Pinolenic acid co-activates free fatty acid receptors 1 and 4 and this signalling reduces GPR119 agonism in mouse colon mucosa

R. Moodaley¹, M. Schindler², D. M. Smith³, H. M. Cox¹. ¹Wolfson Centre for Age-Related Diseases, King's College London, London, United Kingdom, ²Innovative Medicines & Early Development, Cardiovascular & Metabolic Diseases iMed, AstraZeneca Mölndal, Mölndal, Sweden, ³Discovery Sciences, Innovative Medicines & Early Development Biotech Unit, AstraZeneca, Cambridge, United Kingdom.

Introduction: Pinolenic acid (PNO) appears to co-activate free-fatty acid receptors 1 (FFA1) and 4 (FFA4) (1). FFA1 and FFA4 are co-expressed by enteroendocrine L cells (2,3) (alongside unrelated GPR119 (4)) and their stimulation by different triglyceride metabolites release peptide YY (PYY) and glucagon-like peptide 1. FFA1, FFA4 and GPR119 are therefore potential targets for treating obesity and type II diabetes. The aim of this study was to establish the mechanisms of PNO-induced signalling in the presence or absence of a proven GPR119 agonist, PSN632408 (5).

Methods: Mouse (>10 wks; C57BL/6, male and female) colonic mucosal sheets were voltage-clamped (0 mV, at 37^°C), recording changes in epithelial ion transport as a read-out of L cell function (6). Mucosae were pretreated with an FFA1 antagonist (ANT825, 10μM, apically (ap)), or Y₁ + Y₂ antagonists (BIBO3304 (300nM) + BIIE2046 (1μM) basolaterally (bl)), or tetrodotoxin (TTX, 100nM, bl) or vehicles. PNO (1 μM, ap) was added after intermediary vasoactive intestinal polypeptide (30 nM, bl) either alone or with a GPR119 agonist (PSN632408, 10μM, ap). Responses were followed for 20 min and expressed as mean ± 1SEM (μA.cm^-2).

Results: Colonic PNO responses exhibited an EC₅₀ of 298.2 nM (132.8 - 669.7 nM) which was similarly potent and efficacious as the commercial dual FFA1/4 agonist, GW9508 (EC₅₀ of 354.8 nM (191.6 - 656.8 nM)). At 1 μM, PNO responses were significantly inhibited by ANT825 (57.7 ± 6.8 %, P ≤ 0.001, n=5, Student’s t-test), abolished by Y₁ + Y₂ antagonists (99.9 ± 9.9 %, P ≤ 0.001, n=5, Student’s t-test) but were TTX-insensitive (P ≥0.05). Responses to PNO or a maximal concentration of a GPR119 full agonist, PSN632408 were not additive, in fact co-addition of PNO and PSN632408 reduced peak GPR119 responses by 51.5 ± 10.0 % (n=5).

Conclusions: Mucosal PNO responses are partially FFA1-mediated via endogenous L cell derived PYY and independent of enteric nerves, as observed with selective FFA1 agonists (7). Residual PNO responses are likely FFA4-mediated and notably this lipid attenuates coincident GPR119 efficacy, exposing limited L cell signalling in native tissue.

References:

1. Christiansen E et al. (2015) Br. J. Nutr 113, 1677-88

2. Hirasawa A et al (2005) Nat Med 11, 90-4

3. Edfalk S et al. (2008) Diabetes 57, 2280-7

4. Ekberg J et al (2016) Endocrinol 157, 4561-4569

5. Overton H et al. (2006) Cell Metab 3, 167-175

6. Cox HM et al. (2010) Cell Metab 11, 532-42

7. Moodaley R et al (2017) Br.J Pharmacol 174, 4508-4522