Txnip Gene Knockout Ameliorated High Fat Diet-induced Obesity Cardiomyopathy

Objective:Epidemic of obesity accelerates the increase in the number of patients with obesity cardiomyopathy.Thioredoxin interacting protein(TXNIP)has been implicated in the pathogenesis of multiple cardiovascular diseases.However,its specific role in obesity cardiomyopathy is still not well understood.The aim of this study was to investigate the potential role of TXNIP in the pathogenesis of obesity cardiomyopathy.Methods:1.Txnip gene knockout mice in the C57BL/6J background were generated,and txnip knockout(KO)homozygous male mice and wild type(WT)littermate male mice were used and randomly grouped:(1)WT group;(2)KO group;(3)WT-HFD group;(4)KO-HFD group.Each group was fed either with normal diet(ND)or high-fat diet(HFD)at age of 4 weeks and sustained for 24 weeks.The body weight of mice was recorded weekly;2.The changes of cardiac structure and function were examined via echocardiography at the end of feeding;3.Fasting blood glucose(FBG)and oral glucose tolerance test(OGTT)were measured;4.The changes of lipids composition in serum,lipids composition and ATP level in heart tissue were assayed with commercial kits.5.RT-q PCR method was used to detect the m RNA levels of TXNIP,lipid metabolism related molecules-CD36,carnitine palmitoyl transterase-1(CPT1b),long-chain acyl-Co A dehydrogenase(ACADL),medium-chain acyl-Co A dehydrogenase(ACADM),Acyl-coenzyme A oxidase 1(ACOX1),glucose metabolism related molecules-hexokinase(HK),phosphofructokinase(PFK)and pyruvate kinase(PK);6.Western Blot method was used to detect the protein levels of TXNIP,glucose metabolism related molecules-glucose transporter 1 and 4(GLUT1/GLUT4),pyruvate dehydrogenase E1 component’s α subunit(PDHE1α),p-PDHE1α,mitochondria dynamics related molecules-mitofusin-1 protein(MFN1),mitofusin-2 protein(MFN2),optic atrophy 1(OPA1),dynamin-related protein 1(DRP1)and mitochondrial fission 1protein(FIS1);7.The commercial kits were used to detect the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)in heart tissue;8.JC-1 probe was used to detect the mitochondrial membrane potential;9.The activity of mitochondrial respiratory chain complex Ⅴ was detected with the Mitochondrial Respiratory Chain Complex ⅤActivity Assay Kit;10.Transmission electron microscope was used to observe the ultrastructural changes of cardiomyocytes.Results:1.Compared with the groups fed with the ND,it showed excessive body weight gain in both WT and KO mice fed with HFD,but txnip gene knockout had no significant effect on body weight gain;2.The FBG was increased in both WT and KO mice fed with HFD,and txnip gene knockout significantly reduced FBG in both normal and high-fat diet groups.The area under the curve(AUC)of OGTT was increased in both WT and KO mice fed with high-fat diet compared with those fed with normal diet.However,txnip gene knockout significantly reduced the AUC in mice fed with either normal diet or high-fat diet;3.Compared with the WT group,it showed a significant increase in serum TG,NEFAs,TC,HDL-C and LDL-C in WT-HFD mice.However,txnip gene knockout did not show significant effect on the above lipids composition;4.It showed a significant decrease in LVEF and LVFS,and an increase in LVIDs and LVESV in WT-HFD mice compared with the WT mice;However,LVEF and LVFS were significantly restored after txnip gene knockout;5.Long-term HFD promoted TXNIP protein and m RNA expression in heart tissue;6.Txnip gene knockout improved cardiac lipid deposition and ATP reduction induced by a long-term HFD;7.Compared with the WT group,the m RNA levels of cd36,acadl and acox1 in heart tissue were increased in WT-HFD group;TXNIP deletion increased m RNA levels of cd36,cpt1 b,acadl,acadm and acox1 in both normal and high-fat diet groups;8.HFD inhibited SOD activity and increased MDA content in heart tissue,and these changes were significantly reversed by TXNIP deletion;9.Txnip gene knockout significantly increased GLUT1 protein in the membrane in both the normal diet group and the high-fat diet group,reversing the decrease of GLUT1 protein in the membrane caused by high-fat diet.Meanwhile,GLUT1 showed the opposite trend in the cytoplasm.GLUT4 expression was significantly decreased in both cell membrane and cytoplasm in WT-HFD group,and txnip gene knockout reversed this trend;10.Compared with the WT group,the m RNA levels of pfkm and pkm in the heart tissue were decreased in WT-HFD group;TXNIP deletion increased the m RNA levels of hk2 and pfkm in mice fed a nromal diet,along with pfkm and pkm in mice fed a high-fat diet.The ratio of p-PDHE1α/PDHE1α was increased by txnip gene knockout under normal diet and high fat diet.TXNIP deficiency did not alter p-PDHE1α/PDHE1α in mice fed a high-fat diet;11.It showed an integrated spherical or oval shape and clear cristae of mitochondria in WT and KO mice fed with ND.However,we noticed that the heart tissue of mice in WT-HFD group showed an increase in mitochondrial volume density and damaged mitochondria with disarrayed cristae,along with the lipid droplet density as well.These alterations caused by HFD feeding were all reversed by TXNIP deficiency.Txnip gene knockout restored the decreased mitochondrial membrane potential caused by high fat diet.HFD down-regulated the activity of the mitochondria respiratory chain complex Ⅴ,but it was mitigated by txnip gene knockout;12.It showed an increase in the expression of DRP1 protein and a decrease in the expression of MFN2 protein in the heart tissue of mice;TXNIP deletion significantly reversed the above changes.The expressions of MFN1,OPA1 and FIS1 showed no statistical changes.Conclusion:Our results suggested that txnip gene knockout improved mitochondrial dysfunction via reversing the shift from mitochondrial fusion to fission in the context of chronic HFD feeding,and thus promoting cardiac fatty acid oxidation to alleviate chronic HFD-induced lipid accumulation in the heart,and thereby ameliorating the cardiac function in obese mice.In addition,txnip gene knockout promotes cardiac glucose uptake and glycolysis.Our work provides a theoretical basis for TXNIP exerting as a potential therapeutic target for the interventions of obesity cardiomyopathy.