Autophagy In Skeletal Muscle Research Articles

The Effect of Moderate Intensity Continuous Training on Skeletal Muscle Autophagy Biomarkers in Elderly Male Rats

The Effect of Moderate Intensity Continuous Training on Skeletal Muscle Autophagy Biomarkers in Elderly Male Rats

Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle.

The role of autophagy and lysosomal degradation pathway in the regulation of skeletal muscle metabolism was previously studied. However, underlying molecular mechanisms are poorly understood. L-lactate which is utilized as an energetic substrate by skeletal muscle can also augment genes expression related to metabolism and up-regulate those being responsive to reactive oxygen species (ROS). Since ROS is the most important regulator of autophagy in skeletal muscle, we tested if there is a link between cellular lactate metabolism and autophagy in differentiated C2C12 myotubes and the gastrocnemius muscle of male wistar rats. C2C12 mouse skeletal muscle was exposed to 2, 6, 10, and 20mM lactate and evaluated for lactate autophagic effects. Lactate dose-dependently increased autophagy and augmented ROS generation in differentiated C2C12 myotubes. The autophagic effect of lactate deterred in N-acetylcysteine presence (NAC, a ROS scavenger) indicated lactate regulates autophagy with ROS participation. Lactate-induced up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) through ROS was required to regulate the autophagy by lactate. Further analysis about ERK1/2 up- and downstream indicated that lactate regulates autophagy through ROS-mediated the activation of ERK1/2/mTOR/p70S6K pathway in skeletal muscle. The in vitro effects of lactate on autophagy also occurred in the gastrocnemius muscle of male Wistar rats. In conclusion, we provided the lactate-associated regulation evidence of autophagy in skeletal muscle by activating ROS-mediated ERK1/2/mTOR/p70S6K pathway. Since the increase in cellular lactate concentration is a hallmark of energy deficiency, the results provide insight into a skeletal muscle mechanism to fulfill its enhanced energy requirement.

Angiotensin-(1-7) Prevents Lipopolysaccharide-Induced Autophagy via the Mas Receptor in Skeletal Muscle.

Skeletal muscle atrophy, which occurs in lipopolysaccharide (LPS)-induced sepsis, causes a severe muscle function reduction. The increased autophagy contributes to sepsis-induced skeletal muscle atrophy in a model of LPS injection, increasing LC3II/LC3I ratio, autophagy flux, and autophagosomes. Angiotensin-(1-7) (Ang-(1-7)) has anti-atrophic effects via the Mas receptor in skeletal muscle. However, the impact of Ang-(1-7) on LPS-induced autophagy is unknown. In this study, we determined the effect of Ang-(1-7) on sepsis-induced muscle autophagy. C57BL6 wild-type (WT) mice and mice lacking the Mas receptor (KO Mas) were injected with LPS together with the systemic administration of Ang-(1-7) to determine autophagy in skeletal muscle. We also evaluated autophagy and p38 and c-Jun N-terminal kinase (JNK)activation. Our results show that Ang-(1-7) prevents LPS-induced autophagy in the diaphragm, tibialis anterior, and gastrocnemius of WT mice, which is demonstrated by a decrease in the LC3II/LC3I ratio and mRNA levels of lc3b and ctsl. This effect was lost in KO Mas mice, suggesting the role of the Mas receptor. The results in C2C12 cells show that Ang-(1-7) reduces several LPS-dependent effects, such as autophagy (LC3II/LC3I ratio, autophagic flux, and autophagosomes), activation of p38 and JNK, B-cell lymphoma-2 (BCL2) phosphorylation, and disassembly of the Beclin1/BCL2 complex. In conclusion, Ang-(1-7)/Mas receptor reduces LPS-induced autophagy in skeletal muscle. In vitro assays indicate that Ang-(1-7) prevents LPS-induced autophagy and modifies the MAPK signaling and the disassembly of a complex involved at the beginning of autophagy.

Exercise-Induced Autophagy Suppresses Sarcopenia Through Akt/mTOR and Akt/FoxO3a Signal Pathways and AMPK-Mediated Mitochondrial Quality Control.

Exercise training is one of the most effective interventional strategies for sarcopenia in aged people. Nevertheless, the underlying mechanisms are not well recognized. Increasing studies have reported abnormal regulation of autophagy in aged skeletal muscle. Our current study aims to explore the efficiency of exercise interventions, including treadmill exercise, resistance exercise, alternating exercise with treadmill running and resistance exercise, and voluntary wheel running, on 21-month-old rats with sarcopenia and to detect the underlying mechanisms. Results showed the declined mass of gastrocnemius muscle with deficient autophagy and excessive apoptosis as a result of up-regulated Atrogin-1 and MuRF1, declined Beclin1 level and LC3-II/LC3-I ratio, accumulated p62, increased Bax, and reduced Bcl-2 levels, and also exhibited a defective mitochondrial quality control due to declined PGC-1α, Mfn2, Drp1, and PINK1 levels. However, 12-week exercise interventions suppressed the decline in mass loss of skeletal muscle, accompanied by down-regulated Atrogin-1 and MuRF1, increased Beclin1 level, improved LC3-II/LC3-I ratio, declined p62 level, and reduced Bax and increased Bcl-2 level, as well as enhanced mitochondrial function due to the increased PGC-1α, Mfn2, Drp1, and PINK1 levels. Moreover, exercise interventions also down-regulated the phosphorylation of Akt, mTOR, and FoxO3a, and up-regulated phosphorylated AMPK to regulate the functional status of autophagy and mitochondrial quality control. Therefore, exercise-induced autophagy is beneficial for remedying sarcopenia by modulating Akt/mTOR and Akt/FoxO3a signal pathways and AMPK-mediated mitochondrial quality control, and resistance exercise exhibits the best interventional efficiency.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Androgen depletion alters the diurnal patterns to signals that regulate autophagy in the limb skeletal muscle.

Hypogonadism contributes to limb skeletal muscle atrophy by increasing rates of muscle protein breakdown. Androgen depletion increases markers of the autophagy protein breakdown pathway in the limb muscle that persist throughout the diurnal cycle. However, the regulatory signals underpinning the increase in autophagy markers remain ill-defined. The purpose of this study was to characterize changes to autophagy regulatory signals in the limb skeletal muscle following androgen depletion. Male mice were subjected to a castration surgery or a sham surgery as a control. Seven weeks post-surgery, a subset of mice from each group was sacrificed every 4hr over a 24hr period. Protein and mRNA from the Tibialis Anterior (TA) were subjected to Western blot and RT-PCR. Consistent with an overall increase in autophagy, the phosphorylation pattern of Uncoordinated Like Kinase 1 (ULK1) (Ser555) was elevated throughout the diurnal cycle in the TA of castrated mice. Factors that induce the progression of autophagy were also increased in the TA following androgen depletion including an increase in the phosphorylation of c-Jun N-terminal Kinase (JNK) (Thr183/Tyr185) and an increase in the ratio of BCL-2 Associated X (BAX) to B-cell lymphoma 2 (BCL-2). Moreover, we observed an increase in the protein expression pattern of p53 and the mRNA of the p53 target genes Cyclin-Dependent Kinase Inhibitor 1A (p21) and Growth Arrest and DNA Damage Alpha (Gadd45a), which are known to increase autophagy and induce muscle atrophy. These data characterize novel changes toautophagy regulatory signals in the limb skeletal muscle following androgen deprivation.

Effects of acute and chronic strength training on skeletal muscle autophagy in frail elderly men and women.

Aging is associated with alterations in skeletal muscle autophagy, potentially affecting both muscle mass and quality in a negative manner. Strength training with protein supplementation has been reported to improve both muscle mass and quality in frail elderly individuals, but whether improvements are accompanied by alterations in protein quality control is not known. To address this issue, we investigated protein degradation markers in skeletal muscle biopsies (m. vastus lateralis) from twenty-four frail elderly men and women (86±7yr) after acute and chronic (10weeks) strength training with protein supplementation (ST+PRO) or protein supplementation alone (PRO). Acute increases in mRNA expression of genes related to the ubiquitin proteasome system (MuRF-1, MUSA1), autophagy (ATG7, LC3, p62), and mitochondrial fission (DRP1) were observed after the first, but not after the last training session in ST+PRO. Acute changes in gene expression were accompanied by changes in protein levels of both LC3-I and LC3-II. Hence, the acute training-induced activation of proteasomal degradation and autophagy seems to depend on training status, with activation in the untrained, but not trained state. The ten-week training intervention did not affect basal levels of autophagy mRNAs and proteins, and neither markers of the ubiquitin-proteasome system. This suggests that a relatively short period of strength training may not be sufficient to increase the basal rate of protein degradation in frail elderly.

Net protein balance correlates with expression of autophagy, mitochondrial biogenesis, and fat metabolism-related genes in skeletal muscle from older adults.

The mechanisms leading to sarcopenia, the main cause for frailty in older adults, are still unclear. Autophagy and the ubiquitin‐proteasome system (UPS) may play a role in mediating muscle protein breakdown related to sarcopenia. In addition to loss of muscle mass, compromised muscle performance observed in sarcopenic patients has been linked to muscle mitochondria dysfunction. Increased fat deposition and fat cell infiltration in muscle frequently seen in skeletal muscle of older adults may play an additional role for the pathogenesis of sarcopenia. Therefore, the first objective of this study was to understand differences in expression of genes related to autophagy, UPS, mitochondrial biogenesis, and fat metabolism in skeletal muscle of older adults compared with young adults. Our second objective was to determine the correlation between whole body protein kinetics (WBPK) and gene expression with age. Real‐time quantitative PCR was used to determine the relative expression of targeted genes, and hierarchical regression analysis was used to determine if age had a moderating effect on the correlation between expression of targeted genes and WBPK. Increases in the expression of autophagy‐related genes and fat metabolism‐related genes were observed in muscle of older adults compared with young adults. In addition, age enhanced the negative correlations between mitochondrial biogenesis genes and net protein balance. These results suggest that dysregulated gene expression of mitochondrial biogenesis could play a role in muscle loss in older adults.

Mitochondrial dysfunction in skeletal muscle of fukutin-deficient mice is resistant to exercise- and 5-aminoimidazole-4-carboxamide ribonucleotide-induced rescue.

What is the central question of this study? Does fukutin deficiency in skeletal muscle cause mitochondrial dysfunction, and if so, can AMP-activated protein kinase (AMPK) stimulation via 5-aminoimidazole-4-carboxamide ribonucleotide attenuate this through regulation of mitochondrial biogenesis and autophagy? What is the main finding and its importance? Mitochondrial dysfunction is associated with fukutin deficiency and AMPK stimulation may benefit muscle contractility to a greater extent than mitochondrial function. Disruptions in the dystrophin-glycoprotein complex (DGC) are clearly the primary basis underlying various forms of muscular dystrophies and dystroglycanopathies, but the cellular consequences of DGC disruption are still being investigated. Mitochondrial abnormalities are becoming an apparent consequence and contributor to dystrophy disease pathology. Herein, we demonstrate that muscle-specific deletion of the fukutin gene (Myf5/fktn-KO mice (Fktn KO)), a model of secondary dystroglycanopathy, results in ∼30% lower muscle strength (P<0.001) and 16% lower mitochondrial respiratory function (P=0.002) compared to healthy littermate controls (LM). We also observed ∼80% lower expression of the gene for peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) (P=0.004), a primary transcription factor for mitochondrial biogenesis, in Fktn KO mice that likely contributes to the mitochondrial defects. PGC-1α is post-translationally regulated via phosphorylation by AMP-activated protein kinase (AMPK). Treatment with the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to rescue mitochondrial deficits in Fktn KO mice (P=0.458) but did have beneficial (∼30% greater) effects on recovery of muscle contractility following injury in both LM and Fktn KO mice compared to saline treatment (P=0.006). The beneficial effects of AMPK stimulation via AICAR on muscle contractile function may be partially explained by AMPK's other role of regulating skeletal muscle autophagy, a cellular process critical for clearance of damaged and/or dysfunctional organelles. Two primary conclusions can be drawn from this data: (1) fukutin deletion produces intrinsic muscular metabolic defects that likely contribute to dystroglycanopathy disease pathology, and (2) AICAR treatment accelerates recovery of muscle contractile function following injury suggesting AMPK signalling as a possible target for therapeutic strategies.

Effect of exercise and vitamin D on insulin like growth factor-1 level and skeletal muscle autophagy in diabetic rats

Background: diabetic myopathy is one of chronic complications of diabetes mellitus, which is evidenced by muscle weakness and atrophy. In our research we aimed to evaluate the protective role of exercise and vitamin D on diabetic induced changes in the skeletal muscles. Methods: Fourty male Sprague Dawely rats were divided into control, diabetic, exercised diabetic, vitamin D treated diabetic and combined exercise and vitamin D diabetic rats. Fasting blood glucose, fasting insulin, HOMA-IR, serum IGF-1 level as well as the contractile properties of soleus and tibialis anterior muscles were measured. Also western blot analysis for measuring (LC3) and histopathological examination of both muscles were done. Results: in diabetic rats, fasting blood glucose, fasting insulin, HOMA-IR and LC3 (microtubule associated protein-1 light chain-3) expression in muscles were increased whereas serum IGF-1 level was decreased and the contractile properties of skeletal muscles were deteriorated. Vitamin D treatment decreased fasting blood glucose, fasting insulin, HOMA-IR and expression of LC3 but it increased serum IGF-1 with improvement in the skeletal muscles contractility. Also exercised diabetic rats revealed improvement in all biochemical parameters, autophagy marker expression with further improvement in the skeletal muscles contractility. Combining exercise and vitamin D treatment showed a more improvement in all studied parameters and the histopathological examination confirmed our results. Conclusion: regular exercise and vitamin D protect the muscles from diabetic induced lesions by improving glycemic state, serum level of IGF-1and suppressing autophagy which finally reflected on the contractile parameters of the muscles.

Abstract MP101: Functional Maintenance of the Sarcomere Requires Bag3-dependent Autophagy

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone highly expressed in the heart. Clinical studies show BAG3 haploinsufficiency and mutations are associated with heart failure (HF). However, these studies are largely observational and fail to go beyond the observed phenotype and study mechanism. One study in neonatal myocytes suggests a role for BAG3 in structural maintenance of the sarcomere. However, the structural and functional significance of BAG3 in adult myocytes is not known. We found that myofilament BAG3 expression decreases in human heart failure and is associated with impaired myofilament force-generating capacity (F max ). To assess whether rescuing BAG3 levels could restore function, we used a mouse model of HF and treated with BAG3 gene therapy via AAV9. Myofilament function was assessed in skinned cardiomyocytes by force-calcium relationship. HF mice experienced a reduction in F max , but this was fully restored to sham levels by BAG3 gene therapy.To explore mechanism, we used mass spectrometry to identify the BAG3-interactome at the myofilament and found heat shock proteins (HSP) 70 and B8 among the top hits. Immunofluorescence further showed that HSP70, B8, and BAG3 each localized to the sarcomere z-disk. This BAG3-Hsp complex had previously been described to promote ubiquitin-dependent autophagy in skeletal muscle. Notably, in both human HF samples and in the mouse HF model, myofilament ubiquitin levels increased significantly. However, BAG3 gene therapy in HF reduces ubiquitin levels and restores autophagy flux. This suggests that BAG3 serves a role in proteostasis for the sarcomere, which may explain the functional effect of BAG3 gene therapy.To further explore the impact of the BAG3/HSP complex on myofilament function, we used a mouse model with the P209L BAG3 mutation, which had previously been described to disrupt client processing by the complex. We found cardiomyocytes from P209L mice had significantly reduced F max and elevated myofilament ubiquitin levels, suggesting BAG3-dependent autophagy is required to maintain function. Together, our data identify a functional role for BAG3 at the sarcomere and indicate BAG3-mediated autophagy is an important mechanism for maintaining myofilament proteostasis.

Electroacupuncture alleviates polycystic ovary syndrome-like symptoms through improving insulin resistance, mitochondrial dysfunction, and endoplasmic reticulum stress via enhancing autophagy in rats

BackgroundElectroacupuncture (EA), a treatment derived from traditional Chinese medicine, can effectively improve hyperandrogenism and insulin resistance in patients with polycystic ovary syndrome (PCOS), however, its underlying mechanisms remain obscure. This study aimed to investigate whether EA could mitigate PCOS-like symptoms in rats by regulating autophagy.MethodsA rat model of PCOS-like symptoms was established by subcutaneous injection with dehydroepiandrosterone (DHEA), and then EA treatment at acupoints (ST29 and SP6) was carried out for 5 weeks. To inhibit autophagy in rats, intraperitoneal injection with 0.5 mg/kg 3-MA (an autophagy inhibitor) was performed at 30 min before each EA treatment.ResultsEA intervention alleviated PCOS-like symptoms in rats, which was partly counteracted by the combination with 3-MA. Moreover, DHEA-exposure-induced deficient autophagy in skeletal muscle was improved by EA treatment. EA-mediated improvements in insulin resistance, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in PCOS-like rats were counteracted by 3-MA pretreatment. Mechanically, EA attenuated autophagy deficiency-mediated insulin resistance in PCOS-like rats via inactivating mTOR/4E-BP1 signaling pathway.ConclusionsTaken together, our findings indicate that EA treatment ameliorates insulin resistance, mitochondrial dysfunction, and ER stress through enhancing autophagy in a PCOS-like rat model. Our study provides novel insight into the mechanisms underlying the treatment of EA in PCOS, which offers more theoretic foundation for its clinical application.

The Role Of Omi/htra2 During Autophagy In Skeletal Muscle Induced By Exercise Injury

Omi/HtrA2 is known to be a pro-apoptotic protein and also participates in the regulation of autophagy.Previous studies have found that centrifugal exercise can induce an increase in Omi/HtrA2 protein expression in damaged skeletal muscle cells.Omi/HtrA2-Hax1-Beclin1,as the newly discovered autophagy signaling pathway in cells since 2010, has not been reported whether can be specifically expressed in injured skeletal muscle. PURPOSE: This study was designed to explore the role of Omi/HtrA2 during autophagy in skeletal muscle induced by centrifugal exercise injury. METHODS: 168 SD rats were randomly divided into control group (C), dummy control group (D), ucf-101 group (U), exercise group (E) and exercise+ucf-101 group (EU).The U group was injected with ucf-101, a specific inhibitor of Omi/HtrA2;D injection of equal amount of normal saline;for group E with acute heavy load treadmill exercise, the slope was -16°, the speed was 16m/min, and the continuous exercise was 90 min;the EU group performed the same exercise after injection of ucf-101.Soleus muscle was taken from the rats at 0h, 12h, 24h, 48h and 72h after intervention.Ultrastructural changes of autophagosomes in soleus muscle cells were observed by TEM;the expressions of Omi/HtrA2, hax-1 and Beclin1 were detected by Western Blot;the position and content of LC3 were observed by immunofluorescence technique;The binding levels of Hax-1 and Beclin1 were determined by immunoprecipitation. RESULTS:The expression of Omi/HtrA2 protein was increased in group E(0h,2.056±0.114,P<0.05), and the binding of Hax-1 and Beclin1 protein was weakened(0h,0.805±0.095,P<0.05).The expression of Omi/HtrA2 protein was decreased in the U group (0h,0.406±0.178,P<0.01), and the binding of Hax-1 and Beclin1 protein was enhanced (0h,1.536±0.051,P<0.05;24h,8.000±0.197,P<0.01).Compared with group E, autophagy in EU group was decreased, Beclin1 expression was inhibited, and LC3 fluorescence intensity was decreased(Beclin1,U0=0.718±0.039,E0=1.870±0.291,EU0=0.871±0.096). CONCLUSION: A high-load centrifugal exercise can induce autophagy in skeletal muscle, which may enhance the cutting effect of Omi/HtrA2 on Hax-1 through exercise, reduce the binding inhibition level of the latter on Beclin1, and finally enhance autophagy.

Self-Eating for Muscle Fitness: Autophagy in the Control of Energy Metabolism.

Cellular processes that sense and transmit metabolic changes are crucial for adaptation to external signals. In this regard, autophagy provides energy upon nutrient deprivation and represents a quality control mechanism that eliminates damaged organelles or proteins. Here, we review recent findings on the metabolic pathways controlling autophagy in skeletal muscle, a plastic tissue that undergoes major changes in energy demands. We also analyze the implications of autophagy in the regulation of energy metabolism in muscle and how alterations in this process affect energy homeostasis at the whole-body level and the development of metabolic diseases and aging.

Effects of high-load exercise induced skeletal muscle injury on autophagy ultrastructure and Beclin1 and LC3-II / I in rats

To investigate the effects of high-load eccentric exercise on the ultrastructure of autophagy and the autophagy-related proteins Beclin1 and LC3II / I in rats. Forty-eight SD male rats were randomly divided into control group (C, n=8) and high-load eccentric exercise group (E, n=40) after adaptive training. Group E was run downhill for 90 minutes on the running platform, and soleus muscles were collected at 0, 12, 24, 48 and 72 hours after exercise. Transmission electron microscopy was used to observe the ultrastructural changes of skeletal muscle autophagosomes. Western blot was used to detect the expressions of Beclin1 and LC3II / I protein. Immunofluorescence was used to observe the localization and content of LC3. The number of soleus muscle autophagosomes in group E was increased at 0, 12 and 24 hours after exercise, and LC3 autophagic fluorescence was significantly increased (P＜0.01), while autophagic fluorescence at 48 hours after exercise was still increased significantly (P＜0.05). Beclin1 and LC3II / I expression levels were increased after high-load centrifugal intervention (P＜0.05), and were peaked at 12 h～24 h after exercise (P＜0.01), and fully recovered at 72 h after exercise. High-load eccentric exercise can induce ultrastructural changes in skeletal muscle autophagy and increase the expression of autophagy protein. The peak value appears at 12 hours after exercise. The above may be one of the reasons for the decline in skeletal muscle function caused by sports injury.

Comparison of intermittent fasting and exercise on skeletal muscle autophagy activation and fat reduction

To observe the effect of weight control of intermittent fasting in different time course (14 d, 28 d)and explore its effect on skeletal muscle mass and autophagy. Sixty SD rats (male) were randomly divided into sedentary group (Sed), intermittent fasting group (InF) and exercise group (Exe), 20 rats of each group, intervention duration is 14 d and 28 d. Animals of InF group were fasted every other day, animals of Exe group underwent aerobic exercise on treadmill, the weight was recorded every week. The body fat mass was recorded by DEXA and then the fat mass index was calculated. The wet weight of bilateral soleus muscle was weighed and wet weight index was calculated. Immunofluorescence was used to detect laminin and light chain 3 (LC3), which reflect muscle fiber cross-sectional area and autophagosome respectively. Transmission electron microscopy was used to observe the number and morphology of autophagosomes. The expression levels of autophagy related proteins Unc-51 like kinase 1 (ULK1), LC3, sequestosome1 protein (p62), AMP activated protein kinase (AMPKα) and p-AMPKα (Thr172) were detected by Western blot. ① From the 7th day of intervention, the body weight of rats in InF and Exe groups was significantly lower than that in Sed group, and the body weight of InF group was significantly lower than that in Exe group (P＜0.01). After 28 days of intervention, the fat mass index in InF and Exe groups was significantly lower than that in Sed group, the fat mass index of InF group was significantly lower than that in Exe group (P＜0.05).② After 28 days of intervention, the cross-sectional of muscle fibers in Exe group was significantly larger than that in Sed and InF groups (P＜0.01). ③The expressions of AMPKα, p-AMPKα (Thr172) and ULK1 in InF and Exe groups were significantly higher than those in Sed group (P＜0.05). However, at 14 days, only InF group showed the increase of LC3-II/LC3-I ratio and the decrease of p62 level (P＜0.05). The same indicators of Exe group were only significant changed at 28 d. ① Intermittent fasting is superior to exercise in controlling the growth of body weight and body fat in rats. ② In terms of skeletal muscle autophagy activation, the length of intervention required for intermittent fasting (14 days) is shorter than aerobic exercise (28 days).

Nrf2 deficiency promotes the increasing trend of autophagy during aging in skeletal muscle: a potential mechanism for the development of sarcopenia.

This study aims to explore the impact of nuclear factor erythroid 2-related factor 2 (Nrf2) deficiency on skeletal muscle autophagy and the development of sarcopenia. LC3b, P62, Bnip3, Lamp-1, and AMPK protein levels were measured in muscle from young, middle-aged, old Nrf2−/− (knockout, KO) mice and age-matched wild-type (WT) C57/BL6 mice. Autophagy flux was measured in young WT, young KO, old WT, old KO mice, using colchicine as autophagy inhibitor. There was a trend of higher accumulation of LC3b-II, P62, Bnip3, Lamp-1 induced by colchicine in old WT mice compared with young WT mice. Colchicine induced a significantly higher accumulation of LC3b-II, P62, Bnip3, Lamp-1 in KO mice compared with WT mice, both in the young and old groups. AMPK and reactive oxygen species (ROS) were unregulated following Nrf2 KO and increasing age, which was consistent with the increasing trend of autophagy flux following Nrf2 KO and increasing age. Nrf2 KO and increasing age caused decreased cross-sectional area of extensor digitorum longus and soleus muscles. We concluded that Nrf2 deficiency and increasing age may activate AMPK and ROS signals to cause excessive autophagy activation in skeletal muscle, which can be a potential mechanism for the development of sarcopenia.

Sex complicates the effect and treatment of heat stress

Global temperature is continuing to rise, and resultant heat stress (HS) poses a major threat to human health. Using large animal models, we have shown that 12 and 24 h, and 7 d of heat exposure induced oxidative stress and impaired autophagy in oxidative skeletal muscle but not glycolytic muscle. We and others have implicated mitochondrial dysfunction in this process. We hypothesized that treatment with a mitochondrially targeted antioxidant, MitoQ, would decrease oxidative stress and alleviate cellular dysfunction during HS. To address this hypothesis, 3 mo old castrated male pigs (59 ± 1.0m kg; n=8/group) were administered either placebo or MitoQ (40 mg/day) divided into two daily doses every 12 h beginning 48 h prior to environmental treatment and continuing through environmental exposure. Half of the placebo‐ and half of the MitoQ‐treated pigs were assigned to thermoneutral (TN; 20°C) or HS (35°C) conditions for 24 h. The red portion of the semitendinosus (STR) was collected for subsequent analysis. Unexpectedly, malondialdehyde (MDA) concentration, a marker of oxidative stress, was unaffected by treatment or environment. Additionally, LC3A/B‐I was decreased 0.5‐fold (p&lt;0.05) during HS, LC3A/B‐II was similar, and the ratio of LC3A/B‐II/I was increased 1.5‐fold (p&lt;0.05) in HS; p62 was similar between treatment groups. These data suggest increased degradation of autophagosomes in HS males, which may explain maintenance of redox balance but conflicts with previous findings in female pigs; a scenario which raises the possibility of a sex effect on the muscular response to HS. To probe this idea, we compared STR from these HS males to archived HS STR from female pigs treated with a similar experimental design and environmental protocol described above. We confirmed our previous findings of oxidative stress and dysfunctional autophagy in females, and maintenance of redox balance with increased degradation of autophagosomes in males. In total, these data suggest that muscle from female pigs is more susceptible to detrimental effects of environment‐induced hyperthermia than muscle from male pigs.Support or Funding InformationSupported by USDA grant 2017‐05931

Expression of genes related to autophagy and protein breakdown are positively correlated with protein synthesis and protein breakdown in skeletal muscle of healthy adults after a bout of resistance exercise

While it is well‐established that exercise increases muscle protein synthesis rate, it is still unclear that what role of protein breakdown plays in muscle after exercise. Therefore, the objective of this study was to determine if rates of protein synthesis and breakdown correlate with expression of genes related autophagy and protein degradation in skeletal muscle of healthy adults after a bout of resistance exercise. This is a secondary analysis of a previous published study ( Kim et al., 2016). In total, twenty‐three subjects were divided into two groups, rest (n = 12) and exercised (n = 11). Real‐time PCR was used for analyzing gene expression of baseline muscle samples from both groups, and student t test was used to compare significance (p &lt; 0.05) between two groups. Relationships between variables were assessed using Pearson’s Product‐Moment Correlation, and p value lower than 0.05 was considered as significant. Autophagy‐related genes p62, p53, ATG3, ATG5, LC3B, Beclin1, ATF4, and GADD54A, were significantly upregulated (p &lt; 0.05) in skeletal muscle from exercised subjects versus subjects at rest. Two genes involved in ubiquitin‐proteasome pathway, MuRF1 and Atrogin1, were also upregulated (p &lt; 0.05) after exercise. Neither protein synthesis nor protein breakdown rate was correlated with gene expression in skeletal muscle from subjects at rest. However, protein synthesis rate was positively correlated (p &lt; 0.05) with increased expression of p62, p53, ATG3, ATG5, ATG7, LC3B, Beclin1, ATF4, GADD54A, Atrogin‐1 and MuRF1 in exercised muscle. Protein breakdown rates were positively associated (p &lt; 0.05) with increased expression of ATF4, ATG5, and p62 in exercised skeletal muscle. In conclusion, the results provide preliminary evidence that genes related to autophagy and protein breakdown are upregulated after exercise in skeletal muscle from healthy adults and are positively correlated with muscle protein synthesis rate.Support or Funding InformationThis study is funded by an Arkansas Biosciences Institute grant, we would like to thank thegenerous contribution of muscle samples by Dr. Wolfe.

Skeletal Muscle ULK1 and ULK2 Jointly Couple Muscle Mass with Force and Are Required for Survival Under Low Nutrient Availability

The protein kinases Unc‐51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2) have evolved from a single yeast gene, autophagy related 1 (Atg1). Although ULK1 and ULK2 are thought to redundantly stimulate autophagy, other functions are emerging. Interestingly, skeletal muscle expresses both ULK1 and ULK2 at high levels (i.e., &gt;2‐fold the median expression of 91 mouse tissues and cell lines) suggesting an important, yet unknown role for their combined elevated expression in muscle. Therefore, to investigate the joint role of ULK1 and ULK2 on muscle homeostasis and whole‐body metabolism we generated mice with skeletal muscle‐specific knockout of these genes (ULK2/1 mKO). Here, we demonstrate that ULK2/1 mKO mice have larger muscles (~12%) that are actually weaker (~21% decrease in relative force) in comparison with wild type littermates. Loss of ULK2/1 in skeletal muscle also affects whole‐body metabolic homeostasis as indicated by decreased body fat mass (~20% at 7wk of age), attenuated fat mass gains with aging (~40% at 21mo. of age), and life‐threatening inability to maintain euglycemia under fasting conditions (~32% reduction of blood glucose compared to WT). Mechanistically, loss of ULK 2/1 not only impairs skeletal muscle autophagy but also AMPK function as indicated by accumulation of LC3, p62, NBR1, and ubiquitinated proteins, as well as reduced phosphorylation of ACC (a surrogate of AMPK activity), respectively. These results identify a previously unknown role for ULK2 and ULK1 in jointly coupling skeletal muscle size with force. They also reveal skeletal muscle ULK2 and ULK1 as fundamental proteins for survival under conditions of reduced nutrient availability.Support or Funding InformationThis work is funded by:AHA 16SDG30360001 (V.A.L.). and NASA Iowa Space Grant Graduate Fellowship Program