Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance

Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate by the first and rate-limiting enzyme of the hexosamine pathway, glutamine:fructose-6-phosphate amidotransferase.     

Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation

We reported previously that glutamine:F-6-P amidotransferase (GFAT) plays an integral role in the development of insulin resistance by directing the flow of incoming glucose into the hexosamine biosynthesis pathway. To determine whether the enzymatic activity of GFAT is altered during desensitization of the glucose transport system, we treated isolated rat adipocytes with various combinations of insulin, glucose, and glutamine. Treatment with insulin or glucose alone (or in combination) failed to reduce cytosolic GFAT activity after 4 h, whereas combined treatment with all three components elicited a progressive loss of GFAT activity that was rapid (t1/2 of 2 h) and extensive (70% loss). A pronounced loss of GFAT activity was also seen in cells exposed to glucosamine, an agent known to directly enter the hexosamine pathway (55% loss at 4 h, ED50 of 360 microM). Moreover, a close correlation was observed between the induction of desensitization and the loss of GFAT activity as a function of glucose, insulin, glutamine, and glucosamine concentrations. When total intracellular hexosamine products were measured, we found that hexosamine formation was unaltered by insulin or glucose (or a combination) but was elevated by greater than 4-fold in the presence of insulin, glucose, and glutamine (t1/2 of 22 min), a condition known to cause both desensitization and loss of GFAT activity. Additional studies indicated that the loss of GFAT activity under desensitizing conditions is not due to allosteric regulation since removal of potential allosteric factors from the cytosol of desensitized cells by G-25 column chromatography failed to restore enzyme activity. Overall, these studies indicate that 1) GFAT is an insulin-regulated enzyme; however, control of enzyme activity is not due to a direct action of insulin, but rather is mediated by insulin-induced enhancement of glucose uptake; 2) the routing of incoming glucose through the hexosamine pathway and the formation of hexosamine products appears to regulate GFAT activity; and 3) the progressive loss of GFAT activity over several hours is probably not due to allosteric regulation.     

Insulin regulation of pyruvate kinase activity in isolated adipocytes. Crucial role of glucose and the hexosamine biosynthesis pathway in the expression of insulin action.

We recently identified glutamine:fructose-6-phosphate amidotransferase (GFAT) as an insulin-regulated enzyme in adipocytes. Moreover, we found that loss of GFAT activity is not due to a direct action of insulin but rather is mediated by enhanced glucose uptake and the subsequent routing of glucose through the hexosamine biosynthesis pathway. To assess whether other cytosolic enzymes are controlled through formation of hexosamine products, we treated adipocytes for 5 h with physiological concentrations of insulin (ED50 = 0.33 ng/ml), glucose (ED50 = 4.5 mM), and glutamine (ED50 = 4.4 mM) and then measured pyruvate kinase (PK) activity. Combined treatment resulted in a progressive (t 1/2 of 2.5 h) and marked (3-fold) increase in PK activity, whereas omission of one or more of these components failed to alter enzyme activity. Several lines of additional evidence implicated the hexosamine biosynthesis pathway in PK regulation; therefore, it appears that the M2 isoform of pyruvate kinase represents another enzyme regulated by insulin through stimulation of glucose uptake and formation of hexosamine products. Related studies revealed that enhancement of PK activity is dependent upon ongoing mRNA synthesis and de novo protein synthesis and is mediated by an increase in enzyme content. Considered together, these findings provide new insights into the cascade of metabolic events triggered by insulin and implicated a novel metabolic pathway in the pretranslational control of enzyme function.     

The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets

The hexosamine biosynthesis pathway plays a role in the modification of cellular proteins via the provision of substrate for addition of O-linked N-acetylglucosamine (GlcNAc). The relative importance of the GlcNAc modification of proteins to insulin secretion from pancreatic beta-cells has not been investigated and so remains unclear. In the present study, we show that inhibition of the hexosamine biosynthesis pathway decreases insulin secretion from mouse islets in response to a number of secretagogues, including glucose. This impairment in beta-cell function could not be attributed to reduced islet insulin content, altered ATP levels, or cell death and was restored with the addition of N-acetylglucosamine, a substrate that enters the pathway below the point of inhibition. Western blot analysis revealed that decreased islet protein glycosylation paralleled the decrease in insulin secretion following inhibition of the pathway. In conclusion, the data suggest a role for the hexosamine biosynthesis pathway in regulating the secretion of insulin by altering protein glycosylation. This finding may have implications for the development of type 2 diabetes, as chronic increase in flux through the hexosamine biosynthesis pathway may lead to the deterioration of beta-cell function via abnormal protein glycosylation.     

Adipocytes with increased hexosamine flux exhibit insulin resistance, increased glucose uptake, and increased synthesis and storage of lipid

The hexosamine signaling pathway has been shown to serve a nutrient-sensing function. We have previously shown that overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine-fructose-6-phosphate amidotransferase) in adipose tissue of transgenic mice results in skeletal muscle insulin resistance and altered regulation of leptin and adiponectin. To dissect the pathways by which the hexosamine pathway affects fuel storage and energy homeostasis, we have examined the characteristics of adipocytes from these animals. After 3 mo of age, epididymal fat pads from adult transgenic animals are 42% heavier (P = 0.003) and individual adipocytes are 23% larger in diameter (P < 0.05) than those from littermate wild-type controls. Isolated adipocytes from transgenic mice are insulin resistant, with a 2.5-fold increase in the ED50 for stimulation of 2-deoxy-D-glucose uptake. However, maximal insulin-stimulated glucose uptake is increased in transgenic adipocytes by 39% (P < 0.05). This upregulation of glucose uptake was associated with a 41% increase in the expression of GLUT4 mRNA and a 28% increase in GLUT4 protein in transgenics compared with controls (P < 0.05). GLUT1 mRNA and protein did not significantly differ between fasted control and transgenics. Total lipid synthesis was also increased in epididymal adipocytes from transgenic animals by 206% compared with controls (P < 0.05). Fatty acid oxidation was increased 1.6-fold in the transgenic adipocytes (P < 0.05). We conclude that the hexosamine signaling pathway upregulates fat storage in adipocytes in states of carbohydrate excess, in part by increasing GLUT4 and glucose uptake and by augmenting fatty acid synthesis.     

Development and comparison of two 3T3-L1 adipocyte models of insulin resistance: increased glucose flux vs glucosamine treatment

Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C., et al. (1998) J. Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (IR) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated 2-deoxyglucose uptake, however, was reduced by approximately 45% in response to both high glucose/insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. Insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells. Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amidotransferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling.     

Signalling aspects of insulin resistance in skeletal muscle: mechanisms induced by lipid oversupply

A reduced capacity for insulin to elicit increases in glucose uptake and metabolism in target tissues such as skeletal muscle is a common feature of obesity and diabetes. The association between lipid oversupply and such insulin resistance is well established, and evidence for mechanisms through which lipids could play a causative role in the generation of muscle insulin resistance is reviewed. While the effects of lipids may in part be mediated by substrate competition through the glucose-fatty acid cycle, interference with insulin signal transduction by lipid-activated signalling pathways is also likely to play an important role. Thus, studies of insulin resistance in Type 2 diabetes, obesity, fat-fed animals and lipid-treated cells have identified defects both at the level of insulin receptor-mediated tyrosine phosphorylation and at downstream sites such as protein kinase B (PKB) activation. Lipid signalling molecules can be derived from free fatty acids, and include diacylglycerol, which activates isozymes of the protein kinase C (PKC) family, and ceramide, which has several effectors including PKCs and a protein phosphatase. In addition, elevated lipid availability can increase flux through the hexosamine biosynthesis pathway which can also lead to activation of PKC as well as protein glycosylation and modulation of gene expression. The mechanisms giving rise to decreased insulin signalling include serine/threonine phosphorylation of insulin receptor substrate-1, but also direct inhibition of components such as PKB. Thus lipids can inhibit glucose disposal by causing interference with insulin signal transduction, and most likely by more than one pathway depending on the prevalent species of fatty acids.     

Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels: effects of glucose,glutamine, and glucosamine on glycerophosphate dehydrogenase,fatty acid synthase,and acetyl-CoA carboxylase mRNA levels

Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). To determine whether the hexosamine biosynthesis pathway (HBP) mediates glucose regulation of mRNA expression, we treated primary cultured adipocytes for 18 h with insulin (25 ng/ml) and either glucose (20 mm) or glucosamine (2 mm). A ribonuclease protection assay was used to quantitate mRNA levels for FAS, ACC, and glycerol-3-P dehydrogenase (GPDH). Treatment with insulin and various concentrations of d-glucose increased mRNA levels for FAS (280%), ACC (93%), and GPDH (633%) in a dose-dependent manner (ED50 8-16 mm). Mannose similarly elevated mRNA levels, but galactose and fructose were only partially effective. l-glucose had no effect. Omission of glutamine from the culture medium markedly diminished the stimulatory effect of glucose on mRNA expression. Since glutamine is a crucial amide donor in hexosamine biosynthesis, we interpret these data to mean that glucose flux through the HBP is linked to regulation of lipogenesis through control of gene expression. Further evidence for hexosamine regulation was obtained using glucosamine, which is readily transported into adipocytes where it directly enters the HBP. Glucosamine was 15-30 times more potent than glucose in elevating FAS, ACC, and GPDH mRNA levels (ED50 approximately 0.5 mm). In summary: 1) GPDH, FAS, and ACC mRNA levels are upregulated by glucose; 2) glucose-induced up-regulation requires glutamine; and 3) mRNA levels for lipogenic enzymes are up-regulated by glucosamine. Hyperglycemia is the hallmark of diabetes mellitus and leads to insulin resistance, impaired glucose metabolism, and dyslipidemia. We postulate that disease pathophysiology may have a common underlying factor, excessive glucose flux through the HBP.     

Increased hexosamine pathway flux and high fat feeding are not additive in inducing insulin resistance: evidence for a shared pathway

Excess fatty acids and carbohydrates have both been implicated in the pathogenesis of type 2 diabetes, and both can reproduce essential features of the disease including insulin resistance and beta cell failure. It has been proposed that both nutrients may regulate metabolism through a common fuel sensing mechanism, namely hexosamine synthesis. We have previously shown that transgenic overexpression of the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), targeted to muscle and fat, leads to insulin resistance mediated by increased O-linked glycosylation of nuclear and cytosolic proteins. We report here that hexosamine-induced insulin resistance is not additive with that induced by high fat feeding. In control mice fed a high fat diet, glucose disposal rates during euglycemic hyperinsulinemia were decreased by 37% (p < 0.02) compared to mice on a low fat diet. Transgenic mice overexpressing GFA and fed a low fat diet exhibited a 51% decrease in glucose disposal compared to controls on a low fat diet (p < 0.001), but no further decrease was evident in the transgenic mice fed a high fat diet. Decreased glucose disposal rates were mirrored by increases in skeletal muscle levels of the principal end product of the hexosamine pathway, UDP-N-acetyl glucosamine. Serum leptin levels, which are modulated both by feeding and hexosamine flux, also show no additivity in their stimulation by GFA overexpression and high fat feeding. These data are consistent with a shared nutrient sensing pathway for high fat and carbohydrate fluxes and a common pathway by which glucose and lipids induce insulin resistance.     

Reduction of O-GlcNAc protein modification does not prevent insulin resistance in 3T3-L1 adipocytes

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high (25 mM) glucose, provided that insulin (0.6 nM) is included, Akt activation is impaired, and high glucose and insulin act synergistically. Considerable evidence suggests that increased glucose flux via the hexosamine biosynthesis pathway enhances the O-GlcNAc modification (O-GlcNAcylation) of some critical protein(s) that may contribute to insulin resistance. However, whether enhanced protein O-GlcNAcylation is necessary for the development of insulin resistance is unknown. We used two strategies to test this hypothesis. The first strategy was the overexpression of O-GlcNAcase, which removes O-GlcNAc from Ser/Thr of proteins. Cells were infected with O-GlcNAcase-expressing adenovirus (or empty virus) 5 days before they were submitted to protocols that elicit (or not) insulin resistance. O-GlcNAcase was highly expressed and functional as assessed by Western blot, O-GlcNAcase assay, and marked reduction of O-GlcNAcylated proteins. The activity was mainly cytosolic. The second strategy was the expression of O-GlcNAc transferase (OGT) being markedly reduced by transfection of OGT siRNA, resulting in an approximately 90% decrease of nuclear and cytosolic OGT protein expression and similar reduction in O-GlcNAcylated proteins. Nontargeting siRNA had no effect. Preincubation in high glucose with low-dose insulin decreased the acute insulin response of glucose transport by at least 50% and impaired Akt activation. None of these parameters were affected by overexpression of O-GlcNAcase or by OGT knockout. Excess O-GlcNAcylation is one of many factors that can cause insulin resistance. It does not seem to be required for the development of glucose/insulin-induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes.     

Nutrient sensing, leptin and insulin action

The glucose-fatty acid cycle as proposed four decades ago by Randle suggests that insulin resistance develops in consequence of alterations of the metabolic pressure of lipids. The more recently published 'hexosamine pathway theory' and the 'malonyl-CoA hypothesis' depict insulin resistance as a consequence of an imbalance between utilization of lipids and carbohydrates. The latter is finely tuned by entry of fatty acids into the mitochondria and/or by entry of glucose to the hexosamine pathway. A significant body of evidence has also been accumulated which points to the complex effects of leptin, an adipocyte-derived signal of lipid stores, on the storage and metabolism of fats and carbohydrates. These are mediated either directly, through actions on specific tissues, or indirectly, via CNS, endocrine and neural mechanisms. The available literature also provides good evidence that leptin orchestrates the metabolic changes in a number of organs and tissues, and alters nutrient fluxes to favor energy expenditure over energy storage. In this article, the proposed lipopenic effects of leptin as studied in various animal models of diet-induced insulin resistance, and possible regulations of leptin production and action by marine fish oil feeding are reviewed.     

Role of NYGGF4 in insulin resistance

Insulin resistance is a clinical condition that is characterized by reducing glucose uptake in response to insulin. A major factor in the development of insulin resistance syndrome is obesity. NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese subjects. NYGGF4 induced the secretion of FFAs and TNF-α and caused mitochondrial dysfunction, which may cause insulin resistance. This review will summarize the effect of NYGGF4 on the adipogenesis, glucose uptake and mitochondrial dysfunction in vitro, and the possible mechanism and signal pathway of NYGGF4 for insulin resistance.     

Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. A new mechanism of insulin resistance

We have previously shown in primary cultured rat adipocytes that insulin acts at receptor and multiple postreceptor sites to decrease insulin's subsequent ability to stimulate glucose transport. To examine whether D-glucose can regulate glucose transport activity and whether it has a role in insulin-induced insulin resistance, we cultured cells for 24 h in the absence and presence of various glucose and insulin concentrations. After washing cells and allowing the glucose transport system to deactivate, we measured basal and maximally insulin-stimulated 2-deoxyglucose uptake rates (37 degrees C) and cell surface insulin binding (16 degrees C). Alone, incubation with D-glucose had no effect on basal or maximal glucose transport activity, and incubation with insulin, in the absence of glucose, decreased maximal (but not basal) glucose transport rates only 18% at the highest preincubation concentration (50 ng/ml). However, in combination, D-glucose (1-20 mM) markedly enhanced the long-term ability of insulin (1-50 ng/ml) to decrease glucose transport rates in a dose-responsive manner. For example, at 50 ng/ml preincubation insulin concentration, the maximal glucose transport rate fell from 18 to 63%, and the basal uptake rate fell by 89%, as the preincubation D-glucose level was increased from 0 to 20 mM. Moreover, D-glucose more effectively promoted decreases in basal glucose uptake (Ki = 2.2 +/- 0.4 mM) compared with maximal transport rates (Ki = 4.1 +/- 0.4 mM) at all preincubation insulin concentrations (1-50 ng/ml). Similar results were obtained when initial rates of 3-O-methylglucose uptake were used to measure glucose transport. D-glucose, in contrast, did not influence insulin-induced receptor loss. In other studies, D-mannose and D-glucosamine could substitute for D-glucose to promote the insulin-induced changes in glucose transport, but other substrates such as L-glucose, L-arabinase, D-fructose, pyruvate, and maltose were without effect. Also, non-metabolized substrates which competitively inhibit D-glucose uptake (3-O-methylglucose, cytochalasin B) blocked the D-glucose plus insulin effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chronic insulin treatment causes insulin resistance in 3T3-L1 adipocytes through oxidative stress

Insulin resistance and hyperinsulinemia are commonly present in obesity and pre-diabetes, and hyperinsulinemia is both a marker and a cause for insulin resistance. However, the molecular link between hyperinsulinemia and insulin resistance remains elusive. The present study examined the effect of chronic insulin treatment on the reactive oxygen species (ROS) production, insulin signalling and insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The results showed that chronic insulin treatment significantly increased the intracellular generation of superoxide anion, hydrogen peroxide and hydroxyl radical. ROS induced by chronic insulin treatment inhibited insulin signalling and glucose uptake, induced endoplasmic reticulum (ER) stress and JNK activation. Furthermore, these effects were reversed by antioxidants N-acetylcysteine, superoxide dismutase or catalase. These results suggested that ROS, ER stress and JNK pathway are involved in insulin resistance induced by chronic insulin treatment. Therefore, oxidative stress could be a potential interventional target for hyperinsulinemia-induced insulin resistance and related diseases.     

Chronic insulin treatment causes insulin resistance in 3T3-L1 adipocytes through oxidative stress

Insulin resistance and hyperinsulinemia are commonly present in obesity and pre-diabetes, and hyperinsulinemia is both a marker and a cause for insulin resistance. However, the molecular link between hyperinsulinemia and insulin resistance remains elusive. The present study examined the effect of chronic insulin treatment on the reactive oxygen species (ROS) production, insulin signalling and insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The results showed that chronic insulin treatment significantly increased the intracellular generation of superoxide anion, hydrogen peroxide and hydroxyl radical. ROS induced by chronic insulin treatment inhibited insulin signalling and glucose uptake, induced endoplasmic reticulum (ER) stress and JNK activation. Furthermore, these effects were reversed by antioxidants N-acetylcysteine, superoxide dismutase or catalase. These results suggested that ROS, ER stress and JNK pathway are involved in insulin resistance induced by chronic insulin treatment. Therefore, oxidative stress could be a potential interventional target for hyperinsulinemia-induced insulin resistance and related diseases.     

Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons

In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mm glucose for 24 h attenuated the ability of 10 nm insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mm glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased approximately 2- and 15-fold after a 24-h incubation in 20 mm glucose and 1.5 mm glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mm glucose, but not that of 1.5 mm glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, glucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes.     

Glucose-induced insulin resistance of skeletal-muscle glucose transport and uptake.

The ability of glucose and insulin to modify insulin-stimulated glucose transport and uptake was investigated in perfused skeletal muscle. Here we report that perfusion of isolated rat hindlimbs for 5 h with 12 mM-glucose and 20,000 microunits of insulin/ml leads to marked, rapidly developing, impairment of insulin action on muscle glucose transport and uptake. Thus maximal insulin-stimulated glucose uptake at 12 mM-glucose decreased from 34.8 +/- 1.9 to 11.5 +/- 1.1 mumol/h per g (mean +/- S.E.M., n = 10) during 5 h perfusion. This decrease in glucose uptake was accompanied by a similar change in muscle glucose transport as measured by uptake of 3-O-[14C]-methylglucose. Simultaneously, muscle glycogen stores increased to 2-3.5 times initial values, depending on fibre type. Perfusion for 5 h in the presence of glucose but in the absence of insulin decreased subsequent insulin action on glucose uptake by 80% of the effect of glucose with insulin, but without an increase in muscle glycogen concentration. Perfusion for 5 h with insulin but without glucose, and with subsequent addition of glucose back to the perfusate, revealed glucose uptake and transport similar to initial values obtained in the presence of glucose and insulin. The data indicate that exposure to a moderately increased glucose concentration (12 mM) leads to rapidly developing resistance of skeletal-muscle glucose transport and uptake to maximal insulin stimulation. The effect of glucose is enhanced by simultaneous insulin exposure, whereas exposure for 5 h to insulin itself does not cause measurable resistance to maximal insulin stimulation.