MiR-34a-5p Promotes Hepatic Gluconeogenesis By Suppressing SIRT1 Expression

Objective Elevated hepatic gluconeogenesis plays an important role in hyperglycemia in type 2 diabetes.The molecular mechanism of abnormal gluconeogenesis in the liver is helpful for us to further understand diabetes.Micro RNAs(miRNAs)inhibit m RNA translation,reduces the level of target gene protein,and participates in a variety of metabolic diseases,such as obesity,lipid metabolic disorders and diabetes.Mi RNAs play an important role in hepatic glucose output by regulating hepatic gluconeogenesis.It has been found that the level of miR-34a-5p in liver is up-regulated in the liver of animal models with abnormal hepatic gluconeogenesis,such as db/db mice,ob/ob mice and type 1 diabetic mice induced by STZ.The relationship between miR-34a-5p and hepatic gluconeogenesis has not been fully elucidated.The purpose of this study is to explore the effect of miR-34a-5p on gluconeogenesis.In addition,the transcription of miR-34a-5p is regulated by acetyltransferase CREB binding protein(CBP)and p300.We further explore whether A485,the inhibitor of CBP/p300,downregulated hepatic gluconeogenesis through miR-34a-5p.Methods 1.Download miRNAs data sets(GSE13840,GSE17035,and GSE39752)and data were analyzed using an online database,GEO2 R.2.The prediction of miRNA targets was performed with miRDB and Target Scan.3.KEGG pathway analysis was performed for the common target genes predicted by Target Scan and miRDB.4.The application of q RT-PCR was to detect the expression of miR-34a-5p and the key gluconeogenesis enzymes in the livers of db/db and db/m mice.5.q RT-PCR was used to explore the expressions of miR-34a-5p and PCK1 in livers of fed,fasting,and refed C57BL/6 mice.6.To verify the effect of c AMP on miR-34a-5p,q RT-PCR was used to detect the expression of miR-34a-5p and PCK1 in primary mouse hepatocytes.7.The effect of miR-34a-5p on hepatic gluconeogenesis was observed by miR-34a-5p mimic and miR-34a-5p inhibitor.q RT-PCR,Western Blot,and cellular immunofluorescence were used to observe the related molecular mechanism.8.EX-527 was used to study the effect of SIRT1 on hepatic gluconeogenesis.The mechanism of EX-527 regulating hepatic gluconeogenesis was studied by immunoprecipitation technique.9.The toxic effect of EX-527 on primary mouse liver cells was studied by CCK8.10.4-week-old male db/db mice were randomly divided into A485 and control groups.Mice were intraperitoneally injected with A485 for 1 week.Gluconeogenic changes were then analyzed.11.Primary hepatocytes transfected with miR-34a-5p mimic and incubated with A485.q RT-PCR and western blot were used to detect the related molecular mechanism.12.4-week-old male db/db mice were injected with Ad-miR-34a-5p inhibitor and its control Ad-Con via tail vein,the effect of miR-34a-5p on hepatic gluconeogenesis was detected in vivo.Results 1.GEO2 R analysis of the GEO database showed that miR-34a-5p was up-regulated in all three data sets;2.KEGG pathway enrichment analysis showed that miR-34a-5p target genes were related to gluconeogenesis/ glycolysis;3.qRT-PCR results showed that miR-34a-5p was highly expressed in the liver of db/db mice;4.Fasting increased the level of PCK1 expression in the liver of C57BL/6 mice,but did not affect the expression of miR-34a-5p;5.c AMP increased the level of PCK1 expression in hepatic gluconeogenesis,but did not affect the expression of miR-34a-5p;6.The overexpression of miR-34a-5p promoted the expression of hepatic gluconeogenesis-related genes;7.EX-527 promoted hepatic gluconeogenesis and the expression of PEPCK protein,but did not affect the level of PCK1 m RNA;8.EX-527 increased the stability of PEPCK protein by reducing the ubiquitination of PEPCK;9.A485 decreased the expression of miR-34a-5p and PEPCK in the liver of db/db mice,but increased SIRT1 protein level;10.A485 reduced hepatic gluconeogenesis by inhibiting miR-34a-5p.11.Ad-miR-34a-5p inhibitor downregulated hepatic gluconeogenesis in db/db mice in vivo.Conclusions 1.Mi R-34a-5p promotes hepatic gluconeogenesis by targeting SIRT1-regulated PEPCK protein stability.Moreover,we observed an increased level of acetylation and a decreased level of ubiquitin in PEPCK protein in primary mouse hepatocytes treated with EX-527.2.A485 inhibits hepatic gluconeogenesis via inhibiting the miR-34a-5p/SIRT1 pathway.