Pharmacological potential of novel agonists for FFAR4 on islet and enteroendocrine cell function and glucose homeostasis

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Abstract

Background

To investigate the metabolic effects of FFAR4-selective agonists on islet and enteroendocrine cell hormone release and the combined therapeutic effectiveness with DPP-IV inhibitors.

Methods

Insulinotropic activity and specificity of FFAR4 agonists were determined in clonal pancreatic BRIN-BD11 cells. Expression of FFAR4 was assessed by qPCR and western blotting following agonist treatment in BRIN-BD11 cells and by immunohistochemistry in mouse islets. Acute in-vivo effects of agonists was investigated after intraperitoneal (i.p.) or oral administration in lean and HFF-obese diabetic mice.

Results

GSK137647 (1011–104 M) and Compound-A (1010–104 M) stimulated insulin secretion at 5.6 mM (p < 0.05-p < 0.001) and 16.7 mM (p < 0.05-p < 0.001) glucose in BRIN-BD11 cells, with no cytotoxicity effects as assessed by MTT. FFAR4 antagonist (AH-7614) abolished the insulintropic effect of GSK137647 (p < 0.05-p < 0.001), whilst FFAR1 antagonist (GW1100) had no effect. Incubation of BRIN-BD11 cells with GSK137647 and Compound-A increased FFAR4 (p < 0.01) gene expression at 16.7 mM glucose, with a corresponding increase in FFAR4 (p < 0.01) protein concentrations. FFAR4 upregulation was attenuated under normoglycaemic conditions. Immunohistochemistry demonstrated co-localisation of FFAR4 and insulin in mouse islets. Orally administered GSK137647 or Compound-A (0.1 µmol/kgBW) improved glucose tolerance (p < 0.001), increased plasma insulin (p < 0.001), GLP-1 (p < 0.05), GIP (p < 0.05) and induced satiety (p < 0.001) in HFF mice, with glucose-lowering effects enhanced in combination with DPP-IV inhibitor (Sitagliptin) (p < 0.05).

Conclusions

Specific FFAR4 agonism improves glucose tolerance through insulin and incretin secretion, with enhanced DPP-IV inhibition in combination with Sitagliptin.

General significance

These findings have for the first time demonstrated that selective FFAR4 activation regulates both islet and enteroendocrine hormone function with agonist combinational therapy, presenting a promising strategy for the treatment of type-2-diabetes.

Introduction

FFAR4 (GPR120) is a rhodopsin-like G-protein coupled receptor that is activated by unsaturated fatty acids (C16-22) and long chain saturated fatty acids (C14-18) (Hirasawa et al., 2005; Ulven and Christiansen, 2015). The human FFAR4 gene is encoded on chromosome 10.q23.3 (Ichimura et al., 2009). With respect to tissue distribution, FFAR4 is extensively expressed in peripheral tissues, intestines, lungs, spleen and pro-inflammatory macrophages (Milligan et al., 2017). Furthermore, recent studies have demonstrated that FFAR4 is abundantly expressed in the pancreatic islet, with further analysis demonstrating its expression in clonal pancreatic β-cell lines, including MIN6, RINm5f and INS-1E (Dhayal et al., 2008; Gehrmann et al., 2010; BM Moran et al., 2014).

Previously considered as orphan receptors, recent studies have shown FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), FFAR4 (GPR120) and GPR84 to be activated by free fatty acid (FFA) molecules (Ichimura et al., 2009; Moran et al., 2016; Milligan et al., 2017). FFAR3 and FFAR2 exhibit specificity towards short chain fatty acids, GPR84 is activated by medium chain fatty acids, whereas FFAR1 and FFAR4 are activated by long chain fatty acids (Ichimura et al., 2009; Milligan et al., 2017). Furthermore, FFAR4 and FFAR1 share 10% sequence homology and can be activated by similar endogenous ligands (Omega-3-fatty acids), which warrants the utilisation of receptor specific agonists to evaluate the therapeutic potential of FFAR4 (Ulven and Christiansen, 2015; Moran et al., 2016).

FFAR4 has been hypothesised to act as a lipid sensor in the body, and has been proven to have involvement in the regulation of inflammation, adipogenesis, and glucose homeostasis (Hirasawa et al., 2005; Oh et al., 2010; BM Moran et al., 2014). Interestingly, it has been reported that a mutation in the FFAR4 gene (R270H) is linked with the development of obesity. The p.R270H variant impairs the signalling response of FFAR4 upon FFA binding, with subsequent defects observed to intracellular calcium mobilisation and GLP-1 secretion in intestinal cells (Ichimura et al., 2012; Bonnefond et al., 2015). Further studies have demonstrated that FFAR4 knockdown with siRNA impaired the anti-apoptotic effects of omega-3 fatty acids in serum-starved STC-1 cells. Thus, indicating the potential proliferative and anti-apoptotic effects of FFAR4 in pancreatic beta cells (Iakoubov et al., 2007).

Numerous studies have identified the involvement of FFAR4 in the gastrointestinal (GI) tract, including the mediation of glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP) and cholecystokinin (CCK) secretion from intestinal l-cells, K-cells and I-cells, with high FFAR4 expression observed in the intestinal STC-1 and GLUTag cell lines (Hirasawa et al., 2005; Iakoubov et al., 2007; Sankoda et al., 2017). FFAR4 activation has been shown to mediate GLP-1 secretion when tested with its endogenous agonist α-linolenic acid (Hirasawa et al., 2005; Tanaka et al., 2008; Bhaswant et al., 2015). However, other studies suggest that FFAR4 has no role in GLP-1 release (Paulsen et al., 2014). Previous findings have shown FFAR4 to mediate insulin-sensitising and anti-inflammatory properties in peripheral tissues (Oh et al., 2010).

The expression and biological function of FFAR4 in the intestinal tract has been heavily documented, however the role of FFAR4 in pancreatic beta cell function was not investigated until recently (BM Moran et al., 2014). A number of FFAR4 agonists were demonstrated to have regulatory role in glucose dependent insulin secretion in mouse islets, including endogenous docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA) and synthetic GW-9508 (BM Moran et al., 2014; Moran et al., 2016). In addition, these agonists demonstrated insulinotropic and glucose lowering properties in-vivo (BM Moran et al., 2014). However, the selectivity of endogenous FFAR4 agonists (ALA, DHA, EPA) remains uncertain as activation of FFAR1 may contribute to the effects observed, whilst synthetic GW9808 has been shown to exhibit 100-fold greater potency towards FFAR1 over FFAR4 (Briscoe et al., 2003; Christiansen et al., 2015).

Upon activation, FFAR4 primarily couples to Gαq, which stimulates an array of secondary messenger signalling pathways through phospholipase C (PLC), including intracellular calcium and mitogen-activated protein kinases (BM Moran et al., 2014; Milligan et al., 2017). The mechanism of FFAR4 mediated insulin secretion from the pancreatic beta cell is not conclusive; however, studies have shown a range of FFAR4 agonists to induce intercellular calcium release, indicating the potential involvement of inositol trisphosphate on intracellular calcium stores through PLCβ signalling (BM Moran et al., 2014). FFAR4 activation with ALA and DHA leads to the rapid and transient phosphorylation of the receptor of HEK293 cells (Burns et al., 2014). Although FFAR4 has been shown to act predominately through PKC signalling, DHA has also shown to activate G-protein coupled receptor kinase (GPK6) upon FFAR4 phosphorylation, with Thr(347), Ser (350), and Ser(357) shown to be major phosphorylation sites in the C-terminal tail of FFAR4 (Burns et al., 2014).

Recently, Oh et al., have reported an orally available, selective, high affinity, small FFAR4 agonist (Compound A) that exhibits a range of anti-diabetic effects (Oh et al., 2014). Oral administration of Compound A improved glucose tolerance, insulin sensitivity and exerted anti-inflammatory effects on macrophages in high fat fed obese mice (Oh et al., 2014). Sparks et al., recently identified a potent FFAR4 agonist GSK137647 (Sparks et al., 2014) and preliminary in-vitro analysis has demonstrated that GSK137647 augmented insulin secretion in MIN6 cells, with a modest increase in GLP-1 secretion from the NCl-H716 intestinal cell line (Martin et al., 2012; Sparks et al., 2014). In-vivo findings showed that GSK137647 induced GLP-1 release by mouse circumvallate papillae (Martin et al., 2012). The highly selective properties of this agonist suggested that it was suitable to evaluate FFAR4 activation in pancreatic beta cells.

Due to the regulatory role of FFAR4 activation on insulin and incretin secretion (Hirasawa et al., 2005; Tanaka et al., 2008; BM Moran et al., 2014), a promising approach using selective FFAR4 agonists combined with a dipeptidyl peptidase-4 (DPP-IV) inhibitor may offer therapeutic potential (Drucker et al., 2007; Tanaka et al., 2014). The present study has assessed the effect of potent DPP-IV inhibitor (Sitagliptin) in combination with Compound A and GSK137647 on glucose tolerance and insulin secretion in high fat fed (HFF)-induced diabetic mice. This research aims to investigate the acute metabolic effects and of FFAR4 agonist monotherapy and combinational therapy on islet and enteroendocrine cell function, using pancreatic cells and diabetic mice.

Section snippets

. Materials

FFAR4 agonists Compound A and GSK137647 were purchased from Cayman Chemicals (Michigan, USA) and Tocris (Bristol, UK) respectively. Sitagliptin phosphate monohydrate was obtained from Apexbio Technology LLC (Texas, USA). Thiazolyl blue tetrazolium bromide (MTT) was received from Sigma (Poole, UK). Rabbit anti-GPR120 polyclonal IgG antibody (H-155) was purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA) and guinea pig anti-insulin from Abcam (Cambridge, UK).

. Insulin secretion

Generation and

. Determination of FFAR4 agonist selectivity on insulin secretion

The insulinotropic response and specificity of the novel synthetic FFAR4 agonists (Compound A, GSK137647) at 1012–104 mol/l were assessed using clonal pancreatic BRIN-BD11 cells. At 5.6 mM glucose, Compound A at 1010–104 mol/l augmented insulin secretion by 1.2- to 1.9-fold (p<0.05-p<0.001), with a half maximal effective concentration (EC50) of 2.9×107 mol/l, while GSK137647 was more potent (EC50 of 2.2×107 mol/l) with a 1.5- to 2.1-fold increase at 108–104 mol/l (p < 0.05-p

. Discussion

Recent interest in long chain fatty acid receptors has intensified due to identification of their involvement in the maintenance of glucose homeostasis through GPCR signalling. FFAR1 (GPR40) (Itoh et al., 2003), GPR55 (McKillop et al., 2013; McKillop et al., 2016) and GPR119 (BM Moran et al., 2014; McKillop et al., 2016) have been previously reported to regulate islet function and hormone secretion. In particular, orally administered FFAR1 agonist TAK-875 (Fasiglifam) entered stage III clinical

Author contribution

AGMC conducted the investigation, methodology, validation. formal analysis and writing – original draft. MGM conducted the investigation. PRF involved in the writing- review and editing and supervision. AMMK involved in the conceptualization, formal analysis, supervision writing – review and editing.

Acknowledgement

These studies were supported by Diabetes UK. AGMC conducted the investigation, methodology, validation, formal analysis and writing – original draft. MGM conducted the investigation. PRF involved in the writing- review and editing and supervision. AMMK involved in the conceptualization, formal analysis, supervision writing – review and editing.

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