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Thong, Farah S. L., Robert Hudson, Robert Ross, Ian Janssen, and Terry E. Graham. Plasma leptin in moderately obese men: independent effects of weight loss and aerobic exercise. Am J Physiol Endocrinol Metab 279: E307-E313, 2000.--The independent effects of weight loss and exercise on plasma leptin and total (AT), subcutaneous (SAT), and visceral (VAT) adipose tissue were investigated in 52 obese men. Subjects were randomly assigned to four 12-wk protocols: 1) control (C, n = 8), 2) diet-induced weight loss (DWL, n = 14), 3) exercise-induced weight loss (EWL, n = 14), and 4) exercise with weight maintenance (EWS, n = 16). Plasma leptin was unchanged in C (from 7.8 [+ or -] 1.3 to 7.7 [+ or -] 1.0 ng/ml). Equivalent weight loss (7.5 kg) decreased leptin significantly but similarly (DWL, from 8.5 [+ or -] 1.0 to 4.8 [+ or -] 0.6 ng/ml; EWL, from 10.1 [+ or -] 1.0 to 5.0 [+ or -] 0.6 ng/ml). Exercise in the absence of weight loss did not alter leptin levels (from 10.1 [+ or -] 1.3 to 9.2 [+ or -] 1.2 ng/ml). Changes in leptin correlated with changes in AT and SAT (both P [is less than] 0.05) but not in VAT. We conclude that reduction in adipose tissue after weight loss results in a collateral decrease in circulating leptin, and exercise, independent of its effects on weight loss, has no profound influence on leptin secretion. ob gene; caloric restriction; adipose tissue; regional fat distribution; body composition; weight control

Insulin-dependent phosphorylation of Akt target AS160 is required for GLUT4 translocation. Insulin and platelet-derived growth factor (PDGF) (Akt activators) or activation of conventional/novel (c/n) protein kinase C (PKC) and 5' AMP-activated protein kinase (AMPK) all promote a rise in membrane GLUT4 in skeletal muscle and cultured cells. However, the downstream effectors linking these pathways to GLUT4 traffic are unknown. Here we explore the hypothesis that AS160 is a molecular link among diverse signaling cascades converging on GLUT4 translocation. PDGF and insulin increased AS160 phosphorylation in CHO-IR cells. Stimuli that activate c/n PKC or AMPK also elevated AS160 phosphorylation. We therefore examined if these signaling pathways engage AS160 to regulate GLUT4 traffic in muscle cells. Nonphosphorylatable AS160 (4P-AS160) virtually abolished the net surface GLUT4myc gains elicited by insulin, PDGF, K+ depolarization, or 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside but partly, yet significantly, inhibited the effects of 4-phorbol12-myristate-13-acetate. However, the hypertonicity or 2,4-dinitrophenol-dependent gains in surface GLUT4myc were unaffected by 4P-AS160. RK-AS160 (GTPase-activating protein [GAP] inactive) or 4PRK-AS160 (GAP inactive, nonphosphorylatable) had no effect on surface GLUT4myc elicited by all stimuli. Collectively, these results indicate that activation of Akt, c/n PKC, or α2-AMPK intersect at AS160 to regulate GLUT4 traffic, as well as highlight the potential of AS160 as a therapy target to increase muscle glucose uptake. Diabetes 56:414-423, 2007 [ABSTRACT FROM AUTHOR]

Insulin promotes the translocation of glucose transporter 4 (GLUT4) from intracellular pools to the surface of muscle and fat cells via a mechanism dependent on phosphatidylinositol (PtdIns) 3-kinase, actin cytoskeletal remodeling and the v-SNARE VAMP2. The growth factor PDGF-BB also robustly activates PtdIns 3-kinase and induces actin remodeling, raising the question of whether it uses similar mechanisms to insulin in mobilizing GLUT4. In L6 myoblasts stably expressing Myc-tagged GLUT4, neither stimulus affected the rate of GLUT4 endocytosis, confirming that they act primarily by enhancing exocytosis to increase GLUT4 at the cell surface. Although surface GLUT4myc in response to insulin peaked at 10 minutes and remained steady for 30 minutes, PDGF action was transient, peaking at 5 minutes and disappearing by 20 minutes. These GLUT4myc translocation time courses mirrored that of phosphorylation of Akt by the two stimuli. Interestingly, insulin and PDGF caused distinct manifestations of actin remodeling. Insulin induced discrete, long (>5 μm) dorsal actin structures at the cell periphery, whereas PDGF induced multiple short (<5 μm) dorsal structures throughout the cell, including above the nucleus. Latrunculin B, cytochalasin D and jasplakinolide, which disrupt actin dynamics, prevented insulin- and PDGF-induced actin remodeling but significantly inhibited GLUT4myc translocation only in response to insulin (75–85%, P<0.05), not to PDGF (20–30% inhibition). Moreover, transfection of tetanus toxin light chain, which cleaves the v-SNAREs VAMP2 and VAMP3, reduced insulin-induced GLUT4myc translocation by >70% but did not affect the PDGF response. These results suggest that insulin and PDGF rely differently on the actin cytoskeleton and on tetanus-toxin-sensitive VAMPs for mobilizing GLUT4. [ABSTRACT FROM AUTHOR]

Exercise profoundly influences glycemic control by enhancing muscle insulin sensitivity, thus promoting glucometabolic health. While prior glycogen breakdown so far has been deemed integral for muscle insulin sensitivity to be potentiated by exercise, the mechanisms underlying this phenomenon remain enigmatic. We have combined original data from 13 of our studies that investigated insulin action in skeletal muscle either under rested conditions or following a bout of one-legged knee extensor exercise in healthy young male individuals (n = 106). Insulin-stimulated glucose uptake was potentiated and occurred substantially faster in the prior contracted muscles. In this otherwise homogenous group of individuals, a remarkable biological diversity in the glucometabolic responses to insulin is apparent both in skeletal muscle and at the whole-body level. In contrast to the prevailing concept, our analyses reveal that insulin-stimulated muscle glucose uptake and the potentiation thereof by exercise are not associated with muscle glycogen synthase activity, muscle glycogen content, or degree of glycogen utilization during the preceding exercise bout. Our data further suggest that the phenomenon of improved insulin sensitivity in prior contracted muscle is not regulated in a homeostatic feedback manner from glycogen. Instead, we put forward the idea that this phenomenon is regulated by cellular allostatic mechanisms that elevate the muscle glycogen storage set point and enhance insulin sensitivity to promote the uptake of glucose toward faster glycogen resynthesis without development of glucose overload/toxicity or feedback inhibition., (© 2022 by the American Diabetes Association.)

Adiponectin promotes cardioprotection by various mechanisms, and this study used primary cardiomyocytes and the isolated working perfused heart to investigate cardiometabolic effects. We show in adult cardiomyocytes that adiponectin increased CD36 translocation and fatty acid uptake as well as insulin-stimulated glucose transport and Akt phosphorylation. Coimmunoprecipitation showed that adiponectin enhanced association of AdipoR1 with APPL1, subsequent binding of APPL1 with AMPKα2, which led to phosphorylation and inhibition of ACC and increased fatty acid oxidation. Using siRNA to effectively knockdown APPL1 in neonatal cardiomyocytes, we demonstrated an essential role for APPL1 in mediating increased fatty acid uptake and oxidation by adiponectin. Importantly, enhanced fatty acid oxidation in conjunction with AMPK and ACC phosphorylation was also observed in the isolated working heart. Despite increasing fatty acid oxidation and myocardial oxygen consumption, adiponectin increased hydraulic work and maintained cardiac efficiency. In summary, the present study documents several beneficial metabolic effects mediated by adiponectin in the heart and provides novel insight into the mechanisms behind these effects, in particular the importance of APPL1.

Caffeine, an adenosine receptor antagonist, has been studied for decades as a putative ergogenic aid. In the past 2 decades, the information has overwhelmingly demonstrated that it indeed is a powerful ergogenic aid, and frequently theories have been proposed that this is due to alterations in fat and carbohydrate metabolism. While caffeine certainly mobilizes fatty acids from adipose tissue, rarely have measures of the respiratory exchange ratio indicated an increase in fat oxidation. However, this is a difficult measure to perform accurately during exercise, and small changes could be physiologically important. The few studies examining human muscle metabolism directly have also supported the fact that there is no change in fat or carbohydrate metabolism, but these usually have had a small sample size. We combined the data from muscle biopsy analyses of several similar studies to generate a sample size of 16-44, depending on the measure. We examined muscle glycogen, citrate, acetyl-CoA, glucose-6-phosphate, and cyclic adenosine monophosphate (cAMP) in resting samples and in those obtained after 10-15 min of exercise at 70%-85% maximal oxygen consumption. Exercise decreased (p < 0.05) glycogen and increased (p < 0.05) citrate, acetyl-CoA, and glucose-6-phosphate. The only effects of caffeine were to increase (p < 0.05) citrate in resting muscle and cAMP in exercise. There is very little evidence to support the hypothesis that caffeine has ergogenic effects as a result of enhanced fat oxidation. Individuals may, however, respond differently to the effects of caffeine, and there is growing evidence that this could be explained by common genetic variations.

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

Insulin and hypertonicity each increase the content of GLUT4 glucose transporters at the surface of muscle cells. Insulin enhances GLUT4 exocytosis without diminishing its endocytosis. The insulin but not the hypertonicity response is reduced by tetanus neurotoxin, which cleaves vesicle-associated membrane protein (VAMP)2 and VAMP3, and is rescued upon introducing tetanus neurotoxin-resistant VAMP2. Here, we show that hypertonicity enhances GLUT4 recycling, compounding its previously shown ability to reduce GLUT4 endocytosis. To examine whether the canonical soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mechanism is required for the plasma membrane fusion of the tetanus neurotoxin-insensitive GLUT4 vesicles, L6 myoblasts stably expressing myc-tagged GLUT4 (GLUT4myc) were transiently transfected with dominant negative N-ethylmaleimide-sensitive factor (NSF) (DN-NSF) or small-interfering RNA to tetanus neurotoxin-insensitive VAMP (TI-VAMP siRNA). Both strategies markedly reduced the basal level of surface GLUT4myc and the surface gain of GLUT4myc in response to hypertonicity. The insulin effect was abolished by DN-NSF, but only partly reduced by TI-VAMP siRNA. We propose that insulin and hypertonicity recruit GLUT4myc from partly overlapping, but distinct sources defined by VAMP2 and TI-VAMP, respectively.

We have examined the effects of insulin on p38 mitogen-activated protein kinase (MAPK) phosphorylation in human skeletal muscle and the effects of prior exercise hereon. Seven men performed 1-h one-legged knee extensor exercise 3 h before the initiation of a 100-min euglycemic-hyperinsulinemic (600 pmol/l) clamp. Glucose uptake across the legs was measured with the leg balance technique, and muscle biopsies were obtained from the rested and exercised vastus lateralis before and during insulin infusion. Net glucose uptake during the clamp was approximately 50% higher (P < 0.05) in the exercised leg than in the rested leg. Insulin induced a modest sustained 1.2- and 1.3-fold increase (P < 0.05) in p38 MAPK phosphorylation in the rested and exercised legs, respectively. However, p38 phosphorylation was approximately 50% higher (P < 0.05) in the exercised compared with the rested leg before and during insulin infusion. We conclude that a physiological concentration of insulin causes modest but sustained activation of the p38 MAPK pathway in human skeletal muscle. Furthermore, the stimulatory effect of exercise on p38 phosphorylation is persistent for at least 3 h after exercise and remains evident during subsequent insulin stimulation. Because p38 MAPK has been suggested to play a necessary role in activation of GLUT-4 at the cell surface, the present data may suggest a putative role of p38 MAPK in the increased insulin sensitivity of skeletal muscle after exercise.

The caffeine-induced impairment of insulin action is commonly attributed to adenosine receptor (AR) antagonism in skeletal muscle. However, epinephrine, a potent inhibitor of insulin actions, is increased after caffeine ingestion. We tested the hypothesis that the insulin antagonistic effects of caffeine are mediated by epinephrine, and not by AR antagonism, in seven healthy men. On four separate occasions, they received 1) dextrose (placebo, PL), 2) 5 mg/kg caffeine (CAF), 3) 80 mg of propranolol (PR), and 4) 5 mg/kg caffeine + 80 mg of propranolol (CAF + PR) before an oral glucose tolerance test (OGTT). Blood glucose was similar among trials before and during the OGTT. Plasma epinephrine was elevated (P < 0.05) in CAF and CAF + PR. Areas under the insulin and C-peptide curves were 42 and 39% greater (P < 0.05), respectively, in CAF than in PL, PR, and CAF + PR. In the presence of propranolol (CAF + PR), these responses were similar to PL and PR. These data suggest that the insulin antagonistic effects of caffeine in vivo are mediated by elevated epinephrine rather than by peripheral AR antagonism.

Skeletal muscle is the primary site of whole-body glucose disposal and is vital in determining the overall insulin sensitivity and carbohydrate management. Insulin and physical exercise are important stimuli for muscle glucose transport and glycogen metabolism. While it is known that both insulin and contraction stimulate muscle glucose uptake and glycogen metabolism, the post-receptor mechanisms are not completely understood. Local metabolic factors, such as adenosine, have been suggested to play a role in insulin and contraction regulation of carbohydrate metabolism in skeletal muscle. While adenosine has clearly been shown to potentiate insulin-stimulated glucose transport in adipocytes and heart muscle, its role in carbohydrate metabolism in skeletal muscle is less clear, with numerous diverging findings published to date. This review article summarizes findings on the putative roles of adenosine in insulin and exercise-mediated regulation of carbohydrate metabolism and the signalling pathways proposed to be central to these metabolic stimuli in skeletal muscle.

We investigated the effects of caffeine ingestion on skeletal muscle glucose uptake, glycogen synthase (GS) activity, and insulin signaling intermediates during a 100-min euglycemic-hyperinsulinemic (100 microU/ml) clamp. On two occasions, seven men performed 1-h one-legged knee extensor exercise at 3 h before the clamp. Caffeine (5 mg/kg) or placebo was administered in a randomized, double-blind fashion 1 h before the clamp. During the clamp, whole-body glucose disposal was reduced (P < 0.05) in caffeine (37.5 +/- 3.1 micromol x min(-1) x kg(-1)) vs. placebo (54.1 +/- 2.9 micromol x min(-1) x kg(-1)). In accordance, the total area under the curve over 100 min (AUC(0--100 min)) for insulin-stimulated glucose uptake in caffeine was reduced (P < 0.05) by approximately 50% in rested and exercised muscle. Caffeine also reduced (P < 0.05) GS activity before and during insulin infusion in both legs. Exercise increased insulin sensitivity of leg glucose uptake in both caffeine and placebo. Insulin increased insulin receptor tyrosine kinase (IRTK), insulin receptor substrate 1-associated phosphatidylinositol (PI) 3-kinase activities, and Ser(473) phosphorylation of protein kinase B (PKB)/Akt significantly but similarly in rested and exercised legs. Furthermore, insulin significantly decreased glycogen synthase kinase-3alpha (GSK-3alpha) activity equally in both legs. Caffeine did not alter insulin signaling in either leg. Plasma epinephrine and muscle cAMP concentrations were increased in caffeine. We conclude that 1) caffeine impairs insulin-stimulated glucose uptake and GS activity in rested and exercised human skeletal muscle; 2) caffeine-induced impairment of insulin-stimulated muscle glucose uptake and downregulation of GS activity are not accompanied by alterations in IRTK, PI 3-kinase, PKB/Akt, or GSK-3alpha but may be associated with increases in epinephrine and intramuscular cAMP concentrations; and 3) exercise reduces the detrimental effects of caffeine on insulin action in muscle.