| Objective Long-term high fat diet(HFD)causes hepatic fat accumulation,which is a specific manifestation of obesity in the liver.Previous studies have demonstrated that Pescadillo1(PES1)is an important regulator in the disorders of glucolipid metabolism in diabetes,but its role in hepatic fat accumulation is unknown.Metformin(Met)is a conventional first-line glucose-lowering drug in clinical practice,and mainly used in diabetic patients.Some studies in recent years have found that Metformin plays a role in reducing hepatic fat accumulation,but its mechanism of action is not uncertain.Autophagy is closely related to lipid metabolism and is an important pathway for the breakdown and metabolism of accumulated lipids in hepatocyte species.It can break down lipids into free fatty acids by wrapping lipid droplets,forming autophagosomes,and then combining with lysosomes to form autophagic lysosomes.Autophagy reflects the level of triglycerides to a certain extent,and can effectively regulate the glycolipid metabolism in the liver.Autophagy can play the role of removing damaged organelles,renew the organelles,and play a protective role for the liver.What is more,The main experiment of this subject is to explore:1.Whether PES1 is involved in the pathogenesis of hepatic lipid accumulation in mice fed a high-fat diet.2.Whether metformin enhances autophagy and improves hepatic lipid accumulation by downregulating PES1.MethodsNormal healthy male C57BL/6J mice were treated with conventional chow,high-fat chow and high-fat chow plus metformin drug intervention for 12 weeks(8 mice/group).The general characteristics of the mice such as body weight,blood glucose,diet and water intake were observed.And to observe the effect of metformin drug intervention on glucose tolerance and insulin tolerance in mice fed high-fat diet.Total cholesterol(TC)and triglycerides(TG)levels were measured in serum and liver.H&E and oil red O staining were performed to observe the lipid accumulation in the liver.Western blotting was performed to detect the expression of autophagy-related proteins.Mc Ardle 7777 rat hepatocytes were treated with oleic acid(OA)and palmitic acid(PA)in vitro to mimic the effects of HFD in vivo,and then treated with metformin drug.The levels of TC and TG in cells and medium were measured,and the changes on autophagyrelated protein expression after OA+PA and OA+PA+Met treatment of cells were detected by Western blotting.In vitro transfection of Mc Ardle 7777 rat hepatocytes with si RNA knockdown of Pes1 resulted in a significant decrease in TG levels in cells and medium,while TC levels were not significantly altered,and Western blotting assays showed a significant decrease in protein levels of PES1,p62 and a significant increase in Beclin1,LC3 B.The levels of TC and TG in Mc Ardle 7777 rat hepatocytes and culture medium were measured by treatment with metformin on the basis of PES1 overexpression,and the effect of PES1 overexpression plus metformin treatment on autophagy-related proteins was detected by Western blotting.ResultsMetformin intervention for 12 weeks significantly reduced body weight,fasting glucose and glucose tolerance,and liver and serum TG and TG levels in HFD-fed mice,while it had no significant effect on insulin tolerance.Western blotting showed that metformin significantly reduced protein levels of PES1,p62 and elevated Beclin1,LC3 B in the livers of HFD-fed mice.Oil red O staining and H&E staining showed that metformin significantly reduced lipid droplets and fat vacuoles in the liver of HFD-fed mice.OA+PA treatment of Mc Ardle 7777 rat hepatocytes resulted in a significant increase in the levels of TG and TC in the cells and medium,and a significant decrease in the levels of both after metformin treatment on top of this.Western blotting assay showed that metformin treatment decreased the protein levels of PES1,p62 and increased Beclin1,LC3 B in the cells.In vitro transfection of Mc Ardle 7777 rat hepatocytes with si RNA knockdown of Pes1 resulted in a significant decrease in TG levels in cells and medium,while TC levels were not significantly altered,and Western blotting assays showed a significant decrease in protein levels of PES1,p62 and a significant increase in Beclin1,LC3 B.After in vitro transfection of Mc Ardle 7777 rat hepatocytes overexpressing the Pes1 plasmid,the TG levels in cells and medium showed a significant increase,while the TC levels did not change significantly,and Western blotting showed that the levels of PES1,p62 were significantly increased and Beclin1,LC3 B were significantly decreased,while all trends were significantly reversed after treatment with metformin on top of overexpression of PES1.ConclusionsMetformin enhances autophagy,lowers triglyceride levels and improves hepatic lipid accumulation induced by high fat diet through downregulation of PES1. |
