Citrus flavonoids repress the mRNA for stearoyl-CoA desaturase, a key enzyme in lipid synthesis and obesity control, in rat primary hepatocytes.

Citrus flavonoids have been shown to decrease plasma lipid levels, improve glucose tolerance, and attenuate obesity. One possible mechanism underlying these physiological effects is reduction of hepatic levels of the mRNA for stearoyl-CoA desaturase-1 (SCD1), since repression of this enzyme reduces hyperlipidemia and adiposity. Here, we show that citrus flavonoids of two structural classes reduce SCD1 mRNA concentrations in a dose-dependent manner in rat primary hepatocytes. This is the first demonstration of repression of SCD1 by citrus flavonoids, either in vivo or in cultured cells. Furthermore, it is the first use of freshly-isolated hepatocytes from any animal to examine citrus flavonoid action at the mRNA level. This study demonstrates that regulation of SCD1 gene expression may play a role in control of obesity by citrus flavonoids and that rat primary hepatocytes are a physiologically-relevant model system for analyzing the molecular mechanisms of flavonoid action in the liver.

The citrus flavonoids hesperetin and nobiletin differentially regulate low density lipoprotein receptor gene transcription in HepG2 liver cells.

Reduction of plasma cholesterol by citrus flavonoids is associated with effects on specific liver functions related to lipid handling. In previous in vivo studies, polymethoxylated flavones (PMF) reduced plasma cholesterol levels at lower doses than required for flavanones. To delineate hepatic mechanisms that underlie this differential potency, we used HepG2 cells to quantitate effects on expression of the LDL receptor (LDLR) gene. A dose-response analysis showed that 200 micromol/L hesperetin, a flavanone present as a disaccharide in oranges, increased LDLR mRNA levels 3.6- to 4.7-fold of the untreated control. In contrast, nobiletin, a PMF found at the highest concentration in oranges and tangerines, achieved maximal stimulation of 1.5- to 1.6-fold of control at only 5 micromol/L. Transcriptional regulation of the LDLR gene by citrus flavonoids has been implicated but, to our knowledge, not directly demonstrated. Here, using transfection vector constructs containing the upstream region of the LDLR gene, we show differences in both potency and efficacy in the induction of transcription, with peak stimulation of 5.3- to 7.5-fold of control at 150-160 micromol/L hesperetin and 3- to 3.8-fold of control at 10-20 micromol/L nobiletin. Hesperetin sustains induction, whereas nobiletin is inhibitory at high doses, resulting in an inverted-U dose response. The sterol regulatory element (SRE) in the LDLR gene upstream region plays a crucial role, because mutation of this site strongly attenuated induction in response to hesperetin or nobiletin. Thus, citrus flavonoids are likely to act through the SRE-binding proteins, with PMF initially activating these mechanisms at considerably lower concentrations than flavanones.

Flanking sequence composition differentially affects the binding and functional characteristics of glucocorticoid receptor homo- and heterodimers.

The core binding sites for a multitude of transcription factors have been identified and characterized, but these sequences cannot fully account for the nuances of cell-specific and gene-specific control of gene transcription. Many factors may contribute to the precise responsiveness of a gene to a particular transcriptional regulatory protein, including the nucleotides in the proximity of the core binding site for that protein. Here, we examine two flanking sequences bordering a site in the gamma-fibrinogen gene regulatory region that binds a heterodimer of the Xenopus glucocorticoid receptor accessory factor (XGRAF) and the glucocorticoid receptor (GR). Mutation of the upstream flank results in a decrease in the level of XGRAF binding but little change in hormone induction. However, alteration of the downstream flank adjacent to the GR binding site causes a decrease in levels of both GR monomer binding and hormone induction. Conversion of the XGRAF-GR binding site to a full glucocorticoid response element (GRE) alters the role of the flanking sequences. A full GRE in this position requires the wild-type upstream flank to bind GR homodimer and induce transcription to maximal levels. In contrast, mutation of the downstream flank is not detrimental to either the binding or the function of the GR dimer. Thus, flanking sequence composition and dimer partner combine to influence GR function, underscoring the complexities involved in the identification of authentic transcription factor response elements.