Evaluation of the relationship between GPR43 and adiposity in human
© Dewulf et al.; licensee BioMed Central Ltd. 2013
Received: 6 December 2012
Accepted: 10 January 2013
Published: 17 January 2013
GPR43 is a G-protein-coupled receptor that participates in adipocyte differentiation in mice and is over-expressed in adipose tissue of obese mice. The aim of this study was to investigate the implication of GPR43 in adipogenesis in humans and to determine the influence of obesity on its expression in human adipose tissue.
Preadipocytes were isolated from human omental adipose tissue and cultured during 13 days. One PPARγ agonist (troglitazone) and three GPR43 agonists (two physiological and one synthetic) were tested for their ability to induce differentiation. After 13 days, the three GPR43 agonists had no impact on aP2 expression, a marker of adipocyte differentiation, whereas troglitazone led to a huge over-expression of aP2 in these cells but tended to decrease GPR43 expression (p=0.06).
GPR43 and inflammatory markers expression was also quantified in omental adipose tissue from lean and obese individuals. GPR43 expression in total adipose tissue was similar between obese patients and lean subjects and did not correlate with aP2 expression. In contrast, GPR43 expression positively correlated with TNFα mRNA.
Our results suggest the absence of relationship between GPR43 and adipocyte differentiation in humans, unlike what was observed in mice. Furthermore, GPR43 expression is not increased in adipose tissue from obese subjects but could be related to TNFα-related inflammatory processes.
KeywordsHuman GPR43 Adipocyte differentiation Obesity Inflammation
Cluster of differentiation 68
G-protein-coupled receptor 43
Monocyte chemoattractant protein-1
Omental adipose tissue
Peroxisome proliferator-activated receptor gamma
Tumor necrosis factor alpha.
GPR43, also known as free fatty acid receptor 2 (FFA2), is a G-protein-coupled receptor activated by short-chain fatty acids, i.e. acetate and propionate, produced through fermentation of non-digestible carbohydrates [1–3]. It is highly expressed in immune cells but is also present in other tissues (e.g. adipose tissue, spleen, bone marrow, intestine, liver)  and seems implicated in adipose tissue metabolism. GPR43 activation by acetate and propionate in vitro stimulates adipocyte differentiation in 3T3-L1 preadipocytes. 3T3-L1 cells transfected with GPR43 siRNA exhibit decreased expression of peroxisome proliferator-activated receptor gamma (PPARγ) -the master regulator of adipogenesis- and less fat accumulation . Moreover, acetate and propionate inhibit in vitro and in vivo lipolysis by activating GPR43 [5, 6]. In mice fed a high-fat diet, the augmented adiposity and adipocyte enlargement have been associated with an over-expression of PPARγ target genes and GPR43 in subcutaneous adipose tissue. Furthermore, a PPARγ agonist increased GPR43 expression in explants of mouse subcutaneous adipose tissue thus suggesting PPARγ as a driver of GPR43 expression . Altogether, these results suggest a potential link between GPR43 expression and adipocyte differentiation in mice. However, in humans, the implication of this receptor in adipogenesis and its expression in obesity has not been studied yet.
Methods and materials
All chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA), except the synthetic GPR43 agonist [4-chloro-α-(1-methyl ethyl)-N-2-thiazolyl-benzeneacetamide] (CMTB)  (Ambinter, Paris, France) and collagenase A (Roche Diagnostics Belgium, Vilvoorde, Belgium).
Isolation and culture of stromal-vascular cells from human adipose tissue
Omental adipose tissue (OAT) from 4 obese patients undergoing abdominal surgery were fractionated into adipocytes and stromal-vascular cells (SVC) as previously described [9, 10]. Briefly, fat tissue was cut into small pieces and incubated for 15 min in a shaking water bath at 37°C in KREBS-BSA 2% with collagenase A (8.33 mg/g tissue). Digested tissue was filtered and centrifuged at 400 g for 1 min. The infranatant containing the SVC was washed three times. Preadipocytes were grown to confluence in DMEM-F-12 medium (Invitrogen, Life Technologies, Gent, Belgium) with 10% fetal bovine serum (PAA Laboratories, Pasching, Austria), streptomycin 100 μg/ml and penicillin 100 IU/ml (Gibco, Inchinnan, Scotland) at 37°C in humidified 5% CO2 and then differentiated in vitro during 13 days using a chemically defined serum-free medium consisting of DMEM-F-12 (1:1) supplemented with 15 mM HEPES, 15 mM NaHC03, 33 μM biotin, 17 μM panthotenate, 10 μg/ml apotransferrin, 66 nM insulin, 1 μM dexamethasone, 200 pM triiodothyronine, and antibiotics. Isobutylmethylxanthine (500 μM) was added during the first 3 days to induce differentiation. During differentiation, different conditions were tested: control medium (CT), troglitazone (TZD) (10 μM), acetate (10 μM), propionate (10 μM) and CMTB (1 μM). DMSO was used to dilute TZD and CMTB and added to each medium to obtain the same final concentration (0.13%). Cells were then harvested and frozen at −80°C until subsequent mRNA analysis.
Isolation of human adipose tissue
Descriptive characteristics of lean and obese patients
60 ± 4
23.1 ± 0.4
42 ± 3
45.1 ± 1.5
Real-time quantitative PCR
Total RNA was isolated using a TriPure Isolation Reagent Kit (Roche Diagnostics Belgium, Vilvoorde, Belgium), cDNA was prepared by reverse transcription of 100 ng total RNA using a Reverse Transcription System Kit (Promega, Madison, WI, USA) and quantitative PCR was performed as previously described . Primers used to detect the targeted genes are available upon request.
Results are presented as mean ± SEM. Statistical significance of difference was assessed by one-way ANOVA followed by post hoc Tukey’s multiple comparison test when comparing 3 groups or more, or by a Student t-test when comparing 2 groups. Correlations were analyzed using Pearson’s correlation test in GraphPad Prism (version 5.00 for Windows GraphPad Software, San Diego, CA, USA). The level of significance was set at p< 0.05.
Results and discussion
Implication of human GPR43 in adipogenesis
GPR43 Expression in human adipose tissue: link with inflammation
In this first study focusing on GPR43 in human adipose tissue, some data previously shown in mice, i.e. the implication of GPR43 in the process of adipocyte differentiation, have not been confirmed. Indeed, GPR43 does not seem implicated in human adipogenesis as its ligands do not induce differentiation of preadipocytes. Moreover, GPR43 expression is not induced by a PPARγ agonist and is not correlated to aP2 expression, a well-known marker of adipocyte differentiation. However, although GPR43 is not over-expressed in the OAT of all obese individuals, its expression seems associated with TNFα-related inflammatory process. Larger studies are needed in order to evaluate the co-regulation of GPR43 and TNFα expression in human adipose tissue.
We thank Laurence Noel for her technical assistance. Patrice D. Cani is a Research Associate and Laure B. Bindels, a Research Fellow of the FRS-FNRS Belgium.
- Nilsson NE, Kotarsky K, Owman C, Olde B: Identification of a free fatty acid receptor, FFA2R, expressed on leukocytes and activated by short-chain fatty acids. Biochem Biophys Res Commun. 2003, 303: 1047-1052. 10.1016/S0006-291X(03)00488-1.View Article
- Brown AJ, Goldsworthy SM, Barnes AA, Eilert MM, Tcheang L, Daniels D, Muir AI, Wigglesworth MJ, Kinghorn I, Fraser NJ, Pike NB, Strum JC, Steplewski KM, Murdock PR, Holder JC, Marshall FH, Szekeres PG, Wilson S, Ignar DM, Foord SM, Wise A, Dowell SJ: The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. J Biol Chem. 2003, 278: 11312-11319. 10.1074/jbc.M211609200.View Article
- Le Poul E, Loison C, Struyf S, Springael JY, Lannoy V, Decobecq ME, Brezillon S, Dupriez V, Vassart G, Van DJ, Parmentier M, Detheux M: Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation. J Biol Chem. 2003, 278: 25481-25489. 10.1074/jbc.M301403200.View Article
- Swaminath G: Fatty acid binding receptors and their physiological role in type 2 diabetes. Arch Pharm (Weinheim). 2008, 341: 753-761. 10.1002/ardp.200800096.View Article
- Hong YH, Nishimura Y, Hishikawa D, Tsuzuki H, Miyahara H, Gotoh C, Choi KC, Feng DD, Chen C, Lee HG, Katoh K, Roh SG, Sasaki S: Acetate and propionate short chain fatty acids stimulate adipogenesis via GPCR43. Endocrinology. 2005, 146: 5092-5099. 10.1210/en.2005-0545.View Article
- Ge H, Li X, Weiszmann J, Wang P, Baribault H, Chen JL, Tian H, Li Y: Activation of G protein-coupled receptor 43 in adipocytes leads to inhibition of lipolysis and suppression of plasma free fatty acids. Endocrinology. 2008, 149: 4519-4526. 10.1210/en.2008-0059.View Article
- Dewulf EM, Cani PD, Neyrinck AM, Possemiers S, Van HA, Muccioli GG, Deldicque L, Bindels LB, Pachikian BD, Sohet FM, Mignolet E, Francaux M, Larondelle Y, Delzenne NM: Inulin-type fructans with prebiotic properties counteract GPR43 overexpression and PPARgamma-related adipogenesis in the white adipose tissue of high-fat diet-fed mice. J Nutr Biochem. 2011, 22: 712-722. 10.1016/j.jnutbio.2010.05.009.View Article
- Wang Y, Jiao X, Kayser F, Liu J, Wang Z, Wanska M, Greenberg J, Weiszmann J, Ge H, Tian H, Wong S, Schwandner R, Lee T, Li Y: The first synthetic agonists of FFA2: Discovery and SAR of phenylacetamides as allosteric modulators. Bioorg Med Chem Lett. 2010, 20: 493-498. 10.1016/j.bmcl.2009.11.112.View Article
- Halleux CM, Declerck PJ, Tran SL, Detry R, Brichard SM: Hormonal control of plasminogen activator inhibitor-1 gene expression and production in human adipose tissue: stimulation by glucocorticoids and inhibition by catecholamines. J Clin Endocrinol Metab. 1999, 84: 4097-4105. 10.1210/jc.84.11.4097.
- Maury E, Ehala-Aleksejev K, Guiot Y, Detry R, Vandenhooft A, Brichard SM: Adipokines oversecreted by omental adipose tissue in human obesity. Am J Physiol Endocrinol Metab. 2007, 293: E656-E665. 10.1152/ajpendo.00127.2007.View Article
- Gregoire FM, Smas CM, Sul HS: Understanding adipocyte differentiation. Physiol Rev. 1998, 78: 783-809.
- Di Gregorio GB, Yao-Borengasser A, Rasouli N, Varma V, Lu T, Miles LM, Ranganathan G, Peterson CA, McGehee RE, Kern PA: Expression of CD68 and macrophage chemoattractant protein-1 genes in human adipose and muscle tissues: association with cytokine expression, insulin resistance, and reduction by pioglitazone. Diabetes. 2005, 54: 2305-2313. 10.2337/diabetes.54.8.2305.View Article
- Maury E, Brichard SM: Adipokine dysregulation, adipose tissue inflammation and metabolic syndrome. Mol Cell Endocrinol. 2010, 314: 1-16. 10.1016/j.mce.2009.07.031.View Article
- Ouchi N, Parker JL, Lugus JJ, Walsh K: Adipokines in inflammation and metabolic disease. Nat Rev Immunol. 2011, 11: 85-97. 10.1038/nri2921.View Article
- Oh DY, Lagakos WS: The role of G-protein-coupled receptors in mediating the effect of fatty acids on inflammation and insulin sensitivity. Curr Opin Clin Nutr Metab Care. 2011, 14: 322-327. 10.1097/MCO.0b013e3283479230.View Article
- Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, Schilter HC, Rolph MS, Mackay F, Artis D, Xavier RJ, Teixeira MM, Mackay CR: Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature. 2009, 461: 1282-1286. 10.1038/nature08530.View Article
- Sina C, Gavrilova O, Forster M, Till A, Derer S, Hildebrand F, Raabe B, Chalaris A, Scheller J, Rehmann A, Franke A, Ott S, Hasler R, Nikolaus S, Folsch UR, Rose-John S, Jiang HP, Li J, Schreiber S, Rosenstiel P: G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation. J Immunol. 2009, 183: 7514-7522. 10.4049/jimmunol.0900063.View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.