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Am J Physiol Endocrinol Metab 290: E716-E722, 2006. First published November 15, 2005; doi:10.1152/ajpendo.00355.2005
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Activation of the farnesoid X receptor improves lipid metabolism in combined hyperlipidemic hamsters

Stefan Bilz,1 Varman Samuel,1 Katsutaro Morino,1,3 David Savage,1 Cheol Soo Choi,1 and Gerald I. Shulman1,2,3

1Departments of Internal Medicine and 2Cellular and Molecular Physiology and the 3Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut

Submitted 2 August 2005 ; accepted in final form 9 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The transcription factor farnesoid X receptor (FXR) has recently been implicated in the control of hepatic triglyceride production. Activation of FXR may ameliorate hypertriglyceridemia, a cardinal feature of the metabolic syndrome. Because hamsters share many characteristic features of human lipid metabolism, we used a high-fructose-fed hamster model to study the impact of FXR activation with chenodeoxycholic acid (CDCA) on plasma lipoprotein metabolism. Male Syrian hamsters fed a diet containing 60% kcal from fructose for 2 wk developed hypertriglyceridemia and hypercholesterolemia (+120 and +60%, P = 0.005 and 0.0004 vs. controls) due to increased hepatic lipoprotein production. This could be largely attributed to enhanced hepatic de novo lipogenesis, as indicated by increased expression of sterol regulatory element-binding protein-1, fatty acid synthase, and steaoryl-CoA desaturase-1. Lipoprotein analysis demonstrated that the increase in plasma triglycerides occurred in the VLDL density range, whereas increases in VLDL, IDL/LDL, and HDL cholesterol accounted for the elevated plasma cholesterol concentrations. Addition of 0.1% CDCA to the high-fructose diet decreased hepatic de novo lipogenesis and consequently triglyceride production and prevented the increases in plasma triglycerides and cholesterol (–40 and –18%, P = 0.03 and 0.03 vs. high fructose-fed animals). CDCA-treated animals had lower VLDL triglycerides and decreased VLDL and IDL/LDL cholesterol plasma concentrations. These data demonstrate that activation of FXR with CDCA effectively lowers plasma triglyceride and cholesterol concentrations, mainly by decreasing de novo lipogenesis and hepatic secretion of triglyceride-rich lipoproteins. Our studies identify activators of FXR as promising new tools in the therapy of hypertriglyceridemic states, including the insulin resistance syndrome and type 2 diabetes.

chenodeoxycholic acid; triglycerides; lipogenesis

THE FARNESOID X RECEPTOR (FXR, NR1H4) belongs to the family of ligand-activated transcription factors and has recently been identified as a bile acid receptor (19). Activation of the receptor by its natural ligands results in a decrease in endogenous bile acid synthesis from cholesterol through interference with cholesterol 7{alpha}-hydroxylase (Cyp7A1). This negative feedback loop involves other nuclear receptors, among them liver X receptor (LXR), which is a transcriptional regulator of Cyp7A1 (13). Interestingly, a connection between bile acid, cholesterol, and triglyceride metabolism was first noted almost 30 years ago, long before the description of this pathway, when patients with familial hypertriglyceridemia (FHTG) were found to have a defect in ileal bile acid absorption (3). It has also been noted that cholesterol-lowering therapy with bile acid sequestrants, which reduce bile acid flux to the liver, increase serum triglycerides secondarily to hepatic overproduction of very-low-density (VLDL) lipoproteins (2). Thus it was not surprising when liver steatosis and hypertriglyceridemia were described in mice lacking FXR, suggesting that decreased activity of FXR might enhance triglyceride production (30). The associated findings that LXR, which may be regulated by FXR activation, is a strong transcriptional activator of sterol regulatory element-binding protein-1c (Srebp-1c), the major regulator of de novo fatty acid synthesis from carbohydrate precursors, added further evidence for a potentially important role of FXR in the regulation of hepatic triglyceride metabolism (6, 28). A physiological role for FXR in the regulation of triglyceride metabolism is further supported by the observation that the decrease in plasma triglycerides induced by fasting in wild-type mice was blunted in FXR–/– mice (40). Finally, the finding that oral chenodeoxycholic acid (CDCA), a primary bile acid and potent activator of FXR in vitro, lowered plasma triglyceride concentrations and decreased VLDL production in humans indicates that FXR regulates triglyceride metabolism in vivo in various species, and this may, at least in part, result from interference with de novo hepatic lipogenesis (4, 18, 21).

Until recently, de novo lipogenesis has not been regarded as a quantitatively important contributor to the hepatic triglyceride pool, which is a key regulator of hepatic VLDL secretion (9). It has been recognized, however, that insulin-mediated de novo lipogenesis is significantly increased in otherwise insulin-resistant animal models and humans, thereby establishing a vicious cycle of mixed hepatic insulin resistance (9, 12, 29).

To test the hypothesis that activation of FXR decreases hepatic triglyceride production through decreased de novo lipogenesis we treated high-fructose-fed Golden Syrian hamsters with CDCA. This hamster model has previously been shown to develop hypertriglyceridemia and insulin resistance in response to high-fructose feeding (33, 34). Importantly, hamsters are unique and distinctly different from other rodents regarding their hepatic lipid metabolism. Unlike mice and rats, hamsters readily develop hypertriglyceridemia when exposed to high-fat or high-carbohydrate diets, and significant amounts of both cholesterol and triglycerides are carried in lipoproteins of the VLDL and LDL density range. Furthermore, hepatically secreted VLDL contain apolipoprotein (apo)B-100 but not apoB-48 as the main structural apolipoprotein. These features closely resemble human lipoprotein metabolism and make hamsters an attractive model in which to study lipid metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and diet. Male Golden Syrian hamsters weighing 80–90 g were obtained form Charles River and singly caged under controlled temperature (23°C) and lighting (12 h of light, 0700–1900; 12 h of dark, 1900–0700) upon arrival with free access to water and a pelleted standard rodent chow (Teklad 2018S, with 6% calories from fat, 18% calories from protein; Harlan-Teklad, Madison, WI). After being acclimatized for ≥1 wk, animals were allocated to three groups of 8–10 animals: the control group (CON) remained on the standard rodent chow (9), the high-fructose group (HF) received pellets containing 60% calories from fructose (Dyets, Bethlehem, PA), and the third group received the same pelleted HF diet supplemented with 0.1% (wt/wt) CDCA (kindly provided by Dr. Ralph Müller, Falk Pharmaceutical, Freiburg, Germany) corresponding to ~70 mg/kg body wt based on animal weight and food intake (HF + CDCA). Body weight and food intake were assessed every other day. All diets were fed for 2 wk before experiments were performed. All procedures were approved by the Yale University Institutional Animal Care and Use Committee.

Blood and tissue samples. All animals were fasted from 6 PM the day before experiments, and all experiments were performed between 8 and 11 AM the next day. At that time, animals were anesthesized with 4% isoflurane and bled from the retroorbital sinus into tubes containing Na2EDTA (pH 7.4, final concentration 1.2 mg/ml). Tetrahydrolipstatin (THL) was added at a final concentration of 1 µg/ml to inhibit plasma lipases to prevent in vitro breakdown of VLDL triglycerides. A THL stock solution was prepared by dissolving the content of one capsule of Xenical (Hoffmann-La Roche, Nutley, NJ) containing 120 mg of THL in 96% alcohol. Nonsoluble contents were removed by centrifugation. Immediately after bleeding, a small piece was cut from the liver, rinsed with PBS, and stored in RNAlater (Qiagen, Valencia, CA) at –20°C. Thereafter, the liver was removed, rinsed with PBS, weighed, freeze-clamped in liquid nitrogen, and stored at –80°C until further analysis. Plasma glucose was measured on a Beckman Glucose Analyzer II (Beckman, Fullerton, CA) using a glucose oxidase method. Triglycerides, cholesterol, and free fatty acids were measured using colorimetric kits (Diagnostic Chemicals, Oxford, CT; Wako Chemicals, Richmond, VA).

Insulin sensitivity. Insulin sensitivity was calculated from fasting glucose and insulin concentrations by use of the homeostasis model assessment (HOMA): HOMA = glucose (mmol/l) x insulin (µU/ml)/22.5 (20).

Liver triglyceride and cholesterol content. Liver lipids were extracted as described previously (27). Briefly, 2 ml of ice-cold chloroform-methanol (2:1) were added to ~200 mg of powdered liver, homogenized, and gently shaken at room temperature for 3 h. After addition of 200 µl of 1 M H2SO4 and centrifugation at 2,000 rpm for 10 min, the aqueous phase was removed and the organic phase aspirated through the protein disk, dried under a stream of nitrogen, and resuspended in chloroform. Triglyceride and cholesterol concentrations were then measured from dried aliquots as described above.

In vivo hepatic triglyceride production. For the measurement of in vivo hepatic triglyceride production, animals fed the control chow (CON, n = 6), HF diet (n = 5), and HF + CDCA diet (n = 6) had intravenous lines inserted into the right jugular vein in the afternoon before the experiment under sterile conditions. Anesthesia was performed with isoflurane (100% oxygen with 4% isoflurane for anesthesia induction and 1.5–2.5% for maintenance of anesthesia throughout the procedure). Lines were filled with a 1% citrate solution overnight to prevent clotting. At 8 AM, hepatic triglyceride production was measured following the accumulation of triglycerides after the intravenous injection (500 mg/kg body wt) of a 20% solution (wt/vol) of tyloxapol (Sigma) in normal saline (24, 33). Blood samples were taken at 0, 60, and 120 min, and hepatic triglyceride production was estimated from the slope of the increase of plasma triglyceride concentrations. Hepatic triglyceride production was calculated from the difference in triglyceride pool sizes at baseline and after 120 min. Pool sizes were estimated as the product of plasma triglyceride concentrations and plasma volume, which was assumed to be 3.8 ml/100 g body wt (33).

Lipoprotein analysis by gel filtration chromatography. To determine the effects of high-fructose feeding and CDCA on plasma lipoprotein distribution, pooled plasma samples from five to six animals were separated by gel filtration chromatography using two superose 6 HR 10/30 columns in series (Akta FPLC System; Amersham Biosciences, Piscataway, NJ). After equilibration of the columns with 0.15 M NaCl containing 1 mM Na2EDTA, 0.2 ml of plasma filtered through a 0.22-µm PVDF filter were injected onto the columns and eluted at a rate of 0.3 ml/min, and 50 0.5-ml fractions were collected. An aliquot of each fraction was transferred onto a 96-well plate, and triglyceride and cholesterol concentrations were measured using colorimetric kits, as described above, using a plate reader. This method readily separates lipoproteins of the VLDL, intermediate- and low-density lipoprotein (IDL and LDL), and HDL range.

Hepatic gene expression. mRNA levels of genes involved in hepatic lipid metabolism were analyzed by quantitative RT-PCR. Total RNA was isolated from liver with an RNeasy kit (Qiagen) with an on-column DNAse digestion step. cDNA was prepared from 1 µg of RNA with the StrataScript RT-PCR kit (Stratagene, La Jolla, CA) with random primers according to the manufacturer's instructions. The resulting cDNA was diluted, and 1.8-ng aliquots were used in 40-µl PCR reactions using the Brilliant SYBR Green QPCR Master Mix (Stratagene). PCR reactions were run in duplicate and quantitated using the Opticon Real Time PCR Detection System (MJ Research, Waltham, MA). The following primer pairs were designed using an online version of primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi): 18S rRNA (18S, NCBI acc. no. M11188 [GenBank] ): forward 5'-TTCCGATAACGAACGAGACTC T-3', reverse 5'-TGGCTGAACGCCACTTGTC-3'; Srebp-1 (U09103 [GenBank] ): forward 5'-GCACTTTTTGACACGTTTCTTC-3', reverse 5'-CTGTACAGGCTCTCCTGTGG-3'; fatty acid synthase (Fas, AF356086 [GenBank] ): forward 5'-GTGCTAGTGTCAACAAGCAATG-3', reverse 5'-TTCAGGGTACCACTGTATTTGG-3'; steaoryl-CoA desaturase 1 (Scd-1, NM139192): forward 5'-CGTCAGCACCTTCTTGAGATAC-3', reverse 5'-TCACTGGCAGAGTAGTCGTAGG-3'; apoB (M35187 [GenBank] ): forward 5'-TGCTCCCATTTTAACAATGAAC-3', reverse 5'-TAAACTTGTGGTCAACACCTCC-3'; microsomal triglyceride transfer protein (Mtp, U14995 [GenBank] ): forward 5'-ATTCAGCTGCAATCTGGACTAA-3', reverse 5'-ACGAAAGAAGAGTCCACTGTGA-3'; apoC-III (AF356088 [GenBank] ): forward5'-TAATGAGGTAGAGGGGTCCTTG-3', reverse 5'-GTAGCCTTTCAGGGAGGTGA-3'; Srebp-2 (U12330 [GenBank] ): forward 5'-GAGAGCTGTGAATTTTCCAGTG-3', reverse 5'-CTACAGATGATATCCGGACCAA-3'; LDL receptor (LDL-R, M94387 [GenBank] ): forward 5'-CTCCACTCTATCTCCAGCATTG-3', reverse 5'-TTTCAGCCACCAAATTAACATC-3'; Cyp7A1 (L04690 [GenBank] ): forward 5'-AAGACGACATCATCGCTCTTTA-3', reverse 5'-ACAATCCCTATGGCTTCACTTT-3'; hydroxymethylglutaryl-CoA reductase (Hmgcr, M12705 [GenBank] ): forward 5'-GACGGTGACACTTACCATCTGT-3', reverse 5'-CACTGCTCAATACGTCCTCTTC-3'. Normalized gene expressions (NEs) were calculated from pairwise comparisons of the threshold cycles (CTs) of a reference gene (18S rRNA) with the CTs of the target gene according to the formula NE = (Ereference exp CT reference)/(Etarget exp CT target), where Ereference and Etarget are the primer efficiencies and CT reference and CT target the threshold cycles of the reference gene (18S rRNA) and target genes, respectively (22).

Data analysis and statistics. All data are expressed as means ± SE. Student's t-tests using the JMP-IN 4.0.4 statistical software package (SAS Institute, Cary, NC) were used to evaluate the significance of the differences of means between animals fed the CON, HF, and HF + CDCA diets.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Baseline characteristics and metabolic parameters. Baseline weight, weight gain, and caloric intake throughout the study period were comparable between CON, HF, and HF + CDCA hamsters (Table 1), and all diets were well tolerated. Feeding the high-fructose diet resulted in significant increases in fasting plasma triglyceride (+120%, P < 0.01), cholesterol (+60%, P < 0.001), and plasma free fatty acid (+61%, P < 0.05) concentrations. Plasma glucose concentrations remained unchanged, and there was a nonsignificant trend toward increased fasting plasma insulin concentrations and decreased insulin sensitivity when assessed by HOMA (Table 2). Treatment with CDCA significantly reduced the increases in triglyceride, cholesterol, and free fatty acid concentrations compared with the high-fructose-fed hamsters (–40, –18, and –34%, P < 0.01, < 0.05 and < 0.05, respectively; Table 2).


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  		Table 1. Body weight and caloric intake

 

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  		Table 2. Plasma metabolic parameters in overnight-fasted hamsters on different diets

 
Lipoprotein subfraction analysis by gel filtration chromatography. Lipoprotein subfraction analysis by gel filtration chromatography revealed that lipoproteins in the HDL range were the main cholesterol carriers in control animals, although significant amounts of cholesterol could also be detected in the IDL/LDL and VLDL range (Fig. 1A). Triglycerides could be detected only in the VLDL and much less in the IDL/LDL fractions (Fig. 1B). The increase in plasma triglycerides in the HF group could be ascribed to an increase in VLDL and to a lesser extent in IDL/LDL triglycerides (Fig. 1B). Increases in VLDL, IDL/LDL, and HDL cholesterol contributed to the increased total cholesterol concentrations observed in high-fructose-fed hamsters (Fig. 1A). Adding CDCA to the high-fructose chow resulted in a robust decrease in VLDL and also IDL/LDL triglycerides, and the decreased total cholesterol concentrations following CDCA could be accounted for by decreased VLDL and IDL/LDL but not HDL cholesterol concentrations (Fig. 1, A and B).


Figure 1
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  		Fig. 1. Distribution of cholesterol (A) and triglyceride (B) in the plasma of control hamsters (CON, {square}), hamsters fed a high-fructose (HF) diet (bullet), and hamsters fed a HF diet with 0.1% (wt/wt) chenodeoxycholic acid (CDCA; gray circles). Fasting pooled plasma (200 µl) from 5–6 animals per group was analyzed by gel filtration chromatography. Fifty fractions (0.5 ml) were collected. VLDL elutes in fractions 6–13, IDL and LDL in fractions 14–27, and HDL in fractions 28–44.

 
In vivo hepatic triglyceride production. In vivo hepatic triglyceride production was assessed in overnight-fasted hamsters by following the increase of plasma triglycerides after the injection of tyloxapol, which blocks plasma lipolytic activity and thus the breakdown of triglyceride-rich lipoproteins. The weight of hamsters [116 ± 3, 116 ± 2, and 114 ± 2 g for CON, HF, and HF + CDCA animals, P = nonsignificant (NS)] and the weight gain during the 2-wk feeding period (data not shown) were comparable between the groups receiving different diets. A linear increase in plasma triglycerides was observed in all three groups following the injection of tyloxapol (r2 = 0.99, 0.98, and 0.99 for CON, HF, and HF + CDCA groups, respectively; Fig. 2A). The slope of the increase in plasma triglycerides was significantly increased following the high-fructose diet (10.17 ± 0.71 vs. 6.52 ± 0.81 in CON, P < 0.05; Fig. 2A), and this translated into a 46% increase in the triglyceride production rate (0.30 ± 0.04 vs. 0.43 ± 0.04 mg/min, P < 0.05; Fig. 2B). CDCA effectively prevented the increase in triglyceride production rate, which was lowered by 27% compared with high-fructose-fed animals (0.32 ± 0.02 vs. 0.43 ± 0.04 mg/min, P < 0.02 vs. HF; Fig. 2B).


Figure 2
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  		Fig. 2. In vivo production of triglycerides (TG) in CON ({square}), HF (bullet), and HF + CDCA (wt/wt, gray circles) hamsters after iv administration of tyloxapol (500 mg/kg body wt in normal saline). A: slope of the increase in plasma TG was linear and significantly increased by high-fructose feeding (P < 0.05), which was prevented by addition of CDCA to the HF diet (P < 0.05). B: this translated into a significantly increased TG production rate in fructose-fed animals (*P < 0.05 vs. CON) but not in HF + CDCA animals ({dagger}P < 0.05 vs. HF).

 
Tissue lipid concentrations. Liver triglyceride concentrations were slightly, but nonsignificantly, increased by feeding the high-fructose diet and tended to decrease when CDCA was added (7.0 ± 0.5, 8.3 ± 1.4, and 6.1 ± 1.0 mg/g liver for CON, HF, and HF + CDCA animals, respectively; P = NS). However, there was a small but significant increase in liver cholesterol content in high-fructose-fed animals that was prevented by addition of CDCA (2.5 ± 0.1, 3.1 ± 0.1, and 2.6 ± 0.1 mg/g liver for CON, HF, and HF + CDCA animals, respectively, P < 0.05 for CON vs. HF and HF vs. HF + CDCA).

Hepatic gene expression. Hepatic expression of genes involved in the de novo lipogenic pathway, Srebp-1, Fas, and Scd-1, was significantly increased in high-fructose-fed compared with CON animals (1.8-, 2.4-, and 3.8-fold, P < 0.05; Fig. 3A). In contrast, no effects were observed on hepatic mRNA levels of apoB, Mtp, peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), and apoC-III, which are important regulators of hepatic assembly and intravascular catabolism of triglyceride-rich lipoproteins and of Srebp-2 and LDL-R, key factors controlling cholesterol metabolism and catabolism of apoB-containing lipoproteins. Interestingly, the expression of Cyp7A1, the rate-limiting enzyme of cholesterol catabolism to bile acid synthesis, was lowered by 80% in high-fructose-fed animals (Fig. 3B). This was, at least in part, counterbalanced by a 29% decrease in the expression of Hmgcr, the rate-controlling enzyme of de novo cholesterol synthesis (P = 0.06, CON vs. HF). Adding CDCA to the high-fructose diet minimized the increased expression of lipogenic genes (–16, –25, and –52% for Srebp-1, Fas, and Scd-1, P = 0.2, 0.2, and < 0.05, respectively; Fig. 3A), and hepatic levels of Cyp7A1 were further decreased by 60% (P < 0.05; Fig. 3B).


Figure 3
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  		Fig. 3. Hepatic expression of selected genes involved in de novo lipogenesis (A) and hepatic cholesterol metabolism (B). Total RNAs from livers of controls (CON, open bars), HF (filled bars), and HF + CDCA (0.1%, wt/wt, gray bars) hamsters (n = 7–8 animals per group) were extracted and analyzed by quantitative real-time PCR as described in MATERIALS AND METHODS. Srebp-1, sterol regulatory element-binding protein-1; Fas, fatty acid synthase; Scd-1, steaoryl-CoA desaturase 1; Cyp7A1, cholesterol 7{alpha}-hydroxylase. Values are normalized relative to 18S rRNA and are expressed (means ± SE) relative to those of CON, which are arbitrarily set at 1. Statistical differences are indicated: *P < 0.05 vs. CON; {dagger}P < 0.01 vs. CON; {ddagger}P < 0.05 vs. HF.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Syrian hamsters, in contrast to other rodents, are very sensitive to the effects of Western diets and develop combined hyperlipidemia due to overproduction of VLDL triglycerides and insulin resistance even after short periods of feeding a high-fructose diet (33). In agreement, the findings of the present study demonstrate that the increases in both VLDL and IDL/LDL triglyceride and cholesterol concentrations following high-fructose feeding were primarily due to hepatic lipoprotein overproduction, as indicated by a 46% increase in hepatic triglyceride secretion. Although intravascular lipoprotein catabolism was not directly assessed in our studies, hepatic expression of both apoC-III, an important regulator of lipoprotein lipase, and the LDL-R gene was unchanged in fructose-fed animals, making decreased lipoprotein removal an unlikely primary cause of the hyperlipidemia observed. In contrast, hepatic mRNA levels of lipogenic enzymes and Srebp-1, the key lipogenic transcription factor, were clearly increased, identifying enhanced de novo lipogenesis as a main contributor to the development of hypertriglyceridemia. The increase in liver cholesterol may have further enhanced VLDL production in fructose-fed hamsters, since hepatic availability of both triglycerides and cholesterol esters has been shown to drive hepatic VLDL secretion (25). The decreased hepatic expression of Cyp7A1 indicates that the increase in liver cholesterol in fructose-fed animals resulted from decreased cholesterol catabolism to bile acids. Microsomal triglyceride transfer protein (Mtp), which facilitates the transfer of core lipids to nascent VLDL, has been implicated as another pathogenetic factor in the development of hyperlipidemia associated with fructose feeding in hamsters (33). Because hepatic mRNA levels of Mtp were not altered in our high-fructose-fed animals, the previously reported increase in Mtp protein may result from posttranscriptional events. Thus the hypertriglyceridemia observed in fructose-fed hamsters can be largely explained by enhanced hepatic de novo lipogenesis from carbohydrate precursors and makes this model an attractive tool to study interventions targeting this pathway.

Despite the increase in de novo lipogenesis and the resulting hypertriglyceridemia, no increase in liver triglycerides was observed in our study. This might be explained by the efficient triglyceride export system in hamsters and the relatively short feeding period, since an almost 50% increase in liver triglycerides was reported in the same model after 3 wk of high-fructose feeding (5).

CDCA, when added to the high-fructose diet, improved the lipid profile of fructose-fed hamsters by preventing increases of both VLDL and IDL/LDL concentrations. The associated 27% decrease in hepatic triglyceride production is the most likely explanation for this finding and is strongly supported by previous findings in vivo in humans (2). Furthermore, our results suggest that both decreased de novo lipogenesis and decreased availability of free fatty acids may have limited hepatic free fatty acid availability for triglyceride synthesis and export. These findings extend those of a recent study demonstrating that activation of FXR with bile acids or a synthetic agonist decreased plasma and liver triglycerides through a mechanism involving SHP, LXR, and Srebp-1c in mice to the high-fructose-fed hamster model (36). Although hepatic Srebp-1c mRNA levels have been previously described to decrease upon activation of FXR, we saw a nonsignificant 16% reduction of hepatic Srebp-1 mRNA following therapy with CDCA. However, hepatic mRNA levels of Scd-1, an important Srebp-1 target gene in the lipogenic pathway, dropped by 52%. The recent demonstration that the differences in lipogenic gene expression between wild-type and FXR–/– mice cannot be attributed to transcriptional regulation by Srebp-1c together with our findings suggests that activation of FXR may affect hepatic lipogenesis by independent mechanisms (10). A negative effect of FXR activation on direct LXR-mediated lipogenic gene expression could be an alternative explanation (17). Furthermore, feeding the high-fructose diet may have increased lipogenesis by involving carbohydrate response element-binding protein (CHREBP). CHREBP is a recently discovered transcription factor responsible for insulin-independent glucose-induced transcription of lipogenic genes (15, 38). Because CHREBP has not yet been described in hamsters, it is unclear whether it was involved in both enhanced lipogenesis following high-fructose feeding and the triglyceride-lowering effects of CDCA in our model. Several other mechanisms have been implicated in the triglyceride-lowering effects of FXR activation. A decrease in hepatocyte nuclear factor-4 (HNF-4)-mediated Mtp expression was found in Hep G2 cells treated with CDCA (14). Because hepatic Mtp mRNA levels remained unchanged in CDCA-treated animals, transcriptional regulation of Mtp did not account for the triglyceride-lowering effect of FXR activation in our model. PPAR{alpha} is an important transcriptional regulator of enzymes and apolipoproteins involved in both hepatic fatty acid oxidation and intravascular metabolism of triglyceride-rich lipoproteins, and an FXR response element has been recently described in the human PPAR{alpha} promoter (26). Plasma and hepatic levels of apoC-III, a PPAR{alpha} target gene and inhibitor of lipoprotein lipase, decreased in mice and human cell lines following activation of FXR (7, 32). Hepatic levels of both PPAR{alpha} and apoC-III were not affected by FXR activation with CDCA in our study, and it is unlikely that PPAR{alpha} activation in either a ligand-dependent or transcriptional manner accounted for the CDCA-induced lipoprotein changes. Enhanced receptor-mediated lipoprotein catabolism involving both the VLDL and LDL receptor has been implicated in the metabolic effects of FXR activation in human liver cell lines and mice, respectively (23, 31). LDL receptor gene expression was not induced by CDCA in our study, however. Thus decreased hepatic VLDL secretion secondary to decreased de novo lipogenesis is the most robust explanation for the beneficial effects of FXR activation with CDCA in fructose-fed hamsters.

Although CDCA further lowered Cyp7A1 mRNA levels, thereby potentially limiting cholesterol catabolism to bile acids, VLDL and IDL/LDL cholesterol concentrations and hepatic cholesterol content decreased in CDCA-treated animals. Because hepatic cholesterol synthesis did not decrease upon CDCA therapy, when assessed by hepatic mRNA levels of hydroxymethylglutaryl-CoA reductase, enhanced biliary elimination of dietary cholesterol could account for these findings. In fact, bile acid-mediated activation of FXR has been shown to increase hepatic abundance of the ATP-binding cassette (ABC) transporters G5 (ABC-G5) and G8 (ABC-G8), which are located at the apical membranes of hepatocytes and mediate biliary cholesterol secretion (39). Therefore, activation of FXR may allow efficient biliary elimination of cholesterol despite decreased bile acid synthesis.

In addition to its role in the metabolism of hepatically secreted lipoproteins, FXR has recently been implicated in the regulation of HDL metabolism. Activation of FXR with bile acids was associated with an increase in hepatic phospholipid transfer protein (PLTP) mRNA in mice (35). PLTP has an essential role in transferring phospholipids from triglyceride-carrying lipoproteins into HDL, and HDL concentrations are decreased by 50% in PLTP–/– mice (1, 16). Thus FXR activation would be expected to increase plasma HDL concentrations. In contrast, however, the expression of apoA-I, the major protein constituent of HDL, was decreased by FXR activation in a human liver cell line (8). Adding CDCA to the high-fructose diet had no further impact on HDL cholesterol concentrations in our study, suggesting that the overall effect of FXR activation on plasma HDL concentrations may be neutral.

Moreover, FXR has been identified as a novel regulator of glucose metabolism. Hepatic expression of FXR itself was found to be decreased in animal models of diabetes, and a time- and concentration-dependent increase in FXR mRNA abundance was demonstrated in a glucose-treated human liver cell line (11). On the other hand, bile acids decreased hepatic expression of gluconeogenic enzymes in a SHP-dependent manner by interfering with the transcriptional regulation through the forkhead transcription factor Foxo1 and HNF-4 (37). In our study, fasting plasma glucose concentrations were neither increased upon fructose feeding nor lowered by the addition of CDCA.

In summary, the nuclear receptor FXR has been identified as an important regulator of bile acid, triglyceride, and glucose metabolism, mainly through findings obtained in human cell and murine models. Our study extends those findings and suggests that CDCA-mediated activation of the transcription factor FXR decreases hepatic secretion of triglyceride-containing lipoproteins, possibly by interfering with Srebp-1-mediated transcription of Scd-1 and, hence, de novo lipogenesis. Because inadequately enhanced de novo lipogenesis is a hallmark feature of the metabolic syndrome, including type 2 diabetes and primary hypertriglyceridemias, FXR activation may prove to be a new and efficacious strategy in the treatment of these increasingly prevalent diseases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was in part supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK-40936, U24 DK-59635, P30 DK-45735) and the Swiss National Science Foundation (S. Bilz), the Novartis Foundation (S. Bilz), the Martin Hilti Foundation (S. Bilz), and Fonds zur Förderung des Akademischen Nachwuchses of the University Basel (S. Bilz). G. I. Shulman is the recipient of a Distinguished Clinical Scientist Award from the American Diabetes Association and an Investigator of the Howard Hughes Medical Institute.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. I. Shulman, Howard Hughes Medical Institute, Yale Univ. School of Medicine, The Anlyan Center, S269, PO Box 9812, New Haven, CT 06536-8012 (e-mail: gerald.shulman{at}yale.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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