Animals ; DNA-Binding Proteins ; physiology ; Homeostasis ; Humans ; Lipid Metabolism ; Liver ; metabolism ; Liver X Receptors ; NF-kappa B ; physiology ; Nuclear Proteins ; physiology ; Orphan Nuclear Receptors ; Peroxisome Proliferator-Activated Receptors ; physiology ; Receptors, Cytoplasmic and Nuclear ; physiology ; Sterol Regulatory Element Binding Proteins ; physiology ; Transcription Factors ; physiology

OBJECTIVETo investigate the effect of RNA interference (RNAi)-mediated silencing of the SREBP2 on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells.

METHODSShort-hairpin (sh)RNA targeting SREBP2 or negative control (NC) shRNA were transfected into HepG2 cells by a liposomal method. G418-selective culturing was used to obtain the SREBP2 shRNA HepG2 and NC shRNA HepG2 cell lines. The two cell lines were cultured in serum-free medium and left untreated (control) or treated with TNF-a (20 ng/ml), low-density lipoprotein (LDL) loading (100 mug/ml), or a combination LDL plus TNF-a treatment. Lipid accumulation was evaluated by oil red O (ORO) staining. Intracellular cholesterol level was measured by enzymatic assay. The mRNA and protein levels of SREBP2 and its downstream target genes, LDL receptor (LDLr), and HMGCoA reductase, were measured by real-time PCR and Western blotting, respectively.

RESULTSSREBP2 shRNA HepG2 and NC shRNA HepG2 stable cell lines were successfully established. ORO staining and cholesterol quantitative analysis showed that LDL loading significantly increased intracellular cholesterol and that expression of SREBP2 further exacerbated the inflammatory cytokine-induced lipid accumulation, as seen in NC shRNA HepG2 cells. LDL loading of NC shRNA HepG2 decreased the gene and protein expressions of SREBP2, LDLr, and HMGCoA reductase, but the suppressive effect was overridden by inflammatory cytokine. SREBP2 shRNA HepG2 cells showed lower levels of cholesterol accumulation under LDL loading and inflammatory stress conditions. Moreover, the mRNA and protein levels of SREBP2, LDLr, and HMGCoA reductase were much lower than in NC shRNA HepG2 cells under the same conditions.

CONCLUSIONInflammatory cytokine exacerbated cholesterol accumulation in HepG2 via disrupting SREBP2. RNAi-mediated inhibition of SREBP2 expression significantly ameliorated the cholesterol accumulation induced by inflammatory cytokine.

Cholesterol ; metabolism ; Hep G2 Cells ; Humans ; Inflammation ; RNA Interference ; RNA, Small Interfering ; Sterol Regulatory Element Binding Protein 2 ; genetics ; Tumor Necrosis Factor-alpha ; pharmacology

Fatty Liver ; metabolism ; pathology ; Humans ; Inflammation ; Lipidoses ; metabolism ; pathology ; Non-alcoholic Fatty Liver Disease

BACKGROUNDLow-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.

METHODSIntracellular 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.

RESULTSLDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1 beta (IL-1 beta) 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-1 beta 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.

CONCLUSIONSIL-1 beta 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.

Cell Line, Tumor ; Cholesterol ; analysis ; Endoplasmic Reticulum ; metabolism ; Feedback, Physiological ; Humans ; Interleukin-1beta ; pharmacology ; Intracellular Signaling Peptides and Proteins ; analysis ; genetics ; Membrane Proteins ; analysis ; genetics ; Protein Transport ; RNA, Messenger ; analysis ; Receptors, LDL ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sterol Regulatory Element Binding Protein 2 ; analysis ; genetics

BACKGROUNDCardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease. Macrophage death in advanced atherosclerosis 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.

METHODSOil 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.

RESULTSConfocal 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 microg/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.

CONCLUSIONER stress is involved in AcLDL induced apoptosis in THP-1 macrophages with free cholesterol accumulation in the ER.

Apoptosis ; drug effects ; Blotting, Western ; Cell Differentiation ; physiology ; Cell Line ; Cholesterol ; metabolism ; Chromatography, High Pressure Liquid ; Endoplasmic Reticulum ; metabolism ; Humans ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; drug effects ; Microscopy, Confocal ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Transcription Factor CHOP ; genetics

OBJECTIVETo investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and liver fibrosis using a C57BL/6J mouse model of chronic inflammation.

METHODSTwelve male C57BL/6J mice were given a high-fat diet (15.0% fat, 1.25% cholesterol, 0.5% cholic acid) and randomly assigned to the normal control group (n=6; subcutaneously injected with 0.5 mL of isotonic saline, every other day for 14 weeks) or the chronic inflammation model group (n=6; subcutaneously injected with of 0.5 mL of 10% casein, every other day for 14 weeks). At the end of week 14, the animals were sacrificed and blood was collected from the left ventricle for serological analysis of inflammatory markers and lipid profile, including serum amyloid A (SAA), interleukin-6 (IL-6), total cholesterol (TC) and free cholesterol (FC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)). Extracted liver tissues were divided for use in histological analysis (lipid accumulation and fibrosis evaluated by Oil Red O, Sirius red and Masson's trichrome staining) and quantitative fluorescence real-time PCR (to measure b-actin normalized expression of TNF-a MCP1, SREBP-2, LDLr, HMGCoA-r, ATF-6, GRP78, BMP-7, TGF-b, and collagens type I and type IV). Comparisons between groups were made by the two-samples t-test or Satterthwaite t-approximation test, collagen type I and type IV.

RESULTSCompared to the normal control group, the inflammation model group showed elevated serum IL-6 (12.55+/-4.75 vs. 32.41+/-7.42 pg/mL, P less than 0.01), reduced serum TC (14.82+/-1.56 vs. 10.62+/-0.48 mmol/L, P less than 0.01), up-regulated hepatic TNF-a mRNA expression (1.05+/-0.35 vs. 2.12+/-0.72, P less than 0.01), and elevated hepatic TC (12.10+/-2.57 vs. 23.21+/-8.75 mmol/L, P less than 0.05). In addition, the inflammation group showed abnormal lipid deposition, and increased and thickened reticular fibers. The livers of the inflammation group also showed up-regulated mRNA expression of SREBP-2 (normal control: 1.01+/-0.19 vs. 2.63+/-0.13, P less than 0.05), GRP78 (1.07+/-0.47 vs. 2.21+/-0.99, P less than 0.05), TGF-b (1.01+/-0.14 vs. 1.38+/-0.28, P less than 0.05), and collagen type I (1.02+/-0.27 vs. 1.71+/-0.51, P less than 0.05) and down-regulation of BMP-7 (1.01+/-0.15 vs. 0.55+/-0.25, P less than 0.01).

CONCLUSIONActivation of the inflammatory system exacerbates hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice.

Animals ; Cholesterol ; metabolism ; Disease Models, Animal ; Fatty Liver ; metabolism ; pathology ; Inflammation ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVETo investigate if inflammatory stress enhances liver lipid accumulation via SREBPs mediated dysregulation of low density protein receptor (LDLr) expression in apolipoprotein E, scavenger receptors class A and CD36 triple knockout (ApoE/SRA/CD36 KO) mice.

METHODS16 Male ApoE/SRA/CD36 KO mice were subcutaneously injected with 0.5 ml 10% casein or PBS. The mice were fed a Western diet (Harlan, TD88137) containing 21% fat and 0.15% of cholesterol for 14 weeks. Animals were sacrificed and blood samples were collected. The serum amyloid A (SAA), IL-6, total cholesterol (TC), LDL and high density protein (HDL) were assayed. The lipid accumulation in liver was evaluated by Oil Red O staining. The mRNA and protein expression of SREBP-2, SREBPs cleavage activating protein (SCAP) and LDLr were analyzed by Real-Time Polymerase Chain Reaction (RT-PCR) and immunohistochemistry staining.

RESULTSBlood levels of SAA [(26.60+/-3.24) ng/ml vs (14.35+/-1.73) ng/ml, P < 0.01] and IL-6 [(36.37+/-2.20) pg/ml vs (18.02+/-4.87) pg/ml, P < 0.01] were higher, while TC [(7.72+/-1.70) mmol/L vs (13.23+/-3.61)mmol/L, P less than 0.01], LDL-cholesterol [(2.94+/-0.44) mmol/L vs (9.28+/-3.66) mmol/L, P less than 0.01] and HDL cholesterol [(2.24+/-0.63) mmol/L vs (4.13+/-0.42) mmol/L, P less than 0.01] were lower in inflamed mice compared to controls. ORO staining showed that lipid accumulation in the liver was more extensive in inflamed group despite lower blood lipid levels. Meanwhile, Real Time PCR data showed inflammation induced the expression of LDLr (4.56 fold), SCAP (3.14 fold) and SREBP-2 (14.72 fold) in liver. Immunohistochemical staining also indicated increased proteins expression in the liver, which was consistent with mRNA data.

CONCLUSIONSInflammation causes lipid accumulation in liver via disrupting SREBP-2 and LDLr expression.

Animals ; Apolipoproteins E ; genetics ; Cholesterol, LDL ; metabolism ; Fatty Liver ; metabolism ; Inflammation ; metabolism ; Liver ; metabolism ; Male ; Mice ; Mice, Knockout ; Receptors, LDL ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; metabolism

BACKGROUNDDyslipidaemia is a potential independent risk factor for cerebrovascular disease in patients with diabetes. The aim of this study was to investigate dyslipidaemia, treatment and control of dyslipidaemia among diabetic patients with ischemic stroke in a Chinese hospital.

METHODSA 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.

RESULTSDiabetic 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.

CONCLUSIONSMany 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.

Aged ; Aged, 80 and over ; China ; Diabetes Mellitus, Type 2 ; complications ; Dyslipidemias ; complications ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Stroke ; complications ; epidemiology

BACKGROUNDPodocyte 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.

METHODSPodocyte 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.

RESULTSPalmitate 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.

CONCLUSIONPalmitate 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.

Apoptosis ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Heat-Shock Proteins ; analysis ; physiology ; Humans ; Insulin Resistance ; Palmitic Acid ; pharmacology ; Podocytes ; drug effects ; pathology

OBJECTIVETo investigate the role of acyl-coenzyme A: cholesterol acyltransferase inhibitor (ACATI) in apoptosis induced by lipids and whether lipids-induced apoptosis is accompanied by increase of free cholesterol in endoplasmic reticulum (ER), in order to further understand the mechanism of lipids-induced apoptosis in advanced atherosclerosis.

METHODSHuman vascular smooth muscle cells (VSMCs) and phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophages were used. Tritiated thymidine incorporation was applied to detect cell proliferation. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release. 4',6-diamidino-2-phenylindole (DAPI) staining, caspase-3, -7 assay, and Annexin-V/propidium iodide (PI) staining were used to detect apoptosis. High performance liquid chromatography was used in intracellular free cholesterol and cholesterol ester assay. ER free cholesterol was quantified.

RESULTSDifferent lipids had different effects on proliferation and cytotoxicity of VSMCs. 25-hydroxycholesterol (25OHC) had biphasic effects on the proliferation of VSMCs. At low concentration, it stimulated cell proliferation, but turned to proliferation inhibition as concentration reached 15 mug/mL. 25OHC and acetylated low density lipoprotein (AcLDL) could respectively induce apoptosis in human VSMCs and PMA differentiated THP-1 macrophages, which was aggravated by ACATI, accompanied by increase of intracellular free cholesterol content. There was also an increase of cholesterol content in ER with AcLDL-induced apoptosis in THP-1 macrophages.

CONCLUSIONSLipids could induce apoptosis, accompanied by increase of intracellular free cholesterol content, which could be augmented by ACATI, suggesting that insults resulting in ER free cholesterol rise might be the initiator of apoptosis.

Apoptosis ; drug effects ; physiology ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Humans ; Lipids ; physiology ; Sterol O-Acyltransferase ; antagonists & inhibitors