Mercaptoacetate blocks fatty acid-induced GLP-1 secretion in male rats by directly antagonizing GPR40 fatty acid receptors

Abstract

Mercaptoacetate (MA) is an orexigenic agent reported to block fatty acid (FA) oxidation. Recently, however, we reported evidence from isolated nodose ganglion neurons that MA antagonizes the G protein-coupled long- and medium-chain FA receptor GPR40. GPR40 mediates FA-induced secretion of the satietogenic incretin peptide glucagon-like peptide 1 (GLP-1), by enteroendocrine L cells, as well as FA-induced enhancement of glucose-stimulated insulin secretion. Our results in cultured nodose neurons suggest that MA would also block GPR40 in enteroendocrine cells controlling GLP-1 secretion. If so, this would suggest an alternative mechanism by which MA increases food intake. We tested the hypothesis that MA blocks FA-induced GLP-1 secretion in vitro using cultured STC-1 cells (a murine enteroendocrine cell line) and in vivo in adult male rats. In vitro, MA blocked the increase in both cytosolic Ca(2+)and GLP-1 release stimulated by FAs and also reduced (but less effectively) the response of STC-1 cells to grifolic acid, a partial agonist of the GPR120 FA receptor. In vivo, MA reduced GLP-1 secretion following olive oil gavage while also increasing glucose and decreasing insulin levels. The carnitine palmatoyltransferase 1 antagonist etomoxir did not alter these responses. Results indicate that MA's actions, including its orexigenic effect, are mediated by GPR40 (and possibly GPR120) receptor antagonism and not by blockade of fat oxidation, as previously believed. Analysis of MA's interaction with GPR40 may facilitate understanding of the multiple functions of this receptor and the manner in which FAs participate in the control of hunger and satiety.

Keywords: G protein-coupled receptor 120; G protein-coupled receptor 40; fatty acids; glucagon-like peptide-1; mercaptoacetate.

Fig. 1.

Effects of linoleic acid (LA) on cytosolic Ca²⁺ levels ([Ca²⁺]_i) in STC-1 cells. A and B: LA increased [Ca²⁺]_i in STC-1 cells. A: trace showing [Ca²⁺]_i in one individual STC-1 cell perfused with different doses of LA (0.1–5 μM) and a high-potassium (Hi-K) solution (55 mM), indicated by bars above traces. B: averages of peak responses vs. baseline (BL). n = 8–24 cells/treatment. *P < 0.001 vs. BL. C and D: antagonistic effects of mercaptoacetate (MA) on LA-induced [Ca²⁺]_i in STC-1 cells. C: trace of Ca²⁺ levels of one STC-1 cell perfused with LA (0.5 μM) with or without a pretreatment with MA (0.2 mM) applied 2 min before LA (indicated by bars above traces). D: averages of peak responses vs. BL. n = 17 cells/treatment. *P < 0.001 vs. BL. E and F: effects of MA on Hi-K-induced [Ca²⁺]_i in STC-1 cells. E: trace showing one STC-1 cell perfused with Hi-K (55 mM) and LA (1 μM) with or without MA (0.2 mM). F: averages of peak responses vs. BL. n = 22 cells/treatment. *P < 0.001 vs. BL.

Fig. 2.

Effects of G protein-coupled receptor (GPR) 40 agonist GW-9508 (GW) and GPR120 agonist grifolic acid (GA) on [Ca²⁺]_i in STC-1 cells. A and B: MA blocked increases in [Ca²⁺]_i induced by the GPR40 agonist GW in STC-1 cells. A: trace showing responses of one STC-1 cell perfused with different concentrations of GW (1–5 μM) with or without MA (0.2 mM). B: averages of peak responses vs. BL. n = 29 cells/treatment. *P < 0.001 vs. BL. C and D: GA increased [Ca²⁺]_i in STC-1 cells. C: trace showing responses of one STC-1 cell perfused with different concentrations of GA (0.1–1 μM) and LA (10 μM). D: averages of peak responses vs. BL. n = 14–57 cells/treatment. *P < 0.001 vs. BL. E and F: MA blocked increases in [Ca²⁺]_i induced by GA in STC-1 cells. E: antagonistic effects of MA on [Ca²⁺]_i in one STC-1 cell perfused with GA (1 μM), LA (5 μM) with or without pretreatment of MA (0.2 mM). F: averages of peak responses vs. BL. n = 22 cells/treatment. #P < 0.01 and *P < 0.001 vs. BL.

Fig. 3.

LA-induced glucagon-like peptide 1 (GLP-1) secretion from STC-1 cells. GLP-1 release from cultured STC-1 cells 30 min after treatment with LA (100 μM) with or without MA (0.1 mM). Data were normalized based on total protein in each well. *P < 0.05 vs. control (C) or MA + LA. n = 5 wells/treatment.

Fig. 4.

Effects of MA on olive oil-induced GLP-1 and insulin release. A–C: MA reduced GLP-1 (A) and insulin (B) secretion and increased plasma glucose levels (C) in 6-h-fasted rats after olive oil gavage. MA (68 mg/kg ip) or saline vehicle (Sal) (short arrows) was injected 15 min before an olive oil gavage (10 ml/kg body wt; long arrows). D–F: etomoxir (ETO; 10 mg/kg in 0.5% methylcellulose) or vehicle 0.5% methyl cellulose (Veh), gavaged 30 min to 10 ml/kg olive oil gavage, did not reduce olive oil-induced GLP-1 release (D) and had no effect on insulin (E) or blood glucose (F) levels. n = 7–9 rats/group. *P < 0.05 vs. Sal-Sal (or Veh-Sal) and #P < 0.05, Sal-olive vs. MA-olive.