Background Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy. Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum （ER） ...Background Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy. Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum （ER） stress and consequent apoptosis, contributing to the onset of diabetes. We hypothesized that palmitate could induce podocyte apoptosis via ER stress, which initiates or aggravates proteinuria in diabetic nephropathy. Methods Podocyte apoptosis was detected by 4＇,6-diamidio-2-phenylindole （DAPI） stained apoptotic cell count and Annexin V-PI stain. The expressions of ER molecule chaperone glucose-regulated protein 78 （GRP78）, indicators of ER-associated apoptosis C/EBP homologous protein （CHOP）, and Bcl-2 were assayed by Western blotting and real-time PCR. GRP78 and synaptopodin were co-localized by immunofluorescence stain. Results Palmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L （10 hours, 13 hours, and 15 hours compared with 0 hour, P 〈0.001） and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours （compared to control, P 〈0.001）. Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP, and decreased that of Bcl-2. Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to control （P 〈0.001）, with the maximum concentration being 0.75 mmol/L. Palmitate 0.5 mmol/L loading for 3 hours, 8 hours, and 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to 0 hour （P 〈0.001）, with the maximum effect at 3 hours. Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control. Conclusion Palmitate could induce podocyte apoptosis via ER stress, suggesting podocyte apoptosis and consequent proteinuria caused by lipotoxic free fatty acid could be ameliorated by relief of ER stress.展开更多
Background Dyslipidaemia is a potential independent The aim of this study was to investigate dyslipidaemia, with ischemic stroke in a Chinese hospital. risk factor for cerebrovascular disease in patients with diabetes...Background Dyslipidaemia is a potential independent The aim of this study was to investigate dyslipidaemia, with ischemic stroke in a Chinese hospital. risk factor for cerebrovascular disease in patients with diabetes. treatment and control of dyslipidaemia among diabetic patients Methods A total of 1046 type 2 diabetic patients were assigned to diabetes with （n=-522） and diabetes without stroke groups. The two groups were matched by gender, age and diabetes duration. Lipid and lipoprotein profile were measured. Serum level and control of lipids were assessed and classified according to American Diabetes Association （ADA） guidelines and an intensified low density lipoprotein-cholesterol （LDL-C） target recommended in Chinese dyslipidaemia control criteria. Results Diabetic patients suffering stroke displayed not only poorly-controlled lipid and lipoprotein profiles, including the significantly lower proportion of patients achieving intensified LDL-C target of 〈2.07 mmol/L （80 mg/dl）, and high density lipoprotein-cholesterol （HDL-C） target （14.4% vs 21.0%, P=0.005; 45.8% vs 51.9%, P=0.048 respectively）, but also less adherence to therapy prescribed for dyslipidaemia （30.8% vs 41.0%, P=0.001）, when compared with diabetic patients without stroke. For the diabetic women with stroke, situation of dyslipidaemia was worse, with significantly lower serum level of HDL-C and apoA1, higher LDL-C level and higher ratio of apoB/apoA1 when compared with diabetic counterparts without stroke. Conclusions Many diabetic patients with ischemic stroke remain uncontrolled for dyslipidaemia. Intensified LDL-C and overall lipid lowering clinical goals are potential precautions taken against ischemic stroke among diabetic patients in China.展开更多
Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated L...Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line （HepG2） cells under physiological and inflammatory conditions. Methods Intracellular total cholesterol （TC）, free cholesterol （FC） and cholesterol ester （CE） were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein （SREBP）-2 and SREBP cleavage-activating protein （SCAP） mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum （ER） to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies. Results LDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1β （IL-1β） further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1β caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium. Conclusions IL-1β disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.展开更多
Background Cardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease Macrophage death in advanced atherosclerosJs promotes necrosis and plaque destabilization. In vitro...Background Cardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease Macrophage death in advanced atherosclerosJs promotes necrosis and plaque destabilization. In vitro data from peritoneal macrophages show apoptosis triggered through endoplasmic reticulum （ER） stress caused by free cholesterol accumulation plays an important role. Here we used THP-1 cells differentiated by 100 ng/ml of phorbol 12-myristate 13-acetate （PMA） for five days as an in vitro model, to investigate if acetylated low-density lipoprotein （AcLDL） loading could also induce apoptosis and its underlying mechanisms. Methods Oil red O staining was used to examine the lipid droplets. Confocal microscopy was used to visualize the uptake of AcLDL. Hoechst 33258 stain and the caspase 3,7 assay were used to detect apoptosis. High performance liquid chromatography was used in the intracellular free cholesterol （FC） and cholesterol ester （CE） assay. Western blotting was used to demonstrate the protein level. Real-time PCR was used to detect the changes of mRNAs. ER free cholesterol was also assayed. Results Confocal microscopy showed THP-1 cells differentiated by 100 ng/ml of PMA for five days uptake more AcLDL than differentiated for two days. Hoechst 33258 stain showed AcLDL could induce apoptosis in THP-1 macrophages in a time and dose dependent manner. Exposure of THP-1 macrophages to 100 ug/ml of AcLDL for 24 hours resulted in a significant increase in caspase 3,7 activity, a significant increase in FC and CE mass of 1.5 and 2.4-fold, meanwhile, a significant increase in transcription factor C/EBP homologous protein and a decrease in Bcl-2 both in protein and mRNA levels were observed with an 8-fold rise of free cholesterol in the ER. Conclusion ER stress is involved in AcLDL induced apoptosJs in THP-1 macrophages with free cholesterol accumulation in the ER.展开更多
基金This study was supported by the grants from the National Natural Science Foundation of China （No. 30700373） Peking Union Medical College Hospital Foundation （2009）, the National Natural Science Foundation of China （No. 81170665） and the Ministry of Health Sector Fund （No. 200802007）.
文摘Background Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy. Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum （ER） stress and consequent apoptosis, contributing to the onset of diabetes. We hypothesized that palmitate could induce podocyte apoptosis via ER stress, which initiates or aggravates proteinuria in diabetic nephropathy. Methods Podocyte apoptosis was detected by 4＇,6-diamidio-2-phenylindole （DAPI） stained apoptotic cell count and Annexin V-PI stain. The expressions of ER molecule chaperone glucose-regulated protein 78 （GRP78）, indicators of ER-associated apoptosis C/EBP homologous protein （CHOP）, and Bcl-2 were assayed by Western blotting and real-time PCR. GRP78 and synaptopodin were co-localized by immunofluorescence stain. Results Palmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L （10 hours, 13 hours, and 15 hours compared with 0 hour, P 〈0.001） and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours （compared to control, P 〈0.001）. Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP, and decreased that of Bcl-2. Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to control （P 〈0.001）, with the maximum concentration being 0.75 mmol/L. Palmitate 0.5 mmol/L loading for 3 hours, 8 hours, and 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to 0 hour （P 〈0.001）, with the maximum effect at 3 hours. Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control. Conclusion Palmitate could induce podocyte apoptosis via ER stress, suggesting podocyte apoptosis and consequent proteinuria caused by lipotoxic free fatty acid could be ameliorated by relief of ER stress.
基金This work was partially supported by the National Natural Science Foundation of China （No. 30870870, WANG Shao-hua No. 30600206, GUO Yi-jing） and Natural Science Foundation of Jiangsu Province （No. BK2008302, WANG Shao-hua）.Acknowledgment： We would like to express our thanks to the nursing staff of the Division of Endocrinology, Zhongda Hospital of Southeast University, for their help.
文摘Background Dyslipidaemia is a potential independent The aim of this study was to investigate dyslipidaemia, with ischemic stroke in a Chinese hospital. risk factor for cerebrovascular disease in patients with diabetes. treatment and control of dyslipidaemia among diabetic patients Methods A total of 1046 type 2 diabetic patients were assigned to diabetes with （n=-522） and diabetes without stroke groups. The two groups were matched by gender, age and diabetes duration. Lipid and lipoprotein profile were measured. Serum level and control of lipids were assessed and classified according to American Diabetes Association （ADA） guidelines and an intensified low density lipoprotein-cholesterol （LDL-C） target recommended in Chinese dyslipidaemia control criteria. Results Diabetic patients suffering stroke displayed not only poorly-controlled lipid and lipoprotein profiles, including the significantly lower proportion of patients achieving intensified LDL-C target of 〈2.07 mmol/L （80 mg/dl）, and high density lipoprotein-cholesterol （HDL-C） target （14.4% vs 21.0%, P=0.005; 45.8% vs 51.9%, P=0.048 respectively）, but also less adherence to therapy prescribed for dyslipidaemia （30.8% vs 41.0%, P=0.001）, when compared with diabetic patients without stroke. For the diabetic women with stroke, situation of dyslipidaemia was worse, with significantly lower serum level of HDL-C and apoA1, higher LDL-C level and higher ratio of apoB/apoA1 when compared with diabetic counterparts without stroke. Conclusions Many diabetic patients with ischemic stroke remain uncontrolled for dyslipidaemia. Intensified LDL-C and overall lipid lowering clinical goals are potential precautions taken against ischemic stroke among diabetic patients in China.
基金This study was supported by grants from the National Natural Science Foundation of China(Key Program,No.30530360)the National Basic Research Program of China(No.2006CB503907)Royal Free Hospital Special Trustees grant
文摘Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line （HepG2） cells under physiological and inflammatory conditions. Methods Intracellular total cholesterol （TC）, free cholesterol （FC） and cholesterol ester （CE） were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein （SREBP）-2 and SREBP cleavage-activating protein （SCAP） mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum （ER） to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies. Results LDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1β （IL-1β） further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1β caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium. Conclusions IL-1β disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.
基金This study was supported by the grants from the National Natural Science Foundation of China （No. 30700373） and the Royal Free Hospital Special Trustees Grant 115.
文摘Background Cardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease Macrophage death in advanced atherosclerosJs promotes necrosis and plaque destabilization. In vitro data from peritoneal macrophages show apoptosis triggered through endoplasmic reticulum （ER） stress caused by free cholesterol accumulation plays an important role. Here we used THP-1 cells differentiated by 100 ng/ml of phorbol 12-myristate 13-acetate （PMA） for five days as an in vitro model, to investigate if acetylated low-density lipoprotein （AcLDL） loading could also induce apoptosis and its underlying mechanisms. Methods Oil red O staining was used to examine the lipid droplets. Confocal microscopy was used to visualize the uptake of AcLDL. Hoechst 33258 stain and the caspase 3,7 assay were used to detect apoptosis. High performance liquid chromatography was used in the intracellular free cholesterol （FC） and cholesterol ester （CE） assay. Western blotting was used to demonstrate the protein level. Real-time PCR was used to detect the changes of mRNAs. ER free cholesterol was also assayed. Results Confocal microscopy showed THP-1 cells differentiated by 100 ng/ml of PMA for five days uptake more AcLDL than differentiated for two days. Hoechst 33258 stain showed AcLDL could induce apoptosis in THP-1 macrophages in a time and dose dependent manner. Exposure of THP-1 macrophages to 100 ug/ml of AcLDL for 24 hours resulted in a significant increase in caspase 3,7 activity, a significant increase in FC and CE mass of 1.5 and 2.4-fold, meanwhile, a significant increase in transcription factor C/EBP homologous protein and a decrease in Bcl-2 both in protein and mRNA levels were observed with an 8-fold rise of free cholesterol in the ER. Conclusion ER stress is involved in AcLDL induced apoptosJs in THP-1 macrophages with free cholesterol accumulation in the ER.
