Reversal of cellular senescence and insulin resistance in Skeletal muscle cells, via p38 mitogen-activated protein kinase inhibition

Abstract

Abstract Aims/hypothesis; Increased accumulation of senescent cells with ageing is associated with reduced ability of insulin-target tissues to utilise glucose, resulting in increased insulin resistance and glucotoxicity. We investigated the role of senescent-associated secretory phenotype (SASP) within C2C12, skeletal muscle cells on glucose homeostasis and if such effects can be reversed by blocking pro-inflammatory pathways. Methods; C2C12 myotubes were treated with 40% conditioned media from senescent fibroblasts. Indirect glucose uptake was measured, along with glycogen content and cell viability. The effect of SASP on the generation of reactive oxygen species and mitochondrial density in C2C12 myotubes was measured using Mitosox and Mitotracker staining. To assess the effect of blocking pro-inflammatory pathways on cellular senescence, above experiments were repeated with a p38 inhibitor and a western blot was completed using antibodies to NF-κβ in the presence and absence of conditioned media from senescent cells. Results; 40% SASP treatment significantly decreased glucose utilisation and glycogen storage, without reducing cell viability within mature C2C12 myotubes (p = < 0.0001). 40% SASP was successful in inducing oxidative stress and increased mitochondrial density following 48 hours of incubation (p = < 0.0001). Blocking NF-κβ, the pro-inflammatory signalling cascade restored glucose utilisation (p = < 0.01) despite the presence of 40% SASP. Co-incubation of 40% SASP with an NF-κβ inhibitor eliminates excessive reactive oxygen species production and restored mitochondrial density to levels comparable to control treatment. The role of NF-κβ in propagating cellular senescence was confirmed by increased p50 protein expression in C2C12 myotubes cultured in 40% SASP for 48 hours. Conclusion; Our study shows changes in glucose homeostasis in senescent cells is likely to be mediated through SASP and this effect can be reversed by blocking proinflammatory pathways. Targeting these pathways to reduce inflammation could be a potential treatment for individuals experiencing age-related insulin resistance.