Glucose Transporters and Pathophysiologic States

E. Dale Abel

Peter R. Shepherd

Barbara B. Kahn

The effect of insulin to acutely stimulate glucose uptake into muscle and adipose tissue is essential for normal glucose homeostasis. A major pathologic feature of obesity, type 2 diabetes and, to a lesser extent, type 1 diabetes is resistance to this effect of insulin, which has been demonstrated both in vivo using nuclear magnetic resonance (1) and forearm perfusion with indirect calorimetry (2,3) and in vitro in isolated adipocytes (4) and muscle strips (5,6,7). Furthermore, glucose transport is the rate-limiting step for glucose utilization in muscle at most physiologic glucose and insulin levels (8,9,10), as well as in type 2 (1,2), and type 1 diabetes (3). Thus, defects in the insulin-stimulated glucose transport system are likely to be a major cause of peripheral insulin resistance.

Glucose transport into muscle and adipose cells occurs by facilitated diffusion that is mediated primarily by two members of the glucose transporter (GLUT/SLC2A) family of proteins. There are currently 12 identified members of this family that are further subcategorized into three classes (11). The class I molecules GLUT-1 to GLUT-4 have been most extensively characterized. In tissues with insulin-sensitive glucose transport (i.e., muscle and adipose cells), GLUT-4 is the predominant glucose transporter. The large stimulatory effect of insulin in these tissues results from the unique targeting of GLUT-4. In the absence of insulin, GLUT-4 is sequestered in intracellular vesicles; in response to insulin and other stimuli, these vesicles translocate to the plasma membrane (Fig. 62.1). Many studies have addressed the role of glucose transporters in insulin-resistant and other pathophysiologic states. Interest in this area has been fueled by gene “knockout” studies in mice, demonstrating that reduction in GLUT-4 levels can cause insulin resistance and frank diabetes, and by transgenic overexpression studies, showing that increased expression of GLUT-4 in muscle or fat can increase whole-body glucose disposal and ameliorate insulin resistance in transgenic mice. However, in insulin-resistant humans GLUT-4 levels are reduced in adipocytes but not in muscle. The potential contribution of the GLUT-4 reduction in fat has not been clear. However, mice with adipose tissue selective knockout of GLUT-4 develop glucose intolerance and insulin resistance not only in fat but also in skeletal muscle and liver, indicating that a reduction in adipose tissue GLUT-4 expression may contribute to the pathogenesis of insulin resistance and diabetes (12). Although defects in insulin signaling or in the GLUT-4 translocation machinery are more likely causes of insulin resistance in muscle, transgenic studies suggest that strategies that upregulate GLUT-4 expression may have therapeutic benefit in the treatment of insulin resistance. Indeed, the findings that exercise-mediated glucose uptake is normal in muscle from insulin-resistant humans, and that insulin-mediated glucose uptake is increased following exercise (due to increased signal transduction and increased GLUT-4 expression) in insulin-resistant humans, suggest that targeting this GLUT-4 translocation pathway may lead to strategies for overcoming insulin resistance. This chapter will review molecular mechanisms that could potentially account for altered glucose transport in both human and animal models of insulin resistance and enhanced insulin responsiveness. One of the salient themes is that the regulation of glucose transporter gene expression is tissue specific, with much greater changes in expression in adipocytes than in muscle in altered metabolic states. Furthermore, altering glucose transport in either adipocytes or muscle can lead to alterations in insulin action in other target tissues.

Cellular Mechanisms for Stimulation of Glucose Transport: Potential Molecular Defects in Insulin-Resistant States

The stimulatory effect of insulin on glucose transport involves a series of cellular events (13,14) (Fig. 62.1). Dysregulation of any of these steps could result in insulin resistance. The binding of insulin to its receptor generates intracellular signals that regulate GLUT-4 gene transcription and protein levels, stimulate transporter translocation, and modulate the intrinsic activity of the transporter. The nature of the signaling intermediates that mediate these processes is under intense investigation (14,15). It is likely that distinct abnormalities in signaling account for insulin-resistant glucose transport in some pathologic states. Evidence indicates, however, that insulin resistance in muscle in

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many pathophysiologic states probably results from alterations in GLUT-4 vesicle translocation, fusion with the plasma membrane, or intrinsic activity rather than from changes in GLUT-4 gene expression.

Figure 62.1. Sequence of events involved in insulin stimulation of glucose transport in muscle and adipose cells. (1) In the absence of insulin, glucose transporter 4 (GLUT-4) resides within the cell. Other proteins associated with GLUT-4 vesicles include synaptobrevin (vesicle-associated membrane protein, VAMP), insulin-responsive aminopeptide (IRAP), phosphatidylinositol-4 kinase (PI4-kinase), rab4, and secretory carrier-associated membrane proteins (SCAMP). (2) When insulin binds to its receptor in the plasma membrane, it initiates a cascade of signals affecting transcription, translocation, and possibly activation of the glucose transporter. (3) Rab4 dissociates from the vesicle, and the vesicle translocates to the plasma membrane, where it docks and fuses, and glucose transport is activated. (4) GLUT-4 vesicles are endocytosed and recycle through endosomes. (Adapted from Shepherd PR, Kahn BB. Expression of the GLUT4 glucose transporter in diabetes. In:

Draznin B, LeRoith D, eds. Molecular biology of diabetes. Vol II. Totowa, NJ: Humana Press, 1994:535–536

, with permission.)

A uniquely important characteristic of GLUT-4 is its targeting to specific intracellular vesicles distinct from that of GLUT-1 (13). Studies using GLUT-1/GLUT-4 chimeras suggest that specific domains are important for targeting the transporters to their respective vesicles (13). The identification of novel GLUT-4 vesicle cargo proteins such as insulin responsive aminopeptidase (IRAP), which is also localized exclusively in GLUT-4 vesicles (16,17), and cellugyrin (18,19), which associates with GLUT-4 only in recycling endosomes, has begun to shed new insight into the molecular mechanisms that regulate GLUT-4 targeting. There are few studies to date that implicate altered GLUT-4 targeting as a contributor to altered glucose transport in pathophysiologic states. However, two small human studies have described mistargeting of IRAP and GLUT-4 to a high-density microsomal compartment from which GLUT-4 translocation was speculated to be impaired in adipocytes and skeletal muscle obtained from individuals with type 2 diabetes (20,21). Translocation of GLUT-4–containing vesicles to the plasma membrane is the major step leading to the increase in transport in response to a variety of stimuli, including insulin, exercise, hypoxia, other growth factors such as insulin-like growth factor-1 (IGF-1) and in vitro activation of guanine triphosphate binding proteins (22). Colocalization of the small guanine triphosphate binding protein rab4 with the GLUT-4 vesicle and identification of insulin-stimulated translocation of rab4 from low-density microsomes to the cytosol, with the same time course as that of GLUT-4 translocation to the plasma membrane (23), may lead to insights into the vesicle translocation and fusion process.

Translocation time course studies show that after insulin stimulation there is a delay between the arrival of GLUT-4–containing vesicles at the plasma membrane and an increase in glucose transport (24). This implies that the docking or fusion of these vesicles with the plasma membrane may be a regulated step in glucose transport or that the transporters have to be activated once they are inserted in the plasma membrane (Fig. 62.1). Evidence to support this hypothesis includes the observation that isoproterenol and epinephrine impair glucose uptake in adipocytes and skeletal muscle, respectively, without altering GLUT-4 translocation (25,26). There is, however, a reduction in the ability of the exofacial bis-mannose photolabel ATB-BMPA to label GLUT-4 at the plasma membrane, implying that GLUT-4 is not in the correct orientation to bind the photolabel or to transport glucose (25). Such a mechanism may have physiologic relevance in the counterregulatory effects of epinephrine on glucose transport into muscle. Several lines of evidence support the concept that glucose transporter intrinsic activity can be modulated once transporters are appropriately inserted in the plasma membrane and that such alterations may contribute to insulin resistance in some states, such as in muscle of rats after high-fat feeding (27). The mechanisms by which changes in intrinsic activity occur are not known. In 3T3-L1 adipocytes, protein synthesis inhibitors result in a rapid increase in glucose transport with no change in the plasma membrane levels of glucose transporters (28), implicating a role for a rapidly turning over protein in the regulation of intrinsic activity. The observation that vanadate, a phosphatase inhibitor, increases intrinsic activity in rat muscle plasma membrane vesicles (29) suggests that phosphorylation of cellular proteins, but not necessarily of GLUT-4, also may play a role in the regulation of intrinsic activity. Recent studies also implicate a role for insulin mediated stimulation of P38 mitogen-activated protein kinase in the activation of GLUT-4 transporters following insertion in the plasma membrane (30,31). Finally, kinetic data indicate that glucose transporters are constantly recycling between the plasma membrane and the intracellular pool and that insulin increases the rate of exocytosis (24,32). This represents

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another potentially regulated step in glucose transport, and insulin resistance could result from a relative increase in endocytosis versus exocytosis.

Role of Glucose Transporters in Diabetes, Obesity, and Other Insulin-Resistant States in Humans

Muscle is the major site of insulin-stimulated glucose disposal in vivo (33), and in normal subjects GLUT-4 levels in muscle in the basal state correlate positively with whole-body insulin-stimulated glucose disposal (34). Therefore, mutations in GLUT-4 or reductions in GLUT-4 levels in muscle could be expected to result in insulin resistance due to reduction in insulin-stimulated glucose disposal.

Potential Mutations in the Glucose Transporter Genes

Only two mutations in glucose transporter genes have definitively been associated with distinct disease states. Mutations in GLUT-1 cause intractable seizures due to reduced glucose transport across the blood–brain barrier (35), and GLUT-2 mutations cause the rare glycogen storage disease known as Fanconi-Bickel syndrome (36). However, neither of these mutations appears to cause diabetes. Investigations of the possibility that mutations in GLUT-4 could contribute to insulin resistance have revealed only one alteration affecting amino acid sequence: a single nucleotide change that results in the substitution of an isoleucine for valine at codon 383. The Ile383 GLUT-4 allele is present in a small (1%–3%) fraction of the population and does not appear to segregate with non–insulin-dependent diabetes mellitus (NIDDM) (37,38,39). Single-stranded conformational polymorphism and direct sequencing analysis of the GLUT-4 promoter region revealed three genetic variants in 30 NIDDM patients. These variants, however, had no impact on GLUT-4 expression in skeletal muscle (40). Initial reports of a restriction fragment length polymorphism of the GLUT-1 locus that segregated with NIDDM is not generalizable to broad populations (41), but may segregate in obese and overweight women (42).

Regulation of Glucose Transporter 4 Gene Expression

As detailed below, GLUT-4 expression can be altered in vivo, particularly in adipocytes. Insulin-resistant states are generally not associated with impaired GLUT-4 expression in skeletal muscle, although exercise can increase GLUT-4 expression in muscle. The mechanisms regulating GLUT-4 expression are not fully elucidated, but the recent identification of some of the upstream regulatory elements of the GLUT-4 gene (43) has increased our understanding of the mechanisms by which the changes in GLUT-4 expression occur in altered metabolic states, and in response to exercise and adenosine monophosphate (AMP) kinase activation (44,45,46). Obesity and diabetes do not appear to be associated with altered GLUT-4 protein levels in any of the human skeletal muscle groups investigated (Table 62.1). GLUT-4 protein levels were found to be similar to age- and weight-matched control subjects in membranes prepared from vastus lateralis muscle in obese (47,48), type 2 diabetic (47,48), and type 1 diabetic (49) subjects, as well as in quadriceps femoris muscle of type 1 diabetic subjects (50) and gastrocnemius muscle of type 2 diabetic subjects (51). One study reported a small decrease in GLUT-4 levels in rectus abdominus muscle in massively obese nondiabetic and type 2 diabetic subjects (52). Weight reduction, however, did not reverse this small reduction despite improved glucose disposal, indicating that this small decrease did not explain the insulin resistance of obesity or NIDDM (7). In other insulin-resistant states such as gestational diabetes mellitus (48), uremia (53), and pseudoacromegaly (54), muscle GLUT-4 content is also unchanged.

Table 62.1. Regulation of GLUT-4 mRNA and protein levels in humans

Condition	Muscle	Adipocytes
Type 1 DM	↔	ND
Pancreatic transplant in type 1 DM	↓	ND
Type 2 DM	↔	↓
Insulin-resistant relatives of subjects with type 1 DM	↔	ND
Obesity	↔ (or ↓^a)	↓
Gestational DM	↔	↔ or ↓
Aging	↓	ND
Uremia	↔	ND
Polycystic ovary syndrome	ND	↓
Pseudoacromegaly	↔	ND
Exercise	↑	ND
Sulfonylurea therapy	↔	ND
Weight loss	↔	ND
DM, diabetes mellitus; ND, not determined. ^aDecrease in morbidly obese subjects. Adapted from Shepherd PR, Kahn BB. Expression of the GLUT-4 glucose transporter in diabetes. In: Draznin B, LeRoith D, eds. Molecular biology of diabetes. Vol. II. Totowa, NJ: Humana Press, 1994:529.

Because GLUT-4 concentrations vary widely in muscles of normal humans (47,48,49) and those with obesity (47,48) and diabetes (47,48,49), small differences between groups could be obscured by intergroup variation. Hence, longitudinal studies of the same subjects before and after a therapeutic intervention are important. In the studies mentioned above, in which gastric bypass led to weight reduction (7), as well as in studies in which sulfonylurea therapy improved glycemic control in NIDDM subjects (55), improved whole-body glucose disposal was not accompanied by any changes in muscle GLUT-4 content. Although there is little evidence to support the role of altered GLUT-4 levels in muscle in the basal state, recent studies have suggested that the in vivo regulation by insulin of this transporter

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may be deranged in NIDDM and IDDM (56,57). In normal subjects, euglycemic hyperinsulinemia for up to 4 hours resulted in an increase in GLUT-4 messenger RNA (mRNA) and a decrease in GLUT-4 protein concentration in skeletal muscle. These changes did not occur in the diabetic subjects (56,57). However, in IDDM subjects after pancreas transplantation, chronic hyperinsulinemia is associated with 45% lower muscle GLUT-4 content compared with nondiabetic control subjects in the face of markedly increased plasma insulin concentrations (58). This could represent adaptive regulation to prevent hypoglycemia.

Defects in Glucose Transporter 4 Translocation

Although GLUT-4 expression is normal in muscles of obese and diabetic subjects, studies suggest that defects in translocation of GLUT-4 could contribute to the insulin-resistant glucose disposal in muscle. Studies in normal humans show that insulin (59), glucose ingestion (75-g glucose load) (60), or a single bout of exercise (61) results in GLUT-4 translocation to the plasma membrane. In a study that used biotinylated photo-affinity labeling, a 90% reduction in insulin-stimulated GLUT-4 translocation was observed in skeletal muscle from patients with type 2 diabetes. A similar translocation defect was seen in response to hypoxia, thereby suggesting that impaired GLUT-4 translocation is a key mechanism for the reduced glucose uptake that characterizes type 2 diabetes (62). However, in another study of subjects with type 2 diabetes, GLUT-4 recruitment in response to insulin in muscle was impaired, whereas the response to exercise was preserved (61). This suggests that insulin-resistant muscle can respond to other signaling pathways such as 5′-AMP kinase, which plays a role in contraction/exercise-induced GLUT-4 translocation (63).

Studies in Human Adipose Cells

In contrast to muscle, GLUT-4 levels are markedly downregulated in adipose cells in obese human subjects (4), and this reduction is more pronounced in obese subjects with impaired glucose tolerance or overt type 2 diabetes (4). GLUT-4 levels are also decreased in adipocytes from lean and obese patients with polycystic ovarian syndrome when compared with age- and weight-matched controls (64). Insulin-stimulated recruitment of GLUT-4 to the plasma membrane is impaired in adipocytes isolated from gestational diabetics due to changes in expression or subcellular localization of GLUT-4 (65). Until recently, fat was thought to play only a minor role in insulin-stimulated glucose disposal (66). However, recent data indicate a role for adipose tissue in regulating fuel metabolism and appetite through secretion of leptin (67,68) and potentially in insulin action through the secretion of tumor necrosis factor-α (TNF-α) (69) and other recently described adipocytokines such as resistin (70) and adiponectin (71). Recent studies in adipose tissue–selective GLUT-4 knockout mice suggest that reduced adipocyte GLUT-4 content can directly lead to generalized insulin resistance, most likely through the release of novel molecules (12).

Ameliorative Effects of Exercise

Exercise is one of the few states in which GLUT-4 content can be modulated in human muscle. Exercise increases insulin sensitivity in normal and type 2 diabetes subjects independent of changes in body composition (72,73). In middle-aged men with normal glucose tolerance, subjects with impaired glucose tolerance, and subjects with NIDDM (73,74,75,76), exercise is associated with increases in muscle GLUT-4 content and in vivo glucose uptake. The increment in in vivo insulin sensitivity was accounted for by the increase in GLUT-4 in the study of middle-aged men (75), but in those with impaired glucose tolerance, insulin sensitivity increased by 11% despite a 60% increase in skeletal muscle GLUT-4 content (73). This suggests either that GLUT-4 regulation by exercise is fiber type specific, so that the net impact on in vivo glucose uptake is less than the increase in GLUT-4 observed in the muscle sampled; that the increased transporters are not present in or recruited to the plasma membrane; or that factors in addition to GLUT-4 content regulate muscle glucose uptake. An increase in GLUT-4 content could not fully account for the enhanced in vivo glucose uptake observed in athletes by Andersen et al. (76), and no correlation was found between insulin-mediated glucose uptake and GLUT-4 levels in muscle after a hyperinsulinemic clamp. These data suggest that exercise-induced improvements in insulin sensitivity are only partially explained by increases in GLUT-4 expression.

Glucose Transporters in Animal Models of Obesity, Diabetes, and Insulin Resistance

Genetic Models Associated with Hyperinsulinemia

Zucker Rats

Zucker rats carry the autosomal-recessive fa gene, which has subsequently been shown to code for a variant of the leptin receptor. Rats homozygous for the fa gene are obese and progressively develop insulin resistance. Preweaned Zucker (fa/fa) rats (21 days old) reveal hyperresponsive insulin-stimulated glucose uptake in diaphragm muscle that progressively declines, so that by 31 days it is similar to that of lean controls. By 70 days of age, however, glucose uptake in diaphragm muscle is impaired, heralding the development of insulin resistance (77). These changes are not associated with any change in GLUT-4 mRNA or protein in muscle (77). At a time when this insulin-resistant glucose uptake develops in skeletal muscle, heart, and brown adipose tissue, insulin-stimulated glucose disposal remains elevated in white fat (78). Thus, hyperresponsive glucose transport in isolated adipocytes from young Zucker rats persists longer than in muscle, but insulin-resistant glucose uptake in adipocytes eventually ensues in older animals (79). Unlike muscle, in adipocytes of young obese Zucker rats, the insulin hyperresponsiveness

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corresponds with an increase in GLUT-4 protein levels compared with that of lean littermates (79) (Fig. 62.2). Older Zucker (fa/fa) obese rats exhibit insulin resistance and reduced insulin-stimulated glucose utilization in adipocytes and all muscle groups (78,80,81). Levels of GLUT-4 in muscle are the same between lean and obese Zucker rats (80) even as they age (Fig. 62.2). In contrast, by 20 weeks of age, the increase in adipocyte GLUT-4 content seen in young Zucker rats is reversed to levels below those of lean controls (Fig. 62.2), in parallel with the reversal of hyperresponsive, insulin-stimulated glucose transport (79). These changes are summarized in Table 62.2.

Figure 62.2. Altered glucose transporter expression in insulin-resistant Zucker (fa/fa) rats. A: Increased levels of glucose transporter 1 (GLUT-1) and GLUT-4 protein in adipose cells of young (5- and 10-week-old) obese Zucker rats but decreased GLUT-4 levels in older (20-week-old) obese (O) and obese/diabetic (OD) Zucker rats compared with lean controls (L). Obese/diabetic Zucker rats were rendered diabetic with streptozotocin injection and were analyzed 36 hours after diabetes induction. GLUT-4 protein levels were determined by scanning densitometry of autoradiograms of Western blots and are expressed per adipocyte. Values in obese rats are expressed as a percentage of the values in 5-week-old lean Zucker rats. Results are means ± SEM of four separate experiments. (A is reproduced from

Pedersen O, Kahn CR, Kahn BB. Divergent regulation of the GLUT1 and GLUT4 glucose transporters in isolated adipocytes from Zucker rats. J Clin Invest 1992;89:1964

, with permission.) B: Tissue-specific regulation of GLUT-4 protein levels in skeletal muscle (black) and adipose cells (shaded) from 20-week-old Zucker rats. GLUT-4 protein levels were determined by scanning densitometry of autoradiograms of Western blots. Results are means ± SEM. *Difference from control at p = 0.05. (B is reproduced from

Kahn BB, Pedersen O. Suppression of Glut 4 expression in skeletal muscle of rats that are obese from high fat feeding but not from high carbohydrate feeding or genetic obesity. Endocrinology 1993;132:13

, with permission.)

The major cause of insulin resistance in muscle appears to be a defect in translocation of GLUT-4 to the plasma membrane (82,83), as the intrinsic activity and overall level of GLUT-4 in muscle are the same in lean and obese Zucker rats (80,81,82). The translocation defect appears to be confined to translocation signaled by insulin because exercise-stimulated glucose transport in muscle is normal in obese Zucker rats (82). Indeed, there is reduced expression of insulin receptor substrate (IRS)-1/2 and impaired insulin-stimulated activation of phosphatidylinositol-3 (PI-3) kinase and AKT (84,85).

The potential involvement of glucocorticoids in the insulin resistance of obese Zucker rats is suggested by elevated corticosterone concentrations in this model (86,87). Lowering of corticosterone concentrations by adrenalectomy lowers insulin concentrations (87) and partially restores the translocation of GLUT-4 in muscle (86). Chronic vanadate treatment normalizes insulin concentrations in obese Zucker rats without changing fasting glucose concentrations (81). This is accompanied by increased glucose utilization in muscle without altering GLUT-4 expression. These effects are not seen when insulin concentrations are lowered by streptozotocin treatment (80), which results in an increase in blood glucose to diabetic levels. Thus, the insulin concentration alone is not the major determinant of in vivo insulin sensitivity in the Zucker rat.

The phenotype of the fa gene depends on the genetic background on which it is expressed. Males of the ZDF strain of the fa/fa rat (ZDF/drt-fa) have obesity and insulin resistance similar to that of fa/fa rats but are prone to develop overt diabetes associated with a reduced capacity to secrete insulin. In these obese diabetic rats, GLUT-4 protein levels are significantly reduced in skeletal muscle and adipose tissue (88,89). Reduction of hyperglycemia, by decreasing absorption of glucose in the gut with acarbose, results in normalization of GLUT-4 levels and a significant increase in insulin secretion (89). Transfer of the fa gene to a Wistar rat background also results in obesity and overt diabetes, although GLUT-4 mRNA levels in muscle are unchanged from those of lean controls (90).

db/db Diabetic Mouse

The db gene, which codes for a variant of the leptin receptor, is inherited as an autosomal-recessive trait that confers obesity associated with hyperglycemia and hyperinsulinemia in homozygotes. Insulin-stimulated glucose transport into skeletal muscle is impaired in db/db mice (91), but in young (5-week-old) mice, GLUT-4 protein concentrations in skeletal muscle and adipose tissue are similar between obese diabetics and lean controls (92). Despite profound impairment in skeletal muscle glucose uptake in db/db mice in vivo, in vitro insulin-stimulated GLUT-4 translocation (assessed by photolabeling) and insulin-mediated glucose uptake are normal (93). The mechanism for the in vivo muscle insulin resistance is not clear, but may involve glucotoxicity.

ob/ob Obese Mouse

Ob/ob mice have a null mutation in the ob gene that codes for the hormone leptin (94). Mice homozygous for this mutation are obese and display an insulin-resistant phenotype similar to that of the db/db mice, with less severe hyperglycemia due to the genetic background conferring greater islet cell capacity in ob mice. Dysfunctional insulin-stimulated glucose transport appears to play a primary role in the development of insulin resistance in ob/ob mice (95). GLUT-4 protein content in skeletal muscle is similar to control mice. However, basal and

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insulin-stimulated glucose uptake are impaired in soleus and extensor digitorum longus (EDL) muscles (soleus > EDL), but the ability of aminoimidazole-4-carboxamide-1β-riboside (AICAR) to increase glucose uptake is normal (96). Similarities with the hyperinsulinemic but normoglycemic Zucker model are also seen, because ob/ob mice have elevated levels of glucocorticoids (97), and adrenalectomy (98) or vanadate treatment (99) reduces peripheral insulin resistance in ob/ob mice.

Table 62.2. Regulation of GLUT-4 mRNA and protein levels in animal models of altered insulin sensitivity

Animal model	Fasting serum insulin	Fasting serum glucose	GLUT-4 in muscle	GLUT-4 in fat
Obese (fa/fa) Zucker rats
Young	↑	↔	↔	↑↑
Old	↑↑	↔	↔	↓↓
Diabetic Zucker (ZDF/drt) rat	↑	↑↑	↓↓	↓↓
Gold thioglucose-induced obesity in rats	↑↑	↑	↔	↓↓
KK/A^y mice	↑↑	↑	↓↓	↓↓
A^vy/a mice	↑↑	↑	↓↓	↓↓
Brown fat-ablated mice	↑↑	↑	↔	ND
db/db Mice	↑	↑	↔	↔
Neuropeptide Y injection in rats	↑	↔	↔	↑
Ventromedial hypothalamic lesion–induced obesity in rats	↑	↔	↔	↑↑, ↔
High-fat feeding in rats	↑	↔	↔	↓↓
Dexamethasone treatment in rats	↑	↑	↔	ND
Streptozotocin-induced diabetes mellitus in rats and mice	↓	↑↑	↔	↓↓
Spontaneously hypertensive rats	↑	↔	↔	ND
Aged rats	↑	↔	↓↓	↓↓
Hyperthyroid rats	↓	↔	↑	↑
Hypothyroid rats	↔	↔	↓	ND
Metformin treatment of diabetes mellitus in rats	↓	↓	↔	ND
Leptin in rats and mice	↓	↓	ND	ND
Thiazolidinediones in rats	↓	↓	↔	↑
fa/fa, fatty due to mutation in the leptin receptor; ZDF/drt, Zucker diabetic fatty rat; A^vy/a, viable yellow mouse; db/db, genetically obese and diabetic due to mutation in the leptin receptor; ND, not determined. Adapted from Shepherd PR, Kahn BB. Glucose transporters and insulin action: implications for insulin resistance and diabetes mellitus. N Engl J Med 1999;341:248–257, with permission.

Rodent Hypertension

Hypertension is often associated with insulin resistance in humans (100). The spontaneously hypertensive rat (SHR) has been used to study the mechanism of this insulin resistance. Some investigators have taken normal glycemia but elevated insulin levels as an indication of insulin resistance in these animals (101). This conclusion is controversial, however, because insulin-stimulated glucose disposal, as measured by hyperinsulinemic-euglycemic clamp, showed increased sensitivity and normal maximal responsiveness in one study (102) and decreased responsiveness in another study using different conditions (103). Insulin receptor tyrosine kinase activity, GLUT-4 protein content, and GLUT-4 translocation are apparently normal in muscle of spontaneously hypertensive rats (104). Isolated adipocytes from hypertensive rats, however, have lower basal and insulin-stimulated glucose transport capacity, possibly indicating a reduction in transporter number or intrinsic activity (101). Stroke-prone SHRs exhibit impaired insulin-stimulated glucose uptake despite normal insulin stimulated activation of Akt. Interestingly, there are increased levels of flotillin, caveolin, vesicle-associated membrane protein 2 (VAMP-2), and syntaxin 4, suggesting that altered compartmentalization of insulin signaling and GLUT-4 trafficking might represent the mechanism for insulin-resistant glucose uptake in this model (105). This contrasts with observations in another rodent model of hypertension, the Milan rat. These mice do not exhibit in vivo insulin resistance, but a profound decrease in muscle GLUT-4 content, with normal adipocyte GLUT-4 content,

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has been observed (106). Thus, whether genetic models of rodent hypertension will prove to be useful for the study of the association of insulin resistance and hypertension in humans remains to be determined.

Other Genetic Rodent Models of Obesity and Diabetes

Glucose transporters have been studied in several other genetic models of obesity exhibiting hyperinsulinemia and hyperglycemia. Transgenic mice with genetic ablation of brown adipose tissue have marked obesity and insulin resistance (107). Impaired glucose transport is associated with normal GLUT-4 levels in skeletal muscle and precedes the downregulation of GLUT-4 observed in adipocytes (108). In the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of obesity and type 2 diabetes mellitus, GLUT-4 expression is normal in skeletal muscle, but reduced in adipose tissue (109). In the Goto-Kakizaki (GK) rat (type 2 diabetes, less hyperinsulinemia because of a β-cell defect), GLUT-4 levels are normal in muscle and adipose tissue, and impaired glucose uptake occurs on the basis of impaired insulin signaling, which potentially results from glucose toxicity (110,111). Similarly, normal GLUT-4 levels in adipose tissue and muscle in the Tsumura Suzuki obese diabetic mouse are associated with significant impairment in GLUT-4 translocation (112). In contrast, GLUT-4 levels are reduced in fat, quadriceps, and soleus muscles in the KK/A^Y obese mouse (113), in soleus and heart in the A^vy/a viable yellow mouse (88), and in all membrane fractions of hind limb muscle homogenates in the SHR/N-cp obese rat (114) and the LA/N-cp rat (115).

Experimental Models Associated with Hyperinsulinemia

Experimentally Induced Obesity

Obesity can be experimentally induced by surgical (116) or gold thioglucose-induced lesions (117) of the ventromedial hypothalamus, by intraventricular injection of neuropeptide Y (118), or by systemic neonatal injection of monosodium glutamate (119). Gold thioglucose treatment or neuropeptide Y administration induces obesity associated with marked hyperinsulinemia and mild hyperglycemia (117,118). No difference is seen between the skeletal muscle GLUT-4 content of obese and control animals, although there is a significant decrease in GLUT-4 levels in brown and white adipose tissue (117). Changes in adipose cell GLUT-4 content have also been temporally linked to changes in glucose metabolism during the process of developing obesity in rats with surgically induced ventromedial hypothalamus lesions (116) or after neuropeptide Y injection (118). After 1 week, ventromedial hypothalamus-lesioned rats are slightly obese but have normal plasma insulin and glucose concentrations (116). After 1 week of neuropeptide Y infusion, hyperphagia, weight gain, and hyperinsulinemia occur, with insulin-resistant glucose transport in skeletal muscle (118). In both models, adipose tissue is hyperresponsive to insulin in this initial phase, and this correlates with increased GLUT-4 protein levels in adipocytes (116,118). By 6 weeks after ventromedial hypothalamus lesioning, the plasma insulin levels are markedly elevated, indicating insulin resistance (116). The hyperresponsiveness in adipose tissue disappears, and GLUT-4 levels in fat decline to the level of controls. One group (120) has reported that GLUT-4 translocation is intact in fat of animals with hypothalamic obesity induced by monosodium glutamate or gold thioglucose. In contrast to other groups, they also reported a 40% reduction in cardiac and skeletal muscle GLUT-4 content in all membrane fractions (119).

Diet-Induced Obesity

In animal models, high-fat feeding causes obesity associated with marked insulin resistance compared with animals fed a high-carbohydrate diet (121). Plasma insulin levels can be low, normal, or high depending on the amount and composition of the lipid and the duration of the diet. The magnitude of the insulin resistance is also affected by the fatty acid composition of the lipid and correlates with the triglyceride content of skeletal muscle (122). There appear to be multiple mechanisms by which high-fat feeding results in insulin resistance. A high-fat diet (50%–80% of total calories from fat) results in significant reductions of GLUT-4 mRNA and protein in adipose tissue (123). Very high dietary fat content (80%) for prolonged periods also causes downregulation of GLUT-4 expression in skeletal muscle (79). One study in rats showed that translocation of GLUT-4 in muscle remains intact, but the normal increase in GLUT-4 intrinsic activity in response to insulin or exercise is blunted compared with animals made obese by high-carbohydrate feeding (124). This accounted for the decrease in glucose transport in plasma membrane vesicles from the muscles of rats on a modestly high-fat diet (124). In vitro evidence for altered intrinsic activity is seen in the effect of sodium oleate to increase basal glucose uptake in isolated adipocytes without changing GLUT-4 or GLUT-1 levels or subcellular distribution (125). High-fat feeding results in alterations in membrane lipid composition, which could affect transporter translocation, docking and fusion with the plasma membrane, or activation. The change in lipid composition is also hypothesized to alter membrane phosphoinositide composition, which could alter the signal transduction mediated by phosphoinositol derivatives (122). A study has since shown that high-fat feeding induces a defect in GLUT-4 recruitment to the plasma membrane in soleus muscle that is associated with an impaired activation of phosphoinositide-3 kinase activity by insulin (126), and more recent work has suggested that the defect affects activation of Akt and atypical protein kinase C (λ/ζ) activity (127).

An important mechanism that may link diet-induced obesity with impaired glucose uptake in skeletal muscle is increased delivery of free fatty acids (FFAs) to muscle. Increased FFA delivery to muscle leads to impaired insulin-stimulated glucose transport that occurs on the basis of decreased insulin-stimulated IRS-1–associated PI 3-kinase activation (128,129). This is associated with increased activation of protein kinase C θ (PKC-θ) that is believed to occur as a result of FFA-induced accumulation of diacylglycerol (130). PKC-θ in turn leads to

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phosphorylation of IRS-1 on Ser307 (131), leading to impaired insulin signal transduction to PI 3-kinase, Akt, and atypical PKCs (λ/ζ) (132).

Not all models of diet-induced obesity are associated with insulin resistance or alteration in GLUT-4 expression or translocation. For example, obesity induced by overfeeding a mixture of carbohydrate, fat, and protein results in increased fat mass, normal insulin concentrations, and normoglycemia (123). Basal glucose transport is elevated in adipocytes from these animals, but the maximal rate of transport stimulated by insulin is unaltered. Compared with chow-fed lean control rats, GLUT-4 protein levels are unchanged in fat or skeletal muscle (123), and transporter translocation and activation by insulin and exercise remain intact (124).

Hyperinsulinemia Caused by Insulin Infusion

Insulin infusion in the rat results in hyperinsulinemia and hypoglycemia and is associated with insulin resistance in several muscle groups, although insulin responsiveness in fat may actually be increased (133,134). GLUT-4 protein levels are elevated in adipocytes in parallel with the hyperresponsiveness of this tissue. In humans, short-term insulin infusion causes a significant increase in GLUT-4 mRNA levels in skeletal muscle (135), but a decrease in GLUT-4 protein (57). Six hours of euglycemic hyperinsulinemia failed to increase GLUT-4 protein in skeletal muscle and adipose tissue in goats (136). The effect in fat in vivo contrasts with the effect of insulin in vitro in 3T3-L1 adipocytes, where downregulation of GLUT-4 expression is seen (137). The link between GLUT-4 levels and insulin resistance in skeletal muscle is unclear. GLUT-4 protein content is elevated in soleus muscle of insulin-infused hypoglycemic Sprague-Dawley rats (138) but is unchanged in diaphragm and lowered in tibialis muscles of insulin-infused, normoglycemic lean Zucker rats (139). These discrepancies may reflect differences in the fiber types of the muscles studied or a difference in the genetic background of the animals. Theoretically, hypoglycemia or the counterregulatory hormone response elicited by the hypoglycemia could affect GLUT-4 expression in muscle of the Sprague-Dawley rat model above, but these data were not provided.

Hyperglycemia Caused by Glucose Infusion

Glucose infusion induces a state of insulin resistance in humans (10) and a state of hyperinsulinemia and hyperglycemia in rats. Glucose infusion does not change GLUT-4 protein content in rat muscle despite an increase in GLUT-4 mRNA (140). This suggests that GLUT-4 levels are subject to posttranscriptional or translational regulation. The insulin-resistant glucose transport in muscle observed in this model is probably mediated through changes in GLUT-4 translocation or intrinsic activity.

Glucocorticoid Excess

Many genetic rodent models of obesity and insulin resistance exhibit elevated levels of glucocorticoids (86,87,95). The treatment of normal animals with pharmacologic doses of glucocorticoids can also induce hyperinsulinemia and hyperglycemia (141,142). Levels of GLUT-4 are not changed in skeletal muscle of dexamethasone-treated animals, however, indicating that in this model alterations in muscle GLUT-4 content are not the primary mechanism for insulin resistance (141). Incubation of isolated adipocytes with dexamethasone results in rapid redistribution of glucose transporters from the plasma membrane to the intracellular pool (143). Such a translocation defect may be involved in dexamethasone-induced insulin resistance in vivo. One report indicates that glucocorticoid treatment lowers tyrosine phosphorylation of the insulin receptor and its substrate, IRS-1 (142). However, the link between this defect in substrate phosphorylation and resistance to insulin-stimulated glucose disposal is not known.

Growth Hormone Excess

Chronic exposure to elevated concentrations of growth hormone in vivo results in insulin resistance, manifested by marked hyperinsulinemia and mild hyperglycemia (144). Adipocytes chronically exposed to growth hormone in vitro have lowered capacity to transport glucose (145). This is caused by downregulation of GLUT-1 (145). In a transgenic model of growth hormone overexpression, GLUT-4 expression was perturbed, with GLUT-4 mRNA being decreased in skeletal muscle, whereas in fat, GLUT-4 mRNA was significantly increased (144). GLUT-4 protein content was not investigated in this study, so the functional significance of these findings remains in doubt, especially because GLUT-4 mRNA and protein abundance have been shown to change in opposite directions in other insulin-resistant states (141,146).

The importance of normal growth hormone concentrations in the regulation of glucose transporter number in the plasma membrane is suggested by experiments showing that restoration of growth hormone concentrations, but not insulin, IGF-1, or IGF-2 concentrations, could reverse the increase in basal glucose transport seen in isolated adipocytes from hypophysectomized mice (147).

Animal Models of Aging

Aging is associated with mild insulin resistance and hyperinsulinemia (148). Although GLUT-4 protein levels are greatly decreased in both muscle and fat of older rats, the mechanisms for this decrease differ in the two tissues. In fat, the decline in GLUT-4 protein parallels a decrease in mRNA, whereas in muscle, the decline in protein is associated with increasing mRNA, implying decreased translational efficiency or protein stability (146). The decrease in GLUT-4 content in skeletal muscle is fiber type specific, with decreases reported in the heart (149) and epitrochlearis (150) but not in the flexor digitorum brevis (150). The decrement in muscle glucose transport has also been shown to be greater than can be accounted for by changes in GLUT-4 content (150). In adipocytes of older rats, subcellular distribution and translocation of GLUT-4 are intact, suggesting that the insulin resistance can be accounted for

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by the decreased amounts of the transporter in this tissue (151). Aging is not associated with any changes in GLUT-1 abundance in adipocytes. There is a redistribution of the GLUT-1 transporter, with increased abundance in the plasma membrane in the basal state (151). GLUT-1 levels in the plasma membrane are not increased further by insulin treatment, suggesting additional dysregulation in GLUT-1 transporter subcellular trafficking (151,152).

Hypoinsulinemic Models

Experimentally Induced Diabetes

Pancreatectomy (153) or chemical destruction of the pancreatic β-cells with streptozotocin (154) results in hypoinsulinemia, hyperglycemia, and secondary insulin resistance caused by a glucose transport defect (153). In vivo glucose uptake is impaired within 24 hours as a result of impaired whole-body glycolysis. This is followed by impaired suppression of hepatic glucose output by day 3 and impaired insulin-stimulated 2 deoxy-glucose (2DG) uptake into muscle by day 7 (155). GLUT-4 levels in white and brown adipose tissue are markedly downregulated within 48 hours (154). In skeletal muscle, however, impaired glucose transport precedes significant alterations in muscle GLUT-4 content (155). Furthermore, the degree of downregulation of GLUT-4 is quantitatively much greater in fat (more than fivefold by 3 days) (156) versus a 20% decrease in muscle GLUT-4 content by 14 days (Fig. 62.3) (155). Within muscle groups there are differences in the degree of downregulation of GLUT-4, with concentrations decreasing earliest in heart, then in red muscle, and finally in white muscle (157). The decrease in muscle GLUT-4 content does not necessarily correspond to transport measurements, which may actually increase in white muscle (vastus lateralis) despite diminished GLUT-4 content (158) (Table 62.3). Fiber-specific differences in GLUT-4 content may reflect in part fiber-specific differences in GLUT-4 transcription (159). After 7 days of streptozotocin diabetes, GLUT-4 transcription is reduced by 35% and mRNA levels by 50% in red muscle, with no decrease in gene transcription or mRNA levels in white muscle (159). Although decreased transporter number is likely to play an important role in the maintenance of the insulin-resistant state, the initial defect probably involves impaired exposure of plasma membrane transporters to the extracellular milieu or decreased intrinsic activity.

Figure 62.3. Time courses of changes in glucose transporter 4 (GLUT-4) protein content (d) and insulin-stimulated glucose uptake in vivo (s) in soleus, epitrochlearis, and extensor digitorum longus (EDL) muscles 0 to 14 days after streptozotocin (STZ) injection. Each point represents means ± SE for six to eight experiments, expressed as a percentage of the average value of the control. There is no significant change in soleus muscle GLUT-4 content at 14 days (p = 0.56 by analysis of variance). At 7 days GLUT-4 protein levels are not reduced in any of the three muscles, but at 14 days GLUT-4 protein levels are reduced by 20% and 30% in the epitrochlearis and EDL, respectively (p < 0.05). On days 7 and 14, in vivo glucose uptake is reduced by 37% ± 6% and 63% ± 4%, respectively, in soleus (p < 0.01) and by 41% ± 5% and 51% ± 4% in epitrochlearis. In EDL, glucose uptake is decreased by 58% ± 3% at 14 days. There is no correlation between GLUT-4 protein content and maximal in vivo glucose uptake in soleus and EDL. In epitrochlearis a weak relationship (r² = 0.24, p < 0.01) exists between GLUT-4 content and glucose uptake. (Reproduced from

Youn J, Kim J, Buchanan T. Time courses of changes in hepatic and skeletal muscle insulin action and GLUT4 protein in skeletal muscle after STZ injection. Diabetes 1994;43:546

, with permission.)

In streptozotocin-induced diabetic rats, normalization of blood sugar with insulin treatment is associated with rapid restoration of insulin responsiveness and glucose transporter levels in fat and muscle (154,160). Normalizing blood sugar levels with phlorizin, however, which blocks renal tubular reabsorption of glucose, thus enhancing glucose excretion, restores insulin responsiveness in isolated adipocytes without increasing GLUT-4 concentrations in the plasma membrane (153). This suggests that alterations in blood glucose concentrations affect glucose transporter intrinsic activity. Interesting clues to signaling mechanisms that may be involved in the divergent effects of

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insulin- and phlorizin-induced restoration of insulin responsiveness are provided by studies of the 185-kd IRS-1 (161) and GLUT-4 phosphorylation (160) in streptozotocin-induced diabetic rats. In muscle of streptozotocin-induced diabetic rats, insulin-stimulated phosphorylation of IRS-1 was impaired. This impairment was corrected by restoration of glucose concentrations with insulin treatment, which increased intrinsic activity and restored glucose transporter expression, but not when glucose concentrations were restored by phlorizin treatment, which only affects glucose transporter intrinsic activity. This implies that IRS-1 could be involved in signaling of glucose transporter transcription and translation but that it may not be associated with alterations in intrinsic activity (161). Increased phosphorylation of the GLUT-4 transporter is seen in diabetic animals and is associated with diminished cytosolic phosphoserine phosphatase activity. Insulin treatment restores transporter phosphorylation to normal, but phlorizin does not, implying that phosphorylation is not involved in regulation of the intrinsic activity of the transporter (160).

Table 62.3. Discordance between glucose transport and GLUT-4 and GLUT-1 content in specific muscles after 12 to 16 weeks of streptozotocin diabetes mellitus

Muscle	Fiber type	GLUT-4	GLUT-1	In vivo glucose uptake
Soleus	I	↓30%	(↔)	↓ 60%
Plantaris and red gastrocnemius	IIa + IIb	↓45%	(↔)	↔
White gastrocnemius	IIb	↓55%	↔	↑ 100%
Diaphragm	Ia + IIa + IIb	↓40%	↔	↓(60%)
Heart		↓70%	↓60%	↓ 75%
Adapted from Kainulainen H, Breiner M, Schurmann A, et al. In vivo glucose uptake and glucose transporter proteins GLUT1 and GLUT4 in heart and various types of skeletal muscle from streptozotocin diabetic rats. Biochim Biophys Acta 1994;1225:275–282, with permission.

Fasting

Fasting produces hypoinsulinemia and insulin resistance in fat (162). Insulin-resistant glucose uptake into muscle in vivo is seen in anesthetized fasted rats but is milder (L. Rossetti, personal communication) in chronically catheterized, awake rats. In brown and white adipose tissue, GLUT-4 protein levels are significantly reduced, although GLUT-1 levels are unchanged (157,163). In skeletal muscle, however, fasting results in a paradoxic increase in the abundance of both GLUT-1 and GLUT-4 despite insulin-resistant glucose uptake in vivo in the same model (138,157,163). In adipocytes, fewer transporters are recruited to the plasma membrane as a result of the depleted intracellular pool (163). In muscle, however, the translocation is actually increased (164). Refeeding results in normalization of GLUT-4 content in skeletal muscle and increases GLUT-4 levels in fat above control (163). There is a marked increase in glucose utilization by brown adipose tissue and muscle on refeeding, and these changes precede any change in GLUT-4 mRNA (165). These data therefore indicate that the in vivo insulin resistance associated with fasting results largely from changes in the activation, fusion state, or intrinsic activity of the transporter.

Alteration of Glucose Transporters in Transgenic Models

Transgenic mice overexpressing glucose transporters have been created using either the native promoter or a heterologous promoter to target the expression to a single tissue. Transgenic mice expressing the 5′ untranslated region of the human GLUT-4 gene coupled to a reporter gene show expression limited to muscle and fat, identical to that of the endogenous mouse GLUT-4 gene (166,167). The regulatory elements that confer this tissue specificity, as well as hormonal and metabolic regulation of the transgene, reside within 895 bp of the transcription start site (44,168,169). Further analysis has identified a novel binding protein that binds the GLUT-4 promoter between –742 and –712, a MEF2(c) binding site between –473 and –464, and a Kruppel-like factor (KLF15) that binds the GLUT-4 promoter just proximal to and interacts with MEF2(c) as being necessary to confer tissue-specific expression of GLUT-4 (46,170). Overexpression of the GLUT-4 gene product driven by its homologous promoter results in increased systemic glucose clearance and insulin-mediated glucose utilization in muscle in vivo. There is a marked increase in compensatory lipid mobilization and muscle glycogenolysis to maintain substrate availability in order to counteract fasting hypoglycemia (171,172,173). GLUT-4 has also been overexpressed specifically in muscle using muscle-specific promoters, which also results in improved whole-body glucose clearance (174,175). Overexpression of the human GLUT-4 gene selectively in fat using the aP2 promoter results in enhanced in vivo glucose disposal and hyperplastic obesity (176). Basal and insulin-stimulated glucose transport are increased in isolated adipocytes from these transgenic mice (176,177). This results in enhanced whole-body glucose tolerance (177,178). This model demonstrates that the number of GLUT-4 transporters is rate limiting for glucose transport by adipose tissue and that increasing glucose transport into fat can enhance whole-body glucose disposal.

Transgenic mice have also been used to study the link between GLUT-4 levels in muscle and fat and insulin resistance.

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Overexpression of GLUT-4 in muscle and fat initially alleviates the insulin resistance and markedly enhances glycemic control in C57BL/KsJ db/db mice (179). However, in older mice glucose intolerance returns despite elevated skeletal muscle glucose disposal and persistently elevated GLUT-4 levels (93). The mechanism for the attenuation in the ability of overexpressed GLUT-4 to maintain normal glucose homeostasis in the C57BL/KsJ db/db mice may be related to the development of progressive pancreatic atrophy or to progressive hepatic insulin resistance. Transgenic mice overexpressing GLUT-4 in muscle and fat do not develop insulin resistance when fed a high-fat diet (180). While overexpression of GLUT-4 specifically in fat can ameliorate insulin resistance associated with insulin deficiency (181), these mice still become insulin resistant when challenged with a high-fat diet (178). Mice overexpressing either glutamine:fructose-6-phosphate amidotransferase (GFAT) in muscle and adipose tissue (182) or phosphoenolpyruvate carboxykinase in liver (183) both become insulin resistant. GFAT transgenic mice initially exhibit impaired GLUT-4 translocation (184), and both ultimately show a secondary reduction in levels of GLUT-4 in muscle. Overall these findings suggest that increasing GLUT-4 levels, particularly in muscle, may be a strategy to overcome insulin resistance, but may need to be combined with strategies that improve hepatic insulin sensitivity and preserve/maintain islet cell mass.

Mice with genetic ablation of the GLUT-4 gene exhibit surprisingly normal ambient blood glucose concentrations despite glucose intolerance and significant insulin resistance (185). They are growth retarded and exhibit a diminished life span. They develop cardiac hypertrophy, cardiac dysfunction, and severely reduced adipose tissue. This model suggests that GLUT-4 is essential for normal cardiac function. Cardiac selective deletion of GLUT-4 also results in cardiac hypertrophy, but with preserved cardiac function in the absence of hypoxia or ischemia (186). Moreover, these mice have a normal lifespan. Thus, the cardiac dysfunction in GLUT-4 null mice might reflect the combined effects of GLUT-4 deficiency and changes in the delivery of other important cardiac metabolic substrates such as FFAs and lactate that are reduced in these mice. Whether the decrease in adipocyte mass in GLUT-4 null mice occurs as a direct result of loss of GLUT-4 in fat or is secondary either to increased lipolysis or to the effect of chronic illness in these mice is unclear (185). However, adipose selective deletion of GLUT-4 (G4A–/–) does not affect adipose mass (12). Interestingly, adipose GLUT-4 –/– mice develop insulin resistance secondarily in muscle and liver in vivo and become glucose intolerant. This is particularly relevant in light of the consistent observation of reduced adipocyte GLUT-4 content in humans and animals with insulin resistance and diabetes. Thus, the reduction of GLUT-4 expression seen in adipose tissue in obesity and diabetes may play an important role in the pathogenesis of insulin resistance in these states. The mechanism for insulin resistance in muscle and liver of G4A–/– mice appears to be the release of an unidentified adipocyte-secreted factor (187). Interestingly, mice that are heterozygous (+/-) for the GLUT-4 gene deletion express reduced levels of GLUT-4, and 50% to 60% of them become diabetic, providing strong evidence that reduced levels of GLUT-4 can contribute to the pathogenesis of this disease (188). Moreover, diabetes is averted when expression of GLUT-4 is restored specifically in muscle of GLUT-4^+/- mice by crossing these with mice overexpressing GLUT-4 in muscle (175). Similarly, mice with skeletal muscle deletion of GLUT-4 develop glucose intolerance and hyperinsulinemia, caused not only by diminished skeletal muscle glucose uptake, but by the development of hepatic and adipose tissue insulin resistance due, at least in part, to glucose toxicity (189,190).

Overexpression of the human GLUT-1 transporter in skeletal muscle using the myosin light-chain promoter is associated with increased basal glucose transport and muscle glycogen synthesis in vitro, as well as enhanced glucose tolerance (191). Insulin-mediated glucose uptake in vivo is impaired, however, as is the ability of insulin, IGF-1, hypoxia, and muscle contraction to increase glucose transport into skeletal muscle in vitro (192). Photolabeling studies show that impaired GLUT-4– mediated glucose uptake is not due to impaired GLUT-4 translocation in this model (193). Thus, increased basal glucose flux can inhibit the intrinsic activity of GLUT-4 via mechanisms that remain to be elucidated.

Transgenic mice with deletion of proteins in the GLUT-4 vesicle or molecules involved in vesicle docking and fusion have provided additional insights into the regulation of GLUT-4 expression and GLUT-4 translocation in vivo. Mice with targeted ablation of IRAP develop mild insulin resistance that is associated with decreased expression of GLUT-4 (by 45%–85%) in muscle and adipose tissue. Subcellular distribution and translocation to the plasma membrane of this diminished GLUT-4 pool in insulin–stimulated adipocytes is normal (194). The mechanism for the reduction in GLUT-4 expression remains to be elucidated. Mice with a heterozygous null mutation in the t-SNARE protein syntaxin 4 develop impaired glucose tolerance and 50% reduction in skeletal muscle glucose uptake in vivo and in vitro that is associated with reduced insulin stimulated GLUT-4 translocation. Intriguingly, GLUT-4 translocation is normal in adipocytes (195). These data support the critical role for syntaxin 4 in GLUT-4 vesicle docking in vivo and suggest that tissue-specific differences exist in the mechanisms that regulate GLUT-4 translocation in adipose tissue versus skeletal muscle.

Other Models

Exercise-Induced Alterations in Insulin Sensitivity

Muscle contraction and insulin appear to stimulate GLUT-4 translocation by different mechanisms (196), and some data suggest that these two stimuli mobilize distinct pools of GLUT-4–containing vesicles (197). It has recently been demonstrated that GLUT-4 translocation still occurs in response to exercise in muscle from insulin-resistant subjects while insulin-mediated translocation is impaired (61). This raises the prospect that the signaling pathways used by exercise to mediate GLUT-4 translocation might be good targets for drugs designed to treat insulin resistance. Until recently, little was known about the nature of the exercise-induced signaling pathways, but there is growing evidence that the 5′-AMP kinase is involved (63).

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In addition to acute effects of muscle contraction on glucose transporter translocation and intrinsic activity, chronic exercise training increases insulin sensitivity in animals (198). The beneficial effects of chronic exercise training are associated with significant increases in GLUT-4 levels in muscle and fat in normal (198) and insulin-resistant rats (199) that are more pronounced in younger animals (200). Exercise training also increases GLUT-4 levels in muscle of aging humans (201). The elevated GLUT-4 levels in skeletal muscle persist for several days after cessation of training, even after glucose transport rates return to baseline (198,202). There are fiber-specific differences in GLUT-4 content, as well as insulin-stimulated and contraction-mediated increases in glucose transport (199,202). This is exemplified by studies in endurance-trained Zucker rats in which the increase in GLUT-4 content in the plantaris muscle with exercise corresponds with increased insulin- and contraction-stimulated glucose transport. In contrast, in soleus muscle, an increase in GLUT-4 content is associated with an increase in contraction-stimulated glucose transport, whereas insulin-stimulated glucose transport remains unchanged from baseline (199). In skeletal muscle, chronic exercise training produces a marked increase in GLUT-4 protein in the plasma membrane in the basal state and after insulin stimulation (203). The increase in GLUT-4 content in skeletal muscle is associated with increased hexokinase II activity and glycogen synthesis (203). Intrinsic activity is unchanged (199), in contrast to the situation of white adipocytes, in which intrinsic activity might also be increased by exercise (204). Thus, the changes in glucose clearance rates seen with chronic exercise training are at least partially explained by the increased numbers of transporters in the plasma membrane. The presence of additional factors is suggested by the reduction of transport with cessation of training despite persistent elevations of GLUT-4. Chronic administration of AICAR recapitulates many of the changes in GLUT-4 gene expression, glucose uptake, and insulin signaling in muscle that is observed following exercise training (44,96,205), indicating that activation of AMP kinase plays an important role in the adaptive response of skeletal muscle to exercise training.

Physical Inactivity and Denervation

Physical inactivity in rats results in in vivo insulin resistance (206,207), which may be more severe than that observed after streptozocin diabetes (208). Although insulin-mediated glucose disposal into muscle falls within 48 hours of immobilization (206), GLUT-4 expression is only reduced after much longer time points, indicating it is not the cause of the insulin resistance (206,207). Recent studies have shown a reduction in insulin-stimulated Akt1 activity within 24 hours of denervation, which might represent a mechanism for the early insulin resistance that is observed (209).

Denervation of muscle results in insulin resistance that is dependent on muscle fiber type and is not associated with defects in activation of the insulin receptor (210). GLUT-4 protein levels are progressively downregulated in muscle after denervation. This downregulation can be reversed with electrical stimulation in the absence of contraction (204,211). The decline in GLUT-4 levels is not observed when muscle activity is blocked by treating the sciatic nerve with tetrodotoxin, indicating that GLUT-4 expression can be maintained by nerve-derived factors (212) such as neuregulin (213). As is also seen in streptozotocin diabetes, however, insulin resistance develops before the decline in GLUT-4 content (204), indicating that the decrease in GLUT-4 content alone cannot explain the onset of the insulin-resistant state.

Humans with tetraplegia have a form of denervation-induced insulin resistance, and this can be reversed by electrical stimulation of the leg muscles (214). Levels of GLUT-4 in limb muscles are low in tetraplegic subjects, but this markedly increases with electrical stimulation, suggesting that the increased GLUT-4 levels may contribute to improved insulin sensitivity (214).

Oral Antidiabetic Agents

A number of established and potential therapeutic agents act in part by improving insulin sensitivity. The possibility that these effects are mediated through changes in GLUT-4 concentration or function has been investigated. The sulphonylurea glimepiride increases GLUT-4 translocation and decreases its phosphorylation in cultured rat adipocytes (215). The biguanide metformin reverses the downregulation of GLUT-4 translocation to the plasma membrane, as observed in adipocytes cultured in the presence of insulin (216). In vivo administration of metformin to fa/fa Zucker rats is associated with increased insulin-stimulated glucose transport in adipocytes, increased translocation of GLUT-4 and GLUT-1 to the plasma membrane, and increased intrinsic activity of the transporters (217). In the same model, metformin treatment improved in vivo glucose transport without altering GLUT-4 abundance in muscle (218). Application of metformin to cultured L6 myocytes in vivo increased glucose transport without changing GLUT-4 abundance or translocation. The increased transport was accounted for by increased translocation of GLUT-1 to the plasma membrane of metformin-treated cells (219). Metformin potentiated insulin-stimulated glucose uptake into human muscle strips (220) and increased whole-body glucose uptake in NIDDM. Recent studies indicate that the mechanism of action of metformin might be activation of AMP kinase (221). Studies in cultured muscle cells (222) and in muscle biopsy samples obtained from metformin-treated type 2 diabetics (223) show that metformin treatment leads to phosphorylation of threonine 172 within the catalytic (α) subunit of AMP kinase. The mechanisms for this effect remain to be completely elucidated. Metformin does not appear to increase insulin signaling via the PI 3-kinase pathway (224).

Thiazolidinediones (TZDs) are insulin-sensitizing agents that are agonists for peroxisome proliferator–activated receptor-γ (PPARγ) nuclear receptors (225,226), which increase peripheral glucose disposal in insulin-resistant subjects (227) and in animal models of insulin resistance (225,226). Treatment of insulin-resistant rodents with TZDs restores expression and translocation of GLUT-4 in adipocytes from insulin-resistant animals (113,228). TZDs also overcome the TNF-α–induced

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inhibition of insulin-stimulated glucose transport in adipocytes (229). In vivo administration of pioglitazone reverses dexamethasone-induced insulin resistance in rats without changing GLUT-4 levels. TZD treatment of insulin-resistant rats fed high-fat diets and insulinopenic streptozotocin diabetic rats enhances insulin-stimulated glucose uptake in muscle in vivo (113,230). NMR studies in diabetic humans and insulin-resistant rodents have shown that a major mechanism by which TZDs improve muscle insulin sensitivity is by correcting defective glucose transport (231,232). Moreover, TZDs increased insulin-stimulated PI 3-kinase and Akt activity in skeletal muscle and adipocytes from treated type 2 diabetics (224,233) and increased insulin-stimulated activation of atypical PKCs (λ/ζ) in adipocytes and skeletal muscle from insulin-resistant GK rats (110). TZDs also increase GLUT-4 translocation in L6 myocytes and transfected rat adipocytes independently of insulin (234,235), and have recently been shown to increase AMP kinase activity in muscle cells (236). Rosiglitazone reversed the dysregulated overexpression of v- and t-SNARE proteins (cellubrevin, VAMP-2, and syntaxin 4) in skeletal muscle of insulin-resistant Zucker fatty rats (237). Thus, TZDs act to increase glucose uptake by altering multiple signaling pathways that act in concert to increase GLUT-4 translocation (184). Although increased GLUT-4 expression in adipocytes has been observed (233), the effect of TZDs on GLUT-4 expression in muscle is minimal (113,230,238), but an effect on GLUT-1 expression has been observed (239).

Vanadate, a phosphotyrosine phosphatase inhibitor that increases proximal insulin signaling to PI 3-kinase (84), has potent hypoglycemic effects in vivo, and several organo-vanadium compounds are currently being evaluated as potential therapies for insulin resistance (240). Vanadate induces GLUT-4 translocation in rat adipocytes in vitro (241) and increases GLUT-4 intrinsic activity in sarcolemmal vesicles (29). In vivo vanadate increases glucose utilization in the muscle of fa/fa rats without changing GLUT-4 expression (81).

Thyroid Hormone

Thyroid hormone plays an important role in the upregulation of GLUT-4 and the downregulation of GLUT-1, which occur shortly after birth (242). Treatment of normal rats with thyroid hormone is associated with increased GLUT-4 content and increased basal and insulin-stimulated glucose uptake in skeletal muscle (243). GLUT-4 levels in the plasma membrane of both muscle and adipocytes from hyperthyroid rats are greatly elevated in the basal state but are normal in the insulin-stimulated state, indicating a dysregulation in GLUT-4 distribution (244,245). However, incubation of 3T3-L1 adipocytes with thyroid hormone increased GLUT-4 levels in the plasma membrane in the absence of insulin, which were further increased following insulin administration (246). Conversely, hypothyroid rats show reduced GLUT-4 content in muscle. Hypothyroidism is associated with increased GLUT-1 content in rat heart (247). GLUT-4 levels in heart, however, are not altered by either hypothyroidism or hyperthyroidism despite increased cardiac glucose uptake in the hyperthyroid animals (247).

Angiotensin-Converting Enzyme Inhibitors

Large randomized clinical trials have shown that long-term therapy with angiotensin-converting enzyme (ACE) inhibitors reduces the incidence of type 2 diabetes (248). Many studies in humans and animals with insulin resistance or hypertension have demonstrated that ACE inhibition is associated with enhanced whole-body glucose uptake (249,250). Indeed, angiotensin II (ATII) had been shown to antagonize insulin signaling in cardiac muscle (251). However, there is evidence that ACE inhibition will increase glucose transport independently of increasing insulin signaling. Acute or chronic administration of ACE inhibitors to obese Zucker rats (250) and to diabetic KK-Ay mice (252) improved insulin-stimulated glucose uptake in muscle. In the KK-Ay mouse studies, ACE inhibition increased plasma membrane GLUT-4 levels in the basal state to levels observed in insulin-treated control muscle. Furthermore, the effect of ACE inhibition and insulin on GLUT-4 translocation was additive. The increase in GLUT-4 translocation under basal conditions was not associated with any increase in insulin signaling. A similar effect of ACE inhibition to induce GLUT-4 translocation was also observed in L6 muscle cells. The effect is mediated in part by increased bradykinin levels induced by ACE inhibition that in turn increases nitric oxide production by stimulating nitric oxide synthase. A direct role for inhibition of the type 1 ATII receptor as an additional mechanism has been demonstrated in similar studies performed using selective ATII receptor antagonists. In obese Zucker rats, acute ATII receptor blockade increased whole-body insulin sensitivity and insulin-mediated glucose uptake in type I fibers (soleus muscle) but not in type IIb predominant epitrochlearis muscle. Moreover, chronic treatment resulted in additional increases in insulin sensitivity that was associated with increased insulin-mediated glucose uptake in both type I and type II muscles. GLUT-4 protein content was increased in plantaris, soleus, and heart (253). The increase in GLUT-4 in the heart occurs via posttranscriptional mechanisms (254). Similar studies in the OLETF rat showed that the increased muscle glucose uptake was not associated with any increase in insulin-stimulated PI 3-kinase activation (255). The effect of ACE or ATII receptor inhibition to augment glucose transport is seen only in insulin-resistant animals but not in animals with normal insulin sensitivity (256).

Endothelin

The 21–amino acid peptide endothelin (ET-1), which plays an important role in cardiovascular development and homeostasis, was recently shown to stimulate GLUT-4 translocation in 3T3-L1 adipocytes and neonatal cardiomyocytes by activating its cognate receptor (257,258,259). Endothelin also chronically regulates GLUT-1 expression (260). The signaling pathway by which ET-1 acutely stimulates GLUT-4 translocation is distinct from that of insulin-stimulated GLUT-4 translocation involving Gα(q/11), the Ca²⁺ activated tyrosine kinase PYK2, and the small G protein ADP-ribosylation factor 6 (ARF6) (261,262,263). Thus, insulin and endothelin are likely to stimulate functionally

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distinct pools of GLUT-4. Chronic endothelin treatment of 3T3-L1 adipocytes leads to desensitization of insulin-stimulated GLUT-4 translocation by leading to degradation of IRS-1 (264). Endothelin levels have been shown to be elevated in some patients with type 2 diabetes, particularly those with vascular complications (265). However, it remains to be established whether or not endothelin regulates glucose uptake in vivo.

Human Immunodeficiency Virus Protease Inhibitors

Human immunodeficiency virus (HIV) protease inhibitors (HPI) are potent antiretroviral agents used in the management of HIV infection. Use of these agents has been associated with the development of a metabolic syndrome characterized by insulin resistance, lipodystrophy, and dyslipidemia. Administration of the HPI indinavir to normal humans and rats acutely inhibits insulin-stimulated glucose disposal in skeletal muscle (266,267). In isolated skeletal muscle preparations, indinavir inhibited GLUT-4 translocation without altering insulin signaling (268). Studies in adipocytes and in transfected oocytes also suggest that HPIs are selective inhibitors of GLUT-4 but not GLUT-1 intrinsic activity (269,270). Given the pleiotropic effects of HPIs, it is probable that additional mechanisms will be responsible for the full metabolic phenotype observed clinically (271).

Cold Exposure

Cold exposure increases metabolic activity and glucose transport rate acutely in brown fat without changing GLUT-4 levels. With chronic exposure, GLUT-4 protein levels increase in brown fat, although a similar increase is not seen in muscle. This effect is abolished by sympathectomy (272).

Tumor Necrosis Factor

The cytokine TNF-α has been recently proposed to play a role in the insulin resistance of obesity and NIDDM (273). Infusion of TNF-α causes insulin resistance in rats (274). TNF-α expression is elevated in the adipose tissue of multiple experimental models of obesity. Neutralization of TNF-α in one such model, the Zucker (fa/fa) rat, results in improved insulin sensitivity (273). TNF-α mediates insulin resistance at multiple levels and appears to act primarily as a potent inhibitor of the insulin-stimulated tyrosine phosphorylation on the β-subunit of the insulin receptor and of IRS-1. TNF-α has been shown to downregulate GLUT-4 mRNA levels in murine and human adipocyte and myocyte cultures (275,276,277). Insulin still induces GLUT-4 translocation in TNF-α–treated adipocytes, indicating that the primary defect lies with the reduced level of GLUT-4 expression (277). However, TNF-α does not reduce insulin-stimulated glucose transport in muscle or muscle cell cultures in vitro (278,279,280). Furthermore, muscle levels of GLUT-4 are not downregulated in animal models of obesity in which TNF-α expression is increased, and neutralization of TNF-α in these models has no effect on GLUT-4 mRNA expression despite improvements in insulin sensitivity. The role that TNF-α plays in insulin resistance in vivo is currently not clear because studies in transgenic animals in which the TNF-α gene ablated or both forms of the TNF-α receptor were ablated show conflicting results (281,282,283), and the administration of TNF-α–neutralizing monoclonal antibodies in type 2 diabetic patients failed to produce any improvement in insulin resistance (284).

Leptin

Leptin, the protein product of the obese gene (94), is secreted by adipocytes and is thought to act mainly at the hypothalamus as a signal to the brain, indicating energy stores (285). Leptin levels are elevated in obese humans, indicating a state of leptin resistance (286,287). However, leptin may also have direct actions in the periphery to enhance insulin sensitivity. Administration of leptin to either normal (288,289) or genetically obese and diabetic rodents (285) results in a rapid reduction in circulating glucose and insulin levels, and these changes occur before any changes in food intake or body weight occur. Whether this increased insulin sensitivity is due to increased glucose disposal into skeletal muscle and brown adipose tissue is controversial (289). Although some studies indicate that leptin may directly alter glucose transport in muscle and adipocytes (290,291,292) and enhance fatty acid oxidation in skeletal muscle (293), other studies show no direct effect on glucose transport or metabolism in these tissues (293,294,295,296). Therefore, the rapid effects of leptin administration on glucose uptake are most likely mediated through the brain and sympathetic nervous system (289) or through alterations in hepatic glucose flux (297). The long-term effects of hyperleptinemia on glucose metabolism and GLUT-4 expression are currently unknown.

Prenatal Environment

There is growing evidence that low birth weight may increase the risk for developing diabetes in later life. It is currently not clear whether this is a result of defects in pancreatic development or of fetally programmed predisposition to peripheral insulin resistance. Studies in animal models demonstrate that low birth weight caused by fetal malnutrition does not result in altered GLUT-4 levels in either adipocytes (298) or skeletal muscle (299). In insulin-resistant humans who experienced intrauterine growth retardation (IUGR), basal levels of GLUT-4 expression were also unchanged. However, the upregulation of GLUT-4 mRNA observed in insulin-sensitive controls after 3 hours of euglycemic hyperinsulinemia was absent in subjects with IUGR (300). Thus, although programmed insulin resistance caused by low birth weight might not be due to alterations in GLUT-4 expression per se, it remains to be established if abnormal regulation of GLUT-4 expression in response to environmental

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stimuli might contribute to increased insulin resistance in this population.

Summary

Alterations in insulin responsiveness in vivo result from changes in GLUT-4 gene expression, as well as changes in the function of the GLUT-4 protein. GLUT-4 expression undergoes tissue-specific regulation in insulin-resistant states; in many models, greater changes in glucose transporter gene expression are seen in adipose cells than in muscle. Both ambient insulin and ambient glucose concentrations affect GLUT-4, but in different ways. GLUT-4 protein levels are not altered in muscle or fat by glucose infusion or in fat by restoration of elevated glucose concentrations to normal in diabetic rats with phlorizin. In contrast, GLUT-4 content is depressed in both the muscle and fat of hypoinsulinemic animal models of diabetes, and GLUT-4 is restored to normal by insulin treatment. Whereas insulin treatment of normal animals increases GLUT-4 expression in fat, insulin-resistant animals with endogenous hyperinsulinemia and euglycemia do not show major alterations in GLUT-4 protein content in skeletal muscle, the most important site of insulin-stimulated glucose disposal. In contrast, in adipose tissue from these models, GLUT-4 content may be reduced or increased, and these alterations in GLUT-4 tend to parallel changes in insulin responsiveness in isolated adipocytes. The quantitative contribution of adipocyte glucose transport to in vivo glucose disposal is thought to be relatively small. However, downregulation of the GLUT-4 content in adipose tissue may alter other aspects of adipose tissue, including endocrine function, that affect insulin sensitivity. Furthermore, recent data in transgenic mice with overexpression or ablation of GLUT-4 exclusively in fat indicate that adipose tissue plays an important role in in vivo glucose homeostasis.

Defects in insulin-stimulated translocation of glucose transporters in muscle may play a role in some models of insulin resistance, such as the Zucker rat, but are unlikely to be the primary mechanism causing insulin resistance in all models. With high-fat feeding, multiple mechanisms for induction of insulin resistance have been demonstrated. In states such as fasting and streptozotocin diabetes, in vivo circulating or local factors appear to impair the action of GLUT-4 in muscle, either by interfering with membrane docking/fusion or by decreasing transporter intrinsic activity. Glucose transporter intrinsic activity can also be altered in adipose cells, for example, with phlorizin treatment of diabetic rats or refeeding of fasted rats.

Whereas glucose transporter levels are reduced in fat in many insulin-resistant models, glucose transporter levels in muscle are largely unaffected. Even in states in which transporter expression is reduced in muscle, this does not appear to be the primary lesion responsible for the onset of insulin resistance, because resistance develops before glucose transporter content decreases. Once glucose transporter levels are lowered, however, the reductions are likely to influence the progression and severity of the insulin-resistant state. Even though impairment of GLUT-4 expression may not be the primary lesion in most insulin-resistant states, increased GLUT-4 abundance in muscle during exercise training are associated with increased in vivo insulin responsiveness. Therefore, future approaches to overcoming insulin resistance may involve efforts to augment GLUT-4 expression in muscle, as well as attempts to modulate currently unknown factors involved in GLUT-4 translocation, fusion with the plasma membrane, and activation.

Acknowledgments

This work was supported by National Institutes of Health grants DK43051, DK56116, HL62886 and DK02495, and grants from the American Diabetes Association.

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	Diabetes Mellitus: A Fundamental and Clinical Text 3rd Edition
	© 2004 Lippincott Williams & Wilkins

Diabetes Mellitus: A Fundamental and Clinical Text3rd Edition

Diabetes Mellitus: A Fundamental and Clinical Text
3rd Edition