Ketogenic Diet Ameliorates The Lipid Dysregulation In Type 2 Diabetic Mice By Downregulating Hepatic Pescadillo1

Background and aim:Type 2 diabetes mellitus(T2DM),characterized by insulin resistance(IR)and pancreatic ?-cell dysfunction,is a heterogeneous metabolic disorder.And it became a global epidemic over the past few decades.The current treatments for T2DM mainly include surgery,drugs and non-drug methods.Ketogenic diet(KD),has been proposed to attain a remarkable decline of hyperglycemia in T2DM.Due to the high fat content in KD,the bad outcomes by the long-term consumption of KD,including no significant weight loss,lipid accumulation,and liver fibrosis,were also reported in some studies.Previous studies have reported that PES1 is related to various tumors,and lipid metabolism.And the relationship between T2DM and tumors has also been studied in many publications.However,the role of PES1 in regulating T2DM related lipid metabolism and the effect of ketogenic diet(KD)on PES1 have not been reported.The aim of present study is to explore the role of PES1 in effects of KD on diabetic mice and its mediated mechanism.Methods:Normal controls were the subjects with gastric cancer,and diabetic patients were the subjects with gastric cancer plus diabetes.Liver biopsy was taken from the curative surgery.Western blotting and qRT-PCR were utilized to measure the expression of PES1 or PES1 in the livers of the control and case groups.Pearson correlation analysis was applied to evaluate the relationship between hepatic PES1 mRNA levels and plasma lipid profiles.Simultaneously,normal(C57BL/6J)and T2DM mice(KKAy)were fed with normal diet and KD for 16 weeks(n=10-12/group).The general characteristics of mice,such as body weight,fasting plasma glucose,and the intake of diet and drinking water,were observed.And the effect of KD intervention on glucose and insulin tolerance in T2DM mice were also measured.The levels of total cholesterol(TC)and triglycerides(TG)in the serum and liver were measured.Liver lipid accumulation was observed by using hematoxylin-eosin(H?E)and oil red O staining.The expressions of lipid metabolismrelated proteins were detected by Western blotting.McArdle 7777 rat hepatoma cells were treated with ?-hydroxybutyric acid(?-HB)to simulate the effect of KD in vivo.The TC and TG levels in cells and media were measured.The effect of ?-HB on lipid metabolism-related proteins was detected by Western blotting.The expression and localization of PES1 were tested by immunofluorescence.The upstream regulator of PES1 was probed by Chromatin immunoprecipitation(ChIP)experiment.In addition,Pes1 knockdown was performed to detect the levels of TC and TG in cells and media in vitro.And Western blotting was used to detect the effect of PES1 knockdown on lipid metabolism-related proteins.And the levels of TC and TG in cells and media were assessed by overexpression of PES1 plus ?-HB treatment.Western blotting was used to explore the effect of PES1 overexpression plus ?-HB treatment on lipid metabolism-related proteins.CRISPR-cas9 technology was used for conditional knockout(CKO)of Pes1 gene in mouse liver to detect TC and TG in serum and liver.And Western blotting was applied to detect the level of lipid synthesis-related proteins in liver to determine the regulation effect of PES1 on lipid metabolism.ChIP experiment was applied to explore the effect of ?-HB treatment in liver cells on PES1 mediated expression of p300.The Co-immunoprecipitation(CO-IP)experiment was used to probe the binding of p300 to SREBP1c under the conditions of in vitro and in vivo knockdown and overexpression of PES1.Besides,the acetylation of SREBP1c was also explored in the livers of KD-treated mice and the ?-HB-treated cells.Furthermore,the protein expression of NLRP3,Caspase1,and GSDMD was detected by Western blotting under the conditions of KD intervention in mouse liver,?-HBtreatment in cells,PES1 knockdown and overexpression in vitro,and knockout in vivo.The mRNA levels of IL-1? and IL-18 in liver and cells were measured by qRT-PCR.After the knockdown of Caspase1 in cells,the levels of TG and TC in the cells and the media,as well as the mRNA levels of IL-1? and IL-18 were detected in the cells.ChIP experiment was used to explore the effect of ?-HB treatment on the binding of PES1 to Caspase1 promoter.Results:Western blotting and qRT-PCR experiments suggested that the protein of PES 1 or the mRNA of PES1 in the liver of T2DM patients was significantly increased.Correlation analysis showed that the high expression of PES1 gene in the liver of T2DM patients is associated with the high level of plasma TG.After 16 weeks of intervention,KD significantly reduced the fasting blood glucose and insulin resistance of T2DM mice,and the levels of plasma and hepatic TG.Western blotting displayed that KD reduced CHOP,PES1,p300,SREBP1c,FASN and SCD1 in the livers of C57BL/6J and KKAy mice,respectively.However,no significant change in SREBP2 was observed.H?E and oil red O staining showed that fat vacuoles and lipid droplets were improved in the liver of KKAy mice fed by KD.Moreover,?-HB treatment in hepatocytes reduced the levels of TG in cells and media.Meanwhile,Western blotting indicated that ?-HB treatment reduced the protein levels of CHOP,PES1 p300,SREBP1c,FASN and SCD1 in cells.The immunofuorescence showed that ?-HB suppressed PES1 and SREBPlc proteins in nucleus of McArdle cells.After the knockdown Pes1,the levels of TG were decreased.Western blotting suggested that the protein levels of PES 1,p300,SREBP1c,FASN and SCD1 were reduced as well.After the overexpressing of Pes1,the levels of TG were increased.Western blotting showed that the protein levels of PES1,p300,SREBP1c,FASN and SCD1 were also elevated.However,these increases were eliminated by ?-HB treatment.The hepatic and plasma TG were significantly declined in Pesl-CKO mice.And the protein expression of p300,SREBP1c,FASN and SCD1 was dramatically decreased as well.These results are consistent with those of Pes1 knockdown in vitro.The levels of Recombinant NLR Family Pyrin Domain Containing Protein 3(NLRP3),Caspase1,Cleaved-Caspase1,gasdermin-D(GSDMD),and Cleaved-GSDMD were found to be signifcantly inhibited in normal and diabetic mice fed with KD,compared with those fed with SD.Simultaneously,KD led to much less infammation responses in mice than did SD,as indicated by decreased pro-infammatory factor levels(IL-1?and IL-18)in the murine livers,similar to the results of ?-HB treated hepatocytes.Furthermore,the silence or supplementation of Pes1 in vitro elicited the suppression or enhancement of NLRP3,Caspase 1,Cleaved-Caspase 1,GSDMD,Cleaved-GSDMD,IL-1? and IL-18 levels,respectively.More interestingly,the enhancement by overexpression of Pes1 was also impaired with ?-HB treatment in vitro.In addition,consistent with Pes1 silence in vitro,the similar results were observed in Pes1 knockout mice.In addition,after the knockdown of Caspase1,the levels of IL-1? and IL-18,as well as Caspase1 and GSDMD in the cells were dramatically decreased.Interestingly,the levels of TG in cells and media were also decreased.But the levels of TC in cells and media were unchanged.Mechanistically,we found that ?-HB decreased CHOP binding to the Pes1 promoters,resulting in the downregulation of PES1,thereby reducing PES1 binding to p300 and Caspase1 promoters.The inhibition of p300 and Caspase1 expression elicited the dramatic suppression of acetylation of SREBP1c via its interaction with p300,the decreased inflammation response,and the significant decrease in TG.Conclusion:(1)Enhanced hepatic PES1 expression in T2DM is correlated with increased levels of plasma TG.(2)Our current findings demonstrate that KD may ameliorate lipid dysregulation in type 2 diabetic mice by downregulating PES1 mediated acetylation of SREBP1c and inflammation pathways.(3)This study may provide a novel insight to understanding the mechanisms underlying T2DM related lipid dysregulation and contribute a new pharmaceutical target for T2DM treatment.