The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that are bound and activated by oxysterols. These receptors serve as sterol sensors to regulate the transcription of gene products that control intracellular cholesterol homeostasis through catabolism and transport. In this report, we describe a novel LXR target, the sterol regulatory element-binding protein-1c gene (SREBP-1c), which encodes a membrane-bound transcription factor of the basic helix-loop-helix-leucine zipper family. SREBP-1c expression was markedly increased in mouse tissues in an LXR-dependent manner by dietary cholesterol and synthetic agonists for both LXR and its heterodimer partner, the retinoid X receptor (RXR). Expression of the related gene products, SREBP-1a and SREBP-2, were not increased. Analysis of the mouse SREBP-1c gene promoter revealed an RXR/LXR DNA-binding site that is essential for this regulation. The transcriptional increase in SREBP-1c mRNA by RXR/LXR was accompanied by a similar increase in the level of the nuclear, active form of the SREBP-1c protein and an increase in fatty acid synthesis. Because this active form of SREBP-1c controls the transcription of genes involved in fatty acid biosynthesis, our results reveal a unique regulatory interplay between cholesterol and fatty acid metabolism.

Transcription of the gene encoding sterol regulatory elementbinding protein 1c (SREBP-1c) is known to be activated by insulin in the liver. The resultant SREBP-1c protein activates transcription of the genes required for fatty acid synthesis. Here, we use SREBP-1c promoter reporter constructs to dissect the mechanism of insulin activation in freshly isolated rat hepatocytes. The data show that a complete insulin response (increase of 6-to 11-fold) requires two binding sites for liver X receptors (LXRs), which are nuclear receptors that are activated by oxygenated sterols. Disruption of these binding sites did not lower basal transcription but severely reduced the response to insulin. In contrast, disruption of the closely linked binding sites for SREBPs and nuclear factor Y lowered basal transcription drastically but still permitted a 4-to 7-fold increase in response to insulin. Arachidonic acid, an inhibitor of LXR activation, blocked the response to insulin. We conclude that insulin activates the SREBP-1c promoter primarily by increasing the activity of LXRs, possibly through production of a ligand that activates LXRs or their heterodimerizing partner, the retinoid X receptor. Nuclear SREBPs and nuclear factor Y play permissive roles.

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that increase the synthesis of fatty acids as well as cholesterol in animal cells. All three SREBP isoforms (SREBP-1a, -1c, and -2) are subject to feedback regulation by cholesterol, which blocks their proteolytic release from membranes. Previous data indicate that the SREBPs are also negatively regulated by unsaturated fatty acids, but the mechanism is uncertain. In the current experiments, unsaturated fatty acids decreased the nuclear content of SREBP-1, but not SREBP-2, in cultured human embryonic kidney (HEK)-293 cells. The potency of unsaturated fatty acids increased with increasing chain length and degree of unsaturation. Oleate, linoleate, and arachidonate were all effective, but the saturated fatty acids palmitate and stearate were not effective. Downregulation occurred at two levels. The mRNAs encoding SREBP-1a and SREBP-1c were markedly reduced, and the proteolytic processing of these SREBPs was inhibited. When SREBP-1a was produced by a cDNA expressed from an independent promoter, unsaturated fatty acids reduced nuclear SREBP-1a without affecting the mRNA level. There was no effect when the cDNA encoded a truncated version that was not membranebound. When administered together, sterols and unsaturated fatty acids potentiated each other in reducing nuclear SREBP-1. In the absence of fatty acids, sterols did not cause a sustained reduction of nuclear SREBP-1, but they did reduce nuclear SREBP-2. We conclude that unsaturated fatty acids, as well as sterols, can downregulate nuclear SREBPs and that unsaturated fatty acids have their greatest inhibitory effects on SREBP-1a and SREBP-1c, whereas sterols have their greatest inhibitory effects on SREBP-2.

fatty acid synthesis ͉ arachidonic acid ͉ nuclear receptors

sterol regulatory element-binding proteins ͉ statins ͉ oxysterols ͉ fatty acid synthesis ͉ nuclear receptors S terol regulatory element-binding proteins (SREBPs) are a family of three transcription factors that stimulate the synthesis of sterols and unsaturated fatty acids in animal cells (1). The SREBPs are synthesized as membrane-bound proteins, the active portions of which must be released proteolytically to enter the nucleus and activate transcription. Recent data indicate that SREBPs are regulated at multiple levels including (i) the rate of proteolytic cleavage (2) and (ii) the level of mRNA abundance (3-10).The SREBPs are tripartite proteins that are synthesized on endoplasmic-reticulum (ER) membranes (1). The NH 2 -terminal segment of Ϸ480 amino acids is a transcription factor of the basic helix-loop-helix-leucine zipper family. This domain is followed by a membrane-attachment domain of Ϸ90 amino acids consisting of two membrane-spanning segments, separated by a short hydrophilic loop. The COOH-terminal domain of Ϸ600 amino acids performs a regulatory function. The NH 2 -terminal and COOH-terminal domains of SREBPs project into the cytosol, and only the short hydrophilic-loop projects into the ER lumen. The SREBPs form complexes with SREBP cleavage-activating protein, a polytopic membrane protein that escorts the SREBPs from the ER to the Golgi complex (11-13). There, each SREBP is cleaved sequentially by Site-1 protease and Site-2 protease, which release the NH 2 -terminal fragment so that it can enter the nucleus, in which it binds to sterol regulatory elements in the promoters of multiple genes encoding enzymes required for the synthesis of cholesterol and unsaturated fatty acids. When excess cholesterol accumulates in cells, the movement of SREBPs from ER to Golgi is blocked, proteolysis cannot occur, and the transcription of the target genes declines (2, 13).The three SREBPs are encoded by two genes designated SREBP-1 and SREBP-2. The SREBP-1 gene gives rise to two transcripts, SREBP-1a and SREBP-1c (1) that are produced from alternate promoters separated by Ϸ14 kb in the human genome (14) and Ϸ10 kb in the mouse genome (G. Liang, J.L.G., and M.S.B., unpublished observations). The two promoters give rise to two different first exons that are spliced into a common second exon (14-16). Thereafter, the two transcripts are identical. The first exon of the SREBP-1a gene encodes a 42-residue acidic sequence at the NH 2 terminus of the protein that renders it a strong transcriptional activator that induces mRNAs for genes encoding the enzymes for cholesterol and fatty acid biosynthesis as well as the low-density lipoprotein receptor. The SREBP-1c transcript has a shorter acidic activation domain (24 amino acids), and the protein acts selectively to increase mRNAs for enzymes involved in the synthesis of unsaturated fatty acids. The SREBP-2 transcript has a long activation domain (48 amino acids). It is a potent activator of cholesterol synthesis and a weaker activator of fatty acid biosynthesis (1, 17).In ...