A low fish oil inhibits SREBP-1 proteolytic cascade,while a high-fish-oil feeding decreases SREBP-1 mRNA in mice liver: relationship to anti-obesity

Rodents fed fish oil showed less obesity with a reduction of triglyceride synthesis in liver, relative to other dietary oils, along with a decrease of mature form of sterol regulatory element binding protein-1 (SREBP-1) and activation of peroxisome proliferator-activated receptor alpha (PPARalpha). Decrease of mature SREBP-1 protein by fish oil feeding was due to either inhibition of SREBP-1 proteolytic cascade or to decrease of its mRNA. To clarify its mechanism and relation to antiobesity effect, mice were fed fish oil in a range from 10 to 60 energy percent (en%). Fish oil feeding decreased body weight and fat mass in a dose-dependent manner, in parallel with PPARalpha activation and a decrease of SREBP-1 mRNA. However, compared with 0 en% fish oil feeding, 10 en% fish oil feeding decreased mature SREBP-1 protein by 50% with concomitant decreases of lipogenic genes, while precursor SREBP-1 protein rather increased by 1.3-fold. These data suggest that physiological doses of fish oil feeding effectively decrease expression of liver lipogenic enzymes by inhibiting SREBP-1 proteolytic cascade, while substantial decrease of SREBP-1 expression is observed in its pharmacological doses, and that activation of PPARalpha rather than SREBP-1 decrease might be related to the antiobesity effect of fish oil feeding.     

Transcriptional activities of nuclear SREBP-1a, -1c,and -2 to different target promoters of lipogenic and cholesterogenic genes

Recent studies on the in vivo roles of the sterol regulatory element binding protein (SREBP) family indicate that SREBP-2 is more specific to cholesterogenic gene expression whereas SREBP-1 targets lipogenic genes. To define the molecular mechanism involved in this differential regulation, luciferase-reporter gene assays were performed in HepG2 cells to compare the transactivities of nuclear SREBP-1a, -1c, and -2 on a battery of SREBP-target promoters containing sterol regulatory element (SRE), SRE-like, or E-box sequences. The results show first that cholesterogenic genes containing classic SREs in their promoters are strongly and efficiently activated by both SREBP-1a and SREBP-2, but not by SREBP-1c. Second, an E-box containing reporter gene is much less efficiently activated by SREBP-1a and -1c, and SREBP-2 was inactive in spite of its ability to bind to the E-box. Third, promoters of lipogenic enzymes containing variations of SRE (SRE-like sequences) are strongly activated by SREBP-1a, and only modestly and equally by both SREBP-1c and -2. Finally, substitution of the unique tyrosine residue within the basic helix-loop-helix (bHLH) portion of nuclear SREBPs with arginine, the conserved residue found in all other bHLH proteins, abolishes the transactivity of all SREBPs for SRE, and conversely results in markedly increased activity of SREBP-1 but not activity of SREBP-2 for E-boxes. These data demonstrate the different specificity and affinity of nuclear SREBP-1 and -2 for different target DNAs, explaining a part of the mechanism behind the differential in vivo regulation of cholesterogenic and lipogenic enzymes by SREBP-1 and -2, respectively.     

Alternative splicing produces a constitutively active form of human SREBP-1

We identified a novel alternative splicing event that constitutively produces a truncated active form of human sterol regulatory element-binding protein 1 (SREBP-1). A cDNA of this splicing variant (named SREBP-1Δ) contains a translational stop codon-encoding exon sequence between exons 7 and 8. It produces SREBP-1aΔ (470 a.a.) and SREBP-1cΔ (446 a.a.) proteins that lack transmembrane and C-terminal regulatory sequences necessary for localization of SREBP-1 to the endoplasmic reticulum. A luciferase reporter assay showed that SREBP-1aΔ and SREBP-1cΔ transactivated lipogenic gene promoters to the same extent as that induced by N-terminal active fragments of SREBP-1a and SREBP-1c, respectively. SREBP-1Δ mRNA is expressed in human cell lines as well as adipose and liver tissues. Expression levels ranged from 5% to 16% of total SREBP-1 expression. The ratio of SREBP-1Δ expression to total SREBP-1 expression in HepG2 cells was not affected by either insulin or high glucose treatment.     

ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species

Fatty acid synthase (FAS), a key lipogenic enzyme, is expressed in the two major sites of fatty acid production in the body, that is, the liver and the adipose tissue. Surprisingly, the relative contribution of these sites to lipogenesis is highly variable among species. For example, besides the situation in rodents, where liver and fat are equally active, lipogenesis in some mammals such as the pig occurs principally in adipose tissue, whereas in avian species, the liver is the main lipogenic site. We addressed the question concerning the factors determining the site of fatty acid synthesis. We show that the expression of adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein (ADD-1/SREBP-1) mRNA, but not SREBP-2, is linked to FAS protein content or activity in adipose tissues and livers of pig, chicken, and rabbit. Tissue differences in ADD-1/SREBP-1 mRNA expression between species were paralleled by commensurate variations in the nuclear concentration of SREBP-1 protein. Moreover, overexpression of ADD-1/SREBP-1 by adenoviral gene transfer induces FAS in chicken adipocytes, where lipogenesis is normally low. Conversely, the expression of a dominant negative form of ADD-1/SREBP-1 in pig adipocytes downregulates FAS expression.These results reinforce the role of ADD-1/SREBP-1 as a key regulator of lipogenesis, by extending its importance to nonrodent mammals and birds. Furthermore, they establish that differential expression of ADD-1/SREBP-1 is a key determinant of the site of fatty acid synthesis in the body.-Gondret, F., P. Ferré, and I. Dugail. ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. J. Lipid Res. 2001. 42: 106;-113.     

SIRT1 Deacetylates and Inhibits SREBP-1C Activity in Regulation of Hepatic Lipid Metabolism

The SIRT1 deacetylase inhibits fat synthesis and stimulates fat oxidation in response to fasting, but the underlying mechanisms remain unclear. Here we report that SREBP-1c, a key lipogenic activator, is an in vivo target of SIRT1. SIRT1 interaction with SREBP-1c was increased during fasting and decreased upon feeding, and consistently, SREBP-1c acetylation levels were decreased during fasting in mouse liver. Acetylated SREBP-1c levels were also increased in HepG2 cells treated with insulin and glucose to mimic feeding conditions, and down-regulation of p300 by siRNA decreased the acetylation. Depletion of hepatic SIRT1 by adenoviral siRNA increased acetylation of SREBP-1c with increased lipogenic gene expression. Tandem mass spectrometry and mutagenesis studies revealed that SREBP-1c is acetylated by p300 at Lys-289 and Lys-309. Mechanistic studies using acetylation-defective mutants showed that SIRT1 deacetylates and inhibits SREBP-1c transactivation by decreasing its stability and its occupancy at the lipogenic genes. Remarkably, SREBP-1c acetylation levels were elevated in diet-induced obese mice, and hepatic overexpression of SIRT1 or treatment with resveratrol, a SIRT1 activator, daily for 1 week decreased acetylated SREBP-1c levels with beneficial functional outcomes. These results demonstrate an intriguing connection between elevated SREBP-1c acetylation and increased lipogenic gene expression, suggesting that abnormally elevated SREBP-1c acetylation increases SREBP-1c lipogenic activity in obese mice. Reducing acetylation of SREBP-1c by targeting SIRT1 may be useful for treating metabolic disorders, including fatty liver, obesity, and type II diabetes.     

An increase in liver PPARγ2 is an initial event to induce fatty liver in response to a diet high in butter: PPARγ2 knockdown improves fatty liver induced by high-saturated fat

The effects of a diet rich in saturated fat on fatty liver formation and the related mechanisms that induce fatty liver were examined. C57BL/6J mice were fed butter or safflower oil as a high-fat (HF) diet (40% fat calories) for 2, 4, 10, or 17 weeks. Although both HF diets induced similar levels of obesity, HF butter-fed mice showed a two to threefold increase in liver triacylglycerol (TG) concentration compared to HF safflower oil-fed mice at 4 or 10 weeks without hyperinsulinemia. At 4 weeks, increases in peroxisome proliferator-activated receptor γ2 (PPARγ2), CD36, and adipose differentiation-related protein (ADRP) mRNAs were observed in HF butter-fed mice; at 10 weeks, an increase in sterol regulatory element-binding protein-1c (SREBP-1c) was observed; at 17 weeks, these increases were attenuated. At 4 weeks, a single injection of adenoviral vector-based short hairpin interfering RNA against PPARγ2 in HF butter-fed mice reduced PPARγ protein and mRNA of its target genes (CD36 and ADRP) by 43%, 43%, and 39%, respectively, with a reduction in liver TG concentration by 38% in 5 days. PPARγ2 knockdown also reduced mRNAs in lipogenic genes (fatty-acid-synthase, stearoyl-CoA desaturase 1, acetyl-CoA carboxylase 1) without alteration of SREBP-1c mRNA. PPARγ2 knockdown reduced mRNAs in genes related to inflammation (CD68, interleukin-1β, tumor necrosis factor-α, and monocyte chemoattractant protein-1). In conclusion, saturated fatty acid-rich oil induced fatty liver in mice, and this was triggered initially by an increase in PPARγ2 protein in the liver, which led to increased expression of lipogenic genes. Inactivation of PPARγ2 may improve fatty liver induced by HF saturated fat.