GLYCEMIC REGULATlON OF GLUCOSE TRANSPORT IN PERFUSED SKELETAL MUSCLE

Julian Marshall Ranji Mathoo

A thesis submitted in conformity with the requirernents for the Degree of Master of Science

Graduate Department of Physiology University of Toronto

Q Copyright by Julian M. R. Mathoo, 1997

National Library I * m of Canada Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques

395 Wellington Street 395. rue Wellington OttawaON K1A ON4 OttawaON K1AON4 Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, ioan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in ths thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts &om it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

GLYCEMIC REGUWON OF GLUCOSE TRANSPORT IN PERFUSED SKELETAL MUSCLE. MSc. thesis 0 1997, by Julian M. R. Mathoo, Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S i A8.

Glucose is known to regulate its own uptake in certain tissues, independent

of insulin. Adaptive changes to acute hyperglycemia in muscle take place at least

at the level of glucose transporter translocation. The effect of insulin-independent

hypoglycemia on muscle glucose transport has not been investigated.

The effects of acute hypo- and hyperglycernia per se on muscle glucose

transport were investigated in situ using rat hindquarter perfusion. Hypoglycemia

induced an insulin-independent adaptive upregulation glucose uptake and plasma

membrane (PM) GLUT4 content in muscle. In contrast, hyperglycemia

downregulated both muscle glucose uptake and PM GLUM. The effects of

glycemia on glucose uptake were quantitatively similar in oxidative and glycolytic

muscle fibers. It is concluded that glycernia regulates glucose transport in muscle

independent of insulin, partially via changes in PM GLUT4. Hyperglycemia

downregulates PM GLUT4, while hypoglycemia upregulates glucose transport in

muscle in compensation for reduced glucose availability, which may

triggerfaccentuate subsequent hypoglycemia in insulin-treated diabetic patients.

ACKNOWLEDGEMENTS

To my supervisors, Profs. Mladen Vranic, Zhi Qing Shi and Amira Klip, for their

guidance and support throughout the course of my graduate studies. I am grateful

for the opportunity to work on such a broad and challenging project, and affirm to

use the surgical, physiological, biochemical and molecular techniques I have

learned in your laboratories well in a career in science and medicir?e.

To Dr. Michael Wheeler for his guidance and advice as a member of my

supervisory comrnittee.

To Dr. Adria Giacca for insightful scientific discussions and the opportunity to

conduct much of my research in her laboratory.

To Toolsie Ramlal, MayLiza Van Delangeryt, Debbie Bilinski and Loretta Lam for

their scientific and technical advice.

To my fellow graduate students and colleagues in the lab, Shirya Rashid, Masa

Niwa, Hany Sandhu, Kirby Pilch, Dr. Carol Rodgers, Stephanie Weisenthal, Romel

Somwar, Tim Mason and Ban El-Bahrani for everything from scientific advice and

encouragement to 'quarter pool' in the alumni lounge.

iii

To Simon Fisher for being more than just a role model, but a true friend for three

years. Thank you for helping me get into this lab, for scientific and personal advice,

for coffee breaks, and for helping me attain admission to medical school. Thanks

also to Mike Lekas, Richard McCall, Theos Tsakiridis and Tony Rocca. the ghosts

of grad studentç past and present, for their help and encouragement in and outside

of the lab.

To my loving parents, Bharat and Hannelore Mathoo, for their endless support and

encouragement throughout my academic studies.

This work was funded by gants from the Canadian Diabetes Association (CDA) to

Z.Q. Shi, and the Medical Research Council of Canada (MRC) to M. Vranic and

Z.Q. Shi. I would also like to acknowledge the additional personal support in the

form of a studentship from the CDA. a scholarship from the University of Toronto

Open Fellowship program, three teaching assistantships from the Department of

Physiology, and a scholarship awarded but not accepted from the Ontario Graduate

Scholarship (OGS) program.

EXPERIMENTAL PREPARATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Albumin Dialysis 33

Perfusate Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

EXPERIMENTAL PROCEDURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Blood Glucose Measurernents . . . . . . . . . . . . . . . . . . . . . . . . . . 36

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Surgical Procedures 37 Perfusion System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

LABOUATORY METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma Glucose Assay 47

lnsulin Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 . . . . . . . . 2-(3H~-Deoxyglucose-6-~hosphate (2-DG-6-P) Assay 50

Whole Muscle Protein Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

MUSCLE MEMBRANE PREPARATION . . . . . . . . . . . . . . . . . . . . . . . . 54 Membrane Fractionation Procedure . . . . . . . . . . . . . . . . . . . . . . 54 Membrane Protein Determination . . . . . . . . . . . . . . . . . . . . . . . . 56 Western Blot Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

STATlSTlCAL ANALYSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

RESULTS and DISCUSSION

Basal Physical and Metabolic Characteristics of Rats . . . . . . . . . . . . . . 63 Mean Arterial Pressure and Flow Rate . . . . . . . . . . . . . . . . . . . . . . . . . 63 Effect of Glycemia on Muscle Glucose Uptake . . . . . . . . . . . . . . . . . . . 67 Effect of Glycemia on the Metabolic Clearance Rate of Glucose . . . . . 72 Effect of Glycemia on Glucose Transporter Distribution in Muscle . . . . 77

Characterization of Muscle Membranes . . . . . . . . . . . . . . . . . . . 77 GLUTl and GLUT4 Western Blot Results . . . . . . . . . . . . . . . . . 80

Effect of Glycemia on Glucose Uptake in Red and White Skeletal Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

SUMMARY OF RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

IMPLICATIONS and FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . . . . . . . 93

CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

LIST OF TABLES

Table 1 . The Mammalian Facilitative Glucose Transporter (GLUT) Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Table 2 . Basal Metabolic Characteristics of Rats . . . . . . . . . . . . . . . . . . . 63

Table 3 . Protein Yield (pglg muscle) of Subcellular Fractions . . . . . . . . . 78

Table 4 . Characteristics of Skeletal Muscle Membrane Fractions . . . . . . 79

vii

LIST OF FIGURES

F i~u re 1 . The Consequences of Untreated Diabetes Mellitus . . . . . . . . . . . 4

Fiaure 2 . Structural Model of the Human Facilitative GLUT4 Glucose Transporter Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Fiaure 3 . Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Fiaure 4 . Isolation of Hindlimb Circulation . . . . . . . . . . . . . . . . . . . . . . . . . 45

Fiaure 5 . Experimental Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Fiaure 6 . Mean Arterial Pressure Changes During Perfusion Experiments 66 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fiaure 7 . Effect of Glycemia on Glucose Extraction by the Hindlirnb . . . . . 68

Fiaure 8 . Effect of Glycemia on Glucose Clearance by the Hindlimb . . . . . 74

Fiaure 9 . Effect of Glycemia on GLUT4 Distribution . . . . . . . . . . . . . . . . . 81

. Fiaure 10 Effect of Glycemia on GLUTl Distribution . . . . . . . . . . . . . . . . . 82

Laure 11 . Effect of Glycemia on Red 8 White Muscle Glucose Uptake . . . 88

viii

LIST OF ABBREVIATIONS

2-DG 2-Deoxyglucose

2-DG-6-P 2-Deoxyglucoseô-Phosphate

ATP Adenosine Triphosphate

BBB Blood-Brain Barrier

CNS Central Newous System

GLUTI Hep G2IBrainfErythrocyte Glucose Transporter

GLUT4 MusclelFat Specific Glucose Transporter

GU Glucose Uptakelutilization

IDDM Insuiin-Dependent Diabetes Mellitus

IM Interna1 Membranes Fraction

MCR Metabolic Clearance Rate of Glucose

NIDOM Non-lnsulin-Dependent Diabetes Mellitus

NlMGU Non-lnsulin Mediated Glucose Uptake

PAGE Poiyacrylamide Gel Electrophoresis

PM4 Plasma Membrane Fraction4 (25% Sucrose)

PM-II Plasma Membrane Fraction-Il (30% Sucrose)

SDS Sodium Dodecyl Sulphate

SEM Standard Error of the Mean

SR Sarcoplasmic Reticulum

STZ Streptozotocin

TT TransverseTubules

GENERAL INTRODUCTION

DIABETES. HYPERGLYCEMIA AND INSULIN THERAPY

Diabetes mellitus and its complications are thought to be the fourth leading

cause of death by disease in the United States, trailing only AIDS, cardiovascular

disease and cancer, and claiming over 162,000 American lives in 1996 alone (1 ).

According to a report issued by the National Institutes of Health in 1995, there are

approximately 16 million people with diabetes in the U.S., half of whom are

diagnosed, and the incidence is increasing yearly (2). The economic impact of

direct and indirect care is enormous, ranging somewhere between $92-1 05 billion

(U.S.) in 1992 (3,4), approximately 14.6% of ail health care expenditures in the

United States. The probability of developing diabetes appears to double with each

decade of life and with every 20% of excess body weight (5).

Chronic hyperglycernia, or high blood sugar, is the hallmark of diabetes.

This results from either the insufficient secretion of insulin by the P-cells of the islets

of Langerhans (Insulin-Dependent Diabetes Mellitus, or IDDM) or the inability of

secreted insulin to stimulate the cellular uptake of glucose from the blood (Non-

Insulin-Dependent Diabetes Mellitus, or NIDDM). A considerable amount of

evidence from both retrospective (6) and prospective (7-14) clinical studies links

chronic hyperglycemia with the long-term rnicrovascular complications of diabetes,

including retinopathy, neuropathy, and nephropathy, although the development of

nephropathy may be limited to patients with a genetic predisposition to

hypertension (1 5). Chronic hyperglycemia has also been associated with

macrovascular disease (1 6) and impaired cellular immunity (1 7,18). However, in

3

addition to being a manifestation of impaired insulin secretion andlor action, it has

become clear that chronic hyperglycemia is itself a regulator of both, inhibiting

insulin secretion and glucose utilization. Thus, chronic hyperglycemia is not only

a clear indicator of poor metabolic control, but is itself a self-perpetuating regulatory

factor of the diabetic state. This concept has become referred to as glucose toxicity

(1 9-27 )-

The discovery of insulin in 1921 (22) finally provided a treatment modality for

diabetic hyperglycemia and its associated complications. lnsulin acts to decrease

glycemia by acutely stimulating glucose uptake in muscle and fat cells, while

concurrently suppressing glucose production in the liver. By promoting glucose

uptake in adipocytes, which in tum elicits a-glycerol phosphate production and

triglyceride storage, insulin also decreases free fatty acid (FFA) levels in the

circulation. Thus, insulin administration ameliorates two of the most severely

deranged rnetabolic parameters in uncontrolled insulindependent diabetes mellitus

(IDDM), elevated levels of blood glucose and ketone bodies (23). The result is a

decrease in the risk of hyperosmolar coma and death due to diabetic ketoacidosis

(see Figure 1).

Fiaure 1. The Consequences of Uncontrolled Diabetes Mellitus

Dehydratron. volume depletion

In patients with uncontrolled IDDM, the elevated levels of free fatty acids (FFAs) that result frorn increased lipolysis, are converted into ketone bodies in the liver. As a result, these patients exhibit elevated levels of plasma ketone bodies, as well as the characteristic hyperglycemia. Glucose and excess ketone bodies that are cleared by the kidney act as osmotic diuretics that cause excessive excretion of water in the urine. The result is severe dehydration, ketoacidosis, and electrolyte imbalance that often results in coma and death. (Adapted from Fox,S.I. Human Phvsioloay. 5th edition. Wm. C. Brown Publishers, 1996, pp. 591)

HYPOGLYCEMIA: A L ~ M ~ N G FACTOR IN THE TREATMENT OF IDDM

The insulin management of diabetes is, however, not without its

shortcomings. Hypoglycemia, or low blood sugar, is the most serious acute

complication of insulin-treated diabetes. Patients who practice conventional

therapy (defined as 1-2 insulin injections (mixture of long and short acting insulins)

per day, periodic blood or urine glucose measurements without specific numeric

glucose targets, professional health care visits every 3 months) suffer on average

one symptomatic episode of hypoglycemia per week, and 10% of such patients

suffer at least one episode of severe hypoglycemia, often with seizure or coma, per

year (24-26). Patients practising newer intensive therapies (defined as multiple

daily injections or continuous subcutaneous insulin infusion, frequent self-

monitoring of blood glucose levels. at least monthly visits to the health care tearn)

suffer an average of 2 symptomatic hypoglycemic episodes per week, and 25% of

such patients experience at least one episode of severe hypoglycernia per year

(24-26). Incredibly, iatrogenic hypoglycemia accounts for -4% of IDDM deaths

(24,27,28). Clearly, hypoglycemia remains a limiting factor in the management of

IDDM (29). The conventional risk factors and acute complications of hypoglycemia

have been well characterized (30-32), however the chronic implications of

hypog lycemia are not as wel l understood (33-36).

PATHOPHVSIOLOGIC REGULATION OF GLUCOSE TRANSPORT BY GLUCOSE: IMPLICATIONS OF HYPER- AND HYPOGLYCEMIA IN DIABETES

Similar to the regulation of nutrient transport by nutrient availability in

bacteria and yeast (37,38), mammalian glucose transporters are also subject to

substrate availability (39,40). Glucose autoregulation has important implications

in pathophysiological states, such as diabetes meIlitus, where physiological

mechanisms involved in glucoregulation are deranged. In this section, a review of

the pathophysiologic consequences of hyper- and hypoglycernia, as well as the

significance of the glucose autoregulation in these states, is provided.

HYPERGLYCEMIA: PATHOPHYSI~LOGY AND IMPLICATIONS IN DIABETES

Chronic hyperglycemia is not only the primary diagnostic indicator of al1

forms of diabetes, but itself contributes to the progression and long-term

complications of the disease. The results of the Diabetes Control and

Complications Trial (DCCT), a landmark multicenter trial designed to test the

proposition that the complications of diabetes are related to chronically elevated

plasma glucose concentrations, clearly establish the importance of chronic

hyperg lycemia in the development of diabetic retinopathy, neuropathy and

nephropathy (41). In the DCCT, 1,400 patients with IDDM were randomized to

standard or intensive treatment regimens and followed for a mean of 6.5 years. The

result in the intensively treated group, which consistently demonstrated improved

glycemic wntrol over the course of the study, was a dramatic 34-76% reduction in

7

the risk of clinically meaningful diabetic retinopathy (depending on the outcome

measured), a 35% reduction in the incidence of renal impairment, and a 60%

reduction in clinical neuropathy (41). Thus, it is generally believed that chronic

hyperglycernia is toxic to many organs and results in most long-term diabetic

complications. 'Toxic' irnplies progressive, irreversible changes in tissue function.

However, while hyperglycemia clearly has toxic effects on retinal, neural, and renal

tissues, there is a growing amount of evidence to suggest that many peripheral

tissues, particularly skeletal muscle, employ protective mechanisms to shield

themselves from the harmful effects of hyperglycemia and prevent complications

associated with glucose toxicity. Such mechanisms are more aptly referred to as

'adaptive' rather than 'toxic'. Evidence to support a regulatory adaptive effect of

hyperglycernia in muscle has come from exercise studies in diabetic dogs, a

convenient model of hyperglycemia (42-45). In the presence of a small amount of

insulin, glucose clearance (MCR = glucose utilization/plasma glucose) was

inversely correlated to glycernia, but under most conditions glucose utilization by

the muscle was nearly constant, irrespective of glucose concentration (43). In

insul in-deprived, depancreatized dogs, glucose utilization was 70-80% and MC R

only 10-15% that of normal values (45,46). Evidence for adaptive rather than toxic

effects of hyperglycemia has also come from rat models of NIDDM (90%

depancreatized rats), which dis play mild fasting hyperglycemia and abnormal

glucose tolerance due to impaired insulin secretion and glucose utilization (47).

Phlorizin, a pharmacological inhibitor of only rsnal Na+/glucose cotransporters at

8

low concentrations (48), was used to normalize glycemia in these rats by inhibiting

glucose reabsorption in the kidney (i.e. promoting glucosuria). Phlorizin treatment

normalized glucose tolerance and glucose utilization in these rats. Discontinuation

of phlorizin treatment for two weeks resulted in a return to abnormal glucose

tolerance and glucose utilization observed in the diabetic-untreated group.

lmpairsd peripheral glucose clearance was similarly corrected by phlorizin

treatment in alloxan-induced diabetic dogs (49). The results of these studies

suggest that hyperglycemia-induced defects in peripheral glucose utilization are

adaptive, not toxic. responses to increased substrate levels since they are

corrected upon normalization of glycernia.

In order to explain the observed decrease in peripheral glucose utilization

in models of hyperglycemia, it was proposed that hyperglycemia induces this

ada ptive effect b y downreg ulating glucose transporters in muscle (SO), since

skeletal muscle accounts for -95% of postprandial insulin-rnediated glucose uptake

(51 ). Using streptozotocin (STZ)-diabetes as a model of hyperglycemia, it was

discovered that hyperglycemia downregulates both GLUT4 protein and mRNA

expression, independent of insulin, in rat skeletal muscle (50). Using phlorizin to

normalize glucose levels in STZ-diabetic rats without changing plasma insulin

levels normalized GLUT4 content in plasma membranes, but not in interna1

membranes (52). GLUT4 mRNA improved partially (52). lt was concluded that

normalization of glycernia restores the translocation of GLUT4 more quickly than

de novo synthesis of glucose transporters (39,50,52,53). Correction of plasma

9

membrane GLUT4 content in STZ-diabetes with phlorizin is reversed with acute (40

min) restoration of hyperglycemia by intravenous glucose infusion, again

demonstrating an adaptive, not toxic, effect of hyperglycemia on muscle glucose

transport and transporters (Marette A., Klip A., Shi Z.Q., and Vranic M., unpublished

observations). Acute downregulation of glucose clearance is consistent with rat

hindquarter perfusion studies which show that high glucose concentrations c m

cause acute (as rapid as 30 min), concentration-dependent, decreases in insulin-

stimulated glucose transport (54,55).

Recently, however, two groups have reported evidence that seems to

contradict our hypothesis and observations. Galanté et al. (56) found that acute

hyperglycemia (25 mM) induces an upregulation of GLUT4 translocation,

independent of insulin, in C,C,, myotubes, perfused rat skeletal muscle, and muscle

in rats treated with somatostatin to suppress endogenous insulin release. Nolte et

al. (57) observed a twofold increase in glucose uptake in isolated rat epitrochlearis

muscles when exposed to high levels of glucose (20 rnM) relative to low levels of

glucose (8 mM). These results are in contrast to studies both in vivo and in vitro

that suggest hyperglycemia per se acts to downregulate glucose transport and

transporters in skeletal muscle (39,40,58). Since the effects of hyperglycemia on

muscle glucose transport are of great significance to our understanding of

glucoregulation in diabetes, there is clearly a need for more investigation on this

topic.

10

HYPOGLYCEMIA: ACUE AND CHRONIC ~MPUCA~ONS IN INSUUN-TREATED DIABETES

Hypoglycemia is the most serious awte complication of insulin-treated

diabetes, particularly IDDM (59.60). In the DCCT, hypoglycernia was associated

with (as possible, probable, or principle cause) almost 50% of al1 major accidents

inairred by intensively-treated IDDM patients (60), and hypoglycemia accounts for

4% of al1 IDDM deaths (31 ). The major risk factors for iatrogenic hypoglycemia in

lDDM are circumstances resulting in absolute or relative excess of active plasma

insulin (61 ). Absolute excesses of plasma insulin cm result from excessive or III-

timed doses of adrninistered insulin. Risks for relative insulin excess include

missed meals, overnight fasts, excessive physical activity, excessive alcohol

ingestion. and conditions that increase sensitivity to or decrease clearance of

administered insulin (30). However, these conventional risk factors account for only

a minority of clinical hypoglycemic episodes (26). More important contributing

factors are those associated with hypoglycernia-associated autonornic failure.

These include: 1) defective glucose counterregulation; 2) hypoglycemia

unawareness; and 3) elevated glycemic thresholds for symptoms and activation of

glucose counterregulatory systems that effectively lower overall plasma glucose

concentrations (30). Compensatory or physiological defence mechanisms against

hypoglycemia include: 1) cessation of endogenous insulin secretion (62); 2)

glucagon release, which plays a primary role (29,6345); 3) epinephrine and

norepinephrine, which are critical M e n counterregulatory factors (especially

glucagon) are deficient as in most IDDM patients (63,64,66-68); 4) growth hormone

i 1

and cortisol, which are only released after prolonged hypoglycernia (63); and 5)

hepatic glucose autoregulation (69). In type I diabetes, however, the glucagon

secretory response to hypoglycemia becornes deficient in the early course of the

disease (31.70). Patients with long-standing IDDM also acquire impaired

epinephrine secretory responses to hypoglycernia: due presumably to autonomic

neuropathy (30). This hypoglycemia-associated autonornic failure was found to be

specific for the stimulus of hypoglycemia in IDDM patients (71 ).

Most of the research literature available on diabetic hypoglycemia has

foaised on the impairment of hormonal and neural counterregulatory mechanisms.

including a) impairment of a-cell responsiveness to glycemic fluctuations

(49,72,73); b) the strong inhibition of islet a-cell fundion by hyperinsulinemia during

insulin treatment (74-78); c) hypoglycemia cinawareness (34.35.79431 ); and d) roles

of the central and autonornic nervous activities (8285). More recently, however,

much attention has tumed towards better understanding the mechanisms that cause

the increased frequency of severe hypoglycemia observed with intensive insulin

therapies (33.60). In addition to the mechanisms mentioned above, it has been

suggested that recent antecedent hypoglycemia plays a role in the pathogenesiç

of reairrent hypoglycemia by desensitizing the neural glucose sensory apparatus

and retarding the counterregulatory neuroendocrine and hormonal response

(30,33,36,86,87), though it rernains unresolved whether the primary glucose

sensing site is located in the brain or the liver (88-90). It is possible, however. that

in addition to a central defect, deranged glucose fluxes caused by antecedent

12

hypoglycemia may induce peripheral adaptations that may contribute to the

pathogenesis of recurrent hypoglycemia. This has never been examined. There

is ample evidence that hyperglycemia can substantially downregulate glucose

uptake via adaptive changes in glucose transporter translocation in diabetic muscle

(39,52). Though it has not been as intensely studied. there is evidence to suggest

that hypoglycemia may also induce changes in glucose transport and transporter

activity. For example, 24-hour glucopenia in L6 muscle cell culture was shown to

increase glucose uptake and plasma membrane GLUTl and GLUT4 content in the

absence of insulin (91 ). Chronic hypoglycemia increases both glucose transport

(92) and blood-brain barrier (BBB) GLUT1 protein and mRNA expression (93) in

rats. A study on systemic hypoglycemia in humans (94) revealed that glucose

extraction from both muscle and adipose ECF was significantly greater during

hypoglycemia than euglycemia. despite systemic counterregulatory hormone

release and local sympathetic activation (95). This effect was found to be

independent of the observed increase in blood flow to peripheral tissues during

hypoglycemia (95). Similar observations have also been made in the rat (96).

Recurrent antecedent hypoglycemia is now recognized as an important

contributing factor in precipitating subsequent hypoglycemia (33,79,87). It has

been concluded by the DCCT that antecedent hypoglycemia worsens the

hypoglycemia unawareness in intensively-treated IDDM patients and increases the

frequency and severity of subsequent hypoglycemia (60,97). Though the

mechanisms are not fully understood, desensitization of the counterregulatory

13

systems (release of glucagon, catecholamines, etc.) may play a role. Based on the

above evidence of insulin-independent glycernic regulation of glucose transport, it

follows that just as elevated glycemic levels may downregulate glucose transporter

content in plasma membrane of skeletal muscle, reduced glycemic levels may

upregulate glucose transporters. In either situation, the teleological premise is that

a relatively steady rate of tissue glucose uptake is pursued, even in the face of

varying glycemic levels. To achieve this goal, the corresponding adaptive flux in

glucose transporters seems iogical and essential (98), though transporter intrinsic

activity may also be affected. In ce11 culture using rat L6 skeletal cell line, glucose

starvation induœd a parallei upregulation of glucose uptake and plasma membrane

glucose transporters (91 ). More recently, hypoglycemia was shown to enhance

tissue extraction of glucose from the interstitial space of skeletal muscle in humans

(94). The chronic adaptation of brain glucose uptake in response to changes in

circulating glucose has been demonstrated (92). The transport of other substrates

was either unaffected of depressed, suggesting that the increase in brain glucose

uptake is specific. The mechanism for this central adaptation to chronic

hypoglycemia was found to include increased blood-brain barrier (BBB) GLUTl

protein and gene expression (93), and increased neuronal GLUT3 protein

expression (99). Neuronal GLUT3 mRNA expression increases in response to

starvation in mice (1 00). Further studies have been able to demonstrate increased

brain glucose extraction in tightly treated IDDM patients with hypoglycemia

unawareness (101), and an enhancement in brain glucose uptake following

14

induction of repeated hypoglycemia in normal humans (1 02). This evidence tends

to support the hypothesis that antecedent andlor recurrent hypoglycemia may

induce an adaptive upregulation of glucose transport and transporters in peripheral

glucose-consuming tissues. It remains to be explored whether the glucose

transporters are involved in the in vivo hypoglycemic adaptation process in tissues,

especially in skeletal muscle, a major consumer of circulating glucose, and whether

such processes contribute to the pathogenesis of recurrent hypoglycemia. It is

expected that adaptations inducing synthesis and/or translocation of glucose

transporters to the cell membrane would be associated with an increased rate of

glucose uptake. The enhanced activities of glucose transporters and the increased

rate of glucose uptake may render the diabetic individuals more susceptible to the

next hypoglycernic attack. or accentuate the degree of hypoglycemia during

subsequent attacks.

However, evidence that hypoglycernia induces an adaptive upregulation of

glucose transport in peripheral tissues is not universal. Capaldo et al. (103),

performing hyperinsulinernic-hypoglycemic forearm perfusion clamps in humans,

observed that 4 hours of hypoglycernia (-3 mM) induced a severe muscle

resistance to insulin in the forearm. Cohen et al. (104) also performed

hyperinsulinemic-hypoglycemic clamps in humans, and observed that 3 hours of

hypoglycemia (3.410.1 mM) induced a significant decrease in muscle glucose

clearance compared with euglycemia (4.8k0.1 mM). They wncluded that this effect

was due mostly (50%) to a decrease in glycogen synthetic activity in muscle (1 04).

15

Orskov et al. (105), performing similar clamps in humans, concluded that the

decrease in total glucose disposal over 4 hours of hypoglycemia (-2.8 mM) was

due primarily to a reduction of non-oxidative glucose disposal. Al1 of these studies

used insulin to induce hypogl ycemia. The effect hypoglycemia per se, inde pendent

of insulin action, has on glucose transport and transporters in skeletal muscle in

vivo has yet to be determined.

CHARACTERIZATION OF THE MUSCLE GLUCOSE TRANSPORT SYSTEM

The hydrophilic nature of the glucose molecule makes it a poor substrate for

direct transport across cell membranes. Instead, glucose entry into and out of cells

is facilitated by specific carrier proteins, or transporters, that span the hydrophobic

lipid bilayer and allow the transfer of glucose across the cell membrane. There are

currently two major classes of glucose carriers that have been described in

mammalian cells: 1) the ATP-dependent Na+lglucose cotransporter, and 2) the

facilitative glucose transporters of the GLUT family. The Na'lglucose cotransporter

is expressed in the polarized epithelial cells of the small intestine and in the

proximal tubule of the kidney (106,107). The physiological function of this

transporter is to transport glucose 'actively' (Le. requiring energy) against its

concentration gradient from the intestinal lumen into the intestinal cell, or frorn the

lumen of the nephron into the cells of the proximal tubule. The subsequent

facilitative transport of glucose out of these cells allows entry of glucose into the

blood. In contrast to the Na+lglucose cotransporter, rnembers of the facilitative

16

glucose transporter (GLUT) family are expressed in a wide variety of tissues, often

in a tissue-specific manner, and transport glucose down its concentration gradient

by facilitative diffusion. The focus of the remainder of this section will be the GLUT

family of glucose transporters.

THE GLUT FAMILY OF FACILITATIVE GLUCOSE TRANSPORTERS

Facilitative glucose transporters are a farnily of transmembrane

glycoproteins. The members of this family, the GLUTs, share the properties of

being saturable, stereoselective, and bi-directional (1 08). There are currently

seven known members of the GLUT family, GLUTs 1 thru 7, named in the order in

which their cDNAs were cloned (1 09). The tissue specificity and function of the

GLUTs are sumrnarized in Table 1. The protein products of al1 but one have been

identified and sequenced; GLUT6 is a pseudogene and does not encode for a

functional transporter. Each of the GLUT isoforms is encoded by a different gene,

however, based on their cDNA homology and predicted transmembrane

topography, ail isoforms seem to be structurally related. Each contain -500 amino

acid residues and are of molecular weight between 45-55 kDa (1 10,111). The

polypeptide sequences of GLUTs 1 through 5 are 26% identical, share 50-76%

similarity, and 13% of residues represent conservative replacements (1 12).

Table 1. The Mammalian Facilitative Glucose Transporter (GLUT) Family.

Transporter Tissue Specificity Function

GLUTI Blood-brain barrier, Basal glucose uptake in most erythrocytes, al1 known cells (excluding neuronal cells) cultured cells-ubiquitous

GLUT2 Liver, kidney, pancreas, Bidirectional glucose flux in the intestine liver; part of glucose sensing

machinery in the pancreas -

GLUT3 Many tissues, particularly Neuronal glucose uptake neuronal in the rat

GLUT4 Brown and white fat, skeletal Insulin-responsive glucose and cardiac muscle uptake

GLUTS Small intestine, sperm Fructose transport

GLUTG Pseudogene None - -

GLUl7 Liver G-6-P transporter of the endoplasmic reticulum

(From: J. M. Stephens and P.F. Pilch. (1995) The Metabolic Regulation and Vesicular Transport of GLUT4, the Major Insulin-Responsive Glucose Transporter. Endocrine Reviews l6(4): 529-546. )

GLUTI was the first member of the GLUT farnily to be biochemically

identified. This protein was first isolated and purified from erythrocyte membranes

by Kasahara and Hinkle in 1976 (1 13). The cDNA of GLUTI was cloned in 1985

by Mueckler et al. frorn HepG2 cells (1 14). The same group was able to predict the

topography d the protein using sequence analysis of the cDNA clone (1 14). Their

model proposed that about half of the residues were within the 12 membrane

spanning a-helices of the transporter. Both the N- and C- termini were postulated

18

to be located intracellularly, there was an exofacial loop bearing a single N-

glycosylation site between transmembrane domains 1 and 2, and a large

cytoplasmic loop between transmembrane domains 6 and 7. The highest sequence

homology between isofoms exists within the hydrophobie transmembrane domains,

which may confer the sugar transport function (1 09). Thus, the major differences

in the primary structures of the six GLUTs resides in the cytoplasrnic loop, the N-

terminus, and the C-terminus to which the various isofom-specific antibodies are

raised (1 15). This structural model of the GLUTs (see Figure 2) is generally

accepted, however an alternative model involving P-barrels has also been proposed

(1 16).

Fiaure 2. Structural Mode1 of the Human Facilitative GLUT4 Glucose Transporter Protein.

This structural model of the human GLUT4 glucose transporter depicts the 12 putative transmembrane a-helical domains and N-glycosylation site (CHO) comrnon to members of the GLUT family. In addition, this model shows the 12+ amino acid extension of the N-terminus (open circles) that is unique to GLUT4, as well as its divergent COOH-terminus. The dileucine motif near the C-terminus appears to be critical for transporter function. Possible substrate-binding regions are also shown. (Adapted from Czech, M.P. et al. lnsulin Action on Glucose Trans~ort in Diabetes Mellitus, D. LeRoith, S.I. Taylor, and J.M. Olefsky, eds. Lippincott-Raven Publishers, Philadelphia. O 1996)

20

GLUCOSE TRANSPORT AS THE RATE-LIMITING STEP OF MUSCLE GLUCOSE UTILKATION

Skeletal muscle is the main consumer of glucose during the post-prandial

insulin stimulation of glucose uptake (1 17). Studies employing the

hyperinsulinemic-euglycemic clamp technique have shown that under resting

conditions most of this glucose is converted and stored in the form of glycogen

5 118). The rate-limiting step in this pathway of glucose utilization is thought to

be the transport of glucose into muscle cells. Several observations support this

view. 1) levels of intracellular free glucose remain low in both normal and diabetic

muscle regardless of plasma insulin and glucose concentrations (1 19-122); 2)

glucose clearance is constant during both hypo- and hyperinsulinemic-euglycemic

conditions in perfused skeletal muscle (123); and 3) glucose-6-phosphate (G-6-P)

levels remain unchanged in transgenic mice overexpressing GLUTI in skeletal

muscle, even though muscle glycogen and lactate levels increase 10- and 2-fold

respectively (1 24). The importance of glucose transport as the rate-limiting step in

whole-body glucose utilization is realized in metabolic diseases such as obesity and

NIDDM, in which insulin-induced muscle glucose transport is deficient (1 25-728).

Decreased glucose transport in muscle results in decreased whole-body glucose

utilization and the hyperglycemic-hyperinsulinernic pathophysiological state

characteristic of insulin resistance in obesity and NIDDM.

GLUCOSE TRANSPORTER DISTRIBUTION IN SKELETAL MUSCLE

Glucose transport in skeletal muscle is rnediated by two facilitative glucose

transporter isoforms, GLUT4 and GLUTI (1 15,129-1 32). GLUT5 is also expressed

in human but not rat skeletal muscle (1 33,134). However, evidence obtained from

human GLUT5 cDNA transfection of Xenopus oocytes suggests that this transporter

is primarily responsible for fructose uptake into muscle (1 35).

The relative abundance of GLUT1 and GLUT4 in muscle depends on the

developmental stage of the muscle (136). During fetal and early postnatal life,

GLUTI is the predominant isofom expressed. GLUT4 induction occurs during the

perinatal period, during wtiich GLUT4 expression rises whi le G LUT1 expression is

repressed (1 36). By adult life, GLUT4 becomes quantitatively the most important

glucose carrier expressed in skeletal muscle, with GLUTI representing only 5-1 0%

of total glucose carriers (129).

Evidence from subcellular fractionation (1 29-1 31 ,137,138),

immunocytochemical (1 39-1 42), and photolabeling studies (1 43,144) indicate that

GLUTI and GLUT4 show a differential localization in the muscle fiber. Indeed,

immunofluorescence analysis using antibodies specific for GLUTI labelled the

periphery, but not the interior of muscle cells in transverse cryosections of rat

skeletal muscle (1 29). Subcellular fractionation studies demonstrate that GLUTI

carriers are located mainly in a fraction enriched in plasma membrane markers such

as 5'-nucleotidase, ~9~'-ATPase, and Ng IK -ATPase (1 29-1 31 ). Furthermore,

GLUTI appears to be heterogeneously distributed throughout the sarcolemma, but

22

is absent in fractions enriched with T-tubule markers (1 29,137,138). In contrast,

subcellular fractionation (1 29-1 31,145,146) and imrnuno-electron microscopy of

muscle cells (1 39-1 42,147) reveals that under basal conditions, GLUT4 is mainly

associated with intracellular rnernbranous vesicular structures. In rat extensor

digitorum longus muscle, these tubulovesicular elements are found in proximity to

the sarcolemma and T-tubules, as well as in a perinuclear location close to the

Golgi apparatus (147). There is, however, some GLUT4 expressed at the cell

membrane in the fasted basal state; 3% and 8% of total cellular GLUT4 in the

sarcolemma and T-tubules, respectively (1 47).

REGUUTION OF GLUCOSE TRANSPORT IN SKELETAL MUSCLE

Glucose transport in muscle is subject to complex regulation. Indeed,

mechanisms regulating muscle glucose transport are sensitive to a variety of

metabolic factors, including insulinernia, glycernia and exercise. The regu lation of

glucose transport and transporters by insulin and exercise is described below.

Glycernic regulation of glucose transport is discussed separately in the previous

section.

REGULATION B Y INSULIN: TRANSPORTER RECRUITMEN~ & ACTIV~TY

The mechanism by which insulin stimulates glucose uptake in adipose and

muscle tissue has been the focus of much research because of its clinical relevance

to diabetes mellitus. In 1980, independent studies from the laboratories of

23

Cushman (148) and Kono (149) observed that insulin stimulated the insertion of

glucose carr~ers from an intracellular (microsornal) membrane cornpartment to the

plasma membrane in isolated rat adipocytes. The 7- to 10-fold increase in

fundional cell surface transporters resulted in a rapid clearance of glucose (Le. an

increase in V,) from the surroundhg medium. Following the diswvery of the

insulin-stimulated recruitment of transporters to the plasma membrane in

adipocytes, similar studies were perfomed to determine whether an analogous

mechanism exists in muscle. Early studies perfomed on the rat diaphragm

demonstrated that insulin administration induced a 2-fold increase in glucose

transporter content in the plasma membrane with a concomitant decrease in the

intracellular pool of transporters (1 50,157 ). Following the cloning of the GLUT4

isoform of the facilitative glucose transporter (GLUT) family (1 52-1 56), the most

highly expressed glucose transporter in fat and muscle, several groups employing

a myriad of techniques (98.129-1 31.137-1 44.157) were able to establish that

GLUT4 is the glucose transporter that is remited in an insulindependent manner

from an intracellular locus to the cell surface of the muscle fiber. lnsulin promates

the heterogenous insertion of GLUT4 into both the sarcolemrna (1 40-1 42) and T-

tubules (1 29,I38,139,'i58), in regions where the insulin receptor is also

heterogeneously expressed (1 38). Insulin-induced GLUT4 recniitment to the

sarcolemma seems to be localized to regions highly enriched in dystrophin and

deficient in clathrin (1 38). The pastulate of transporter recniitment or translocation

24

is now widely accepted as the primary mechanism of insulin-dependent transport

activation in skeletal muscle (1 59).

However, in addition to the recniitrnent hypothesis, the possibility that insulin

may also affect the "intrinsic" or "specific" activity of glucose transporters has been

proposed. This suggestion has been raised to explain the fact that increases in

plasma membrane glucose transporter number are not always directly proportional

to increases in insulin-stimulated glucose transport (160,161 ). 'Intrinsic activity' can

be defined as the number of glucose molecules transferred per transporter

molecule per unit time, and can only be determined in studies in which both

transport activity and transporter number are measured in the same preparation.

The intrinsic functional activity of a transporter may be altered due to changes in

the affinity of the transporter for its substrate (Le. changes in &), andlor in the

velocity of transport of substrate through the transporter independent of any

substrate mass effect (Le. changes in V,, at constant concentrations of substrate,

designated v). As a result, the intrinsic activity of transporters has been calculated

as the ratio of the observed vlK,,, (162).

Insulin-induced biochernical modifications of glucose transporters, such as

phosphorylation or transporter couplinglinteraction with other proteins, could

conceivably influence either v andlor &, and thereby affect the intrinsic activity of

glucose transporters. There is evidence that both GLUTI and GLUT4 contain

phosphorylation sites and are phosphorylated in cultured tells (163-1 65), however,

the phosphorylation state of neither glucose transporter is altered upon acute

25

insulin stimulation (l63,165,166). With respect to glucose transporter regulation

by protein-protein interaction, there is evidence that GLUTI interacts with other

proteins that could influence transporter activity (167). but this type of regulation

has not been demonstrated to occur in response to insulin. In addition. there is

some evidence to suggest that GLUT4 may also interact with cytosolic proteins that

affect the immunoreactivity of intracellular GLUT4, which could conceivably

influence transporter function (1 68,A 69). The effect of changes in intrinsic activity

on insulin-stimulated glucose transport is still unresolved and rernains the focus of

much study.

REGULA TION B Y INSULIN: POST-RECEPTOR SIGNALLING

Another major area of research is the identification of the insulin-regulated

signalling pathway(s) involved in the translocation of GLUT4 vesicles. lnsulin

initiates a number of signalling cascades in muscle that mediate its metabolic and

mitogenic effects. All of these begin with the activation and autophosphorylation

of the insulin receptor, followed by the tyrosine (Tyr) phosphorylation of its three

known substrates: the insulin receptor substrates 1 and 2 (IRS-1 & -2) and shc

(1 ?O). Each of these substrates is Tyr phosphorylated at sites surrounded by highly

conserved amino acid residues that represent potential binding sites for src-

homology 2 (SH2)-containing proteins (1 71 ). Thus, these substrates, in their

phosphorylated form, associate with other signaling molecules through protein-

protein interactions to forrn, and often activate (1 72,173), large, multicomponent

26

signaling apparatuses that mediate insulin-stimulated downstream events. In this

way, the insulin receptor operates differently from other receptor protein tyrosine

kinases (RPTKs) in that it promotes the activation of SH2-containing proteins by

specific association with receptor substrates rather than with the activated receptor

itself.

IRS-1 is the only insulin receptor substrate to date that has been studied for

its potential role in glucose transport. The majority of current evidence suggests

that IRS-1 does play an important role in mediating insulin action on glucose uptake

(1 74-1 77), though this observation is not universal (1 78). In contrast, studies

utilizing wortmannin and LY294002, microbial metabolite and synthetic inhibitors

of phosphatidylinositol 3-kinase (PI 3kinase), respectively, have clearly established

PI 3-kinase as a critical signal transduction effector molecule in the insulin-

stimulated glucose transport pathway of muscle and fat cells (1 79-1 81 ). PI 3-kinase

is a heterodimeric enzyme composed of a p85 (M, 85,000) regulatory subunit and

a p l 10 (M, 11 0,000) catalytic subunit. The p85 subunit contains two SH2 domains,

which bind preferentially in vifro to four phosphopeptides derived from the IRS-1

sequence surrounding Tyr 460,608, 939, and 987 (1 82). This would suggest that

Tyr phosphorylated IRS-1 acts as a docking protein for the binding and activation

of PI 3-kinase via its SH2 domains. Indeed, the majority of PI 3-kinase after insulin

stimulation is associated with IRS-1 (1 72,173,183).

In addition to the direct activation of PI 3-kinase by IRS-1, PI 3-kinase

activation by interaction with the 21 kDa membrane associated GTP-binding protein

27

p2qR" (Ras), of the Ras-MAPK,, signalling pathway (170), has also been

postulated. Results mainly from Ras overexpression studies indicate that Ras may

attenuate or even mirnic the action of insulin on glucose uptake (184-186).

However, the majority of data suggests that this signalling mechanism of glucose

transport is not essential for the rapid stimulation of glucose transport by insulin

(1 87-1 92).

The essential role of PI 3kinase in insulin-stimulated glucose transport and

GLUT4 translocation is established. However, information regarding downstream

events in this cascade is still limited. Recently, two downstream targets of PI 3-

kinase have been identified, protein kinase B (PKB) (1 93,194) and p70 S6 kinase

(180,195,196). PKB, also known as Akt, is a serine/threonine kinase whose

catalytic domain closely resernbles that of protein kinase C (PKC) and CAMP-

dependent protein kinase (1 97-1 99). Studies have shown that insulin-induced

activation of PKB is prevented in the presence of wortmannin (1 93,2OOl2Ol ), and

that transfection of constitutively active PUB in rat and cultured adipocytes induces

GLUT4 translocation to the cell membrane (202,203). Thus, early evidence

suggests a potential role for PKB in the PI 3-kinase pathway of insulin-stimulated

glucose uptake. The mechanism of PKB activation by PI 3-kinase is not

determined, though there is evidence that 1) insulin activates PKB by an as yet

unidentified upstream PKB kinase (193,200,204-206), and 2) that the natural

products of PI 3-kinase phosphorylation, PI-3-Pl PI-3,4-P, and PI-3,4,5-P may be

wfactors of PKB that may aid in its activation by an unknown kinase (204,205). In

28

contrast to PKB, p70 S6 kinase activation does not appear to participate in the

acute stimulation of glucose transport and GLUT4 translocation by insulin (207).

REGULA TION B Y &ERCISE

Glucose uptake during exercise is closely regulated by both hormonal and

non-hormonal mechanisms. Exercise rapidiy stimulates the rate at which glucose

is transported into muscle cells (208,209). Kinetic analysis studies in vivo (21 0) and

in vitro (208,211-2l3) suggest that the effect of exercise on muscle glucose

transport is a consequence of an enhancement of the maximal velocity (V,,,) for

this proœss, while glucose transporter afinity for glucose (k) remains unchanged.

Since glucose transport is the rate limiting step of glucose utilization in muscle,

except perhaps during the onset of exercise (214) and during heavy exercise (21 5),

the predorninant means by which exercise stimulate glucose uptake is by increasing

the number and activity of glucose transporters (GLUT4) in the plasma membrane

of muscle (1 31,216-21 9). Interestingly, exercise recruits GLUT4 to the plasma

membrane from an intracellular compartment distinct from the one that is

upregulated in response to insulin (1 31,216). This is an important observation

because it suggests that there are at least two distinct intracellular pools of glucose

transporters in muscle, one that is sensitive to insulin and one that is sensitive to

exercise. This observation also leaves open the possibility that other pools of

glucose transporters may exist in muscle that are sensitive to other stimuli,

29

including glycemia. Such a discovery would have great implications in our

understanding and treatment of diabetes mellitus.

OUTLINE OF OBJECTIVES

As reviewed above, glucose transport in muscle is regulated by both

hormonal and non-hormonal mechanisms. Most of the available literature has

focused on the regulation of muscle glucose transport by insulin and exercise.

However, glucose also acts to regulate its own uptake in muscle cells, and there is

increasing interest in the implications of this glucose autoregulation for the

pathogenesis and treatment of diabetes. The objectives of this study were to

assess the acute effects of hypo- and hyperglycemia per se on muscle glucose

transport. In particular, they were:

1) to determine whether hypoglycemia can acutely affect glucose transport

andor transporter regulation, independent of insulin, in perfused rat skeletal muscle

2) to determine whether hyperglycemia per se acutely up- or downregulates

glucose transport andfor transporters in perfused skeletal muscle.

The experimental model and protocols used to fulfill these objectives are

outlined in the following section. This is followed by a description and discussion

of the results obtained. Conclusions will then be presented based on the findings

of this study.

GENERAL MATERIALS AND METHODS

EXPERJMENTAL ANIMALS

CHOICE OF ANIMAL MODEL

The primary focus of this study was to determine whether glycemia per se

actuely regulates glucose transport and/or transporters, in the absence of insulin,

in normal skeletal muscle. While diabetes is a convienient model for the study of

the effects of insulin-deficiency and hyperglycemia on muscle glucose transport in

vivo, there is no equivalent model for the study of the insulin-independent effects

of hypoglycemia in vivo, since insulin is normally required to induce hypoglycemia

in animal and human rnodels. Instead, hyperinsulinemic-hypoglycemic and

euglycemic clamp studies are cornmonly performed, in which insuiin infusion rates

remain constant regardless of the level of glycemia, and are, therefore, considered

to be independent of any effects observed. However, the large doses of insulin

infused during these studies are themselves powerful stimulators of glucose

transport and transporter activities, and, therefore, may mask the effects of

hypoglycemia andfor other factors that may regulate glucose transport

wincidentally, though independently of insuiin. To circumvent this possibility, the

in situ rat hindlimb perfusion model was chosen over in vivo alternatives to study the

insulin-independent effects of glycemia on muscle glucose transport because

hindlimb perfusion allows the study of muscle metabolism independent of both

hormonal and neural regulation. This model of experimentation was first described

by Ruderman et a/. in 1971 (220), and has since been validated by a number of

groups (reviewed in (22 1 )).

ANIMAL CARE AND MAINTENANCE

Male specific pathogen-free Sprague-Dawley rats weighing between 300-400

g were used for experimentation. These rats were obtained from Charles River

(Charles River, ON), and housed in pairs in the Department of Comparative

Medicine, University of Toronto. Until the time of experimentation, rats were fed

standard rat chow (Agway) and supplied drinking water ad libitum. All procedures

herein were in accordance with the Canadian Council on Animal Care standards

and were approved by the Animal Care Cornmittee of the University of Toronto.

rn Bovine Serum Albumin (BSA) - Initial fractionation by cold alcohol precipitation, Fraction V (minimum 96% pure) (Sigma Diagnostics, St. Louis, MO) Spectra/Por@ molecularporous membrane tubing (Spectrum Medical Industries, Inc., Houston, TX)

rn 0.3% Sodium sulfide (Na,S) solution 0.2% Sulfuric acid solution

METHODS

The purpose of albumin dialysis is to enhance the purity of commercial

albumin used in the preparation of perfusate. Dialysis is a method of separating

possible impurities, such as free fatty acids (FFAs), from molecules of albumin,

wfiich are relatively larger in size. This requires the use of a thin semi-permeable

34

membrane whose pores are too small to allow the passage of protein particles, but

large enough to permit the passage of smaller impurities. This membrane is used

to separate a solution of albumin from distilled water. As a result, impurities diffuse

down a concentration gradient and across the membrane, while a pure solution of

albumin is retained.

To ensure that the membrane was itself free of impurities (ie. glycerol, etc.).

80 cm strips of spectralPo$ dialysis membrane were first subjected to a membrane

preparation protocol. Cold tap water was first allowed to run over the membrane

for at least 3-4 hours. The membrane was then soaked in 0.3% Na,S solution,

heated to 80°C for 1 minute, and washed in a distilled water bath heated to 60°C

for 2 minutes. Membranes were then allowed to soak in 0.2% sulfuric acid at room

temperature for 4-5 minutes, followed by at least 3 washes in warm distilled water

(60°C).

A known amount (see Perfusate Preparation below) of bovine serum alburnin

(BSA) was dissolved slowly in distilled water to avoid excessive foaming. The

concentrated BSA solution was added to the treated strips of membrane, which

were then placed in a stirred. cold distilled water bath (4°C) for at least 36 hours.

The distilled water was replaced at 3, 12-16, and 24-30 hours to ensure efficient

dialysis.

O Dialyzed Bovine Serum Albumin (see Albumin Dialysis above) NaCI, KCI, NaH,PO,, Na,SO,, CaCI,, NaHCO, (BDH Inc., Toronto, ON)

rn MgCI, (Caledon Laboratories Ltd., Georgetown ON) pH Meter, Model E632 (Metrohm Ltd., Herisan, Switzerland) VacuCap@GO 0.45 prn Bottletop Vacuum filter units (Gelrnan Sciences, Ann Arbor, Michigan) sterilized 500 ml glass flasks 50% Dextrose solution (Abbott Laboratories Ltd., Montreal, PQ)

METHODS

The electrolyte composition of perfusate was as follows (concentrations in

mM): 115 NaCI, 5.9 KCI, 1.2 MgCI,, 1.2 Na6 P B , 1.2 Ng SQ , 2.5 CaÇl , 25

NaHCO, (222). The final concentration of BSA was 5% (w/v). To achieve these

concentrations, chernicals were added to the concentrated dialyzed BSA solution

(see Alburnin Dialysis above) in the following amounts:

1 C hemical Information 1 Amount Needed for: 1 - - - - . -- - - -- - - -

1 Concentration 1 Cornpound 1 MW 1 1 L 1 2 L 1 3 L 1 4 L 1 5%

115 mM

5.9 rnM

1.2 mM

1.2 mM

1.2 mM

2.5 mM

25 mM ,'

BSA

NaCl

KCI

Mg CI2 NaH,PO,

Na,SO,

CaCI,

NaHCO,

-

58 -44

74.55

203.30

137.99

142.04

147.02

84.01

50 g 6.72 g

0.440 g

0.244 g

0.166 g

0.171 g

0.3689

2.101 g

100 g

13.44 g

0.880 g

0.488 g

0.331 g

0.341 g

0.735 g

4.201 g

150 g

20.16 g

1.320 g

0.732 g

0.498 g

0.51 3 g

1.104 g

6.303 g

200 g

26.88 g

1.760 g

0.976 g

0.662 g

0.682 g

1.470 g

8.402 g

36

The pH of the perfusate was adjusted to 7.45, and the volume brought up to the

final desired volume with double distilled water. The perfusate was then filtered into

sterile 500 ml glass Rasks using high flow rate vacuum filters (vacuCapQ 60 0.45

prn Bottletop Vacuum filter units; Gelman Sciences, Ann Arbor, Michigan).

Perfusate was stored at -20°C until needed.

Glucose was not added to the perfusate until the time of experimentation.

The perfusate was thawed (38°C) and 50% Dextrose solution (Abbott Laboratories

Ltd., Montreal, PQ) was added as required:

1 Protocol 1 Perfusate 1 Total Volume 1 ~ l u c o s e ~ e ~ u i r e d 1 50% Dextrose Added 1 I LOW (2 mM) Glucose Perfusate 1 500 ml 1 180 mg 1 0.36 ml I

The Dextrose was allowed at least 5 minutes to mix with the perfusate before

glucose concentrations were confirmed using a Beckrnan Glucose Analyzer 2

(Beckman, F ullerton, CA) (see Plasma Glucose Assay below).

NORMAL (5.5 mM) Glucose Perfusate

NORMAL (6.5 mM) Glucose Perfusate

HlGH (20 mM) Glucose Perfusate

EXPERIMENTAL PROCEDURES

MA TERIALS

200 ml

500 ml

500 ml

Scalpel Blade a Glucorneter ~lite@ Blood Glucose Meter (Miles Canada Inc., Etobicoke, ON)

Glucorneter Hite@ Test Strips (Miles Canada Inc., Etobicoke, ON)

198 mg

585 mg

1800 mg

0.40 ml

1.17 ml

3.60 ml

METHOD

Before any administration of anaesthesia. the blood glucose concentration

of the animal was detennined. Using a sharp scalpel blade. the 'tail-ni&' method

was used to obtain -5 pl of blood from the end of the animal's tail. Capillary action

at the end of the Test Stnp draws a small amount of blood (3-5 pl) into the reaction

chamber. The reaction chamber contains a small amount of the enzyme glucose

oxidase, which catalyzes a readion that uses oxygen to convert glucose to gluconic

aud (see Plasma Glucose Assay below). Since oxygen consumption is proportional

to the amount of glucose in the blood sample. the Glucorneter ~l i te ' uses electrode

sensor technology to measure the rate of oxygen consurnption during the reaction

to detemine the blood glucose concentration. Measuring time is 60 seconds. and

blood glucose is dispiayed in millimoles per litre (mM).

~ a r s " Electronic Scale (Marantz Scales International. Thomhill. ON) ~travet* (Acepromazine Maleate; Ayerst Laboratories. Montreal. PQ) et ale an" (Ketamine HCI), ~omnGto~ (Sodium Pentobarbitol; MTC Pharmaceuticals. Cambridge. ON) ~ompun" (Xylazine HCI; Miles Canada Inc.. Etobicoke. ON) 1 cc Tuberculin syringes Needles 23G, 1 incb Sodium Ruoride (Fisher Scientific, Markham, ON) Hair Clippers Masking Tape

Heating Pad (~Purnp@ Model TP400; Gaymar Industries Inc., Orchard Park, NY) Topical lodine solution Scalpel Blade Mosquito Forceps, Haemostats Suture material, black braided silk 3-0 Gauze sponges Saline (0.9% NaCl solution) Hepaleang (Heparin; Wyeth-Ayerst Canada Inc., Toronto, ON) 1 % Lidocaine HCI (Abbott Laboratones Ltd., Montreal, PQ) Angiocaths@ 14G and 16G (8ecton Dickinson Vascular Access, Sandy, Utah)

Rats were weighed using an electronic scale. A regimen of balanced

anaesthesia was used. The use of balanced anaesthesia minimizes the risk of

overdose while maximizing the efficiency and duration of anaesthesia. An

intraperitoneal (i. p.) injection of 0.75 rnglkg Acepromazine maleate, 0.1 5 mglkg

Xylazine HCI and 150 mglkg Ketamine HCI was administered in the lower left

quadrant of the abdomen to minimize the risk of visceral penetration. Once an

acceptable level of anaesthesia was achieved, as evidenced by the absence of a

pedal reflex, the abdomen of the rat was shaved using a pair of hair clippers. Any

excess fur was removed using masking tape. The rat was then secured to a

heating pad (38°C) with masking tape to prevent the risk of hypothermia during

surgery. A 1 ml sample of blood was taken before surgery using the cardiac

puncture technique for basal plasma insulin determination. The sample was

collected in a microfuge tube containing approximately 2.5 mg of sodium fluoride,

to prevent glucose degradation, and dried heparin (50 U) to prevent coagulation.

39

It was then subjected to centrifugation to separate the plasma and cellular

compartrnents. Plasma samples were collected and stored at -20°C for further

analysis.

The surgical procedures of the hindlimb preparation were performed simi larl y

to those of McDermott et al. (222). with minor modifications as outlined below.

Following sterilization of the abdomen with topical iodine solution, a midline

laparotomy was performed to expose the abdominal viscera. The rat was then

subjected to partial evisceration of lower abdominal organs. Tight 3-0 silk ligatures

were placed around the proximal duodenum and distal sigmoid colon, and the

intestines between the ligatures were excised. Ligatures were then tied around the

left and right spermatic cords and both testes were removed. Tight ligatures were

also placed around the seminal vesicles, bladder and prostate to exclude them from

the circulation. To concentrate flow to the hindlimb and minimize loss of perfusate

through collateral circulation, the following vessels (arteries and veins) were ligated:

renal, superior and inferior mesenteric, testicular, ileolumbar, median sacral and

intemal iliac vessels (see Figure 4). Two loose ligatures were placed around each

of the abdominal aorta and inferior vena cava, just below the level of the inferior

rnesenteric artery. One loose ligature was also placed around both the abdominal

aorta and inferior vena cava below the left renal vessels, and another around both

the left common iliac artery and vein (see Figure 4).

To prevent coagulation in the hindlimb vasculature before the onset of

perfusate flow, 2000 U of heparin (~epalean~; Wyeth-Ayerst Canada Inc., Toronto,

40

ON) was injected systemically before cannulation via the inferior vena cava (223).

2-3 minutes after the injection of heparin, the abdominal aorta was cannulated

between the two loose ligatures using a 16-gauge ~ngiocath" (Becton Dickinson

Vascular Access, Sandy, Utah) such that the tip of the catheter was -2-3 mm above

the aortic bifurcation. The inferior ligature was tied around the ~ n g i o c a t h ~ to seal

the vessel, and the superior ligature to occlude superior arterial circulation and

secure the Angiocathg in place. 1-2 draps of 1 % Lidocaine were used to dilate the

aorta in order to facilitate cannulation. The period of ischemia before initiation of

perfusate fiow was generally c l 5 seconds. Perfusate Row was initiated immediatel y

upon aortic cannulation and gradually increased to 17.5 mllmin/leg (see Perfusion

System below). The inferior vena Gava was then cannulated in a similar fashion

using a 14-gauge ~ngiocath". Once the perfusion catheters were inserted and

secured, the perfusion period is commenced and the rat sacrificed by intracardiac

injection of 20 mg sodium pentobarbital. The ligature placed around both the aorta

and vena Gava was then tied to ensure closure of the perfusion system. The first

70-75 ml of effluent was discarded to minimize red blood cell contamination of the

perfusion system, at which point the perfusate was recirculated for the duration of

the experirnent. The mass of perfused hindquarter muscle was estirnated as 16.6%

of body weight (224).

Shaking Water Bath. Model 125 (Fisher Scientific, USA) Peristaltic Pump, Model 302s (Watson-Marlow Ltd., Falmouth, UK) Membrane Oxygenator, ModeIl201 44-MH (Radnoti Glass Technology Inc., Monovia, CA) 95% 0,/5% CO, gas (Canox Ltd., Mississauga, ON) Sodium fluoride (Fisher Scientific, Markham, ON) Hepaleang (Heparin; Wyeth-Ayerst Canada Inc., Toronto, ON) Heating Pad ( ~ / ~ u m p @ Model TP4OO; Gaymar Industries Inc., Orchard Park. NY) Pressure Transducer Gilson Physiograph Microcentrifuge. ~ i k r o " 12-24 (Hettich, Tuttlingen, Germany) 2-(1 ,2-3H(N)]-deoxy-D-glucose (Dupont NEN Products, Boston, MA)

Hindlimbs were perfused with 500 ml of a standard cell-free perfusate

containing physiological concentrations of electrolytes and 5% defatted and

diaiyzed bovine serum alburnin (see Perfusate Preparation above). Glucose

concentrations were 2, 6.5, and 20 mM glucose during the 2-hour glycernic

conditioning periods of the LOW, NORMAL, and HlGH glucose protocols

respectively, and 5.5 mM glucose during the following 30-minute period in each

protocol. The perfusate was maintained at 38OC in a shaking water bath and

oxygenated through an artificial lung, supplied with a gaseous mixture of 95%0,/5%

CO,, as described by McDermott et al. (222). A constant flow rate of 17.5

mllminlleg was maintained throughout the perfusion protocols, yielding mean

arterial pressures between 80-90 mmHg. In a set of control experiments,

42

oxygenation of the perfusate was confirmed using an A B L ~ @ Acid-Base Laboratory

(Radiometer, Copenhagen, Denmark), measuring typical arterial pO, levels

between 380-385 rnmHg. Perfusate pressure in the arterial lines was monitored by

a pressure transducer connected to a physiograph, or by a mercury manometer.

The tissue was maintained at physiological temperatures (38°C) by wrapping it in

a heating pad.

Arterial and venous perfusate was sampled (1 ml samples) every 10 minutes

for glucose rneasurements. Arterial samples (1 ml) collected every 30 minutes were

also used for insulin measurements. All samples collected for glucose and insulin

measurements were collected in microfuge tubes containing approxirnately 2.5 mg

of sodium fluoride, to prevent glucose degradation, and dried heparin (50 U per 1 .O

ml sample) to prevent coagulation. Samples were centrifuged to separate any

blood cells frorn the perfusate. Perfusate sarnples were then assayed for glucose

content (see Plasma Glucose Assay below), and stored at -20°C.

At the end of the two hour conditioning period, muscle was excised frorn the

left leg for western blot analysis of glucose transporter distribution (rnixed

quadriceps muscle). Excised muscle was immediately freeze-clamped with

aluminum clamps pre-cooled in liquid N,, and stored at -80°C until needed. The

loose ligature around both the left cornmon iliac artery and vein (see Surgical

Procedures above) was then tied to direct perfusion solely to the right hindlimb.

Flow rate was adjusted to maintain constant flow of 17.5 mllminlleg.

43

During the following 30 minute perfusion of the right hindlimb, glycemia was

normalized in each protocol using 200 ml of 5.5 mM glucose perfusate. A 15

minute equilibration ensued to allow perfusion pressure and the metabolic profile

of the hindlimb to stabilize. Afler the 15 minute equilibration period. 35 pCi of 2-

[ l ,2-3H(~)]deoxy-D-glucoçe (Dupont NEN Products, Boston, MA) was added to the

perfusate and mixed. After 30 minutes of perfusion with 5.5 mM glucose perfusate.

the hindlimb was perfused for 2 minutes with a bicarbonate buffer composed of

(concentrations in mM) 11 5 NaCI, 5.9 KCI, 1.2 MgCl,. 1.2 NaH2P0,, 1.2 NqSO,,

2.5 CaCI,, 25 NaHCO, , and 5.5 mM glucose. The purpose of this short perfusion

was to remove protein and 2-[3H]deoxyglucose (2-DG) in the vasculature that could

interfere with total muscle protein and 2-DG uptake assays. After the short 2

minute perfusion, the soleus and EDL muscles were excised. -100 mg of each

muscle sample was placed in 0.5 ml of 1 N NaOH for analysis of 2-[3H]-

deoxyglucose-&phosphate (2-DG-6-P) content (see 2-[3H]-DG-6-P Assay below).

The remaining portion of each muscle sample was quickly freeze-clamped with

aluminurn clamps pre-cooled in liquid N,. Frozen muscle samples were then stored

at -80°C for further analysis of total protein content.

Fiaure 3. Exparimental Setup

A.A. I I LE'.<

Perfusate containing glucose, physiological concentrations of electrolfies anci5% dialyzed albumTn i s mixed and hëated (38i~) inashaking waferbath. A peristaltic pump pumps the perfusate through the lines at physiological pressures measured by a pressure transducer connected to a physiograph. The perfusate is pumped through an artificial lung heated to 38°C and supplied with a 95% 045% CO, gas mixture. The oxygenated perfusate then enters the arterial circulation via the abdominal aorta (A.A.), perfuses through the tissue, exits via the inferior vena cava (I.V.C.), and is recirculated back to the source of perfusate. The tissue is wrapped in a heating pad to maintain body temperature (38°C). Arterial and venous perfusate samples are taken at 10 min intervals throughout the experiment.

Fiaure 4. Isolation of Hindlimb Circulation

Su p. Mssenteric Artery & Vein

Interna1 lliac

Heart

Aorta

Inf. Mesenteric Artery & Vein

Renal Artery & Vein

Cornmon lliac Artery & Vein

Femoral Artery

In order to isolate the hindlimb vasculature for perfusion, tight ligatures were placed around the following vessels (arteries and veins) as shown: renal, superior and inferior mesenteric, and interna1 iliac vessels. Not shown are ligatures around (arteries and veins): testicular, ileolumbar, and median sacral vessels. A tight ligature was also placed around both the abdominal aorta and inferior vena cava above the point of cannulation to prevent any backflow of perfusate to the superior portion of the rat. Also depicted is the loose ligature placed around the left cornmon iliac artery and vein for Iigation after the Zhour glycemic conditioning period.

Fiaure 5. Experimental Protocol

Lef€ & Rght Legs Right Leg

2-hov Conditioninci Perfusion:

L w Glucose (2mM) Normal Glucose (6.5mM) High Glucose (20mM)

30min Perfusion:

Nomial Glucose (5.5mM) 2-pHI-oGt

T i m (min)

Three protocols are used to simulate hypo-, eu-, and hyperglycemic conditions. Either LOW (2mM). NORMAL (6.5mM), or HlGH (20mM) glucose perfusate is perfused through both the ieft and right legs for a 2-hour 'glycernic conditioning' period. At the 2-hour mark, mixed muscle sarnples are excised from the left leg, freeze-clamped, and stored at -80°C for further Western Blot analysis of glucose transporter translocation. The left cornrnon iliac artery & vein are then ligated and the right leg perfused for an additional 30 minute period with perfusate containing NORMAL (5.5mM) glucose and 35pCi of 2-[3~]-deoxyglucose (2-DG). Red (soleus) and white (extensor digitorum longus) muscle samples are then excised and assayed for 2-DG-6-P content.

LABORATORY METHODS

- Beckman Glucose Analyzer 2 (Beckman, Fullerton, CA) Beckman Certified Glucose Reagent (1 40 Ufml glucose oxidase, 5% denatured alcohol, 1 O-* M potassium iodide, catalase, ammonium molybdate; Beckman, Mississauga, ON) - Beckman Certifieci Glucose Standard (150 mg/dl glucose, 50 rng/dl urea nitrogen; Beckman, Mississauga, ON)

Glucose concentrations of both plasma and perfusate sarnples were

rneasured using a calibrated Beckman Glucose Analyzer 2 (Beckman, Fullerton,

CA), which utilizes the glucose oxidase method (225) of glucose determination. A

10 pl sample of plasma or perfusate is injected into an enzyme reagent solution

containing oxygen and glucose oxidase. D-glucose in the sarnple reacts with

dissolved oxygen in the solution according to the following reaction catalyzed by

glucose oxidase:

D -Glucose + O, t ii,O Glucose OxicIc~se , Gluconic ncid + &OS

In the reaction, oxygen is consumed at the same rate (1:l ratio) as glucose, which

together form gluconic acid. Thus, the signal generated by the polarographic

oxygen sensor in the reaction chamber is directly proportional to the concentration

of glucose in the sample. Results are obtainable within 30 seconds of addition of

sample, and are acwrate to f 3 mgldl. The analyzer was calibrated before use and

after approxirnately every tenth sarnple using the 150 rngldl Beckman Certified

Glucose Standard.

O Purified Rat Insulin (NOVO, Copenhagen, Denmark) Wright's Antiserum, 1 : 950,000 dilution (University of Indiana, Indianapolis,

O

IN) Glycine Buffer (0.2 MI pH 8.8) Normal Sheep Serum (Grand Island Biological, Grand Island, NY)

O Bovine Serum Albumin (BSA; Sigma Diagnostics, St. Louis, MO) O 1251-labelled Porcine lnsulin (NEN Life Science Research Products,

Mississauga, ON) Cobra II@ Gamma Counter, Model D5005 (Canberra Packard, Mississauga, ON)

METHOOS

The insulin radioirnmunoassay (RIA) was perfomed as described by Herbert

et al. (226) for detemination of plasma and perfusate insulin levels. The principle

behind the RIA is the competition between labelled (radioactive) and non-labelled

hormone for a specific amount of antibody, as first described by Berson and Yalow

(227). Unbound insulin, both labelled and unlabelled, is then removed from the

sarnple by addition of charcoal, which binds the free hormone and sediments to the

bottom of the sample tube. Antibody-bound insulin remains in solution, and is

removed from the sample by aspiration of the supernatant. The radioactivity of the

pellet is then counted. The higher the concentration of unlabelled insulin in the

sarnple, the higher the amount of radioactive insulin left in the pellet. A standard

49

cuve is constructed, using purified rat insulin (NOVO, Copenhagen. Denmark) in

concentrations ranging between 0-4000 pglml. to quantify a relationship between

the amount of insulin in a sample and the number of counts recorded in the pellet.

Two hundred and fQ microlitres of WrighYs antisenim (University of Indiana,

Indianapolis, IN) was added to 200 pl samples of insulin standard, plasma. or

perfusate (in duplicates). Tubes were then incubated for 24 hours at 4%

Subsequently. 500 pl of 125~-labelled insulin was added (-12000 cpmltube), after

which tubes were inwbated for 2 more days at 4%. To detenine non-specific

binding (NSB), glycine buffer was added to several samples or empty tubes and

incubated with labelled insulin without antibody. Total counts per minute (cpm)

were determined in 3 tubes containing only 1251-insulin. Normal sheep serum (NSS)

and charcoal were then added to al1 tubes, except those designated for total cprn.

The sheep semm was used to provide the optimal amount of protein required to

prevent adsorption of the antibody-bound '251-insulin by the charcoal. Tubes were

incubated for 30 minutes at 4'C, and then œntnfuged for 15 minutes at 800 x g and

4°C. The supernatant was Vien aspirated, and the pellet was counted for 4 minutes

using a gamma counter (Canberra Packard, Mississauga. ON). Standard sample

values were calwlated from the following equations:

Percent non-specific binding (NSB) values were calculated and used as a

check for the validity of the assay. Values for % free taken from the standards were

used to fom the standard cuwe from which sample values were intrapolated.

1 N NaOH solution 1 N HCI solution Analytical Balance, AE 200 (Mettler Instrument Corporation. Hightstown. N J) AO" Shaking Water Bath, Model 40601 5 (American Opticai, Buffalo, NY) 6% HCIO, solution Titrated 0.3 N Ba(OH), and 5% (wlv) ZnSO, solutions (Sigma Diagnostics, St. Louis. MO) Microcentrifuge, Mikro" 72-24 (Hettich, Tuttlingen, Germany) Glass scintillation vials (Fisher Scientific, USA) Ready protein@ Scintillation cocktail (Beckman Instruments. Fullerton, CA) B-Scintillation counter (Canberra Packard, Mississauga, ON)

METHOOS

Phosphorylated 2-[3~]deoxyglucose. or 2-DGô-P, was assayed in soleus

and extensor digitormm longus (EDL) muscle samples extracted from the right

hindlimb according to Ferré et al. (228). Briefly. -100 mg of fresh muscle sample

was digeste in a pre-weighed plastic tube containing 0.5 mi of 1 N NaOH solution

for immediate enzyme inactivation and muscle digestion. The tube was re-weighed,

and the wet muscle mass was detemined. The sample was then piaced in a 60°C

shaking water bath for at least 45 minutes, until the muscle sample was completely

51

dissolved. 0.5 ml of 1 N HCI was then added to the sample to neutralize the

solution.

This rnethod of 2-[3~]-DG-6-~ determination utilizes the fact that both 2-DG

and 2-DG-6-P are soluble in 6% HCIO, , but only 2-DG is soluble in the Somogyi

reagent (equal volumes of titrated 0.3 N Ba(OH), and 5% (wiv) ZnSO,) (229). 2-

DG-6-P is instead adsorbed on the BaSO,/Zn(OH), precipitate (230). Thus, by

detenining the difference in radioactive counts in both the 6% HCIO, and Somogyi

solutions, an estimate of the amount of 2-DG-6-Pl in counts per minute (cpm), can

be calculated.

Two hundred microlitres of the neutralized sample was added to 1 ml of 6%

HCIO,. Another 200 pl of the neutralized sample was added to a mixture of 0.5 ml

of 0.3 N Ba(OH), and 0.5 ml of 5% (wlv) ZnSQ . The samples were then mixed

throughly and centrifuged to pellet the precipitate formed by the reaction. Eight

hundred microlitres of supernatant from each sample was then placed in a glass

scintillation via1 with 10 ml of Ready Protein" Scintillation cocktail (Beckman

Instruments, Fullerton, CA). Vials were well shaken and were counted for 10

minutes each in a B-Scintillation counter (Canberra Packard, Mississauga. ON). A

glass scintillation via1 containing only scintillation cocktail was used as a

background control. 2-DG-6-P content was determined as follows:

2 -DG- 6 - P (cpnr) = 6% HClO, son~pfe (cpm) - Somogyi sumpie (cpni)

52

2-DG-6-P content was expressed as cpmlmg protein (see Whole Muscle Protein

Assay below).

Ice Dry lce Homogenization Buffer (1 O mM Tris-HCI, 30 mM NaCl, 1 mM Dithiothreitol, 0.1 mM PMSF; pH 7.5) Analytiml Balance, A€ 200 (Mettler Instrument Corporation, Hightstown, NJ) Polytron@ Hornogenizer (Brinkmann Instruments Inc., Toronto, ON) IEC PR-6000 Refrigerated Centrifuge (DamonllEC Division, Needharn Heights, Massachusetts) BCA Protein AssayB Kit (Pierce Chernical Co.. USA) Stock Protein Standard, 500 pglrrtl Bovine Semm Albumin (BSA) AO@' Shaking Water Bath, Model406015 (American Optical, Buffalo, NY) Disposable Cuvettes DUg-64 Spectrophotometer (Beckrnan Instruments Inc., Fullerton, CA)

Red (soleus) and white (extensor digitorum longus) muscle sarnples

obtained, freeze-clamped and stored at -80" C following the 30 minute perfusion

with 5.5 mM glucose perfusate were used to determine whole muscle protein

content. Samples were transported on dry ice to maintain tissue integrity. Cold

homogenization bde r (1 ml) was placed in a plastic tube and weighed on a digital

scale. Approximately 100 mg of muscle was immersed into the homogenization

buffer and the tube quickly re-weighed to determine the mass of the sample. The

muscle was then homogenized at a high speed for at least 20 seconds using a

53

Polytron" homogenizer (Brinkmann Instruments Inc., Toronto, ON). Cold

homogenization buffer (4 ml) was then added to bring the total volume of the

sample to 5 ml. Samples were then centrifuged at 2300 rpm for 10 minutes to

separate connective tissue from the homogenate. The supernatant was collected

and kept on ice for protein determination.

Total protein content was determined using the BCA Protein ~ s s a y @ kit

(Pierce Chernical Co., USA). The bicinchoninic acid (BCA) protein assay is a highly

sensitive method for the spectrophotometric determination of protein concentration

(231). Briefly, 5 pl of supernatant (-1-2 mg proteiniml) was added to 45 pl of

ddH,O. A standard curve was set up consisting of 0, 1 0, 20, 30, 40 and 50 pg BSA

from a standard stock solution of 500 pg/ml. Double distilled H,O was added to the

standards to a final volume of 50 pl. Working BCA Protein Assay" Reagent (0.5 ml)

was added to al1 samples and standards, mixed, and incubated in a shaking 37°C

waterbath for 30 minutes. Following incubation, standards and samples were

transferred to plastic disposable cuvettes and optical density (00) measured at 562

nm. A standard curve was constructed and the protein concentrations of the

sarnples intrapolated from the curve.

MUSCLE MEMBRANE PREPARATlON

MEMBRANE FRACTIONATION PROCEDURE

MATERIALS

Ice Hornogenization Buffer (20 mM NaHCO,, 250 mM sucrose, 5 mM NaN,, 0.1 mM PMSF; pH 7.0) Dilution Buffer (20 mM NaHCO,, 5 mM NaN,, 0.1 mM PMSF; pH 7.0) Analytical Balance, AE 200 (Mettler Instrument Corporation, Hightstown, NJ) Polytrona Homogenizer (Brinkmann Instruments Inc., Toronto, ON) Beckman 50 ml Polycarbonate Ultracentrifugation t