Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.

Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Akt can be inducibly expressed in adult skeletal muscle and thereby demonstrate that acute activation of Akt is sufficient to induce rapid and significant skeletal muscle hypertrophy in vivo, accompanied by activation of the downstream Akt/p70S6 kinase protein synthesis pathway. Upon induction of Akt in skeletal muscle, there was also a significant decrease in adipose tissue. These findings suggest that pharmacologic approaches directed toward activating Akt will be useful in inducing skeletal muscle hypertrophy and that an increase in lean muscle mass is sufficient to decrease fat storage.

Glucagon supports glucose homeostasis by stimulating hepatic gluconeogenesis, in part by promoting the uptake and conversion of amino acids into gluconeogenic precursors. Genetic disruption or pharmacologic inhibition of glucagon signaling results in elevated plasma amino acids and compensatory glucagon hypersecretion involving expansion of pancreatic α cell mass. Recent findings indicate that hyperaminoacidemia triggers pancreatic α cell proliferation via an mTOR-dependent pathway. We confirm and extend these findings by demonstrating that glucagon pathway blockade selectively increases expression of the sodium-coupled neutral amino acid transporter Slc38a5 in a subset of highly proliferative α cells and that Slc38a5 controls the pancreatic response to glucagon pathway blockade; most notably, mice deficient in Slc38a5 exhibit markedly decreased α cell hyperplasia to glucagon pathway blockade-induced hyperaminoacidemia. These results show that Slc38a5 is a key component of the feedback circuit between glucagon receptor signaling in the liver and amino-acid-dependent regulation of pancreatic α cell mass in mice.

SUMMARY Yi et al. (2013) recently reported that angiopoietin-like protein 8 (ANGPTL8) was the long-sought “betatrophin” that could control pancreatic beta cell proliferation. However, studies of Angptl8−/− mice revealed profound reduction of triglyceride levels, but no abnormalities in glucose homeostasis (Wang et al. 2013). We now report that Angptl8−/− mice undergo entirely normal beta cell expansion in response to insulin resistance resulting from either a high fat diet, or from the administration of the insulin receptor antagonist S961. Furthermore, overexpression of ANGPTL8 in livers of mice doubles plasma triglyceride levels, but does not alter beta cell expansion nor glucose metabolism. These data indicate ANGPTL8 does not play a role in controlling beta cell growth, nor can it be given to induce such expansion. The findings that plasma triglyceride levels are reduced by Angptl8 deletion and increased following ANGPTL8 overexpression support the possibility that inhibition of ANGPTL8 represents a therapeutic strategy for hypertriglyceridemia.

Growth and differentiation factor 8 (GDF8) is a TGF-β superfamily member, and negative regulator of skeletal muscle mass. GDF8 inhibition results in prominent muscle growth in mice, but less impressive hypertrophy in primates, including man. Broad TGF-β inhibition suggests another family member negatively regulates muscle mass, and its blockade enhances muscle growth seen with GDF8-specific inhibition. Here we show that activin A is the long-sought second negative muscle regulator. Activin A specific inhibition, on top of GDF8 inhibition, leads to pronounced muscle hypertrophy and force production in mice and monkeys. Inhibition of these two ligands mimics the hypertrophy seen with broad TGF-β blockers, while avoiding the adverse effects due to inhibition of multiple family members. Altogether, we identify activin A as a second negative regulator of muscle mass, and suggest that inhibition of both ligands provides a preferred therapeutic approach, which maximizes the benefit:risk ratio for muscle diseases in man.

BackgroundLoss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases.MethodsWe generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Smad2/3 signaling assays. REGN1033 was tested in mice for the ability to induce skeletal muscle hypertrophy and prevent atrophy induced by immobilization, hindlimb suspension, or dexamethasone. The effect of REGN1033 on exercise training was tested in aged mice. Messenger RNA sequencing, immunohistochemistry, and ex vivo force measurements were performed on skeletal muscle samples from REGN1033-treated mice.ResultsThe human monoclonal antibody REGN1033 is a specific and potent myostatin antagonist. Chronic treatment of mice with REGN1033 increased muscle fiber size, muscle mass, and force production. REGN1033 prevented the loss of muscle mass induced by immobilization, glucocorticoid treatment, or hindlimb unweighting and increased the gain of muscle mass during recovery from pre-existing atrophy. In aged mice, REGN1033 increased muscle mass and strength and improved physical performance during treadmill exercise.ConclusionsWe show that specific myostatin antagonism with the human antibody REGN1033 enhanced muscle mass and function in young and aged mice and had beneficial effects in models of skeletal muscle atrophy.

Antagonizing glucagon action represents an attractive therapeutic option for reducing hepatic glucose production in settings of hyperglycemia where glucagon excess plays a key pathophysiological role. We therefore generated REGN1193, a fully human monoclonal antibody that binds and inhibits glucagon receptor (GCGR) signaling in vitro. REGN1193 administration to diabetic ob/ob and diet-induced obese mice lowered blood glucose to levels observed in GCGR-deficient mice. In diet-induced obese mice, REGN1193 reduced food intake, adipose tissue mass, and body weight. REGN1193 increased circulating levels of glucagon and glucagon-like peptide 1 and was associated with reversible expansion of pancreatic α-cell area. Hyperglucagonemia and α-cell hyperplasia was observed in fibroblast growth factor 21-deficient mice treated with REGN1193. Single administration of REGN1193 to diabetic cynomolgus monkeys normalized fasting blood glucose and glucose tolerance and increased circulating levels of glucagon and amino acids. Finally, administration of REGN1193 for 8 weeks to normoglycemic cynomolgus monkeys did not cause hypoglycemia or increase pancreatic α-cell area. In summary, the GCGR-blocking antibody REGN1193 normalizes blood glucose in diabetic mice and monkeys but does not produce hypoglycemia in normoglycemic monkeys. Thus, REGN1193 provides a potential therapeutic modality for diabetes mellitus and acute hyperglycemic conditions.

Inactivating mutations in the insulin receptor results in extreme insulin resistance. The resulting hyperglycemia is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes. We used the insulin receptor antagonist S961 to induce severe insulin resistance, hyperglycemia, and ketonemia in mice. Using this model, we show that glucagon receptor (GCGR) inhibition with a monoclonal antibody normalized blood glucose and β-hydroxybutyrate levels. Insulin receptor antagonism increased pancreatic β-cell mass threefold. Normalization of blood glucose levels with GCGR-blocking antibody unexpectedly doubled β-cell mass relative to that observed with S961 alone and 5.8-fold over control. GCGR antibody blockage expanded α-cell mass 5.7-fold, and S961 had no additional effects. Collectively, these data show that GCGR antibody inhibition represents a potential therapeutic option for treatment of patients with extreme insulin-resistance syndromes.