Human muscle signaling responses to 3-day head-out dry immersion

To date little is known about catabolic NO-dependent signaling systems in human skeletal muscle during early stages of gravitational unloading. The goal of the study was to analyze signaling pathways that determine the initial development of proteolytic events in human soleus muscle during short-term gravitational unloading (simulated microgravity). Gravitational unloading was simulated by 3-day head-out dry immersion. Before and after the immersion the samples of soleus muscle were taken under local anesthesia, using biopsy technique. The content of desmin, IRS-1, phospho-AMPK, total and phospho-nNOS in soleus of 6 healthy men was determined using Western-blotting before and after the dry-immersion. Three days of the dry immersion resulted in a significant decrease in desmin, phospho-nNOS and phospho-AMPK as compared to the pre-immersion values. The results of the study suggest that proteolytic processes in human soleus at the early stage of gravitational unloading are associated with inactivation of nNOS. Reduction in AMPK phosphorylation could serve as a trigger event for the development of primary atrophic changes in skeletal muscle.     

Effects of Exercise on AMPK Signaling and Downstream Components to PI3K in Rat with Type 2 Diabetes

Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague–Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK) and downstream components of phosphatidylinositol 3-kinase (PI3K) signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC). Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKC)ζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB)/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr³⁰⁸) and (Ser⁴⁷³) and AS160 phosphorylation. Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1) mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of components of the AMPK and downstream to PIK3 (aPKC, Akt), and improve GLUT4 trafficking in skeletal muscle. These data help explain the mechanism how exercise regulates glucose homeostasis in diabetic rats.     

Salicylate acutely stimulates 5′-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscles

Salicylate (SAL) has been recently implicated in the antidiabetic effect in humans. We assessed whether 5′-AMP-activated protein kinase (AMPK) in skeletal muscle is involved in the effect of SAL on glucose homeostasis. Rat fast-twitch epitrochlearis and slow-twitch soleus muscles were incubated in buffer containing SAL. Intracellular concentrations of SAL increased rapidly (<5 min) in both skeletal muscles, and the Thr¹⁷² phosphorylation of the α subunit of AMPK increased in a dose- and time-dependent manner. SAL increased both AMPKα1 and AMPKα2 activities. These increases in enzyme activity were accompanied by an increase in the activity of 3-O-methyl-d-glucose transport, and decreases in ATP, phosphocreatine, and glycogen contents. SAL did not change the phosphorylation of insulin receptor signaling including insulin receptor substrate 1, Akt, and p70 ribosomal protein S6 kinase. These results suggest that SAL may be transported into skeletal muscle and may stimulate AMPK and glucose transport via energy deprivation in multiple muscle types. Skeletal muscle AMPK might be part of the mechanism responsible for the metabolic improvement induced by SAL.     

Effects of support stimulation on human soleus fiber characteristics during exposure to "dry" immersion

In this study the model of 7-day dry immersion (DI) was used. 17 male volunteers (23-29 years old) were divided in 2 groups: (i) 7-day DI without support (DI, n=9), (ii) 7-day DI using support stimulation (DIS, n=8). Support stimulator device exerted pressure of 0.2 +/- 0.15 kg/cm2 upon the plantar support zones simulating the walking pattern 6 times a day for 20 minutes of every hour: 10 minutes at a speed of 75 steps/min and 10 minutes at a speed of 120 steps/min. M. soleus biopsy was performed before and immediately after DI. The m. soleus fiber myosin heavy chain (MHC) profile, myofiber cross-sectional area (CSA) and total protein concentration were analyzed in frozen serial sections. In addition, NO-synthase 1 (NOS1) levels indicative of normal muscle cell signaling were analyzed by western blotting in 4 persons in each group. After dry immersion, percentage of muscle fibers containing type I MHC decreased by 6% (p<0.05) in group DI, but was not changed significantly in group DIS. Percentage of the type IIa fibers was significantly altered in none of the groups. Type I fiber CSA decreased by 24.4% (p<0.05) in group DI. No significant changes of type I fiber CSA were found in group DIS. CSA of the type IIa fibers significantly altered in none of the groups. The total protein concentration was found increased by 17.6% in group DI and by 21% in group DIS. The increased total protein content in group DI suggests a diminution of fiber CSA attributed to the loss of non-protein component of fibers. NOS1 decreased by 35.6% in group DI and increased by 58.1% in group DIS. We conclude that 7 days in dry immersion lead to reduction in the type I muscle fiber percentage, loss of the non-protein component and decline in NOS1. These changes were clearly prevented by the support stimulation protocol applied during the DI period.     

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from AMPK alpha2 knockout (KO), AMPK gamma3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser473 phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr172 (P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46) (P < 0.01) in WT, AMPK alpha2 KO, and AMPK gamma3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46). In conclusion, functional alpha2 and gamma3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.     

AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4.

This study was designed to determine whether the reductions in GLUT-4 seen in 3-day-denervated muscles can be prevented through chemical activation of 5'-AMP-activated protein kinase (AMPK). Muscle AMPK can be chemically activated in rats using subcutaneous injections with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). In this study, the tibial nerve was sectioned on one side; the other was sham operated but without nerve section. Acute injections of AICAR resulted in significantly increased AMPK activity in denervated gastrocnemius but not soleus muscles. Acetyl-CoA carboxylase activity, a reporter of AMPK activation, declined in both gastrocnemius and soleus in both denervated and contralateral muscles. Three days after denervation, GLUT-4 levels were significantly decreased by approximately 40% in gastrocnemius muscles and by approximately 30% in soleus muscles. When rats were injected with AICAR (1 mg/g body wt) for 3 days, the decline in GLUT-4 levels was prevented in denervated gastrocnemius muscles but not in denervated soleus muscles. The extent of denervation-induced muscle atrophy was similar in AICAR-treated vs. saline-treated rats. These studies provide evidence that some effects of denervation may be prevented by chemical activation of the appropriate signaling pathways.     

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.     

Ketogenic essential amino acids replacement diet ameliorated hepatosteatosis with altering autophagy-associated molecules

Ketogenic amino acid (KAA) replacement diet has been shown to cure hepatic steatosis, a serious liver disease associated with diverse metabolic defects. In this study, we investigated the effects of KAA replacement diet on nutrition sensing signaling pathway and analyzed whether induction of hepatic autophagy was involved. Mice are fed with high fat diet (HFD) or KAA replacement in high-fat diet (30% fat in food; HFD)-fed (HFD^KAAR) and sacrificed at 8, 12, 16 weeks after initiation of experimental food. Hepatic autophagy was analyzed in protein expression of several autophagy-associated molecules and in light chain-3 green fluorescent protein (LC-3 GFP) transgenic mice. HFD^KAAR showed increased AMP-activated protein kinase (AMPK) phosphorylation and enhanced liver kinase B1 (LKB1) expression compared to control HFD-fed mice. The KAA-HFD-induced activation of AMPK was associated with an increased protein expression of sirtuin 1 (Sirt1), decreased forkhead box protein O3a (Foxo3a) level, and suppression of mammalian target of rapamycin (mTOR) phosphorylation compared with the HFD-fed mice. The intervention study revealed that a KAA-replacement diet also ameliorated all the established metabolic and autophagy defects in the HFD-fed mice, suggesting that a KAA-replacement diet can be used therapeutically in established diseases. These results indicate that KAA replacement in food could be a novel strategy to combat hepatic steatosis and metabolic abnormalities likely involvement of an induction of autophagy.     

Diminished muscle growth in the obese Zucker rat following overload is associated with hyperphosphorylation of AMPK and dsRNA-dependent protein kinase

Previous data have suggested that insulin-resistant skeletal muscle may exhibit a diminished ability to undergo hypertrophy and that this result may be mediated, at least in part, from decrements in mammalian target of rapamycin (mTOR) signaling (Katta A, Kundla S, Kakarla SK, Wu M, Fannin J, Paturi S, Liu H, Addagarla HS, Blough ER. Am J Physiol Regul Integr Comp Physiol 299: R1666-R1675, 2010). Herein, we attempt to extend these observations by determining if this attenuation in muscle growth is associated with alterations in AMP-activated protein kinase (AMPK) signaling, an upstream mediator of mTOR, and changes in the activation of dsRNA-dependent protein kinase (PKR), which functions as an inhibitor of protein synthesis and potential mediator of protein degradation. Compared with that observed in lean Zucker (LZ) rats, the phosphorylation of AMPKα at Thr172 was higher after 3 wk of overload in the insulin-resistant obese Zucker (OZ) soleus (P < 0.05). This change in AMPKα phosphorylation was accompanied by increases in the amount of phosphorylated PKR (Thr446), elevations in the PKR-dependent phosphorylation of eukaryotic initiation factor (eIF)-2α (Ser51), augmented p38 MAP kinase (Thr180/Tyr182) phosphorylation, and increases in the amount of protein ubiquitination (P < 0.05). Taken together, these results suggest that the diminished hypertrophic response we observe in the OZ rat may be mediated, at least in part, by the hyperactivation of AMPK- and PKR-related signaling.     

Translation initiation factor eIF3a regulates glucose metabolism and cell proliferation via promoting small GTPase Rheb synthesis and AMPK activation

Eukaryotic translation initiation factor 3 subunit A (eIF3a), the largest subunit of the eIF3 complex, has been shown to be overexpressed in malignant cancer cells, potentially making it a proto-oncogene. eIF3a overexpression can drive cancer cell proliferation but contributes to better prognosis. While its contribution to prognosis was previously shown to be due to its function in suppressing synthesis of DNA damage repair proteins, it remains unclear how eIF3a regulates cancer cell proliferation. In this study, we show using genetic approaches that eIF3a controls cell proliferation by regulating glucose metabolism via the phosphorylation and activation of AMP-activated protein kinase alpha (AMPKα) at Thr¹⁷² in its kinase activation loop. We demonstrate that eIF3a regulates AMPK activation mainly by controlling synthesis of the small GTPase Rheb, largely independent of the well-known AMPK upstream liver kinase B1 and Ca²⁺/calmodulin-dependent protein kinase kinase 2, and also independent of mammalian target of rapamycin signaling and glucose levels. Our findings suggest that glucose metabolism in and proliferation of cancer cells may be translationally regulated via a novel eIF3a–Rheb–AMPK signaling axis.     

Diminished overload-induced hypertrophy in aged fast-twitch skeletal muscle is associated with AMPK hyperphosphorylation.

Skeletal muscle mass declines with age, as does the potential for overload-induced fast-twitch skeletal muscle hypertrophy. Because 5'-AMP-activated protein kinase (AMPK) activity is thought to inhibit skeletal muscle protein synthesis and may therefore modulate muscle mass and hypertrophy, the purpose of this investigation was to examine AMPK phosphorylation status (a marker of AMPK activity) and its potential association with the attenuated overload-induced hypertrophy observed in aged skeletal muscle. One-week overload of fast-twitch plantaris and slow-twitch soleus muscles was achieved in young adult (8 mo; n = 7) and old (30 mo; n = 7) Fischer344 x Brown Norway male rats via unilateral gastrocnemius ablation. Significant (P < or = 0.05) age-related atrophy (as measured by total protein content) was noted in plantaris and soleus control (sham-operated) muscles. In fast-twitch plantaris muscles, percent hypertrophy with overload was significantly attenuated with age, whereas AMPK phosphorylation status as determined by Western blotting [phospho-AMPK (Thr172)/total AMPK] was significantly elevated with age (regardless of loading status). There was also a main effect of loading on AMPK phosphorylation status in plantaris muscles (overload > control). Moreover, a strong and significant negative correlation (r = -0.82) was observed between AMPK phosphorylation status and percent hypertrophy in the overloaded plantaris muscles of all animals. In contrast to the plantaris, overload-induced hypertrophy of the slow-twitch soleus muscle was similar between ages, and AMPK phosphorylation in this muscle was also unaffected by age or overload. These data support the possibility that an age-related elevation in AMPK phosphorylation may partly contribute to the attenuated hypertrophic response observed with age in overloaded fast-twitch plantaris muscle.     

Passive stretch reduces calpain activity through nitric oxide pathway in unloaded soleus muscles

Unloading in spaceflight or long-term bed rest induces to pronounced atrophy of anti-gravity skeletal muscles. Passive stretch partially resists unloading-induced atrophy of skeletal muscle, but the mechanism remains elusive. The aims of this study were to investigate the hypotheses that stretch tension might increase protein level of neuronal nitric oxide synthase (nNOS) in unloaded skeletal muscle, and then nNOS-derived NO alleviated atrophy of skeletal muscle by inhibiting calpain activity. The tail-suspended rats were used to unload rat hindlimbs for 2 weeks, at the same time, left soleus muscle was stretched by applying a plaster cast to fix the ankle at 35° dorsiflexion. Stretch partially resisted atrophy and inhibited the decreased protein level and activity of nNOS in unloaded soleus muscles. Unloading increased frequency of calcium sparks and elevated intracellular resting and caffeine-induced Ca(2+) concentration ([Ca(2+)]i) in unloaded soleus muscle fibers. Stretch reduced frequency of calcium sparks and restored intracellular resting and caffeine-induced Ca(2+) concentration to control levels in unloaded soleus muscle fibers. The increased protein level and activity of calpain as well as the higher degradation of desmin induced by unloading were inhibited by stretch in soleus muscles. In conclusion, these results suggest that stretch can preserve the stability of sarcoplasmic reticulum Ca(2+) release channels which prevents the elevated [Ca(2+)]i by means of keeping nNOS activity, and then the enhanced protein level and activity of calpain return to control levels in unloaded soleus muscles. Therefore, stretch can resist in part atrophy of unloaded soleus muscles.     

Possible CaMKK-dependent regulation of AMPK phosphorylation and glucose uptake at the onset of mild tetanic skeletal muscle contraction

The Ca(2+)/calmodulin (CaM) competitive inhibitor KN-93 has previously been used to evaluate 5'-AMP-activated protein kinase (AMPK)-independent Ca(2+)-signaling to contraction-stimulated glucose uptake in muscle during intense electrical stimulation ex vivo. With the use of low-intensity tetanic contraction of mouse soleus and extensor digitorum longus (EDL) muscles ex vivo, this study demonstrates that KN-93 can potently inhibit AMPK phosphorylation and activity after 2 min but not 10 min of contraction while strongly inhibiting contraction-stimulated 2-deoxyglucose uptake at both the 2- and 10-min time points. These data suggest inhibition of Ca(2+)/CaM-dependent signaling events upstream of AMPK, the most likely candidate being the novel AMPK kinase CaM-dependent protein kinase kinase (CaMKK). CaMKK protein expression was detected in mouse skeletal muscle. Similar to KN-93, the CaMKK inhibitor STO-609 strongly reduced AMPK phosphorylation and activity at 2 min and less potently at 10 min. Pretreatment with STO-609 inhibited contraction-stimulated glucose uptake at 2 min in soleus, but not EDL, and in both muscles after 10 min. Neither KN-93 nor STO-609 inhibited 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside-stimulated glucose uptake, AMPK phosphorylation, or recombinant LKB1 activity, suggestive of an LKB1-independent effect. Finally, neither KN-93 nor STO-609 had effects on the reductions in glucose uptake seen in mice overexpressing a kinase-dead AMPK construct, indicating that the effects of KN-93 and STO-609 on glucose uptake require inhibition of AMPK activity. We propose that CaMKKs act in mouse skeletal muscle regulating AMPK phosphorylation and glucose uptake at the onset of mild tetanic contraction and that an intensity- and/or time-dependent switch occurs in the relative importance of AMPKKs during contraction.     

Regulation of M1 Muscarinic Receptor-Mediated Signaling in Intact Cells by Exogenous,but Not Endogenously Produced,Nitric Oxide

Regulation of nitric oxide (NO) formation is critical to ensure maintenance of appropriate cellular concentrations of this labile, signaling molecule. This study investigated the role exogenous and endogenously produced NO have in feeding back to regulate NO synthesis in intact cells. Two NO donors inhibited activation of neuronal NO synthase (nNOS) in response to the muscarinic receptor agonist carbachol in Chinese hamster ovary (CHO) cells stably transfected with the M₁ muscarinic receptor and nNOS. The presence of the NO scavenger [2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide · potassium salt] (C-PTIO) potentiated carbachol-induced activation of nNOS in transfected CHO cells. C-PTIO also potentiated nNOS activity in response to the Ca²⁺ ionophore ionomycin. In contrast, the NO scavenger oxyhemoglobin depressed carbachol- and ionomycin-induced NO formation. These discrepant results suggest that it is unlikely that endogenously produced NO induces feed back inhibition at the level of nNOS activation itself. Exogenous sources of NO inhibited carbachol-induced inositol phosphates formation. However, endogenously produced NO did not appear to feed back to regulate phosphoinositide hydrolysis as there was no difference in [³H]inositol phosphates formation between cells that do or do not express nNOS. There was also no change in carbachol-induced [³H]inositol phosphates formation in the presence or absence of a NOS inhibitor or the NO scavenger C-PTIO. A decrease in the carbachol-mediated transient Ca²⁺ peak was observed in cells that express nNOS as compared to cells lacking the enzyme, suggesting that endogenous NO might inhibit receptor mediated Ca²⁺ signaling. This conclusion, however, was not supported by the lack of ability of a NOS inhibitor to modulate carbachol-induced Ca²⁺ elevations. Taken together, these results highlight differences in the regulation of the nNOS activation cascade by endogenous vs. exogenous sources of NO.     

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.     

Exercise improves glucose uptake in murine myotubes through the AMPKα2-mediated induction of Sestrins

Exercise training increases insulin sensitivity. Over the past decades, considerable progress has been made in understanding the molecular basis for this important effect of physical exercise. However, the underlying mechanism is still not fully described. Recent studies have revealed that the stress responsive protein family Sestrins (SESNs) may play an important role in improving insulin sensitivity of skeletal muscle under exercise training. In this study, we aim to better understand the relationship between SESNs and AMPK in response to exercise training and the possible mechanism by which SESNs mediate glucose metabolism. We used wild type, AMPKα2^+/? and AMPKα2^?/? C57BL/6 mice to reveal the pathway by which 6?weeks of exercise training induced SESNs. We explored the mechanism through which SESNs regulated glucose metabolism in vitro by overexpressing or inhibiting SESNs, and inhibiting AMPK or autophagy in myotubes. We found that a 6-week exercise training regime improved oxidative metabolism, activated the insulin signaling pathway and increased the level of SESN2 and SESN3 in an AMPKα2-dependent manner. Overexpression of SESN3 or SESN2 and SESN3 together increased glucose uptake, activated the insulin signaling pathway, and promoted GLUT4 translocation in myotubes. Although inhibition of SESNs had no effect on glucose uptake, SESNs could reverse reduced glucose uptake following autophagy inhibition, and may be downstream effectors of AMPK responses in myotubes. Taken together our data show that SESNs are induced by AMPKα2 after exercise training, and SESNs, specifically SESN3, play a key role in exercise training-mediated glucose metabolism in skeletal muscle.