DHA缓解肝X受体激活所致HepG2细胞的甘油三酯积聚

目的探讨DHA对肝X受体激动剂T0901317诱导的HepG2细胞甘油三酯积聚的影响。方法体外培养HepG2细胞,以50μmol／LDHA、10μmol／LT0901317分别处理细胞以及50μmloL／LDHA和10μmol／LT0901317共同处理细胞48h。油红0染色观察细胞内脂质沉积;氯仿-甲醇抽提细胞总脂质,酶法定量检测细胞甘油三酯含量;实时定量PCR检测与脂肪酸代谢相关基因如SREBP-1c、FAS、SCD-1、PPARa和CD36的mRNA水平。结果与对照组相比,10μmol／LT0901317处理48h后,HepG2细胞内的油红O染色脂滴增多,甘油二酯浓度升高了50％;脂肪酸合成基因：SREBP-1c、FAS和SCD-1及脂肪酸吸收基因CD36的mRNA水平分别升高了9．9、5．2、2．2和1．5倍,而脂肪酸降解基因PPARoz的mRNA无变化。DHA与T0901317共同处理的HepG2细胞内脂滴明显减少;甘油三酯含量比70901317处理组降低了15％：SREBP—1c、FAS、SCD-1和CD36的mRNA水平比T0901317处理组分别降低了92％、31％、46％和60％,而PPARa的mRNA水平比T0901317处理组升高了30％。结论DHA通过降低脂肪酸合成和吸收基因的表达并升高脂肪酸降解基因的表达缓解肝x受体激活所致HepG2细胞内甘油三酯积聚。     

S-Allyl cysteine attenuates free fatty acid-induced lipogenesis in human HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway

S-Allyl cysteine (SAC), a nontoxic garlic compound, has a variety of pharmacological properties, including antioxidant and hepatoprotective properties. In this report, we provide evidence that SAC prevented free fatty acid (FFA)-induced lipid accumulation and lipotoxicity in hepatocytes. SAC significantly reduced FFA-induced generation of reactive oxygen species, caspase activation and subsequent cell death. Also, SAC mitigated total cellular lipid and triglyceride accumulation in steatotic HepG2 cells. SAC significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells. Additionally, SAC down-regulated the levels of sterol regulatory element binding protein-1 (SREBP-1) and its target genes, including ACC and fatty acid synthase. Use of a specific inhibitor showed that SAC activated AMPK via calcium/calmodulin-dependent kinase kinase (CaMKK) and silent information regulator T1. Our results demonstrate that SAC activates AMPK through CaMKK and inhibits SREBP-1-mediated hepatic lipogenesis. Therefore, SAC has therapeutic potential for preventing nonalcoholic fatty liver disease.     

Hepatic n-3 polyunsaturated fatty acid depletion promotes steatosis and insulin resistance in mice: genomic analysis of cellular targets

Patients with non-alcoholic fatty liver disease are characterised by a decreased n-3/n-6 polyunsaturated fatty acid (PUFA) ratio in hepatic phospholipids. The metabolic consequences of n-3 PUFA depletion in the liver are poorly understood. We have reproduced a drastic drop in n-3 PUFA among hepatic phospholipids by feeding C57Bl/6J mice for 3 months with an n-3 PUFA depleted diet (DEF) versus a control diet (CT), which only differed in the PUFA content. DEF mice exhibited hepatic insulin resistance (assessed by euglycemic-hyperinsulinemic clamp) and steatosis that was associated with a decrease in fatty acid oxidation and occurred despite a higher capacity for triglyceride secretion. Microarray and qPCR analysis of the liver tissue revealed higher expression of all the enzymes involved in lipogenesis in DEF mice compared to CT mice, as well as increased expression and activation of sterol regulatory element binding protein-1c (SREBP-1c). Our data suggest that the activation of the liver X receptor pathway is involved in the overexpression of SREBP-1c, and this phenomenon cannot be attributed to insulin or to endoplasmic reticulum stress responses. In conclusion, n-3 PUFA depletion in liver phospholipids leads to activation of SREBP-1c and lipogenesis, which contributes to hepatic steatosis.     

The role of LXRα in goose primary hepatocyte lipogenesis

In this study, we investigate the role of liver X receptor alpha (LXR alpha) in lipogenesis in geese in order to understand the differences in hepatic steatosis mechanisms between mammals and waterfowl. Primary goose hepatocytes were isolated and treated with the LXR alpha agonist T0901317. Triglyceride (TG) accumulation, acetyl-CoA carboxylase alpha (ACC alpha) and fatty acid synthase (FAS) activities, and gene expression levels of LXR alpha, sterol regulatory element-binding proteins-1 (SREBP-1), FAS, ACC alpha and lipoprotein lipase (LPL) were measured in primary hepatocytes. We found a dose-dependent up-regulation of TG accumulation, ACC, and FAS activities and the mRNA levels of LXR alpha, SREBP-1, FAS, ACC alpha, and LPL genes in the presence of To-901317. We also found that binding of nuclear SREBP-1 to ACC alpha SRE sequence was induced by To-901317 (P < 0.05). In conclusion, LXR alpha is involved in the induction of the lipogenic pathway through activation of SREBP-1 and its target genes in goose primary hepatocytes.     

Diets enriched in menhaden fish oil, seal oil, or shark liver oil have distinct effects on the lipid and fatty-acid composition of guinea pig heart

The purpose of this investigation was to determine whether diets supplemented with oils from three different marine sources, all of which contain high proportions of long-chain n-3 polyunsaturated fatty acids (PUFA), result in qualitatively distinct lipid and fatty acid profiles in guinea pig heart. Albino guinea pigs (14 days old) were fed standard, nonpurified guinea pig diets (NP) or NP supplemented with menhaden fish oil (MO), harp seal oil (SLO) or porbeagle shark liver oil (PLO) (10%, w/w) for 4-5 weeks. An n-6 PUFA control group was fed NP supplemented with corn oil (CO). All animals appeared healthy, with weight gains marginally lower in animals fed the marine oils. Comparison of relative organ weights indicated that only the livers responded to the diets, and that they were heavier only in the marine-oil fed guinea pigs. Heart total cholesterol levels were unaffected by supplementing NP with any of the oils, whereas all increased the triacylglycerol (TAG) content. The fatty-acid profiles of totalphospholipid (TPL), TAG and free fatty acid (FFA) fractions of heart lipids showed that feeding n-3 PUFA significantly altered the proportions of specific fatty-acid classes. For example, all marine-oil-rich diets were associated with increases in total monounsaturated fatty acids in TPL (p < 0.05), and with decreases in total saturates in TAG (p < 0.05). Predictably, the n-3 PUFA enriched regimens significantly increased the cardiac content of n-3 PUFA and decreased that of n-6 PUFA, although the extent varied among the diets. As a result, n-6/n-3 ratios were significantly lower in all myocardial lipid classes of marine-oil-fed guinea pigs. Analyses of the profiles of individual PUFA indicated that quantitatively, the fatty acids of the three marine oils were metabolized and/or incorporated into TPL, TAG and FFA in a diet-specific manner. In animals fed MO-enriched diets in which eicosapentaenoic acid (EPA) > docosahexacnoic acid (DHA), ratios of DHA /EPA in the hearts were 1.2, 2.2 and 1.5 in TPL, TAG and FFA, respectively. In SLO-fed guinea pigs in which dietary EPA DHA, ratios of DHA/EPA were 0.9, 3.4 and 2.1 in TPL, TAG and FFA, respectively. Feeding NP + PLO (DHA/EPA = 4.8), resulted in values for DHA/EPA in cardiac tissue of 2.1, 10.6 and 2.9 in TPL, TAG and FFA, respectively. In the TAG and FFA, proportions of n-3 docosapentaenoic acid (n-3 DPA) were equal to or higher than EPA in the SLO- and PLO-fed animals. The latter group exhibited the greatest difference between the DHA/n-3 DPA ratio in the diet and in cardiac TAG and FFA fractions (7, 3.4 and 3.1, respectively). Quantitative analysis indicated that 85% of the n-3 PUFA were in TPL, 7-11% were in TAG, and 2-6% were FFA. Specific patterns of distribution of EPA, DPA and DHA depended on the dietary oil. Both the qualitative and quantitative results of this study demonstrated that in guinea pigs, n-3 PUFA in different marine oils are metabolized and/or incorporated into cardiac lipids in distinct manners. In support of the concept that the diet-induced alterations reflect changes specifically in cardiomyocytes, we observed that direct supplementation of cultured guinea pig myocytes for 2-3 weeks with EPA or DHA produced changes in the PUFA profiles of their TPL that were qualitatively similar to those observed in tissue from the dietary study. The factors that regulate specific deposition of n-3 PUFA from either dietary oils or individual PUFA are not yet known, however the differences that we observed could in some manner be related to cardiac function and thus their relative potentials as health-promoting dietary fats.     

Highly purified eicosapentaenoic acid prevents the progression of hepatic steatosis by repressing monounsaturated fatty acid synthesis in high-fat/high-sucrose diet-fed mice

Eicosapentaenoic acid (EPA) is a member of the family of n-3 polyunsaturated fatty acids (PUFAs) that are clinically used to treat hypertriglyceridemia. The triglyceride (TG) lowering effect is likely due to an alteration in lipid metabolism in the liver, but details have not been fully elucidated. To assess the effects of EPA on hepatic TG metabolism, mice were fed a high-fat and high-sucrose diet (HFHSD) for 2 weeks and were given highly purified EPA ethyl ester (EPA-E) daily by gavage. The HFHSD diet increased the hepatic TG content and the composition of monounsaturated fatty acids (MUFAs). EPA significantly suppressed the hepatic TG content that was increased by the HFHSD diet. EPA also altered the composition of fatty acids by lowering the MUFAs C16:1 and C18:1 and increasing n-3 PUFAs, including EPA and docosahexaenoic acid (DHA). Linear regression analysis revealed that hepatic TG content was significantly correlated with the ratios of C16:1/C16:0, C18:1/C18:0, and MUFA/n-3 PUFA, but was not correlated with the n-6/n-3 PUFA ratio. EPA also decreased the hepatic mRNA expression and nuclear protein level of sterol regulatory element binding protein-1c (SREBP-1c). This was reflected in the levels of lipogenic genes, such as acetyl-CoA carboxylase α (ACCα), fatty acid synthase, stearoyl-CoA desaturase 1 (SCD1), and glycerol-3-phosphate acyltransferase (GPAT), which are regulated by SREBP-1c. In conclusion, oral administration of EPA-E ameliorates hepatic fat accumulation by suppressing TG synthesis enzymes regulated by SREBP-1 and decreases hepatic MUFAs accumulation by SCD1.     

The liver X receptor ligand T0901317 down-regulates APOA5 gene expression through activation of SREBP-1c

Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X receptor (LXR) ligand-mediated effect on plasma triglyceride levels. Following treatment with the LXR ligand T0901317, we found that APOA5 mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5 promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease of APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.     

Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice

Elongation of very long chain fatty acids (ELOVL)5 is one of seven mammalian fatty acid condensing enzymes involved in microsomal fatty acid elongation. To determine the in vivo substrates and function of ELOVL5, we generated Elovl5(-/-) mice. Studies using liver microsomal protein from wild-type and knockout mice demonstrated that the elongation of gamma-linolenic (C18:3, n-6) to dihomo-gamma-linolenic (C20:3, n-6) and stearidonic (C18:4, n-3) to omega3-arachidonic acid (C20:4, n-3) required ELOVL5 activity. Tissues of Elovl5(-/-) mice accumulated the C18 substrates of ELOVL5 and the levels of the downstream products, arachidonic acid (C20:4, n-6) and docosahexaenoic acid (DHA, C22:6, n-3), were decreased. A consequence of decreased cellular arachidonic acid and DHA concentrations was the activation of sterol regulatory element-binding protein (SREBP)-1c and its target genes involved in fatty acid and triglyceride synthesis, which culminated in the development of hepatic steatosis in Elovl5(-/-) mice. The molecular and metabolic changes in fatty acid metabolism in Elovl5(-/-) mice were reversed by dietary supplementation with arachidonic acid and DHA. These studies demonstrate that reduced ELOVL5 activity leads to hepatic steatosis, and endogenously synthesized PUFAs are key regulators of SREBP-1c activation and fatty acid synthesis in livers of mice.     

Estrogen and n-3 polyunsaturated fatty acid supplementation have a synergistic hypotriglyceridemic effect in ovariectomized rats

The n-3 polyunsaturated fatty acids (PUFAs), EPA and DHA, as well as estrogen have been shown to decrease circulating levels of triglyceride (TG), but their underlying mode of action is unclear. The purpose of this study was to determine the effects of n-3 PUFA consumption and estrogen injection on TG metabolism. Rats (n = 48) were fed a modified AIN-93G diet with 0, 1, or 2 % EPA + DHA relative to the total energy intake during 12 weeks. At 8 weeks, rats were ovariectomized (OVX), and after a 1-week recovery, rats were injected with either 17β-estradiol-3-benzoate (E₂) or corn oil for the last 3 weeks. The n-3 PUFA consumption and E₂ injection independently decreased the hepatic expressions of sterol regulatory element-binding protein 1, acetyl-CoA carboxylase 1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2) (P < 0.05). There were interactions between n-3 PUFA consumption and E₂ injection on hepatic expression of FAS and DGAT2. In addition, n-3 PUFA consumption and E₂ injection up-regulated the expression of AMP-activated protein kinase (AMPK), phosphorylated AMPK, peroxisomal proliferator-activated receptor α, and carnitine palmitoyltransferase 1 in liver and skeletal muscle. E₂ injection increased the expression of estrogen receptor α and β in skeletal muscle and liver, but n-3 PUFA consumption increased the expression of both receptors only in skeletal muscle. The present study suggests that the hypotriglyceridemic effects of n-3 PUFA consumption and E₂ injection could be due to the down-regulation of hepatic TG synthesis and up-regulation of TG oxidation in liver and skeletal muscle in OVX rats.