HDAC5 Inhibits Hepatic Lipogenic Genes Expression by Attenuating the Transcriptional Activity of Liver X Receptor

It has been well-demonstrated that liver X receptor (LXR), a member of the nuclear receptor superfamily of ligand-activated transcription factors, acts as a cholesterol sensor to modulate several key genes involved in hepatic lipogenesis [1,2]. For instance, LXR binds to the promoter region of sterol regulatory element-binding protein-1c (SREBP-1c) and carbohydrate-responsive element-binding protein (ChREBP), two master transcriptional regulators of fatty acids and triglycerides synthesis, to promote the expression of both genes [3,4]. Therefore, ablation of LXR in mice reduced hepatic mRNA expression level of SREBP-1c and its target genes, resulting in decreased serum triglycerides levels [5]. Consistently, administration with LXR agonists in rodents can lead to dramatic increases in hepatic and circulating triglycerides [6,7]. Besides, activation of LXR by T0901317 in high-fat fed rats was associated with restored muscle GLUT4 expression and insulin-stimulated AS160 phosphorylation, suggesting a protective role for LXR in the insulin sensitivity [8].

The transcriptional activities of nuclear receptors are usually tightly regulated by the balance between coactivators and corepressors [9]. Although recent studies have revealed several coactivators of LXR [10,11,12], little is known regarding corepressors of LXR and their physiological roles in LXR-mediated gene transcription. Histone deacetylases (HDACs) are shown to regulate mRNA transcription by catalyzing deacetylation reactions [13]. Recent studies have demonstrated that HDACs play fundamental roles in many biological events, including cell proliferation, differentiation, apoptosis, tumorigenesis and metabolism [14,15]. For instance, HDAC3 was shown to control the circadian rhythm of hepatic lipogenesis [16,17]. As a result, depletion of HDAC3 in mouse liver up-regulated lipogenic genes and resulted in severe liver steatosis [16], establishing HDAC3 as a critical epigenomic modifier that integrated signals from the circadian clock in the regulation of hepatic lipids metabolism.

In the present study, we investigated the roles of HDAC5, a member of class II histone deacetylases, and found that HDAC5 could inhibit lipogenic genes expression through inhibition of LXR activity.

Male C57BL/6 lean and db/db mice aged 6 weeks were purchased from the Shanghai Laboratory Animal Company (SLAC, Shanghai, China). High-fat-diet (HFD)-induced obese mice were maintained with free access to a high-fat chow (D12492, Research Diets, New Brunswick, New Jersey, USA) and drinking water. The HFD contains 60 Kcal% fat, 20 Kcal% carbohydrate and 20 Kcal% protein. The normal diet (ND) contains 10 Kcal% fat, 70 Kcal% carbohydrate and 20 Kcal% protein.

Human embryonic kidney (HEK) 293T cell lines and HepG2 cell lines were maintained in DMEM (Invitrogen, USA) medium, supplemented with 10% fetal bovine serum (GIBOCO, USA) and 1% penicillin/streptomycin in 5% CO₂ atmosphere. To determine the cellular triglyceride (TG) content, cells were extracted by 0.5% NP40 and analyzed using commercial kits (Biovision, USA) according to the manufacturer's instructions. For luciferase assays, 293T cells were placed in 12-well plates and transfected with indicated reporter and expression vectors in duplicate wells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA).

Total RNA from tissues and cells was extracted using the RNA Isolation Kit (Takara, Dalian, China) according to the manufacturer's instructions. Quantitative real-time PCR was performed by using an Applied Biosystems 7300 Real-time PCR System and a TaqMan Universal PCR Master Mix. Expression levels of the target genes were normalized to that of the β-actin.

Cells or tissues were harvested and lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM 2-Mercaptoethanol, 2% w/v SDS, 10% glycerol). After centrifugation at 10000× g for 10 min at 4°C, proteins in the supernatants were quantified and separated by 10% SDS PAGE. Western blot assay was performed using anti-HDAC5 (ab55403, Abcam), LXR (ab41902, Abcam) or HSP90 antibody (ab13495, Abcam). HSP90 was determined as a loading control.

The data shown represent the mean ± standard error (SE) values of three independent experiments. Significance was analyzed using Student's t-test (* p<0.05, ** p<0.01, *** p<0.001).

Firstly, C57BL/6 mice were fed with a high-fat-diet or normal diet for 12 weeks. Then, the expression levels of HDAC5 were analyzed by quantitative real-time PCR and Western blots, respectively. As a result, we found that hepatic HDAC5 was down-regulated in high-fat-diet-induced obese mice (Fig. 1A-1C). Next, leptin receptor deficient mice db/db mice were used. Consistently, the mRNA and protein levels of HDAC5 were also decreased in db/db mice, compared with age-matched lean mice (Fig. 2A-2C).

To further investigate the pathophysiological conditions contributing to the hepatic up-regulation of HDAC5 in obesity, the effects of over-nutrient were determined. Therefore, HepG2 cells were treated with high glucose or palmitate, which did not affect cell viability as shown by MTT assays (Fig. 3A-3B). As a result, we found that the mRNA levels of HDAC5 were inhibited by high glucose and palmitate (Fig. 3C-3D). On the other hand, neither insulin, nor the glucocorticoid analog dexamethasone regulated HDAC5 expression in HepG2 cells (Fig. 3E-3F).

The above clues drove us to ask whether HDAC5 plays a role in lipids metabolism. To this end, endogenous HDAC5 was depleted in HepG2 cells by using small interfering RNA (siRNA) (Fig. 4A-4B). Gene expression analysis showed that SREBP-1c and ChREBP were increased (Fig. 4C). Their down-stream target genes, including fatty acid cynthase (FASN), stearoyl-CoA desaturase-1 (SCD-1) and liver pyruvate kinase (LPK), were also up-regulated with HDAC5 knockdown (Fig. 4D), whereas genes involved in fatty acid oxidation were unaffected (Fig. 4E). In agreement, cellular triglycerides contents were increased by HDAC5 depletion (Fig. 4F). Taken together, these data suggest that HDAC5 plays a role in the regulation of lipogenic genes expression.

Given the critical role of HDAC5 in the regulation of lipogenic genes expression, we performed cotransfection and luciferase reporter assays to evaluate the roles of HDAC5 on the regulation of LXR activity. As shown in the Fig. 5A and 5B, overexpression of HDAC5 inhibited the transcriptional activity of LXR on a synthetic LXRE promoter in a dose-dependent manner, both in the absence or presence of GW3965, a specific LXR ligand. Besides, we observed that HDAC5 repressed the transactivation of both the SREBP-1c and ChREBP promoters (Fig. 5C-5D). Importantly, HDAC5 did not affect the transcription of SREBP-1c and ChREBP promoters containing mutations in the LXRE, indicating that its corepressor function, at least in these cases, is specifically dependent on LXR recruitment to the LXRE (Fig. 5E and 5F). Thus, these data demonstrates that HDAC5 inhibited transcriptional activity of LXR to repress lipogenic genes expression.

Finally, we speculated that HDAC5 could interact with LXR in hepatocytes. Therefore, Flag-HDAC5 and His-LXR were constructed and co-transfected into HEK293T cells. Then, co-immunoprecipitation experiments were then performed. Immunoprecipitation showed that LXR could be pull-downed by HDAC5 (Fig. 6A). To further examine the interaction of endogenous HDAC5 and LXR, mouse liver nuclear extracts were used for co-immunoprecipitation assay. As a result, LXR could be pull-downed by HDAC5 using antibodies against HDAC5, compared with IgG antibody (Fig. 6B), suggesting the interaction of these two proteins

In the present study, we for the first time, uncovered the roles of HDAC5 in the regulation of hepatic lipogenesis. Our results showed that HDAC5 was down-regulated in livers from obese mice, at least in part, due to over-nutrition. Interestingly, a recent study found that hypothalamic HDAC5 expression was also reduced in high-fat diet-induced obese mice when compared with chow-fed lean controls [18]. Besides, prolonged fasting induced a decrease in HDAC5 gene expression and an increase after acute refeeding with high-fat diet [18], suggesting that dietary lipids might be important for the regulation of HDAC5 in the hypothalamus. Therefore, these two studies may be conceptually relevant, although the precise mechanisms for the down-regulation of HDAC5 remain to be determined.

Previous studies have demonstrated that HDAC5 provides critical mechanisms for regulating glucose homeostasis. Raichur S et al. reported that knockdown of HDAC5 in human primary muscle cells increased glucose uptake and was associated with increased GLUT4 expression and promoter activity [19]. Besides, McGee SL et al showed that compensatory regulation of HDAC5 in muscle maintained metabolic adaptive responses and metabolism in response to energetic stress [20]. Moreover, 4-Phenylbutyric acid was shown to increases GLUT4 gene expression in C2C12 myotubes through suppression of HDAC5 [21]. Therefore, modulation of HDAC5 expression or activity may be an effective therapeutic strategy to improve muscle metabolism in these diseases. However, the roles of HDAC5 in the regulation of hepatic lipid metabolism remains poorly understood.

Here, we found that HDAC5 could act as a novel corepressor for LXR to inhibit its transcriptional activity. Given that LXR was shown to promote lipogenesis and hepatosteatosis [20], activation of HDAC5 might be a new strategy to fight against fatty liver and related metabolic diseases. Besides, knockdown of HDAC5 phenotypically matches that of the liver specific HDAC3 knockout mouse [15]. Given that HDACs usually recruit other co-repressors to inhibit gene transcription, we speculate that HDAC5 and HDAC3 might form a complex to repress the transcriptional activity of certain transcription factors, including LXR. Further studies are still needed to clarify this question.

In summary, HDAC5 could act as a novel co-repressor of LXR. Down-regulation of HDAC5 might promote lipogenesis and hepatosteatosis via an increase of LXR transactivity.

None.