Enzymes required for de novo lipogenesis are induced in mammalian liver after a meal high in carbohydrates. In addition to insulin, increased glucose metabolism initiates an intracellular signaling pathway that transcriptionally regulates genes encoding lipogenic enzymes. A cis-acting sequence, the carbohydrate response element (ChoRE), has been found in the promoter region of several of these genes. ChREBP (carbohydrate response element-binding protein) was recently identified as a candidate transcription factor in the glucose-signaling pathway. We reported that ChREBP requires the heterodimeric partner Max-like factor X (Mlx) to bind to ChoRE sequences. In this study we provide further evidence to support a direct role of Mlx in glucose signaling in the liver. We constructed two different dominant negative forms of Mlx that could dimerize with ChREBP but block its binding to DNA. When introduced into hepatocytes, both dominant negative forms of Mlx inhibited the glucose response of a transfected ChoRE-containing promoter. The glucose response was rescued by adding exogenous wild type Mlx or ChREBP, but not MondoA, a paralog of ChREBP that can also form a heterodimer with Mlx. Furthermore, dominant negative Mlx blocked the induction of glucose-responsive genes from their natural chromosomal context under high glucose conditions. In contrast, genes induced by the insulin and thyroid hormone-signaling pathways were unaffected by dominant negative Mlx. Mlx was present in the glucoseresponsive complex of liver nuclear extract from which ChREBP was purified. In conclusion, Mlx is an obligatory partner of ChREBP in regulating lipogenic enzyme genes in liver.

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Objective: To investigate the differential effects of acute central administration of distinct fatty acids (FA) on food intake, body weight and energy metabolism. Design: Male Sprague-Dawley rats were treated with bolus intracerebroventricular injections of control hydroxypropylb-cyclodextrin (HPB) or HPB complexed with 30 nmol of saturated palmitic acid (PA), monounsaturated oleic acid (OA) or polyunsaturated o-3 docosahexaenoic acid (DHA). Food intake, body weight, neuropeptide expression and various serum parameters were assessed. Results: When compared with controls, rats injected with either OA or DHA had significantly reduced food intake and body weight for 48 h following injections. No significant changes in food intake or body weight were observed in the PA group. In conjunction with reduced food intake, hypothalamic anorexigenic pro-opiomelanocortin (POMC) gene expression was significantly augmented in the OA and DHA groups, with essentially no changes observed in the PA group. Changes in serum measures of energy metabolism also changed coinciding with the observed differences in food intake. Moreover, central administration of SHU9119, a melanocortin-4-rececptor (MC4R) antagonist, completely abolished the anorexigenic actions of OA, suggesting a role for OA-induced augmentation of hypothalamic POMC expression in mediating its central inhibition of food intake. Conclusions: The hypothalamus differentially senses FA and, specifically, that OA and DHA, but not PA, reduce food intake and body weight, which may be mediated through POMC/MC4R signaling.

In the liver, induction of genes encoding enzymes involved in de novo lipogenesis occurs in response to increased glucose metabolism. ChREBP (carbohydrate-response-element-binding protein) is a basic helix-loop-helix/leucine zipper transcription factor that regulates expression of these genes. To evaluate the potential role of ChREBP phosphorylation in its regulation, we used MS to identify modified residues. In the present paper, we report the detection of multiple phosphorylation sites of ChREBP expressed in hepatocytes, several of which are only observed under high-glucose conditions. Mutation of each of these serine/threonine residues of ChREBP did not alter its ability to respond to glucose. However, mutation of five N-terminal phosphoacceptor sites resulted in a major decrease in activity under high-glucose conditions. These phosphorylated residues are located within a region of ChREBP (amino acids 1-197) that is critical for glucose regulation. Mutation of Ser(56) within this region to an aspartate residue resulted in increased nuclear accumulation and activity under high-glucose conditions. Together, these data suggest that ChREBP activity is regulated by complex multisite phosphorylation patterns involving its N-terminal regulatory region.

The Spot 14 (S14) gene is rapidly up-regulated by signals that induce lipogenesis such as enhanced glucose metabolism and thyroid hormone administration. Previous studies in S14 null mice show that S14 is required for normal lipogenesis in the lactating mammary gland, but not the liver. We speculated that the lack of a hepatic phenotype was due to the expression of a compensatory gene. We recently reported that this gene is likely an S14 paralog that we named S14-Related (S14-R). S14-R is expressed in the liver, but not in the mammary gland. If S14-R compensates for the absence of S14 in the liver, we hypothesized that, like S14, S14-R expression should be glucose responsive. Here, we report that hepatic S14-R mRNA levels increase with high-carbohydrate feeding in mice or within 2 h of treating cultured hepatocytes with elevated glucose. A potential carbohydrate response element (ChoRE) was identified at position -458 of the S14-R promoter. Deletion of or point mutations within the putative S14-R ChoRE led to 50-95% inhibition of the glucose response. Gel-shift analysis revealed that the glucose-activated transcription complex carbohydrate responsive element-binding protein/Max-like protein X (Mlx) binds to the S14-R ChoRE. Finally, S14-R glucose induction is completely blocked when a dominant-negative form of Mlx is overexpressed in primary hepatocytes. In conclusion, our results indicate that the S14-R gene is a glucose-responsive target of carbohydrate responsive element-binding protein/Mlx and suggest that the S14-R protein is a compensatory factor, at least partially responsible for the normal liver lipogenesis observed in the S14 null mouse.