Activation of farnesoid X receptor promotes triglycerides lowering by suppressing phospholipase A2 G12B expression
Qingli Liu a, Meng Yang a, Xuekun Fu a, Renzhong Liu a, Caijun Sun b, Haobo Pan a, Chi-Wai Wong c, Min Guan a
Abstract
As a novel mediator of hepatic very low-density lipoproteins (VLDL) secretion, phospholipase A2 G12B (PLA2G12B) is transcriptionally regulated by hepatocyte nuclear factor-4 alpha (HNF-4a). Farnesoid X receptor (FXR) plays a critical role in maintaining bile acids and triglycerides (TG) homeostasis. Here we report that FXR regulates serum TG level in part through PLA2G12B. Activation of FXR by chenodeoxycholic acid (CDCA) or GW4064 significantly decreased PLA2G12B expression in HepG2 cells. PLA2G12B expression was transcriptionally repressed due to an FXR-mediated up-regulation of small heterodimer partner (SHP) which functionally suppresses HNF-4a activity. We found that hepatic PLA2G12B expression was suppressed and serum TG level reduced in high fat diet mice treated with CDCA. Concurrently, CDCA treatment lowered hepatic VLDL-TG secretion. Our data demonstrate that activation of FXR promotes TG lowering, not only by decreasing de novo lipogenesis but also reducing hepatic secretion of TG-rich VLDL particles in part through suppressing PLA2G12B expression.
Keywords:
Triglyceride
Very low-density lipoprotein
Phospholipase A2 G12B
Farnesoid X receptor
1. Introduction
Non-alcoholic fatty liver disease (NAFLD) is believed to be a precursor to developing metabolic syndrome (Lonardo et al., 2015). Both hyperlipidemia and hypolipidemia are considered strong risk factors for NAFLD (Cave et al., 2016; Lonardo et al., 2015; Nonalcoholic Fatty Liver Disease Study et al., 2015). Inappropriate regulation of hepatic production and secretion of very low-density lipoproteins (VLDL) can lead to hyperlipidemia or hypolipidemia (Tao et al., 2010; Wiegman et al., 2003). A number of genes, including apolipoprotein-B (ApoB) and microsomal triglyceride transfer protein (MTP), are involved in hepatic synthesis of ApoB containing VLDL particles (Raabe et al.,1999; Wang et al., 2012). We previously reported that, in addition to MTP, secreted phospholipase A2 G12B (PLA2G12B) is a novel mediator of triglyceride metabolism in the liver (Guan et al., 2011). Hepatic VLDL production is significantly compromised in PLA2G12B-knockout mice. Specifically, PLA2G12B-null mice accumulate triglyceride, cholesterol, and fatty acids in the liver and develop severe hepatosteatosis over time even on regular diet.
Farnesoid X receptor (FXR, NR1H4), a bile acid sensing nuclear receptor, is known to exert significant effects on lipids, carbohydrate and cholesterol metabolisms (Calkin and Tontonoz, 2012; Duran-Sandoval et al., 2005; Ma et al., 2006; Evans et al., 2009; Mencarelli and Fiorucci, 2010). For instance, chenodeoxycholic acid (CDCA), a primary bile acid and potent activator of FXR (Bavner et al., 2005; Chiang, 2000), decreases plasma triglycerides level and reduces VLDL production in hyperlipidemic hamsters and human, indicating that FXR regulates lipid metabolism in various species in vivo (Bilz et al., 2006; Miller and Nestel, 1974). Decreased de novo hepatic lipogenesis and reduced hepatic triglycerides-rich VLDL assembly and secretion are both thought to be effected upon FXR activation. How FXR activation suppresses these pathways is believed to be in part mediated by transcriptional induction of a negative regulator small heterodimer partner (SHP) (Boulias et al., 2005). As an atypical negative nuclear receptor, SHP functionally interacts with and suppresses the activities of a number of heterodimeric nuclear receptors including hepatocyte nuclear factor-4 alpha (HNF-4a) (Hirokane et al., 2004; Yamagata et al., 2004).
We previously demonstrated that PLA2G12B is transcriptionally regulated by HNF-4a (Guan et al., 2011). As the HNF-4a mediated MTP expression is reduced by bile acids in HepG2 cells via SHP (Hirokane et al., 2004), it is likely that PLA2G12B is subjected to similar regulation. This prompted us to study if the bile acids induced lipid-lowering effect involves repressing hepatic PLA2G12B transcription; thereby, lowering VLDL assembly and secretion. Herein, we demonstrated that FXR agonists downregulate PLA2G12B expression by impairing HNF-4a transcriptional activity through SHP. We further found that CDCA lowers PLA2G12B and MTP expression levels leading to decreased hepatic VLDL assembly and secretion in hyperlipidemic mice.
2. Materials and methods
2.1. Animals and diet
C57BL/6J male mice were obtained at 6 weeks of age from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). All mice were maintained on a fixed cycle environment (12 h of light and 12 h of dark) and given free access to water and food. Body weight (bw) was measured every week. Mice were randomly divided into 3 groups of 5e6 mice. One group was fed standard chow diet (CD, n ¼ 5), and two groups were placed on high-fat diet (HFD, n ¼ 6) containing 23.1% protein, 25.9% carbohydrate and 34.9% fat (Testdiet 58Y1, Saint Louis, MO) for 12 weeks. For treatment with bile acids, one group of HFD mice were gavaged with chenodeoxycholic acid (Sigma-Aldrich C9377, Saint Louis, MO) at 200 mg/kg once a day for 3 weeks before sacrifice (HFD þ CDCA, n ¼ 6). The vehicle used in this study consisted of vitamin E-TPGS 10%, PEG400 20% and 70% water. All animal study protocols were designed under the guidance of the Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Animal Research Committee of the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.
2.2. Cloning and plasmids
Expression constructs of human PGC-1a and mouse HNF-4a were described previously (Guan et al., 2011; Wang et al., 2009). A fragment of human FXR was inserted into pcDNA3.1 (Invitrogen, Carlsbad, CA) to generate pcDNA3.1-FXR expression construct. A dominant negative FXR transactivation-deficient point mutant, FXR (W469A) (Ananthanarayanan et al., 2001), was generated using QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Genomic fragment encompassing mouse PLA2G12B promoter extending from 1 to 1102 bp relative to the transcription initiation site was subcloned into the pGL3 vector (Promega, Madison, WI) to generate pGL3-PLA2G12B reporter plasmid as previously described. To generate expression plasmid pCMV-BD-SHP, human SHP fragment obtained by reverse transcription-PCR using total RNA from HepG2 cells was ligated into pCMV-BD vector (Promega).
2.3. siRNA gene silencing
FXR small interference RNA (FXR-siRNA) targeting human FXR (cDNA sequence 50-CAAGTGACCTCGACAACAA-30) and control siRNA (scrambled-siRNA) were chemically synthesized and annealed to duplex by Ribobio (Guangzhou, China). For gene silencing with small interference RNA, cells were transfected with FXR-siRNA or scrambled-siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.
2.4. Cell culture, transient transfection, and luciferase assays
HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (Hycolne, Logan, UT) supplemented with 10% fetal bovine serum (Corning 35-076-cv, Corning, NY) at 37 C in 5% CO2. For quantitative RT-PCR and western blot analysis, medium was replaced with DMEM containing 0.5% FBS in the presence or absence of CDCA or GW4064 (Sleleck S2782, Houston, TX) at the indicated concentrations and further incubated for 24 h. For dualluciferase report assays, 70e80% confluent cells were seeded at 2 104/per well onto 96-wells for 24 h. Transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. Simply, medium was replaced with aMEM (phenol red-free) with 10% FBS (charcoal stripped) before transfection. pRL-TK vector was transfected in each experiment as an internal control. pGL3-PLA2G12B, pCMV-BD-SHP, and pcDNA4HNF-4a plasmids or the corresponding empty expression vectors were transfected into HepG2 cells. For gene silencing with small interference RNA, cells were cotransfected with pGL3-PLA2G12B and FXR-siRNA or scrambled-siRNA. After 24 h, cells were harvested or treated with CDCA or GW4064 at the indicated concentrations and further incubated for 24 h in a-MEM (phenol red-free) supplemented with 0.5% FBS (charcoal stripped). Luciferase activity was measured with a dual-luciferase reporter assay system (Promega E1960) and microplate luminometer (Promega GloMax96 E6521) and normalized for Renilla-luciferase activity in the same sample.
2.5. Quantitative RT-PCR
mRNA levels of genes involved in hepatic lipid metabolism were analyzed by quantitative RT-PCR. Total RNA was extracted using trizol reagent (Invitrogen). cDNA was prepared from RNA with a cDNA synthesis kit (Thermo K1622, Waltham, MA) and oligo (dT) primers according to the manufacturer’s protocol. Primer pairs were designed according to Primer Bank using published sequence data obtained from the National Center for Biotechnology Information database. Relative efficiency of primers were validated before use. RT-PCR reactions were performed using SYBR Green (Toyobo, Osaka, Japan) and analyzed by a Real-Time PCR System (Bio-Rad CFX96, Hercules, CA) using DDCt threshold cycle method. Relative gene expression was normalized to glyceraldehyde-3phosphate dehydrogenase (GAPDH) level. Primer sequences are listed in Table 1.
2.6. Western blots
Total protein was extracted from cells or frozen liver samples. Cells or homogenized liver samples were lysed with cell lysis buffer and treated by ultrasonic probes, then centrifuged at 4 C, 12,000g for 10 min. Protein fractions were quantified by a BCA protein assay kit (Pierce, Waltham, MA) according to the manufacturer’s instructions. Equal amounts of protein extract were separated on a 12% SDS-PAGE gel and electrotransferred to 0.45 mm PVDF membranes using a Bio-Rad wet transfer tank. After blocking with 5% nonfat-milk at room temperature, membranes were incubated with anti-b-Actin (Beyotime AA128, Shanghai, China), antiPLA2G12B (Abbioetc 252133, San Diego, CA) or anti-MTP (Santa cruz sc-33116, Dallas, TX) antibodies overnight. After an incubation with HRP-conjugated mouse antibody (Santa cruz sc-2031) or rabbit antibody (Santa cruz sc-2030), blots were developed using an enhanced chemiluminescence kit (Millipore WBLUR0500, Billerica, MA) and exposed in ChemiDoc XRS chemiluminescence imaging system (Bio-Rad). Band intensities were quantified by Quantity One software. Relative protein expression was normalized synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. to b-Actin level.
2.7. Determination of intracellular triglyceride accumulation
Huh7 cells were cultured in DMEM supplemented with 10% FBS. Cells were seeded on a 12-well plate and then transfected with FXR-siRNA or scramble-siRNA for 12 h. Oleic acid (100 mM) was added for another 12 h. Medium was then replaced with DMEM containing CDCA (100 mM) or GW4064 (5 mM) and further incubated for 12e24 h. Intracellular triglyceride was extracted and measured using a triglyceride assay kit (GPO-POD; Applygen technologies Inc., Beijing, China). Protein concentrations in cell lysates were measured using a BCA Protein Assay Kit (Pierce). Intracellular TG content was expressed as millimoles of lipid per milligram of cellular protein. For lipid droplet analysis, Huh7 cells were fixed for 15 min with 4% paraformaldehyde, washed 3 times with PBS and stained for 10 min with 4,4-difluoro-1,3,5,7,8pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY493/503, Invitrogen) and DAPI (D1306, Thermo) for 3 min. Images were obtained using an Olympus fluorescent microscope (BX53, Tokyo, Japan).
2.8. Blood and hepatic chemistry analysis, hepatic histopathology
All mice were fasted overnight before being sacrificed. Blood glucose was measured with ACCU-CHEK blood glucose meter (Roche, Mannheim, Germany). Serum total cholesterol (TC) and triglycerides (TG) were measured using commercial kits (Applygen technologies Inc. and Beihai biotechnology Inc., Shanghai, China). Livers were collected, fixed in 10% neutral buffered formalin or snap frozen in liquid nitrogen for storage at 80 C until further analysis. Hepatic lipids were extracted as previously described and used to measure TG and TC by specific kits according to colorimetric methods as described. Fixed livers were embedded in paraffin and sliced sections were stained with hematoxylin and eosin (H&E). Frozen liver slides were selected to perform oil red O staining.
2.9. Hepatic VLDL secretion analysis
Hepatic VLDL secretion analysis was performed as previously described (Guan et al., 2011). After a fasting period of 12 h, all mice received 15% (w/v) Triton WR1339 (Sigma-Aldrich) (500 mg/kg body weight in 0.9% NaCl) through retro-orbital injection. Tail blood samples were taken at 30 min intervals and serum TG content was measured using commercial kits as described above. Hepatic secretion rate was calculated from the slope of the curve and expressed as mmol/h. Plasma from each group was collected in heparinized tubes, pooled and centrifuged at d ¼ 1.006 g/ml, 48,000 rpm, 4 C in ultracentrifuge (Beckman OPTIMAL-80XP, Brea, CA) for 6 h. VLDL particles were isolated and delipidated with ethanol/ether (1:1). Precipitates were collected by centrifugation at 1000 g for 15 min. Precipitated apolipoproteins were subjected to electrophoresis on 6% SDS-PAGE for ApoB (Santa cruz sc-11795) western blot analysis as previously described. Mean diameters of VLDL particles were determined using a particle analyzer (Malvern Zetasizer Nano ZS, Worcestershire, UK).
2.10. Statistical analysis
The data shown are means ± SD. Differences between treatment groups were evaluated using two-tailed Student’s t-test. P < 0.05 was considered to be statistically significant.
3. Results
3.1. Overexpression of SHP or activating FXR represses PLA2G12B promoter
We previously demonstrated that PLA2G12B is transcriptionally regulated by HNF-4a (Guan et al., 2011). Since SHP functionally suppresses HNF-4a activity, we first examined if SHP would affect PLA2G12B expression. We utilized a PLA2G12B-luciferase reporter (pGL3-PLA2G12B) harboring a bona fide HNF-4a response element located 1200 bp upstream of the mouse PLA2G12B promoter to investigate whether this promoter region is sufficient to confer responsiveness to SHP. In transiently transfected 293T cells, the expression of pGL3-PLA2G12B luciferase reporter was significantly induced by overexpression of HNF-4a and its coactivator PGC-1a. This HNF-4a/PGC-1a-mediated induction was dramatically suppressed by overexpression of SHP in a dose dependent manner (Fig. 1A), indicating that SHP can functionally suppress the activity of HNF-4a on PLA2G12B promoter. We next tested if elevating the expression level of SHP via activating FXR with either a bile acid CDCA or an FXR specific agonist GW4064 would affect PLA2G12Bluciferase reporter expression. We took advantage of a hepatoma cell line HepG2 with endogenous expression of FXR. CDCA and GW4064 induced the expression of endogenous FXR and SHP in HepG2 cells (Fig. 2B). In HepG2 cells, activation of FXR by these agonists indeed dose-dependently suppressed the HNF-4a/PGC1a-induced PLA2G12B-luciferase reporter (Fig. 1B and C), suggesting that the FXR agonist-induced SHP expression is responsible for the suppression on PLA2G12B.
We then utilized a dominant negative mutant of FXR W469A (Ananthanarayanan et al., 2001; Chen et al., 2003) to further test the role of FXR on PLA2G12B-luciferase reporter. While wild type FXR in the presence of CDCA or GW4064 suppressed PLA2G12Bluciferase reporter expression, the dominant negative mutant FXR W469A actually elevated the expression of PLA2G12B-luciferase reporter in the absence of agonists and negated the agonistsuppressed expression (Fig. 1D). Besides, knocking down the expression of FXR by siRNA blunted the CDCA- or GW4064suppressed PLA2G12B-luciferase reporter expression (Fig. 1E). Collectively, these data strongly implicate FXR having a regulatory role on the expression of PLA2G12B.
3.2. FXR agonists reduce PLA2G12B expression in HepG2
To confirm the transcriptional regulation of FXR on endogenous PLA2G12B, we next treated HepG2 cells for 24 h with different concentrations of CDCA (25 and 100 mM) or GW4064 (2 and 10 mM). Expression levels of certain lipid metabolism-related genes were quantified by real-time PCR. We first determined the effects of FXR agonists on genes involved in lipogenesis. We found that both CDCA and GW4064 reduced mRNA expression levels of SCD-1, FASN, and ACACa (Fig. 2A); whereas, SHP and OSTb were dose-dependently induced (Fig. 2B). Importantly, mRNA expression levels of PLA2G12B and MTP were dose-dependently suppressed by either CDCA or GW4064 (Fig. 2C). To investigate whether endogenous PLA2G12B and MTP protein levels were also reduced by FXR agonists, western blot analyses were performed using HepG2 cells treated with CDCA or GW4064 for 24 h. Consistent with their declined mRNA expression levels, PLA2G12B and MTP protein levels were reduced with the addition of FXR agonists (Fig. 2D). Our results recapitulated the FXR regulation on MTP as previously reported; importantly, we established that another VLDL metabolism-related gene PLA2G12B is also subjected to regulation by FXR.
Since both MTP and PLA2G12B, two essential genes for VLDL production and secretion, are transcriptionally repressed by activation of FXR, we next assessed the impact of FXR agonists on intracellular triglycerides accumulation in vitro. Suppressing VLDL production and secretion is expected to raise intracellular triglycerides level; particularly, in the presence of exogenously supplemented oleic acid (Mahdessian et al., 2014; Ye et al., 2009). Huh7 cells were either transfected with scramble-siRNA or FXR-siRNA and then treated with oleic acid in the presence of FXR agonists. We found that both CDCA and GW4064 trended to elevate intracellular triglycerides levels and this FXR-agonist induced accumulation was blunted by FXR-siRNA (Fig. 3), indicating that VLDL production and secretion is suppressed by activation of FXR.
3.3. FXR ligand CDCA significantly improves hyperlipidemia in HFD mice
We previously generated PLA2G12B-knockout mice and found that PLA2G12B is a novel mediator of hepatic VLDL secretion. The findings that activation of FXR represses PLA2G12B expression prompted us to investigate the relationship between FXR and PLA2G12B in vivo using a hyperlipidemia mouse model. As expected, after high-fat diet (HFD) feeding for 12 weeks, liver TG and TC as well as serum TG, TC and blood glucose levels of HFD-mice increased significantly compared to chow diet group (CD-mice) (Fig. 4A and B). We then treated HFD-mice with CDCA (HFD þ CDCA-mice) at 200 mg/kg for 3 weeks. Data from blood biochemistry analysis revealed that treatment of HFD-mice with CDCA significantly reduced serum TG by 37% and TC by 27% compared to HFD mice on vehicle control (Fig. 4B). Besides, blood glucose was also moderately reduced. Taken together, we recapitulated that activation of FXR by CDCA in HFD-mice lowers blood lipids. To further analyze whether activation of FXR in vivo regulates hepatic lipids levels, liver samples were extracted for TG and TC measurements. Consistent with previous reports (Bilz et al., 2006; Evans et al., 2009; Watanabe et al., 2004), hepatic TG and TC levels of mice were increased by feeding high-fat diet and tended to decrease when CDCA was added (Fig. 4A). Histological analysis of liver samples was performed to further confirm the changes of TG in HFD þ CDCA-mice hepatocytes. Results of H&E and oil red O staining showed that the amount of fat droplets in HFD þ CDCAmice hepatocytes were reduced noticeably compared to HFD-mice, indicating that CDCA treatment reduced neutral lipid accumulation in the liver of HFD þ CDCA-mice (data not shown). These data suggest that administration of CDCA to HFD mice markedly results in a reduction of TG and TC in blood and liver by mechanisms requiring FXR activity.
3.4. CDCA treatment lowers hepatic triglycerides-rich VLDL secretion through decreased PLA2G12B and MTP expression in HFD mice
Hepatic secretion of TG and cholesterol in the form of VLDL-TG is responsible for transporting endogenous lipids into the serum. PLA2G12B and MTP are both critical mediators of VLDL secretion in the liver. We asked if the protective effect of CDCA might be mediated through regulating hepatic PLA2G12B and MTP expression. To test if CDCA treatment lowers hepatic triglycerides-rich VLDL secretion, hepatic TG production in vivo was assessed. After overnight-fasting, mice were treated with Triton WR1339 to prevent breakdown of TG-rich lipoproteins. Accumulation of serum TG was measured over time (Fig. 4C). We found that the secretion rate of serum VLDL-triglycerides was enhanced by 39% in HFD mice compared to control mice and lowered 17% by CDCA treatment (Fig. 4D). Since each VLDL particle contains one molecule of ApoB, we found that both ApoB100 and ApoB48 within the VLDL fraction were increased in mice after high fat diet (Fig. 4E); however, the amounts of ApoB100 and ApoB48 were reduced significantly in HFD þ CDCA-mice by western blot analysis (Fig. 4E). Consistently, after treatment with CDCA, the average diameter of HFD þ CDCAmice serum VLDL particle was moderately decreased compared to HFD group (Fig. 4F), indicating less lipid packaged into the VLDL particles. Parallel to the reduction of hepatic VLDL production, we found that treatment of CDCA markedly reduced the hepatic expression levels of PLA2G12B as well as MTP of HFD þ CDCA-mice (Fig. 4G). In all, our data suggested that CDCA treatment lowers hepatic triglycerides-rich VLDL secretion through decreasing both PLA2G12B and MTP expression in hyperlipidemic mice.
4. Discussion
Bile acids have emerged as key regulators of metabolism via membrane receptors such as GPBAR1 and nuclear receptors such as FXR (Porez et al., 2012; Trabelsi et al., 2015). FXR activation can improve hepatic insulin sensitivity and decrease hepatosteatosis by inhibiting lipogenesis (Cipriani et al., 2010; Fiorucci et al., 2010). Activation of FXR by agonists (CDCA, GW4064 or WAY-362450) also significantly reduced blood triglycerides, cholesterol and glucose levels in wild-type mice, diabetic db/db mice, KK-Ay and highfructose diet hamsters but not in FXR knockout mice respectively (Bilz et al., 2006; Evans et al., 2009; Watanabe et al., 2004; Zhang et al., 2006), establishing the importance of FXR in lipid metabolism. Besides these evidence from animal models, studies in patients with gallstones found that CDCA treatment resulted in decreased blood TG levels and VLDL production (Schoenfield and Lachin, 1981).
These FXR-mediated beneficial effects may be mediated by decreased hepatic lipid synthesis and enhanced peripheral clearance of VLDL (Cariou, 2008). Previous studies focused upon the effect of CDCA treatment on hepatic de novo lipogenesis. Our data as well as others highlighted the repression of SCD-1, FASN and ACACa genes in HepG2 cells upon FXR activation. A reduction in hepatic VLDL production and secretion by FXR activation could also underlie the mechanism by which CDCA reduces serum lipids. In our current study, we focused on the impact of FXR activation on VLDL production and secretion from the liver. HNF-4a is a key physiological PLA2G12B transcriptional regulator and that PLA2G12B is a novel mediator of triglyceride metabolism in the liver (Guan et al., 2011; Li et al., 2014). We used both an endogenous ligand CDCA and a synthetic FXR-specific agonist GW4064 to demonstrate that activation of FXR inhibits PLA2G12B and MTP expression. Specifically, PLA2G12B was transcriptionally repressed via FXR mediated up-regulation of SHP which interferes with HNF-4a and PGC-1a. Concordantly, hepatic VLDL secretion was decreased by CDCA treatment in part through down-regulation of PLA2G12B expression in the liver. Our current data provides further experimental evidence that CDCA treatment reduces VLDL-TG secretion rate; namely, FXR activation reduces VLDL particle number and size with less TG packaged-VLDL secretion from the liver, resulting in lipidlowering in HFD fed mice.
Our analysis points to a regulation in which bile acids through FXR induces SHP to affect hepatic PLA2G12B expression. In parallel to a similar FXR-SHP regulation on MTP, FXR and SHP coordinately regulate two HNF-4a target genes that are rate limiting for VLDL production and secretion (Hirokane et al., 2004). This FXR-SHPMTP/PLA2G12B regulatory cascade can also explain a number of previous results linking VLDL secretion to TG levels in the serum of FXR or SHP knockout mice. FXR knockout mice show increased serum TG levels and FXR agonist GW4046 or CDCA decreased serum TG in several animal models. However, this effect was compromised in SHP knockout mice (Watanabe et al., 2004). Importantly, mutations in the SHP gene in Japanese patients lead to hypertriglyceridemia which may be resulted from a failure to suppress VLDL production and secretion (Nishigori et al., 2001). Collectively, we conclude that SHP-attenuated VLDL secretion via suppressing MTP/PLA2G12B expression can contribute to inhibitory effects of FXR agonists on lowing serum TG.
Intriguingly, several recent studies reported that intestinal FXR signaling is involved in metabolic regulation (Fang et al., 2015; Jiang et al., 2015; Trabelsi et al., 2015). Interestingly, MTP and PLA2G12B are both expressed in the intestine which might be targets of gutbiased FXR modulation (Iqbal et al., 2014; Mera et al., 2015). Since bile acids are not suitable for long term medical use in humans because of severe hepatotoxicity (Song et al., 2011), selective FXR modulators with gut-targeting selectivity and improved safety profiles are suggested to be options as treatments of metabolic diseases (Fang et al., 2015). We recently identified from traditional Chinese remedies natural compounds such as oleanolic acid and andrographolide that can function as modulators of FXR, highlighting their potentials for treating metabolic syndrome (Liu and Wong, 2010; Liu et al., 2014). It would be informative if said natural compounds with poor bioavailability are indeed gut-targeting FXR modulators that impact on the FXR-SHP-MTP/PLA2G12B regulatory cascade to affect lipid metabolism.
5. Conclusion
Our data show that down-regulating PLA2G12B expression is part of the regulation by which activation of FXR suppresses hepatic VLDL-TG secretion. Our results also raise a question whether potent FXR agonists would suppress hepatic VLDL-TG secretion potentially contributing to fatty liver development.
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