Essential roles of bile acid receptors FXR and TGR5 as metabolic regulators

Abstract

Alterations of bile acid (BA) metabolism in type II diabetes (T2D) have revealed a link between BAs and metabolic homeostasis. The BA receptors farnesoid X receptor (FXR) and G-protein coupled BA receptor 1 (TGR5) both have shown to regulate lipid, glucose, and energy metabolism, rendering them potential therapeutic targets for T2D therapy. Astonishingly, BA signaling is vital as it has known to be a positive factor for beneficial improvements in vertical sleeve gastrectomy surgery, in turn making these BA receptors beneficial tools in therapeutic targets for T2D. These metabolic regulators: FXR and TGR5 are essential modulators in the metabolic system, which affect the human machinery and will be the focus of this review.

Keywords:

• bile acid
• FXR
• TGR5

Introduction

Type II diabetes (T2D) has escalated dramatically in party by the increase in obesity and its correlation with metabolic disorders. Aside from their given function of lipid emulsification and absorption, bile acids (BAs) are responsible for homeostasis of the glucose/lipid metabolism and energy expenditure to control whole body energy metabolism. Thus, the fact that BAs are endogenous ligands for the nuclear BA receptor farnesoid X receptor (FXR) and the G-protein coupled BA receptor 1 (TGR5) creates an avenue for new perspectives on the physiological roles of BA signaling in monitoring the complete metabolic system. Thus, it is pertinent to develop novel therapeutic approaches targeting FXR and TGR5 to solve metabolic conundrums such as obesity, insulin resistance, and eventually T2D.

FXR: nuclear BA receptor

As a member of the nuclear receptor superfamily, FXR contains both DNA and ligand binding domains (Forman et al. 1995). A heterodimer is formed with retinoid X receptor (RXR) by FXR when binding is initiated by endogenous ligands, the binding with BAs initiates the RXR/FXR heterodimer binding to the FXR response element which is located in the promoter regions of FXR target genes. An abundant expression of FXR has been reported in the hepatic liver as well as adrenal glands, kidney, and intestinal tract (Forman et al. 1995; Lee et al. 2006; Modica et al. 2008, 2010; Zhang et al. 2014), where it functions as an intracellular sensor of BAs to control BA synthesis in the liver (Makishima et al. 1999; Lu et al. 2000; Chiang 2002; Li-Hawkins et al. 2002; Wang et al. 2002). Recent discoveries have demonstrated that FXR is responsible to regulate hepatic glucose production, lipid metabolism, liver regeneration, intestinal homeostasis, function of pancreatic β cells, and urine concentration in the kidney (Edwards et al. 2002; Huang et al. 2006; Jiang et al. 2007; Popescu et al. 2010; Renga et al. 2010; Zhang et al. 2004, 2006, 2014).

FXR and BA metabolism

FXR regulates endogenous levels of BAs by cholesterol catabolism. Synthesis of BAs is restricted to hepatocytes from which two pathways are involved: key enzymes, cholesterol-7α-hydroxylase (CYP7A1), and sterol-27-hydroxylase (CYP27A1), respectively (Chiang 2002; Russell 2003). BAs are conjugated to amino acids (taurine or glycine) upon synthesis which will then be secreted into the bile canaliculi (Chiang 2002; Russell 2003; Li & Chiang 2013); BAs make their ways down the small intestine and reach the ileum, ~95% are recycled and transported back to the liver via enterohepatic circulation (Chiang 2002; Russell 2003; Li & Chiang 2013). This leaves ~5% of the BAs excluded in each enterohepatic cycle; therefore, the liver is compelled to compensate an equivalent amount to maintain a constant pool size of BAs. As a result, a stringent-regulated process is required to control cholesterol/bile aid metabolism. The increase of binding BAs to FXR enhances transcriptional activation in FXR target gene expression dependent on an increase intracellular BA levels. FXR is responsible for induction of small heterodimer partner, which suppresses gene expression of CYP7A1 to reduce the rate of hepatic BA synthesis via negative feedback (counterbalance; Lu et al. 2000; Li-Hawkins et al. 2002; Wang et al. 2002). BA synthesis has another means of regulation, and that is through activation of FXR in enterocytes. Upon BA activation in enterocytes of small intestine, fibroblast growth factor 15 (FGF15) expression rises through enhanced FXR transcriptional activity (Inagaki et al. 2005; Song et al. 2009). Succeedingly, FGF15 binding to fibroblast growth factor receptor 4 (FGFR4) in hepatocytes promotes activation of the c-Jun N-terminal kinases (JNK) pathway, which in turn suppresses CYP7A1 gene expression to reduce BA synthesis (Song et al. 2009). Because FXR is also a gene regulator of BA secretion and transporter, it would be safe to say that FXR is responsible for BA synthesis and metabolism.

FXR and glucose/lipid metabolism

Recent studies have revealed FXR as a regulator to control carbohydrate metabolism, and it has also been demonstrated to control the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK; Stayrook et al. 2005). This enzyme located in the liver is vital because it catalyzes a crucial step in hepatic gluconeogenesis. Impaired glucose tolerance and insulin resistance in FXR-null mice revealed the important role of hepatic FXR on glucose homeostasis (Claudel et al. 2005; Duran-Sandoval et al. 2005; Ma et al. 2006). Aside from regulating PEPCK and glucose-6-phosphatase (G6pc), the FXR activation also raises glycogen levels in hepatocytes via enhancing downstream insulin receptor signaling (Zhang et al. 2006).

An extended amount of research using FXR-null mice showed that FXR affects lipid metabolism. Previous discoveries have found these FXR-null mice exhibited elevated levels of plasma triglycerides and cholesterol (Lambert et al. 2003; Zhang et al. 2006). In addition, these FXR-null mice also exhibited higher than normal levels of lipoprotein (HDL) cholesterol in plasma which correlates with a reduced hepatic expression of scavenger receptor class B member 1, a receptor known to make clearance of HDL cholesterol from the blood (Lambert et al. 2003). When activated with FXR agonist, wild type mice exhibited less plasma triglycerides (Lambert et al. 2003; Zhang et al. 2006); furthermore, FXR revealed its influence by regulating a set of genes involved in lipoprotein metabolism including SREBP-1c, phospholipid transfer protein, SCD-1, the very low density lipoprotein receptor, apolipoprotein C-II, and apolipoprotein E (Lambert et al. 2003).

Therefore, FXR is a key regulator of lipid metabolism to control fatty acid and triglyceride synthesis and cholesterol homeostasis.

FXR and liver regeneration

Morphologic reconstruction and sequential changes in gene expressions induced by specific stimuli play important roles in liver regeneration. The adaptive liver regeneration responses are due to a highly complex network of signal transductions. Recent studies have shown BAs as key metabolic signaling modulators to regulate FXR downstream pathway during liver regeneration after a 70% partial hepatectomy (PHx; Huang et al. 2006; Fan et al. 2014); the regenerative effect has been debilitated in FXR-null mice, suggesting that FXR is an integral transcriptional factor to regulate BA signaling to promote the reconstruction of the liver.

FXR and intestinal homeostasis

Bile duct ligation has been shown to reduce BA levels in the distal small intestine, leading to bacterial overgrowth in small intestine and eventually bacterial translocation. Notable studies have indicated that FXR plays a protective role to control intestinal homeostasis by which the intestinal tract avoids damage and inflammation from bacterial overgrowth (Inagaki et al. 2006). Bile duct ligation is a model to show a compromised BA homeostasis as well as low traits of FXR activation in intestine. Animals with bile duct ligation clearly exhibited bacterial overgrowth in intestine and bacterial translocation into other tissues. However, FXR agonist treatment attenuated mucosal injury and reduced bacterial growth in the intestine. Given that FXR regulates gene expressions involved in epithelial barriers and antimicrobial peptides, such as inducible nitric oxide synthase (iNOS), occludin, and interleukin 18 (IL-18), FXR may play a key role to protecting against bacteria-mediated intestinal inflammation.

FXR and pancreatic β cells physiology

It was previously reported that FXR is present in pancreatic β cells and FXR activation in β cells has an impact on systemic glucose homeostasis (Popescu et al. 2010; Renga et al. 2010; Dufer et al. 2012b). Usually, FXR is translocated into nucleus when activated by ligand. Interestingly, previous study reported that localization of FXR in the β cells is context-dependent. In lean mice, FXR is predominantly localized in the cytosol, whereas FXR is mainly localized in the nucleus in obese mice (Popescu et al. 2010). Moreover, FXR activation has been shown to protect human islets from lipotoxicity and increase insulin secretion of pancreatic β cells (Renga et al. 2010).

In addition, FXR plays a role as a cytosolic and non-genomic effector to control K_ATP currents in pancreatic β cells (Dufer, Hörth, Krippeit-Drews, et al. 2012; Dufer, Horth, Wagner, et al. 2012). FXR activation is responsible for insulin augmentation and secretion by increasing Ca²⁺-influx to reduce the K_ATP current in β cells. In contrast, FXR-null mice showed a reduction of insulin content and glucose-stimulated insulin secretion (Dufer, Horth, Wagner, et al. 2012). Altogether, FXR activation may be an important therapeutic approach for treating T2D to restore glucose-dependent insulin secretion in pancreatic β cells.

FXR and kidney homeostasis

The kidney regulates water and sodium secretion though a counter-current system which expels urine. Although roles of FXR in the renal area are not directly understood, concentration of urine is due to in part the roles of FXR in renal tubes and renal collecting ducts. Identification of the ilea apical sodium and BA transporter in the kidney and localization of the FXR target genes OSTα and OSTβ on the basolateral surface of renal tubular cells are consistent with the idea that FXR-mediated BA signaling may control the function of these transporters to maintain kidney homeostasis. With FXR agonist treatment, FXR inhibited expression of lipogenic genes, including SREBP-1c. Besides lipogenic gene regulation, FXR also regulated TGF-β and IL-6 gene expression, suggesting that FXR modulates renal lipid metabolism, fibrosis, and inflammation in the kidney (Jiang et al. 2007). In addition, a recent study has shown FXR as a participant in water and sodium homeostasis in the kidney (Zhang et al. 2014). FXR orchestrates urine volume in mice. FXR activation reduces urine volume and increases urine osmolality in animals through induction of apical aquaporin 2 (AQP2; Zhang et al. 2014). In contrast, with a reduction in osmolality, there was an increased urine output in FXR-null mice, suggesting the ability of FXR to concentrate urine became attenuated in FXR-null mice. Altogether, these findings reveal a unique mechanism in the maintenance of renal water homeostasis by FXR-mediated BA signaling.

TGR5: a membrane BA receptor

TGR5 is a member of the rhodopsin-like superfamily of G protein-coupled receptor that transduces signals through Gs protein. Numerous studies have shown that lithocholic acid (LCA) and taurolithocholic acid (TLCA) are potent endogenous ligands to activate TGR5 at EC₅₀ of 600 and 300 nM, respectively. The receptor becomes activated though other BAs such as cholic acid, deoxycholic acid, and chenodeoxycholic acid with higher micromolar concentrations. Expression of TGR5 varies from organ to organ; endocrine glands, adipocytes, muscles, immune organs, spinal cord, and the enteric nervous system (Duboc et al. 2014). TGR5 stimulates cAMP synthesis and activation of the mitogen-activated protein kinase (MAPK) pathway (Duboc et al. 2014). The increase in intracellular cAMP activates protein kinase A, which phosphorylates cAMP response element binding protein to transactivate its target gene expressions by binding to cAMP response element in the target promoter regions. Previous studies have shown that TGR5 plays a key role in the regulation of energy expenditure (Watanabe et al. 2006); TGR5 is now recognized as a potential target for the treatment of metabolic disorders, including as simple as obesity, insulin resistance, and eventually T2D. Moreover, TGR5 also has been shown to regulate inflammatory cytokine levels in monocytes, suggesting that TGR5 is now largely involved to regulate immune responses in human pathologies.

TGR5 and energy expenditure in brown adipose tissue

Previous reports describe how TGR5 is associated with energy expenditure in brown adipose tissue (BAT) by increasing basal metabolic rate (Watanabe et al. 2006, 2012). The increase in energy expenditure prevents diet-induced weight gain and insulin resistance in mice. Given that TGR5 is independent of FXR activity, Taurocholic acid (TCA)-mediated TGR5 activation increases intracellular cAMP level to induce expression of type II iodothyronine deiodinase (Dio2) in BAT. The downstream occurs by induction of Dio2 gene expression in BAT, D2 converts thyroxine (T4) to tri-iodothyronine (T3), resulting in an increased energy expenditure without the changes in circulating thyroid hormone levels. In addition to Dio2, several genes involved in thermogenesis and energy expenditure were increased in BAT by BA treatment: peroxisome proliferator-activated receptor γ coactivator-1α (PCG1α) and 1β (PGC1β), uncoupling protein-1 (UCP1) and uncoupling protein-3 (UCP3), and straight-chain acyl-CoA oxidase 1. The increase of energy expenditure by BA was not observed in D2 knockout mice. These findings highlight the role of a TGR5-cAMP-D2-thyroid hormone axis to regulate energy homeostasis.

TGR5 and glucose homeostasis

TGR5 has been demonstrated to stimulate the secretion of glucagon-like peptide (GLP) 1, a member of the incretins family (Katsuma et al. 2005; Thomas et al. 2009). Upon minutes of consuming nutrition, intestinal enteroendocrine cells secrete insulinotropic gastrointestinal hormones into the bloodstream. The incretin then mediates insulin secretion which accounts for least 50% of the total insulin secretion after an oral glucose load. BAs have been shown to induce GLP1 secretion via TGR5 activation from rodent colon and enteroendocrine cell lines through a cAMP-dependent mechanism. Eventually, treatment of synthetic ligand for TGR5 also induce GLP1 secretion in enteroendocrine cells to stimulate insulin secretion in pancreatic β cells, resulting in a decrease of blood glucose level in animal model (Thomas et al. 2009). These studies suggest that TGR5 activation restores insulin secretion to reduce blood glucose level via modulating secretion of incretins in high fat diet-induced obese mouse model.

TGR5 in immune cells: macrophages and Kupffer cells

TGR5 has been recently reported to be expressed in macrophage and reduce inflammation in adipose tissue using macrophage-specific TGR5 knockout mouse model (Perino et al. 2014). This recent study reported that adipose tissue from obese mice lacking TGR5 in macrophages exhibited enhanced inflammation, increased chemokine expression, and higher macrophage numbers in adipose tissue, leading to insulin resistance. Overall, the roles of TGR5 to modulate chemokine expression would be therapeutic target to prevent obesity-related metabolic syndromes, such as insulin resistance.

It has been speculated that hepatocytes lack TGR5 expression, there are two different cell types that exhibit TGR5 expression in the liver: sinusoidal endothelisal cells (SECs) and Kupffer cells. Exposure to high levels of LCA and TLCA induces cAMP-dependent activation to induce gene expression of endothelial nitric oxide (NO) synthase (eNOS) and the production of NO in SECs. Thus, the increase of NO protects the hepatic endothelial cells from oxidative stress and lipid peroxidation, suggesting that TGR5 plays a key role to regulate intrahepatic microcirculation (Keitel et al. 2007).

The roles of TGR5 in Kupffer cells, a type of liver-resident macrophage, are still controversial. Previous study has shown that TGR5 activation reduced cytokine release and inflammatory processes to attenuate immunoactivity in Kupffer cells (Keitel et al. 2008). The authors showed that TGR5 activation inhibited lipopolysaccharide induced cytokine expression via cAMP-dependent pathway in Kupffer cells. In contrast, a recent study showed that TGR5 mediates the BA-induced pro-inflammatory cytokine production in Kupffer cells through JNK-dependent pathway (Lou et al. 2014). The authors showed that TGR5 activation alone induced the expression of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in Kupffer cells. This induction of IL-1β and TNF-α was blunted in JNK-null mice. Thus, the roles of TGR5 in Kupffer cells to modulate cytokine expression still remain unclear.

TGR5 and intestinal homeostasis

Recent study has revealed the involvement of TGR5 to maintain epithelial barrier layers in intestinal tract (Cipriani et al. 2011). As opposed to wild type mice, TGR5-null mice exhibited an abnormal morphology of colonic mucous cells and altered architecture of epithelial tight junctions expression, resulting in increased intestinal permeability and susceptibility to intestinal inflammation. TGR5-null mice developed severe colitis in response to DSS, an experimental model of colitis at early stage of life. These studies suggest that TGR5 regulates intestinal barrier structure to maintain intestinal homeostasis.

FXR and TGR5 as therapeutic targets in human pathologies

For many years, the molecular mechanisms of bariatric surgery for beneficial effects have not been clear. Sleeve gastrectomy surgery has been shown to limit the stomachs capacity, resulting in weight loss; in obese mouse models, there has been sufficient evidence suggesting that a surgical weight loss procedure requires the FXR-mediated BA signaling to exhibit a reduction in body weight gains and improvements in glucose tolerance (Ryan et al. 2014). Altogether, these changes reconstitute the gut bacterial composition to stimulate host metabolic functions (Ryan et al. 2014). It has been shown that patients with bariatric surgery usually exhibit higher circulating BA levels which are most definitely correlated to the secretion of GLP-1 (Liu et al. 2011; Rabiee et al. 2011; Gerhard et al. 2013; Kohli et al. 2013). Also, obese patients consistently exhibit a decreased postprandial BA response and GLP-1 secretion as opposed to subjects with normal body weight. Given that TGR5 participates in GLP-1 secretion in response to BAs, TGR5 would be an ideal target for intervening in metabolic disorders.

Both FXR and TGR5 have provided a novel therapeutic approach to improve metabolic syndromes for which numerous studies have revealed their beneficial effects on many physiological processes. The development of novel synthetic agonists for FXR and TGR5 is likely to bring beneficiary responses; these receptors may be useful therapeutic targets for metabolic disorders.