FXR Antagonist FLG249 Lowers Hepatic Triacylglycerol and Serum Cholesterol Level in High-Fat Diet-Induced Obese Mice

Abstract

Farnesoid X receptor (FXR) is a nuclear receptor that regulates the synthesis and enterohepatic circulation of bile acids (BAs). It also regulates lipid and carbohydrate metabolism, making FXR ligands potential therapeutic agents for systemic and/or hepatic metabolic disorders. We previously synthesized a series of FXR antagonists and showed that oral administration of FLG249 reduced the expression of several FXR target genes in the mouse ileum. Here, we investigated the effects of FLG249 on lipid metabolism in mice fed a high-fat diet (HFD). When FLG249 was administered for 4 weeks to HFD-induced obese mice, it altered the expression of genes related to BA metabolism, ceramide synthesis and fatty acid β-oxidation, improving lipid metabolism in the liver and ileum without decreasing body weight. These findings suggest that FLG249 has the potential to be a low toxicity pharmaceutical compound and likely acts as a nonsteroidal FXR antagonist to improve lipid metabolism disorders.

INTRODUCTION

Farnesoid X receptor (FXR) is a nuclear receptor that regulates the enterohepatic circulation of bile acids (BAs) by controlling the expression of genes involved in BA synthesis, biliary BA secretion, and transintestinal BA transport to the liver via portal blood circulation.¹⁾ FXR is also responsible for the regulation of specific target genes involved in biological processes in glucose and lipid homeostasis; thereby, it has become an important drug target for the treatment of many FXR-mediated diseases.

Activation of FXR in the liver decreases the expression of sterol regulatory element binding protein-1c (SREBP-1c), a transcription factor that plays a critical role in lipid synthesis through small heterodimer partner (SHP).²⁾ This results in the suppression of triacylglycerol (TG) synthesis and a decrease in very-low-density lipoprotein (VLDL) secretion into the blood. In addition, activation of FXR has inhibitory effects on gluconeogenesis and promotes glycogen synthesis through the fibroblast growth factor 15/19 (FGF15/19) pathway, indicating its role in regulating lipid and carbohydrate metabolism. Based on these findings, various FXR agonists have been developed, including obeticholic acid (OCA).^3,4)

Activation of the FXR–SHP pathway in the liver and the FXR–FGF15 pathway in the intestine also suppresses cholesterol (Ch) catabolism by downregulating the expression of cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for BA biosynthesis. In fact, activation of FXR induces the expression and secretion of FGF15/19 in the ileum and suppresses the expression of CYP7A1 in the liver.⁵⁾ For these reasons, various FXR antagonists have been developed to improve Ch metabolism.³⁾ FXR antagonists have also shown efficacy in improving cholestasis and metabolic disorders in animal models.^6,7) Furthermore, in humans, a positive correlation between body mass index and intestinal FXR expression level has been observed.⁶⁾

High-fat diets (HFDs) have been shown to activate FXR in the intestines of rodent models.^8,9) However, a study revealed that when HFD-fed mice were administered antioxidant tempo and antimicrobial agents, tauro-β-muricholic acid (T-β-MCA) was increased in the intestines and competitively inhibited FXR activation by suppressing ceramide synthesis in intestinal tissue. This inhibition leads to the suppression of SREBP-1c-dependent fatty acid synthesis in the liver and to improvement in lipid and glucose metabolism.^6,8–11) It has been also reported that long-term administration of glycine-β-muricholic acid (G-β-MCA), which is less susceptible to hydrolysis than T-β-MCA, exhibits similar effect and reduces body weight, liver weight, and hepatic TG content.⁶⁾

Regarding fatty acid β-oxidation, a recent study demonstrated that administration of the FXR agonist OCA to mice caused a decrease in acylcarnitine levels, accompanied by a decrease in the expression of fatty acid β-oxidation-related genes.¹²⁾ Similarly, when OCA was administered to intestine-specific Fxr-null mice, the degree of reduction in acylcarnitine levels was lower. These results, together with additional gene expression analyses, show that activation of the intestinal FXR–FGF15 pathway induced by FXR agonists suppresses fatty acid β-oxidation in the liver by repressing hepatic peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) via inactivation of hepatic cAMP response element-binding protein (CREB).¹²⁾ Thus, intestine-selective FXR antagonists could be useful therapeutic agents for metabolic disorders.

In previous studies, we reported the development of FXR ligands with benzimidazole scaffold.^13–20) Among these series of developments, FLG249 was identified as an orally administered FXR antagonist¹⁶⁾ (Fig. 1). The compound showed high FXR antagonist activity and receptor selectivity in both luciferase and time-resolved fluorescence energy transfer assays.¹⁶⁾ Although FLG249 tends to distribute in the ileum and liver and regulates the expression of some FXR target genes in the mouse ileum, the effects of FLG249 on animal models remain unclear.

Fig. 1. Structure of the Nonsteroidal FXR Antagonist FLG249

In this study, we investigated the effects of FLG249 on lipid metabolism by evaluating the effects on genes related to Ch, ceramide metabolism and fatty acid β-oxidation in HFD-fed mice.

MATERIALS AND METHODS

Chemical

FLG249, (3-[(1S)-1-(1-cyclopropyl-6-fluoro-benzimidazol-2-yl)-2-[1-(2-methyl-propanoyl)-4-piperidyl]-ethyl]-1-(5)imidazolidine-2,4-dione), was synthesized in our laboratory and the purity is more than 99%.¹⁶⁾ For oral administration of FLG249 to mice, it was dissolved in a 20% (w/v) 2-hydroxypropyl-β-cyclodextrin solution as a concentration of 1.25 mg/mL.

Mice and Dietary Treatment

Six-week-old specific pathogen-free male mice (C57BL/6N) were purchased from The Jackson Laboratory Japan, Inc. (Yokohama, Japan). The animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication) and approved by the Institutional Animal Care and Use Committee at Hiroshima International University (Permit. No. AE21-020). Mice were housed for 1-week acclimation under a 12 : 12-h light/dark cycle, controlled temperature (22–24 °C), and humidity (50–60%) with free access to water and food. The mice were fed an HFD (HFD-60, 60% kcal from fat; Oriental Yeast Co., Ltd., Tokyo, Japan) for 13 weeks. The food was changed 2–3 times a week. After 13 weeks, 12 mice were randomly divided into two groups, with six mice per group and three mice per cage. Mice were orally dosed with vehicle (HFD) or FLG249 at 10 mg/kg twice daily (HFD + FLG249) for 4 weeks while concurrently on an HFD. Mice fed a normal diet (ND) served as the control group (n = 6). During FLG249 administration, the amount of food intake was measured. After 4 weeks, mice were weighed and then euthanized by cervical dislocation after which whole blood, liver, ileum, gallbladder bile, and feces were collected. The liver was weighed and whole blood was centrifuged at 2000 × g for 10 min at 4 °C. The resulting supernatant (serum) was transferred to a clean microcentrifuge tube.

Measurement of Serum and Hepatic Lipid Levels and Alanine Transaminase (ALT) Activity

Total serum Ch and TG levels were measured using commercial enzyme kits (Thermo Fisher Scientific, Waltham, MA, U.S.A. and FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The frozen liver samples (approximately 100 mg) were homogenized, and lipids in the liver were extracted with a chloroform/methanol (2 : 1) solution under acidity with acetic acid. After the solvent was distilled in a nitrogen atmosphere, the residue was dissolved in a 1% Triton-X 100 solution and used for the measurement of total Ch and TG levels with enzymatic chemistry kits. Serum ALT activity was measured by Alanine Transaminase Colorimetric Activity Assay Kit (FUJIFILM Wako Pure Chemical Corporation).

Measurement of Fecal and Biliary BAs

After freeze-drying the collected feces, fecal BAs were extracted with a 50% ethanol solution. After solvent distillation, the residue was dissolved in distilled water and subjected to solid-phase extraction. Methyl ester and trimethylsilyl derivatization treatments were performed according to conventional methods and analyzed by GC–MS.²¹⁾ Nordeoxycholic acid was used as an internal standard for the quantification of BAs. To analyze the BAs in bile, solid-phase extraction was performed followed by alkaline hydrolysis to deconjugate the conjugated BAs. After extraction under acidic hydrochloric acid, the extract was derivatized as described above and analyzed by GC–MS. The total amount of deoxycholic acid, α-muricholic acid, cholic acid, ursodeoxycholic acid, and β-muricholic acid detected was considered total BAs.

Quantitative PCR

Tissue samples (approximately 20 mg) preserved in Allprotect Tissue Reagent (QIAGEN, Venlo, the Netherlands) were homogenized, and total RNA was extracted and purified using the ReliaPrep™ RNA Miniprep System (Promega, Madison, WI, U.S.A.). cDNA was synthesized from total RNA with the ReverTra Ace^® qPCR RT Master Mix (TOYOBO, Osaka, Japan). Quantitative PCR (qPCR) was conducted using the THUNDERBIRD^® SYBR™ qPCR Mix (TOYOBO) in the StepOne™ Real-Time PCR System (Applied Biosystems, Waltham, MA, U.S.A.). The primer sequences used for qPCR are listed in Supplementary Table 1. The mRNA Ct values for these genes were normalized to 18s and expressed as a relative increase or decrease in the tissues from ND mice to those in HFD and HFD + FLG249 mice.

Statistical Analysis

Statistical analysis was assessed using the ANOVA, followed by Tukey’s test. p < 0.05 was considered significant. All values are reported as the mean ± standard deviation (S.D.).

RESULTS

Effects of FLG249 on Physiological and Biochemical Parameters

FLG249 is an orally active and nonsteroidal FXR antagonist with unique profiles, such as a propensity for ileum distribution and a significant change in expression level of three FXR target genes in the mouse ileum.¹⁶⁾ To evaluate the potential impact of FLG249 as a pharmaceutical compound, we examined its effects on an HFD-induced obesity mouse model. By administering HFD to mice for 13 weeks, significant weight gain was observed compared to mice receiving an ND (48.3 ± 2.7 vs. 34.0 ± 2.4 g). Then FLG249 was orally administered for 4 weeks concurrent with an HFD diet. Food intake was checked for each cage, but no clear increase or decrease in food intake was observed during FLG249 administration (ND: 30.4 ± 3.3, HFD: 31.2 ± 8.1, HFD + FLG249: 33.2 ± 7.79 kcal/cage/d). The weight was slightly decreased after administration of FLG249, but there was no significant difference between the two HFD groups with or without administration of FLG249 (Fig. 2A). HFD intake increased liver weight compared to the ND group, which was significantly reduced by additional FLG249 treatment to the same extent as the ND group (Fig. 2B). In addition, serum ALT activity did not change significantly in any groups (Fig. 2C).

Fig. 2. FLG249 Reduced Liver Weight without Changing Body Weight and Serum ALT Levels in HFD-Induced Obese Mice

Mice fed a HFD for 13 weeks were orally administered FLG249 at a dose of 10 mg/kg in twice a day while concurrently on an HFD diet for an additional 4 weeks (HFD + FLG249, n = 6). Mice orally administered vehicle served as the control group (normal diet (ND) and HFD, n = 6). (A) Body weight was measured after the FLG249 administration period. (B) Liver weights were measured from mice euthanized by cervical dislocation. (C) Serum alanine transaminase (ALT) levels were measured by Alanine Transaminase Colorimetric Activity Assay Kit (FUJIFILM). * p < 0.05 vs. ND, ^# p < 0.05 vs. HFD.

The effects of FLG249 on lipid metabolism were evaluated. Hepatic TG was markedly decreased by FLG249 treatment to the same level as the ND group, while the hepatic Ch level remained unchanged in the three groups (Figs. 3A, B), suggesting that FLG249 improved TG metabolism in the liver of HFD-induced obese mice. Regarding blood lipid levels, FLG249 did not affect TG level in HFD-induced obese mice but significantly decreased the increased blood Ch level caused by HFD (Figs. 3C, D). To further evaluate the effects of FLG249 on Ch metabolism, fecal and biliary BA levels were investigated. Total BA level in the gallbladder bile, which was decreased by HFD, was restored to the level of the ND group by FLG249 administration (Fig. 3E), whereas total BA excretion in feces was increased due to the HFD and notably reduced by FLG249 (Fig. 3F). These results suggest that administration of FLG249 improves lipid metabolism.

Fig. 3. FLG249 Altered the Lipid Profiles of HFD-Induced Obese Mice

Mice were euthanized and serum, liver, gallbladder bile, and feces were obtained. (A, B) TG and Ch profiles in the liver (C, D) TG and Ch profiles in the serum. (E, F) BA content in the gallbladder bile and feces. n = 6 mice per group. * p < 0.05 vs. ND, ^# p < 0.05 vs. HFD.

Effects of FLG249 on the Expression of FXR Target Genes in the Liver and Ileum

As shown in Fig. 3, FLG249 improved the lipid profile from HFD loading. Therefore, we examined the effects of HFD and FLG249 administration on the expression of genes related to BA and lipid metabolism in the liver and ileum (Fig. 4). In the liver, administration of FLG249 markedly increased the expression of Cyp7a1, which is involved in the stimulation of BA synthesis (Fig. 4A); this increase corresponded with an increase in the amount of total BAs in the gallbladder bile (Fig. 3E). There was no significant effect on the expression of other genes in HFD-fed mice. In the ileum, FLG249 treatment significantly decreased the expression levels of Shp and Fgf15, consistent with the increased expression of Cyp7a1 in the liver (Fig. 4B). The expression levels of Fxr and Srebp1c were nearly unchanged. On the other hand, expression of the apical sodium-dependent BA transporter (Asbt) was notably increased to the same level as the ND group by FLG249 treatment (Fig. 4B), which was correlated with a decrease in BA excretion into feces as shown in Fig. 3F. These results suggest that FLG249 improves Ch metabolism by selectively acting on FXR in the ileum, in accordance with a previous study.¹⁶⁾

Fig. 4. FLG249 Changed the Expression Levels of Genes Regulated by FXR in the Liver and Ileum

The effects of FLG249 on FXR-targeted genes in the liver (A) and ileum (B) of ND, HFD and HFD + FLG249 mouse groups were analyzed by qPCR. n = 6 mice per group. * p < 0.05 vs. ND, ^# p < 0.05 vs. HFD.

Effects of FLG249 on the Expression of Ceramide Metabolism-Related Genes in the Liver and Ileum

Regarding TG metabolism, several studies have shown that antagonism toward FXR or its deletion causes an increase in SREBP-1c expression and TG accumulation.^22,23) By contrast, administration of FLG249 to HFD-induced obese mice significantly reduced hepatic TG level (Fig. 3A). To determine the underlying mechanism, the expression levels of genes involved in ceramide metabolism were investigated in the liver and ileum since increased ceramide in the liver stimulates SREBP-1c expression.⁶⁾ In the liver, HFD increased the expression levels of ceramide synthase 6 (CerS6), which is responsible for the de novo ceramide synthesis pathway, and Δ⁴-desaturase, sphingolipid 2 (Degs2), which catalyzes the hydroxylation/desaturation reaction of ceramide. These expression levels were significantly decreased by administration of FLG249 (Fig. 5A). Similar results were obtained for the expression levels of sphingomyelin phosphodiesterase 3/4 (Smpd3/4), which generates ceramide from sphingomyelin, and alkaline ceramidase 2 (Acer2), which is involved in the decomposition of ceramide. In addition, the expression levels of serine palmitoyltransferase long chain base subunit 1 (Sptlc1), which catalyzes the initial reaction of the sphingolipid biosynthesis pathway, and Degs1, Smpd1, and sphingomyelin synthase 1 (Sgms1), which synthesize sphingomyelin from ceramide, were significantly reduced by FLG249. Unfortunately, FLG249 had little effect on the expression levels of genes related to ceramide metabolism in the ileum (Fig. 5B). A decrease in FXR activity in the intestinal tract reportedly causes inhibition of ceramide synthesis in intestinal tissues and a decrease in its blood concentration, which in turn inhibits SREBP1c-dependent fat synthesis in the liver.^8,9,11) However, the present results were inconsistent with a previous report.⁶⁾ In this study, the expression level of hepatic Srebp1c tended to decrease (Fig. 4A). These results suggest that FLG249 may directly act on the liver and suppress ceramide synthesis by downregulating the expression of genes related to ceramide metabolism, resulting in a decrease in hepatic TG.

Fig. 5. FLG249 Altered the Expression of Genes Related to Ceramide Metabolism

The effects of FLG249 on gene expression related to ceramide metabolism in the liver (A) and ileum (B) were evaluated using qPCR. n = 6 mice per group. * p < 0.05 vs. ND, ^#p < 0.05 vs. HFD.

Effect of FLG249 on the Expression of Fatty Acid β-Oxidation Pathway-Related Genes in the Liver

Previous studies have revealed an important role of the intestinal FXR–FGF15 pathway related to inhibition of hepatic fatty acid β-oxidation.¹²⁾ In this study, as administration of FLG249 significantly reduced hepatic TG level, we evaluated the effect of FLG249 on gene expression related to fatty acid β-oxidation pathway in the liver of HFD-induced obese mouse (Fig. 6). The expression levels of several genes involved in β-oxidation, including peroxisome proliferator-activated receptor alpha (Pparα), were increased by HFD. This suggests that the β-oxidation system in the liver is enhanced by HFD.

Fig. 6. FLG249 Affected the Expression of Genes Related to Hepatic Fatty Acid β-Oxidation

Relative expression of genes for fatty acid β-oxidation and pro-inflammatory cytokine in the liver of ND, HFD and HFD + FLG249 groups were determined by qPCR. n = 6 mice per group. * p < 0.05 vs. ND, ^# p < 0.05 vs. HFD.

Fatty acid binding protein 1 (FABP1) and cluster of differentiation 36 (CD36) are involved in the transport of long-chain fatty acids in hepatocytes.^24,25) FLG249 did not alter these expression levels. Acyl-CoA synthetase long-chain family member 1 (ACSL1) catalyzes the esterification of fatty acids to form fatty acyl-CoA, whereas acyl-CoA thioesterase 1 (ACOT1) subsequently converts acyl-CoA to fatty acids and CoA. Therefore, the expression of these two enzymes might affect the balance of cytoplasmic concentrations of acyl-CoA and fatty acids. While Acsl1 mRNA levels did not change, Acot1 mRNA was crucially reduced to the level of ND group by FLG249 treatment (Fig. 6), suggesting that the acyl-CoA pool is increased by FLG249 treatment.

PPARα directly regulates the transcription of genes involved in mitochondrial/peroxisomal fatty acid uptake and β-oxidation. PGC1α is a coactivator that cooperates with PPARα to regulate fatty acid β-oxidation by increasing the expression of several genes in the β-oxidation pathway. The hepatic nuclear factor 4 alpha (HNF4α) transcription factor regulates lipid mobilization and β-oxidation. As shown in Fig. 6, Pparα mRNA level was not affected, but Pgc1α and Hnf4α mRNA levels were significantly increased by FLG249. Since PGC1α is involved in the regulation of carnitine palmitoyltransferase expression by cAMP in combination with HNF4α and CREB,^26,27) the increased expression of Hnf4α and Pgc1α is considered indicative of enhancement of fatty acid β-oxidation.

Carnitine palmitoyltransferase 1A (Cpt1a), carnitine palmitoyltransferase 2 (Cpt2), carnitine-acylcarnitine translocase (Cact), and carnitine acetyltransferase (Crat) genes encode four members of the carnitine acyltransferase family, which are important for translocation of acyl-CoA into the mitochondria matrix through their inner membranes. Among these, Cpt1a and Cact expression was markedly enhanced with FLG249 administration (Fig. 6). As CPT1 is a rate-limiting enzyme for the transport of acyl-CoA into the mitochondrial matrix, an increase in its expression might contribute to the facilitation of fatty acid β-oxidation.

Acetyl-CoA carboxylase α (ACACA) inhibits the β-oxidation of mitochondrial fatty acids by inhibiting the transfer of acyl groups from acyl-CoA to carnitine. Uncoupling protein 2 (UCP2) also has the activity of uncoupling mitochondrial oxidative phosphorylation. FLG249 had no significant effect on the mRNA levels of Acacα and Ucp2 compared to the HFD group.

Regarding fatty acid β-oxidation enzymes in the mitochondria, the acyl-CoA dehydrogenase long chain (Acadl) gene encodes a key enzyme catalyzing the initial dehydrogenation reaction of long chain fatty acid. The mRNA expression of Acadl was markedly increased with FLG249 (Fig. 6). The cooperative increase in Acot1, Cpt1a and Acadl expression suggests that fatty acid β-oxidation in mitochondria is facilitated by FLG249. The hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (Hadhα) gene, which encodes enzymes that operate the dehydrogenation reaction in the β-oxidation pathway, showed little significant change upon administration of FLG249. In addition, the solute carrier 16A family of monocarboxylate transporter (Slc16a), which is involved in ketone body import, and Na⁺-dependent carnitine transport by organic cation transporter (Octn) gene, which encodes a transporter involved in carnitine uptake, did not change in HFD-fed mice. FLG249 notably increased the mRNA expression level of reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex 9 (Ndufα9) encoding a subunit of mitochondrial respiratory chain complex I. Very-low-density lipoprotein receptor (VLDLR), which is involved in the production of TG-rich lipoproteins, was decreased by FLG249 administration, possibly due to a decrease in the amount of TG in the liver.

A previous study demonstrated that activation of the intestinal FXR–FGF15 pathway by OCA suppresses fatty acid β-oxidation.¹²⁾ However, the results of the present study suggest the possibility that FLG249 promotes fatty acid β-oxidation in the liver by inhibiting the FXR–FGF15 pathway in the ileum and improving inflammatory conditions by hepatic TG accumulation.

DISCUSSION

FXR has garnered interest as an attractive pharmacological target due to its control of Ch, TG, and glucose metabolism.²⁸⁾ Several types of FXR antagonists have been reported including natural antagonists, compound-T3 (T3), G-β-MCA, and N-phenylbenzamide analogs.³⁾ Recent evidence suggests that intestine-specific FXR antagonists exhibit remarkable metabolic improvements and slow the progression of metabolic dysfunction-associated steatohepatitis.^29,30) However, none have been successful in clinical trials to date. We previously reported that the short-term administration of FLG249 in normal mice significantly decreased the expression levels of FXR target genes in the ileum, but not in the liver.¹⁶⁾ In a previous report from another group, long-term administration of G-β-MCA to HFD-fed mice resulted in significant weight loss and decreased hepatic TG content.⁶⁾ Therefore, we administered FLG249 to HFD-induced obese mice to evaluate the efficacy of newly discovered FXR antagonist.

Previously, when FLG249 was administered orally to C57BL/6N mice, it remained in the ileum for at least 6 h. Additionally, administration of 10 mg/kg/d changed the expression level of target genes.¹⁶⁾ Based on these observations, we used this dosage in the current study. When FLG249 was administered at a dose of 10 mg/kg/d for 4 weeks, all mice survived and their food intake remained constant. In addition, serum ALT levels were not changed among three groups (Fig. 2C). These findings indicated that FLG249 did not have severe toxicity under our experimental condition. When FLG249 was administered to HFD-induced obese mice, no significant weight loss was observed, but liver weight was significantly reduced (Figs. 2A, B). These results suggest that FLG249 has little effect on systemic lipid metabolism, and longer term administration may decrease body weight.

The important findings of this study are that FLG249 administration significantly reduced liver weight (Fig. 2B), hepatic TG content, and serum Ch (Figs. 3A, D). There are two possibilities by which FLG249 may decrease hepatic TG level. It has been shown that a decrease in FXR activity in the intestinal tract is accompanied by the suppression of ceramide synthesis in intestinal tissues and a decrease in its blood concentration.¹¹⁾ Furthermore, SREBP-1c-dependent fat synthesis in the liver is suppressed through a decrease in ceramide synthesis.^6,8–11) Based on these reports, we examined the expression levels of genes related to ceramide metabolism in the ileum and found no significant changes in most genes except Sgms1 (Fig. 5B). By contrast, in the liver, the expression levels of these genes were significantly decreased or tended to decrease upon FLG249 administration (Fig. 5A). Although the expression level of Srebp1c showed a tendency to decrease, it was not statistically significant in the liver and ileum (Fig. 4). In this study, we evaluated the therapeutic effects of oral administration of FLG249 for 4 weeks to established obese mice fed an HFD. Meanwhile, Jiang et al. treated with an HFD and drug administration at the same time.⁶⁾ Differences in HFD and drug administration periods may have influenced the expression levels of ceramide synthesis-related genes in the ileum. We considered the possibility FLG249 lowers hepatic TG content through a different mechanism than T-β-MCA and G-β-MCA.

Another possible mechanism why which FLG249 reduces hepatic TG level might be related to the increased expression of genes involved in mitochondrial fatty acid β-oxidation. Activation of the ileum FXR–FGF15 pathway by the FXR agonist OCA has been shown to downregulate hepatic PGC1α signaling and suppress fatty acid β-oxidation through inactivation of hepatic CREB.¹²⁾ In this study, FLG249 administration decreased the expression of ileum Fgf15 and increased the expression of hepatic Hnf4α and Pgc1α, suggesting that fatty acid β-oxidation increases in the liver (Figs. 4B, 6). Furthermore, the mRNA expression of Acot1 decreased with a decrease in Fgf15 expression, consistent with previous reports.^12,31) Along with the increased expression of Pgc1α, the mRNA expression of key fatty acid β-oxidation-associated genes including Cpt1a, Cact and Acadl was also significantly increased in response to FLG249 treatment. These results suggest that FLG249 suppresses the activation of intestinal FXR and reduces the expression of Fgf15, thereby promoting fatty acid β-oxidation in the liver and reducing the amount of TG in the liver. Although PPARα may be involved, FLG249 has not been found to have the ability to activate PPARα.¹⁶⁾ It has also been reported that PPARα suppresses BA synthesis,³²⁾ but the increased expression of Cyp7a1 suggests that BA synthesis was not suppressed in this study. Considering that FLG249 acts predominantly in the intestinal tract, it is thought that FLG249 has little effect on hepatic PPARα.

Regarding Ch metabolism, FLG249 significantly decreased serum Ch level, although it did not affect the level of Ch in the liver (Figs. 3B, D). This result was similar to the effects observed with synthetic FXR antagonist T3 administration.^33,34) The expression levels of liver Shp and bile salt export pump (Bsep) were not altered by FLG249, but the expression levels of ileal Shp and Fgf15 were significantly suppressed. Additionally, the expression level of liver Cyp7a1 was significantly increased. These findings suggest that the decrease in serum Ch levels is a result of FLG249 antagonizing FXR in the ileum, leading to decreased Fgf15 expression and increased Cyp7a1 expression in the liver, which in turn increases Ch clearance from the bloodstream. As a result, BA levels in the gallbladder bile were significantly increased compared to the HFD group. As the expression level of ileal Asbt was also significantly increased, FLG249 treatment resulted in a significant decrease in BA excretion into the feces, suggesting an increase in the BA pool size.

In this study, FLG249 improved lipid abnormalities in HFD-induced obese mice by predominantly inhibiting the ileum FXR–FGF15 pathway (Figs. 3, 4, 6). Additionally, the increased expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in HFD-fed mice tended to decrease by FLG249. These cytokines are thought to be involved in the earliest events of liver injury and recruitment of inflammatory cells, and trigger fibrosis in the liver. Overall, our study demonstrates the potential benefits of using FXR antagonists for the treatment of metabolic diseases. Promoting fatty acid β-oxidation, such as with PPARα or PPARδ agonists, has been shown to reduce lipid accumulation.³⁵⁾ Unlike other FXR antagonists, FLG249 is thought to improve TG metabolism by acting predominantly on the intestinal tract. Therefore, FLG249 may be a potential therapeutic agent that reduces hepatic lipid accumulation in a PPAR-independent manner. The analysis of metabolic changes with FLG249 will be the subject of further research.

Acknowledgments

The research project was supported by the Scholarship Fund for Young/Women Researchers. Funding was provided by The Promotion and Mutual Aid Corporation for Private Schools of Japan (PMAC). We thank Mr. D. De Stefano for editing and proofreading this article.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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