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

Abstract

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 long-chain 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.

Figure 1.

FFAs stimulate NT secretion from neuroendocrine cells. (a) STC-1 cells were stained with NT antibody and observed by confocal microscope using the 60× oil objective. (b) STC-1 cells were treated with or without PMA (100 nM) or sodium oleate (0.25 mM) for 30 minutes; media were collected and NT EIA performed. *P < 0.05 vs DMSO. (c) BON cells were treated with or without different FFAs at various dosages for 3 hours; media were collected and NT EIA performed. *P < 0.05 vs control; ^†P < 0.05 vs 10 and 30 μM of OA; ^‡P < 0.05 vs 10 μM of POA; ^#P > 0.05 vs 10 μM of LA; ^&P > 0.05 vs 30 μM of LA. (d–f) BON, QGP-1, and STC-1 cells were treated with or without different concentrations of DHA or 10 nM PMA (a positive control) for 3 hours; media (upper panels) and cells (lower panels) were analyzed by NT EIA and western blot, respectively. (d) BON cells: *P < 0.05 vs DMSO; ^†P < 0.05 vs 10, 30 μM DHA; (e) QGP-1 cells: *P < 0.05 vs DMSO; ^†P < 0.05 vs 10, 30 μM DHA; (f) STC-1 cells: *P < 0.05 vs DMSO; ^†P < 0.05 vs 10 μM DHA; ^‡P < 0.05 vs 30 μM DHA. All data represent mean ± SD. Experiments were repeated at least three times. BA, butyric acid; DMSO, dimethyl sulfoxide; PA, palmitic acid.

Figure 2.

FFAR1 and FFAR4 are involved in stimulation of NT release. (a) Total RNA was isolated from BON and QGP-1 cells and qPCR performed targeting human FFAR1 and FFAR4. GAPDH was used as the internal control. (b) STC-1 cells were treated with or without FFAR1 A3 (10 μM), FFAR4 A3 (10 μM), cpdA (10 μM), and OA (0.5 mM) for 1 hour. FSK (10 μM) was used as a negative control. (c) BON cells were treated with or without GW 9508 or TUG 891 in various dosages for 3 hours. PMA (10 nM) was used as a positive control. Media were collected and NT EIA performed (upper panel: *P < 0.05 vs DMSO; ^†P < 0.05 vs 0.1, 1.0 μM GW; ^‡P < 0.05 vs 0.1, 1.0 μM TUG). Cells were lysed and western blotting analysis performed (lower panel). (d, e) BON cells were pretreated with (d) FFAR1 A3 (upper panels: *P < 0.05 vs DMSO; ^†P < 0.05 vs FFAR1 A3 alone) or (e) TUG 891 (upper panels: *P < 0.05 vs DMSO; ^†P < 0.05 vs DHA; ^‡P < 0.05 vs TUG alone) for 30 minutes, followed by DHA (5 μM) with or without the agonists for 3 hours. Media were collected and NT EIA performed (upper panels); cells were lysed and western blotting analysis performed (lower panels). All data represent mean ± SD. Experiments were repeated at least three times. A3, agonist III; DMSO, dimethyl sulfoxide; FSK, forskolin; GW, GW 9508; TUG, TUG 891.

Figure 3.

Double knockdown of FFAR1 and FFAR4 decreased, but overexpression of FFAR4 increased, NT release. (a) BON cells were doubly transfected with FFAR1 (30 nM) and FFAR4 (5 nM) siRNAs and grown for 24 hours. Cells were collected, total RNA extracted, and qPCR performed targeting FFAR1 and FFAR4. *P < 0.05 vs NTC siRNA. (b) BON cells, doubly transfected with FFAR1 and FFAR4 siRNAs, as in (a), were treated with DHA (100 μM) or TUG 891 (10 μM) for 3 hours. Media were collected and NT EIA performed (upper panel); cells were lysed for western blotting analysis (lower panel). Upper panels: *P < 0.05 vs DMSO in NTC siRNA; ^†P < 0.05 vs DHA in NTC siRNA; ^‡P < 0.05 vs TUG 891 in NTC siRNA. (c) BON/NEG and BON/FFAR4-HA cell lines were treated with or without GW 9508 and TUG 891 for 3 hours. Media were collected and NT EIA performed (upper panel) and cells lysed for western blotting analysis (lower panel). Upper panels: *P < 0.05 vs DMSO in BON/NEG; ^†P < 0.05 vs GW 9508 in BON/NEG; ^‡P < 0.05 vs TUG 891 in BON/NEG. All data are mean ± SD. Experiments were repeated at least three times. DMSO, dimethyl sulfoxide; GW, GW 9508; NTC, nontargeting control; TUG, TUG 891.

Figure 4.

NT release in vivo. (a) SIs of C57BL/6 mice were divided into four fragments. Mucosa was scraped, total RNA isolated, and qPCR performed targeting mouse NT. (b) Jejunal perfusions were performed and plasma NT measured by NT EIA; TDC (vehicle), n = 8; DHA, n = 9. *P < 0.05 vs TDC. (c) Scraped mucosa from (b) were analyzed by western blot (left panel). Intensity of p-ERK was analyzed by ImageJ software and normalized with total ERK. (d) Ileal perfusions were performed and plasma NT measured by NT EIA. TDC (vehicle), n = 8; DHA, n = 9. (e) Scraped mucosa from (d) were analyzed by western blot (left panel). Intensity of p-ERK (middle panel) and p-AMPK (right panel) was analyzed by ImageJ software and normalized with total ERK or AMPK. (f) C57BL/6 mice were gavaged with saline or cpdA for 1 hour, followed by saline or OO for 30 minutes. Plasma NT was measured by NT EIA. N = 3 mice per group. *P < 0.05 vs saline alone; ^†P < 0.05 vs olive oil alone; ^‡P < 0.05 vs cpdA alone. All data represent mean ± SD. OO, olive oil; SI, small intestine; TDC, taurodeoxycholate.

Figure 5.

MAPK/ERK signaling is involved in FFA-stimulated NT secretion. (a) STC-1 cells were pretreated with various dosages of PD for 30 minutes, followed by 0.5 mM OA for 1 hour. Media were collected and NT EIA performed (upper panel) and cells lysed for western blotting analysis (lower panel). Upper panel: *P < 0.05 vs DMSO alone; ^†P < 0.05 vs OA in DMSO; ^‡P < 0.05 vs DMSO alone; ^#P < 0.05 vs 1, 5 nM PD. (b) Both BON/NTCsh and BON/ERK1sh cells were treated with or without DHA (100 μM) or LA (100 μM) for 1 hour. PMA (10 nM) was used as a positive control. Media were collected and NT EIA performed (upper panel) and cells lysed for western blotting analysis (lower panel). Upper panel: *P < 0.05 vs DMSO in BON/NTCsh; ^†P < 0.05 vs PMA in BON/NTCsh; ^‡P < 0.05 vs DHA in BON/NTCsh; ^#P < 0.05 vs LA in BON/NTCsh. All data represent mean ± SD. Experiments were repeated at least three times. DMSO, dimethyl sulfoxide.

Figure 6.

Inhibition of AMPK signaling further increased NT secretion stimulated by GW 9508 or TUG 891. (a,b) BON cells were pretreated with or without CC (10 μM) for 30 minutes, followed with or without PD (10 nM) for another 30 minutes and then by combined treatment of (a) GW 9508 (10 μM) or (b) TUG 891 (10 μM) with CC or PD for 3 hours. Media were collected and NT EIA performed (upper panels) and cells lysed for western blotting analysis (lower panels). Upper panels: *P < 0.05 vs DMSO; ^†P < 0.05 vs GW 9508 or TUG 891 alone; ^‡P < 0.05 vs GW 9508 or TUG 891 plus CC. (c,d) BON cells were pretreated with CC (10 μM) for 30 minutes and then with or without AICAR (1 mM) for another 30 minutes, followed by combined treatment of (c) GW 9508 (10 μM) or (d) TUG 891 (10 μM) with AICAR or CC for 3 hours. Media were collected and NT EIA performed. *P < 0.05 vs DMSO; ^†P < 0.05 vs GW 9508 or TUG alone; ^‡P < 0.05 vs GW 9508 or TUG 891 plus AICAR. Experiments were repeated at least three times. (e) Summary of signaling downstream of FFAR1 and FFAR4 in regulation of NT release. DHA and FFAR1 or FFAR4 agonists activate FFAR1 and FFAR4 and increase NT secretion through activation of ERK1/2. PD compound inhibits NT secretion by inhibiting MEK/ERK1/2 signaling. Inhibition of MEK/ERK or AMPK releases the inhibitory regulation of each other. DMSO, dimethyl sulfoxide; GW, GW 9508; TUG, TUG 891.