SREBP-2 Gene Research Articles

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2−/− mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2−/− mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.

Delayed Hepatic Adaptation to Weaning in ACBP−/− Mice Is Caused by Disruption of the Epidermal Barrier

We previously reported that mice deficient in acyl-CoA-binding protein (ACBP) display a delayed metabolic adaptation to weaning. This includes a delayed activation of the hepatic lipogenic gene program, which may result from hepatic accumulation of triacylglycerol and/or cholesteryl esters in the late suckling period. To further investigate the basis for this phenotype, we generated mice deficient in ACBP in hepatocytes (Alb-ACBP(-/-)) and keratinocytes (K14-ACBP(-/-)). Surprisingly, the delayed adaptation to weaning, including hepatic lipid accumulation, is caused by ACBP deficiency in the skin rather than in the liver. Similarly to ACBP(-/-) mice, K14-ACBP(-/-) mice exhibit an increased transepidermal water loss, and we show that the hepatic phenotype is caused specifically by the epidermal barrier defect, which leads to increased lipolysis in white adipose tissue. Our data demonstrate that an imperfect epidermal barrier leads to profound suppression of the hepatic SREBP gene program and lipid accumulation in the liver.

Association between SREBP-1 gene expression in mammary gland and milk fat yield in Sarda breed sheep

SummaryThe aim of this study was to examine the expression patterns of SREBP-1 gene in milk somatic cells and its association with milk fat yield during early lactation in Sarda breed sheep. A sample of 20 Sarda ewes, aged between 4 and 5 years, in their third to fourth lactation were chosen. From each ewe 28 days after lambing milk yield was measured, and a 160 ml milk sample for the RNA extraction and to test somatic cells count and lactose, fat and protein contents were collected. From the obtained RNA, total cDNA was synthesized and the quantitative PCR was performed. The fat, proteins and lactose content showed many differences among the animals, but these variations were no correlated with the milk yield. The SREBP-1 gene expression resulted higher in the high milk fat producing ewes. The correlation analysis showed that the SREBP-1 expression level is directly related to the amount of milk fat (g/die) (P < 0.001), while the total RNA obtained from each sample was found to be related to the somatic cells number (P < 0.001). Instead the expression of this gene showed no relations with the concentration of fat in milk. Our data highlight that in sheep SREBP-1 gene is expressed in the mammary gland during early lactation. Moreover, the positive relationship between SREBP-1 gene expression and the milk fat yield suggests that SREBP-1 gene is required for the lipid synthesis in the sheep mammary gland.

Hepatic SREBP-2 and cholesterol biosynthesis are regulated by FoxO3 and Sirt6

Cholesterol homeostasis is crucial for cellular function and organismal health. The key regulator for the cholesterol biosynthesis is sterol-regulatory element binding protein (SREBP)-2. The biochemical process and physiological function of SREBP-2 have been well characterized; however, it is not clear how this gene is epigenetically regulated. Here we have identified sirtuin (Sirt)6 as a critical factor for Srebp2 gene regulation. Hepatic deficiency of Sirt6 in mice leads to elevated cholesterol levels. On the mechanistic level, Sirt6 is recruited by forkhead box O (FoxO)3 to the Srebp2 gene promoter where Sirt6 deacetylates histone H3 at lysines 9 and 56, thereby promoting a repressive chromatin state. Remarkably, Sirt6 or FoxO3 overexpression improves hypercholesterolemia in diet-induced or genetically obese mice. In summary, our data suggest an important role of hepatic Sirt6 and FoxO3 in the regulation of cholesterol homeostasis.

Epigenetic signature in human development of vascular lesions: effects of maternal hypercolesterolemia

Background and purpose: It is well established that human vascular lesions begin very early stage in life with the development of fatty streak lesions in the arteries of fetuses. Although several studies have demonstrated that maternal cholesterol may exert detrimental effects on the number and degree of vascular lesions in fetuses, however, how cholesterol affects in utero programming is still unclear. Epigenetics migh promote pathogenic mechanisms by which gene-environment interactions could occurr. The aim of this study was to verify whether maternal hypercholesterolemia during pregnancy affects epigenetic signature in fetal aortic tissues. Methods: Human fetal aortas (n=42) were analyzed by immunohistochemistry and dichotomized in their respective high and low maternal cholesterol during pregnancy. Immunohistochemistry in fetal arteries was performed with the following antibodies: Anti-trimethyl-Histone H3, (Lys4, Lys9, Lys27), and Anti-CD68 antibody. Methyl DNA immunoprecipitation was performed by MeDIP kit (DIAGENODE). Results: The data indicated a strong signal >75% of positive cells with histone trimethyl lys 27 antibody present in 27% of the biggest fetal aortic lesion area (>120mm2/section) (p>0.039). Molecular analysis of specific cholesterol regulatory circuit gene expression revealed hypermethylation in promoter and 3' UTR of SREBP2 and LDL receptor gene in aortas from hypercholesterolemic mothers compared to normocholesterolemic group (p<0.004). Conclusions: Our data indicate that epigenetic mechanisms may contribute to the development of vascular lesions in humans. Maternal hypercholesterolemia during pregnancy may exacerbate the triggering of in promoter and 3' UTR of SREBP2 and LDL receptor gene. The understanding of epigenetic mechanisms during fetal programming by this approach could lead to potential applications in clinical practice through the development of epigenetic biomarkers for early diagnosis of vascular disease and/or novel drugs adversing such epigentic mechanisms.

Multi-Analytic Approach Elucidates Significant Role of Hormonal and Hepatocanalicular Transporter Genetic Variants in Gallstone Disease in North Indian Population

ObjectiveCholesterol gallstone disease (CGD) is a multifactorial and multistep disease. Apart from female gender and increasing age being the documented non-modifiable risk factor for gallstones the pathobiological mechanisms underlying the phenotypic expression of CGD appear to be rather complex, and one or more variations in genes could play critical roles in the diverse pathways further progressing to cholesterol crystal formation. In the present study we performed genotyping score, Multifactor dimensionality reduction (MDR) and Classification and Regression Tree analysis (CART) to identify combinations of alleles among the hormonal, hepatocanalicular transporter and adipogenesis differentiation pathway genes in modifying the risk for CGD.DesignThe present case-control study recruited total of 450 subjects, including 230 CGD patients and 220 controls. We analyzed common ESR1, ESR2, PGR, ADRB3, ADRA2A, ABCG8, SLCO1B1, PPARγ2, and SREBP2 gene polymorphisms to find out combinations of genetic variants contributing to CGD risk, using multi-analytical approaches (G-score, MDR, and CART).ResultsSingle locus analysis by logistic regression showed association of ESR1 IVS1-397C>T (rs2234693), IVS1-351A>G (rs9340799) PGR ins/del (rs1042838) ADRB3-190 T>C (rs4994) ABCG8 D19H (rs11887534), SLCO1B1 Exon4 C>A (rs11045819) and SREBP2 1784G>C (rs2228314) with CGD risk. However, the MDR and CART analysis revealed ESR1 IVS1-397C>T (rs2234693) ADRB3-190 T>C (rs4994) and ABCG8 D19H (rs11887534) polymorphisms as the best polymorphic signature for discriminating between cases and controls. The overall odds ratio for the applied multi-analytical approaches ranged from 4.33 to 10.05 showing an incremental risk for cholesterol crystal formation. In conclusion, our muti-analytical approach suggests that, ESR1, ADRB3, in addition to ABCG8 genetic variants confer significant risk for cholesterol gallstone disease.

Silent information regulator 1 inhibition induces lipid metabolism disorders of hepatocytes and enhances hepatitis C virus replication

Hepatic steatosis is an important histopathological feature of chronic hepatitis C virus (HCV) infection. Silent information regulator 1 (SIRT1) plays key role in regulation of hepatic lipid metabolism. We investigated the possible effect of HCV replication on lipid metabolism of hepatocytes and expression of SIRT1 using Huh-7.5 cells harboring HCV replicon. The level of reactive oxygen species (ROS) and malondialdehyde (MDA), the activity of superoxide dismutase (SOD), and the value of nicotinamide adenine dinucleotide (NAD(+) )/NADH was detected. The level of triacylglycerol (TG), total cholesterol (TC) and fatty acid β-oxidation rate was detected. The activity and expression levels of SIRT1 and expression of its downstream lipid-metabolism genes were measured. In replicon cells, the level of ROS and MDA increased, SOD activity and the value of NAD(+) /NADH decreased, then the activity and expression level of mRNA and protein of SIRT1 reduced. Inhibition of SIRT1 decreased phosphorylation of forkhead box O1 (FoxO1), which not only upregulated SREBP-1c, FAS, ACC, SREBP-2, HMGR and HMGS genes and increased fatty acid synthesis; but also downregulated PPAR-α and CPT1A genes and decreased fatty acid β-oxidation. Interferon treatment restored aforementioned changes. SIRT1 activator improved lipid metabolism disorders by an increase in fatty acid β-oxidation and a decrease in TG and TC synthesis and inhibited HCV replication. HCV replication decreasing NAD(+) /NADH ratio may downregulate the activity and the expression of SIRT1, then change the expression profile of lipid metabolism-related genes, thereby cause lipid metabolism disorders of hepatocytes and promote HCV replication. Treatment with SIRT1 activator ameliorates lipid metabolic disorders and inhibits HCV replication.

Effect of Dai-Saiko-To (Da-Chai-Hu-Tang) on LDL-Receptor Gene Expression in Human Hepatoma Cell Line (HepG2)

We previously reported that Dai-saiko-to (Da-Chai-Hu-Tang), a traditional Japanese kampo medicine, increased LDL receptor mRNA expression in the liver of the hypercholesterolemic rabbits. In this study, we focused on LDL receptor gene expression in a human hepatoma cell line (HepG2) treated with Dai-saiko-to extract and the extracts of eight herbs presented in Dai-saiko-to. Dai-saiko-to extract significantly increased LDL receptor gene and SREBP2 gene expression compared with the control. The extracts of four herbs, Bupleurum root, Pinellia tuber, Scutellaria root and Peony root significantly increased the LDL receptor gene expression. Whereas, Jujube, Immature orange, Ginger and Rhubarb extracts did not change the gene expression. These results suggest that Dai-saiko-to increased the expression of the cholesterol transport gene (LDL receptor) regulated by SREBP2 gene in the human hepatoma cell line. The pharmacological activity of Dai-saiko-to against hypercholesterolemia and atheromatous lesions related for these four herbal components.

日本短角種の枝肉皮下脂肪中の脂肪酸組成にSCD, FASN, SREBP-1 およびGHの各遺伝子多型が及ぼす影響

日本短角種の枝肉皮下脂肪中の脂肪酸組成に<I>SCD, FASN, SREBP-1</I> および<I>GH</I>の各遺伝子多型が及ぼす影響

Polymorphism of SREBP1 is associated with beef fatty acid composition in Simmental bulls

The sterol regulatory element binding factor 1 gene (SREBP1) plays an important role in the biosynthesis of fatty acids and cholesterol, and in lipid metabolism. The objective of this study was to investigate the effect of genetic polymorphisms of SREBP1 on the fatty acid composition of muscle and carcass traits in Simmental bulls and Snow Dragon black cattle. The 84-bp insertion/deletion (indel) in intron 5 of the bovine SREBP1 gene was genotyped by polymerase chain reaction to investigate its associations with traits. The results showed that the 84-bp indel in intron 5 was significantly associated with palmitoleic acid (C16:1), stearic acid (C18:0), saturated fatty acids (SFA), triglycerides (TAG), and the C16 index in Simmental bulls (P < 0.05). Cattle with the LL genotype had higher palmitic acid (C16:1), triglycerides, and C16 index but lower stearic acid (C18:0) and SFA compared to those with the LS genotype (P < 0.05). In conclusion, the 84-bp indel of SREBP1 could be used as a genetic marker for selecting Simmental breeding stock for healthier fatty acid composition.

Novel single nucleotide polymorphisms of bovine SREBP1 gene is association with fatty acid composition and marbling score in commercial Korean cattle (Hanwoo)

The sterol regulatory element binding factor 1 (SREBP1) gene plays an important role in the biosynthesis of fatty acids. The aim of this study was to investigate the association between SNPs within SREBP1 gene and fatty acid composition of muscle fat and marbling score in a commercial population of Korean cattle. Among 10 SNPs in dbSNP of bovine SREBP1 gene, we identified 7 polymorphic SNPs and 84-bp ins/del variation (SREBP1-INT5 SNP) in commercial Korean cattle (n = 513). The SREBP1-9 SNP showed a significant effect on marbling score, monounsaturated fatty acids and C18:1n-9 in the muscle fat of commercial Korean cattle. However, SREBP1-INT5 SNP showed no effect on any fatty acid composition. Therefore, two polymorphisms in SREBP1 may improve flavored beef and meat quality in commercial Korean cattle.

Intracellular lipid accumulation inhibitory effect of Weissella koreensis OK1-6 isolated from Kimchi on differentiating adipocyte

To investigate the intracellular lipid accumulation inhibitory effect of spent culture medium extract and the cytoplasmic fraction of Weissella koreensis OK1-6 cells isolated from kimchi in differentiating 3T3-L1 cells. Differentiating 3T3-L1 cells were treated with either cytoplasmic fraction of W.koreensis OK1-6 cells or its spent media for 4days. Both the spent culture medium extract and cytoplasmic fraction of W.koreensis OK1-6 cells significantly decreased the triglyceride concentration and intracellular lipid accumulation in the treated groups compared with the control group. The mRNA expression levels of C/EBP-α, one of the major transcriptional factors involved in adipocyte differentiation, were significantly less expressed in 3T3-L1 cells treated with the spent medium and cytoplasmic fraction. The expressions of aP2, fatty acid synthase (FAS) and SREBP1 genes were also decreased significantly. These results suggested that W.koreensis OK1-6 could play a crucial role in preventing intracellular lipid accumulation by down-regulating the expression of adipocyte-specific genes C/EBPα, aP2, SREBP1 and FAS. These results may contribute to nutraceutical and food industries in developing probiotic-based therapies for the treatment and prevention of obesity.

Effect of RNAi-mediated silencing of SREBP2 gene on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells

To investigate the effect of RNA interference (RNAi)-mediated silencing of the SREBP2 on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells. Short-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. SREBP2 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. Inflammatory cytokine exacerbated cholesterol accumulation in HepG2 via disrupting SREBP2. RNAi-mediated inhibition of SREBP2 expression significantly ameliorated the cholesterol accumulation induced by inflammatory cytokine.

Activation of Pregnane X Receptor by Pregnenolone 16 α-carbonitrile Prevents High-Fat Diet-Induced Obesity in AKR/J Mice

Pregnane X receptor (PXR) is known to function as a xenobiotic sensor to regulate xenobiotic metabolism through selective transcription of genes responsible for maintaining physiological homeostasis. Here we report that the activation of PXR by pregnenolone 16α-carbonitrile (PCN) in AKR/J mice can prevent the development of high-fat diet-induced obesity and insulin resistance. The beneficial effects of PCN treatment are seen with reduced lipogenesis and gluconeogenesis in the liver, and lack of hepatic accumulation of lipid and lipid storage in the adipose tissues. RT-PCR analysis of genes involved in gluconeogenesis, lipid metabolism and energy homeostasis reveal that PCN treatment on high-fat diet-fed mice reduces expression in the liver of G6Pase, Pepck, Cyp7a1, Cd36, L-Fabp, Srebp, and Fas genes and slightly enhances expression of Cyp27a1 and Abca1 genes. RT-PCR analysis of genes involved in adipocyte differentiation and lipid metabolism in white adipose tissue show that PCN treatment reduces expression of Pparγ2, Acc1, Cd36, but increases expression of Cpt1b and Pparα genes in mice fed with high-fat diet. Similarly, PCN treatment of animals on high-fat diet increases expression in brown adipose tissue of Pparα, Hsl, Cpt1b, and Cd36 genes, but reduces expression of Acc1 and Scd-1 genes. PXR activation by PCN in high-fat diet fed mice also increases expression of genes involved in thermogenesis in brown adipose tissue including Dio2, Pgc-1α, Pgc-1β, Cidea, and Ucp-3. These results verify the important function of PXR in lipid and energy metabolism and suggest that PXR represents a novel therapeutic target for prevention and treatment of obesity and insulin resistance.

FoxO1 Inhibits Sterol Regulatory Element-binding Protein-1c (SREBP-1c) Gene Expression via Transcription Factors Sp1 and SREBP-1c

Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

Central Role of SREBP-2 in the Pathogenesis of Osteoarthritis

BackgroundRecent studies have implied that osteoarthritis (OA) is a metabolic disease linked to deregulation of genes involved in lipid metabolism and cholesterol efflux. Sterol Regulatory Element Binding Proteins (SREBPs) are transcription factors regulating lipid metabolism with so far no association with OA. Our aim was to test the hypothesis that SREBP-2, a gene that plays a key role in cholesterol homeostasis, is crucially involved in OA pathogenesis and to identify possible mechanisms of action.Methodology/Principal FindingsWe performed a genetic association analysis using a cohort of 1,410 Greek OA patients and healthy controls and found significant association between single nucleotide polymorphism (SNP) 1784G>C in SREBP-2 gene and OA development. Moreover, the above SNP was functionally active, as normal chondrocytes’ transfection with SREBP-2-G/C plasmid resulted in interleukin-1β and metalloproteinase-13 (MMP-13) upregulation. We also evaluated SREBP-2, its target gene 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR), phospho-phosphoinositide3-kinase (PI3K), phospho-Akt, integrin-alphaV (ITGAV) and transforming growth factor-β (TGF-β) mRNA and protein expression levels in osteoarthritic and normal chondrocytes and found that they were all significantly elevated in OA chondrocytes. To test whether TGF-β alone can induce SREBP-2, we treated normal chondrocytes with TGF-β and found significant upregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13. We also showed that TGF-β activated aggrecan (ACAN) in chondrocytes only through Smad3, which interacts with SREBP-2. Finally, we examined the effect of an integrin inhibitor, cyclo-RGDFV peptide, on osteoarthritic chondrocytes, and found that it resulted in significant upregulation of ACAN and downregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13 expression levels.Conclusions/SignificanceWe demonstrated, for the first time, the association of SREBP-2 with OA pathogenesis and provided evidence on the molecular mechanism involved. We suggest that TGF-β induces SREBP-2 pathway activation through ITGAV and PI3K playing a key role in OA and that integrin blockage may be a potential molecular target for OA treatment.

Dietary soy protein induces hepatic lipogenic enzyme gene expression while suppressing hepatosteatosis in obese female Zucker rats bearing DMBA-initiated mammary tumors

Fatty liver is associated with obesity and breast cancer. We used an obese rat model of mammary cancer to examine whether hepatosteatosis is modifiable by diet and associated with altered expression of hepatic lipogenic enzyme genes, thyroid hormone system genes and cholesterol metabolism-related genes. Beginning at the age of 5weeks, lean and obese female Zucker rats were fed high-isoflavone soy protein- or casein (control protein)-containing diets. Rats were euthanized at 200days of age [corresponding to 147days after administration of carcinogen to induce mammary tumors; (Hakkak et al. in, Oncol Lett 2:29-36, 2011)]. Obese rats had a greater degree of liver steatosis than lean rats. Obese casein-fed rats had marked steatosis with small foci of mononuclear infiltration, whereas obese soy protein-fed rats had a significantly lower steatosis index. Comparisons between lean and obese casein-fed rats showed that obesity was associated with significant reductions in hepatic mRNA abundance for Glucose 6-Phosphate Dehydrogenase (G6PD), 6-Phosphogluconate Dehydrogenase (6PGD), Thyroid Receptor Alpha 1 (TRα1), Thyroid Receptor Beta 1 (TRβ1) and Iodothyronine Deiodinase 1 (DIO1). The soy protein diet was associated with increased expression of Fatty Acid Synthase (FASN), Malic Enzyme 1 (ME1), 6PGD, Sterol Regulatory Element Binding Protein-1c (SREBP-1c) and SREBP-2 genes in the livers of obese but not lean rats. Western blot analysis showed a significant induction of ME1 protein expression in the livers of obese, soy protein-fed rats, which paralleled the increased serum insulin level in this group. Long-term soy protein consumption can counter hepatic steatosis while coincidently promoting hepatic lipogenic gene expression, the latter likely a consequence of elevated serum insulin. We suggest that elevations in serum insulin, hepatic lipogenesis and cholesterol synthesis all contributed to the increased tumorigenesis previously observed for the obese, soy protein-fed rats.

Abstract 5169: Roles of lipogenesis in cancer stem-like cells at early stage of breast cancer

Abstract Ductal carcinoma in situ (DCIS) is considered an early stage of breast cancer and it is the precursor of invasive breast carcinoma. However, the transition from normal breast epithelial cells to DCIS is still not clearly elucidated. In order to determine the role of lipogenesis in normal to DCIS transition, we investigated the roles of SREBP1, a major transcription regulator of lipogenesis and its downstream lipogenic genes such as ACLY, ACC1 and FAS, by isolating cancer stem-like cells (CSCs) from DCIS.com cell line which forms typical DCIS lesions in animals by using the well-established cell surface markers, CD24−/CD44+/ESA+. We found that lipid synthesis was significantly upregulated in CSCs from DCIS.com in contrast to the corresponding population from normal breast epithelial cells (MCF10A). Furthermore, we found that this elevation of lipogenesis is indeed caused by the overexpression of SREBP1 which co-ordinately induces the overexpression of the downstream lipogenic genes. The CSCs obtained from DCIS.com were also found to be more tumorigenic than the parental cells as shown by the limiting dilution analysis. To determine the role of elevated level of SREBP1 and lipogenesis in the transition of normal breast epithelial cells to DCIS phenotype, we overexpressed SREBP1 in MCF10A and examined the transforming properties. Our in vitro data showed that MCF10A-SREBP1 cells indeed expressed a significantly higher level of SREBP1 and other lipogenic genes and this elevated level of lipogenesis resulted in the increased cell proliferation, increased mammospheres forming ability and enhanced cellular growth in 3D culture. These data suggest that increased lipogenesis in breast CSCs plays critical roles in DCIS formation. We also examined the effects of Resveratrol on CSCs from DCIS.com. Resveratrol significantly reduced the total lipid content by inhibiting SREBP1 and in turn other lipogenic genes and induced a series of pro-apoptotic genes such as DAPK2 and BNIP3 causing the death of CSCs from DCIS. The mammosphere forming ability of CSCs from DCIS was also blocked by Resveratrol. We also observed that Resveratrol indeed significantly suppressed the formation of DCIS by inhibiting lipogenesis and by upregulating DAPK2 and BNIP3 in our animal model of human DCIS. Collectively, our results indicate that lipogenic genes ACLY, ACC-1, FAS and SREBP1 are significantly up regulated in early stage of breast tumorigenesis and they confer proliferative and growth advantages to these cells. Lipogenesis targeting effect of Resveratrol on CSCs from DCIS provides us with a strong rationale to use this agent for chemo-prevention against DCIS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5169. doi:1538-7445.AM2012-5169

Mir-33 regulates cell proliferation and cell cycle progression

Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. microRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene expression, acting predominantly at posttranscriptional level. Recent work from our group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the Srebp genes, regulate cholesterol and fatty acid metabolism in concert with their host genes. Here, we show that hsa-miR-33 family members modulate the expression of genes involved in cell cycle regulation and cell proliferation. MiR-33 inhibits the expression of the cyclin-dependent kinase 6 (CDK6) and cyclin D1 (CCND1), thereby reducing cell proliferation and cell cycle progression. Overexpression of miR-33 induces a significant G1 cell cycle arrest in Huh7 and A549 cell lines. Most importantly, inhibition of miR-33 expression using 2’fluoro/methoxyethyl-modified (2’F/MOE-modified) phosphorothioate backbone antisense oligonucleotides improves liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these results suggest that Srebp/miR-33 locus may cooperate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration.

Cholesterol-lowering effect of dietary Lupinus angustifolius proteins in adult rats through regulation of genes involved in cholesterol homeostasis

In the absence of a clear indication from previous studies, a rat study was designed to evaluate a possible hypolipidaemic effect of Lupinus angustifolius (blue lupin) proteins. Rats were fed for 28days Nath’s hypercholesterolaemic diets containing 20% casein or blue lupin proteins. After 14 and 28days of dietary treatment, blue-lupin-fed rats had markedly lower plasma total cholesterol levels than rats fed casein (−53.0% and −55.3%, respectively, p<0.0005). No significant differences were instead observed for triglyceride and HDL-cholesterol levels between the two groups. Lupin-protein-fed rats displayed higher hepatic mRNA levels of SREBP-2, a major transcriptional regulator of intracellular cholesterol levels, and CYP7A1, the rate-limiting enzyme in bile acid biosynthesis (p<0.05). In conclusion, the present study demonstrates a marked cholesterol-lowering activity of proteins from L. angustifolius in rats. Moreover, blue lupin proteins appear to affect cellular lipid homeostasis by up-regulating SREBP-2 and CYP7A1 genes.