The G-protein-coupled receptor GPR40 directly mediates long-chain fatty acid-induced secretion of cholecystokinin

Abstract

Background & aims: Long-chain fatty acid receptors G-protein-coupled receptor 40 (GPR40) (FFAR1) and GPR120 have been implicated in the chemosensation of dietary fats. I cells in the intestine secrete cholecystokinin (CCK), a peptide hormone that stimulates digestion of fat and protein, but these cells are rare and hard to identify. We sought to determine whether dietary fat-induced secretion of CCK is directly mediated by GPR40 expressed on I cells.

Methods: We used fluorescence-activated cell sorting to isolate a pure population of I cells from duodenal mucosa in transgenic mice that expressed green fluorescent protein under the control of the CCK promoter (CCK-enhanced green fluorescent protein [eGFP] bacterial artificial chromosome mice). CCK-eGFP cells were evaluated for GPR40 expression by quantitative reverse transcription polymerase chain reaction and immunostaining. GPR40(-/-) mice were bred with CCK-eGFP mice to evaluate functional relevance of GPR40 on long-chain fatty acid-stimulated increases in [Ca(2+)]i and CCK secretion in isolated CCK-eGFP cells. Plasma levels of CCK after olive oil gavage were compared between GPR40(+/+) and GPR40(-/-) mice.

Results: Cells that expressed eGFP also expressed GPR40; expression of GPR40 was 100-fold greater than that of cells that did not express eGFP. In vitro, linoleic, oleic, and linolenic acids increased [Ca(2+)]i; linolenic acid increased CCK secretion by 53% in isolated GPR40(+/+) cells that expressed eGFP. In contrast, in GPR40(-/-) that expressed eGFP, [Ca(2+)]i response to linoleic acid was reduced by 50% and there was no significant CCK secretion in response to linolenic acid. In mice, olive oil gavage significantly increased plasma levels of CCK compared with pregavage levels: 5.7-fold in GPR40(+/+) mice and 3.1-fold in GPR40(-/-) mice.

Conclusions: Long-chain fatty acid receptor GPR40 induces secretion of CCK by I cells in response to dietary fat.

Figure 1

Validation of cholecystokinin (CCK)–enhanced green fluorescent protein (eGFP) transgene specificity and fluorescence-activated cell sorting (FACS) purification. All eGFP cells were CCK immunoreactive in (A) duodenal tissue, (B) dispersed single cells (20×), and (C) post-sorted cells (20×). FACS gating for GFP expression in singly dispersed cells from (D) CCK-eGFP⁻ mice (negative control), (E) presorted, and (F) post-sorted singly dispersed cells from CCK-eGFP⁺ mice.

Figure 2

Verification of fluorescence-activated cell sorting (FACS) purification of cholecystokinin (CCK)–enhanced green fluorescent protein (eGFP) cells by gene expression using Taqman reverse transcriptase polymerase chain reaction. Expression of CCK messenger RNA was high relative to β-actin (×10³) in CCK-eGFP cells with nearly undetectable contaminating transcripts for enterocyte (alkaline phosphatase [AKP3]) and goblet cell (calcium/chloride channel [CLCA3]) markers compared to a simultaneously sorted non-eGFP population (n = 5–6 separate cell isolations).

Figure 3

Gene expression of putative fatty-acid sensors in acutely isolated cholecystokinin (CCK)–enhanced green fluorescent protein (eGFP) cells. Taqman reverse transcriptase polymerase chain reaction results for G-protein–coupled receptor (GPR) 40 and GPR120 gene transcripts in the CCK-eGFP and non-eGFP cell population relative to housekeeping gene β-actin (×10³). Statistical significance was determined by paired t test between the ΔCt values of the GFP and non-GFP cell populations. n = 4–5 separate cell isolations. *P < .05, **P < 001.

Figure 4

G-protein–coupled receptor 40 (GPR40) expression in cholecystokinin (CCK)–enhanced green fluorescent protein (eGFP) cells of primary duodenal villi. GPR40 immunoreactivity (red) was present in CCK-eGFP cells (green) from (A) GPR40^+/+ mice, but not (B) GPR40^−/− mice or (C) in the absence of primary antibody (negative control). (D) GPR40 immunoreactivity was present in rare eGFP-negative cells in GPR40^+/+ mice. Images in each panel (63×) include CCK-eGFP only (top left; green), α-GPR40 or AlexaFluor 633 secondary antibody only (top right; red), transmitted light (bottom left), and merged panels (bottom right).

Figure 5

Long-chain fatty acid (LCFA) stimulated release of [Ca²⁺]_i in fluorescence-activated cell sorting (FACS) isolated cholecystokinin (CCK-)–enhanced green fluorescent protein (eGFP) cells. (A) G-protein–coupled receptor 40 (GPR40)^+/+ CCK-eGFP cells exhibit a dose-dependent response to linoleic acid (1–100 μM; n = 3–12 cells). (B) GPR40^+/+ CCK-eGFP cell response to various LCFAs (all 10 μM; n = 6–7 cells). Representative (C) confocal images and (D) time-courses of isolated Quest Rhod4-loaded CCK-eGFP cell in response to various LCFAs. [Ca²⁺]_i response to (E) linoleic acid (10 μM) in GPR40^+/+ vs GPR40^−/− CCK-eGFP cells (n = 10 cells, respectively), and (F) an increasing dose of the GPR40-selective agonist MEDICA 16 (0–50 μM) in GPR40^+/+ CCK-eGFP cells (n = 3–12 cells). Changes in [Ca²⁺]_i are expressed as mean ± standard error of mean of maximum percent increase in the fluorescence intensity (FI) of Quest Rhod4, relative to baseline. Cont, vehicle control. *P < .05; **P ≤ .001; ***P < .0001.

Figure 6

Fatty-acid stimulated cholecystokinin (CCK) secretion from acutely isolated CCK- enhanced green fluorescent protein (eGFP) cells with or without targeted disruption of G-protein–coupled receptor 40 (GPR40). CCK secretion in response to linolenic acid (4 uM) and methyl linolenate (4 uM) in (A) GPR40^+/+ (black bars) and (B) GPR40^−/− (clear bars) CCK-eGFP cells. KCl (50 mM) was used as a positive control. n = 6–12 separate cell preparations with each experiment performed in triplicate and assayed in duplicate. *P < .05 compared to baseline or between linolenic acid vs methyl linolenate. NS = not significant.

Figure 7

Effect of G-protein–coupled receptor 40 (GPR40) expression on cholecystokinin (CCK) secretion in response to olive oil gavage. Time course of plasma CCK in GPR40^+/+ (n = 7) and GPR40^−/− mice (n = 6) gavaged with 0.5 mL olive oil. Values are presented as mean ± standard error of mean. Significance was determined by 2-way analysis of variance. *P < .05. Experiment was repeated in duplicate.