Failure of leptin to affect basal and insulin-stimulated glucose metabolism of rat skeletal muscle in vitro

Summary Studies on different isolated tissues have provided evidence that leptin may directly modulate cellular glucose handling. The present study was performed to elucidate leptin's action on basal and insulin-stimulated glucose metabolism in native muscle tissue, which under physiological circumstances is the quantitatively most important target tissue of insulin. Isolated rat soleus muscle strips were incubated for 1 h in the absence or presence of leptin (0, 1, 10, or 100 nmol/l) under basal or insulin-stimulated conditions (10 nmol/l). No effects of leptin were found on the rates of ³H-2-deoxy-glucose transport (basal: control, 314 ± 14; 1 nmol/l leptin, 320 ± 17; 10 nmol/l leptin, 314 ± 13; 100 nmol/l leptin, 322 ± 16; insulin-stimulated: control, 690 ± 33; 1 nmol/l leptin, 691 ± 29; 10 nmol/l leptin, 665 ± 26; 100 nmol/l leptin, 664 ± 27; cpm · mg^–1· h^–1; NS vs respective control) and on net glucose incorporation into glycogen (basal: control, 1.75 ± 0.18; 1 nmol/l leptin, 2.01 ± 0.13; 10 nmol/l leptin, 1.92 ± 0.11; 100 nmol/l leptin, 1.81 ± 0.13; insulin-stimulated: control, 5.98 ± 0.40; 1 nmol/l leptin, 5.93 ± 0.30; 10 nmol/l leptin, 5.46 ± 0.25; 100 nmol/l leptin, 5.85 ± 0.30; μmol · g^–1· h^–1; NS vs respective control). In parallel, leptin failed to affect rates of aerobic and anaerobic glycolysis as well as muscle glycogen content. Further experiments revealed that the inability of leptin to directly affect muscle glucose handling prevailed independently of muscle fiber type (soleus and epitrochlearis muscle), of ambient insulin concentrations (0–30 nmol/l), and of leptin exposure time (1 h or 6 h). Thus, our findings fail to support speculations about a physiological role of direct insulin-mimetic or insulin-desensitizing effects of leptin on skeletal muscle tissue. [Diabetologia (1998) 41: 524–529] Received: 7 October 1997 and in revised form: 23 January 1998     

糖代谢与骨骼肌的关系

骨骼肌是体内胰岛素刺激下摄取葡萄糖的主要组织,在糖代谢平衡中发挥着重要的作用.病理状态下的骨骼肌这种代谢调节能力下降.肌糖原及循环中葡萄糖是维持骨骼肌细胞正常代谢及功能的主要物质.糖代谢紊乱尤其是高血糖对骨骼肌的代谢、结构及功能等都有明显影响,包括高血糖导致骨骼肌胰岛素抵抗、对肌糖原代谢的影响、肌萎缩以及血管异常等,肌组织病变反过来又影响代谢的控制,使病情加重.     

Anti-insulin receptor antibodies inhibit insulin binding and stimulate glucose metabolism in skeletal muscle

Summary Autoantibodies against the insulin receptor are found in the serum of some patients with severe insulin resistance. The effects of one of these sera on insulin binding and on glucose transport and metabolism were investigated in the isolated mouse soleus muscle. Preincubation of muscles with the patient's serum resulted in an inhibition of subsequent¹²⁵I-insulin binding (half-maximal effect at 1500 dilution) and in a two to three-fold stimulation of glucose transport and metabolism (half-maximal effect at 12000 dilution). The insulin-like effects were blocked by anti-human IgG, but not by antiinsulin antibodies. The magnitude of the serum effects on 2-deoxyglucose uptake and glycolysis was similar to that of insulin, but the effect on glycogen synthesis was smaller than that of insulin. It is suggested that the patient's serum and insulin promote glucose transport and glycolysis through a common pathway, but act differently on glycogen synthesis.     

Two potentially arrhythmogenic mechanisms of adrenaline action in cardiac muscle

The effects of rate changes and 'premature' stimulation on the slow inward current of cardiac muscle were studied in the frog atrium. The effects of adrenaline under these conditions was investigated. It was found that adrenaline markedly accelerates the repriming kinetics of the slow inward channel. Adrenaline at low concentrations, sufficient to augment this current, also increases the effect of rate changes. Thus there is a larger second inward current 'staircase' in the presence of adrenaline. It is proposed that the combination of these effects is a possible mechanism by which adrenaline can induce cardiac disorders of rhythm.     

Effect of physical training on glucose tolerance and on glucose metabolism of skeletal muscle in anaesthetized normal rats

Summary The effect of physical training on glucose tolerance in vivo and skeletal muscle glucose metabolism in vitro was investigated in normal rats. Treadmill running for 10 days up to 240 min/day led to a decrease of basal and glucose-stimulated plasma insulin levels without major alterations of the IV glucose tolerance (1 g/kg body weight). Swim training of two weeks' duration, i. e. exercise up to 2×75 min/ day, which did not induce significant changes in body composition, skeletal muscle glycogen levels or citrate synthase activity, resulted in a significant improvement of IV glucose tolerance and substantial reductions of basal and glucose-stimulated plasma insulin levels. Associated with this apparent improvement of insulin sensitivity in vivo, significant increases of the insulin-stimulated glucose uptake (+ 55%) and lactate oxidation (+ 78%) in vitro were found on perfusion of the isolated hindquarter of swim-trained animals. It is suggested that mild physical training can improve glucose tolerance and insulin sensitivity in normal rats, at least in part, due to an increase of insulin sensitivity of skeletal muscle glucose metabolism.     

Role of skeletal muscle-fibre type in regulation of glucose metabolism in middle-aged subjects with impaired glucose tolerance during a long-term exercise and dietary intervention

AIM: The aim of this study was to investigate the role of skeletal muscle fibre type in the regulation of glucose metabolism in middle-aged obese subjects with impaired glucose tolerance (IGT) during a 2-year exercise and dietary intervention. METHODS: Muscle biopsies (musculus vastus lateralis) were taken from 22 subjects belonging to the intervention group of the Finnish Diabetes Prevention Study [1]. According to their myosin heavy chain (MHC) profile at the baseline, the subjects were divided into two groups: IGT(slow) (n=10) with a high proportion of MHC I isoforms and IGT(fast) (n=12) with a high proportion of MHC II isoforms in the vastus lateralis muscle. The intervention consisted of dietary counselling, strength and power training and/or aerobic exercise. The amount of exercise was the same in both groups; the exercise frequency was 5.1+/-2.7 h/week in the IGT(slow) and 5.1+/-2.8 h/week in the IGT(fast) group. RESULTS: Fasting glucose (p<0.05), 2-h glucose (p<0.05), fasting insulin (p<0.05), haemoglobin A1c (HbA(1c)) (p<0.01) and insulin resistance (p<0.05) [homeostasis model assessment for insulin resistance (HOMA-IR)] decreased in the IGT(fast) group, whereas only the 2-h glucose and HbA(1c) concentrations decreased in the IGT(slow) group. The amount of the glycogen synthase kinase-3-alphabeta (GSK-3-alphabeta) decreased in the IGT(fast) group (p<0.05). Exercise training increased the lactate dehydrogenase (LDH) (p<0.01), LDH-1 (p<0.05) and citrate synthase (CS) (p<0.05) activities in the vastus lateralis muscle in the IGT(slow) group, but only the CS activity (p<0.05) in the IGT(fast) group. CONCLUSIONS: The glucose metabolism improved both in the IGT(slow) and IGT(fast) group during the 2-year exercise and dietary intervention. The change was more prominent in the IGT(fast) group than in the IGT(slow) group, associated with the decrease of the GSK-alphabeta protein expression in skeletal muscle. The exercise training improved both glycolytic and oxidative capacity in the vastus lateralis muscle. The glycolytic capacity improved in the IGT(slow) group and the oxidative capacity in both groups.     

Effects of adrenaline on whole-body glucose metabolism and insulin-mediated regulation of glycogen synthase and PKB phosphorylation in human skeletal muscle

In the present study, we investigated the effect of adrenaline on insulin-mediated regulation of glucose and fat metabolism with focus on regulation of skeletal muscle PKB, GSK-3, and glycogen synthase (GS) phosphorylation. Ten healthy subjects (5 men and 5 women) received a 240-minute intravenous infusion of adrenaline (0.05 μg/[kg min]) or saline; after 120 minutes, a hyperinsulinemic-euglycemic clamp was added. Adrenaline infusion increased blood glucose concentration by approximately 50%, but the hyperinsulinemic clamp normalized blood glucose within 30 minutes. Glucose infusion rate during the last hour was approximately 60% lower during adrenaline infusion compared with saline (4.3 ± 0.5 vs 11.2 ± 0.6 mg/kg lean body mass per minute). Insulin increased PKB Ser⁴⁷³, PKB Thr³⁰⁸, and GSK-3β Ser⁹ phosphorylation in skeletal muscles; coinfusion of adrenaline did not influence insulin-stimulated PKB and GSK-3 phosphorylation. Adrenaline alone did not influence phosphorylation of PKB and GSK-3β. Insulin increased GS fractional activity and decreased GS Ser⁶⁴¹ and Ser^{645,649,653,657} phosphorylation. In the presence of adrenaline, insulin did neither activate GS nor dephosphorylate GS Ser⁶⁴¹. Surprisingly, GS Ser⁷ phosphorylation was not influenced by adrenaline. Adrenaline increased plasma lactate concentration; and muscle glycogen content was reduced in skeletal muscle the day after adrenaline infusion, supporting that insulin does not stimulate glycogen synthesis in skeletal muscles when adrenaline is present. In conclusion, adrenaline did not influence basal or insulin-stimulated PKB and GSK-3β phosphorylation in muscles, but completely blocked insulin-mediated GS activation and Ser⁶⁴¹ dephosphorylation. Still, insulin normalized adrenaline-mediated hyperglycemia.     

Effect of work-induced hypertrophy on muscle glucose metabolism in lean and obese mice

Summary The effect of work-induced hypertrophy (without any concomitant change in circulating parameters) on skeletal muscle metabolism was studied in lean mice and in goldthioglucose obese-mice. Soleus muscle was functionally overloaded in one leg by tenotomy of gastrocnemius muscle 4 days before muscle isolation, muscle in the other leg being used as control. Basal deoxyglucose uptake and glycolysis were markedly increased in overloaded muscles compared with control muscles, together with a ten-fold increase in fructose 2–6 bisphosphate content. In the presence of maximally effective insulin concentrations, deoxyglucose uptake and glycolysis were identical in overloaded and control muscles of lean mice, while the effects of overload and insulin were partly additive in muscles of goldthioglucose-obese mice. The sensitivity to insulin and insulin binding to muscles were not modified in overloaded muscles. Insulin-stimulated glycogenogenesis was decreased by about 50% probably due to a lower amount of glycogen synthase in overloaded than in control muscles. Thus, in muscles of goldthioglucose-obese mice work-induced hypertrophy increased the response to maximal insulin concentrations without modifying the altered insulin sensitivity and decreased insulin binding.     

Contrasting short- and long-term effects of weight loss on lipoprotein levels

The short- and long-term effects of weight loss on high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol levels were examined in 42 women who completed a 14-session behavioral weight-loss program. Lipid values were determined from samples taken before treatment, after treatment, and at six-month follow-up. There were significant changes in plasma lipid levels, but the short- and long-term effects differed. Both total and LDL cholesterol levels decreased during treatment and remained lower at follow-up. However, HDL cholesterol level and the HDL/LDL ratio did not change during treatment but increased significantly above pretreatment levels at follow-up. Furthermore, long-term changes in lipoprotein levels were significantly correlated with changes in the body-mass index even after correction for initial values. These results show that weight loss can, in the long term, have a potentially beneficial impact on lipoprotein levels in women.     

Reprogramming of glucose metabolism in hepatocellular carcinoma: Progress and prospects

Hepatocellular carcinoma(HCC) is one of the most lethal cancers, and its rate of incidence is rising annually. Despite the progress in diagnosis and treatment, the overall prognoses of HCC patients remain dismal due to the difficulties in early diagnosis and the high level of tumor invasion, metastasis and recurrence. It is urgent to explore the underlying mechanism of HCC carcinogenesis and progression to find out the specific biomarkers for HCC early diagnosis and the promising target for HCC chemotherapy. Recently, the reprogramming of cancer metabolism has been identified as a hallmark of cancer. The shift from the oxidative phosphorylation metabolic pathway to the glycolysis pathway in HCC meets the demands of rapid cell proliferation and offers a favorable microenvironment for tumor progression. Such metabolic reprogramming could be considered as a critical link between the different HCC genotypes and phenotypes. The regulation of metabolic reprogramming in cancer is complex and may occur via genetic mutations and epigenetic modulations including oncogenes, tumor suppressor genes, signaling pathways, noncoding RNAs, and glycolytic enzymes etc. Understanding the regulatory mechanisms of glycolysis in HCC may enrich our knowledge of hepatocellular carcinogenesis and provide important foundations in the search for novel diagnostic biomarkers and promising therapeutic targets for HCC.     

In vitro insulin action on erythrocyte glucose metabolism in normal and diabetic rats

Summary Alloxan-induced diabetes in rats significantly impaired the capacity of the erythrocytes to metabolise glucose in vitro to either lactic acid or CO₂. Both these metabolic activities were initially insensitive to insulin in normal as well as in diabetic animals; but became responsive when these cells were subjected to insulin and glucose starvation for 1 h through incubation in their absence. This action of insulin in starved cells showed concentration dependence and required preincubation with the hormone prior to addition of glucose.     

Potential role of skeletal muscle glucose metabolism on the regulation of insulin secretion

Pancreatic beta cells sense glucose flux and release as much insulin as required in order to maintain glycaemia within a narrow range. Insulin secretion is regulated by many factors including glucose, incretins, and sympathetic and parasympathetic tones among other physiological factors. To identify the mechanisms linking obesity‐related insulin resistance with impaired insulin secretion represents a central challenge. Recently, it has been argued that a crosstalk between skeletal muscle and the pancreas may regulate insulin secretion. Considering that skeletal muscle is the largest organ in non‐obese subjects and a major site of insulin‐ and exercise‐stimulated glucose disposal, it appears plausible that muscle might interact with the pancreas and modulate insulin secretion for appropriate peripheral intracellular glucose utilization. There is growing evidence that muscle can secrete so‐called myokines that can have auto/para/endocrine actions. Although it is unclear in which direction they act, interleukin‐6 seems to be a possible muscle‐derived candidate protein mediating such inter‐organ communication. We herein review some of the putative skeletal muscle‐derived factors mediating this interaction. In addition, the evidence coming from in vitro, animal and human studies that support such inter‐organ crosstalk is thoroughly discussed.     

Regulation of glucose and fatty acid metabolism in skeletal muscle during contraction

The glucose-fatty acid cycle explains the preference for fatty acid during moderate and long duration physical exercise. In contrast, there is a high glucose availability and oxidation rate in response to intense physical exercise. The reactive oxygen species (ROS) production during physical exercise suggests that the redox balance is important to regulate of lipids/carbohydrate metabolism. ROS reduces the activity of the Krebs cycle, and increases the activity of mitochondrial uncoupling proteins. The opposite effects happen during moderate physical activity. Thus, some issues is highlighted in the present review: Why does skeletal muscle prefer lipids in the basal and during moderate physical activity? Why does glucose-fatty acid fail to carry out their effects during intense physical exercise? How skeletal muscles regulate the lipids and carbohydrate metabolism during the contraction-relaxation cycle?     

Involvement of phosphoinositide 3-kinase in insulin stimulation of MAP-kinase and phosphorylation of protein kinase-B in human skeletal muscle: implications for glucose metabolism

Summary Isolated skeletal muscle from healthy individuals was used to evaluate the role of phosphoinositide 3-kinase (PI 3-kinase) in insulin signalling pathways regulating mitogen activated protein kinase (MAP-kinase) and protein kinase-B and to investigate whether MAP-kinase was involved in signalling pathways regulating glucose metabolism. Insulin stimulated glycogen synthase activity ( ≈ 1.7 fold), increased 3-o-methylglucose transport into human skeletal muscle strips ( ≈ 2 fold) and stimulated phosphorylation of the p42 ERK-2 isoform of MAP-kinase. This phosphorylation of p42 ERK2 was not blocked by the PI 3-kinase inhibitors LY294002 and wortmannin although it was blocked by the MAP-kinase kinase (MEK) inhibitor PD 98059. However, PD98059 (up to 20 μmol/l) did not block insulin activation of glycogen synthase or stimulation of 3-o-methylglucose transport. Wortmannin and LY294002 did block insulin stimulation of protein kinase-B (PKB) phosphorylation and stimulation of 3-o-methylglucose transport was inhibited by wortmannin (IC₅₀≈ 100 nmol/l). These results indicate that MAP-kinase is activated by insulin in human skeletal muscle by a PI 3-kinase independent pathway. Furthermore this activation is not necessary for insulin stimulation of glucose transport or activation of glycogen synthase in this tissue. [Diabetologia (1997) 40: 1172–1177]     

Lifestyle changes and lipid metabolism gene expression and protein content in skeletal muscle of subjects with impaired glucose tolerance

Aims/hypothesis Skeletal muscle of pre-diabetic patients is characterised by a diminished capacity to handle fatty acids. A diminished content of several enzymes involved in fatty-acid transport and oxidation have been suggested to underlie these defects. The aim of this study was to investigate whether the combination of dietary advice, increased physical activity and weight loss improves lipid metabolic gene and protein expression in skeletal muscle of subjects with impaired glucose tolerance.Methods Before and after 1 year of a lifestyle-intervention programme, expression of several genes and proteins involved in lipid metabolism were measured in vastus lateralis muscle biopsies from subjects in the intervention (n=7) and control group (n=6).Results After 1 year the intervention group had an improved glycaemic control and reduced body fat compared to the control group. Significant differences were observed for acetyl CoA-carboxylase 2 and uncoupling protein 2 expression (ACC2: –16.8±12.4% vs +51.5±32.3% for the intervention and control group respectively; p<0.05) (UCP2: –26.9±10.3% vs +10.5±6.2% for the intervention and control group respectively; p<0.05). Change in 3-hydroxyacyl-CoA dehydrogenase protein content tended to be different between groups (+3.2±1.1 vs –0.9±1.9 U/mg.ww for the intervention and control group, p=0.07).Conclusions/interpretation Lifestyle changes leading to an improved glycaemic control and reduced adiposity, resulted in a down-regulation of ACC-2 and UCP2 expression and in an increase in HAD protein content, reflecting a better capacity to utilise fatty acids.Abbreviations FFM Fat free mass - CPT-2 carnitine palmitoyl transferase-2 - CPT-1m carnitine palmitoyl transferase-1 muscle form - ACC-2 acetyl Co-enzyme A carboxylase-2 - LPL lipoprotein lipase - PPAR peroxisome proliferator-activated receptor- - UCP-2 uncoupling protein 2 - UCP-3 uncoupling protein 3 - FAT/CD36 fatty acid translocase/CD36 - FABPc cytosolic fatty acid binding protein - CS citrate synthase - HAD 3-hydroxyacyl-coA dehydrogenase - MJ megajoule     

Failure of GLP-l(7–36)amide to affect glycogenesis in rat skeletal muscle

Summary Glucagon-like peptide-1(7–36)amide has been described as exerting potent glycogenic action and as stimulating glycolysis in skeletal muscle. We exposed isolated rat soleus muscle strips to various concentrations of glucagon-like peptide-1(7–36) amide (10⁻¹¹–10⁻⁶ mol/l) or insulin (10⁻¹⁰–10⁻⁷ mol/l) and determined the respective effects on glucose metabolism. Insulin markedly increased the rate of glucose incorporation into glycogen with a maximal effect at 10⁻⁸ mol/l insulin (348 ± 46 % of intraindividual control experiment, p < 0.005), while glucagon-like peptide-1(7–36)amide was without an effect (e. g. 10⁻¹¹ mol/l, 96 ± 10 %; 10⁻⁹ mol/l, 104 ± 9 %; 10⁻⁷ mol/l, 121 ± 13 %; not significant). Likewise, glucagon-like peptide-1(7–36)amide did not affect the rate of ³H-2-deoxy-glucose transport or glycogen content of soleus muscle strips. The rates of aerobic or anaerobic glycolysis were also not increased. The findings were independent of peptide source and of employed muscle size. Our results do not suggest any effect of glucagon-like peptide-1(7–36)amide on skeletal muscle glucose metabolism and, hence, are in contrast to data derived from similar experiments by others. [Diabetologia (1995) 38: 864–867] Received: 29 December 1994 and in revised form: 13 March 1995     

Effect of glucose and insulin on triacylglycerol metabolism in isolated normal and diabetic skeletal muscle

The effects of insulin and glucose on triacylglycerol (TG) metabolism in normal and diabetic isolated skeletal muscle were investigated in this study. Intracellular TG was continuously synthesized and hydrolyzed in both normal and diabetic skeletal muscle. In the absence of insulin and glucose, normal and diabetic skeletal muscle TG content and synthesis were decreased. In contrast, in the presence of insulin and glucose, the normal and diabetic TG contents were unchanged and triacylglycerol synthesis was increased as compared with the respective control values. However, insulin and glucose increased intramuscular TG content to a greater extent than could be accounted for by their stimulation of TG synthesis, indicating that insulin and glucose appear to inhibit TG hydrolysis in diabetic muscle, as well as in normal muscle. In addition, these data suggest that diabetes causes a defect in the ability of insulin and glucose to stimulate TG synthesis, as the increase in diabetic muscle TG synthesis in the presence of insulin and glucose was less than in normal muscle.     

The effect of dietary creatine supplementation on skeletal muscle metabolism in congestive heart failure

Aims To assess the effects of dietary creatine supplementation onskeletal muscle metabolism and endurance in patients with chronicheart failure. Methods A forearm model of muscle metabolism was used, with a cannulainserted retrogradely into an antecubital vein of the dominantforearm. Maximum voluntary contraction was measured using handgripdynanometry. Subjects performed handgrip exercise, 5s contractionfollowed by 5s rest for 5min at 25%, 50%, and 75% of maximumvoluntary contraction or until exhaustion. Blood was taken atrest and 0 and 2min after exercise for measurement of lactateand ammonia. After 30min the procedure was repeated with fixedworkloads of 7kg, 14kg and 21kg. Patients were assigned to creatine20g daily or matching placebo for 5 days and returned after6 days for repeat study. Results Contractions (median (25th, 75th interquartiles)) until exhaustionat 75% of maximum voluntary contraction increased after creatinetreatment (8 (6, 14) vs 14 (8, 17), P=0·025) with nosignificant placebo effect. Ammonia per contraction at 75% maximumvoluntary contraction (11·6µmol/l/contraction (8·3,15·7) vs 8·9µmol/l/contraction (5·9,10·8), P=0·037) and lactate per contraction at75% maximum voluntary contraction (0·32mmol/l/contraction(0·28, 0·61) vs 0.27mmol/l/contraction (0·19,0·49), P=0·07) fell after creatine but not afterplacebo. Conclusions Creatine supplementation in chronic heart failure augments skeletalmuscle endurance and attenuates the abnormal skeletal musclemetabolic response to exercise.     

Distinct effects of short- and long-term leptin treatment on glucose and fatty acid uptake and metabolism in HL-1 cardiomyocytes

Alterations in cardiac glucose and fatty acid metabolism are possible contributors to the pathogenesis of heart failure in obesity. Here we examined the effect of leptin, the product of the obese (ob) gene, on metabolism in murine cardiomyocytes. Neither short-term (1 hour) nor long-term (24 hours) treatment with leptin (60 nmol/L) altered basal or insulin-stimulated glucose uptake and oxidation, glycogen synthesis, insulin receptor substrate 1 tyrosine, Akt, or glycogen synthase kinase 3beta phosphorylation. Extracellular lactate levels were also unaffected by leptin. However, leptin increased basal and insulin-stimulated palmitate uptake at both short and long exposure times and this corresponded with increased cell surface CD36 levels and elevated fatty acid transport protein 1 (FATP1) and CD36 protein content. Whereas short-term leptin treatment increased fatty acid oxidation, there was a decrease in oxidation after 24 hours. The former corresponded with increased acetyl coenzyme A carboxylase phosphorylation and the latter with increased expression of this enzyme. The discrepancy between uptake and oxidation of fatty acids led to a transient decrease in intracellular lipid content with lipid accumulation ensuing after 24 hours. In summary, we demonstrate that leptin did not alter glucose uptake or metabolism in murine cardiomyocytes. However, fatty acid uptake increased while oxidation decreased over time leading to intracellular lipid accumulation, which may lead to lipotoxic damage in heart failure.