Protease cleavage of RNF20 facilitates coronavirus replication via stabilization of SREBP1.

COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.

Chemical constituents from the stem bark of Albizia julibrissin and their SREBP-1c inhibitory activity.

One new lignan, julibrissinoside II, along with thirteen known compounds, was isolated from the stem bark of Albizia julibrissin. The structure of julibrissinoside II was determined on the basis of extensive spectroscopic methods, including NMR and CD spectroscopic data. The isolated compounds were tested for their SREBP-1c inhibitory activity at different concentrations using mouse hepatocyte AML12 cell lines. Among them, linoleic acid (2) and 3-O-methylfisetin (4) showed significant SREBP-1c inhibitory activity at the concentration of 100 µM.

ZHX2 inhibits SREBP1c-mediated de novo lipogenesis in hepatocellular carcinoma via miR-24-3p.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Lipogenesis has been considered as a critical player in HCC initiation and progression. However, the underlying mechanism is still not fully understood. Here, we identified zinc fingers and homeoboxes 2 (ZHX2), an HCC-associated tumor suppressor, as an important repressor of de novo lipogenesis. Ectopic expression of ZHX2 significantly inhibited de novo lipogenesis in HCC cells and decreased expression of FASN, ACL, ACC1, and SCD1. In accordance with this, ZHX2 was negatively associated with SREBP1c, the master regulator of de novo lipogenesis, in HCC cell lines and human specimens. Results from silencing and overexpression demonstrated that ZHX2 inhibited de novo lipogenesis and consequent HCC progression via repression of SREBP1c. Furthermore, treatment with the SREBP1c inhibitor fatostatin dampened the spontaneous formation of tumors in liver-specific Zhx2 knockout mice. Mechanistically, ZHX2 increased expression of miR-24-3p transcriptionally, which targeted SREBP1c and led to its degradation. In conclusion, our data suggest a novel mechanism through which ZHX2 suppresses HCC progression, which may provide a new strategy for the treatment of HCC. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lipid Inhibitory Effect of (-)-loliolide Isolated from Sargassum horneri in 3T3-L1 Adipocytes: Inhibitory Mechanism of Adipose-Specific Proteins.

Sargassum horneri (S. horneri) is a well-known brown seaweed widely distributed worldwide. Several biological activities of S. horneri have been reported. However, its effects on lipid metabolism and the underlying mechanisms remain elusive. In the present study, we examined the inhibitory effect of the active compound "(-)-loliolide ((6S,7aR)-6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1-benzofuran-2(4H)-one (HTT))" from S. horneri extract on lipid accumulation in differentiated adipocytes. MTT assays demonstrated that (-)-loliolide is not toxic to 3T3-L1 adipocytes in a range of concentrations. (-)-loliolide significantly reduced intracellular lipid accumulation in the differentiated phase of 3T3-L1 adipocytes as shown by Oil Red O staining. Western blot analysis revealed that (-)-loliolide increased the expression of lipolytic protein phospho-hormone-sensitive lipase (p-HSL) and thermogenic protein peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1). Additionally, (-)-loliolide decreased expression of adipogenic and lipogenic proteins, including sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-α (C/EBP-α), and fatty acid-binding protein 4 (FABP4) in 3T3-L1 adipocytes. These results indicate that (-)-loliolide from S. horneri could suppress lipid accumulation via regulation of antiadipogenic and prolipolytic mechanisms in 3T3-L1 cells. Considering the multifunctional effect of (-)-loliolide, it can be useful as a lipid-lowering agent in the management of patients who suffer from obesity.

Influence of Genistein on Hepatic Lipid Metabolism in an In Vitro Model of Hepatic Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels.

Beneficial Effects of SREBP Decoy Oligodeoxynucleotide in an Animal Model of Hyperlipidemia.

Hyperlipidemia is a chronic disorder that plays an important role in the development of cardiovascular diseases, type II diabetes, atherosclerosis, hypertension, and non-alcoholic fatty liver disease. Hyperlipidemias have created a worldwide health crisis and impose a substantial burden not only on personal health but also on societies and economies. Transcription factors in the sterol regulatory element binding protein (SREBP) family are key regulators of the lipogenic genes in the liver. SREBPs regulate lipid homeostasis by controlling the expression of a range of enzymes required for the synthesis of endogenous cholesterol, fatty acids, triacylglycerol, and phospholipids. Thereby, SREBPs have been considered as targets for the treatment of metabolic diseases. The aim of this study was to investigate the beneficial functions and the possible underlying molecular mechanisms of SREBP decoy ODN, which is a novel inhibitor of SREBPs, in high-fat diet (HFD)-fed hyperlipidemic mice. Our studies using HFD-induced hyperlipidemia animal model revealed that SREBB decoy ODN inhibited the increased expression of fatty acid synthetic pathway, such as SREBP-1c, FAS, SCD-1, ACC1, and HMGCR. In addition, SREBP decoy ODN decreased pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-8, and IL-6 expression. These results suggest that SREBP decoy ODN exerts its anti-hyperlipidemia effects in HFD-induced hyperlipidemia mice by regulating their lipid metabolism and inhibiting lipogenesis through inactivation of the SREPB pathway.

Influences of sterol regulatory element binding protein-1c silencing on glucose production in HepG2 cells treated with free fatty acid.

BACKGROUND: Elevation of exogenous free fatty acid (FFA) level leads to insulin resistance (IR) in liver, IR is manifested by elevated hepatic glucose production. We aim to study whether inhibition of endogenous fatty acid synthesis could decrease hepatic glucose production. METHODS: Low-passage HepG2 cells derived from human liver tissue were cultured in medium supplemented with FFA to induce IR, the influences of sterol regulatory element binding protein-1c (SREBP-1c) silencing on glucose production of HepG2 cells were investigated, and genes responsible for fatty acid and glucose metabolism were detected by real-time PCR. RESULTS: Compared with HepG2 cells cultured in normal growth medium, glucose production of HepG2 cells treated by FFA was significantly increased {[(0.28 ± 0.01) vs (0.83 ± 0.02)] umol.ug- 1 protein, n = 6 wells, P < 0.01}; the mRNA expression of phosphoenolpyruvate carboxylase kinase (PEPCK) and glucose-6-phosphatase (G6PC) in HepG2 cells increased by more than 5-fold and 3-fold, respectively; the mRNA expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) increased by approximately 4-fold and 1.1-fold, respectively; the mRNA expression of carnitine palmitoyltransferase-1 (CPT-1) changed slightly. Compared with the scrambled siRNA control, glucose production of HepG2 cells treated by FFA significantly increased after SREBP-1c silencing {[(0.018 ± 0.001) vs (0.028 ± 0.002)] umol.ug- 1 protein, n = 6 wells, P < 0.01}; the mRNA expression of PEPCK and G6PC increased by approximately 1.5-fold and 5-fold, respectively, but the mRNA expression of FAS, SCD1 and CPT-1 changed slightly. CONCLUSIONS: SREBP-1c silencing further augmented glucose production of HepG2 cells treated by FFA significantly, genes responsible for fatty acid synthesis and gluconeogenesis played an important role in this process. SREBP-1c functions not only as a lipid regulator but also plays an important role in regulation of glucose metabolism.

Crocin ameliorates hepatic steatosis through activation of AMPK signaling in db/db mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity, type 2 diabetes and other metabolic disorders worldwide. Crocin is a carotenoid compound possessing various pharmacological activities. In the present study, we aimed to investigate the effect on fatty liver under diabetic and obese condition and to examine the possible role of AMP-activated protein kinase (AMPK) signaling. METHODS: db/db mice were administrated with crocin and injected with LV-shAMPK or its negative control lentivirus. Metabolic dysfunction, lipogenesis and fatty acid-oxidation in liver were evaluated. RESULTS: In db/db mice, we found that oral administration of crocin significantly upregulated the phosphorylation of AMPK and downregulated the phosphorylation of mTOR in liver. Crocin reduced liver weight, serum levels of alanine aminotransferase, alanine aminotransferase, and liver triglyceride content, and attenuated morphological injury of liver in db/db mice. Crocin inhibited the mRNA expression of lipogenesis-associated genes, including sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor γ, fatty acid synthase, stearoyl-CoA desaturase 1, and diacylglycerol acyltransferase 1, and increased the mRNA expression of genes involved in the regulation of ß-oxidation of fatty acids, including PPARα, acyl-CoA oxidase 1, carnitine palmitoyltransferase 1, and 3-hydroxy-3-methylglutaryl-CoA synthase 2. Moreover, treatment of crocin resulted in a amelioration of general metabolic disorder, as evidenced by decreased fasting blood glucose, reduced serum levels of insulin, triglyceride, total cholesterol, and non-esterified fatty acid, and improved glucose intolerance. Crocin-induced protective effects against fatty liver and metabolic disorder were significantly blocked by lentivirus-mediated downregulation of AMPK. CONCLUSIONS: The results suggest that crocin can inhibit lipogenesis and promote ß-oxidation of fatty acids through activation of AMPK, leading to improvement of fatty liver and metabolic dysfunction. Therefore, crocin may be a potential promising option for the clinical treatment for NAFLD and associated metabolic diseases.

Long noncoding RNA lncARSR promotes hepatic lipogenesis via Akt/SREBP-1c pathway and contributes to the pathogenesis of nonalcoholic steatohepatitis.

Non-alcoholic fatty liver disease and steatohepatitis (NAFLD and NASH) account for the majority of liver disease in industrialized countries. However, the pathogenesis still unclear. Long non-coding RNAs (lncRNAs) has been reported to be involved in various pathophysiological processes. Here, we reported a novel role of lncARSR in hepatic lipogenesis in NAFLD. The expression of lncARSR was induced both in NAFLD patients and mouse model, as well as in hepatocytes treated with fatty acid. Moreover, overexpression of lncARSR enhanced while knockdown of lncARSR ameliorated hepatic lipid accumulation in vivo and in vitro. Furthermore, the expression of genes related to fatty acid synthesis and oxidation increased with lncARSR overexpression in vivo. Mechanistically, we identified that lncARSR regulated hepatic lipogenesis via upregulating SREBP-1c, the key regulatory molecule involved in lipogenesis. Knockdown of SREBP-1c by shRNA blocked the effect of lncARSR on lipogenesis. Furthermore, we demonstrated that lncARSR regulated SREBP-1c expression by PI3K/Akt pathway. In conclusion, our data indicated that lncARSR potentially contributes to the hepatic steatosis in NAFLD, which may be a new therapeutic target against NAFLD.

Apigetrin inhibits adipogenesis in 3T3-L1 cells by downregulating PPARγ and CEBP-α.

BACKGROUND: Apigetrin, a flavonoid found in many plant leaves and seeds, has been known to possess antimutagenic, anti-cancer, antioxidant and anti-inflammatory properties. Here, we are investigating the effect of the apigetrin on adipocytes differentiation in 3T3-L1 adipocytes, and elucidating the mechanism of its action. METHODS: Lipids accumulation was measured by Oil Red O staining and cell cycle was analyzed by flow cytometry. The antioxidant effect of apigetrin was evaluated against hydrogen peroxide. The expression of various genes, involved in adipogenesis and inflammation, was studied by real-time PCR. RESULTS: Our results showed that apigterin treatment inhibited significantly lipid accumulation without effect on cell viability at 100 µM, and it exerted the anti-adipogenic effect during the early stages of differentiation. Flow cytometry analysis showed that apigenin-7-O-glucoside (Ap7G) inhibited cell proliferation during mitotic clonal expansion and caused cell cycle delay. Quantitative PCR analysis revealed that the mRNA levels of C/EBP-α, PPAR-γ, SREBP-1c and FAS were suppressed after apigetrin treatment at 100 µM. Moreover, the mRNA level of pro-inflammatory genes (TNF-α and IL-6) were suppressed after apigterin treatment, at high concentration preadipocyte cells. CONCLUSION: Taken together, these results indicated that apigenin-7-O-glucoside inhibits adipogenesis of 3T3-L1 preadipocytes at early stage of adipogenesis.

Transcriptional and Translational Modulation of myo-Inositol Oxygenase (Miox) by Fatty Acids: IMPLICATIONS IN RENAL TUBULAR INJURY INDUCED IN OBESITY AND DIABETES.

The kidney is one of the target organs for various metabolic diseases, including diabetes, metabolic syndrome, and obesity. Most of the metabolic studies underscore glomerular pathobiology, although the tubulo-interstitial compartment has been underemphasized. This study highlights mechanisms concerning the pathobiology of tubular injury in the context of myo-inositol oxygenase (Miox), a tubular enzyme. The kidneys of mice fed a high fat diet (HFD) had increased Miox expression and activity, and the latter was related to phosphorylation of serine/threonine residues. Also, expression of sterol regulatory element-binding protein1 (Srebp1) and markers of cellular/nuclear damage was increased along with accentuated apoptosis and loss of tubular brush border. Similar results were observed in cells treated with palmitate/BSA. Multiple sterol-response elements and E-box motifs were found in the miox promoter, and its activity was modulated by palmitate/BSA. Electrophoretic mobility and ChIP assays confirmed binding of Srebp to consensus sequences of the miox promoter. Exposure of palmitate/BSA-treated cells to rapamycin normalized Miox expression and prevented Srebp1 nuclear translocation. In addition, rapamycin treatment reduced p53 expression and apoptosis. Like rapamycin, srebp siRNA reduced Miox expression. Increased expression of Miox was associated with the generation of reactive oxygen species (ROS) in kidney tubules of mice fed an HFD and cell exposed to palmitate/BSA. Both miox and srebp1 siRNAs reduced generation of ROS. Collectively, these findings suggest that HFD or fatty acids modulate transcriptional, translational, and post-translational regulation of Miox expression/activity and underscore Miox being a novel target of the transcription factor Srebp1. Conceivably, activation of the mTORC1/Srebp1/Miox pathway leads to the generation of ROS culminating into tubulo-interstitial injury in states of obesity.

Sterol regulatory element-binding proteins are transcriptional regulators of the thyroglobulin gene in thyroid cells.

The genes encoding sodium/iodide symporter (NIS) and thyroid peroxidase (TPO), both of which are essential for thyroid hormone (TH) synthesis, were shown to be regulated by sterol regulatory element-binding proteins (SREBP)-1c and -2. In the present study we tested the hypothesis that transcription of a further gene essential for TH synthesis, the thyroglobulin (TG) gene, is under the control of SREBP. To test this hypothesis, we studied the influence of inhibition of SREBP maturation and SREBP knockdown on TG expression in FRTL-5 thyrocytes and explored transcriptional regulation of the TG promoter by reporter gene experiments in FRTL-5 and HepG2 cells, gel shift assays and chromatin immunoprecipitation. Inhibition of SREBP maturation by 25-hydroxycholesterol and siRNA-mediated knockdown of either SREBP-1c or SREBP-2 decreased mRNA and protein levels of TG in FRTL-5 thyrocytes. Reporter gene assays with wild-type and mutated TG promoter reporter truncation constructs revealed that the rat TG promoter is transcriptionally activated by nSREBP-1c and nSREBP-2. DNA-binding assays and chromatin immunoprecipitation assays showed that both nSREBP-1c and nSREBP-2 bind to a SREBP binding motif with characteristics of an E-box SRE at position -63 in the rat TG promoter. In connection with recent findings that NIS and TPO are regulated by SREBP in thyrocytes the present findings support the view that SREBP are regulators of essential steps of TH synthesis in the thyroid gland such as iodide uptake, iodide oxidation and iodination of tyrosyl residues of TG. This moreover suggests that SREBP may be molecular targets for pharmacological modulation of TH synthesis.

Sterol regulatory element-binding protein 1 inhibitors decrease pancreatic cancer cell viability and proliferation.

Sterol regulatory element-binding protein1 (SREBP1) is a key regulatory factor that controls lipid homeostasis. Overactivation of SREBP1 and elevated lipid biogenesis are considered the major characteristics in malignancies of prostate cancer, endometrial cancer, and glioblastoma. However, the impact of SREBP1 activation in the progression of pancreatic cancer has not been explored. The present study examines the effect of suppression of SREBP1 activation by its inhibitors like fatostatin and PF429242 besides analyzing the impact of inhibitory effects on SREBP1 downstream signaling cascade such as fatty acid synthase (FAS), hydroxymethylglutaryl-CoA reductase (HMGCoAR), stearoyl-CoA desaturase-1 (SCD-1), and tumor suppressor protein p53 in MIA PaCa-2 pancreatic cancer cells. Both fatostatin and PF429242 inhibited the growth of MIA PaCa-2 cells in a time and concentration-dependent manner with maximal inhibition attained at 72 h time period with IC50 values of 14.5 µM and 24.5 µM respectively. Detailed Western blot analysis performed using fatostatin and PF429242 at 72 h time point led to significant decrease in the levels of the active form of SREBP1 and its downstream signaling proteins such as FAS, SCD-1 and HMGCoAR and the mutant form of tumor suppressor protein, p53, levels in comparison to the levels observed in vehicle treated control group of MIA PaCa-2 pancreatic cells over the same time period. Our in vitro data suggest that SREBP1 may contribute to pancreatic tumor growth and its inhibitors could be considered as a potential target in the management of pancreatic cancer cell proliferation.

Glu-Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes.

A Glu-Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization-mass (ESI-MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography-ESI-MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.

Suppression of adipocyte differentiation and lipid accumulation by stearidonic acid (SDA) in 3T3-L1 cells.

BACKGROUND: Increased consumption of omega-3 (ω-3) fatty acids found in cold-water fish and fish oil has been reported to protect against obesity. A potential mechanism may be through reduction in adipocyte differentiation. Stearidonic acid (SDA), a plant-based ω-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids; however, its role in adipocyte differentiation is unknown. This study was designed to evaluate the effects of SDA on adipocyte differentiation in 3T3-L1 cells. METHODS: 3T3-L1 preadipocytes were differentiated in the presence of SDA or vehicle-control. Cell viability assay was conducted to determine potential toxicity of SDA. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) quantification in differentiated 3T3-L1 adipocytes. Adipocyte differentiation was evaluated by adipogenic transcription factors and lipid accumulation gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Fatty acid analysis was conducted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). RESULTS: 3T3-L1 cells treated with SDA were viable at concentrations used for all studies. SDA treatment reduced lipid accumulation in 3T3-L1 adipocytes. This anti-adipogenic effect by SDA was a result of down-regulation of mRNA levels of the adipogenic transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBPα, C/EBPß), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol-regulatory element binding protein-1c (SREBP-1c). SDA treatment resulted in decreased expression of the lipid accumulation genes adipocyte fatty-acid binding protein (AP2), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD-1), lipoprotein lipase (LPL), glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK). The transcriptional activity of PPARγ was found to be decreased with SDA treatment. SDA treatment led to significant EPA enrichment in 3T3-L1 adipocytes compared to vehicle-control. CONCLUSION: These results demonstrated that SDA can suppress adipocyte differentiation and lipid accumulation in 3T3-L1 cells through down-regulation of adipogenic transcription factors and genes associated with lipid accumulation. This study suggests the use of SDA as a dietary treatment for obesity.

Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation.

Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome.

Luteolin Inhibits Proliferation and Induces Apoptosis of Human Placental Choriocarcinoma Cells by Blocking the PI3K/AKT Pathway and Regulating Sterol Regulatory Element Binding Protein Activity.

Luteolin is a natural compound known for its anticancer effects on various human cancers by regulating signal transduction cascades. However, the effects of luteolin on human placental choriocarcinoma are not known. Results of the present study revealed that luteolin decreased viability of JAR and JEG-3 cells, which are valuable placental models, in a dose-dependent manner, and it induced apoptosis and loss of mitochondrial membrane potential in JAR and JEG-3 cells. The results also suggested that the PI3K/AKT pathway was inhibited by luteolin treatment of JAR and JEG-3 cells in a dose- and time-dependent manner. Next, we established effects of luteolin in the presence of pharmacological inhibitors of PI3K/AKT, ERK1/2 MAPK, and mTOR on proliferation of JAR and JEG-3 cells. In addition, these inhibitors were used to verify phosphorylation of AKT, GSK3beta, and ERK1/2 and to confirm mechanisms regulated by luteolin in JAR and JEG-3 cells. We also determined levels of SREBP1 and SREBP2 expression to investigate regulatory functions of luteolin in lipid metabolism in JAR and JEG-3 cells. Expression levels of both SREBP1 and SREBP2 mRNAs were significantly reduced, but only SREBP1 protein was influenced by luteolin. We compared viability of JAR and JEG-3 cells in response to luteolin alone or in combination with other chemotherapeutic drugs (etoposide, cisplatin, and paclitaxel) and found that luteolin has synergistic effects with the conventional chemotherapeutic drugs as an anticancer agent. Collectively, these results showed that luteolin plays an important role in the treatment of human choriocarcinoma cells by inhibiting the PI3K/AKT/mTOR/SREBP cascade and expression of lipogenic genes.

Co-regulation of SREBP-1 and mTOR ameliorates lipid accumulation in kidney of diabetic mice.

SREBP-1 and mTOR have been proved to involve in renal lipid metabolism of diabetes mellitus. In the present study, we investigated the effect of co-regulation of SREBP-1 and mTOR on renal lipid metabolism using diabetic mice and cultured renal tubular cells. The results showed that compared with those in high glucose-stimulated HKC cells single transfected with shRNA-SREBP-1 vector, the level of SREBP-1 protein were significantly reduced by 64.1% followed by decreased FASN mRNA, ACC mRNA, ADRP protein and lipid droplets in HKC cells co-transfected with shRNA-SREBP-1 vector and kinase-dead mTOR vector. Furthermore, diabetic mice co-injected with shRNA-SREBP-1 vector and kinase-dead mTOR vector showed that renal SREBP-1 protein, FASN mRNA and ACC mRNA were respectively decreased by 34.6%, 45.9%, 22.0% in comparison with those in diabetic mice single injected with shRNA-SREBP-1 vector accompanied by reduced ADRP protein and triglyceride content. In the end our study suggests that co-regulation of SREBP-1 and mTOR in kidney of diabetic mice is more effective in lowering renal lipogenesis than only regulation of SREBP-1.

Allyl isothiocyanate suppresses the proteolytic activation of sterol regulatory element-binding proteins and de novo fatty acid and cholesterol synthesis.

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of genes involved in fatty acid and cholesterol synthesis. In this study, we used a stable cell line that expresses a luciferase reporter gene driven by an SRE-containing fatty acid synthase promoter to identify allyl isothiocyanate (AITC), one of the major isothiocyanates in cruciferous vegetables, as a novel SREBP inactivator. We found that AITC downregulated the proteolytic processing of SREBPs and the expression of their target genes in human hepatoma Huh-7 cells. Furthermore, AITC reduced the de novo synthesis of both fatty acids and cholesterol. Our results indicate a novel physiological function of AITC in lipid metabolism regulation.

Hepatic differentiated embryo-chondrocyte-expressed gene 1 (Dec1) inhibits sterol regulatory element-binding protein-1c (Srebp-1c) expression and alleviates fatty liver phenotype.

Hepatic steatosis, characterized by ectopic hepatic triglyceride accumulation, is considered as the early manifestation of non-alcoholic fatty liver diseases (NAFLD). Increased SREBP-1c level and activity contribute to excessive hepatic triglyceride accumulation in NAFLD patients; however, negative regulators of Srebp-1c are not well defined. In this study, we show that Dec1, a critical regulator of circadian rhythm, negatively regulates hepatic Srebp-1c expression. Hepatic Dec1 expression levels are markedly decreased in NAFLD mouse models. Restored Dec1 gene expression levels in NAFLD mouse livers decreased the expression of Srebp-1c and lipogenic genes, subsequently ameliorating the fatty liver phenotype. Conversely, knockdown of Dec1 expression by an adenovirus expressing Dec1-specific shRNA led to an increase in hepatic TG content in normal mouse livers. Correspondingly, expression levels of lipogenic genes, including Srebp-1c, Fas, and Acc, were increased in livers of mice with Dec1 knockdown. Moreover, a functional lipogenesis assay suggested that Dec1 overexpression repressed lipid synthesis in primary hepatocytes. Finally, a luciferase reporter gene assay indicates that DEC1 inhibits Srebp-1c gene transcription via the E-box mapped to the promoter region. Chromatin immunoprecipitation confirmed that DEC1 proteins bound to the identified E-box element. Our studies indicate that DEC1 is an important regulator of Srebp-1c expression and links circadian rhythm to hepatic lipogenesis. Activation of Dec1 can alleviate the nonalcoholic fatty liver phenotype.