REVIEWS Beyond intestinal soap—bile acids in metabolic control Folkert Kuipers, Vincent W. Bloks and Albert K. Groen Abstract | Over the past decade, it has become apparent that bile acids are involved in a host of activities beyond their classic functions in bile formation and fat absorption. The identification of the farnesoid X receptor (FXR) as a nuclear receptor directly activated by bile acids and the discovery that bile acids are also ligands for the membrane-bound, G‑protein coupled bile acid receptor 1 (also known as TGR5) have opened new avenues of research. Both FXR and TGR5 regulate various elements of glucose, lipid and energy metabolism. Consequently, a picture has emerged of bile acids acting as modulators of (postprandial) metabolism. Therefore, strategies that interfere with either bile acid metabolism or signalling cascades mediated by bile acids may represent novel therapeutic approaches for metabolic diseases. Synthetic modulators of FXR have been designed and tested, primarily in animal models. Furthermore, the use of bile acid sequestrants to reduce plasma cholesterol levels has unexpected benefits. For example, treatment of patients with type 2 diabetes mellitus (T2DM) with sequestrants causes substantial reductions in plasma levels of glucose and HbA1c. This Review aims to provide an overview of the molecular mechanisms by which bile acids modulate glucose and energy metabolism, particularly focusing on the glucose-lowering actions of bile acid sequestrants in insulin resistant states and T2DM. Kuipers, F. et al. Nat. Rev. Endocrinol. advance online publication 13 May 2014; doi:10.1038/nrendo.2014.60

Introduction

Department of Pediatrics, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700RB Groningen, Netherlands (F.K., V.W.B., A.K.G.). Correspondence to: F.K. [email protected]

Bile acids are amphipathic steroids that are synthesized from cholesterol exclusively in the liver. Bile acids and bile-acid-like molecules are present in mam­m als and in nonmammalian species, such as fish and nematode worms. The immediate products of the biosynthetic pathways that convert water-insoluble cholesterol into water-soluble molecules with detergent properties are referred to as primary bile acids (Figure 1). These molecules are conjugated to either taurine (2‑­ aminoethanesulphonic acid) or glycine to increase hydrophilicity, secreted into the bile and then dis­char­ ged into the intestine. The chemical diversity of the bile acid pool present in the body is increased by conversion of primary bile acids into secondary bile acids by intestinal bacteria (Figure 1). Efficient active absorption of primary bile acids in the terminal ileum and pas­sive absorption of secondary bile acids from the colon, followed by hepatic uptake from the portal blood and resecre­tion into bile, results in the accumulation of a pool of bile acids within the body. This bile acid pool cycles between the liver and the intestine (enterohepatic circulation) with a frequency that is partially determined by the pattern of food intake. The existence of such a circulating pool ensures Competing interests F.K. declares that part of the original research referred to in this article was supported by TiPharma (T1-106) and that he received an unrestricted research grant from Daiichi Sankyo to evaluate the effects of colesevelam on bile acid metabolism in humans and animals. V.W.B. and A.K.G. declare no competing interests.

the presence of adequate bile acid concentrations (in the millimolar range) at sites of physiological actions; namely, in the bile canaliculi to promote bile formation, in the gallbladder to prevent cholesterol crystallization and in the intestinal lumen to facilitate the absorption of dietary fat and fat-soluble vitamins.1 For example, a bile acid pool with an average size of ~2 g that cycles ~10 times each day requires that the liver and intestine can transport ~20 g of bile acids every 24 h.2 Highly effective hepatic and intestinal transport systems have evolved to ac­commodate this flux. The individual components of the bile acid transport machinery, which comprise transporter proteins for uptake and excretion localized at the basolateral and apical membrane domains of hepatocytes and enterocytes (Figure 2), have been identified.3–5 Defects in these components have been implicated in human heritable diseases with severe phenotypes, such as progressive familial intrahepatic cholestasis (PFIC) types 1–3. Approximately 5% of the bile acids escape intestinal reabsorption and are lost in the faeces. This loss of ~0.6 g per day in humans is accurately compensated for by de novo bile acid synthesis in the liver to maintain the pool size and represents a major determinant of cholesterol turnover.6 The detergent properties of bile acids are determined by the number and orientation of the hydroxyl groups and the presence or absence of an amino acid moiety (Figure 1) and are crucial for biological functions.3–5,7,8 However, these properties might also impose a risk to cells exposed to high concentrations

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REVIEWS Key points ■■ Bile acids are amphipathic steroids derived from cholesterol that serve important physiological functions such as bile formation and intestinal fat absorption that are dependent on their detergent nature ■■ The discovery of farnesoid X receptor (FXR) and G-protein coupled bile acid receptor 1 (TGR5) as bile acid receptors that regulate glucose, lipid and energy metabolism has highlighted bile acids as key players in metabolic control ■■ Modulators of bile acid receptors have been developed as potential treatments for cholestatic liver diseases and metabolic diseases; organ-specific and/or gene-cluster-selective modulators are expected in the near future ■■ Type 2 diabetes mellitus (T2DM) is accompanied by a shift in primary bile acid synthesis towards cholic acid and a corresponding increase in the secondary bile acid deoxycholic acid ■■ Beneficial effects of bile acid sequestrants on glucose metabolism in patients with T2DM could reflect changed compartmentalization of the bile acid pool that modifies intestinal bile acid signalling

of bile acids. For example, bile acids can cause inflammation, apoptosis and liver cell necrosis. Consequently, protective mechanisms aimed at lowering intracellular bile acid concentrations, such as downregulation of cellular uptake systems and upregulation of detoxifying biotransformation reactions, become active during accumulation of bile acids. A method for ‘sensing’ bile acids is, therefore, required to ensure both the initiation of mechanisms to protect cells and tight regulation of the size of the bile acid pool. The discovery in 1999 that bile acids act as natural ligands for the transcription factor farnesoid X receptor (FXR; also known as bile acid receptor or nuclear receptor subfamily 1 group H member 4) provided a mechanistic framework for bile acid sensing.9–11 The expression of many genes that encode proteins involved in bile acid synthesis, transport and metabolism is directly controlled by bile acids through activation of FXR.5,12 Furthermore, bile acids were found to serve specific, hormone-like functions in the control of glucose, lipid and energy metabolism. The activation of FXR by bile acids modulates the expression of key metabolic genes; bile acids also influence metabolism through signalling via the membrane-bound, G‑protein coupled bile acid receptor 1 (also known as TGR5). Bile acids can be considered as metabolic integrators that are particularly active postprandially. This role could have additional dimensions through interactions between indi­vidual bile acids and the microbiome, which is another important factor in the maintenance of health.13–15 The microbiome plays a part in controlling the composition of the bile acid pool through deconjugation and dehydroxylation reactions, and hence also modulates bile acid signalling functions.16–18 Conversely, bile acids can inhibit bacterial growth and so influence the composition of the microbiome.19 Altered bile acid signalling might contribute to the development and/or worsening of components of the metabolic syndrome (hepatic steato­sis, low-grade inflammation, hypertriglyceridae­ mia, low levels of HDL cholesterol or hyperglycaemia).20 Conse­quently, interference with bile acid metabolism or with bile acid signalling cascades could represent novel t­reatment options.

This Review aims to provide a condensed overview of the modes of action by which bile acids can modulate glucose and energy metabolism, particularly focusing on clinically relevant aspects such as the benefi­cial metabolic effects of bariatric surgery and the glucose-­ lowering effects of bile acid sequestrants in type 2 di­abetes mellitus (T2DM).

Bile acids modulate metabolism Signal transduction pathways As shown in Figure 2, bile acids have key roles in signal transduction pathways that involve FXR, fibroblast growth factor 19 (FGF19) and TGR5. Once activated by bile acids, FXR promotes the release of circulating FGF19 from the ileum, which contributes to the regulation of hepatic bile acid synthesis and gallbladder function,21 as well as postprandial energy metabolism.22–24 In addition, an FXR-independent mode of bile acid signalling has been identified.25,26 This pathway involves bile-acidinduced activation of TGR5.27,28 Specific roles for bileacid mediated TGR5 signalling in the control of energy and glucose metabolism (for example, promotion of glucagon-like peptide 1 [GLP‑1] release by intestinal L cells) and in the regulation of enterohepatic circulation dynamics (for example, gallbladder function and intestinal motility) have been identified.27–32 FXR FXR is a member of the nuclear receptor superfamily of transcription factors that comprises 48 family members in humans.33 Activation by its ligand causes FXR to form a heterodimeric complex with another nuclear receptor family member, retinoic X receptor α (RXR), and bind to specific DNA elements within the promoter regions of target genes. Once bound to DNA, activated FXR recruits transcriptional co-regulators and the RNA polymerase machinery to initiate gene transcription.34,35 As shown in Figure 2, SHP, which encodes the SHP protein (small heterodimer partner, also known as nuclear receptor subfamily 0 group B member 2) is a key target gene of FXR. Indeed, most of the suppressive effects of FXR on genes encoding components of the bile acid synthesis cascade, as well as genes involved in metabolism, are predominantly mediated through activation of SHP.36–39 Notably, the SHP protein lacks a DNA-binding domain and functions as a transcriptional repressor by binding directly to a number of nuclear receptors such as liver receptor homolog 1 (LRH‑1, also known as nuclear receptor subfamily 5 group A member 2) to control expression of cholesterol 7α‑hydroxylase (also known as cholesterol 7α‑monooxygenase or CYP7A1; Figure 2). Expression of the FXR protein is high in organs that constitute the enterohepatic circulation (liver and intes­tine), but it is also present in white adipose tissue, kidney, adrenal gland, stomach, pancreas, endothelial cells, vascular smooth muscle cells and cells of the immune system.40–47 This distribution pattern indicates a broad spectrum of biological functions that seems to potentially involve cell types that lack high concentrations of bile acids (for example, adipocytes). Yet, one

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REVIEWS Cholesterol

Cholic acid

O

OH

OH

HO

Taurocholic acid O OH HO

NH

OH

O

S

O

OH OH

HO

Liver CDCA 3α,7αOH

Cholesterol (5-cholesten-3β-ol)

CA 3α,7α,12αOH

Primary bile acids αMCA 3α,6β,7αOH

Secondary bile acids

βMCA 3α,6β,7βOH

HCA 3α,6α,7αOH

ωMCA 3α,6α,7βOH

LCA 3αOH

MDCA 3α,6βOH

HDCA 3α,6αOH

UDCA 3α,7βOH

DCA 3α,12αOH

Intestine

Figure 1 | Schematic overview of primary and secondary bile acid species. The conversion of cholesterol into bile acids in the liver involves multiple enzymatic steps.137,138 The initial products of this cascade are the primary bile acids (CA and CDCA in humans and CA, αMCA and βMCA in rodents). Primary bile acids are conjugated to either taurine or glycine, secreted into the bile and stored in the gallbladder to be discharged into the intestinal lumen upon ingestion of a meal. The chemical diversity of the pool of bile acids is enhanced by the actions of intestinal bacteria to form secondary bile acids (DCA and LCA in humans). Abbreviations: αMCA, α‑muricholic acid (3α,6β,7α-trihydroxy‑5β-cholanoic acid); βMCA, β‑muricholic acid (3α,6β,7β-trihydroxy‑5β-cholanoic acid); ωMCA, ω‑muricholic acid (3α,6α,7β-trihydroxy‑5β-cholanoic acid); CA, cholic acid (3α,7α,12α-trihydroxy‑ 5β-cholanoic acid); CDCA, chenodeoxycholic acid (3α,7α-dihydroxy‑5β-cholanoic acid); DCA, deoxycholic acid (3α,12α-dihydroxy‑5β-cholanoic acid); HCA, hyocholic acid (3α,6α,7α-trihydroxy‑5β-cholanoic acid); HDCA, hyodeoxycholic acid (3α,6αdihydroxy‑5β-cholanoic acid); LCA, lithocholic acid (3α-hydroxy‑5β-cholanoic acid); MDCA, murideoxycholic acid (3α,6β-dihydroxy‑5β-cholanoic acid); UDCA, ursodeoxycholic acid (3α,7β-dihydroxy‑5β-cholanoic acid).

of the most intriguing phenotypic characteristics of whole-body FXR-deficient mice is the presence of small, in­sulin-resistant adipocytes.42 This phenotype is probably attributable to a role of FXR in the process of adipo­ cyte differentiation. 44,48 FXR deficiency also prevents weight gain and the development of hyperglycaemia in both the leptin-deficient ob/ob mouse model and in lean C57BL/6N mice during ageing.49–52 Two features of FXR biology require specific attention in the context of this Review. First, not all bile

acids are equally effective in the activation of FXR. The ranking of bile acids in this respect is, from the highest to the lowest potency: chenodeoxycholic acid, deoxycholic acid, lithocholic acid and cholic acid;9,10 the very hydrophilic (rodent-specific) bile acid α‑muricholic acid might actually exert antagonistic effects.17,53 Mouse studies have demonstrated the metabolic consequences of selective changes in bile acid composition.18,54,55 For instance, Haeusler et al. noted that a relative deficiency of 12α‑hydroxylated bile acids (cholic and deoxycholic acids) in mice contributes to a diabetic lipid phenotype through impaired FXR signalling. 54 This observation implies that changes in the composition of the bile acid pool that might occur in disease states, such as T2DM (discussed later), or as a consequence of dietary changes,56 can influence bile acid signalling in the body. Likewise, the dynamics of the enterohepatic circulation, as determined by gallbladder emptying or intestinal motility, is another relevant parameter that is influenced by disease and diet.57 Second, the expression and activity of FXR are modulated by metabolic status. The mRNA levels of FXR are markedly reduced in the livers of rodent models of type 1 diabetes mellitus and T2DM, whereas FXR gene expression in hepatocytes is modulated by glucose, possibly via intermediates of the pentose phosphate pathway.58 Another mode of FXR dysregulation that can occur in metabolic disease is post-translational modification of the protein.34 Thus, phosphorylation of FXR by protein kinase C promotes its transcriptional activity.59 The activity of FXR is also regulated by glucose fluxes in hepatocytes through direct O‑GlcNAcylation catalysed by the hexosamine biosynthetic pathway, which increases FXR protein stability and transcriptional activity.60 Finally, FXR is a target of the NAD-dependent protein deacetylase sirtuin‑1 that removes acetyl groups from modified lysine residues in histones and transcription factors.61 Acetylation increases FXR stability but inhibits its ability to heterodimerize with RXR, bind DNA and induce gene expression. In two well-established models of obesity and insulin resistance (ob/ob mice and mice fed a high-fat diet), elevated levels of acetylated FXR were detected in the liver, a situation that could be reversed by treatment with the sirtuin‑1 activator resveratrol.61 FGF19 A number of the physiological effects of bile acids are now known to be mediated by FGF19 (or its counterpart FGF15 in mice and rats). Expression of FGF19 is almost exclusively restricted to the terminal ileum, which corres­ponds to the site where bile acids are actively taken up by the ileal sodium/bile acid cotransporter (ASBT; Figure 2). Bile acids induce ileal expression of the FGF19 gene through FXR.62 In addition, fat-soluble vitamins A and D regulate FGF15 gene expression in mice via the vitamin D3 receptor (VDR) and retinoid-responsive nuclear receptors. 63 Diet1 was identified as another modulator of FGF15 production. The Diet1 protein is expressed in enterocytes of the small intestine and mutations in its gene confer resistance to hyper­lipidaemia

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REVIEWS Hepatocyte HNF4A

FGF15/19

β-Klotho

LXR

CYP7A1

FGFR4

Cholesterol

PXR

SHP Metabolic effects

BA

BA ABCB11

BA

NTCP

FXR

Enterocyte

BA

OSTα/β

SHP FXR BA

ASBT

BA resin

Diet1 FGF15/19

Metabolic effects

FGF15/19

BA

VDR

L-cell BA

GLP-1 Metabolic effects

GLP-1

TGR5 BA resin

Figure 2 | Schematic overview of bile acid signalling within the enterohepatic circulation. Bile acids are actively secreted from hepatocytes into the bile canaliculi by the ATP-dependent transporter ABCB11. Most bile acids are reabsorbed in the terminal ileum via ASBT. Within ileal enterocytes, bile acids stimulate production of FGF19 (FGF15 in mice). Upon reaching the liver, FGF19 activates signalling pathways that repress bile acid synthesis. The presence of a resin (sequestrant) in the intestinal lumen prevents ileal bile acid uptake and FGF19 production. Bile acids in the portal circulation are taken up by the liver via NTCP for re-secretion into bile. As hepatic extraction is not complete, part of the intestine-derived bile acids spill over to the systemic circulation to act on peripheral organs and tissues via FXR-initiated or TGR5-initiated signalling pathways. Bile acids present in the lower gastrointestinal tract (unbound and sequestrant-bound) are able to activate TGR5 on L cells to promote production and secretion of GLP‑1. Abbreviation: BA, bile acid.

and atherosclerosis in mice, which suggests that FGF15/ FGF19 action constitutes an important control point in the relationship between bile acid metabolism and lipid homeostasis.64 FGF19 is excreted from the enterocytes into the portal circulation by an unknown route and travels to the liver, where it exerts pleiotropic effects on hepatic bile acid, lipid and glucose metabolism. These effects require binding of FGF19 to FGF receptor 4 (FGFR4), which is localized at the plasma membrane of the hepatocytes. Interaction of FGFR4 with the accessory protein β‑klotho is necessary to activate signal­ling through an incompletely elucidated signal transduc­tion path­way, probably involving SHP to suppress CYP7A1, the rate-controlling enzyme in bile acid synthe­s is (Figure 2).62,65,66 Indeed, a postprandial rise in plasma levels of FGF19 precedes a reduction in the plasma levels of C4, a marker of hepatic bile acid synthesis, which highlights a potential causal relationship.67

The role of the FXR–FGF15 axis in the control of bile acid synthesis was confirmed in numerous rodent studies. 68,69 Nonetheless, the contributions of direct (FXR) and indirect (FGF15) control remain unclear, as does the level of coordination between these factors.70 Unexpectedly, FGF15-deficient mice are glucose intolerant and store less glycogen in their livers than do wild-type mice, whereas intravenous administration of FGF19 to overnight fasted wild-type mice stimulates hepatic glycogen synthesis. 23 Supplementation with FGF19 induces phosphorylation and inactivation of the α and β subunits of glycogen synthase kinase, which in turn leads to increased hepatic glycogen synthesis and increased glucose disposal. This effect is insulin-­ independent, which might explain why mice with defective insulin signalling retain the ability to store glycogen in their livers.71 Thus, increased ileal bile acid signalling induces an insulin-like effect with respect to glycaemic control but does not promote lipogenesis owing to the involvement of different signal transduction pathways.21 Of note, reduced plasma levels of FGF19 have been reported among patients with T2DM.72,73 TGR5 The TGR5 receptor is highly expressed in liver cells other than hepatocytes, including Kupffer cells and cholangiocytes, and also in gallbladder epithelial cells and immune cells.30 In addition, TGR5 is expressed in brown adipose tissue, the enteric nervous system, the central nervous system, muscle and in the small intestinal and colonic enteroendocrine L cells that produce GLP‑1 (Figure 2).31 Activation of TGR5 by bile acids, or by other endogenous ligands or synthetic agonists,74,75 triggers internalization of the receptor, increased intra­cellular levels of cAMP and activation of protein kinase A, which in turn leads to increased phosphorylation of target proteins. The consequences of TGR5 activation are cell-type-specific and comprise anti-inflammatory effects, gallbladder relaxation, increased intestinal motility, increased energy expenditure in brown adipose tissue and improved glucose metabolism and insulin sensitivity. The addition of cholic acid to a high-fat diet resulted in elevated plasma levels of bile acids that attenuated diet-induced obesity and insulin resistance in mice.76 Activation of TGR5 in brown adipose tissue by elevated circulating bile acid levels was postulated to increase energy expenditure through activation of the type 2 iodothyronine deiodinase that, in turn, leads to increased levels of active T4 and induction of genes involved in energy metabolism. These effects were absent in mice fed normal chow and the identity of the per­missive factor associated with intake of the high-fat diet has remained elusive. Suppression of bile acid synthesis and reduction of the bile acid pool using the FXR agonist GW4064 was associated with reduced energy expenditure, pronounced weight gain and glucose intolerance in mice fed a high-fat diet.77 Counter-intuitively, treatment with the bile acid sequestrant colestimide improved metabolic control in mice fed a high-fat diet through stimulation of energy expenditure, a change that

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REVIEWS coincided with activation of thermogenesis in brown adipose tissue.78 A role for TGR5 in energy homeostasis is further supported by observations that female (but not male) TGR5-deficient mice become more obese than wildtype controls when fed a high-fat diet.79 Further­more, treatment with a TGR5 agonist reduces the develop­ ment of obesity in wild-type mice under the same diet­ ary conditions.80 Analysis of TGR5 expression in human pe­r thyroid adipose tissue biopsy samples (obtained dur­ing thyroid surgery) showed that TGR5 expression was positive­ly correlated to the resting metabolic rate.81

Bariatric surgery and metabolic control Insights into the metabolic actions of bile acids have sparked interest in their potential role in (patho)physio­ logy, for instance in mediating the beneficial effects of bariatric surgery, which has become an important therapeutic option for morbid obesity. Evidence is accumulating that the metabolic improvements that occur as a result of the various surgical procedures available are not merely caused by mechanical restriction of meal size or malabsorption of macronutrients. A putative link to human energy metabolism was provided by a study that found plasma bile acid levels to be appreciably higher among patients with obesity after bariatric surgery than in weight-matched control individuals, suggesting that bile acids could contribute to improved metabolic control after weight-loss surgery.82 Nonethe­ less, a study that compared the metabolic consequences of two bariatric procedures to reduce morbid obesity reported that gastric bypass resulted in elevated fasting and post­prandial levels of bile acids and increased TGR5 si­gnalling when compared with gastric banding.83 The increased levels of bile acids associated with gas­ tric bypass did not, however, predict changes in glu­ cose homeostasis or energy metabolism. Steinert et al. com­pared time-dependent effects of gastric bypass and sleeve gastrectomy in patients with morbid obe­s­ity.84 Pre­surgery levels of plasma bile acids were lower in patients with obesity than in a cohort of healthy indi­ vi­duals. In both patient groups, marked increases in GLP‑1 levels and improved glycaemic control were evi­ dent at 1 week and 3 months after surgery. Yet, fasting and postprandial levels of bile acids were appreciably elevated only at 1 year after surgery, suggesting that an increased pool of circulating bile acids was not causal in this respect. In support of these findings, Brufau et al.85 compared hypermetabolic patients (indi­viduals in whom energy expenditure is high relative to body surface) with liver cirrhosis and elevated plasma bile acid levels to control individuals matched for sex, age and BMI and found that neither total nor individual plasma bile acid levels corre­lated with energy expenditure. However, another study that compared small groups of patients with a­lcohol-induced cirrhosis of the liver and healthy individuals did find a positive relationship between fasting plasma bile acid levels and energy metabolism.86 Clearly, the relevance of ele­ vated levels of circulating bile acids in the control of

energy metabolism after bariatric surgery remains to be fully established. Conversely, some data indicate that altered bile acid kin­etics owing to the surgically induced anatomical changes within the enterohepatic c­irculation—particularly upon Roux-en‑Y gastric bypass surgery—might be of physiological relevance. Indeed, statistically significantly elevated plasma FGF19 levels have been reported in patients with obesity after gastric bypass surgery,87–89 which is indicative of enhanced bile acid signalling in the terminal ileum. Elevated levels of FGF19 might improve the metabolic status of patients through effects on carbo­hydrate metabolism in the liver and white adipose tissue (reviewed elsewhere90). In addition, increased FGF19 levels could also act in the brain to induce insulin-­independent plasma glucose lowering.91 Likewise, enhanced intestinal bile acid signalling through TGR5, owing to the presence of elevated bile acid concentrations in the distal part of the intestinal tract, might be involved.27,28,31 In this scenario, excess bile acids present in the intestinal lumen activate TGR5 associated with L cells, which leads to secretion of GLP‑1 into the circulation to improve intestinal motility, as well as hepatic and pancreatic function to reduce insulin resistance.92 Indeed, gastric bypass surgery is associated with a dramatic increase in the secretion of GLP‑1 (as well as other gut hormones, such as peptide YY).93 The observed changes in the levels of gut hormones might contribute to a sustained improvement of glucose metabolism under such conditions.94

Targeting bile acid metabolism Given the new insights into bile acid functions discussed here, strategies for treatment of metabolic disease and cholestatic liver disease have been defined and tested in model systems and in selected patient groups (reviewed elsewhere27,95–102).

Bile acids Bile acids have been used for decades in the treatment of gallstones (chenodeoxycholic acid and ursodeoxycholic acid), cholestatic liver diseases (ursodeoxycholic acid) and genetic conditions that affect bile acid synth­ esis (cholic acid and chenodeoxycholic acid). Data on the overall metabolic consequences of such treatments are few and mainly descriptive in nature. 5 However, treatment of gallstones with chenodeoxycholic acid has been reported to reduce not only the levels of plasma tri­glycerides (a positive effect) but also levels of HDL cholesterol (a potentially negative effect), probably in part through FXR-mediated modulation of the expression of target genes, such as APOA1, APOC1, APOC3 and CETP. For bile acids and most of the synthetic compounds (for example, FXR agonists, discussed later) tested in animal models, it should be stressed that beneficial effects were only apparent under conditions of compromised health status (for example, in mouse models of genetic or diet-induced obesity and insulin resistance).27 Evidently, interference with bile acid signalling is effective only when the metabolic and signal

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REVIEWS Bile acid synthesis

Bile acid pool size

Baseline

Colesevelam

Baseline

Colesevelam

1,769 μmol per day

2,983 μmol per day

4,706 μmol

3,955 μmol

2,226 μmol per day

4,259 μmol per day

4,816 μmol

4,902 μmol

Controls

T2DM

CA

CDCA

DCA

Figure 3 | The effects of T2DM and sequestration on bile acid kinetics. The synthesis rates (CA, CDCA) and the pool sizes (CA, CDCA, DCA) of the major bile acid species in humans were quantified by stable isotope dilution techniques in male individuals with T2DM and control individuals without diabetes mellitus matched for age, sex and BMI, prior to treatment (baseline) and after 8 weeks of treatment with the bile acid sequestrant colesevelam. Data from Brufau et al.115 Abbreviations: CA, cholic acid (3α,7α,12α-trihydroxy‑5β-cholanoic acid); CDCA, chenodeoxycholic acid (3α,7α-dihydroxy‑5β-cholanoic acid); DCA, deoxycholic acid (3α,12α-dihydroxy‑5β-cholanoic acid); T2DM, type 2 diabetes mellitus.

transduction pathways are balanced at a new set point in response to a prevailing insulin-resistant, nutrient-rich state and/or when altered bile acid metabolism per se is causative in disease development.

FXR agonists A number of clinical studies have been initiated with synthetic FXR agonists, such as obeticholic acid (6α-­ethylchenodeoxycholic acid, also known as INT‑747), as potential therapies for T2DM and nonalcoholic steato­ hepatitis. 27,97 For example, a phase 2 double-blind ­placebo-controlled proof-of-concept study tested the effects of obeticholic acid in patients with both T2DM and nonalcoholic fatty liver disease.103 Administration of obeticholic acid (25 mg or 50 mg daily) for 6 weeks was well-tolerated, increased insulin sensitivity and reduced markers of liver inflammation and fibrosis. Extensive discussion of these findings is beyond the scope of this Review. Bile acid sequestrants During the past few years, bile acid sequestration, an ‘old trick’ to lower plasma LDL cholesterol levels for prevention of cardiovascular diseases, has showed unexpected beneficial effects among patients with hyperlipidaemia and T2DM. These effects may be explained by the role of bile acids in metabolic control. Interruption of the entero­hepatic circulation of bile acids by either ileal bypass surgery or administration of bile acid sequestrants, such as cholestyramine, colestipol and colesevelam, is effective in prevention of coronary heart disease by lowering LDL cholesterol.104,105 Withdrawal of bile acids from the circulation is also associated with slightly

elevated plasma levels of triglycerides and HDL cholesterol.20 Bile acid sequestrants in general, and the secondgeneration compound colesevelam in particular, also improve glycaemic control in patients with T2DM.106–113 Administration of colesevelam plus common anti­dia­ betic agents such as metformin caused a mean reduction in fasting blood glucose levels of –0.83 mmol/l and a drop in mean HbA1c level of 0.5%.114 The original studies that were the subject of a Cochrane review on this topic114 provided the basis for approval of colesevelam by the FDA as an adjunct therapy for glycaemic control in patients with T2DM in 2008. Data on the long-term effects of colesevelam on microvascular and macro­vascular complications and cardiovascular risk in patients with T2DM are still awaited. Mechanisms of action—clinical insights The mode of action by which sequestrants improve gly­ caemic control remains to be elucidated. Bile acid sequestration might act by reducing the size or changing the composition of the bile acid pool and/or by al­tering the kinetics of enterohepatic bile acid cycling. One study addressed this issue by quantitatively assessing bile acid metabolism in patients with T2DM using state-ofthe-art stable isotope techniques and quantifying the effects of colesevelam on bile acid metabolism in these patients.115 Male participants with T2DM and a group of control individuals matched for age, sex and BMI were studied at baseline and after 2 weeks and 8 weeks of treatment (Figure 3). The patients with T2DM had an increased cholic acid synthesis rate, an increased input rate of deoxycholic acid, a correspondingly increased deoxycholic acid pool size and a trend toward a smaller chenodeoxycholic acid pool size; however, the total bile acid pool size was unchanged. A preponderance of deoxycholic acid among patients with T2DM was also found in a study that used multiple experimental methods to detect metabolites in blood samples after fasting.116 These data are concordant with those from previous studies that reported altered bile acid metabolism in patients with T2DM;117–121 however, these earlier studies were restricted to measures of bile acid composition alone or total bile acid synthesis without data on individual bile acid species. As expected (but never before quantified), the effects of colesevelam on bile acid metabolism were both profound and specific for the type of bile acid.115 Treatment with colesevelam doubled the synthesis of cholic acid in both the diabetic and control groups, whereas chenodeoxycholic acid synthesis was stimulated in both groups to a lesser extent (Figure 3). The reduction of fasting and postprandial levels of FGF19 observed in both groups upon sequestrant therapy might have contributed to the differential derepression of bile acid synthesis. The cholic acid pool size was doubled after treatment in patients and controls, whereas the chenodeoxycholic acid and deoxycholic acid pool sizes were statistically significantly decreased. The differences in individual pool sizes balanced each other so that, unexpectedly, no statistically significant differences in total bile acid pool size were

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REVIEWS induced. As chenodeoxycholic acid and deoxycholic acid are more hydrophobic than cholic acid, colesevelam increased the hydrophilicity of the circulating bile acid pool (which, in theory, led to a decrease in the activity of bile acids on both FXR and TGR5). Also in this study,115 treatment with colesevelam reduced insulin resistance (as determined by HOMA) and fasting glucose and HbA1c levels. However, no relationships between any of the kinetic parameters of bile acid metabolism and changes in glucose metabolism or markers of insulin resistance were found. A double-blind randomized placebo-controlled study evaluated a 12‑week course of colesevelam therapy in a group of patients with T2DM who were also being treated with diet and exercise, sulphonylurea, metformin or a combination thereof.122 Stable isotope techniques were used to quantify relevant parameters of glucose metabolism in vivo. Compared with placebo, treatment with colesevelam improved the following measures of glycaemic control: plasma glucose levels, HbA1c levels, fasting plasma glucose clearance and glycolytic disposal of oral glucose. However, colesevelam did not alter gluco­neogenesis or the rate at which glucose levels increased in blood after an oral glucose load, which is a measure of the rate of intestinal absorption. Fasting endogenous glucose production and glycogenolysis both remained unchanged in the colesevelam group but showed an increase in the group assigned placebo. Treatment with colesevelam increased total levels of GLP‑1 (in line with previous animal experiments123) and improved β‑cell function; however, plasma insulin levels, hepatic glucagon levels and insulin resistance (as determined by HOMA) remained unchanged. Consistent with previous findings, 67,115 circulating levels of FGF19 were found to be reduced after bile acid sequestration. In another study, the effects of colesevelam on fasting and postprandial glucose metabolism were evaluated among patients with T2DM who were receiving monotherapy with metformin.124 Colesevelam decreased both fasting and postprandial plasma levels of glucose and HbA1c in the absence of any changes in the levels of insulin. Surprisingly, postprandial GLP‑1 concentrations were not altered by colesevelam in this study. Endogenous glucose production and glucose disposal remained unchanged; however, the rate at which meal-derived glucose appeared in the bloodstream was decreased by colesevelam, which suggests either increased splanchnic uptake of ingested glucose or delayed intestinal absorption. The partly differing outcomes of these two studies of colesevelam122,124 might be related to differences in adjuvant therapies and/or in analytical approaches. Mechanisms of action—experimental insights Animal studies have been performed to compare the effects of sequestration on the kinetics of bile acids and glu­cose in lean mice versus obese and diabetic (db/db) mice.125,126 Administration of colesevelam reduced intestinal bile acid absorption by 30% and stimulated hepatic bile acid

synthesis (particularly that of cholic acid) by twofold to threefold both in lean and obese mice.125 The size of the circulating bile acid pool was not reduced in response to colesevelam—meaning that increased faecal loss was accurately compensated for by hepatic synthesis— whereas plasma bile acid levels were lower in treated mice than in untreated mice.125 Colesevelam reduced the levels of plasma glucose and insulin resistance and increased the metabolic clearance of glucose (by 37%) in the db/db mice, but did not affect gluconeogenesis and total hepatic or endogenous glucose production in either of the mouse strains evaluated. Improved metabolic clearance of glucose in treated db/db mice was accompanied by normalization of strongly elevated acylcarnitine concentrations in muscle and plasma, particularly of the acylcarnitines containing saturated palmitic acid (C16:0) and stearic acid (C18:0) fatty acid species. Normalization of these markers of inefficient mitochondrial fatty acid oxidation might reflect an improved capability to switch to the utilisation of glucose.126 In contrast to the situation described for bariatric surgery, FGF19 is clearly unlikely to be involved in the meta­bolic improvement observed upon bile acid sequestrant therapy in humans and mice. Increased release of GLP‑1 from ileal L cells induced by fatty acids (that reach the ileum owing to defective micellar solubilization and impaired absorption) has been proposed to underpin the mechanism for improved glycaemic control in res­ponse to colesevelam in patients with T2DM.127 Although this mechanism probably contributes to the observed effects, it is now evident that bile acids can induce GLP‑1 secretion from the luminal side of the intestinal cells in the ileum and colon, even when bound to a sequestrant.27,53,93 Indeed, colonic administration of cholic acid in mice and healthy humans produces elevated levels of GLP‑1 in the circulation.128,129 Likewise, mice that lack the ileal bile acid uptake protein ASBT (Figure 2) exhibit improved glycaemic control when fed a high-fat diet that is associated with elevated plasma GLP‑1 levels, as do mice treated with an ASBT inhibitor.130 Clearly, in both situations, there is overflow of bile acids into the colon owing to defective uptake from the ileum. Induction of intestinal GLP‑1 expression by high levels of sequestrant-bound bile acids and unabsorbed fatty acids could contribute to the metabolic improvements observed in db/db mice. 125 Intriguingly, high expression levels of hepatic FGF21 were also found in these mice in response to colesevelam.126 Overexpression of FGF21 in the livers of diabetic rodents improves glucose clearance owing to the actions of FGF21 on muscle and adipose tissue.131–133 In the aforementioned studies,125,126 no quantitative effects of bile acid sequestration on hepatic glucose fluxes were found. However, Potthoff et al.128 reported that treatment of diet-induced obese mice with colesevelam reduced hepatic glucose production by suppression of hepatic glycogenolysis. This effect was partly ascribed to activation of TGR5 and release of GLP‑1 because it could be blocked by a GLP‑1 antagonist. Accordingly, the ability of colesevelam to reduce glycaemia, spare hepatic

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REVIEWS glycogen and induce secretion of GLP‑1 was compromised in TGR5-deficient mice. 128 Thus, although the influence of bile acid sequestration on glycaemic control is evident in metabolically compromised humans and animals, the underlying mechanism(s) seem to vary in accordance with the prevailing metabolic status.

Future perspectives The area of bile acid research has become tremendously active in the past few years. A number of beneficial and adverse metabolic effects have been linked to interference with bile acid homeostasis and signalling mediated by bile acids. Interestingly, both increased (following bariatric surgery) and decreased (upon use of sequestrants) plasma bile acid levels were shown to be associated with beneficial effects on energy metabolism and glycaemic control in humans. The exact reason for this apparent contradiction is not yet clear but may involve differential effects on the dynamics of enterohepatic cycling of the different bile acid species by these interventions. In this respect, more insight into the metabolic actions of the individual bile acid species in vivo is required. From a therapeutic point of view, development of organ-­selective and/or gene-cluster-specific modulators of FXR is warranted. Agonists that selectively target intestinal FXR may be particularly useful for treatment of obesity-­associated disorders. Lately, the interaction between intestinal microbiota and bile acid metabolism has become en vogue and data indicate that, in addition to the so-called short-chain fatty acids, bile acids may act as prime signal transducing molecules between microbiota and host metabolism.134 Many of these studies have been carried out in animal models; it is important to note that major differences in bile acid metabolism exist across the animal kingdom that have to be taken into account when interpreting the results. In the current Review, we have focused on the interactions of bile acids with FXR and TGR5 and ignored other nuclear receptors such as pregnane X receptor, constitutive androstane receptor and VDR that also interact with certain bile acid species. The interaction between the various nuclear 1.

2.

3. 4.

5.

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Hofmann, A. F. The enterohepatic circulation of bile acids in mammals: form and functions. Front. Biosci. 14, 2584–2598 (2009). Stellaard, F., Sackmann, M., Sauerbruch, T. & Paumgartner, G. Simultaneous determination of cholic acid and chenodeoxycholic acid pool sizes and fractional turnover rates in human serum using 13C-labeled bile acids. J. Lipid Res. 25, 1313–1319 (1984). Meier, P. J. & Stieger, B. Bile salt transporters. Annu. Rev. Physiol. 64, 635–661 (2002). Trauner, M., Wagner, M., Fickert, P. & Zollner, G. Molecular regulation of hepatobiliary transport systems: clinical implications for understanding and treating cholestasis. J. Clin. Gastroenterol. 39, S111–S124 (2005). Lefebvre, P., Cariou, B., Lien, F., Kuipers, F. & Staels, B. Role of bile acids and bile acid receptors in metabolic regulation. Physiol. Rev. 89, 147–191 (2009). Brufau, G., Groen, A. K. & Kuipers, F. Reverse cholesterol transport revisited: contribution of biliary versus intestinal cholesterol excretion.

receptor networks in determining the overall metabolic activity of bile acids in vivo deserves further attention.

Conclusions Evidence is accumulating that bile acids play a crucial but complex part in coordinating the whole-body res­ ponse to ingestion of food. The physiological and chemical properties of bile acids are essential for effective uptake of dietary fat and fat-soluble vitamins. By activating signal transduction pathways via the receptors FXR and TGR5, bile acids also contribute to the integration of an adequate postprandial response. These pathways pro­vide promising targets for pharmacological intervention in metabolic diseases. However, in view of the complexity of the metabolic networks involved, the dynamic nature of the enterohepatic circulation and its constituents, and the fact that signalling pathways mediated by bile acids can simultaneously exert health-­promoting and adverse effects, a ‘one size fits all’ approach will not be successful. Clearly, both pathway-specific and celltype-­s elective interference will be required to ensure t­herapies are effective. In conclusion, bile acids should not be considered as merely ‘intestinal soap’ but rather as integrators and mod­ulators of key metabolic responses. Exposure to high levels of bile acids in fetal life (for example, in the off­ spring of mothers with cholestasis during preg­nancy) can program susceptibility to metabolic dis­ease later in life.135,136 These observations underscore the important relationship between bile acids and the control of metabolism. Review criteria PubMed was searched for relevant topics, using the search terms “bile acids” or “bile salts” in combination with “sequestrants”, “resins”, “type 2 diabetes”, “glycemic control”, “glucose-lowering mechanisms”, “energy metabolism”, “FXR”, “TGR5”, “FGF15”, “FGF19”, “microbiome” and “metabolic control”, without publication time constraints. References cited in this article include both original research and reviews by experts in the field.

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Author contributions F.K. researched the data for the article, provided substantial contribution to discussions of the content, contributed equally to writing the article and reviewed and/or edited the manuscript before submission. V.W.B. researched the data for the article and reviewed and/or edited the manuscript before submission. A.K.G. provided substantial contribution to discussions of the content, contributed equally to writing the article and reviewed and/or edited the manuscript before submission.

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