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.

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.

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.

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.

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.

Metabolic responses to exercise after fasting.

Fasting before exercise increases fat utilization and lowers the rate of muscle glycogen depletion. Since a 24-h fast also depletes liver glycogen, we were interested in blood glucose homeostasis during exercise after fasting. An experiment was conducted with human subjects to determine the effect of fasting on blood metabolite concentrations during exercise. Nine male subjects ran (70% maximum O2 consumption) two counterbalanced trials, once fed and once after a 23-h fast. Plasma glucose was elevated by exercise in the fasted trial but there was no difference between fed and fasted during exercise. Lactate was significantly higher (P less than 0.05) in fasted than fed throughout the exercise bout. Fat mobilization and utilization appeared to be greater in the fasted trial as evidenced by higher plasma concentrations of free fatty acids, glycerol, and beta-hydroxybutyrate as well as lower respiratory exchange ratio in the fasted trial during the first 30 min of exercise. These results demonstrate that in humans blood glucose concentration is maintained at normal levels during exercise after fasting despite the depletion of liver glycogen. Homeostasis is probably maintained as a result of increased gluconeogenesis and decreased utilization of glucose in the muscle as a result of lowered pyruvate dehydrogenase activity.

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.

Protein degradation during endurance exercise and recovery.

During endurance exercise, there is a net breakdown of body protein and the amino acids so mobilized are available for increased rates of oxidation and gluconeogenesis. At least part of the net loss of protein is due to a decrease in the rate of protein synthesis during exercise. Liver protein degradation is increased during exercise as a result of autophagy and proteolysis of cell material inside the secondary lysosomes. The rate of degradation of contractile proteins is decreased during exercise but is increased during the recovery period if the exercise is of high intensity and of long duration. Preliminary evidence suggests that the rate of degradation of non-contractile proteins in muscle may be increased at the same time that contractile protein degradation is decreased.

Glyceride metabolism in the myopathic hamster.

Analysis of plasma lipids of 30- and 185-day-old BIO 82.62 myopathic hamsters and age-matched normal controls revealed a decrease in only the concentration of cholesteryl esters of 185-day-old diseased animals. Measurement of lipoprotein lipase (LPL) activity in heart, muscle, and adipose tissue showed no difference between the activity of the enzyme in the heart and muscle of the cardiomyopathic hamsters and that of the age-matched controls. In adipose tissue, however, LPL activity was depressed in the diseased animals in both age groups. No difference was found in the activity of hormone sensitive lipase. Incorporation of sn[U-14C] glycerol-3-phosphate into total lipids was found to be depressed in homogenates of heart, muscle, and adipose tissue but unchanged in liver homogenates of diseased animals. It was concluded that the decrease in the capacity to synthesize glycerides, rather than limiting substrate concentrations, could be the cause of the decrease in the lipid content in some tissues of the cardiomyopathic hamster.

Lipid content and fatty acid composition of heart and muscle of the BIO 82.62 cardiomyopathic hamster.

Microscopic and analytical studies of the lipids in the heart and muscle of the BIO 82.62 myopathic hamsters and age-matched normal animals at the average ages of 33, 67, and 108 days were performed. Microscopic examinations did not show increased lipid depositions in the hearts of the diseased animals as was found in the BIO 14.6 strain. No consistent differences in the lipid content of the cardiomyopathic hamsters (BIO 82.62) and age-matched controls were observed in the three age groups except in the cholesterol content of muscle. Cholesterol increased significantly (P less than 0.01) in the 67 and 108 day old animals. This increase elevated the cholesterol/phospholipid ratio. Analysis of the fatty acid composition of triglycerides showed that the cardiomyopathic hamsters store more saturated fatty acids in both heart and muscle than do their normal counterparts. The abundance of more saturated fatty acids may imply that either the desaturation mechanism is altered in the diseased animals or that unsaturated fatty acids are preferentially utilized in other processes.

The influence of glycogen level on hepatic glucose efflux in the anesthetized rat.

This study was initiated to study the effect of a number of physiological conditions on the flux of metabolites across the liver. It was observed that there was a rather high rate of hepatic glycogen depletion in the anesthetized rat and this preparation was used to investigate the relationship between the rate of hepatic glycogenolysis and the level of glycogen in the liver. To produce a range of liver glycogen levels, rats were either ad libitum fed or 8- or 24-hr fasted. In addition, half of each group was anesthetized for either 10 or 70 min. Glucose flux across the liver was determined by measuring blood flow and glucose concentrations in the portal vein, hepatic artery, and hepatic vein. The rate of glycogen depletion and glucose efflux were greatest in the ad libitum fed animals and lowest in the 24-hr fasted rats. In all three groups glucose efflux was lower after 70 min of anesthesia than after 10 min. The results of this study demonstrate that anesthesia causes a rather high rate of hepatic glycogenolysis and the rate of glycogenolysis is decreased as the liver glycogen level is lowered.

Effect of an acute dose of ethanol on rat liver microsomal mixed function oxygenase components and membrane lipid composition.

The lipid composition of rat liver microsomes was not altered in response to a single acute dose of ethanol relative to isocaloric glycerol-treated controls. Microsomal cytochrome P-450 was transiently elevated, the elevation paralleling blood ethanol levels. Microsomal NADPH oxidase was transiently elevated relative to the activity in isocaloric glycerol-treated controls.

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.

Protein metabolism during endurance exercise.

After reviewing all the available results from our laboratory and numerous reports in the literature concerning changes that have occurred in various aspects of protein metabolism during exercise, a number of conclusions can be drawn with some degree of confidence. During exercise, protein synthesis is depressed and this change leaves amino acids available for catabolic processes. The rate of leucine oxidation appears to be increased during exercise, and there is a movement of amino acids, mostly in the form of alanine, from muscle to liver where the rate of gluconeogenesis is increased as a result of exercise. These changes in protein metabolism are probably physiologically significant in at least three ways: amino acid conversion to citric acid cycle intermediates enhances the rate of oxidation of acetyl-CoA generated from glucose and fatty acid oxidation; increased conversion of amino acids to glucose helps to prevent hypoglycemia; oxidation of some amino acids may provide energy for muscular contraction.

Influence of fasting on glycogen depletion in rats during exercise.

We recently observed that a 24-h fasted group of rats could run longer than an ad libitum fed control group before becoming exhausted. Because of the demonstrated importance of glycogen levels and free fatty acid availability during endurance exercise, we have investigated several parameters of carbohydrate and lipid metabolism in exercised and nonexercised rats that were either fed ad libitum or fasted for 24 h. A 24-h fast depleted liver glycogen, lowered plasma glucose concentration, decreased muscle glycogen levels, and increased free fatty acid and beta-hydroxybutyrate concentrations in plasma. During exercise the fasted group had lower plasma glucose concentration, higher plasma concentration of free fatty acids and beta-hydroxybutyrate, and a lower muscle glycogen depletion rate than did the ad libitum fed group. Since fasted rats were able to continue running even when plasma glucose had dropped to levels lower than those of fed-exhausted rats, it seems unlikely that blood glucose level, per se, is a factor in causing exhaustion. These results suggest that fasting increases fatty acid utilization during exercise and the resulting "glycogen sparing" effect may result in increased endurance.

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.

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.

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.

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.