Effect Of Ectopic Fat Deposition On Renal Tubule Glucose Transporter And Its Mechanism

Part 1: Effects of lipid deposition on renal tubule glucose transportersBackgrounds: The kidney maintains systemic glucose homeostasis by reabsorbing glucose through its proximal tubules.Studies have confirmed that visceral lipid deposition is an important driver of many metabolic disorders.Previous experiments of our group found that renal lipid accumulation in rats could increase glucose reabsorption by upregulating SGLT2 molecule,while other renal tubule glucose reabsorption molecules were not further explored.Therefore,this study aims to further investigate the correlation between lipid deposition and the expression of SGLT1,GLUT1,GLUT2 and ATPα1 protein molecules through animal experiments and cell experiments.Methods: Six week-old C57BL/6 male mice were put into two groups,randomly.one group was fed with normal diet(Control,CON),the other group was fed a high fat diet(HFD),and continued feeding for 12 weeks to establish the mouse obesity model.When the weight of the HFD group exceeds 20% of the weight of the CON group,the model is determined to be successful.Intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were used in two groups.At the same time,the 24-hour urine of mice was collected by the metabolic cage for quantitative determination of urine glucose.Blood samples were collected to detect serum lipids,creatinine,urea nitrogen and other biochemical indexes.The renal cortex was retained and its lipid content was evaluated by oil red O staining and quantitative determination of free fatty acid(FFA).Immunofluorescence assay,Western blot assay(WB)and quantitative real-time polymerase chain reaction(q PCR)were used to detect the expression levels of glucose transporters in the kidney.In vitro,HK2 cells were treated with palmitic acid(PA).The intracellular lipid deposition,glucose uptake activity and the expression of key molecules for glucose reabsorption were detected.Results: 1.The weight of mice was dramatically higher in the HFD group than that in the CON group(P < 0.0001).The blood glucose of mice in the two groups was monitored for 12 consecutive weeks,and the difference in average blood glucose between the two groups is not significant(P=0.056).2.The results of IPGTT and IPITT indicated that the mice in the HFD group had abnormal glucose tolerance,but failed to be diagnosed as diabetes.3.Oil red O staining showed obvious lipid deposition in renal tubules of mice in the HFD group,while no lipid droplets were observed in renal tubules of mice in the CON group.The quantitative determination of FFA in renal cortex of HFD mice demonstrated that the FFA content in renal cortex of HFD mice was dramatically higher than that of CON mice(P <0.0001).4.Immunofluorescence staining,q PCR and WB trails revealed that the expression levels of SGLT2,GLUT1 and ATPα1 in HFD group were higher than those in CON group.However,there was no significant difference in the expression of SGLT1 and GLUT2 between the two groups.5.Pearson correlation analysis demonstrated that FFA content in renal cortex was significantly positively correlated with SGLT2,GLUT1 and ATPα1 protein expression levels.6.In vitro experiments also confirmed that the activity of glucose uptake and intracellular lipid deposition were significantly enhanced after PA treatment in HK2 cells,and the expression of SGLT2,GLUT1 and ATPα1 proteins were significantly up-regulated after PA intervention in HK2 cells.Conclusions: Our results showed that although renal lipid deposition did not change the expression levels of GLUT2 and SGLT1 molecules,it up-regulated the expression levels of SGLT2,GLUT1 and ATPα1 molecules in renal tubular epithelium,leading to increased renal glucose reabsorption,which provided a new direction for the involvement of lipid metabolism disorders in the occurrence and development of diabetes.Part Ⅱ: PPAR-α regulates lipid metabolism and affects renal tubule glucose transportersBackgrounds: The content of Free fatty acids(FFA)in obese people is usually increased,and palmitic acid(PA)is the most abundant saturated FFA in circulation.The intake of a large amount of saturated fatty acids(especially PA)will increase the concentration of Free fatty acids in the blood and lead to inflammation.The results of the first part of animal and cell studies indicate that renal tubule lipid deposition is an important factor causing changes in glucose transporter expression.However,whether improved lipid deposition can reverse the upregulation of glucose transporter is worth further investigation.PPAR-α is a ligand activated nuclear hormone receptor transcription factor,which is located in the proximal tubules of the kidney.It can detect free fatty acids and their derivatives in the cytoplasm and transfer them to the nucleus to regulate the expression of fatty acid oxidation genes.So far,combined with previous research conclusions,we planned to observe the changes of SGLT2 and GLUT1 proteins after reversing lipid deposition with fenofibrate,a PPAR-α agonist,in vitro.Methods: Immunofluorescence staining and WB assay were used to detect the expression of PPAR-α in mice of CON group and HFD group.Human proximal tubular epithelial cells(HK2 cells)were send to normal group and PA intervention group.Immunofluorescence staining and WB assay were used to detect the changes of PPAR-α expression in HK2 cells of the two groups.In addition,intracellular lipid deposition was assessed after both PA and fenofibrate were administered to HK2 cells,and the expression levels of SGLT2,GLUT1,and PPAR-α were again detected by immunofluorescence.Results: 1.Immunofluorescence staining results of the two groups of mice showed that PPAR-α was expressed in the proximal renal tubules of normal mice,and the mean fluorescence intensity of PPAR-α in HFD group was lower than that in CON group,and the results verified by WB test at the protein level were consistent with the immunofluorescence results.Pearson correlation analysis showed that PPAR-α was negatively correlated with FFA,GLUT1 and SGLT2.2.After the intervention of 200 mol/L PA in HK2 cells,WB trails found that the expression of PPAR-α in the PAtreated group was decreased sharply at the protein level(P<0.05),the immunofluorescence staining results revealed that PPAR-α was located in normal HK2 cells,and the mean fluorescence of the PA treated HK2 cells was significantly decreased compared with control group.3.After the intervention of PA and fenofibrate,the intracellular lipid contents were decreased significantly.Immunofluorescence staining results demonstrated that the expression of PPAR-α was up-regulated,and the expression of SGLT2 and GLUT1 was significantly decreased compared with the PA treated group.Conclusion: The PPAR-α agonist fenofibrate reversed the lipid deposition induced by PPAR-α down-regulation,and then up-regulate the expression of SGLT2 and GLUT1.