FFAR4 Is Involved in Regulation of Neurotensin Release From Neuroendocrine Cells and Male C57BL/6 Mice

RE SE AR CH A RT IC LE FFAR4 Is Involved in Regulation of Neurotensin Release From Neuroendocrine Cells and Male C57BL/6 Mice Jing Li,1,2 Jun Song,1,2 Xian Li,1,2 Stephanie B. Rock,1,2 Heather F. Sinner,1,2 Heidi L. Weiss,2 Todd Weiss,2 Courtney M. Townsend Jr.,3 Tianyan Gao,2 and B. Mark Evers1,2 1 Neurotensin (NT), a 13 amino-acid peptide, is predominantly released from enteroendocrine cells of the small bowel in response to fat ingestion. Free fatty acid receptors (FFARs) FFAR1 and FFAR4 regulate secretion of gut hormones and insulin. Here, we show that docosahexaenoic acid, a longchain fatty acid, has the most dramatic effect on NT release. FFAR1 agonists slightly stimulate and FFAR4 agonists dramatically stimulate and amplify NT secretion. Double knockdown of FFAR1 and FFAR4 decreases NT release, whereas overexpression of FFAR4, but not FFAR1, increases NT release. Administration of cpdA, an FFAR4 agonist, but not TAK-875, a selective FFAR1 agonist, increases plasma NT levels and further increases olive oil–stimulated plasma NT levels. Inhibition of MAPK kinase (MEK)/ERK1/2 decreased fatty acid–stimulated NT release but increased AMP-activated protein kinase (AMPK) phosphorylation. In contrast, inhibition of AMPK further increased NT secretion and ERK1/2 phosphorylation mediated by FFAR1 or FFAR4. Our results indicate that FFAR4 plays a more critical role than FFAR1 in mediation of fat-regulated NT release and in inhibitory crosstalk between MEK/ERK1/2 and AMPK in the control of NT release downstream of FFAR1 and FFAR4. (Endocrinology 159: 2939–2952, 2018) N eurotensin (NT), a tridecapeptide initially identified and characterized by Carraway and Leeman (1), is released from N cells of the small bowel in response to intraluminal fats (2–6). NT affects glucose homeostasis through a glucose-sensitive promotion of insulin and pancreatic polypeptide secretion (7–9). We have shown that NT-deficient mice are protected from obesity, hepatic steatosis, and insulin resistance associated with consumption of high levels of fat (10). Moreover, in human longitudinal studies among nonobese subjects, high levels of pro-NT denoted a doubling of the risk of obesity developing later in life. Importantly, our findings directly link NT with increased fat absorption and obesity, and suggest that NT may provide a prognostic marker of future obesity and a potential target for prevention and treatment. Therefore, a better understanding of the molecular mechanisms regulating NT secretion is required to delineate the effects of NT during physiologic and pathologic conditions. Free fatty acids (FFAs) can be classified according to their chain length as short-chain fatty acids (carbon chain length, 1 to 6); medium-chain fatty acids (MCFAs; carbon chain length, 7 to 12); and long-chain fatty acids (LCFAs; carbon chain length .12) (11). Physiologic ISSN Online 1945-7170 Copyright © 2018 Endocrine Society Received 22 March 2018. Accepted 10 May 2018. First Published Online 22 May 2018 Abbreviations: ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; ALA, a-linolenic acid; AMPK, AMP-activated protein kinase; BON/NEG, BON negative control vector; BON/NTCsh, BON nontargeting control small hairpin RNA; [Ca2+]i, intracellular calcium; CC, compound C; DHA, docosahexaenoic acid; EIA, enzyme immunoassay; ERK1sh, ERK 1 small hairpin RNA; FA, fatty acid; FBS, fetal bovine serum; FFA, free fatty acid; FFAR, free fatty acid receptor; FFAR4-HA, hemagglutinintagged free fatty acid receptor 4; GLP-1, glucagon-like peptide-1; HA, hemagglutinin; LA, lauric acid; LCFA, long-chain fatty acid; MCFA, medium-chain fatty acid; MEK, MAPK kinase; NT, neurotensin; NTCsh, nontargeting control small hairpin RNA; OA, oleic acid; p-AMPK, phosphorylated AMP–activated protein kinase; PD, PD 0325901; p-ERK1/2, phosphorylated ERK1/2; PMA, phorbol 12-myristate 13-acetate; POA, palmitoleic acid; qPCR, quantitative PCR; siRNA, small interfering RNA. doi: 10.1210/en.2018-00284 Endocrinology, August 2018, 159(8):2939–2952 https://academic.oup.com/endo 2939 Department of Surgery, University of Kentucky, Lexington, Kentucky 40536; 2Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536; and 3Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555 2940 Li et al FFAR1/4 Regulation of Neurotensin Secretion glucose-stimulated insulin secretion in pancreatic b-cells to maintain glucose homeostasis (30, 31). We have shown that activation of AMPK stimulates NT secretion from endocrine cells through the inhibition of mTORC1 and negative feedback activation of ERK1/2 (32, 33). In the present study, we established that docosahexaenoic acid (DHA), a polyunsaturated LCFA, has the greatest effect on stimulation of NT secretion and the involvement of FFAR1 and FFAR4 in NT release in vitro and in vivo. Furthermore, ERK1/2 is functionally involved as a downstream effector of FFAR1 and FFAR4 signaling. Importantly, we describe an inhibitory crosstalk model between MAPK and AMPK signaling cascades in which both signaling pathways can serve in an inhibitory role to control the release of NT mediated by FFAR1 and FFAR4 activation. Materials and Methods Reagents Phorbol 12-myristate 13-acetate (PMA), sodium oleate, oleic acid (OA), a-linolenic acid (ALA), DHA, palmitoleic acid (POA), lauric acid (LA), palmitic acid, and butyric acid were obtained from Sigma-Aldrich (St. Louis, MO). Potent and selective FFAR1 (GW 9508) and FFAR4 (TUG 891) agonists were obtained from Tocris (Minneapolis, MN); GPR40 agonist III and cpdA from MilliporeSigma (Billerica, MA); and Fasiglifam (TAK-875), a selective FFAR1 agonist, from Selleckchem (Houston, TX). Phospho-AMPKa (Thr172) (34), AMPKa (35), phospho-ERK1/2 (Thr202/Tyr204) (36), ERK1/2 (37), phospho-acetyl-CoA carboxylase (ACC; Ser79) (38), and ACC (39) antibodies were obtained from Cell Signaling Technology (Danvers, MA); NT antibody (40) from Abcam (Cambridge, MA); b-actin antibody (41) and sodium taurodeoxycholate from Sigma-Aldrich; and Alexa Fluor 488 Secondary antibody (42) from Thermo Fisher Scientific (Grand Island, NY). ON-TARGETplus SMARTpool (FFAR1 and FFAR4) and ON-TARGETplus Nontargeting Control Pool small interfering RNA (siRNA) were purchased from GE Dharmacon (Lafayette, CO); ERK1 short hairpin RNA (ERK1sh) and the nontargeting control short hairpin RNA (NTCsh) in bacterial glycerol stock from Sigma-Aldrich; hemagglutinin (HA)tagged FFAR4 (FFAR4-HA) and the negative control lentiviral vectors from GeneCopoeia (Rockville, MD); and compound C (CC; AMPK inhibitor) and PD 0325901 [PD; MAPK kinase (MEK) inhibitor] from Cayman (Ann Arbor, MI). Cell culture, transfection, and treatment The BON cell line was derived from a human pancreatic carcinoid tumor (male), which has been characterized (43, 44). BON cells were maintained in a 1:1 mixture of DMEM and nutrient mixture, F12K, supplemented with 5% fetal bovine s (...truncated)