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CELLULAR MECHANISMS OF INSULIN RESISTANCE

The stimulation of glucose metabolism by insulin, requires that the hormone must first bind to specific receptors that are present on the cell surface of all insulin target tissues (1,274-277). After insulin has bound to and activated its receptor, "second messengers" are generated and these second messengers initiate a series of events involving a cascade of phosphorylation-dephosphorylation reactions (1,274-280) that eventually result in the stimulation of intracellular glucose metabolism. The initial step in glucose metabolism involves activation of the glucose transport system, leading to influx of glucose into insulin target tissues, primarily muscle (1,281,282). The free glucose, which has entered the cell, subsequently is metabolized by a series of enzymatic steps that are under the control of insulin. Of these, the most important are glucose phosphorylation (catalyzed by hexokinase), glycogen synthase (which controls glycogen synthesis), and phosphofructokinase (PFK) and PDH (which regulate glycolysis and glucose oxidation, respectively).

Insulin Receptor/Insulin Receptor Tyrosine Kinase

The insulin receptor is a glycoprotein consisting of two a-subunits and two ß-subunits linked by disulfide bonds (1,274-277) . The a-subunit of the insulin receptor is entirely extracellular and contains the insulin-binding domain. The ß-subunit has an extracellular domain, a transmembrane domain, and an intracellular domain that expresses insulin-stimulated kinase activity directed towards its own tyrosine residues (1,274-277). Insulin receptor phosphorylation of the ß-subunit, with subsequent activation of insulin receptor tyrosine kinase, represents the first step in the action of insulin on glucose metabolism (274-277). Mutagenesis experiments have shown that insulin receptors devoid of tyrosine kinase activity are completely ineffective in mediating insulin stimulation of cellular metabolism (283,284). Similarly, mutagenesis of any of the three major phosphorylation sites (at residues 1158, 1163, and 1162) impairs insulin receptor kinase activity, resulting in a decrease in the acute metabolic and growth promoting effects of insulin (283,285).

Insulin Receptor Signal Transduction

Following activation, insulin receptor tyrosine kinase phosphorylates specific intracellular proteins, of which at least nine have been identified (282). Four of these belong to the family of insulin-receptor substrate proteins: IRS-1, IRS-2, IRS-3, IRS-4 (the others include Shc, Cbl, Gab-1, p60dok, and APS). In muscle IRS-1 serves as the major docking protein that interacts with the insulin receptor tyrosine kinase and undergoes tyrosine phosphorylation in regions containing amino acid sequence motifs (YXXM or YMXM) that, when phosphorylated, serve as recognition sites for proteins containing src-homology 2 (SH2) domains (where y = tyrosine, M = methionine, and x - any amino acid) (274,275). Mutation of these specific tyrosines severely impairs the ability of insulin to stimulate glycogen and DNA synthesis, establishing the important role of IRS-1 in insulin signal transduction (282). In liver, IRS-2 serves as the primary docking protein that undergoes tyrosine phosphorylation and mediates the effect of insulin on hepatic glucose production, gluconeogenesis and glycogen formation (287). In adipoctes, Cbl represents another substrate which is phosphorylated following its interaction with the insulin receptor tyrosine kinase and which is required for stimulation of GLUT 4 transloctaion. Phosphorylation of Cbl occurs when the CAP/Cbl complex associates with flotillin in caveolae, or lipid rafts, containing insulin receptors (288,289).

In muscle, the phosphorylated tyrosine residues on IRS-1 mediate an association between the two SH2 domains of the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase), leading to activation of the enzyme (274-284,290,291) . PI3-kinase is a heterodimeric enzyme comprised of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. The latter catalyzes the 3-prime phosphorylation of phosphatidylinositol (PI), PI-4-phosphate, and PI-4,5-diphosphate, resulting in the stimulation of glucose transport (274-277). Activation of PI3-kinase by phosphorylated IRS-1 also leads to activation of glycogen synthase (274,275), via a process that involves activation of PKB/Akt and subsequent inhibition of kinases such as GSK-3 (292) and activation of protein phosphatase 1 (PP1) (293). Inhibitors of PI3-kinase impair glucose transport (274-277,294) by interfering with the translocation of GLUT 4 transporters from their intracellular location (281,282) and block the activation of glycogen synthase (295) and hexokinase (HK)-II expression (296). The action of insulin to increase protein synthesis and inhibit protein degradation also is mediated by PI-3 kinase and involves the activation of mTOR (297,298). mTOR controls translation machinery by phosphorylation and activation of p70 ribosomal S6 kinase (p70rsk) (297) and phosphorylation of initiation factors (299). Insulin also promotes hepatic triglyceride synthesis via increasing the transcription factor steroid regulatory element-binding protein (SREBP)-1c (300) and this lipogenic effect of insulin also appears to be mediated via the PI3-kinase pathway (274).

Other proteins with SH2 domains, including the adapter protein Grb2 and Shc, also interact with IRS-1 and become phosphorylated following exposure to insulin (274-276,301). Grb2 and Shc serve to link IRS-1/IRS-2 to the mitogen-activated protein (MAP) signaling pathway , which plays an important role in the generation of transcription factors (274,275). Following the interaction between IRS-1/IRS-2 and Grb2 and Shc, Ras is activated, leading to the stepwise activation of Raf, MEK, and ERK. Activated ERK than translocates into the nucleus of the cell where it catalyzes the phosphorylation of transcription factors which promote cell growth, proliferation, and differentiation (274-276,301-303). Blockade of the MAP kinase pathway prevents the stimulation of cell growth by insulin but has no effect on the metabolic actions of the hormone (304-306).

Under anabolic conditions insulin stimulates glycogen synthesis by simultaneously activating glycogen synthase and inhibiting glycogen phosphorylase (307-309). The effect of insulin is mediated via the PI3 kinase pathway which inactivates kinases such as glycogen synthase kinase-3 and activates phosphatases, particularly protein phosphatase 1 (PP1). It is believed that PP1 is the primary regulator of glycogen metabolism (307-310). In skeletal muscle, PP1 associates with a specific glycogen-binding regulatory subunit, causing dephosphorylation (activation) of glycogen synthase. PP1 also phosphorylates (inactivates) glycogen phosphorylase. The precise steps that link insulin receptor tyrosine kinase/PI 3-kinase activation to stimulation of PP1 have yet to be defined. Some evidence suggests that p90 ribosomal S6-kinase may be involved in the activation of glycogen synthase (274). Akt also has been shown to phosphorylate and thus inactivate GSK-3 (292). This decreases glycogen synthase phosphorylation, leading to activation of the enzyme (292). A number of studies have convincingly demonstrated that inhibitors of PI3-kinase inhibit glycogen synthase activity and abolish glycogen synthesis (274,293,310). From the physiological standpoint, it makes sense that activation of glucose transport and glycogen synthase should be linked to the same signaling mechanism in order to provide a coordinated stimulation of intracellular glucose metabolism.

Insulin Signal Transduction Defects in Type 2 Diabetes

Insulin Receptor Number and Affinity

Both receptor and postreceptor defects have been shown to contribute to insulin resistance in type 2 diabetic individuals. Some, but not all studies have demonstrated a modest 20-30% reduction in insulin binding to monocytes and adipocytes from type 2 diabetic patients (1,311-316). This reduction is due to a decreased number of insulin receptors without change in insulin receptor affinity. In addition to the decreased number of cell-surface receptors, a variety of defects in insulin receptor internalization and processing have been described (314,315). However, some caution should be employed in interpreting these studies. Muscle and liver, not adipocytes, represent the major tissues responsible for the regulation of glucose homeostasis in vivo and insulin biding to solubilized receptors obtained from skeletal muscle biopsies and liver has been shown to be normal in obese and lean diabetic individuals when expressed per milligram of protein (312,313,316-318). Moreover, a decrease in insulin receptor number cannot be demonstrated in over half of type 2 diabetic subjects (319,320), and it has been difficult to demonstrate a correlation between reduced insulin binding and the severity of insulin resistance (321,322). The insulin receptor gene has been sequenced in a large number of type 2 diabetic patients from diverse ethnic populations using denaturing-gradient gel electrophoresis or single-stranded conformational polymorphism analysis, and, with very rare exceptions (323), physiologically significant mutations in the insulin receptor gene have not been observed (324,325). This excludes a structural gene abnormality in the insulin receptor as a cause of common type 2 diabetes mellitus.

Insulin Receptor Tyrosine Kinase Activity

Insulin receptor tyrosine kinase activity has been examined in a variety of cell types (skeletal muscle, adipocytes, hepatocytes, and erythrocytes) from normal-weight and obese diabetic subjects. Most (278,301,312,313,320,326-328), but not all (317,329) investigators have found reduced tyrosine kinase activity that cannot be explained by alterations in insulin receptor number or insulin receptor binding. However, near-normalization of the fasting plasma glucose concentration, (by weight loss) has been reported to correct the defect in insulin receptor tyrosine kinase activity (330). This observation suggests that the defect in tyrosine kinase is acquired and results from some combination of hyperglycemia, defective intracellular glucose metabolism, hyperinsulinemia, and insulin resistance - all of which improved after weight loss. A glucose-induced reduction in insulin receptor tyrosine kinase activity has been demonstrated in rat fibroblast culture in vitro (331). Insulin receptor tyrosine kinase activity assays are performed in vitro, and the results of these assays could provide misleading information with regard to insulin receptor function in vivo. To circumvent this problem, investigators have employed the euglycemic hyperinsulinemic clamp in combination with muscle biopsies and anti-phosphotyrosine immunoblot analysis (301). Such analysis yields a "snap shot" of the insulin-stimulated tyrosine phosphorylation state of the receptor in vivo. The results of these studies have demonstrated a substantial decrease in insulin receptor tyrosine phosphorylation in both obese nondiabetic and type 2 diabetic subjects (301,328). When insulin-stimulated insulin receptor tyrosine phosphorylation was examined in normal-glucose-tolerant or impaired-glucose-tolerant individuals at high risk of developing type 2 diabetes, a normal increase in tyrosine phosphorylation of the insulin receptor has been observed (332). These observations are consistent with the concept that impaired insulin receptor tyrosine kinase activity in type 2 diabetic patients is acquired secondarily to hyperglycemia or some other metabolic disturbance.

Insulin Signaling (IRS-1 and PI-3 kinase) Defects

A physiologic increase in the plasma insulin concentration stimulates tyrosine phosphorylation of the insulin receptor and IRS-1 in lean healthy subjects to 150-200% of basal values (280,301,328,332,333). In obese nondiabetic subjects, the ability of insulin to activate these two early insulin receptor signaling events in muscle is reduced, while in type 2 diabetics insulin has no significant stimulatory effect on either insulin receptor or IRS-1 tyrosine phosphorylation (301) . The association of p85 protein and PI3-kinase activity with IRS-1 also is greatly reduced in obese non-diabetic and type 2 diabetic subjects compared to lean healthy subjects (301,328-334) . Insulin also failed to increase the association of the p85 subunit of PI3-kinase with IRS-2 in muscle, indicating that type 2 diabetes is characterized by a combined defect in IRS-1 and IRS-2 function (301,328). The decrease in insulin stimulation of the association of the p85 regulatory subunit of PI3-kinase with IRS-1 is closely correlated with the impairment in muscle glycogen synthase activity and in vivo insulin-stimulated glucose disposal (301). Defective regulation of PI3-kinase gene expression by insulin also has been demonstrated in skeletal muscle and adipose tissue of type 2 diabetic subjects (335). In animal models of diabetes, an 80% decrease in IRS-1 phosphorylation and a greater than 90% reduction in insulin-stimulated PI3-kinase activity have been reported (336).

In the insulin resistant, normal glucose tolerant offspring of two type 2 diabetic parents, IRS-1 tyrosine phosphorylation and the association of p85 protein/PI3-kinase activity with IRS-1 are markedly decreased despite normal tyrosine phosphorylation of the insulin receptor; these insulin signaling defects are correlated closely with the severity of insulin resistance, measured with the euglycemic insulin clamp technique (332). In summary, a defect in the association of PI3-kinase with IRS-1 and its subsequent activation appears to be a characteristic abnormality in type 2 diabetics, is closely correlated with in vivo muscle insulin resistance, and is unrelated to a disturbance in insulin receptor tyrosine phosphorylation. Several groups (337,338) have reported that a common mutation in the IRS-1 gene (Gly 972 Arg) is associated with type 2 diabetes, insulin resistance, and obesity, but the physiologic significance of this mutation remains to be established (339).

The profound insulin resistance of the PI3-kinase signaling pathway contrasts markedly with the ability of insulin to stimulate MAP kinase pathway activity in insulin-resistant type 2 diabetic and obese non-diabetics individuals (301,328). Hyperinsulinemia increases MEK1 activity and ERK1/2 phosphorylation and activity to the same extent in lean healthy as in insulin resistant obese nondiabetic and type 2 diabetic patients (301,328). This finding of selective insulin resistance is similar to that recently observed in vasculature of Zucker fatty rats (340). Two possible reasons for this difference are alternate insulin signaling pathways and differential signal amplification. With regard to the former, the MAP kinase pathway can be activated either through Grb2/Sos interaction with IRS-1/IRS-2 or with Shc. Because IRS-1 tyrosine phosphorylation is dramatically reduced in the diabetics, it is possible that insulin activation of the MAP kinase pathway in vivo primarily occurs through Shc activation. There is evidence from in vitro studies to support this concept (341). Like ERK and MEK activity, insulin increased Shc phosphorylation to the same extent in lean and obese nondiabetic and type 2 diabetic subjects (301). These results indicate that, in type 2 diabetes, insulin induces sufficient activation of the insulin receptor tyrosine kinase to increase Shc phosphorylation normally. It also is possible that differential signal amplification in the PI3-kinase and MAP kinase pathways can explain their differing susceptibilities to the effects of insulin resistance.

Maintenance of insulin stimulation of the MAP kinase pathway in the presence of insulin resistance in the PI3-kinase pathway may be important in the development of insulin resistance. ERKs can phosphorylate IRS-1 on serine residues (342), and serine phosphorylation of IRS-1 and the insulin receptor itself has been implicated in desensitizion insulin receptor signaling (343). Continued ERK activity, when IRS-1 function already is impaired, could lead to a worsening of insulin resistance. Thus, diabetic and obese subjects have inappropriately high MAP kinase activity. One also could postulate that insulin resistance in the metabolic (PI3-kinase) pathway, with its compensatory increase in beta cell function and hyperinsulinemia, leads to excessive stimulation of the MAP kinase pathway in vascular tissue (301,302). This would result in the proliferation of vascular smooth muscle cells, increased collagen formation, and increased production of growth factors and inflammatory cytokines, possibly explaining the accelerated rate of atherosclerosis in type 2 diabetic individuals.

Glucose Transport

Activation of the insulin signal transduction system in insulin target tissues leads to the stimulation of glucose transport. The effect of insulin is brought about by the translocation of a large intracellular pool of glucose transporters (associated with low-density microsomes) to the plasma membrane (281,282,344). There are five major, different facilitative glucose transporters with distinctive tissue distributions (281,282,345,346) (Table 2). GLUT4, the insulin regulatable transporter, is found in insulin-sensitive tissues (muscle and adipocytes), has a Km of ~5 mmol/l which is close to that of the plasma glucose concentration, and is associated with HK-II (347-349). In adipocytes and muscle, its concentration in the plasma membrane increases markedly after exposure to insulin, and this increase is associated with a reciprocal decline in the intracellular GLUT4 pool. GLUT1 represents the predominant glucose transporter in the insulin-independent tissues (brain and erythrocytes), but also is found in muscle and adipocytes. It is located primarily in the plasma membrane, where its concentration changes little after the addition of insulin. It has a low Km (~1 mmol/l) and is well suited for its function, which is to mediate basal glucose uptake. It is found in association with HKI (347-349). GLUT2 predominates in the liver and pancreatic ß-cells, where it is found in association with a specific hexokinase, HKIV (347-350). In the ß-cell, HKIV is referred to as glucokinase (350,351). GLUT2 has a high Km, (~15-20 mmol/l) and, as a consequence, the glucose concentration in cells expressing this transporter rises in direct proportion to the increase in plasma glucose concentration. This characteristic allows these cells to respond as glucose sensors. In summary, each tissue has a specific glucose transporter and associated hexokinase, which allow it uniquely to carry out its specialized function to maintain whole-body glucose economy.

Glucose transport activity in type 2 diabetic patints uniformly has been found to be decreased in adipocytes (281,282,320,351,352) and muscle (281,282,354-356). In adipocytes from type 2 diabetic human and rodent models of diabetes, there is a severe reduction in GLUT4 mRNA and protein, and the ability of insulin to elicit a normal translocation response and to activate the GLUT4 transporter after its insertion into the cell membrane is impaired (281,282,320,353,357). In contrast, muscle tissue obtained from lean and obese type 2 diabetic subjects exhibits normal or increased levels of GLUT4 mRNA expression and normal levels of GLUT4 protein (358-361). Moreover, acute (2- 4-h) physiological hyperinsulinemia does not increase the number of GLUT4 transporters in muscle in either healthy or type 2 diabetic subjects (358-361). Several studies have demonstrated an increase in muscle GLUT4 mRNA levels in response to insulin in control subjects (333,360), but not in diabetics (360), suggesting insulin resistance at the level of gene transcription. However, the physiological significance of the blunted increase in muscle GLUT4 mRNA levels in type 2 diabetic subjects is unclear, since both basal and insulin-stimulated GLUT4 protein levels are normal. Large populations of type 2 diabetics have been screened for mutations in the GLUT4 gene (362,363). Such mutations are very uncommon and, when detected, have been of questionable physiologic significance.

The results summarized above indicate that the gene (GLUT4) encoding the major insulin-responsive glucose transporter and its transcriptional/translational regultion are not impaired in type 2 diabetes. However, in contrast to the normal expression of GLUT4 protein and mRNA in muscle of type 2 diabetic subjects, every study that has examined adipose tissue has reported reduced basal and insulin-stimulated GLUT4 mRNA levels, decreased GLUT4 transporter number in all subcellular fractions, diminished GLUT4 translocation, and impaired intrinsic activity of GLUT4 (281,282,353,361,364). These observations demonstrate that GLUT4 expression in humans is subject to tissue-specific regulation. Although insulin does not increase GLUT 4 expression in muscle, it stimulates the translocation of GLUT4 transporters from their intracellular location to the cell membrane (354,365,366). In type 2 diabetic humans, the ability of insulin to stimulate GLUT4 translocation in muscle is impaired (354,367). Using a novel triple-tracer technique, the in vivo dose-response curve for the action of insulin on glucose transport in forearm skeletal muscle has been examined in nondiabetic and type 2 diabetic subjects (368-370). Insulin-stimulated inward muscle glucose transport is severely impaired in type 2 diabetic subjects who are studied under euglycemic conditions . The defect in glucose transport cannot be overcome by repeating the insulin clamp at each subject's normal fasting glucose (hyperglycemia) level . Since the number of GLUT4 transporters in the muscle of diabetic subjects is normal (358-361), impaired GLUT4 translocation (281,354,367) and decreased intrinsic activity of the glucose transporter (366,371) must be responsible for the defect in muscle glucose transport. Impaired in vivo muscle glucose transport in type 2 diabetics also has been demonstrated using MRI (372) and PET (373).

Glucose Phosphorylation

Glucose phosphorylation and glucose transport are tightly coupled phenomena (374). Isoenzymes of hexokinase (HKI-HKIV) catalyze the first committed intracellular step of glucose metabolism, the conversion of glucose to glucose-6-phosphate (G-6-P) (347-350,375) (Table 2). HKI, HKII, and HKIII are single-chain peptides that have a number of properties in common, including a very high affinity for glucose and product inhibition by G-6-P. HKIV, also called glucokinase, has a lower affinity for glucose and is not inhibited by G-6-P. Glucokinase (HKIVB) is believed to be the glucose sensor in the ß-cell, while HKIVL plays an important role in the regulation of hepatic glucose metabolism.

In both rat (375-377) and human (333,348,378-380)skeletal muscle, HKII transcription is regulated by insulin. HKI also is present in human skeletal muscle, but it is not regulated by insulin (378). In response to physiological euglycemic hyperinsulinemia, HKII cytosolic activity, protein content, and mRNA levels increase by 50-200% in healthy non-diabetic subjects (378,380) and this is associated with the translocation of hexokinase II from the cytosol to the mitochondria (381). In contrast, insulin has no effect on HK-I activity, protein content, or mRNA levels (378).

In forearm muscle, insulin-stimulated glucose transport (measured with the triple tracer technique) has been shown to be markedly impaired in lean type 2 diabetics (370) . However, since the rate of intracellular glucose phosphorylation was impaired to an even greater extent , insulin caused an increase in the intracellular free glucose concentration. By performing the insulin clamp at each diabetic's normal level of fasting hyperglycemia, normal rates of whole-body glucose disposal and a normal rate of glucose influx into muscle was elicited . However, the rate of intracellular glucose phosphorylation increased only modestly ; consequently, there was a dramatic rise in the free glucose concentration within the intracellular space that is accessible to glucose. These observations indicate that in type 2 diabetic individuals, while both glucose transport and glucose phosphorylation are severely resistant to the action of insulin, impaired glucose phosphorylation (HKII) appears to be the rate-limiting step for insulin action. A similar pattern of impaired muscle glucose phosphorylation and transport is present in the insulin-resistant, normal glucose-tolerant offspring of two diabetic parents (382). These results are consistent with dose-response studies using PET to evaluated glucose phosphorylation and transport in skeletal muscle of type 2 diabetics (373). They also are consistent with 31P-NMR studies (383) which demonstrate that, during hyperinsulinemia, muscle G-6-P concentrations decline in type 2 diabetic versus control subjects. However, subsequent studies using 31P-NMR in combination with 1-14C-glucose suggest that the defect in insulin-stimulated muscle glucose transport exceeds the defect in glucose phosphorylation and is responsible for the decline in muscle glucose-6-P concentration (372). Because of methodologic differences, the results of the triple tracer (370) and MRI (372) studies cannot be reconciled at present. Nonetheless, observations from these studies are consistent in demonstrating that the defects in glucose phosphorylation and glucose transport in muscle are established early in the natural history of type 2 diabetes and cannot be explained by glucose toxicity (91). Clear evidence that HKII activity is crucial for glucose uptake derives from studies in transgenic mice who overexpress HKII. In this model, HKII overexpressiion increased both insulin- and exercise-stimulated muscle glucose uptake (384).

In healthy nondiabetic subjects, physiologic hyperinsulinemia for as little as 2-4 hours increases muscle HKII activity, gene transcription, and translation (333,378). In lean type 2 diabetics insulin-stimulated HKII activity and mRNA levels are markedly reduced compared to controls (383,385) . Decreased basal muscle HKII activity and mRNA levels (385) and impaired insulin-stimulated HKII activity(379,380,386,387) in type 2 diabetic subjects have been reported by other investigators. A decrease in insulin-stimulated muscle HKII activity also has been described in subjects with IGT (388). Because of its central role in insulin-mediated muscle glucose metabolism, several groups have looked for point mutations in the HKII gene in individuals with type 2 diabetes (388-390). Although several nucleotide substitutions have been found, none have been located close to the glucose and ATP binding sites and none have been associated with insulin resistance. Thus, an abnormality in the HKII gene is unlikely to explain the inherited insulin resistance in common variety type 2 diabetes mellitus.

Glycogen Synthesis

After glucose is phosphorylated by hexokinsae II, it either can be converted to glycogen or enter the glycolytic pathway. Of the glucose that enters the glycolytic pathway, ~90% is oxidized. At low physiologic plasma insulin concentrations, glycogen synthesis and glucose oxidation are of approximately equal quantitative importance. With increasing plasma insulin concentrations, glycogen synthesis predominates (18,391). If the rate of glucose oxidation (determined by indirect calorimetry) is subtracted from the rate of whole-body insulin-mediated glucose disposal (determined from the insulin clamp), the difference represents nonoxidative glucose disposal (or glucose storage) (17,360), which primarily reflects glycogen synthesis (1,4,162,216,392). Glucose conversion to lipid accounts for <5% of total body glucose disposal (18,198,199) and less than 5-10% of the glucose taken up by muscle is released as lactate (5,393,394). Reduced insulin-stimulated glycogen synthesis is a characteristic finding in all insulin-resistant states, including obesity, diabetes, and the combination of obesity plus diabetes (1,4,18,43,59,159,162,218,219,377,393-395). Impaired glycogen synthesis also represents the major cause of insulin resistance in obese subjects with normal or only slightly impaired glucose tolerance (1,4,162,218,393,395,396). Thus, the inability of insulin to promote glycogen synthesis is a characteristic and early defect in the development of insulin resistance in both obesity and type 2 diabetes. The emergence of overt diabetes with fasting hyperglycemia is associated with a major reduction in insulin-mediated nonoxidative glucose disposal (glycogen synthesis) in all ethnic groups (1,4,18,162,377,396). Impaired glycogen synthesis also has been demonstrated in the normal-glucose-tolerant offspring of two diabetic parents (43,397), in the first-degree relatives of type 2 diabetic individuals (41,398,399), and in the normoglycemic twin of a monozygotic twin pair in which the other twin has type 2 diabetes (101).

Using NMR imaging spectroscopy, a decrease in insulin-stimulated incorporation of [1H, 13C]-glucose into muscle glycogen of type 2 diabetic subjects has been demonstrated directly (215) . In type 2 diabetics, there was a marked lag in the onset of insulin-stimulated glycogen synthesis , that was similar to the delay in insulin-mediated leg muscle glucose uptake . The rate of glycogen synthesis in type 2 diabetic subjects was decreased by ~50%, paralleling the decrease in total glucose uptake by leg muscle (3) , and impaired muscle glycogen synthesis accounted for essentially all of the defect in whole body glucose disposal.

In summary, an abundance of convincing evidence demonstrates that impaired glycogen synthesis is the major metabolic defect in normal glucose tolerant obese subjects, in individuals with IGT, and in patients with overt diabetes. Moreover, numerous studies have documented that the earliest detectable metabolic abnormality responsible for the insulin resistance in normal glucose tolerant individuals who are destined to develop type 2 diabetes is impaired glycogen synthesis (4,41,43,101,382,392,399,400).

Glycogen synthase is the key insulin-regulated enzyme which controls the rate of muscle glycogen synthesis (307,308,310,379,401,402). Insulin enhances glycogen synthase activity by stimulating a cascade of phosphorylation dephosphorylation reactions (307,308,361-363,403) (see above discussion of insulin receptor signal transduction), which ultimately lead to the activation of PP1 (also called glycogen synthase phosphatase) (307,308,310,402). The regulatory subunit (G) of PP1 has two serine phosphorylation sites, called site 1 and site 2. Phosphorylation of site 2 by cAMP-dependent kinase (PKA) inactivates PP1, while phosphorylation of site 1 by insulin activates PP1, leading to the stimulation of glycogen synthase (307,308,402,404). Phosphorylation of site 1 of PP1 by insulin in muscle is catalyzed by insulin-stimulated protein kinase 1 (ISPK-1) (309,405), which is part of a family of serine/threonine protein kinases termed ribosomal S6-kinases. Because of their central role in muscle glycogen formation, considerable attention has focused on the three enzymes glycogen synthase, PP1, and ISPK-1 in the pathogenesis of insulin resistance in individuals with type 2 diabetes.

Glycogen synthase exists in an active (dephosphorylated) and an inactive (phosphorylated) form (307-310). Under fasting conditions, total glycogen synthase activity in type 2 diabetic subjects is reduced and the ability of insulin to activate glycogen synthase is severely impaired (301,384,406-410). An impaired ability of insulin to activate glycogen synthase also has been demonstrated in the normal glucose tolerant relatives of type 2 diabetic individuals (400). Insulin-mediated activation of glycogen synthase and insulin-stimulated glycogen synthase gene expression have been shown to be impaired in cultured myocytes and fibroblasts from type 2 diabetic subjects (411,412). Studies in insulin-resistant nondiabetic and diabetic Pima Indians have documented that the ability of insulin to activate muscle PP1 (glycogen synthase phosphatase) is severely reduced (413). PP1 dephosphorylates glycogen synthase, leading to its activation. Therefore, a defect in PP1 appears to play an important role in muscle insulin resistance (309).

The effect of insulin on glycogen synthase gene transcription and translation in vivo has been studied extensively. Most studies (378,414,415) have shown that insulin does not increase glycogen synthase mRNA or protein expression in human muscle studied in vivo. However, glycogen synthase mRNA expression is decreased in muscle of type 2 diabetic patients (415,416), explaining in part the decreased glycogen synthase activity in this disease. However, the major abnormality in glycogen synthase regulation in type 2 diabetes and other insulin resistant conditions is its lack of dephosphorylation and activation by insulin as a result of insulin receptor signaling abnormalities (see previous discussion). The glycogen synthase gene (417) has been the subject of intensive investigation. An association between glycogen synthase gene markers and type 2 diabetes has been demonstrated in Japanese, French, Finnish, and Pima Indian populations. However, DNA sequencing has revealed either no mutations (418) or rare nucleotide substitutions (419,420) that cannot explain the defect in insulin-stimulated glycogen synthase. Nonetheless, the association between the glycogen synthase gene and type 2 diabetes mellitus (418) suggests that another gene close to the glycogen synthase gene may be involved in the development of type 2 diabetes. The genes encoding the catalytic subunits of PP1 (421) and ISPK-1 (422) have been examined in insulin-resistant Pima Indians and Danes with type 2 diabetes. Several silent nucleotide substitutions were found in the PP1 and ISPK-1 genes in the Danish population; the mRNA levels of both genes were normal in skeletal muscle (422). No structural gene abnormalities in the catalytic subunit of PP1 were detected in Pima Indians (422). Thus, neither abnormalities in the PP1 and ISPK-1 genes nor abnormalities in their translation can explain the impaired enzymatic activities of glycogen synthase and PP1 that have been observed in vivo. Similarly, there is no evidence that an alteration in glycogen phosphorylase plays any role in the abnormality in glycogen formation in type 2 diabetes (423).

In summary, glycogen synthase activity is severely impaired in patients with type 2 diabetes mellitus and in insulin-resistant normal glucose tolerant individuals who are predisposed to develop type 2 diabetes. However, the defect cannot be explained by an abnormality in the genes encoding glycogen synthase or is promoter or by other key genes - PP1 or ISPK-1 - involved in the regulation of glycogen synthase activity.

Glycolysis/Glucose Oxidation

Glucose oxidation accounts for ~90% of total glycolytic flux, while anaerobic glycolysis accounts for the other 10% (393,394). Two enzymes, phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH), play pivotal roles in the regulation of glycolysis and glucose oxidation, respectively. In type 2 diabetic individuals the glycolytic/glucose oxidative pathway has been show to be impaired in many individuals with type 2 diabetes (393,394). Although one study suggested that the activity of PFK is modestly reduced in muscle biopsies from type 2 diabetic subjects (424), the majority of evidence indicates that the activity of PFK is normal (407,412,417). Insulin has no effect on muscle PFK activity, mRNA levels, or protein content in either nondiabetic or diabetic individuals (417). PDH is a key insulin-regulated enzyme whose activity in muscle is acutely stimulated by a physiological increment in the plasma insulin concentration (415). Three previous studies have examined PDH activity in type 2 diabetic patients. Insulin-stimulated PDH activity is decreased in isolated subcutaneous human adipocytes from patients with type 2 diabetes mellitus (425) and in skeletal muscle from type 2 diabetic subjects undergoing euglycemic hyperinsulinemic clamps (426). However, when type 2 diabetic patients had muscle biopsies during hyperglycemic hyperinsulinemic clamps, activation of PDH by insulin was normal (409), in concert with normalized rates of muscle glucose uptake. These results suggest that insulin stimulation of PDH activity is influenced by glycolytic flux.

Both obesity and type 2 diabetes mellitus are associated with accelerated FFA turnover and oxidation (1,4,18,162), which would be expected, according to the Randle cycle (232), to inhibit PDH activity and consequently glucose oxidation (see prior discussion). Thus, any observed defect in glucose oxidation or PDH activity could be acquired secondarily to increased FFA oxidation and feedback inhibition of PDH by elevated intracellular levels of acetyl-CoA and reduced availability of NAD. Consistent with this observation, the rates of basal and insulin-stimulated glucose oxidation have been shown to be normal in the normal glucose tolerant offspring of two diabetic parents (43) and in the first degree relatives of type 2 diabetic subjects (41,423), while it is decreased in overtly diabetic subjects (1,4,393,394,427). Studies examining PHD activity in muscle tissue from lean diabetic subjects with mild fasting hyperglycemia are needed before the role of this enzyme in the development of insulin resistance in type 2 diabetes can be established or excluded.

In summary, postbinding defects in insulin action primarily are responsible for the insulin resistance in type 2 diabetes. Diminished insulin binding, when present, is small, occurs in individuals with IGT or very mild diabetes, and results secondarily from downregulation of the insulin receptor by chronic sustained hyperinsulinemia. In type 2 diabetic patients with overt fasting hyperglycemia, postbinding defects are responsible for the insulin resistance. A number of postbinding defects have been documented, including diminished insulin receptor tyrosine kinase activity, insulin signal transduction abnormalities, decreased glucose transport, reduced glucose phosphorylation, and impaired glycogen synthase activity. The glycolytic/glucose oxidative pathway appears to be largely intact and, when defects are observed, they appear to be acquired secondarily to enhanced FFA/lipid oxidation. From the quantitative standpoint, impaired glycogen synthesis represents the major pathway responsible for the insulin resistance in type 2 diabetes, and family studies suggests that a defect in the glycogen synthetic pathway represents the earliest detectable abnormality in type 2 diabetes. Recent studies link the impairment in glycogen synthase activation to a defect in the ability of insulin to phosphorylate IRS-1, causing a reduced association of the p85 subunit of PI 3-kinase with IRS-1 and decreased activation of the enzyme (PI 3-kinase).