Resistance exercise preconditioning prevents disuse muscle atrophy by inhibiting apoptosis and protein degradation via SESN2 in C57BL/6J mice.

AIM: To compare the effects of different exercise preconditioning in the context of skeletal muscle atrophy and to investigate the potential involvement of Sestrin2 (SESN2), a stress-inducible protein that can be regulated by exercise, in exercise preconditioning on preventing disuse muscle atrophy. METHODS: Eight-week-old male C57BL/6J mice were randomly assigned to sedentary groups (SD), aerobic exercise groups (AE), resistance exercise groups (RE), and combined exercise groups (CE) with or without 7 days of immobilization. The duration of the exercise intervention was 10 weeks. The effects of different exercise preconditioning to prevent muscle atrophy were analyzed by evaluating skeletal muscle function and mass. Additionally, to investigate the potential underlying mechanism of exercise-induced protection of skeletal muscle, wild-type and SESN2--/-- mice were randomly divided into sedentary group and resistance exercise preconditioning group. C2C12 cells were treated with SESN2 adenoviruses and MK2206 (an AKT inhibitor) for 48 h to elucidate the underlined mechanism. RESULTS: RE was more effective in preserving skeletal muscle function, muscle mass and maintaining skeletal muscle protein homeostasis than AE and CE under immobilized condition. Importantly, exercise performance, muscle mass to body weight ratio, and the cross-sectional area of muscle fibers were significantly lower in SESN2-/- mice than wild-type mice after resistance exercise preconditioning. Mechanistically, the absence of SESN2 led to activation of the ubiquitin-proteasome system and induction of apoptosis. In vitro experiments showed that MK2206 treatment mitigated the regulatory effects of overexpression-SESN2 on protein hydrolysis and apoptosis. CONCLUSION: RE was more effective than AE or CE in preventing disuse muscle atrophy. SESN2 mediated the protective effects of resistance exercise preconditioning on skeletal muscle atrophy.

Resistance exercise alleviates dexamethasone-induced muscle atrophy via Sestrin2/MSTN pathway in C57BL/6J mice.

AIM: It has long been recognized that resistance exercise can substantially increase skeletal muscle mass and strength, but whether it can protect against glucocorticoid-induced muscle atrophy and its potential mechanism is yet to be determined. This study aimed to investigate the protective effects of resistance exercise in dexamethasone-induced muscle atrophy and elucidate the possible function of exercise-induced protein Sestrin2 in this process. METHODS: Eight-week-old male C57BL/6J mice carried out the incremental mouse ladder exercise for 11 weeks. Two weeks before the end of the intervention, mice were daily intraperitoneally injected with dexamethasone. Body composition, muscle mass, and exercise performance were examined to evaluate muscle atrophy. In vitro, C2C12 cells were used for RT-qPCR, Western Blot, and immunofluorescence experiments to elucidate the potential mechanism. RESULTS: Our results showed that long-term resistance exercise is an effective intervention for dexamethasone-induced muscle atrophy. We also found that Sestrin2 plays a vital role in dexamethasone-induced muscle atrophy. In both animal (P = .0006) and cell models (P = .0266), dexamethasone intervention significantly reduced the protein expression of Sestrin2, which was increased (P = .0112) by resistance exercise. Inversely, overexpression of Sestrin2 improved (P < .0001) dexamethasone-induced myotube cell atrophy by reducing the activation of the ubiquitin-proteasome pathway via inhibiting Forkhead box O3 (FoxO3a) and myostatin (MSTN)/small mother against decapentaplegic (Smad) signaling pathways. CONCLUSION: Taken together, our results indicated that Sestrin2 may serve as an effective molecule that mimics the protective effect of resistance exercise on dexamethasone-induced muscle atrophy.

Globular adiponectin ameliorates insulin resistance in skeletal muscle by enhancing the LKB1-mediated AMPK activation via SESN2.

Adiponectin has been demonstrated to be a mediator of insulin sensitivity; however, the underlined mechanisms remain unclear. SESN2 is a stress-inducible protein that phosphorylates AMPK in different tissues. In this study, we aimed to validate the amelioration of insulin resistance by globular adiponectin (gAd) and to reveal the role of SESN2 in the improvement of glucose metabolism by gAd. We used a high-fat diet-induced wild-type and SESN2-/- C57BL/6J insulin resistance mice model to study the effects of six-week aerobic exercise or gAd administration on insulin resistance. In vitro study, C2C12 myotubes were used to determine the potential mechanism by overexpressing or inhibiting SESN2. Similar to exercise, six-week gAd administration decreased fasting glucose, triglyceride and insulin levels, reduced lipid deposition in skeletal muscle and reversed whole-body insulin resistance in mice fed on a high-fat diet. Moreover, gAd enhanced skeletal muscle glucose uptake by activating insulin signaling. However, these effects were diminished in SESN2-/- mice. We found that gAd administration increased the expression of SESN2 and Liver kinase B1 (LKB1) and increased AMPK-T172 phosphorylation in skeletal muscle of wild-type mice, while in SESN2-/- mice, LKB1 expression was also increased but the pAMPK-T172 was unchanged. At the cellular level, gAd increased cellular SESN2 and pAMPK-T172 expression. Immunoprecipitation experiment suggested that SESN2 promoted the formation of complexes of AMPK and LKB1 and hence phosphorylated AMPK. In conclusion, our results revealed that SESN2 played a critical role in gAd-induced AMPK phosphorylation, activation of insulin signaling and skeletal muscle insulin sensitization in mice with insulin resistance.

Aerobic exercise ameliorates insulin resistance in C57BL/6 J mice via activating Sestrin3.

Skeletal muscle insulin resistance (IR) is closely linked to hyperglycemia and metabolic disorders. Regular exercise enhances insulin sensitivity in skeletal muscle, but its underlying mechanisms remain unknown. Sestrin3 (SESN3) is a stress-inducible protein that protects against obesity-induced hepatic steatosis and insulin resistance. Regular exercise training is known to increase SESN3 expression in skeletal muscle. The purpose of this study was to explore whether SESN3 mediates the metabolic effects of exercise in the mouse model of high-fat diet (HFD)-induced IR. SESN3-/- mice exhibited severer body weight gain, ectopic lipid accumulation, and dysregulation of glucose metabolism after long-term HFD feeding compared with the wild-type (WT) mice. Moreover, we found that SESN3 deficiency weakened the effects of exercise on reducing serum insulin levels and improving glucose tolerance in mice. Exercise training increased pAKT-S473 and GLUT4 expression, accompanied by enhanced pmTOR-S2481 (an indicator of mTORC2 activity) in WT quadriceps that were less pronounced in SESN3-/- mice. SESN3 overexpression in C2C12 myotubes further confirmed that SESN3 played an important role in skeletal muscle glucose metabolism. SESN3 overexpression increased the binding of Rictor to mTOR and pmTOR-S2481 in C2C12 myotubes. Moreover, SESN3 overexpression resulted in an elevation of glucose uptake and a concomitant increase of pAKT-S473 in C2C12 myotubes, whereas these effects were diminished by downregulation of mTORC2 activity. Taken together, SESN3 is a crucial protein in amplifying the beneficial effects of exercise on insulin sensitivity in skeletal muscle and systemic glucose levels. SESN3/mTORC2/AKT pathway mediated the effects of exercise on skeletal muscle insulin sensitivity.

Integration of ATAC-Seq and RNA-Seq Analysis to Identify Key Genes in the Longissimus Dorsi Muscle Development of the Tianzhu White Yak.

During the postnatal stages, skeletal muscle development undergoes a series of meticulously regulated alterations in gene expression. However, limited studies have employed chromatin accessibility to unravel the underlying molecular mechanisms governing muscle development in yak species. Therefore, we conducted an analysis of both gene expression levels and chromatin accessibility to comprehensively characterize the dynamic genome-wide chromatin accessibility during muscle growth and development in the Tianzhu white yak, thereby elucidating the features of accessible chromatin regions throughout this process. Initially, we compared the differences in chromatin accessibility between two groups and observed that calves exhibited higher levels of chromatin accessibility compared to adult cattle, particularly within ±2 kb of the transcription start site (TSS). In order to investigate the correlation between alterations in chromatin accessible regions and variations in gene expression levels, we employed a combination of ATAC-seq and RNA-seq techniques, leading to the identification of 18 central transcriptional factors (TFs) and 110 key genes with significant effects. Through further analysis, we successfully identified several TFs, including Sp1, YY1, MyoG, MEF2A and MEF2C, as well as a number of candidate genes (ANKRD2, ANKRD1, BTG2 and LMOD3) which may be closely associated with muscle growth and development. Moreover, we constructed an interactive network program encompassing hub TFs and key genes related to muscle growth and development. This innovative approach provided valuable insights into the molecular mechanism underlying skeletal muscle development in the postnatal stages of Tianzhu white yaks while also establishing a solid theoretical foundation for future research on yak muscle development.

Combined cell grafting and VPA administration facilitates neural repair through axonal regeneration and synaptogenesis in traumatic brain injury.

Neuronal regeneration and functional recovery are severely compromised following traumatic brain injury (TBI). Treatment options, including cell transplantation and drug therapy, have been shown to benefit TBI, although the underlying mechanisms remain elusive. In this study, neural stem cells (NSCs) are transplanted into TBI-challenged mice, together with olfactory ensheathing cells (OECs) or followed by valproic acid (VPA) treatment. Both OEC grafting and VPA treatment facilitate the differentiation of NSCs into neurons (including endogenous and exogenous neurons) and significantly attenuate neurological functional defects in TBI mice. Combination of NSCs with OECs or VPA administration leads to overt improvement in axonal regeneration, synaptogenesis, and synaptic plasticity in the cerebral cortex in TBI-challenged mice, as shown by retrograde corticospinal tract tracing, electron microscopy, growth-associated protein 43 (GAP43), and synaptophysin (SYN) analyses. However, these beneficial effects of VPA are reversed by local delivery of N-methyl-D-aspartate (NMDA) into tissues surrounding the injury epicenter in the cerebral cortex, accompanied by a pronounced drop in axons and synapses in the brain. Our findings reveal that increased axonal regeneration and synaptogenesis evoked by cell grafting and VPA fosters neural repair in a murine model of TBI. Moreover, VPA-induced neuroprotective roles are antagonized by exogenous NMDA administration and its concomitant decrease in the number of neurons of local brain, indicating that increased neurons induced by VPA treatment mediate axonal regeneration and synaptogenesis in mice after TBI operation. Collectively, this study provides new insights into NSC transplantation therapy for TBI.

Exercise improves high-fat diet-induced metabolic disorder by promoting HDAC5 degradation through the ubiquitin-proteasome system in skeletal muscle.

Histone deacetylase 4/5 (HDAC4/5) are essential for regulating metabolic gene expression; AMPKα2 regulates HDAC4/5 activity and the expression of MuRF1 during exercise. In this study, we used wild-type and AMPKα2-/- mice to explore the potential regulatory relationship between AMPKα2 and HDAC4/5 expression during exercise. Firstly, we fed C57BL/6J mice with high-fat diet for 8 weeks to assess the effects of high-fat diet on skeletal muscle metabolism and HDAC4/5 expression. We then performed a 6-week treadmill exercise on both wild-type and AMPKα2-/- mice. After exercise, the expressions of HDAC4/5 were examined in both gastrocnemius and soleus. The citrate synthase activity and proteins involved in skeletal muscle oxidative process were assessed. To determine the relationship of HDAC4/5 and skeletal muscle oxidative capacity, citrate synthase activity was assessed after silencing HDAC4/5. Moreover, HDAC5 ubiquitination and the association of MuRF1 to HDAC5 were also investigated. Our results showed that 6-week exercise increased the skeletal muscle oxidative capacity and decreased HDAC4/5 expression only in soleus. HDAC5 silencing increased C2C12 cell oxidative capacity. Proteasome inhibition by MG132 abolished exercise-induced HDAC5 degradation mediated by MuRF1-ubiquitin-proteasome system. However, the ubiquitin-proteasome system (UPS) did not dominantly account for exercise-induced HDAC4 degradation. Exercise upregulated MuRF1-HDAC5 association in wild-type mice but not in AMPKα2-/- mice. Our results revealed that 6-week exercise increased the skeletal muscle oxidative capacity and promoted HDAC5 degradation in soleus through the UPS, MuRF1-mediated HDAC5 ubiquitination. Although AMPKα2 played a partial role in regulating MuRF1 expression and HDAC5 ubiquitination, exercise-induced HDAC5 degradation did not fully depend on AMPKα2.

Exercise protects intestinal epithelial barrier from high fat diet- induced permeabilization through SESN2/AMPKα1/HIF-1α signaling.

Over-nutrition and a sedentary lifestyle are associated with increased intestinal permeability. This condition promotes obesity and associated metabolic disorders. Sestrin2 (SESN2) is a stress-inducible protein thought to promote the survival and recovery of epithelial cells and act as a positive regulator in exercise-induced improvements of glycolipid metabolism. Here we aimed to test the hypothesis that chronic exercise can protect intestinal barrier function against high-fat diet induced permeabilization through SESN2. WT and SESN2-/- mice were randomly assigned to five groups, fed with either normal chow or high fat diet (HFD), and provided with or without exercise training for 15-week. Metabolic parameters, fecal microbiota composition, and intestinal barrier integrity were assessed. The role of the gut microbiota was investigated by administering a mixture of broad-spectrum antibiotics (ABX). Fifteen-week HFD feeding induced dysmetabolism, dysbiosis and gut barrier dysfunctions in the WT mice. These effects were exaggerated in SESN2-/- mice. Chronic aerobic exercise significantly reversed HFD-induced pathologic changes, while SESN2 ablation weakened the protective effects of exercise. ABX did not abolish the differences in gut barrier function between WT and SESN2-/- mice. We speculated that SESN2 may protect intestinal integrity partly independent of gut microbiome. Combining ex vivo and in vivo approaches, we demonstrated that SESN2/pAMPK-Thr172/HIF-1α pathway may play an important role in exercise- improved intestinal permeability. Taken together, our study demonstrated that HFD and SESN2-KO have synergistic effects on intestinal homeostasis. SESN2 is crucial in exercise-improved intestinal permeability.

Alterations of Lysine Acetylation Profile in Murine Skeletal Muscles Upon Exercise.

Objective: Regular exercise is a powerful tool that enhances skeletal muscle mass and strength. Lysine acetylation is an important post-translational modification (PTM) involved in a broad array of cellular functions. Skeletal muscle protein contains a considerable number of lysine-acetylated (Kac) sites, so we aimed to investigate the effects of exercise-induced lysine acetylation on skeletal muscle proteins. Methods: We randomly divided 20 male C57BL/6 mice into exercise and control groups. After 6 weeks of treadmill exercise, a lysine acetylation proteomics analysis of the gastrocnemius muscles of mice was performed. Results: A total of 2,254 lysine acetylation sites in 693 protein groups were identified, among which 1,916 sites in 528 proteins were quantified. The enrichment analysis suggested that protein acetylation could influence both structural and functional muscle protein properties. Moreover, molecular docking revealed that mimicking protein deacetylation primarily influenced the interaction between substrates and enzymes. Conclusion: Exercise-induced lysine acetylation appears to be a crucial contributor to the alteration of skeletal muscle protein binding free energy, suggesting that its modulation is a potential approach for improving exercise performance.

Sestrin2 ablation attenuates the exercise-induced browning of white adipose tissue in C57BL/6J mice.

AIM: With exercise, white adipose tissues (WAT) are readily convertible to a "brown-like" state, altering from lipid-storing to energy-catabolizing function, which counteracts obesity and increases insulin sensitivity. Sestrin2 (SESN2) is a stress-inducible protein that can regulate the cold-induced increase of uncoupling protein 1 (UCP1), which is paramount for the thermogenic capacity of brown-like WAT. This study aimed to elucidate the necessity of SESN2 in mediating exercise-induced browning of WAT. METHODS: We used 8-week, male wild-type and SESN2 knockout C57BL/6J mice to explore the potential role of SESN2 in the exercise-induced WAT browning process. Over a 3-week intervention (sedentary versus treadmill exercise, normal chow versus 60% high-fat diet), we examined the exercise-induced alterations of the browning phenotype in different depots of white fat. In vitro, 3T3-L1 pre-adipocytes and primary adipocytes were used to determine the potential mechanism. RESULTS: Our data revealed that SESN2 was required for the exercise-induced subcutaneous WAT (scWAT) browning. This may be mediated by higher fibronectin type III domain containing 5 (FNDC5) contents in scWAT locally, rather than skeletal muscle FNDC5 expression and circulating serum irisin levels. SESN2 ablation significantly impaired the exercise-improved glucose metabolism, where browning of scWAT may serve as an essential pathway. Moreover, SESN2 ablation significantly attenuated the exercise-promoted respiratory exchange ratio and indexes of energy metabolism (oxygen uptake and energy expenditure). CONCLUSION: Taken together, our results provided evidence that SESN2 is a key integrating factor in driving the diverse metabolic benefits conferred by aerobic exercise.

Exercise improves lipid metabolism disorders induced by high-fat diet in a SESN2/JNK-independent manner.

SESN2 and JNK are emerging powerful stress-inducible proteins in regulating lipid metabolism. The aim of this study was to determine the underlying mechanism of SESN2/JNK signaling in exercise to improve lipid disorder induced by high-fat diet (HFD). Our data showed that HFD and SESN2 knockout resulted in abnormalities including elevated body weight, increased fat mass, serum total cholesterol, lipid biosynthesis related proteins, and a concomitant increase of pJNK-Thr183/Tyr185. The above changes were reversed by exercise training. SESN2 silencing or JNK inhibition in palmitate-treated C2C12 further confirmed that SESN2 and JNK play a vital role in lipid biosynthesis. Rescue experiment further demonstrated that SESN2 reduced lipid biosynthesis through inhibition of JNK. SESN2/JNK signaling axis regulates lipid biosynthesis in both animal and cell models with abnormalities of lipid metabolism induced by HFD or palmitate treatment. This study provided evidence that exercise ameliorated lipid metabolic disorder induced by HFD feeding or by SESN2 knockout. SESN2 may improve lipid metabolism through inhibition JNK expression in skeletal muscle cells, providing a molecular mechanism that may represent an attractive target for the treatment of lipid disorder. Novelty: Exercise improved lipid disorder induced by HFD feeding and SESN2 knockout. SESN2 and JNK play a vital role in lipid biosynthesis in vivo and in vitro. SESN2 suppressed JNK to improve lipid metabolism in skeletal muscle cells.

Aerobic Exercise Improves Mitochondrial Function in Sarcopenia Mice Through Sestrin2 in an AMPKα2-Dependent Manner.

Sarcopenia, the age-related loss of skeletal muscle mass and function, contributes to high morbidity and mortality in the older population. Regular exercise is necessary to avoid the initiation and progression of sarcopenia, in which the underlying molecular mechanism is still not clear. Our data revealed that the outcomes induced by sarcopenia, including muscle mass and strength loss, decreased cross-sectional area of gastrocnemius fiber, chronic inflammation, and increased dysfunctional mitochondria, were reversed by regulation exercise. Knockout or silencing of Sestrin2 (Sesn2) resulted in imbalanced mitochondrial fusion and fission, mitochondrial biogenesis, and mitophagy damage in vivo and in vitro, which was attenuated by aerobic exercise or overexpression of Sesn2. Moreover, we found that the effects of Sesn2 on mitochondrial function are dependent on AMP-activated protein kinase α2 (AMPKα2). This study indicates that aerobic exercise alleviates the negative effects resulting from sarcopenia via the Sesn2/AMPKα2 pathway and provides new insights into the molecular mechanism by which the Sesn2/AMPKα2 signaling axis mediates the beneficial impact of exercise on sarcopenia.

Scriptaid/exercise-induced lysine acetylation is another type of posttranslational modification occurring in titin.

Titin serves important functions in skeletal muscle during exercise, and posttranslational modifications of titin participate in the regulation of titin-based sarcomeric functions. Scriptaid has exercise-like effects through the inhibition of HDAC and regulatory acetylation of proteins. However, it remains mostly unclear if exercise could result in titin's acetylation and whether Scriptaid could regulate acetylation of titin. We treated C57BL/6 mice with 6-wk treadmill exercise and 6-wk Scriptaid administration to explore Scriptaid's effects on mice exercise capacity and whether Scriptaid administration/exercise could induce titin's acetylation modification. An exercise endurance test was conducted to explore their effects on mice exercise capacity, and proteomic studies were conducted with gastrocnemius muscle tissue of mice from different groups to explore titin's acetylation modification. We found that Scriptaid and exercise did not change titin's protein expression, but they did induce acetylation modification changes of titin. In total, 333 acetylated lysine sites were identified. Exercise changed the acetylation levels of 33 lysine sites of titin, whereas Scriptaid changed acetylation levels of 31 titin lysine sites. Exercise treatment and Scriptaid administration shared 11 lysine sites. In conclusion, Scriptaid increased exercise endurance of mice by increasing the time mice spent running to fatigue. Acetylation is a common type of posttranslational modification of titin, and exercise/Scriptaid changed the acetylation levels of titin and titin-interacting proteins. Most importantly, titin may be a mediator through which Scriptaid and exercise modulate the properties and functions of exercise-induced skeletal muscle at the molecular level.NEW & NOTEWORTHY Scriptaid administration increased mouse exercise endurance. Acetylation is another type of posttranslational modification of titin. Scriptaid/exercise changed acetylation levels of titin and titin-interacting proteins. Titin may mediate exercise-induced skeletal muscle properties and functions.

Effect of exercise and butyrate supplementation on microbiota composition and lipid metabolism.

The composition and activity of the gut microbiota depend on the host genome, nutrition, and lifestyle. Exercise and sodium butyrate (NaB) exert metabolic benefits in both mice and humans. However, the underlying mechanisms have not been fully elucidated. This study aimed to examine the effect of exercise training and dietary supplementation of butyrate on the composition of gut microbiota and whether the altered gut microbiota can stimulate differential production of short-chain fatty acids (SCFAs), which promote the expression of SESN2 and CRTC2 to improve metabolic health and protect against obesity. C57BL/6J mice were used to study the effect of exercise and high-fat diet (HFD) with or without NaB on gut microbiota. Bacterial communities were assayed in fecal samples using pyrosequencing of 16S rRNA gene amplicons. Western blot was performed using relevant antibodies to detect the protein expressions in liver and HepG2 cell extracts. Exercise and butyrate administration significantly reversed metabolic dysfunctions induced by HFD (P < 0.05). The number of Firmicutes and the proportion of Firmicutes to Bacteroidetes order were predominant in all HFD groups (P = 0.001). Exercise and butyrate supplementation significantly inhibited the relative abundance of lipopolysaccharide-producing phyla (P = 0.001). SESN2 and CRTC2 expression in the liver of mice were significantly increased after exercise (P < 0.05) and/or supplementation of butyrate (P < 0.05). Exercise enhances butyrate-producing fecal bacteria and increases butyrate production and consequently improves lipid metabolism through the butyrate-SESN2/CRTC2 pathway. Excess butyrate may reduce the proportion of probiotics and reverse the metabolic effects.

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.

Exercise-induced GLUT4 transcription via inactivation of HDAC4/5 in mouse skeletal muscle in an AMPKα2-dependent manner.

Abnormal glucose metabolism induces various metabolic disorders such as insulin resistance and type 2 diabetes. Regular exercise improved glucose uptake and enhanced glucose oxidation by increasing GLUT4 transcription in skeletal muscle. However, the regulatory mechanisms of GLUT4 transcription in response to exercise are poorly understood. AMPK is a sensor of exercise and upstream kinase of class II HDACs that act as transcriptional repressors. We used 6-week treadmill exercise or one single-bout exercise wild type or AMPKα2-/- C57BL/6J mice to explore how HDACs regulate GLUT4 transcription and the underlying molecular mechanisms mediated by AMPK in the physiologic process of exercise. We demonstrate that regular physical exercise significantly enhanced GLUT4 transcription by inactivating HDAC4/5 in skeletal muscle by ChIP experiment. HDAC4 coordinately regulated with HDAC5 represses transcriptional activity of GLUT4 promoter in C2C12 myotubes by Luciferase assay. If either HDAC4 or HDAC5 is silenced via RNAi technology, the functional compensation by the other will occur. In addition, a single-bout of exercise decreased HDAC4/5 activity in skeletal muscle of wild type but not in AMPKα2-/- mice, suggesting an AMPKα2-dependent manner. Those findings provide new insight into the mechanisms responsible for AMPKα2-dependent regulation of GLUT4 transcription after exercise.

Sestrin 2 induces autophagy and attenuates insulin resistance by regulating AMPK signaling in C2C12 myotubes.

Impaired insulin-stimulated glucose uptake in skeletal muscle serves a critical role in the development of insulin resistance (IR), whereas the precise mechanism of the process remains unknown. Recently, the evolutionarily conserved, stress-inducible protein Sestrin2 (Sesn2) has been proposed to play a protective role against obesity-induced IR and diabetes. Activation of Sesn2 may activate AMP-activated protein kinase (AMPK) accompanied by suppression of mammalian target of rapamycin (mTOR), which may ultimately lead to autophagy induction. In view of the potential protective effects of autophagy on the physiological and the pathological regulatory processes via the regulation of energy homeostasis and metabolism, we investigated the effects of Sesn2 on the components of the insulin signaling pathway and insulin-stimulated glucose uptake in palmitate-induced insulin-resistant C2C12 myotubes. We showed that Sesn2 effectively restored the impaired insulin signaling. Moreover, autophagic activity decreased in response to palmitate, whereas Sesn2 significantly reversed the palmitate-suppressed autophagic signaling in this context. Our findings further revealed that Sesn2-induced autophagy contributed to restore the impaired insulin signaling through the activation of AMPK signal. Even in the presence of palmitate, Sesn2 up-regulation maintained insulin sensitivity and glucose metabolism via AMPK-dependent autophagic activation.

AMP-activated protein kinase-mediated expression of heat shock protein beta 1 enhanced insulin sensitivity in the skeletal muscle.

Activation of AMP-activated protein kinase (AMPK) has been viewed as an important target for the treatment of insulin resistance. Here, by proteomic analysis, we found that expression of heat shock protein beta-1 (HSPB1) was induced by the AMP analog 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside in palmitate-induced insulin-resistant cells. Overexpression of AMPKα2, or activation of AMPKα via acute/chronic exercise training, increased HSPB1 expression in the skeletal muscle. In AMPKα2-/- mice, HSPB1 expression was downregulated in the quadriceps muscles. Exercise did not increase HSPB1 expression in AMPKα2-/- mice. Moreover, overexpression of HSPB1 enhanced insulin sensitivity in palmitate-induced insulin-resistant cells and restored metabolic phenotypes associated with defective AMPK. Finally, HSPB1 was required for AMPK-mediated activation of the class IIa histone deacetylases and glucose uptake in the skeletal muscle. Our results demonstrate that AMPK-mediated HSPB1 expression enhanced insulin sensitivity in the skeletal muscle.

AMPK binds to Sestrins and mediates the effect of exercise to increase insulin-sensitivity through autophagy.

OBJECTIVE: Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying the metabolic effects of exercise are not completely understood. We know that autophagy plays an important role in maintaining cellular homeostasis. In this study, we aimed to better understand the metabolic effects of autophagy in skeletal muscle and the role of Sestrins/AMPK in mediating the beneficial effects of physical exercise through autophagy. MATERIAL/METHODS: We used wild type and AMPKα2(-/-) C57BL/6 mice as animal models to elucidate the role of AMPK in autophagy activation and the metabolism-promoting effects of acute and regular exercise. C2C12 myotubes were used to study the metabolic effects of autophagy in vitro. RESULTS: Autophagy promotes glucose uptake in skeletal muscle. A single bout of exercise increased the activity of autophagy in the skeletal muscle of wild type mice but not of AMPKα2(-/-) mice. This difference was associated with increased amounts of both Sestrin2 and Sestrin3 coimmunoprecipitated with AMPKα2. Long-term physical exercise significantly increased the basal level of muscle autophagy and protein expression of Sestrin2 and Sestrin3 in both normal chow and high-fat diet-fed mice. CONCLUSION: We believe that exercise-induced AMPK and Sestrins interaction may be involved in the beneficial metabolic effects of exercise by activating autophagy. This interaction provides a molecular mechanism that is a potential target in metabolic syndromes.