Identification of a free fatty acid receptor, FFA2R, expressed on leukocytes and activated by short-chain fatty acids

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Abstract

Short-chain fatty acids (SCFAs) have long been known to exert cellular effects on blood leukocytes. Acetate, propionate, and butyrate represent the most capable SCFA, inducing calcium mobilization which subsequently regulates leukocyte function in the immune system. We have cloned the previously described putative orphan G-protein coupled receptor, GPR43, and have functionally identified SCFA as the activating ligands. Acetate and propionate were found to be the two most potent ligands, although butyrate, formate, and valerate (in this order of potency) also were able to induce receptor activation. Both the human and mouse receptor homologues were found to share the same pattern of ligand activation. This finding, together with a high degree of amino acid sequence similarity between the mouse and human homologues, indicates an evolutionary conserved function. Upon ligand stimulation, the receptor mobilized intracellular calcium in both a recombinant system as well as in human granulocytes. We found the human gene to be predominantly expressed in peripheral blood leukocytes and, to a lesser extent, in spleen. We suggest the designation FFA2R to this second receptor activated by free fatty acids. The first-described FFAR, now named FFA1R, is activated by medium- to long-chain free fatty acids.

Section snippets

Materials and methods

Cloning. Cloning of the GPR43 ORF (open reading frame) from genomic (human and mouse) DNA was performed by PCR using the following primers:
Human ORF:

  • forward: 5-ATTGCGGCCGCAGGATGCTGCCGGACTGGAAGAGC-3;

  • reverse: 5-ATTGCGGCCGCACTGCTACTCTGTAGTGAAGTCCGA-3.


Murine ORF:
  • forward: 5-CGGAATTCCGGGGGACTCTCTACTCGGTGACAA-3;

  • reverse: 5-ATTGCGGCCGCCAGAATGACCCCAGACTGGCACAGT-3.


PCR conditions were: 96 °C for 3 min, 96 °C for 45 s; 58 °C for 1 min; and 72 °C for 1 min (30 cycles). The PCR products were restricted with

SCFA-induced activation of human GPR43

The previously identified putative GPCR ORF, GPR43 (GenBank Accession No. AF024690) [23] was cloned and stably expressed in the recombinant reporter cell line, HFF11, which is the second-generation reporter cell line [19]. A spectrum of SCFAs activated the receptor, but failed to induce any response in sham-transfected reporter cells. High concentrations, in the millimolar range, of SCFA, such as acetate (carbon chain length=C2), propionate (C3), and butyrate (C4), activated the reporter cells

Concluding remarks

What the actual physiological significance is and how SCFA activation of FFA2R affects the immune system, primarily the granulocytes, remain to be further investigated. SCFA have a multitude of cellular and immunoregulatory effects and, in fact, all related functions of PMNC, except cell adhesion, appear to be modulated by SCFA [25]. We have identified the previous orphan GPR43 as a receptor that is functionally activated by SCFA—most efficiently by acetate and propionate, but not by

Acknowledgements

This work was supported by GS Development, the Segerfalk Foundation, Crafoord Foundation, Ingabritt and Arne Lundberg Foundation, Kock Foundation, Swedish Society for Medical Research, Royal Physiographic Society, and Swedish Research Council.

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