To elucidate the physiological role of sterol regulatory element-binding protein-1 (SREBP-1), the hepatic mRNA levels of genes encoding various lipogenic enzymes were estimated in SREBP-1 gene knockout mice after a fasting-refeeding treatment, which is an established dietary manipulation for the induction of lipogenic enzymes. In the fasted state, the mRNA levels of all lipogenic enzymes were consistently low in both wild-type andSREBP-1 ^−/− mice. However, the absence of SREBP-1 severely impaired the marked induction of hepatic mRNAs of fatty acid synthetic genes, such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase, that was observed upon refeeding in the wild-type mice. Furthermore, the refeeding responses of other lipogenic enzymes, glycerol-3-phosphate acyltransferase, ATP citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase, and S14 mRNAs, were completely abolished inSREBP-1 ^−/− mice. In contrast, mRNA levels for cholesterol biosynthetic genes were elevated in the refedSREBP-1 ^−/− livers accompanied by an increase in nuclear SREBP-2 protein. When fed a high carbohydrate diet for 14 days, the mRNA levels for these lipogenic enzymes were also strikingly lower in SREBP-1 ^−/− mice than those in wild-type mice. These data demonstrate that SREBP-1 plays a crucial role in the induction of lipogenesis but not cholesterol biosynthesis in liver when excess energy by carbohydrates is consumed.

Hormone-sensitive lipase (HSL) is known to mediate the hydrolysis not only of triacylglycerol stored in adipose tissue but also of cholesterol esters in the adrenals, ovaries, testes, and macrophages. To elucidate its precise role in the development of obesity and steroidogenesis, we generated HSL knockout mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutant HSL allele (HSL−/−) were superficially normal except that the males were sterile because of oligospermia. HSL−/− mice did not have hypogonadism or adrenal insufficiency. Instead, the testes completely lacked neutral cholesterol ester hydrolase (NCEH) activities and contained increased amounts of cholesterol ester. Many epithelial cells in the seminiferous tubules were vacuolated. NCEH activities were completely absent from both brown adipose tissue (BAT) and white adipose tissue (WAT) in HSL−/− mice. Consistently, adipocytes were significantly enlarged in the BAT (5-fold) and, to a lesser extent in the WAT (2-fold), supporting the concept that the hydrolysis of triacylglycerol was, at least in part, impaired in HSL−/− mice. The BAT mass was increased by 1.65-fold, but the WAT mass remained unchanged. Discrepancy of the size differences between cell and tissue suggests the heterogeneity of adipocytes. Despite these morphological changes, HSL−/− mice were neither obese nor cold sensitive. Furthermore, WAT from HSL−/− mice retained 40% of triacylglycerol lipase activities compared with the wild-type WAT. In conclusion, HSL is required for spermatogenesis but is not the only enzyme that mediates the hydrolysis of triacylglycerol stored in adipocytes.

In an attempt to identify transcription factors which activate sterol-regulatory element-binding protein 1c (SREBP-1c) transcription, we screened an expression cDNA library from adipose tissue of SREBP-1 knockout mice using a reporter gene containing the 2.6-kb mouse SREBP-1 gene promoter. We cloned and identified the oxysterol receptors liver X receptor (LXRα) and LXRβ as strong activators of the mouse SREBP-1c promoter. In the transfection studies, expression of either LXRα or -β activated the SREBP-1c promoter-luciferase gene in a dose-dependent manner. Deletion and mutation studies, as well as gel mobility shift assays, located an LXR response element complex consisting of two new LXR-binding motifs which showed high similarity to an LXR response element recently found in the ABC1 gene promoter, a reverse cholesterol transporter. Addition of an LXR ligand, 22(R)-hydroxycholesterol, increased the promoter activity. Coexpression of retinoid X receptor (RXR), a heterodimeric partner, and its ligand 9-cis-retinoic acid also synergistically activated the SREBP-1c promoter. In HepG2 cells, SREBP-1c mRNA and precursor protein levels were induced by treatment with 22(R)-hydroxycholesterol and 9-cis-retinoic acid, confirming that endogenous LXR-RXR activation can induce endogenous SREBP-1c expression. The activation of SREBP-1c by LXR is associated with a slight increase in nuclear SREBP-1c, resulting in activation of the gene for fatty acid synthase, one of its downstream genes, as measured by the luciferase assay. These data demonstrate that LXR-RXR can modify the expression of genes for lipogenic enzymes by regulating SREBP-1c expression, providing a novel link between fatty acid and cholesterol metabolism.

Previous studies have demonstrated that polyunsaturated fatty acids (PUFAs) suppress sterol regulatory element-binding protein 1c (SREBP-1c) expression and, thus, lipogenesis. In the current study, the molecular mechanism for this suppressive effect was investigated with luciferase reporter gene assays using the SREBP-1c promoter in HEK293 cells. Consistent with previous data, the addition of PUFAs to the medium in the assays robustly inhibited the SREBP-1c promoter activity. Deletion and mutation of the two liver X receptor (LXR)-responsive elements (LXREs) in the SREBP-1c promoter region eliminated this suppressive effect, indicating that both LXREs are important PUFA-suppressive elements. The luciferase activities of both SREBP-1c promoter and LXRE enhancer constructs induced by co-expression of LXRα or -β were strongly suppressed by the addition of various PUFAs (arachidonic acid > eicosapentaenoic acid > docosahexaenoic acid > linoleic acid), whereas saturated or mono-unsaturated fatty acids had minimal effects. Gel shift mobility and ligand binding domain activation assays demonstrated that PUFA suppression of SREBP-1c expression is mediated through its competition with LXR ligand in the activation of the ligand binding domain of LXR, thereby inhibiting binding of LXR/retinoid X receptor heterodimer to the LXREs in the SREBP-1c promoter. These data suggest that PUFAs could be deeply involved in nutritional regulation of cellular fatty acid levels by inhibiting an LXR-SREBP-1c system crucial for lipogenesis.

Obesity is a common nutritional problem often associated with diabetes, insulin resistance, and fatty liver (excess fat deposition in liver). Leptin-deficientLep ^ob/Lep ^ob mice develop obesity and those obesity-related syndromes. Increased lipogenesis in both liver and adipose tissue of these mice has been suggested. We have previously shown that the transcription factor sterol regulatory element-binding protein-1 (SREBP-1) plays a crucial role in the regulation of lipogenesis in vivo. To explore the possible involvement of SREBP-1 in the pathogenesis of obesity and its related syndromes, we generated mice deficient in both leptin and SREBP-1. In doubly mutant Lep ^ob/ob × Srebp-1 ^−/− mice, fatty livers were markedly attenuated, but obesity and insulin resistance remained persistent. The mRNA levels of lipogenic enzymes such as fatty acid synthase were proportional to triglyceride accumulation in liver. In contrast, the mRNA abundance of SREBP-1 and lipogenic enzymes in the adipose tissue ofLep ^ob/Lep ^ob mice was profoundly decreased despite sustained fat, which could explain why the SREBP-1 disruption had little effect on obesity. In conclusion, SREBP-1 regulation of lipogenesis is highly involved in the development of fatty livers but does not seem to be a determinant of obesity inLep ^ob/Lep ^ob mice.