Maximal Insulin-stimulated Glucose Transport Research Articles

Insulin action in adipose tissue in type 1 diabetes

Background:Insulin action has been reported to be normal in type 1 diabetic patients. However, some studies have reported an insulin resistance state in these patients. The aim of this study was to investigate insulin resistance in a group of type 1 diabetic patients. We studied the insulin action in adipose tissue and analyzed the effects of duration of disease, body mass index (BMI), and glycosylated hemoglobin on insulin action at the receptor and postreceptor levels in adipocytes.Methods:Nine female type 1 diabetic patients with different durations of disease and eight nondiabetic female patients of comparable age and BMI were studied. 125I-insulin binding and U-[14C]-D-glucose transport was measured in a sample of subcutaneous gluteus adipose tissue obtained by open surgical biopsy from each subject.Results:The duration of disease was negatively correlated with both 125I-insulin binding capacity (r = −0.70, P < 0.05) and basal and maximum insulin-stimulated glucose transport (r = −0.87, P < 0.01, and r = −0.88, P < 0.01, respectively). Maximum specific 125I-insulin binding to the receptors in adipocytes was higher in the group of patients with a shorter duration of disease (P < 0.01). Basal and maximum insulin-stimulated glucose transport was significantly higher in the group with less than 5 years of disease (P < 0.01). No correlation was found between BMI and insulin action.Conclusion:Female type 1 diabetic patients have normal insulin action. There is a high glucose uptake in the early phase of the disease, although a longer duration of disease appears to be a contributing factor to a decrease in insulin action in these patients, and involving both receptor and postreceptor mechanisms.

Oral administration of a PPAR-δ agonist to rodents worsens, not improves, maximal insulin-stimulated glucose transport in skeletal muscle of different fibers

Agonists targeting the nuclear receptor peroxisome proliferator-activated receptors (PPAR)-delta may be potential therapeutic agents for insulin-resistant related conditions, as they may be able to stimulate fatty acid (FA) oxidation and attenuate the accumulation of harmful lipid species in skeletal muscle. Several reports have demonstrated that PPAR-delta agonists improve whole body insulin sensitivity. However, whether these agonists exert their direct effects on glucose and FA metabolism in skeletal muscle, and specifically with different fiber types, is unknown. This study was undertaken to determine the effects of oral treatment with the PPAR-delta agonist, GW 501516, in conjunction with the administration of a high-saturated-fat diet on insulin-stimulated glucose transport in isolated oxidative (soleus) and glycolytic (epitrochlearis) rodent skeletal muscle in vitro. High-fat feeding significantly decreased maximal insulin-stimulated glucose transport in soleus, but not epitrochlearis muscle, and was associated with increased skeletal muscle diacylglycerol and ceramide content. Unexpectedly, treatment with the PPAR-delta agonist significantly reduced insulin-stimulated glucose transport in both soleus and epitrochlearis muscles, regardless of dietary fat content. The reduction in insulin-stimulated glucose transport induced by the agonist was associated with large increases in total muscle fatty acid translocase (FAT)/CD36protein content, but not diacylglycerol or ceramide contents. Agonist treatment did not alter the protein content of PPAR-delta, GLUT4, or insulin-signaling proteins (IRS-1, p85 PI3-K, Akt). Agonist treatment led to a small, but significant increase, in the oxidative capacity of glycolytic but not oxidative muscle. We propose that chronic treatment with the PPAR-delta agonist GW 501516 may induce or worsen insulin resistance in rodent skeletal muscle by increasing the capacity for FA transport across the sarcolemma without a sufficient compensatory increase in FA oxidation. However, an accumulation of diacylglycerol and ceramide, while associated with diet-induced insulin resistance, does not appear to be responsible for the agonist-induced reduction in insulin-stimulated glucose transport.

Adiponectin resistance precedes the accumulation of skeletal muscle lipids and insulin resistance in high-fat-fed rats

High-fat (HF) diets can induce insulin resistance (IR) by altering skeletal muscle lipid metabolism. An imbalance between fatty acid (FA) uptake and oxidation results in intramuscular lipid accumulation, which can impair the insulin-signaling cascade. Adiponectin (Ad) is an insulin-sensitizing adipokine known to stimulate skeletal muscle FA oxidation and reduce lipid accumulation. Evidence of Ad resistance has been shown in obesity and following chronic HF feeding and may contribute to lipid accumulation observed in these conditions. Whether Ad resistance precedes and is associated with the development of IR is unknown. We conducted a time course HF feeding trial for 3 days, 2 wk, or 4 wk to determine the onset of Ad resistance and identify the ensuing changes in lipid metabolism and insulin signaling leading to IR in skeletal muscle. Ad stimulated FA oxidation (+28%, P < or = 0.05) and acetyl-CoA carboxylase phosphorylation (+34%, P < or = 0.05) in control animals but failed to do so in any HF-fed group (i.e., as early as 3 days). By 2 wk, plasma membrane FA transporters and intramuscular diacylglycerol (DAG) and ceramide were increased, and insulin-stimulated phosphorylation of both protein kinase B and protein kinase B substrate 160 was blunted compared with control animals. After 4 wk of HF feeding, maximal insulin-stimulated glucose transport was impaired compared with control. Taken together, our results demonstrate that an early loss of Ad's stimulatory effect on FA oxidation precedes an increase in plasmalemmal FA transporters and the accumulation of intramuscular DAG and ceramide, blunted insulin signaling, and ultimately impaired maximal insulin-stimulated glucose transport in skeletal muscle induced by HF diets.

Polycystic Ovary Syndrome Is Associated with Tissue-Specific Differences in Insulin Resistance

The potential differential contributions of skeletal muscle and adipose tissue to whole body insulin resistance were evaluated in subjects with polycystic ovary syndrome (PCOS). Forty-two PCOS subjects and 15 body mass index-matched control subjects were studied. Insulin action was evaluated by the hyperinsulinemic/euglycemic clamp procedure. Isolated adipocytes and cultured muscle cells were analyzed for glucose transport activity; adipocytes, muscle tissue, and myotubes were analyzed for the expression and phosphorylation of insulin-signaling proteins. Fifty-seven per cent of the PCOS subjects had impaired glucose tolerance and the lowest rate of maximal insulin-stimulated whole body glucose disposal compared to controls (P < 0.01). PCOS subjects with normal glucose tolerance had intermediate reduction in glucose disposal rate (P < 0.05 vs. both control and impaired glucose tolerance subjects). However, rates of maximal insulin-stimulated glucose transport (insulin responsiveness) into isolated adipocytes were comparable between all three groups, whereas PCOS subjects displayed impaired insulin sensitivity. In contrast, myotubes from PCOS subjects displayed reduced insulin responsiveness for glucose uptake and normal sensitivity. There were no differences between groups in the expression of glucose transporter 4 or insulin-signaling proteins or maximal insulin stimulation of phosphorylation of Akt in skeletal muscle, myotubes, or adipocytes. Individuals with PCOS display impaired insulin responsiveness in skeletal muscle and myotubes, whereas isolated adipocytes display impaired insulin sensitivity but normal responsiveness. Skeletal muscle and adipose tissue contribute differently to insulin resistance in PCOS. Insulin resistance in PCOS cannot be accounted for by differences in the expression of selected signaling molecules or maximal phosphorylation of Akt.

Atypical Antipsychotic Drugs Directly Impair Insulin Action in Adipocytes: Effects on Glucose Transport, Lipogenesis, and Antilipolysis

Treatment with second-generation antipsychotics (SGAs) has been associated with weight gain and the development of diabetes mellitus, although the mechanisms are unknown. We tested the hypothesis that SGAs exert direct cellular effects on insulin action and substrate metabolism in adipocytes. We utilized two cultured cell models including 3T3-L1 adipocytes and primary cultured rat adipocytes, and tested for effects of SGAs risperidone (RISP), clozapine (CLZ), olanzapine (OLZ), and quetiapine (QUE), together with conventional antipsychotic drugs butyrophenone (BUTY), and trifluoperazine (TFP), over a wide concentration range from 1 to 500 microM. The effects of antipsychotic drugs on basal and insulin-stimulated rates of glucose transport were studied at 3 h, 15 h, and 3 days. Both CLZ and OLZ (but not RISP) at doses as low as 5 microM were able to significantly decrease the maximal insulin-stimulated glucose transport rate by approximately 40% in 3T3-L1 cells, whereas CLZ and RISP reduced insulin-stimulated glucose transport rates in primary cultured rat adipocytes by approximately 50-70%. Conventional drugs (BUTY and TFP) did not affect glucose transport rates. Regarding intracellular glucose metabolism, both SGAs (OLZ, QUE, RISP) and conventional drugs (BUTY and TFP) increased basal and/or insulin-stimulated glucose oxidation rates, whereas rates of lipogenesis were increased by CLZ, OLZ, QUE, and BUTY. Finally, rates of lipolysis in response to isoproterenol were reduced by the SGAs (CLZ, OLZ, QUE, RISP), but not by BUTY or TFP. These experiments demonstrate that antipsychotic drugs can differentially affect insulin action and metabolism through direct cellular effects in adipocytes. However, only SGAs were able to impair the insulin-responsive glucose transport system and to impair lipolysis in adipocytes. Thus, SGAs directly induce insulin resistance and alter lipogenesis and lipolysis in favor of progressive lipid accumulation and adipocyte enlargement. These effects of SGAs on adipocytes could explain, in part, the association of SGAs with weight gain and diabetes.

Activation of the A1adenosine receptor increases insulin-stimulated glucose transport in isolated rat soleus muscle

The A1 adenosine receptor (A1AR) has been suggested to participate in insulin- and contraction-stimulated glucose transport in skeletal muscle, but the qualitative and quantitative nature of the effect are controversial. We sought to determine if A1AR is expressed in rat soleus muscle and then characterize its role in glucose transport in this muscle. A1AR mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. To examine the role of adenosine in 3-O-methylglucose transport, isolated muscles were exposed to adenosine deaminase and alpha,beta-methylene adenosine diphosphate to remove endogenous adenosine and were left unstimulated (basal) or stimulated with insulin. To assess the functional participation of A1AR in 3-O-methylglucose transport, muscles were incubated with A1-selective agonist and (or) antagonist in the absence of endogenous adenosine and with or without insulin. A1AR mRNA was expressed in soleus muscle and A1AR was present at the plasma membrane. Removal of endogenous adenosine reduced glucose transport in response to 100 microU/mL insulin (approximately 50%). The A1-selective agonist, N6-cyclopentyladenosine, increased submaximal (100 microU/mL) insulin-stimulated glucose transport in a dose-dependent manner (0.001-1.0 micromol/L). This stimulatory effect was inhibited by the A1-selective receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine in a concentration-dependent manner (0.001-1.0 micromol/L). However, neither activation nor inhibition of A1AR altered basal or maximal (10 mU/mL) insulin-stimulated glucose transport. Our results suggest that adenosine contributes approximately 50% to insulin-stimulated muscle glucose transport by activating the A1AR. This effect is limited to increasing insulin sensitivity, but not to either basal or maximal insulin-stimulated glucose uptake in rat soleus muscle.

Globular adiponectin resistance develops independently of impaired insulin-stimulated glucose transport in soleus muscle from high-fat-fed rats

High-fat (HF) diets induce insulin resistance and alter lipid metabolism, although controversy exists regarding the impact of saturated vs. polyunsaturated fats. Adiponectin (Ad) stimulates fatty acid (FA) oxidation and improves insulin sensitivity in humans and rodents, due in part to the activation of AMP-activated protein kinase (AMPK) and subsequent deactivation of acetyl coenzyme A carboxylase (ACC). In genetically obese, diabetic mice, this acute stimulatory effect on AMPK in muscle is lost. The ability of a HF diet to induce skeletal muscle Ad resistance has not been examined. The purpose of this study was to determine whether Ad's effects on FA oxidation and AMPK/ACC would be reduced following different HF diets, and if this coincided with the development of impaired maximal insulin-stimulated glucose transport. Rats were fed a control (10% kcal fat, CON), high unsaturated fat (60% kcal safflower oil, SAFF), or high saturated fat diet (60% kcal lard, LARD) for 4 wk. Following the dietary intervention, glucose transport, lipid metabolism, and AMPK/ACC phosphorylation were measured in the presence and absence of globular Ad (gAd, 2.5 microg/ml) in isolated soleus muscle. LARD rats showed reduced rates of maximal insulin-stimulated glucose transport compared with CON and SAFF (+68 vs. +172 and +184%, P < or = 0.001). gAd increased pACC (+25%, P < or = 0.01) and FA oxidation (+28%, P < or = 0.05) in CON rats, but not in either HF group. Thus 4 wk of HF feeding results in the loss of gAd stimulatory effect on ACC phosphorylation and muscle FA oxidation, and this can occur independently of impaired maximal insulin-stimulated glucose transport.

A role of lipin in human obesity and insulin resistance: relation to adipocyte glucose transport and GLUT4 expression

The mouse lipin gene, Lpin1, is important for adipose tissue development and is a candidate gene for insulin resistance. Here, we investigate the adipose tissue expression levels of the human LPIN1 gene in relation to various clinical variables as well as adipocyte function. LPIN1 gene expression was induced at an early step in human preadipocyte differentiation in parallel with peroxisome proliferator-activated receptor gamma. Lipin mRNA levels were higher in fat cells than in adipose tissue segments but showed no difference between subcutaneous and omental depots. Moreover, LPIN1 expression levels were reduced in obesity, improved following weight reduction in obese subjects, and were downregulated in women with the metabolic syndrome. With respect to adipocyte function, adipose LPIN1 gene expression was strongly associated with both basal and insulin-mediated subcutaneous adipocyte glucose transport as well as mRNA levels of glucose transporter 4 (GLUT4). We show that body fat accumulation is a major regulator of human adipose LPIN1 expression and suggest a role of LPIN1 in human preadipocyte as well as mature adipocyte function.

Atypical Antipsychotic Drugs Directly Impair Insulin Action in Adipocytes: Effects on Glucose Transport, Lipogenesis, and Antilipolysis

Treatment with second-generation antipsychotics (SGAs) has been associated with weight gain and the development of diabetes mellitus, although the mechanisms are unknown. We tested the hypothesis that SGAs exert direct cellular effects on insulin action and substrate metabolism in adipocytes. We utilized two cultured cell models including 3T3-L1 adipocytes and primary cultured rat adipocytes, and tested for effects of SGAs risperidone (RISP), clozapine (CLZ), olanzapine (OLZ), and quetiapine (QUE), together with conventional antipsychotic drugs butyrophenone (BUTY), and trifluoperazine (TFP), over a wide concentration range from 1 to 500 microM. The effects of antipsychotic drugs on basal and insulin-stimulated rates of glucose transport were studied at 3 h, 15 h, and 3 days. Both CLZ and OLZ (but not RISP) at doses as low as 5 microM were able to significantly decrease the maximal insulin-stimulated glucose transport rate by approximately 40% in 3T3-L1 cells, whereas CLZ and RISP reduced insulin-stimulated glucose transport rates in primary cultured rat adipocytes by approximately 50-70%. Conventional drugs (BUTY and TFP) did not affect glucose transport rates. Regarding intracellular glucose metabolism, both SGAs (OLZ, QUE, RISP) and conventional drugs (BUTY and TFP) increased basal and/or insulin-stimulated glucose oxidation rates, whereas rates of lipogenesis were increased by CLZ, OLZ, QUE, and BUTY. Finally, rates of lipolysis in response to isoproterenol were reduced by the SGAs (CLZ, OLZ, QUE, RISP), but not by BUTY or TFP. These experiments demonstrate that antipsychotic drugs can differentially affect insulin action and metabolism through direct cellular effects in adipocytes. However, only SGAs were able to impair the insulin-responsive glucose transport system and to impair lipolysis in adipocytes. Thus, SGAs directly induce insulin resistance and alter lipogenesis and lipolysis in favor of progressive lipid accumulation and adipocyte enlargement. These effects of SGAs on adipocytes could explain, in part, the association of SGAs with weight gain and diabetes.

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling.

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport in skeletal muscle.

Insulin signal transduction and glucose transport in human adipocytes: effects of obesity and low calorie diet.

We examined insulin signal transduction at the level of insulin receptor substrates (IRS) 1 and 2, phosphatidylinositol (PI) 3-kinase and glucose transport in isolated subcutaneous adipocytes from obese and lean women. Glucose transport and insulin signalling were investigated in isolated adipocytes from six obese women (BMI 36-43 kg/m(2)) (before and after 11 days of very low calorie diet) and from six lean women (BMI 22-26 kg/m(2)). Insulin sensitivity of glucose transport was reduced in adipocytes from obese women (p<0.05), with further reductions in basal and maximal insulin-stimulated glucose transport after a very low calorie diet (p<0.05). In obese women, IRS-1 associated PI 3-kinase activity was markedly impaired (p<0.05), whereas, IRS-2 associated PI 3-kinase activity was normal. IRS-1 associated PI 3-kinase activity remained blunted after a very low calorie diet, whereas IRS-2 associated PI 3-kinase activity was increased. GLUT4 protein was reduced by 37% in obese versus lean subjects (p<0.05), and decreased further after a very low calorie diet (from 19+/-4 to 14+/-4 arbitrary units; p<0.05). IRS-1 signalling to PI 3-kinase is a site of insulin resistance in adipocytes from obese women, whereas insulin action on IRS-2 is normal. Thus, IRS-1 and IRS-2 undergo differential regulation in adipocytes from obese insulin resistant subjects. Finally, a very low calorie diet is associated with a further impairment in glucose transport in adipose tissue. The defect in glucose transport after a very low calorie diet occurs independent of further defects in insulin signalling at the level of the PI 3-kinase.

Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice.

Decreased GLUT4 expression, impaired insulin receptor (IR), IRS-1, and pp60/IRS-3 tyrosine phosphorylation are characteristics of adipocytes from insulin-resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/-)) as a novel model of diabetes to study the development of insulin signaling defects in adipocytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/-) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin-stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin-stimulated tyrosine phosphorylation of IR, IRS-1, and pp60/IRS-3 were noted with no changes in the cellular content of IR, IRS-1, and p85 in N/H adipocytes. Insulin-stimulated protein tyrosine phosphorylation was further decreased to 12-23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin-stimulated, IRS-1-associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/-) adipocytes. However, total insulin-stimulated PI3 kinase activity was normal in H/H GLUT4(+/-) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hyperglycemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate-limiting factor in mediating maximal insulin-stimulated glucose uptake in GLUT4(+/-) adipocytes.

Secretion of tumor necrosis factor-alpha shows a strong relationship to insulin-stimulated glucose transport in human adipose tissue.

Some animal models suggest that tumor necrosis factor (TNF)-alpha is a key component in obesity-linked insulin resistance because it inhibits insulin receptor signaling and glucose transport in insulin-sensitive tissues. However, in vivo data in humans have given conflicting results regarding the relationship between circulating TNF-alpha levels and insulin sensitivity. In the present study, the potential local role of TNF-alpha on insulin action in human subcutaneous adipose tissue was studied in 42 obese women (BMI 39+/-10 kg/m2). We found a strong inverse correlation between adipose TNF-alpha secretion and maximum insulin-stimulated glucose transport in adipocytes that was independent of fat cell volume, age, and BMI (P < 0.001, r = 0.58). As much as one-third of the variation in insulin-stimulated glucose transport could be accounted for by variations in TNF-alpha secretion. There was no significant correlation (r = 0.11) between secretion of adipose plasminogen activator inhibitor 1 and glucose transport. Furthermore, subcutaneous adipose tissue of 4 obese women (BMI 40+/-4) incubated with TNF-A for 24 h showed a one-third concentration-dependent inhibition of insulin-stimulated glucose transport (P < 0.01). In conclusion, adipose TNF-alpha may be an important specific and local factor in adipose tissue that influences the ability of insulin to stimulate glucose transport in human fat cells, at least in obese women.

Divergent regulation of Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats.

To determine whether impaired Akt (protein kinase B or rac) activation contributes to insulin resistance in vivo, we examined the expression, phosphorylation, and kinase activities of Akt1 and Akt2 isoforms in insulin target tissues of insulin-resistant obese Zucker rats. In lean rats, insulin (10 U/kg i.v. x 2.5 min) stimulated Akt1 activity 6.2-, 8.8-, and 4.4-fold and Akt2 activity 5.4-, 9.3-, and 1.8-fold in muscle, liver, and adipose tissue, respectively. In obese rats, insulin-stimulated Akt1 activity decreased 30% in muscle and 21% in adipose tissue but increased 37% in liver compared with lean littermates. Insulin-stimulated Akt2 activity decreased 29% in muscle and 37% in liver but increased 24% in adipose tissue. Akt2 protein levels were reduced 56% in muscle and 35% in liver of obese rats, but Akt1 expression was unaltered. Phosphoinositide 3-kinase (PI3K) activity associated with insulin receptor substrate (IRS)-1 or phosphotyrosine was reduced 67-86% in tissues of obese rats because of lower IRS-1 protein levels and reduced insulin receptor and IRS-1 phosphorylation. In adipose tissue of obese rats, in spite of an 86% reduction in insulin-stimulated PI3K activity, activation of Akt2 was increased. Maximal insulin-stimulated (100 nmol/l) glucose transport was reduced 70% in isolated adipocytes, with a rightward shift in the insulin dose response for transport and for Akt1 stimulation but normal sensitivity for Akt2. These findings suggest that PI3K-dependent effects on glucose transport in adipocytes are not mediated primarily by Akt2. Akt1 and Akt2 activations by insulin have a similar time course and are maximal by 2.5 min in adipocytes of both lean and obese rats. We conclude that 1) activation of Akt1 and Akt2 in vivo is much less impaired than activation of PI3K in this insulin-resistant state, and 2) the mechanisms for divergent alterations in insulin action on Akt1 and Akt2 activities in tissues of insulin-resistant obese rats involve tissue- and isoform-specific changes in both expression and activation.

Muscle fiber type-specific defects in insulin signal transduction to glucose transport in diabetic GK rats.

To determine whether defects in the insulin signal transduction pathway to glucose transport occur in a muscle fiber type-specific manner, post-receptor insulin-signaling events were assessed in oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skeletal muscle from Wistar or diabetic GK rats. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/l) glucose transport was significantly decreased, compared with that of Wistar rats. In EDL muscle from GK rats, maximal insulin-stimulated glucose transport was normal, while the submaximal response was reduced compared with that of Wistar rats. We next treated diabetic GK rats with phlorizin for 4 weeks to determine whether restoration of glycemia would lead to improved insulin signal transduction. Phlorizin treatment of GK rats resulted in full restoration of insulin-stimulated glucose transport in soleus and EDL muscle. In soleus muscle from GK rats, submaximal and maximal insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and IRS-1-associated phosphatidylinositol (PI) 3-kinase activity were markedly reduced, compared with that of Wistar rats, but only submaximal insulin-stimulated PI 3-kinase was restored after phlorizin treatment. In EDL muscle, insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI-3 kinase were not altered between GK and Wistar rats. Maximal insulin-stimulated Akt (protein kinase B) kinase activity is decreased in soleus muscle from GK rats and restored upon normalization of glycemia (Krook et al., Diabetes 46:2100-2114, 1997). Here, we show that in EDL muscle from GK rats, maximal insulin-stimulated Akt kinase activity is also impaired and restored to Wistar rat levels after phlorizin treatment. In conclusion, functional defects in IRS-1 and PI 3-kinase in skeletal muscle from diabetic GK rats are fiber-type-specific, with alterations observed in oxidative, but not glycolytic, muscle. Furthermore, regardless of muscle fiber type, downstream steps to PI 3-kinase (i.e., Akt and glucose transport) are sensitive to changes in the level of glycemia.

Exercise training and the glucose transport system in obese SHHF/Mcc-facprats

The effects of a similar exercise training stimulus on maximal insulin-stimulated (MIS) plasma membrane glucose transporter number and glucose transport were determined in lean and obese SHHF/Mcc-facp rats. Six-week-old lean and obese male rats were randomly divided into four groups: lean sedentary (LSed), obese sedentary (OSed), lean exercise (LEx), and obese exercise (OEx). An 8- to 12-wk treadmill running program equalized daily muscular work for LEx and OEx. Plasma membranes were isolated from control and MIS muscles of mixed fiber types. MIS significantly increased glucose transport (3.4- and 2.8-fold) in LSed and OSed, respectively. MIS significantly increased glucose transporter number (2.5-fold) in LSed, but there was no increase in glucose transporter number in OSed. Peak oxygen uptake and citrate synthase activity were increased a similar amount for LEx and OEx groups, demonstrating a similar training stimulus. MIS significantly and similarly increased glucose transport in LEx and OEx (4.4- and 5.1-fold, respectively). The effects of MIS on plasma membrane glucose transporter number in the exercise-trained rats were similar to the responses observed in the sedentary lean and obese groups. MIS significantly increased glucose transporter number (2.6-fold) in LEx, whereas there was no increase in glucose transporter number in OEx. The reduction in MIS glucose transport in OSed appears to be related to a defect in the processes associated with the translocation of glucose transporters to the plasma membrane. Exercise training of the obese rats apparently did not alter this defect. Similar increases in peak oxygen uptake, citrate synthase, and MIS glucose transport in LEx and OEx groups suggest that insulin resistance does not limit the ability of the glucose transport system to adapt to exercise training in the obese male SHHF/Mcc-facp rats.

Glucose transport and GLUT4 protein distribution in skeletal muscle of GLUT4 transgenic mice

The aim of the present investigation was to determine whether the subcellular distribution and insulin-stimulated translocation of the GLUT4 isoform of the glucose transporter are affected when GLUT4 is overexpressed in mouse skeletal muscle, and if the overexpression of GLUT4 alters maximal insulin-stimulated glucose transport and metabolism. Rates of glucose transport and metabolism were assessed by hind-limb perfusion in GLUT4 transgenic (TG) mice and non-transgenic (NTG) controls. Glucose-transport activity was determined under basal (no insulin), submaximal (0.2 m-unit/ml) and maximal (10 m-units/ml) insulin conditions using a perfusate containing 8 mM 3-O-methyl-D-glucose. Glucose metabolism was quantified by perfusing the hind limbs for 25 min with a perfusate containing 8 mM glucose and 10 m-units/ml insulin. Under basal conditions, there was no difference in muscle glucose transport between TG (1.10 +/- 0.10 mumol/h per g; mean +/- S.E.M.) and NTG (0.93 +/- 0.16 mumol/h per g) mice. However, TG mice displayed significantly greater glucose-transport activity during submaximal (4.42 +/- 0.49 compared with 2.69 +/- 0.33 mumol/h per g) and maximal (11.68 +/- 1.13 compared with 7.53 +/- 0.80 mumol/h per g) insulin stimulation. Nevertheless, overexpression of the GLUT4 protein did not alter maximal rates of glucose metabolism. Membrane purification revealed that, under basal conditions, plasma-membrane (approximately 12-fold) and intracellular-membrane (approximately 4-fold) GLUT4 protein concentrations were greater in TG than NTG mice. Submaximal insulin stimulation did not increase plasma-membrane GLUT4 protein concentration whereas maximal insulin stimulation increased this protein in both NTG (4.1-fold) and TG (2.6-fold) mice. These results suggest that the increase in insulin-stimulated glucose transport following overexpression of the GLUT4 protein is limited by factors other than the plasma-membrane GLUT4 protein concentration. Furthermore, GLUT4 overexpression is not coupled to glucose-metabolic capacity.

Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling.

(+/-)-5-([4-[2-Methyl-2(pyridylamino)ethoxy]phenyl]methyl) 2,4-thiazolidinedione (BRL 49653) is a new potent antidiabetic agent that improves insulin sensitivity in animal models of NIDDM. In C57BL/6 obese (ob/ob) mice, BRL 49653, included in the diet for 8 days, improved glucose tolerance. The half-maximal effective dose was 3 mumol/kg diet, which is equivalent to approximately 0.1 mg/kg body wt. Improvements in glucose tolerance were accompanied by significant reductions in circulating triacylglycerol, nonesterified fatty acids, and insulin. The insulin receptor number of epididymal white adipocytes prepared from obese mice treated with BRL 49653 (30 mumol/kg diet) for 14 days was increased twofold. The affinity of the receptor for insulin was unchanged. In the absence of added insulin, the rates of glucose transport in adipocytes from untreated and BRL 49653-treated obese mice were similar. Insulin (73 nmol/l) produced only a 1.5-fold increase in glucose transport in adipocytes from control obese mice, whereas after BRL 49653 treatment, insulin stimulated glucose transport 2.8-fold. BRL 49653 did not alter the sensitivity of glucose transport to insulin. The increase in insulin responsiveness was accompanied by a 2.5-fold increase in the total tissue content of the glucose transporter GLUT4. Glucose transport in adipocytes from lean littermates was not altered by BRL 49653. To establish the contribution of changes in glucose transporter trafficking to the BRL 49653-mediated increase in insulin action, the cell-impermeant bis-mannose photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos++ +-4-yloxy) -2-[2-3H]-propylamine was used to measure adipocyte cell-surface-associated glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute and chronic exercise on skeletal muscle glucose transport in aged rats.

The purpose of this study was to investigate the effects of acute and chronic exercise on skeletal muscle glucose transport in aged rats by using an isolated sarcolemmal membrane preparation. In 24-mo-old female Fischer 344 rats, a maximum dose of insulin increased glucose transport from 43 +/- 6 to 82 +/- 6 pmol.mg protein-1.15 s-1. A 45-min bout of exhaustive treadmill running increased glucose transport to the same maximum level (88 +/- 5 pmol.mg protein-1.15 s-1). Eight weeks of progressive exercise training resulted in a 65% increase in succinic dehydrogenase activity in hindlimb muscles and a 55% increase in total cellular GLUT-4 content. Despite these biochemical adaptations, there was no change in either basal or maximum insulin-stimulated glucose transport between control (43 +/- 6 and 82 +/- 6 pmol.mg protein-1.15 s-1, respectively) and trained (42 +/- 2 and 82 +/- 8 pmol.mg protein-1.15 s-1, respectively) animals. When hindlimb muscle succinate dehydrogenase activity and GLUT-4 content were compared for both the combined sedentary and trained groups, a significant correlation (r = 0.68) was obtained. This study demonstrates that the skeletal muscle glucose transport system of 24-mo-old rats is fully stimulated by acute exercise and that, although GLUT-4 levels are increased in aged animals after exercise training, this does not result in an enhancement of maximal insulin-stimulated glucose transport. Thus increases in GLUT-4 are not sufficient to improve muscle insulin responsiveness with training.

Growth hormone reduces glucose transport but not GLUT-1 or GLUT-4 in adult and old rats

The primary purpose of this study was to investigate the influence of administration of recombinant-derived human growth hormone (rhGH) to adult male rats of several ages (9, 20, and 31 mo) on skeletal muscle glucose transport. Rats were injected with rhGH (0.7 mg/kg) or vehicle twice daily for 10 days. The rhGH treatment led to a doubling of circulating insulin-like growth factor I levels at each age. Skeletal muscle glucose transport activity was evaluated in isolated epitrochlearis muscle with use of 3-O-methylglucose at three insulin concentrations (0, 100, and 20,000 microU/ml). The results indicate that, after 10 days of rhGH administration, 1) an approximately 20-30% reduction in basal glucose transport activity was evident in muscles from every age group, 2) the ability of a submaximally effective insulin concentration (100 microU/ml) to increase glucose transport activity above basal values was not significantly reduced in any age group, 3) maximal insulin-stimulated glucose transport activity (with 20,000 microU/ml) was significantly reduced (approximately 40%) by rhGH treatment only in the oldest rats, and 4) the alterations in glucose transport activity occurred despite no change in skeletal muscle GLUT-1 or GLUT-4 protein levels.