. The pellet from [D] was resuspended into

TES buffer and centrifuged again at 48,254 x g for 30 min at

4C. This washed plasma membrane pellet was resuspended into

TES buffer, frozen in liquid nitrogen and stored at -200C.

The pellet resulting from the [B], representing the high

density membrane fraction, was resuspended into TES buffer

and centrifuged at 48,254 x g for 30 min at 40C. This washed

pellet was resuspended into 0.3 ml TES, and stored in a








manner similar to the plasma membrane fraction. The

supernatant from [B] was centrifuged at 212,000 x g for 75

min at 40C. The pellet, representing the low density

membrane fraction, was resuspended in TES buffer and

centrifuged at 212,000 x g for 75 min at 40C. This washed

pellet was resuspended into 0.3 ml TES, frozen in liquid

nitrogen, and stored at -200C. For Western blot analysis,

samples were diluted into sample dilution buffer containing 6

M urea and 10% B-mercaptoethanol before separation by SDS-

PAGE and transfer to nitrocellulose as described above.


Cell Surface Biotinylation


The modification of proteins with membrane impermeable

chemicals has recently proven to be an effective tool in the

study of membrane proteins, including the vitronectin

receptor (Nesbitt and Horton, 1992), Thy-i and lymphocyte

surface marker proteins (Arni et al., 1992), surface proteins

of 3T3-L1 fibroblasts (Hare and Taylor, 1991), surface

proteins of 3T3-L1 adipocytes (Hare and Taylor, 1992),

insulin receptors (Levy-Toledano et al., 1993), and tumor

necrosis factor receptor (Hsu and Chao, 1993). Several

membrane-impermeant modifiers are now commercially available,

with different functional groups to confer different chemical

reactivities. Many of these modifiers are conjugated to

biotin, a small molecule that has a tight affinity for

avidin. Biotin has many advantages as a molecular tag; it is

small and in many cases does not abolish biological activity








of ligands or enzymes, it can be conjugated to many different

types of compounds and so is quite versatile in its uses, and

finally, it can be readily detected by binding of avidin or

streptavidin. The binding between avidin and biotin is the

tightest binding described, with a dissociation constant of

approximately 10-15. Avidin and streptavidin can be detected

by several methods; one method utilizes radioactively labeled

avidin or streptavidin, a second utilizes fluorescently

labeled avidin or streptavidin that can be visualized by

illuminating with fluorescent light, a third method utilizes

enzyme-avidin or streptavidin conjugates which can be

detected by incubation with the proper substrate which can

lead to the production of color or light.

In this work, a membrane impermeant biotin probe which

exhibits reactivity towards free amine groups was utilized.

Once the cell surface was derivitized with the biotin probe,

the membrane fraction was solubilized and biotinylated

proteins recovered with an immobilized form of streptavidin.

Streptavidin was chosen over avidin because of avidin's

extremely high isoelectric point (>10) which can lead to non-

specific interactions. The isoelectric point of streptavidin

is approximately 5, and exhibits much lower non-specific

reactivity in immunoaffinity assays. Once the biotinylated

proteins are recovered, unbiotinylated proteins are removed

and the specifically bound proteins analyzed by SDS-PAGE and

Western blotting.








Specifically, 3T3-L1 adipocytes were rinsed in PBS at

40C and maintained at 4*C. When the cells were equilibrated

to 40C, the PBS was removed and the cells washed in PBS, pH

8.5 at 40C. Cells were then incubated in 2.0 ml of the same

solution in the presence or absence of 0.5 mg/ml sulfo-NHC-

LC-biotin for 1 hour. The solution was then aspirated and

the cells washed in TES to quench any remaining biotin probe.

A membrane fraction was then prepared and solubilized in the

extraction buffer used for immunoprecipitation. After the

removal of insoluble material by centrifugation, the

clarified supernatant was incubated in the presence of 50 pl

streptavidin-agarose for 6 hours at 40C with end-over-end

rotation. The complexes were then washed 5 times in

extraction buffer to remove non-specific interactions.

Specifically bound proteins were released by incubating the

complexes in sample dilution buffer containing 6 M urea and

10% B-mercaptoethanol at 370C for 30 min. The released

proteins were then analyzed by SDS-PAGE and a modified

Western blotting technique.

For the detection of the biotinylated proteins, the gel

was transferred to nitrocellulose as described before.

Following transfer, the blot was briefly stained in amido

black (0.2% w/v in 40% methanol, 10% acetic acid) and

destined. Non-specific binding sites were blocked by

incubating the blot in 5% non-fat dry milk (NFDM) in Tris-

buffered saline containing 0.1% Tween-20 (TBS-T) for 1 hour

at room temperature. The blot was then washed in TBS-T








briefly before a streptavidin-horseradish peroxidase

(streptavidin-HRP) conjugate was added in 0.5% NFDM/TBS-T at

a final concentration of 2 x 10-5 mg/ml for 1 hour at room

temperature. The blot was then extensively washed at room

temperature with TBS-T to remove the unbound and non-

specifically bound streptavidin-HRP. The blot was then

incubated in the ECL Enhanced Chemiluminescent Western

blotting detection reagents according to the manufacturer's

instructions. The developed blot was then exposed to film

for a period of 10 sec 10 min.


Plasma Membrane Fragment Isolation


Another technique which has recently become popular for

the analysis of processes occurring at the plasma membrane is

the isolation of plasma membrane fragments. In this

technique, cells that are plated onto glass coverslips are

adhered to the surface by incubating the cells in poly-L-

lysine of high molecular weight (<100,000 M.W.). The

mechanism of adherence by poly-L-lysine is not exactly known,

but it is thought that the highly positive nature of the

poly-L-lysine polymer acts to bind the negatively charged

plasma membrane and the negatively charged glass surface in

an ionic interaction. Gentle sonication is then used to

remove parts of the cell that are not adhered to the surface,

and results in large fragments of the plasma membrane left on

the glass coverslip. This procedure has been used

extensively for the characterization of the assembly of








clathrin-coated pits which occurs at the inside of the plasma

membrane surface (Lin et al., 1992; Lin et al., 1994) and the

purification of caveolin (Chang et al., 1991). Recently, it

has also been modified for use in the 3T3-L1 adipocytes for

analyzing the translocation of the GLUT1 glucose transporter

to the plasma membrane in response to insulin treatment

(Robinson and James, 1992). This procedure, therefore, can

be used to generate pure plasma membrane fragments which are

free from contamination of endoplasmic reticulum and golgi

membrane fractions.

3T3-L1 cells were grown on 22 mm round glass coverslips

(Fisher) and differentiated as described earlier. Once fully

differentiated, cells were incubated in the presence or

absence of glucose for the indicated period of time. The

cells were then washed 3 times in PBS at 40C. The glass

coverslip was removed and placed into a clean 35 mm tissue

culture before being incubated in the same solution

containing 0.5 mg/ml poly-L-lysine (M.W. > 100,000) for 1

minute. The monolayer was then washed three times at 40C

with 3.0 ml 1/3X sonication buffer [1X sonication buffer; 70

mM KC1, 30 mM HEPES, pH 7.5, 5 mM MgC12, 3 mM EGTA]. This

solution was aspirated and replaced with 1 ml sonication

buffer at 4"C. The monolayer was then immediately placed

under a 1/2-inch tapped flat horn at a distance of 5 mm and

sonicated for approximately 2 sec at power 1.5 on a Heat

Systems Sonicator. The glass coverslip was then washed three

times at 40C in PBS. All last traces of PBS were carefully





42

removed from the coverslip and tissue culture dish. Sample

dilution buffer (50 pl) was added directly to the coverslip

and the glass scraped with a rubber scraper. The sample

dilution buffer was then recovered and loaded onto a mini

SDS-PAGE gel for separation. The presence of GLUT1 and GRP78

was detected by Western blotting as described above.













CHAPTER 3
CHARACTERIZATION OF GLUT1 GLYCOFORMS


Introduction


Glucose deprivation results in the appearance of a lower

molecular weight protein recognized by anti-GLUTI antiserum

in several cell lines, including 3T3-C2 fibroblasts (Haspel

et al., 1991), 3T3-L1 fibroblasts (Reed et al., 1990), 3T3-L1

adipocytes (Reed et al., 1990; Tordjman et al., 1990; Kitzman

et al., 1994), and normal rat kidney cells (Haspel et al.,

1990). The appearance of this low molecular weight protein

can be prevented by incubation with fructose, suggesting that

sugar metabolism is required to prevent its appearance

(Haspel et al., 1986; Kitzman et al., 1994). In normal rat

kidney cells and 3T3-C2 cells, the migration of this protein

was not altered upon digestion by endoglycosidase F and

therefore was interpreted to be unglycosylated (Haspel et

al., 1986; Haspel et al., 1990). In 3T3-L1 adipocytes,

however, the lower molecular weight protein is glycosylated,

but with a smaller oligosaccharide (Kitzman et al., 1994).

Careful examination of this protein and its relationship to

the normal GLUT1 protein in 3T3-L1 adipocytes has not been

reported. This section describes the characterization of the

normal GLUT1 protein as well as the lower molecular weight

protein that appears in glucose-deprived 3T3-L1 adipocytes.








These analyses will be performed with a combination of

Western blotting, glycosylation inhibitors, tryptic digests,

and glycosidase digestions. The goal of this section is to

test the hypothesis that the lower molecular weight protein

detected by Western blot in glucose-deprived cells is a

second form of the GLUT1 transporter that arises not from

alterations in the core protein, but through alterations in

glycosylation.


Results


Characterization of Anti-GT1 Antiserum


In order to confirm that the anti-GTl antiserum

specifically recognized the GLUT1 carboxy-terminal peptide,

dot blots and Western blots were performed. In Figure 3-1 A,

varying amounts of the GT1 peptide (CEELFHPLGADSQV) and GT4

peptide (CSTELEYLGPDEND) were blotted onto dry

nitrocellulose, dried, and detected with the anti-GT1

antiserum. This result indicates that the anti-GT1 antiserum

at a dilution of 1:1000 can detect as little as 0.1 ng GT1

peptide. The antiserum, however, did not recognize the

carboxy-terminal GLUT4 peptide when blotted in a similar

manner, demonstrating that the reaction was specific for the

GLUT1 peptide. Note that the GLUT1 peptide and the GLUT4

peptide share little identity. The observation that the

anti-GLUT1 antiserum does not recognize the GLUT4 peptide














-4
E-) E-1
I 0
o I
0 -H i-H
0 0)
E0 H






0 0r
W.4Jr
0C 0




'0 *4m 0 e



S-4-4
0 ,QE-












H 00 *) mc
o 00





* 0 0 0



0 >-1 0 p
a W0





0 t-),4'






4)n m
CD
0p U -H










00 3
- 0 C Ul
iN0c10
-4 m 0






U 04a '









R0.4 -
-4 00 V
r m0. '
















0)
8


Cn


+


+


m



LO'O.0

.0o

.0
9





0.
a L


0.
0)


sunwwl



SId


I I I
S i z


& -








reduces the possibility that this antiserum will recognize

the other transporter normally expressed in the 3T3-L1

adipocytes.

The anti-GTl antiserum was then tested in a traditional

Western blot to confirm that the correct protein from 3T3-L1

adipocytes would be recognized. Equal amounts (30 pg) of

membrane protein from normal and glucose-deprived (48 hours)

cells were resolved by SDS-PAGE and transferred to

nitrocellulose. GLUT1 was then detected by Western blot.

The anti-GTl antiserum recognizes a wide band of average

molecular weight of 46 kDa, consistent with the recognition

of a similar band with other anti-GLUT1 antiserum (Haspel et

al., 1985; Haspel et al., 1986; Kitzman et al., 1994) (Figure

3-1 B). It should be noted that the migration of

glycoproteins in SDS-PAGE can deviate from its true molecular

weight according to the size of the oligosaccharide and the

percentage of acrylamide in the resolving gel (Leach et al.,

1980). It is therefore assumed that, despite slightly

different migration, the protein recognized by this antiserum

and other antisera are recognizing the same protein. In

glucose-deprived cells, the lower molecular weight protein,

which migrates at 37 kDa (p37), is also recognized by the

anti-GTl antiserum. The pre-immune serum (PIS) does not

detect either of these bands and shows no other cross-

reactivity to other membrane proteins from 3T3-L1 adipocytes.








Glucose Deprivation and GLUT1 Protein Expression


The effect of glucose deprivation on the expression of

GLUT1 protein was analyzed by Western blotting. 3T3-L1

adipocytes were incubated in glucose-free medium for a total

of 48 hours, and a membrane fraction was prepared at the

times indicated. Membrane proteins (25 pg/lane) were then

resolved by SDS-PAGE and transferred to nitrocellulose. The

anti-GT1 antiserum was then used to probe the protein blot in

order to visualize both the relative amount and molecular

weight of the GLUT1 transporter protein. The normal

glycoform of the GLUT1 transporter from 3T3-L1 adipocytes is

observed at time 0; it migrates with an average molecular

weight of approximately 46 kDa (p46) and exhibits the wide

heterogeneous glycosylation expected (Figure 3-2 A). For the

first 15 hours of glucose deprivation, both the level and the

migration of this protein is relatively unchanged. After 15

hours, however, the appearance of the second immunoreactive

protein at 37 kDa (p37) is observed. After 48 hours of

glucose-deprivation, the total level of immunoreactive

protein is increased by 2-fold. This increase is solely

attributable to the accumulation of the 37 kDa protein

(Figure 3-2 B), since the normal GLUT1 protein does not

change significantly during this period. Also in this

figure, the effect of cycloheximide, a protein synthesis

inhibitor, on the accumulation of p37 was analyzed. The

Western blot indicates that cycloheximide, even when added
































Figure 3-2 Effect of Cycloheximide on GLUT1 Expression
During Glucose Deprivation 3T3-L1 adipocytes were incubated
in glucose-free medium for up to 48 hours and membrane
fractions prepared at the times indicated. Panel A Membrane
proteins probed for GLUT1 by Western blot with anti-GTl
antiserum. The right panel shows cells incubated with
cycloheximide (20 pM) added 12 hours after the withdrawal of
glucose. These samples were then analyzed in an identical
manner. Panel B Densitometry of GLUT1 protein from panel A.
These results are representative of at least 4 independent
experiments.



















A Glucose Deprivation, hours
0 3 6 9 15 24 36 48 15 24 36 48




Is I -p37


-cycloheximide +cycloheximide
at 12 hours


B o -l


12 24 36 48
Glucose Deprivation, hours








twelve hours after the onset of glucose deprivation,

prevented the appearance of p37 (Figure 3-2 A, right panel).

It has been generally accepted that p37 is a second GLUT1

protein (Haspel et al., 1986; Haspel et al., 1990; Tordjman

et al., 1991; Kitzman et al., 1994), although a careful

examination of both proteins has not been reported. The

following sections will describe experiments suggesting that

p37 is another form of the GLUT1 transporter only synthesized

in glucose-deprived cells.


Tryptic Digest of GLUT1 Transporter


To determine whether p46 and p37 arise from the same

protein, tryptic digests were performed with the expectation

that peptides generated by proteolysis would be identical if

both p37 and p46 derived from the same protein. It should be

noted that although many tryptic peptides can be generated,

only those retaining the carboxy-terminal region against

which the antiserum was generated could be recognized in the

Western blot. Membrane proteins from normal or glucose-

deprived (48 hours) cells were first isolated. These

membrane proteins were then incubated in the presence of

trypsin (10 Mg) for the times indicated, when the reaction

was stopped with a 10-fold excess concentration of soybean

trypsin inhibitor. These samples were then resolved by 10%

SDS-PAGE and transferred to nitrocellulose. GLUT1 proteins

were then detected with the anti-GTl antiserum in a Western

blot. GLUT1 peptides generated by partial trypsin treatment
































Figure 3-3 Tryptic Digest of p46 and p37 3T3-L1 adipocytes
were incubated in the presence (+Glucose) or absence (-
Glucose) of glucose for 48 hours before a total membrane
fraction was prepared. These membrane proteins were
incubated trypsin (10 pg) for the times indicated before the
reaction was stopped with excess trypsin inhibitor. The
digested membrane proteins were then probed for GLUT1 by
Western blot with anti-GTl antiserum. Molecular weight
standards are indicated. This result is representative of at
least two independent experiments.




























+Glucose
Incubation time, min 0 0.5 1 2 3 4 5 6 10


66-
45- Aa.I.. ---


31-

21-

14-

-Glucose


--p46









- p46
- p37








migrated at relative molecular weights of 25 kDa, 21 kDa, and

17 kDa (Figure 3-3 A and 3-3 B). These peptides,

corresponding to the carboxy-terminal half of the GLUT1

transporter which do not contain the glycosylation site, are

identical in control and glucose-deprived cells. No

additional peptides were observed from glucose-deprived

cells, as would be expected if that protein were of different

length or sequence. Together these data suggest that the

core protein of the normal GLUT1 protein and p37 protein is

very similar.


Effect of Tunicamycin Treatment on GLUT1 Protein


Tryptic digest experiments suggested that the protein

from which GLUT1 and p37 arise is very similar; therefore,

the possibility that the difference in relative molecular

weight between p46 and p37 was a difference in glycosylation

was tested. To prevent N-linked glycosylation of GLUT1, 3T3-

L1 adipocytes were treated with various concentrations (0-10

pg/ml) of the N-linked glycosylation inhibitor tunicamycin

dissolved in 20 mM potassium hydroxide for 24 hours.

Membrane proteins were then isolated and probed for GLUT1 by

Western blot using the anti-GLUT1 antiserum. The normal form

of the transporter, p46, (lane KOH, or 20 mM potassium

hydroxide alone) is drastically different from the

transporter from tunicamycin-treated cells, which migrates at

a relative molecular weight of 36 kDa (p36), in agreement

with the predicted molecular weight from the amino acid















I 0) H


0 4J WH )
o 0 (a -

- iO-4 0
0 d H '4


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0 0 0-H
U 0 .oC
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r e 4 J


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H *O H


000



00 ) 0



WM & 3E-1
0 4) WO t.

*-4 -H 0 4
am S 0

o E




0 Q)4 r-.4
4H -0 0

Cl H



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ZH 0 d


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*1o c4 r
('44-4 to0
C V 44r. -



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sequence (Figure 3-4, + glucose). The next species, p37,

present in the glucose-deprived cells, migrates slightly but

distinctly above the core protein with a difference in

molecular weight of approximately 1-2 kDa (Figure 3-4 -

glucose). The GLUT1 protein resulting from tunicamycin

treatment is identical whether it is derived from normal or

glucose-deprived adipocytes, supporting the conclusion that

the core protein in glucose-deprived adipocytes is identical

in size, and that differences in molecular weight between p46

and p37 arise from differences in glycosylation.

The production of unglycosylated GLUT1 protein provided

an opportunity to follow GLUT1 processing if the effect of

tunicamycin could be removed. A previous report demonstrated

that the processing of the insulin receptor in 3T3-L1

adipocytes could be followed after tunicamycin washout

(Ronnet et al., 1988). To this end, adipocytes were exposed

to tunicamycin (1 pg/ml) for 24 hours in either the presence

or absence of glucose. Following this treatment, the medium

was changed into medium without tunicamycin for 24 hours.

The production of the unglycosylated transporter p36 is

confirmed after exposure to tunicamycin for 24 hours (Figure

3-5). However, after the medium is changed into medium

without tunicamycin, no reduction in the level of the

unglycosylated transporter is observed. The inclusion of 25

mM N-acetylglucosamine was added in order to compete with the

action tunicamycin for the glycosylation process. The

results from this experiment indicate that even with the

































Figure 3-5 GLUT1 Expression Following Tunicamycin Washout
Normal or glucose-deprived (24 hours) 3T3-L1 adipocytes were
incubated in the presence of tunicamycin (1 pg/ml) for 24
hours. The tunicamycin-containing medium was then removed
and replaced with either medium containing tunicamycin (1
pg/ml), normal medium, or glucose-free medium containing 25
mM N-acetylglucosamine, for 24 hours. Membrane proteins were
then probed for GLUT1 by Western blot with anti-GTl
antiserum.













2 C
+ J <


4+ 3I


45- ago"
I a 0


- p46
p-p37
-----p36








inclusion of N-acetylglucosamine, the production of

unglycosylated transporter could not be prevented. Together,

these data indicate that although tunicamycin resulted in the

generation of unglycosylated GLUT1, the effect of tunicamycin

could not be removed and therefore elimated tunicamycin as a

useful tool in the analysis of GLUT1 processing. The

inability to remove tunicamycin in these experiments is not

consistent with the previous report on the processing of the

insulin receptor, but it may lie in the processing of a

multi-subunit protein such as the insulin receptor is

fundamentally different from a monomeric glycoprotein like

GLUT1.


Glycosidase Digestion of GLUT1 Glycoforms


In order to characterize the type of oligosaccharide

present on p46 and p37 GLUT1, membrane proteins from normal

or glucose-deprived cells were digested with either

endoglycosidase H, which cleaves high mannose

oligosaccharides, or N-Glycosidase F, which cleaves all N-

linked oligosaccharides. For comparison of these glycoforms,

membrane proteins from the LEC1 CHO cell line were also

tested. The LEC1 CHO cell line is a glycosylation mutant

that is deficient in N-acetylglucosamine transferase I, the

first committed step towards the production of hybrid and

complex type glycoproteins (Stanley and Chaney, 1985). As a













Em 0

of a-


o e4)


10 (

O 'OH
w -1 44 to


S0 4- -d







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4 d- Q501




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Sd ai


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0 -1 r.

























I V V MW
> 40 i m

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ma ) a0







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0



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+ I

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I +


+ I

I I


I +


+ I


I I


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LU








result, glycoproteins in this cell line are terminated at the

high mannose stage, and remain endoglycosidase H sensitive.

Figure 3-6 shows a Western blot of these membrane proteins

probed for GLUT1 using the anti-GLUTI antiserum. The

migration of the normal GLUT1 glycoform present in 3T3-L1

adipocytes was not altered by treatment with endoglycosidase

H, indicating that this GLUT1 glycoform is not a high mannose

type glycoprotein. The migration of p46 was altered,

however, by digestion with N-Glycosidase F, confirming

earlier observations that GLUT1 is an N-linked glycoprotein

(Kitzman, et al., 1993). The molecular weight of the N-

Glycanase F digested GLUT1 protein is 36 kDa, in agreement

with the molecular weight predicted by amino acid sequence

and the molecular weight of GLUT1 from tunicamycin-treated

adipocytes. In glucose-deprived adipocytes, neither the

normal nor the low molecular weight GLUT1 glycoform was

sensitive to digestion by endoglycosidase H, indicating that

neither is a high mannose glycoprotein. Once again, the

migration of the normal and low molecular weight GLUT1 was

resolved to the core 36 kDa protein upon digestion with N-

Glycanase F. This data confirms earlier observations that

the difference between the normal and low molecular weight

GLUT1 glycoforms is due to differences in glycosylation and

not changes in the primary sequence of the protein. To

confirm that the endoglycosidase H in this assay was active,

membrane proteins from the LEC1 CHO cell line were also

tested. The GLUT1 glycoform present in these cells (p38),








which was predicted to be a high mannose glycoprotein, was

sensitive to digestion with endoglycosidase H and was

resolved to a 36 kDa core protein. Together, these data

indicate that the low molecular weight GLUT1 present in

glucose-deprived 3T3-L1 adipocytes is glycosylated, but with

a relatively small oligosaccharide (approx. 1 kDa). While

this oligosaccharide is N-linked, it is not a high mannose

structure as might be expected for a normal processing

intermediate prematurely terminated due to the lack of

glucose. This oligosaccharide on the low molecular weight

GLUT1 from glucose-deprived 3T3-L1 adipocytes likely

represents an alternative oligosaccharide generated in the

absence of glucose.


Specificity and Efficiency of GLUT1 Immunoprecipitation


The procedure for immunoprecipitation of GLUT1 from 3T3-

L1 adipocytes was assessed using metabolically labeled

control or glucose-deprived adipocytes. A wide band

migrating at 46 kDa (p46) was immunoprecipitated from

membrane extracts of control cells using purified anti-GT1

antibody (-GT1, + glucose) (Figure 3-7). In contrast, a 37

kDa protein was immunoprecipitated from cells deprived of

glucose for 36 hours (-GT1, glucose). The specificity of

the immunoprecipitation procedure was verified by

immunoprecipitating GLUT1 in the presence of 1 pg of the GT1

peptide. Inclusion of this peptide completely blocked the

immunoprecipitation of p46 GLUT1 in control cells, as well as













mo




S r. o0
U Wa) rO
8 BS





(d DU aO






4O ,0 40-
F a) ao





























r-i O ,-o
m fu 4)
0H 1 l 0 H





*.al g'g
















\o ., I + v.
-,1 4
4.3 44


0- 4 :3 r r -4





) V *r i' 0) 0



0
0 4) a.r -43 H.

A4 U



o -. M H
-1 4 W2 Eo


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+) a)o 0 .




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P4 u Z-rq > I + a






























'C +





I





Sk

0' +


+r



a)r



0r


r- (O








p37 GLUT1 in glucose-deprived cells (+GT1, glucose). In a

similar experiment, a GLUT4 carboxy-terminal peptide

(CSTELEYLGPDEND) did not block the immunoprecipitation of

GLUT1 (+GT4, glucose). GLUT1 protein of either molecular

weight was not observed in membrane extracts

immunoprecipitated with Protein A Sepharose alone, with

preimmune serum, or with 5 pg IgG from an unrelated antisera

(-Ab, PIS, IRR, glucose). GLUT1 protein was also not

observed when extracts were immunoprecipitated with an equal

amount of the unbound antibody fraction (flow through) from

peptide purification of the same antiserum, indicating that

the purification step removed the majority of the GLUT1

specific IgG (FT, glucose).


Conclusions


This chapter described experiments designed to

demonstrate that the second immunoreactive protein that

appears during glucose deprivation of 3T3-L1 adipocytes is a

second glycoform of GLUT1. Tryptic digest of membrane

proteins from normal and glucose-deprived cells indicates

that peptides detected with the anti-GTl antiserum migrated

in an identical fashion. I interpret these data to suggest

that there were no gross alterations in the length of the

GLUT1 core protein. The expression of a different GLUT1

protein, therefore, could not account for the difference in

molecular weight between p46 and p37.








The effect of tunicamycin on the relative molecular

weight of the transporter supports this conclusion. These

experiments indicate that the core protein generated in

tunicamycin-treated cells, regardless of the presence or

absence of glucose, migrated at the same molecular weight (36

kDa). This type of experiment analyzes the GLUT1 protein

before it has an opportunity to be glycosylated. Enzymatic

deglycosylation of p37 and p46, where the oligosaccharide is

removed after glycosylation, also results in the same protein

migrating at 36 kDa, consistent with the tunicamycin

experiment. Additional information about the type of

oligosaccharide on p37 was obtained. Although p37 was

sensitive to digestion with N-Glycosidase F, indicating that

it was an N-linked glycoprotein, p37 was not sensitive to

endoglycosidase H, indicating that this oligosaccharide was

not of the high mannose class. In order to confirm that the

endoglycosidase H reaction was working correctly, digestion

of GLUT1 derived from the LEC1 CHO cell line was performed

and verified that the GLUT1 from this glycosylation mutant

was a high mannose type glycoprotein, despite its similar

migration to p37. Earlier studies utilizing glucose-deprived

CHO cells indicated that an alternative glycosylation pathway

exists, where the cell synthesized a truncated

oligosaccharide core, GlcNAc2MansGlc3, that was subsequently

added to proteins synthesized in the endoplasmic reticulum

(Rearick, et al., 1981). This alternative oligosaccharide

was shown to be endoglycosidase H resistant. The








endoglycosidase H insensitivity and size of the

oligosaccharide on p37 are consistent with this type of post-

translational modification. The biosynthesis and processing

of the transporter in the absence of glucose will be

addressed in more detail in Chapter 4.

Together, these data provide evidence that p37 is an

alternatively glycosylated form of GLUT1. The protein

component is likely unaltered in the absence of glucose.

Furthermore, the difference in relative molecular weight

between p46 GLUT1 and p37 GLUT1 resides in a difference in

the size of the oligosaccharide. p37 GLUT1 is glycosylated

in glucose-deprived adipocytes, although with an

oligosaccharide drastically reduced in size. This

oligosaccharide is likely not a processing intermediate

prematurely terminated in the absence of glucose, due to the

observation that the small oligosaccharide is approximately

the same size as a high mannose core but is endoglycosidase H

insensitive. The next chapter will describe experiments

designed to elucidate biosynthesis, processing, and turnover

of these GLUT1 glycoforms in normal and glucose-deprived 3T3-

L1 adipocytes.













CHAPTER 4
REGULATION OF GLUT1 BIOSYNTHESIS, PROCESSING AND TURNOVER BY
GLUCOSE IN 3T3-L1 ADIPOCYTES


Introduction


The biosynthesis of integral membrane glycoproteins

involves a complex journey through multiple membrane

compartments containing specifically localized enzymes.

During the translation of polypeptides through the

endoplasmic reticulum, a preformed oligosaccharide is

transferred by the enzyme oligosaccharyltransferase to

asparagine residues on the protein when they occur in the

recognition sequence Asn-X-Ser/Thr (Voet and Voet, 1990).

The transfer of oligosaccharides is regulated by a variety of

factors, including cell type, conformation of the protein

during translation, nutritional status, and distance from the

recognition site to the inside face of the endoplasmic

reticulum (Kornfeld and Kornfeld, 1980).

The pre-assembled oligosaccharide core is synthesized on

both the cytsolic and lumenal faces of the endoplasmic

reticulum. Sugar residues are added sequentially to a lipid

carrier molecule, dolichol pyrophosphate, generating a

growing oligosaccharide core. The first seven sugars, two N-

acetylglucosamine residues and five mannose residues, are

donated from their respective GDP nucleotide derivatives








generating the oligosaccharide dolichol-PP-GlcNAc2Man5. The

second seven sugars, 4 mannose residues and 3 glucose

molecules, are added from their respective dolichol phosphate

derivatives. The completed core oligosaccharide, dolichol-

PP-GlcNAc2MangGlc3, is transferred cotranslationally to the

growing polypeptide chain in the endoplasmic reticulum, and

is identical regardless of the glycoprotein in question.

Cleavage and processing of this oligosaccaride, however,

varies widely depending upon the type of cell, the individual

glycoprotein, and the final target of the glycoprotein.

Processing of the oligosaccharide begins immediately in

the endoplasmic reticulum upon the attachment of the core

oligosaccharide. Two specific glucosidases (glucosidase I

and glucosidase II) remove the first and second two glucose

residues, respectively, generating Asn-GlcNAc2Mang. Recently,

reglucosylation of the core oligosaccharide in the

endoplasmic reticulum has been described, although the exact

purpose of this enzymatic reaction has not yet been clearly

elucidated (Kornfeld R. reference). When the glycoprotein

has moved from the endoplasmic reticulum into the cis golgi,

mannosidases begin the sequential removal of four of the nine

mannose residues, eventually resulting in Asn-GlcNAc2Mans.

For glycoproteins destined to be of the high mannose class,

additional mannose units are added onto this oligosaccharide.

For complex type glycoproteins, however, a variety of other

sugars are then added onto the oligosaccharide, often

creating large and highly branched structures. These








modifications occur in the medial golgi, trans golgi, and the

trans golgi network. Some of the later sugar modifications

catatalyzed are the addition of N-acetylglucosamine, sialic

acid, fucose and nueraminic acid. A third type of

glycoprotein, the hybrid type, exhibits characteristics of

both high mannose and complex oligosaccharides, and often

contains a bisecting N-acetylglucosamine residue (Kornfeld

and Kornfeld, 1980). Sulfation, acetylation, and

phosphorylation of sugars are other types of oligosaccharide

modifications that are possible. For lysosomal proteins,

specific mannose sugars are phosphorylated, and this serves

to act as the appropriate recognition mechanism for targeting

to the lysosomes (Kaplan et al., 1977; Goldberg and Kornfeld,

1981).

The glucose-dependent regulation of glucose transport

has been described in many cell types (for review see Klip et

al., 1994). In each of these cell types, a common result of

glucose deprivation is an increase in the maximal velocity

(Vnx) of glucose transport. However, the mechanisms) (i.e.,

transcriptional, post-transcriptional, and post-

translational) responsible for this stimulation vary

according to cell type. Several investigators have

postulated that one component of the transport upregulation

might be an alteration of transporter turnover, particularly

GLUT1 (Yamada et al., 1983; Germinario et al., 1982; Walker

et al., 1989; Shawver et al., 1987; Haspel et al., 1991). In

particular, if the degradation of the glucose transporter








were inhibited in glucose-deprived cells while the synthesis

of the transporter remained constant, the end result would be

an increased number of GLUT1 molecules available for glucose

transport (increasing the Vmx). Few studies, however, have

investigated the processing and turnover of GLUT1 directly

because of the technical difficulties involved (Haspel et

al., 1985). The primary goal of this chapter is to define

the processing pathway of the GLUT1 transporter and determine

the kinetics of GLUT1 turnover in both the presence and

absence of glucose.

In 3T3-L1 adipocytes, glucose deprivation does not

result in a significant change in the level of GLUT1 protein

(as measured by Western blotting) over the period in which

transport induction is observed (Kitzman et al., 1994).

Beyond this time, we and others have reported the

accumulation of a lower molecular weight GLUT1 species which

results in a 2-fold increase in the total GLUT1 mass by 48

hours of glucose deprivation. (Reed et al., 1990; Kitzman et

al., 1994; Haspel et al., 1991). The role of this second

GLUT1 protein in transport stimulation has been investigated

and found not to be responsible for the activation of

transport during glucose deprivation (Kitzman et al., 1994).

However, the origin and characteristics of this lower

molecular weight GLUT1 and its relationship to the normal

GLUT1 glycoform were unknown. Several scenarios are

possible. One possibility is that the low molecular weight

GLUT1 glycoform is a normal intermediate in GLUT1 processing








that accumulates during glucose deprivation. In this case,

the transporter in glucose-deprived adipocytes would be

processed in the same manner as in normal cells, but due to

the inactivation of a processing enzyme or the depletion of a

critical precursor, a premature GLUT1 glycoform becomes the

terminal glycoform. This hypothesis assumes that the core

oligosaccharide produced in glucose-deprived cells is the

same as in normal cells. Alternatively, the low molecular

weight GLUT1 glycoform could arise from the glycosylation of

the core protein with a different oligosaccharide that is

produced only in the absence of glucose. In this scenario,

the oligosaccharide is aberrant before being added to the

protein, and further processing is not observed because the

aberrant oligosaccharide is not suitable substrate for the

processing enzymes found in the golgi apparatus. A third,

less likely, possibility is that the low molecular weight

GLUT1 transporter is a degradation product, that arises in

glucose-deprived cells due to the action of proteases on the

normal transporter, reducing its apparent molecular weight to

37 kDa. The latter is not likely due to the identical

molecular weight that the transporter exhibits either when

incubated in the presence of tunicamycin, when treated with

N-Glycanase F to remove the oligosaccharide, or compared to

the expected molecular weight of the core transporter protein

given by amino acid sequence. To address these issues,

existing methods of GLUT1 immunoprecipitation were modified

to improve recovery and reduce non-specific interactions.








This has allowed a clear model of the processing and turnover

of the metabolically labeled transporter to be obtained in

both normal and glucose-deprived adipocytes.


Results



Synthesis and Glycosylation of GLUT1 in Control and Glucose-
deprived 3T3-L1 Adipocytes


To determine if glucose plays a role in regulating the

synthetic rate of GLUT1, control and glucose-deprived

adipocytes were methionine and cysteine depleted before being

metabolically labeled for 10 to 60 min. GLUT1 was then

immuoprecipitated from solubilized membrane extracts and

analyzed by SDS-PAGE and fluorography. Figure 4-1 A shows

the fluorograph of the GLUT1 immunoprecipitates. Two GLUT1

species were synthesized in control cells during the first 10

min of labeling; a 36 kDa protein, and a glycosylated

intermediate migrating at 42 kDa. The 36 kDa protein is

likely the core protein based on the predicted molecular

weight of the core GLUT1 protein and tunicamycin experiments

(see Chapter 3). When normal adipocytes were labeled for an

additional 10 min, the mature GLUT1 protein (46 kDa) was

observed along with p36 and p42. No further GLUT1 proteins

were observed out to 60 minutes of labeling. When glucose-

deprived cells were labeled for 10 min, only the 37 kDa GLUT1

protein was observed. The 36 kDa core GLUT1 protein is

likely present but obscured due to the width of the p37
































Figure 4-1 Biosynthesis of GLUT1 in Normal and Glucose-
Deprived 3T3-L1 Adipocytes Panel A Cells, cultured with or
without glucose for 36 hours, were metabolically labeled with
200 pCi/ml Tran35S-Label for the times indicated as described
in Chapter 2. GLUT1 was then immunoprecipitated from
membrane extracts and analyzed by SDS-PAGE and fluorography.
Panel B Densitometry of total GLUT1 during labeling. Total
GLUT1 intensity was obtained in all experiments by summing
the appropriate bands after densitometry of each species
separately (i.e. p36+p42+p46). Inset, radioactivity in
membrane fraction during labeling relative to 10 min label.
This result is representative of at least two independent
experiments.

















A
Glucose + + + + -



45- -p46
p- p42
S9P36 P37

10 20 30 60
Labeling Time, min

B


S10

ioo- 1 6
S100 6


S0 10 20 30 40 50 60

" 0 Control
0







0 10 20 30 40 50 60

Labeling Time, minControl
- 50- Glucose-deprived


I-
-J


0.
0 10 20 30 40 50 60

Labeling Time, min








protein. Lighter exposures provided no further

discrimination between p37 and any other GLUT1 protein. When

glucose-deprived adipocytes were labeled for 20 min or more,

no additional GLUT1 species were observed. The relative

synthetic rate of GLUT1 in normal and glucose-deprived

adipocytes was measured by adding the densitometric intensity

of all GLUT1 bands at each time point. Although the

intensity of total GLUT1 protein in glucose-deprived cells

was greater than that of control cells at every time point

(Figure 4-1 B), this effect was not specific to GLUT1 when

taking into account an equal increase in radioactivity of the

membrane fraction from glucose-deprived cells (Figure 4-1 B,

inset). This phenomenon, therefore, could represent an

increase in the specific activity of the methionine and

cysteine pools in glucose-deprived cells. Taken together,

these data demonstrate that the synthetic rate of GLUT1 was

unaffected by glucose availability despite the differences in

glycosylation.


Regulation of GLUT1 Processing by Glucose


To examine the processing pathway of GLUT1 in control

and glucose-deprived adipocytes, normal or glucose-deprived

adipocytes were metabolically labeled for 10 min and then

chased for a total of 60 min. Cells were harvested for

immunoprecipitation during the indicated times during the

chase period. GLUT1 was then immunoprecipitated from these

membrane extracts and analyzed by SDS-PAGE and fluorography.
































Figure 4-2 Processing of GLUT1 in Normal and Glucose-
Deprived 3T3-L1 Adipocytes Cells incubated with (+ glucose)
or without (- glucose) glucose for 36 hours were labeled with
500 pCi/ml Tran35S-Label for 10 min and then chased for a
total of 60 min. GLUT1 was immunoprecipitated from membrane
extracts of each and analyzed by SDS-PAGE and fluorography.
Panel A Control adipocytes chased in the presence of
glucose. Panel B Glucose-deprived adipocytes chased in the
absence of glucose. Panel C Glucose-deprived cells chased
in the presence of glucose. Shown is the result of a single
experiment.














A Glucose + + + + +

45- i J '. p46
.II8 -p42
--p36


B Glucose -

45- I

4-p37


C Glucose + + + +

45 -

m .4- -p37
Chase Time, min 0 10 20 30 60








In control cells, the 36 kDa and 42 kDa GLUT1 precursors

synthesized during the pulse disappeared concomitant with the

emergence of the mature GLUT1 at 46 kDa after 20 min of chase

(Figure 4-2 A). In contrast, p37 synthesized in glucose-

deprived cells was unchanged in either molecular weight or

intensity when chased in the absence of glucose (Figure 4-2

B). Although this data indicated that p37 GLUT1 was not

processed in glucose-deprived cells, it did not rule out the

possibility that p37 could be processed if glucose were

present. This hypothesis was tested by labeling glucose-

deprived (36 hours) adipocytes for 10 min and then chasing

for 60 minutes in the presence of 25 mM glucose. It was

found that like the experiment that contained no glucose

during the chase, p37 GLUT1 remained unchanged (Figure 4-2

C). These results demonstrate that although 3T3-L1

adipocytes have the capacity to glycosylate GLUT1 in the

absence of glucose, the transporter cannot be further

processed due to either the structure of the oligosaccharide

or the location of GLUT1 along the processing pathway.


Synthesis of GLUT1 Glycoforms During Glucose Deprivation and
Glucose Refeedinq


To determine the time required for glucose deprivation

to affect GLUT1 glycosylation, normal adipocytes were placed

into glucose-free medium and labeled for 1 hour at specific

times during the next 36 hours of glucose deprivation. GLUT1

was then immunoprecipitated from solubilized membrane












S1-




0l .4 .3
.. -o
I "
























r(Oo,




+ 0


0
+ ^ N ) X



+ co 8
I o ~5



C) 0]







(0 ID
0:*M a 6*


Ssl!un








extracts and analyzed by SDS-PAGE and fluorography. This

type of experiment provides a "snapshot" of what GLUT1

glycoform(s) are being synthesized during glucose-

deprivation. Despite glucose withdrawal, the normal p46

GLUT1 glycoform was the only GLUT1 protein synthesized during

the first 12 hours of glucose deprivation (Figure 4-3 A).

Beyond 12 hours of glucose deprivation, however, the

synthesis of GLUT1 shifted to the p37 glycoform.

Densitometry of the GLUT1 bands indicated that the total

amount (addition of all bands) of GLUT1 in glucose-deprived

cells remained nearly constant (Figure 4-3 B). To examine

the reversibility of this effect, cells glucose-deprived for

36 hours were placed into medium containing 25 mM glucose and

labeled for 1 hour during various points during the

refeeding. As expected, p37 was the only form synthesized in

the glucose-deprived cells. Within 6 h of refeeding,

however, the normal p46 GLUT1 glycoform was the only GLUT1

protein synthesized (Figure 4-3 C). Furthermore, the

readdition of glucose resulted in a 3.5 fold increase in

GLUT1 (Figure 4-3 D) despite only a 40% increase in total

membrane radioactivity (data not shown). To examine this

rapid recovery in more detail, we performed the same

experiment on glucose-deprived (36 hours) cells and placed

them into glucose containing medium. Cells were then labeled

every hour for the first 6 hours of refeeding. Surprisingly,

within 1 h of glucose readdition, only the normal p46

















m0

ai

01
U W4)









0
I
0))





o (Cgo
o o'-
44



3 0 4 4
H .0 4 1-










a-4 C
,4i V








0 a

a*H 44 >



O 00
4-4
"age



0o


0 Q)


0 0 il
S0.
o 0.a
4 -I C) a)





0 -If p -P


oa) e





m 'a C


















+ .44 IL

coo
o + Co
.0
+
+ a
4&-

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glycoform was synthesized; no metabolically labeled p37 was

observed (Figure 4-4).


GLUT1 Half-Life in Control and Glucose-Deprived Adipocytes


To determine the glucose-dependent regulation of GLUT1

turnover in 3T3-L1 adipocytes, control and glucose-deprived

cells were metabolically labeled for 1 hour and then chased

in either the presence or absence of glucose. Cells were

harvested at various times during the chase period for

immunoprecipitation of GLUT1. GLUT1 immunoprecipitates were

then analyzed by SDS-PAGE and fluorography. The normal GLUT1

glycoform (p46) in control adipocytes exhibited a half-life

of approximately 14 h (Figure 4-5 A, B). When normal

adipocytes were labeled for 1 hour and then chased in the

absence of glucose, the turnover of GLUT1 was similar for

approximately the first 12 hours to that of cells chased in

the presence of glucose. Thereafter, the turnover of GLUT1

was significantly inhibited, exhibiting a turnover time of

greater than 50 hours. Likewise, the aberrant GLUT1

glycoform (p37) synthesized in glucose-deprived cells was not

significantly degraded when chased in the absence of glucose

(Figure 4-6 A, B). Additionally, a second GLUT1 glycoform,

although not present during the initial labeling period (time

= 0) appeared during the chase period. The turnover of this

GLUT1 glycoform, which exhibited a relative migration of































Figure 4-5 Turnover GLUT1 in Glucose-Deprived 3T3-L1
Adipocytes Control adipocytes were labeled with 200 pCi/ml
Tran35S-Label for 1 hour and then chased in the presence (+)
or absence (-) of glucose for a total of 48 hours. GLUT1 was
then immunoprecipitated from membrane extracts and analyzed
by SDS-PAGE and fluorography. Panel A Fluorograph of GLUT1
immunoprecipitates. Panel B Densitometry of total GLUT1
immunoprecipitates from panel A. Results shown are
representative of at least 4 independent experiments.




Full Text
This work is dedicated to my wife Laura and my daughter
Amelia.


CONCLUSIONS AND FUTURE DIRECTIONS
147
Conclusions 147
Future Directions 154
LIST OF REFERENCES 158
BIOGRAPHICAL SKETCH 165
vii


48
Glucose Deprivation and GLUT1 Protein Expression
The effect of glucose deprivation on the expression of
GLUT1 protein was analyzed by Western blotting. 3T3-L1
adipocytes were incubated in glucose-free medium for a total
of 48 hours, and a membrane fraction was prepared at the
times indicated. Membrane proteins (25 pg/lane) were then
resolved by SDS-PAGE and transferred to nitrocellulose. The
anti-GTl antiserum was then used to probe the protein blot in
order to visualize both the relative amount and molecular
weight of the GLUT1 transporter protein. The normal
glycoform of the GLUT1 transporter from 3T3-L1 adipocytes is
observed at time 0; it migrates with an average molecular
weight of approximately 46 kDa (p46) and exhibits the wide
heterogeneous glycosylation expected (Figure 3-2 A). For the
first 15 hours of glucose deprivation, both the level and the
migration of this protein is relatively unchanged. After 15
hours, however, the appearance of the second immunoreactive
protein at 37 kDa (p37) is observed. After 48 hours of
glucose-deprivation, the total level of immunoreactive
protein is increased by 2-fold. This increase is solely
attributable to the accumulation of the 37 kDa protein
(Figure 3-2 B), since the normal GLUT1 protein does not
change significantly during this period. Also in this
figure, the effect of cycloheximide, a protein synthesis
inhibitor, on the accumulation of p37 was analyzed. The
Western blot indicates that cycloheximide, even when added


CHAPTER 7
CONCLUSIONS AND FUTURE DIRECTIONS
Conclusions
The experiments described in this work analyzed the
effects of glucose deprivation, a specific type of nutrient
deprivation that induces large increases in glucose uptake,
on the biosynthesis, turnover, and targeting of the GLUT1
transporter. This work is important because the mechanism(s)
responsible for the upregulation of transport in response to
glucose deprivation is not known. A clear picture of the
action of glucose deprivation on the processing and targeting
of the GLUT1 transporter must be considered when formulating
a hypothesis to reconcile the 10-fold increase in glucose
transport activity with a 2-fold increase in total
transporter protein level and a previously undetermined
population of GLUT1 transporters at the cell surface.
In addition to the normal GLUT1 protein (p46), a second
protein recognized by anti-GLUTl peptide antiserum was
observed after 15 hours of glucose deprivation.
Cycloheximide prevented the appearance of p37 in glucose-
deprived cells, indicating that protein synthesis was
required. Several lines of evidence shown in Chapter 3
strongly suggests that p37 is a second GLUT1 protein. First,
trypsin treatment of the GLUT1 protein and the p37 protein in
147


Jp ^
Glucose +-+-+-
97
66
45


137
consistent with the regulation of GRP78 in these cells
(Kitzman et al., submitted for review). Again, although
cross contamination is evident in the plasma membrane
fraction, GKP78 is most prevalent in the high density
fraction.
The middle blot shows the same fractions probed for
GLUT4. The antiserum used for this blot was generated
against a GLUT4 carboxy-terminal peptide in the same manner
as the GLUT1 antiserum, and shows no cross reactivity towards
the GLUT1 peptide. The subfractionation of this protein is
less clear. The GLUT4 protein can be detected in significant
amounts in both the high density and low density fractions.
It is also present in the plasma membrane, but to a lower
extent.
When membrane fractions from control adipocytes were
probed for GLUT1, it was detectable in all fractions, but
most prevalent in the high density and plasma membrane
fractions (lower blot, figure 6-1). In fractions obtained
from cells deprived of glucose for 48 hours, the 46 kDa and
the 37 kDa GLUT1 glycoforms were present in all fractions.
The ratio of p46 to p37 GLUT1 present in the homogenate was
taken as a measure of the relative amount of each glycoform,
since the homogenate represents the sum of all fractions.
The ratio of p46 to p37 GLUT1 in the low density, high
density and plasma membrane fractions were unchanged (Figure
6-2). This demonstrates that there is no specific difference


131
glycosylation in targeting GLUT1 to the functional
compartment (i.e., plasma membrane) is not clear. The mature
GLUT1 transporter in 3T3-L1 adipocytes and other cell types
at the cell surface is a complex-type glycoprotein. However,
GLUT1 from the mutant cell line LEC1 CHO resides at the cell
surface even though the oligosaccharide is altered by a
glycosylation mutation (Haspel et al., 1986). Clearly, the
type of oligosaccharide present or endoglycosidase
sensitivity alone cannot be used to predict or define protein
localization. The glucose-deprivation model, coupled with
techniques designed to detect cell surface molecules, may
provide information regarding the effects of alternative
glycosylation on GLUT1 targeting. The goal of the
experiments described in this chapter is to determine if the
alternatively glycosylated GLUT1 glycoform synthesized in
glucose-deprived cells reaches the plasma membrane. This
will be done using a combination of three different
approaches; subcellular fractionation, cell surface
biotinylation, and plasma membrane fragment isolation.
Results
Subfractionation of Control and Glucose-Deprived 3T3-L1
Adipocytes
To determine the relative amount of cross contamination
of the subfractionation procedure for the 3T3-L1 adipocytes,
membrane fractions were probed for the presence of proteins


117
Glycogen Content by Cell Type
Cell Type
nmol qlucose/106 cells
3T3-L1 adipocytes, control
0.537 0.097
3T3-L1 adipocytes, glucose-deprived
0.055 0.001
CHO-K1 cells, control
0.005 0.0001
Table 5-1 Glycogen Content by Cell Type Glycogen was
extracted from cells cultured in 10 cm plates. Glycogen was
then hydrolyzed and assayed for glucose. These data,
expressed as the average standard deviation, are
representative of three independent experiments assayed in
triplicate for 3T3-L1 cells, and four independent experiments
measured in triplicate for CHO-K1 cells.


105
initial rate of 0.052 pmol glucose/106 cells/hour and a half
time of 6 hours (Figure 5-2, inset).
Glycogen Accumulation During Refeedinq
To confirm that the pathways for the synthesis and
breakdown of glycogen had not been permanently impaired by
glucose deprivation, glucose-deprived adipocytes (24 hr) were
cultured in the presence of 25 mM glucose for 24 hours and
the glycogen measured during the refeeding period. The
glycogen pool in adipocytes glucose-deprived for 24 h
exhibited a greatly decreased glycogen pool (Figure 5-3).
Unlike glycogen depletion, glycogen levels increased linearly
until normal levels were reached at approximately 24 hours of
refeeding. This observation demonstrates that glucose-
deprived cells, despite the absence of glucose, remain
competent to synthesize glycogen when given appropriate
substrate.
Effect of Fructose on Glycogen Depletion in Glucose-Deprived
Adipocytes
Fructose prevents alterations in GLUT1 glycosylation in
glucose-deprived cells (Haspel et al., 1986; Haspel et al.,
1991, Kitzman et al., 1994), demonstrating that it can
substitute for glucose and provide adequate substrate for
protein glycosylation. Given this similarity in metabolism,
we hypothesized that fructose would likewise be able to
substitute for glucose in the generation of glycogen. To


ACKNOWLEDGMENTS
I would like to acknowledge the people without whose
help and encouragement this project could not have been
completed. I would first like to acknowledge the members of
my committee, Dr. Charles Allen, Dr. Christopher West, Dr.
Michael Kilberg, and Dr. John Gander, who graciously agreed
to help partway through the completion of this thesis. A
debt of thanks goes to Dr. Susan Frost, whose ability to
simultaneously allow her students the freedom to investigate
problems in their own fashion and yet maintain their focus
constitute only two of the many admirable qualities I hope to
bring to my own lab.
I would also like to acknowledge the assistance given to
me on numerous occasions by Dr. Ron Laine and Marc Malandro.
My discussions with them have been invaluable in overcoming
obstacles that arise during any thesis. I would also like to
thank Maxine Fisher for always reminding me to cultivate
personal relationships between scientists which are essential
for a successful career.
X would also like to thank my parents, Joseph and
Florence, for the freedom to always pursue my ambitions with
their constant support. Lastly, I would like to thank my
wife Laura for her unending support and encouragement during
iii


138
in the targeting of the p46 and p37 GLUT1 glycoforms as
measured by subfractionation.
Cell Surface Biotinylation of Control and Glucose-deprived
3T3-L1 Adipocytes.
An alternative method of determining the localization of
the low molecular weight glucose transporter was undertaken
in order to confirm the results from cellular
subfractionation. The composition of biotinylated membrane
proteins was determined by biotinylating either control (fed)
or glucose-deprived (48 hours) adipocytes. This time of
glucose deprivation was chosen in order to allow for ample
accumulation of the low molecular weight transporter and
localization. A membrane extract was then prepared from each
sample and biotinylated membrane protein recovered with
streptavidin-agarose. These proteins were resolved by 7.5%
SDS-PAGE and transferred to nitrocellulose. The spectrum of
biotinylated protein was then detected by incubation with a
streptavidin-horseradish peroxidase conjugate. In cells that
were mock treated with the biotinlyation reagent, only two
bands are observed, which migrate at relative molecular
weights of 120 kDa and 68 kDa (Figure 6-3 A). These likely
represent proteins that contain an integral biotin molecule
as a cofactor subfractionating with the particulate fraction
of the homogenate. Possible candidates for these two
proteins include holocarboxylase synthetase (64 kDa),
pyruvate carboxylase (127 kDa), or the biotin containing


DO
Densitometric ratio p46/p37
o
b
o
Ul
on
iv)
b
Homog
HDM
LDM
PM
>
Intenstiy Relative to Homog
o
o
no
o
CO
o
o
cn
o
cn
o
b
b
b
b
b
b
b
136
+Glucose -Glucose


Figure 6-2 Subcellular Fractionation of GLUT1 in Normal and
Glucose-deprived 3T3-L1 adipocytes Densitometry of results
presented in Figure 6-1. Panel A Densitometry of GRP78,
GLUT4, and GLUT1 in subcellular fractions. Each lane is
expressed relative to the densitometry of the corresponding
protein in the homogenate. Panel B Ratio of densitometric
intensity of p46 to p37 bands present in each subcellular
fraction. Results shown are representative of at least two
experiments.


153
studied in the liver for its role in providing other tissues
with carbohydrate when needed, the role of glycogen in the
maintenance of the cell's own metabolism has received much
less attention. These observations point to the importance
that glycogen plays in a cell's own adaptation as a buffer in
times of glucose deprivation.
Finally, the targeting of p37 GLUT1 was analyzed
utilizing three separate techniques. The targeting of the
low molecular weight GLUT1 glycoform in glucose-deprived
cells has been determined in normal rat kidney cell by
immunofluoresence, but is unknown in the 3T3-L1 adipocytes.
Likewise, the contribution that the low molecular weight
transporter makes to the total transport activity in glucose-
deprived cells is unknown. This is an important question
because for the transporter to have physiological function,
it must be present at the cell surface. The data presented
in this work demonstrates that p37 GLUT1 can be detected at
the cell surface, and therefore, if retaining activity, can
contribute to the total transport activity. In subcellular
subfractionation experiments, the normal and low molecular
weight forms of the GLUT1 transporter were present in all
fractions, although most prevalent in the plasma membrane and
high density fraction. An important determination in these
experiments, however, is that the ratio of the glycoforms is
similar in each fraction, indicating that there was no
specific purification of p37 GLUT1 in comparison to p46 GLUT1
in any particular fraction. This strongly suggest that there


34
incubated in a boiling water bath for 15 min. The sample was
removed and cooled briefly before the addition of 3.5 ml 95%
EtOH. After mixing, the samples were heated in a boiling
water bath for 3 min and then cooled to room temp. The
precipitate was collected in a clinical centrifuge at 1300 x
g for 5 min at room temperature. The clear supernatant was
removed by aspiration and the precipitate washed in 1 ml 95%
EtOH by resuspension. The suspension was transferred to a
microcentrifuge tube and recollected at 13,300 x g for 5 min.
The supernatant was aspirated and the precipitate stored at
-20C. When all samples were collected, the precipitate was
dissolved in 0.2 ml 2N h2S04 and incubated in a boiling water
bath for 2 hours. The samples were cooled before the
addition of 0.15 ml 2N NaOH and 0.65 ml H20. The hydrolysates
were used for glucose determination as described below.
Glucose Assay
The assay chosen for the determination of glucose
released from glycogen is based upon the following reaction
mechanism. Glucose is first phosphorylated by hexokinase in
the presence of ATP. Glucose-6-phosphate is then oxidized in
the presence of NAD+ by glucose-6-phosphate dehydrogenase,
forming 6-phosphogluconate. This results in the production
of NADH in direct proportion to the initial concentration of
glucose. To perform the assay, 0.1 ml glycogen hydrolysate
was diluted with 0.4 ml H2S04 / NaOH/ H20 mixture (2/1.5/6.5).
Samples of this solution (0.1 ml) were assayed in triplicate.


this period of immense personal and professional growth,
love and involvement have enriched during this experience
beyond measure.
Her
rv


47
reduces the possibility that this antiserum will recognize
the other transporter normally expressed in the 3T3-L1
adipocytes.
The anti-GTl antiserum was then tested in a traditional
Western blot to confirm that the correct protein from 3T3-L1
adipocytes would be recognized. Equal amounts (30 pg) of
membrane protein from normal and glucose-deprived (48 hours)
cells were resolved by SDS-PAGE and transferred to
nitrocellulose. GLUT1 was then detected by Western blot.
The anti-GTl antiserum recognizes a wide band of average
molecular weight of 46 kDa, consistent with the recognition
of a similar band with other anti-GLUTl antiserum (Haspel et
al., 1985; Haspel et al., 1986; Kitzman et al., 1994) (Figure
3-1 B). It should be noted that the migration of
glycoproteins in SDS-PAGE can deviate from its true molecular
weight according to the size of the oligosaccharide and the
percentage of acrylamide in the resolving gel (Leach et al.,
1980). It is therefore assumed that, despite slightly
different migration, the protein recognized by this antiserum
and other antisera are recognizing the same protein. In
glucose-deprived cells, the lower molecular weight protein,
which migrates at 37 kDa (p37), is also recognized by the
anti-GTl antiserum. The pre-immune serum (PIS) does not
detect either of these bands and shows no other cross
reactivity to other membrane proteins from 3T3-L1 adipocytes.


Figure 4-3 Time Course biosynthesis p37 GLUT1 Panel A Control cells were placed into
glucose-free medium and labeled for 1 hour with 200 pCi/ml Tran35S-Label at the times
indicated. GLUT1 was immunoprecipitated from membrane extracts and analyzed by SDS-PAGE and
fluorography. Panel B Densitometry of total GLUT1 during glucose deprivation. Panel C
Glucose-deprived (36 hours) cells were placed into medium containing glucose and labeled for
1 hour with 200 pCi/ml Tran35S-Label at times indicated. GLUT1 was then analyzed as in A.
Panel D Densitometry of total GLUT1 during glucose refeeding. These results are
representative of at least three independent experiments.


7
an asparagine residue located between the first and second
membrane spanning region (Carruthers, 1990). Although the
complete oligosaccharide seguence is not known, some
characteristics have been elucidated. All members of the
glucose transporter family can be digested with N-glycanase F
or endoglycosidase F to release the oligosaccharide,
indicating N-linked glycosylation (Haspel et al., 1986;
Kitzman et al., 1994). The oligosaccharide is usually a
large structure that accounts for approximately 10 kDa of
migration by SDS-PAGE, or 20% of its total relative molecular
weight. Additionally, GLUT proteins migrate as diffuse bands
when analyzed by SDS-PAGE, but whether this heterogeneity
results from differences in the oligosaccharide structure
(known as microheterogeneity), or from different charges
associated with the terminal sugar chains, is not known.
Regulation of Glucose Transport
The regulation of glucose transport has received much
attention due to the central role the transporter plays in
the utilization of glucose. Glucose transport is regulated by
many factors, including hormonal influence and cellular
stress. The influence of hormones on the transport process
has been extensively reviewed elsewhere (Czech et al., 1992;
Czech, 1995) and will not be discussed in this work. Rather,
X will highlight the role of cellular stress, particularly
nutrient deprivation, on the regulation of glucose
transporter expression.


129
There has also been an intense investigation
specifically into the role of glycosylation in the process of
organic solute transport. As early as 1979, Olden et al.
found that the treatment of chick embryo fibroblasts (CEF)
with the N-linked glycosylation inhibitor tunicamycin had a
drastic effect on several transport processes, including
glucose transport, uridine transport, and amino acid
transport. However, tunicamycin treatment had no effect on
other processes that occur at the plasma membrane, such as
protein secretion or adenylate cyclase activity. These
results indicate an important role of oligosaccharides in
certain transport systems.
A structural and functional analysis of the
oligosaccharide on the glycine transporter has recently been
reported (Nunez and Aragon, 1994). The glycine transporter
expressed in the brain was found to contain at least one and
possibly two N-linked oligosaccharides which could be
released with N-Glycanase F treatment. Reconstitution of the
solubilized transporter that was treated with
endoglycosidases demonstrated that although sialidase
treatment had no effect on the reconstituted activity, N-
Glycanase F treatment severely inhibited transport. These
observations point to the importance of oligosaccharides in
the transport of at least one organic solute.
Not all transporters, however, are affected by changes
in glycosylation status as severely as the glycine
transporter. The cystic fibrosis conductance regulator


141
subunits of propionyl CoA carboxylase and methycrotonyl CoA
carboxylase.
In cells modified with the membrane impermeant
biotinylation reagent, a wide spectrum of proteins was
observed. These differences represent proteins present at
the cell surface in one state and not in the other. Proteins
could be brought to, or removed from, the cell surface by
several means; translocation of the protein from an
intracellular location to the cell surface, new synthesis of
plasma membrane proteins that are specific to either the fed
or deprived state, or endocytosis and trafficking to the
lysosomal compartment.
Cell surface biotinylation of either fed or glucose-
deprived cells was again carried out and biotinylated
proteins recovered by capture with streptavidin agarose. The
biotinylated proteins were then resolved by SDS-PAGE and
transferred to nitrocellulose. The presence of GLUT1
glycoforms was then determined by performing a traditional
Western blot on the pool of biotinylated proteins. The
normal glycoform of the GLUT1 transporter, migrating at 46
kDa, is present at the cell surface in the fed state, as
expected (Figure 6-3 B). In cells that had been glucose-
deprived for 48 hours prior to cell surface biotinylation,
both GLUT1 glycoforms, the 46 kDa as well as the low
molecular weight glycoform, 37 kDa, were observed. These
data clearly indicate that the low molecular weight GLUT1
glycoform synthesized in glucose-deprived 3T3-L1 adipocytes


Figure 3-1 Characterization of GLUT1 Antiserum Panel A Various amounts of the GT1 or GT4
peptides were blotted onto dry nitocellulose and detected by Western blot with the anti-GTl
antiserum. Panel B Membrane proteins (30 pg) from fed or glucose-deprived 3T3-L1
adipocytes (48 hours) were probed by Western blot with the anti-GTl antiserum. Molecular
weight standards, along with the positions of p46 and p37, are indicated.


38
of ligands or enzymes, it can be conjugated to many different
types of compounds and so is quite versatile in its uses, and
finally, it can be readily detected by binding of avidin or
streptavidin. The binding between avidin and biotin is the
tightest binding described, with a dissociation constant of
approximately 10"15. Avidin and streptavidin can be detected
by several methods; one method utilizes radioactively labeled
avidin or streptavidin, a second utilizes fluorescently
labeled avidin or streptavidin that can be visualized by
illuminating with fluorescent light, a third method utilizes
enzyme-avidin or streptavidin conjugates which can be
detected by incubation with the proper substrate which can
lead to the production of color or light.
In this work, a membrane impermeant biotin probe which
exhibits reactivity towards free amine groups was utilized.
Once the cell surface was derivitized with the biotin probe,
the membrane fraction was solubilized and biotinylated
proteins recovered with an immobilized form of streptavidin.
Streptavidin was chosen over avidin because of avidin's
extremely high isoelectric point (>10) which can lead to non
specific interactions. The isoelectric point of streptavidin
is approximately 5, and exhibits much lower non-specific
reactivity in immunoaffinity assays. Once the biotinylated
proteins are recovered, unbiotinylated proteins are removed
and the specifically bound proteins analyzed by SDS-PAGE and
Western blotting.


5
Plasma
Membrane
Figure 1-1 Predicted Secondary Structure of GLUT! The
predicted topology of the GLUT1 transporter, based on
hydropathy values (Mueckler et al., 1985). The amino- and
carboxy-terminal ends of the transporter are designated by
[N] and [C], respectively. The intracellular- and
extracellular-facing sides of the transporter are designated
by [IN] and [OUT], respectively. The boxes represent
membrane spanning regions and the branched chain represents
oligosaccharide attached to the extracellular loop between
membrane spanning regions 1 and 2.


74
that accumulates during glucose deprivation. In this case,
the transporter in glucose-deprived adipocytes would be
processed in the same manner as in normal cells, but due to
the inactivation of a processing enzyme or the depletion of a
critical precursor, a premature GLUT1 glycoform becomes the
terminal glycoform. This hypothesis assumes that the core
oligosaccharide produced in glucose-deprived cells is the
same as in normal cells. Alternatively, the low molecular
weight GLUTl glycoform could arise from the glycosylation of
the core protein with a different oligosaccharide that is
produced only in the absence of glucose. In this scenario,
the oligosaccharide is aberrant before being added to the
protein, and further processing is not observed because the
aberrant oligosaccharide is not suitable substrate for the
processing enzymes found in the golgi apparatus. A third,
less likely, possibility is that the low molecular weight
GLUTl transporter is a degradation product, that arises in
glucose-deprived cells due to the action of proteases on the
normal transporter, reducing its apparent molecular weight to
37 kDa. The latter is not likely due to the identical
molecular weight that the transporter exhibits either when
incubated in the presence of tunicamycin, when treated with
N-Glycanase F to remove the oligosaccharide, or compared to
the expected molecular weight of the core transporter protein
given by amino acid sequence. To address these issues,
existing methods of GLUTl immunoprecipitation were modified
to improve recovery and reduce non-specific interactions.


93
mature form of GLUT1 that migrated as a broad band at
approximately 46 kDa was identified. Two precursor forms
were distinguished from the mature GLUT1. These represent
the core protein (36 kDa) and an intermediate (42 kDa) in the
processing pathway consistent with analysis of GLUT1
processing in a cell-free system (Mueckler and Lodish, 1985).
When metabolically labeled GLUT1 was immunoprecipitated from
glucose-deprived cells, a single 37 kDa protein was observed.
It should be noted that not all cells appear competent to
glycosylate GLUT1 in the absence of glucose. The lower
molecular weight GLUT1 observed in glucose-deprived rat
kidney cells, for example, migrates at the same molecular
weight as GLUT1 from these cells incubated with tunicamycin
or GLUTl transporter isolated from these cells treated with
endoglycosidase (Haspel, et al., 1991). Glycosylation
therefore represents another cell type-specific aspect of the
regulation of GLUTl expression by glucose.
The GLUTl glycoform in glucose-deprived 3T3-L1
adipocytes is interesting from several perspectives. First,
it is intriguing that over 12 hours are required for its
appearance. This indicates that sufficient sugar is
available to support normal glycosylation for this period of
glucose deprivation. Yet, it is unlikely that any "free"
sugar would be available to support core oligosaccharide
biosynthesis due to the high rate of glucose utilization.
However, glycogen breakdown may provide sufficient substrate
for this process during the early phase of deprivation (see


Samples, as well as the assay buffer, were pre-warmed to
37C. Assay reagent (1.0 ml) was mixed with 0.1 ml sample
and incubated at 37C for 30 min. The absorbance of the
samples was then measured at 340 nm.
Subfractionation of 3T3-L1 Adipocytes
Subfractionation is a procedure for separating and
purifying the different compartments of the cell. This
usually involves breaking the cell open while attempting to
maintain the integrity of the intracellular organelles. When
these individual organelles are then subjected to centrifugal
force, these organelles separate from each other on the basis
of both their size and density. In this manner, the
localization of proteins can be determined. One disadvantage
of this technique, is that the difference in density and size
between different cell organelles is relatively small. This
allows incomplete separation of these organelles resulting in
cross-contamination; that is, that a part of the golgi
fraction may be present in the plasma membrane, and so forth.
The following section describes a protocol used to separate
fractions enriched in plasma membrane, high density
microsomes, and low density microsomes. The high density
membrane fraction is usually enriched in proteins contained
in the endoplasmic reticulum and the golgi apparatus. The
low density membrane fraction is enriched in proteins
characteristic of endosomes, lysosomes, and the trans-golgi
network. Although all subfractionation procedures exhibit


104
A
E
c
o
3
CO
-4
CO
0)
O
cz
CO
.a
o
C/5
.Q
<
0 10 20 30
B
Incubation time, min
Figure 5-1 Time Course and Concentration Curve of Glucose
Assay Panel A A glucose solution (20 pg/ml) was incubated
with assay reagent for a total of 30 min at 37C. Samples
were removed at the indicated times and the absorbance
measured at 340 nm. Panel B Various concentrations of
glucose, as indicated, were incubated with assay reagent for
30 min at 37C. The absorbance of the solutions was then
measured at 340 nm and plotted as a function of the amount of
glucose in the sample.


69
\
endoglycosidase H insensitivity and size of the
oligosaccharide on p37 are consistent with this type of post-
translational modification. The biosynthesis and processing
of the transporter in the absence of glucose will be
addressed in more detail in Chapter 4.
Together, these data provide evidence that p37 is an
alternatively glycosylated form of GLUT1. The protein
component is likely unaltered in the absence of glucose.
Furthermore, the difference in relative molecular weight
between p46 GLUT1 and p37 GLUT1 resides in a difference in
the size of the oligosaccharide. p37 GLUT1 is glycosylated
in glucose-deprived adipocytes, although with an
oligosaccharide drastically reduced in size. This
oligosaccharide is likely not a processing intermediate
prematurely terminated in the absence of glucose, due to the
observation that the small oligosaccharide is approximately
the same size as a high mannose core but is endoglycosidase H
insensitive. The next chapter will describe experiments
designed to elucidate biosynthesis, processing, and turnover
of these GLUT1 glycoforms in normal and glucose-deprived 3T3-
L1 adipocytes.


92
approximately 44 kDa, was identical to that of the p37 GLUT1.
When glucose-deprived adipocytes were labeled for 1 hour and
then chased in medium containing glucose, the turnover of
GLUT1 (both p44 and p37 glycoforms) returned so that it was
similar to that of p46 in control adipocytes.
Conclusions
In this report, we used immunoprecipitation to analyze
the effect of glucose deprivation on the biosynthesis,
processing, and turnover of GLUT1 in 3T3-L1 adipocytes.
Although several studies have previously reported the
appearance of a low molecular weight form of GLUT1 resulting
from the withdrawal of glucose, the relationship between this
form and the normal molecular weight species has not been
investigated. Thus this data has extended these prior
studies in several ways. First, these experiments
demonstrate that p37 GLUT1 is a unique, newly synthesized
protein generated only in the absence of glucose. Second,
the data shows that p37 is not a precursor to the normal form
of GLUT1 (p46), eliminating the possibility that p37 GLUT1 is
a normal processing intermediate prematurely terminated by
the absence of glucose. Finally, the data establishes that
in glucose-deprived 3T3-L1 adipocytes, p37 GLUT1 is post-
translationally processed by a pathway unique from that of
p46 GLUT1 resulting from the attachment of an abbreviated and
likely aberrant oligosaccharide. These conclusions are based
on the following observations. In control cells, a single


TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
LIST OF FIGURES viii
ABSTRACT X
OVERVIEW 1
GLUT Proteins and Glucose Transport 1
GLUT Protein Family 1
Glucose Transporter Structure 4
Regulation of Glucose Transport 7
Regulation of Glucose Transport Activity by Glucose
Deprivation 8
Regulation of Glucose Transporter Protein 8
3T3-L1 Adipocytes 10
MATERIALS AND METHODS 16
Materials 16
Methods 17
Cell Culture 17
Antibody Production 18
Peptide Purification of Anti-GTl Antibody 21
Membrane Fraction Isolation 23
Markwell Assay for Protein Determination 26
Gel Electrophoresis 27
Electrotransfer and Western Blotting 27
Metabolic Radiolabeling of 3T3-L1 Adipocytes 28
GLUT1 Immunoprecipitation 30
Endoglycosidase Digestions 32
Glycogen Isolation 33
Glucose Assay 34
Subfractionation of 3T3-L1 Adipocytes 35
Cell Surface Biotinylation 37
Plasma Membrane Fragment Isolation 40
CHARACTERIZATION OF GLUT1 GLYCOFORMS 43
Introduction 43
Results 44
Characterization of Anti-GTl Antiserum 44
Glucose Deprivation and GLUT1 Protein Expression 48
Tryptic Digest of GLUT1 Transporter 51
Effect of Tunicamycin Treatment on GLUT1 Protein 54
Glycosidase Digestion of GLUT1 Glycoforms 60
v


108
Incubation time, hours
Figure 5-3 Glycogen Content of Glucose-Deprived 3T3-L1
Adipocytes During Refeeding Control or glucose-deprived (24
hours) cells were placed into glucose-containing medium for a
total of 24 hours. Glycogen was extracted at the times
indicated and assayed. This result, expressed as the average
the standard deviation, is a single experiment
representative of at least three independent experiments
measured in triplicate.


6
1984; Cairns et al., 1987; Davies et al., 1987). Utilization
of peptide-specific antibodies directed against amino or
carboxyl terminal ends of GLUT1, in conjunction with tryptic
digestion, allowed the analysis of the predicted
intracellular and extracellular faces of the transporter,
using an antiserum that recognized GLUT1 in its native form
and a preparation of erythrocyte membranes of known
orientation, it was determined that the carboxy terminus
resided intracellularly because the transporter was
recognized only in vesicles which were predominantly "inside
out". Furthermore, tryptic digests of the transporter
resulted in two large fragments of only slightly different
molecular weights, supporting the hypothesis that the major
cleavage site in the protein is near the middle of the large
intracellular loop between membrane spanning regions 6 and 7.
Antiserum specific for the amino terminus of the transporter
recognized a large diffuse band, suggesting that this
fragment contained the oligosaccharide, confirming that the
putative N-linked glycosylation site in the first
extracellular domain was utilized. Labeling the erythrocyte
transporter with the competitive ligand cytochalasin B before
proteolysis demonstrated that the binding site for this
ligand resided in the carboxy terminal half of the protein
(Holman and Rees, 1987).
Apart from the amino acid sequence, a second aspect that
deserves review is the glycosylation of the glucose
transporters. All glucose transporters are glycosylated at


Figure 5-6 Glycogen Depletion and GLUT1 Glvcosvlation Cells were pre-incubated in either
25 mM glucose or 25 mM fructose for 24 hours before complete sugar withdrawal for 36 hours.
At the times indicated, a total membrane fraction was collected for GLUT1 analysis by
Western blotting. Equal protein (50 pg/lane) was resolved by 7.5% SDS-PAGE. Western blot
of GLUT1 from glucose deprived cells pre-incubated in the presence of glucose or fructose
probed with an anti-GLUTl peptide antibody. Shown is a single result representative of at
least four independent experiments.


110
synthesis. Adipocytes were incubated in the presence or
absence of glucose, fructose, or insulin (or a combination of
these) for 24 hours before glycogen was determined.
Incubation with fructose in the absence of glucose resulted
in the depletion of the glycogen pool, as expected (Figure 5-
5). Insulin treatment, in conjunction with fructose,
provided some protection from total glycogen depletion.
Whether this effect stems from partial inhibition of glycogen
phosphorylase or partial enhancement of glycogen synthesis is
not known. This contrasts the effect of insulin on normal
cells, where the level of glycogen is increased approximately
2-fold in comparison to control cells. Insulin had no effect
on the level of glycogen in glucose-deprived cells that were
not provided fructose.
To determine whether partial inhibition of phosphorylase
a by addition of an allosteric inhibitor like glucose, could
prevent glycogen depletion, cells were incubated with a
combination of fructose and 3-0-methylglucose (20 mM and 5
mM, respectively) for 24 hours before glycogen determination.
3-O-methylglucose is a glucose analog that cannot be
metabolized once it enters the cell. 3-O-methylglucose
mimics glucose, but cannot provide substrate for glycogen
biosynthesis. 3-O-methylglucose had no effect by itself in
either glucose-deprived or normal (glucose-fed) adipocytes
(Figure 5-5 B). Likewise, no significant difference in the
glycogen content of cells incubated in a combination of 3-O-
methylglucose and fructose was observed in comparison to


96
all of the required characteristics, confirmation of this
hypothesis would require purification of the transporter in
significant quantities and subsequent carbohydrate
sequencing.
Finally two previous reports attempting to measure the
half-life of GLUT1 by direct methods have proven
contradictory. Haspel et al., (1985) observed a half-life of
90 min for GLUT1 in 3T3-L1 cells. In contrast, Sargeant and
Paquet (1991) studied the turnover of GLUT1 in these cells
and observed a half-life of approximately 19 hours. Our data
showing a half-life of 14 hours is more consistent with the
latter. In neither of these earlier papers was the effect of
glucose deprivation measured. Thus our study describes an
aspect of GLUT1 regulation not previously identified. Even
though glucose deprivation had no specific effect on the rate
of GLUT1 synthesis (despite the difference in glycosylation),
the effect on GLUT1 turnover was striking. Clearly, the
degradation of p37 was significantly inhibited.
Interestingly, so was the turnover of the total pool of
membrane protein. P46 turnover is affected only after 12h of
glucose deprivation concurrent with the onset of abnormal or
deficient glycosylation as indicated by the appearance of
p37. Thus, the loss of glucose itself is not the signal for
the inhibition of turnover. Rather, the depletion of a
specific glycoprotein, required for degradation, may impair
turnover. This is supported by that fact that glucose


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145
in fed cells, and the normal and p37 GLUT1 protein was
observed in glucose-deprived cells (Figure 6-4). Likewise,
GRP78 was present in both total membrane fractions, but GRP78
was upregulated in glucose-deprived cells. In the plasma
membrane fragments, the normal GLUT1 protein is observed in
fed cells, as expected, in glucose-deprived cells, both
GLUT1 proteins are observed in the plasma membrane fragments,
in agreement with the subfractionation and cell surface
biotinylation experiments. The relative level of cross
contamination of the endoplasmic reticulum in this experiment
is concluded to be insignificant, due to the inability to
detect GRP78 in the plasma membrane fragments. These results
clearly support the conclusions from the other techniques
presented here that the targeting of the alternatively-
glycosylated GLUT1 transporter is not affected either by the
absence of glucose or by the altered state of glycosylation.
Conclusions
The data presented here indicate that the glucose
transporter GLUT1 belongs to the class of glycoproteins whose
targeting is not affected by changes in glycosylation or
glucose deprivation. This characteristic sets it apart from
some other membrane proteins, such as the viral coat proteins
of the Sindbis virus and Stomatitis viruses, whose activity
and targeting is severely affected by any changes in
glycosylation and/or glucose deprivation. The results
presented here do contrast, however, with previous


122
oligosaccharide. During our study of the alternative
oligosaccharide on p37, it became apparent that the
oligosaccharide generated in glucose-deprived CHO cells
exhibited many of the same characteristics. One major
difference in this response, however, was the kinetics at
which this process took place. Our observations in 3T3-L1
adipocytes suggested that the alternative oligosaccharide was
not present on GLUT1 until after 12 hours following the
withdrawal of glucose while only three hours of glucose
deprivation were required for the same effect in CHO cells.
The concentration of glucose inside cells is thought to be
extremely low; we therefore hypothesized that the 3T3-L1
adipocyte used some other carbohydrate source to delay the
generation of the alternative oligosaccharide. The breakdown
of glycogen could represent such a source, but the glycogen
content in the 3T3-L1 adipocyte and its response to glucose
deprivation had not been previously measured.
Rather than measure glycogen directly, it was
precipitated and hydrolyzed to free glucose, which could then
be measured by an NAD-linked assay. This procedure indicates
that 3T3-L1 cells contain approximately 0.5 pmol glucose/106
adipocytes. Following the withdrawal of glucose, the
glycogen pool decreased in a first order, time-dependent
manner with an initial rate of 0.052 pmol/106 adipocytes/hr
and a half time of 6 hours. Glucose-l-phosphate released by
the breakdown of glycogen is likely used to provide
glycolytic energy and lactic acid, since adipocytes are not


128
Stomatitis Virus and the Sindbis Virus expressed in
tunicamycin treated cells are synthesized, but are devoid of
any carbohydrate (Leavitt et al., 1977). The unglycosylated
proteins could not be detected in the plasma membrane, while
their glycosylated counterparts could easily be detected at
the cell surface. Additionally, the solubility of these
proteins was drastically affected; while glycosylated viral
proteins could be solubilized in the non-ionic detergent
Triton X-100, their unglycosylated counterparts could only be
solubilized with a combination of a strong denaturant, 6M
urea, and Triton X-100. Dialysis of the urea from these
samples resulted in the immediate reprecipitation of the
viral proteins.
The role of oligosaccharides in the proper targeting of
glycoproteins is not yet clear. Truncation of
oligosaccharides, such as occurs in the cases of glucose
deprivation and in the mutation of certain cell lines, can
also drastically affect the localization of a glycoprotein.
When hemagglutinin is expressed in a CHO mutant cell line
that synthesizes GlcNAc2Man5 instead of the normal
oligosaccharide core of GlcNAc2Man9Glc3, the cell surface
expression becomes temperature sensitive (Hearing et al.,
1989). The cause of this temperature sensitivity was that
the stability of conformations and trimerization of the
subunits were adversely affected by the altered
glycosylation.


103
and plotted as a function of time. The reaction, as assessed
by maximal NADH concentration, was complete within 30 minutes
(Figure 5-1 A). For the remainder of the experiments
described in this chapter, the reaction was incubated for 30
minutes before measuring the absorbance.
The linearity of the assay was assessed by performing
the assay on a range of glucose concentrations. The
absorbance of the solutions were then plotted as a function
of the concentration of the solution. This experiment
indicates that the glucose assay is linear over a wide range
of glucose concentrations, and is linear up to an absorbance
of 2.0 (Figure 5-1 B).
Effect of Glucose-Deprivation on Glycogen Content in 3T3-L1
Adipocytes
To determine whether glucose deprivation affects the
level of glycogen in adipocytes, glycogen was isolated from a
10 cm dish of adipocytes that was either fed or glucose-
deprived over a 24 hour period. Glycogen was then measured
by the NAD-linked glucose assay. Normal 3T3-L1 adipocytes
routinely contained 0.4 0.6 pmol glucose/106 cells.
Although glycogen granules have been observed in 3T3-L1
adipocytes, this is to my knowledge the first quantitative
measurement of glycogen content. Upon the withdrawal of
glucose, the amount of glycogen decreased in a first order,
time-dependent fashion (Figure 5-2). This process had an


94
chapter 5). Only when the glycogen pool is depleted would
p37 accumulate. Yet, in the face of extended glucose
deprivation with no apparent source of glucose for
oligosaccharide synthesis, GLUT1 (p37) is still glycosylated,
albeit in abbreviated form. It is possible that carbohydrate
is scavenged from the degradation of other glycoproteins
whose function is not needed under these conditions.
Alternatively, the lipid-linked oligosaccharide pool may not
be completely depleted between 12 and 36 hours of deprivation
and thus provides continuous core oligosaccharide.
The oligosaccharide structure on p37 form is currently
unknown, although it is evidently not the same as that on
GLUT1 from LEC1 cells based on endoglycosidase H sensitivity.
However, previous studies provide some clues as to the type
of oligosaccharide that might be generated in the absence of
glucose (Rearick et al., 1981). In CHO cells deprived of
glucose, the synthesis of lipid-linked oligosaccharide shifts
from a normal Glc3Man9GlcNAc2 to an alternative structure,
GlC3Man5GlcNAc2. The transfer of this oligosaccharide to
protein acceptors occurred normally, although the resulting
glycoproteins were endoglycosidase H insensitive.
The cause of this shift in oligosaccharide biosynthesis
in glucose-deprived CHO cells has been investigated. Chapman
and Calhoun (1988) determined that the activities of the
enzymes required for the biosynthesis of GlcNAc2Man5 through
GlcNAc2Man9Glc3 were present and were unchanged in glucose-
deprived cells. Furthermore, in vitro assays of cell


159
Chapman, A.E., and Calhoun, J.C. (1988) Arch. Biochem.
Biophvs. 260, 320-333.
Chin, J.J., Jucg, E.K., Jung, C.Y. (1986) J. Biol. Chem. 261
7101-7104.
Clancy, B.M., and Czech, M.P. (1990) J. Biol. Chem. 265,
12434-12443.
Coleman, R.A., Reed, B.C., Mackall, J.C., Student, A.K.,
Lane, M.D., Bell, R.M. (1978) J. Biol. Chem. 253. 7256-
7261.
Czech, M.P. (1995) Ann. Rev. Nutr 15. 441-71.
Czech, M.P., Clancy, B., Pessino, A., Woon, C-W., Harrison,
S.A. (1992) TIBS 17. 197-201.
Dankert, J.R., Shiver, J.W., Esser, A.F. (1985) Biochemistry
24, 2754.
Davies, A., Marren, K., Cairns, M.T., Baldwin, S.A., (1987)
J. Biol. Chem. 262, 3502-3509.
Froesch, E.R., and Ginsberg, J.L. (1962) J. Biol. Chem. 237.
3317-3324.
Frost, S.C. and Lane, M.D. (1985) J. Biol. Chem. 260. 2546-
2652.
Fuegeas, J-P., Neel, D., Pavia, A.A., Laham, A., Goussault,
Y., and Derappe, C. (1990) Biochm. Biophvs. Acta 1030.
60-64.
Fuegeas, J.P., Neel, D., Goussault, Y., Derappe, C. (1991)
Biochim. Biophvs. Acta 1066. 59-62.
Fung, K.P., Choy, Y.M., Chan, T.W., Lam, W.P., and Lee, C.Y.
(1986) Biochem. Biophvs. Res. Commun. 134. 1231-1237.
Germinarlo, R.J., Rockman, H., Oliveira, M., Manuel, S.,
Taylor, M. (1982) J. Cell. Phvs. 112. 367-372.
Gould, G.W. and Bell, G.I. (1990) TIBS 15, 17-23.
Green, H., and Kehinde, O. (1974) Cell 1. 113-116.
Green, H., and Meuth, M. (1974) Cell 3. 127-133.
Hare, J.F., Taylor, K. (1991) Proc. Natl. Acad. Sci. U.S.A.
88, 5902-5906.


40
briefly before a streptavidin-horseradish peroxidase
(streptavidin-HRP) conjugate was added in 0.5% NFDM/TBS-T at
a final concentration of 2 x 105 mg/ml for 1 hour at room
temperature. The blot was then extensively washed at room
temperature with TBS-T to remove the unbound and non-
specifically bound streptavidin-HRP. The blot was then
incubated in the ECL Enhanced Chemiluminescent Western
blotting detection reagents according to the manufacturer's
instructions. The developed blot was then exposed to film
for a period of 10 sec 10 min.
Plasma Membrane Fragment Isolation
Another technique which has recently become popular for
the analysis of processes occurring at the plasma membrane is
the isolation of plasma membrane fragments. In this
technique, cells that are plated onto glass coverslips are
adhered to the surface by incubating the cells in poly-L-
lysine of high molecular weight (<100,000 M.W.). The
mechanism of adherence by poly-L-lysine is not exactly known,
but it is thought that the highly positive nature of the
poly-L-lysine polymer acts to bind the negatively charged
plasma membrane and the negatively charged glass surface in
an ionic interaction. Gentle sonication is then used to
remove parts of the cell that are not adhered to the surface,
and results in large fragments of the plasma membrane left on
the glass coverslip. This procedure has been used
extensively for the characterization of the assembly of


}
CHAPTER 4
REGULATION OF GLUT1 BIOSYNTHESIS, PROCESSING AND TURNOVER BY
GLUCOSE IN 3T3-L1 ADIPOCYTES
Introduction
The biosynthesis of integral membrane glycoproteins
involves a complex journey through multiple membrane
compartments containing specifically localized enzymes.
During the translation of polypeptides through the
endoplasmic reticulum, a preformed oligosaccharide is
transferred by the enzyme oligosaccharyltransferase to
asparagine residues on the protein when they occur in the
recognition seguence Asn-X-Ser/Thr (Voet and Voet, 1990).
The transfer of oligosaccharides is regulated by a variety of
factors, including cell type, conformation of the protein
during translation, nutritional status, and distance from the
recognition site to the inside face of the endoplasmic
reticulum (Kornfeld and Kornfeld, 1980).
The pre-assembled oligosaccharide core is synthesized on
both the cytsolic and lumenal faces of the endoplasmic
reticulum. Sugar residues are added sequentially to a lipid
carrier molecule, dolichol pyrophosphate, generating a
growing oligosaccharide core. The first seven sugars, two N-
acetylglucosamine residues and five mannose residues, are
donated from their respective GDP nucleotide derivatives
70


13
during glucose deprivation (Kitzman et al., 1994). Up-
regulation of glucose transport by glucose deprivation was
concentration-dependent, with a transport activation
threshold of approximately 0.5 mM. Additionally, transport
activation was completely reversible upon the re-addition of
glucose, indicating that down-regulation of transport was
also glucose-dependent (Kitzman et al., 1994). The
dependence of transport activation upon glucose or one of the
metabolites of glucose was tested by analyzing the up-
regulation of transport in the presence of several other
sugars; some of which are not transported by glucose
transporters, some of which are only ligands of the
transporter, and some which are transported but not
metabolized (Haspel et al., 1990; Kitzman et al., 1994).
Incubation of glucose-deprived 3T3-L1 adipocytes with
fructose, galactose, or maltose, none of which are
internalized by the glucose transporter, although maltose (a
disaccharide of glucose) is bound by the transporter, had no
effect on the up-regulation of transport activity. 3-0-
methylglucose, which is transported by the glucose
transporter but not metabolized, and 2-deoxyglucose, which is
only phosphorylated, completely abolish transport activation
(Kitzman et al., 1994). These observations suggest that
neither the binding of the transporter nor the metabolism of
glucose is required for prevention of transport activation;
rather, they suggest that a molecule that is either glucose


IS
determination of the glycosylation state of p37 and the
sensitivity of its appearance to cycloheximide, little else
is known about the regulation of the glucose transporter
protein by glucose. It should be noted that there is one
report where glucose deprivation resulted in an increase in
the level of the normal transporter protein, but the
discrepancy between this and other reports may lie in the
fact that equal numbers of cell equivalents were not analyzed
(Tordjman et al., 1990). The hypothesis that p37 is a second
GLUT1 protein synthesized in glucose-deprived 3T3-L1
adipocytes evokes several important questions regarding its
genesis, processing, turnover, and targeting. The goal of
this work is to describe how the expression of GLUT1 is
altered in the absence of glucose. This includes how the
transporter is processed to its final form, how it is
degraded, and to determine whether its altered glycosylation
state affects its targeting to the plasma membrane.


Figure 3-5 GLUT1 Expression Following Tunicamvcin Washout
Normal or glucose-deprived (24 hours) 3T3-L1 adipocytes were
incubated in the presence of tunicamycin (1 pg/ml) for 24
hours. The tunicamycin-containing medium was then removed
and replaced with either medium containing tunicamycin (1
pg/ml), normal medium, or glucose-free medium containing 25
mM N-acetylglucosamine, for 24 hours. Membrane proteins were
then probed for GLUT1 by Western blot with anti-GTl
antiserum.


157
The result would be hydrolases, which are required for
protein degradation, that are present but inactivated because
of reduced activity at elevated pH. A third possibility is
that the internalization of plasma membrane protein is
inhibited so that newly synthesized membrane glycoproteins
are targeted to the plasma membrane but are not degraded
because they are not trafficked to the lysosomal compartment.
A closer examination of these questions could include the
determination of amino acids transport rates to determine if
other transport processes are affected by glucose
deprivation, and examinations of lysosomal hydrolase
activities.
In summary, several important facets of glycoprotein
expression which require glucose for their proper expression
were elaborated in this work. A further analysis of the
above questions will enhance the understanding of the role
that carbohydrates play in important cellular functions aside
from the generation of metabolic energy.


80
A
Glucose + + + + +
^-p46
*-p42
*-p36
Chase Time, min o 10 20 30 60


78
protein. Lighter exposures provided no further
discrimination between p37 and any other GLUT1 protein. When
glucose-deprived adipocytes were labeled for 20 min or more,
no additional GLUT1 species were observed. The relative
synthetic rate of GLUT1 in normal and glucose-deprived
adipocytes was measured by adding the densitometric intensity
of all GLUT1 bands at each time point. Although the
intensity of total GLUTl protein in glucose-deprived cells
was greater than that of control cells at every time point
(Figure 4-1 B), this effect was not specific to GLUTl when
taking into account an equal increase in radioactivity of the
membrane fraction from glucose-deprived cells (Figure 4-1 B,
inset). This phenomenon, therefore, could represent an
increase in the specific activity of the methionine and
cysteine pools in glucose-deprived cells. Taken together,
these data demonstrate that the synthetic rate of GLUTl was
unaffected by glucose availability despite the differences in
glycosylation.
Regulation of GLUTl Processing by Glucose
To examine the processing pathway of GLUTl in control
and glucose-deprived adipocytes, normal or glucose-deprived
adipocytes were metabolically labeled for 10 min and then
chased for a total of 60 min. Cells were harvested for
immunoprecipitation during the indicated times during the
chase period. GLUTl was then immunoprecipitated from these
membrane extracts and analyzed by SDS-PAGE and fluorography.


152
that the mechanisms are responsible for the inhibition of
membrane protein turnover and the alteration of protein
glycosylation are not activated until this lag period
elapses. This work demonstrates that the source of
carbohydrate utilized to sustain these processes following
the withdrawal of glucose is glycogen. The glycogen stores
in 3T3-L1 adipocytes are depleted in a time and concentration
dependent manner in response to glucose deprivation. This is
an effect which fructose, which can be converted into almost
all the metabolites of glucose, is unable to prevent.
Likewise, insulin, an activator of glycogen biosynthesis, is
unable to prevent the depletion of glycogen in the absence of
glucose. These two observations suggest that carbohydrate
present in the cell, even if it derives from fructose, is
utilized for pathways other than the generation of glycogen.
Indeed, the observation that fructose can sustain the normal
glycosylation of GLUT1 in the absence of glucose supports
this hypothesis. A direct link between glycogen and protein
glycosylation was obtained through the analysis of glycogen-
depleted, but glycosylation-competent adipocytes generated by
a 24 hour incubation with fructose, in these cells, glucose
deprivation results in alterations in GLUT1 glycosylation
more rapidly than cell which have normal glycogen levels.
Furthermore, the CHO cell line, a cell line which normally
stores very little glycogen, can sustain normal protein
glycosylation in the face of glucose deprivation for less
than 3 hours. Although glycogen turnover has been intensely


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 GLUTl GLUCOSE TRANSPORTER EXPRESSION BY GLUCOSE
IN 3T3-L1 ADIPOCYTES
By
ROBERT JOSEPH MCMAHON
DECEMBER, 1995
Chairperson: Susan C. Frost
Major Department: Biochemistry and Molecular Biology
The regulation of GLUTl expression by glucose was
analyzed in the 3T3-L1 adipocyte. Glucose deprivation of
3T3-L1 adipocytes for more than 12 hours resulted in the
accumulation of a second GLUTl glycoform (p37). 48 hours of
glucose deprivation resulted in a 2-fold increase in total
GLUTl, a reflection of the accumulation of p37.
Metabolically labeled GLUTl migrated as a wide band of
approximately 46 kDa. In contrast, metabolically labeled
GLUTl protein in glucose-deprived adipocytes migrated at 37
kDa. Although at least 12 hours of glucose-deprivation were
required to affect GLUTl glycosylation, glucose-deprived
cells quickly recovered the ability to correctly glycosylate
nascent GLUTl upon the readdition of glucose.
x


CHAPTER 6
GLUT1 TARGETING IN NORMAL AND GLUCOSE-DEPRIVED 3T3-L1
ADIPOCYTES
Introduction
In order for a protein to fulfill its physiological
role, it needs to reach a suitable location where its
activity is appropriate. For membrane glycoproteins such as
the glucose transporter, biosynthesis and targeting is a
complex pathway that could involve interactions with many
proteins, including glycosidases, proteases, kinases, and GTP
binding proteins, all while being carried along a route of
membrane traffic that is also regulated. As demonstrated in
a previous chapter (Chapter 4), the biosynthesis of the
transporter involves glycosylation and oligosaccharide
processing before attaining its mature form. The attachment
of oligosaccharide, however, does not indicate that the
protein has reached its intended locale; indeed, the
modification of oligosaccharides can have large effects on
the localization and activity of a glycoprotein (Schwarz and
Datema, 1982).
Different oligosaccharides are often present on the same
glycosylation site for any given glycoprotein. A protein
that contains variants of oligosaccharides at the same site
is said to have several glycoforms (Rademacher et al., 1988).
126


2
be transported against its concentration gradient if
transport is coupled to that of another solute that has a
concentration gradient of greater magnitude. The third
mechanism of transport is termed active transport. In this
process, the cleavage of high energy phosphoryl bonds
(usually in the form of ATP) is coupled to changes in the
conformation of the transport protein that result in the
translocation of the solute across the plasma membrane.
Since energy is expended in this process, solutes can be
transported against their concentration gradients. Examples
of this include the Na+, K+ ATPase and the Ca2+ ATPase.
Due to a large volume of scientific study, the transport
of glucose has served as a paradigm for the facilitated
transport of small, neutral organic solutes. The most well
understood glucose transport system exists in the mammalian
erythrocyte. The partial purification of a glucose
transporter protein was facilitated by its high concentration
in the erythrocyte plasma membrane. The purified transporter
from erythrocyte ghosts migrated as a wide band with an
average molecular weight of 55 kDa (Kasahara and Hinkle,
1977; Baldwin et al., 1979). Screening a cDNA expression
library with antiserum against the purified transporter
resulted in the isolation of a clone coding for a protein
capable of saturable, D-stereoisomer specific, glucose
transport (Mueckler et al., 1985; Birnbaum et al., 1986).
The study of glucose transport then underwent another
expansion when it was discovered that the transport of


+ Glucose
- Glucose
0.25
-s p46
-e p36
Tunicamycin, pg/ml
-e p46
g P37 p36


33
for 20 min at 37C. Then, for N-glycosidase F digestions,
the samples were brought to 150 mM Tris-HCl, pH 8.0, 1% NP-
40, protease inhibitors as above, 1.25 U N-glycosidase F and
then incubated at 37"C for 2 hours. For endoglycosidase H
digestions, the denatured protein sample was brought to 75 mM
sodium citrate, pH 5.5, protease inhibitors as above, and 2.5
mU endoglycosidase H and incubated at 37C for 2 hours. An
equal volume of 2X sample dilution buffer was added and
loaded onto an 8% SDS-PAGE gel for separation.
Glycogen Isolation
Glycogen, a polymerized form of glucose, is stored by
most cells (Voet and Voet, 1990). Glycogen breakdown
provides a source of fuel for energy production by glycolysis
as well as other pathways. The determination of glycogen in
this work is not made directly; rather the glycogen is
hydrolyzed with acid to produce free glucose which can then
be assayed directly by the method described below. The
results of this assay are therefore represented as the amount
of glucose contained in glycogen. The procedure used was
based on a previously described method for the isolation of
glycogen from liver (Pfleiderer 1963). After rinsing in
phosphate-buffered saline, pH 7.2 (PBS) at 4C, cells were
scraped into a 15 ml polypropylene tube in 1 ml PBS. The
cells were sonicated for 10 sec on power 1.5 at 50% duty
cycle on a Branson Sonifier 450. KOH (2.0 ml, 30% w/v) was
added and the mixture mixed vigorously. The sample was then


A
B
Glucose
(Tv


Glucose
45
+ + + + + +
0 1 2 3 4 5 6
Incubation time, hrs
p46
p37
00


151
suspect because of glycosylation alterations. If proteins
present at the onset of glucose-deprivation were degraded and
the newly synthesized glycoproteins exhibited reduced or no
activity, the cell would be unable to sustain itself for any
extended period of time. Upon returning glucose to the
culture medium, however, the cells quickly regain the ability
to glycosylate protein normally. In summary, the effects of
glucose deprivation on the processing and turnover of
glycoproteins like GLUT1 is to adapt by sustaining protein
glycosylation with truncated oligosaccharides, and preserving
glycoprotein function by inhibiting the turnover of proteins
following the withdrawal of glucose.
Several observations in this work indicated that there
was a finite period in which the cell was able to sustain
completely normal protein glycosylation and protein turnover
in the absence of glucose. The first of these observations
stems from experiments analyzing the synthesis of GLUT1 over
the course of glucose deprivation. These experiments
demonstrated that the normal glycoform of GLUT1 was
synthesized during the fist 12 hours of glucose deprivation.
This indicates that the substrates and pathways required for
the biosynthesis of normal core oligosaccharide are present
despite the absence of any extracellular glucose. The second
observation is derived from experiments analyzing GLUT1
turnover in the presence and absence of glucose. In a
likewise fashion, the turnover of GLUT1 was not affected for
the first 12 hours of glucose deprivation. This suggests


Figure 5-8 GLUT1 Glvcosvlation and Glucose Deprivation in CHO cells CH0-K1 cells were
grown to confluence and then placed into DMEM/10% dialyzed FBS supplemented with 0.1 mM
proline and 5 mM glucose. A total membrane fraction was prepared at the times indicated and
assayed for GLUT1 protein by Western blotting. Left Panel, Western blot of membrane
proteins from glucose-deprived CHO-K1 cells (50 pg/lane) probed with the anti-GTl antisera.
Right Panel, Western blot of membrane proteins (50 pg/lane) from 3T3-L1 adipocytes incubated
in medium containing the indicated glucose concentration for 24 hours and probed with the
anti-GTl antiserum. Shown is a single result representative of at least three independent
experiments.


163
Sargeant, R.J., and Paquet, M.R. (1993) Biochem. J. 290. 913
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Schwarz, R.T., and Datema, R. (1982) The Glvcoconiuqates
Academic Press, New York 47-75.
Shanahan, M.F., D'artel-Ellis, J. (1984) J. Biol. Chem. 259.
13878-13884.
Shawver, L.K., Olson, S.A., White, M., Weber, M.J. (1987)
Mol. Cell. Biol. 7, 2112-2118.
Sigel, M.B., Sinha, Y.N., VanderLaan, W.P. (1983) Meth.
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Silverman, M. (1991) Ann. Rev. Biochem. 60, 757-794.
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Tordjman, K.M., Weingang, K. A., and Mueckler, M. (1990)
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Tordjman, K.M., Weingang, K.A., James, D.E., Mueckler, M.M.
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Voet, D. and Voet, J. (1990) Biochemistry. John Wiley and
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Walker, P.S., Donovan, J.A., Van Ness, B.G., Fellows, R.E.,
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Biol. Chem. 264. 6587-6595.


Figure 4-5 Turnover GLUT1 in Glucose-Deprived 3T3-L1
Adipocytes Control adipocytes were labeled with 200 pCi/ml
Tran35S-Label for 1 hour and then chased in the presence (+)
or absence (-) of glucose for a total of 48 hours. GLUT1 was
then immunoprecipitated from membrane extracts and analyzed
by SDS-PAGE and fluorography. Panel A Fluorograph of GLUT1
immunoprecipitates. Panel B Densitometry of total GLUT1
immunoprecipitates from panel A. Results shown are
representative of at least 4 independent experiments.


28
hours at 4C in 25 mM Tris-base (unadjusted pH), 192 mM
glycine, 20% methanol. The blot was stained briefly in amido
black (0.2% in 40% methanol, 10% acetic acid) and destained
(40% methanol, 10% acetic acid). The blot was blocked in
Tris-buffered saline/0.1% Tween-20 (TBS-T) containing 5% non
fat dry milk (NFDM) for 30 min at room temperature. The blot
was agitated constantly with an orbital shaker throughout the
procedure. The blot was then incubated in the same solution
containing a 1:500 dilution of the anti-GTl antiserum for 1
hour at room temperature. After extensively washing the blot
in TBS-T, the blot was incubated in 1:50,000 dilution of goat
anti-rabbit antibody coupled to horseradish peroxidase in
TBS-T/5% NFDM for 1 hour at room temperature. The blot was
then extensively washed in TBS-T before before being
visualized with Amersham's Enhanced Chemiluminescence
detection reagents following manufacturer's directions.
Light emissions from the blot were captured on Hyperfilm MP
(Amersham). Band intensity was quantitated in the linear
range of the film on a Visage Bioscan video densitometer.
Metabolic Radiolabelinq of 3T3-L1 Adipocytes
In order to characterize individual proteins for their
turnover and processing, they need to be metabolically
labeled for a defined period of time. Usually, this is
accomplished with the use of radioactively labeled amino
acids, such as 35S labeled methionine and/or cysteine. In
order to determine the time required for the degradation of


A
Glucose
C
Glucose
co
OJ


Figure 4-6 Turnover GLUT1 in Glucose-Refed 3T3-L1 Adipocytes
Glucose-deprived cells were labeled with 200 pCi/ml Tran35S-
Label for 1 hour and then chased in the presence (+) or
absence (-) of glucose. GLUT1 was immunoprecipitated from
membrane extracts and analyzed by SDS-PAGE and fluorography.
Panel A Fluorograph of GLUT1 immunoprecipitates. Panel B
Densitometry of total GLUT1 immunoprecipitates during the
chase period from panel A. Results shown are representative
of at least 4 independent experiments.


The regulation of GLUT1 turnover by glucose was analyzed
by metabolic labeling and pulse-chase analysis. GLUT1 in
normal adipocytes exhibited a half-life of 14 hours. In
contrast, the turnover of the transporter in glucose-deprived
adipocytes was greater than 50 hours. The inhibition of
transporter turnover was readily reversed upon the re
addition of glucose.
The glycogen content of 3T3-L1 adipocytes was assessed
as a potential carbohydrate source for protein glycosylation.
Normal 3T3-L1 adipocytes contained 0.537 0.097 pmol
glucose/106 adipocytes, which decreased in a first order
fashion upon glucose deprivation (0.0.052 pmol glucose/106
cells/hr, t-x/2 = 6 hours). Fructose was unable to prevent the
depletion of glycogen in glucose-deprived adipocytes. In
glycogen-depleted cells, alterations in GLUTl glycosylation
occurred more rapidly upon glucose deprivation than in
adipocytes with normal glycogen levels. Chinese Hamster
Ovary cells (CHO), which contain only 1% of the glycogen of
3T3-L1 adipocytes, exhibited the most rapid alteration in
GLUTl glycosylation, occurring within 3 hours of glucose
deprivation.
The targeting of the alternatively glycosylated GLUTl
transporter was assessed by plasma membrane fragment
isolation, cell surface biotinylation, and subcellular
fractionation. All three techniques detected both GLUTl
glycoforms (p46 and p37) at the plasma membrane, indicating
that the targeting of the GLUTl protein was not affected by
xi


112
Figure 5-5 Effect of Insulin and 3-0-methvlqlucose on
Glycogen 3T3-L1 Adipocytes were incubated in the indicated
sugars in the presence or absence of insulin (1 pM) for 24
hours before glycogen determination. All sugars were
prepared at 25 mM except for the combination of fructose and
3-0-methylglucose, where the concentrations were prepared at
20 mM and 5 mM, repectively. Each result, reported as the
average the standard deviation, represents two samples
measured in triplicate. Glc, glucose; None, no sugar; Frc,
fructose; Ins, insulin; 3-0-Meglc, 3-0-Methylglucose.


77
A
B
Glucose + + + + -
10 20 30 60
Labeling Time, min


o
r~
c
GLUT4
GRP78

Homog
I
\
HDM
LDM
PM
Homog
1
HDM
LDM
f
PM

134


10
et al., 1986; Kitzman et al., 1994). Furthermore, the
migration of the normal GLUT1 protein and the low molecular
weight protein were found to be identical upon digestion with
N-Glycanase F, indicating that resultant core proteins were
of identical molecular weight. Most investigators have now
accepted the assumption that the second protein is indeed a
different form of the GLUT1 transporter (Haspel et al., 1986;
Haspel et al., 1990; Kitzman, et al., 1993).
3T3-L1 Adipocytes
White adipose tissue plays an important role in the
storage of triglyceride resulting from excess blood glucose.
With this important role in the disposal of glucose, the
transport system for glucose is highly regulated. Many
studies involving the transport of glucose utilize adipocytes
isolated from fat pads, but these cells exhibit several
disadvantages, including the propensity to dedifferentiate
once isolated, and the inability to culture these cells for
extended periods of time. For these reasons, the work
described herein utilized the 3T3-L1 adipocyte, an adipocyte
model cell line, to study glucose transporter regulation.
In 1974, Green and Kehinde isolated several subclones of
mouse fibroblasts 3T3 cells that exhibited the propensity to
accumulate large lipid droplets once confluent. This
phenomenon could be prevented or reversed by passing and
reculturing the cells at low density. Extensive subcloning
of these cells resulted in the establishment of the 3T3-L1


21
precipitate. The reaction mixture was then allowed to stand
upright overnight at 4C to collect precipitate, which
represents the KLH-peptide conjugate.
A polyclonal antiserum to this conjugate was then
generated by injection of the KLH-GT1 conjugate (200 pg)
emulsified in Freund's Complete Adjuvant into the popliteal
lymph node of a New Zealand white rabbit (Sigel et al.,
1983). The rabbit was boosted by intradermal injection with
an equal amount of the GT1-KLH conjugate emulsified in
Freund's Incomplete Adjuvant 28 days later, and then again
two weeks later. Serum (designated as anti-GTl) was
collected every week by laceration of the medial ear vein.
Additional boost injections were administered approximately
every 6 weeks. This crude antiserum was divided into 1 ml
aliquots and stored at -20C.
Antiserum against a carboxy-terminal peptide of the
insulin-responsive glucose transporter GLUT4 (CSTELEYLGPDEND)
and an amino-terminal peptide of the glucose-regulated
protein GRP78 (EEEDLLEDVGTVC) were generated in the same
manner. This antiserum were used unpurified for Western
blotting.
Peptide Purification of Anti-GTl Antibody
Although the crude antiserum generated against the KLH-
GT1 conjugate recognized the peptide, a fraction of the whole
serum containing the total IgG was purified using a
modification of a previously described method (Dankert et


68
1
The effect of tunicamycin on the relative molecular
weight of the transporter supports this conclusion. These
experiments indicate that the core protein generated in
tunicamycin-treated cells, regardless of the presence or
absence of glucose, migrated at the same molecular weight (36
kDa). This type of experiment analyzes the GLUT1 protein
before it has an opportunity to be glycosylated. Enzymatic
deglycosylation of p37 and p46, where the oligosaccharide is
removed after glycosylation, also results in the same protein
migrating at 36 kDa, consistent with the tunicamycin
experiment. Additional information about the type of
oligosaccharide on p37 was obtained. Although p37 was
sensitive to digestion with N-Glycosidase F, indicating that
it was an N-linked glycoprotein, p37 was not sensitive to
endoglycosidase H, indicating that this oligosaccharide was
not of the high mannose class. In order to confirm that the
endoglycosidase H reaction was working correctly, digestion
of GLUT1 derived from the LEC1 CHO cell line was performed
and verified that the GLUT1 from this glycosylation mutant
was a high mannose type glycoprotein, despite its similar
migration to p37. Earlier studies utilizing glucose-deprived
CHO cells indicated that an alternative glycosylation pathway
exists, where the cell synthesized a truncated
oligosaccharide core, GlcNAc2Man5Glc3i that was subsequently
added to proteins synthesized in the endoplasmic reticulum
(Rearick, et al., 1981). This alternative oligosaccharide
was shown to be endoglycosidase H resistant. The


9
as intensely studied as the regulation of transport activity,
progress has been made in elucidating the regulation of GLUT1
protein by glucose. The regulation of transporter expression
can be categorized into two classes; the first, where the
level of the transporter changes in response to glucose
deprivation, and the second, where a second transporter form
accumulates. Glucose deprivation results in an increase in
GLUT1 protein levels in some cell types, including the
fibroblast cell line 3T3-C2 and the kidney cell line NRK
(Haspel et al., 1986; Haspel et al., 1991). This effect,
however, is not conserved in all cell types; in 3T3-L1
adipocytes, there is no change in the level of normal GLUT1
transporter (Reed et al., 1990; Kitzman et al., 1994). With
extended periods of glucose deprivation, an additional
protein recognized by anti-GLUTl antiserum accumulates. This
phenomenon is observed in several other cell types, including
the 3T3-L1 fibroblasts, normal rat kidney cells, and a Wilm's
tumor cell line (Haspel et al., 1986, Haspel et al., 1990;
Shawver et al., 1987; Haspel et al., 1986, Haspel et al.,
1990; Kitzman et al., 1994). This second protein migrates as
analyzed by SDS-PAGE approximately 10 kDa lower than the
normal GLUT1 protein. The glycosylation state of this
protein was investigated by treatment with endoglycosidase F
or N-Glycanase F, which remove all N-linked oligosaccharides.
The migration of this second protein can be altered slightly
upon this treatment, indicating that like the normal GLUT1
protein, it is modified with N-linked oligosaccharide (Haspel


67
\
p37 GLUT1 in glucose-deprived cells (+GT1, glucose). In a
similar experiment, a GLUT4 carboxy-terminal peptide
(CSTELEYLGPDEND) did not block the immunoprecipitation of
GLUT1 (+GT4, glucose). GLUT1 protein of either molecular
weight was not observed in membrane extracts
immunoprecipitated with Protein A Sepharose alone, with
preimmune serum, or with 5 pg IgG from an unrelated antisera
(-Ab, PIS, IRR, glucose). GLUT1 protein was also not
observed when extracts were immunoprecipitated with an equal
amount of the unbound antibody fraction (flow through) from
peptide purification of the same antiserum, indicating that
the purification step removed the majority of the GLUT1
specific IgG (FT, glucose).
Conclusions
This chapter described experiments designed to
demonstrate that the second immunoreactive protein that
appears during glucose deprivation of 3T3-L1 adipocytes is a
second glycoform of GLUT1. Tryptic digest of membrane
proteins from normal and glucose-deprived cells indicates
that peptides detected with the anti-GTl antiserum migrated
in an identical fashion. I interpret these data to suggest
that there were no gross alterations in the length of the
GLUT1 core protein. The expression of a different GLUT1
protein, therefore, could not account for the difference in
molecular weight between p46 and p37.


4
tissue-specific expression, developmental regulation, and
hormonal regulation, lends itself well to being capable of
delivering glucose to different tissues with varying
requirements for carbohydrate.
Glucose Transporter Structure
Although GLUT proteins exhibit a high degree of tissue-
specific expression, analysis of the primary sequences of the
cloned transporters reveals a high degree of homology. The
GLUT family exhibits approximately 68% overall homology; the
highest degree occurs in the membrane spanning regions, and
the lowest conservation occurs in the amino and carboxy
terminal ends and the large intracellular loop between
membrane spanning regions 6 and 7 (Carruthers, 1990). The
predicted topology of the transporter is depicted in Figure
1. The disposition of the transporter in the membrane was
based upon hydrophobicity and hydrophilicity measurements
which predict putative membrane spanning domains (Mueckler et
al., 1985). This model predicts that the GLUT proteins
exhibit 12 membrane spanning regions, and places both the
amino and carboxy terminal ends facing the cytoplasm
(Mueckler et al., 1985). Infrared spectroscopy and circular
dichroism have revealed a high degree of a-helical nature in
the transporter protein, which likely represents the membrane
spanning regions (Chin et al., 1986; Alvarez et al., 1987).
This topological model has received a significant amount of
experimental support (Shanahan et al., 1984; Cairns et al.,


155
The determination of the transport activity of p37 GLUTl
is a serious undertaking, a process which would require the
separation of GLUTl proteins which on many levels are very
similar. Reconstitution of the purified transporters into an
active form in artificial liposomes would then be required
for activity determination. Although not completed in this
work, an outline of the required steps for this process is
provided. The first part of this procedure would involve the
purification of p37 GLUTl glycoform away from the normal
GLUTl glycoform. It should be noted that there is very
little GLUT4 present in the cell at periods when there is
significant levels of the low molecular weight GLUTl
transporter, but steps should be taken to ensure that GLUT4
does not copurify with the low molecular weight GLUTl. A
solubilized membrane fraction from glucose-deprived cells
would be prepared as a starting material. From this, a
reasonable next purification step would be the large scale
isolation of both GLUTl glycoforms from the remainder of the
membrane fraction by affinity chromatography over an anti-
GLUT1 peptide column with the antiserum generated in this
work. Once the two GLUTl glycoforms have been purified,
affinity chromatography over an appropriate lectin column may
provide enough discrimination between the normal and low
molecular weight GLUTl transporter to obtain a fraction which
is enriched in the low molecular weight transporter and free
in the normal GLUTl glycoform. A possible alternative is the
omission of the peptide purification step preceding the


54
migrated at relative molecular weights of 25 kDa, 21 kDa, and
17 kDa (Figure 3-3 A and 3-3 B). These peptides,
corresponding to the carboxy-terminal half of the GLUT1
transporter which do not contain the glycosylation site, are
identical in control and glucose-deprived cells. No
additional peptides were observed from glucose-deprived
cells, as would be expected if that protein were of different
length or sequence. Together these data suggest that the
core protein of the normal GLUT1 protein and p37 protein is
very similar.
Effect of Tunicamvcin Treatment on GLUT1 Protein
Tryptic digest experiments suggested that the protein
from which GLUT1 and p37 arise is very similar; therefore,
the possibility that the difference in relative molecular
weight between p46 and p37 was a difference in glycosylation
was tested. To prevent N-linked glycosylation of GLUT1, 3T3-
L1 adipocytes were treated with various concentrations (0-10
pg/ml) of the N-linked glycosylation inhibitor tunicamycin
dissolved in 20 mM potassium hydroxide for 24 hours.
Membrane proteins were then isolated and probed for GLUT1 by
Western blot using the anti-GLUTl antiserum. The normal form
of the transporter, p46, (lane KOH, or 20 mM potassium
hydroxide alone) is drastically different from the
transporter from tunicamycin-treated cells, which migrates at
a relative molecular weight of 36 kDa (p36), in agreement
with the predicted molecular weight from the amino acid


32
Dried gels were exposed to Amersham Hyperfilm typically for 4
days.
Endoqlvcosidase Digestions
Endoglycosidases refer to a class of enzymes that
catalyze the cleavage of oligosaccharide chains at specific
sugar residues. These enzymes are often useful for the
characterization of oligosaccharides on glycoproteins. For
the analysis of the oligosaccharide on GLUT1, two specific
endoglycosidases were chosen. The first, N-Glycanase F,
cleaves between the two GlcNAc residues adjacent to the
asparagine residue on the protein. This enzyme can be used
to determine if glycoproteins are post-translationally
modified with an N-linked oligosaccharide. This enzyme will
not cleave oligosaccharides attached to serine or threonine
resides (O-linked). The second endoglycosidase,
endoglycosidsase H, also cleaves between the two GlcNAc
residues but is specific for the high mannose type of N-
linked oligosaccharide. This enzyme is useful for not only
determining if a protein contains an N-linked
oligosaccharide, but can be used to distinguish among high
mannose type and complex type oligosaccharides. Like N-
Glycanase F, this enzyme will not cleave oligosaccharides
attached in an O-linked manner.
Membrane protein (20 pg) from control or glucose-
deprived adipocytes and 10 pg from LEC1 CHO cells were
denatured in 50 mM BME/0.5% SDS in a final volume of 50 pi


149
GLUT1, the oligosaccharide on GLUT1 from LEC1 CHO cells was a
high mannose structure. These observations strongly suggest
that glucose-deprived adipocytes synthesize a truncated
oligosaccharide in the absence of glucose, which is
transferred to protein, but at least in some cases, is not
further processed. These observations are consistent with
the alternative glycosylation pathway described for glucose-
deprived CHO cells (Rearick, et al., 1981). In this cell
line, the withdrawal of glucose results in the biosynthesis
of a truncated oligosaccharide which replaces the normal core
oligosaccharide. The reason this occurs is probable due to
the specific depletion of dolichol-linked mannose residues,
which are responsible for the production of GlcNAc2Man6_9
(Chapman and Calhoun, 1988).
A clear model of the processing of the transporter in
glucose-deprived cells has not been previously reported. The
data presented here demonstrate that in contrast to normal
adipocytes, the transporter is cotranslationally modified
with an N-linked oligosaccharide which is processed through
an intermediate form (p42) before final maturation. The
transporter in glucose-deprived cells, however, is not
processed after glycosylation. The inability to process p37
GLUT1 occurs in either the presence or absence of glucose.
The alterations in the transporter processing pathway occur
in the absence of any specific effect on the biosynthetic
rate of the transporter protein; although GLUT1 is
synthesized at a slightly higher rate in glucose-deprived


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.
Associate Professor of
Biochemistry and Molecular
Biology
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.
iichael S. Kilberg
Professor of Biochemistry
and Molecular Biology
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.
Charles M. Allen
Professor of Biochemistry
and Molecular Biology
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.
(Mr
Christopher West
Associate Professor of
Anatomy and Cell Biology


127
There is evidence that individual glycoforms of a
glycoprotein exhibit different activities. In a study of
bovine pancreatic ribonuclease B, characterized by the
presence of five major glycoforms, capillary electrophoresis
allowed the purification of these individual glycoforms in
sufficient quantities to test the stability (protease
resistance) and activity in comparison with the
unglycosylated form. In stability experiments, it was found
that oligosaccharides afforded protection of susceptible
protease cleavage sites; all glycoforms exhibited increased
protease resistance in comparison to the unglycosylated form.
Furthermore, although the three-dimensional structure of the
protein was not significantly altered in any of the
glycoforms, the exchange rate for amide protons
(demonstrating flexibility) was considerably different;
glycoforms containing large oligosaccharides were less
flexible than the unglycosylated counterpart. Finally, a
four-fold variability in activity was found between
glycoforms, demonstrating that the activity of any given
glycoprotein sample is represented by an average of all the
individual activities of the glycoforms present.
Glycosylation effects are not limited to soluble
glycoproteins; there is growing evidence that glycosylation
state affects stability, targeting, and activity of integral
membrane proteins, including transporters. The proper
targeting of virus proteins requires appropriate
glycosylation. Integral membrane proteins from the Vesicular


99
cannot be acted upon by glycogen phosphorylase. Instead, a-
(l-4)-glucan 6-glucosyltransferase removes three of the final
four glucose residues from a branch point, transferring them
to the other branch. This final glucose residue is removed
by a separate enzyme, Amylo-a-(l-6)-glucosidase, which
releases free glucose. The linear part of the glycogen
particle is then once again a substrate for glycogen
phosphorylase until the next branch point is reached.
In contrast to the glycogen breakdown, the synthesis of
glycogen is carried out by a separate series of enzymes (Voet
and Voet, 1990). The first step in glycogen synthesis is the
conversion of glucose-6-phosphate to glucose-1-phosphate by
phosphoglucomutase. Glucose-l-phosphate, however, cannot be
added directly onto the termini of the glycogen particle;
rather, it must be activated to UDP-glucose, utilizing UTP
and the enzyme UDP-glucose pyrophosphorylase. The activated
nucleotide derivative of glucose can then be added onto the
non-reducing termini of the glycogen particle by glycogen
synthase. Branch points on the glycogen particle are
produced by the action of amylo-(1,4-l,6)-
glucosyltransferase. This results in the movement of a
glucose residue from the non-reducing termini of one branch
to a more interior position, connecting it with an a-(l-6)
glycosidic bond.
The regulation of the enzymes responsible for glycogen
breakdown and biosynthesis is tightly controlled in an
inverse manner by hormones and other factors in a


109
Incubation time, hours
Figure 5-4 Effect of Fructose on Glycogen Depletion Cells
were incubated in either complete medium (DMEM/10% FBS),
glucose-free medium (glucose-free DMEM/10% dialyzed FBS), or
fructose-containing medium (glucose-free medium supplemented
with 25 mM fructose) for 24 hours. Glycogen was then
extracted, hydrolyzed to glucose, and assayed. The results,
expressed as the average the standard deviation, is
representative of two independent experiments measured in
triplicate.


148
glucose-deprived cells produced identical peptides when
detected with an GLUT1 carboxy-terminal peptide antibody. In
addition, this investigator's laboratory has demonstrated
that during glucose-deprivation, no additional mRNA species
were observed with low stringency northern blot analysis.
These observations make any explanation of the genesis of p37
involving alternative splicing, truncations, or additions to
the GLUT1 mRNA/protein unlikely. Furthermore, removal of fl
unked oligosaccharides from membrane proteins of glucose-
deprived adipocytes resolves both p46 and p37 to 36 kDa,
indicating that (i) both proteins are glycosylated, (ii) both
core proteins are of identical size, and (iii), the core
protein is consistent with the predicted molecular weight of
the GLUT1 core protein.
Characterization of the oligosaccharide on GLUT1 in both
normal and glucose-deprived adipocytes was undertaken by
glycosidase digestion. Enzymatic deglycosylation indicated
that the normal GLUT1 protein was modified with a large,
heterogeneous, complex type of N-linked oligosaccharide. p37
GLUT1, however, was modified with a small and less
heterogeneous oligosaccharide. Furthermore, resistance to
endoglycosidase H indicated that the oligosaccharide on p37
GLUT1 was not a complex type of structure. For comparison,
the oligosaccharide on p37 GLUT1 was compared to another
oligosaccharide of similar size on GLUT1 from the
glycosylation mutant LEC1 CHO cell line. These experiments
demonstrated that, in contrast to the oligosaccharide on p37



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Figure 6-4 Plasma Membrane Fragments 3T3-L1 adipocytes were incubated in the presence or
absence of glucose for 48 hours before plasma membrane fragments were isolated as described
in Chapter 2. Fragments were scraped into sample dilution buffer and resolved along with
total membrane samples by 7.5% mini SDS-PAGE. The membrane proteins were then probed by
Western blot for GRP78 or GLUT1 as indicated.


100
phosphorylation/dephosphorylation cascade (reviewed by Hue
and Hers, 1976, 1983). Conditions that result in the
activation of glycogen breakdown inhibit glycogen synthesis,
and activation of glycogen synthase is concomitant with an
inhibition of glycogen degradation. Hormones such as
glucagon and epinephrine, which are released when the level
of circulating glucose reaches a critical minima, stimulate
the breakdown of glycogen by activation through a signal
transduction cascade involving adenylate cyclase. Adenylate
cyclase activation, which results in the production of cyclic
AMP (cAMP), activates cAMP-dependent protein kinase. cAMP-
dependent protein kinase inactivates glycogen synthase via
phosphorylation, and indirectly activates glycogen breakdown
by increasing the phosphorylation of phosphorylase kinase.
Activated glycogen phosphorylase kinase in turn activates
glycogen phosphorylase.
A second mechanism by which glycogen degradation can be
stimulated is through the action of the calcium ion, Ca2+
(reviewed by Hers, 1983). Release of calcium ions, such as
occurs in skeletal muscle contraction, allows binding of
calcium to a regulatory subunit of phosphorylase kinase.
Even in the absence of the cAMP-dependent protein kinase
activation, the binding of calcium ions to phosphorylase
kinsae preactivates the glycogen degradation pathway.
The level of GLUT1 transporter remains constant during
glucose deprivation (Kitzman et al., 1994). With extended
glucose deprivation (12 48 h), however, an alternatively


CHAPTER 5
REGULATION OF GLYCOGEN CONTENT AND GLYCOSYLATION OF GLUT1
GLUCOSE TRANSPORTER
Introduction
Glycogen, a polymerized storage form of glucose, is
maintained in most mammalian cells as cytosolic particles
approximately 2500 nm in diameter (Voet and Voet, 1990).
This particle, complete with the associated enzymes required
for both the synthesis and degradation of glycogen, serves as
a rapidly mobilized form of fuel by which energy can be
derived through glycolysis. The glucose units in a glycogen
particle are arranged by a-(l-4) glycosidic bonds. This
linkage forms linear strands with branch points consisting of
glucose molecules every 10 12 glucose units linked by an a-
(1 - 6) bond.
The level of glycogen is regulated by two separate
enzymes, glycogen phosphorylase and glycogen synthase (Voet
and Voet, 1990). The degradation of glycogen is catalyzed by
the enzyme glycogen phosphorylase, which removes by
phosphorolysis a glucose residue from the outermost non
reducing termini of length n, generating glucose-l-phosphate
and a glycogen branch of length n-1. Glucose-l-phosphate can
then feed into glycolysis by conversion into glucose-6-
phosphate by phosphoglucomutase. Branch points, however,
98


Relative GLUT1 Intensity
Glucose
+
+
+ +
+
0 6 12 24 36 48
Chase Time, hrs
Chase Time, hrs


57
sequence (Figure 3-4, + glucose). The next species, p37,
present in the glucose-deprived cells, migrates slightly but
distinctly above the core protein with a difference in
molecular weight of approximately 1-2 kDa (Figure 3-4 -
glucose). The GLUT1 protein resulting from tunicamycin
treatment is identical whether it is derived from normal or
glucose-deprived adipocytes, supporting the conclusion that
the core protein in glucose-deprived adipocytes is identical
in size, and that differences in molecular weight between p46
and p37 arise from differences in glycosylation.
The production of unglycosylated GLUT1 protein provided
an opportunity to follow GLUT1 processing if the effect of
tunicamycin could be removed. A previous report demonstrated
that the processing of the insulin receptor in 3T3-L1
adipocytes could be followed after tunicamycin washout
(Ronnet et al., 1988). To this end, adipocytes were exposed
to tunicamycin (1 pg/ml) for 24 hours in either the presence
or absence of glucose. Following this treatment, the medium
was changed into medium without tunicamycin for 24 hours.
The production of the unglycosylated transporter p36 is
confirmed after exposure to tunicamycin for 24 hours (Figure
3-5). However, after the medium is changed into medium
without tunicamycin, no reduction in the level of the
unglycosylated transporter is observed. The inclusion of 25
mM N-acetylglucosamine was added in order to compete with the
action tunicamycin for the glycosylation process. The
results from this experiment indicate that even with the


17
Pierce. All other chemicals were obtained through either
Fisher or Sigma.
Methods
Cell Culture
Cell culture and differentiation of 3T3-L1 fibroblasts
were performed as previously described (Frost and Lane,
1985). Cells were seeded in plastic tissue culture dishes
and cultured in D-MEM containing 10% calf serum (CS) for
seven days, refeeding every other day until confluent. After
seven days, the cells were incubated in D-MEM containing 10%
fetal bovine serum (FBS), 1 pg/ml insulin, 0.5 mM
methylisobutylxanthine, and 0.25 mM dexamethasone (Kuri-
Haruch and Green, 1978; Rubin et al., 1978; Reed and Lane,
1980). Two days later, the cells were incubated in D-MEM/10%
FBS containing 1 pM insulin only. Thereafter, the cells were
fed with D-MEM/10% FBS alone. Cells were used for
experiments between days 8 and 12 post-differentiation. To
prepare glucose-deprived 3T3-L1 adipocytes, the normal
culture medium was aspirated and replaced with glucose-free
D-MEM containing 10% dialyzed FBS for the indicated periods
of time.
CHO-K1 cells, obtained from American Type Cell Culture,
Rockville, MD, and LEC1 CHO cells, obtained as a kind gift of
Dr. Pamela Stanley, (Albert Einstein College of Medicine, NY,
NY) were seeded onto plastic tissue culture dishes (100 cm2


81
In control cells, the 36 kDa and 42 kDa GLUT1 precursors
synthesized during the pulse disappeared concomitant with the
emergence of the mature GLUT1 at 46 kDa after 20 min of chase
(Figure 4-2 A). In contrast, p37 synthesized in glucose-
deprived cells was unchanged in either molecular weight or
intensity when chased in the absence of glucose (Figure 4-2
B). Although this data indicated that p37 GLUT1 was not
processed in glucose-deprived cells, it did not rule out the
possibility that p37 could be processed if glucose were
present. This hypothesis was tested by labeling glucose-
deprived (36 hours) adipocytes for 10 min and then chasing
for 60 minutes in the presence of 25 mM glucose. It was
found that like the experiment that contained no glucose
during the chase, p37 GLUT1 remained unchanged (Figure 4-2
C). These results demonstrate that although 3T3-L1
adipocytes have the capacity to glycosylate GLUT1 in the
absence of glucose, the transporter cannot be further
processed due to either the structure of the oligosaccharide
or the location of GLUT1 along the processing pathway.
Synthesis of GLUT1 Glvcoforms During Glucose Deprivation and
Glucose Refeedinq
To determine the time required for glucose deprivation
to affect GLUT1 glycosylation, normal adipocytes were placed
into glucose-free medium and labeled for 1 hour at specific
times during the next 36 hours of glucose deprivation. GLUT1
was then immunoprecipitated from solubilized membrane


11
(Lipid accumulating subclone 1) cell line in which
essentially all cells accumulate fat droplets (Green and
Meuth, 1974).
After observation of the onset of lipogenesis during the
differentiation of 3T3-L1 cells, it was established that the
3T3-L1 adipocytes express the proteins necessary to carry out
the metabolism characteristic of true adipose tissue.
Lipoprotein lipase (Spooner et al., 1979), fatty acid
synthase (Student et al., 1980; Wise et al., 1980),
acquisition of hormonal sensitivity, including to insulin
(Rubin et al., 1977; Resh et al., 1982), enzymes involved in
phosphatidylcholine and phosphoethanolamine biosynthesis
(Coleman et al., 1978), stearoyl CoA desaturase and fatty
acid binding protein (Cook et al., 1988) are all expressed
specifically during the onset of differentiation of 3T3-L1
cells. The large body of information regarding the
metabolism of the 3T3-L1 adipocyte has established this cell
line as a valid model for both the differentiation and
physiology of true adipose tissue.
3T3-L1 adipocytes express two members of the glucose
transporter family; GLUT1, the basal transporter, and GLUT4,
the insulin-sensitive glucose transporter. It has been
estimated by cell surface labeling techniques in normal and
insulin-treated adipocytes that each cell expresses
approximately 1 x 106 and 0.3 x 106 GLUT1 and GLUT4
transporter molecules, respectively (Calderhead et al.,
1990). The 3T3-L1 cell line has been used extensively in the


146
observations on the targeting of the totally unglycosylated
GLUT1 transporter. Mutagenized GLUT1 transporter missing the
N-linked glycosylation site appears to be targeted to the
plasma membrane, as evidence by its ability to be cell
surface labeled by the bis-mannose ligand [3H]2-N-4-(1-azi-
2,2,2,trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-
propylamine. The labeling of the oligosaccharide-deficient
transporter, however, was greatly reduced in comparison to
labeling of the wild-type transporter, suggesting that some
of the unglycosylated transporter may be retained
intracellularly (Asano et al., 1991). In agreement with the
latter conclusion, the localization of the oligosaccharide-
deficient transporter was analyzed by immunofluoresence. A
significant proportion of the mutated transporter was
retained intracellularly, whereas the localization of the
wild-type transporter was overwhelmingly plasma membrane
specific (Asano et al., 1993). in comparison to these
studies, it may be suggested that, assuming that the
alternatively glycosylated transporter is active,
glycosylation of the GLUT1 transporter with a truncated
oligosaccharide in the absence of glucose may allow the
transporter to retain proper targeting, and thus rescue the
functionality of the transporter. This may allow for the
cell to prepare itself for the return of nutrient or to
scavenge what nutrient is present.


CHAPTER 1
OVERVIEW
GLUT Proteins and Glucose Transport
GLUT Protein Family
The maintenance of intracellular metabolism relies upon
the ability of the cell to incorporate solutes that cannot
readily diffuse across the plasma membrane. The uptake of
organic solutes, such as sugars and amino acids, is
accomplished by specific integral membrane proteins that
mediate the movement of molecules from the extracellular
environment to the cytosol. The transport of solutes across
the plasma membrane is accomplished by one of three
mechanisms (Voet and Voet, 1990). The first is passive
diffusion across the plasma membrane, a slow process due to
the low solubility of organic molecules in the hydrophobic
membrane interior. The second transport mechanism, termed
facilitated transport, is mediated by specific integral
membrane proteins that undergo conformational changes that
allow the solute access to the cytosol. The direction of
flux in this type of transport is determined solely by the
concentration gradient of the solute. Facilitated transport
describes many transport systems, including many salt, sugar,
and amino acid transporters. In some instances, solutes can
1


Figure 3-7 immunoprecipitation of GLUT1 from Normal and Glucose-Deprived 3T3-L1 Adipocytes
Cells, cultured with or without glucose for 36 hours, were metabolically labeled with 200
pCi/ml Tran35S-Label for 1 hour as described in Experimental Procedures. GLUT1 was
immunoprecipitated from a total membrane fraction as indicated, separated by SDS-PAGE, and
visualized by fluorography. -Ab, no antibody; PIS, pre-immune serum; IRR, unrelated serum;
-GT1, immunoprecipitated in the absence of peptide; +GT1, inclusion of 1 pg GT1 peptide;
+GT4, inclusion of 1 pg GT4 peptide; FT, unbound antibody fraction from peptide
purification.


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.
John Gander
Professor of Microbiology
and Cell Science
This thesis was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
December, 1995
Dean, Graduate School


23
inert substrate. The GT1 peptide (2 mg) was covalently
coupled to a Sulfolink matrix through the additional cysteine
residue on the GT1 peptide following the manufacturer's
instructions. Sulfolink matrix consists of 6% cross-linked
beaded agarose with a terminal iodoacetyl group that confers
reactivity with sulfhydryl groups under mild (neutral pH)
conditions. An additional advantage of the Sulfolink matrix
is that the linkage between the peptide and the matrix is
irreversible; other matrices utilizing sulfhydryl chemistry
can be reversed under reducing conditions. Anti-GTl
antibodies were purified using the following method. The
total IgG fraction (15 mg each batch) purified from whole
serum was incubated with the peptide column and rotated end-
over-end at room temperature for 2 h. Unbound IgG was washed
from the column with PBS, monitoring the eluate by absorbance
at 280 nm until a baseline was reached. Specifically bound
IgG was then eluted with 0.1 M glycine, pH 3.0, by collecting
1 ml fractions. The eluate was immediately neutralized with
0.1 ml 1M Tris-Base. Peak fractions, as assessed by
absorbance at 280 nm, were pooled and dialyzed against PBS
for 12 h at 4C. The final IgG was stored at -20C as a 0.5
mg/ml solution in PBS.
Membrane Fraction Isolation
To facilitate the detection of integral membrane proteins
such as the glucose transporter, it is often desirable to


160
Hare, J.F., Taylor, K. (1992) Arch. Biochem. Biophvs. 293,
416-423.
Harlow, E and Lane, D. (1988) Antibodies: A Laboratory Manual
Cold Spring Harbor Laboratory, New York, 55-57.
Haspel, H.C., Birnbaum, M.J., Wilk, E.W., Rosen, O.M. (1985)
J. Biol. Chem. 260. 7219-7225.
Haspel, H.C., Mynarcik, D.C., Ortiz, P.A., Honkanen, R.A.,
and Rosenfeld, M.G. (1991) Mol. Endo. 5. 61-72.
Haspel, H.C., Revillame, J., and Rosen, O.M. (1988) J. Cell.
Phvs. 136. 361-366.
Haspel, H.C., Wilk, E.W., Birnbaum, M.J., Cushman, S.W., and
Rosen, O.M. (1986) J. Biol. Chem. 261. 6778-6789.
Hearing, J., Gething, M-J., and Sambrook, J. (1989) J. Cell.
Biol. 108, 355-365.
Hendershot, L.M., Ting, J. and Lee, A.S. (1988) Mol. Cell.
Biol. 8, 4250-4256.
Hers, H.G. (1976) Ann. Rev. Biochem. 45, 167-189.
Hers, H.G. and Hue, L. (1983) Ann. Rev. Biochem. 52, 617-653.
Holman, G.D., and Rees, W.D., (1987) Biochim. Biophvs. Acta
897. 395-405.
HSU, K.C., Chao, M.V. (1993) J. Biol. Chem. 268. 16430-16436.
Kao, F-T., and Puck, T.T. (1968) Proc. Nat. Acad. Sci. U.S.A.
M, 1275-1281.
Karnelli, E., Zarnowski, M.J., Hissin, P.J., Simpson, I.A.,
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4777.
Kasahara, M., Hinkle, P.C. (1977) J. Biol. Chem. 253. 7384-
7390.
Rayano, T., Fukumoto, H., Eddy, R., Fan, Y.S., Beyers, M.G.,
et al., (1988) J, Biol. Chem. 263. 15245-15248.
Kitzman, H.H., McMahon, R.J., Williams, M.G., and Frost, S.C.
(1994) J. Biol. Chem. 268. 1320-1325.
Kletzein, R.F. and Perdue, J.F. (1975) J. Biol. Chem. 250.
593-600.


8
Regulation of Glucose Transport Activity by Glucose
Deprivation
Glucose deprivation, a specific type of nutrient
deprivation, augments glucose uptake in a variety of cell
types, including glial cells (Walker et al., 1989), avian
fibroblasts (Martineau et al., 1972; Kletzein and Purdue
1975; Pessin et al., 1982; Yamada et al., 1983), mammalian
fibroblasts (Germinarlo et al., 1982; Haspel et al., 1986,
Ortiz and Haspel, 1993), muscle cells (Walker et al., 1989;
Koivisto et al., 1991; Maher and Harrison, 1991), and
adipocytes (van Putten and Krans, 1985; Reed et al., 1990;
Tordjman et al., 1990). Several characteristics of this
enhanced uptake are common among these cell types. Glucose
uptake enhancement results from an increase in the of the
transport process with only a small change in the affinity
(Km) (Kletzien and Purdue, 1975; van Putten and Krans, 1975).
Transport activation also contains a protein synthesis-
dependent component, since exposure to cycloheximide
attenuates this response (Tordjman et al., 1990; Kitzman et
al., 1994). Metabolism or metabolites of glucose are not
reguired since incubation with 3-O-methyl glucose and 2-
deoxyglucose (2-deoxyglucose is only phosphorylated) can
prevent transport activation (Kitzman et al., 1994).
Regulation of Glucose Transporter Protein
Glucose deprivation also regulates glucose transport by
regulating the GLUT1 transporter protein level. Although not


41
clathrin-coated pits which occurs at the inside of the plasma
membrane surface (Lin et al., 1992; Lin et al., 1994) and the
purification of caveolin (Chang et al., 1991). Recently, it
has also been modified for use in the 3T3-L1 adipocytes for
analyzing the translocation of the GLUT1 glucose transporter
to the plasma membrane in response to insulin treatment
(Robinson and James, 1992). This procedure, therefore, can
be used to generate pure plasma membrane fragments which are
free from contamination of endoplasmic reticulum and golgi
membrane fractions.
3T3-L1 cells were grown on 22 mm round glass coverslips
(Fisher) and differentiated as described earlier. Once fully
differentiated, cells were incubated in the presence or
absence of glucose for the indicated period of time. The
cells were then washed 3 times in PBS at 4C. The glass
coverslip was removed and placed into a clean 35 mm tissue
culture before being incubated in the same solution
containing 0.5 mg/ml poly-L-lysine (M.W. > 100,000) for 1
minute. The monolayer was then washed three times at 4C
with 3.0 ml 1/3X sonication buffer [IX sonication buffer; 70
mM KC1, 30 mM HEPES, pH 7.5, 5 mM MgCl2, 3 mM EGTA]. This
solution was aspirated and replaced with 1 ml sonication
buffer at 4C. The monolayer was then immediately placed
under a 1/2-inch tapped flat horn at a distance of 5 mm and
sonicated for approximately 2 sec at power 1.5 on a Heat
Systems Sonicator. The glass coverslip was then washed three
times at 4C in PBS. All last traces of PBS were carefully


+Glc
TunTun
TunD-MEM
Tun5*- GIcNAc
-Glc
cn
vo


Figure 4-1 Biosynthesis of GLUT1 in Normal and Glucose-
Deprived 3T3-L1 Adipocytes Panel A Cells, cultured with or
without glucose for 36 hours, were metabolically labeled with
200 pCi/ml Tran35S-Label for the times indicated as described
in Chapter 2. GLUT1 was then immunoprecipitated from
membrane extracts and analyzed by SDS-PAGE and fluorography.
Panel B Densitometry of total GLUT1 during labeling. Total
GLUT1 intensity was obtained in all experiments by summing
the appropriate bands after densitometry of each species
separately (i.e. p36+p42+p46). Inset, radioactivity in
membrane fraction during labeling relative to 10 min label.
This result is representative of at least two independent
experiments.


154
is no specific trafficking difference of the glucose
transporter in glucose-deprived adipocytes, regardless of
glycosylation status. The subfractionation technique,
however, suffers from cross contamination of cell fractions;
In order to support these conclusions, cell surface
biotinylation and plasma membrane fragment isolation were
also utilized. These techniques also indicated that both p37
and p46 GLUT1 glycoforms were present at the plasma membrane.
These results, which determined the localization of the
glucose transporter in glucose-deprived adipocytes, represent
an important contribution to the discussion of the total
transport activity present at the surface of glucose-deprived
adipocytes.
Future Directions
Several aspects of the regulation of GLUT1 expression by
glucose are still unknown. The first is the determination of
the activity inherent in the p37 GLUT1 glycoform. Although
unlikely, the alternative oligosaccharide could confer
increased activity to GLUT1 in comparison to the normal GLUT1
glycoform. This would result in p37 GLUT1 contributing
significant activity to the total observed glucose uptake
Alternatively, p37 GLUT1 may have no or greatly reduced
activity due to altered glycosylation, despite being targeted
correctly to the plasma membrane. In this scenario, the
additional GLUT1 protein would not contribute significantly
to the total observed transport activity.


14
itself or a molecule closely resembling glucose is the
regulatory factor controlling transport activity.
Another effect of glucose deprivation in 3T3-L1
adipocytes is the appearance of a second protein recognized
by anti-GLUTl antiserum that migrates at a relative molecular
weight of 37 kDa, in contrast to the normal GLUT1 transporter
which migrates at a relative molecular weight of 46 kDa (Reed
and Vang, 1990; Kitzman et al., 1994). The migration of
these two proteins, when treated with N-Glycanase F, is
identical (36 kDa) (Haspel et al., 1986; Kitzman et al.,
1994). This led several groups to conclude that the p37
protein is a second GLUT1 transporter which contains a
different oligosaccharide than the normal transporter. Like
transport activation, the appearance of p37 was prevented by
exposure to cycloheximide, even when added 12 hours after the
withdrawal of glucose, indicating that the appearance of p37
required new protein synthesis (Kitzman et al., 1994). The
threshold glucose concentration required to elicit the
accumulation of p37 was 0.005 mM. The difference in glucose
concentration (approximately 100-fold) for the activation of
glucose transport and the appearance of p37 strongly suggests
that p37 is not involved in, or responsible for, the
activation of glucose uptake (Kitzman et al., 1994). In
agreement with this conclusion, incubation of glucose-
deprived cells with fructose prevents the appearance of p37
while allowing the activation of glucose transport (Haspel et
al., 1986, Kitzman et al., 1994). Aside from the


121
O 6 12 18 24
Incubation time, hours
Figure 5-9 GLUT1 Glvcosvlation and Glucose Deprivation in
CHO cells CHO-K1 cells were grown to confluence and then
placed into DMEM/10% dialyzed FBS supplemented with 0.1 mM
proline and 5 mM glucose. A total membrane fraction was
prepared at the times indicated and assayed for GLUT1 protein
by Western blotting. Densitometric analysis of GLUT1 protein
from glucose-deprived CHO cells from Figure 5-8.


150
adipocytes, membrane protein in general was likewise
upregulated in synthetic rate. These observations suggests
that either the alternative oligosaccharide placed on GLUT1
cannot be processed because it is not recognized as a
substrate by the processing enzymes, or that the aberrant
GLUT1 glycoform is trafficked beyond the processing enzymes
before they have opportunity to act upon it. In either case,
it is reasonable to conclude that adipocytes, like the CHO
cell line, adapt to the withdrawal of glucose by economizing
on oligosaccharide biosynthesis in order to be able to put a
carbohydrate chain on protein, which may preserve critical
activity of glycoproteins.
A careful analysis of the effect of glucose deprivation
on the turnover of the transporter had not been previously
analyzed due to technical difficulties. Experiments shown
here which overcome these difficulties indicate that the
major effect of glucose deprivation is the inhibition of
membrane protein turnover, including the GLUT1 transporter.
This effect of turnover inhibition is separate from the
effects observed for the alterations in the processing
pathway, because the turnover of the normal GLUT1 glycoform
is inhibited in the same manner as p37 GLUT1. This
observation can, like the alteration is oligosaccharide
biosynthesis, also be interpreted as a protective adaptation
to nutrient withdrawal; to inhibit the turnover of proteins
already present preserves the functionality of the cell in a
period when the activity of newly synthesized proteins is


72
\
modifications occur in the medial golgi, trans golgi, and the
trans golgi network. Some of the later sugar modifications
catatalyzed are the addition of N-acetylglucosamine, sialic
acid, fucose and nueraminic acid. A third type of
glycoprotein, the hybrid type, exhibits characteristics of
both high mannose and complex oligosaccharides, and often
contains a bisecting N-acetylglucosamine residue (Kornfeld
and Kornfeld, 1980). Sulfation, acetylation, and
phosphorylation of sugars are other types of oligosaccharide
modifications that are possible. For lysosomal proteins,
specific mannose sugars are phosphorylated, and this serves
to act as the appropriate recognition mechanism for targeting
to the lysosomes (Kaplan et al., 1977; Goldberg and Kornfeld,
1981).
The glucose-dependent regulation of glucose transport
has been described in many cell types (for review see Klip et
al., 1994). In each of these cell types, a common result of
glucose deprivation is an increase in the maximal velocity
(vmax) f glucose transport. However, the mechanism(s) (i.e.,
transcriptional, post-transcriptional, and post-
translational) responsible for this stimulation vary
according to cell type. Several investigators have
postulated that one component of the transport upregulation
might be an alteration of transporter turnover, particularly
GLUT1 (Yamada et al., 1983; Germinarlo et al., 1982; Walker
et al., 1989; Shawver et al., 1987; Haspel et al., 1991). In
particular, if the degradation of the glucose transporter


19
Some small peptides are poorly immunogenic; therefore,
in order to ensure antigenicity, they are often coupled to
larger proteins, termed immune carriers, (Harlow and Lane,
1988). Covalent conjugation of small proteins or peptides to
these immune carriers results in an antigen large enough to
elicit an immune response. Antiserum prepared in this manner
contain antibodies against the immune carrier and the
antigen. The proteins typcially chosen as immune carriers
are BSA, ovalbumin, or keyhole limpet hemocyanin. Careful
choice of the immune carrier can result in antiserum which
recognizes only the peptide because the immune carrier is
absent from the experimental system. Antisera prepared
against synthetic peptides have several advantages over
antisera generated against whole proteins (Harlow and Lane,
1988). First, the antibody can be generated without knowing
the entire sequence of the protein or purifying the protein.
Second, the antiserum can be generated in such a way that it
will be specific for specific regions of the protein of
interest. Finally, peptide-specific antibodies have a higher
potential of being conformation specific.
For the preparation of an antiserum against GLUT1, a
peptide (CEELFHPLGADSQV) that corresponds to amino acid
residues 480-492 of the GLUT1 sequence was chosen as the
antigenic region. This peptide, designated GT1, was
synthesized by the Protein Chemistry Core facility at the
University of Florida and verified by amino acid analysis.
An additional cysteine was added to the amino terminus of the


124
that had no glycogen would not be able to delay the
alteration in GLUT1 glycosylation during glucose deprivation.
We therefore incubated adipocytes in the presence of fructose
for 24 hours in order to first deplete the glycogen pool.
When these cells were then deprived of glucose, the
alteration in GLUT1 glycosylation was evident 12 hours
earlier than in cells that had a normal glycogen level.
These data strongly support the hypothesis that the glycogen
pool can be used, at least in part, for the production of
oligosaccharides in the absence of glucose, demonstrating the
importance of this pathway for maintaining cellular
metabolism.
Finally, the effect of glucose deprivation on the
glycosylation of GLUT1 in CHO cells was determined. The
glycogen content of CHO cells was measured in a manner
identical to that of the 3T3-L1 cells, and was found to be
approximately 1% of that in 3T3-L1 adipocytes, and only 10%
of that contained in glucose-deprived adipocytes. With the
extremely low amount of glycogen normally stored in these
cells, we predicted that the glycosylation of GLUT1 would be
altered rapidly upon the withdrawal of glucose. The data
demonstrates that the alternatively glycosylated glycoform of
GLUT1 appeared within 3 hours of glucose deprivation, the
most rapid adaptation yet described for this process.
In summary, we have demonstrated that the glycogen
contained in 3T3-L1 cells serves as a buffer for N-linked
oligosaccharide production in times of glucose stress. This


164
Whitesell, R.R., Regen, D.M., Pelletier, D., and Abumrad,
N.A. (1990) Diabetes 39, 1228-1234.
Wise, L.S., Sui, H.S., Rubin, C.S. (1984) J. Biol. Chem. 259,
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Yang, J., and Holman, G.D. (1993) J. Biol. Chem. 268. 4600-
4603.


123
gluconeogenic. Importantly, a part of this flux is drawn off
into the hexosamine pathway that generates precursors for the
production of oligosaccharides. The process of glycogen
depletion is completely reversible, supported by the
observation that glucose-deprived adipocytes, once given
glucose in the culture medium, accumulate glycogen in a
linear manner until normal levels are reached within 24
hours. These observations suggest that glycogen is utilized
for the adipocyte's own requirements during glucose stress
and that the pathways for the synthesis and breakdown of
glycogen remain intact whether the cells are incubated in the
presence or absence of glucose.
The surprising observation in this study is the
inability of the cells to use fructose to generate glycogen
in the absence of glucose. Given the previous observation
that fructose could prevent the alterations in GLUT1
glycosylation in the absence of glucose, we presumed that the
similarity in metabolism between these two sugars would also
allow fructose to substitute for glucose in the generation of
carbons for glycogen. Fructose, however, had no effect on
the depletion of glycogen in the absence of glucose,
suggesting that fructose may be preferentially used for other
pathways than glycogen production. This observation, however,
provided us with a method of depleting the cellular glycogen
content while maintaining the normal glycosylation of GLUT1.
We surmised that if glycogen was used for protein
glycosylation during periods of glucose deprivation, a cell


51
twelve hours after the onset of glucose deprivation,
prevented the appearance of p37 (Figure 3-2 A, right panel).
It has been generally accepted that p37 is a second GLUT1
protein (Haspel et al., 1986; Haspel et al., 1990; Tordjman
et al., 1991; Kitzman et al., 1994), although a careful
examination of both proteins has not been reported. The
following sections will describe experiments suggesting that
p37 is another form of the GLUT1 transporter only synthesized
in glucose-deprived cells.
Tryptic Digest of GLUT1 Transporter
To determine whether p46 and p37 arise from the same
protein, tryptic digests were performed with the expectation
that peptides generated by proteolysis would be identical if
both p37 and p46 derived from the same protein. It should be
noted that although many tryptic peptides can be generated,
only those retaining the carboxy-terminal region against
which the antiserum was generated could be recognized in the
Western blot. Membrane proteins from normal or glucose-
deprived (48 hours) cells were first isolated. These
membrane proteins were then incubated in the presence of
trypsin (10 pg) for the times indicated, when the reaction
was stopped with a 10-fold excess concentration of soybean
trypsin inhibitor. These samples were then resolved by 10%
SDS-PAGE and transferred to nitrocellulose. GLUT1 proteins
were then detected with the anti-GTl antiserum in a western
blot. GLUT1 peptides generated by partial trypsin treatment


97
readdition quickly restores normal GLUT1 glycosylation and
turnover.


37
manner similar to the plasma membrane fraction. The
supernatant from [B] was centrifuged at 212,000 x g for 75
min at 4C. The pellet, representing the low density
membrane fraction, was resuspended in TES buffer and
centrifuged at 212,000 x g for 75 min at 4C. This washed
pellet was resuspended into 0.3 ml TES, frozen in liquid
nitrogen, and stored at -20C. For Western blot analysis,
samples were diluted into sample dilution buffer containing 6
M urea and 10% B-mercaptoethanol before separation by SDS-
PAGE and transfer to nitrocellulose as described above.
Cell Surface Biotinylation
The modification of proteins with membrane impermeable
chemicals has recently proven to be an effective tool in the
study of membrane proteins, including the vitronectin
receptor (Nesbitt and Horton, 1992), Thy-1 and lymphocyte
surface marker proteins (Arni et al., 1992), surface proteins
of 3T3-L1 fibroblasts (Hare and Taylor, 1991), surface
proteins of 3T3-L1 adipocytes (Hare and Taylor, 1992),
insulin receptors (Levy-Toledano et al., 1993), and tumor
necrosis factor receptor (Hsu and Chao, 1993). Several
membrane-impermeant modifiers are now commercially available,
with different functional groups to confer different chemical
reactivities. Many of these modifiers are conjugated to
biotin, a small molecule that has a tight affinity for
avidin. Biotin has many advantages as a molecular tag; it is
small and in many cases does not abolish biological activity


29
the labeled protein, the label is then "chased" by incubating
the cells in a large excess of unlabeled amino acids in order
to dilute the specific activity of the intracellular amino
acid pools (Harlow and Lane, 1988). Immunoprecipitation and
recovery of the protein of interest during this period allows
the analysis of the radioactivity present in the initially
labeled protein pool and its disappearance due to degradation
as a function of time. The same strategy can be used for
very short labeling periods where the protein is labeled, and
then the processing or glycosylation of the protein can then
be followed by observing shifts in relative molecular weight
as assessed by gel electrophoresis. This section describes a
metabolic labeling procedure optimized for the labeling and
immunoprecipitation of GLUT1 expressed at endogenous levels
in 3T3-L1 adipocytes for subsequent gel electrophoresis and
fluorography.
Cells were incubated in 8 ml methionine- and cysteine-
free D-MEM without added serum for 1 hour to deplete the
intracellular pools of methionine and cysteine. The
depletion medium was aspirated and the cells incubated in 2
ml methionine- and cysteine-free DMEM containing 200 pCi/ml
Tran35S-Label for the specific times indicated. To initiate
the chase periods, the labeling medium was aspirated and
replaced with 8 ml complete DMEM/10% FBS containing 2 mM
methionine and 2 mM cysteine. These conditions were
established to generate an adequate signal for fluorography
while minimizing the time of methionine and cysteine


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R.A. (1993) Am. J. Phvs 265. C688-C694.


A
+ +
Glucose
B
Streptavidin-HRP
+ +
Glucose
Anti-GLUT1


Figure 6-3 Cell Surface Biotinylation 3T3-L1 adipocytes were incubated in the presence or
absence of glucose for 48 hours before being biotinylated with NHS-LC-biotin. Biotinylated
proteins were recovered with streptavidin-agarose and resolved by 7.5% SDS-PAGE. Panel A
Biotinylated proteins were detected in a modified Western blot using streptavidin-HRP.
Panel B GLUT1 in the biotinylated proteins pool was detected by Western blot using anti-GTl
antiserum. The positions of p46 and p37 GLUT1 are indicated, along with molecular weight
standards. This result is representative of at least three experiments.


Figure 4-2 Processing of GLUT1 in Normal and Glucose-
Deprived 3T3-L1 Adipocytes Cells incubated with (+ glucose)
or without (- glucose) glucose for 36 hours were labeled with
500 pCi/ml Tran35S-Label for 10 min and then chased for a
total of 60 min. GLUT1 was immunoprecipitated from membrane
extracts of each and analyzed by SDS-PAGE and fluorography.
Panel A Control adipocytes chased in the presence of
glucose. Panel B Glucose-deprived adipocytes chased in the
absence of glucose. Panel C Glucose-deprived cells chased
in the presence of glucose. Shown is the result of a single
experiment.


Figure 3-2 Effect of Cvcloheximide on GLUT1 Expression
During Glucose Deprivation 3T3-L1 adipocytes were incubated
in glucose-free medium for up to 48 hours and membrane
fractions prepared at the times indicated. Panel A Membrane
proteins probed for GLUT1 by Western blot with anti-GTl
antiserum. The right panel shows cells incubated with
cycloheximide (20 pM) added 12 hours after the withdrawal of
glucose. These samples were then analyzed in an identical
manner. Panel B Densitometry of GLUT1 protein from panel A.
These results are representative of at least 4 independent
experiments.


106
Figure 5-2 Glycogen Content of 3T3-L1 Adipocytes During
Glucose-Deprivation. Cells cultured in 10 cm dishes were
incubated in either complete medium (dmem/10% FBS) or
glucose-free medium (glucose-free DMEM/10% dialyzed FBS) for
24 hours. Glycogen was extracted from cells at the times
indicated as described in Chapter 2. Glycogen was then
hydrolyzed to glucose and assayed enzymatically. The
results, expressed as the average the standard error of the
mean, is representative of three independent experiments
measured in triplicate.


60
inclusion of N-acetylglucosamine, the production of
unglycosylated transporter could not be prevented. Together,
these data indicate that although tunicamycin resulted in the
generation of unglycosylated GLUT1, the effect of tunicamycin
could not be removed and therefore elimated tunicamycin as a
useful tool in the analysis of GLUT1 processing. The
inability to remove tunicamycin in these experiments is not
consistent with the previous report on the processing of the
insulin receptor, but it may lie in the processing of a
multi-subunit protein such as the insulin receptor is
fundamentally different from a monomeric glycoprotein like
GLUT1.
Glvcosidase Digestion of GLUT1 Glvcoforms
In order to characterize the type of oligosaccharide
present on p46 and p37 GLUT1, membrane proteins from normal
or glucose-deprived cells were digested with either
endoglycosidase H, which cleaves high mannose
oligosaccharides, or N-Glycosidase F, which cleaves all N-
linked oligosaccharides. For comparison of these glycoforms,
membrane proteins from the LEC1 CHO cell line were also
tested. The LEC1 CHO cell line is a glycosylation mutant
that is deficient in N-acetylglucosamine transferase I, the
first committed step towards the production of hybrid and
complex type glycoproteins (Stanley and Chaney, 1985). As a


5-2 Glycogen Content and Glucose Deprivation 106
5-3 Glycogen Content and Glucose Refeeding 108
5-4 Glycogen Content in Fructose-Incubated Adipocytes. 109
5-5 Effect of Insulin and 3-O-methylglucose on Glycogen
Content 112
5-6 Glycogen Depletion and GLUT1 Glycosylation 114
5-7 Glycogen Depletion and GLUT1 Glycosylation 116
5-8 GLUT1 Glycosylation and Glucose Deprivation in CHO
cells 119
5-9 GLUT1 Glycosylation and Glucose Deprivation in CHO
cells 121
6-1 Subcellular Fractionation of 3T3-L1 Adipocytes 133
6-2 Subcellular Fractionation of 3T3-L1 Adipocytes 135
6-3 Cell Surface Biotinylation 139
6-4 Plasma Membrane Fragment Isolation 143
ix


BIOGRAPHICAL SKETCH
Robert Joseph McMahon was born in Flemington, New
Jersey, in 1968. He was the last of the line in a family
that included three brothers and two sisters. He was raised
in Clinton, New Jersey, until he attended college at the
Florida Institute of Technology in Melbourne, Florida. There
he graduated with a bachelor's degree in molecular biology
before attending the University of Florida to pursue his
doctorate. He married Laura Gisella Michelassi on May 23,
1992 in Gainesville, Florida. They have one daughter, Amelia
Frances, born on June 23, 1995.
165


CH0-K1
Time, h 0 3 6 9 12 24 24
Glucose
3T3-L1
25 5 0 Glucose, mM
120


CHAPTER 2
MATERIALS AND METHODS
Materials
Dulbecco's Modified Eagle's Medium (D-MEM), Modified
Earle' Medium, a modification (a-MEM), and D-MEM base powder
was obtained from Life Technologies, Inc. Calf serum (Cat.
No. 1100-90) and fetal bovine serum (FBS) (Cat. No. 1020-75)
were obtained from Intergen. Glucose-free FBS was prepared
by dialyzing against phosphate buffered saline, pH 7.4, (PBS)
for 48 hours at 4C, with a molecular weight cutoff of 13,000
(dialysis tubing obtained from SpectraPor). L-methionine, L-
cysteine, L-proline, D-glucose, D-fructose, D-xylose, 3-0-
methylglucose, methylisobutylxanthine, dexamethasone, protein
A Sepharose, Keyhole Limpet Hemocyanin, Sephadex G-100,
Freund's Complete and Incomplete Adjuvant, polyoxyethylene 9
lauryl ether (C12E9), octanoic acid, bovine serum albumin,
poly-L-lysine (M.W. > 150,000), and anti-rabbit IgG
conjugated horseradish peroxidase were obtained from Sigma.
Insulin was a generous gift of Dr. Ronald Chance of Eli Lilly
Corp. Tran35S-Label (1100 Ci/mmol) was obtained from ICN.
Aquacide was obtained from Calbiochem. Streptavidin-agarose,
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-
MBS), the Sulfolink matrix, and sulfosuccinimidyl-6-
(biotinamido) hexanoate (NHS-LC-Biotin) were obtained from
16


118
of time that the cell can tolerate glucose deprivation before
alterations in glycosylation begin.
GLUT1 Glvcosvlation from Glucose-Deprived CHO Cells
To support our conclusion that cells that cannot store
glycogen are not able to maintain protein glycosylation in
the absence of glucose, CHO cells were placed into glucose-
containing or glucose-free medium for 24 h while assessing
GLUT1 glycosylation by Western blot. To be consistent with
the measurement in the 3T3-L1 cells, the appearance of the
low molecular weight GLUT1 species in glucose-deprived CHO
cells was taken as a measurement of the time required for the
alternative glycosylation of GLUT1. As seen in Figures 5-8
and 5-9, a low molecular weight GLUT1 species also migrating
at 37 kDa, as in the 3T3-L1 adipocytes, is detectable within
3 hours of glucose deprivation, appearing much more rapidly
than in normal or glycogen-depleted 3T3-L1 adipocytes.
Conclusions
The glycosylation of the GLUT1 glucose transporter
during glucose deprivation differs according to cell type.
The goal of this section was to determine if the response of
two different cell types towards GLUT1 glycosylation during
glucose deprivation was due in part to the amount of glycogen
stored in each cell. In the 3T3-L1 adipocyte, the GLUT1
transporter has been previously found to be glycosylated
during glucose deprivation, but with a relatively small


30
depletion based on previous reports of possible alterations
in protein turnover with prolonged amino acid depletion
(Haspel et al., 1985).
GLUT1 Immunoprecipitation
Immunoprecipitation is a procedure by which a particular
protein can be isolated from a crude mixture of proteins by
incubation and binding of specific antibodies. Once this
immunocomplex is formed, it is captured on a substrate,
commonly protein A Sepharose, so that it can be removed from
solution. Proteins associated in a non-specific manner can
then be removed by washing the protein A Sepharose-
immunocomplex. Finally, the immunoprecipitated protein can
be released by destroying the interaction between the
antibody and the protein A, usually with strong denaturing
solutions such as urea, low pH, SDS, or a combination of
these conditions (Harlow and Lane, 1988).
GLUT1 was immunoprecipitated from the total membrane
fraction in the following manner. A total membrane fraction
was obtained by first washing the monolayer in cold PBS. The
cells were then scraped into TES buffer and homogenized by 20
strokes in a 10 ml Potter-Elvejhem flask with a teflon
pestle. The homogenate was then centrifuged at 212,000 x g
for 1 hour at 4C to collect a total membrane pellet. The
final membrane pellet was then homogenized with 20 strokes of
a 2 ml Potter-Elvehjem homogenizing flask with a teflon
pestle in 1 ml extraction buffer [PBS containing 1 mM EDTA,


22
al., 1985). The eventual purpose of this technique was to
produce an antibody fraction that could be used for the
purification of peptide specific antibodies.
First, the pH of approximately 15 ml whole rabbit serum
was adjusted to 5 with 3M acetic acid. While vigorously
stirring, 0.75 ml octanoic acid (1 part in 20) was added
dropwise; the solution was then stirred an additional 30 min
at room temperature. The precipitate was collected at 41,000
x g for 30 min at 4C and the pellet discarded. The
supernatant was then removed and its volume determined. An
equal volume of ammonium sulfate (saturated solution in
water) was added and stirred overnight at 4C. The
precipitate was then collected by centrifugation at 41,000 x
g for 30 min at 4C. The pellet, consisting of primarily
immunoglobulin, was dissolved in PBS and the concentration of
this solution was estimated by measuring its absorbance at
280 nm (using a milligram extinction coefficient of 1.4).
The ammonium sulfate was then removed by dialysis against
three changes of PBS over 48 hours at 4C with a molecular
weight cutoff of 13,000 daltons.
The purification of peptide specific antibodies from the
total antibody fraction provides an advantage of reducing
non-specific proteinantibody interactions that could
complicate the interpretation of data gathered by
immunoprecipitation. Peptide-specific antibodies were
purified from the total IgG fraction by affinity
chromatography over a column of GT1 peptide immobilized on an


Figure 3-3 Tryptic Digest of p46 and p37 3T3-L1 adipocytes
were incubated in the presence (+Glucose) or absence (-
Glucose) of glucose for 48 hours before a total membrane
fraction was prepared. These membrane proteins were
incubated trypsin (10 pg) for the times indicated before the
reaction was stopped with excess trypsin inhibitor. The
digested membrane proteins were then probed for GLUT1 by
Western blot with anti-GTl antiserum. Molecular weight
standards are indicated. This result is representative of at
least two independent experiments.


Figure 3-6 Glvcosidase Digestion of GLUT1 Glvcoforms Membrane protein from normal or
glucose-deprived 3T3-L1 adipocytes (20 pg/lane) or LEC1 CHO cells (10 pg/lane) were
denatured and treated with the indicated glycosidase for 2 hours at 37C. Membrane proteins
were then probed by Western blot with anti-GTl antiserum. The position of p46, p37, and p36
GLUT1 are indicated along with molecular weight standards. This result is representative of
at least three independent experiments.


50
A
Glucose Deprivation, hours
0 3 6 9 15 24 36 48 15 24 36 48
45-
-cycloheximide +cycloheximide
at 12 hours
t p46
p37


either the withdrawal of glucose or the alteration in N-
linked glycosylation.
Xll


3
glucose was not accomplished by the same protein in every
cell type. Screening cDNA libraries from other tissues at
low stringency led to the cloning of additional glucose
transporters, which in total are now known as the GLUT
protein family (GLUcose Transporter), a group of structurally
and functionally related integral membrane glycoproteins that
mediate the tissue-specific uptake of glucose (reviewed by
Gould and Bell, 1990). The expression of GLUT1 is virtually
ubiquitous; in most tissues, it is thought to be responsible
for a basal, constitutive transport activity. GLUT2 is
expressed predominantly in liver and is characterized by a
relatively high affinity constant; its role has been ascribed
to equilibrating glucose between the hepatocyte and blood
plasma according to the extracellular glucose concentration.
GLUT3 is expressed primarily in fetal muscle and brain
tissue. GLUT4 is expressed in insulin-sensitive tissues such
as heart, muscle, and adipose. The localization of GLUT4 can
be altered upon the binding of insulin, where the transporter
is translocated from an intracellular compartment to the
plasma membrane. GLUTS is a recently cloned transporter that
is characterized by a high affinity for fructose; its role in
glucose transport and utilization has not yet been
elucidated. GLUT 6, a recently cloned gene, is a non-
expressed pseudogene. GLUT7, a component of the glucose-6-
phosphatase system, is localized in the endoplasmic reticulum
of the hepatocyte where its role is vital in gluconeogenesis.
The complex arrangement of transporter family, exhibiting


162
Mueckler, M., Caruso, C.( Baldwin, S., Pnico, M., Blench,
I., et al., (1985) Science 229, 942-945.
Mueckler, M., Lodish, H.F. (1986) Cell 44. 629-637.
Nesbitt, S.A., Horton, M.A. (1992) Anal. Biochem. 206. 267-
272.
Novikoff, A.B., Novikoff, P.M., Rosen, O.M., and Rubin, C.S.
(1980) J. Cell. Biol. 87, 180-196.
Nunez, E. and Aragon, C. (1994) J. Biol. Chem. 269, 16920-
16924.
Olden, K., Pratt, R., Jaworski, C., and Yamada, K. M. (1979)
Proc. Natl. Acad. Sci. U.S.A. 76, 791-795.
Pessin, J.E., Tillotsin, L.G., Yamada, K., Gitomer, W.,
Carter-Su, C., Mora, R., Isselbacher, K.J., Czech, M.P.
(1982) Proc Natl Acad Sci U.S.A. 79, 2286-2290.
Peterson, G.L. (1979) Anal. Biochem. 100, 201-205.
Pfleiderer, G. (1963) Methods of Enzymatic Analysis Academic
Press, NY, 91-103.
Rademacher, T.W., Parekh, R.B., and Dwek, R.A. (1988) Ann.
Rev. Biochem. 57, 785-838.
Rearick, J.I., Chapman, A., and Kornfeld, S. (1981) J. Biol.
Chem. 256. 6255-6261.
Reed, B.C., Shade, D., Alperovich, F., and Vang M. (1981)
Arch. Biochem. Biophvs. 279. 261-274.
Reed, B.C., Shade, D., Alperovich, F., Vang, M. (1990) J.
Biol. Chem. 279. 261-274.
Resh, M.D. (1983) Biochem. 22, 2781-2784.
Robinson, L.J., and James, D.E. (1992) Am. J. Phvs. 263,
E383-E393.
Ronnet, G. V-, Knutson, V. P., Kohanski, R. A., Simpson, T.
L., and Lane, M. D. (1984) J. Biol. Chem. 259, 4566-
4575.
Rubin, C.S., Hirsch, A., Fung, C., Rosen, O.M. (1978) J.
Biol. Chem. 253. 7570-7578.


24
E
c
o
oo
C\J
4
03
c
o
-t
o
tc
o
c
CO
.Q
o
c/)
<
Fraction Number
Figure 2-1 Elution Profile of Peptide-Purified anti-GTl
Antibody IgG from a total antibody fraction was incubated
with the anti-GTl column for 2 hours at room temperature.
Unbound antibody was washed from the column with PBS, and
specifically bound antibody was eluted with 0.1 M glycine, pH
3.0. The absorbance of 1 ml fractions collected during the
elution was then measured at 280 nm. This profile is
representative of at least ten elution profiles performed on
this column.


Time, h
Fructose
Glucose
0
6 12 18 24 36 60
115


31
2% Ci2E9, 0.1% SDS and protease inhibitor cocktail as above].
Any insoluble material was then removed by centrifugation in
a microcentrifuge for 5 min at 4C. The supernatant was then
recovered, frozen in liquid nitrogen, and stored at -20C.
In order to eliminate non-specific interactions between
membrane proteins and immunoglobulin, thawed extracts
(containing between 1.5 2 mg membrane protein) were
incubated with an unrelated non-immune antiserum (5 pi) and
collected with a 50% suspension of Protein A Sepharose (25
pi) for 1 h at 4C. Samples were centrifuged briefly in a
microcentrifuge to pellet the protein A Sepharose pellet and
the pre-cleared supernatants transferred to new
microcentrifuge tubes. Extracts were adjusted to equal
protein concentration in 1 ml with additional extraction
buffer and incubated with the anti-GTl antibody (5 pg) for 3
hours at 4C with end-over-end rotation. The 50% Protein A
Sepharose suspension in PBS was then added (25 pi) for 2
hours to collect the immunoprecipitates. The Protein A
Sepharose was then washed 3 times with extraction buffer,
followed by 4 times for 10 min with extraction buffer
containing 1M NaCl. Immunoprecipitates were released by
incubation in 0.1 ml sample dilution buffer containing 6 M
urea and 10% J3ME for 15 min at 37C. The supernatants were
then loaded onto an 8% SDS-PAGE gel and run overnight at 50
V. For fluorography, the gels were fixed in 10% TCA/40% MeOH
for 30 min, soaked in water for 30 min, and then soaked in 1M
sodium salicylate for 1 h before drying at 60C under vacuum.


CHAPTER 3
CHARACTERIZATION OF GLUT1 GLYCOFORMS
Introduction
Glucose deprivation results in the appearance of a lower
molecular weight protein recognized by anti-GLUTl antiserum
in several cell lines, including 3T3-C2 fibroblasts (Haspel
et al., 1991), 3T3-L1 fibroblasts (Reed et al., 1990), 3T3-L1
adipocytes (Reed et al., 1990; Tordjman et al., 1990; Kitzman
et al., 1994), and normal rat kidney cells (Haspel et al.,
1990). The appearance of this low molecular weight protein
can be prevented by incubation with fructose, suggesting that
sugar metabolism is required to prevent its appearance
(Haspel et al., 1986; Kitzman et al., 1994). In normal rat
kidney cells and 3T3-C2 cells, the migration of this protein
was not altered upon digestion by endoglycosidase F and
therefore was interpreted to be unglycosylated (Haspel et
al., 1986; Haspel et al., 1990). In 3T3-L1 adipocytes,
however, the lower molecular weight protein is glycosylated,
but with a smaller oligosaccharide (Kitzman et al., 1994).
Careful examination of this protein and its relationship to
the normal GLUT1 protein in 3T3-L1 adipocytes has not been
reported. This section describes the characterization of the
normal GLUT1 protein as well as the lower molecular weight
protein that appears in glucose-deprived 3T3-L1 adipocytes.
43


64
which was predicted to be a high mannose glycoprotein, was
sensitive to digestion with endoglycosidase H and was
resolved to a 36 kDa core protein. Together, these data
indicate that the low molecular weight GLUT1 present in
glucose-deprived 3T3-L1 adipocytes is glycosylated, but with
a relatively small oligosaccharide (approx. 1 kDa). While
this oligosaccharide is N-linked, it is not a high mannose
structure as might be expected for a normal processing
intermediate prematurely terminated due to the lack of
glucose. This oligosaccharide on the low molecular weight
GLUT1 from glucose-deprived 3T3-L1 adipocytes likely
represents an alternative oligosaccharide generated in the
absence of glucose.
Specificity and Efficiency of GLUT1 Immunoprecipitation
The procedure for immunoprecipitation of GLUT1 from 3T3-
L1 adipocytes was assessed using metabolically labeled
control or glucose-deprived adipocytes. A wide band
migrating at 46 kDa (p46) was immunoprecipitated from
membrane extracts of control cells using purified anti-GTl
antibody (-GT1, + glucose) (Figure 3-7). In contrast, a 37
kDa protein was immunoprecipitated from cells deprived of
glucose for 36 hours (-GT1, glucose). The specificity of
the immunoprecipitation procedure was verified by
immunoprecipitating GLUT1 in the presence of 1 pg of the GT1
peptide. Inclusion of this peptide completely blocked the
immunoprecipitation of p46 GLUT1 in control cells, as well as


25
partially purify a cellular fraction which is enriched in
membrane protein. This affords the advantage of preparing
relatively concentrated preparations of the membrane protein
of interest, and furthermore, reduces non-specific
interactions in procedures such as Western blotting and
immunoprecipitation. To this end, GLUT1 was routinely
partially purified by preparing a cell lysate fraction which
was depleted of cytosolic and nuclear proteins. To prepare
this fraction, the culture medium was aspirated and the cell
monolayer rinsed in phosphate-buffered saline (PBS) at 4C.
Cells were then scraped into 5 ml TES buffer [20 mM Tris-HCl,
pH 7.4, 1 mM EDTA, 250 mM sucrose, and protease inhibitors
(20 pg/ml each of aprotonin, leupeptin, pepstatin, TPCK,
TLCK, and 1 mM PMSF)] and homogenized by 20 strokes in a 10
ml Potter-Elvejhem homogenizing flask with a teflon pestle.
The homogenate was centrifuged at 1300 x g for 5 min at 4C
to remove nuclei and unbroken cells. The supernatant was
centrifuged at 212,000 x g for 1 hour at 4C to pellet the
remaining particulate fraction. The supernatant, containing
cytosolic proteins, was discarded and the final membrane
pellet resuspended in TES buffer (usually 0.3 0.5 ml) by
homogenization. The resuspended membrane fraction was
quickly frozen by submersion in liquid nitrogen and then
stored until use at -20C.


125
conclusion is based upon the following observations; (i) the
glycogen pool in 3T3-L1 adipocytes is depleted during periods
of glucose deprivation; (ii) prior depletion of the glycogen
pool before glucose-deprivation results in a more rapid
alteration in GLUT1 glycosylation than in cells with normal
glycogen stores; (iii) a cell line in which the glycosylation
of GLUT1 is altered very quickly upon glucose deprivation
contains very little glycogen. Glycogen, therefore, serves
as an important buffer not only for the generation of energy
via glycolysis, but the production of structural and
regulatory molecules in the form of oligosaccharides.


42
removed from the coverslip and tissue culture dish. Sample
dilution buffer (50 pi) was added directly to the coverslip
and the glass scraped with a rubber scraper. The sample
dilution buffer was then recovered and loaded onto a mini
SDS-PAGE gel for separation. The presence of GLUT1 and GRP78
was detected by Western blotting as described above.


89
Glucose + + + + + +
45
B
Chase Time, hrs


87
glycoform was synthesized; no metabolically labeled p37 was
observed (Figure 4-4).
GLUT1 Half-Life in Control and Glucose-Deprived Adipocytes
To determine the glucose-dependent regulation of GLUT1
turnover in 3T3-L1 adipocytes, control and glucose-deprived
cells were metabolically labeled for 1 hour and then chased
in either the presence or absence of glucose. Cells were
harvested at various times during the chase period for
immunoprecipitation of GLUT1. GLUT1 immunoprecipitates were
then analyzed by SDS-PAGE and fluorography. The normal GLUT1
glycoform (p46) in control adipocytes exhibited a half-life
of approximately 14 h (Figure 4-5 A, B). When normal
adipocytes were labeled for 1 hour and then chased in the
absence of glucose, the turnover of GLUT1 was similar for
approximately the first 12 hours to that of cells chased in
the presence of glucose. Thereafter, the turnover of GLUT1
was significantly inhibited, exhibiting a turnover time of
greater than 50 hours. Likewise, the aberrant GLUT1
glycoform (p37) synthesized in glucose-deprived cells was not
significantly degraded when chased in the absence of glucose
(Figure 4-6 A, B). Additionally, a second GLUT1 glycoform,
although not present during the initial labeling period (time
= 0) appeared during the chase period. The turnover of this
GLUT1 glycoform, which exhibited a relative migration of


Specificity and Efficiency of GLUT1
Immunoprecipitation 64
Conclusions 67
REGULATION OF GLUT1 BIOSYNTHESIS, PROCESSING AND
TURNOVER BY GLUCOSE IN 3T3-L1 ADIPOCYTES 70
Introduction 70
Results 75
Synthesis and Glycosylation of GLUT1 in Control and
Glucose-deprived 3T3-L1 Adipocytes 75
Regulation of GLUT1 Processing by Glucose 78
Synthesis of GLUT1 Glycoforras During Glucose
Deprivation and Glucose Refeeding 81
GLUT1 Half-Life in Control and Glucose-Deprived
Adipocytes 87
Conclusions 92
REGULATION OF GLYCOGEN CONTENT AND GLYCOSYLATION OF
GLUT1 GLUCOSE TRANSPORTER 98
Introduction 98
Results 102
Time Course and Concentration Curve of Glucose
Assay 102
Effect of Glucose-Deprivation on Glycogen Content
in 3T3-L1 Adipocytes 103
Glycogen Accumulation During Refeeding 105
Effect of Fructose on Glycogen Depletion in
Glucose-Deprived Adipocytes 105
Effect of Insulin and 3-0-methylglucose on the
Glycogen Level in Glucose-Deprived 3T3-L1
Adipocytes Incubated with Fructose 107
Prior Depletion of the Cellular Glycogen Pool
Results in a More Rapid Alteration of
Glycosylation in the Absence of Glucose Ill
Glycogen Content in the CHO Cell Line 113
GLUT1 Glycosylation from Glucose-Deprived CHO
Cells 118
Conclusions 118
GLUT1 TARGETING IN NORMAL AND GLUCOSE-DEPRIVED 3T3-L1
ADIPOCYTES 126
Introduction 126
Results 131
Subfractionation of Control and Glucose-Deprived
3T3-L1 Adipocytes 131
Cell Surface Biotinylation of Control and Glucose-
deprived 3T3-L1 Adipocytes 138
Conclusions 145
vi


27
Na+, K+ tartrate, 1.0% SDS to 1 part 4% CuS04]), was added,
mixed well, and incubated at room temperature for 10 minutes.
Folin Phenol (0.1 ml, IN) was then added to each sample and
mixed well immediately after addition. The samples were
incubated for 45 min at room temperature for color
development. The absorbance of the samples was then
determined at 650 nm. The protein concentration of the
unknown samples was then determined from the standard curve.
Gel Electrophoresis
Gel electrophoresis was performed essentially as
originally described (Laemmli et al., 1970). Protein samples
were diluted with an equal volume of sample dilution buffer
consisting of 4% SDS, 6M urea, 10% B-mercaptoethanol, 0.15
mg/ml bromophenol blue, 40% glycerol and 20 mM Tris-base, pH
6.6. Gels were run at approximately 45 V overnight at room
temperature.
Electrotransfer and Western Blotting
The detection of proteins by the western blotting method
requires that the protein sample be transferred to a
substrate, usually nitrocellulose. The transfer buffer and
conditions closely follow the original procedure outlined by
Towbin et al., (1979). The electrophoresis gel was removed
from the gel apparatus and soaked in transfer buffer for 30
min at room temperature prior to transfer. Resolved proteins
were then transferred to nitrocellulose at 250 mA for 2.5


132
whose localization has been well established. The first
protein marker, glucose-regulated protein 78 (GRP78), is a
chaperone localized to the endoplasmic reticulum, where it is
involved in the folding of integral membrane proteins, as
well as secretory proteins (for review see Lee, 1992). The
localization of this protein has been established by
subfractionation, immunofluoresence, and immunoelectron
microscopy. A second protein was used, the insulin-sensitive
glucose transporter GLUT4, which is translocated from an
intracellular site to the plasma membrane upon insulin
binding. In the absence of insulin, the localization of this
protein is primarily intracellular (Gould and Bell, 1993).
The localization of GLUT1 is approximately 20% at the plasma
membrane (Fisher and Frost, submitted for review), although
there is constant cycling between an intracellular site and
the plasma membrane (Yang and Holman, 1993). 3T3-L1
adipocytes were incubated for 48 hours in either the presence
or absence of glucose. The cells were then subfractionated
to obtain a plasma membrane, high density, and low density
membrane fraction. Equal amounts of protein were resolved by
7.5% SDS-PAGE and transferred to nitrocellulose. The blots
were then probed for the presence of the three marker
proteins. In the first blot, GRP78 was detected (Figures 6-1
and 6-2). GRP78 is most prevalent in the high density
fraction, indicating enrichment of ER proteins, although
there is some cross contamination in the plasma membrane, in
the starved state, the amount of GRP78 is upregulated,


18
for most experiments) and grown to confluence in a-MEM medium
containing 10% FBS. Cells were used between days 1 and 5
post-confluence. For glucose deprivation of the CHO-K1 cells
and the LEC1 CHO cells, the a-MEM medium was aspirated and
replaced with glucose-free DMEM containing 10% dialyzed FBS.
This medium was supplemented with 0.1 mM proline in order to
satisfy the auxotrophic requirement of the CHO cell line .
Incubation of all cells with other sugars (i.e., xylose,
fructose, 3-O-methylglucose) was performed by adding the
appropriate amount of crystalline sugar in glucose-free DMEM
containing 10% dialyzed FBS to obtain a final concentration
of 25 mM.
Antibody Production
The detection of GLUT1 transporter protein was
accomplished through the generation of a polyclonal antiserum
against a peptide corresponding to the carboxy terminus of
the amino acid sequence. This region was chosen because of
its charged nature and its low degree of identity compared to
the same region in other members of the GLUT family.
Although use of this crude antiserum is adequate for the
detection of GLUT1 in Western blotting applications, it was
necessary to purify the GLUTl-specific antibodies for use in
immunoprecipitation experiments. The following sections
describe the generation of the anti-GLUTl antiserum and the
purification of the peptide-specific antibodies.


73
were inhibited in glucose-deprived cells while the synthesis
of the transporter remained constant, the end result would be
an increased number of GLUT1 molecules available for glucose
transport (increasing the V^). Few studies, however, have
investigated the processing and turnover of GLUT1 directly
because of the technical difficulties involved (Haspel et
al., 1985). The primary goal of this chapter is to define
the processing pathway of the GLUT1 transporter and determine
the kinetics of GLUT1 turnover in both the presence and
absence of glucose.
In 3T3-L1 adipocytes, glucose deprivation does not
result in a significant change in the level of GLUT1 protein
(as measured by Western blotting) over the period in which
transport induction is observed (Kitzman et al., 1994).
Beyond this time, we and others have reported the
accumulation of a lower molecular weight GLUT1 species which
results in a 2-fold increase in the total GLUT1 mass by 48
hours of glucose deprivation. (Reed et al., 1990; Kitzman et
al., 1994; Haspel et al., 1991). The role of this second
GLUT1 protein in transport stimulation has been investigated
and found not to be responsible for the activation of
transport during glucose deprivation (Kitzman et al., 1994).
However, the origin and characteristics of this lower
molecular weight GLUT1 and its relationship to the normal
GLUT1 glycoform were unknown. Several scenarios are
possible. One possibility is that the low molecular weight
GLUT1 glycoform is a normal intermediate in GLUT1 processing




75
This has allowed a clear model of the processing and turnover
of the metabolically labeled transporter to be obtained in
both normal and glucose-deprived adipocytes.
Results
Synthesis and Glvcosvlation of GLUT1 in Control and Glucose-
deprived 3T3-L1 Adipocytes
To determine if glucose plays a role in regulating the
synthetic rate of GLUT1, control and glucose-deprived
adipocytes were methionine and cysteine depleted before being
metabolically labeled for 10 to 60 min. GLUT1 was then
immuoprecipitated from solubilized membrane extracts and
analyzed by SDS-PAGE and fluorography. Figure 4-1 A shows
the fluorograph of the GLUT1 immunoprecipitates. Two GLUT1
species were synthesized in control cells during the first 10
min of labeling; a 36 kDa protein, and a glycosylated
intermediate migrating at 42 kDa. The 36 kDa protein is
likely the core protein based on the predicted molecular
weight of the core GLUT1 protein and tunicamycin experiments
(see Chapter 3). When normal adipocytes were labeled for an
additional 10 min, the mature GLUT1 protein (46 kDa) was
observed along with p36 and p42. No further GLUT1 proteins
were observed out to 60 minutes of labeling. When glucose-
deprived cells were labeled for 10 min, only the 37 kDa GLUT1
protein was observed. The 36 kDa core GLUT1 protein is
likely present but obscured due to the width of the p37


116
O 12 24 36
Glucose deprivation, hours
Figure 5-7 Glycogen Depletion and GLUT1 Glvcosvlation Cells
were pre-incubated in either 25 mM glucose or 25 mM fructose
for 24 hours before complete sugar withdrawal for 36 hours.
At the times indicated, a total membrane fraction was
collected for GLUT1 analysis by Western blotting. Equal
protein (50 pg/lane) was resolved by 7.5% SDS-PAGE.
Densitometric analysis of GLUT1 protein from Figure 5-6,
expressed relative to p46 GLUT1 at time 0.


53
+Glucose
Incubation time, min 0 0.5 1 23 4 5 610
- -Glucose


Endo H
N-G F
45
Glucose-
fed
II
Glucose-
deprived
Glucose-
fed
II
II
It
+
- +
- +
+
- ¡
1
1
i
3T3-L1
LEC1 CHO
- p46

O


142
reaches the plasma membrane, the appropriate location for the
transporter.
Another method that has recently become popular for the
study of membrane proteins and the cellular processes that
occur at the plasma membrane was also used. The isolation of
plasma membrane fragments is potentially a powerful tool to
analyze cell surface proteins, because no modification of the
surface is required, and a very pure plasma membrane fraction
can be obtained. This technique has also proved useful in
the study of transporter targeting, where the application of
this method resulted in the largest fold difference ever
reported for the translocation of the insulin-sensitive
glucose transporter GLUT4 from an intracellular site to the
plasma membrane (Robinson and James, 1992).
Once again, the GRP78 protein was used as a marker to
measure the relative amount of cross-contamination of the
endoplasmic reticulum in the plasma membrane. Adipocytes
grown on glass coverslips were incubated in the presence or
absence of glucose for 48 hours, adhered with poly-L-lysine,
and sonicated as described in Chapter 2. The plasma membrane
fragments were then solubilized in sample dilution buffer and
resolved on 7.5% mini-SDS PAGE gels. Plasma membrane
proteins were probed for GLUT1 and GRP78 with the appropriate
antiserum. Samples of the total membrane fractions from
identically incubated cells were also probed to determine
which proteins are present in the total cell. In the total
membrane fraction, only the normal GLUT1 protein was observed


36
some cross-contamination, this procedure will still provide
useful information in conjunction with other procedures to
analyze protein targeting.
Plasma membrane, high density, and low density membrane
fractions were isolated with the following subfractionation
procedure developed by Fisher and Frost (in preparation).
3T3-L1 adipocytes cultured in 10 cm tissue culture plates (5
plates per condition) were washed in PBS at 4C before being
scraped into 20 ml TES buffer. The cells were passed by a
ball bearing in a homogenizing block with a 0.0025 inch
clearance ten times at 4C. The homogenate was centrifuged
at 17,369 x g for 15 min at 4C [A]. The supernatant from
[A] was centrifuged at 48,254 x g for 30 min at 4C [B].
The pellet from [A] was resuspended into 25 ml TES buffer and
layered over 11.5 ml sucrose cushion of TES containing 1.12M
sucrose. This step gradient was centrifuged at 95,133 x g
for 65 min at 4C in a swinging bucket rotor [C]. The
interface from the step gradient, representing the plasma
membrane enriched fraction, was centrifuged at 48,254 x g for
30 min at 4C [D]. The pellet from [D] was resuspended into
TES buffer and centrifuged again at 48,254 x g for 30 min at
4C. This washed plasma membrane pellet was resuspended into
TES buffer, frozen in liquid nitrogen and stored at -20C.
The pellet resulting from the [B], representing the high
density membrane fraction, was resuspended into TES buffer
and centrifuged at 48,254 x g for 30 min at 4C. This washed
pellet was resuspended into 0.3 ml TES, and stored in a


Figure 4-4 Biosynthesis of t>37 GLUT1 During Glucose-Refeeding Panel A Glucose-deprived
adipcoytes (36 hours) were placed into glucose-containing medium and labeled at every hour
during the first six hours of refeeding. GLUT1 was then immunoprecipitated from membrane
extracts of each sample and analyzed by SDS-PAGE and fluorography.


REGULATION OF EXPRESSION OF THE GLUCOSE TRANSPORTER GLUT1 BY
GLUCOSE IN 3T3-L1 ADIPOCYTES
By
ROBERT JOSEPH MCMAHON
A THESIS 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
1995


95
extracts from glucose-deprived cells indicated that when
given the appropriate substrates, all the enzymes required
for the synthesis of the complete core oligosaccharide were
intact. There appeared to be no regulation of
oligosaccharide biosynthesis at the enzyme level; the levels,
therefore, of critical intermediates in the biosynthetic
pathway for the generation of core oligosaccharide were
measured. The levels of dolichol-P-mannose were present were
similar in both normal and glucose-deprived cells, but the
level of GDP-mannose, the precursor that donates the mannose
residue to elongate the core oligosaccharide chain from
GlcNAc2Man5 to GlcNAc2Man9, were drastically reduced.
Additionally, the levels of dolichol-P-mannose were slightly
elevated in glucose-deprived cells, suggesting that the cell
preferentially utilizes mannose for the production of
dolichol linked precursors rather than nucleotide precursors.
This situation may be beneficial in glucose-deprived cells
because it many help ensure that oligosaccharide, albeit a
truncated version of the complete core oligosaccharide, is
generated for modification of protein.
Using the data presented here and the previous
observation mentioned above, it could be hypothesized that
3T3-L1 adipocytes might also generate a similar
oligosaccharide in the absence of glucose, resulting in a
GLUTl protein that was both only slightly greater in
molecular weight than the core protein, and insensitive to
digestion by endoglycosidase H. Although p37 GLUTl displays


Figure 6-1 Subcellular Fractionation of 3T3-L1 Adipocytes
Cells were incubated in the presence or absence of glucose
for 48 hours before subcellular fractionation as described in
Chapter 2. Membrane proteins (50 pg/lane) were resolved by
7.5% SDS-PAGE and probed by Western blot for GRP78, GLUT4,
and GLUT1 as indicated. Results shown are representative of
at least two experiments.


39
Specifically, 3T3-L1 adipocytes were rinsed in PBS at
4C and maintained at 4C. When the cells were equilibrated
to 4C, the PBS was removed and the cells washed in PBS, pH
8.5 at 4C. Cells were then incubated in 2.0 ml of the same
solution in the presence or absence of 0.5 mg/ml sulfo-NHC-
LC-biotin for 1 hour. The solution was then aspirated and
the cells washed in TES to quench any remaining biotin probe.
A membrane fraction was then prepared and solubilized in the
extraction buffer used for immunoprecipitation. After the
removal of insoluble material by centrifugation, the
clarified supernatant was incubated in the presence of 50 pi
streptavidin-agarose for 6 hours at 4C with end-over-end
rotation. The complexes were then washed 5 times in
extraction buffer to remove non-specific interactions.
Specifically bound proteins were released by incubating the
complexes in sample dilution buffer containing 6 M urea and
10% B-mercaptoethanol at 37C for 30 min. The released
proteins were then analyzed by SDS-PAGE and a modified
Western blotting technique.
For the detection of the biotinylated proteins, the gel
was transferred to nitrocellulose as described before.
Following transfer, the blot was briefly stained in amido
black (0.2% w/v in 40% methanol, 10% acetic acid) and
destained. Non-specific binding sites were blocked by
incubating the blot in 5% non-fat dry milk (NFDM) in Tris-
buffered saline containing 0.1% Tween-20 (TBS-T) for 1 hour
at room temperature. The blot was then washed in TBS-T


130
(CFTR), the protein responsible for chloride ion transport
across epithelial tissues, was recently analyzed for the
effects of oligosaccharide modifications on the its targeting
and activity (Morris et al., 1993). Several different forms
of the CFTR transporter were generated using inhibitors of
enzymes involved in oligosaccharide processing. Treatment of
cells with swainsonine, which inhibits a-glucosidase, or
deoxymannojirimycin and deoxynojirimycin, which inhibit
mannosidases, all resulted in a reduction in the molecular
weight of the CFTR protein when expressed in a human colon
cell line. The Cl" conductance and conductance stimulated by
cyclic AMP, however, was unaffected. Likewise, treatment
with these glycosidase inhibitors had no effect the apical
targeting of the CFTR protein in polarized cells. These data
demonstrate that some transporters like CFTR are unaffected
by changes in the oligosaccharide. It should be noted,
however, that an unglycosylated form of CFTR was not tested.
The unglycosylated form of the CFTR protein could have
exhibited altered targeting.
Studies directed towards understanding the role of
glycosylation in the expression of the glucose transporter
have provided contradictory results. Cells expressing a
GLUT1 transporter with a mutated glycosylation site, retained
some glucose transport activity (Asano et al., 1991).
However, treatment of GLUTl-containing vesicles with exo- and
endoglycosidases resulted in a loss of transport activity
(Fuegeas et al., 1990). Additionally, the role of


71
\
generating the oligosaccharide dolichol-PP-GlcNAc2Man5. The
second seven sugars, 4 mannose residues and 3 glucose
molecules, are added from their respective dolichol phosphate
derivatives. The completed core oligosaccharide, dolichol-
PP-GlcNAc2Man9Glc3, is transferred cotranslationally to the
growing polypeptide chain in the endoplasmic reticulum, and
is identical regardless of the glycoprotein in question.
Cleavage and processing of this oligosaccaride, however,
varies widely depending upon the type of cell, the individual
glycoprotein, and the final target of the glycoprotein.
Processing of the oligosaccharide begins immediately in
the endoplasmic reticulum upon the attachment of the core
oligosaccharide. Two specific glucosidases (glucosidase I
and glucosidase II) remove the first and second two glucose
residues, respectively, generating Asn-GlcNAc2Man9. Recently,
reglucosylation of the core oligosaccharide in the
endoplasmic reticulum has been described, although the exact
purpose of this enzymatic reaction has not yet been clearly
elucidated (Kornfeld R. reference). When the glycoprotein
has moved from the endoplasmic reticulum into the cis golgi,
mannosidases begin the sequential removal of four of the nine
mannose residues, eventually resulting in Asn-GlcNAc2Man5.
For glycoproteins destined to be of the high mannose class,
additional mannose units are added onto this oligosaccharide.
For complex type glycoproteins, however, a variety of other
sugars are then added onto the oligosaccharide, often
creating large and highly branched structures. These


113
glycosylation during glucose deprivation. Remember that
fructose, while not preventing glycogen depletion in the
absence of glucose, maintains the normal, heterogeneous
glycosylation of GLUT1. All sugar was then withdrawn for a
total of 36 hours. The glycosylation of GLUT1 was assessed
by western blot at specific intervals during the sugar
deprivation. The appearance of the low molecular weight
GLUT1 (p37) was taken as a measure of the time required for
the alteration in glycosylation to occur. In cells
maintained in the presence of fructose prior to glucose
deprivation, p37 reached higher levels more rapidly than
adipocytes cultured in glucose prior to glucose deprivation
(Figures 5-6, 5-7).
Glycogen Content in the CHO Cell Line
Previous studies in the CHO cell line demonstrated that
only 5 min of glucose deprivation were required to affect
oligosaccharide biosynthesis (Rearick et al., 1981). If our
hypothesis is correct, the rapid change in glycosylation in
glucose-deprived CHO cells would be a result of very low
glycogen content. We therefore measured the glycogen level
in CHO cells in a manner similar to that in the 3T3-L1 cells.
As seen in Table 5-1, the amount of glycogen in CHO cells is
less than 1% of the amount in glucose-fed 3T3-L1 adipocytes
and less than 10% of that in glucose-deprived adipocytes.
This observation, therefore, is consistent with our
hypothesis that the level of glycogen determines the amount


156
lectin chromatography step if it could be ensured that no
other protein (like the glucose transporter in the
endoplasmic reticulum) which could transport glucose would
significantly contribute to any activity presented by the low
molecular weight GLUT1 glycoform. It should be noted that
the separation of p37 from p46 GLUT1 would not be possible by
size exclusion chromatography, due to insufficient resolution
(Dr. Nancy Denslow, personal communication). Following the
isolation of p37 GLUT1, the transporter would then be
reconstituted into artificial liposomes, possibly consisting
of lysolecithin, by sonication and freeze-thaw cycles.
Apart from the determination of the activity of the p37
GLUT1 transporter, the mechanisms by which the withdrawal of
glucose inhibits the turnover of membrane protein are
unknown. It was discussed earlier that this could possibly
be a protective mechanism by which the cell protects cellular
function when the activity of newly synthesized glycoproteins
is jeopardized. Several scenarios could explain the
inhibition of membrane turnover in the absence of glucose.
One explanation is that a glycoprotein required for the
degradation of membrane protein is inactivated in glucose-
deprived cells either because of altered glycosylation,
altered protein expression, or both. A direct involvement of
a glycoprotein, however, need not be invoked; a second
possibility is that a transporter or membrane protein which
is integral to the maintenance of a low intralysosomal pH is
inactivated by one or more of the mechanisms discussed above.


Total
Membranes
+
PM
Fragments
GRP78
ag
GLUT1
+
Glucose
144


107
test this hypothesis, adipocytes were incubated in 25 mM
fructose instead of glucose for a total of 24 hours.
Incubation of cell with fructose instead of glucose had no
effect upon the extent of glycogen depletion or the rate at
which this depletion occurred (Figure 5-4). The inability of
fructose to prevent the glycogen depletion may result from
preferential utilization of fructose for other processes in
the absence of glucose.
Effect of Insulin and 3-O-methvlqlucose on the Glycogen Level
in Glucose-Deprived 3T3-L1 Adipocytes Incubated with Fructose
The observation that fructose was not able to substitute
for glucose in the generation of glycogen could be explained
by several scenarios. The cells might preferentially utilize
fructose for purposes other than the biosynthesis of glycogen
when extracellular glucose is absent. Alternatively,
fructose may provide substrate for glycogen synthesis but the
flux of carbons favors net glycogen breakdown rather than
glycogen synthesis. Glucose itself may play a role in the
balance of glycogen synthesis and breakdown through the
partial inhibition of phosphorylase a; the absence of glucose
in its allosteric binding site might prevent the cell from
accumulating glycogen because the pathway for glycogen
degradation would be partially activated. The ability of
insulin to activate glycogen synthesis provided an
opportunity to determine whether insulin could overcome the
inability of the cells to use fructose carbons for glycogen


102
glycogen could provide carbohydrate for protein glycosylation
in the absence of glucose. Although glycogen granules have
been visualized by electron microscopy (Novikoff et al.,
1975), the glycogen content has never been measured in 3T3-L1
adipocytes. The goal of the following experiments is to
determine the level of glucose stored in the form of glycogen
in 3T3-L1 adipocytes and assess its ability to provide
substrate for protein glycosylation. The data presented here
indicate that glycogen provides a pool of carbohydrate that
can be used during periods of glucose stress, allowing normal
protein glycosylation while glucose is absent. The
differences in the time of glucose deprivation required to
result in aberrant glycosylation among different cell types
is likely related to cell type-specific differences in the
ability to store significant amounts of glycogen.
Results
Time Course and Concentration Curve of Glucose Assay
Glucose was released from glycogen by acid hydrolysis
and measured by an NAD-linked assay as described in Chapter
2. Figure 5-1 shows the experimental conditions that
optimized glucose quantitation. For a standard curve, a
stock solution of glucose in the same solution as the
glycogen hydrolysates, i.e. H2SO4/NaOH/H20, was prepared. The
reaction was incubated at 37C for up to 30 min. The
absorbance was then measured at 340 nm at specific intervals


63
result, glycoproteins in this cell line are terminated at the
high mannose stage, and remain endoglycosidase H sensitive.
Figure 3-6 shows a Western blot of these membrane proteins
probed for GLUT1 using the anti-GLUTl antiserum. The
migration of the normal GLUT1 glycoform present in 3T3-L1
adipocytes was not altered by treatment with endoglycosidase
H, indicating that this GLUT1 glycoform is not a high mannose
type glycoprotein. The migration of p46 was altered,
however, by digestion with N-Glycosidase F, confirming
earlier observations that GLUT1 is an N-linked glycoprotein
(Kitzman, et al., 1993). The molecular weight of the N-
Glycanase F digested GLUT1 protein is 36 kDa, in agreement
with the molecular weight predicted by amino acid sequence
and the molecular weight of GLUT1 from tunicamycin-treated
adipocytes. In glucose-deprived adipocytes, neither the
normal nor the low molecular weight GLUT1 glycoform was
sensitive to digestion by endoglycosidase H, indicating that
neither is a high mannose glycoprotein. Once again, the
migration of the normal and low molecular weight GLUT1 was
resolved to the core 36 kDa protein upon digestion with N-
Glycanase F. This data confirms earlier observations that
the difference between the normal and low molecular weight
GLUT1 glycoforms is due to differences in glycosylation and
not changes in the primary sequence of the protein. To
confirm that the endoglycosidase H in this assay was active,
membrane proteins from the LEC1 CHO cell line were also
tested. The GLUT1 glycoform present in these cells (p38),


LIST OF FIGURES
Figure page
1-1 Predicted Secondary Structure of Glucose Transporters. .4
2-1 Elution of GTl-Specific Antibodies 23
3-1 Characterization of Anti-GTl Antiserum 45
3-2 Glucose Deprivation and GLUT1 Expression 49
3-3 Tryptic Digest of p46 and p37 52
3-4 Effect of Tunicamycin on Expression of GLUT1 55
3-5 GLUT1 Expression Following Tunicamycin Washout 58
3-6 Glycosidase Digestion of GLUT1 Glycoforms 61
3-7 Immunoprecipitation of GLUT1 from Normal and Glucose-
Deprived 3T3-L1 Adipocytes 65
4-1 Biosynthesis of GLUT1 in Normal and Glucose-Deprived
Adipocytes 76
4-2 Processing of GLUT1 in Normal and Glucose-Deprived
Adipocytes 79
4-3 Time Course of p37 GLUT1 Biosynthesis in Glucose-
Deprived Adipocytes 82
4-4 Biiosynthesis of p37 GLUT1 During Glucose-Refeeding. ..85
4-5 Turnover of GLUT1 in Glucose-Deprived Adipocytes 88
4-6 Turnover of GLUTl in Glucose-Refed Adipocytes 90
5-1 Time Course and Concentration Curve of Glucose Assay. 104
viii


Figure 3-4 Effect of Tunicamvcin on Expression of GLUT1 Control (tGlucose) or glucose-
deprived (24 hours) (-Glucose) 3T3-L1 adipocytes were incubated with tunicamycin as
indicated for 24 hours. Membrane proteins were then probed for GLUT1 by Western blot with
anti-GTl antiserum. Positions of the normal GLUT1 protein (p46), the lower molecular weight
GLUT1 (p37), and the unglycosylated form of GLUT1 (p36) are indicated along with molecular
weight standards.


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Ill
fructose alone. To eliminate the possibility that osmotic
pressure differences accounted for the changes in glycogen
content, glucose-deprived adipocytes were incubated in the
presence of 25 mM xylose instead of glucose for 24 hours.
Xylose is a sugar that is not transported by mammalian cells.
Xylose, in the absence of glucose, had no effect on the level
of glycogen (Figure 5-5). These data suggest that either 3-
O-methylglucose cannot bind to the allosteric site of
phosphorylase kinase, or that if 3-0-methylglucose does
regulate phosphorylase kinase allosterically, that carbons
from fructose are shunted into pathways other than glycogen
biosynthesis. Furthermore, hormonal activation of glycogen
biosynthesis is not able to overcome the depletion of
glycogen in the presence of fructose alone, again indicating
that fructose is utilized for other purposes in the absence
of glucose.
Prior Depletion of the Cellular Glycogen Pool Results in a
More Rapid Alteration of Glvcosvlation in the Absence of
Glucose
Chapter 3 demonstrated that 3T3-L1 adipocytes maintain
normal protein glycosylation for at least 12 h in the absence
of glucose. Because the level of free glucose inside
adipocytes is thought to be low, the source of carbohydrate
utilized for glycosylation was unclear. To determine whether
glycogen could be used for protein glycosylation, cells were
incubated in the presence of fructose for 24 h to deplete the
intracellular glycogen level before assessing GLUT1


26
Markwell Assay for Protein Determination
Several assays have been developed for the determination
of protein concentration in biological samples. All protein
determinations, however, suffer the limitation that certain
compounds, such as detergents or urea, can interfere with the
accuracy of the assay. For the determination of protein in
samples containing membranes, the sample must be extracted
with detergent in order to obtain a true reflection of the
protein content. The most popular method, a procedure
developed by Lowry, has come into wide use because of its
ease and sensitivity (Lowry et al., 1951). The Lowry protein
assay, however, is interfered by the presence of detergents,
especially sodium dodecyl sulfate (SDS) (Peterson, 1979).
Protein determinations in this work were most commonly
performed on membrane fractions, necessitating the routine
use of the Markwell modification of the Lowry assay for
protein determination. The Markwell assay is a modification
of the Lowry protein determination which is compatible with
certain detergents, including SDS.
To generate a standard curve, samples of purified bovine
serum albumin (BSA) (0-100 pg) were diluted to a total of 0.1
ml with water. Aliquots of the unknown samples (usually 5 -
25 pi) were also diluted to 0.1 ml water. If the unknown
protein samples were in buffer, an equal volume of that
buffer was also added to the protein standards. Assay
solution (1 ml of [100 parts 2.0% Na2C03, 0.4% NaOH, 0.16%


84
extracts and analyzed by SDS-PAGE and fluorography. This
type of experiment provides a "snapshot" of what GLUT1
glycoform(s) are being synthesized during glucose-
deprivation. Despite glucose withdrawal, the normal p46
GLUT1 glycoform was the only GLUT1 protein synthesized during
the first 12 hours of glucose deprivation (Figure 4-3 A).
Beyond 12 hours of glucose deprivation, however, the
synthesis of GLUT1 shifted to the p37 glycoform.
Densitometry of the GLUT1 bands indicated that the total
amount (addition of all bands) of GLUT1 in glucose-deprived
cells remained nearly constant (Figure 4-3 B). To examine
the reversibility of this effect, cells glucose-deprived for
36 hours were placed into medium containing 25 mM glucose and
labeled for 1 hour during various points during the
refeeding. As expected, p37 was the only form synthesized in
the glucose-deprived cells. Within 6 h of refeeding,
however, the normal p46 GLUT1 glycoform was the only GLUT1
protein synthesized (Figure 4-3 C). Furthermore, the
readdition of glucose resulted in a 3.5 fold increase in
GLUT1 (Figure 4-3 D) despite only a 40% increase in total
membrane radioactivity (data not shown). To examine this
rapid recovery in more detail, we performed the same
experiment on glucose-deprived (36 hours) cells and placed
them into glucose containing medium. Cells were then labeled
every hour for the first 6 hours of refeeding. Surprisingly,
within 1 h of glucose readdition, only the normal p46


101
glycosylated GLUT1 glycoform accumulates (Tordjman et al.,
1990; Kitzman et al., 1994), a reflection of reduced
precursor levels used for oligosaccharide biosynthesis
(Chapman and Calhoun, 1987). The biosynthesis and processing
of this transporter have been elucidated in Chapter 4.
The exact structure of the oligosaccharide on the
alternatively glycosylated GLUT1 glycoform is not known,
although Chapter 3 demonstrated that it is a small, N-linked,
endoglycosidase H-resistant structure that may be similar to
the dolichol-linked oligosaccharide species identified in
glucose-deprived CHO cells (Rearick, et al., 1981). It was
intriguing that in glucose-deprived CHO cells, the shift to
the synthesis of the alternative lipid-linked oligosaccharide
structure occurred within 5 minutes of glucose deprivation.
In 3T3-L1 adipocytes, at least 12 h of glucose deprivation
are required to observe alternatively glycosylated GLUT1.
The large difference in the time of glucose deprivation
required for the lipid-linked oligosaccharides to be
synthesized in CHO cells and the appearance of alternatively
glycosylated GLUT1 in 3T3-L1 adipocytes indirectly suggested
that the oligosaccharide biosynthesis pathways in CHO cells
were more sensitive to the absence of glucose. Because the
intracellular concentration of free glucose is presumed to be
exceedingly low in adipocytes (Froesch and Ginsberg, 1962;
Whitesell et al., 1990) The adipocyte, therefore, must have
an alternative source of carbohydrate for oligosaccharide
biosynthesis. I therefore tested the hypothesis that


44
These analyses will be performed with a combination of
Western blotting, glycosylation inhibitors, tryptic digests,
and glycosidase digestions. The goal of this section is to
test the hypothesis that the lower molecular weight protein
detected by Western blot in glucose-deprived cells is a
second form of the GLUT1 transporter that arises not from
alterations in the core protein, but through alterations in
glycosylation.
Results
Characterization of Anti-GTl Antiserum
In order to confirm that the anti-GTl antiserum
specifically recognized the GLUT1 carboxy-terminal peptide,
dot blots and Western blots were performed. In Figure 3-1 A,
varying amounts of the GT1 peptide (CEELFHPLGADSQV) and GT4
peptide (CSTELEYLGPDEND) were blotted onto dry
nitrocellulose, dried, and detected with the anti-GTl
antiserum. This result indicates that the anti-GTl antiserum
at a dilution of 1:1000 can detect as little as 0.1 ng GT1
peptide. The antiserum, however, did not recognize the
carboxy-terminal GLUT4 peptide when blotted in a similar
manner, demonstrating that the reaction was specific for the
GLUT1 peptide. Note that the GLUT1 peptide and the GLUT4
peptide share little identity. The observation that the
anti-GLUTl antiserum does not recognize the GLUT4 peptide


12
study of glucose transport regulation, including regulation
by insulin (reviewed by Czech, 1995), cholera toxin and
dibutyryl cyclic AMP (Clancy and Czech, 1990), cadmium, and
tumor necrosis factor a (Stephens et al., 1992).
In particular, the 3T3-H adipocyte has been used to
investigate the effects of glucose deprivation on glucose
transport activity and glucose transporter expression. The
withdrawal of glucose from the culture medium of 3T3-L1
adipocytes results in a 10-fold increase in the rate of
glucose transport, consistent with the responses of other
cell types to glucose deprivation (van Putten and Krans,
1985, Kitzman et al., 1994). Furthermore, the transport
activation results from an increase in the of the
transport system, with little change in the affinity of the
transporter for glucose (Km)((van Putten and Krans, 1985).
Other groups have since confirmed this result (Tordjman et
al., 1990; Kitzman et al., 1994). An increase in the maximal
velocity of transport without a change in the Km suggests
that there is either a greater number of transporter
molecules at the cell surface available for transport, or
that the activity of the transporter has been increased,
possibly by allosteric or covalent modification. Transport
activation, beginning at 3 hours post glucose withdrawal and
reaching a maximum at 24 hours, is protein synthesis-
dependent as assessed by cycloheximide sensitivity (Tordjman
et al., 1985, Kitzman et al., 1994). Additionally,
cycloheximide attenuated transport activation at any time


20
peptide in order to facilitate covalent coupling to the
immune carrier. A keyhole limpet hemocyanin-GTl conjugate
was generated by using a thiol-specific cross-linking
reagent, sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide ester
(sulfo-MBS). Keyhole limpet hemocyanin (KLH) (60 mg) was
dissolved in 10 ml 10 mM potassium phosphate, pH 7.3 and
dialyzed at 4C against the same for 48 hours. The
dialysate was concentrated to approximately 4 ml by covering
the dialysis bag in a liberal amount of Aquacide and wrapping
in foil (Calbiochem). The concentrated dialysate was divided
into microcentrifuge tubes and centrifuged in microcentrifuge
tube for 10 minutes to remove any protein aggregates. The
concentration of protein in the supernatant was estimated by
measuring the absorbance at 280 nm using the milligram
extinction coefficient for KLH of 1.6. KLH (6 mg) from a 12
mg/ml solution in 10 mM potassium phosphate, pH 7.3 was
reacted at room temperature with 0.6 mg sulfo-MBS for 45 min.
The KLH-MBS conjugate was separated from free sulfo-MBS by
passing the reaction mixture over a 10 ml G-100 column pre
equilibrated in 50 mM potassium phosphate, pH 6.0. The KLH-
MBS conjugate is excluded in the void volume, and the free
sulfo-MBS is retarded in the column. Ten fractions (1 ml
each) were collected and measured at 280 nm. Peak fractions,
containing KLH-MBS were pooled and reacted with the GT1
peptide (6 mg) for 3 hours at room temperature with end-over-
end rotation. The pH of the reaction mixture was adjusted
with HC1 in order to produce the maximum amount of