Souradip Mallick, National Institute of Technology, Rourkela
Type 2 diabetes (T2D) is a systemic and progressive disease characterized by hyperglycemia causing dysfunction in beta cells and peripheral insulin resistance affecting more than 370 million people worldwide. Insulin mediates its physiological effects through the binding to the insulin receptor (INSR) at the plasma membrane level of target cells. There are two INSR isoforms, A and B. The B isoform differentially expressed in insulin-responsive tissues such as the liver, adipose tissue, and skeletal muscle. Insulin binding to INSR activates insulin receptor tyrosine kinase leading to the subsequent phosphorylation of the insulin receptor substrate 1(IRS-1). Phosphorylated IRS1 recruits and activates phosphatidylinositol-3 kinase leading ultimately via multiple signaling molecules including AKT and RAC1 to the translocation of the glucose transporter type 4(GLUT-4) to the plasma membrane and the subsequent glucose uptake. The reduction in membrane INSR levels explains the insulin resistance triggered by obesity. It was observed that more than 80% of patients with T2D are oversized.
Mitsugumin 53(MG53) is a novel regulator of insulin signaling in skeletal muscle regulating cell membrane repair machinery. During myogenesis, its expression is enhanced and promotes membrane repair by interacting with dysferlin-1 and caveolin-3. Intravenous injection of recombinant MG53 improves skeletal muscle damage. It has been reported that MG53 regulates the insulin signaling pathway in vitro and more importantly in vivo. MG53-deficient mice are resistant to diet-induced obesity and glucose intolerance. This was associated with obesity, hypertension, and dyslipidemia
The expression of MG53 was determined by qPCR across various mice models of insulin resistance. It was not regulated in a consistent manner across models and muscle types (gastrocnemius versus soleus). Its expression is significantly lower in gastrocnemius from both HFD and ob/ob mice while no change in soleus.
To detect the role of MG53 in insulin signaling in vitro, loss of function experiments was performed using small interfering RNA in differentiated C2C12 cells. Transfection of RNAi targeting MG53 led to a robust and reproducible gene knock-down compared to siCTL or Mock transfected cells which resulted in a complete reduction in MG53 protein levels in C2C12 cells.
HEK293 cells were selected as a simple cellular system with a good response to insulin and devoid of endogenous MG53 expression. MG53 behaves as a glucose-responsive myokine able to control peripheral insulin sensitivity by allosterically regulating the insulin receptor. Treatment with recombinant human MG53 did not result in significant inhibition of insulin-mediated AKT phosphorylation in both C2C12 cells and HSMMs.
Transcription and translation start sites as well as the Ring and B-Box Zn finger domains were deleted leading to the absence of mg53 transcription. This approach led to the generation of mg53 -/- mice in C5BL/6J genetic background. These mice have no difference compared to wild type mice even though mg53 -/- mice were born at less than the expected ratio. Hence it was concluded that MG53 is not a critical regulator of an insulin signaling pathway in skeletal muscle.
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