Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of hi...Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of high glucose(HG)and free fatty acid(FFA)and determined its association with TGF-beta-activated kinase 1(TAK1).Methods HK-2 cells were exposed to a combination of HG and FFA.USP19 mRNA expression was detected by quantitative RT-PCR(qRT-PCR),and protein analysis was performed by immunoblotting(IB).Cell growth was assessed by Cell Counting Kit-8(CCK-8)viability and 5-ethynyl-2′-deoxyuridine(EdU)proliferation assays.Cell cycle distribution and apoptosis were detected by flow cytometry.The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation(Co-IP)assays and IB.Results In HG+FFA-challenged HK-2 cells,USP19 was highly expressed.USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells.Moreover,USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1(PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species(ROS)generation in HK-2 cells.Mechanistically,USP19 stabilized the TAK1 protein through deubiquitination.Importantly,increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.Conclusion The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1,providing a potential therapeutic strategy for combating DN.展开更多
As the consumption of fructose and saturated fatty acids(FAs) has greatly increased in western diets and is linked with an increased risk of metabolic syndrome,the aim of this study was to investigate the effects of a...As the consumption of fructose and saturated fatty acids(FAs) has greatly increased in western diets and is linked with an increased risk of metabolic syndrome,the aim of this study was to investigate the effects of a mod-erate(10 weeks) and a prolonged(30 weeks) high fructose and saturated fatty acid(HFS) diet on plasma FA com-position in rats.The effects of a few weeks of HFS diet had already been described,but in this paper we tried to es-tablish whether these effects persist or if they are modified after 10 or 30 weeks.We hypothesized that the plasma FA profile would be altered between 10 and 30 weeks of the HFS diet.Rats fed with either the HFS or a standard diet were tested after 10 weeks and again after 30 weeks.After 10 weeks of feeding,HFS-fed rats developed the metabolic syndrome,as manifested by an increase in fasting insulinemia,total cholesterol and triglyceride levels,as well as by impaired glucose tolerance.Furthermore,the plasma FA profile of the HFS group showed higher proportions of monounsaturated FAs like palmitoleic acid [16:1(n-7)] and oleic acid [18:1(n-9)],whereas the proportions of some polyunsaturated n-6 FAs,such as linoleic acid [18:2(n-6)] and arachidonic acid [20:4(n-6)],were lower than those in the control group.After 30 weeks of the HFS diet,we observed changes mainly in the levels of 16:1(n-7)(decreased) and 20:4(n-6)(increased).Together,our results suggest that an HFS diet could lead to an adaptive response of the plasma FA profile over time,in association with the development of the metabolic syndrome.展开更多
目的:探究高糖高脂对成骨细胞增殖和凋亡的影响。方法:以完全培养基为对照组培养基,以含20.0mM葡萄糖(D-glucose)、0.4mM棕榈酸(Palmitic acid,PA)及0.5%牛血清白蛋白(Bovine ser um albumin,BSA)的完全培养基为实验组模拟高糖高脂环境...目的:探究高糖高脂对成骨细胞增殖和凋亡的影响。方法:以完全培养基为对照组培养基,以含20.0mM葡萄糖(D-glucose)、0.4mM棕榈酸(Palmitic acid,PA)及0.5%牛血清白蛋白(Bovine ser um albumin,BSA)的完全培养基为实验组模拟高糖高脂环境,分别处理成骨细胞0-48h后,显微镜下观察细胞形态,采用CCK-8检测细胞增殖活性,Annex in V/PI双染流式检测细胞凋亡率,Western blotting检测caspase-3及cleaved caspase-3蛋白水平。结果:成骨细胞在高糖高脂环境下培养24h时观察到细胞皱缩变圆,胞间间隙增宽;CCK-8检测提示高糖高脂培养成骨细胞24h时与同时刻对照组相比出现增殖抑制(F_(24h)=24.489,P=0.001),48h后增殖活性(0.027±0.022)较同时刻对照组差异有统计学意义(F_(48h)=272.440,P<0.001);Annex in V/PI双染流式检测显示,24h时高糖高脂组细胞凋亡率为9.68%±3.33%,36h时细胞凋亡率上升至12.89%±3.40%,可达对照组(1.96±1.32)约6.6倍;Western blotting检测发现健康成骨细胞极少表达cleaved caspase-3,高糖高脂可时间依赖性上调cleaved caspase-3蛋白水平,24h时cleaved caspase-3相对蛋白表达量(0.349±0.068)约为对照组(0.146±0.033)2.4倍,36h时(0.468±0.136)达对照组3.2倍(F=9.603,P=0.001)。结论:高糖高脂可时间依赖性抑制成骨细胞增殖,促进凋亡。展开更多
基金supported by Natural Science Foundation of Shaanxi Province(No.2023-JC-YB-743 and No.2021JQ-905).
文摘Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of high glucose(HG)and free fatty acid(FFA)and determined its association with TGF-beta-activated kinase 1(TAK1).Methods HK-2 cells were exposed to a combination of HG and FFA.USP19 mRNA expression was detected by quantitative RT-PCR(qRT-PCR),and protein analysis was performed by immunoblotting(IB).Cell growth was assessed by Cell Counting Kit-8(CCK-8)viability and 5-ethynyl-2′-deoxyuridine(EdU)proliferation assays.Cell cycle distribution and apoptosis were detected by flow cytometry.The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation(Co-IP)assays and IB.Results In HG+FFA-challenged HK-2 cells,USP19 was highly expressed.USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells.Moreover,USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1(PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species(ROS)generation in HK-2 cells.Mechanistically,USP19 stabilized the TAK1 protein through deubiquitination.Importantly,increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.Conclusion The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1,providing a potential therapeutic strategy for combating DN.
文摘As the consumption of fructose and saturated fatty acids(FAs) has greatly increased in western diets and is linked with an increased risk of metabolic syndrome,the aim of this study was to investigate the effects of a mod-erate(10 weeks) and a prolonged(30 weeks) high fructose and saturated fatty acid(HFS) diet on plasma FA com-position in rats.The effects of a few weeks of HFS diet had already been described,but in this paper we tried to es-tablish whether these effects persist or if they are modified after 10 or 30 weeks.We hypothesized that the plasma FA profile would be altered between 10 and 30 weeks of the HFS diet.Rats fed with either the HFS or a standard diet were tested after 10 weeks and again after 30 weeks.After 10 weeks of feeding,HFS-fed rats developed the metabolic syndrome,as manifested by an increase in fasting insulinemia,total cholesterol and triglyceride levels,as well as by impaired glucose tolerance.Furthermore,the plasma FA profile of the HFS group showed higher proportions of monounsaturated FAs like palmitoleic acid [16:1(n-7)] and oleic acid [18:1(n-9)],whereas the proportions of some polyunsaturated n-6 FAs,such as linoleic acid [18:2(n-6)] and arachidonic acid [20:4(n-6)],were lower than those in the control group.After 30 weeks of the HFS diet,we observed changes mainly in the levels of 16:1(n-7)(decreased) and 20:4(n-6)(increased).Together,our results suggest that an HFS diet could lead to an adaptive response of the plasma FA profile over time,in association with the development of the metabolic syndrome.
文摘目的:探究高糖高脂对成骨细胞增殖和凋亡的影响。方法:以完全培养基为对照组培养基,以含20.0mM葡萄糖(D-glucose)、0.4mM棕榈酸(Palmitic acid,PA)及0.5%牛血清白蛋白(Bovine ser um albumin,BSA)的完全培养基为实验组模拟高糖高脂环境,分别处理成骨细胞0-48h后,显微镜下观察细胞形态,采用CCK-8检测细胞增殖活性,Annex in V/PI双染流式检测细胞凋亡率,Western blotting检测caspase-3及cleaved caspase-3蛋白水平。结果:成骨细胞在高糖高脂环境下培养24h时观察到细胞皱缩变圆,胞间间隙增宽;CCK-8检测提示高糖高脂培养成骨细胞24h时与同时刻对照组相比出现增殖抑制(F_(24h)=24.489,P=0.001),48h后增殖活性(0.027±0.022)较同时刻对照组差异有统计学意义(F_(48h)=272.440,P<0.001);Annex in V/PI双染流式检测显示,24h时高糖高脂组细胞凋亡率为9.68%±3.33%,36h时细胞凋亡率上升至12.89%±3.40%,可达对照组(1.96±1.32)约6.6倍;Western blotting检测发现健康成骨细胞极少表达cleaved caspase-3,高糖高脂可时间依赖性上调cleaved caspase-3蛋白水平,24h时cleaved caspase-3相对蛋白表达量(0.349±0.068)约为对照组(0.146±0.033)2.4倍,36h时(0.468±0.136)达对照组3.2倍(F=9.603,P=0.001)。结论:高糖高脂可时间依赖性抑制成骨细胞增殖,促进凋亡。
