Effect of a Low-Protein Diet Supplemented with Ketoacids on Skeletal Muscle Atrophy and Autophagy in Rats with Type 2 Diabetic Nephropathy

A low-protein diet supplemented with ketoacids maintains nutritional status in patients with diabetic nephropathy. The activation of autophagy has been shown in the skeletal muscle of diabetic and uremic rats. This study aimed to determine whether a low-protein diet supplemented with ketoacids improves muscle atrophy and decreases the increased autophagy observed in rats with type 2 diabetic nephropathy. In this study, 24-week-old Goto-Kakizaki male rats were randomly divided into groups that received either a normal protein diet (NPD group), a low-protein diet (LPD group) or a low-protein diet supplemented with ketoacids (LPD+KA group) for 24 weeks. Age- and weight-matched Wistar rats served as control animals and received a normal protein diet (control group). We found that protein restriction attenuated proteinuria and decreased blood urea nitrogen and serum creatinine levels. Compared with the NPD and LPD groups, the LPD+KA group showed a delay in body weight loss, an attenuation in soleus muscle mass loss and a decrease of the mean cross-sectional area of soleus muscle fibers. The mRNA and protein expression of autophagy-related genes, such as Beclin-1, LC3B, Bnip3, p62 and Cathepsin L, were increased in the soleus muscle of GK rats fed with NPD compared to Wistar rats. Importantly, LPD resulted in a slight reduction in the expression of autophagy-related genes; however, these differences were not statistically significant. In addition, LPD+KA abolished the upregulation of autophagy-related gene expression. Furthermore, the activation of autophagy in the NPD and LPD groups was confirmed by the appearance of autophagosomes or autolysosomes using electron microscopy, when compared with the Control and LPD+KA groups. Our results showed that LPD+KA abolished the activation of autophagy in skeletal muscle and decreased muscle loss in rats with type 2 diabetic nephropathy.     

Autophagy inhibition induces atrophy and myopathy in adult skeletal muscles

Autophagy is required for cellular survival and for the clearance of damaged proteins and altered organelles. Excessive autophagy activation contributes to muscle loss in different catabolic conditions. However, the function of basal autophagy for homeostasis of skeletal muscle was unknown. To clarify this issue we have generated conditional and inducible knockout mice for the critical gene Atg7, to block autophagy specifically in skeletal muscle. Atg7 null muscles reveal an unexpected phenotype which is characterized by muscle atrophy, weakness and features of myofiber degeneration. Morphological, biochemical, and molecular analyses of our autophagy knockout mice show the presence of protein aggregates, abnormal mitochondria, accumulation of membrane bodies, sarcoplasmic reticulum distension, vacuolization, oxidative stress and apoptosis. Moreover, autophagy inhibition does not protect skeletal muscles from atrophy during denervation and fasting, but instead promotes greater muscle loss. In conclusion, autophagy plays a critical role for myofiber maintenance and its activation is crucial to avoid accumulation of toxic proteins and dysfunctional organelles that, in the end, would lead to atrophy and weakness.     

Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles

Autophagy is a catabolic process that provides the degradation of altered/damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagic flux is fundamental for the homeostasis of skeletal muscles in physiological conditions and in response to stress. Defective as well as excessive autophagy is detrimental for muscle health and has a pathogenic role in several forms of muscle diseases. Recently, we found that defective activation of the autophagic machinery plays a key role in the pathogenesis of muscular dystrophies linked to collagen VI. Impairment of the autophagic flux in collagen VI null (Col6a1–/–) mice causes accumulation of dysfunctional mitochondria and altered sarcoplasmic reticulum, leading to apoptosis and degeneration of muscle fibers. Here we show that physical exercise activates autophagy in skeletal muscles. Notably, physical training exacerbated the dystrophic phenotype of Col6a1–/– mice, where autophagy flux is compromised. Autophagy was not induced in Col6a1–/– muscles after either acute or prolonged exercise, and this led to a marked increase of muscle wasting and apoptosis. These findings indicate that proper activation of autophagy is important for muscle homeostasis during physical activity.     

Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice

Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1-null (col6a1^−/−) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1^−/− mice. Systemic administration of spermidine in col6a1^−/− mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.     

Autophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity

Physical activity has been recently documented to play a fundamental physiological role in the regulation of autophagy in several tissues. It has also been reported that autophagy is required for exercise itself and for training-induced adaptations in glucose homeostasis. These autophagy-mediated metabolic improvements are thought to be largely dependent on the activation of the metabolic sensor PRKAA1/AMPK. However, it is unknown whether these important benefits stem from systemic adaptations or are due solely to alterations in skeletal muscle metabolism. To address this we utilized inducible, muscle-specific, atg7 knockout mice that we have recently generated. Our findings indicate that acute inhibition of autophagy in skeletal muscle just prior to exercise does not have an impact on physical performance, PRKAA1 activation, or glucose homeostasis. However, we reveal that autophagy is critical for the preservation of mitochondrial function during damaging muscle contraction. This effect appears to be gender specific affecting primarily females. We also establish that basal oxidative stress plays a crucial role in mitochondrial maintenance during normal physical activity. Therefore, autophagy is an adaptive response to exercise that ensures effective mitochondrial quality control during damaging physical activity.     

Autophagy-Associated Atrophy and Metabolic Remodeling of the Mouse Diaphragm after Short-Term Intermittent Hypoxia

Background

Short-term intermittent hypoxia (IH) is common in patients with acute respiratory disorders. Although prolonged exposure to hypoxia induces atrophy and increased fatigability of skeletal muscle, the response to short-term IH is less well known. We hypothesized that the diaphragm and limb muscles would adapt differently to short-term IH given that hypoxia stimulates ventilation and triggers a superimposed exercise stimulus in the diaphragm.

Methods

We determined the structural, metabolic, and contractile properties of the mouse diaphragm after 4 days of IH (8 hours per day, 30 episodes per hour to a FiO2 nadir=6%), and compared responses in the diaphragm to a commonly studied reference limb muscle, the tibialis anterior. Outcome measures included muscle fiber size, assays of muscle proteolysis (calpain, ubiquitin-proteasome, and autophagy pathways), markers of oxidative stress and mitochondrial function, quantification of intramyocellular lipid and lipid metabolism genes, type I myosin heavy chain (MyHC) expression, and in vitro contractile properties.

Results

After 4 days of IH, the diaphragm alone demonstrated significant atrophy (30% decrease of myofiber size) together with increased LC3B-II protein (2.4-fold) and mRNA markers of the autophagy pathway (LC3B, Gabarapl1, Bnip3), whereas active calpain and E3 ubiquitin ligases (MuRF1, atrogin-1) were unaffected in both muscles. Succinate dehydrogenase activity was significantly reduced by IH in both muscles. However, only the diaphragm exhibited increased intramyocellular lipid droplets (2.5-fold) after IH, along with upregulation of genes linked to activated lipid metabolism. In addition, although the diaphragm showed evidence for acute fatigue immediately following IH, it underwent an adaptive fiber type switch toward slow type I MyHC-expressing fibers, associated with greater intrinsic endurance of the muscle during repetitive stimulation in vitro.

Conclusions

Short-term IH induces preferential atrophy in the mouse diaphragm together with increased autophagy and a rapid compensatory metabolic adaptation associated with enhanced fatigue resistance.     

Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF

Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation.     

Emerging role of autophagy in mediating widespread actions of ADIPOQ/adiponectin

Autophagy can dictate changes in cell metabolism via numerous mechanisms. ADIPOQ/adiponectin has been extensively characterized to have beneficial metabolic effects, both via INS/insulin-sensitizing and INS-independent actions. Our recent work examined the regulation of skeletal muscle autophagy by ADIPOQ and the functional significance. We showed that ADIPOQ directly stimulates autophagic flux in cultured skeletal muscle cells via an AMPK-dependent signaling pathway leading to phosphorylation of ULK1 (Ser555). Pharmacological inhibition of autophagy or overexpressing an inactive mutant of ATG5 to create an autophagy-deficient cell model reduces INS sensitivity. A high-fat diet (HFD) does not induce skeletal muscle autophagy in Adipoq knockout (Ad-KO) mice, whereas it does in wild-type (WT) mice, although ADIPOQ replenishment in Ad-KO mice stimulates autophagy. Changes in skeletal muscle autophagy correlate well with peripheral INS sensitivity and glucose metabolism. Thus, ADIPOQ stimulates autophagic flux in skeletal muscle, which likely represents one important mechanism mediating multiple favorable metabolic effects.     

Impaired autophagy contributes to muscle atrophy in glycogen storage disease type II patients

The autophagy-lysosome system is essential for muscle cell homeostasis and its dysfunction has been linked to muscle disorders that are typically distinguished by massive autophagic buildup. Among them, glycogen storage disease type II (GSDII) is characterized by the presence of large glycogen-filled lysosomes in the skeletal muscle, due to a defect in the lysosomal enzyme acid α-glucosidase (GAA). The accumulation of autophagosomes is believed to be detrimental for myofiber function. However, the role of autophagy in the pathogenesis of GSDII is still unclear. To address this issue we monitored autophagy in muscle biopsies and myotubes of early and late-onset GSDII patients at different time points of disease progression. Moreover we also analyzed muscles from patients treated with enzyme replacement therapy (ERT). Our data suggest that autophagy is a protective mechanism that is required for myofiber survival in late-onset forms of GSDII. Importantly, our findings suggest that a normal autophagy flux is important for a correct maturation of GAA and for the uptake of recombinant human GAA. In conclusion, autophagy failure plays an important role in GSDII disease progression, and the development of new drugs to restore the autophagic flux should be considered to improve ERT efficacy.     

Time-Point Dependent Activation of Autophagy and the UPS in SOD1G93A Mice Skeletal Muscle

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by a selective loss of motor neurons together with a progressive muscle weakness. Albeit the pathophysiological mechanisms of the disease remain unknown, growing evidence suggests that skeletal muscle can be a target of ALS toxicity. In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue. In this study we investigated the activation of autophagy and the UPS as well as apoptosis in the skeletal muscle from SOD1G93A mice along disease progression. Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages. The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy. Furthermore, no apoptosis activation was observed along disease progression. The combined data provided clear evidence for the first time that there is a time-point dependent activation of autophagy and UPS in the skeletal muscle from SOD1G93A mice.     

Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles

Autophagy is a catabolic process that provides the degradation of altered/damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagic flux is fundamental for the homeostasis of skeletal muscles in physiological conditions and in response to stress. Defective as well as excessive autophagy is detrimental for muscle health and has a pathogenic role in several forms of muscle diseases. Recently, we found that defective activation of the autophagic machinery plays a key role in the pathogenesis of muscular dystrophies linked to collagen VI. Impairment of the autophagic flux in collagen VI null (Col6a1–/–) mice causes accumulation of dysfunctional mitochondria and altered sarcoplasmic reticulum, leading to apoptosis and degeneration of muscle fibers. Here we show that physical exercise activates autophagy in skeletal muscles. Notably, physical training exacerbated the dystrophic phenotype of Col6a1–/– mice, where autophagy flux is compromised. Autophagy was not induced in Col6a1–/– muscles after either acute or prolonged exercise, and this led to a marked increase of muscle wasting and apoptosis. These findings indicate that proper activation of autophagy is important for muscle homeostasis during physical activity.     

Autophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity

Physical activity has been recently documented to play a fundamental physiological role in the regulation of autophagy in several tissues. It has also been reported that autophagy is required for exercise itself and for training-induced adaptations in glucose homeostasis. These autophagy-mediated metabolic improvements are thought to be largely dependent on the activation of the metabolic sensor PRKAA1/AMPK. However, it is unknown whether these important benefits stem from systemic adaptations or are due solely to alterations in skeletal muscle metabolism. To address this we utilized inducible, muscle-specific, atg7 knockout mice that we have recently generated. Our findings indicate that acute inhibition of autophagy in skeletal muscle just prior to exercise does not have an impact on physical performance, PRKAA1 activation, or glucose homeostasis. However, we reveal that autophagy is critical for the preservation of mitochondrial function during damaging muscle contraction. This effect appears to be gender specific affecting primarily females. We also establish that basal oxidative stress plays a crucial role in mitochondrial maintenance during normal physical activity. Therefore, autophagy is an adaptive response to exercise that ensures effective mitochondrial quality control during damaging physical activity.     

Myostatin induces autophagy in skeletal muscle in vitro

Myostatin is an important regulator of muscle mass that contributes to the loss of muscle mass in a number of chronic diseases. Myostatin is known to activate the expression of components of the ubiquitin-proteosomal pathway but its effect on the autophagic pathway is not known. We therefore analysed the effect of myostatin and TGF-β on autophagy in C2C12 cells by determining the effect of these proteins on LC3 processing, autophagosome formation and autophagy gene expression. Both myostatin and TGF-β increased LC3II expression and turnover as well as autophagosome formation (marked by the formation of puncta in LC3-GFP transfected cells). Myostatin also significantly increased the expression of ATG-4B and ULK-2 mRNA while TGF-β caused a trend towards an increase in these genes. We conclude that myostatin and TGF-β increase autophagy in skeletal muscle cells.     

Proteomic profiling reveals oxidative phosphorylation pathway is suppressed in longissimus dorsi muscle of weaned piglets fed low-protein diet supplemented with limiting amino acids

Feeding high-protein diets in animals can lead to a decrease of nitrogen utilization efficiency, and then promote the environmental pollution. Recently, more reports have demonstrated that lowering protein level in diets supplemented with specific amino acids can address these problems. However, the whole proteome alteration in the skeletal muscle of weaned piglets fed low-protein diets is poorly understood. Here, we applied the isobaric tags for relative and absolute quantification approach to investigate this alteration. We fed weaned piglets with normal protein diet (20% crude protein) and low-protein diet supplemented with lysine, methionine, threonine, and tryptophan (17% crude protein) for 25 days. Then proteomic profiling of skeletal muscles was performed. In total, 1354 proteins were quantified in the porcine skeletal muscle proteome. 132 proteins were identified as differentially expressed proteins between the two groups. Differentially expressed proteins were significantly enriched in various nutrient metabolism including lipid, carbohydrate, and amino acid metabolism. Interestingly, a total of 20 differentially expressed proteins, which are involved in the oxidative phosphorylation pathway, were all down-regulated by the low-protein diet feeding. Further immunoblotting confirmed the down-regulations of MT-ATP8, COX2, NDUFA6, and SDHB, four selected proteins among these 20 proteins. Meanwhile, the ATP level in the low-protein diet group was also reduced. These findings for the first time reveal that oxidative phosphorylation pathway is suppressed in longissimus dorsi muscle of weaned piglets fed low-protein diet supplemented with limiting amino acids, which may provide new insights into further formula design and the choice of limiting amino acids in diets.     

Exercise protects against doxorubicin-induced markers of autophagy signaling in skeletal muscle

Doxorubicin (DOX) is an effective antitumor agent used in cancer treatment. Unfortunately, DOX is also toxic to skeletal muscle and can result in significant muscle wasting. The cellular mechanism(s) by which DOX induces toxicity in skeletal muscle fibers remains unclear. Nonetheless, DOX-induced toxicity is associated with increased generation of reactive oxygen species, oxidative damage, and activation of the calpain and caspase-3 proteolytic systems within muscle fibers. It is currently unknown if autophagy, a proteolytic system that can be triggered by oxidative stress, is activated in skeletal muscles following DOX treatment. Therefore, we tested the hypothesis that systemic administration of DOX leads to increased expression of autophagy markers in the rat soleus muscle. Our results reveal that DOX administration results in increased muscle mRNA levels and/or protein abundance of several important autophagy proteins, including: Beclin-1, Atg12, Atg7, LC3, LC3II-to-LCI ratio, and cathepsin L. Furthermore, given that endurance exercise increases skeletal muscle antioxidant capacity and protects muscle against DOX-induced oxidative stress, we performed additional experiments to determine whether exercise training before DOX administration would attenuate DOX-induced increases in expression of autophagy genes. Our results clearly show that exercise can protect skeletal muscle from DOX-induced expression of autophagy genes. Collectively, our findings indicate that DOX administration increases the expression of autophagy genes in skeletal muscle, and that exercise can protect skeletal muscle against DOX-induced activation of autophagy.     

Increased autophagy accelerates colchicine-induced muscle toxicity

Colchicine treatment is associated with an autophagic vacuolar myopathy in human patients. The presumed mechanism of colchicine-induced myotoxicity is the destabilization of the microtubule system that leads to impaired autophagosome-lysosome fusion and the accumulation of autophagic vacuoles. Using the MTOR inhibitor rapamycin we augmented colchicine’s myotoxic effect by increasing the autophagic flux; this resulted in an acute myopathy with muscle necrosis. In contrast to myonecrosis induced by cardiotoxin, myonecrosis induced by a combination of rapamycin and colchicine was associated with accumulation of autophagic substrates such as LC3-II and SQSTM1; as a result, autophagic vacuoles accumulated in the center of myofibers, where LC3-positive autophagosomes failed to colocalize with the lysosomal protein marker LAMP2. A similar pattern of central LC3 accumulation and myonecrosis is seen in human patients with colchicine myopathy, many of whom have been treated with statins (HMGCR/HMG-CoA reductase inhibitors) in addition to colchicine. In mice, cotreatment with colchicine and simvastatin also led to muscle necrosis and LC3 accumulation, suggesting that, like rapamycin, simvastatin activates autophagy. Consistent with this, treatment of mice with four different statin medications enhanced autophagic flux in skeletal muscle in vivo. Polypharmacy is a known risk factor for toxic myopathies; our data suggest that some medication combinations may simultaneously activate upstream autophagy signaling pathways while inhibiting the degradation of these newly synthesized autophagosomes, resulting in myotoxicity.     

Impaired autophagy contributes to muscle atrophy in glycogen storage disease type II patients

The autophagy-lysosome system is essential for muscle cell homeostasis and its dysfunction has been linked to muscle disorders that are typically distinguished by massive autophagic buildup. Among them, glycogen storage disease type II (GSDII) is characterized by the presence of large glycogen-filled lysosomes in the skeletal muscle, due to a defect in the lysosomal enzyme acid α-glucosidase (GAA). The accumulation of autophagosomes is believed to be detrimental for myofiber function. However, the role of autophagy in the pathogenesis of GSDII is still unclear. To address this issue we monitored autophagy in muscle biopsies and myotubes of early and late-onset GSDII patients at different time points of disease progression. Moreover we also analyzed muscles from patients treated with enzyme replacement therapy (ERT). Our data suggest that autophagy is a protective mechanism that is required for myofiber survival in late-onset forms of GSDII. Importantly, our findings suggest that a normal autophagy flux is important for a correct maturation of GAA and for the uptake of recombinant human GAA. In conclusion, autophagy failure plays an important role in GSDII disease progression, and the development of new drugs to restore the autophagic flux should be considered to improve ERT efficacy.     

Pre- and postnatal protein undernutrition increases hepatic carnitine palmitoyltransferase I activity and decreases enzyme sensitivity to inhibitors in the suckling rat.

Rats were pair-fed isocaloric diets containing either 25% (control diet) or 6% protein (low-protein diet) during the 5 weeks prior to conception and through the gestation and lactation periods; then, carnitine palmitoyltransferase I (CPT-I) activity was determined in liver and skeletal muscle mitochondria isolated from the corresponding pups. Maternal protein undernutrition increased the activity of hepatic CPT-I all along the suckling period, whereas the activity of the skeletal muscle enzyme was unaffected. Moreover, the sensitivity of hepatic CPT-I to inhibition by both malonyl-CoA and 4-hydroxyphenylglyoxylate was decreased in the low-protein group. These alterations in the properties of hepatic CPT-I may be involved in the appearance of hyperketonemia in the rat pup upon maternal administration of low-protein diets.     

The role of autophagy in the pathogenesis of glycogen storage disease type II (GSDII)

Regulated removal of proteins and organelles by autophagy-lysosome system is critical for muscle homeostasis. Excessive activation of autophagy-dependent degradation contributes to muscle atrophy and cachexia. Conversely, inhibition of autophagy causes accumulation of protein aggregates and abnormal organelles, leading to myofiber degeneration and myopathy. Defects in lysosomal function result in severe muscle disorders such as Pompe (glycogen storage disease type II (GSDII)) disease, characterized by an accumulation of autophagosomes. However, whether autophagy is detrimental or not in muscle function of Pompe patients is unclear. We studied infantile and late-onset GSDII patients and correlated impairment of autophagy with muscle wasting. We also monitored autophagy in patients who received recombinant α-glucosidase. Our data show that infantile and late-onset patients have different levels of autophagic flux, accumulation of p62-positive protein aggregates and expression of atrophy-related genes. Although the infantile patients show impaired autophagic function, the late-onset patients display an interesting correlation among autophagy impairment, atrophy and disease progression. Moreover, reactivation of autophagy in vitro contributes to acid α-glucosidase maturation in both healthy and diseased myotubes. Together, our data suggest that autophagy protects myofibers from disease progression and atrophy in late-onset patients.