Regulation of lipogenesis via BHLHB2/DEC1 and ChREBP feedback looping

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Abstract

BHLHB2/DEC1 is a transcription factor implicated in cell proliferation, apoptosis, and metabolism, and is also known to play an important role in the regulation of the mammalian circadian rhythm. However, its precise role in metabolism remains unclear. We investigated the link between BHLHB2 and ChREBP, a glucose-activated transcription factor involved in the regulation of lipogenesis. Glucose stimulation and overexpression of dominant active ChREBP induced Bhlhb2 mRNA expression in rat hepatocytes. Deletion studies showed that ChoRE (−160 to −143 bp) in the mouse Bhlhb2 promoter region is functional in vivo. Overexpression of BHLHB2 inhibited glucose and ChREBP-mediated induction of rat Fasn and liver pyruvate kinase (Lpk) mRNA. ChIP assay demonstrated that BHLHB2 bound to ChoRE in the Fasn, Lpk, and Bhlhb2 promoter regions in vivo. In conclusion, BHLHB2 and ChREBP constitute a novel feedback loop involved in the regulation of lipogenesis.

Section snippets

Materials and methods

Animals, isolation of rat primary hepatocytes, and cell culture. The protocols for all animal experiments were approved by the Institutional Animal Care and Use Committee of Gunma University Medical School (code no. 08–025). Rat primary hepatocytes were isolated from 6-week-old male Wister rats by the collagenase perfusion methods [4]. Isolated hepatocytes were suspended with DMEM supplemented with 10% fetal calf serum (FCS), 100 nM insulin, 100 nM dexamethasone (dex), 10 nM triiodothronine (T3),

Glucose activation of Bhlhb2 gene expression

Increases in Bhlhb2 mRNA have been reported for diabetic and insulin-resistant patients, and insulin is known to increase Bhlhb2 expression [5], [8]; however, whether glucose activates Bhlhb2 expression remains unclear. Mouse, rat, and human proximal Bhlhb2 promoters contain the conserved ChoRE, which is composed of one perfect E-box and one imperfect E-box, separated by 5-bp spaces (Fig. 1A). Lpk and Fasn are well known to be glucose-response genes targeted by ChREBP [1].

We therefore used Lpk

Discussion

In this study, we show that the glucose-activated transcription factor, ChREBP, regulates mouse Bhlhb2 gene expression by directly binding to ChoRE in the mouse Bhlhb2 promoter. BHLHB2 competes with ChREBP for binding to ChoRE and suppresses the transactivities of Fasn, Lpk, and Bhlhb2 promoter mediated by ChREBP. These data indicate that BHLHB2 and ChREBP coordinately regulate de novo lipogenesis in the rat liver.

We also show that CHREBP induces Bhlhb2 mRNA expression by binding to ChoRE in

Acknowledgments

This work was supported by a Grant in Aid for Scientific Research from the Japan Society for the Promotion of Science (K. Iizuka), the ONO Medical Research Foundation (K. Iizuka) and in part, by a New Energy and Industrial Technology Development Organization grant to Y. Horikawa.

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