The Molecular Mechanistic Study Of Purβ In The Regulation Of Hepatic Glucose Metabolism

The liver is an important organ that regulates glucose homeostasis.In the process of starvation,hypoglycemia can promote the secretion of glucagon by islet α cells.Glucagon binds to the glucagon receptor on the liver,promotes the breakdown of liver glycogen,increases the gluconeogenesis and stabilizes blood glucose.Obese and type 2 diabetes patients show enhanced glucagon signaling and increased liver gluconeogenesis,but the detailed mechanism is not entirely clear.Purβ is a purine-rich element binding protein that can bind to purine-rich single-or double-stranded DNA or RNA.In recent years,the transcriptional function of Purβhas been gradually explored.Whether Purβ regulates hepatic glucose metabolism is unclear.In this thesis,the molecular mechanism of Purβ in the regulation of hepatic glucose metabolism was studied in depth.The main findings are as follows:1.Purβ mRNA and protein levels were significantly increased under starvation or glucagon induction,suggesting that Purβ is most likely involved in hepatic glucose metabolism.To test this hypothesis,primary hepatocytes were isolated and infected with Ad-Purβ or Ad-shPURB to overexpress or knock down Purβ in hepatocytes,and then HGP assays were performed.The results show that overexpression of Purβ can promote gluconeogenesis in hepatocytes,and knockdown of Purβ can inhibit cellular gluconeogenesis.Specific knockdown of Purβ in the liver of db / db mice significantly reduced hyperglycemia in db / db mice,and alleviated glucose intolerance in db / db mice,indicating that hepatic Purβ is a key molecule that can regulate glucose metabolism.2.Overexpression or knockdown of Purβ did not change insulin-induced AKT phosphorylation,and specific knockdown of Purβ in the liver of db / db mice did not increase insulin sensitivity.These data suggest that Purβ regulates hepatic glucose metabolism independent of insulin signaling pathway.3.The expression of G6 Pase and PEPCK was significantly reduced in Purβ knockdown hepatocytes or db / db mice,which was most likely due to decreased pCREB level,indicating that Purβ not only inhibited the Glucagon / cAMP / PKA / CREB signaling pathway,but also inhibited gluconeogenesis.Conversely,the expression of G6 Pase and PEPCK was significantly increased in hepatocytes overexpressing Purβ,likely due to increased p-CREB levels.These data suggest that Purβ regulates glucose metabolism through the glucagon signaling pathway.4.RNA sequencing analysis was performed in Purβ-overexpressing and PurβKD hepatocytes,which showed that Adcy 6 may be a possible target gene of Purβ.The expression of liver-enriched Adcy6 was upregulated by overexpression of Purβ and downregulated by knockdown of Purβ.Similar to Purβ,hepatic ADCY6 expression could also be induced by fasting and glucagon.Hepatic ADCY6 expression was also abnormally elevated in the two obese mouse models.In addition,the results of luciferase assay and ChIP assay demonstrate that Purβ directly binds to the Adcy6 promoter(-1558 to-1850 region)and increases its transcription.In summary,Purβ is a previously undiscovered liver gluconeogenesis regulator.Purβ was shown to positively regulate HGP by directly binding to the promoter of the Adcy6 gene,increasing its expression and thereby enhancing the glucagon/ cAMP/PKA/CREB signaling pathway.Thereby enhancing liver gluconeogenesis and promoting the occurrence of hyperglycemia in obese patients.Purβ might serve as an important drug target for the treatment of hyperglycemia in patients with type 2 diabetes.