Intervention Of Empagliflozin Or Combined With Metformin In Type 2 Diabetic Mice Ameliorates The Glucose And Lipid Homeostasis

Objective:To explore the protective effect of Empagliflozin or combined with Metforminon on diabetic dyslipidemia in addition to its anti-hyperglycemia in type 2 diabetic mice.Methods:1.Annimal treatments.Thirty-two db/db mice were randomly grouped and used forfour treatments(n=8/group): 10 ml/kg/day Nitrosol(0.5%)(group DM),250 mg/kg/day Metformin monotherapy(group DMT1),250 mg/kg/day Metformin plus 10 mg/kg/day Empagliflozin combined therapy(group DMT2),10 mg/kg/day Empagliflozin monotherapy(group DMT3),eight db/m mice as a vehicle group(group NC)received Natrosol(0.5%).The treatment periodwas 8 weeks.2.Measurement of murine plasma glucose and lipid parametersFasting plasma glucose(FPG)and body weight of mice were recorded weekly and liver weight was also recorded after mice being sacrificed.Mice hemoglobin A1c(HbA1c)and high-density lipoprotein cholesterol(HDL-C)were measured by ELISA.Plasma total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and triglyceride(TG)were determined using autoanalyzer.Hepatic TC and TG were determined by colorimetric method.Extracting RNA and protein from hepatic tissue homogenate,and then expressionof ApoA1,ApoB100 mRNA and protein were measured by qRT-PCR and westernblot,respectively.3.Hepatic tissue paraffin sectioning and staining,then the histology of hepatic tissues was observed.4.Statistical analysesData are expressed as mean ± standard deviation(SD).The following statistical tests were performed using SPSS19.0: one-way ANOVA and S-N-K(Student-Newman-Keuls).The graphs were maked by GraphPad Grism 5.P<0.05 were considered statistically significant.Results:1.Compared with the DM group,FPGlevels were significantly decreased in the groups of DMT1,DMT2 and DMT3 after three-weeks treatments(P<0.05).There was no difference in the four groups(NC,DMT1,DMT2 and DMT3)(P>0.05).At the end of this study,FPG levels in DM group were significantly higher than those in DMT1,while FPG levels in DMT1 group were higher than those in DMT2 and DMT3.Compared with DM group,HbA1 c levels in the groups of DMT1,DMT2 and DMT3 were significantly decreased(P<0.05).2.After three-weeks treatments,body weight of mice in group DM was significantly increased,compared with the groups of NC,DMT1,DMT2 and DMT3(P<0.001).At the end of study,liver weights of mice in groups of DMT2 and DMT3 were significantly decreased,compared with the groups of DM and DMT1(P<0.01,P<0.05,respectively).3.Plasma TC levels in db/db mice(groups of DM,DMT1,DMT2,DMT3)werehigher than those in db/m mice(NC group)(P<0.001),and significantly decreased by Empagliflozin or combined with Metformin in groups of DMT2 and DMT3(P<0.05).There was no difference in the levels of hepatic TC between db/m mice and db/db mice(P>0.05).HDL-C levels in group of DMT2 and DMT3 were significantly increased,compared with groups of NC,DM,DMT1(P<0.05).There was also no difference on LDL-C levels in the five groups(P>0.05).Plasma TG levels in groups of DM and DMT3 were significantly higher than those in NC group(P<0.05),but there was no difference in the four groups of db/db mice(P>0.05).HepaticTG levels in groups of DM,DMT1,DMT2,DMT3 were significantly higher than those in group NC(P<0.01),and the levels in groups of DMT2,DMT3 were significantly decreased,compared with groups of DM and DMT1(P<0.05).4.After the intervention by Empagliflozin or combined with Metformin,hepatic ApoA1 gene expressions in groups of DMT2 and DMT3 were significantly increased,compared with groups of DM and DMT1(P<0.05),but no difference was founded in hepatic ApoB100 gene expression in the five groups(P>0.05).5.Hepatic ApoA1 protein levels in db/db mice were decreased,but significantly increased in the diabetic groups intervened by Empagliflozin or combined with Metformin,whereas there were no difference in hepatic ApoB100 protein levels in the five groups.6.H&E staining of murine livers showed: A large number of hepatocytes appeared edema in groups of DM and DMT1.Furthermore,many hepatocytes were founded steatosis.But the pathologic changes were attenuated by Empagliflozin or combined with Metformin intervention.Conclusions:1.Metformin or Empagliflozin as well as their combination have good anti-hyperglycemia effects,but the effect of Empagliflozin monotherapy or combined with Metformin might be more stable.2.Metformin or Empagliflozin as well as their combination may derease body weight,while Empagliflozin monotherapy or combined with Metformin may decrease liver weight.3.Empagliflozin monotherapy or combined with Metformin may decrease plasma TC levels and hepatic TG levels,while increase plasma HDL-C levels by prompting the expression of ApoA1 mRNA and protein.As a result,hepatocytes were protected from edema and steatosis.4.Empagliflozin monotherapy or combined with Metformin may improve lipid metabolism in type 2 diabetic mice.Nevertheless,our study did not show the effects of Empagliflozin combined with Metformin was better than those of Empagliflozin monotherapy.