Activation of the insulin signal transduction system in insulin target tissues stimulates glucose transport via a mechanism that involves translocation of a large intracellular pool of glucose transporters (associated with low-density microsomes) to the plasma membrane and their subsequent activation after insertion into the cell membrane [261,262]. There are five major facilitative glucose transporters with distinctive tissue distributions [263,264] (Table 25.2). GLUT4, the insulin regulatable transporter, is found in insulin-sensitive tissues (muscle and adipocytes), has a Km of ∼5mmol L−1 which is close to that of the plasma glucose concentration, and is associated with hexokinase (HK)-II [263,264]. In adipocytes and muscle, GLUT4 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 is the predominant glucose transporter in the insulin-independent tissues (brain and erythrocytes), but is also found in muscle and adipocytes. GLUT1 is located primarily in the plasma membrane, where its concentration is unchanged following exposure to insulin. It has a low Km (∼1mmol L−1) and is well suited for its function, which is to mediate basal glucose uptake. It is found in association with HKI . GLUT2 is the predominant transporter in liver and pancreatic β cells, where it is found in association with a specific hexokinase, HKIV or glucokinase . GLUT2 has a very high Km (∼15–20mmol L−1), which allows the glucose concentration in cells expressing this transporter to rise in direct proportion to the increase in plasma glucose concentration. This unique characteristic allows these cells to function as glucose sensors.
In adipocytes and muscle of type 2 diabetic patients glucose transport activity is severely impaired [220,249,261,262,267,268]. In adipocytes from human and rodent models of T2DM,GLUT4 mRNA and protein content are markedly reduced, and the ability of insulin to elicit a normal translocation response and to activate the GLUT4 transporter after insertion into the cell membrane is decreased. In contrast to adipocytes, muscle tissue from lean and obese type 2 diabetic subjects exhibits normal levels of GLUT4 mRNA expression and normal levels of GLUT4 protein, thus demonstrating that transcriptional and translational regulation of GLUT4 is not impaired [269,270]. 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 and decreased GLUT4 transporter number in all subcellular fractions. These observations demonstrate that GLUT4 expression in humans is subject to tissue-specific regulation. 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 type 2 diabetic subjects and insulin-stimulated inward muscle glucose transport has been shown to be severely impaired [36,203,271]. Impaired in vivo muscle glucose transport in type 2 diabetics has also been demonstrated using MRI and PET . Since the number of GLUT4 transporters in the muscle of diabetic subjects is normal, impaired GLUT4 translocation and decreased intrinsic activity of the glucose transporter are responsible for the defect in muscle glucose transport. Large populations of type 2 diabetics have been screened for mutations in the GLUT4 gene . Such mutations are very uncommon and, when detected, have been of questionable physiologic significance.
Glucose phosphorylation and glucose transport are tightly coupled phenomena .Hexokinase isoenzymes (HK-I–HK-IV) catalyze the first committed step of glucose metabolism, the intracellular conversion of free glucose to glucose-6-phosphate [263–265,275] (G-6-P) (Table 25.2). HK-I, HK-II, and HK-III are single-chain peptides that have a very high affinity for glucose and demonstrate product inhibition by (G-6-P). HK-IV, also called glucokinase, has a lower affinity for glucose and is not inhibited by G-6-P. Glucokinase (HK-IVB) represents the glucose sensor in the β cell, while HK-IVL in the liver plays a central role in the regulation of hepatic glucose metabolism.
In human skeletal muscle, HK-II transcription is regulated by insulin, whereas HK-ImRNA and protein levels are not affected by insulin [276–278]. In response to physiologic euglycemic hyperinsulinemia of 2–4 h duration, HK-II cytosolic activity, protein content, and mRNA levels increase by 50–200% in healthy nondiabetic subjects and this is associated with the translocation of HK-II from the cytosol to the mitochondria. In forearm muscle, insulin-stimulated glucose transport (measured with the triple tracer technique) is markedly impaired in lean type 2 diabetics [36,203,271]. However, the rate of intracellular glucose phosphorylation is impaired to an even greater extent, resulting in an increase 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 (HK-II) appears to be the rate-limiting step for insulin action. Studies using 31P-NMR in combination with 1-14C-glucose have also demonstrated that both insulin-stimulated muscle glucose transport and glucose phosphorylation are impaired in type 2 diabetic subjects, but results from this study suggest that the defect in transport exceeds the defect in phosphorylation . Because of methodologic differences, the results of the triple tracer [36,203,271] and MRI  studies cannot be reconciled at present. Nonetheless, these studies are consistent in demonstrating that abnormalities in both muscle glucose phosphorylation and glucose transport are well established early in the natural history of T2DM and cannot be explained by glucose toxicity.
In healthy nondiabetic subjects, a physiologic increase in the plasma insulin concentration for as little as 2–4 hours increases muscle HK-II activity, gene transcription, and translation . In lean type 2 diabetics the ability of insulin to augment HK-II activity and mRNA levels is markedly reduced compared to controls . Decreased basal muscle HK-II activity and mRNA levels and impaired insulin-stimulated HK-II activity in type 2 diabetic subjects have been reported by other investigators [278,279]. A decrease in insulin-stimulated muscle HK-II activity has also been described in subjects with IGT . Several groups have looked for point mutations in the HKII gene in individuals with T2DM and, although several nucleotide substitutions have been found, none are close to the glucose and ATP binding sites and none have been associated with insulin resistance [280–282]. Thus, an abnormality in the HKII gene is unlikely to explain the inherited insulin resistance in common variety T2DM mellitus.
Following phosphorylation by HK-II, glucose either can be converted to glycogen or enter the glycolytic pathway. Of the glucose that enters the glycolytic pathway, ∼90% is oxidized and the remaining 10% is released as lactate. At low physiologic plasma insulin concentrations, glycogen synthesis and glucose oxidation contribute approximately equally to glucose disposal. However, with increasing plasma insulin concentrations, glycogen synthesis predominates [1–3,283]. Impaired insulin-stimulated glycogen synthesis is a characteristic finding in all insulin-resistant states including obesity, IGT, diabetes, and diabesity in all ethnic groups and accounts for the majority of the defect in insulin-mediated whole body glucose disposal [1–3,12,162,284–287]. Impaired glycogen synthesis also has been documented in the normal glucose-tolerant offspring of two diabetic parents, in the first-degree relatives of type 2 diabetic individuals, and in the normoglycemic twin of a monozygotic twin pair in which the other twin has T2DM [162,288,289].
Glycogen synthase is the key insulin-regulated enzyme, which controls the rate of muscle glycogen synthesis [241, 243,278,289–291]. Insulin actives glycogen synthase by stimulating a cascade of phosphorylation-dephosphorylation reactions, which ultimately lead to the activation of PP1 (also called glycogen synthase phosphatase). The regulatory subunit of PP1 has two serine phosphorylation sites. 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. Phosphorylation of site 1 of PP1 by insulin in muscle is catalyzed by insulin-stimulated protein kinase 1 (ISPK-1). Because of their central role in muscle glycogen formation, the three enzymes—glycogen synthase, PP1, ISPK-1—have been extensively studied in the individuals with T2DM.
Glycogen synthase exists in an active (dephosphorylated) and an inactive (phosphorylated) form [241–243]. Under basal conditions, total glycogen synthase activity in type 2 diabetic subjects is reduced and the ability of insulin to activate glycogen synthase is severely impaired [210,292–294]. The ability of insulin to stimulate glycogen synthase is also diminished in the normal glucose-tolerant, insulin-resistant relatives of type 2 diabetic individuals . In insulin-resistant nondiabetic and diabetic Pima Indians activation of muscle PP1 (glycogen synthase phosphatase) by insulin is severely reduced . Since PP1 dephosphorylates glycogen synthase, leading to its activation, the defect in PP1 plays an important role in the muscle insulin resistance of T2DM.
The effect of insulin on glycogen synthase gene transcription and translation in vivo has been studied extensively. Most studies have demonstrated that insulin does not increase glycogen synthase mRNA or protein expression in human muscle [276,297,298]. However, glycogen synthase mRNA and protein levels are decreased in muscle of type 2 diabetic patients, explaining in part the decreased glycogen synthase activity [298,299]. The major abnormality in glycogen synthase regulation in T2DM is its lack of dephosphorylation and activation by insulin, as a result of insulin receptor signaling abnormalities (see previous discussion).
The glycogen synthase gene has been the subject of intensive investigation, and DNA sequencing has revealed either no mutations or rare nucleotide substitutions that cannot explain the defect in insulin-stimulated glycogen synthase activity [300–302]. The genes encoding the catalytic subunits of PP1 and ISPK-1 have been examined in Pima Indians and Danes with T2DM [303,304]. Several silent nucleotide substitutions were found in the PP1 and ISPK-1 genes in the Danish population, but the mRNA levels of both genes were normal in skeletalmuscle.No structural gene abnormalities in the catalytic subunit of PP1 were detected in Pima Indians. Thus, neither mutations 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 T2DM .
In summary, glycogen synthase activity is severely impaired in type 2 diabetic individuals, but the molecular etiology of the defect remains to be determined.
Glucose oxidation accounts for ∼90% of total glycolytic flux, while anaerobic glycolysis accounts for the other 10%. 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 shown to be impaired . Although one study has suggested that PFK activity is modestly reduced in muscle biopsies from type 2 diabetic subjects , most evidence indicates that the activity of PFK is normal [293,298]. Insulin has no effect on muscle PFK activity, mRNA levels, or protein content in either nondiabetic or diabetic individuals . PDH is a key insulin-regulated enzyme whose activity in muscle is acutely stimulated by insulin . In type 2 diabetic patients, insulin-stimulated PDH activity is decreased in human adipocytes and in skeletal muscle [307,308].
Obesity and T2DM are associated with accelerated FFA turnover and oxidation [1–3,12,309], which would be expected, according to the Randle cycle , to inhibit PDH activity and consequently glucose oxidation. Therefore, it is likely that the observed defects in glucose oxidation and PDH activity are acquired secondary to increased FFA oxidation and feedback inhibition of PDH by elevated intracellular levels of acetyl-CoA and reduced availability of NAD. Consistent with this scenario, the rates of basal and insulin-stimulated glucose oxidation are not reduced in the normal glucose-tolerant offspring of two diabetic parents and in the first-degree relatives of type 2 diabetic subjects, while it is decreased in overtly diabetic subjects.