Pharmacological potential of novel agonists for FFAR4 on islet and enteroendocrine cell function and glucose homeostasis
Graphical abstrcat
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 10–12–10–4 mol/l were assessed using clonal pancreatic BRIN-BD11 cells. At 5.6 mM glucose, Compound A at 10–10–10–4 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 2.9×10–7 mol/l, while GSK137647 was more potent (EC50 of 2.2×10–7 mol/l) with a 1.5- to 2.1-fold increase at 10–8–10–4 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.
References (40)
- et al.
Mechanisms of enhanced insulin secretion and sensitivity with n-3 unsaturated fatty acids
J. Nutr. Biochem.
(2015) - et al.
Differential effects of fat and sucrose on body composition in A/J and C57BL/6 mice
Metabolism
(1998) - et al.
The orphan g protein-coupled receptor GPR40 is activated by medium and long chain fatty acids
J. Biol. Chem
(2003) - et al.
Mechanisms of homologous and heterologous phosphorylation of FFA receptor 4 (GPR120): GRK6 and PKC mediate phosphorylation of Thr(3)(4)(7), Ser(3)(5)(0), and Ser(3)(5)(7) in the C-terminal tail
Biochem. Pharmacol.
(2014) - et al.
Free fatty acid receptors act as nutrient sensors to regulate energy homeostasis
Prostaglandins Other Lipid Mediat.
(2009) - et al.
Lipid-mediated release of GLP-1 by mouse taste buds from circumvallate papillae: putative involvement of GPR120 and impact on taste sensitivity
J. Lipid Res.
(2012) - et al.
GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects
Cell
(2010) - et al.
Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120)
Bioorg. Med. Chem. Lett.
(2014) - et al.
Alteration of the glucagon axis in GPR120 (FFAR4) knockout mice
J. Biol. Chem.
(2014) - et al.
Novel GPR40 agonist AS2575959 exhibits glucose metabolism improvement and synergistic effect with sitagliptin on insulin and incretin secretion
Life Sci.
(2014)
The gut hormone peptide YY regulates appetite
Ann. N. Y. Acad. Sci.
Contribution of the low-frequency, loss-of-function p.R270H mutation in FFAR4 (GPR120) to increased fasting plasma glucose levels
J. Med. Genet.
High-fat diets: modeling the metabolic disorders of human obesity in rodents
Obesity (Silver Spring)
Activity of dietary fatty acids on FFA1 and FFA4 and characterisation of pinolenic acid as a dual FFA1/FFA4 agonist with potential effect against metabolic diseases
Br. J. Nutr.
Nature of action of Sitagliptin, the dipeptidyl peptidase-IV inhibitor in diabetic animals
Indian J. Pharmacol.
Structural requirements for the cytoprotective actions of mono-unsaturated fatty acids in the pacreatic beta-cell line, BRIN-BD11
Br. J. Pharmacol.
Sitagliptin
Nat. Rev. Drug. Discov.
Abnormal plasma glucose and insulin responses in heterozygous lean (ob/+) mice
Diabetologia
Role of metabolically generated reactive oxygen species for lipotoxicity in pancreatic β-cells
Diabetes Obes. Metab.
Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120
Nat. Med.
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