Molecular Mechanism Of Adenine Nucleotide-Mediated Gluconeogenesis Under Hepatic Stress

The liver produces glucose through glycogenolysis and gluconeogenesis.Gluconeogenesis is a physiological function necessary for survival,but the abnormal increase of gluconeogenesis in the liver significantly promotes diabetic fasting hyperglycemia.The transcription factor FOXO1,which is closely related to the stress response,plays a dominant role in activating hepatic gluconeogenesis.Insulin inhibits hepatic gluconeogenesis by phosphorylating FOXO1 under physiological conditions.Disruption of Foxo1 in a liver-specific manner preserves normal gluconeogenesis function in mice with targeting insulin signaling.The expression of the Foxo1 gene is elevated in the livers of diabetic mice and insulin resistance patients.Suppression of Foxo1 transcription may be an insulin-independent strategy for the treatment of diabetes mellitus.Therefore,it is important to elucidate the transcriptional regulation mechanism of Foxo1 in type 2 diabetes.First,varieties of stress simulation models were established to explore the changes of adenine nucleotides under stress.It was found that mice under various conditions such as toxin stress(capsaicin and carbon tetrachloride),hypoxia stress,injury stress,and starvation stress,showed two common characteristics:first,the level of hepatic gluconeogenesis was increased in the liver of mice under stress;second,the concentration of plasma 5'-AMP,adenosine and uric acid increased accompanied by the increase of intracellular adenosine and uric acid.The activation of hepatic gluconeogenesis and the change trend of purine metabolites in type 2 diabetic mice were similar to the characteristics of mice under stress.These results suggested that gluconeogenesis under stress may have a similar mechanism with enhanced hepatic gluconeogenesis in type 2 diabetes.Transcriptome analysis suggested that 5'-AMP stimulated the Foxo pathway.Treatment of 5'-AMP increased Foxo1 transcription and the gene expression of downstream gluconeogenesis-related genes phosphoenolpyruvate carboxykinase(Pepck)and glucose-6-phosphatase(G6Pc)in the liver.5'-AMP treatment resulted in higher blood glucose changes using pyruvic acid,glycerol,lactic acid,fructose,or glutamine as gluconeogenesis carbon sources.Transcription inhibition could block the increase in Foxo1 gene levels caused by 5'-AMP.5'-AMP could not affect the degradation of Foxo1 m RNA.The results indicated that 5'-AMP upregulated Foxo1expression at the post-transcriptional level.5'-AMP is degraded into adenosine by CD73 on the cell membrane.Theophylline,a nonspecific adenosine receptor antagonist,could not inhibit 5'-AMP stimulated Foxo1 transcription in cultured hepatocytes.Dipyridamole,an adenosine transcription inhibitor,could block the increase in Foxo1gene levels caused by 5'-AMP.These results suggested that 5'-AMP stimulated Foxo1expression by increasing intracellular adenosine concentration.Adenosine is an inhibitor of S-adenosine homocysteine(SAH)hydrolase.The increase of adenosine resulted in the decrease of intracellular S-adenosylmethionine(SAM)/SAH ratio,which reduced the potential of intracellular methylation.By using betaine and cycloleucine in mice,we were able to change the ratio of mouse SAM/SAH and thereby changed the expression of Foxo1.In type 2 diabetic mice,the use of betaine increased the SAM/SAH ratio and reduced the expression of Foxo1.The reduced SAM/SAH ratio induced by 5'-AMP treatment resulted in the change of histone H3K9 methylation level in the liver,especially in the promoter of Foxo1,G6Pc,and Pepck,which may be related to the increased transcription of these genes.Transcriptomic profiling further revealed that the expression of hepatic circadian clock genes changed significantly in mice under stress and treated with 5'-AMP,manifested by the increase of Per1.Quantitative PCR analysis showed high expression of the Per1 in mice treated with carbon tetrachloride or 5'-AMP and db/db mice.The absence of Per1 in mice decreased glucose production.However,the absence of Per1did not affect the expression of Foxo1 and the related gluconeogenesis-related genes Pepck and G6Pc.Further examination demonstrated the expression of pyruvate transporter genes(Mpc1 and Mpc2)increased,and the expression of lactate dehydrogenase(Ldha)decreased in the liver of Per1^-/-mice.In wild-type mice,liver pyruvate had a rhythmic activity that varies between day and night.The absence of Per1 in mice decreased the concentration of hepatic pyruvate,increased the concentration of lactic acid,and lost rhythmicity.These results suggested that the effect of Per1 on gluconeogenesis was independent of Foxo1 signaling,and reducing the expression of the Per1 gene under stress may be a new strategy to improve gluconeogenesis.Conclusion:(1)Increased plasma concentration of 5'-AMP,adenosine and uric acid,and increased intracellular adenosine and uric acid were typical features of various stress responses including type 2 diabetes;(2)Adenosine enhanced gluconeogenesis by promoting Foxo1 transcription;(3)Under stress,adenosine stimulated the expression of circadian clock gene Per1,and the decrease of Per1 reduced the level of gluconeogenesis independently of Foxo1 signaling.