[Withdrawn] Regulation of insulin resistance by myostatin
Date of Issue2013
School of Biological Sciences
Myostatin (Mstn) is a myokine that belongs to the TGF-β super family. Genetic analysis has shown that Mstn is a potent inhibitor of skeletal muscle growth. Recent findings have also identified a potential role for Mstn in muscle metabolism, as inactivation of Mstn results in increased insulin sensitivity and resistance to high fat diet-induced obesity. However, the exact signaling mechanism by which Mstn regulates carbohydrate metabolism in muscle is not yet known. Therefore, main aim of this thesis was to identify the role of Mstn in carbohydrate metabolism, for which several lines of investigations were undertaken. In vitro and in vivo studies indicated that high levels of glucose and fatty acid (palmitate) promote the expression of Mstn. We have identified that ChREBP and SREBP1c transcription factors induced by high levels of glucose and fat respectively are critical for the activation of the Mstn promoter during high glucose and high fat diet feeding of mice. Additionally, elevated Mstn levels were found to activate the IRS1 specific E3 ligase, Cblb (Casitas B-lineage lymphoma proto-oncogene b) which degraded IRS1 protein to induce insulin resistance. We have also confirmed that this mechanism is highly conserved from mouse to human since, increased circulatory Mstn in humans also increased signaling of the Mstn-Cblb pathway resulting in reduced IRS1 protein in primary myoblasts established from insulin resistant subjects. Pharmacological inhibition of Mstn signaling in insulin resistant primary myoblasts using sActRIIB or Mstn siRNA reduced the Mstn-Cblb signaling pathway and improved glucose uptake in insulin resistant myoblasts. Therefore, collectively these results supported the notion that high glucose and fat intake promotes insulin resistance through activating Mstn via both ChREBP and SREBP1c transcription factors. We undertook efforts to identify novel mediators involved in the development of insulin resistance in skeletal muscle. Using microarray, we have identified Pid1 (Phosphotyrosine interacting domain containing protein 1) as a protein that is induced during insulin resistance in myoblasts from human subjects. Molecular analysis revealed that Pid1 is an early marker of insulin resistance in adipose and liver tissues, whereas in skeletal muscle Pid1 is up regulated during later stages of obesity and diabetes. Furthermore, Pid1 is up-regulated by Mstn in skeletal muscle. Mstn-mediated up-regulation of Pid1 was found to be via NF-kB signaling. While the over-expression of Pid1 in human myoblasts affected insulin mediated glucose uptake and insulin signaling, the inhibition of Pid1 expression by shRNA in human myoblasts improved insulin signaling and increased glucose uptake. Overall this particular study suggested Pid1 as a novel target of Mstn to induce insulin resistance in peripheral tissues. PPARβ/δ (Peroxisome proliferator-activated receptor β/δ) is member of the family of important regulators of fat metabolism and is involved in obesity and type 2 diabetes. Muscle specific activation of PPARβ/δ protects mice from developing obesity and type 2 diabetes, when fed with high fat diet. Furthermore, overwhelming evidence suggested that treatment with PPARβ/δ agonists increased myogenesis and muscle growth, however very little is known about mechanism behind PPARβ/δ mediated increase in myogenesis. We performed microarray in myotubes and discovered that activation of PPARβ/δ induced Gasp-1 (Growth and differentiation factor associated protein-1), a potent antagonists of Mstn. We further confirmed that the presence of a PPAR responsive element (PPRE) within the 1.5kb proximal Gasp-1 promoter region was responsible for PPARβ/δ mediated up-regulation of Gasp-1. Gasp-1 in turn interacted with Mstn and inhibited the activity of Mstn. The inactivation of Mstn by PPARβ/δ mediated up-regulation of Gasp-1 increased myogenesis. hGASP-1 protein improved myogenecity of human myoblasts with increased proliferation and differentiation, independent of PPARβ/δ-mediated transcriptional activation. Moreover, lack of hGASP-1 inhibited human myoblasts proliferation and differentiation. Collectively, the results show that PPARβ/δ enhances myogenesis by modulating Mstn activity via Gasp-1. Finally, taken together the results demonstrate a very significant role of Mstn in glucose and fat metabolism in muscle which involves regulation of key molecules like Cblb, Pid1 and PPARs.
DRNTU::Science::Biological sciences::Molecular biology