Development of insulin resistance in 3T3-L1 adipocytes

MISSING IMAGE

Material Information

Title:
Development of insulin resistance in 3T3-L1 adipocytes
Physical Description:
xiii, 156 leaves : ill. ; 29 cm.
Language:
English
Creator:
Thomson, Michael James, 1972-
Publication Date:

Subjects

Subjects / Keywords:
Research   ( mesh )
Insulin Resistance   ( mesh )
Adipocytes   ( mesh )
Monosaccharide Transport Proteins   ( mesh )
Insulin -- pharmacology   ( mesh )
Glucose -- pharmacology   ( mesh )
Glucosamine -- pharmacology   ( mesh )
Department of Biochemistry and Molecular Biology thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Biochemistry and Molecular Biology -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1999.
Bibliography:
Bibliography: leaves 145-155.
Statement of Responsibility:
by Michael James Thomson.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 030406754
oclc - 51619713
System ID:
AA00025275:00001

Full Text













DEVELOPMENT OF INSULIN RESISTANCE IN 3T3-L1 ADIPOCYTES


By

MICHAEL JAMES THOMSON

















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

UNIVERSITY OF FLORIDA


1999






























This is dedicated to God, my parents, my dearest Amy, and

Moxie.
















ACKNOWLEDGMENTS


I would sincerely like to thank the members of my

committee Drs. Michael Kilberg, Charles Allen, Thomas

O'Brien, Mark Atkinson, and my advisor Dr. Susan Frost for

their guidance throughout the course of this project. In

addition, I would like to thank the members of the lab and

my friends for their help throughout the time I have spent

here and for making this such an enjoyable period in my

life. My most heartfelt thanks goes to my parents, without

whose love and support I would never have achieved such an

undertaking. Last, but certainly not least, I would like to

thank my dearest Amy, whose love and companionship over the

last several months has not only marked a highlight in my

graduate career, but in my life as well.
















TABLE OF CONTENTS

page

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

LIST OF TABLES .......................................... vii

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

ABSTRACT .................................................. xi

CHAPTERS

1 INTRODUCTION ......................................... 1

Overview of Diabetes ..................................... 1
Insulin Resistance....................................... 2
Role of Adipose in Insulin Resistance ............... 3
Role of the Hexosamine Biosynthetic
Pathway in Insulin Resistance .................. 6
3T3-L1 Adipocytes ........................................ 8
GLUT Transporter Family................................. 9
Characteristics of GLUT Family...................... 9
Translocation of GLUT4............................. 12
Insulin Receptor and Signaling......................... 13
3T3-L1 Adipocytes: A Model for
Insulin Resistance.................................. 17

2 MATERIALS AND METHODS.................................. 18

Materials ............................................ 18
Methods .............................................. 19
Cell Culture ........................................... 19
Chronic Insulin Treatment and
Insulin Washout................................. 20
Glucose Transport Assay............................ 21
Subcellular Fractionation of
3T3-L1 Adipocytes............................... 21
Markwell Assay for Protein Determination........... 22
Gel Electrophoresis................................ 24
Electrotransfer and Western Blotting............... 25
RNA Isolation and Northern Blotting................ 26
Glycogen Analysis ................................... 27









Glycogen Synthesis .................................. 30
Production and Characterization of
GLUT4 Antibody................................... 30
Peptide Purification of GLUT4 Antibody............. 36
Preparation of Total Membranes ................... .. 39
Metabolic Labeling of 3T3-LI Adipocytes............ 40
Immunoprecipitation of GLUT4....................... 41
ATP-Luciferase Assay............................... 43
Leptin Assay........................................ 44

3 DEVELOPMENT OF INSULIN RESISTANCE .................... 49

Introduction ......................................... 49
Results .............................................. 50
Insulin-resistant Glucose
Transport Activity.............................. 50
Role of Glucose and Glucosamine.................... 58
Effects of Various Inhibitors on the
Development of Insulin Resistance............... 61
Effect of Chronic Insulin on GLUT4
Expression and Translocation.................... 69
Effect of Glucose Deprivation on
GLUT4 Expression................................ 77
Effect of Chronic Insulin on
Glut4 mRNA Expression........................... 77
Reversal of Insulin Resistance..................... 80
Conclusions .......................................... 82

4 MECHANISMS OF DECREASED GLUT4 LEVELS IN
INSULIN-RESISTANT CELLS ............................ 88

Introduction ......................................... 88
Results .............................................. 89
Specificity and Efficiency of
GLUT4 Immunoprecipitation........................ 89
Synthesis of GLUT4 in
Insulin-resistant Cells......................... 90
Degradation of GLUT4 in
Insulin-resistant Cells......................... 94
Effects of Cycloheximide on the
Loss of GLUT4 .................................... 99
Effects of Protease Inhibitors on
GLUT4 Expression................................ 99
Conclusions .......................................... 102









5 ROLE OF THE HEXOSAMINE BIOSYNTHETIC
PATHWAY IN THE DEVELOPMENT OF
INSULIN RESISTANCE................................ 107

Introduction ......................................... 107
Results ........ ............................... .. ... ... .. 110
Effects of Fructose Feeding on the
Glucose Transport System....................... .. 110
Effect of Fructose Feeding on
Glycogen Metabolism............................. 117
Conclusions .......................................... 123

6 CONCLUSIONS AND FUTURE DIRECTIONS .................... 129

Conclusions .......................................... 129
Future Directions ..................................... 132

APPENDICES

A GLUCOSE DEPRIVATION AND GLUT TRANSPORTERS............. 137

B LEPTIN AND INSULIN RESISTANCE......................... 141

REFERENCES .............................................. 145

BIOGRAPHICAL SKETCH...................................... 156
















LIST OF TABLES


Table


5-1 Glycogen Synthesis in 3T3-L1 Adipocytes............. 124


page
















LIST OF FIGURES


Figure page

1-1 Overview of the Hexosamine Biosynthetic Pathway.... 7

1-2 Predicted Secondary Structure of GLUT
Transporters ...................................... 10

2-1 Subfractionation of 3T3-Ll Adipocytes............... 23

2-2 Fluctuations in Glycogen Levels Over a Week........ 29

2-3 Specificity of GLUT4 Antiserum for
GLUT4 Peptide ..................................... 34

2-4 Specificity of GLUT4 Antiserum for
GLUT4 Protein..................................... 35

2-5 Elution Profile of Peptide-Purified
anti-GLUT4 Antibody.............................. 38

2-6 ATP Standard Curve................................... 45

2-7 Leptin Standard Curve............................... 48

3-1 Effect of Chronic Insulin on Glucose
Transport in 3T3-L1 Adipocytes .................. 53

3-2 Time Course of Acute Insulin Stimulation............ 54

3-3 Development of Insulin Resistance ................... 57

3-4 Effects of Glucose and Glucosamine on
Insulin Resistance............................... 60

3-5 Effects of Actinomycin and Cycloheximide on
Insulin Resistance............................... 62

3-6 Effects of Nikkomycin Z on Insulin Resistance...... 64

3-7 Effects of Tunicamycin on Insulin Resistance....... 66









3-8 Effects of E-64 on Insulin Resistance............... 67

3-9 Effects of E-64 on Glycogen Synthesis............... 68

3-10 Effects of Acute and Chronic Insulin on
Total GLUT Protein Levels........................ 70

3-11 Subfractionation of Insulin-Resistant
3T3-Ll Adipocytes.............................. 72-73

3-12 Effects of Chronic Insulin on GLUT4
Expression........................................ 75

3-13 Effects of Chronic Insulin on GLUT4
Expression with Larger Samples ................... 76

3-14 Effect of Glucose Deprivation on
GLUT4 Expression................................. 78

3-15 Effect of Chronic Insulin Treatment on
GLUT4 mRNA Levels................................ 79

3-16 Reversal of Insulin Resistance...................... 81

3-17 Reversal of Insulin Resistance in the
Presence of Cycloheximide........................ 83

4-1 Titration of Peptide-Purified GLUT4 Antibody
for Immunoprecipitation.......................... 91

4-2 Specificity of the Peptide-Purified GLUT4
Antibody in Immunoprecipitation.................. 92

4-3 Efficiency of GLUT4 Immunoprecipitation............. 93

4-4 Effect of Chronic Insulin on the
Synthesis of GLUT4 ............................... 96

4-5 Effect of Chronic Insulin on the
Degradation of GLUT4............................. 98

4-6 Degradation of Total Protein in
Cells Treated with Chronic Insulin............... 100

4-7 Effect of Cycloheximide on the Loss of GLUT4....... 101

4-8 Effects of Protease Inhibitors on
GLUT4 Expression................................ 104









5-1 Effects of Fructose on the Development of
Insulin Resistance............................... 112

5-2 Effect of Fructose on GLUT4 Transporter Levels..... 114

.5-3 Translocation of GLUT4 in Cells Treated with
Fructose .......................................... 116

5-4 Effects of Fructose on ATP Levels ................... 119

5-5 Effect of Fructose and Chronic Insulin on
Glycogen Levels.................................. 120

5-6 Effects of Fructose and Chronic Insulin on
Glycogen Synthesis............................... 122

A-l Effect of Glucose Deprivation and Chronic
Insulin on the Aberrant Glycosylation of
GLUT1 and GLUT4 .................................. 138

A-2 Effect of Glucose Deprivation and Chronic
Insulin on Glycogen Levels...................... 140

B-i Leptin Levels ....................................... 144
















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

DEVELOPMENT OF INSULIN RESISTANCE IN 3T3-Ll ADIPOCYTES

By

Michael James Thomson

August 1999


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

Insulin resistance is a manifestation of both diabetes

mellitus and obesity. However, the mechanism is still not

clearly identified. My goal was to determine if an in vitro

system, 3T3-Ll adipocytes, could serve as a model for

insulin resistance. This study describes a procedure that,

indeed, allows the evaluation of the development of insulin

resistance in 3T3-Ll adipocytes. Under these conditions, it

has been shown that the concentration of insulin required

for 50% desensitization of glucose transport activity is 100

pM and maximal desensitization could be achieved with 1 nM.

This demonstrates for the first time that 3T3-Ll adipocytes

develop insulin resistance in response to physiologically

relevant concentrations of insulin. Glucose (or

glucosamine), in addition to insulin, was required to









establish desensitization. The expression of GLUT4 protein

decreased by 50% with exposure to 10 nM insulin. The dose-

dependent loss of GLUT4 was similar to the dose-dependence

for insulin-resistant transport activity. Translocation in

the presence of acute insulin was apparent, but the extent

of recruitment directly reflected the decrease in GLUT4

protein. GLUT4 mRNA also declined, but the K50 was

approximately 5 nM. Together, these data suggest that the

loss of GLUT4 protein underlies the cause of

desensitization. This decrease in GLUT4 protein was found

to be a combination of both decreased synthesis and

accelerated degradation.

Glucose, through its metabolism via the hexosamine

pathway, has been implicated in the regulation of insulin-

sensitive glucose transport. Surprisingly, fructose, an

alternate substrate for this pathway, does not appear to

support the development of insulin-resistant transport. To

study this apparent anomaly, we examined the levels and

subcellular distribution of GLUT4 in 3T3-L1 adipocytes in

response to chronic and acute insulin in the presence of

fructose or glucose. The results indicate that cells

exposed to either glucose or fructose, with chronic insulin,

down-regulate the expression of GLUT4. However, cells

exposed to fructose and chronic insulin and subsequently

stimulated with insulin, reveal levels of GLUT4 in the









plasma membrane which are similar to controls. While the

mechanism underlying this apparent "enhancement" of GLUT4

translocation is unknown, it explains the observation that

the insulin-stimulated glucose transport activity in cells

exposed to fructose does not reflect the insulin-resistant

state.
















CHAPTER 1
INTRODUCTION


Overview of Diabetes


Diabetes is one of the leading causes of death and

disability in the United States. An estimated 16 million

people in the United States have diabetes mellitus and about

half of these people do not know that they have the

disorder. Of these 16 million, 127,000 are under the age of

19. Each year, an additional 650,000 people are diagnosed

with diabetes. In 1992, diabetes contributed to the deaths

of more than 169,000 people and cost $92 billion in direct

and indirect costs. In total, diabetes accounted for 1 in 7

health care dollars spent (1).

Diabetes results when the pancreas produces little or

no insulin or the body's cells do not respond to insulin.

As a result, glucose accumulates in the blood, leading to

kidney overload and glucose excretion into the urine. There

are several types of diabetes: type I, type II, and

gestational diabetes. Type I, or insulin-dependent diabetes

mellitus (IDDM), is considered an autoimmune disease during

which the immune system attacks and destroys the insulin-

producing beta cells of the pancreas. Over time, this leads









to an inability of the pancreas to produce insulin. Type

II, or non-insulin-dependent diabetes mellitus (NIDDM), is

the most prevalent and least understood form of diabetes.

About 90-95% of the people with diabetes have type II. This

form usually develops in adults over 40 and is most common

in adults over 55. Type II diabetes is also associated with

obesity in that 80% of the people with this form of diabetes

are overweight. With this form, the pancreas generally

produces insulin, but the body does not respond to insulin

effectively, a condition known as insulin resistance.

Gestational diabetes develops during pregnancy and generally

disappears afterwards. However, women who develop diabetes

during pregnancy have a greater risk of developing type II

diabetes later in life.


Insulin Resistance


Insulin resistance is one of the determining

characteristics of type II diabetes mellitus (NIDDM) and

obesity and is believed to be the underlying pathology (1).

NIDDM is characterized by both hyperglycemia and

hyperinsulinemia, whereas obesity manifests only

hyperinsulinemia (2). Complications of insulin resistance

and diabetes include retinopathy (3), nephropathy (4),

neuropathy (5), and artherosclerosis (6). The

hyperlipidemia (7), hypercoagulation of platelets (8), and









artherosclerosis associated with diabetes can lead to

coronary artery disease, which is the leading cause of

mortality among diabetics (6).

Insulin resistance is defined as a decreased biological

response to physiological concentrations of insulin in

insulin-responsive tissues (1). In adipocytes, this is

characterized by a reduction in insulin-stimulated glucose

transport activity (9). As of yet, the exact mechanism

leading to the onset of insulin resistance has yet to be

elucidated. However, several mechanisms have been proposed

including decreased autophosphorylation of the insulin

receptor (2,10-13), down-regulation of the insulin-

responsive glucose transporter GLUT4 (2,14), altered

translocation of GLUT4 to the plasma membrane from its

intracellular storage vesicles (15-17), and the actions of

the intermediates or products of the hexosamine biosynthetic

pathway (18-21).


Role of Adipose in Insulin Resistance


As mentioned, many obese subjects also exhibit insulin

resistance. However, when these patients are placed on

calorie-restrictive diets to decrease their weight, insulin

resistance is reversed (22,23). This suggests that adipose

plays an important role in insulin resistance. Another

condition which shows the importance of adipose in insulin









resistance is the metabolic syndrome X. This syndrome

manifests diabetes, hypertension, and dyslipidemia with

obesity as the underlying cause.

Adipose also secretes several factors which may be

involved in insulin resistance. For instance, evidence

exists that adipose tissue of obese mice have elevated TNF-

a mRNA levels and secretion of TNF-c protein (24-26).

Also, administration of TNF-a to Zucker fa/fa rats causes

reduced autophosphorylation of the insulin receptor as well

as decreased insulin-stimulated phosphorylation of IRS-1 in

both muscle and adipose tissue. Neutralization of TNF-a by

the addition of soluble TNF-a receptors prevents these

effects (10) and increases insulin sensitivity (24). It has

also been shown that TNF-a down-regulates GLUT4 mRNA levels

in adipocyte and myocyte cultures (24,27,28) as well as

reduces insulin-stimulated [3H]2-deoxyglucose transport into

3T3-L1 adipocytes (11). These studies provide evidence that

TNF-a plays a role in inducing insulin resistance in

adipose as well as muscle. One can imagine that increased

expression of TNF-a by adipose feeds back directly on

adipose by an autocrine loop mechanism, while its effects on

muscle may occur through a paracrine mechanism (25).

However, it has been shown in db/db mice that the elevated

levels of plasma TNF-a are well below those required to









support insulin resistance in cultured cell systems (24,25).

Therefore, it is very plausible that other proteins or

factors may be secreted by the adipose tissue which induce

insulin resistance.

Fatty acids have also been shown to be involved in the

induction of insulin resistance. In 1963, Randle et al.

(29) hypothesized that increased serum levels of free fatty

acids could interfere with glucose uptake and metabolism in

muscles, resulting in insulin resistance. This has come to

be known as the Randle hypothesis. Since the presentation

of this hypothesis, it has been shown by several groups,

including Randle's, that elevated serum free fatty acids

interfere with glucose utilization in vitro and in vivo (30-

33). In further support of this hypothesis, Bierman et al.

(34) have shown that type II diabetics have elevated fasting

levels of free fatty acids. In contrast, the absence of

white adipose tissue can also lead to diabetes as shown by

studies using a transgenic mouse model (35). These mice

express a dominant negative protein, called A-ZIP/F, which

prevents the DNA binding of B-ZIP transcription factors of

both the C/EBP and Jun families, which are necessary for

white adipose differentiation and formation. These mice are

diabetic, with fatty livers and elevated serum glucose,

insulin, and surprisingly, free fatty acids and

triglycerides. Interestingly, when fat is transplanted into









these mice, they lose the diabetic phenotype. This might

argue against the participation of adipose in the

development of insulin resistance. However, the fact that

obesity leads to insulin resistance demonstrates the

significance of adipose, suggesting that a small amount of

fat is beneficial, but a large excess is detrimental.


Role of the Hexosamine Biosynthetic Pathway in Insulin
Resistance


Marshall and colleagues (18) proposed that the

hexosamine biosynthetic pathway was involved in the

induction of insulin resistance in adipocytes because of the

in vitro requirement for glucose, glutamine, and insulin

(15,20). Figure 1-1 represents a diagram of this pathway.

In this pathway, glucose is transported into the cell by

either GLUT1, the constitutive glucose transporter, and/or

by GLUT4, the insulin-responsive transporter, where it is

rapidly phosphorylated to glucose 6-phosphate. This is then

isomerized to fructose 6-phosphate and the majority (97%) of

this is shunted into glycolysis and oxidative metabolism.

However, 2-3% of the fructose 6-phosphate enters the

hexosamine biosynthetic pathway through the rate-limiting

enzyme, glutamine:fructose 6-phosphate amidotransferase

(GFAT). GFAT transfers the amide group from glutamine to

fructose 6-phosphate to form glucosamine 6-phosphate.

Through several other reactions, UDP-N-acetylglucosamine

















Glucose-6-P


Glucosamine-6-P


Fructose-6-P 97 Glycolytic/TCA
PDfhliwaw


Glutamine
GFAT 1
4, Glutamate

> Glucosamine-6-P


UDP-N-Acetylglucosamine


glycolipids
glycosylated proteins
GPI-linked proteins


*. Insulin
Resistance


Figure 1-1. Overview of the Hexosamine Biosynthetic
Pathway.


= =,i i *f,









is formed, which is used in the synthesis of glycolipids,

glycoproteins, and GPI-linked proteins. It was also shown

that glucosamine was at least 40 times more potent than

glucose in inducing insulin resistance (18). This

phenomenon can be explained by appreciating the fact that

glucosamine, which is transported into cells by the same

transporter as glucose, bypasses GFAT through direct

phosphorylation by hexokinase (18,21). The role of this

pathway was also confirmed by the use of two inhibitors of

GFAT: O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-

oxonorleucine (DON), which are well established glutamine

analogs that irreversibly inhibit reactions in which

glutamine is an amide donor. In isolated rat adipocytes,

administration of these compounds blocked the development of

insulin-resistant glucose transport (18). Therefore, it was

hypothesized that products and/or intermediates of this

pathway are responsible for inducing insulin resistance.


3T3-Ll Adipocytes


The 3T3-Lls derive from murine embryonic fibroblasts

that, under the appropriate cell culture conditions,

differentiate into adipocytes (36,37). In the fibroblast

state, cells can be propagated and/or frozen under cryogenic

conditions. When these cells differentiate into adipocytes,

they exhibit the morphological and biochemical









characteristics of adipocytes. Differentiation is a

terminal process at which point the cells no longer divide.

Rather, the cells accumulate lipid droplets and express

lipogenic enzymes such as glycerophosphate acyltransferase

and malic enzyme (38), ATP-citrate lyase, acetyl Co-A

carboxylase, and fatty acid synthetase (39). These cells,

once differentiated, also express increased numbers of

insulin-binding sites (40). As adipocytes, these cells

express two isoforms of the glucose transporter family:

GLUT1, the constitutive glucose transporter, and GLUT4, the

insulin-sensitive glucose transporter (41,42). This feature

is characteristic of authentic adipocytes (43). In

addition, GLUT4 is only expressed in these cells following

differentiation (42).


GLUT Transporter Family


Characteristics of GLUT Family


GLUT1 and GLUT4 are members of a larger group of

proteins responsible for the facilitated transport of

glucose into cells (44). The members of this family were

discovered using a variety of molecular biology techniques

and named in the order in which they were identified, GLUT1-

5 (for GLUcose Tranporters). Figure 1-2 shows the predicted

secondary structure of these transporters, based on

hydropathy plots, as they might appear in the plasma




























45 1 :1 z? 0 1 PM




N ^c
IN









Figure 1-2. Predicted Secondary Structure of GLUT
Transporters.









membrane of the cell. These proteins are integral membrane

proteins consisting of twelve membrane spanning domains.

Both the amino and carboxy termini are located on the

intracellular side of the membrane. A large intracellular

loop between the sixth and seventh membrane spanning domains

divides the two halves of the protein. There is also a

single N-linked glycosylation site in the first

extracellular loop. The overall amino acid homology within

this family of proteins is 68% (45) and the amino acid

sequence identity between GLUT1 and GLUT4 is 63% (42).

The members of this family have differential tissue

distribution: GLUT1 is ubiquitously expressed at high levels

in fetal tissues, erythrocytes, brain, kidney, colon, and

adipocytes; GLUT2 is expressed in liver, P-cells, kidney,

and small intestine; GLUT3 is expressed in many tissues

including brain, placenta, and kidney; GLUT4 is expressed in

skeletal muscle, heart, and adipocytes; and GLUT5, a

proposed fructose transporter as well, is expressed in the

small intestine (46). The Km'S of these transporters also

differ. The Km's of GLUT1, 2, 3, and 4 for 3-0-

methylglucose, a none metabolizable form of glucose, are 21,

42, 10, and 2 mM, respectively, based on the oocyte

expression system (5). The high Km of GLUT2 is rationalized

by its localization to tissues involved in glucose

homeostasis (liver) and glucose sensing (P-cells).









Therefore, the flux of glucose into these tissues would be

expected to vary in a linear fashion over the typical range

of blood glucose levels (5). Following a meal, the very low

Km associated with GLUT4 would ensure that it operates close

to its Vmax even at decreasing blood glucose levels to ensure

maximum uptake of glucose from the blood into insulin-

responsive tissues (5).


Translocation of GLUT4


GLUT4, also referred to as the insulin-responsive

glucose transporter, resides in the trans-Golgi network of

vesicles that translocate to the plasma membrane in response

to insulin stimulation (47-49). This translocation

increases the number of glucose transporters on the cell

surface, and along with the lower Km of GLUT4, increases the

flux of glucose into the cell. Several groups have been

credited with cloning GLUT4 (42,50-53), which has ultimately

enabled studies investigating the regulation of its

translocation.

The basis for understanding translocation comes from

Rothman and colleagues (54) who proposed the SNARE hypothesis

for vesicular trafficking. This theory proposes that a

unique vesicle-bound molecule (v-SNARE) specifically

recognizes and binds to a unique receptor molecule (t-SNARE)

on the target membrane. Three different v-SNARES (VAMP-l,









VAMP-2, and cellubrevin) and eight different t-SNARES

(Syntaxin-l, Syntaxin-2, Syntaxin-3, Syntaxin-4, Syntaxin-6,

Syntaxin-7, SNAP-25, and Syndet) have been described thus

far (55). VAMP-2 is associated with GLUT4 containing

vesicles and is the v-SNARE that is responsible for binding

to the t-SNARES Syntaxin-4 and Syndet, the murine homologue

of SNAP-25 (56,57). In addition, the protein Muncl8c has

been shown to regulate the translocation of GLUT4 by

inhibiting the association of VAMP-2 with Syntaxin-4 (58).

Insulin relieves this inhibition by inducing the

dissociation of Muncl8c from Syntaxin-4 allowing VAMP-2 to

bind to Syntaxin-4. However, it is not known yet how

insulin stimulates the association and dissociation of these

molecules. In addition, other molecules may be involved in

GLUT4 translocation as investigations continue in this

exciting area.


Insulin Receptor and Signaling


Insulin action is mediated by a specific cell-surface

receptor. The insulin receptor is a heterotetramer

consisting of two c- and two P-subunits. Each a- and 3-

subunit is connected by a disulfide bond to form an a/P

dimer and each a/P dimer is then attached via a single

disulfide bond between the a-subunits to form the tetramer.

The a-subunit has a molecular mass of 135,000 Da and is









located completely on the outer surface of the plasma

membrane and contains the insulin binding site. The

3-subunit has a molecular mass of 95,000 Da and contains a

single transmembrane domain. The intracellular domain of

this subunit contains a juxtamembrane domain, an ATP binding

domain, and several tyrosine residues that are capable of

being phosphorylated. The intracellular domain also

contains a tyrosine kinase that is activated upon binding of

insulin presumably via propagation of a conformational

change from the a-subunit, which is transmitted through the

transmembrane domain of the 3-subunit. Once the tyrosine

kinase has been activated, it is capable of

autophosphorylating tyrosines in specific regions of the

intracellular domain of the P-subunit including the

juxtamembrane domain, the regulatory domain, and the carboxy

terminal tail (1). This tyrosine kinase also phosphorylates

selected proteins on tyrosine residues within the cell such

as IRS-1. The phosphorylated IRS-1 recognizes and binds to

the src homology-2 (SH2) domains of various signal

transduction proteins, two of which are Grb2 and PI 3-

kinase. The activation of these molecules sets up divergent

signaling pathways within the cell.

Activation of Grb allows it to bind to son-of-sevenless

(SOS) through an SH3 domain. The Grb2/SOS complex then

activates p21ras which has been shown to bind directly to









Raf-l serine/threonine kinase. This leads to the activation

of MAP kinase kinase which phosphorylates and activates MAP

kinase which activates transcription factors and increases

gene expression (59). It should be noted that

pharmacological concentrations of insulin are required for

activation of gene transcription. This brings doubt to the

physiological relevance of this path to insulin action (60).

The unique aspect of insulin action is its short-term

regulation of metabolic events including increased glucose

transport, increased glycogen synthesis, increased lipid

synthesis, and decreased lipolysis (1). Recent evidence has

shown that the PI 3-kinase cascade is the major pathway for

GLUT4 translocation and thus the stimulation of glucose

transport (61,62). PI 3-kinase is activated when the

phosphorylated IRS-1 binds to the SH2 domain of its p85a

regulatory subunit, this allows the pll0 subunit to become

active. Activated PI 3-kinase leads to the activation of

the serine/threonine kinase Akt (also known as protein

kinase B). Although Akt is involved in the specific

stimulation of GLUT4 translocation (62), the exact mechanism

by which this occurs is not known. However, inhibitor

studies has shown the importance of the PI 3-kinase pathway

over the p21ras pathway. For instance, wortmannin, an

inhibitor of PI 3-kinase, blocks the stimulation of glucose

transport by insulin (63). In contrast, inhibition of









endogenous p21ras has no effect on insulin-stimulated GLUT4

translocation (61).

The insulin receptor and the insulin signaling

cascade(s) are important in insulin resistance because it

has been shown that defects in autophosphorylation of the

insulin receptor leads to insulin-resistant cells (2,10-13).

This leads to a decrease in the phosphorylation of IRS-1

(2,10-13) which in turn leads to a reduction in signaling

cascades. This effect, however, may primarily be due to

secondary effects of TNF-x and the pharmacological doses of

insulin used. Genetic defects in the insulin receptor also

result in decreased autophosphorylation of the receptor and

decreased phosphorylation of IRS-1. This is only a minor

cause for the development of insulin resistance, as it does

not account for the induction of insulin resistance in

individuals with normal insulin receptors and those

individuals who develop insulin resistance as a result of

becoming obese. However, there is evidence in 3T3-L1

adipocytes that chronic insulin treatment alone can lead to

impaired insulin receptor signaling and down-regulation of

IRS-1 expression and phosphorylation (64). Again, these

studies were performed using pharmacological concentrations

of insulin.









3T3-Ll Adipocytes: A Model for Insulin Resistance


Many investigators have used the 3T3-L1 adipocyte cell

line to investigate insulin action. The majority of these

studies have used pharmacological doses of insulin to define

mechanisms. Under these conditions, insulin acts as a

growth factor sharing many of the mitogenic signaling paths

elicited by other growth factors (60). The metabolic

effects of insulin, observed at physiological

concentrations, are unique to insulin and cannot be

reproduced by other cellular stimuli (65-67). Thus, it

became important to determine the development of insulin

resistance under physiological conditions, the goal of this

research. I show that 1.) insulin-stimulated transport

activity decreases in response to physiological insulin; 2.)

GLUT4 expression is reduced resulting in fewer transporters

for mediating glucose uptake; 3.) insulin specifically

modulates the turnover of GLUT4; and 4.) translocation of

GLUT4 is not defective. These studies are complemented by

the roles of alternative hexoses on the development of

insulin resistance of both transport and glycogen turnover.

The appendices include studies on the effects of glucose

deprivation and chronic insulin on GLUT1 and leptin levels.
















CHAPTER 2
MATERIALS AND METHODS


Materials


Dulbecco's modified Eagle's medium (DMEM) (Cat. No.

12100-061) and glutamine-, glucose-free DMEM (Cat. No.

23800-022) were obtained from Life Technologies, Inc. Fetal

bovine serum (FBS) (Cat. No. 1020-75) and calf serum (Cat.

No. 1100-90) were obtained from Intergen. Glucose-free FBS

was prepared by dialyzing FBS against phosphate-buffered

saline (PBS), pH 7.4, for 48 hours at 4C using dialysis

tubing with a molecular weight cutoff of 13,000 Da. Bovine

serum albumin (Cat. No. A-7030) was purchased from Sigma.

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

Lilly Corp. L-Glutamine, D-glucosamine, D-fructose,

dexamethasone, and methylisobutylxanthine were obtained from

Sigma and D-glucose was obtained from Fisher. ProMix-35 S-

Label (1000 Ci/mmol) (Cat. No. SJQ0079), 2-deoxy-D-[2,6 3H]

glucose (45 Ci/mmol) (Cat. No. TRK672), D-[U-4C]-glucose

(310 mCi/mmol) (Cat. No. CFB96), and D-[U-14C]-fructose (321

mCi/mmol) (Cat. No. CFB47) were obtained from Amersham.

SulfoLink Kit (Cat. No. 20405) was purchased from Pierce.

Protein A-Sepharose was obtained from Sigma. Anti-rabbit









IgG conjugated horseradish peroxidase was purchased from

Sigma. Luciferin/luciferase (Cat. No. L9134) was obtained

from Sigma. Mouse leptin RIA Kit (Cat. No. ML-82K) was

obtained from Linco. All other reagents were of the highest

quality available.


Methods


Cell Culture


Cells were grown and differentiated as previously

described (68). Briefly, 3T3-L1 fibroblasts were seeded on

polystyrene tissue culture dishes and fed every other day

with DMEM containing 10% calf serum (CS) for seven days

until confluence was reached. The fibroblasts were then

induced to differentiate into adipocytes by feeding with

DMEM containing 10% fetal bovine serum (FBS), 1 |jg/mL

insulin, 0.5 mM methylisobutylxanthine, and 0.25 mM

dexamethasone (69). Two days later, the cells were refed

with DMEM containing 1 [tg/mL insulin. Following this

treatment, cells were maintained by feeding with DMEM

containing FBS every other day. Cells were used 8-12 days

following differentiation.









Chronic Insulin Treatment and Insulin Washout


Fully differentiated 3T3-L1 adipocytes were incubated

in DMEM containing 10% FBS and specific concentrations of

insulin ranging from 1 pM to 10 nM for 12 h at 37C in a 7%

C02 incubator. Care was taken to reduce the loss of insulin

in solution, particularly at low concentrations. Thus,

insulin dissolved in 0.01 N HCl was added to DMEM containing

10% FBS to give a final concentration of lOnM insulin. The

remaining insulin concentrations were achieved by serial

dilution into DMEM containing 10% FBS. In addition,

solutions were stored in plastic containers to prevent

insulin binding which occurs in glass bottles. Finally,

cells were refed every 2 h as adipocytes rapidly degrade

insulin.

After chronic treatment, an insulin washout procedure

was performed with the goal of rapidly returning the cells

to a basal state. Specifically, adipocytes were removed

from the C02 incubator and placed in a 37C waterbath.

Plates were washed three times with 3 mL of Krebs' Ringer

Phosphate buffer (KRP), pH 7.4, containing 0.1% BSA and 5 mM

glucose, every 20 min over 60 min. The final wash at 60 min

was performed with KRP alone. The cells were then assayed

according to the particular experimental protocol as

described below. After determining the optimal

concentration of insulin to induce insulin resistance in









these cells, a concentration of 10 nM insulin was chosen for

chronic insulin treatment for the remaining experiments (see

Chapter 1). This concentration was chosen as it can be

removed from the cells by the washout procedure, but the

medium does not have to be replaced every 2 h to maintain

effective insulin concentrations.


Glucose Transport Assay


Glucose transport activity was performed as described

previously (68). Briefly, medium was removed from 3T3-LI

adipocytes grown in 35-mm tissue culture dishes by washing

three times with 3 mL of KRP buffer. Cells were then

incubated in 1 mL KRP with or without 1 JM insulin for 10

min. This was then followed by addition of 200 JiM [3H]2-

deoxyglucose (0.2 jiCi). After 10 min, transport was

terminated by washing the cells three times with 3 mL of

ice-cold phosphate-buffered saline (PBS). Cells were then

air-dried and lysed with 0.1% SDS and duplicate aliquots of

300 jiL were taken for counting by liquid scintillation. The

rate of 2-deoxyglucose transport is reported as nmoles/106

cells/min.


Subcellular Fractionation of 3T3-L1 adipocytes


The subcellular fractionation technique allowed the

subcellular localization of GLUT4. Plasma membrane (PM),









low density membrane (LDM), and high density membrane (HDM)

fractions were isolated by a modification (70) of a

technique described by Weber et al. (71). The major

modification of this technique is the use of a steel block

homogenizer and tungsten ballbearing originally designed by

Balch and Rothman (72). The complete procedure is outlined

in Figure 2-1. Briefly, control or insulin-treated cells

were scraped into TES buffer (10 mM Tris-HCl, pH 7.4, 1 mM

EDTA, and 250 mM sucrose) at 18C. The cells were then

passed over a tungsten ball ten times in the steel block

homogenizer (at 18C) with a clearance of 0.0025 inch. A

crude plasma membrane fraction was collected at 17,000 X g

for 15 min at 4C. Purified membranes were collected from

this fraction by sucrose gradient centrifugation (71). LDM

and HDM fractions were collected by differential

centrifugation in 300 JiL TES. Membrane fractions were

stored at -20C. Protein was determined by the method of

Markwell et al. (73).


Markwell Assay for Protein Determination


The Lowry procedure is widely used for the assay of

soluble proteins (74). However, detergents, used to

solubilize membranes for the release of integral membrane

proteins, and common compounds such as sucrose and EDTA used

in buffers for the isolation of membranes can interfere with









Scrape 5, 10-cm plates
at 18C with 4 mL TES each


Homogenize in steel block at 18C
(subsequent steps at 4C)

SS-34
12K, 15min


Pellet

I SS-34
12K, 20min


Pellet


Supernatant


Pellet


Sucrose Cushion
SW-28
23K, 65min

Interface


SS-34
20K, 30min


HDM


SS-34
20K, 30min



Pellet

SS-34
20K, 30min


PM


SS-34
20K, 30min

Supernatant
Supernatant


I Ti70.1
65K,75min
W
Pellet

Ti70.1
65K,60min


LDM


Figure 2-1. Subfractionation of 3T3-L1 Adipocytes.
Overview of subcellular fractionation procedure.









protein determination by the Lowry method (73). Therefore,

the Markwell procedure, which is a modification of the Lowry

procedure, was used for the analysis of membrane proteins in

our specific extraction buffer, which contains both sucrose

and EDTA (73).

A standard curve (0-100 )'g) was generated by diluting a

solution of BSA (1 mg/mL) in water to a final volume of 0.1

mL. Ten jiL of membranes suspended in TES was added to 0.1

mL of water. Ten [tL of TES was also added to the standard

curve. One mL of a solution containing 2.0% Na2CO3, 0.4%

NaOH, 0.16% Na+ K+ tartrate, and 1% SDS was added to the

standard curve and unknown samples, mixed, and allowed to

stand for 10 min. Folin reagent (0.1 mL of 1 N) was added,

mixed gently, and allowed to stand for 45 min. The

absorbance of the solution was measured at 650 nm and the

protein concentration of the samples was determined by

comparing them to the standards. A second order equation

was used to solve for the constants and the quadratic

equation was used to determine the protein concentration of

the unknown samples.


Gel Electrophoresis


Electrophoresis was performed as described by Laemmli

et al. (75). Equal amounts of membrane protein (50 j.g) were

mixed with half the volume of 2X Laemmli sample dilution









buffer (4% SDS, 6 M urea, 10% P-mercaptoethanol, 0.15 mg/mL

bromophenol blue, 40% glycerol and 20 mM Tris-base, pH 6.6).

Proteins were separated on 10% SDS-polyacrylamide gels by

running the gels for 15 h at 40 V.


Electrotransfer and Western Blotting


Following electrophoresis, the proteins were

transferred to nitrocellulose (pore size 0.45 pm) at 200 mA

for 2 h in transfer buffer (150 mM glycine, 20 mM Tris-base,

20% methanol, pH 8.2) using the method described by Clancy

and Czech (76). Nitrocellulose membranes were then immersed

in blocking buffer (20 mM Tris-base, 137 mM NaCl, 0.1%

Tween-20, pH 7.5, and 5% non-fat dry milk) for 1 h at room

temperature. Membranes were then incubated in blocking

buffer containing a 1:1000 dilution of either GLUT4 antisera

or GLUT1 antisera for 1 h at room temperature. Three washes

for 1 min followed by two washes for 5 min were performed

with buffer in the absence of non-fat dry milk. Membranes

were then incubated with a 1:100,000 dilution of horse-

radish peroxidase conjugated goat anti-rabbit IgG in

blocking buffer for 1 h. Four 1-min and three 5-min washes

were performed with buffer in the absence of non-fat dry

milk to remove excess secondary antibody. The protein-

antibody complex was visualized using enhanced

chemiluminescence.









RNA Isolation and Northern Blotting


RNA isolation and northern blotting was performed by

Martin Williams. Total cellular RNA was isolated by the

guanidinium thiocyanate procedure (77). Twenty jig of total

RNA was loaded onto a 1% formaldehyde-agarose gel and the

gel was run for 12-16 h at 40 V with constant buffer

recirculation. The RNA was then electrophoretically

transferred to an uncharged nylon membrane and cross-linked

with UV light for 3.5 min. GLUT4 probe was then generated

from cDNA generously provided by Dr. Maureen Charron (Albert

Einstein College of Medicine). The insert was labeled by

primer extension (78). Briefly, the DNA (0.5 jig) was

denatured by boiling at 100C for 5 min. After cooling for

3 min, the insert was incubated with a 6-base primer,

deoxynucleotides, one of which was labeled ([32PJ]dATP, dCTP,

dTTP, dGTP), and DNA polymerase. Free nucleotides were

removed by gel chromatography over Sephadex G-50. The

eluted labeled DNA was used directly to probe the RNA blot.

Hybridization was performed for 12-16 h in a buffer

containing 1% bovine serum albumin, 1 mM EDTA, 0.5 M sodium

phosphate, pH 7.2, and 1% SDS at 60C. The membranes were

washed under high stringency conditions (3 X 10-min washes

in 1 mM EDTA, 40 mM sodium phosphate, pH 7.2, and 1% SDS at

65C) and exposed to film for various lengths of time.









Relative intensity of each band was quantified by video

densitometry within the linear range of the film using the

Biolmage Visage 110.


Glycogen Analysis


Total glycogen was isolated as described previously

(79) based on a method described by Pfleiderer (80). Cells

grown on 10-cm tissue culture dishes were washed at 4C with

PBS, pH 7.2, and scraped into a 15-mL polypropylene tube in

1 mL PBS. The cells were then sonicated for 10 s on power 2

at 50% duty cycle (Branson Sonifier 450). Two mL of 30%

(w/v) KOH was added and the sample was mixed on a vortex

mixer. The sample was then boiled in a water bath for 15

min. After cooling to room temperature, 3.5 mL of 95% EtOH

were added. The samples were mixed and heated in a boiling

water bath for 3 min and cooled again to room temperature.

The precipitated glycogen was collected by centrifugation at

1,300 X g for 5 min at room temperature. The supernatant

was removed by aspiration and the glycogen pellet washed by

resuspension in 1 mL 95% EtOH. This suspension was

transferred to a 1 mL microfuge tube and the glycogen

collected by centrifugation at 13,300 X g for 5 min at 4C.

The supernatant was removed by aspiration and the

precipitate was stored at -20C.









Glucose was released from glycogen by acid hydrolysis.

Specifically, 0.2 mL 2 N H2S04 was added to the glycogen

pellet and incubated in a boiling water bath for 2 h. The

samples were cooled to room temperature and 0.15 mL 2 N NaOH

and 0.65 mL H20 were added. The concentration of glucose in

the hydrolysate was then determined using a hexokinase-based

glucose kit (Glucose HK Kit) from Sigma. For samples fed

with glucose, 0.01 mL of hydrolysate was diluted with 0.99

mL of H2S04/NaOH/H20 mixture (2:1.5:6.5). Samples deprived

of glucose were diluted by adding 0.1 mL of hydrolysate to

0.9 mL of the H2SO4/NaOH/H20 mixture. Duplicate aliquots of

0.1 mL were added to 1 mL of prewarmed assay reagent and

incubated at 37C for 30 min. The absorbance of the samples

was then measured at 340 nm.

To limit the variability in experiments dealing with

glycogen, glycogen levels were monitored during the week in

which the 3T3-L1 adipocytes were fully differentiated. As

these cells are fed every 48 h during their normal

maintenance feeding schedule and they begin to enter

glycogen deprivation on the tail end of this time, it was

hypothesized that glycogen levels would fluctuate between

feedings. Upon collecting glycogen from the cells each day

of the week, at the same time of day, I found that glycogen

levels did indeed fluctuate. As Figure 2-2 shows, glycogen

levels were highest the day after they were fed and lowest
















S1.5
0
'I

^ 1.04
0 I
0


-- 0.5

0.0


1 2 3 4 5

A Day t

Fed Fed


Figure 2-2. Fluctuations in Glycogen Levels Over a Week.
Following differentiation, cells were maintained on their
feeding schedule and fed with DMEM containing 10% FBS on the
days indicated. Cells were collected at the same time every
day during the week and glycogen isolated by ethanol
precipitation. Following acid hydrolysis, glucose present
in glycogen was determined by a commercially available kit
(Sigma). Data represent the average S.D. of duplicate
samples within a single experiment (n=2).









on the days of a scheduled feeding as they became glucose

deprived. In addition, glycogen levels increased over the

course of the week from day 1 to day 5. This shows the

importance of performing a glycogen experiment the day

following a feeding and also performing the experiment at

the same time during the week (usually day 2 or 3 following

a feeding) to obtain consistent data.


Glycogen Synthesis


Cells adherent to 35-mm dishes were extensively washed

with KRP containing 0.1% BSA and 5 mM glucose as described

under "Insulin Washout" to return the cells to a basal level

of glucose transport. These cells were then labeled with 2

iCi/plate of 14C-U-[D-glucose] in 1.5 mL KRP containing 5 mM

glucose (giving a final specific activity of 284 pCi/mmol)

with or without 1 [tM insulin for 1 h. Glycogen was then

collected as described above and the pellet dissolved in 300

P.L of water. Radioactivity present was determined by liquid

scintillation.


Production and Characterization of GLUT4 Antibody


Hydrophilicity of a peptide is a criterion for

selecting a suitable sequence for antibody production

because hydrophilic peptides are more likely to be soluble

for coupling reactions and also more likely to be exposed on









the surface of the native protein (81). We chose to

synthesize a peptide corresponding to the last 13 amino

acids of the carboxy terminus GLUT4 due to both its

hydrophilicity and divergence from other members of this

transporter family and specifically GLUT1, which is also

expressed in 3T3-Ll adipocytes. The carboxy-terminal tail of

this transporter also makes it a good sequence for antibody

production because the predicted secondary structure shows

that the tail is probably exposed (42). However, a peptide

of only 13 amino acids is too small to elicit an immune

response if injected into a rabbit alone. Therefore, the

peptide is typically covalently linked to a larger protein

known as an immune carrier such as keyhole limpet hemacyanin

(KLH) or bovine serum albumin (BSA) (81). Although KLH and

BSA are the two most common immune carriers, others used are

ovalbumin, mouse serum albumin, or rabbit serum albumin

(81). As the peptide used to generate GLUT4 was injected

into a rabbit, KLH was used as the carrier.

Antiserum against GLUT4 was generated using a peptide

(CSTELEYLGPDEND) corresponding to amino acids 498-510 of the

GLUT4 sequence (underlined). This peptide was generated by

the Protein Chemistry Core facility at the University of

Florida and verified by amino acid analysis. An N-terminal

cysteine was added to allow its conjugation to KLH. This

conjugation was performed by using a thiol-specific cross-









linking reagent, sulfo-m-maleimidobenzoyl-N-

hydroxysuccinimide ester (sulfo-MSB). KLH (60 mg) was

dissolved in 10 mL of 10 mM potassium phosphate, pH 7.3, and

dialyzed at 4C against 10 mM potassium phosphate for 48 h.

The dialysate was concentrated by coating the dialysis

tubing with a liberal amount of Aquacide and wrapping in

foil. Protein aggregates were removed from the concentrated

dialysate by centrifugation in a microfuge for 10 min. The

concentration of protein in the dialysate was determined by

measuring its absorbance at 280 nm using the milligram

extinction coefficient of KLH (1.6). Six mg of KLH from a

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

added to 0.6 mg sulfo-MSB and incubated at room temperature

for 45 min. The KLH-MSB conjugate was separated from free

sulfo-MSB by size exclusion chromatography on a G-100 column

pre-equilibrated with 50 mM potassium phosphate, pH 6.0.

The free sulfo-MSB is retarded in the column and the KLH-MSB

conjugate flows through. Ten, 1 mL fractions were collected

and measured at 280 nm. KLH-MSB containing fractions were

pooled and reacted with 6 mg of the GLUT4 peptide for 3 h at

room temperature with rotation. The pH of the mixture was

adjusted with HCl to produce the maximum amount of

precipitate. The mixture was then allowed to stand upright

overnight at 4C to allow the precipitate, which represents

the KLH-peptide conjugate, to settle.









The KLH-GLUT4 conjugate (200 4g) dissolved in Freund's

Complete Adjuvant was then injected into the popliteal lymph

node of a New Zealand white rabbit (82). The rabbit was

boosted 28 days later by intradermal injection of an equal

amount of KLH-GLUT4 conjugate and Freund's Incomplete

Adjuvant. This procedure was repeated 2 weeks later. Test

bleeds were collected every week by laceration of the medial

ear vein. Serum was isolated following coagulation of the

red blood cells. Additional boosts were performed every 6

weeks. The antiserum was divided into 1 mL aliquots and

stored at -20C.

To demonstrate the specificity of the GLUT4 antiserum

to GLUT4 protein, dot blots were first performed. GLUT1 or

GLUT4 peptide was spotted onto separate nitrocellulose

sheets and the western blot procedure followed in which both

sheets were incubated with 1:1000 dilution of the GLUT4

antiserum. Figure 2-3 shows that the GLUT4 antiserum is

specific for the GLUT4 peptide as it does not cross react

with the GLUT1 peptide. Next, the GLUT4 antiserum was shown

to be specific for the GLUT4 protein on a western blot, as

shown in Figure 2-4. The reactivity of the GLUT4 serum with

GLUT4 protein was completely prevented by competition with

GLUT4 peptide but not GLUT1 peptide, showing that the

interaction was specific to GLUT4 and not due to non-

specific binding.



















1 2.5 5 jIg GLUT1 peptide


1 2.5 5 Vig GLUT4 peptide


Figure 2-3. Specificity of GLUT4 Antiserum for GLUT4
Peptide. Panel A, 1, 2.5, and 5 [tg of GLUT1 peptide was
blotted onto nitrocellulose and allowed to dry. Membranes
were blocked and probed with a 1:1000 dilution of the GLUT4
antiserum as described under "Electrotransfer and Western
Blotting". The protein-antibody complex was visualized by
enhanced chemiluminescence. Panel B, GLUT4 peptide was
blotted onto nitrocellulose and treated as in panel A.













4
o 00
o 0
9q


GLUT4 -


1 2 3 4


Figure 2-4. Specificity of GLUT4 Antiserum for GLUT4
Protein. Equal protein (50 [tg) from the LDM fraction of
control cells was loaded per lane and subjected to SDS-PAGE.
Proteins were transferred to nitrocellulose and western blot
analysis performed under the following conditions: lane 1,
pre-immune serum; lane 2, 1:1000 dilution of GLUT4
antiserum; lane 3, GLUT4 antiserum competed with 10 pig of
GLUT1 peptide; and lane 4, GLUT4 antiserum competed with 10
jg of GLUT4 peptide.









Peptide Purification of GLUT4 Antibody


In order to study the turnover and synthesis of GLUT4,

metabolic labeling with 35S-cysteine and -methionine was

performed followed by immunoprecipitation. In order to

prevent non-specific interactions during

immunoprecipitation, GLUT4-specific IgG was purified from

the anti-GLUT4 rabbit serum using a modification of the

technique described by Dankert et al. (83). Total IgG was

purified from 15 mL of serum by adjusting the pH to 5 with 3

M acetic acid and adding 0.75 mL of capryllic acid while

vigorously stirring. After stirring for 30 min, the

precipitate was collected by centrifugation at 41,000 X g

for 30 min at 4C. The supernatant was removed and its

volume measured. An equal volume of saturated ammonium

sulfate was added to the supernatant and stirred overnight

at 4C. The next day, the solution was divided equally

between two 15-mL corex tubes and the precipitate was

collected by centrifugation at 41,000 X g for 30 min at 4C.

The supernatant was discarded and each pellet was

resuspended in 0.5 mL of PBS containing 0.1% sodium azide.

The ammonium sulfate was removed from the samples by

dialyzing against three changes of PBS containing 0.1%

sodium azide over 48 h using tubing with a molecular weight

cut off of 13,000 Da. The concentration of the total IgG

solution was determined by measuring the absorbance of the









solution at 280 nm using a milligram extinction coefficient

of 1.4.

GLUT4 antibody was purified from the total IgG solution

by peptide chromatography. For this technique, we used a

SulfoLink Kit from Pierce. This kit contains a column

composed of 6% agarose beads cross-linked to iodoacetate.

The acetyl group binds irreversibly to free sulfhydryl

groups. Two mg of the GLUT4 peptide were bound to this

matrix through the terminal cysteine residue following the

manufacturer's instructions. GLUT4-specific antibody was

then purified by placing 2-3 mL of the total IgG solution on

the column and rotating at room temperature for 2 h. The

column was then washed with 16 mL of PBS and the GLUT4

antibody was eluted with 8 mL of 0.1 M glycine, pH 3.0. One

mL fractions were collected in tubes containing 100 )iL 1 M

Tris-base. The absorbance of the fractions was measured at

280 nm and fractions containing antibody were pooled.

Figure 2-5 shows a typical elution profile of peptide-

purified anti-GLUT4 antibody. The concentration of the

resulting antibody solution was determined by its absorbance

at 280 nm using a milligram extinction coefficient of 1.4.

The purified GLUT4 antibody was then stored as 0.1 mL

aliquots at -20C.















0.5

0.4

0 0.3

0.2

0.1

0.0 IMP7
01 "57
o~o ^ VA ^ y/ ^Ty

0 1 22 3 4 5 6 7 8

Fraction




Figure 2-5. Elution Profile of Peptide-Purified anti-GLUT4
Antibody. Total IgG was added to the column and rotated for
2 h. Unbound antibody was washed from the column using PBS.
GLUT4 antibody was eluted using 0.1 M glycine, pH 3.0. One
mL fractions were collected and neutralized with 1 M Tris-
base. Absorbance of the fractions was measured at 280 nm
and the concentration determined using a milligram
extinction coefficient of 1.4.









Preparation of Total Membranes


3T3-L1 adipocytes grown in 10-cm tissue culture dishes

were washed three times with 4 mL of KRP at 37C. Cells

were then scraped into 4 mL of TES buffer (10 mM Tris-HCl,

pH 7.4, 1 mM EDTA, 250 mM sucrose, and 20 [.g/mL PMSF) on

ice. The cells were homogenized using 20 strokes of a

teflon pestle in a 10 mL Potter-Elvejhem flask. The

homogenate was then centrifuged at 1,300 X g for 5 min to

remove nuclei and unbroken cells. The supernatant was

placed into polycarbonate tubes and membranes were collected

by centrifugation at 212,000 X g for 1 h in a Ti70.1 rotor

at 4C. The supernatant was discarded. For

immunoprecipitation, the pellet was resuspended in 1 mL of

extraction buffer (PBS containing 2% C12E9, 0.1% SDS,

1 mM EDTA, and 20 tg/mL PMSF) using 10 strokes of a teflon

pestle in a 2 mL Potter-Elvejhem homogenizing flask.

Insoluble material was then removed by centrifugation at

13,300 X g in a microfuge for 5 min. The supernatant was

transferred to a separate microfuge tube and protein

concentration was determined as described above. For

western blotting, the pellet was resuspended in 300 |JL of

TES and protein concentration was determined as described

above.









Metabolic Labeling of 3T3-L1 Adipocytes


To measure the synthesis and turnover of proteins,

radiolabeling of proteins with [35S]cysteine/methionine was

performed to "mark" a population of proteins within a

specific period of time. The accumulation of radiolabel

into proteins was determined to measure synthesis. To

measure turnover, cells were pulsed with radiolabel followed

by an extended chase with nonradiolabeled medium. The

disappearance of the labeled protein was then monitored. To

examine synthesis of GLUT4, cells were incubated in complete

DMEM with 10% FBS in the presence or absence of 10 nM

insulin for 11 h. The medium was then changed to cysteine-

/methionine-free DMEM, also in the presence or absence of 10

nM insulin, for 1 h. This medium was then aspirated and 2

mL of the cysteine-/methionine-free DMEM containing 400 iCi

of [35S]cysteine/methionine 10 nM insulin was added to

each plate. The plates were incubated at 37C for 10, 20,

30, 45, or 60 min at which time total membranes were

collected and extracted as described above.

Immunoprecipitation was performed on this solution.

Degradation of GLUT4 was examined by first incubating

cells in cysteine-/methionine-free DMEM for 1 h. Following

aspiration of this medium, 2 mL of the same medium

containing 400 piCi of [35S]cysteine/methionine were added to









each plate and incubated at 37C for 3 h. This medium was

aspirated and replaced with complete DMEM containing 10% FBS

with or without 10 nM insulin for the chase. This medium

was refreshed every 12 h and total membranes were collected

at 0, 6, 12, 24, 48, and 72 h. Total membranes were

collected and immunoprecipitation performed.


Immunoprecipitation of GLUT4


Immunoprecipitation provides a means of studying the

turnover of a particular protein. This technique depends on

the existence of an antibody specific for the protein of

interest. Addition of the antibody to a mixture of proteins

results in specific antibody-protein complex formation.

This complex can then be collected by the addition of

Sepharose beads conjugated with protein A, a bacterial

protein that binds to the heavy chain of IgG. The beads are

precipitated by a short centrifugation and the bound

proteins are analyzed. Although in these studies this

technique was employed to study the dynamics of GLUT4,

protein-protein interactions can be determined by analyzing

proteins which co-immunoprecipitate with the protein of

interest.

To examine the synthesis of GLUT4, extracted membrane

protein of specific concentration (1.5 mg) was brought to 1

mL with additional extraction buffer. To investigate the









degradation of GLUT4, equal volumes (750 pL) of each sample,

which represents equal cell number, were brought to 1 mL

instead of equal protein. This was done as the total cell

protein content increases over extended periods of chronic

insulin treatment. Non-specific interactions with protein

A-Sepharose were reduced by preincubation of extracted

material with 50 pL of a 50% slurry of protein A-Sepharose

at 4C for 1 h. The sepharose beads were removed by brief

centrifugation. Five Vg of peptide-purified GLUT4 antibody

was added to the supernatants. The samples were rotated

overnight at 4C. The next morning, 25 pL of a 50% slurry

of protein A-Sepharose were added to each sample and

rotation was continued for an additional 2 h at 4C. The

sepharose bead complexes were collected by brief

centrifugation. The supernatant was discarded. The beads

were then washed three times with 1 mL of extraction buffer

for 10 min followed by four, 10-min washes with 1 mL of

extraction buffer containing 1 M NaCl. Samples were then

washed with 1 mL TES buffer. The final pellet was

resuspended in 25 pL of TES buffer. Sample dilution buffer

(30 pL) was added to the beads and incubated at 37C for 30

min. Proteins in the entire sample were resolved on a 10%

polyacrylamide gel by electrophoresis as described above.

The gel was then fixed in 100 mL of 10% (w/v)

trichloroacetic acid and 60% (v/v) methanol for 30 min. The









solvent was removed by soaking the gel in water for 30 min.

The gel was then soaked in 100 mL of 1 M sodium salicylate

for 1 h. The gel was dried under vacuum onto 3MM Whatman

paper and juxtaposed to x-ray film at -80C. The P

particles emitted by the 35S bombard the ring structure of

the sodium salicylate causing it to fluoresce and this

emission exposes the x-ray film.


ATP-Luciferase Assay


ATP levels were measured in the 3T3-L1 adipocytes by

using an ATP-luciferin/luciferase assay with the assistance

of Dr. James Gardner. The cleavage of luciferin by

luciferase requires ATP and is the basis of the assay. When

luciferin is cleaved, light is emitted and the intensity of

this light correlates with the amount of ATP in the sample.

Cells (grown in 35-mm dishes) were first rinsed three

times with 3 mL of KRP at 37C. One mL of 8% perchloric

acid at 4C was added to each plate and the cells scraped

directly into 1.5-mL eppendorf tubes. The samples were

neutralized by the addition of 3 M KOH containing 0.5 M

triethanolamine base dropwise until a pH of 6.0 was

obtained. The samples were kept on ice during this

procedure and the volume of the samples before and after the

addition of KOH was measured to calculate the volume of KOH

added. The precipitate formed was removed by centrifugation









in a microfuge for 5 min at 4C. The supernatant was

diluted 1:250.

Ten iL of the 1:250 diluted sample or ATP standard was

added to a plastic 12 x 75 mm tube. To this, 500 pL of

assay buffer (60 mM Tris-Acetate, pH 7.75, 10 mM MgCI2, 1 mM

KC1l, 1.5 mM EDTA, 2.5 mM 3-mercaptoethanol, 0.4 mg/mL

luciferin/luciferase stock) were added. The V-

mercaptoethanol was added after the initial solution was

brought to pH 7.75. The luciferin/luciferase stock was

prepared by adding 50 mg of luciferin/luciferase to 2.5 mL

of 50% glycerol. This was divided into 300 pL aliquots and

stored in liquid nitrogen and added to the assay buffer just

prior to use. Following addition of the assay buffer to the

sample, the tube was flicked for 10 sec then placed in the

luminometer and the fluorescence measured for 20 sec. ATP

in the samples was calculated by comparison against the

standard curve seen in Figure 2-6.


Leptin Assay


Cells (5, 10-cm plates) were fed with serum-free DMEM

for 12 h. The medium was then collected, pooled from the

five plates (a total of 40 mL), and concentrated using the

"Centriplus 3" centrifugal concentrating tubes from Amicon,

which have a molecular weight cut-off of 3,000 Da. The













16000 -

S12000 -

8000 -

S 4000 -

0 I
0 1 2 3

pmol ATP



Figure 2-6. ATP Standard Curve. Ten [tl of specific
concentrations of ATP were added to 12 x 75 mm plastic
tubes. The luciferin/luciferase assay buffer was added, the
tube was flicked for 10 sec., and the fluorescence measured
in a luminometer for 20 sec.









medium was separated equally into four tubes and

concentrated by centrifugation at 3,000 x g (4,900 rpm) for

4.8 h at 4C in an SS-34 rotor. The retained solution was

collected from the four concentrators and pooled. The

pooled solution was then placed into a single new tube and

concentrated further by centrifugation at equal force and

time. The retained solution was collected again and brought

to 4 mL using PBS.

The leptin radioimmunoassay (RIA) was performed as per

the manufacturer's instructions. Briefly, 100 [iL of assay

buffer (0.05 M PBS pH 7.4 containing 0.025 M EDTA, 0.1%

sodium azide, 0.05% Triton X-100 and 1% RIA grade BSA) were

added to 12 x 75 mm borosilicate glass tubes. Leptin

standards (100 4L) and unknown samples (100 ptL) were added

to the appropriate tubes. Next, mouse leptin antibody (100

|LL) was added to the tubes. The tubes were mixed on a

vortex incubated overnight at 4C. The next morning, 125s-

mouse leptin (100 pL) was added to all the tubes, mixed, and

incubated overnight at 4C. The following morning, 1 mL of

cold precipitating reagent was added to all the tubes and

mixed. The tubes were incubated for 20 min at 4C and the

precipitate collected by centrifugation for 15 min at 3,000

x g (5,000 rpm) in an SS-34 rotor at 4C. The supernatant

was decanted immediately and the tubes drained for 1 min.

Excess fluid was blotted from the lip of the tubes and the






47


pellet was counted in a gamma counter. Leptin levels

present in the samples were calculated by comparing against

the standard curve seen in Figure 2-7.














100







1



1 0I , , ,I I II
1 10


Leptin (ng/mL)

Figure 2-7. Leptin Standard Curve. Mouse leptin antibody
was added to specific concentrations of leptin provided in
the kit and incubated overnight at 40C. 125I-mouse leptin
was added and incubated at 4C overnight. One mL of cold
precipitating reagent was added and incubated for 20 min at
40C. The precipitate was collected by centrifugation, the
supernatant was decanted and the radioactivity present in
the pellet determined by a gamma counter.
















CHAPTER 3
DEVELOPMENT OF INSULIN RESISTANCE


Introduction


Insulin resistance has been studied in animal models,

freshly isolated cells, and immortalized cells. Using a

cell line has several advantages. One of these advantages

is the ease of harvesting cultured cells. Another is the

consistency and reproducibility of data, the lack of which

plagues in vivo studies.

Although 3T3-Ll adipocytes have been the focus of many

insulin action studies, in each case, pharmacological doses

of insulin were used. In contrast to other classes of

hormones, insulin does not exhibit biphasic effects, thus

the rationale for using pharmacological doses. However,

caution must be taken as insulin at high concentrations in

cell culture activates the mitogenic paths held in common

with other growth factors, the relevance of which is

questioned (60). That said, with long term exposure to

pharmacological doses of insulin, GLUT4 expression (both

mRNA and protein) is reduced (14). This reduction is caused

by both down-regulation of transcription and enhanced

turnover of mRNA. However, the concentration of insulin









required to affect a 50% change in expression of message was

reported as 23 nM. This level of insulin is at least 2

orders of magnitude higher than the concentration of

circulating insulin in humans (2). No comparable dose-

response studies have examined the development of insulin-

resistant glucose transport activity or GLUT4 expression in

these cells. Therefore, I have developed a procedure that

has allowed the measurement of glucose transport after

chronic exposure (12 h) to physiological concentrations of

insulin.


Results


Insulin-resistant Glucose Transport Activity


Chronic insulin exposure elevates glucose transport

activity in 3T3-LI adipocytes. This can be prevented by

protein synthesis inhibitors like cycloheximide (Risch and

Frost, unpublished data). More recent studies have shown

that pharmacological doses of insulin induces GLUT1

transcription (14) leading to elevated GLUT1 protein and

thus transport activity. In the face of elevated GLUT1,

insulin-sensitive glucose transport activity can not be

observed. While this may imply insulin resistance, elevated

basal transport activity complicates interpretation. My

first challenge was to develop a protocol to reestablish

basal transport activity after chronic exposure to insulin.









I relied initially on studies by Garvey et al. (15). These

investigators showed that incubation of cultured rat

adipocytes with insulin for 12 h was sufficient to complete

the desensitization process, resulting in a 50% reduction in

insulin-sensitive glucose transport activity. Therefore,

cells were first treated with either 10 nM or 1 [iM insulin

for 12 h. The cells were then washed in KRP buffer

containing 5 mM glucose and 0.1% defatted BSA at 20-min

intervals over a 140-min time course. At specific times

during this washout period, cells were rinsed in KRP alone

and glucose transport activity determined in a 2-min pulse.

Figure 3-1 A shows the comparison between control cells

(washed in an identical manner) and those treated

chronically with either 1 tM or 10 nM insulin. Cells

treated chronically with 1 ItM insulin showed significantly

elevated transport at the start of washout (time 0) but

never achieved basal levels despite the extensive time of

washing. However, those cells treated with 10 nM insulin

returned to basal levels within 60 min of washing. When

these latter cells were subsequently restimulated with an

acute insulin challenge following the 60-min wash, the rate

of glucose transport was reduced by 50% compared to control

cells (Figures 3-1 B and 3-2). In addition to the decreased

rate, the cells were less sensitive to insulin in that the


















Figure 3-1. Effect of Chronic Insulin on Glucose Transport
in 3T3-L1 Adipocytes. Panel A, fully differentiated 3T3-L1
adipocytes were incubated in DMEM containing 10% FBS in the
absence of added insulin (0) or with 1 |LM (A) or 10 nM (U)
insulin for 12 h. Plates were washed as described in
Chapter 2 under "Insulin Washout Procedure". Glucose
transport activity was measured in KRP (in the absence of
bovine serum albumin and glucose) by the addition of 200 jiM
[3H]2-deoxyglucose (0.2 jiCi). After 2 min, transport was
terminated by the addition of ice-cold phosphate-buffered
saline. Cells were lysed with a 0.1% solution of SDS and
duplicate aliquots of 300 jiL were taken for quantitating
radioactivity. Panel B, cells were incubated as above in
the absence (control) or presence (chronic) of 10 nM
insulin. Removal of insulin was accomplished within 60 min
(see panel A), then 1 (JM insulin was added back, or not, for
10 min, and glucose transport activity was determined.
Panel C, cells were incubated for 12 h in the absence
(control) or presence (chronic) of 10 nM insulin. The
cells were washed for 60 min to remove insulin. In order
to maintain the low levels of insulin in solution, glucose
transport activity was measured in KRP containing 0.1% BSA.
Therefore, following the final wash at 60 min, 950 [iL of KRP
containing 0.1% BSA were added to each plate. Various
concentrations of insulin, dissolved in 0.1%, were then
added for 10 min for acute stimulation. Glucose transport
activity was measured by the addition of [3H]2-deoxyglucose.
Each panel represents the average S.E. of two independent
experiments (n=4).














0.5


rD.S
tO


Q -
- 0


^^o
g 2


1.5

1.0


0.5

0.0
Acute Insulin


0 50 100 150
Time of Washout (min)


- +


-


Control Chronic


100 -c/ 1
80 control
60 -
40 -chronic



0 0.01 1 100
Insulin Concentration (nM)


B


f// /

v7n //. / /_













-^ 2.0

ot
+-

^ ~1.5- r


--X0 1.0


o0.5


00 I I.
0 5 10 15

Time (min)





Figure 3-2. Time Course of Acute Insulin Stimulation.
Cells were incubated in DMEM containing 10% FBS 10 nM
insulin for 12 h. Plates were then washed as described in
Chapter 2 under "Insulin Washout Procedure". Cells were
then stimulated for specific times with 1 [tM insulin and
glucose transport activity determined. Data represent the
average S.D. of duplicate samples within a single
experiment (n=2). control; N chronic insulin.









dose-response curve to acute insulin challenge was shifted

to the right by an order of magnitude (Figure 3-1 C). This

is the first demonstration of "true" insulin-resistant

glucose transport activity in 3T3-L1 adipocytes because of

the ability to reestablish the basal state.

To determine if physiological insulin could establish

the insulin-resistant state, cells were exposed to specific

concentrations of insulin for 12 h. Cells were refed every

2 h to maintain the extracellular insulin levels,

particularly important at the lower concentrations of

insulin as these cells possess the ability to rapidly

degrade insulin. As shown in Figure 3-3 A, the

concentration of insulin that elicits a 50% reduction in

insulin-sensitive glucose transport was approximately 100

pM. This is extremely interesting because the fasting level

of insulin in non-diabetic humans is about 40 pM while that

in obese individuals is about 70 pM and individuals with

non-insulin-dependent diabetes have insulin levels around

200 pM (84). Insulin as low as 1 nM was sufficient to

completely desensitize the transport system. At 10 nM,

maximal desensitization was achieved whether the cells were

refed every 2 h or not. Thus, we chose a concentration of

10 nM to examine the time required for the development of

insulin resistance. As shown in Figure 3-3 B, the

phenomenon of desensitization was completely established

























Figure 3-3. Development of Insulin Resistance. Panel A,
cells were incubated for 12 h with specific concentrations
of insulin as indicated. During this incubation, the
medium was replaced every 2 h. The cells were then washed
for 60 min and glucose transport activity determined
following acute (10 min) stimulation with 1 iM insulin. The
"fractional difference" was determined by subtracting the
glucose uptake rate at 10 nM insulin from the glucose
uptake rate at each point divided by the difference in
uptake rates between 0 and 10 nM insulin. Panel B, cells
were incubated with 10 nM insulin for specific times.
Medium was replaced every 2 h. At appropriate times, the
cells were washed and acutely stimulated with insulin, and
glucose transport activity was measured. The fractional
difference in activity was determined as in panel A. Data
represent the average S.E. of three independent
experiments (n=6).







A
1.0
o = 0.8
0.6-
0.4 -
S0.2 -

u^ 0.0-

0- 0J

Insulin Concentration (nM)

B

z' 1.0
I 0.8-
3 0.6-
0.4-
0.2-
n nn


0 2 4 6 8 10 12


Time (h)









within 8 h of the initial exposure to insulin. This result

is similar to that described in isolated adipocytes (21)


Role of Glucose and Glucosamine


Marshall and his colleagues (15,20,21) have shown in a

series of elegant experiments the requirement of glucose and

glutamine, as well as insulin, for the expression of insulin

resistance in isolated rat adipocytes implicating the N-

acetylglucosamine biosynthetic pathway in this phenomenon.

To test if the same is true in 3T3-L1 adipocytes, I

performed similar experiments. One complication in my

experiments that was not encountered in isolated adipocytes

is the time-dependent activation of glucose transport

activity in the absence of glucose (85-88). This difference

between rat adipocytes and 3T3-Ll adipocytes may result from

higher glycogen stores in the former (89,90) compared with

3T3-Ll adipocytes (79), which provides a metabolic buffer

from external glucose deprivation. We therefore minimized

the time that the cells were exposed to glucose-free medium

but suffered in that only 75% of maximal desensitization was

achieved in these experiments. Importantly, though, the

basal rates of transport were not affected such that true

resistance could be evaluated. Figure 3-4 shows that in the

absence of glucose, insulin was unable to induce

desensitization. Either glucose, in the presence of

























Figure 3-4. Effects of Glucose and Glucosamine on Insulin
Resistance. Panel A, cells were incubated for 6 h in DMEM
containing specific concentrations of glucose in the
absence (control) or presence (chronic) of 10 nM insulin.
Cells were washed and glucose transport activity was
determined in the presence of 1 gM insulin. Panel B, cells
were incubated for 6 h in glucose-free and glutamine-free
DMEM containing specific concentrations of glucosamine in
the absence (control) or presence (chronic) of 10 nM
insulin. Washes were performed on the cells as described
earlier, and glucose transport activity following acute
stimulation with 1 iM insulin was determined. Data
represent the average S.E. of two independent experiments
(n=4). Basal glucose transport activity in control and
glucose-deprived cells was 0.167 0.02 and 0.167 0.01
nmol/106 cells/min, respectively.





A
S1.4

|1.0
I ~0.8
0.6
Q 0.4 -- Control
N, 0.2 -- Chronically treated
m^ 0.0 -------
S0 5 10 15 20

Glucose Concentration (mM)
B
. 1.4


|1.0
S 0.8
o 0.6
0.4- Control
<^ 0.2 -U- Chronically treated
m 0.0 -------
S0.0 0.5 1.0 1.5 2.0

Glucosamine Concentration (mM)









glutamine (Figure 3-4 A), or glucosamine, in the absence of

both glucose and glutamine (Figure 3-4 B), provided

appropriate substrate for the development of insulin

resistance.


Effects of Various Inhibitors on the Development of Insulin
Resistance


Inhibitors of DNA transcription, protein synthesis,

glycosylation and proteases were used to characterize the

development of insulin resistance in these cells. Cells

incubated for 12 h with 4 j.M actinomycin D, an inhibitor of

DNA transcription, exhibited an increase in basal glucose

transport activity and a reduction in insulin-stimulated

glucose transport in both control and chronically-treated

cells (Figure 3-5). The underlying mechanism for this

elevation is unknown. Given elevated basal activity, the

effect of actinomycin D on insulin-sensitive transport is

difficult to determine, although changes in the presence and

absence of acute insulin were fairly small. In contrast,

studies in isolated rat adipocytes (91) showed that

incubation with up to 200 nM actinomycin D for 18 h actually

decreased basal levels. However, in these studies,

actinomycin D prevented the development of insulin

resistance. It is believed that actinomycin D prevented

insulin resistance in these studies by inhibiting GFAT

activity, as insulin resistance induced by incubation with











1.2 T
S1.0


Q- 0.8
006

0.6


0.2-
o.o ^^-^ ^ ^--2^^

acute insulin + + + + + +
actinomycin + + +
cycloheximide + + + +
I I
control chronic






Figure 3-5. Effects of Actinomycin and Cycloheximide on
Insulin Resistance. 3T3-L1 adipocytes were incubated in the
presence or absence of 4 gM actinomycin D or 20 IJM
cycloheximide also in the presence (chronic) or absence
(control) of 10 nM insulin for 12 h. Insulin was removed
from the cells by washing as described previously. Basal
and insulin-stimulated rates of glucose transport were
measured by incubating the cells in KRP with 1 jiM insulin,
or not, for 10 min. The data represent the average S.E.
of two independent experiments (n=4).









glucosamine, which bypasses GFAT, could not be prevented by

actinomycin D (91). As Figure 3-5 shows, in 3T3-L1

adipocytes, 20 (JM cycloheximide had no effect on basal

transport, but inhibited insulin-stimulated glucose

transport in both control and chronically-treated cells.

Therefore, cycloheximide did not appear to prevent insulin

resistance. Studies in isolated rat adipocytes also showed

that cycloheximide was unable to prevent insulin resistance

(92).

To further test the involvement of the hexosamine

biosynthetic pathway in the development of insulin

resistance, the effects of nikkomycin Z on insulin-resistant

glucose transport activity were tested. Nikkomycin Z is a

peptide-nucleoside antibiotic that competitively inhibits

chitin synthase in fungi and insects due to its structural

similarity to UDP-N-acetylglucosamine (93). I hypothesized

that this compound might prevent the onset of insulin

resistance by competing for enzymes which use UDP-N-

acetylglucosamine in mammalian cells. As Figure 3-6 shows,

neither concentration of nikkomycin Z tested had an effect

on glucose transport. Although I have no evidence that

nikkomycin is taken up by these cells, the concentrations

used are in far excess of 0.1 tM, the Ki of nikkomycin for


chitin synthase (94).













S0.8



0.6


d) 0.4 ,,

hx J- -/ ^ / T







acute insulin + +
20M Nikkomycin +
200 iM Nikkomycin + + +
I I
control chronic






Figure 3-6. Effects of Nikkomycin Z on Insulin Resistance.
Cells were incubated in the presence or absence of specific
concentrations of nikkomycin Z also in the presence
(chronic) or absence (control) of 10 nM insulin for 12 h.
Plates were washed to remove insulin and basal and insulin-
stimulated rates of glucose transport were measured as
previously described. Data represent the average + S.D. of
duplicate samples within a single experiment (n=2).









Because the hexosamine biosynthetic pathway provides

substrate for glycoprotein synthesis, I tested the effects

of tunicamycin on the development of insulin resistance. As

shown in Figure 3-7, 2.5 J.g/mL tunicamycin, which inhibits

N-linked glycosylation, decreased the insulin-stimulated

rate of glucose transport in both control and chronically-

treated cells. This is similar to the effect of

cycloheximide and again makes it difficult to determine if

tunicamycin was able to prevent the decrease in glucose

transport in insulin-resistant cells.

It has been hypothesized by Knutson et al. (95) that a

proteolytic fragment of the P-subunit of the insulin

receptor is involved in the development of insulin

resistance. To test this, I used a thiol protease inhibitor

(E-64) to prevent cleavage of the 3-subunit (95). As is

shown in Figure 3-8, 100 |JM E-64 had no effect on glucose

transport activity in control or chronically-treated cells.

As the Knutson group showed the largest effect of E-64 on

glycogen synthesis, I also investigated this insulin-

sensitive process. As Figure 3-9 shows, 100 tM E-64 had no

effect on rates of glycogen synthesis in control or insulin-

resistant cells.













1.0 -

< E 0.8 -
0 r.f /

S 0.6 "0.




//
gr 0.4 E I I
//" I I11 I -




tunicamycin a i t p

Control Chronic







Figure 3-7. Effects of Tunicamycin on Insulin Resistance.
Cells were incubated in the presence or absence of 2.5 Ig/mL
tunicamycin also in the presence (chronic) or absence
(control) of 10 nM insulin for 12 h. Cells were washed to
remove insulin and basal and insulin-stimulated rates of
glucose transport were measured as previously described.
Data represent the average + S.D. of duplicate samples
within a single experiment (n=2).











1.2


1.0
0.8


c(

I
o _
o '-


acute insulin


E-64


- +


+ +


control


c +
chronic


Figure 3-8. Effects of E-64 on Insulin Resistance. Cells
were incubated in the presence or absence of 100 iM E-64
also in the presence (chronic) or absence (control) of 10 nM
insulin for 12 h. Cells were then washed to remove insulin
and basal and insulin-stimulated rates of glucose transport
were determined as previously described. Data represent the
average S.E. of two independent experiments (n=4).


0.6
0.4
0.2
0.0


Jigj

















-
c,)0

c,)0^


40


30


20


10


0


acute insulin
chronic insulin
E-64


+ + + +


+ +


+ +
+ + + +


Figure 3-9. Effects of E-64 on Glycogen Synthesis. Cells
were incubated in the presence or absence of 100 [M E-64
10 nM insulin for 12 h. Plates were then washed to remove
insulin and labeled with 14C-U-[D-glucose] (2 tCi/plate) in
KRP containing 5 mM glucose in the presence or absence of 1
gM insulin for 1 h. Glycogen was then collected as
described and radioactivity counted by liquid scintillation.
Data represent a single experiment.









Effect of Chronic Insulin on GLUT4 Expression and
Translocation


A total membrane fraction revealed that insulin-

resistant cells (i.e. cells exposed to 10 nM insulin for 12

h) expressed 2.4-fold less GLUT4 than control cells while

GLUT1 increased by 2.2-fold (Figure 3-10). Another

important point to make as shown by Figure 3-10 is that

acute insulin stimulation did not alter the total levels of

GLUT1 or GLUT4. Only chronic insulin treatment affected the

total amounts of these proteins. To examine the subcellular

distribution of these changes, I used a subcellular

fractionation technique recently developed in this lab (70)

to isolate three membrane fractions: plasma membrane (PM),

low density membranes (LDM), and high density membranes

(HDM). The LDM consists primarily of the small endosomal

storage vesicles containing GLUT4 and the HDM consists

primarily of endoplasmic reticulum and Golgi. Figure 3-11

shows the distribution of GLUT4 and GLUT1 among these three

membrane fractions in control and insulin-resistant cells.

Each set went through the washout procedure prior to

membrane fractionation. Control cells, which were

stimulated acutely with 1 jiM insulin, showed redistribution

of both GLUT4 and GLUT1; GLUT4 increased by about 6-fold in

the PM (Figure 3-11 A and B) while GLUT1 increased by about

2-fold (Figure 3-11 C and D). These data are similar to


















Acute Insulin
Chronic Insulin


GLUT1 -



GLUT4 -


+ +


Figure 3-10. Effects of Acute and Chronic Insulin on Total
GLUT Protein Levels. Equal protein of the PM, LDM, and HDM
fractions of Figure 3-11 were combined and subjected to SDS-
PAGE and transferred to nitrocellulose. Immunoblot
detection of GLUT1 and GLUT4 was carried out using C-
terminal specific antibodies. The protein-antibody complex
was visualized by enhanced chemiluminescence.

























Figure 3-11. Subfractionation of Insulin-Resistant 3T3-L1
Adipocytes. Cells were treated for 12 h in the absence
(control) or presence (chronic) of 10 nM insulin and
subsequently washed. Following acute stimulation with 1 (JM
insulin, PM, LDM, and HDM were collected as described in
Chapter 2. SDS-polyacrylamide gels of equal protein (70 [tg)
transferred to nitrocellulose allowed immunoblot detection
of GLUT1 and GLUT4 using C-terminal specific antibodies.
The protein-antibody complex was visualized by enhanced
chemiluminescence. Bands were quantitated by video
densitometry. Panel A, immunoblot of membrane fractions
probed with anti-GLUT4 antibody; panel B, densitometry of
GLUT4 immunoblot; panel C, immunoblot of membrane fractions
probed with anti-GLUTi antibody; panel D, densitometry of
GLUT1 immunoblot. I] control; M control + acute insulin; S
chronic insulin treatment; M chronic insulin treatment +
acute insulin. Data represent a single experiment. A
duplicate experiment gave similar results.













A PM LDM HDM
Chronic Insulin + + + + + +
Acute Insulin + + + + + +
GLUT4 i--.n



B GLUT4 Levels
50

S40 -

30 p





PM LDM HDM
S20 | X rN
H /, ^V./N
10 o- XNl k
rOl N\ I l
PM LDM HDM


















PM LDM HDM


Chronic Insulin
Acute Insulin

GLUT1 -*


+ +
+ +


GLUT1 Levels


100


80

r 60

* S 40

20

0


1 ^^ ^,
,'R-^


PM LDMHD


+ +


+ +


+ +


+ +









those that analyzed translocation using the cell surface

photolabel, ATB[2-3H]BMPA [2-N-4-(l-azi-2,2,2-

trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-

propylamine] (16). Cells chronically exposed to insulin

(followed by washout) showed levels of GLUT4 in the plasma

membrane equivalent to that of controls (Figure 3-11 A and

B). While acute insulin challenge stimulated translocation,

the amount of GLUT4 was reduced by about 50% compared with

controls. This reflects the down-regulation of GLUT4 and

correlates well with the loss of insulin-stimulated glucose

transport activity. Cells chronically exposed to 10 nM

insulin showed a 2-fold increase in the level of GLUT1 in

the PM after washout compared with controls (Figure 3-11 C

and D), despite the equivalent rates of glucose transport.

Acute insulin challenge stimulated translocation but to a

much more limited degree than in control cells.

As the LDM fraction reflects the loss of GLUT4, we used

this fraction to examine the dose-dependent loss in cells

chronically treated with specific concentrations of insulin.

As shown in Figure 3-12 A, the level of GLUT4 decreased over

time in response to increasing insulin. Figure 3-13 shows a

similar experiment with a larger sample (5, 10-cm plates per

condition compared to 1, 10-cm plate). This larger sample

allows for the somewhat more accurate quantitation of the

LDM fraction. The numbers listed below the blot in Figure















Insulin
[nM]


GLUT4 -4


0 W


0


00
I-


0 0.1 1 10 100 1000
Insulin [nM]


Figure 3-12. Effects of Chronic Insulin on GLUT4
Expression. Panel A, cells were incubated with specific
concentrations of insulin for 12 h (fed with fresh medium
every 2 h). Cells were then washed for 60 min and
subfractionated to isolate the LDM fraction. Proteins were
separated by SDS-polyacrylamide gel electrophoresis and
subsequently transferred to nitrocellulose membrane (70 p[g
of protein were loaded per lane). The membrane was then
probed for GLUT4 and visualized by enhanced
chemiluminescence. Panel B, GLUT4 bands were quantitated by
densitometry. Fractional difference in GLUT4 expression was
calculated from five independent experiments performed as in
panel A. Data represent the average S.E.



















Insulin [nM] 0 (j K- N


GLUT4 --


100 93


67 37


Figure 3-13. Effects of Chronic Insulin on GLUT4 Expression
with Larger Samples. Cells were incubated as described in
Figure 3-12 with the exception that 5, 10-cm plates were
used per condition compared to 1, 10-cm plate in Figure 3-
12. The LDM fraction was collected and treated as described
in Figure 3-12. The numbers listed below the figure are the
percentages of GLUT4 present in each sample compared to the
0 nM insulin control based on densitometric analysis.









3-13 are the percentages of GLUT4 in each sample compared to

the 0 nM insulin control as determined by densitometric

analysis. The dose dependence of this down-regulation

(Figure 3-12 B and Figure 3-13) yielded a K50 of about

600pM, slightly higher than the K50 of insulin-resistant

glucose transport, but a technically more difficult

parameter to measure.


Effect of Glucose Deprivation on GLUT4 Expression


Based on the observation that glucose deprivation

prevented the loss in insulin sensitivity (see Figure 3-4

A), we examined the expression of GLUT4 in the LDM fraction

of cells exposed to glucose-free medium. Figure 3-14 shows

that glucose deprivation blocked the loss of GLUT4 in

chronically treated cells. Thus, this shows for the first

time that glucose is important in regulating the expression

of GLUT4 in response to chronic insulin.


Effect of Chronic Insulin on GLUT4 mRNA Expression


These studies were conducted by Martin Williams to

evaluate the underlying mechanism of the reduction in GLUT4

protein. The level of GLUT4 mRNA was measured after

exposure to specific concentrations of insulin. As shown in

Figure 3-15 A, the level of GLUT4 mRNA decreases with

increasing insulin concentration. However, the





















Glucose + +
Chronic Insulin +


GLUT4 --


Figure 3-14. Effect of Glucose Deprivation on GLUT4
Expression. Cells were maintained in medium for 12 h in the
absence or presence of 10 nM insulin and/or 25 mM glucose.
The LDM fraction was isolated and GLUT4 analyzed by
immunoblot analysis. Data are representative of three
independent experiments.














Insulin
[nM]


GLUT 4 -



Actin -.


W ...qw.e


B

a.)
0


S0.0 i 1. .i ... 1 iil lw
0.1 1 10 100 1000
Insulin [nM]


Figure 3-15. Effects of Chronic Insulin Treatment on GLUT4
mRNA Levels. Panel A, cells were incubated for 12 h in the
presence of specific concentrations of insulin (refed every
2 h). Cells were then washed three times with 8 mL KRP at
which time RNA was extracted using the phenol:chloroform
extraction method. Twenty jig of total RNA were loaded onto
a 1% formaldehyde-agarose gel and subsequently transferred
to a nylon membrane. The membranes were probed with a 32P-
labeled cDNA for GLUT4. Panel B, densitometric analysis
represented as fractional difference in sensitivity. Data
shown represent a single experiment replicated three times.









concentration of insulin required to elicit a 50% loss in

GLUT4 mRNA was about 5 nM (Figure 3-15 B), which is 15 times

greater than that required for the equivalent loss of

insulin-sensitive glucose transport activity and 10-times

that for GLUT4 expression.


Reversal of Insulin Resistance


To determine if the effects of chronic insulin on 3T3-

Ll adipocytes could be reversed, insulin-resistant cells

were incubated with complete medium following the washout

procedure. Both basal and insulin-stimulated rates of

glucose transport were measured at specific times. Eight

hours after insulin removal, glucose transport activity of

previously insulin-resistant cells was equal to that of

controls (Figure 3-16). An interesting point is that the

reversal appears to occur between 4-8 h, as no improvement

in insulin-sensitive transport occurs before that time.

Also, the rate of basal transport between control cells and

resistant cells is identical over this time while only the

insulin-stimulated rates are changing. This shows that the

increase in insulin-stimulated transport seen in resistant

cells over this time is not due to increasing basal rates,

but improved insulin sensitivity.

To determine if this reversal in insulin resistance

required protein synthesis, cycloheximide was included in













a.)
M 2.0-


SI 1-5
- 0
S 1.0


Q 0.5
cIS~

rr 0.0 I
0 2 4 6 8

Time (h)





Figure 3-16. Reversal of Insulin Resistance. Cells were
incubated in the presence (chronic) or absence (control) of
10 nM insulin for 12 h. Plates were washed to remove
insulin and refed with fresh medium lacking insulin. At
specific times, basal and insulin-stimulated rates of
glucose transport were determined. Data represent the
average S.E. of two independent experiments (n=4).
control; U control + acute insulin; A chronic; V chronic +
acute insulin.









this experiment. As is shown in Figure 3-17, cycloheximide

completely blocked the reversal of insulin-resistant glucose

transport activity. It should be noted that cycloheximide

decreased the insulin-stimulated rate of glucose transport

in control cells (closed squares versus inverted triangles).

However, the cycloheximide-induced block in reversal is

considerably larger (open squares versus inverted

triangles). This suggests that the reversal of insulin-

resistant glucose transport is dependent upon protein

synthesis. It is not clear, however, if this reversal is

due to the synthesis and recovery of GLUT4, as those studies

have not yielded reliable results.


Conclusions


In the above studies, I have tested the hypothesis that

3T3-L1 adipocytes can serve as a model for studying the

development of insulin resistance under conditions that

might be realized in a physiological setting. Support for

this hypothesis has been gained from the following

observations. Chronic exposure to physiological levels of

insulin decreased the ability of an insulin challenge to

stimulate glucose transport. Interestingly, postprandial

concentrations of insulin in normal, obese, and diabetic

humans (84) plot along the inflection in the dose-response

curve between no change in insulin responsiveness and that









S2.0

+1.5



0-.
0^


>1.


~ ~0.5

0.0
0 2 4 6 8


Time (h)

-Control
U Control + acute ins
A Control + cyclo
Control + cyclo + acute ins
Chronic
Chronic + acute ins
Chronic + cyclo
-v-- Chronic + cyclo + acute ins





Figure 3-17. Reversal of Insulin Resistance in the Presence
of Cycloheximide. Cells were incubated and washed as
described in the previous figure. Plates were then refed
with fresh medium 20 jM cycloheximide. At specific times,
basal and insulin-stimulated rates of glucose transport were
determined. Data represent the average S.D. of duplicate
samples within a single experiment (n=2).









of maximal resistance. Thus, I have shown for the first

time that 3T3-L1 adipocytes develop insulin resistance in

response to physiologically relevant concentrations of

insulin. I have extended previous work by demonstrating

that insulin challenge of resistant cells stimulates

translocation, although the extent of recruitment is

suppressed relative to controls due to the reduction in the

total expression of GLUT4. Together, these data suggest

that the loss of GLUT4 protein underlies the inability of

3T3-L1 adipocytes to respond to insulin after chronic

exposure. This mimics the clinical manifestation of human

obesity and non-insulin-dependent diabetes where loss of

GLUT4 protein has been observed in adipose tissue (96),

although not in muscle (97).

It should be pointed out that transporter expression

differs in adipose tissue relative to 3T3-Ll adipocytes. In

isolated rat adipocytes, GLUT4 represents 97% of the GLUT

transporter pool (43). In 3T3-Ll adipocytes, GLUT4

represents only 33% of the pool (98), indicating the

substantially higher expression of GLUT1 relative to GLUT4

in this cell line. In control 3T3-L1 adipocytes, the PM

fraction contains about 25% of the GLUT1 pool. Chronic

insulin treatment increases the total pool of GLUT1, which

in turn doubles the GLUT1 content of the PM fraction.

Despite this 2-fold increase in GLUT1 in the PM of resistant









cells, no difference is observed in basal transport activity

(after washout) compared with controls. Resistant cells

treated acutely with insulin show little additional change

in GLUT1 in the PM. This argues that GLUT1 plays but a

small role in insulin-resistant glucose transport. In

contrast, only 3% of the GLUT4 pool resides in the PM of

either control or resistant cells (again, after washout).

When insulin is added acutely, GLUT4 content in the PM

reveals significant translocation; resistant cells show 50%

less GLUT4 following translocation compared to controls

reflecting the difference in the total pool. Again, this

suggests that GLUT4 expression determines insulin

resistance, even with the elevated levels of GLUT1 in the

3T3-L1 adipocyte cell line.

There are some differences between these data and those

reported previously on 3T3-L1 adipocytes. Flores-Riveros et

al. (14) reported that the concentration of insulin required

to induce a 50% change in GLUT4 mRNA was 23 nM in contrast

to the value I calculated, which was about 5 nM, close to

the Kd for the insulin receptor (99). This difference can

be explained by my refeeding protocol during chronic insulin

treatment to maintain the level of extracellular insulin,

particularly important at low hormone concentration, in the

face of extensive degradation of insulin by these cells. In

addition, cells in this study were exposed to insulin for









only 12 h compared to the 24-h exposure in the Flores-

Riveros study (14), which further lessens the impact of

insulin degradation. This temporal difference (24 versus 12

h of insulin treatment) also accounts for the smaller

magnitude of the increase in GLUT1 and decrease in GLUT4 in

our study relative to previous studies (14,76).

Importantly, the down-regulation of GLUT4 mRNA occurs at

insulin concentrations that are not likely to persist in the

physiological state. These insulin concentrations also do

not correlate with those required for the development of

insulin-resistant glucose transport or decrease in GLUT4

protein. Thus, GLUT4 expression appears to be regulated

transcriptionally, but this regulation may not be relevant

to insulin resistance.

Other studies in 3T3-Ll adipocytes have shown varying

results in transporter protein expression. Tordjman et al.

(100) and Koska et al. (16) showed that chronic insulin

treatment did not affect total GLUT4 protein expression

while studies of Flores-Riveros et al. (14) and Clancy and

Czech (76) showed a marked decrease. These latter data along

with my studies are consistent with the accelerated turnover

of GLUT4 in the presence of chronic insulin as measured by

Sargeant and Paquet (101), which will be discussed in the

next chapter. Ricort et al. (64,102) showed a small

decrease in the expression of GLUT4 but also very little









translocation to the PM with acute insulin stimulation.

These authors interpreted their data to mean that GLUT4

translocation was blocked, which clearly differs from our

studies. Kozka et al. (16) interpreted their cell surface

ligand binding experiments similarly, even though they

demonstrated a 50% reduction in cell surface GLUT4, which

would agree with my studies. I can only speculate as to the

cause for the different results. In both of these latter

studies, the loss in the GLUT4 pool was determined by

analyzing homogenate protein, revealing only modest changes

in expression. As the translocatable GLUT4 resides in the

LDM fraction, it may be that the loss was substantially

underestimated. Neither study separated the LDM fraction

from the HDM fraction; thus, this possibility can not be

evaluated. Finally, it is important to point out that my

experiments are the first to show that basal transport

activity can be achieved after chronic insulin treatment,

which allowed me to evaluate true insulin resistance. Data

collected under these conditions are consistent with the

hypothesis that the onset of insulin resistance (i.e.

depressed insulin-sensitive glucose transport) is a

reflection of the reduced GLUT4 pool, not a defect in

translocation. In addition, this decrease in insulin-

sensitive glucose transport can be reversed within 8 h and

is protein synthesis dependent.