Liver and muscle-fat type glucose transporter gene expression in obese and diabetic rats

In order to investigate the regulation of glucose transporter gene expression in the altered metabolic conditions of obesity and diabetes, we have measured mRNA levels encoding GLUT2 in the liver and GLUT4 in the gastrocnemius muscle from various insulin resistant animal models, including Zucker fatty, Wistar fatty, and streptozocin(STZ)-treated diabetic rats. Northern blot analysis revealed that GLUT2 mRNA levels were significantly (P less than 0.001) elevated in 14 wk Zucker fatty and Wistar fatty rats relative to lean littermates but were similar in these two groups at 5 wk of age. Furthermore, there was significant increase (P less than 0.01) in GLUT2 mRNA levels in STZ diabetic rats at 3 wk after treatment. GLUT4 mRNA levels were not significantly different between control and insulin resistant rats in all animal models. These results indicate that neither hyperinsulinemia nor hyperglycemia affects GLUT4 mRNA levels in the muscle. However, GLUT2 mRNA levels in the liver were elevated in obesity and diabetes, although this regulatory event occurred independently from circulating insulin or glucose concentrations.     

Fucoxanthin promotes translocation and induction of glucose transporter 4 in skeletal muscles of diabetic/obese KK-A(y) mice

Fucoxanthin (Fx) isolated from Undaria pinnatifida suppresses the development of hyperglycemia and hyperinsulinemia of diabetic/obese KK-A(y) mice after 2 weeks of feeding 0.2% Fx-containing diet. In the soleus muscle of KK-A(y) mice that were fed Fx, glucose transporter 4 (GLUT4) translocation to plasma membranes from cytosol was promoted. On the other hand, Fx increased GLUT4 expression levels in the extensor digitorum longus (EDL) muscle, although GLUT4 translocation tended to increase. The expression levels of insulin receptor (IR) mRNA and phosphorylation of Akt, which are in upstream of the insulin signaling pathway regulating GLUT4 translocation, were also enhanced in the soleus and EDL muscles of the mice fed Fx. Furthermore, Fx induced peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), which has been reported to increase GLUT4 expression, in both soleus and EDL muscles. These results suggest that in diabetic/obese KK-A(y) mice, Fx improves hyperglycemia by activating the insulin signaling pathway, including GLUT4 translocation, and inducing GLUT4 expression in the soleus and EDL muscles, respectively, of diabetic/obese KK-A(y) mice.     

Hyperglycemia, maturity-onset obesity, and insulin resistance in NONcNZO10/LtJ males, a new mouse model of type 2 diabetes

As a new mouse model of obesity-induced diabetes generated by combining quantitative trait loci from New Zealand Obese (NZO/HlLt) and Nonobese Nondiabetic (NON/LtJ) mice, NONcNZO10/LtJ (RCS10) male mice developed type 2 diabetes characterized by maturity onset obesity, hyperglycemia, and insulin resistance. To metabolically profile the progression to diabetes in preobese and obese states, a 2-h hyperinsulinemic euglycemic clamp was performed and organ-specific changes in insulin action were assessed in awake RCS10 and NON/LtJ (control) males at 8 and 13 wk of age. Prior to development of obesity and attendant increases in hepatic lipid content, 8-wk-old RCS10 mice developed insulin resistance in liver and skeletal muscle due to significant decreases in insulin-stimulated glucose uptake and GLUT4 expression in muscle. Transition to an obese and hyperglycemic state by 13 wk of age exacerbated insulin resistance in skeletal muscle, liver, and heart associated with organ-specific increases in lipid content. Thus, this polygenic mouse model of type 2 diabetes, wherein plasma insulin is only modestly elevated and obesity develops with maturity yet insulin action and glucose metabolism in skeletal muscle and liver are reduced at an early prediabetic age, should provide new insights into the etiology of type 2 diabetes.     

Effect of iridoid glucoside on plasma lipid profile, tissue fatty acid changes, inflammatory cytokines, and GLUT4 expression in skeletal muscle of streptozotocin-induced diabetic rats

The present study was designed to examine the antihyperlipidaemic potential of iridoid glucoside isolated from Vitex negundo leaves in STZ-induced diabetic rats. The levels of cholesterol (TC), triglycerides, lipoproteins, free fatty acids, phospholipids, fatty acid composition, proinflammatory cytokines, muscle glycogen content, and glucose transporter 4 (GLUT4) expression were estimated in control and diabetic rats. Oral administration of iridoid glucoside at a dose of 50 mg/kg body weight per day to STZ-induced diabetic rats for a period of 30 days resulted in a significant reduction in plasma and tissue (liver and kidney) cholesterol, triglycerides, free fatty acids, and phospholipids. In addition, the decreased plasma levels of high-density lipoprotein-cholesterol and increased plasma levels of low density lipoprotein- and very low density lipoprotein-cholesterol in diabetic rats were restored to near normal levels following treatment with iridoid glucoside. The fatty acid composition of the liver and kidney was analyzed by gas chromatography. The altered fatty acid composition in the liver and kidney of diabetic rats was also restored upon treatment with iridoid glucoside. Moreover, the elevated plasma levels of proinflammatory cytokines and decreased levels of muscle glycogen and GLUT4 expression in the skeletal muscle of diabetic rats were reinstated to their normal levels via enhanced secretion of insulin from the remnant β cells of pancreas by the administration of iridoid glucoside. The effect produced by iridoid glucoside on various parameters was comparable with that of glibenclamide, a well-known antihyperglycemic drug.     

Metformin and exercise reduce muscle FAT/CD36 and lipid accumulation and blunt the progression of high-fat diet-induced hyperglycemia

Derangements in skeletal muscle fatty acid (FA) metabolism associated with insulin resistance in obesity appear to involve decreased FA oxidation and increased accumulation of lipids such as ceramides and diacylglycerol (DAG). We investigated potential lipid-related mechanisms of metformin (Met) and/or exercise for blunting the progression of hyperglycemia/hyperinsulinemia and skeletal muscle insulin resistance in female Zucker diabetic fatty rats (ZDF), a high-fat (HF) diet-induced model of diabetes. Lean and ZDF rats consumed control or HF diet (48 kcal %fat) alone or with Met (500 mg/kg), with treadmill exercise, or with both exercise and Met interventions for 8 wk. HF-fed ZDF rats developed hyperglycemia (mean: 24.4 +/- 2.1 mM), impairments in muscle insulin-stimulated glucose transport, increases in the FA transporter FAT/CD36, and increases in total ceramide and DAG content. The development of hyperglycemia was significantly attenuated with all interventions, as was skeletal muscle FAT/CD36 abundance and ceramide and DAG content. Interestingly, improvements in insulin-stimulated glucose transport and increased GLUT4 transporter expression in isolated muscle were seen only in conditions that included exercise training. Reduced FA oxidation and increased triacylglycerol synthesis in isolated muscle were observed with all ZDF rats compared with lean rats (P < 0.01) and were unaltered by therapeutic intervention. However, exercise did induce modest increases in peroxisome proliferator-activated receptor-gamma coactivator-1alpha, citrate synthase, and beta-hydroxyacyl-CoA dehydrogenase activity. Thus reduction of skeletal muscle FAT/CD36 and content of ceramide and DAG may be important mechanisms by which exercise training blunts the progression of diet-induced insulin resistance in skeletal muscle.     

A high-fat diet catalyzes progression to hyperglycemia in mice with selective impairment of insulin action in Glut4-expressing tissues

Insulin resistance impairs postprandial glucose uptake through glucose transporter type 4 (GLUT4) and is the primary defect preceding type 2 diabetes. We previously generated an insulin-resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in the muscle, adipose, and neuronal subpopulations. However, the rate of diabetes in GIRKO mice remained low prior to 6 months of age on normal chow diet (NCD), suggesting that additional factors/mechanisms are responsible for adverse metabolic effects driving the ultimate progression of overt diabetes. In this study, we characterized the metabolic phenotypes of the adult GIRKO mice acutely switched to high-fat diet (HFD) feeding in order to identify additional metabolic challenges required for disease progression. Distinct from other diet-induced obesity (DIO) and genetic models (e.g., db/db mice), GIRKO mice remained leaner on HFD feeding, but developed other cardinal features of insulin resistance syndrome. GIRKO mice rapidly developed hyperglycemia despite compensatory increases in β-cell mass and hyperinsulinemia. Furthermore, GIRKO mice also had impaired oral glucose tolerance and a limited glucose-lowering benefit from exendin-4, suggesting that the blunted incretin effect contributed to hyperglycemia. Secondly, GIRKO mice manifested severe dyslipidemia while on HFD due to elevated hepatic lipid secretion, serum triglyceride concentration, and lipid droplet accumulation in hepatocytes. Thirdly, GIRKO mice on HFD had increased inflammatory cues in the gut, which were associated with the HFD-induced microbiome alterations and increased serum lipopolysaccharide (LPS). In conclusion, our studies identified important gene/diet interactions contributing to diabetes progression, which might be leveraged to develop more efficacious therapies.     

GLUT4 and UBC9 protein expression is reduced in muscle from type 2 diabetic patients with severe insulin resistance

Aims

Subgroups of patients with type 2 diabetes mellitus demand large insulin doses to maintain euglycemia. These patients are characterized by severe skeletal muscle insulin resistance and the underlying pathology remains unclear. The purpose of this study was to examine protein expression of the principal glucose transporter, GLUT4, and associated proteins in skeletal muscle from type 2 diabetic patients characterized by severe insulin resistance.

Methods

Seven type 2 diabetic patients with severe insulin resistance (mean insulin dose 195 IU/day) were compared with seven age matched type 2 diabetic patients who did not require insulin treatment, and with an age matched healthy control group. Protein expression of GLUT4 and associated proteins was assessed in muscle and fat biopsies using standard western blotting techniques.

Results

GLUT4 protein expression was significantly reduced by ∼30 pct in skeletal muscle tissue from severely insulin resistant type 2 diabetic subjects, compared with both healthy controls and type 2 diabetic subjects that did not require insulin treatment. In fat tissue, GLUT4 protein expression was reduced in both diabetic groups. In skeletal muscle, the reduced GLUT4 expression in severe insulin resistance was associated with decreased ubiquitin-conjugating enzyme 9 (UBC9) expression while expression of GLUT1, TBC1D1 and AS160 was not significantly different among type 2 diabetic patients and matched controls.

Conclusions

Type 2 diabetic patients with severe insulin resistance have reduced expression of GLUT4 in skeletal muscle compared to patients treated with oral antidiabetic drugs alone. GLUT4 protein levels may therefore play a role in the pathology behind type 2 diabetes mellitus among subgroups of patients, and this may explain the heterogeneous response to insulin treatment. This new finding contributes to the understanding of the underlying mechanisms for the development of extreme insulin resistance.     

Nutritionally induced diabetes in desert rodents as models of type 2 diabetes: Acomys cahirinus (spiny mice) and Psammomys obesus (desert gerbil)

The dietary effects of hyperglycemia increasingly result in type 2 diabetes in humans. Two species, the spiny mice (Acomys cahirinus) and the desert gerbil (Psammomys obesus), which have different metabolic responses to such effects, are discussed. Spiny mice exemplify a pathway that leads to diabetes without marked insulin resistance due to low supply of insulin on abundant nutrition, possibly characteristic of a desert animal. They respond with obesity and glucose intolerance, beta-cell hyperplasia, and hypertrophy on a standard rodent diet supplemented with fat-rich seeds. The accompanying hyperglycemia and hyperinsulinemia are mild and intermittent but after a few months, the enlarged pancreatic islets suddenly collapse, resulting in loss of insulin and ketosis. Glucose and other secretagogues produce only limited insulin release in vivo and in vitro, pointing to the inherent disability of the beta-cells to respond with proper insulin secretion despite their ample insulin content. On a 50% sucrose diet there is marked lipogenesis with hyperlipidemia without obesity or diabetes, although beta-cell hypertrophy is evident. P.obesus is characterized by muscle insulin resistance and the inability of insulin to activate the insulin signaling on a high-energy (HE) diet. Insulin resistance imposes a vicious cycle of Hyperglycemia and compensatory hyperinsulinemia, leading to beta-cell failure and increased secretion of proinsulin. Ultrastructural studies reveal gradual disappearance of beta-cell glucokinase, GLUT 2 transporter, and insulin, followed by apoptosis of beta-cells. Studies using the non-insulin-resistant HE diet-fed animals maintained as a control group are discussed. The insulin resistance that is evident to date in the normoglycemic state on a low-energy diet indicates sparing of glucose fuel in muscles of a desert-adapted animal for the benefit of glucose obligatory tissues. Also discussed are the effect of Psammomys age on the disabetogenicity of the HE diet; the impaired function of several components of the insulin signal transduction pathway in muscles, which reduces the availability of GLUT4 transporter; the testing of several antidiabetic modalities for the prevention of nutritional diabetes in Psammomys; and various complications related to the diabetic condition.     

Curcumin prevents liver fat accumulation and serum fetuin-A increase in rats fed a high-fat diet

Fetuin-A is synthesized in the liver and is secreted into the bloodstream. Clinical studies suggest involvement of fetuin-A in metabolic disorders such as visceral obesity, insulin resistance, diabetes, and fatty liver. Curcumin is extracted from the rhizome Curcuma longa and has been shown to possess potent antioxidant, anticarcinogenic, anti-inflammatory, and hypoglycemic properties. In this study, we investigated the effect of curcumin treatment on serum fetuin-A levels as well as hepatic lipids and prooxidant–antioxidant status in rats fed a high-fat diet (HFD). Male Sprague–Dawley rats were divided into six groups. Group 1 was fed control diet (10 % of total calories from fat). Groups 2 and 3 were given curcumin (100 and 400 mg/kg bw/day, respectively ) by gavage for 8 weeks and were fed control diet. Group 4 was fed with HFD (60 % of total calories from fat). Groups 5 and 6 received HFD together with the two doses of curcumin, respectively. Curcumin treatment appeared to be effective in reducing liver triglycerides and serum fetuin-A levels. These findings suggest that the reduction of fetuin-A may contribute to the beneficial effects of curcumin in the pathogenesis of obesity.     

Fructose transport and metabolism in adipose tissue of Zucker rats: Diminished GLUT5 activity during obesity and insulin resistance

Fructose is a major dietary sugar, which is elevated in the serum of diabetic humans, and is associated with metabolic syndromes important in the pathogenesis of diabetic complications. The facilitative fructose transporter, GLUT5, is expressed in insulin-sensitive tissues (skeletal muscle and adipocytes) of humans and rodents, where it mediates the uptake of substantial quantities of dietary fructose, but little is known about its regulation. We found that GLUT5 abundance and activity were compromised severely during obesity and insulin resistance in Zucker rat adipocytes. Adipocytes from young obese (fa/fa), highly insulin-responsive Zucker rats contained considerably more plasma membrane GLUT5 than those from their lean counterparts (1.8-fold per microgram membrane protein), and consequently exhibited higher fructose transport (fivefold) and metabolism (threefold) rates. Lactate production was the preferred route for fructose metabolism in these cells. As the rats aged and become more obese and insulin-resistant, adipocyte GLUT5 surface density (12-fold) and fructose transport (10-fold) and utilisation rates (threefold) fell markedly. The GLUT5 loss was more dramatic in adipocytes from obese animals, which developed a more marked insulin resistance than lean counterparts. The decline of GLUT5 levels in adipocytes from older, obese animals was not a generalised effect, and was not observed in kidney, nor was this expression pattern shared by the 1 subunit of the Na⁺/K⁺ ATPase. Our findings suggest that plasma membrane GLUT5 levels and thus fructose utilisation rates in adipocytes are dependent upon cellular insulin sensitivity, inferring a possible role for GLUT5 in the elevated circulating fructose observed during diabetes, and associated pathological complications. (Mol Cell Biochem 261: 23–33, 2004)     

Protein-rich extract of <Emphasis Type="Italic">Musca domestica</Emphasis> larvae alleviated metabolic disorder in STZ-induced type 2 diabetic rat model via hepatoprotective and pancreatic β-cell protective activities

The study was designed to explore the beneficial effect of Musca domestica larvae extract (MDLE) on a metabolic disorder using a diabetic rat model. Streptozotocin-induced diabetic rats were treated with or without MDLE. Blood glucose, insulin levels, lipid profiles, and oxidative stress markers were measured. The morphological changes in the pancreas and liver were determined, as well as insulin expression. The expression of glucose transporter 4 (GLUT4), phospho-adenosine monophosphate-activated protein kinase (p-AMPK)/total AMPK, superoxide dismutase 1 (SOD1), catalase (CAT), and peroxisome proliferator-activated receptor gamma (PPARγ) were detected. Compared with untreated diabetic rats, MDLE-treated rats had decreased urine volume, food intake, and water intake, along with significantly lower levels of blood glucose, malondialdehyde (MDA), plasma triglycerides, low-density lipoprotein (LDL), and total cholesterol. MDLE-treated rats also had higher levels of SOD activity, high-density lipoprotein (HDL), and insulin. MDLE treatment partially restored the β-cell population, improved the liver necrosis and islet cell damage, reversed the decreased expression of GLUT4, phospho-AMPK, SOD1, and CAT in the liver, skeletal muscle and pancreatic tissue, and also increased the expression of PPARγ in the liver and adipose tissue in diabetic rats. In conclusion, the obtained results suggest that MDLE could possibly be used pharmacologically as an adjuvant for the treatment of diabetes.     

Fargesin improves lipid and glucose metabolism in 3T3-L1 adipocytes and high-fat diet-induced obese mice

This study examined the effects of fargesin, a neolignan isolated from Magnolia plants, on obesity and insulin resistance and the possible mechanisms involved in these effects in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese mice. Fargesin promoted the glucose uptake in 3T3-L1 adipocytes. In HFD-induced obese mice, fargesin decreased the body weight gain, white adipose tissue (WAT), and plasma triglyceride, non-esterified fatty acid and glucose levels, and improved the glucose tolerance. Fargesin increased glucose transporter 4 (GLUT4) protein expression and phosphorylation of Akt, AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in both 3T3-L1 adipocytes and WAT of HFD-induced obese mice. Fargesin also decreased the mRNA expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase-1 (CPT-1), uncoupling protein-2 (UCP-2) and leptin in WAT. Taken together, the present findings suggest that fargesin improves dyslipidemia and hyperglycemia by activating Akt and AMPK in WAT. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Divergence between GLUT4 mRNA and protein abundance in skeletal muscle of insulin resistant rats

Hyperglycemia and skeletal muscle insulin resistance coexist in uncontrolled type 2 diabetes mellitus. Similar defects in insulin action were observed in glucose-infused, normal rats, a model of glucose toxicity. In these rats insulin-stimulated glucose uptake by skeletal muscle was decreased due to a post-receptor defect. We investigated whether the impaired glucose uptake resulted from a decrease in the abundance of the predominant muscle glucose transporter (GLUT4) mRNA and/or protein. GLUT4 protein abundance in the hyperglycemic rats was not different from the control group despite a 50% decrease in muscle glucose uptake. GLUT4 mRNA abundance was 2.5-fold greater in the hyperglycemic rats as compared to the control animals. We conclude that the coexistence of hyperglycemia and hyperinsulinemia results in (1) a defect in GLUT4 compartmentalization and/or functional activity and (2) a divergence between GLUT4 mRNA levels and translation.     

Increased muscular dehydroepiandrosterone levels are associated with improved hyperglycemia in obese rats

This study was undertaken to assess the effects of dehydroepiandrosterone (DHEA) administration and exercise training on muscular DHEA and 5α-dihydrotestosterone (DHT) levels and hyperglycemia in diet-induced obese and hyperglycemic rats. After 14 wk of a high-sucrose diet, obese male Wistar rats were assigned randomly to one of three 6-wk regimens: control, DHEA treatment, or exercise training (running at 25 m/min for 1 h, 5 days/wk; n = 10 each group). Results indicate that either 6 wk of DHEA treatment or exercise training significantly attenuated serum insulin and fasting glucose levels compared with the control group. Plasma and muscle concentrations of DHEA and DHT and expression levels of 5α-reductase were significantly higher in the DHEA-treated and exercise-training groups. Moreover, both DHEA administration and exercise training upregulated GLUT4 translocation with concomitant increases in protein kinase B and protein kinase Cζ/λ phosphorylation. Muscle DHEA and DHT concentrations closely correlated with blood glucose levels (DHEA treatment: r = -0.68, P < 0.001; exercise training: r = -0.65, P < 0.001), serum insulin levels, and activation of the GLUT4-regulated signaling pathway. Thus, increased levels of muscle sex steroids may contribute to improved fasting glucose levels via upregulation of GLUT4-regulated signaling in diet-induced obesity and hyperglycemia.     

Prevention mechanisms of glucose intolerance and obesity by cacao liquor procyanidin extract in high-fat diet-fed C57BL/6 mice

In this study, we investigated whether cacao liquor procyanidin (CLPr) extract, which consists of 4.3% catechin, 6.1% epicatechin, 39.4% procyanidins and others, ameliorated hyperglycemia and obesity in C57BL/6 mice fed a control or high-fat diet for 13 weeks. CLPr suppressed high-fat diet-induced hyperglycemia, glucose intolerance and fat accumulation in white adipose tissue. CLPr also promoted translocation of glucose transporter 4 (GLUT4) and phosphorylation of AMP-activated protein kinase α (AMPKα) in the plasma membrane of skeletal muscle and brown adipose tissue. Phosphorylation of AMPKα was also enhanced in the liver and white adipose tissue. CLPr up-regulated the gene and protein expression levels of uncoupling protein (UCP)-1 in brown adipose tissue and UCP-3 in skeletal muscle. These results indicate that CLPr is a beneficial food material for the prevention of hyperglycemia and obesity. Activation of AMPKα, translocation of GLUT4 and up-regulation of UCP expression in skeletal muscle and adipose tissue are involved in the molecular mechanisms by which CLPr prevents hyperglycemia and obesity.     

Resistance training restores metabolic alterations induced by monosodium glutamate in a sex-dependent manner in male and female rats

Despite resistance exercises being associated with health outcomes, numerous issues are still unresolved and further research is required before the exercise can faithfully be prescribed as medicine. The goal of this study was to investigate whether there are sex differences in resistance training effects on metabolic alterations induced by monosodium glutamate (MSG), a model of obesity, in male and female rats. Male and female Wistar rats received MSG (4 g/kg body weight/day, s.c.) from postnatal day 1 to 10. After 10 days from MSG administration, the rats were separated into two groups: MSG-sedentary and MSG-exercised. At postnatal day 60, the animals started a resistance training protocol in an 80 degrees inclined vertical ladder apparatus and performed it for 7 weeks. Control rats received saline solution and were divided in saline-sedentary and saline-exercised. Resistance training restored all plasma biochemical parameters (glucose, cholesterol, triglycerides, aspartate aminotransferase, and alanine aminotransferase) increased in male and female rats treated with MSG. The MSG administration induced hyperglycemia associated with a decrease in the skeletal muscle glucose transporter 4 (GLUT4) levels and accompanied by deregulation in proteins, G-6Pase, and tyrosine aminotransferase, involved in hepatic glucose metabolism of male and female rats. MSG induced dyslipidemia and lipotoxicity in the liver and skeletal muscle of male rats. Regarding female rats, lipotoxicity was found only in the skeletal muscle. The resistance training had beneficial effects against metabolic alterations induced by MSG in male and female rats, through regulation of proteins (GLUT2, protein kinase B, and GLUT4) involved in glucose and lipid pathways in the liver and skeletal muscle.     

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body¹. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes^2,3.The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.     

Insulin and glucose transporter gene expression in obesity and diabetes.

In order to determine the role of insulin and glucose transporter gene expression in the development of diabetes in obesity, we examined insulin and GLUT2-liver type and GLUT4-muscle-fat type glucose transporter mRNA levels in obese and diabetic rats. Ventromedial hypothalamus-lesioned (VMH), Zucker fatty (ZF), and Wistar fatty (WF) rats were used as models. VMH and ZF rats are most frequently used as models for simple obesity. In contrast, WF rats, which have been established by transferring the fa gene of ZF rats to Wistar Kyoto rats, develop both obesity and diabetes. Pancreatic insulin content of VMH rats at 10 weeks after the operation and of ZF rats at 5 and 14 weeks of age was significantly higher than that of controls. On the other hand, insulin content of WF rats at 5 and 14 weeks of age was not significantly different from that of lean littermates. The insulin mRNA levels of VMH rats were increased progressively and were significantly higher than those in sham-operated animals at 4 and 10 weeks after the operation. In ZF rats, the insulin mRNA levels at 5 and 14 weeks of age were significantly higher than those of their lean littermates. In WF rats, by contrast, the insulin mRNA levels were similar to those of lean littermates at 5 and 14 weeks of age. The insulin mRNA levels of WF rats were about 40% of that of ZF rats at 14 weeks of age. On the other hand, at 14 weeks of age, the GLUT2 mRNA levels of liver were significantly higher in ZF and WF rats than those in their respective littermates, but not at 5 weeks of age. The GLUT4 mRNA levels of skeletal muscle in both ZF and WF rats were not significantly different from those of controls. It is suggested that the inability of WF rats to augment insulin gene expression in response to a large demand for insulin is associated with the occurrence of diabetes, and that the activation of GLUT2 mRNA without the activation of GLUT4 mRNA is common to obesity with and without diabetes.     

Expression of GLUT4 glucose transporter protein in adipose tissue and skeletal muscle from streptozotocin-induced diabetic pregnant rats.

Depletion of GLUT4, the primary glucose transporter protein in adipose tissue and skeletal muscle, is reported to contribute to insulin resistance in pregnancy or diabetes. To examine this phenomenon, the expression of GLUT4 protein was assessed by Western blotting in streptozotocin-induced diabetic pregnant rats. In adipose tissue, relative to control, it was decreased by 30% in the normal pregnant group (p<0.001), by 37% in the diabetic nonpregnant group (p<0.01) and by 65% in the diabetic pregnant group (p<0.001). On the other hand, no significant variation was evident among the groups in skeletal muscle. To assess the mechanisms responsible for depletion of GLUT4 protein in adipose tissue, we quantitated levels of GLUT4 mRNA with a RNase protection assay. It was decreased by 44% in the normal pregnant group (p<0.05) and by 55% in the diabetic pregnant group (p<0.05), but not altered in the diabetic nonpregnant group. These results suggest that the depletion of GLUT4 protein in adipose tissue is a factor contributing to insulin resistance in pregnancy or diabetes, especially when the two states exist in combination.     

Adipose-specific overexpression of GLUT4 reverses insulin resistance and diabetes in mice lacking GLUT4 selectively in muscle

Adipose tissue plays an important role in glucose homeostasis and affects insulin sensitivity in other tissues. In obesity and type 2 diabetes, glucose transporter 4 (GLUT4) is downregulated in adipose tissue, and glucose transport is also impaired in muscle. To determine whether overexpression of GLUT4 selectively in adipose tissue could prevent insulin resistance when glucose transport is impaired in muscle, we bred muscle GLUT4 knockout (MG4KO) mice to mice overexpressing GLUT4 in adipose tissue (AG4Tg). Overexpression of GLUT4 in fat not only normalized the fasting hyperglycemia and glucose intolerance in MG4KO mice, but it reduced these parameters to below normal levels. Glucose infusion rate during a euglycemic clamp study was reduced 46% in MG4KO compared with controls and was restored to control levels in AG4Tg-MG4KO. Similarly, insulin action to suppress hepatic glucose production was impaired in MG4KO mice and was restored to control levels in AG4Tg-MG4KO. 2-deoxyglucose uptake during the clamp was increased approximately twofold in white adipose tissue but remained reduced in skeletal muscle of AG4Tg-MG4KO mice. AG4Tg and AG4Tg-MG4KO mice have a slight increase in fat mass, a twofold elevation in serum free fatty acids, an approximately 50% increase in serum leptin, and a 50% decrease in serum adiponectin. In MG4KO mice, serum resistin is increased 34% and GLUT4 overexpression in fat reverses this. Overexpression of GLUT4 in fat also reverses the enhanced clearance of an oral lipid load in MG4KO mice. Thus overexpression of GLUT4 in fat reverses whole body insulin resistance in MG4KO mice without restoring glucose transport in muscle. This effect occurs even though AG4Tg-MG4KO mice have increased fat mass and low adiponectin and is associated with normalization of elevated resistin levels.