OBJECTIVETo investigate the effects of hypoxia on lipid metabolism in the normal human hepatic cell line L02 and to investigate the underlying molecular mechanisms.

METHODSL02 cells were exposed to hypoxic conditions (experimental groups: at 1% O2, 5% CO2, 94% N2 for 3, 6, 12, 24, or 48 hours) or normoxic conditions (control group: at 21% O2). Lipid droplet accumulation and triglyceride content were measured in each group by oil red O staining and biochemical assay, respectively. The mRNA expression levels of hypoxia-inducible factor (HIF)-2a and sterol regulatory element binding protein (SREBP)-1c were detected by reverse transcription-polymerase chain reaction. Protein expression levels of HIF-2a, adipose differentiation-related protein (ADRP), and Fas were tested by Western blot analysis.

RESULTSLipid droplet accumulation and the triglyceride content were significantly higher in the hypoxia group than the normoxia group. In addition, the hypoxia groups had significantly down-regulated mRNA expression of SREBP-1c (12h: 0.236+/-0.043, 24 h: 0.287+/-0.044, 48 h: 0.342+/-0.049 vs. normoxia: 0.503+/-0.037; F = 28.37, P less than 0.01) and FAS protein (12 h: 0.562+/-0.054, 24 h: 0.674+/-0.062, 48 h: 0.682+/-0.057 vs normoxia: 0.857+/-0.069; F = 16.08, P less than 0.01). In normoxic cells, little or no expression of HIF-2a protein was detected by Western blot. In hypoxic cells, HIF-2a protein expression peaked at 6h (0.973+/-0.067). ADRP protein expression was significantly higher in hypoxia groups than in the normoxia group (12 h: 0.319+/-0.043, 24 h: 0.732+/-0.056 and 48 h: 0.873+/-0.066 vs. 0.211+/-0.019; all, P less than 0.05.

CONCLUSIONExposure to hypoxic conditions might induce lipidosis in normal human hepatic cells by stimulating HIF-2a and ADRP expression.

Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Hypoxia ; Cell Line ; Down-Regulation ; Hepatocytes ; metabolism ; Humans ; Lipid Metabolism ; Membrane Proteins ; metabolism ; Oxygen ; metabolism ; Perilipin-2 ; Sterol Regulatory Element Binding Protein 1 ; metabolism

OBJECTIVETo investigate the relationship between SREBP-1c and the risk of liver disease associated with the triacylglyceride lipase PNPLA3 I148M variant using a human hepatoma cell line model transfected with recombinant lentiviruses.

METHODSHuh7 cells were transfected with control lentivirus or lentivirus containing the PNPLA3 I148M variant (variant). The two cell groups were compared to assess differences in triglyceride content (using oil red O staining), levels of triglyceride and cholesterol (using automated biochemical analyzer), expression of SREBP-lc mRNA (using fluorescence quantitative PCR), and expression of SREBP-1c protein (using western blot.

RESULTSCells expressing the PNPLA3 I148M variant showed higher triglyceride content (0.54+/-0.03 mmol/L vs. control cells: 0.23+/-0.02 mmol/L; t=22.58, P<0.001), cholesterol level (0.28+/-0.03 mmol/L vs. control cells: 0.13+/-0.02 mmol/L; t =11.83, P<0.001), SREBP-1cmRNA expression (13.59+/-0.60 vs. 11.81+/-0.82; [The abstract and text in the paper say variant increases, but the data shown says the higher value is in the control cells. Please correct to properly express the data.] P=0.001), and SREBP-1c protein expression. The level of SREBP-1c was positively correlated with serum triglyceride in the cells expressing the PNPLA3 I148M variant (r=0.912, P<0.01).

CONCLUSIONThe risk of liver disease associated with the PNPLA3 I148M variant, which increases lipogenesis, may involve SREBP-1c and a pathway that increases triglycerides.

Cell Line, Tumor ; Humans ; Lipase ; Liver Diseases ; Membrane Proteins ; Risk Factors ; Sterol Regulatory Element Binding Protein 1 ; Triglycerides

Animals ; Fasting ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Sterol Regulatory Element Binding Protein 1 ; metabolism ; Transcription Factors ; deficiency ; metabolism

PURPOSE: The relatively low incidence of breast cancer in Asian countries with cultures which traditionally eat a large amount of soy is worth noticing in research fields. Genistein is a isoflavone phytoestrogen found in soy and its consumption may have a role in cancer etiology. We have established a hypothesis that a diet high in soy consumption is related to a low incidence of breast cancer. Fatty acid synthase (FAS) is a multi-protein enzyme responsible for de novo biosynthesis of fatty acids. Recent studies have demonstrated that high levels of FAS occurs in a subset of human cancers, such as breast cancer, ovarian cancer, and prostate cancer. High level of FAS are associated with a poor prognosis. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate genes involved in lipid metabolism, including FAS. Recent studies show that expression of SREBP1c is correlates with FAS expression. The aim of this study is to investigate the effect of genistein on the expression of FAS in breast cancer cells. METHODS: We performed immunofluorescent staining to examine the expression of FAS under different concentration of genistein. RT-PCR was also performed to investigate the mRNA expression of FAS and SREBP1c in different conditioned breast cancer cells treated with different concentration of FAS inhibitor and genistein. RESULTS: By immunofluorescent staining, the FAS expression after treatment with the FAS inhibitor, C75, decreased at a micron10 M concentration. However the expression of FAS decreased at all concentrations of genistein (0.5, 1, 5, 10 micronM). The mRNA levels of FAS and SREBP1c after treatment with C75 decreased constantly according to time and concentration. However the effect was noted only after 12 hr. The mRNA level of FAS and SREBP1c following treatment with genistein decreased at only a 10 micronM concentration (p<0.005). CONCLUSION: Genistein may down regulate FAS expression in breast cancer cells through modulation of SREBP-1c. This finding may account for the relatively low incidence of breast cancer in Asians who consume a large amount of soy in their diet.
Asian Continental Ancestry Group ; Breast Neoplasms* ; Breast* ; Diet ; Fatty Acids ; Genistein* ; Humans ; Incidence ; Lipid Metabolism ; Ovarian Neoplasms ; Phytoestrogens ; Prognosis ; Prostatic Neoplasms ; RNA, Messenger ; Sterol Regulatory Element Binding Protein 1 ; Sterol Regulatory Element Binding Proteins ; Transcription Factors

The polytopic transmembrane protein, Niemann-Pick type C1 Like 1 (NPC1L1), is the key point of exogenous cholesterol absorption and plays an important role in cholesterol metabolism. However, the molecular mechanism of NPC1L1's role in cholesterol uptake remains unclear. NPC1L1 expression is highly regulated by a variety of molecular actors. Nuclear receptors regulate NPC1L1 expression through its promoter region. Polyunsaturated fatty acids down-regulates NPC1L1 expression by the way of sterol regulatory element binding protein 2 (SREBP2). In addition, curcumin and sphingosine-phosphate take part in the regulation of NPC1L1 expression. NPC1L1 has been recognized as an essential protein for sterol absorption and is the molecular target of ezetimibe. Moreover, inhibition of the expression of NPC1L1 has been shown to have beneficial effects on components of the metabolic syndrome. The recent progress in the structure, function and regulation of NPC1L1 is reviewed.
Azetidines ; pharmacology ; Biological Transport ; Cholesterol ; metabolism ; Ezetimibe ; Fatty Acids ; metabolism ; Humans ; Membrane Proteins ; metabolism ; Metabolic Syndrome ; physiopathology ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; metabolism

OBJECTIVE: To investigate effects of berberine (BBR) on cholesterol synthesis in HepG2 cells with free fatty acid (FFA)-induced steatosis and to explore the underlying mechanisms. METHODS: A steatosis cell model was induced in HepG2 cell line fed with FFA (0.5 mmol/L, oleic acid:palmitic acid = 2:1), and then treated with three concentrations of BBR; cell viability was assessed with cell counting kit-8 assays. Lipid accumulation in cells was observed through oil red O staining and total cholesterol (TC) content was detected by TC assay. The effects of BBR on cholesterol synthesis mediators were assessed by Western blotting and quantitative polymerase chain reaction. In addition, both silent information regulator 1 (SIRT1) and forkhead box transcription factor O1 (FoxO1) inhibitors were employed for validation. RESULTS: FFA-induced steatosis was successfully established in HepG2 cells. Lipid accumulation and TC content in BBR groups were significantly lower (P < 0.05, P < 0.01), associated with significantly higher mRNA and protein levels of SIRT1(P < 0.05, P < 0.01), significantly lower sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy 3-methylglutaryl-CoA reductase levels (P < 0.05, P < 0.01), as well as higher Acetyl-FoxO1 protein level (P < 0.05, P < 0.01) compared to the FFA only group. Both SIRT1 inhibitor SIRT1-IN-1 and FoxO1 inhibitor AS1842856 blocked the BBR-mediated therapeutic effects. Immunofluorescence showed that the increased SIRT1 expression increased FoxO1 deacetylation, and promoted its nuclear translocation. CONCLUSION BBR can mitigate FFA-induced steatosis in HepG2 cells by activating SIRT1-FoxO1-SREBP2 signal pathway. BBR may emerge as a potential drug candidate for treating nonalcoholic hepatic steatosis.
Berberine/pharmacology* ; Cholesterol ; Forkhead Box Protein O1/genetics* ; Humans ; Non-alcoholic Fatty Liver Disease/drug therapy* ; Sirtuin 1/genetics* ; Sterol Regulatory Element Binding Proteins

BACKGROUND/OBJECTIVES: Reducing the number of adipocytes by inducing apoptosis of mature adipocytes as well as suppressing differentiation of preadipocytes plays an important role in preventing obesity. This study examines the anti-adipogenic and pro-apoptotic effect of red pepper seed water extract (RPS) prepared at 4℃ (RPS4) in 3T3-L1 cells. MATERIALS/METHODS: Effect of RPS4 or its fractions on lipid accumulation was determined in 3T3-L1 cells using oil red O (ORO) staining. The expressions of AMP-activated protein kinase (AMPK) and adipogenic associated proteins [peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding proteins α (C/EBP α), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC)] were measured in 3T3-L1 cells treated with RPS4. Apoptosis and the expression of Akt and Bcl-2 family proteins [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad), Bcl-2 like protein 4 (Bax), Bal-2 homologous antagonist/killer (Bak)] were measured in mature 3T3-L1 cells treated with RPS4. RESULTS: Treatment of RPS4 (0–75 µg/mL) or its fractions (0–50 µg/mL) for 24 h did not have an apparent cytotoxicity on pre and mature 3T3-L1 cells. RPS4 significantly suppressed differentiation and cellular lipid accumulation by increasing the phosphorylation of AMPK and reducing the expression of PPAR-γ, C/EBP α, SREBP-1c, FAS, and ACC. In addition, all fractions except ethyl acetate fraction significantly suppressed cellular lipid accumulation. RPS4 induced the apoptosis of mature adipocytes by hypophosphorylating Akt, increasing the expression of the pro-apoptotic proteins, Bak, Bax, and Bad, and reducing the expression of the anti-apoptotic proteins, Bcl-2 and p-Bad. CONCLUSIONS: These finding suggest that RPS4 can reduce the numbers as well as the size of adipocytes and might useful for preventing and treating obesity.
3T3-L1 Cells* ; Acetyl-CoA Carboxylase ; Adipocytes* ; Adipogenesis ; AMP-Activated Protein Kinases ; Apoptosis Regulatory Proteins ; Apoptosis* ; Capsicum* ; Humans ; Lipid Metabolism ; Lymphoma ; Obesity ; Phosphorylation ; Sterol Regulatory Element Binding Protein 1 ; Water*

BACKGROUND/AIMS: Hepatic steatosis is caused by an imbalance between free fatty acids (FFAs) uptake, utilization, storage, and disposal. Understanding the molecular mechanisms involved in FFAs accumulation and its modulation could drive the development of potential therapies for Nonalcoholic fatty liver disease. The aim of the current study was to explore the effects of picroside II, a phytoactive found in Picrorhiza kurroa, on fatty acid accumulation vis-à-vis silibinin, a known hepatoprotective phytoactive from Silybum marianum. METHODS: HepG2 cells were loaded with FFAs (oleic acid:palmitic acid/2:1) for 20 hours to mimic hepatic steatosis. The FFAs concentration achieving maximum fat accumulation and minimal cytotoxicity (500 μM) was standardized. HepG2 cells were exposed to the standardized FFAs concentration with and without picroside II pretreatment. RESULTS: Picroside II pretreatment inhibited FFAs-induced lipid accumulation by attenuating the expression of fatty acid transport protein 5, sterol regulatory element binding protein 1 and stearoyl CoA desaturase. Preatreatment with picroside II was also found to decrease the expression of forkhead box protein O1 and phosphoenolpyruvate carboxykinase. CONCLUSIONS: These findings suggest that picroside II effectively attenuated fatty acid accumulation by decreasing FFAs uptake and lipogenesis. Picroside II also decreased the expression of gluconeogenic genes.
Fatty Acid Transport Proteins ; Fatty Acids, Nonesterified ; Hep G2 Cells* ; Lipogenesis ; Milk Thistle ; Non-alcoholic Fatty Liver Disease ; Phosphoenolpyruvate ; Picrorhiza ; Stearoyl-CoA Desaturase ; Sterol Regulatory Element Binding Protein 1

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

Peroxisome proliferator activated receptor (PPAR) gamma coactivator-1alpha (PGC-1alpha) may be implicated in cholesterol metabolism since PGC-1alpha co-activates estrogen receptor alpha (ERalpha) transactivity and estrogen/ERalpha induces the transcription of LDL receptor (LDLR). Here, we show that overexpression of PGC-1alpha in HepG2 cells represses the gene expression of LDLR and does not affect the ERalpha-induced LDLR expression. PGC-1alpha suppressed the LDLR promoter-luciferase (pLR1563-luc) activity regardless of cholesterol or functional sterol-regulatory element-1. Serial deletions of the LDLR promoter revealed that the inhibition by PGC-1alpha required the LDLR promoter regions between -650 bp and -974 bp. Phosphorylation of PGC-1alpha may not affect the suppression of LDLR expression because treatment of SB202190, a p38 MAP kinase inhibitor, did not reverse the LDLR down-regulation by PGC-1alpha. This may be the first report showing the repressive function of PGC-1alpha on gene expression. PGC-1alpha might be a novel modulator of LDLR gene expression in a sterol-independent manner, and implicated in atherogenesis.
Base Sequence ; Cell Line, Tumor ; Cholesterol/metabolism ; Estrogen Receptor alpha/metabolism ; Gene Expression Regulation ; Heat-Shock Proteins/*genetics/*metabolism ; Humans ; Molecular Sequence Data ; Promoter Regions, Genetic ; RNA, Messenger/genetics ; Receptors, LDL/*genetics/*metabolism ; Sterol Regulatory Element Binding Protein 2/metabolism ; Transcription Factors/*genetics/*metabolism ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors