Study On The Mechanism Of Empagliflozin Causes Browning In WAT Of KKAy Mice By Promoting Mitochondrial Fusion

Objective:Sodium glucose co-transpo rter 2(SGLT2)inhibitors have become pro mising new hypoglycemic agnts for the treatment of type 2 diabetes.SGLT2 inhibitors can reduce hyperglycmia and weight loss.Recent clinical studies have shown that long term use of Empagliflozin(Empa)can cause an increase in heat production and activate white fat brown-like changes,but these potential mechanisms for the effects of Empa on energy omeostasis and obesity have not been established._Therefore,we investigate the effect and mechanism of Empa on KKAy mice and preadipocyte cell line(3T3-L1)in white fat brownin.Methods:We used KKAy mice as our model.The mice were orally administered with Empa or normal saline for 8 weeks.The C57BL/6J mouse model served as a non-diabetic control.3T3-L1 preadipocytes were used in vitro to induce adipogenesis and intervention with Empa(4μmol/L).At 16 weeks in vivo,blood glucose,body weight and rectal temperature were measured in four groups of After 16 weeks of feeding,pWAT and eWAT were isolated from the mices,hematoxylin staining was performed to observe changes in tissue morphology.Immunohistochemistry was used to observe whether uocoupling protein-1(UCP1)was increased.Mature 3T3-L1 preadipocytes were induced in vitro for oil red O staining,and the changes of lipid droplets after Empa intervention were observed.At the same time,qRT-PCR,western blot and immunofluorescenee were used to observe the expression of heat,lipid metabolism,mitochondrial biosynthesis,mitochondrial kinetics and mitochondrial oxidative phosphorylation in and induced mature in vivo.In addition,we used AMPK activators and inhibitors to investigate the effects of AMPK/P GC-1αsignaling pathway in vitro.We used PGC-1α siRNA for cell transfection.The western blot method was to observe that Empa promoted mitochondral fusion through AMPK/P GC-lα/Mfn2 signaling pathway.Results:_In Empa reduced the body weight of KKAy mice,and the pWAT andeWAT staining showed a decrease in fat volume in the DM+Empa group.ln vitro,the 3T3-L1 preadipocytes showed that the DM+Empa group had a small lipid droplet.In vitro and in vivo experiments confirmed that Empa up-regulated the mitochondrial biosynthesis factors(Dio2,PGC1α,CytoC and TFAM),browning factors(UCP1,PRDM16 and Irisin)in pWAT and eWAT.Key enzymes of ETC(Ndufabl,sdhd,cox7a1 and cox8b)and TCA(CS,Idh3a and Ogdh).Mitochondrial enzyme activity(Complex Ⅰ and KGDH activity)can be seen increased in the DM+Empa group.At the same time,Empa can increase the expression of oxidative respirato ry chain complex Ⅰ,Ⅱ and Ⅲ protesin.The expression of Mfn2 protein is increased in terms of mitochondrial dynamics.It was shown that the fluorescence of Mfn2 was decreased in the DM group.Furthermore,it was further confirmed that administration of AMPK activators and inhibitors can increase and decrease the expression of downstream factors of the AMPK/PGC-1α signaling pathway and affect mitochondrial biosynthesis in vitro.PGC-1α siRNA transfects fat The cells were found to significantly inhibit induced PGC1α activation,accompanied by decreased expression of Mfn2 and TFAM.Conclusions:1.Empa promotes mitochondrial oxidative phosphorylation,increases tricarboxylic acid cycle and electronic respiratory chain function.2.Empa promotes mitochondrial fusion through the AMPK/PGC-1α signaling pathway,increases mitochondrial homeostasis,and promotes browning of white fat,thereby increasing endogenous heat generation,improving lipid metabolism,and reducing body weight.3.The effect of Empa on the browning of white fat is independent of the hypoglycemic effect.