Metabolomic Analysis For The Effects Of α-ketoglutarate Supplementation On Skeletal Muscle Cells In Different Energy States

Skeletal muscle is the largest organ of the human body.It is particularly important to restore muscle strength and function when the muscle is damaged or insufficient muscle energy supply occurs during long-term exercise training or pathological processes.As the meeting point of organic carbon and nitrogen metabolism and also a key intermediate in the TCA cycle,α-ketoglutarate(AKG)has shown pleiotropic effects in clinical and animal experiments and can improve muscle performance.Given that glucose is the main energy source for maintaining the normal physiological functions of skeletal muscles,the energy state of muscles is closely related to muscle function.It can be expected that the effect of supplementing AKG in improving muscle performance largely depends on the glucose level in skeletal muscle.However,it is not yet clear what would be the metabolic difference of AKG on skeletal muscle in different energy states.In this article,we used normal medium(Nor)and low-glucose medium(Low)to mimic two different energy states,and we evaluated the effects of AKG on mouse C2C12 myoblasts and myotubes in normal medium(Nor-A)and low-glucose medium(Low-A).NMR-based metabolomics analysis was performed to screen the metabolic pathways that significantly changed by AKG supplementation,and the effects of AKG supplementation on C2C12 myoblasts and myotubes under different energy states were compared.Firstly,by culturing C2C12 myoblasts under normal and low-glucose conditions with or without AKG supplementation,we found that AKG enhanced the proliferation and differentiation of myoblasts.Secondly,we continued to supplement AKG during the differentiation stage from myoblasts to myotubes,and found that AKG promoted the formation and hypertrophy of myotubes.Two parts metabolomics analysis showed that AKG promoted the proliferation and differentiation of skeletal muscle cells by regulating glutamine and glutamate metabolism,oxidative stress and energy metabolism in two energy states.Under normal proliferation conditions,AKG up-regulated intracellular glutamine levels,changed cell energy status,and maintained cellular antioxidant capacity.Under energy-deficient conditions,AKG can be used as a metabolic substrate to reduce the dependence on glutamine of skeletal muscle cells,greatly enhanced the antioxidant capacity and increased the intracellular ATP content,thereby ensuring the growth and metabolism of cells in an insufficient energy condition.This article discussed the differences between AKG-induced metabolic changes and underlying metabolic mechanisms in two energy states of skeletal muscle cells(myoblasts and myotubes).Our work may benefit the further development of AKG supplements in the sports and therapeutic fields,thereby promote muscle recovery and energy supply in sports training,and delay muscle loss in people with wasting diseases.