Edward B. Tapscott

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 leptins 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 μg/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.

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/obmice, 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 insulins 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 insulins effects on lipid metabolism but only attenuated leptins 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 insulins 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.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 insulins 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 insulins effects on lipid metabolism but only attenuated leptins 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 insulins 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.

Mechanism for Improved Insulin Sensitivity after Gastric Bypass Surgery

CONTEXT Surgical treatments of obesity have been shown to induce rapid and prolonged improvements in insulin sensitivity. OBJECTIVE The aim of the study was to investigate the effects of gastric bypass surgery and the mechanisms that explain the improvement in insulin sensitivity. DESIGN We performed a cross-sectional, nonrandomized, controlled study. SETTING This study was conducted jointly between the Departments of Exercise Science and Physiology at East Carolina University in Greenville, North Carolina. SUBJECTS Subjects were recruited into four groups: 1) lean [body mass index (BMI) < 25 kg/m(2); n = 93]; 2) weight-matched (BMI = 25 to 35 kg/m(2); n = 310); 3) morbidly obese (BMI > 35 kg/m(2); n = 43); and 4) postsurgery patients (BMI approximately 30 kg/m(2); n = 40). Postsurgery patients were weight stable 1 yr after surgery. MAIN OUTCOME MEASURES Whole-body insulin sensitivity, muscle glucose transport, and muscle insulin signaling were assessed. RESULTS Postsurgery subjects had insulin sensitivity index values that were similar to the lean and higher than morbidly obese and weight-matched control subjects. Glucose transport was higher in the postsurgery vs. morbidly obese and weight-matched groups. IRS1-pSer(312) in the postsurgery group was lower than morbidly obese and weight-matched groups. Inhibitor kappaBalpha was higher in the postsurgery vs. the morbidly obese and weight-matched controls, indicating reduced inhibitor of kappaB kinase beta activity. CONCLUSIONS Insulin sensitivity and glucose transport are greater in the postsurgery patients than predicted from the weight-matched group, suggesting that improved insulin sensitivity after bypass is due to something other than, or in addition to, weight loss. Improved insulin sensitivity is related to reduced inhibitor of kappaB kinase beta activity and enhanced insulin signaling in muscle.

Measurement of in vivo protein synthesis in rats during an exercise bout

Abstract The purpose of the present investigation was twofold: (i) to develop a simple and reliable method for measuring muscle protein synthesis in vivo and (ii) to use the method to study the effect of exercise on muscle protein synthesis. The method that has been developed is based on the principle that [ 3 H]tyrosine is released at a fairly constant rate from a subcutaneously injected emulsion of water and sesame oil. Incorporation of label into muscle protein and [ 3 H]tyrosine specific activity are linear for at least 60 min. Thus, removing muscle samples at two time periods (30 and 60 min) allows the rate of muscle protein synthesis to be determined. In a previous study, we found that swimming rats for 1 hr resulted in an 18% reduction in the rate of protein synthesis. In the present study, 1 hr or running resulted in a 30% depression while running to exhaustion caused a 71% depression in muscle protein synthesis. These results indicate that muscle protein synthesis changes in response to the intensity and the duration of an exercise bout.

Changes in muscle mitochondrial lipid composition resulting from training and exhaustive exercise

Abstract This study was undertaken to determine if the changes in mitochondrial structure and function that occur in muscle with exhaustive exercise could be caused by alterations in lipid composition of mitochondrial membranes. Further, the effect of training on lipid composition was studied to ascertain if lipid changes accompany the adaptation in the level of mitochondrial protein. Training decreased free fatty acids and triglycerides. Exhaustion of untrained animals resulted in increases of total phospholipid and phosphatidyl choline while exhaustion of trained rats caused a lowering of total phospholipid and phosphatidyl choline. Alterations in membrane lipid composition are most likely not the cause of changes in mitochondrial structure and function after exhaustive exercise since mitochondrial yield and lipid levels did not change in concert; i.e. muscle mitochondrial yield was decreased in both untrained and trained rats while total phospholipids were increased in untrained rats and decreased in trained rats as a result of exhaustive exercise. Although the physiological significance of the effects observed remains to be determined, this study does demonstrate that the lipid composition of mitochondria is not a constant parameter but can change in response to a chronic (training) or acute (exhaustive exercise) physiological condition.

Changes in body fat and lipogenic enzyme activities in rats after termination of exercise training.

Summary Exercise training lowered the body fat content of rats but, after training was terminated, there was a rapid increase in body weight and carcass fat. After 2 weeks of detraining, there were increases in the activities of liver and adipose tissue fatty acid synthetase, adipose glucose-6-phos-phate dehydrogenase, adipose citrate cleavage enzyme, and adipose malic enzyme. The increase in the activities of lipogenic enzymes may play a role in the rapid deposition of fat after termination of exercise training.

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 agematched 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<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.

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 decresed (P<0.002) glucose uptake (μmol g−1 h−1) with maximal insulin stimulation, while training significantly increased uptake (P<0.01) at both insulin concentrations. Glycogen synthesis was also increased by training (P<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.

Metabolic profiling of muscle contraction in lean compared with obese rodents.

Interest in the pathophysiological relevance of intramuscular triacylglycerol (IMTG) accumulation has grown from numerous studies reporting that abnormally high glycerolipid levels in tissues of obese and diabetic subjects correlate negatively with glucose tolerance. Here, we used a hindlimb perfusion model to examine the impact of obesity and elevated IMTG levels on contraction-induced changes in skeletal muscle fuel metabolism. Comprehensive lipid profiling was performed on gastrocnemius muscles harvested from lean and obese Zucker rats immediately and 25 min after 15 min of one-legged electrically stimulated contraction compared with the contralateral control (rested) limbs. Predictably, IMTG content was grossly elevated in control muscles from obese rats compared with their lean counterparts. In muscles of obese (but not lean) rats, contraction resulted in marked hydrolysis of IMTG, which was then restored to near resting levels during 25 min of recovery. Despite dramatic phenotypical differences in contraction-induced IMTG turnover, muscle levels of diacylglycerol (DAG) and long-chain acyl-CoAs (LCACoA) were surprisingly similar between groups. Tissue profiles of acylcarnitine metabolites suggested that the surfeit of IMTG in obese rats fueled higher rates of fat oxidation relative to the lean group. Muscles of the obese rats had reduced lactate levels immediately following contraction and higher glycogen resynthesis during recovery, consistent with a lipid-associated glucose-sparing effect. Together, these findings suggest that contraction-induced mobilization of local lipid reserves in obese muscles promotes beta-oxidation, while discouraging glucose utilization. Further studies are necessary to determine whether persistent oxidation of IMTG-derived fatty acids contributes to systemic glucose intolerance in other physiological settings.

Effect of Exercise on Liver Protein Loss and Lysosomal Enzyme Levels in Fed and Fasted Rats

Abstract Exhaustive treadmill running caused a significant decrease in hepatic protein levels. The rate of protein loss was 0.23 g/hr. This protein loss was accompanied by an increase in the activities of free (nonsedimentable) cathepsin D and arylsulfatase, suggesting the involvement of lysosomes. A 24-hr fast lowered the liver protein levels by about 20%. Plots of liver protein levels versus the time it took to reach exhaustion showed that the absolute rate of protein loss was the same for both the fed and the fasted rats. Since the initial protein levels were lower in the fasted rats, their relative rate of protein loss was greater. The level of activity of free (nonsedimentable) cathepsin D and arylsulfatase increased as the time to exhaustion increased in both the fed and fasted animals. The increase was severalfold greater in the fasted animals suggesting that fasting alters the structure of lysosomes in a way that enhances the increase in lysosomal fragility caused by exercise.