Diabetes Mellitus: A Fundamental and Clinical Text
3rd Edition
© 2004 Lippincott Williams & Wilkins
←↑→
63
Glucose Toxicity: Effect of Chronic Hyperglycemia on Insulin Action
Luciano Rossetti
The value of glycemic control in the prevention and amelioration of the microvascular complications of type 1 diabetes mellitus (DM) is now well established in prospective clinical trials (1,2,3,4,5,6,7,8). A considerable amount of epidemiologic and animal data also support the link between chronic hyperglycemia and long-term microvascular complications of DM, including retinopathy, neuropathy, and nephropathy (9,10,11,12,13,14). In addition to microvascular complications, hyperglycemia may also contribute to macrovascular disease (15) and impaired cellular immunity (16,17) in DM.
It has become clear that chronic hyperglycemia also has a deleterious effect on both insulin secretion (18,19,20,21,22,23) and insulin action (24,25), a concept that has been referred to as glucose toxicity (18,26,27). In this regard, chronic hyperglycemia not only represents a hallmark of DM, but is itself a self-perpetuating regulatory factor that contributes to poor metabolic control.
In this chapter, I focus on the effect of chronic hyperglycemia on insulin action. The ensuing discussion reviews our current understanding of the pathophysiology, biochemistry, and clinical significance of glucose-induced insulin resistance. The deleterious impact of hyperglycemia on insulin secretion and the microvasculature is discussed in Chapters 10,11,90,91, and 94.
Hyperglycemia and Tissue Sensitivity to Insulin
Skeletal muscle insulin resistance is present in conventionally treated patients with type 1 (28,29) and type 2 DM (30,31,32,33,34). In the latter, the defect in insulin action is thought to be multifactorial and at least partially inherited (35,36,37,38,39,40,41,42), whereas in the former, it is likely to be acquired (29). In an editorial, Unger and Grundy (18) suggested that chronic hyperglycemia may be partially responsible for the defect in insulin-mediated glucose disposal in patients with types 1 and 2 DM. Preliminary evidence in support of this hypothesis was derived from several observations in animals and humans:
  • Insulin resistance in type 1 DM is a consequence of poor metabolic control.
  • Tight metabolic control leads to some improvement in insulin sensitivity.
  • Defects in insulin secretion lead to the development of insulin resistance.
In type 1 DM, insulin sensitivity appears to be a consequence of poor metabolic control and is well correlated with glycosylated hemoglobin levels (43,44,45,46,47,48,49). Moderately and poorly controlled patients with type 1 DM have some degree of peripheral (skeletal muscle) insulin resistance (28,29,43,44,45,46,47,48,49). However, insulin sensitivity is normal during remission (44,45,46,47,49), suggesting that insulin resistance in type 1 DM is an acquired and reversible defect. Yki-Jarvinen et al. (29), in a comprehensive analysis of insulin resistance in patients with type 1 DM, found a significant correlation between the degree of insulin resistance and the glycosylated hemoglobin concentration.
Near-normal glycemic control in diabetic humans, however it is achieved (e.g., diet, insulin therapy, sulfonylureas), leads to some improvement in insulin sensitivity (44,45,46,47,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70). If hyperglycemia contributes to the insulin resistance observed in human DM, normalization of the plasma glucose profile, regardless of the means, would be expected to lead to an improvement in tissue sensitivity. Several studies have shown a significant improvement in insulin sensitivity in diabetic patients after institution of intensified insulin therapy (44,45,46,47,49,50,51,52,53,54,55,56,57,58). Using the insulin clamp technique, a number of investigators have examined the effect of diet (weight loss) (63,64,65,66,67), sulfonylureas (68,69,70,71,72), and insulin therapy (50,53,54,55,56,57,73,74) on insulin action in patients with type 1 and type 2 DM. Strict glycemic control with insulin in patients with type 1 DM (44,45,46,47,49) uniformly improved insulin sensitivity, whereas a similar degree of glycemic control had a less consistent effect on improving insulin action in subjects with type 2 DM (50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74). Furthermore, in patients with type 2 DM, partial but not complete reversal of insulin resistance was seen (26). At first glance, these results in type 2 DM might be interpreted as evidence against the glucose toxicity hypothesis. However, a primary abnormality that is inherited in type 2 DM is insulin resistance (35,36,37,38,39,40,41,42), and the defect in insulin action becomes maximally manifest early in the natural history of type 2 DM (38,39,40,41,42). Thus, even when another severely insulin-resistant state, such as
P.940

obesity, is superimposed on type 2 DM, the insulin resistance is only slightly greater than with DM alone or obesity alone (75). Therefore, if the insulin resistance due to glucose toxicity is superimposed on the insulin resistance of obesity and on that inherited in type 2 DM, a significant additional deterioration in insulin action is not expected. Thus, normalization of the plasma glucose concentration and removal of glucose toxicity is not expected to produce a major improvement in insulin sensitivity. Conversely, in type 1 DM in humans and in experimental animal models of DM—where the insulin resistance is likely to be entirely acquired—glycemic control, however it is achieved, is expected to lead to an improvement in insulin-mediated glucose disposal.
From the aforementioned studies, however, it cannot be ascertained whether it is the insulin replacement per se, the correction of the hyperglycemia, or the normalization of other nonglucose (e.g., lipid, amino acid/protein) metabolic abnormalities that is responsible for the improvement of insulin action. In addition, chronic sustained hyperinsulinemia has also been shown to downregulate insulin receptor number and to result in an impairment in postreceptor events that regulate insulin-mediated glucose disposal (76,77). This may explain, in part, why intensified insulin treatment in subjects with type 2 DM fails to normalize completely the defect in insulin action (50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74).
In several animal models, an initial defect in insulin secretion leads to the development of insulin resistance (24,78,79,80,81,82,83,84). In animal models, a primary reduction in β-cell mass and consequent moderate to severe hypoinsulinemia and hyperglycemia lead to impaired insulin action in vivo (24,78,79,80,81,82) and at the cellular level in vitro (81,82,83,84,85). In particular, skeletal muscle insulin resistance has been clearly demonstrated in rodents and dogs made diabetic by pharmacologic (streptozotocin, alloxan) or surgical reduction of the β-cell mass (24,78,79,80,81,82). After 90% partial pancreatectomy in rats, a moderate increase in the fed plasma glucose concentrations to approximately 300 mg/dL induced a 30% decrease in the ability of insulin to promote peripheral glucose disposal (24,81,82). Because fasting plasma insulin and free fatty acid concentrations were similar to control levels in this model, it could be argued that the insulin resistance was at least partially due to the sustained increase in the plasma glucose concentrations (81,82).
However, it is not clear whether insulin deficiency or some other metabolic derangement that occurs as a result of the insulinopenia is responsible for defects in insulin action. A similar argument can be made concerning the beneficial effects of weight loss, sulfonylureas, and insulin therapy on tissue sensitivity to insulin.
A more rigorous test of the glucose toxicity hypothesis was provided by studies in which:
  • Experimentally induced prolonged hyperglycemia led to peripheral insulin resistance in patients with type 1 DM and in normal animals.
  • Lowering the plasma glucose concentration in diabetic animals without altering circulating hormones and other substrate levels normalized tissue sensitivity to insulin.
Prolonged hyperglycemia resulted in peripheral insulin resistance in patients with type 1 DM and normal animals (86,87,88,89,90,91,92). If the previously mentioned findings have relevance to the development of insulin resistance in humans, we would expect that sustained, physiologic hyperglycemia would lead to the development of insulin resistance. In fact, if the deleterious effect of hyperglycemia on muscle insulin sensitivity is a universal biologic phenomenon, then it should be reproducible in vivo by the induction of sustained hyperglycemia in humans and animals. Indeed, severe reduction in insulin-mediated muscle glucose uptake after prolonged elevations in the glucose levels has been shown in intact rats and in perfused hindquarters. In particular, Hager et al. (86) showed that 72-hour glucose infusions in healthy rats caused sustained hyperglycemia and hyperinsulinemia and the onset of severe skeletal muscle insulin resistance. In a series of reports, Richter and colleagues (87,88,89) induced defective insulin stimulation of hindlimb glucose uptake and glycogen synthesis after prolonged exposure to glucose and insulin. Considerable evidence suggests that this glucose-induced desensitization of muscle glucose uptake may operate in humans as well. This question has been examined in well-controlled patients with type 1 DM after prolonged (24–48 hours) experimental hyperglycemia (90,91,92). In particular, Vuorinen-Markkola et al. (91) studied patients with type 1 DM who were receiving chronic subcutaneous insulin infusion therapy. Subjects participated in two euglycemic insulin clamp procedures. During the initial study, the plasma glucose concentration was maintained at the basal level (128 ± 7 mg/dL) for 24 hours before the insulin clamp; before the second study, the plasma glucose concentration was elevated to 360 ± 5 mg/dL for 24 hours. As can be seen in Fig. 63.1, as little as 24 hours of hyperglycemia was sufficient to induce a 35% decline in the rate of insulin-mediated glucose disposal. Similar results were also obtained by Fowelin et al. (92). Thus, moderate whole-body and skeletal muscle insulin resistance were induced within 24 hours of hyperglycemia with moderate impairment of both glucose uptake and nonoxidative glucose disposal.
Lowering the plasma glucose concentration in diabetic animals without altering circulating hormones and substrate levels (other than glucose) normalized tissue sensitivity to insulin. There is one agent that fulfills these requirements: phlorizin. Phlorizin is a potent inhibitor of renal tubular glucose transport and blocks proximal tubular glucose reabsorption when the plasma glucose concentration is increased above the basal level. Thus, it leads to a normalization of the plasma glucose without causing hypoglycemia or altering plasma insulin, amino acid, free fatty acid, or other substrate/hormone concentrations (24,93,94). Furthermore, in the doses used in the current study, phlorizin had no effect on gut or muscle glucose transport.
We provided experimental proof in a diabetic animal model of type 2 DM that hyperglycemia per se plays a central role in the development of insulin resistance after insulin deficiency (24). Four groups of chronically catheterized, awake, unstressed rats were studied: group 1, sham-operated controls; group 2, 90% surgically pancreatectomized rats; group 3, 90% pancreatectomized rats treated with phlorizin to normalize the plasma glucose profile; and group 4, diabetic rats treated with phlorizin for 6 weeks and subsequently restudied 2 weeks after discontinuation of the phlorizin. Groups 1 to 3 were studied 6 weeks after pancreatectomy or sham pancreatectomy. The pancreatectomized
P.941

rats had mild fasting hyperglycemia and an abnormal meal tolerance test compared with the control rats (24). Insulin secretion in response to the mixed meal was markedly impaired in the diabetic group. Phlorizin treatment normalized the fasting plasma glucose concentration and the postmeal glucose profile without any change in plasma insulin or other hormone/substrate levels. When phlorizin was discontinued, glucose intolerance returned, and the plasma insulin response remained markedly deficient.
Figure 63.1. Rates of total (bar) and oxidative (solid area of the bar) tissue glucose uptake during hyperglycemic and euglycemic clamp studies in patients with type 1 diabetes mellitus after 24 hours of normoglycemia or hyperglycemia. During the euglycemic clamp studies, the rates of glucose disposal were significantly lower after 24-hour hyperglycemia than normoglycemia. This decrease was entirely accounted for by a severe impairment in nonoxidative glucose disposal (open area of the bar). (Reproduced from
Vuorinen-Markkola H, Koivisto VA, Yki-Jarvinen H. Mechanisms of hyperglycemia-induced insulin resistance in whole body and skeletal muscle of type 1 diabetic patients. Diabetes 1992;41:571
, with permission.)
If hyperglycemia is an important regulator of insulin action in vivo, the diabetic animals should be insulin resistant and phlorizin would be expected to improve the defect in insulin-mediated glucose disposal even though insulin secretion remained markedly deficient. To examine this question, we performed euglycemic insulin clamp studies in conscious rats. As can be seen in Fig. 63.2, this is precisely what was observed. Not only was insulin sensitivity improved in phlorizin-treated diabetic rats, it was completely returned to normal. As further proof of the deleterious effect of hyperglycemia on insulin-mediated glucose disposal, discontinuation of phlorizin therapy was associated with a return of the insulin resistance. Because phlorizin failed to enhance insulin sensitivity in sham-operated rats (24), the improvement in insulin action in diabetic rats cannot be attributed to a nonspecific effect of phlorizin.
Our results also demonstrated an inverse relationship between the defect in total-body (primarily muscle) glucose uptake and the plasma glucose level during the meal tolerance test (24). Thus, correction of hyperglycemia with phlorizin resulted in the complete normalization of tissue sensitivity to insulin in diabetic rats without any change in basal or meal-stimulated insulin levels (24,93,94). Furthermore, the close inverse relationship between the postmeal plasma glucose concentrations and the insulin-mediated glucose uptake during insulin clamp studies suggests the presence of a link between the severity of the hyperglycemia and the degree of peripheral insulin resistance (24).
Figure 63.2. Rates of whole-body insulin-mediated tissue glucose uptake during +80 mU/mL (solid bars) and +160 mU/mL (open bars) euglycemic insulin clamp studies performed in four groups of awake, unstressed, chronically catheterized rats: sham-operated controls (Con), partially (90%) pancreatectomized diabetic rats (Panx), and partially pancreatectomized diabetic rats treated with phlorizin for 6 weeks (+Phlor) and again after discontinuation of the phlorizin for 2 weeks (- Phlor). *p < 0.01 versus Con. (Reproduced from
Rossetti L, Smith D, Shulman GI, et al. Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J Clin Invest 1987;79: 1510
, with permission.)
Although correction of hyperglycemia with phlorizin is capable of normalizing meal tolerance and total-body insulin-mediated glucose disposal in diabetic animals, it cannot be concluded a priori that the intracellular pathways of glucose metabolism are intact. To examine this question, we contrasted the effects of vanadate (an insulinomimetic agent) and phlorizin on whole-body and muscle glucose metabolism in 90% pancreatectomized rats (94). As previously described (24), diabetic rats manifested a 30% reduction in whole-body tissue sensitivity to insulin, which was corrected completely by phlorizin treatment. A similar improvement was observed with vanadate (Fig. 63.3). However, phlorizin, unlike vanadate, did not correct the severe impairment in muscle glycogen synthesis (Fig. 63.3). Consistent with this, the maximum velocity (Vmax) of muscle glycogen synthase was reduced in diabetic rats and returned to normal with vanadate but not with phlorizin (94). These results indicate that correction of hyperglycemia in diabetic rats normalizes the defect in glucose transport but does not improve the intracellular abnormality in glycogen synthesis. Thus, the glucose toxicity effect in muscle appears to be directed specifically against the glucose transport system.
In summary, a variety of experimental designs, using both in vivo and in vitro techniques, have demonstrated that chronic, physiologic hyperglycemia is capable of inducing a state of insulin resistance.
Biochemical Mechanisms of Glucose Toxicity
Glucose-Induced Desensitization of Glucose Uptake
At the cellular level, DM in both animals (85,95,96,97) and humans (98,99) is associated with a reduction in insulin-stimulated glucose transport activity in muscle and adipose cells.
P.942

Glucose transport in isolated cell systems is substrate (i.e., glucose) regulated (100,101,102,103,104,105,106,107). In vitro, glucose starvation of fibroblasts (100), 3T3-L1 preadipocytes (101), and cultured and intact muscle cells (102) results in increased basal and insulin-stimulated glucose transport, whereas hyperglycemia leads to a downregulation of glucose transport activity (101,103). Garvey et al. (103) demonstrated the synergistic effect of insulin and glucose in causing desensitization of the glucose transport system in primary cultures of adipose cell. Thus, incubation of muscle cells and adipocytes with a high medium glucose concentration (10–20 mM) leads to a progressive decline in glucose transport activity, loss of cytochalasin B binding activity in both plasma and low-density microsomal membrane fractions, and the development of insulin resistance (100,101,102,103,104). Conversely, glucose starvation leads to an upregulation of the glucose transport system in cultured muscle cells, adipocytes, and fibroblasts (100,104,105,106,107). The studies of Sasson and Cerasi (102,104) are particularly relevant to the preceding discussion. When rat soleus muscle or myocytes were incubated for 24 hours in a high medium glucose concentration, a progressive decline in insulin-mediated glucose transport was demonstrated. This effect of hyperglycemia was both dose and time dependent and fully reversible (102).
Figure 63.3. Rates of whole-body insulin-mediated tissue glucose uptake (top) and of muscle glycogen synthesis (bottom) during +700 mU/mL euglycemic insulin clamp studies performed in four groups of awake, unstressed, chronically catheterized rats: sham-operated controls (Con; open bar), partially (90%) pancreatectomized diabetic rats (Panx; solid bar), partially pancreatectomized diabetic rats treated with vanadate for 4 weeks (Van; widely cross-hatched bar), and partially pancreatectomized diabetic rats treated with phlorizin for 4 weeks (Phlor; gray-shaded bar). *p < 0.01 versus Con. (Reproduced from
Rossetti L, Laughlin MR. Correction of chronic hyperglycemia with vanadate, but not with phlorizin, normalizes in vivo glycogen repletion and in vitro glycogen synthase activity in diabetic skeletal muscle. J Clin Invest 1989;84:892
, with permission.)
However, because several characteristics of the glucose transport system (e.g., responsiveness to insulin, abundance of glucose transporter species) may be altered in cultured cells, caution should be used in extrapolating these results to skeletal muscle in the intact organism. To define the mechanisms responsible for the insulin resistance observed after chronic hyperglycemia in partially pancreatectomized diabetic rats, we measured 3-O-methylglucose transport in isolated adipose cells 6 weeks after pancreatectomy (85). In vivo, 3 to 4 weeks’ normalization of the plasma glucose profile with phlorizin restored insulin-stimulated 3-O-methylglucose transport in adipose cells to normal levels in diabetic rats (85), and these changes paralleled the normalization of whole-body insulin-mediated glucose uptake (24,85,94). Our results indicate that insulin-stimulated glucose transport is diminished in diabetic rats and that the defect in glucose transport is restored to normal with phlorizin treatment. Moreover, the improvement in glucose transport activity was closely correlated with the improvement in in vivo insulin sensitivity.
In summary, strong experimental support for a secondary form of insulin resistance after a sustained elevation in the extracellular glucose levels is available in both humans and animals in vivo and in a variety of isolated cell systems. Although decreased glucose uptake is a common end point in all experimental settings, a potential role of sustained elevations in hexose phosphate levels on the onset and evolution of the desensitization process has also been proposed. In particular, it is intriguing that the onset of glucose toxicity at the level of glucose transport/phosphorylation is accelerated by the concomitant presence of high insulin. This may indicate that enhanced glucose disposal rather than high glucose per se is the primary cause of the downregulation of glucose uptake. An early defect in intracellular glucose disposal—due to different mechanisms depending on the experimental setting (e.g., glycogen “saturation” in the prolonged glucose infusion studies and hypoinsulinemia in diabetic animal models)—may also exacerbate the deleterious effects of high glucose and insulin by amplifying the glucose-induced “signal.” Defective skeletal muscle glycogen synthase activity is a common feature of both diabetic and nondiabetic models of glucose toxicity. Thus, it will be particularly important to examine whether defective insulin action on muscle glycolysis, glycogenolysis, or glycogen synthesis and the consequent increase in hexose phosphate concentrations can contribute to the onset and development of the desensitization process.
Time-Dependent Changes in Insulin Action: Onset and Reversal of Glucose Toxicity
It is conceivable that the acquisition of defects in glucose metabolism and in particular in insulin-mediated glucose uptake after a sustained increase in extracellular glucose levels proceeds in a sequential manner. Several investigators have attempted to address this important issue in culture cells, perfused organ systems, and intact animals and humans. Traxinger and Marshall (108) examined the time required for the development of desensitization of the glucose transport system and for its recovery in primary culture of adipose cells exposed to high glucose and insulin. They described a strong correlation between rates of recovery and desensitization in this cell system with an average half-life of 3 hours for the induction of insulin resistance and of 3.3 hours for the recovery of insulin responsiveness. Marshall et al. (109) also proposed that the desensitization of the glucose transport system, mediated by the glucosamine (GlcN) pathway, develops through an early stage characterized by impaired translocation of glucose transporters in response to insulin and later effects on glucose transporter gene expression. In perfused rat hindquarters, Richter, Hansen, and colleagues (87,88,89) examined the time course of the glucose-induced decrease in insulin responsiveness. The appearance of insulin resistance in
P.943

this system was extremely rapid (<2 hours) and related to glucose in a dose-dependent manner, and its extent was enhanced by a concomitant elevation in the perfusate insulin levels (87,88,89). Glucose-mediated desensitization of hindquarter glucose uptake was obtained even in the absence of insulin and of a significant decline in glycogen synthase activity, suggesting a primary effect on the glucose transport system (87).
Unfortunately, extrapolation of these in vitro data to the in vivo condition may be misleading. In fact, the disposition of glucose in response to insulin stimulation under these experimental conditions is often characterized by a marked decrease in insulin’s action on oxidative pathways and an enhanced disposal in anaerobic glycolysis (110,111). In addition, in cultured cell systems, the relative role of the various glucose transporter species involved in the response to insulin may differ from those operating in vivo. These limitations are particularly relevant when examining the time courses and relative contribution of different metabolic/biochemical alterations to the glucose-induced desensitization.
Some information on the development of peripheral insulin resistance is also available in diabetic animal models. The time course of the onset of insulin resistance and the underlying molecular mechanisms have often been shown to be divergent in different insulin target tissues; in particular, discrepancies were apparent between adipose cells and skeletal muscle (112,113). In adipose cells, decreased glucose transporter type 4 (GLUT-4) messenger RNA (mRNA) and protein have been consistently reported as early as 3 days after streptozotocin-induced DM in rats (113,114,115,116,117). However, near normalization of the plasma glucose concentration by phlorizin treatment was equally ineffective in restoring GLUT-4 mRNA to normal levels after 3 days (118) and after 3 weeks (85), suggesting that the decrease in GLUT-4 gene expression in these diabetic models was the result of insulin deficiency rather than of chronic hyperglycemia. In skeletal muscle, we reported a severe impairment in insulin-mediated glucose uptake but normal skeletal muscle GLUT-4 protein 7 days after the induction of insulinopenic DM by streptozotocin (113). Decreased skeletal muscle GLUT-4 protein was shown only 14 days poststreptozotocin (113,119). Although the latter study did not distinguish between the consequences of hyperglycemia, hypoinsulinemia, and other associated metabolic alterations, it underscores the sequential appearance of biochemical and molecular defects in insulin-sensitive tissues. Dimitrakoudis et al. (120) showed a significant increase in GLUT-4 protein in a skeletal muscle plasma membrane fraction only 2 days after normalization of the fasting plasma glucose concentration by phlorizin in streptozotocin diabetic rats. However, it is not known whether a reversal of the hyperglycemic state of such a short duration has any beneficial effect on insulin stimulation of skeletal muscle glucose uptake. Finally, a moderate impairment in insulin-mediated glucose uptake (18%–24% decrease) was demonstrated in patients with type 1 DM after just 24 hours of sustained hyperglycemia (90,91,92).
In summary, large variations have been reported in the time course of the onset of metabolic defects after sustained elevation of glucose levels. These differences are mostly due to the selection of the experimental system and perhaps to the concomitant insulin levels. In vivo, although decreased insulin stimulation of glucose uptake is an early effect of severely insulinopenic DM, there is no information on the time interval required for its reversal after correction of hyperglycemia. The definition of the progressive appearance and reversal of glucose-induced metabolic alterations appears to be germane to an understanding of the mechanisms of glucose-induced insulin resistance that are operating in vivo. For example, if an early defect in intracellular glucose disposal precedes the onset of defective insulin-mediated glucose uptake, it may suggest that interplay between increased glucose availability (hyperglycemia) and defective disposal through insulin-sensitive pathways is the most plausible explanation for the in vivo expression of glucose-induced insulin resistance. It is particularly important to examine whether defective insulin action on muscle glycogenolysis and the consequent increase in hexose phosphate concentrations precedes the onset of desensitization of the glucose transport system.
Studies have attempted to define the mechanisms by which prolonged glucose and insulin infusions lead to decreased insulin-mediated glucose uptake in skeletal muscle. Hagar et al. (86) demonstrated a severe downregulation in skeletal muscle insulin-stimulated glucose uptake in 72-hour glucose-infused rats, in the absence of any alteration in insulin binding. Similarly, Hansen et al. (89) induced marked skeletal muscle insulin resistance in glucose-infused hindquarters in the absence of a detectable alteration in muscle insulin receptor tyrosine kinase activity. Consistent with these observations, we could not demonstrate any alteration in muscle and liver insulin receptor tyrosine kinase activity in insulin-resistant diabetic rats, and no change after normalization of plasma glucose concentrations and insulin-mediated glucose disposal by phlorizin treatment (121). In vitro studies in a primary culture of adipose cells demonstrated complete dissociation between the insulin effect on glucose uptake (decreased) and that on protein metabolism (stimulated) after 7-hour incubations with glucose, insulin, and amino acids (122,123). Together, these studies appear to suggest that the glucose-induced desensitization of muscle glucose uptake should be attributed to factors other than impaired early insulin signal transduction.
A common interpretative problem with these in vivo studies is the difficulty in discerning the relative role (primary or secondary) of the marked downregulation of glucose transport and glycogen synthase
P.944

activity in the sequence of events that leads to the desensitization of the glucose transport system. Hansen et al. (89) have argued for the primary role of the glucose transport defect because both skeletal muscle free glucose and glucose-6-phosphate levels were not increased in the glucose-infused hindquarters compared with the control study. Similarly, Vuorinen-Markkola et al. (91) showed a marked decrease in insulin action on skeletal muscle glucose uptake and glycogen storage in muscle biopsies from patients with type 1 DM infused with glucose for 24 hours in the absence of any detectable increase in free glucose and glucose-6-phosphate. Using a completely different experimental approach (i.e., the reversal of glucose-induced insulin resistance in phlorizin-treated diabetic rats), we reached a similar conclusion (85,94). In fact, surgical reduction of β-cell mass and the consequent hypoinsulinemia and hyperglycemia caused the onset of defects in both skeletal muscle glucose transport/phosphorylation and glycogen synthase activity. However, normalization of the plasma glucose concentration by phlorizin treatment restored insulin-mediated glucose uptake to normal levels without improving the decreased Vmax of skeletal muscle glycogen synthase (94). We reported that 3 to 4 weeks’ normalization of the plasma glucose profile with phlorizin can restore insulin-stimulated 3-O-methylglucose transport in adipose cells to normal levels in diabetic rats (85). These effects on glucose transport activity occurred in the absence of alterations in the expression of the two species of glucose transporters present in adipose cells (i.e., GLUT-4 and GLUT-1) or in their translocation to the plasma membrane in response to insulin. Thus, we suggested that it might result from changes in the functional activity of glucose transporters. Similarly, we were unable to demonstrate any recovery of GLUT-4 protein and mRNA in skeletal muscle of diabetic rats chronically treated with phlorizin (B.B. Kahn and L. Rossetti, unpublished observations). Consistent with our findings, Sivitz et al. (118) reported no change in GLUT-4 mRNA in adipose cells from streptozotocin diabetic rats after near-normalization of the plasma glucose concentration by phlorizin treatment, and concluded that “the relative glycemic state does not influence GLUT-4 mRNA expression in vivo.” Dimitrakoudis et al. (120) reported that, in phlorizin-treated streptozotocin diabetic rats, although GLUT-4 protein in skeletal muscle tends to return toward normal levels in the plasma membrane fraction, it does not change in the intracellular pool. Although the latter study did not examine the effect of insulin stimulation on GLUT-4 translocation, it suggests that an increased translocation of GLUT-4 to the plasma membrane is a potential mechanism for the improved skeletal muscle glucose uptake after correction of hyperglycemia by phlorizin treatment.
Thus, defective insulin stimulation of the glucose transport system has been proposed as the major cellular manifestation of prolonged hyperglycemia. Although glucose-mediated alterations in the abundance of GLUT-4 protein have not been firmly established, defects in the translocation or functional activity of glucose transporters have been proposed.
The Glucosamine Hypothesis
Using primary cultures of adipose cells, Marshall and colleagues (124) suggested that the desensitization of the glucose transport system in cells incubated with high levels of glucose and insulin required the metabolism of glucose/hexose phosphates in a quantitatively minor pathway of intracellular glucose utilization (i.e., the hexosamine biosynthesis pathway; Fig. 63.4), which is initiated by the conversion of fructose-6-phosphate to glucosamine-6-phosphate by the enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). They first showed that insulin had a permissive effect on glucose-induced desensitization that was independent of changes in insulin receptor binding (83,103). The researchers suggested that it might act by promoting glucose uptake and metabolism and that the latter was indispensable for the development of glucose-induced desensitization. The potential role of the hexosamine biosynthesis pathway was suggested by the observation that, in addition to glucose and insulin, the amino acid glutamine (donor of amido groups, which are needed for GFAT activity) was also required for the onset of desensitization in primary culture of adipose cells (125). Strong evidence for this “hexosamine hypothesis” was provided by experiments inducing desensitization of the glucose transport system after prolonged incubations with GlcN in the absence of glucose, and the prevention of desensitization in the presence of glucose and insulin by the inhibition of GFAT (124). Further experimental support for this hypothesis comes from studies indicating that the metabolism of glucose through the hexosamine biosynthesis pathway may mediate some of insulin’s effects on pyruvate kinase (126) and glycogen synthase (127,128). Thus, in these isolated cell systems, hyperactivity of the hexosamine biosynthesis pathway caused, and its inhibition prevented, the development of desensitization of the glucose transport system. Whether this negative feedback system is operating in the skeletal muscle of intact animals is not known.
In summary, some evidence supports the notion that the hexosamine biosynthesis pathway functions as a regulatory pathway capable of desensitizing the glucose transport system to insulin in adipose tissue and skeletal muscle. This would represent an attractive unifying hypothesis for the presence of defective insulin action in glucose uptake in insulin-resistant states. In fact, increased routing of glucose carbons through the GlcN pathway could result from a sustained elevation in intracellular fructose-6-phosphate concentrations because of either increased glucose availability or decreased disposal through alternative major pathways (i.e., glycolysis or glycogen synthesis), or both. Thus, the mechanism by which hyperglycemia causes the impairment of insulin-stimulated glucose transport may
P.945

unveil a more fundamental feedback control mechanism that downregulates cellular glucose uptake in response to a sustained increase in the intracellular availability of hexose phosphates. If such a regulatory pathway was operating in skeletal muscle in vivo and was capable of desensitizing the glucose transport system to insulin, it would help explain the universal presence of defective insulin action on glucose transport/phosphorylation in insulin-resistant states.
Figure 63.4. Schematic representation of the common steps in the intracellular metabolism of glucose and glucosamine. In skeletal muscle and adipose cells, glucose enter the cells through the action of glucose transporters (mostly GLUT-4 in the presence of insulin) and is then rapidly phosphorylated to glucose-6-phosphate by the action of low-Km hexokinases. Most of the glucose-6-phosphate is metabolized through glycogen synthesis, glycolysis, and pentose phosphate shunt (not shown). However, approximately 1% to 3% of the incoming glucose is used for the formation of glucosamine-6-phosphate through the action of the enzyme glutamine: fructose-6-phosphate amidotransferase. Glucosamine is also transported by the glucose transporter system and then directly enters the hexosamine biosynthetic pathway at the level of glucosamine-6-phosphate. Further metabolism of glucosamine-6-phosphate ultimately leads to the formation of uridine diphosphate (UDP)-N-acetylglucosamine, which is the precursor for the formation of sialic acid and oligosaccharide side chains of proteins and lipids. (Reproduced from
Rossetti L, Hawkins M, Chen W, et al. In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J Clin Invest 1995; 96:132
, with permission.)
We therefore aimed to investigate whether the hexosamine biosynthetic pathway may function as a regulatory pathway capable of desensitizing the glucose transport system to insulin in skeletal muscle in vivo. For this purpose, we examined whether glucose-induced desensitization of insulin-mediated glucose uptake can be induced in the absence of sustained hyperglycemia through increased exogenous availability of glucosamine-6-phosphate, and whether this effect is modulated by concomitant chronic hyperglycemia. We monitored glucose uptake, glycolysis, and glycogen synthesis during insulin clamp studies in 6-hour fasted conscious rats in the presence of a sustained (7-hour) increase in GlcN availability (129).
The GlcN hypothesis, as proposed by Marshall and colleagues, is based on the observation that approximately 1% to 3% of the glucose metabolized in insulin-sensitive tissues is through the GlcN biosynthetic pathway (Fig. 63.4). Because the glucose concentrations required to induce insulin resistance in short-term studies is approximately 20 mM and the affinity of GlcN for the glucose transport system is approximately fourfold lower than that of glucose, it can be calculated that extracellular GlcN concentrations of 0.8 to 2.4 mM are required to simulate the glucose flux through the GlcN pathway under conditions that cause maximal desensitization of the glucose transport system to insulin stimulation. Similarly, the initial glucose flux during glucose “desensitization protocols” (high glucose + high insulin) is approximately 420 mmol/kg/min, of which 8 mmol/kg/min (approximately 2%) may enter the GlcN pathway. Taking into account the lower affinity of GlcN versus glucose for the glucose transport system, it can be calculated that the infusion of GlcN at a rate of approximately 32 mmol/kg/min would be required to reproduce the flux of glucose to glucosamine-6-phosphate during glucose-induced insulin resistance. Based on these estimates, we chose to increase the plasma GlcN concentrations to approximately 1.2 mM by infusing GlcN at a rate of 30 mmol/kg/min. These circulating levels of GlcN closely approximate the concentration of GlcN (1 mM for 5 hours) required for maximal desensitization of the glucose transport system in isolated adipose cells (124), but are lower than the GlcN concentration (10 mM for 60–180 minutes) that induced decreased insulin-mediated glucose transport in isolated muscle (127).
The effect of GlcN infusions on insulin-mediated glucose uptake and metabolism (Figs. 63.5 and 63.6) was then examined (129). Plasma GlcN concentrations were increased to approximately 1.2 mM during prolonged euglycemic hyperinsulinemic clamp studies. The infusion of GlcN was associated with a time-dependent decline in the rates of glucose uptake and glycogen synthesis during the insulin clamp studies (129). After approximately 5 hours, GlcN infusion caused an approximately 32% decrease in insulin-mediated glucose uptake, despite no impairment of muscle glycogen synthase kinetics or increase in glucose-6-phosphate concentrations. Both glycogen synthesis and glycolysis were significantly impaired during the second clamp study compared with the first or with saline control studies (Fig. 63.6). However, although glycolysis was significantly decreased by 22%, the decline in glycogen synthesis was more severe and accounted for approximately 80% of the decreased Rd. In additional studies, we established that the GlcN-induced decrease in Rd reached a maximal effect by 5 hours, and that the time required for a half-maximal effect of the amino sugar on Rd was approximately 3 hours (129). The proportionally greater decrease in glycogen synthesis suggests that a step beyond glucose transport/phosphorylation also is involved in GlcN-induced insulin resistance. Because muscle glycogen synthase was normally activated by insulin, it is likely that the marked decrease in muscle uridine diphosphate (UDP)-glucose concentrations contributed to the impairment of glycogen synthesis (Fig. 63.7). Thus, increased GlcN availability can induce peripheral insulin resistance in vivo in conscious nondiabetic rats in the absence of prolonged exposure to high glucose and insulin.
Can the increased routing of glucose in the GlcN pathway cause further desensitization of glucose uptake in diabetic rats? We reasoned that if the impaired insulin action on skeletal muscle
P.946

glucose uptake in diabetic rats is due to a chronic increase in the flux of glucose carbons through the GlcN pathway, the short-term effects of GlcN infusion on insulin-mediated glucose uptake may be blunted in chronically hyperglycemic diabetic rats versus nondiabetic rats. Similarly, we hypothesized that the ability of GlcN infusions to generate peripheral insulin resistance may be restored in diabetic rats by normalizing the plasma glucose concentration with phlorizin. Thus, we examined the short-term regulation of glucose uptake and intracellular glucose disposal by the enhanced carbon flux through the GlcN pathway in chronically hyperglycemic diabetic rats and in phlorizin-treated diabetic rats (129). GlcN infusions generated similar plasma GlcN and muscle UDP-N-acetylglucosamine concentrations in all groups. However, peripheral glucose uptake, glycolysis, and glycogen synthesis were not significantly affected by increased GlcN availability in diabetic rats (Figs. 63.5 and 63.6). Long-term normalization of the plasma glucose concentrations by phlorizin treatment restored insulin-mediated glucose uptake, but not glycogen synthesis, to normal in diabetic rats. Correction of hyperglycemia also restored the marked effects of the GlcN infusion on insulin-mediated glucose uptake, glycolysis, and glycogen synthesis (Figs. 63.5 and 63.6). These alterations in glucose fluxes occurred in the absence of significant changes in the kinetics of muscle glycogen synthase and hexokinase and in the concentration of glucose-6-phosphate (129).
Figure 63.5. Rates of glucose disappearance (top) and of glucose infusion (bottom) in control rats (CON), 90% pancreatectomized diabetic rats (PANX), and phlorizin-treated diabetic rats (PHLOR) in the presence of similar concentrations of glucosamine. Results obtained during identical insulin clamp studies performed during the first 2 hours and the last 2 hours of the 7-hour glucosamine infusion. *p < 0.01 for 5 to 7 hours versus 0 to 2 hours. (Reproduced from
Rossetti L, Hawkins M, Chen W, et al. In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J Clin Invest 1995;96:132
, with permission.)
Figure 63.6. Rates of glycolysis (top) and glycogen synthesis (bottom) in control rats (CON), 90% pancreatectomized diabetic rats (PANX), and phlorizin-treated diabetic rats (PHLOR) in the presence of similar concentrations of glucosamine. Results obtained during identical insulin clamp studies performed during the first 2 hours and the last 2 hours of the 7-hour glucosamine infusion. *p < 0.01 for 5 to 7 hours versus 0 to 2 hours. (Reproduced from
Rossetti L, Hawkins M, Chen W, et al. In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J Clin Invest 1995;96: 132
, with permission.)
Figure 63.7. Skeletal muscle concentrations of uridine diphosphate (UDP)-N-acetylglucosamine (UDP GlcNA) and UDP-glucose at the end of insulin clamp studies in combination with saline and glucosamine (GlcN) infusions. (Reproduced from
Rossetti L, Hawkins M, Chen W, et al. In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J Clin Invest 1995;96: 132
, with permission.)
Because a sustained increase in the availability of GlcN caused a marked impairment in glucose uptake, glycogen synthesis, and glycolysis in the absence of significant elevations in muscle glucose-6-phosphate concentrations, GlcN is likely to act at an early stage of glucose uptake. Although most previous studies point toward impairment of the glucose transport system as the major mechanism of action, an alteration in the phosphorylation of glucose cannot be excluded.
The aforementioned studies demonstrate that increased GlcN availability in vivo impairs insulin’s ability to stimulate glucose uptake and glycogen synthesis in normoglycemic but not in chronically hyperglycemic conscious rats. These observations indicate that increased flux through the GlcN pathway can generate marked insulin resistance in skeletal muscle in vivo and that this effect is not additive to the insulin resistance induced by chronic hyperglycemia. Because the ability of prolonged GlcN infusions to induce peripheral insulin resistance is lost in diabetic rats and is restored in phlorizin-treated diabetic rats, the deleterious effects of chronic hyperglycemia and GlcN infusions on peripheral insulin resistance do not appear to be additive and they may act on a common pathway. This supports the hypothesis that increased flux through the GlcN pathway in skeletal muscle may play an important role in glucose-induced insulin resistance in vivo.
Decreased Disposal of Hexose Phosphates Leads to Defective Insulin Stimulation of Glucose Uptake: Is this a Manifestation of Glucose Toxicity?
The glucose–fatty acid cycle as first proposed by Randle et al. (130) is centered on substrate competition between carbohydrates and lipids, which ultimately leads to decreased rate of hexose phosphate (particularly fructose-6-phosphate) disposal when the availability of fatty acids is increased. Several hypotheses have been advanced regarding the specific biochemical events involved in this cycle (reviewed in reference 131). The increased formation of acetyl-coenzyme A (CoA) results in inhibition
P.947

of pyruvate dehydrogenase and stimulation of pyruvate carboxylase. Increased concentrations of cytosolic citrate inhibit the activity of phosphofructokinase and enhance the activity of acetyl-CoA carboxylase. The latter effect combined with the expanded acetyl-CoA pool results in marked increase in malonyl-CoA, which in turn inhibits carnitine palmitoyltransferase-1 and fatty acid oxidation and results in the accumulation of long chain acyl-CoAs (LC-CoAs) and tissue triglyceride. Although it is now well established that increased tissue levels of malonyl-CoA, LC-CoAs, and triglyceride are correlated with insulin resistance in humans and in animal models (131), the biochemical steps responsible for decreased insulin stimulation of glucose uptake under these conditions remain to be delineated. We have proposed that the inhibition of phosphofructokinase plays a central role by shifting fructose-6-phosphate from the glycolytic to the hexosamine biosynthetic pathway (132). Increased carbon flux in this pathway has been shown to induce defects in insulin action on glucose uptake and GLUT-4 translocation/function (129,133,134,135,136). This may suggest the existence of redundant energy-sensing mechanisms in skeletal muscle, which may interact at several levels. For example, the malonyl-CoA fuel-sensing and the hexosamine pathways may be concomitantly activated when glucose and fatty acid fluxes are increased. They may share common biochemical targets, such as selective isoforms of protein kinase C (131,137,138), and may engage in cross-talk through alterations in common effectors such as cytosolic citrate (131).
Overall, regardless of the signaling events involved, “substrate competition” leading to decreased utilization of hexose phosphates is a common mechanism by which insulin resistance at the level of glucose transport/phosphorylation can be acquired.
Intracellular Glucose Toxicity: Implications for Type 2 Diabetes Mellitus
Available evidence points to a multifactorial origin of insulin resistance in type 2 DM. However, despite some unique features, there are some biochemical/metabolic characteristics that are common to most people once the syndrome of insulin resistance is fully expressed. In particular, impairment in the ability of insulin to promote skeletal muscle glucose uptake is a common end point of these conditions, one that ultimately requires the impairment of insulin action on glucose transport or phosphorylation.
We and others have suggested that the similarity of all insulin resistance syndromes, regardless of their primary cause, might be due to the deleterious effects of chronic hyperglycemia per se on insulin action, particularly on skeletal muscle glucose transport. The latter hypothesis may explain the evolution of syndromes of insulin resistance, which are associated with some degree of hyperglycemia. However, peripheral insulin resistance is also a feature of people with normal glucose tolerance. In particular, defects in insulin action on skeletal muscle glucose transport/phosphorylation and on glucose oxidation, glucose storage, and glycogen synthase have been shown in these subjects. A common primary defect in insulin activation of both skeletal muscle glucose transport and glycogen synthase remains a viable etiologic hypothesis. However, the variety of pathophysiologic conditions (e.g., nondiabetic siblings of diabetic parents, obesity, hypertension) and ethnic backgrounds (e.g., European and American whites, Pima Indians, Mexican Americans) involved appears to indicate that the primary genetic or acquired defects are likely to be different between and within groups.
A potential explanation is that either a primary cause of moderate glucose intolerance (e.g., β-cell or hepatic defect) or a primary impairment in a major pathway of intracellular glucose disposal (e.g., glycogen synthesis or glycolysis) may underlie the full expression of the insulin resistance syndrome through a common mechanism.
The GlcN biosynthetic pathway functions as a regulatory pathway in skeletal muscle in vivo and it is capable of desensitizing the glucose transport system to insulin. Thus, a unifying hypothesis for the universal presence of defective insulin action on glucose transport/phosphorylation in insulin-resistant states may be advanced. Increased routing of glucose carbons through the GlcN pathway could result from a sustained elevation in intracellular fructose-6-phosphate concentrations due to either increased glucose availability or decreased disposal through alternative major pathways, or both (i.e., glycolysis and glycogen synthesis; Fig. 63.8).
Thus, the mechanism by which hyperglycemia causes impaired insulin action on glucose transport/phosphorylation may shed light on a more fundamental feedback control system
P.948

that downregulates cellular glucose uptake in response to a sustained increase in the intracellular availability of hexose phosphates.
Figure 63.8. Unifying hypothesis for the pathogenesis of insulin resistance in type 2 diabetes mellitus. Genetic or acquired alterations in carbohydrate metabolism may generate decreased insulin-mediated uptake of glucose in skeletal muscle through a common biochemical mechanism. In fact, either a primary cause of moderate glucose intolerance (e.g., B-cell or hepatic defect) or a primary impairment in a major pathway of intracellular glucose disposal (e.g., glycogen synthesis or glycolysis) is likely to result in a sustained elevation in intracellular hexose phosphate concentrations (intracellular glucose toxicity). This in turn leads to the increased routing of glucose carbons (fructose-6-phosphate) into the hexosamine biosynthesis pathway because of either increased glucose availability (high insulin and/or glucose) or decreased disposal through alternative major pathways, or both (i.e., glycolysis and glycogen synthesis).
References
1. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977.
2. Reichard P, Nilsson B-Y, Rosenqvist U. The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus. N Engl J Med 1993;329:304.
3. Job D, Eshwege E, Guyot-Argenton C, et al. Effect of multiple daily insulin injection on the course of retinopathy. Diabetes 1976;25: 463.
4. Lauritzen T, Frost-Larsen K, Larsen HW, et al. Steno Study Group: two year experience with continuous subcutaneous insulin infusion in relation to retinopathy and neuropathy. Diabetes 1985;34(suppl 3):74.
5. Deckert T, Lauritzen T, Parving H, et al. Steno Study Group: effect of two years of strict metabolic control on kidney function in long-term insulin dependent-diabetics. Diabetic Nephropathy 1983;2:6.
6. Feldt-Rasmussen B, Mathiesen ER, Deckert T. Effect of two years of strict metabolic control on progression of incipient nephropathy in insulin-dependent diabetes. Lancet 1986;2:1300.
7. Dahl-Jorgensen K, Brichmann-Hanssen O, Hanssen KF, et al. Effect of near normoglycemia for two years on progression of early diabetic retinopathy, nephropathy, and neuropathy: the Oslo Study. BMJ 1986;293:1194.
8. Rosenstock J, Friberg T, Raskin P. The effect of glycemic control on the microvascular complications in patients with type I diabetes mellitus. Am J Med 1986;81:1012.
9. Raskin P, Pietri A, Unger R, et al. The effect of diabetic control on skeletal muscle capillary basement membrane width in patients with type I diabetes mellitus. N Engl J Med 1983;309:1546.
10. Krolewski AS, Canessa M, Warram JH, et al. Predisposition to hypertension and susceptibility to renal disease in insulin-dependent diabetes mellitus. N Engl J Med 1988;319:140.
11. Engerman R, Bloodworth JMB, Nelson S. Relationship of microvascular disease in diabetes to metabolic control. Diabetes 1977;26:760.
12. Mauer SM, Steffes MW, Sutherland DER, et al. Studies of the rate of regression of the glomerular lesions in diabetic rats treated with pancreatic islet transplantation. Diabetes 1975;24:280.
13. Johnsson SL. Retinopathy and nephropathy in diabetes mellitus: comparison of the effect of two forms of treatment. Diabetes 1960;9:1.
14. Pirart J. Diabetes mellitus and its degenerative complications: a prospective study of 4,400 patients observed between 1947 and 1973. Diabetes Metab 1977;3:97.
15. Eschwege E, Richard JL, Thibult N, et al. Coronary heart diseased mortality in relation with diabetes, blood glucose and plasma insulin levels: the Paris Prospective Study, ten years later. Horm Metab Res 1985;15(suppl):41.
16. Hostetter MK. Handicaps to host defenses: effect of hyperglycemia on C3 and Candida albicans. Diabetes 1990;39:271.
17. Rayfield EJ, Ault MJ, Keusch GT, et al. Infection and diabetes: the case for glucose control. Am J Med 1982;72:439.
18. Unger RH, Grundy S. Hyperglycaemia as an inducer as well as a consequence of impaired islet cell function and insulin resistance: implications for the management of diabetes. Diabetologia 1985; 28:119.
19. Bonner-Weir S, Trent DF, Weir GC. Partial pancreatectomy in the rat and subsequent defect in glucose-induced insulin release. J Clin Invest 1983;71:1544.
20. Rossetti L, Shulman GI, Zawalich W, et al. Effect of chronic hyperglycemia on in vivo insulin secretion in partially pancreatectomized rats. J Clin Invest 1987;80:1037.
21. Leahy JL, Cooper HE, Weir GC. Chronic hyperglycemia is associated with impaired glucose influence on insulin secretion. J Clin Invest 1986;77:908.
22. Leahy JL, Bonner-Weir S, Weir GC. Minimal chronic hyperglycemia is a critical determinant of impaired insulin secretion after an incomplete pancreatectomy. J Clin Invest 1988;81:1407.
23. Robertson RP. Type II diabetes, glucose “non-sense,” and islet cell desensitization. Diabetes 1989;38:1501.
24. Rossetti L, Smith D, Shulman GI, et al. Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J Clin Invest 1987;79:1510.
25. Yki-Jarvinen H, Helve E, Koivisto VA. Hyperglycemia decreases glucose uptake in type I diabetes. Diabetes 1987;36:892.
26. Rossetti L, Giaccari A, DeFronzo RA. Glucose toxicity. Diabetes Care 1990;13:610.
27. Yki-Jarvinen H. Glucose toxicity. Endocr Rev 1992;13:415.
28. DeFronzo RA, Hendler R, Simonson D. Insulin resistance is a prominent feature of insulin dependent diabetes. Diabetes 1982;31:795.
29. Yki-Jarvinen, Koivisto VA. Natural history of insulin resistance in type I diabetes. N Engl J Med 1986;315:224.
30. Himsworth HP, Kerr RB. Insulin-sensitive types of diabetes mellitus. Clin Sci 1942;4:120.
31. DeFronzo RA, Diebert D, Hendler R, et al. Insulin sensitivity and insulin binding in maturity onset diabetes. J Clin Invest 1979;63: 939.
32. Kolterman OG, Gray RS, Griffin J, et al. Receptor and postreceptor defects contribute to the insulin resistance in noninsulin-dependent diabetes mellitus. J Clin Invest 1981;68:957.
33. Rizza RA, Mandarino LJ, Gerich JE. Mechanism and significance of insulin resistance in noninsulin-dependent diabetes mellitus. Diabetes 1981;30:990.
34. DeFronzo RA, Gunnarsson R, Bjorkman O, et al. Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin dependent (type II) diabetes mellitus. J Clin Invest 1985;76:149.
35. Kobberling J, Tillil H. Empirical risk figures for first-degree relatives of non-insulin-dependent diabetics. In: Kobberling J, Tsttersal R, eds. The genetics of diabetes mellitus. London: Academic, 1982:201.
36. Newman B, Selby JV, King MC, et al. Concordance for type 2 (non–insulin-dependent) diabetes mellitus in male twins. Diabetologia 1987;30:763.
37. Barner AH, Eff C, Leslie RD, et al. Diabetes in identical twins: a study of 2 pairs. Diabetologia 1981;20:87.
38. Lillioja S, Mott DM, Zawadski JK, et al. In vivo insulin action is a familial characteristic in nondiabetic Pima Indians. Diabetes 1987;36: 1329.
39. Lillioja S, Bogardus C. Obesity and insulin resistance: lessons learned from the Pima Indians. Diabetes Metab Rev 1988;4:515.
40. Eriksson J, Franssila-Kallunki A, Edstrand A, et al. Early metabolic defects in persons at increased risk of non–insulin-dependent diabetes mellitus. N Engl J Med 1989;321:337.
41. Vaag A, Henriksen JE, Beck-Nielsen H. Decreased insulin activation of glycogen synthase in skeletal muscle in young nonobese Caucasian first-degree relatives of patients with non-insulin-dependent diabetes mellitus. J Clin Invest 1992;89:782.
42. Gulli G, Ferrannini E, Stern M, et al. The metabolic profile of NIDDM is fully established in glucose-tolerant offspring of two Mexican-American NIDDM parents. Diabetes 1992;41:1575.
43. DeFronzo RA, Simonson D, Ferrannini E. Hepatic and peripheral insulin resistance: a common feature of insulin-independent and non–insulin-dependent diabetes. Diabetologia 1982;23:313.
44. Beck-Nielsen H, Richelsen B, Hasling C, et al. Improved in vivo insulin effect during continuous subcutaneous insulin infusion in patients with IDDM. Diabetes 1984;33:832.
45. Yki-Jarvinen H, Koivisto VA. Continuous subcutaneous insulin infusion therapy decreases insulin resistance in type I diabetes. J Clin Endocrinol Metab 1984;58:659.
46. Yki-Jarvinen H, Koivisto VA. Insulin sensitivity in newly diagnosed type I diabetics after ketoacidosis and after three months of insulin therapy. J Clin Endocrinol Metab 1984;59:371.
P.949

47. Lager I, Lonnroth P, Von Schenck H, et al. Reversal of insulin resistance in type I diabetes after treatment with continuous subcutaneous insulin infusion. BMJ 1983;287:1001.
48. Kurszynska YT, Petranyi G, Home PD, et al. Muscle enzyme activity and insulin sensitivity in type I (insulin-dependent) diabetes mellitus. Diabetologia 1986;29:699.
49. Gray RS, Cowan P, Duncan LJP, et al. Reversal of insulin resistance in type I diabetes following initiation of insulin treatment. Diabet Med 1986;3:18.
50. Garvey WT, Olefsky JM, Griffin J, et al. The effect of insulin treatment on insulin secretion and insulin action in type II diabetes mellitus. Diabetes 1985;34:222.
51. Foley J, Kashiwagi A, Verso MA, et al. Improvement in in vitro insulin action after one month of insulin therapy in obese noninsulin-dependent diabetics. J Clin Invest 1983;72:1901.
52. Nankervis A, Proietto J, Aitken P, et al. Differential effects of insulin therapy on hepatic and peripheral insulin sensitivity in type 2 (non–insulin-dependent) diabetes. Diabetologia 1982;23:320.
53. Greenfield M, Lardinois C, Doberne L, et al. Metabolic effects of intensive insulin therapy in patients with non-insulin-dependent diabetes (NIDDM). Diabetes 1982;31(suppl 2):58A.
54. Andrews WJ, Vasquez B, Nagulesparan M, et al. Insulin therapy in obese, non–insulin-dependent diabetes induces improvements in insulin action and secretion that are maintained for two weeks after insulin withdrawal. Diabetes 1984;33:634.
55. Scarlett JA, Garvey RS, Griffin J, et al. Insulin treatment reverses the insulin resistance of type II diabetes mellitus. Diabetes Care 1982;5: 353.
56. Gormley MJJ, Hadden DR, Woods R, et al. One month’s insulin treatment of type II diabetes: the early and medium-term effects following insulin withdrawal. Metabolism 1986;35:1029.
57. Hidaka H, Nagulesparan M, Klimes I, et al. Improvement of insulin secretion but not insulin resistance after short term control of plasma glucose in obese type II diabetics. J Clin Endocrinol Metab 1982;54: 217.
58. Scarlett JA, Kolterman OG, Ciaraldi TP, et al. Insulin treatment reverses the postreceptor defect in adipocyte 3-O-methylglucose transport in type II diabetes mellitus. J Clin Endocrinol Metab 1983;56: 1195.
59. Savage PJ, Bennion LO, Flock EV, et al. Diet-induced improvement of abnormalities in insulin and glucagon secretion and in insulin receptor binding in diabetes mellitus. J Clin Endocrinol Metab 1979; 48:999.
60. Henry RR, Scheaffer L, Olefsky JM. Glycemic effects of intensive caloric restriction and isocaloric refeeding in noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1985;61:917.
61. Henry RR, Wiest-Kent TA, Scheaffer L, et al. Metabolic consequences of very-low-calorie diet therapy in obese non-insulin-dependent diabetic and nondiabetic subjects. Diabetes 1986;35:155.
62. Nagulesparan N, Savage PJ, Bennion LJ, et al. Diminished effect of caloric restriction on control of hyperglycemia with increasing duration of type II diabetes mellitus. J Clin Endocrinol Metab 1981;53:560.
63. Henry RR, Wallace P, Olefsky JM. Effects of weight loss on mechanisms of hyperglycemia in obese non–insulin dependent diabetes mellitus. Diabetes 1986;35:990.
64. Beck-Nielsen H, Pedersen O, Sorensen NS. Effects of dietary changes on cellular insulin binding and in vivo insulin sensitivity. Metabolism 1980;29:482.
65. Laakso M, Uusitupa M, Takala J, et al. Effects of hypocaloric diet and insulin therapy on metabolic control and mechanisms of hyperglycemia in obese non–insulin-dependent diabetic subjects. Metabolism 1988;37:1092.
66. Zawadski JK, Bogardus C, Foley JE. Insulin action in obese non–insulin-dependent diabetics and in their isolated adipocytes before and after weight loss. Diabetes 1987;36:227.
67. Walshe K, Andrews WJ, Sheridan B, et al. Three months energy restricted diet does not induce peripheral insulin resistance in new diagnosed non insulin dependent diabetics. Horm Metabol Res 1987; 19:197.
68. Kolterman OG, Grag RS, Shapiro G, et al. The acute and chronic effects of sulfonylurea therapy in type II diabetes. Diabetes 1984; 33:346.
69. Firth RG, Bell PM, Rizza RA. Effects of tolazamide and exogenous insulin on insulin action in patients with non–insulin-dependent diabetes mellitus. N Engl J Med 1986;314:1280.
70. Mandarino LJ, Gerich JE. Prolonged sulfonylurea administration decreases insulin resistance and increases insulin secretion in non–insulin-dependent diabetes mellitus: evidence for improved insulin action at a postreceptor site in hepatic as well as extrahepatic tissues. Diabetes Care 1984;7(suppl 1):89.
71. Simonson DC, Ferrannini E, Bevilacqua S, et al. Mechanism of improvement in glucose metabolism after chronic glyburide therapy. Diabetes 1984;33:838.
72. Groop L, Schalin C, Franssila-Kalolunki A, et al. Characteristics of non-insulin dependent diabetic patients with secondary failure to oral antidiabetic therapy. Am J Med 1989;87:183.
73. Castillo M, Scheen AJ, Paolisso G, et al. The addition of glipizide to insulin therapy in type II diabetic patients with secondary failure to sulfonylureas is useful only in the presence of a significant residual insulin secretion. Acta Endocrinol (Copenhagen) 1987;116:364.
74. Yki-Jarvinen H, Nikkila E, Eero H, et al. Clinical benefits and mechanisms of a sustained response to intermittent insulin therapy in type 2 diabetic patients with secondary drug failure. Am J Med 1988; 84:185.
75. Hollenbeck CB, Chen Y-DI, Reaven GM. A comparison of the relative effects of obesity and non–insulin-dependent diabetes mellitus on in vivo insulin-stimulated glucose utilization. Diabetes 1984;33: 622.
76. Garvey WT, Olefsky JM, Marshall S. Insulin receptor down-regulation is linked to an insulin-induced postreceptor defect in the glucose transport system in rat adipocytes. J Clin Invest 1985;76:22.
77. Rizza RA, Mandarino LJ, Genest J, et al. Production of insulin resistance by hyperinsulinemia in man. Diabetologia 1985;28:70.
78. Reaven GM, Sageman WS, Swenson RS. Development of insulin resistance in normal dogs following alloxan-induced insulin deficiency. Diabetologia 1977;13:459.
79. Dall’Aglio E, Chang H, Hollenbeck CB, et al. In vivo and in vitro resistance to maximal insulin stimulated glucose disposal in insulin deficiency. Am J Physiol 1985;249:E312.
80. Bevilacqua S, Barrett EJ, Smith D, et al. Hepatic and peripheral insulin resistance following streptozotocin-induced insulin deficiency in the dog. Metabolism 1985;34:817.
81. Rossetti L, Giaccari A. Relative contribution of glycogen synthesis and glycolysis to insulin-mediated glucose uptake. A dose-response euglycemic clamp study in normal and diabetic rats. J Clin Invest 1990;85:1785.
82. Rossetti L, Hu M. Skeletal muscle glycogenolysis is more sensitive to insulin than is glucose transport/phosphorylation. Relation to the insulin-induced inhibition of hepatic glucose production. J Clin Invest 1993;92:2963.
83. Levy J, Gavin JR III, Fausto A, et al. Impaired insulin action in rats with non-insulin dependent diabetes. Diabetes 1984;33:901.
84. Kurszynska YT, Home PD. Liver and muscle insulin sensitivity, glycogen concentration, and glycogen synthase activity in a rat model of non-insulin dependent diabetes. Diabetologia 1988;31: 304.
85. Kahn BB, Shulman GI, DeFronzo RA, et al. Normalization of blood glucose in diabetic rats with phlorizin treatment reverses insulin resistant glucose transport in adipose cells without restoring glucose transporter gene expression. J Clin Invest 1991;87:561.
86. Hager SR, Jochen AL, Kalkhoff RK. Insulin resistance in normal rats infused with glucose for 72h. Am J Physiol 1991;260:E353.
87. Richter EA, Hansen BF, Hansen SA. Glucose-induced insulin resistance of skeletal muscle glucose transport and uptake. Biochem J 1988;252:733.
88. Richter EA, Hansen SA, Hansen BF. Mechanisms limiting glycogen storage in muscle during prolonged insulin stimulation. Am J Physiol 1988;255:E621.
P.950

89. Hansen BF, Hansen SA, Ploug T, et al. Effects of glucose and insulin on development of impaired insulin action in muscle. Am J Physiol 1992;262:E440.
90. Yki-Jarvinen H, Helve E, Koivisto VA. Hyperglycemia decreases glucose uptake in type 1 diabetes. Diabetes 1987;36:892.
91. Vuorinen-Markkola H, Koivisto VA, Yki-Jarvinen H. Mechanisms of hyperglycemia-induced insulin resistance in whole body and skeletal muscle of type 1 diabetic patients. Diabetes 1992;41:571.
92. Fowelin J, Attvall S, Von Schenck H, et al. Effect of prolonged hyperglycemia on growth hormone levels and insulin sensitivity in insulin-dependent diabetes mellitus. Metabolism 1993;42:387.
93. Rossetti L, Shulman GI, Zawalich W, et al. Effect of chronic hyperglycemia on in vivo insulin secretion in partially pancreatectomized rats. J Clin Invest 1987;80:1037.
94. Rossetti L, Laughlin MR. Correction of chronic hyperglycemia with vanadate, but not with phlorizin, normalizes in vivo glycogen repletion and in vitro glycogen synthase activity in diabetic skeletal muscle. J Clin Invest 1989;84:892.
95. Karnieli E, Hissin PJ, Simpson IA, et al. A possible mechanism of insulin resistance in the rat adipose cell in streptozotocin-induced diabetes mellitus: depletion of intracellular glucose transport systems. J Clin Invest 1981;68:811.
96. Kasuga MY, Akanuma Y, Iwamoto K, et al. Insulin binding and glucose metabolism in adipocytes from streptozotocin-diabetic rats. Am J Physiol 1978;235:E175.
97. Kobayashi M, Olefsky JM. Effects of streptozotocin-induced diabetes on insulin binding, glucose transport, and intracellular glucose metabolism in isolated rat adipocytes. Diabetes 1979;28:87.
98. Dohm GL, Tapscott EB, Pories WJ, et al. An in vitro human muscle preparation suitable for metabolic studies: decreased insulin stimulation of glucose transport in muscle from morbidly obese and diabetic subjects. J Clin Invest 1988;82:486.
99. Garvey WT, Huecksteadt TP, Matthaei S, et al. Role of glucose transporters in the cellular insulin resistance of type II non–insulin-dependent diabetes mellitus. J Clin Invest 1988;81:1528.
100. Haspal HC, Vilk EW, Birnbaum MT, et al. Glucose deprivation and hexose transporter polypeptides of murine fibroblasts. J Biol Chem 1986;261:6778.
101. Van Putten JPM, Krans HMJ. Glucose as a regulator of insulin-sensitive hexose uptake in 3T3 adipocytes. J Biol Chem 1985;260:7996.
102. Sasson S, Cerasi E. Substrate regulation of the glucose transport system in rat skeletal muscle. J Biol Chem 1986;261:16827.
103. Garvey WT, Olefsky JM, Matthaei S, et al. Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes: a new mechanism of insulin resistance. J Biol Chem 1987;262:189.
104. Sasson S, Edelson D, Cerasi E. In vitro autoregulation of glucose utilization in rat soleus muscle. Diabetes 1987;36:1041.
105. Kletzien RF, Perdue JF. Induction of sugar transport in chick embryo fibroblasts by hexose starvation: evidence for transcriptional regulation of transport. J Biol Chem 1985;250:593.
106. Ullrey D, Gammon BMT, Kalckar HM. Uptake patterns and transport enhancements in cultures of hamster cells deprived of carbohydrates. Arch Biochem Biophys 1975;167:410.
107. Ullrey DB, Kalckar HM. The nature of regulation of hexose transport in cultured mammalian fibroblasts: aerobic “repressive” control by D-glucosamine. Arch Biochem Biophys 1981;209:168.
108. Traxinger RR, Marshall S. Recovery of maximal insulin responsiveness after induction of insulin resistance. J Biol Chem 1989;264: 8156.
109. Marshall S, Garvey WT, Traxinger RR. New insights into the metabolic regulation of insulin action and insulin resistance: role of glucose and amino acids. FASEB J 1991;5:3031.
110. Crettaz M, Prentky M, Zaninetti D, et al. Insulin resistance in soleus muscle from obese Zucker rats. Biochem J 1980;186:525.
111. Berger M, Hagg SA, Goodman MN, et al. Glucose metabolism in perfused skeletal muscle. Biochem J 1976;158:191.
112. Charron MJ, Kahn BB. Divergent molecular mechanisms for insulin resistant glucose transport in muscle and adipose cells in vivo. J Biol Chem 1990;265:7994.
113. Kahn BB, Rossetti L, Lodish HF, Charron MJ. Decreased in vivo glucose uptake but normal expression of GLUT1 and GLUT4 in skeletal muscle. J Clin Invest 1991;87:2197.
114. Garvey WT, Huecksteadt TP, Birnbaum MT. Pretranslational suppression of an insulin-responsive glucose transporter in rats with diabetes mellitus. Science 1989;245:60.
115. Sivitz WI, DeSautel SL, Kayano T, et al. Regulation of glucose transporter messenger RNA in insulin-deficient states. Nature 1989; 340:72.
116. Berger JC, Biswas P, Vicario P, et al. Decreased expression of the insulin-responsive glucose transporter in diabetes and fasting. Nature 1989;340:70.
117. Kahn BB, Charron MJ, Lodish HF, et al. Differential regulation of two glucose transporters in adipose cells from diabetic and insulin treated diabetic rats. J Clin Invest 1989;84:404.
118. Sivitz WI, DeSautel SL, Kayano T, et al. Regulation of glucose transporter messenger RNA levels in rat adipose tissue by insulin. Mol Endocrinol 1990;4:583.
119. Kahn BB, Flier JS. Regulation of glucose transporter gene expression in vivo and in vitro glucose. Diabetes Care 1991;87:561.
120. Dimitrakoudis D, Ramlal T, Rastogi S, et al. Glycemia regulates the glucose transporter number in the plasma membrane of the rat skeletal muscle. Biochem J 1992;284:341.
121. Cordera R, Andraghetti G, DeFronzo RA, et al. Effect of in vivo vanadate treatment on insulin receptor tyrosine kinase activity in partially pancreatectomized diabetic rats. Endocrinology 1990;126: 2177.
122. Marshall S, Monzon R. Amino acid regulation of insulin action in isolated adipocytes. J Biol Chem 1989;264:2037.
123. Marshall S. Kinetics of insulin action on protein synthesis in isolated adipocytes: ability of glucose to selectively desensitize the glucose transport system without altering insulin stimulation of protein synthesis. J Biol Chem 1989;264:2029.
124. Marshall S, Bacote V, Traxinger RR. Discovery of a metabolic pathway mediating desensitization of the glucose transport system: role of hexosamine biosynthesis in the induction of insulin resistance. J Biol Chem 1992;266:4706.
125. Traxinger RR, Marshall S. Role of aminoacids in modulating glucose-induced desensitization of the glucose transport system. J Biol Chem 1989;264:20910.
126. Traxinger RR, Marshall S. Insulin regulation of pyruvate kinase activity in isolated adipocytes: crucial role of glucose and the hexosamine biosynthesis pathway in the expression of insulin action. J Biol Chem 1992;267:9718.
127. Robinson KA, Sens DA, Buse MG. Pre-exposure to glucosamine induces insulin resistance of glucose transport and glycogen synthesis in isolated rat skeletal muscles. Diabetes 1993;42:1333.
128. Crook ED, Daniels MC, Smith TM, et al. Regulation of insulin-stimulated glycogen synthase activity by overexpression of glutamine:fructose-6-phosphate amidotransferase in rat-1 fibroblasts. Diabetes 1993;42:1289.
129. Rossetti L, Hawkins M, Chen W, et al. In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J Clin Invest 1995;96:132.
130. Randle PJ, Garland PB, Hales CN, et al. The glucose fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963;1:785.
131. Ruderman NB, Saha AK, Vavvas D, et al. Malonyl-CoA, fuel sensing, and insulin resistance. Am J Physiol 1999;276:E1.
132. Hawkins M, Barzilai N, Hu M, et al. Role of the glucosamine pathway in fat-induced insulin resistance. J Clin Invest 1997;99: 2173.
133. Hawkins M, Angelov I, Liu R, et al. The tissue concentration of UDP-N-acetylglucosamine modulates the stimulatory effect of insulin on skeletal muscle glucose uptake. J Biol Chem 1997;272: 4889.
134. Baron A, Zhu J-S, Zhu J-H, et al. Glucosamine induces insulin resistance in vivo by affecting GLUT4 translocation in skeletal muscle. J Clin Invest 1995;96:2792.
P.951

135. Hebert L, Daniels M, Zhou J, et al. Overexpression of glutamine: fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance. J Clin Invest 1996;98:930.
136. Cooksey RC, Hebert E, Zhu J-H, et al. Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione. Endocrinology 1999;140:1151.
137. Pillay TS, Xiao S, Olefsky JM. Glucose-induced phosphorylation of the insulin receptor. J Clin Invest 1996;97:613.
138. Filippis A, Clark S, Proietto J. Increased flux through the hexosamine biosynthesis pathway inhibits glucose transport acutely by activation of protein kinase C. Biochem J 1997;324:978.
←↑→