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

To investigate the effect of HBV on the expression of Sterol regulatory element binding proteins( SREBP ) in the hepatocyte of patients with chronic hepatitis B (CHB) combined with hepatic fatty change. 55 cases diagnosed as CHB combined with hepatic fatty change in our department were selected and liver biopsies were carried out. The patients were dividied into 3 groups, group A: HBV DNA is less than or equal to 1000 copies/ml(15 cases), group B: 1000 copies/ml less than HBV DNA less than 100000 copies/ml (18 cases) and group C: HBV DNA is more than or equal to 100000 copies/ml (22 cases). 10 patients with HBV DNA in less than or equal to 1000 copies/ml after antiviral therapy with Nucleoside analogues were seen as group C1 (before treatment) and group C2 (after treatment) respectively; 12 patients with HBV DNA is more than or equal to 100000 copies/ml after antiviral therapy were classified as group C3 (before treatment) and group C4 (after treatment). Lipid droplets in the hepatic tissue were observed with oil red staining. Real time PCR were performed to detect the expressions of SREBP-1c and SREBP-2 mRNA in the liver. The protein expressions of SREBP-1c and SREBP-2 were detected with immunohistochemistry staining. Statistic data were analysed with SPSS11.5 software. (1) Red integrated optical densities (IOD) reflected by lipid drops in group A, B and C are 1004.27+/-218.63, 1937.01+/-401.47 and 4133.79+/-389.28 respectively, the degree of oil red O in each group was different (F = 385.69, P is less than to 0.01), which is increased as HBV DNA load increasing; Red IOD in group C1, C2 and C3, C4 are 4020.84+/-326.64, 1012.02+/-244.89, 4189.18+/-329.21 and 4121.76+/-304.09 respectively. Compared with group C1, the degree of oil red O in group C2 is decreased and the difference is statistically significant (t = 22.55, P is less than to 0.01); However, the difference of the degree of oil red O between group C4 and C3 is not statistically significant. (2) Compared with group A, the expressions of SREBP-1c mRNA in group B and C are raised by 1.218+/-0.130 and 1.798+/-0.118 times respectively, among group A, B, C, the expressions of SREBP-1c mRNA are statistically significant different ( F = 297.47, P is less than to 0.01). The expressions of SREBP-2 mRNA in group B and C are decreased by 0.956+/-0.118 and 0.972+/-0.153 times as compared to group A. However, the difference of SREBP-2 mRNA expression among the 3 groups is not statistically significant ( F = 0.568, P is more than to 0.05). Compared with group C1, SREBP-1c mRNA in group C2 is decreased by 0.714+/-0.081 folds (t=11.224, P is less than to 0.01), while SREBP-2 mRNA in group C2 is raised by1.034+/-0.155 times(t=0.692, P is more than to 0.05). SREBP-1c mRNA and SREBP-2 mRNA in group C4 are raised by 1.012+/-0.206 times and decreased by 0.998+/-0.183 times as compared to group C3 without difference found (t=0.196 or 0.031, P is more than to 0.05). (3) the expressions of SREBP-1c protein in group A, B and C are 36257.21+/-5709.79, 50413.47+/-4989.28 and 71025.83+/-6047.13 respectively, and the difference is statistically significant among the 3 groups (F = 178.26, P is less than to 0.01); the expressions of SREBP-2 protein in group A, B and C are 32913.52+/-3951.21, 32625.91+/-4025.06 and 34173.44+/-5316.25 respectively, but the difference is not statistically significant among the 3 groups ( F = 0.562, P is more than to 0.05), SREBP-1c protein levels in group C1, C2, C3, C4 are 69832.16+/-4941.36, 48735.47+/-5471.41, 70871.69+/-5083.14 and 68913.32+/-5343.22 respectively, the difference of SREBP-1c protein levels between group C1 and C2 is statistically significant (t=10.260, P is less than to 0.01); while the difference between group C3 and group C4 is not statistically significant(t=1.558, P is more than to 0.05). The expressions of SREBP-2 protein in group C1, C2, C3 and C4 are 33 980.21+/-4081.80, 34011.50+/-3859.27, 33610.12+/-4761.10 and 32915.66+/-5023.61 respectively, the difference of SREBP-2 protein levels in group C1 and group C2 is not statistically significant (t=0.038, P is more than to 0.05) and same result exists between group C3 and group C4 (t=0.459, P is more than to 0.05). HBV DNA may participate in the hepatic steatosis formation through interfering with the SREBP-1c expression.
Fatty Liver ; metabolism ; Hepatitis B, Chronic ; metabolism ; Hepatocytes ; metabolism ; Humans ; Sterol Regulatory Element Binding Protein 1 ; metabolism

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

OBJECTIVETo explore the roles of intracellular cholesterol metabolism in neuroendocrine (NE) differentiation of prostate cancer based on an androgen-independent prostate cancer NE cell model induced by androgen deprivation.

METHODSLNCaP cells were cultured in androgen-depleted medium, and NE phenotypes were identified by observing the changes in cell morphology, molecular markers (SgIII, NSE and CgA) and cell proliferation. The expression and distribution of cholesterol and Sg III were determined by immunofluorescence staining. The expressions of the key genes LDL-R, SREBP-1 and SREBP-2 involved in cholesterol synthesis and uptake were detected by semi-quantitative RT-PCR.

RESULTSThe LNCaP cells showed shrinking bodies and extending axons after androgen deprivation, and all the molecular markers, such as Sg III, NSE and CgA, significantly increased in a time-dependent manner, while the cell proliferation was obviously inhibited (P < 0.05). The cholesterol distribution in the LNCaP cells after NE differentiation presented remarkable aggregation at the axon terminals. However, there were no significant differences in the expression of cholesterol between the two types of cells, nor in the changes of the expressions of key genes LDL-R, SREBP-1 and SREBP-2 involved in cholesterol synthesis and uptake (P > 0.05).

CONCLUSIONTransient androgen depletion could successfully induce NE differentiation of LNCaP cells, and the intracellular cholesterol could re-distribute into axon terminals to enhance the formation of neurosecretory granules.

Androgens ; pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Cholesterol ; metabolism ; Humans ; Male ; Neurosecretory Systems ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; Receptors, LDL ; metabolism ; Sterol Regulatory Element Binding Protein 1 ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; metabolism

BackgroundA high consumption of fructose leads to hepatic steatosis. About 20-30% of triglycerides are synthesized via de novo lipogenesis. Some studies showed that endoplasmic reticulum stress (ERS) is involved in this process, while others showed that a lipotoxic environment directly influences ER homeostasis. Here, our aim was to investigate the causal relationship between ERS and fatty acid synthesis and the effect of X-box binding protein-1 (XBP-1), one marker of ERS, on hepatic lipid accumulation stimulated by high fructose.

MethodsHepG2 cells were incubated with different concentrations of fructose. Upstream regulators of de novo lipogenesis (i.e., carbohydrate response element-binding protein [ChREBP] and sterol regulatory element-binding protein 1c [SREBP-1c]) were measured by polymerase chain reaction and key lipogenic enzymes (acetyl-CoA carboxylase [ACC], fatty acid synthase [FAS], and stearoyl-CoA desaturase-1 [SCD-1]) by Western blotting. The same lipogenesis-associated factors were then evaluated after exposure of HepG2 cells to high fructose followed by the ERS inhibitor tauroursodeoxycholic acid (TUDCA) or the ERS inducer thapsigargin. Finally, the same lipogenesis-associated factors were evaluated in HepG2 cells after XBP-1 upregulation or downregulation through cell transfection.

ResultsExposure to high fructose increased triglyceride levels in a dose- and time-dependent manner and significantly increased mRNA levels of SREBP-1c and ChREBP and protein levels of FAS, ACC, and SCD-1, concomitant with XBP-1 conversion to an active spliced form. Lipogenesis-associated factors induced by high fructose were inhibited by TUDCA and induced by thapsigargin. Triglyceride level in XBP-1-deficient group decreased significantly compared with high-fructose group (4.41 ± 0.54 μmol/g vs. 6.52 ± 0.38 μmol/g, P < 0.001), as mRNA expressions of SREBP-1c (2.92 ± 0.46 vs. 5.08 ± 0.41, P < 0.01) and protein levels of FAS (0.53 ± 0.06 vs. 0.85 ± 0.05, P = 0.01), SCD-1 (0.65 ± 0.06 vs. 0.90 ± 0.04, P = 0.04), and ACC (0.38 ± 0.03 vs. 0.95 ± 0.06, P < 0.01) decreased. Conversely, levels of triglyceride (4.22 ± 0.54 μmol/g vs. 2.41 ± 0.35 μmol/g, P < 0.001), mRNA expression of SREBP-1c (2.70 ± 0.33 vs. 1.00 ± 0.00, P < 0.01), and protein expression of SCD-1 (0.93 ± 0.06 vs. 0.26 ± 0.05, P < 0.01), ACC (0.98 ± 0.09 vs. 0.43 ± 0.03, P < 0.01), and FAS (0.90 ± 0.33 vs. 0.71 ± 0.02, P = 0.04) in XBP-1s-upregulated group increased compared with the untransfected group.

ConclusionsERS is associated with de novo lipogenesis, and XBP-1 partially mediates high-fructose-induced lipid accumulation in HepG2 cells through augmentation of de novo lipogenesis.

Endoplasmic Reticulum Stress ; physiology ; Fatty Liver ; Fructose ; metabolism ; Hep G2 Cells ; Humans ; Lipogenesis ; physiology ; Liver ; Sterol Regulatory Element Binding Protein 1 ; X-Box Binding Protein 1 ; physiology

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

Obesity occurs when a person's calorie intake exceeds the amount of energy burns, which may lead to pathologic growth of adipocytes and the accumulation of fat in the tissues. In this study, the effect and mechanism of pear pomace extracts on 3T3-L1 adipocyte differentiation and apoptosis of mature adipocytes were investigated. The effects of pear pomace extract on cell viability and the anti-adipogenic and proapoptotic effects were investigated via MTT assay, Oil red O staining, western blot analysis and apoptosis assay. 3T3-L1 preadipocytes were stimulated with DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 microg/ml insulin and 1 microM dexamethasone for differentiation to adipocytes. 3T3-L1 cells were cultured with PBS or water extract of pear pomace. Water extract of pear pomace effectively inhibited lipid accumulations and expressions of PPAR-gamma and C/EBPalpha in 3T3-L1 cells. It also increased expression of p-AMPK and decreased the expression of SREBP-1c and FAS in 3T3-L1 cells. The induction of apoptosis was observed in 3T3-L1 cells treated with pear pomace. These results indicate that pear pomace water extract inhibits adipogenesis and induces apoptosis of adipocytes and thus can be used as a potential therapeutic substance as part of prevention or treatment strategy for obesity.
1-Methyl-3-isobutylxanthine ; 3T3-L1 Cells ; Adipocytes* ; Adipogenesis* ; Apoptosis* ; Blotting, Western ; Burns ; Cell Survival ; Dexamethasone ; Insulin ; Obesity ; Pyrus* ; Sterol Regulatory Element Binding Protein 1 ; Water*

BACKGROUND/OBJECTIVES: Citrus flavonoids have a variety of physiological properties such as anti-oxidant, anti-inflammation, anti-cancer, and anti-obesity. We investigated whether bioconversion of Citrus unshiu with cytolase (CU-C) ameliorates the anti-adipogenic effects by modulation of adipocyte differentiation and lipid metabolism in 3T3-L1 cells. MATERIALS/METHODS: Glycoside forms of Citrus unshiu (CU) were converted into aglycoside forms with cytolase treatment. Cell viability of CU and CU-C was measured at various concentrations in 3T3L-1 cells. The anti-adipogenic and lipolytic effects were examined using Oil red O staining and free glycerol assay, respectively. We performed real time-polymerase chain reaction and western immunoblotting assay to detect mRNA and protein expression of adipogenic transcription factors, respectively. RESULTS: Treatment with cytolase decreased flavanone rutinoside forms (narirutin and hesperidin) and instead, increased flavanone aglycoside forms (naringenin and hesperetin). During adipocyte differentiation, 3T3-L1 cells were treated with CU or CU-C at a dose of 0.5 mg/ml. Adipocyte differentiation was inhibited in CU-C group, but not in CU group. CU-C markedly suppressed the insulin-induced protein expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) as well as the mRNA levels of CEBPalpha, PPARgamma, and sterol regulatory element binding protein 1c (SREBP1c). Both CU and CU-C groups significantly increased the adipolytic activity with the higher release of free glycerol than those of control group in differentiated 3T3-L1 adipocytes. CU-C is particularly superior in suppression of adipogenesis, whereas CU-C has similar effect to CU on stimulation of lipolysis. CONCLUSIONS: These results suggest that bioconversion of Citrus unshiu peel extracts with cytolase enhances aglycoside flavonoids and improves the anti-adipogenic metabolism via both inhibition of key adipogenic transcription factors and induction of adipolytic activity.
3T3-L1 Cells* ; Adipocytes ; Adipogenesis ; Blotting, Western ; Cell Survival ; Citrus* ; Flavonoids ; Glycerol ; Lipid Metabolism ; Lipolysis ; Metabolism ; PPAR gamma ; RNA, Messenger ; Sterol Regulatory Element Binding Protein 1 ; Transcription Factors

OBJECTIVETo investigate the hepatic expression of sterol regulatory element-binding proteins-1c (SREBP-1c) in patients with nonalcoholic fatty liver disease (NAFLD).

METHODSNAFLD patients were confirmed using clinical and pathological methods. The expressions of protein and mRNA of SREBP-1 c in the livers were detected by immunohistochemistry, reverse transcription and polymerase chain reactions. The expression of protein of fatty acid synthases (FAS) controlled by SREBP-1c was observed by immunofluorescence. Levels of triglyceride and cholesterol in serum were measured.

RESULTSThe serum levels of triglyceride and cholesterol were positively correlated with hepatic adipose degeneration (r=0.71 or 0.70, P less than 0.05). The levels of SREBP-1c protein in the livers of NAFLD patients were significantly higher than those of the control group (2.19+/-0.31 vs 1.15+/-0.20, t=11.06, P less than 0.05), and the more severe the fatty degeneration, the stronger their expression (F=24.54, P less than 0.01). The expressions of the SREBP-1C mRNA in the livers showed the same changes (0.69+/-0.02 vs 0.40+/-0.02, t= -14.63, P less than 0.05). The expression of FAS was enhanced significantly in the livers of the NAFLD patients.

CONCLUSIONEnhanced expression of SREBP-1 c and FAS maybe a factor causing hepatic lipopexia in NAFLD patients.

Adult ; Fatty Acid Synthases ; metabolism ; Fatty Liver ; metabolism ; Female ; Humans ; Liver ; metabolism ; Male ; Middle Aged ; Sterol Regulatory Element Binding Protein 1 ; metabolism

Adult ; Case-Control Studies ; Fatty Liver ; genetics ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Sterol Regulatory Element Binding Protein 1 ; genetics