Zhaoyong Hu

Regulation of Muscle Protein Degradation: Coordinated Control of Apoptotic and Ubiquitin-Proteasome Systems by Phosphatidylinositol 3 Kinase

Muscle proteolysis from catabolic conditions, including chronic kidney disease, requires coordinated activation of both the apoptotic and ATP-ubiquitin-proteasome systems (Ub-Psome), including upregulation of components of the Ub-Psome system. Activation of the apoptotic system is required because caspase-3 initially cleaves myofibrils, yielding substrates for the Ub-Psome system plus a characteristic 14-kD actin fragment. The authors studied insulin deficiency, a model of accelerated muscle atrophy, to understand how regulation of the apoptotic and the Ub-Psome systems could be coordinated. As expected, phosphatidylinositol 3 kinase activity (PI3K) was suppressed in muscle; in addition to decreased insulin, the mechanism includes IRS-1 phosphorylation at serine-307. Caspase-3 activity was also increased, and the authors linked it to a low PI3K-induced activation of the apoptotic system that includes a conformational change in Bax and release of cytochrome C. Coordinated atrogin-1/MAFbx expression is required as a critical factor for Ub-Psome system-dependent muscle proteolysis in diabetes and other catabolic states. The mechanism that regulates atrogin-1/MAFbx expression is unknown. Atrogin-1/MAFbx expression increased when the authors suppressed PI3K activity in muscle cells. The forkhead transcriptional factor, a downstream substrate of PI3K, stimulated atrogin-1/MAFbx promoter transcriptional activity markedly. The authors found in diabetic muscle that mRNA of the forkhead transcriptional factor, its nuclear translocation, and binding to the atrogin-1/MAFbx promoter were increased. When PI3K activity is low, both apoptotic and Ub-Psome pathways are activated coordinately to cause muscle proteolysis. This mechanism could increase muscle atrophy in conditions with impaired insulin responsiveness.

Chronic Kidney Disease Causes Defects in Signaling through the Insulin Receptor Substrate/Phosphatidylinositol 3-Kinase/Akt Pathway: Implications for Muscle Atrophy

Complications of chronic kidney disease (CKD) include depressed responses to insulin/IGF-1 and accelerated muscle proteolysis as a result of activation of caspase-3 and the ubiquitin-proteasome system. Experimentally, proteolysis in muscle cells occurs when there is suppression of phosphatidylinositol 3-kinase (PI3-K) activity. Postreceptor signaling through the insulin receptor substrate (IRS)/PI3-K/Akt pathway was evaluated in muscles of acidotic, CKD and pair-fed control rats under physiologic conditions and in response to a dose of insulin that quickly stimulated the pathway. Basal IRS-1-associated PI3-K activity was suppressed by CKD; IRS-2-associated PI3-K activity was increased. The basal level of activated Akt in CKD muscles also was low, indicating that the higher IRS-2-associated PI3-K activity did not compensate for the reduced IRS-1-associated PI3-K activity. Insulin treatment overcame this abnormality. The low IRS-1-associated PI3-K activity in muscle was not due to a decrease in IRS-1 protein, but there was a higher amount of the PI3-K p85 subunit protein without a concomitant increase in the p110 catalytic subunit, offering a potential explanation for the lower IRS-1-associated PI3-K activity. Eliminating the acidosis of CKD partially corrected the decrease in basal IRS-1-associated PI3-K activity and protein degradation in muscle. It is concluded that in CKD, acidosis and an increase in the PI3-K p85 subunit are mechanisms that contribute to suppression of PI3-K activity in muscle, and this leads to accelerated muscle proteolysis.

IL-6 and Serum Amyloid A Synergy Mediates Angiotensin II–Induced Muscle Wasting

Animal studies suggest that increased levels of circulating angiotensin II (AngII) could contribute to the loss of lean body mass in chronic kidney disease, but the mechanism by which this occurs is unclear. Here, AngII infusion increased circulating IL-6 and its hepatic production in wild-type mice, suggesting that AngII-induced inflammation may trigger muscle loss. AngII infusion also stimulated the suppressor of cytokine signaling (SOCS3) in muscle, which led to loss of insulin receptor substrate 1 (IRS-1), thereby impairing insulin/IGF-1 signaling and enhancing protein degradation. All of these responses to AngII were suppressed in IL-6-deficient mice. Recombinant human IL-6 (rhIL-6) treatment of cultured myotubes only minimally increased SOCS3, however, suggesting the contribution of other mediators. Because AngII increases hepatic serum amyloid A (SAA) expression in an IL-6-dependent manner, we treated wild-type mice with rhIL-6 and an SAA1-overexpressing adenovirus; the combination led to a significantly greater increase in SOCS3 and decrease in IRS-1 compared with either rhIL-6 or SAA1 alone. We observed similar effects on SOCS3 and IRS-1 when we treated cultured muscle myotubes with rhIL-6 and SAA1. Taken together, these results suggest an interorgan response to high levels of AngII: Hepatic production of IL-6 and SAA increases, and these mediators act synergistically to impair insulin/IGF-1 signaling, which promotes muscle proteolysis. Targeting the high levels of IL-6 and SAA in catabolic disorders might be a therapeutic approach to prevent muscle wasting.

Pharmacological inhibition of myostatin suppresses systemic inflammation and muscle atrophy in mice with chronic kidney disease

Chronic kidney disease (CKD) and several other catabolic conditions are characterized by increased circulating inflammatory cytokines, defects in IGF‐1 signaling, abnormal muscle protein metabolism, and progressive muscle atrophy. In these conditions, no reliable treatments successfully block the development of muscle atrophy. In mice with CKD, we found a 2‐ to 3‐fold increase in myostatin expression in muscle. Its pharmacological inhibition by subcutaneous injections of an anti‐myostatin peptibody into CKD mice (IC50 ~1.2 nM) reversed the loss of body weight (≈5–7% increase in body mass) and muscle mass (~10% increase in muscle mass) and suppressed circulating inflammatory cytokines vs. results from CKD mice injected with PBS. Pharmacological myostatin inhibition also decreased the rate of protein degradation (16.38±1.29%;P<0.05), increased protein synthesis in extensor digitorum longus muscles (13.21±1.09%; P<0.05), markedly enhanced satellite cell function, and improved IGF‐1 intracellular signaling. In cultured muscle cells, TNF‐α increased myostatin expression via a NF‐κB‐dependent pathway, whereas muscle cells exposed to myostatin stimulated IL‐6 production via p38 MAPK and MEK1 pathways. Because IL‐6 stimulates muscle protein breakdown, we conclude that CKD increases myostatin through cytokine‐activated pathways, leading to muscle atrophy. Myostatin antagonism might become a therapeutic strategy for improving muscle growth in CKD and other conditions with similar characteristics.—Zhang, L., Rajan, V., Lin, E., Hu, Z., Han, H.Q., Zhou, X., Song, Y., Min, H., Wang, X., Du, J., Mitch, W. E. Pharmacological inhibition of myostatin suppresses systemic inflammation and muscle atrophy in mice with chronic kidney disease. FASEB J. 25, 1653–1663 (2011). www.fasebj.org

Endogenous glucocorticoids and impaired insulin signaling are both required to stimulate muscle wasting under pathophysiological conditions in mice

Muscle wasting is associated with a number of pathophysiologic conditions, including metabolic acidosis, diabetes, sepsis, and high angiotensin II levels. Under these conditions, activation of muscle protein degradation requires endogenous glucocorticoids. As the mechanism(s) underlying this dependence on glucocorticoids have not been identified, we analyzed the effects of glucocorticoids on muscle wasting in a mouse model of acute diabetes. Adrenalectomized, acutely diabetic mice given a physiologic dose of glucocorticoids exhibited decreased IRS-1-associated PI3K activity in muscle and progressive muscle atrophy. These responses were related to increased association of PI3K with the glucocorticoid receptor (GR). In mice with muscle-specific GR deletion (referred to as MGRKO mice), acute diabetes minimally suppressed IRS-1-associated PI3K activity in muscle and did not cause muscle atrophy. However, when a physiologic dose of glucocorticoids was given to mice with muscle-specific IR deletion, muscle protein degradation was accelerated. Fluorescence resonance energy transfer and an in vitro competition assay revealed that activated GRs competed for PI3K, reducing its association with IRS-1. Reexpression of WT GRs or those with a mutation in the nuclear localization signal in the muscle of MGRKO mice indicated that competition for PI3K was a prominent mechanism underlying reduced IRS-1-associated PI3K activity. This nongenomic influence of the GR contributes to activation of muscle protein degradation. We therefore conclude that stimulation of muscle proteolysis requires 2 events, increased glucocorticoid levels and impaired insulin signaling.

Development of a Diagnostic Method for Detecting Increased Muscle Protein Degradation in Patients with Catabolic Conditions

Muscle atrophy in catabolic illnesses is due largely to accelerated protein degradation. Unfortunately, methods for detecting accelerated muscle proteolysis are cumbersome. The goal of this study was to develop a method for detecting muscle protein breakdown and assess the effectiveness of anticatabolic therapy. In rodent models of catabolic conditions, it was found that accelerated muscle protein degradation is triggered by activation of caspase-3. Caspase-3 cleaves actomyosin/myofibrils to form substrates for the ubiquitin-proteasome system and leaves a characteristic 14-kD actin fragment in the insoluble fraction of a muscle lysate. Muscle biopsies were obtained from normal adults and three groups of patients: 14 who were undergoing hip arthroplasty, 28 hemodialysis patients who were participating in exercise programs, and seven severely burned patients. In muscle of patients who were undergoing hip arthroplasty, the 14-kD actin fragment level was correlated (r = 0.787, P < 0.01) with the fractional rate of protein degradation. In muscle of hemodialysis patients who were undergoing endurance exercise training, the 14-kD actin fragment decreased to values similar to levels in normal adults; strength training did not significantly decrease the actin fragment. Severely burned patients had increased muscle protein degradation and actin fragment levels, but the two measures were not significantly correlated. The experimental results suggest that the 14-kD actin fragment in muscle biopsies is increased in catabolic states and could be used in conjunction with other methods to detect and monitor changes in muscle proteolysis that occur in patients with mild or sustained increases in muscle proteolysis.

Transcription factor FoxO1, the dominant mediator of muscle wasting in chronic kidney disease, is inhibited by microRNA-486

Chronic kidney disease (CKD) accelerates muscle protein degradation by stimulating the ubiquitin proteasome system through activation of the E3 ligases, Atrogin-1/MaFbx and MuRF-1. Forkhead transcription factors (FoxO) can control the expression of these E3 ligases, but the contribution of individual FoxOs to muscle wasting is unclear. To study this we created mice with a muscle-specific FoxO1 deletion. The absence of FoxO1 blocked 70% of the increase in E3 ligases induction by CKD as well as the proteolysis and loss of muscle mass. Thus, FoxO1 has a role in controlling ubiquitin proteasome system-related proteolysis. Since microRNA (miR)-486 reportedly dampens FoxO1 expression and its activity, we transfected a miR-486 mimic into primary cultures of myotubes and found this blocked dexamethasone-stimulated protein degradation without influencing protein synthesis. It also decreased FoxO1 protein translation and increased FoxO1 phosphorylation by down-regulation of PTEN phosphatase, a negative regulator of p-Akt. To test its efficacy in vivo, we electroporated miR-486 into muscles and found expression of the E3 ligases was suppressed and muscle mass increased despite CKD. Thus, FoxO1 is a dominant mediator of CKD-induced muscle wasting and miR-486 coordinately decreases FoxO1 and PTEN to protect against this catabolic response.

PTEN Inhibition Improves Muscle Regeneration in Mice Fed a High-Fat Diet

OBJECTIVE Mechanisms impairing wound healing in diabetes are poorly understood. To identify mechanisms, we induced insulin resistance by chronically feeding mice a high-fat diet (HFD). We also examined the regulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) during muscle regeneration because augmented IGF-1 signaling can improve muscle regeneration. RESEARCH DESIGN AND METHODS Muscle regeneration was induced by cardiotoxin injury, and we evaluated satellite cell activation and muscle maturation in HFD-fed mice. We also measured PIP3 and the enzymes regulating its level, IRS-1–associated phosphatidylinositol 3-kinase (PI3K) and PTEN. Using primary cultures of muscle, we examined how fatty acids affect PTEN expression and how PTEN knockout influences muscle growth. Mice with muscle-specific PTEN knockout were used to examine how the HFD changes muscle regeneration. RESULTS The HFD raised circulating fatty acids and impaired the growth of regenerating myofibers while delaying myofiber maturation and increasing collagen deposition. These changes were independent of impaired proliferation of muscle progenitor or satellite cells but were principally related to increased expression of PTEN, which reduced PIP3 in muscle. In cultured muscle cells, palmitate directly stimulated PTEN expression and reduced cell growth. Knocking out PTEN restored cell growth. In mice, muscle-specific PTEN knockout improved the defects in muscle repair induced by HFD. CONCLUSIONS Insulin resistance impairs muscle regeneration by preventing myofiber maturation. The mechanism involves fatty acid–stimulated PTEN expression, which lowers muscle PIP3. If similar pathways occur in diabetic patients, therapeutic strategies directed at improving the repair of damaged muscle could include suppression of PTEN activity.

Adiponectin promotes monocyte-to-fibroblast transition in renal fibrosis

Bone marrow-derived fibroblasts may contribute substantially to the pathogenesis of renal fibrosis through the excessive production and deposition of extracellular matrix. However, the mechanisms underlying the accumulation and activation of these fibroblasts are not understood. Here, we used a mouse model of tubulointerstitial fibrosis to determine whether adiponectin, which is elevated in CKD and is associated with disease progression, regulates monocyte-to-fibroblast transition and fibroblast activation in injured kidneys. In wild-type mice, the expression of adiponectin and the number of bone marrow-derived fibroblasts in the kidney increased after renal obstruction. In contrast, the obstructed kidneys of adiponectin-knockout mice had fewer bone marrow-derived fibroblasts. Adiponectin deficiency also led to a reduction in the number of myofibroblasts, the expression of profibrotic chemokines and cytokines, and the number of procollagen-expressing M2 macrophages in injured kidneys. Consistent with these findings, adiponectin-deficiency reduced the expression of collagen I and fibronectin. Similar results were observed in wild-type and adiponectin-knockout mice after ischemia-reperfusion injury. In cultured bone marrow-derived monocytes, adiponectin stimulated the expression of α-smooth muscle actin (SMA) and extracellular matrix proteins and activated AMP-activated protein kinase (AMPK) in a time- and dose-dependent manner. Furthermore, specific activation of AMPK increased the expression of α-SMA and extracellular matrix proteins, while inhibition of AMPK attenuated these responses. Taken together, these findings identify adiponectin as a critical regulator of monocyte-to-fibroblast transition and renal fibrosis, suggesting that inhibition of adiponectin/AMPK signaling may represent a novel therapeutic target for fibrotic kidney disease.

PTEN expression contributes to the regulation of muscle protein degradation in diabetes

OBJECTIVE—Conditions accelerating muscle proteolysis are frequently associated with defective phosphatidylinositol 3-kinase (PI3K)/Akt signaling and reduced PI3K-generated phosphatidylinositol 3,4,5-triphosphate (PIP3). We evaluated the control of muscle protein synthesis and degradation in mouse models of type 1 and 2 diabetes to determine whether defects besides PI3K/Akt activities affect muscle metabolism. RESEARCH DESIGN AND METHODS—We evaluated the expression and activity of PTEN, the phosphatase converting PIP3 to inactive phosphatidylinositol 4,5-bisphosphate, and studied how PTEN influences muscle protein in diabetic wild-type mice and in mice with partial deficiency of PTEN+/−. RESULTS—In acutely diabetic mice, muscle PTEN expression was decreased. It was increased by chronic diabetes or insulin resistance. In cultured C2C12 myotubes, acute suppression of PI3K activity led to decreased PTEN expression, while palmitic acid increased PTEN in myotubes in a p38-dependent fashion. To examine whether PTEN affects muscle protein turnover, we studied primary myotubes cultures from wild-type and PTEN+/− mice. The proteolysis induced by serum deprivation was suppressed in PTEN+/− cells. Moreover, the sizes of muscle fibers in PTEN+/− and wild-type mice were similar, but the increase in muscle proteolysis caused by acute diabetes was significantly suppressed by PTEN+/−. This antiproteolytic response involved higher PIP3 and p-Akt levels and a decrease in caspase-3–mediated actin cleavage and activation of the ubiquitin-proteasome system as signified by reduced induction of atrogin-1/MAFbx or MurF1 (muscle-specific RING finger protein 1). CONCLUSIONS—Changes in PTEN expression participate in the regulation of muscle proteolytic pathways. A decrease in PTEN could be a compensatory mechanism to prevent muscle protein losses.