Pretranslational suppression of a glucose transporter protein causes insulin resistance in adipocytes from patients with non-insulin-dependent diabetes mellitus and obesity.

A major portion of insulin-mediated glucose uptake occurs via the translocation of GLUT 4 glucose transporter proteins from an intracellular depot to the plasma membrane. We have examined gene expression for the GLUT 4 transporter isoform in subcutaneous adipocytes, a classic insulin target cell, to better understand molecular mechanisms causing insulin resistance in non-insulin-dependent diabetes mellitus (NIDDM) and obesity. In subgroups of lean (body mass index [BMI] = 24 +/- 1) and obese (BMI = 32 +/- 2) controls and in obese NIDDM (BMI = 35 +/- 2) patients, the number of GLUT 4 glucose transporters was measured in total postnuclear and subcellular membrane fractions using specific antibodies on Western blots. Relative to lean controls, the cellular content of GLUT 4 was decreased 40% in obesity and 85% in NIDDM in total cellular membranes. In obesity, cellular depletion of GLUT 4 primarily involved low density microsomes (LDM), leaving fewer transporters available for insulin-mediated recruitment to the plasma membrane (PM). In NIDDM, loss of GLUT 4 was profound in all membrane subfractions, PM, LDM, as well as high density microsomes. These observations corresponded with decrements in maximally stimulated glucose transport rates in intact cells. To assess mechanisms responsible for depletion of GLUT 4, we quantitated levels of mRNA specifically hybridizing with human GLUT 4 cDNA on Northern blots. In obesity, GLUT 4 mRNA was decreased 36% compared with lean controls, and the level was well correlated (r = + 0.77) with the cellular content of GLUT 4 protein over a wide spectrum of body weight. GLUT 4 mRNA in adipocytes from NIDDM patients was profoundly reduced by 86% compared with lean controls and by 78% relative to their weight-matched nondiabetic counterparts (whether expressed per RNA, per cell, or for the amount of CHO-B mRNA). Interestingly, GLUT 4 mRNA levels in patients with impaired glucose tolerance (BMI = 34 +/- 4) were decreased to the same level as in overt NIDDM. We conclude that, in obesity, insulin resistance in adipocytes is due to depletion of GLUT 4 glucose transporters, and that the cellular content of GLUT 4 is determined by the level of encoding mRNA over a wide range of body weight. In NIDDM, more profound insulin resistance is caused by a further reduction in GLUT 4 mRNA and protein than is attributable to obesity per se. Suppression of GLUT 4 mRNA is observed in patients with impaired glucose tolerance, and therefore, may occur early in the evolution of diabetes. Thus, pretranslational suppression of GLUT 4 transporter gene expression may be an important mechanism that produces and maintains cellular insulin resistance in NIDDM.

Glucosamine induces insulin resistance in vivo by affecting GLUT 4 translocation in skeletal muscle. Implications for glucose toxicity.

Glucosamine (Glmn), a product of glucose metabolism via the hexosamine pathway, causes insulin resistance in isolated adipocytes by impairing insulin-induced GLUT 4 glucose transporter translocation to the plasma membrane. We hypothesized that Glmn causes insulin resistance in vivo by a similar mechanism in skeletal muscle. We performed euglycemic hyperinsulinemic clamps (12 mU/kg/min + 3H-3-glucose) in awake male Sprague-Dawley rats with and without Glmn infusion at rates ranging from 0.1 to 6.5 mg/kg/min. After 4h of euglycemic clamping, hindquarter muscles were quick-frozen and homogenized, and membranes were subfractionated by differential centrifugation and separated on a discontinuous sucrose gradient (25, 30, and 35% sucrose). Membrane proteins were solubilized and immunoblotted for GLUT 4. With Glmn, glucose uptake (GU) was maximally reduced by 33 +/- 1%, P < 0.001. The apparent Glmn dose to reduce maximal GU by 50% was 0.1 mg/kg/min or 1/70th the rate of GU on a molar basis. Control galactosamine and mannosamine infusions had no effect on GU. Relative to baseline, insulin caused a 2.6-fold increase in GLUT 4 in the 25% membrane fraction (f), P < 0.01, and a 40% reduction in the 35%f, P < 0.05, but had no effect on GLUT 4 in the 30% f, P= NS. Addition of Glmn to insulin caused a 41% reduction of GLUT 4 in the 25%f, P < 0.05, a 29% fall in the 30%f, and prevented the reduction of GLUT 4 in the 35% f. The 30%f membranes were subjected to a second separation with a 27 and 30% sucrose gradient. Insulin mobilized GLUT 4 away from the 30%f, P < 0.05, but not the 27% f. In contrast, Glmn reduced GLUT 4 in the 27%f, P < 0.05, but not the 30%f. Thus Glmn appears to alter translocation of an insulin-insensitive GLUT 4 pool. Coinfusion of Glmn did not alter enrichment of the sarcolemmal markers 5'-nucleotidase, Na+/K+ATPase, and phospholemman in either 25, 30, or 35% f. Thus Glmn completely blocked movement of Glut 4 induced by insulin. Glmn is a potent inducer of insulin resistance in vivo by causing (at least in part) a defect intrinsic to GLUT 4 translocation and/or trafficking. These data support a potential role for Glmn to cause glucose-induced insulin resistance (glucose toxicity).

Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance.

Insulin resistance is instrumental in the pathogenesis of type 2 diabetes mellitus and the Insulin Resistance Syndrome. While insulin resistance involves decreased glucose transport activity in skeletal muscle, its molecular basis is unknown. Since muscle GLUT4 glucose transporter levels are normal in type 2 diabetes, we have tested the hypothesis that insulin resistance is due to impaired translocation of intracellular GLUT4 to sarcolemma. Both insulin-sensitive and insulin-resistant nondiabetic subgroups were studied, in addition to type 2 diabetic patients. Biopsies were obtained from basal and insulin-stimulated muscle, and membranes were subfractionated on discontinuous sucrose density gradients to equilibrium or under nonequilibrium conditions after a shortened centrifugation time. In equilibrium fractions from basal muscle, GLUT4 was decreased by 25-29% in both 25 and 28% sucrose density fractions and increased twofold in both the 32% sucrose fraction and bottom pellet in diabetics compared with insulin-sensitive controls, without any differences in membrane markers (phospholemman, phosphalamban, dihydropyridine-binding complex alpha-1 subunit). Thus, insulin resistance was associated with redistribution of GLUT4 to denser membrane vesicles. No effects of insulin stimulation on GLUT4 localization were observed. In non-equilibrium fractions, insulin led to small GLUT4 decrements in the 25 and 28% sucrose fractions and increased GLUT4 in the 32% sucrose fraction by 2.8-fold over basal in insulin-sensitive but only by 1.5-fold in both insulin-resistant and diabetic subgroups. The GLUT4 increments in the 32% sucrose fraction were correlated with maximal in vivo glucose disposal rates (r = +0.51, P = 0.026), and, therefore, represented GLUT4 recruitment to sarcolemma or a quantitative marker for this process. Similar to GLUT4, the insulin-regulated aminopeptidase (vp165) was redistributed to a dense membrane compartment and did not translocate in response to insulin in insulin-resistant subgroups. In conclusion, insulin alters the subcellular localization of GLUT4 vesicles in human muscle, and this effect is impaired equally in insulin-resistant subjects with and without diabetes. This translocation defect is associated with abnormal accumulation of GLUT4 in a dense membrane compartment demonstrable in basal muscle. We have previously observed a similar pattern of defects causing insulin resistance in human adipocytes. Based on these data, we propose that human insulin resistance involves a defect in GLUT4 traffic and targeting leading to accumulation in a dense membrane compartment from which insulin is unable to recruit GLUT4 to the cell surface.

Glucose and insulin chronically regulate insulin action via different mechanisms in BC3H1 myocytes. Effects on glucose transporter gene expression.

We previously reported that, in primary cultured adipocytes, chronic exposure to glucose plus insulin impairs the insulin-responsive glucose transport system. In this study, we examined regulation of glucose transport in BC3H1 myocytes as a model for muscle and found important differences between BC3H1 cells and adipocytes. In myocytes, chronic glucose exposure per se (25 mM) decreased basal glucose transport activity by 78% and insulin's acute ability to maximally stimulate transport by 68% (ED50 approximately 2.5 mM; T1/2 approximately 4 h). D-Mannose and 3-O-methyl-glucose diminished transport rates with approximately 100 and 50% of the potency of D-glucose, respectively, whereas L-glucose, D-fructose, and D-galactose were inactive. Chronic glucose exposure also reduced cell surface insulin binding by 30% via an apparent decrease in receptor affinity, and this effect was associated with a comparable rightward shift in the insulin-glucose transport dose-response curve. In other studies, persistent stimulation with 15 nM insulin also decreased maximally stimulated glucose transport activity, which was independent and additive to the regulatory effect of glucose. Moreover, glucose and insulin-induced insulin resistance via different mechanisms. Glucose (25 mM) reduced the number of cellular glucose transporter proteins by 84% and levels of GLUT1 transporter mRNA by 50% (whether normalized to total RNA or CHO-B mRNA). In contrast, chronic insulin exposure led to a 2.1-fold increase in GLUT1 mRNA but did not alter cellular levels of transporter protein. Cotreatment with glucose prevented the insulin-induced rise in GLUT1 mRNA. BC3H1 cells did not express GLUT4 mRNA that encodes the major transporter isoform in skeletal muscle. In conclusion, in BC3H1 myocytes 1) glucose diminished insulin sensitivity by decreasing insulin receptor binding affinity and decreased basal and maximally insulin-stimulated glucose transport rates via cellular depletion of glucose transporters and suppression of GLUT1 mRNA; 2) chronic insulin exposure exerted an independent and additive effect to reduce maximal transport activity; however, insulin increased levels of GLUT1 mRNA and did not alter the cellular content of glucose transporters; and 3) although BC3H1 cells are commonly used as a model for skeletal muscle, studies examining glucose transport should be interpreted cautiously due to the absence of GLUT4 expression. Nevertheless, the data generally support the idea that, in non-insulin-dependent diabetes mellitus, hyperglycemia and hyperinsulinemia can induce or exacerbate insulin resistance in target tissues.

Multiple defects in the adipocyte glucose transport system cause cellular insulin resistance in gestational diabetes. Heterogeneity in the number and a novel abnormality in subcellular localization of GLUT4 glucose transporters.

Mechanisms causing cellular insulin resistance in gestational diabetes mellitus are not known. We, therefore, studied isolated omental adipocytes obtained during elective cesarean sections in nondiabetic (control) and GDM gravidas. Cellular insulin resistance was attributed to impaired stimulation of glucose transport; compared with control subjects, basal and maximally insulin-stimulated transport rates (per surface area) were reduced 38 and 60% in GDM patients, respectively. To determine underlying mechanisms, we assessed the number, subcellular distribution, and translocation of GLUT4, the predominant insulin-responsive glucose transporter isoform. The cellular content of GLUT4 was decreased by 44% in GDM patients as assessed by immunoblot analysis of total postnuclear membranes. However, GDM patients segregated into two subgroups; half expected profound (76%) cellular depletion of GLUT4 and half had GLUT4 levels in the normal range. Cellular GLUT4 was negatively correlated with adipocyte size in the control subjects and GDM patients with normal GLUT4 (r = 0.60), but fell way below this continuum in GDM patients with low GLUT4, indicating that heterogeneity was not caused by differences in obesity. All GDM. distribution. In basal cells, increased amounts of GLUT4 were detected in membranes fractionating with (such that the plasma membrane GLUT4 level in GDM (such that the plasma membrane GLUT4 level in GDM patients was equal to that observed in insulin-stimulated cells from control subjects). Furthermore, insulin stimulation induced translocation of GLUT4 from low-density microsomes to plasma membranes in control subjects but did not alter subcellular distribution in GDM patients. In other experiments, cellular content of GLUT1 was normal in GDM patients, and GLUT1 did not undergo insulin-mediated recruitment to plasma membranes in either control subjects or GDM patients. A faint signal was detected for GLUT3 only in low-density microsomes and only with one of two different antibodies. In GDM, we conclude that insulin resistance in adipocytes involves impaired stimulation of glucose transport and arises from a heterogeneity of defects intrinsic to the glucose transport effector system. GLUT4 content in adipocytes is profoundly depleted in approximately 50% of GDM patients, whereas all patients are found to exhibit a novel abnormality in GLUT4 subcellular distribution. This latter defect is characterized by accumulation of GLUT4 in membranes cofractionating with plasma membranes and high-density microsomes in basal cells and absence of translocation in response to insulin. The data suggest that abnormalities in cellular traffic or targeting relegate GLUT4 to a membrane compartment from which insulin cannot recruit transporters to the cell surface and have important implications regarding skeletal muscle insulin resistance in GDM and NIDDM.

Gene expression of GLUT4 in skeletal muscle from insulin-resistant patients with obesity, IGT, GDM, and NIDDM.

In obesity, impaired glucose tolerance (IGT), non-insulin-dependent diabetes mellitus (NIDDM), and gestational diabetes mellitus (GDM), defects in glucose transport system activity, contribute to insulin resistance in target tissues. In adipocytes from obese and NIDDM patients, we found that pretranslational suppression of the insulin-responsive GLUT4 glucose transporter isoform is a major cause of cellular insulin resistance; however, whether this process is operative in skeletal muscle is not clear. To address this issue, we performed percutaneous biopsies of the vastus lateralis in lean and obese control subjects and in obese patients with IGT and NIDDM and open biopsies of the rectus abdominis at cesarian section in lean and obese gravidas and gravidas with GDM. GLUT4 was measured in total postnuclear membrane fractions from both muscles by immunoblot analyses. The maximally insulin-stimulated rate of in vivo glucose disposal, assessed with euglycemic glucose clamps, decreased 26% in obesity and 74% in NIDDM, reflecting diminished glucose uptake by muscle. However, in vastus lateralis, relative amounts of GLUT4 per milligram membrane protein were similar (NS) among lean (1.0 +/- 0.2) and obese (1.5 +/- 0.3) subjects and patients with IGT (1.4 +/- 0.2) and NIDDM (1.2 +/- 0.2). GLUT4 content was also unchanged when levels were normalized per wet weight, per total protein, and per DNA as an index of cell number. Levels of GLUT4 mRNA were similarly not affected by obesity, IGT, or NIDDM whether normalized per RNA or for the amount of an unrelated constitutive mRNA species. Because muscle fibers (types I and II) exhibit different capacities for insulin-mediated glucose uptake, we tested whether a change in fiber composition could cause insulin resistance without altering overall levels of GLUT4. However, we found that quantities of fiber-specific isoenzymes (phopholamban and types I and II Ca(2+)-ATPase) were similar in all subject groups. In rectus abdominis, GLUT4 content was similar in the lean, obese, and GDM gravidas whether normalized per milligram membrane protein (relative levels were 1.0 +/- 0.2, 1.3 +/- 0.1, and 1.0 +/- 0.2, respectively) or per wet weight, total protein, and DNA. We conclude that in human disease states characterized by insulin resistance, i.e., obesity, IGT, NIDDM, and GDM, GLUT4 gene expression is normal in vastus lateralis or rectus abdominis. To the extent that these muscles are representative of total muscle mass, insulin resistance in skeletal muscle may involve impaired GLUT4 function or translocation and not transporter depletion as observed in adipose tissue.

Muscle Rad expression and human metabolism: potential role of the novel Ras-related GTPase in energy expenditure and body composition.

Ras associated with diabetes (Rad), a new ras-related GTPase, was recently identified by subtractive cloning as an mRNA in skeletal muscle that is overexpressed in NIDDM. To better understand its metabolic significance, we measured skeletal muscle Rad expression in well-characterized insulin sensitive (IS) and insulin resistant (IR) subjects with normal glucose tolerance and in untreated NIDDM patients. We found no differences in expression of Rad mRNA levels among IS, IR, and NIDDM groups using a ribonuclease protection assay (0.22 +/- 0.06, 0.13 +/- 0.01, and 0.16 +/- 0.02 relative units, respectively; NS) and no differences in Rad protein expression using a specific anti-peptide Rad antibody (1.05 +/- 0.18, 1.14 +/- 0.08, and 1.08 +/- 0.21 units/mg protein, respectively; NS). However, Rad protein levels were positively correlated with BMI (r = 0.43, P = 0.03) and percentage body fat (r = 0.55, P < 0.005), two independent measures of obesity, and negatively correlated with resting metabolic rate (r = 0.49, P = 0.01). In multiple regression analyses, percentage body fat and resting metabolic rate independently accounted for 30 and 10% of individual variability in muscle Rad protein expression. In conclusion, Rad expression in skeletal muscle is not altered as a function of insulin resistance or NIDDM in humans. However, these data, for the first time, implicate a role for Rad in regulating body composition and energy expenditure and provide a framework for studies designed to elucidate Rad's cellular functions.

Potential role for insulin and cycloheximide in regulating the intrinsic activity of glucose transporters in isolated rat adipocytes.

We examined the hypothesis that insulin stimulation of cellular glucose transport may involve a protein synthesis-dependent regulation of glucose transporter (GTer) activity independent of GTer translocation to the cell surface. Rat adipocytes were isolated, incubated with or without 10 micrograms/ml (36 microM) cycloheximide (CHX) for 60 min, and then with or without 7 nM insulin for 30 min. Glucose transport rates were assessed in intact cells, and both glucose transport rates and GTer levels were assessed in subcellular fractions of membrane vesicles before and after reconstitution into artificial liposomes. GTer functional and intrinsic activities were calculated as the ratio between these transport rates and GTer levels in native and reconstituted membranes, respectively. Insulin increased functional activity by 340% in native plasma membrane (PM) vesicles and intrinsic activity by 60% in reconstituted membranes (from 54 +/- 4 to 86 +/- 4 molecules transported per GTer/sec, P < 0.02). CHX preincubation of cells did not interfere with the insulin effect to stimulate glucose transport rate in either intact cells or in native PMs; it did, however, reduce PM GTer levels by 27-30%, but not affecting those in the intracellular pool. However, CHX additively increased the insulin-stimulated intrinsic activity of PM GTers by 67%. Relative reconstitution efficiencies, assessed by immunoblotting both native and reconstituted membranes against specific antibodies, were similar for GLUT 1 and GLUT 4. Although insulin did not alter this efficiency, CHX slightly decreased it for GLUT 4. Our data suggest that insulin stimulation of glucose transport may involve, as part of its mechanism, modulation of the GTer intrinsic activity. We further hypothesize that CHX effects on increasing this activity state of GTer may involve as yet unknown protein synthesis-dependent regulator(s).

Adipocytes exhibit abnormal subcellular distribution and translocation of vesicles containing glucose transporter 4 and insulin-regulated aminopeptidase in type 2 diabetes mellitus: implications regarding defects in vesicle trafficking.

Insulin resistance in type 2 diabetes is due to impaired stimulation of the glucose transport system in muscle and fat. Different defects are operative in these two target tissues because glucose transporter 4 (GLUT 4) expression is normal in muscle but markedly reduced in fat. In muscle, GLUT 4 is redistributed to a dense membrane compartment, and insulin-mediated translocation to plasma membrane (PM) is impaired. Whether similar trafficking defects are operative in human fat is unknown. Therefore, we studied subcellular localization of GLUT4 and insulin-regulated aminopeptidase (IRAP; also referred to as vp165 or gp160), which is a constituent of GLUT4 vesicles and also translocates to PM in response to insulin. Subcutaneous fat was obtained from eight normoglycemic control subjects (body mass index, 29 +/- 2 kg/m2) and eight type 2 diabetic patients (body mass index, 30 +/- 1 kg/m2; fasting glucose, 14 +/- 1 mM). In adipocytes isolated from diabetics, the basal 3-O-methylglucose transport rate was decreased by 50% compared with controls (7.1 +/- 2.9 vs. 14.1 +/- 3.7 mmol/mm2 surface area/min), and there was no increase in response to maximal insulin (7.9 +/- 2.7 vs. 44.5 +/- 9.2 in controls). In membrane subfractions from controls, insulin led to a marked increase of IRAP in the PM from 0.103 +/- 0.04 to 1.00 +/- 0.33 relative units/mg protein, concomitant with an 18% decrease in low-density microsomes and no change in high-density microsomes (HDM). In type 2 diabetes, IRAP overall expression in adipocytes was similar to that in controls; however, two abnormalities were observed. First, in basal cells, IRAP was redistributed away from low-density microsomes, and more IRAP was recovered in HDM (1.2-fold) and PM (4.4-fold) from diabetics compared with controls. Second, IRAP recruitment to PM by maximal insulin was markedly impaired. GLUT4 was depleted in all membrane subfractions (43-67%) in diabetes, and there was no increase in PM GLUT4 in response to insulin. Type 2 diabetes did not affect the fractionation of marker enzymes. We conclude that in human adipocytes: 1) IRAP is expressed and translocates to PM in response to insulin; 2) GLUT4 depletion involves all membrane subfractions in type 2 diabetes, although cellular levels of IRAP are normal; and 3) in type 2 diabetes, IRAP accumulates in membrane vesicles cofractionating with HDM and PM under basal conditions, and insulin-mediated recruitment to PM is impaired. Therefore, in type 2 diabetes, adipocytes express defects in trafficking of GLUT4/IRAP-containing vesicles similar to those causing insulin resistance in skeletal muscle.

Mechanisms of enhanced insulin sensitivity in endurance-trained athletes: effects on blood flow and differential expression of GLUT 4 in skeletal muscles.

Exercise is associated with increased insulin sensitivity. To better understand mechanisms that could be responsible for this association, we studied seven controls and seven endurance-trained athletes. A 600 mU/m2.min hyperinsulinemic euglycemic glucose clamp with the limb balance technique assessed insulin sensitivity as whole body glucose uptake (WBGU) and leg glucose uptake (LGU). Indirect calorimetry and hemodynamic measurements, such as leg blood flow (LBF) and cardiac output, were performed at baseline and maximal insulin stimulation. The content of the glucose transporter GLUT 4 and muscle fiber type were evaluated in three muscle groups: vastus lateralis, gastrocnemius, and biceps. Athletes exhibited 35% higher WBGU and 30% higher LGU than controls. Basal LBF (liters per min) was higher in athletes, but the difference was not statistically significant. After insulin stimulation, LBF was 31% higher in athletes than controls (P = 0.05). Indirect calorimetry revealed that athletes had a 44% higher rate of nonoxidative glucose metabolism than controls (P = 0.01). GLUT 4 levels in vastus were 90% (P < 0.05) greater in athletes, whereas smaller differences were noted between athletes and controls in biceps and gastrocnemius. Importantly, the vastus lateralis GLUT 4 content was correlated with WBGU (r = 0.60; P < 0.05) and LGU (r = 0.62; P < 0.05). Relative numbers of oxidative fibers were increased in vastus from athletes and were positively correlated with maximal oxygen consumption (VO2 max), but GLUT 4 content could not be correlated with oxidative fiber content in individual controls or athletes. We conclude that in humans 1) endurance training enhances insulin's ability to increase LBF; 2) GLUT 4 is differentially expressed as a function of muscle group and is up-regulated by exercise in a muscle-specific manner; 3) in vastus lateralis, GLUT 4 levels are well correlated with insulin-stimulated rates of both WBGU and LGU; and 4) GLUT 4 content and in vivo insulin sensitivity do not vary as a function of fiber type composition. Thus, blood flow and GLUT 4 expression in muscle are important mechanisms that mediate greater insulin sensitivity in athletes.

Gene expression of epithelial glucose transporters: the role of diabetes mellitus.

The functions of absorption of dietary glucose by the small intestine and reabsorption of filtered glucose by the renal proximal tubule are strikingly similar in their organization and in the way they adapt to uncontrolled diabetes mellitus. In both cases, transepithelial glucose and Na+ fluxes are augmented. The epithelial adaptations to hyperglycemia of uncontrolled diabetes are accomplished by increasing the glucose transport surface area and the number of the efflux glucose transporter GLUT2 located in the basolateral membrane. The signals that modify the size of the epithelium and the overexpression of basolateral GLUT2 are not known. It was speculated that high glucose levels and enhanced Na+ flux may be important factors in the signaling event that culminates in a renal and intestinal epithelium that is modified to transport higher rates of glucose against a higher extracellular level of glucose.

Molecular adaptations of GLUT1 and GLUT2 in renal proximal tubules of diabetic rats.

The renal reabsorption of glucose is mediated by two major classes of transporters. Initially, luminal glucose is concentrated in tubules by Na(+)-glucose cotransporters (Na(+)-GLUT). Afterwards, glucose reaches the blood space through facilitative glucose transporters, low-Michaelis constant (Km) GLUT1 and high-Km GLUT2. Hence, the transtubular flux of glucose could be impaired in hyperglycemia because the outwardly directed glucose gradient, from tubule to blood, is potentially lowered. However, in diabetic rats, transtubular glucose flux is not reduced but increased. In this work the molecular mechanism underlying this adaptation was examined. We tested the hypothesis that upregulation of renal tubular high-Km GLUT2 gene may compensate for the decrease in the tubule to blood glucose gradient. In rat tubules, GLUT1 protein and mRNA steady-state levels were reduced, and GLUT2 protein and mRNA levels were increased in rats after 2, 3, and 4 wk of uncontrolled streptozotocin-induced diabetes. These molecular adaptations were associated with augmented facilitative glucose flux. In summary, changes in GLUT1 and GLUT2 gene expression are important to the preservation of renal glucose reabsorption in hyperglycemia.

Glucose transporters of rat proximal tubule: differential expression and subcellular distribution.

In the late proximal tubule, glucose reabsorption progressively lowers the concentration of luminal glucose, and concentrative glucose influx increases to ensure complete glucose reabsorption. The change in glucose influx is effected by luminal Na(+)-dependent glucose transporters (Na(+)-GLUT), which exhibit higher Na(+)-to-glucose stoichiometric ratios in the late proximal tubule. In this work, the corresponding changes in the axial distribution of basolateral glucose efflux transporters (GLUTs) were examined. mRNAs encoding high-affinity facilitative basolateral transporter GLUT1, low-affinity GLUT2, and apical Na(+)-GLUT were identified in mixed populations of proximal convoluted and straight tubules. The organization of the cognate proteins was also appraised on Western blots. GLUT1 was present in glomeruli, proximal convoluted, and straight tubules, GLUT2 was only expressed in the proximal convoluted tubule, and Na(+)-GLUT was present in both proximal convoluted and straight segments. GLUT1 and GLUT2 were confined to the basolateral membrane, whereas Na(+)-GLUT was preferentially localized to the brush-border membrane. These data are consistent with the idea that glucose influx in early and late proximal tubule is achieved through Na(+)-GLUT, that GLUT1 and GLUT2 are responsible for glucose efflux in the early proximal tubule, and that in the late proximal tubule, where transcellular glucose flux is lower, only GLUT1 mediates glucose efflux.

Isolation by preparative free-flow electrophoresis and aqueous two-phase partition from rat adipocytes of an insulin-responsive small vesicle fraction with glucose transport activity.

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 microU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristic responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.

Identification of Rad's effector-binding domain, intracellular localization, and analysis of expression in Pima Indians.

In order to characterize the endogenous gene product for rad (ras-related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109-121, 178-195, 254-271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP-binding activity was inhibited by the addition of N-ethylmaliemide, GTP, GTP gamma S, and GDP beta S but not ATP or dithiothreitol. Moreover, cytosol-dependent rad-GTPase activity was stimulated by the peptide corresponding to amino acids 109-121. Antibodies corresponding to this epitope inhibited cytosol-dependent rad-GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP-binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109-121) may be involved in regulating rad-GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis.