Role Of CIRP/AKT Signaling Pathway In Glucose Metabolism Of Gastrocnemius Muscle In Cold Exposed Mice

The climatic conditions are closely related to the development of animal husbandry.Ambient low temperature is one of the important factors hindering the development of animal husbandry.Especially in the cold regions of northern China,farmed animals are highly vulnerable to cold stimulation,resulting in abnormal energy metabolism and slow growth.Skeletal muscle heat production during acute cold exposure is the main source of increased body heat production.Cold-induced RNA binding protein(CIRP)is a stress-responsive protein that can be induced in response to low temperatures,which involves complex signaling pathways associated with a variety of cellular physiological processes.To determine the effect of CIRP on skeletal muscle glucose metabolism under cold exposure.Reveal that CIRP plays a regulatory role in skeletal muscle glucose metabolism in resisting cold exposure,so that the body maintains normal body temperature against cold stress,which not only lays a theoretical foundation for further exploration of the molecular regulation mechanism of cold stress,but also provides a theoretical basis for the prevention and control of animal husbandry chilling.In order to study the effects of acute cold exposure on skeletal muscle glucose metabolism and CIRP protein expression in mice,ICR mice were selected as the experimental subjects,which were randomly divided into normal temperature control group,cold exposure 2 h group,cold exposure 4 h group and cold exposure 8 h group.In order to clarify the effect of acute cold exposure on skeletal muscle glucose metabolism in mice,the blood glucose,glucagon,insulin and the expression of CIRP,AKT signaling pathway-related proteins in skeletal muscle were detected.The results showed that after acute cold exposure,the blood glucose level of the mice was significantly reduced,glucagon was significantly increased,and insulin levels were regulated during cold exposure.Acute cold exposure caused an increase in CIRP protein in skeletal muscle of mice,and increased AKT,GSK3?,PFKFB2 protein phosphorylation and Glut4 protein expression levels,and decreased GS phosphorylation.In addition,acute cold exposure significantly increased the ratio of Bcl-2 to Bax protein,while Cleaved Caspase-3protein expression did not change significantly.To investigate the effect of CIRP on glucose metabolism in myoblasts of C2C12 mice,we used CRP overexpressing lentivirus to infect C2C12 mouse myoblasts,and placed them at 32 °Cwith wild-type cells for sub-low temperature treatment to detect CIRP protein expression,AKT Signaling pathway and expression of apoptosis-related proteins.The results showed that in wild-type cells,mild hypothermia treatment significantly increased CIRP protein expression in C2C12 cells,and protein expression levels of AKT,GSK3?,PFKFB2 phosphorylation and Glut4 were significantly increased,and GS phosphorylation levels were significantly decreased.Mild hypothermia treatment also increased the expression of Cleaved Caspase-3 protein and the ratio of Bcl-2 and Bax protein expression in C2C12 cells.In addition,compared with the wild-type cells in the blank group,AKT,GSK,PFKFB2 phosphorylation and Glut4 protein expression levels were up-regulated and the phosphorylation level of GS was significantly reduced after infection with CIRP overexpressing lentivirus.Among the cells infected with CIRP overexpressing lentivirus,the expression level of Cleaved Caspase-3 protein was significantly lower than that of wild type cells.The ratio of Bcl-2 and Bax protein expression was significantly higher than that of wild type cells.To further validate the regulatory relationship between CIRP and AKT under cold exposure conditions and the effect of AKT signaling pathway on glucose metabolism changes induced by acute cold exposure in mice,we designed CIRP in vitro interference and AKT in vivo inhibition assay.4 h was selected as the optimal time for cold exposure and mild hypothermia treatment,and CIRP protein expression,AKT signaling pathway and apoptosis-related protein expression were detected.In vitro interference assay results showed that CIRP si RNA significantly reduced the phosphorylation level of AKT in C2C12 cells.In vivo inhibitor test results showed that injection of wortmannin significantly reduced GSK3?,PFKFB-2 protein phosphorylation and Glut4 protein expression levels.In addition,after the injection of wortmannin,the expression of Cleaved Caspase-3 protein was significantly increased,while the ratio of Bcl-2 to Bax protein expression was significantly reduced.The results of this study indicate that acute cold exposure can increase the expression of CIRP protein,thereby regulating skeletal muscle glucose metabolism through AKT signaling pathway to maintain cell energy balance under cold exposure conditions,thereby slowing the apoptosis of skeletal muscle cells induced by acute cold exposure.