Leptin opposes insulin's effects on fatty acid partitioning in muscles isolated from obese ob/ob mice.

Because muscle triacylglycerol (TAG) accumulation might contribute to insulin resistance in leptin-deficient ob/ob mice, we studied the acute (60- to 90-min) effects of leptin and insulin on [14C]glucose and [14C]oleate metabolism in muscles isolated from lean and obese ob/ob mice. In ob/ob soleus, leptin decreased glycogen synthesis 36-46% (P < 0.05), increased oleate oxidation 26% (P < 0.05), decreased oleate incorporation into TAG 32% (P < 0.05), and decreased the oleate partitioning ratio (oleate partitioned into TAG/CO2) 44% (P < 0.05). Insulin decreased oleate oxidation 31% (P < 0.05), increased oleate incorporation into TAG 46% (P < 0.05), and increased the partitioning ratio 125% (P < 0.01). Adding leptin diminished insulin's antioxidative, lipogenic effects. In soleus from lean mice, insulin increased the partitioning ratio 142%, whereas leptin decreased it 51%, as previously reported (Muoio, D. M. , G. L. Dohm, F. T. Fiedorek, E. B. Tapscott, and R. A. Coleman. Diabetes 46: 1360-1363, 1997). The phosphatidylinositol 3-kinase inhibitor wortmannin blocked insulin's effects on lipid metabolism but only attenuated leptin's effects. Increasing glucose concentration from 5 to 10 mM did not affect TAG synthesis, suggesting that insulin-induced lipogenesis is independent of increased glucose uptake. These data indicate that leptin opposes insulin's promotion of TAG accumulation in lean and ob/ob muscles. Because acute leptin exposure does not correct insulin resistance in ob/ob muscles, in vivo improvements in glucose homeostasis appear to require other long-term factors, possibly TAG depletion.

Regulation of glucose transporters GLUT-4 and GLUT-1 gene transcription in denervated skeletal muscle.

Because GLUT-4 expression is decreased whereas GLUT-1 expression is increased in denervated skeletal muscle, we examined the effects of denervation on GLUT-4 and GLUT-1 gene transcription. The right hindlimb skeletal muscle of male transgenic mice containing sequential truncations (2,400, 1,639, 1,154, and 730 bp) of the human GLUT-4 promoter linked to the chloramphenacol acyl transferase (CAT) gene was denervated, and the contralateral hindlimb was sham operated. RNase protection analysis revealed that after 72 h denervation decreased CAT mRNA and GLUT-4 mRNA levels 64-85%, respectively (P < 0.05), in the gastrocnemius muscles. In contrast, denervation of the right hindlimb of male rats increased GLUT-1 gene transcription and GLUT-1 mRNA levels by 94 and 213%, respectively (P < 0.05). In conclusion, GLUT-4 transcription is decreased but GLUT-1 transcription is increased in denervated skeletal muscle, suggesting that the effects of denervation on GLUT-4 and GLUT-1 expression are, in part, transcriptionally mediated. Furthermore, these data indicate that a DNA sequence regulated by denervation is located within 730 bp of the 5'-flanking promoter region of the human GLUT-4 gene.

Vanadate stimulation of 2-deoxyglucose transport is not mediated by PI 3-kinase in human skeletal muscle.

Glucose transport in mammalian skeletal muscle is stimulated by insulin, hypoxia and tyrosine protein phosphatase inhibitors such as vanadate. However, it is unknown whether the vanadate signaling mechanism shares a common or separate pathway with insulin or hypoxia. Therefore, experiments were conducted on incubated human muscle strips to compare the effects of vanadate with insulin and hypoxia stimulated 2-deoxyglucose transport (2-DOG). We also used the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin to examine whether PI 3-kinase is a common step by which each stimulate glucose transport. Results demonstrate that whereas the effects of vanadate and hypoxia were additive with insulin stimulated glucose transport, the effect of vanadate plus hypoxia was not. In addition, wortmannin significantly (P < 0.05) reduced insulin but not vanadate or hypoxia stimulated 2-DOG transport. Moreover, PI 3-kinase activity was significantly elevated (P < 0.05) in the presence of insulin but not vanadate. In conclusion, these data suggest that vanadate and hypoxia stimulate glucose transport via a similar signaling pathway which is distinct from insulin and that the vanadate signaling pathway is not mediated by PI 3-kinase in human skeletal muscle.

Transcriptional regulation of hexokinase II in denervated rat skeletal muscle.

Hexokinase II protein is augmented in denervated skeletal muscle; therefore, we determined if hexokinase II gene transcription rates and mRNA levels are increased with denervation. The right hindlimb skeletal muscles of male rats were denervated while the left hindlimbs were sham operated. Seventy-two h following surgery, rats were sacrificed and the gastrocnemius and soleus muscles were harvested for nuclear and RNA isolation. Nuclear run-on and ribonuclease protection analyses indicated that denervation increased hexokinase II transcription rates and mRNA levels 42% and 88%, respectively (p < 0.05). Total hexokinase activity rose 23% in denervated gastrocnemius muscle. In conclusion, the increase in hexokinase II gene transcription and mRNA may account for the increase in hexokinase II protein and the subsequent rise in total hexokinase activity in denervated rat skeletal muscle.

Leptin directly alters lipid partitioning in skeletal muscle.

Leptin, an adipocyte-derived hormone that directly regulates both adiposity and energy homeostasis, decreases food intake and appears to partition metabolic fuels toward utilization and away from storage. Because skeletal muscle expresses the leptin receptor and plays a major role in determining energy metabolism, we studied leptin's effects on glucose and fatty acid (FA) metabolism in isolated mouse soleus and extensor digitorum longus (EDL) muscles. One muscle from each animal served as a basal control. The contralateral muscle was treated with insulin (10 mU/ml), leptin (0.01-10 microg/ml), or insulin plus leptin, and incorporation of [14C]glucose or [14C]oleate into CO2 and into either glycogen or triacylglycerol (TAG) was determined. Leptin increased soleus muscle FA oxidation by 42% (P < 0.001) and decreased incorporation of FA into TAG by 35% (P < 0.01) in a dose-dependent manner. In contrast, insulin decreased soleus muscle FA oxidation by 40% (P < 0.001) and increased incorporation into TAG by 70% (P < 0.001). When both hormones were present, leptin attenuated both the antioxidative and the lipogenic effects of insulin by 50%. Less pronounced hormone effects were observed in EDL muscle. Leptin did not alter insulin-stimulated muscle glucose metabolism. These data demonstrate that leptin has direct and acute effects on skeletal muscle.

Skeletal muscle content of membrane glycoprotein PC-1 in obesity. Relationship to muscle glucose transport.

Membrane glycoprotein PC-1, an inhibitor of insulin signaling, produces insulin resistance when overexpressed in cells transfected with PC-1 cDNA. In the present study, we determined whether PC-1 plays a role in the insulin resistance of skeletal muscle in obesity. Rectus abdominus muscle biopsies were taken from patients undergoing elective surgery. Subjects included both NIDDM patients (n = 14) and nondiabetic patients (n = 34) across a wide range of BMI values (19.5-90.1). Insulin-stimulated glucose transport was measured in incubated muscle strips, and PC-1 content, enzymatic activity, and insulin receptor content were measured in solubilized muscle extracts. Increasing BMI correlated with both an increase in the content of PC-1 in muscle (r = 0.55, P < 0.001) and a decrease in insulin stimulation of muscle glucose transport (r = -0.58, P = 0.008). NIDDM had no effect on either PC-1 content or glucose transport for any given level of obesity. Insulin stimulation of muscle glucose transport was negatively related to muscle PC-1 content (r = -0.68, P = 0.001) and positively related to insulin receptor content (r = 0.60, P = 0.005). Multivariate analysis indicated that both skeletal muscle PC-1 content and insulin receptor content, but not BMI, were independent predictors of insulin-stimulated glucose transport. Muscle PC-1 content accounted for 42% and insulin receptor content for 17% of the variance in glucose transport values. These studies raise the possibility that increased expression of PC-1 and a decreased insulin receptor content in skeletal muscle may be involved in the insulin resistance of obesity.

Hindlimb perfusion induces GLUT-1 and immediate early gene expression in skeletal muscle.

The purpose of the present study was to test the suitability of the rat hindlimb perfusion technique for studying the acute regulation of the GLUT-1 and GLUT-4 glucose transporter genes in adult skeletal muscle. To further examine the stability of the technique, we also monitored the transcription rate and mRNA content of selected immediate early genes. Nuclei and total RNA were isolated from red and white hindlimb muscle from perfused (2 h) and nonperfused control animals. Although GLUT-4 transcription and mRNA content remained stable, perfusion elicited a marked 3.5-fold increase in GLUT-1 mRNA in red and 2.2-fold increase in white skeletal muscle in the absence of any detectable change in transcription. In contrast to both GLUT-1 and GLUT-4, transcription originating from the c-fos and c-myc immediate early genes increased from 2.0- to 2.7-fold with perfusion in both red and white skeletal muscle, whereas transcription of the beta-actin gene decreased by 40-60%. Both c-fos and c-myc mRNA levels also increased with perfusion, whereas beta-actin mRNA remained unchanged. These findings clearly demonstrate that the current method of performing the hindlimb perfusion technique rapidly and dramatically alters the regulation of selected genes in skeletal muscle.

Liver glucokinase: decreased activity in patients with type II diabetes.

Because of the demonstration of a genetic linkage between glucokinase and Type II diabetes, and the central role of glucokinase on glucose metabolism, we studied glucokinase activity in the liver of patients with and without Type II diabetes. Glucokinase activity was decreased by about 50% in obese subjects with diabetes (n = 12) compared with (p < 0.01) lean controls (n = 9) and (p < 0.05) obese controls (n = 10). There was no difference between lean and obese controls. Fifty percent of subjects with diabetes had lower liver glucokinase activity than the lowest value of the controls. These data further support the important role that glucokinase plays in the pathogenesis of Type II diabetes.

Stimulation of glucose uptake by insulin-like growth factor II in human muscle is not mediated by the insulin-like growth factor II/mannose 6-phosphate receptor.

Although the growth-promoting effects of insulin-like growth factor II (IGF-II) have been intensively studied, the acute actions of this hormone on glucose metabolism have been less well evaluated, especially in skeletal muscle of humans. We and other groups have shown that IGFs reduce glycaemic levels in humans and stimulate glucose uptake in rat muscle. The purpose of the present study was to evaluate the effect of IGF-II on glucose transport in muscle of normal and obese patients with and without non-insulin-dependent diabetes mellitus (NIDDM), as well as to identify the receptor responsible for this action. 2-Deoxyglucose transport was determined in vitro using a muscle-fibre strip preparation. IGF-II were investigated in biopsy material of rectus abdominus muscle taken from lean and obese patients and obese patients with NIDDM at the time of surgery. In the lean group, IGF-II (100 nM) stimulated glucose transport 2.1-fold, which was slightly less than stimulation by insulin (2.8-fold) at the same concentration. Binding of IGF-II was approx. 25% of that of insulin at 1 nM concentrations of both hormones. Obesity with or without NIDDM significantly reduced IGF-II-stimulated glucose uptake compared with the lean group. In order to explore which receptor mediated the IGF-II effect, we compared glucose uptake induced by IGF-II and two IGF-II analogues: [Leu27]IGF-II, with high affinity for the IGF-II/Man 6-P receptor but markedly reduced affinity for the IGF-I and insulin receptors, and [Arg54,Arg55]IGF-II was similar to that of IGF-II, whereas [Leu27]IGF-II had a very diminished effect. Results show that IGF-II is capable of stimulating muscle glucose uptake in lean but not in obese subjects and this effect seems not to be mediated via an IGF-II/Man 6-P receptor.

Effect of moderate obesity on glucose transport in human muscle.

We have shown that maximally stimulated glucose transport is reduced in in vitro incubated muscle of morbidly obese subjects. To investigate the possibility that a "threshold" of obesity exists, above which glucose transport is significantly decreased, hormone (insulin, IGF-I, or IGF-II) stimulation of glucose transport was correlated with body mass index using muscle biopsies from a group of 30 lean to obese females with BMI ranging from 16 to 40. There was a significant negative relationship between stimulation for glucose transport and BMI (R = 0.765). These data suggest there is no obesity threshold for insulin resistance in skeletal muscle but a continuous decline in glucose transport below a BMI of approximately 30 kg/m2, after which insulin and the IGFs no longer stimulate glucose transport.

Increased muscle glucose uptake in response to chronic glyburide treatment is not related to changes in glucose transporter (GLUT4) protein.

1. The mechanism of action of glyburide (a sulfonylurea) on muscle has been investigated by measuring glucose uptake and glucose transporter (GLUT4) protein levels after chronic glyburide treatment. 2. A dietary induced insulin resistant rat model (4 wk of high-fat, high-sucrose feeding) was given glyburide (2 mg/kg/day) for 10 days and glucose uptake was measured in a perfused hindquarter preparation. 3. Protein levels of the GLUT4 glucose transporter were determined by Western analysis. 4. After 7 days of treatment, rats fed glyburide had lower blood glucose concentrations 2 hr (72 +/- 5 vs 103 +/- 12 mg/dl) and 24 hr (97 +/- 7 vs 123 +/- 7 mg/dl) after glyburide administration with no difference in serum insulin levels compared to vehicle treated animals. 5. Glucose uptake was approx doubled in basal state (0 insulin) in response to glyburide (2.8 +/- 0.4 vs 1.7 +/- 0.2 mumol/g per hr). 6. Maximal insulin (100 nM) stimulated glucose uptake tended to be higher in the glyburide treated group, but did not reach statistical significance (8.0 +/- 0.7 vs 7.0 +/- 0.6 mumol/g per hr). 7. Western analysis revealed no significant effect of glyburide on the GLUT4 protein level in skeletal muscle. 8. These results suggest that glyburide alters glucose uptake through some mechanism other than alterations in the level of the GLUT4 glucose transporter protein.

Contractile activity restores insulin responsiveness in skeletal muscle of obese Zucker rats.

Both insulin and contraction stimulate glucose transport in skeletal muscle. Insulin-stimulated glucose transport is decreased in obese humans and rats. The aims of this study were (1) to determine if contraction-stimulated glucose transport was also compromised in skeletal muscle of genetically obese insulin-resistant Zucker rats, and (2) to determine whether the additive effects of insulin and contraction previously observed in muscle from lean subjects were evident in muscle from the obese animals. To measure glucose transport, hindlimbs from lean and obese Zucker rats were perfused under basal, insulin-stimulated (0.1 microM), contraction-stimulated (electrical stimulation of the sciatic nerve) and combined insulin-(+)contraction-stimulated conditions. One hindlimb was stimulated to contract while the contralateral leg served as an unstimulated control. 2-Deoxyglucose transport rates were measured in the white gastrocnemius, red gastrocnemius and extensor digitorum longus muscles. As expected, the insulin-stimulated glucose transport rate in each of the three muscles was significantly slower (P < 0.05) in obese rats when compared with lean animals. When expressed as fold stimulation over basal, there was no significant difference in contraction-induced muscle glucose transport rates between lean and obese animals. Insulin-(+)contraction-stimulation was additive in skeletal muscle of lean animals, but synergistic in skeletal muscle of obese animals. Prior contraction increased insulin responsiveness of glucose transport 2-5-fold in the obese rats, but had no effect on insulin responsiveness in the lean controls. This contraction-induced improvement in insulin responsiveness could be of clinical importance to obese subjects as a way to improve insulin-stimulated glucose uptake in resistant skeletal muscle.

Restoration of insulin responsiveness in skeletal muscle of morbidly obese patients after weight loss. Effect on muscle glucose transport and glucose transporter GLUT4.

A major defect contributing to impaired insulin action in human obesity is reduced glucose transport activity in skeletal muscle. This study was designed to determine whether the improvement in whole body glucose disposal associated with weight reduction is related to a change in skeletal muscle glucose transport activity and levels of the glucose transporter protein GLUT4. Seven morbidly obese (body mass index = 45.8 +/- 2.5, mean +/- SE) patients, including four with non-insulin-dependent diabetes mellitus (NIDDM), underwent gastric bypass surgery for treatment of their obesity. In vivo glucose disposal during a euglycemic clamp at an insulin infusion rate of 40 mU/m2 per min was reduced to 27% of nonobese controls (P less than 0.01) and improved to 78% of normal after weight loss of 43.1 +/- 3.1 kg (P less than 0.01). Maximal insulin-stimulated glucose transport activity in incubated muscle fibers was reduced by approximately 50% in obese patients at the time of gastric bypass surgery but increased twofold (P less than 0.01) to 88% of normal in five separate patients after similar weight reduction. Muscle biopsies obtained from vastus lateralis before and after weight loss revealed no significant change in levels of GLUT4 glucose transporter protein. These data demonstrate conclusively that insulin resistance in skeletal muscle of mobidly obese patients with and without NIDDM cannot be causally related to the cellular content of GLUT4 protein. The results further suggest that morbid obesity contributes to whole body insulin resistance through a reversible defect in skeletal muscle glucose transport activity. The mechanism for this improvement may involve enhanced transporter translocation and/or activation.

Insulin responsiveness in skeletal muscle is determined by glucose transporter (Glut4) protein level.

Glucose transport in skeletal muscle is mediated by two distinct transporter isoforms, designated muscle/adipose glucose transporter (Glut4) and erythrocyte/HepG2/brain glucose transporter (Glut1), which differ in both abundance and membrane distribution. The present study was designed to investigate whether differences in insulin responsiveness of red and white muscle might be due to differential expression of the glucose transporter isoforms. Glucose transport, as well as Glut1 and Glut4 protein and mRNA levels, were determined in red and white portions of the quadriceps and gastrocnemius muscles of male Sprague-Dawley rats (body wt. approx. 250 g). Maximal glucose transport (in response to 100 nM-insulin) in the perfused hindlimb was 3.6 times greater in red than in white muscle. Red muscle contained approx. 5 times more total Glut4 protein and 2 times more Glut4 mRNA than white muscle, but there were no differences in the Glut1 protein or mRNA levels between the fibre types. Our data indicate that differences in responsiveness of glucose transport in specific skeletal muscle fibre types may be dependent upon the amount of Glut4 protein. Because this protein plays such an integral part in glucose transport in skeletal muscle, any impairment in its expression may play a role in insulin resistance.

Insulin resistance induced by high-fat feeding is only partially reversed by exercise training.

Diets high in saturated fat and simple carbohydrate result in an insulin-resistant state, while training increases insulin sensitivity. Insulin resistance was induced by feeding a high-fat, high-sucrose (HFS) diet to 4-week-old female Sprague-Dawley rats. A control diet (low-fat, complex-carbohydrate) was fed to another group for comparison. During the 4-week dietary treatment, half of each group was trained by treadmill running (2 h day-1, 6 days week-1m 30 m min-1, 0% grade). At the end of this 4-week experimental period, hindquarter perfusions were performed at either basal (0) or maximal (100 nM) insulin concentrations to determine glucose uptake, glycogen synthesis, total glycogen content and the activity of several enzymes. Insulin (100 nM) significantly increased glucose uptake and glycogen synthesis in all four groups (CON-UN, CON-TR, HFS-UN, HFS-TR, where CON, UN and TR refer to control, untrained and trained respectively). HFS feeding significantly decreased (P less than 0.002) glucose uptake (mumol g-1 h-1) with maximal insulin stimulation, while training significantly increased uptake (P less than 0.01) at both insulin concentrations. Glycogen synthesis was also increased by training (P less than 0.05) at both insulin concentrations, but accounted for only 25-28% of the glucose uptake. Although training improved the insulin resistance caused by the HFS diet, glucose uptake in the HFS-TR group was still significantly lower than the CON-TR group. Changes in glycogen synthesis are not great enough to account for the decrease or increase in glucose uptake found in the HFS-fed or trained animals.

Differences in glucose transport rates between perfused and in vitro incubated muscles.

In vitro incubated muscles are a convenient preparation for glucose transport studies, but it is not known how closely they reflect the in vivo condition. Perfused muscle preparations more closely resemble the in vivo condition, and thus to validate the use of in vitro incubated muscles, we have compared glucose transport rates in the two preparations. 3-O-Methylglucose transport rates in incubated soleus (SOL) and extensor digitorum longus (EDL) muscle strips were compared to transport rates obtained in SOL and EDL muscles removed from perfused hindquarters. Male Sprague-Dawley rats (250 g) were used for both procedures. SOL muscles showed an average 25% higher transport rate than EDL muscles at all insulin concentrations examined (0-100 nM) in the perfused system. This difference was diminished in the incubated muscles, SOL being 15% greater than EDL, but the relationship between the two muscles was maintained. Basal transport was lower and maximal transport was higher in the perfused muscles compared to the incubated muscles. This resulted in significantly higher fold stimulation in the perfused vs. incubated muscles (15 vs. 2.5 in the SOL, and 9.8 vs. 2.3 in the EDL). We conclude that in vitro muscle preparations may be convenient for showing relative differences between experimental treatments, but absolute transport rates and insulin stimulation must be interpreted with caution.

Effect of a high-fat-sucrose diet on in vivo insulin receptor kinase activation.

Insulin-stimulated glucose uptake into muscle is depressed by high-fat-sucrose (HFS) feeding of rats. To investigate the mechanism of this insulin resistance, the in vivo activation of the insulin receptor kinase in liver and muscle of control and HFS-fed rats was determined. Rats were injected with glucose and insulin and killed 0, 5, 15, and 30 min after injection. Insulin binding was not changed in partially purified receptors from muscle of HFS rats. In control rats insulin receptor kinase activity was maximally stimulated threefold in liver at 5 min and fourfold in muscle at 15 min after insulin-glucose injection. The insulin-stimulated tyrosine kinase activity of receptors isolated from the liver of rats fed the HFS diet was decreased by 30% in comparison with the controls. In contrast, receptors isolated from muscle did not show any difference in basal or insulin-stimulated kinase activity between HFS-fed and control rats. Decreased in vivo activation of the insulin receptor kinase may be at least partially responsible for insulin resistance in liver. Because insulin binding and insulin stimulation of receptor kinase were normal in muscle of HFS-fed animals, it is concluded that the insulin resistance of glucose uptake into muscle is caused by a defect distal to the insulin receptor.

Changes in glucose transporters in muscle in response to exercise.

The mechanism underlying the increase in glucose uptake in response to muscular contraction is not known, although it has been established that the change does not require insulin. It is our hypothesis that exercise, like insulin, stimulates translocation of glucose transporters to the plasma membrane. To test this hypothesis an experiment was performed to determine whether glucose transporters are translocated from an intracellular membrane to the plasma membrane during exercise. Untrained male rats weighing approximately 250 g were exercised by treadmill running for 2 h at 25 m/min. They were killed immediately after completion of exercise, and the gastrocnemius and quadriceps muscles were quickly removed. Sedentary animals were treated in the same way. Plasma and intracellular membranes were isolated by sucrose density gradient centrifugation and cytochalasin B binding assays were performed. Exercise resulted in a redistribution of glucose transporters from the intracellular membrane to the plasma membrane. The ratio of cytochalasin B binding sites in the membrane fractions (intracellular/plasma membrane) was 3.2 +/- 0.6 in rested animals and 1.3 +/- 0.3 after exercise. The concentration of glucose transporters was increased in the plasma membrane (from 19.8 +/- 1.8 to 30.4 +/- 3.9 pmol/mg protein) and decreased in the intracellular membrane (from 20.7 +/- 3.0 to 10.8 +/- 1.1 pmol/mg protein) in response to exercise. These results suggest that at least part of the increase in glucose uptake that occurs during exercise is the result of a redistribution of glucose transporters to the plasma membrane.

Effect of carbohydrate ingestion on exercise endurance and metabolism after a 1-day fast.

Fasting before an exercise event has been demonstrated to decrease endurance. The purpose of this study was to investigate whether this decrement in performance after fasting could be reversed by ingestion of a carbohydrate solution before and during exercise. Nine fit male subjects ran to exhaustion at approximately 70% VO2max in two counterbalanced trials. The subjects were fasted for 21 h before both trials, and the trials were arranged so that the subjects ingested either a carbohydrate (CHO) or placebo (PL) solution. Although ratings of perceived exertion were significantly lower in the CHO trial, there were no differences in endurance time to exhaustion in the two trials (102 +/- 8 min in the PL trial and 106 +/- 8 min in the CHO trial). There were no differences between trials for the VO2, heart rate, and blood lactate concentrations. As expected, the blood glucose and insulin concentrations were higher in the CHO trial. The respiratory exchange ratio was significantly higher in the CHO trial at 40 min of exercise and tended to be higher at all other times, suggesting a greater reliance on carbohydrate and less on fat as an energy source. This seemed to be confirmed by the significantly lower plasma glycerol concentration, which suggested less fat mobilization in the CHO trial. Ingestion of a glucose polymer solution increased carbohydrate utilization in fasted subjects, but exercise performance was not improved.

An in vitro human muscle preparation suitable for metabolic studies. Decreased insulin stimulation of glucose transport in muscle from morbidly obese and diabetic subjects.

We have developed an in vitro muscle preparation suitable for metabolic studies with human muscle tissue and have investigated the effects of obesity and non-insulin-dependent diabetes mellitus (NIDDM) on glucose transport. Transport of 3-O-methylglucose and 2-deoxyglucose was stimulated approximately twofold by insulin in muscle from normal nonobese subjects and stimulation occurred in the normal physiological range of insulin concentrations. In contrast to insulin stimulation of 3-O-methylglucose and 2-deoxyglucose transport in muscle from normal, nonobese subjects, tissue from morbidly obese subjects, with or without NIDDM, were not responsive to insulin. Maximal 3-O-methylglucose transport was lower in muscle of obese than nonobese subjects. Morbidly obese patients, with or without NIDDM, have a severe state of insulin resistance in glucose transport. The novel in vitro human skeletal muscle preparation herein described should be useful in investigating the mechanism of this insulin resistance.