Gastroenterology

Gastroenterology

Volume 140, Issue 3, March 2011, Pages 903-912.e4
Gastroenterology

Basic—Alimentary Tract
The G-Protein−Coupled Receptor GPR40 Directly Mediates Long-Chain Fatty Acid−Induced Secretion of Cholecystokinin

https://doi.org/10.1053/j.gastro.2010.10.012Get rights and content

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 [Ca2+]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 [Ca2+]i; linolenic acid increased CCK secretion by 53% in isolated GPR40+/+ cells that expressed eGFP. In contrast, in GPR40−/− that expressed eGFP, [Ca2+]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.

Section snippets

Experimental Animals

Transgenic mice with CCK promoter-driven enhanced green fluorescent protein (eGFP) were developed by the GenSat Bacterial Artificial Chromosomes (BAC) Transgenic project14 and obtained from the Mutant Mouse Regional Resource Center (Davis, CA).

A GPR40-targeted knockout mouse was developed by replacing the GPR40 coding region with a 21-nucleotide DNA fragment encoding genes for 9 amino acids of influenza hemagglutinin antigen, eGFP, and neomycin (Supplementary Figure 1A). eGFP was inserted with

Validation of Purity of CCK-eGFP Cells From the CCK-eGFP BAC Transgenic Mouse

Rare eGFP-expressing cells from CCK-eGFP BAC transgenic mice were sparsely distributed along the epithelium of the duodenal mucosa, displaying typical teardrop enteroendocrine cell morphology and co-immunoreactivity with CCK (Figure 1A). Roughly 0.1%−0.2% of the preparation of singly dispersed duodenal mucosal cells were eGFP+ cells, as determined by flow cytometric analysis (data not shown). Enzymatically dispersed eGFP+ cells continued to be immunoreactive with CCK antiserum (Figure 1B and E

Discussion

In the present study, we utilized BAC transgenic mice expressing CCK-promoter driven eGFP to isolate a pure population of intestinal I cells and identify the expression of GPR40 on a gene and protein level. Furthermore, to show that GPR40 is a LCFA receptor directly mediating fatty acid−induced CCK secretion, we obtained in vitro functional data from these isolated cells and demonstrated that targeted deletion of GPR40 markedly reduces [Ca2+]i signaling and CCK secretion in response to LCFAs.

References (40)

  • J.T. McLaughlin et al.

    Fatty acids stimulate cholecystokinin secretion via an acyl chain length-specific, Ca2+-dependent mechanism in the enteroendocrine cell line STC-1

    J Physiol

    (1998)
  • R. Guimbaud et al.

    Intraduodenal free fatty acids rather than triglycerides are responsible for the release of CCK in humans

    Pancreas

    (1997)
  • D.K. Covington et al.

    The G-protein-coupled receptor 40 family (GPR40-GPR43) and its role in nutrient sensing

    Biochem Soc Trans

    (2006)
  • A. Hirasawa et al.

    Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120

    Nat Med

    (2005)
  • T. Tanaka et al.

    Free fatty acids induce cholecystokinin secretion through GPR120

    Naunyn Schmiedebergs Arch Pharmacol

    (2008)
  • J. McLaughlin

    Long-chain fatty acid sensing in the gastrointestinal tract

    Biochem Soc Trans

    (2007)
  • A. Salehi et al.

    Free fatty acid receptor 1 (FFA(1)R/GPR40) and its involvement in fatty-acid-stimulated insulin secretion

    Cell Tissue Res

    (2005)
  • S. Edfalk et al.

    Gpr40 is expressed in enteroendocrine cells and mediates FFA stimulation of incretin secretion

    Diabetes

    (2008)
  • S. Gong et al.

    A gene expression atlas of the central nervous system based on bacterial artificial chromosomes

    Nature

    (2003)
  • M. Kebede et al.

    The fatty-acid receptor GPR40 plays a role in insulin secretion in vivo after high-fat feeding

    Diabetes

    (2008)
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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by the Veterinary Scientists Training Program, University of California, Davis School of Veterinary Medicine (APL), National Institutes of Health DK41004 (HER), and National Institute of Diabetes, Digestive, and Kidney Diseases Intramural funding.

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