Effect Of Phospho-FoxO1on Lipid Accumulation In HK2Cells

Objective: Diabetic nephropathy is one of the chronic complications ofdiabetes, and is characterized by mesangial cell proliferation and hypertrophy,extracellular matrix accumulation, glomerular sclerosis, renal tubularepithelial-mesenchymal-transition, renal tubular interstitial fibrosis and footcell apoptosis. Diabetic nephropathy makes great damage to human health andmany factors are involved in the pathogenesis of this disease includinghyperglycemia, high blood perfusion, inflammatory factors, immune injury,oxidative stress and lipid metabolism abnormality. The previous studies haverevealed that abnormal lipid metabolism in renal intrinsic cells plays animportant role in the development of diabetic nephropathy. Our previousresearch has testified that high glucose can induce increased ADRP and FASby activating PI3K/Akt signal pathway in human renal tubular cells. However,LY294002known as an inhibitor of PI3K/Akt pathway can block theactivation of Akt and lower cellular lipid droplets. The above study proposedthat PI3K/Akt pathway was involved in renal lipid accumulation of diabetes.However, the exact mechanism has not been fully elucidated. At present, anumber of genes have been confirmed to be regulated by activated Akt asdown-stream targets such as Bad, FoxO1, mTOR, PRAS40and GSK-3β.Among these, Forkhead transcription factors of O class1(FoxO1) is reportedto widely exist in various tissue and cell and controlled by PI3K/Akt signalpathway. In detail, activated Akt can phosphorylate FoxO1and then weakenthe transcription function of FoxO1by translocation from nucleus to cellularplasma. A series of studies on the relationship between FoxO1and diabetesrevealed that high glucose could enhance the expression of phospho-FoxO1and decrease nuclear FoxO1in the intrinsic cells of kidney via activated Akt.LY294002can cause decreased phospho-FoxO1and increase nuclear FoxO1.These data suggested that FoxO1as down-stream gene of PI3K/Akt pathway participated in the pathogenesis and development of diabetes. Nevertheless, itis needed to be further confirmed whether FoxO1participates in lipidmetabolism of diabetic nephropathy.Fatty acid synthase (FAS) is a key enzyme of fatty acid synthesis that isused to catalyze acetyl CoA and malonyl CoA and then compound long-chainfatty acids. Adipose differentiation-related protein (ADRP) is a sensitivemarker of lipid droplets and is commonly used to detect early and small lipiddroplet. Again, Oil Red O staining is recognized as a specific method topresent cellular lipid droplet. Therefore, FAS, ADRP and Oil Red O stainingwere chosen as methods to assess cellular lipid metabolism.In the present study, we observe the expression of FoxO1, p-FoxO1,ADRP protein and FAS mRNA and explore the relationship between FoxO1,p-FoxO1and diabetic renal tubular lipid deposition using highglucose-stimulated in vitro cultured-human renal tubular cells respectivelytransfected with wild-type FoxO1plasmid and Ser256sites mutated FoxO1plasmid.Methods: Human tubular epithelial cells (HK2) were cultured at37℃ina humidified incubator containing5%CO2in DMEM medium with penicillin(100KU/L), streptomycin (100mg/L) and10%fetal bovine serum (FBS).①The competent DH5a bacteria is prepared with method of CaCl2. Therecombinant wild-type FoxO1plasmid and Ser256sites mutated FoxO1plasmid are respectively transformed into competent DH5a for plasmidamplification and extraction.②HK2cells were randomly divided into twogroups: normal control group (glucose5.5mmol/L) and high glucose group(glucose25mmol/L). At48hours after the treatment of high glucose, Westernblot was used todetect FoxO1, p-FoxO1(Ser256) and ADRP proteinexpression. Real-time PCR was used to detect the expression of FAS mRNA.The cellular lipid droplet formation was evaluated by the method of Oil Red Ostaining.③In order to examine the direct effect of FoxO1and p-FoxO1onlipid droplets formation, HK2cells were randomly divided into4groups:untransfected control group, pcDNA3.1vector control group, recombinant wild-type FoxO1plasmid group and recombinant Ser256sites mutated FoxO1plasmid group. Transient transfection was carried out by Lipofectamine2000according to the manufacturer ’s instruction. After48hours culture, lipiddroplets were examined by Oil Red O staining and the cells were collected forthe extraction of protein and total RNA. FoxO1, p-FoxO1and ADRP proteinexpression were detected by Western blot. FAS mRNA was detected byReal-time PCR.Results:1. High glucose modulates FoxO1, p-FoxO1, ADRP proteins and FASmRNA expression in HK2cells.The expression of FoxO1and p-FoxO1were detected by the method ofWestern blot. No marked difference in FoxO1expression between normalcontrol group and high glucose group was found and the IOD ratios of blotswere respectively0.64±0.10and0.69±0.06. It was revealed that p-FoxO1(theinactivated type of FoxO1) showed higher expression in high glucose groupthan that in normal control group and the relative expression values were0.43±0.04and0.23±0.04respectively. The difference was statisticallysignificant (P<0.05). It is tested that the relative expression values of ADRPwere0.66±0.22and1.19±0.20in normal control group and high glucosegroup (P<0.05). The expression of FAS mRNA was detected by the method ofReal-time PCR and the result showed that FAS mRNA expression level wassignificantly elevated by2.73times in high glucose group compared withnormal control group.2. Effect of recombinant pcDNA3.1-wt FoxO1plasmid on lipid dropletformation in HK2cellsFrom the results of Western blot normalized by β-actine, IOD ratios ofFoxO1protein was0.64±0.10for normal control group,0.64±0.14forpcDNA3.1group,1.07±0.15for pcDNA3.1-wt FoxO1group. FoxO1proteinexpression was up-regulated by transfection of pcDNA3.1-wt FoxO1plasmid.Meanwhile, compared with normal control group, the phospho-FoxO1proteinexpression was increased by1.91times in pcDNA3.1-wt FoxO1plasmid transfection group. ADRP is a kind of sensitive marker of lipid droplet.According to Western blot, IOD ratio of ADRP protein in pcDNA3.1-wtFoxO1group was1.92±0.35that is as1.56times as that in normal controlgroup. We detected the FAS mRNA expression in HK2cells transfected withwt FoxO1specific plasmid by Real-time PCR, and found that FAS mRNA inHK2cells transfected with pcDNA3.1-wt FoxO1plasmid was significantlyhigher than that in untransfected HK2cells. From the Oil Red O staining,pcDNA3.1-wt FoxO1plasmid transfection in HK2cells results in more lipiddroplet formation than that in normal control HK2cells.3. Effect of recombinant specific pcDNA3.1-mutation FoxO1(S256A)plasmid on lipid droplets formation in HK2cellsThe expression of FoxO1, phospho-FoxO1and ADRP were detected bythe method of Western blot. From the results we can see that compared withnormal control group, the total FoxO1protein expression was up-regulated bytransfection of pcDNA3.1-mutation FoxO1(S256A) plasmid and the valueswere1.05±0.31. Meanwhile, we can see that phospho-FoxO1showed lowerexpression in pcDNA3.1-mutation FoxO1(S256A) plasmid transfection groupthan that in normal control group. According to the result of Western blot,IOD ratio of ADRP protein in pcDNA3.1-mutation FoxO1(S256A) plasmidgroup was0.73±0.14, and compared with normal control group, the ADRPprotein was significantly inhibited by31.8％. The FAS mRNA was examinedusing the method of Real-time PCR. Compared with normal control group, theFAS mRNA expression level in HK2cells transfected withpcDNA3.1-mutation FoxO1(S256A) plasmid was significantly inhibited by36.5％. From the Oil Red O staining, over-expression of mutation of Ser256site of FoxO1in HK2cells results in less lipid droplet formation than that innormal control HK2cells.Conclusions:1. High glucose can lead to more lipid droplet formation in HK2cells bysimultaneously enhancing the expression of phospho-FoxO1, FAS and ADRP,which indicates that phospho-FoxO1may be involved in lipid accumulation in renal tubular cells of diabetes.2. Increased p-FoxO1by transfection of specific pcDNA3.1-wt FoxO1plasmid in high glucose-stimulated HK2cells plays an important role in lipiddroplet formation by up-regulating FAS mRNA expression.3. The transfection of recombinant specific pcDNA3.1-mutation FoxO1(S256A) plasmid could significantly inhibit the lipid formation, whichsuggests high glucose can not stimulate the expression of phospho-FoxO1once the gene mutation happens at Ser256phosphorylation sites.