Colesevelam ameliorates non-alcoholic steatohepatitis and obesity in mice

Background Obesity, non-alcoholic fatty liver disease (NAFLD) and its more advanced form non-alcoholic steatohepatitis (NASH) are important causes of morbidity and mortality worldwide. Bile acid dysregulation is a pivotal part in their pathogenesis. The aim of this study was to evaluate the bile acid sequestrant colesevelam in a microbiome-humanized mouse model of diet-induced obesity and steatohepatitis. Methods Germ-free C57BL/6 mice were associated with stool from patients with NASH and subjected to 20 weeks of Western diet feeding with and without colesevelam. Results Colesevelam reduced Western diet-induced body and liver weight gain in microbiome-humanized mice compared with controls. It ameliorated Western diet-induced hepatic inflammation, steatosis, fibrosis and insulin resistance. Colesevelam increased de novo bile acid synthesis and decreased hepatic cholesterol content in microbiome-humanized mice fed a Western diet. It further induced the gene expression of the antimicrobials Reg3g and Reg3b in the distal small intestine and decreased plasma levels of LPS. Conclusions Colesevelam ameliorates Western diet-induced steatohepatitis and obesity in microbiome-humanized mice.


Introduction
The prevalence of obesity (body mass index ≥ 30 kg/m 2 ) is 19.5% in countries of the Organization for Economic Cooperation and Development [1]. One out of 4 subjects suffers from non-alcoholic fatty liver disease (NAFLD) globally [2]. Non-alcoholic steatohepatitis (NASH), a more advanced and serious form of NAFLD, is now the second most common indication for liver transplantation in the United States after alcohol-associated liver disease [2][3][4]. Lifestyle modifications, including a well-balanced diet and daily physical exercise, are the primary treatment options for obesity, NAFLD and NASH [5]. Despite these lifestyle modifications, obesity and NAFLD/NASH oftentimes progress. Effective pharmacologic treatment options are currently lacking. Bile acid sequestrants bind bile acids in the intestinal lumen, thereby inhibiting their enterohepatic circulation, and induce de novo synthesis of bile acids from cholesterol in the liver [6,7]. The bile acid sequestrant colesevelam, when administered orally, ameliorates bile acid malabsorption-associated diarrhea in patients with Crohn's disease [8] and reduces hemoglobin A1c and lowdensity lipoprotein-cholesterol in patients with type 2 diabetes mellitus [9,10].
The aim of this study was to evaluate the effects of colesevelam in a microbiome-humanized mouse model of Western diet-induced steatohepatitis and obesity.

Mice
Male C57BL/6 germ-free mice were bred at the University of California San Diego (UCSD). Fecal microbiota transplantation with non-pooled samples from 2 patients with non-alcoholic steatohepatitis (NASH) ( Table 1) was performed at the age of 5-6 weeks and repeated 2 weeks later as described [11]. Briefly, mice were gavaged with 100 μL of a stool suspension, which was prepared by dissolving 1 g stool in 30 mL Luria-Bertani (LB) medium containing 15% glycerol under anaerobic conditions. Two weeks after the second gavage, mice were placed for 20 weeks on an irradiated Western-style fast-food diet ("Western diet") (TD.200289; containing 41.9% kcal from fat, 43.0% kcal from carbohydrate, 15.2% from protein, 4.6 kcal/g) with or without 2% colesevelam (obtained from Apothecon Pharmaceuticals). As a control, mice were fed for 20 weeks with an irradiated low-fat control diet ("chow diet") (TD.110637; containing 13.0% kcal from fat, 67.9% kcal from carbohydrate, 19.1% kcal from protein, 3.6 kcal/g) with or without 2% colesevelam. Diets were manufactured by Teklad Diets, Madison, WI. We have replenished the diet every 2 weeks and quantitated the daily amount of diet consumed by dividing the amount of diet consumed over the 2 weeks by 14 and by the respective weight of the mice at the time of replenishment. The areas under the curve of diet consumption per body weight were used to determine differences between the groups. For the subcutaneous adipose tissue measurement, the white adipose tissue between the skin and the peritoneum in the lower abdomen below  [29]. ALP alkaline phosphatase, ALT alanine aminotransferase, AST aspartate aminotransferase, GGT gamma-glutamyl transferase, HDL highdensity lipoprotein, INR international normalized ratio, LDL lowdensity lipoprotein, NAFLD non-alcoholic fatty liver disease, NASH non-alcoholic steatohepatitis  Activity Score  5  7  Steatosis grade  1  3  Ballooning  2  2  Inflammation grade  2  2  Fibrosis stage  3  2 the rib cage was quantitated after an anterior-median abdominal dissection through the skin and subsequent dissection through the skin in the transverse plane below the rib cage. For the mesenteric adipose tissue measurement, the white adipose tissue located between stomach, intestine, pancreas, and spleen was quantitated. Mice were randomly assigned to the different groups at the beginning of the study. Animals were maintained on a 12 h:12 h light-dark cycle in Sentry SPP systems (Allentown, NJ) under gnotobiotic conditions [12]. All manipulations were performed during the light cycle. After 16 weeks of feeding, an insulin tolerance test was performed by injecting insulin (Novolin N NPH; Novo Nordisk Inc., Princeton, NJ) at a dose of 1 mU/g body weight intraperitoneally. Prior to the insulin tolerance test, mice were fasted for 10 h. Tail vein blood for measuring blood glucose levels was obtained before injection (t = 0 min) and at t = 30, 60, 90, 120 min after insulin injection. The areas under the curve of the blood glucose levels were used to determine differences between the groups.
To determine lipid accumulation, liver sections were embedded in OCT compound, and 10 μm frozen sections were cut and stained with Oil Red O (Sigma-Aldrich).

Biochemical analysis
Hepatic triglyceride levels were measured using the Triglyceride Liquid Reagents Kit (Pointe Scientific). For fecal triglyceride quantitation, fecal samples were mixed with phosphate-buffered saline (PBS) in a 1:40 dilution and centrifuged at 12,000 g for 5 min to separate solid particles. Triglyceride concentrations in supernatants were determined

Colesevelam improves Western diet-induced obesity and liver weight gain in microbiome-humanized mice
Germ-free C57BL/6 mice were associated with fecal microbiota from two patients with NASH (Table 1) and subjected to feeding of a Western diet over 20 weeks. Colesevelam treatment resulted in lower absolute body weight and reduced body weight gain compared with Western diet-fed control mice (Fig. 1A-B). Administration of the bile acid sequestrant decreased subcutaneous and mesenteric white fat gain in relation to controls (Fig. 1C-D). Further, liver weight and liver weight-to-body weight ratio were decreased in Western diet-fed mice receiving colesevelam relative to Western diet-fed control mice (Fig. 1E-F). These changes occurred despite increased food intake of the Western diet in colesevelam-treated mice versus their controls (Fig. 1G). The fecal triglyceride content was not significantly different between the Western diet-fed mouse groups (Fig. 1H).
Colesevelam did not result in apparent loose stools in the mice.

Colesevelam ameliorates Western diet-induced hepatic inflammation, steatosis, and insulin resistance in microbiome-humanized mice
Colesevelam reduced hepatic inflammation as evidenced by lower hepatic mRNA expression of tumor necrosis factoralpha (Tnf-α), chemokine C-C motif ligand-2 (Ccl2), and adhesion G protein-coupled receptor E1 (Adgre1), also known as F4/80, ( Fig. 2A-C) in comparison with Western diet-fed controls. Additionally, it reduced liver steatosis in Western diet-fed mice, as indicated by significantly lower hepatic triglycerides and fewer and smaller fat droplets on staining in relation to Western diet-fed mice not administered the bile acid sequestrant (Fig. 2D-F). Colesevelam also improved insulin sensitivity in Western diet-fed mice, as determined by insulin tolerance test (Fig. 2G).

Colesevelam reduces Western diet-induced hepatic fibrosis in microbiome-humanized mice
Twenty weeks of Western diet feeding resulted in liver fibrosis in microbiome-humanized mice, as determined by increased hepatic mRNA expression of genes involved in fibrosis, including transforming growth factor-beta 1 (Tgfb1), tissue inhibitor of metalloproteinase-1 (Timp1), and collagen type I alpha 1 (Col1a1) (Fig. 3A-C). These changes were attenuated when mice were treated with Fig. 1 Colesevelam decreases Western diet-induced body and liver weight gain despite increased food consumption in microbiomehumanized mice. Germ-free C57BL/6 mice were colonized with feces from two patients with NASH and subjected to 20 weeks of Western diet-feeding with or without colesevelam (n = 13-15 per group) or chow diet with or without colesevelam (n = 5 per group). a Absolute body weight. b Body weight gain relative to start of feeding experiment.  Mann-Whitney test (c, f-g). *p < 0.05. Ccl2, chemokine C-C motif ligand-2; Tnf-α, tumor necrosis factor-alpha colesevelam. Reduced liver fibrosis was confirmed by reduced total hepatic hydroxyproline content and by Sirius Red staining of liver sections in Western dietfed mice relative to their Western diet-fed counterparts ( Fig. 3D-E). A total of two patients with NASH were chosen as stool donors. One of the NASH patients had type 2 diabetes mellitus; however, we did not observe significant differences in body weight and liver disease markers such as liver weight-to-body weight ratio, hepatic triglycerides, gene expression of inflammatory and fibrosis markers including Tnf-α and Tgfb1, respectively, between mice that received stool from the NASH patient with type 2 diabetes mellitus and mice that received stool from the NASH patient without type 2 diabetes mellitus (data not shown).

Colesevelam increases genes involved in de novo bile acid synthesis in microbiome-humanized mice fed a Western diet
Colesevelam induced enzymes of the classic bile acid synthesis pathway, including the rate-limiting enzymes cytochrome P450 7a1 (Cyp7a1) and 8b1 (Cyp8b1), after 20 weeks of Western diet feeding compared with mice not given colesevelam (Fig. 4A-B). However   cytochrome P450 27a1 (Cyp27a1) and 7b1 (Cyp7b1) (Fig. 4C-D). Hepatic cholesterol, the substrate for de novo bile acid synthesis, was reduced in mice administered colesevelam in relation to their controls after 20 weeks of Western diet feeding (Fig. 4E). The sequestrant did not alter total plasma bile acid levels after Western diet or chow diet feeding over 20 weeks, although the former group had significantly higher levels than the latter (Fig. 4F). Bile acid sequestrants are known to decrease intestinal farnesoid X receptor (Fxr) signaling by luminal binding of bile acids, which modulates hepatic Cyp7a1 expression [14]. We, therefore, assessed the intestinal gene expression of Fxrdependent genes small heterodimer partner (Shp) and fibroblast growth factor 15 (Fgf15), which were both significantly decreased in colesevelam-treated mice in relation to their Western diet-fed counterparts not treated with the bile acid binder (Fig. 4G-H).

Colesevelam decreases plasma lipopolysaccharide (LPS) levels and induces the gene expression of intestinal antimicrobials in microbiome-humanized mice fed a Western diet
LPS is known to contribute to the development of liver steatosis, inflammation, and fibrosis in rodent models of dietinduced steatohepatitis [15,16]. We, therefore, quantitated the LPS plasma levels in our mouse model, and found that colesevelam significantly decreased LPS concentrations in Western diet-fed mice (Fig. 5A). To explain the difference in LPS levels, we next determined the gene expression of major intestinal tight junction proteins, but did not detect significant differences in the expression of Occludin, Claudin 1, and Claudin 4 between Western diet-fed mice treated with colesevelam and those not treated with the bile acid sequestrant (Fig. 5B-D). We then assessed intestinal antimicrobials, and found that colesevelam treatment was associ- regenerating islet-derived protein 3 gamma and beta (Reg3g and Reg3b) in Western diet-fed mice (Fig. 5E-F).

Discussion
NAFLD and NASH are leading causes of morbidity, liver cirrhosis, and liver transplantation [2][3][4]. Here, we demonstrate that colesevelam decreases Western diet-induced weight gain, hepatic inflammation, steatosis, and fibrosis, and insulin resistance in mice colonized with stool from patients with NASH. This was associated with increased expression of de novo bile acid synthesis enzymes, lower plasma LPS levels, and increased intestinal expression of the antimicrobials Reg3g and Reg3b.
In the present study, we demonstrate that colesevelam reduces Western diet-induced obesity and associated metabolic dysregulation in mice, as indicated by a significantly lower body weight gain and better insulin response compared with control mice that do not receive the bile acid binder. It has been shown that colesevelam improves metabolic responses, such as lowering hepatic glucose production by suppressing hepatic glycogenolysis as well as increasing insulin sensitivity, in diet-induced obese rodents by activation of the intestinal Takeda G protein-coupled receptor-5 (Tgr-5) and glucagon-like peptide-1 (Glp-1) release [7,19]. These beneficial metabolic effects are consistent with studies in humans, where 12 and 24 weeks of treatment with colesevelam significantly improved hemoglobin A1c and low-density lipoprotein-cholesterol in patients with type 2 diabetes mellitus [9,10].
Further, we show that colesevelam reduces liver weight and hepatic steatosis after 20 weeks of Western diet feeding relative to controls. This is in contrast to a study in patients with NASH, which showed that treatment with 3.75 g of colesevelam daily over 24 weeks resulted in significant worsening of liver fat per magnetic resonance imaging-proton-density-fat-fraction (MRI-PDFF) as well as conventional magnetic resonance spectroscopy (MRS) [20]. Liver biopsy on the other hand did not detect any effect of treatment [20]. Contrary to these findings, treatment with another bile acid sequestrant Colestimide (3 g daily) over 24 weeks resulted in a significant improvement of hepatic steatosis and visceral fat in patients with NASH [21]. It is unclear how colesevelam deteriorated liver fat content in the prior study given several studies demonstrating beneficial metabolic effects of bile acid sequestrants and colesevelam in particular in rodent and human studies. Colesevelam was also shown to be protective in other experimental liver diseases such as cholestatic liver disease [22] or ethanol-induced liver disease in rodents [23]. Moreover, the bile acid sequestrant sevelamer was found to improve diet-induced steatohepatitis in rodents as well [14,24], whereas another bile acid binder, cholestyramine, given over 8 weeks did not improve hepatic steatosis in ob/ob mice [25]. The difference in the hepatic phenotype could possibly be explained by a difference in treatment duration (e.g., 20 weeks as in our model versus 8 weeks in the ob/ob mouse model [25]). It could be that a significant depletion of hepatic cholesterol secondary to bile acid sequestrant use is required -as in our study -to achieve improvement of diet-induced steatohepatitis [26]. Similarly, this could explain why in the human studies above colesevelam deteriorated [20] whereas colestimide improved human NASH [21]. Although both bile acid binders were administered over 24 weeks, it could be that a longer treatment duration with colesevelam would have been necessary to result in depletion of hepatic cholesterol with an associated possible amelioration of NASH [26]. Further, there are genetic, metabolic, and microbiome-related differences between rodents and humans, which could partially account for the different effects between the species. Additionally, it is important to note that metabolism is different between different liver diseases, disease stages, and associated other conditions including metabolic syndrome or diabetes mellitus. The inconsistent and sometimes contradictory results in preclinical and clinical studies about bile acid sequestrants in NASH could explain the lack of ongoing clinical trials of bile acid binders in NASH.
Colesevelam was previously found to suppress ileal Fgf15 expression due to its binding of bile acids, which subsequently derepresses hepatic Cyp7a1 expression via the Fxr-Fgf15-Cyp7a1 axis in a mouse model of high-fat dietinduced obesity [7], which we confirmed in our microbiomehumanized mice. Furthermore, it induces the other enzyme of the classic de novo bile acid synthesis pathway Cyp8b1 in relation to the Western diet-fed control mice. Conversely, the enzymes of the alternative/acidic bile acid synthesis pathway are not induced by colesevelam compared with Western dietfed controls. The main primary bile acids synthesized via the hepatic de novo bile acid synthesis pathways are cholic acid and chenodeoxycholic acid; their ratio is determined by the expression of Cyp8b1 [27]. The increased expression of Cyp8b1 in the colesevelam group relative to its Western diet-fed counterpart not treated with colesevelam likely skews the production in favor of cholic acid. This is consistent with studies in humans where colesevelam treatment preferentially increases cholic acid levels but decreases chenodeoxycholic acid levels [28]. Colesevelam binds intestinal bile acids, inhibits the enterohepatic circulation of bile acids and induces conversion of hepatic cholesterol into bile acids [6,7], which explains the significantly lower hepatic cholesterol content in Western diet-fed colesevelam-treated mice versus Western diet-fed controls despite similar total plasma bile acid levels. This liver cholesterol lowering effect is likely very important for the protection against dietinduced liver disease [26]. Additionally, we demonstrated that colesevelam decreases plasma LPS levels in Western diet-fed mice, which might at least partially be the result of increased intestinal expression of antimicrobial proteins. Furthermore, colesevelam likely reduces plasma LPS levels by directly binding intestinal LPS, as bile acid sequestrants are known to bind luminal LPS in the intestine [24]. As LPS contributes to the development of liver steatosis, inflammation, and fibrosis in diet-induced steatohepatitis [15,16], this LPS lowering effect represents another mechanism how colesevelam improves diet-induced liver disease.

Conclusion
In conclusion, colesevelam decreases Western diet-induced weight gain, hepatic inflammation, steatosis, and fibrosis, and insulin resistance in mice colonized with stool from patients with NASH, indicating a potential benefit of bile acid sequestration in obesity and NASH. However, larger clinical trials with bile acid sequestrants are required to better evaluate therapeutic effects in patients with obesity and NASH.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.