Investigation of Spexin Effects on Insulin‐Dependent Pathway Genes in High‐Glucose‐Induced Insulin‐Resistant C2C12 Myotubes

ABSTRACT

Diabetes is a global health concern, with type 2 diabetes mellitus (T2DM) being the most common form. T2DM is characterized by insulin resistance and impaired glucose uptake in skeletal muscle. Spexin shows hypoglycaemic effects, but its mechanism of action, especially in skeletal muscles, remains elusive. This study aims to investigate the effects of spexin on key genes associated with the insulin‐dependent pathway in C2C12 cells. The differentiation of C2C12 myoblasts to C2C12 myotubes was confirmed using the fusion index and gene expression of myogenic factor 5 ( Myf5 ), myosin heavy chain ( MyHC ), and myogenic regulatory factor 4 ( MRF4 ) analysis. Cytotoxicity of spexin at varying concentrations (100 nM, 200 nM, 400 nM, 800 nM, and 1000 nM) was assessed via MTS assay. Insulin resistance–like conditions were induced in C2C12 myotubes using a high‐glucose medium and characterized at the mRNA level by quantitative reverse transcription PCR (RT‐qPCR) of peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha ( PGC‐1α ), glucose transporter 4 ( GLUT4 ), and hexokinase II ( HKII ) genes. Fusion index analysis evaluated high‐glucose effects on myogenesis. RT‐qPCR examined spexin's effects on insulin receptor substrate 1 ( IRS‐1 ), phosphatidylinositol 3‐kinase ( PI3K ), and GLUT4 expression over different time points. Spexin concentrations at 1000 nM were not cytotoxic. High‐glucose incubation did not affect myogenesis yet was associated with downregulation of PGC‐1α and GLUT4 genes, consistent with a transcriptional profile typically observed in insulin‐resistant states. Spexin‐treated C2C12 cells statistically significantly downregulated the expression of IRS‐1 after 2 h of incubation. The effects of spexin on IRS‐1 , PI3K , and GLUT4 gene expression were comparable to metformin‐treated in insulin resistance C2C12 myotubes.