Crucial role for LKB1 to AMPKα2 axis in the regulation of CD36-mediated long-chain fatty acid uptake into cardiomyocytes

Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKα1 versus AMPKα2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKα2-kinase-dead, AMPKα2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKα2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKα2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKα2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKα2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.     

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.     

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.     

5-amino-imidazole carboxamide riboside acutely potentiates glucose-stimulated insulin secretion from mouse pancreatic islets by KATP channel-dependent and -independent pathways

AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic beta-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 microM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K(+) (K(ATP)) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5mM) AICAR did not affect K(ATP) activity or intracellular ([Ca(2+)](i)). AICAR also did not inhibit (86)Rb(+) efflux from islets isolated from Sur1(-/-) mice that lack K(ATP) channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K(ATP) channel-dependent and -independent pathways.     

Effects of AICAR and exercise on insulin-stimulated glucose uptake, signaling, and GLUT-4 content in rat muscles.

Physical activity is known to increase insulin action in skeletal muscle, and data have indicated that 5'-AMP-activated protein kinase (AMPK) is involved in the molecular mechanisms behind this beneficial effect. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) can be used as a pharmacological tool to repetitively activate AMPK, and the objective of this study was to explore whether the increase in insulin-stimulated glucose uptake after either long-term exercise or chronic AICAR administration was followed by fiber-type-specific changes in insulin signaling and/or changes in GLUT-4 expression. Wistar rats were allocated into three groups: an exercise group trained on treadmill for 5 days, an AICAR group exposed to daily subcutaneous injections of AICAR, and a sedentary control group. AMPK activity, insulin-stimulated glucose transport, insulin signaling, and GLUT-4 expression were determined in muscles characterized by different fiber type compositions. Both exercised and AICAR-injected animals displayed a fiber-type-specific increase in glucose transport with the most marked increase in muscles with a high content of type IIb fibers. This increase was accompanied by a concomitant increase in GLUT-4 expression. Insulin signaling as assessed by phosphatidylinositol 3-kinase and PKB/Akt activity was enhanced only after AICAR administration and in a non-fiber-type-specific manner. In conclusion, chronic AICAR administration and long-term exercise both improve insulin-stimulated glucose transport in skeletal muscle in a fiber-type-specific way, and this is associated with an increase in GLUT-4 content.     

Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice

Bioactive components from bitter melon (BM) have been reported to improve glucose metabolism in vivo, but definitive studies on efficacy and mechanism of action are lacking. We sought to investigate the effects of BM bioactives on body weight, muscle lipid content and insulin signaling in mice fed a high-fat diet and on insulin signaling in L6 myotubes. Male C57BL/6J mice were randomly divided into low-fat diet control (LFD), high-fat diet (HFD) and HFD plus BM (BM) groups. Body weight, body composition, plasma glucose, leptin, insulin and muscle lipid profile were determined over 12 weeks. Insulin signaling was determined in the mouse muscle taken at end of study and in L6 myotubes exposed to the extract. Body weight, plasma glucose, insulin, leptin levels and HOMA-IR values were significantly lower in the BM-fed HFD group when compared to the HFD group. BM supplementation significantly increased IRS-2, IR β, PI 3K and GLUT4 protein abundance in skeletal muscle, as well as phosphorylation of IRS-1, Akt1 and Akt2 when compared with HFD (P<.05 and P<.01). BM also significantly reduced muscle lipid content in the HFD mice. BM extract greatly increased glucose uptake and enhanced insulin signaling in L6 myotubes. This study shows that BM bioactives reduced body weight, improved glucose metabolism and enhanced skeletal muscle insulin signaling. A contributing mechanism to the enhanced insulin signaling may be associated with the reduction in skeletal muscle lipid content. Nutritional supplementation with this extract, if validated for human studies, may offer an adjunctive therapy for diabetes.     

Role of the nitric oxide pathway in AMPK-mediated glucose uptake and GLUT4 translocation in heart muscle

AMP-activated protein kinase (AMPK) is a serine-threonine kinase that regulates cellular metabolism and has an essential role in activating glucose transport during hypoxia and ischemia. The mechanisms responsible for AMPK stimulation of glucose transport are uncertain, but may involve interaction with other signaling pathways or direct effects on GLUT vesicular trafficking. One potential downstream mediator of AMPK signaling is the nitric oxide pathway. The aim of this study was to examine the extent to which AMPK mediates glucose transport through activation of the nitric oxide (NO)-signaling pathway in isolated heart muscles. Incubation with 1 mM 5-amino-4-imidazole-1-beta-carboxamide ribofuranoside (AICAR) activated AMPK (P < 0.01) and stimulated glucose uptake (P < 0.05) and translocation of the cardiomyocyte glucose transporter GLUT4 to the cell surface (P < 0.05). AICAR treatment increased phosphorylation of endothelial NO synthase (eNOS) approximately 1.8-fold (P < 0.05). eNOS, but not neuronal NOS, coimmunoprecipitated with both the alpha(2) and alpha(1) AMPK catalytic subunits in heart muscle. NO donors also increased glucose uptake and GLUT4 translocation (P < 0.05). Inhibition of NOS with N(omega)-nitro-l-arginine and N(omega)-methyl-l-arginine reduced AICAR-stimulated glucose uptake by 21 +/- 3% (P < 0.05) and 25 +/- 4% (P < 0.05), respectively. Inhibition of guanylate cyclase with ODQ and LY-83583 reduced AICAR-stimulated glucose uptake by 31 +/- 4% (P < 0.05) and 22 +/- 3% (P < 0.05), respectively, as well as GLUT4 translocation to the cell surface (P < 0.05). Taken together, these results indicate that activation of the NO-guanylate cyclase pathway contributes to, but is not the sole mediator of, AMPK stimulation of glucose uptake and GLUT4 translocation in heart muscle.     

Glucose, insulin, and leptin signaling pathways modulate nitric oxide synthesis in glucose-inhibited neurons in the ventromedial hypothalamus

Glucose-sensing neurons in the ventromedial hypothalamus (VMH) are involved in the regulation of glucose homeostasis. Glucose-sensing neurons alter their action potential frequency in response to physiological changes in extracellular glucose, insulin, and leptin. Glucose-excited neurons decrease, whereas glucose-inhibited (GI) neurons increase, their action potential frequency when extracellular glucose is reduced. Central nitric oxide (NO) synthesis is regulated by changes in local fuel availability, as well as insulin and leptin. NO is involved in the regulation of food intake and is altered in obesity and diabetes. Thus this study tests the hypothesis that NO synthesis is a site of convergence for glucose, leptin, and insulin signaling in VMH glucose-sensing neurons. With the use of the NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein in conjunction with the membrane potential-sensitive dye fluorometric imaging plate reader, we found that glucose and leptin suppress, whereas insulin stimulates neuronal nitric oxide synthase (nNOS)-dependent NO production in cultured VMH GI neurons. The effects of glucose and leptin were mediated by suppression of AMP-activated protein kinase (AMPK). The AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) increased both NO production and neuronal activity in GI neurons. In contrast, the effects of insulin on NO production were blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Furthermore, decreased glucose, insulin, and AICAR increase the phosphorylation of VMH nNOS, whereas leptin decreases it. Finally, VMH neurons express soluble guanylyl cyclase, a downstream mediator of NO signaling. Thus NO may mediate, in part, glucose, leptin, and insulin signaling in VMH glucose-sensing neurons.     

5'-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside

Exercise is known to increase insulin sensitivity and is an effective form of treatment for the hyperglycemia observed in type 2 diabetes. Activation of 5'-AMP-activated protein kinase (AMPK) by 5-aminoimidazole-4-carboxamide riboside (AICAR), exercise, or electrically stimulated contraction leads to increased glucose transport in skeletal muscle. Here we report the first evidence of a direct interaction between AMPK and the most upstream component of the insulin-signaling cascade, insulin receptor substrate-1 (IRS-1). We find that AMPK rapidly phosphorylates IRS-1 on Ser-789 in cell-free assays as well as in mouse C2C12 myotubes incubated with AICAR. In the C2C12 myotubes activation of AMPK by AICAR matched the phosphorylation of IRS-1 on Ser-789. This phosphorylation correlates with a 65% increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity in C2C12 myotubes preincubated with AICAR. The binding of phosphatidylinositol 3-kinase to IRS-1 was not affected by AICAR. These results demonstrate the existence of an interaction between AMPK and early insulin signaling that could be of importance to our understanding of the potentiating effects of exercise on insulin signaling.     

Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated.

Fatty acid oxidation in muscle has been reported to be diminished when insulin and glucose levels are elevated. This study was designed to determine whether activation of AMP-activated protein kinase (AMPK) will prevent inhibitory effects of insulin and glucose on the rate of fatty acid oxidation. Rat hindlimbs were perfused with medium containing 0, 0.3, or 60 nM insulin with or without 2 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). Glucose uptake was stimulated four- to fivefold by inclusion of insulin in the medium. Insulin attenuated the increase in AMPK caused by AICAR both in perfused hindlimbs and in isolated epitrochlearis muscles. The activation constant for citrate activation of acetyl-CoA carboxylase (ACC) was significantly increased in response to AICAR, and the increase was slightly attenuated if insulin was present in the perfusion medium. Insulin stimulated an increase in malonyl-CoA content of the muscles in the absence of AICAR. Malonyl-CoA was decreased to approximately the same value in AICAR-perfused muscle, regardless of insulin concentration. Muscle glucose 6-phosphate and citrate were significantly increased in response to AICAR and insulin. The rate of palmitate oxidation tended to decrease in response to insulin and in the absence of AICAR. AICAR increased palmitate oxidation to approximately the same level regardless of the insulin concentration or the rate of glucose uptake into the muscle. The rate of palmitate oxidation showed a curvilinear relationship as a function of muscle malonyl-CoA content, with half-maximal inhibition at approximately 0.6 nmol/g. We conclude that AMPK activation can prevent high rates of glucose uptake and glycolytic flux from inhibiting palmitate oxidation in predominantly fast-twitch muscle under these conditions.     

AICAR and hyperosmotic stress increase insulin-stimulated glucose transport.

Sensitivity of glucose transport to stimulation by insulin has been shown to occur concomitant with activation of the AMP-activated protein kinase (AMPK) in skeletal muscle, suggesting a role of AMPK in regulation of insulin action. The purpose of the present study was to evaluate a possible role of AMPK in potentiation of insulin action in muscle cells. The experimental model involved insulin-responsive C2C12 myotubes that exhibit a twofold increase in glucose transport in the presence of insulin. Treatment of myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), followed by a 2-h recovery, augmented the ability of insulin to stimulate glucose transport. Similarly, incubation in hyperosmotic medium, another AMPK-activating treatment, acted synergistically with insulin to stimulate glucose transport. Furthermore, the increase in insulin action caused by hyperosmotic stress was prevented by inclusion of compound C, an AMPK inhibitor, in hyperosmotic medium. In addition, iodotubercidin, a general kinase inhibitor that is effective against AMPK, also prevented the combined effects of insulin and hyperosmotic stress on glucose transport. The new information provided by these data is that previously reported AICAR effects on insulin action are generalizable to myotubes, hyperosmotic stress and insulin synergistically increase glucose transport, and AMPK appears to mediate potentiation of insulin action.     

Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of ∼20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min⁻¹) and was decreased during the steady-state responses to insulin (0.18 min⁻¹), AICAR (0.16 min⁻¹), and A-769662 (0.24 min⁻¹). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.     

Acetyl-l-carnitine inhibits TNF-α-induced insulin resistance via AMPK pathway in rat skeletal muscle cells

In this study, we demonstrated effects of acetyl-l-carnitine (ALC) on insulin resistance induced by tumor necrosis factor-α (TNF-α) in rat L6 cells. TNF-α downregulated insulin-stimulated glucose uptake and increased Serine 307 phosphorylation of insulin receptor substrate-1 (IRS-1). However, the treatment of ALC improved insulin-stimulated glucose uptake via AMP-activated protein kinase (AMPK) activation in a dose-dependent manner. Together, our data suggest that ALC inhibits TNF-α-induced insulin resistance through AMPK pathway in skeletal muscle cells.     

AICAR reverses ketone body mediated insulin resistance in isolated oxidative muscle

Recently it was demonstrated that the ketone body β-hydroxybutyrate (BOH) inhibits insulin-mediated glucose transport in isolated oxidative muscle, which was associated with decreased phosphorylation of Akt/protein kinase B. The purpose of the present study was to determine if activation of AMP-dependent protein kinase by the pharmacological activator AICAR could reverse the insulin resistance induced by BOH. Isolated mouse soleus muscle was incubated in vitro in the absence or presence of 5 mM BOH for ∼20 h. Following prolonged incubation, insulin increased 2-deoxyglucose glucose (2-DG) uptake 3-fold, but in the presence of BOH most of the insulin response was lost (only ∼30% remained). Addition of 2 mM AICAR during the last 2 h of prolonged incubation increased the insulin response in the presence of BOH to ∼80% of the normal insulin effect on 2-DG uptake. The AICAR-mediated reversal of the insulin resistance was not associated with a restoration of the insulin effect on Akt/protein kinase B phosphorylation. However, AICAR enhanced the insulin-induced phosphorylation of the Akt substrate, AS160. In conclusion, these data demonstrate that AICAR reverses the negative effect of BOH on insulin-mediated glucose uptake and this is attributed to activation of a late step in insulin signaling.     

Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways

AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by approximately 80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway.     

Artemisia princeps Extract Promoted Glucose Uptake in Cultured L6 Muscle Cells via Glucose Transporter 4 Translocation

Artemisia princeps is a familiar plant as a food substance and medicinal herb. In this study, we evaluated the effects of an ethanol extract of A. princeps (APE) on glucose uptake in differentiated L6 muscle cells. Treatment with APE elevated deoxyglucose uptake, and translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane in L6 myotubes occurred. The PI3K inhibitor LY294002 attenuated glucose uptake induced by APE. Phosphorylation of the Ser⁴⁷³ residue of Akt was not observed, but phosphorylation of PI3K, Akt (Thr³⁰⁸), and atypical PKC was. In addition, APE stimulated phosphorylation of AMP-activated protein kinase (AMPK) at a level similar to 5′-amino-5-imidazolecarboxamide-riboside (AICAR). These results indicate that APE stimulates glucose uptake by inducing GLUT4 translocation, which is in part mediated by combination of the PI3K-dependent atypical PKC pathway and AMPK pathways.     

The evolution of insulin resistance in muscle of the glucose infused rat

Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser⁴⁷³) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3β (Ser⁹) and AS160 (Thr⁶⁴²) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.