SUMMARY Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT), and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic ER stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

Diets rich in saturated fatty acids have long been associated with increased plasma cholesterol concentrations and hence increased risk of cardiovascular disease. More recently, they have also been suggested to promote the development of non-alcoholic fatty liver disease. While there is now considerable evidence to suggest that polyunsaturated fatty acids exert many of their effects through regulating the activity of transcription factors, including peroxisome proliferator activated receptors, sterol regulatory binding proteins (SREBPs) and liver X receptor, our understanding of how saturated fatty acids act is still limited. Here we review the potential mechanisms whereby saturated fatty acids modulate hepatic lipid metabolism thereby impacting on the synthesis, storage and secretion of lipids. Evidence is presented that their effects are, at least partly, mediated through modulation of the activity of the SREBP family of transcription factors.

Cholesterol is a precursor for the biosynthesis of steroid hormones, bile acids, and vitamin D, and is a critical determinant of cell membrane permeability and fl uidity ( 1, 2 ). Cholesterol biosynthesis and homeostasis are regulated by the sterol regulatory element-binding protein (SREBP) transcription factor family. SREBPs are basic-helix-loophelix-leucine zipper transcription factors, which are activated in response to low cellular sterol levels by a series of protein cleavage/transport events ( 3-5 ). There are three SREBP isoforms, which originate from two genes. Srebf1 encodes SREBP-1a and SREBP-1c, which have distinct promoters and 5 ′ exons. Srebf2 encodes SREBP-2. SREBP-1c is the predominant isoform in metabolic tissues such as liver and adipose tissue, but has a relatively weak transcriptionactivation domain compared with the other SREBPs. There is overlap in the activities of the SREBP isoforms, but it is generally held that SREBP-1c primarily targets genes implicated in fatty acid synthesis, whereas SREBP-2 preferentially regulates genes involved in cholesterol synthesis ( 5-8 ). SREBP-1a is a potent activator of both triglyceride and cholesterol biosynthetic pathways, but is expressed at low levels in metabolic tissues, making the physiological role of the protein unclear ( 9 ). SREBP-1c, itself, is regulated by SREBPs, indicating a high degree of cross-talk among SREBP proteins, making it diffi cult to assign distinct physiological functions to individual SREBP proteins.Abstract Cholesterol and fatty acid biosynthesis are regulated by the sterol regulatory element-binding proteins (SREBPs), encoded by Srebf1 and Srebf2 . We generated mice that were either defi cient or hypomorphic for SREBP-2. SREBP-2 defi ciency generally caused death during embryonic development. Analyses of Srebf2؊ / ؊ embryos revealed a requirement for SREBP-2 in limb development and expression of morphogenic genes. We encountered only one viable Srebf2 ؊ / ؊ mouse, which displayed alopecia, attenuated growth, and reduced adipose tissue stores. Hypomorphic SREBP-2 mice (expressing low levels of SREBP-2) survived development, but the female mice exhibited reduced body weight and died between 8 and 12 weeks of age. Male hypomorphic mice were viable but had reduced cholesterol stores in the liver and lower expression of SREBP target genes. Reduced SREBP-2 expression affected SREBP-1 isoforms in a tissue-specifi c manner. In the liver, reduced SREBP-2 expression nearly abolished Srebf1c transcripts and reduced Srebf1a mRNA levels. In contrast, adipose tissue displayed normal expression of SREBP target genes, likely due to a compensatory increase in Srebf1a expression. Our results establish that SREBP-2 is critical for survival and limb patterning during development. Reduced expression of SREBP-2 from the hypomorphic allele leads to early death in females and reduced cholesterol content in the liver, but not in adipose tissue.

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Dietary lipids are thought to regulate the expression of lipogenic enzymes, primarily through modulation of the activity of sterol regulatory element binding proteins (SREBP) (1) . The present study investigated the effects of dietary cholesterol and fat on the tissue-specific expression of SREBP and of the lipogenic enzymes acetyl-CoA carboxylase-1 (ACC) and fatty acid synthase (FAS).Thirty-two male hamsters were divided into four groups of eight animals that were fed: low-cholesterol low-fat chow (LC/LF); chow supplemented with 0.2 % cholesterol (w/w; HC/LF); chow supplemented with fat (HF/LC); chow supplemented with cholesterol and fat (HF/HC). High-fat diets were supplemented with 15 % fat (w/w) blended to produce a fatty acid composition similar to that of a typical 'Western' diet. After 4 weeks animals were killed and mRNA isolated from perirenal adipose tissue and liver. cDNA was prepared and SREBP isoform (1a, 1c and 2), ACC and FAS mRNA were quantified by quantitative real-time PCR. Tissue mRNA concentrations were normalized to TATA-box binding protein mRNA, which was unaffected by diet. Data was analysed by two-way ANOVA with dietary fat and cholesterol as independent factors.Neither dietary cholesterol nor fat had any significant effect on mRNA concentrations for any of the SREBP isoforms in adipose tissue. By contrast dietary cholesterol significantly raised hepatic SREBP1a (P = 0.006) and 1c (P = 0.019) mRNA concentrations but decreased SREBP2 (P < 0.001). There was no significant interaction between cholesterol and fat in their effect on hepatic SREBP mRNA. In adipose tissue both ACC (P = 0.006) and FAS (P = 0.003) mRNA concentrations were decrease by dietary fat but unaffected by cholesterol. Both cholesterol and fat reduced hepatic expression of these lipogenic enzymes with significant interactive effects on both ACC (P = 0.019) and FAS (P = 0.02) mRNA. In liver, but not adipose tissue, there was a highly significant correlation between SREBP2 mRNA concentration and that of both ACC (r 0.65, P < 0.001) and FAS (r 0.68, P < 0.001).It is concluded that dietary cholesterol and fat may regulate hepatic lipogenic gene expression through modulating the expression of SREBP2. However, in adipose tissue the reduction in ACC and FAS gene expression must be mediated by other factors.