All-trans-retinoic acid ameliorates hepatic steatosis in mice by a novel transcriptional cascade

Abstract

Mice deficient in small heterodimer partner (SHP) are protected from diet-induced hepatic steatosis resulting from increased fatty acid oxidation and decreased lipogenesis. The decreased lipogenesis appears to be a direct consequence of very low expression of peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2), a potent lipogenic transcription factor, in the SHP(-/-) liver. The current study focused on the identification of a SHP-dependent regulatory cascade that controls PPAR-γ2 gene expression, thereby regulating hepatic fat accumulation. Illumina BeadChip array (Illumina, Inc., San Diego, CA) and real-time polymerase chain reaction were used to identify genes responsible for the linkage between SHP and PPAR-γ2 using hepatic RNAs isolated from SHP(-/-) and SHP-overexpressing mice. The initial efforts identify that hairy and enhancer of split 6 (Hes6), a novel transcriptional repressor, is an important mediator of the regulation of PPAR-γ2 transcription by SHP. The Hes6 promoter is specifically activated by the retinoic acid receptor (RAR) in response to its natural agonist ligand, all-trans retinoic acid (atRA), and is repressed by SHP. Hes6 subsequently represses hepatocyte nuclear factor 4 alpha (HNF-4α)-activated PPAR-γ2 gene expression by direct inhibition of HNF-4α transcriptional activity. Furthermore, we provide evidences that atRA treatment or adenovirus-mediated RAR-α overexpression significantly reduced hepatic fat accumulation in obese mouse models, as observed in earlier studies, and the beneficial effect is achieved by the proposed transcriptional cascade.

Conclusions: Our study describes a novel transcriptional regulatory cascade controlling hepatic lipid metabolism that identifies retinoic acid signaling as a new therapeutic approach to nonalcoholic fatty liver diseases.

Fig. 1. mRNA levels of hepatic Hes6, PPARγ, and Fsp27 in the suggested transcriptional cascade upon various challenges

(A) RNAs isolated from livers of SHP^−/− and WT mice after 22 weeks WestD regimen were processed for qPCR to quantify the expression of the indicated genes. (B) Hepa1-6, a mouse hepatoma cell line, cells were infected with adenovirus expressing SHP (AdSHP) or GFP (AdGFP) for 3 days. Isolated RNAs were quantified for the indicated genes using qPCR. n = 3. (C) Hepatic RNAs from WT mice infected with AdSHP or AdGFP for 7 days were prepared and quantified for the indicated genes using qPCR. n = 3, *; p < 0.05 vs WT counterparts, #; p < 0.03 vs chow-fed control.

Fig. 2. Regulation of Hes6 promoter by SHP and atRA

(A) Hes6(2.7kb)luc activities by cotransfection of RAR plus RXR or RXR alone with treatment of 1μM of the indicated ligands. (B) Repression of RAR-mediated Hes6(2.7kb)luc activity by SHP. (C) Top panel, mHes6luc reporter plasmids. open bars; potential RAR-RXR response elements (RARE), black bars; reported HNF4α binding sites (23). Bottom left, atRA responsiveness of each deletion reporter. Bottom right, The luciferase activities of Hes6(1.2kb)luc containing mutations on each RARE with cotransfection of RAR/RXR in the presence or absence of 1μM atRA. (E) ChIP analysis on Hes6 promoter region from 2 mouse livers using indicated antibodies. 0.3kb upstream covers HNF4α and RAR binding sites. 2.5kb upstream for negative control.

Fig. 3. Effect of atRA on hepatic lipid homeostasis

(A) Hepa1-6 cells (left) or C57BL/6 mice (right) were treated with 1μM or 15mg/kg, respectively, of atRA or vehicle for 24hrs. mRNA levels of the indicated genes were isolated from cells or mouse liver and quantified using qPCR and plotted with SEM. n=3, *; p < 0.05 (B) A group of mice were challenged with WestD for 2 month and were daily gavaged with corn oil containing vehicle or atRA (15mg/kg) for 7 days while on the WestD regimen. Their livers were collected and processed for indicated stainings (left) or TG and total cholesterol measurement (right). (C) Their body weights and serum glucose levels were measured before and after atRA treatment, plotted and compared with those of vehicle treated control mice. Graphs are presented as means ± SEM (n=4). *; p < 0.05, ***; p < 0.005.

Fig. 4. Effect of atRA on brown adipocyte function

(A) Brown adipose tissues were isolated from the atRA or vehicle-treated mice described in Fig. 3. Gross morphology and weight to body weight ratio are presented at left panel. Their fixed tissue sections were processed for hematoxilin and eosin staining. A representative image from each group is shown (right panel). (B) Total RNAs were isolated from brown adipose tissues and quantified for the expression level of indicated genes using real time PCR (n=4). *; p < 0.05, ***; p < 0.005.

Fig. 5. Amelioration of hepatic lipid accumulation in diet-induced obese mice by RAR-α

(A) WT mice were fed WestD for 2 months and infected with 1×10⁹ pfu of Adnull or AdRAR for 7 days (4 mice per group). Their body weights and fasting serum glucose levels were measured before and after the viral injection and plotted with standard deviations. (B) Livers were collected for Oil Red O staining, TG and cholesterol contents. (C) Hepatic gene expression was examined using real time PCR with the indicated primers and plotted with SEM. *; p < 0.05, **; p < 0.01, ***; p < 0.005.

Fig. 6. Transcriptional regulatory network in atRA/SHP-controlled fat mobilization in hepatocyte

In the suggested fat mobilization pathway, SHP represses Hes6 expression via RAR. Decreased level of Hes6 then derepresses HNF4α-regulated PPARγ2 expression. Eventually, the overexpressed PPARγ2 induces fat accumulation via turning on lipogenic programs in the liver. On the contrary, RAR ligand atRA results in fat utilization via shutting down PPARγ2 expression. Purple and orange represent protein and gene, respectively. Arrow indicates promoter region binding and activation.