By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Wiley Online Library will be unavailable on Saturday 7th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 08.00 EDT / 13.00 BST / 17:30 IST / 20.00 SGT and Sunday 8th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 06.00 EDT / 11.00 BST / 15:30 IST / 18.00 SGT for essential maintenance. Apologies for the inconvenience.
Thyroid hormone-responsive SPOT 14 homolog promotes hepatic lipogenesis, and its expression is regulated by Liver X receptor α through a sterol regulatory element-binding protein 1c–dependent mechanism in mice
Department of Physiology and Pathophysiology, Peking University Health Science Center, Key Laboratory of Cardiovascular Science of the Ministry of Education, Beijing, China
Department of Pathophysiology, Hebei United University, Tangshan, China
Department of Physiology and Pathophysiology, Peking University Health Science Center, Key Laboratory of Cardiovascular Science of the Ministry of Education, Beijing, China
Address reprint requests to: Youfei Guan, M.D., Ph.D., Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xueyuan Road, Beijing 1000191, ChinaJan-Åke Gustafsson, PhD Center for Nuclear Receptors and Cell Signaling, UniversityofHouston, 3013 Cullen Blv, 77204, Houston,Texas, USA, Tel:(001)832-842-8803, E-mail: email@example.com. E-mail: firstname.lastname@example.org fax: +86 10-8280-14471;
Potential conflict of interest: Nothing to report.
This work was supported by the Ministry of Science and Technology (2012CB517504/2011ZX09102, to Y.G.; 2009CB941603, to J.Y. and G.X.; 2010CB912503, to Y.G.; and 2011CB707703 and 2013CB733802, to F.W.) and the Natural Science Foundation (30870905/81030003; to Y.G.). This work was also supported by National Natural Science Foundation of China (30900499; to J.W.) and the Specialized Research Fund for the Doctoral Program of Higher Education (20090001120042; to J.W.). J.Å.G. is grateful to the Swedish Research Council and the Robert A. Welch Foundation for support.
The protein, thyroid hormone-responsive SPOT 14 homolog (Thrsp), has been reported to be a lipogenic gene in cultured hepatocytes, implicating an important role of Thrsp in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Thrsp expression is known to be regulated by a variety of transcription factors, including thyroid hormone receptor, pregnane X receptor, and constitutive androstane receptor. Emerging in vitro evidence also points to a critical role of liver X receptor (LXR) in regulating Thrsp transcription in hepatocytes. In the present study, we showed that Thrsp was up-regulated in livers of db/db mice and high-fat-diet–fed mice, two models of murine NAFLD. Hepatic overexpression of Thrsp increased triglyceride accumulation with enhanced lipogenesis in livers of C57Bl/6 mice, whereas hepatic Thrsp gene silencing attenuated the fatty liver phenotype in db/db mice. LXR activator TO901317 induced Thrsp expression in livers of wild-type (WT) and LXR-β gene-deficient mice, but not in LXR-α or LXR-α/β double-knockout mice. TO901317 treatment significantly enhanced hepatic sterol regulatory element-binding protein 1c (SREBP-1c) expression and activity in WT mice, but failed to induce Thrsp expression in SREBP-1c gene-deficient mice. Sequence analysis revealed four LXR response-element–like elements and one sterol regulatory element (SRE)-binding site within a −2,468 ∼+1-base-pair region of the Thrsp promoter. TO901317 treatment and LXR-α overexpression failed to induce, whereas overexpression of SREBP-1c significantly increased Thrsp promoter activity. Moreover, deletion of the SRE site completely abolished SREBP-1c–induced Thrsp transcription. Conclusion: Thrsp is a lipogenic gene in the liver that is induced by the LXR agonist through an LXR-α–mediated, SREBP-1c–dependent mechanism. Therefore, Thrsp may represent a potential therapeutic target for the treatment of NAFLD. (Hepatology 2013;58:617–628)
Nonalcoholic fatty liver disease (NAFLD) is a common component of metabolic syndrome, which has become an epidemic worldwide as a result of improved living conditions, excessive food intake, and sedentary lifestyles. NAFLD comprises a spectrum of liver pathology, including bland steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. NAFLD is estimated to be present in up to 20% of the general population in the United States and ∼15% in China. NAFLD is mostly accompanied by obesity, type 2 diabetes, and dyslipidemia. Although the underlying mechanisms remain unclear, NAFLD is considered to represent a state of fat accumulation, mainly triglycerides (TGs), in hepatocytes, and its pathogenesis is associated with hepatic insulin resistance and enhanced liver lipogenesis. To date, effective therapeutic strategies for prevention of disease progression in patients with NAFLD are still lacking.
Hepatic lipid accumulation results from an imbalance between lipid availability and lipid disposal. Several metabolic nuclear receptors (NRs) and transcription factors, such as peroxisome proliferator-activated receptors (PPARs), farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), have been reported to be critical for hepatic lipid homeostasis by controlling circulating lipid uptake, de novo lipogenesis, free fatty acid oxidation, and TG-rich lipoprotein secretion in the liver.[4, 5] Although liver X receptors (LXRs), comprising LXR-α and LXR-β, mainly act as intracellular cholesterol sensors whose activation leads to protection from cholesterol overload, their role in the regulation of fatty acid and TG metabolism is becoming clearer. LXR isoform nonselective agonist TO901317 induces fatty liver and promotes the secretion of large, TG-rich very-low-density lipoprotein particles in mice, largely through the induction of lipogenic genes, including sterol regulatory element-binding protein 1c (SREBP-1c), carbohydrate-responsive element-binding protein, stearoyl-CoA (coenzyme A) desaturase 1, and fatty acid synthase (FAS). It has recently been reported that LXR activation may also regulate gene expression of thyroid hormone-responsive SPOT 14 homolog (Thrsp), a gene abundantly present in lipogenic tissues, where it is rapidly up-regulated by lipogenic stimuli, including thyroid hormone and a high-carbohydrate diet. Previous studies demonstrate that Thrsp expression is directly under transcriptional regulation by the NRs, thyroid hormone receptor (TR) and CAR,[12, 13] and it may play an important role in lipogenic processes in the mammary gland. However, Thrsp-null mice display a greater rate of hepatic de novo lipogenesis when exposed to long-term treatment with thyroid hormone or a diet promoting lipogenesis, possibly because of compensation by a paralog of THRSP (MID1IP1, also named S14-R or Mig12). Therefore, it remains unclear whether Thrsp promotes lipogenesis in the liver.
In the present study, our aim was to elucidate the role of Thrsp in hepatic lipid homeostasis and the mechanism by which LXRs up-regulate Thrsp expression. We provide evidence that hepatic overexpression of Thrsp enhances lipogenesis in livers of C57Bl/6 mice and that hepatic knockdown of Thrsp attenuates liver steatosis in db/db mice. Thrsp expression is induced by LXR agonist TO901317 through an LXR-α–mediated, SREBP-1c–dependent mechanism.
Materials and Methods
TO901317 was purchased from Cayman Chemicals (Ann Arbor, MI). TRIzol was purchased from Invitrogen (Carlsbad, CA). Reverse-transcription and probe-labeling kits were purchased from Promega (Madison, WI). α-32P-labeled deoxycytidine triphosphate (dCTP) and γ-32P-labeled deoxyadenosine triphosphate (dATP) were purchased from Amersham Biosciences (Amersham, UK). Quantitative real-time polymerase chain reaction (qPCR) was performed in PTC-200 (MJ Research Inc., St. Bruno, Quebec, Canada) with reagents obtained from BioTeke (Beijing, China). Antibodies (Abs) against SREBP-1 (2A4), FAS (H-300), and eIF5 (C-14) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and Thrsp was obtained from BD Biosciences (San Jose, CA). Horseradish-peroxidase–coupled secondary Abs were purchased from Zhongshan Golden Bridge (Beijing, China). Transient transfection reagent was purchased from Vigorous (Beijing, China).
Animals and Treatment
Male db/db mice (8-12 weeks of age), age-matched and sex-matched db/m mice on a C57BKS background (The Jackson Laboratory, Bar Harbor, ME), and C57Bl/6 mice fed with a high-fat diet (HFD) (Diet #MD45%fat; Mediscience Ltd, China), with the detailed nutritional information in Supporting Table 1, were used for 3 months to determine Thrsp expression in the liver. Eight-week-old male C57Bl/6 mice (The Jackson Laboratory) were used to determine the role of hepatic overexpression of Thrsp in liver lipid metabolism. SREBP-1c–null mice were purchased from The Jackson Laboratory (stock#-004365). LXR-α/β–null mice were generated by Dr. J.-Å. Gustafsson (Karolinska Institutet, Huddinge, Sweden). Mice were treated with TO901317 at a dose of 5 mg/kg/day for 3 days. Adenoviruses were injected through the tail vein at a dose of 5 × 108 plague-forming unit (pfu) for each mouse. To knock down hepatic Thrsp in db/db mice, a mixture of three sets of stealth short interfering RNA (siRNA) against mouse Thrsp complementary DNA (cDNA) coding sequence was synthesized by Invitrogen (sense1, 5’-UUGGGAUAGCGUUUCGUUAGCACUU-3’, antisense1, 5’-AAGUGCUAACGAAACGCUAUCCCAA-3’; sense2, 5’-UUCUCAGCCUCGCUGGUUUCGUUGC-3’, antisense2, 5’-GCAACGAAACCAGCGAGGCUGAGAA-3’; sense3, 5’-AUUUCCUGGUAUUUCCGCGUCACCU-3’, antisense3, 5’-AGGUGACGCGGAAAUACCAGGAAAU-3’). The siRNA cocktail mixture was administrated to db/db mice through tail vein injection at 2.5 mg/kg body weight in 100 μL of sterile saline, as previously described. The same amount of scrambled sequences from Invitrogen was used as a control. Three days later, hepatic tissues were collected for Oil Red O staining, real-time PCR, and histological assays. Study protocols and use of animals were reviewed and approved by the animal care and use review committee of Peking University Health Science Center (Beijing, China).
RNA Extraction, Northern Blotting Analysis, and qPCR
Total RNA from mouse livers was isolated by the use of TRIzol reagent. For northern blotting, mouse cDNA probes were prepared by real-time PCR, and PCR products with expected sizes (274 base pairs [bp] for Thrsp and 449 bp for glyceraldehyde 3-phosphate dehydrogenase) were confirmed by sequencing. Probes were labeled with α-32P-dCTP through use of a DNA-labeling kit (Promega), and northern blotting was performed. For real-time PCR, 2 μg of each RNA sample were reverse transcribed and cDNA samples were then used as templates for qPCR, as previously described. Primers for the various genes were kindly provided by Prof. Yong Liu. β-actin was used as an internal control.
Western Blotting Analysis
To determine expression levels of selected proteins, 100 μg of liver protein was separated by way of 15% or 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Western blotting analysis was performed as previously described.
Cell-Culture, Transient Transfection, and Luciferase Assays
pCMX-hLXR-α overexpression vector was a gift from Dr. B.M. Forman. Expression vectors encoding human SREBP-1c(N) and SREBP-2(N) were obtained from Prof. Y. Chang. Thrsp reporter genes (−2,931/+22 bp, −1,015/+22 bp, −608/+22 bp, −316/+22 bp, and −75/+22 bp) were prepared using several sets of 5’ and 3’ primers by PCR to generate various segments of the sequences between −2,931 and +22 bp in mouse Thrsp gene promoter. These fragments were then cloned into the pGL3-luciferase plasmid (Promega) and sequenced to confirm orientation and sequence. The adenovirus containing an entire coding sequence of mouse Thrsp cDNA (Ad-Thrsp) was prepared by the SinoGenoMax Company (Beijing, China) (Supporting Fig. 1). Transient transfections were performed on HepG2 cells, and transfected cells were treated with drugs for 24 hours after 24 hours of transfection before luciferase activities were assayed.
Hepatic Lipid Uptake Measurement
[(18)F]fluoro-6-thia-heptadecanoic acid was used as a tracer to evaluate hepatic fatty acid uptake in adenovirus encoding green fluorescent protein (Ad-GFP)-treated mice and Ad-Thrsp-treated mice. Three days after injection of Ad-GFP or Ad-Thrsp by tail vein, mice were administered with the 18F radiotracer (0.1 mL, 3.7 MBq) by tail vein injection. All mice were sacrificed at 30 minutes postinjection. Blood and liver were collected, wet weighed, and measured using a gamma-counter. Results were presented as a percentage of injected dose per gram of tissue. Micro-positron emission tomography imaging for each group was achieved at the same time point.
Probes corresponding to nucleotides −156 to −71 bp of the Thrsp promoter, which contains a putative SRE (5’-TCACCTGATA-3’), were chemically synthesized and labeled with γ-32P-dATP by use of a DNA-labeling kit (Promega). The isotope-labeled probe was incubated with liver nuclear extract (24 μg). After a 20-minute incubation at room temperature, samples were resolved on 5% polyacrylamide gel in 0.5× Tris/borate/ethylenediaminetetraacetic acid at 45 mA for 2 hours at 4°C. For the competition assays, unlabeled oligonucleotides were added to the reactions at ∼250-fold molar excess. Sequences of the oligonucleotides are as follows: Thrsp −156 to −71 bp, 5’-GTC CCT GGG TAG ATG GAT CAC CTG ATA CAG ACA CTG GGG ACC AAA CGC TGG GAT TGG CTC AAA ACA GGG CTG TGT TGC TCC AAT GG -3’ (sense); 5’- CCA TTG GAG CAA CAC AGC CCT GTT TTG AGC CAA TCC CAG CGT TTG GTC CCC AGT GTC TGT ATC AGG TGA TCC ATC TAC CCA GGG AC-3’ (antisense).
Data are presented as mean ± standard error. Analysis involved the use of analysis of variance and the Student t test. P < 0.05 was considered significant.
Increased Hepatic Thrsp Protein Expression in db/db Mice and HFD-Fed Mice
To determine the role of Thrsp in hepatic lipid metabolism, Thrsp expression in livers of db/db mice and mice fed an HFD was evaluated. Hepatic Thrsp protein levels were increased 3.1-fold in livers of db/db mice, as compared with db/m mice (Fig. 1A). Similarly, Thrsp protein expression was increased in livers of mice fed with an HFD for 12 weeks (Fig. 1B). These findings suggest that Thrsp may play an important role in the regulation of liver lipid homeostasis and the pathogenesis of NAFLD.
Hepatic Thrsp Overexpression Promotes Liver Lipogenesis in C57Bl/6 Mice
To determine the role of Thrsp in lipid metabolism in the liver, C57Bl/6 mice were intravenously injected with Ad-Thrsp or Ad-GFP as control. Animals were sacrificed 3 days postinjection. Hepatic Thrsp levels were significantly increased in livers with Ad-Thrsp infection (Fig. 2D). Oil Red O staining revealed enhanced hepatic lipid accumulation in mice transfected with Ad-Thrsp (Fig. 2A). Consistently, experimental animal computed tomography scan study further showed that the fatty liver ratio was increased after overexpression of Thrsp for 3 days (Supporting Fig. 2B). Liver TG content was also consistently and significantly increased in Ad-Thrsp-infected mice (Fig. 2B). Thrsp overexpression slightly elevated hepatic cholesterol content (Fig. 2C). Although plasma TG levels were significantly increased in Thrsp-overexpressing mice, no change was found in total plasma cholesterol levels (Supporting Fig. 3). Efficacy of Thrsp overexpression was confirmed in HepG2 cells transfected with Ad-Thrsp (Supporting Fig. 1).
To elucidate the mechanisms by which hepatic Thrsp overexpression leads to fatty liver, the expression of the genes involved in hepatic lipogenesis, fatty acid uptake and oxidation, and gluconeogenesis were measured. In Ad-Thrsp-infected mouse livers, western blotting and qPCR analysis revealed a prominent elevation of FAS (by ≈1.5-fold at the protein level and ≈6-fold at the messenger RNA [mRNA] level) (Fig. 2D,E). Furthermore, FAS and acetyl-CoA carboxylase (ACC) activity were significantly increased in Ad-Thrsp-infected mouse livers (Supporting Fig. 2C,D). Hepatic overexpression of Thrsp also resulted in an approximately 3.6-fold increase in SREBP-1c expression, ≈2-fold increase in diacylglycerol O-acyltransferase (DGAT)1 expression, and ≈3-fold increase in DGAT2 expression (Fig. 2E). Thrsp overexpression also caused a considerable increase in the expression of SREBP-2 (by ≈2-fold) (Fig. 2E), which may be responsible for the slight elevation in hepatic cholesterol levels observed (Fig. 2C). Expression of CD36/fatty acid translocase, a key protein involved in regulating the uptake of fatty acid across the plasma membrane, was significantly decreased by nearly 90% (Fig. 2E), implying a decrease in hepatic fatty acid uptake. This was further supported by an in vivo lipid uptake study showing a reduced lipid uptake in livers with Thrsp overexpression (Supporting Fig. 4A,B). Quantitative analysis of genes involved in fatty acid oxidation further showed that the expression of PPARα, acyl-CoA oxidase, and peroxisomal ketothiolase was significantly up-regulated with little change in medium-chain and long-chain fatty acyl CoA dehydrogenase levels after overexpressing Thrsp (Fig. 2E and Supporting Fig. 5). These findings suggest increased β oxidation in Thrsp-overexpressed livers. Interestingly, expression levels and activities of glucose-6-phosphatase (G6pase), a key gluconeogenic enzyme, were significantly increased in Thrsp-overexpressed livers (Fig. 2E and Supporting Fig. 2E), suggesting that Thrsp may increase the release of glucose from the liver and contribute to type 2 diabetes.
Hepatic Thrsp Gene Silencing Attenuates Liver Steatosis in db/db Mice
To further confirm the lipogenic effect of Thrsp in the liver, hepatic Thrsp expression was silenced by a siRNA-based approach. As expected, liver Thrsp expression was decreased approximately 2-fold at the protein level in db/db mice with Thrsp gene knockdown (Supporting Fig. 6A). Hepatic Thrsp gene silencing significantly reduced hepatic TG content (Supporting Fig. 6B). Thrsp gene knockdown also significantly ameliorated liver steatosis of db/db mice, as evidenced by morphological changes and Oil Red O staining (Supporting Fig. 6C). Consistently, expression of FAS was also markedly reduced after Thrsp gene knockdown (Supporting Fig. 6A). In addition, coinciding with the amelioration of fatty liver, serum alanine aminotransferase activity and hepatic interleukin-1 expression were significantly reduced in db/db mice with hepatic Thrsp gene knockdown (Supporting Fig. 7A,B), suggesting a hepatoprotective effect of Thrsp knockdown on the liver.
To determine the role of Thrsp in LXR-induced hepatic lipogenesis, we transfected murine primary hepatocytes with si-Thrsp or scrambled siRNA. Six hours after transfection, cells were treated with TO901317 or dimethyl sulfoxide for 36 hours. TO901317 significantly increased the expression of Thrsp, which was abolished by si-Thrsp transfection (Supporting Fig. 8A). TO901317-induced lipid deposition in hepatocytes was significantly attenuated, but not completely abolished, by the knockdown of Thrsp (Supporting Fig. 8B). TO901317 treatment increased the expression of SREBP-1, FAS, and ACC, and Thrsp gene silencing markedly reduced ACC expression with a declining trend in SREBP-1 and FAS expression (Supporting Fig. 8C). These results imply that Thrsp mediates, at least in part, the lipogenic effects of LXR activation in hepatocytes and further supports our conclusion that Thrsp promotes hepatic lipogenesis.
Hepatic Thrsp Expression Is Up-Regulated in Mice Treated With the LXR Agonist, TO901317
Because activation of the NR, LXR, also leads to enhanced lipogenesis in the liver, and Thrsp expression is regulated by several NRs,[12, 13, 23] we sought to determine whether LXRs might regulate the expression of Thrsp. Mice were treated with TO901317 (5 mg/kg/day) for 3 days, and TG levels were examined. In line with previous reports, TO901317 treatment resulted in significantly enlarged livers (Supporting Fig. 9A), with increased intracellular lipid accumulation revealed by Oil Red O staining (Supporting Fig. 9B). LXR activation also significantly increased liver TG content (Supporting Fig. 9C), with no effect on cholesterol content (Supporting Fig. 9D). To determine the effect of LXR activation on hepatic Thrsp expression, northern blotting and western blotting assays were utilized. Thrsp expression was significantly up-regulated in TO901317-treated livers at both the mRNA (Fig. 3A,B) and protein levels (Fig. 3C,D).
LXR-α, but Not LXR-β, Mediates TO901317-Induced Thrsp Expression in the Liver
TO901317 is a synthetic agonist for both LXR-α and LXR-β. It also activates other NRs, including PXR and FXR.[24, 25] We next determined whether TO901317-induced Thrsp up-regulation is LXR dependent. TO901317 treatment resulted in a significant increase in hepatic Thrsp expression in wild-type (WT) mice (Fig. 4A,B), but not in LXR-α/β double-knockout (KO) mice. To further identify the LXR isoform responsible for TO901317-induced Thrsp expression, we treated both LXR-α KO mice and LXR-β KO mice with TO901317. TO901317 treatment led to a significant increase in Thrsp expression in LXR-β KO mice, but not in LXR-α KO mice, suggesting that LXR-α is required for TO901317-induced Thrsp up-regulation in the liver (Fig. 4C,D).
SREBP-1c Is Required for LXR Agonist-Induced Hepatic Thrsp Expression
SREBP-1c, as a direct LXR target gene, mediates several lipogenic effects of LXRs. To further characterize the mechanism by which TO901317-activated LXR-α receptor increases Thrsp expression, hepatic SREBPs were measured in livers of mice receiving TO901317 treatment. Both precursor and mature forms of SREBP-1, but not SREBP-2, were significantly induced by LXR activation (Fig. 5A). This was further supported by the findings of the gel-shift assay, in which LXR activation resulted in a significant increase in binding of SREBP(s) to the SRE site (−156 to −71 bp) in the Thrsp promoter in TO901317-treated mouse liver (Fig. 5B). Because Thrsp transcription was reported to be regulated by SREBP-1, we then tested the possibility that LXR-α activation-mediated up-regulation of Thrsp is SREBP-1 dependent. There was a significant reduction of Thrsp levels at baseline in SREBP-1c KO mice, compared to the WT mice (Fig. 5C,D). Induction of hepatic Thrsp expression by the LXR agonist, TO901317, was almost completely abolished in SREBP-1c KO mice (Fig. 5C,D), suggesting that SREBP-1c plays a critical role in LXR-α–mediated Thrsp up-regulation. It was also noticed that basal hepatic TG content was decreased in vehicle-treated SREBP-1c KO mice, compared to WT mice. TO901317-induced hepatic TG accumulation was significantly reduced in SREBP-1c KO mice, as compared to that in WT mice (Fig. 5E).
Transcriptional Regulation of Thrsp by LXR-α Is Not LXR Response Element, but Steroid Regulatory Element, Dependent
To further characterize the molecular mechanism mediating LXR-α–induced Thrsp transcription, the mouse Thrsp promoter, ranging from −3,000 to +22 bp was analyzed by the Transcription Element Search System. Four potential LXR response elements (LXREs) and one steroid regulatory element (SRE) were identified (Fig. 6A). The ∼3-kilobase (kb) mouse Thrsp promoter DNA was amplified by PCR. Different deletion fragments were generated by PCR using the ∼3-kb mouse Thrsp promoter DNA fragment as the template and then cloned into the pGL3-basic vector (Fig. 6B). Luciferase activity assay showed that deletion of all four potential LXRE sites had little effect on SREBP-1c-induced Thrsp promoter activity, but ablation of the proximal region containing the potential SRE site almost completely abolished promoter activity (Fig. 6B). This finding suggests that the SRE site within the Thrsp promoter is responsible for SREBP-1c–induced up-regulation of Thrsp expression. It also suggests that SREBP-1 may be essential for basal Thrsp expression, which is consistent with the finding that basal levels of Thrsp expression were significantly lower in SREBP-1c−/− mice than in WT mice (Fig. 5C). By using the full-length Thrsp promoter (−2,931/+22 bp)-driven luciferase construct, we determined the effect of TO901317 treatment and overexpression of LXR-α, SREBP-1, and SREBP-2 on luciferase activity. Reporter activity was not induced either by TO901317 treatment or by LXR-α overexpression (Fig. 6C). However, reporter activity was significantly increased by both SREBP-1c(N) and SREBP-2(N) overexpression, with SREBP-1c(N) being more potent than SREBP-2(N) (Fig. 6C). This finding is consistent with increased binding of SREBPs to the SRE site of the Thrsp promoter in TO901317-treated mouse liver (Fig. 5B). Together, these results demonstrate that LXR-induced Thrsp transcription occurs by an SREBP-dependent mechanism.
In the present study, we provide direct evidence that specific overexpression of Thrsp in livers of C57Bl/6 mice significantly promotes hepatic lipogenesis, and that hepatic knockdown of Thrsp markedly attenuates the fatty liver phenotype in db/db mice, suggesting that Thrsp is an important lipogenic factor in the liver. Hepatic Thrsp expression is induced by the LXR agonist, TO901317, which is mediated by LXR-α and dependent on the downstream transcriptional factor, SREBP-1. Our findings suggest that Thrsp may be involved in LXR activator-induced fatty liver, and it may represent a therapeutic target for the treatment of NAFLD.
Since Thrsp was discovered three decades ago, a number of studies have reported that it acts as a transducer of hormone-related and nutrient-related signals to genes involved in lipid metabolism. Recently, accumulating evidence has suggested that Thrsp may also play an important role in the induction of lipogenic enzymes, particularly by carbohydrate feeding and thyroid hormone administration, with the biochemical mechanism uncharacterized.[11, 14] Some recent studies demonstrate that Thrsp may work as a cofactor regulating TR-dependent and p53-dependent transcriptional activation, providing a clue to its biochemical function.[28, 29] In the present study, we provide direct evidence that hepatic overexpression of Thrsp significantly increased liver lipid accumulation in normal mice and that hepatic knockdown of the Thrsp gene resulted in a significant improvement of liver steatosis in db/db mice. The underlying mechanisms may include the induction of the lipogenic transcriptional factor, SREBP-1c, accompanied by a significant increase of FAS, DGAT1, and DGAT2, key enzymes involved in fatty acid and TG biosynthesis. We also noticed that expression and activity of G6pase, a key gluconeogenic enzyme, is significantly increased, suggesting that Thrsp may play a role in glucose homeostasis in the liver as well.
LXRs are critical transcriptional factors in controlling hepatic lipid metabolism and their agonists have a number of potential therapeutic implications, including antiatherosclerotic action, antidiabetic properties, and protection against renal lipotoxicity. However, the side effect of LXR agonists in inducing hepatic steatosis and hypertriglyceridemia limits their clinical use. Multiple mechanisms may be involved in these unwanted effects. LXR activation was reported to enhance hepatic uptake of free fatty acids by up-regulation of CD36, a major hepatic fatty acid transporter, which is a direct target of LXR. In addition, LXR can significantly up-regulate FAS expression directly or by induction of its target gene, SREBP-1c, thereby mediating de novo lipogenesis in the liver. The present study revealed that the lipogenic Thrsp gene is also under the direct control of SREBP-1c, which is induced by LXR activation in the liver. Together with our finding that Thrsp gene silencing attenuates LXR agonist-induced lipid accumulation in primary mouse hepatocytes and previous reports that Thrsp may promote lipogenesis in vitro,[11, 23] the present findings reveal that induction of Thrsp expression may contribute, at least in part, to increased lipogenesis by LXRs and provide novel insight into LXR-elicited fatty liver and hypertriglyceridemia. However, although Thrsp is involved in LXR-induced hepatic lipogenesis, it appears to have little effect on LXR-induced fatty acid uptake.
The present study also addressed whether LXR-α and LXR-β have similar regulatory effects on Thrsp expression in the liver. Although both isoforms share significant similarity at the amino acid sequence level and both are thought to be essential for the regulation of hepatic lipid metabolism, LXR-α and LXR-β have been found to exert overlapping, but not identical, functions.[36, 37] By using isoform-specific gene KO mice, we investigated whether LXR-α and LXR-β exert different effects on Thrsp expression in the liver. Induction of Thrsp by nonselective LXR agonist TO901317 was completely abolished in mice deficient for both LXR isoforms, indicating that TO901317-induced Thrsp up-regulation is LXR dependent. The finding that TO901317 up-regulated Thrsp expression in LXR-β–deficient, but not LXR-α–deficient, mice further revealed that activation of the LXR-α isoform is responsible for TO901317-induced Thrsp expression. These findings indicate that LXR-α, but not LXR-β, has an essential role in the regulation of Thrsp transcription.
Thrsp is reported to be involved in liver steatosis induced by PXR, which is another receptor for TO901317. However, whether PXR-mediated Thrsp expression is involved in the steatotic effects induced by other PXR activators, such as rifampicin, nifedipine, and carbamazepine, remains uncharacterized. In contrast to LXR-α, which induces Thrsp expression by the SREBP-1c–dependent pathway, PXR can up-regulate Thrsp expression by directly binding to TRE in the Thrsp promoter. Because LXR-α/β double-KO mice exhibited a complete abrogation of TO901317-induced Thrsp expression, it is unlikely that PXR is responsible for this process. Although SREBP-1c gene deficiency significantly reduced basal and TO901317-induced Thrsp expression, Thrsp levels in TO901317-treated, SREBP-1c–null mouse livers tended to increase, indicating that minor regulatory mechanism(s) other than LXR and PXR pathways may be involved.
In conclusion, the present study provides direct evidence that Thrsp is a lipogenic gene in the liver. LXR activation promotes Thrsp expression through an LXR-α–mediated, SREBP-1c–dependent mechanism (Fig. 7). Thrsp may represent a potential therapeutic target for the treatment of NAFLD.
The authors thank T. Guan for his assistance in editing the manuscript.