Er-Miao-Fang And Berberine Restrain Hepatic Gluconeogenesis Via Regulation Of Protein Acetylation

Nowadays the incidence of diabetes is increasing globally due to nutirent-rich food,and emerges as a challenge to human health.Traditional Chinese Medicine(TCM)interprets the pathological basis of metabolic disorders including diabetes from different aspects,regarding phlegm stagnancy,blood stasis,irascibility,spleen deficiency and internal toxin as the main pathological factors.According to the theory of TCM,therapeutical stratigies are desinged to invigorate spleen and reduce phlegm,promote blood circulation and remove blood stasis,clear liver and heat,purge fire and detoxify are confirmed to have beneficial effects in the management of metabolic disordors.The Er-Miao-Fang(EMF,the ethanol extract of Er-Miao-San.In order to distinguish from Er-Miao-San,here we called Er-Miao-Fang)is a traditional Chinese medical prescription consisting of two herbs namely Cortex Phellodendri and Rhizoma Atractylodis,exerting the ability of strengtheniing the spleen in transportation and clearing heat with the removal of dampness.The prescription has positive effect on the regulation of glucose metabolic homeostasis.Enhanced hepatic gluconeogenesis is responsible for the elevated levels of fasting blood glucose in metabolic diseases like diabetes,but it is only recently that the role of disregulated gluconeogensis in the development of diabetes has attracted attention.Protein functions can be regulated at different levels,and lysine acetylation modification is involved in the regulation of metabolic enzyme activity,implicated in the regulation of glucose,lipid and energy metabolism.Therefore,selectively targeting gluconeogenic enzymes to prevent excessive hepatic gluose output may be a potential therapeutical stratigy for the treatment of diabetes and the development of new drug.Aims:Peroxisome proliferator-activated receptor-γcoactivator-1α(PGC-1α)is a co-factor in the transcriptional regulation of gluconeogenic enzymes,while mitochondrial pyruvate carrier(MPC)mediates mitochondrial transport of pyruvate,providing the substrate for hepatic glucsoe production.Based on this,this study investigated the effects of EMF and its main active alkaloid component berberine on hepatic gluconeogenesis with focus on the regulation of PGC-1αand MPC1 protein acetylation,aiming to elucidate the molecular pathways and mechanism underlying their action.Methods:Animal models in fasted mice and high-fat diet(HFD)-fed mice,and cultured cell model in isolated hepatocyte treated with palmitate(PA)were established.With the combination of animal and cell experiments,we investigated inhibitory effects of EMF and berberine on hepatic gluconeogenesis,lipid ectopic deposition and their regulatory pathway.Moreover,the molecular mechanisms by which EMF and berberine regulate PGC-1αand MPC1 acetylation to restrain hepatic glucose production were investigated.Results:EMF and berberine lowed fasting blood glucose by inhibiting hepatic gluconeogenesis in HFD-fed and fasted mice.EMF and berberine downregulated the levels of fasting blood free fatty acid,and reduced hepatic lipid ectopic accumulation in HFD mice.Subsequently,we explored the molecular mechanism of EMF and berberine on the improvement of hepatic gluconeogenesis from the aspect of PGC-1αand MPC1protein acetylation.The results showed that EMF reduced the ratio of NAD~+/NADH in the liver,thereby inhibiting deacetylase Sirtuin 1 SIRT1 activity located in the cytoplasm.Besides,EMF promoted protein phosphorylation levels of adenosine monophosphate-activated protein kinase(AMPK)and its downstream target acetyl-co A carboxylase(ACC)in a time-and dose-dependent manner under physiological condition.EMF increased the contents of acetyl-Co A required for GCN5-mediated PGC-1αprotein acetylation in hepatocytes through AMPK/ACC signaling and inhibited PGC-1αactivity via promoting protein acetylation,and thus inhibiting hepatic gluconeogenesis.Berberine reduced the levels of acetyl Co A in hepatocytes by limiting fatty acid oxidation,thus preserved pyruvate dehydrogenase(PDH)activity with suppression of pyruvate carboxylase(PC),diminishing the impact of acetyle Co A to promote mitochondrial pyruvate oxidation.In addition,berberine inhibited hepatocyte mitochondrial respiration and down-regulated NAD~+/NADH in the liver tissue,and thus increased the acetylation level of MPC1 by inhibiting the mitochondrial deacetylase SIRT3 activity.As expected,berberine impaired MPC1 stability to promote protein degradation,resultantly reduced hepatic glucose output via blocking MPC1-mediated mitochondrial pyruvate transport.Conclusion:EMF as well as berberine inhibited lipid ectopic deposition in the liver of HFD mice and improved hepatic lipid metabolism,contributing to reducing hepatic gluconeogenesis.EMF improved PGC-1αacetylation by regulating AMPK/ACC signaling and inhibited PGC-1αprotein activity,thereby reducing hepatic glucose output.These results demonstrated the special role of AMPK in the regulation of liver metabolism by EMF.Berberine inhibited MPC1 protein activity via preserving the acetylation of MPC1,and thus blocked the transport of pyruvate into mitochondria for hepatic gluconeogenesis.We demonstrated that berberine could reduce hepatic glucose production independent of glucagon signaling.In conclusion,this study not only provide new insight into the theoretical basis of EMF,but also suggeste a novel therapeutic strategy for the treatment of metabolic diseases such as diabetes based on the regulation of protein acetylation.