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1.  Exercise Protects against Diet-Induced Insulin Resistance through Downregulation of Protein Kinase Cβ in Mice 
PLoS ONE  2013;8(12):e81364.
Physical exercise is an important and effective therapy for diabetes. However, its underlying mechanism is not fully understood. Protein kinase Cβ (PKCβ) has been suggested to be involved in the pathogenesis of obesity and insulin resistance, but the role of PKCβ in exercise-induced improvements in insulin resistance is completely unknown. In this study, we evaluated the involvement of PKCβ in exercise-attenuated insulin resistance in high-fat diet (HFD)-fed mice. PKCβ-/- and wild-type mice were fed a HFD with or without exercise training. PKC protein expression, body and tissue weight change, glucose and insulin tolerance, metabolic rate, mitochondria size and number, adipose inflammation, and AKT activation were determined to evaluate insulin sensitivity and metabolic changes after intervention. PKCβ expression decreased in both skeletal muscle and liver tissue after exercise. Exercise and PKCβ deficiency can alleviate HFD-induced insulin resistance, as evidenced by improved insulin tolerance. In addition, fat accumulation and mitochondrial dysfunction induced by HFD were also ameliorated by both exercise and PKCβ deficiency. On the other hand, exercise had little effect on PKCβ-/- mice. Further, our data indicated improved activation of AKT, the downstream signal molecule of insulin, in skeletal muscle and liver of exercised mice, whereas PKCβ deficiency blunted the difference between sedentary and exercised mice. These results suggest that downregulation of PKCβ contributes to exercise-induced improvement of insulin resistance in HFD-fed mice.
doi:10.1371/journal.pone.0081364
PMCID: PMC3857188  PMID: 24349059
2.  Phospho-Bcl-xL(Ser62) plays a key role at DNA damage-induced G2 checkpoint 
Cell Cycle  2012;11(11):2159-2169.
Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G2 checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G2 arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G2 arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G2 checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G2 checkpoint, phospho-Bcl-xL(Ser62) stabilizes G2 arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.
doi:10.4161/cc.20672
PMCID: PMC3368867  PMID: 22617334
Bcl-xL; cdk1(cdc2); cell cycle checkpoint; DNA damage; nucleolus
3.  Autophagy in Myf5+ progenitors regulates energy and glucose homeostasis through control of brown fat and skeletal muscle development 
EMBO Reports  2013;14(9):795-803.
Autophagy in Myf5+ progenitors regulates energy and glucose homeostasis through control of brown fat and skeletal muscle development
Atg7 deletion in Myf5+ progenitors blocks autophagy in brown adipose tissue and muscle, affecting their differentiation and function. Knockout mice have higher body temperatures and glucose intolerance, underscoring the importance of autophagy in these processes.
Macroautophagy (MA) regulates cellular quality control and energy balance. For example, loss of MA in aP2-positive adipocytes converts white adipose tissue (WAT) into brown adipose tissue (BAT)-like, enhancing BAT function and thereby insulin sensitivity. However, whether MA regulates early BAT development is unknown. We report that deleting Atg7 in myogenic Myf5+ progenitors inhibits MA in Myf5-cell-derived BAT and muscle. Knock out (KO) mice have defective BAT differentiation and function. Surprisingly, their body temperature is higher due to WAT lipolysis-driven increases in fatty acid oxidation in ‘Beige' cells in inguinal WAT, BAT and muscle. KO mice also present impaired muscle differentiation, reduced muscle mass and glucose intolerance. Our studies show that ATG7 in Myf5+ progenitors is required to maintain energy and glucose homeostasis through effects on BAT and muscle development. Decreased MA in myogenic progenitors with age and/or overnutrition might contribute to the metabolic defects and sarcopenia observed in these conditions.
doi:10.1038/embor.2013.111
PMCID: PMC3790054  PMID: 23907538
Autophagy; Myf5+ progenitors; brown fat
4.  Exercise-induced muscle glucose uptake in mice with graded, muscle-specific GLUT-4 deletion 
Physiological Reports  2013;1(3):e00065.
To investigate the importance of the glucose transporter GLUT-4 for muscle glucose uptake during exercise, transgenic mice with skeletal muscle GLUT-4 expression approximately 30–60% of normal (CON) and approximately 5–10% of normal (KO) were generated using the Cre/Lox system and compared with wild-type (WT) mice during approximately 40 min of treadmill running (KO: 37.7 ± 1.3 min; WT: 40 min; CON: 40 min, P = 0.18). In WT and CON animals, exercise resulted in an overall increase in muscle glucose uptake. More specifically, glucose uptake was increased in red gastrocnemius of WT mice and in the soleus and red gastrocnemius of CON mice. In contrast, the exercise-induced increase in muscle glucose uptake in all muscles was completely abolished in KO mice. Muscle glucose uptake increased during exercise in both red and white quadriceps of WT mice, while the small increases in CON mice were not statistically significant. In KO mice, there was no change at all in quadriceps muscle glucose uptake. No differences in muscle glycogen use during exercise were observed between any of the groups. However, there was a significant increase in plasma glucose levels after exercise in KO mice. The results of this study demonstrated that a reduction in skeletal muscle GLUT-4 expression to approximately 10% of normal levels completely abolished the exercise-induced increase in muscle glucose uptake.
doi:10.1002/phy2.65
PMCID: PMC3835018  PMID: 24303141
exercise; GLUT4; muscle glucose uptake
5.  Exercise–induced BCL2–regulated autophagy is required for muscle glucose homeostasis 
Nature  2012;481(7382):511-515.
Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes1. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control2. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism3. Moreover, in animal models, autophagy protects against diseases such as cancer, neuro-degenerative disorders, infections, inflammatory diseases, ageing and insulin resistance4-6. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)- induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.
doi:10.1038/nature10758
PMCID: PMC3518436  PMID: 22258505
6.  Acute Exercise Induces FGF21 Expression in Mice and in Healthy Humans 
PLoS ONE  2013;8(5):e63517.
Fibroblast growth factor 21 (FGF21) plays an important role in the regulation of energy homeostasis during starvation and has an excellent therapeutic potential for the treatment of type 2 diabetes in rodents and monkeys. Acute exercise affects glucose and lipid metabolism by increasing glucose uptake and lipolysis. However, it is not known whether acute exercise affects FGF21 expression. Here, we showed that serum FGF21 level is increased in mice after a single bout of acute exercise, and that this is accompanied by increased serum levels of free fatty acid, glycerol and ketone body. FGF21 gene expression was induced in the liver but not in skeletal muscle and white adipose tissue of mice after acute exercise, and further, the gene expression levels of hepatic peroxisome proliferator-activated receptor α (PPARα) and activating transcription factor 4 (ATF4) were also increased. In addition, we observed increased FGF21 level in serum of healthy male volunteers performing a treadmill run at 50 or 80% VO2max. These results suggest that FGF21 may also be associated with exercise-induced lipolysis in addition to increased catecholamines and reduced insulin.
doi:10.1371/journal.pone.0063517
PMCID: PMC3646740  PMID: 23667629
7.  Muscle-Specific IRS-1 Ser→Ala Transgenic Mice Are Protected From Fat-Induced Insulin Resistance in Skeletal Muscle 
Diabetes  2008;57(10):2644-2651.
OBJECTIVE—Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo.
RESEARCH DESIGN AND METHODS—To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser302, Ser307, and Ser612 mutated to alanine (Tg IRS-1 Ser→Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo.
RESULTS—Tg IRS-1 Ser→Ala mice were protected from fat-induced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser→Ala mice displayed a significant increase in insulin-stimulated IRS-1–associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates.
CONCLUSIONS—These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fat-induced insulin resistance in skeletal muscle in vivo.
doi:10.2337/db06-0454
PMCID: PMC2551673  PMID: 18633112
8.  Liver autophagy contributes to the maintenance of blood glucose and amino acid levels 
Autophagy  2011;7(7):727-736.
Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.
doi:10.4161/auto.7.7.15371
PMCID: PMC3149698  PMID: 21471734
amino acid; autophagy; liver; gluconeogenesis; insulin; phosphoenolpyruvate carboxykinase
9.  Treadmill Exercise Induces Neutrophil Recruitment into Muscle Tissue in a Reactive Oxygen Species-Dependent Manner. An Intravital Microscopy Study 
PLoS ONE  2014;9(5):e96464.
Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process.
doi:10.1371/journal.pone.0096464
PMCID: PMC4010495  PMID: 24798414
10.  Chronic Caloric Restriction and Exercise Improve Metabolic Conditions of Dietary-Induced Obese Mice in Autophagy Correlated Manner without Involving AMPK 
Journal of Diabetes Research  2013;2013:852754.
Aim. To investigate the role of AMPK activation and autophagy in mediating the beneficial effects of exercise and caloric restriction in obesity. Methods. Dietary-induced obesity mice were made and divided into 5 groups; one additional group of normal mice serves as control. Mice in each group received different combinations of interventions including low fat diet, caloric restriction, and exercise. Then their metabolic conditions were assessed by measuring serum glucose and insulin, serum lipids, and liver function. AMPK phosphorylation and autophagy activity were detected by western blotting. Results. Obese mice models were successfully induced by high fat diet. Caloric restriction consistently improved the metabolic conditions of the obese mice, and the effects are more prominent than the mice that received only exercise. Also, caloric restriction, exercise, and low fat diet showed a synergistic effect in the improvement of metabolic conditions. Western blotting results showed that this improvement was not related with the activation of AMPK in liver, skeletal muscle, or heart but correlates well with the autophagy activity. Conclusion. Caloric restriction has more prominent beneficial effects than exercise in dietary-induced obese mice. These effects are correlated with the autophagy activity and may be independent of AMPK activation.
doi:10.1155/2013/852754
PMCID: PMC3671310  PMID: 23762877
11.  Autophagy Signaling in Skeletal Muscle of Infarcted Rats 
PLoS ONE  2014;9(1):e85820.
Background
Heart failure (HF)-induced skeletal muscle atrophy is often associated to exercise intolerance and poor prognosis. Better understanding of the molecular mechanisms underlying HF-induced muscle atrophy may contribute to the development of pharmacological strategies to prevent or treat such condition. It has been shown that autophagy-lysosome system is an important mechanism for maintenance of muscle mass. However, its role in HF-induced myopathy has not been addressed yet. Therefore, the aim of the present study was to evaluate autophagy signaling in myocardial infarction (MI)-induced muscle atrophy in rats.
Methods/Principal Findings
Wistar rats underwent MI or Sham surgeries, and after 12 weeks were submitted to echocardiography, exercise tolerance and histology evaluations. Cathepsin L activity and expression of autophagy-related genes and proteins were assessed in soleus and plantaris muscles by fluorimetric assay, qRT-PCR and immunoblotting, respectively. MI rats displayed exercise intolerance, left ventricular dysfunction and dilation, thereby suggesting the presence of HF. The key findings of the present study were: a) upregulation of autophagy-related genes (GABARAPL1, ATG7, BNIP3, CTSL1 and LAMP2) was observed only in plantaris while muscle atrophy was observed in both soleus and plantaris muscles, and b) Cathepsin L activity, Bnip3 and Fis1 protein levels, and levels of lipid hydroperoxides were increased specifically in plantaris muscle of MI rats.
Conclusions
Altogether our results provide evidence for autophagy signaling regulation in HF-induced plantaris atrophy but not soleus atrophy. Therefore, autophagy-lysosome system is differentially regulated in atrophic muscles comprising different fiber-types and metabolic characteristics.
doi:10.1371/journal.pone.0085820
PMCID: PMC3888434  PMID: 24427319
12.  Activation of autophagy is required for muscle homeostasis during physical exercise 
Autophagy  2011;7(12):1405-1406.
Skeletal muscle fibers of collagen VI null (Col6a1−/−) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.
doi:10.4161/auto.7.12.18315
PMCID: PMC3288013  PMID: 22082869
lysosome; metabolism; physiology; stress; vacuole
13.  Systemic regulation of autophagy in Caenorhabditis elegans 
Autophagy  2009;5(4):565.
When no supply of environmental nutrients is available, cells induce autophagy, thereby generating a source of emergency metabolic substrates and energy to maintain the basal cellular activity needed for survival. This autophagy response to starvation has been well characterized in various multicellular organisms, including worms, flies and mice. Although prosurvival effects of autophagy in response to starvation are well known in animals, the mechanisms by which animals regulate and coordinate autophagy systemically remain elusive. Using C. elegans as a model system, we found that specific amino acids could regulate starvation-induced autophagy, and that MGL-1 and MGL-2, Caenorhabditis elegans homologs of metabotropic glutamate receptors, were involved. MGL-1 and MGL-2 specifically acted in AIY and AIB neurons, respectively, to modulate the autophagy response in other tissues such as pharyngeal muscle. Our recent study suggests that the autophagy response to starvation, previously thought to be cell-autonomous, can be systemically regulated, and that there is a specific sensor for monitoring systemic amino acids levels in Caenorhabditis elegans.
PMCID: PMC2819339  PMID: 19270490
autophagy; starvation; metabotropic glutamate receptor; amino acid response; Caenorhabditis elegans; hormesis
14.  Hepatic Glucagon Action Is Essential for Exercise-Induced Reversal of Mouse Fatty Liver 
Diabetes  2011;60(11):2720-2729.
OBJECTIVE
Exercise is an effective intervention to treat fatty liver. However, the mechanism(s) that underlie exercise-induced reductions in fatty liver are unclear. Here we tested the hypothesis that exercise requires hepatic glucagon action to reduce fatty liver.
RESEARCH DESIGN AND METHODS
C57BL/6 mice were fed high-fat diet (HFD) and assessed using magnetic resonance, biochemical, and histological techniques to establish a timeline for fatty liver development over 20 weeks. Glucagon receptor null (gcgr−/−) and wild-type (gcgr+/+) littermate mice were subsequently fed HFD to provoke moderate fatty liver and then performed either 10 or 6 weeks of running wheel or treadmill exercise, respectively.
RESULTS
Exercise reverses progression of HFD-induced fatty liver in gcgr+/+ mice. Remarkably, such changes are absent in gcgr−/− mice, thus confirming the hypothesis that exercise-stimulated hepatic glucagon receptor activation is critical to reduce HFD-induced fatty liver.
CONCLUSIONS
These findings suggest that therapies that use antagonism of hepatic glucagon action to reduce blood glucose may interfere with the ability of exercise and perhaps other interventions to positively affect fatty liver.
doi:10.2337/db11-0455
PMCID: PMC3198076  PMID: 21885872
15.  FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway 
Autophagy  2012;8(12):1712-1723.
Forkhead box O (FOXO) transcriptional protein family members, including FOXO1 and FOXO3, are involved in the modulation of autophagy. However, whether there is redundancy between FOXO1 and FOXO3 in the ability to induce autophagy remains unclear. In this study, we showed that FOXO3 induced a transcription-dependent autophagy, and FOXO1 was required for this process. Overexpression of wild-type FOXO3 (WT) or FOXO3 (3A), which harbors alanine mutations at residues Thr32, Ser253 and Ser315, but not transcription-inactive FOXO3 (∆DB3A), significantly induced autophagy in the human embryonic kidney cell line HEK293T and mouse embryonic fibroblast (MEF) cell lines. Interestingly, depletion of FOXO1 by siRNA attenuated FOXO3-induced autophagy. Our data also showed that FOXO3 overexpression did not increase the expression of FOXO1 at the protein level, although FOXO3 was capable of binding the promoter region of FOXO1 and inducing an increase in the transcription of FOXO1 mRNA. Furthermore, our results showed that FOXO3 promoted the translocation of FOXO1 from the nucleus to the cytoplasm, resulting in an increase in FOXO1-induced autophagy. Moreover, our results supported a mechanism whereby FOXO3 dramatically increased the expression of the class I PtdIns3K catalytic subunit PIK3CA, leading to an increase in AKT1 activity, which resulted in the phosphorylation and nuclear export of FOXO1. To the best of our knowledge, our data are the first to suggest that FOXO1 plays a central role in FOXO3-induced autophagy.
doi:10.4161/auto.21830
PMCID: PMC3541283  PMID: 22931788
FOXO1; FOXO3; autophagy; PIK3CA; AKT1
16.  Deficiency of the Transcriptional Repressor B Cell Lymphoma 6 (Bcl6) Is Accompanied by Dysregulated Lipid Metabolism 
PLoS ONE  2014;9(6):e97090.
The transcriptional repressor B-cell Lymphoma 6 (Bcl6) was recently identified in a profile of genes regulated in adipocytes, suggesting a relationship between Bcl6 and metabolic regulation. As a representative target gene repressed by Bcl6, Suppressor of Cytokine Signaling (Socs) 2 expression was elevated in Bcl6 deficient (KO) mice, including metabolic tissues liver, adipose tissue and muscle, as well as in spleen and thymus. Bcl6 occupied the Socs2 promoter in wild-type, but not Bcl6 KO mice, suggesting direct regulation of Socs2 by Bcl6 in vivo. Mice deficient in Bcl6 were found to exhibit multiple features of dysregulated lipid metabolism. Adipose tissue mass was dramatically reduced or absent in Bcl6 KO mice. Further, hepatic and serum triglycerides were low. Bcl6 deficiency was accompanied by decreased hepatic expression of Stearoyl-CoA desaturase 1 (Scd1) and Fatty acid synthase (Fasn) genes which encode lipogenic enzymes. Expression of the gene for the transcription factor Carbohydrate-Responsive Element Binding Protein (Chrebp), which regulates expression of lipogenic genes, was also reduced in liver of Bcl6 KO mice. Bcl6 deficiency disrupted fasting-induced increases in hepatic triglyceride deposition, but not decreases in lipogenic gene expression. Taken together, these findings suggest that in addition to its well-recognized roles in immune regulation, Bcl6 plays a role in regulatory events of lipid metabolism, and that in the absence of Bcl6, lipid metabolism in liver and adipose tissue is dysregulated.
doi:10.1371/journal.pone.0097090
PMCID: PMC4043531  PMID: 24892698
17.  Obesity impairs skeletal muscle AMPK signaling during exercise: role of AMPKα2 in the regulation of exercise capacity in vivo 
Objective
Skeletal muscle AMP-activated protein kinase (AMPK)α2 activity is impaired in obese, insulin resistant individuals during exercise. We determined whether this defect contributes to the metabolic dysregulation and reduced exercise capacity observed in the obese state.
Design
C57BL/6J wild-type (WT) mice and/or mice expressing a kinase dead AMPKα2 subunit in skeletal muscle (α2-KD) were fed chow or high fat (HF) diets from 3–16 weeks (wks) of age. At 15wks mice performed an exercise stress test to determine exercise capacity. In WT mice, muscle glucose uptake and skeletal muscle AMPKα2 activity was assessed in chronically catheterized mice (carotid artery/jugular vein) at 16wks. In a separate study, HF-fed WT and α2-KD mice performed 5wks of exercise training (from 15–20wks of age) to test whether AMPKα2 is necessary to restore work tolerance.
Results
HF-fed WT mice had reduced exercise tolerance during an exercise stress test, and an attenuation in muscle glucose uptake and AMPKα2 activity during a single bout of exercise (p<0.05 vs. chow). In chow-fed α2-KD mice running speed and time were impaired ~45% and ~55%, respectively (p<0.05 vs. WT chow); HF feeding further reduced running time ~25% (p<0.05 vs. α2-KD chow). In response to 5wks of exercise training, HF-fed WT and α2-KD mice increased maximum running speed ~35% (p<0.05 vs. pre-training) and maintained body weight at pre-training levels, whereas body weight increased in untrained HF WT and α2-KD mice. Exercise training restored running speed to levels seen in healthy, chow-fed mice.
Conclusion
HF feeding impairs AMPKα2 activity in skeletal muscle during exercise in vivo. While this defect directly contributes to reduced exercise capacity, findings in HF-fed α2-KD mice show that AMPKα2-independent mechanisms are also involved. Importantly, α2-KD mice on a HF-fed diet adapt to regular exercise by increasing exercise tolerance, demonstrating that this adaptation is independent of skeletal muscle AMPKα2 activity.
doi:10.1038/ijo.2010.220
PMCID: PMC3049903  PMID: 21079619
obesity; muscle glucose uptake; exercise tolerance; exercise training; diet
18.  Combinatory effects of siRNA‐induced myostatin inhibition and exercise on skeletal muscle homeostasis and body composition 
Physiological Reports  2014;2(3):e00262.
Abstract
Inhibition of myostatin (Mstn) stimulates skeletal muscle growth, reduces body fat, and induces a number of metabolic changes. However, it remains unexplored how exercise training modulates the response to Mstn inhibition. The aim of this study was to investigate how siRNA‐mediated Mstn inhibition alone but also in combination with physical activity affects body composition and skeletal muscle homeostasis. Adult mice were treated with Mstn‐targeting siRNA and subjected to a treadmill‐based exercise protocol for 4 weeks. Effects on skeletal muscle and fat tissue, expression of genes, and serum concentration of proteins involved in myostatin signaling, skeletal muscle homeostasis, and lipid metabolism were investigated and compared with Mstn−/− mice. The combination of siRNA‐mediated Mstn knockdown and exercise induced skeletal muscle hypertrophy, which was associated with an upregulation of markers for satellite cell activity. SiRNA‐mediated Mstn knockdown decreased visceral fat and modulated lipid metabolism similar to effects observed in Mstn−/− mice. Myostatin did not regulate its own expression via an autoregulatory loop, however, Mstn knockdown resulted in a decrease in the serum concentrations of myostatin propeptide, leptin, and follistatin. The ratio of these three parameters was distinct between Mstn knockdown, exercise, and their combination. Taken together, siRNA‐mediated Mstn knockdown in combination with exercise stimulated skeletal muscle hypertrophy. Each intervention or their combination induced a specific set of adaptive responses in the skeletal muscle and fat metabolism which could be identified by marker proteins in serum.
The aim of this study was to investigate how siRNA‐mediated Mstn inhibition alone but also in combination with physical activity affects body composition and skeletal muscle homeostasis. Adult mice were treated with Mstn‐targeting siRNA and subjected to a treadmill‐based exercise protocol for 4 weeks. SiRNA‐mediated Mstn knockdown in combination with exercise stimulated skeletal muscle hypertrophy and resulted in reduced body fat content.
doi:10.1002/phy2.262
PMCID: PMC4002242  PMID: 24760516
Exercise; follistatin; myostatin; RNA interference
19.  Dissociation of Bcl-2–Beclin1 Complex by Activated AMPK Enhances Cardiac Autophagy and Protects Against Cardiomyocyte Apoptosis in Diabetes 
Diabetes  2013;62(4):1270-1281.
Diabetic cardiomyopathy is associated with suppression of cardiac autophagy, and activation of AMP-activated protein kinase (AMPK) restores cardiac autophagy and prevents cardiomyopathy in diabetic mice, albeit by an unknown mechanism. We hypothesized that AMPK-induced autophagy ameliorates diabetic cardiomyopathy by inhibiting cardiomyocyte apoptosis and examined the effects of AMPK on the interaction between Beclin1 and Bcl-2, a switch between autophagy and apoptosis, in diabetic mice and high glucose–treated H9c2 cardiac myoblast cells. Exposure of H9c2 cells to high glucose reduced AMPK activity, inhibited Jun NH2-terminal kinase 1 (JNK1)–B-cell lymphoma 2 (Bcl-2) signaling, and promoted Beclin1 binding to Bcl-2. Conversely, activation of AMPK by metformin stimulated JNK1–Bcl-2 signaling and disrupted the Beclin1–Bcl-2 complex. Activation of AMPK, which normalized cardiac autophagy, attenuated high glucose–induced apoptosis in cultured H9c2 cells. This effect was attenuated by inhibition of autophagy. Finally, chronic administration of metformin in diabetic mice restored cardiac autophagy by activating JNK1–Bcl-2 pathways and dissociating Beclin1 and Bcl-2. The induction of autophagy protected against cardiac apoptosis and improved cardiac structure and function in diabetic mice. We concluded that dissociation of Bcl-2 from Beclin1 may be an important mechanism for preventing diabetic cardiomyopathy via AMPK activation that restores autophagy and protects against cardiac apoptosis.
doi:10.2337/db12-0533
PMCID: PMC3609561  PMID: 23223177
20.  Prophylactic effects of swimming exercise on autophagy-induced muscle atrophy in diabetic rats 
Laboratory Animal Research  2012;28(3):171-179.
Diabetes decreases skeletal muscle mass and induces atrophy. However, the mechanisms by which hyperglycemia and insulin deficiency modify muscle mass are not well defined. In this study, we evaluated the effects of swimming exercise on muscle mass and intracellular protein degradation in diabetic rats, and proposed that autophagy inhibition induced by swimming exercise serves as a hypercatabolic mechanism in the skeletal muscles of diabetic rats, supporting a notion that swimming exercise could efficiently reverse the reduced skeletal muscle mass caused by diabetes. Adult male Sprague-Dawley rats were injected intraperitoneally with streptozotocin (60 mg/kg body weight) to induce diabetes and then submitted to 1 hr per day of forced swimming exercise, 5 days per week for 4 weeks. We conducted an intraperitoneal glucose tolerance test on the animals and measured body weight, skeletal muscle mass, and protein degradation and examined the level of autophagy in the isolated extensor digitorum longus, plantaris, and soleus muscles. Body weight and muscle tissue mass were higher in the exercising diabetic rats than in control diabetic rats that remained sedentary. Compared to control rats, exercising diabetic rats had lower blood glucose levels, increased intracellular contractile protein expression, and decreased autophagic protein expression. We conclude that swimming exercise improves muscle mass in diabetes-induced skeletal muscle atrophy, suggesting the activation of autophagy in diabetes contributes to muscle atrophy through hypercatabolic metabolism and that aerobic exercise, by suppressing autophagy, may modify or reverse skeletal muscle wasting in diabetic patients.
doi:10.5625/lar.2012.28.3.171
PMCID: PMC3469845  PMID: 23091517
Autophagy; diabetes; muscle atrophy; swimming exercise; prophylactic effect
21.  Autophagy in proximal tubules protects against acute kidney injury 
Kidney international  2012;82(12):1271-1283.
Autophagy is induced in renal tubular cells during acute kidney injury, however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia-reperfusion induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule-specific autophagy-related gene 7 knockout mouse model shown to be defective in both basal and cisplatin induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia-reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms.
doi:10.1038/ki.2012.261
PMCID: PMC3491167  PMID: 22854643
autophagy; Atg7; cisplatin; ischemia-reperfusion; acute kidney injury
22.  Toll-Like Receptor 4 Knockout Mice Are Protected against Endoplasmic Reticulum Stress Induced by a High-Fat Diet 
PLoS ONE  2013;8(5):e65061.
The purpose of this study was to investigate whether toll-like receptor 4 (TLR4) is implicated in the development of endoplasmic reticulum stress (ER stress) observed after a high-fat diet (HFD) in liver, skeletal muscle and adipose tissue. TLR4−/− and C57BL/6J wild-type mice (WT) were fed with chow or HFD (45% calories from fat) during 18 weeks. An oral glucose tolerance-test was performed. The animals were sacrificed in a fasted state and the tissues were removed. TLR4 deletion protected from body weight gain and glucose intolerance induced by HFD whereas energy intake was higher in transgenic mice suggesting larger energy expenditure. HFD induced an ER stress in skeletal muscle, liver and adipose tissue of WT mice as assessed by BiP, CHOP, spliced and unspliced XBP1 and phospho-eIF2α. TLR4−/− mice were protected against HFD-induced ER stress. Then, we investigated the main signaling downstream of TLR4 namely the NF-κB pathway, expecting to identify the mechanism by which TLR4 is able to activate ER stress. The mRNA levels of cytokines regulated by NF-κB namely TNFα, IL-1β and IL-6, were not changed after HFD and phospho-IκB-α (ser 32) was not changed. Our results indicate that TLR4 is essential for the development of ER stress related to HFD. Nevertheless, the NFκ-B pathway does not seem to be directly implicated. The reduced fat storage in TLR4−/− mice could explain the absence of an ER stress after HFD.
doi:10.1371/journal.pone.0065061
PMCID: PMC3669084  PMID: 23741455
23.  Autophagy in Muscle of Glucose-Infusion Hyperglycemia Rats and Streptozotocin-Induced Hyperglycemia Rats via Selective Activation of m-TOR or FoxO3 
PLoS ONE  2014;9(2):e87254.
Autophagy is a conserved process in eukaryotes required for metabolism and is involved in diverse diseases. To investigate autophagy in skeletal muscle under hyperglycemia status, we established two hyperglycemia-rat models that differ in their circulating insulin levels, by glucose infusion and singe high-dose streptozotocin injection. We then detected expression of autophagy related genes with real-time PCR and western blot. We found that under hyperglycemia status induced by glucose-infusion, autophagy was inhibited in rat skeletal muscle, whereas under streptozotocin-induced hyperglycemia status autophagy was enhanced. Meanwhile, hyperglycemic gastrocnemius muscle was more prone to autophagy than soleus muscle. Furthermore, inhibition of autophagy in skeletal muscle in glucose-infusion hyperglycemia rats was mediated by the m-TOR pathway while m-TOR and FoxO3 both contributed to enhancement of autophagy in gastrocnemius muscle in streptozotocin-induced hyperglycemia rats. These data shows that insulin plays a relatively more important role than hyperglycemia in regulating autophagy in hyperglycemia rat muscle through selectively activating the m-TOR or FoxO3 pathway in a fiber-selective manner.
doi:10.1371/journal.pone.0087254
PMCID: PMC3911944  PMID: 24498304
24.  Identification of ROCK1 kinase as a critical regulator of Beclin1 mediated autophagy during metabolic stress 
Nature communications  2013;4:2189.
The Ser/Thr Rho kinase 1 (ROCK1) is known to play major roles in a wide range of cellular activities, including those involved in tumor metastasis and apoptosis. Here we identify an indispensable function of ROCK1 in metabolic stress-induced autophagy. Applying a proteomics approach, we characterize Beclin1, a proximal component of the PI(3)kinase class III lipid-kinase complex that induces autophagy, as an interacting partner of ROCK1. Upon nutrient deprivation, activated ROCK1 promotes autophagy by binding and phosphorylating Beclin1 at Thr119. This results in the specific dissociation of the Beclin1-Bcl-2 complex, without affecting the Beclin1-UVRAG interaction. Conversely, inhibition of ROCK1 activity increases Beclin1-Bcl-2 association, thus reducing nutritional stress-mediated autophagy. Genetic knockout of ROCK1 function in mice also leads to impaired autophagy as evidenced by reduced autophagosome formation. These results show that ROCK1 acts as a prominent upstream regulator of Beclin1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy.
doi:10.1038/ncomms3189
PMCID: PMC3740589  PMID: 23877263
25.  Regulation of Op18 during Spindle Assembly in Xenopus Egg Extracts 
The Journal of Cell Biology  2001;153(1):149-158.
Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.
PMCID: PMC2185528  PMID: 11285281
microtubule dynamics; spindle assembly; phosphorylation; Plx1; chromatin

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