Effects Of O-GlcNAcylation Modification On Glucose Metabolism In Gastrocnemius And Liver Of Acute Cold-Exposed Mice And Piglets

Ambient low temperature is one of the main stressors faced by animals in the cold regions of northern China.Long-term low feeding temperature or acute cold exposure has a great impact on animal growth performance,muscle characteristics and meat quality,seriously endangering the development of animal husbandry in the northern cold regions.After cold exposure,the body maintains normal body temperature by enhancing its own metabolic activity and skeletal muscle contraction.Skeletal muscle accounts for 40% to 60% of the body weight and is an important organ for energy storage and utilization.The function and energy level of skeletal muscle during cold exposure play an important role in the normal functioning of the body’s exercise capacity.Studies have shown that O-Glc NAcylation can affect the growth and development of skeletal muscle and its physiological functions.O-Glc NAcylation as one of the post-translational modification types of proteins,has important biological functions and can regulate various biological processes to maintain normal physiological functions of cells,such as signal transduction,proteasome activity,apoptosis,transcription and translation.It has been reported that O-Glc NAcylation can play a role of "nutrition and stress receptors" in the body’s stress and metabolic regulation.Then,the role of O-Glc NAcylation in the regulation of energy metabolism in the body under acute cold exposure conditions is still unclear.Analyze the response of piglet skeletal muscle and liver tissue to cold stress,and then clearly regulate the signal transduction mechanism of glucose metabolism,which will help to solve the cold resistance and mortality of young animals in the northern alpine region.Higher realities are of great significance to improving the survival rate of young animals and ensuring the sustainable and healthy development of animal husbandry.To investigate the effects of acute cold exposure on body behavior,this study used C57BL/6 mice as the research object to establish a cold stress model.Mice were divided into cold exposure test group（cold exposure 2 h,4 h,6 h,8 h,10 h and 12 h,4℃）and normal temperature control group（28℃）.Before the cold stimulation,each group of mice was weighed and recorded one by one,and the body weight,body temperature measurement and open field test were performed immediately after the cold stimulation.The results showed that the exercise distance and standing frequency of the cold-exposed group were significantly lower than those of the normal-temperature control group in the open field test,the exercise capacity was decreased,the spontaneous activity were significantly weakened（P<0.05）,and the total exercise time and rest time were compared.With the prolongation of cold exposure time,the body temperature of the mice was significantly lower than that of the control group（P<0.01）,and the body weight was not significantly different from the control group（P>0.05）.To investigate the effect of O-Glc NAcylation on glucose metabolism in gastrocnemius and liver of C57BL/6 mice under acute cold exposure conditions,this study collected cold exposure for 2 h,4 h,6 h,8 h,10 h and 12 h.The gastrocnemius,liver and blood of mice were tested for plasma glucose,insulin,glucagon levels,ATP,ADP and AMP contents,and O-Glc NAc,OGT,Akt expression in tissues.The expression levels of glucose metabolism and apoptosis-related proteins were detected.The results showed that acute cold exposure caused a significant decrease in plasma glucose levels（P<0.05）,a significant increase in glucagon levels（P<0.01）,and insulin levels show fluctuation regulation（P>0.05）,ATP/（ADP+AMP）contents showed temporal fluctuations（P>0.05）,promoted O-Glc NAcylation and expression of OGT protein in gastrocnemius and liver tissues,and promoted Akt protein activation.Induction of Akt downstream glucose metabolismassociated protein AS160（glucose transport key protein）,Glut4（glucose transport key protein）,GSK3β（glycogen synthesis key protein）,GS（glycogen synthesis）and PFKFB2（glycolysis key protein）phosphorylation.Before and after cold exposure the ratio of apoptosis-related protein Bcl-2/Bax was significantly different（P<0.05）,while Cleaved-Caspase-3（P>0.05）was no significant change.The gastrocnemius and liver tissues of cold-exposed mice showed a sequential change in response.To investigate the effects of skeletal muscle OGT^-/-conditional knockout on glucose metabolism in gastrocnemius and liver of C57BL/6 mice under acute cold exposure conditions,this study constructed skeletal muscle-specific OGT^-/-knockout mice.Wild-type and skeletal muscle-specific OGT^-/-knockout mice were given cold stimulation at 4℃ for 0 h and 4 h,collecting gastrocnemius,liver and blood samples.Detecting plasma glucose,insulin levels,the levels of glycolytic intermediates FDP and end products PA,and O-Glc NAcylation,OGT protein expression,IRS-1/Akt pathway and downstream glucose metabolism-related proteins and Bcl-2,Bax,Caspase-3 and other apoptosis-related proteins.The results showed that blood glucose and insulin levels were significantly lower in skeletal muscle-specific OGT^-/-knockout mice than wild-type mice underacute cold exposure（P<0.05）.FDP and PA levels in gastrocnemius muscle were extremely high significantly elevated（P<0.01）,but there was no significant change in the liver（P>0.05）.It promoted the expression of IRS-1/Akt and downstream glucose metabolism-related proteins,and enhance the body’s glucose metabolism and systemic insulin sensitivity,as while promote apoptosis associated protein expression was significantly increased,cytoprotective effects of weakening.In order to further explore the effects of cold exposure on the gastrocnemius and liver of piglets,and make it more suitable for production practice,this study selected the commonly culture breeding variety "Du × Chang × Bai" ternary hybrid piglets as the research object,collected gastrocnemius,liver and blood samples.Detecting blood glucose levels,ATP,ADP and AMP contents,O-Glc NAc,OGT,Akt,and glucose metabolism and apoptosis-related proteins.There was no significant change in plasma glucose levels in piglets after acute cold exposure（P>0.05）,but it could promote the expression of O-Glc NAcylation and OGT protein in gastrocnemius and liver,promote the activation of Akt protein.Furthermore,phosphorylation of glucose metabolismrelated proteins downstream of Akt was induced,and the gastrocnemius and liver tissues of coldexposed piglets showed a continuous increase in response to cold stress.In summary,the global O-Glc NAcylation levels in the gastrocnemius and liver of C57BL/6 mice changes dynamically during acute cold exposure,and the body’s glucose metabolism and insulin sensitivity are regulated by the IRS-1/Akt signaling pathway.It is also involved in regulating the expression of apoptosis-related proteins and achieving cytoprotective effects under stress conditions.O-Glc NAcylation can also induce Akt activation in gastrocnemius and liver during acute cold exposure of piglets,regulate downstream glucose metabolism and expression of apoptosis-related proteins,and achieve cytoprotective effects under glucose regulation and stress conditions.