Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c (original) (raw)
Cholic acid lowers serum TGs in KK-Ay mice. To study the TG-lowering effect of bile acids in vivo, we first explored the effect of feeding CA on TG homeostasis in KK-Ay mice. KK is an inbred strain that develops type 2 diabetes mellitus with only mild obesity, even after maturity (27). Introduction of the lethal yellow mutation in the agouti gene (Ay) (28) causes overt type 2 diabetes and massive obesity with a relatively late onset (29). These KK-Ay mice are characterized by a severe prolonged hyperinsulinemia, hyperglycemia, and hyperlipidemia. Under basal conditions the elevation of TGs is only moderate (192 ± 16 mg/dl), but can be further increased by a high-fat diet (273 ± 14 mg/dl, Figure 1A). This elevation of serum TG levels on a high-fat diet is not observed in normal C57BL/6J mice (data not shown and ref. 30), making the KK-Ay mice a unique model to study diet-induced hypertriglyceridemia. In addition, the elevation of serum TG is progessive and dependent on the age of the KK-Ay mice. At 7 weeks of age the hypertriglyceridemia is moderate (192 ± 16 mg/dl, Figure 1A), whereas at 12 weeks of age, it is severe (453 ± 24 mg/dl, Figure 1C).
CA lowers serum TGs in KK-Ay mice. (A) Food intake of KK-Ay mice during 1 week on the diets as described in the figure. Serum levels of TGs, total cholesterol (Chol), and FFA in KK-Ay mice after 1 week on the different diets (age 7 weeks, n = 5). Kcal, kilocalories; BW, body weight. (B) TG and cholesterol lipoprotein profiles after size-exclusion chromatography of serum pools from five animals. The quantification of the VLDL TGs is shown in an inset. (C) Serum TGs in KK-Ay (top, age 12 weeks, n = 4) and ob/ob (bottom, age 12 weeks, n = 4) mice after 0, 1, 3, and 7 days of treatment with the synthetic FXR agonist GW4064. *P < 0.05; **P < 0.01 throughout the figures.
Male KK-Ay mice were given normal chow or high-fat diets with or without 0.5% CA for 1 week. The CA-containing diets were well tolerated, and food intake was not affected during a 1-week (Figure 1A) and a 3-week study (not shown). Interestingly, CA robustly lowered circulating TG levels in both KK-Ay mice on a normal-chow (51%) and on a high-fat diet (65%, Figure 1A). Total plasma cholesterol was also decreased (24% on a normal-chow diet and 35% on a high-fat diet, Figure 1A). CA feeding decreased plasma FFAs, particularly in mice on the high-fat diet (Figure 1A). High-performance liquid chromatography analysis of lipoproteins demonstrated that CA feeding decreased plasma TGs primarily by decreasing VLDL TGs, and decreased cholesterol primarily by decreasing HDL cholesterol. CA feeding increased LDL cholesterol levels (Figure 1B).
To verify whether the observed effects of CA were mediated through FXR, we treated chow-fed KK-Ay and ob/ob mice during 1 week with the synthetic FXR agonist GW4064. GW4064 potently lowered serum TG levels in both the KK-Ay and ob/ob mice (Figure 1C), suggesting that the observed effects are mediated by FXR. In addition, this experiment shows that the TG-lowering effect of FXR activation is not restricted to KK-Ay mice, a model for diet-induced hypertriglyceridemia, but also occurs in another model of hypertriglyceridemia, the ob/ob mouse.
CA lowers hepatic TG levels. Administration of CA to KK-Ay mice for 3 weeks also changed liver morphology. Livers of chow-fed KK-Ay mice have a pale color, suggestive of increased lipid storage. Livers of animals that were treated with CA were less pale and had a more normal reddish appearance (Figure 2A). This effect of bile acids on hepatosteatosis was more pronounced in mice fed high-fat diets, which showed an impressive return to a normal morphology. H&E-stained sections of the livers of animals on the high-fat diet with CA showed much lower levels of unstained inclusions (Figure 2A). Staining of these liver sections with Oil Red O (Sigma-Aldrich) demonstrated that CA-treated animals accumulated less neutral lipids, an effect coherent with both gross morphological appearance and H&E staining (Figure 2A). Consistent with the morphological appearance, livers of CA-treated animals contained significantly lower amounts of TGs in animals on chow or high-fat diet (Figure 2B). In contrast, CA feeding increased hepatic cholesterol, particularly in the animals fed high-fat diets (Figure 2C).
CA lowers hepatic TGs in KK-Ay mice. (A) Liver morphology, H&E-stained liver sections (HE), and Oil Red O_stained liver sections (OR) in animals treated with the indicated diets for 3 weeks. When sacrificed, the mice were 9 weeks old. (B) Liver TG and (C) cholesterol content after 3 weeks on the different diets (age 9 weeks, n = 5). (D) In vivo VLDL secretion from the livers of mice on the different diets (age 7 weeks, n = 4) or after treatment with GW4064 (age 12 weeks, n = 4). C, control diet; C + CA, control diet with CA; HF, high-fat diet; HF + CA, high-fat diet with CA.
To assess whether the decrease in liver TG content is associated with decreased export, we measured VLDL production in KK-Ay mice after 1 week of CA feeding. CA feeding significantly decreased liver VLDL production on both a chow and high-fat diet (Figure 2D). In a similar experiment, a 1-week administration of GW4064 also significantly lowered VLDL secretion in KK-Ay mice (Figure 2D), again suggesting that this effect is mediated via FXR.
CA decreases expression of SREBP-1c and other lipogenic genes. To better understand the molecular mechanism underlying the TG-lowering effect of bile acids, we used quantitative RT-PCR to measure hepatic mRNA levels of several important proteins involved in lipid homeostasis in KK-Ay and C57BL/6J mice after 1 and 7 days of CA supplementation of a chow diet (Table 1). No major changes were observed in the expression of several transcription factors involved in liver lipid and bile acid homeostasis, such as LXRα, LRH-1 (NR5A2), FXR, and SREBP-2. Expression of the LDL receptor (LDL-R) and genes involved in cholesterol biosynthesis had a tendency to decrease, whereas genes involved in encoding cholesterol transport proteins (ABCA1 and ABCG5) were slightly increased, although these differences never reached statistical significance. Expression of genes encoding enzymes involved in fatty acid and TG biosynthesis, such as AceCS, ME, and SCD-1, was significantly reduced by CA feeding. This reduction is strongest after 1 day of treatment and is mitigated somewhat after 7 days of treatment. To verify whether an increase in fatty acid β-oxidation contributed to the TG-lowering effects in our studies, we measured the hepatic expression levels of liver carnitine palmitoyltransferase I (CPT-I), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD). In C57BL/6J mice, the expression of MCAD and LCAD was significantly decreased after 1 and 7 days of treatment with CA. Interestingly, in KK-Ay mice there was no change in the expression of these genes. Thus increased expression of genes involved in β-oxidation of fatty acids is not responsible for the decrease in serum TG. Aside from these differences in genes involved in β-oxidation, no major differences were observed between the results obtained in KK-Ay and C57BL/6J mice.
CA decreases expression of SREBP-1c and other lipogenic genes
Interestingly, the changes in expression of genes involved in lipogenesis were paralleled by changes in SREBP-1c expression (Table 1). Furthermore, expression of SHP showed an opposite pattern, with a robust induction upon treatment with CA. SHP is an FXR target gene that represses the activity of several nuclear receptors, including LRH-1 and LXR, which are essential for the transcription of cholesterol 7α-hydroxylase (CYP7A1; see Table 1), the rate-limiting enzyme in bile acid biosynthesis. This FXR-mediated SHP induction underlies the negative-feedback regulation of bile acid biosynthesis (17, 18, 23, 31–33). The effect of CA on lipogenesis seems to be specific for the liver, since in white adipose tissue, none of the lipogenic genes were lowered in expression in response to CA treatment (data not shown).
The activity of the SREBP-1c promoter is attenuated by bile acids and SHP. We examined the ability of CDCA to lower expression of endogenous SREBP-1c and its target genes in vitro in mouse primary hepatocytes. The SREBP-1c promoter had previously been shown to be regulated by LXR, an effect that contributes to the TG-raising activity of LXR agonists (10, 11). We confirmed the induction of endogenous SREBP-1c expression by RXR and LXR ligands and showed that this expression decreased dose-dependently by increasing amounts of CDCA. The expression of the lipogenic target genes of SREBP-1c decreased likewise (Figure 3A). This reduction was most robust for SCD-1, but expression of AceCS and ME was also reduced. SHP expression was increased by the addition of CDCA. These results demonstrate that, both in vivo and in vitro, the expression of endogenous SREBP-1c, as well as the lipogenic enzymes that are regulated by SREBP-1c, is significantly affected by bile acids.
Bile acids and SHP decrease expression from the SREBP-1c promoter. (A) Expression of SREBP-1c and several of its target genes in mouse liver primary hepatocyte cultures. The presence of ligands for LXR (22(R)-hydroxycholesterol, 20 µM) and RXR (LG100268, 1 µM) is indicated by a + sign. FXR was activated by the addition of CDCA to the medium (50 µM and 200 µM). (B) Activity of the mouse SREBP-1c promoter in the McA-RH7777 cell line after the addition of 200 µM CDCA or 200 µM CA to the medium. Cells were tested in the absence or presence of cotransfected LXRα and ligands for RXR and LXR at the concentrations specified in A. (C) Schematic representation of the different constructs of the mouse SREBP-1c promoter used in transfection assays. Binding sites for LXR are displayed as ovals. The nucleotide numbering is relative to the SREBP-1c start codon. (D) Sequence comparison of the LXRREs in the human and mouse SREBP-1c promoters. The GenBank accession numbers for the human SREBP-1c promoter sequence are NT_010718 or AC122129. (E) Activity of the mSREBP-1c reporters in McA-RH7777 cells transfected either with an empty expression vector or with the indicated combinations of expression vectors for mouse LRH-1, mouse RXRα, human LXRα, mouse SHP in the presence (black bars) or absence (white bars) of LXR (22(R)-hydroxycholesterol; 20 µM) and RXR agonists (LG100268; 1 µM).
To examine this potential role of bile acids in the regulation of SREBP-1c expression, we cloned the mouse SREBP-1c promoter and generated a luciferase reporter construct (S1, Figure 3C). McA-RH7777 rat hepatoma cells were cotransfected with this construct and a LXRα expression vector and treated with LXR and RXR agonists in the presence or absence of CA or CDCA. SREBP-1c promoter activity was induced by transfection of LXRα and/or the addition of its ligands. This increase was attenuated when cells were incubated with either of the bile acids (Figure 3B). Furthermore, induction of SREBP-1c promoter activity by LXR and RXR agonists does not depend on the overexpression of LXR, showing that McA-RH7777 cells have endogenous LXR activity.
The effect of CA on the expression of lipogenic genes, as well as the opposite changes in gene expression of SREBP-1c and SHP, prompted us to analyze the contribution of the FXR-SHP cascade to the regulation of SREBP-1c gene expression. We hence generated a number of reporter constructs containing 5′ nested deletions (S1–S5, Figure 3C) or point mutations in the two previously characterized LXR response elements (LXRREs; S6, Figure 3, C and D). As expected, SREBP-1c promoter activity is induced by overexpression of the RXR/LXRα heterodimer in rat hepatoma McA-RH7777 cells in the presence of a natural LXR agonist and a synthetic RXR ligand (10, 11, 34). When the LXRRE sites are mutated or deleted, basal activity of the promoter is markedly reduced (S5 and S6, Figure 3E). It is probable that this effect is conserved between mice and humans, since SREBP-1c expression is also induced by an LXR agonist in human hepatoma cells and primary hepatocytes (34, 35). In addition, the two LXRREs are highly conserved between the mouse and the human SREBP-1c promoter (Figure 3D). Transfection of LRH-1 induced SREBP-1c promoter activity and improved the magnitude of induction by ligand-activated LXR, suggesting that LRH-1 acts as a competence factor for LXR as was reported for a number of genes (17, 18, 36) (Figure 3E). The induction of the SREBP-1c promoter by LRH-1 was lost after deletion of LXRREa (S4, Figure 3E). The finding that the promoter region between –327 and –276 contained no consensus LRH-1 response element (LRH-1RE) suggested that the induction might be mediated via LXRREa. Mutation of the 5′ extension of this LXRRE, which does not disrupt the LXRRE consensus sequence itself, did lead to the loss of response to LRH-1 (S7, Figure 3E), which is in line with the suggestion that the LXRREa might mediate the response of SREBP-1c to LRH-1. Interestingly, cotransfection with SHP potently attenuated the induction of the SREBP-1c reporter in the presence of LRH-1, LXR, and RXR (Figure 3E). From this we conclude that SHP regulates the SREBP-1c promoter. Due to technical problems (the loss of basal promoter activity after the mutagenesis of the LXRREs), we cannot attribute this effect with certainty to a particular site in the promoter.
CA attenuates LXR agonist-induced lipogenesis in vivo. To study the inhibition of the SREBP-1c promoter by CA in more detail, we fed C57BL/6J mice chow or chow supplemented with 0.5% CA for 1 day. Mice were gavaged with the LXR agonist T0901317 or vehicle, and the different diets were continued for one more day, after which the mice were sacrificed. We confirmed the previously observed induction of liver weight and serum TGs by LXR agonists on a chow diet (10, 11, 34). Coadministration of CA completely prevented these effects (Figure 4A). As expected, both LXR (SREBP-1c, CYP7A1, ABCA1, ABCG5, ABCG8, and ANGPTL3) and SREBP-1c (ME, ACC1, and ACC2) target genes were induced by administration of the LXR ligand. CA coadministration increased SHP expression and prevented the induction of SREBP-1c and its target genes. Interestingly, some LXR target genes were downregulated by CA (SREBP-1c, CYP7A1, and ANGPTL3), whereas others (ABCA1, ABCG5, and ABCG8) were not (Figure 4B). This led us to conclude that not all LXR target genes are responsive to inhibition by SHP, suggesting that besides LXR there might be additional factor(s) targeted by CA to explain their efficient downregulation.
CA attenuates LXR agonist_induced lipogenesis in vivo. (A) Liver weight and serum TGs after feeding a CA-containing diet and coadministration of the LXR agonist T0901317 (age 11 weeks, n = 6). (B) Hepatic expression levels of SREBP-1c, CYP7A1, ME, ACC1, ACC2, ABCA1, ABCG5, ABCG8, ANGPTL3, and SHP as determined using quantitative RT-PCR (n = 4).
Attenuation of the TG-lowering effects of FXR agonists in SHP–/– mice. To critically test the role of SHP in the lowering of TG biosynthesis, we administered either a diet containing 0.5% CA or the synthetic FXR agonist GW4064 to wild-type and SHP-null mice and measured serum TGs after 0, 3, and 7 days. CA and GW4064 significantly lowered serum TGs in the SHP+/+ mice. CA seemed more potent in decreasing serum TGs, an effect that may be attributed to the poor pharmacokinetic profile of GW4064. More importantly, this decrease in serum TGs was completely abolished in the SHP–/– mice (Figure 5A). To determine whether this attenuation of the TG-lowering effects of FXR agonists in SHP–/– mice is paralleled by similar changes at the molecular level, we measured the hepatic expression of SREBP-1c, ME, CYP7A1, and ANGPTL3 in animals sacrificed after a 1-day treatment with the different FXR agonists. Expression of all four genes was significantly reduced in the SHP+/+ mice that received CA or GW4064 (Figure 5B). This is in sharp contrast with the results obtained in the SHP–/– mice, in which no decrease in this expression was detected (Figure 5B). The attenuation of CYP7A1 downregulation in SHP–/– mice upon FXR agonist administration is in apparent contrast with earlier studies (23). This is probably a reflection of the differences in experimental approach (1 day vs. 7 days and 0.5% CA vs. 1% CA treatment) that favored activation of secondary regulatory pathways in the earlier studies (23). These data further support that SHP plays an essential role in the SREBP-1c–mediated downregulation of lipogenesis in mice treated with bile acids.
SHP is essential for bile acid_mediated downregulation of lipogenesis. (A) SHP_/_ and SHP+/+ mice were fed a diet containing 0.5% CA or received GW4064 by oral gavage, and serum TG levels were measured after 0, 3, and 7 days (age 9 weeks, n = 5). (B) In two separate experiments, mice received CA or GW4064 as just described and were sacrificed after 1 day. Hepatic expression levels of SREBP-1c, CYP7A1, ME, and ANGPTL3 were determined using quantitative RT-PCR (age 9 weeks, n = 5).
LXR is essential for SHP-mediated lowering of TGs. To determine whether LXR is essential for the TG-lowering effect of bile acids, we used LXR_α/LXR_β double-knockout mice (LXR_α/β_–/–). Wild-type and LXR_α/β_–/– mice were fed with a diet containing 0.5% CA for 3 days, after which we measured serum TGs (Figure 6A) and hepatic gene expression of SREBP-1c, ME, CYP7A1, and SHP (Figure 6B). As a result of the loss of LXR transactivation in the LXR_α/β_–/– mice, the basal level of expression of the LXR (SREBP-1c and CYP7A1) and SREBP-1c (ME) target genes is significantly reduced, causing a lower basal serum TG level (Figure 6, A and B). In wild-type animals, CA treatment decreased serum TG subsequent to a decrease in the expression SREBP-1c and ME. The decrease in serum TGs, SREBP-1c, and ME is not observed in the LXR_α/β_–/– mice (Figure 6, A and B). Though less pronounced in LXR_α/β_–/– mice, SHP was induced significantly in both wild-type and LXR_α/β_–/– animals. These experiments demonstrate that LXR is essential for the SHP-mediated lowering of TGs in vivo.
LXR is essential for SHP-mediated lowering of TGs. (A) Serum TGs in wild-type and LXR_α/β_/__ mice after treatment with 0.5% CA (age 10_14 weeks, n = 6). (B) Hepatic expression levels of SREBP-1c, CYP7A1, ME, and SHP in wild-type and LXR_α/β_/__ mice after treatment with CA as determined using quantitative RT-PCR. # denotes P = 0.09 (n = 6). (C) A FXR-SHP-SREBP-1c regulatory cascade. Schematic representation of the proposed role of SHP in mediating the effects of FXR agonists on SREBP-1c expression and lipogenesis.