GPR120 promotes adipogenesis through intracellular calcium and extracellular signal-regulated kinase 1/2 signal pathway

https://doi.org/10.1016/j.mce.2016.06.009Get rights and content

Highlights

  • DHA, ALA, LA showed the different effects on adipogenesis and GPR120 was related to the pro-adipogenic function of ALA.

  • TUG-891, a selective agonist of GPR120, promoted adipogenesis in a GPR120-dependent manner.

  • TUG-891 increased expression of PPARγ in a GPR120-dependent manner.

  • Ca2+-ERK1/2 signaling contributed to the GPR120-induced lipogenesis.

Abstract

Numerous researches have demonstrated that GPR120 (also called FFAR4) exerts novel functions in insulin resistance and adipogenesis. However, the molecular mechanism of GPR120-mediated adipogenic differentiation is still unclear. This study was aimed to interpret the relevant function mechanism of GPR120 in the differentiation of 3T3-L1 adipocytes. The results showed that GPR120 expression was dramatically increased along with the adipogenic differentiation of 3T3-L1 adipocytes and the adipogenic ability was significantly inhibited in shGPR120-transfected cells. TUG-891, a selective agonist of GPR120, promoted the intracellular triglyceride accumulation in a dose-dependent manner and did not enhance adipogenesis in shGPR120-transfected cells. Markedly, TUG-891 increased the activation of PPARγ in a GPR120-dependent pathway as assessed by luciferase reporter assay. Furthermore, in the adipogenic differentiation process of 3T3-L1 adipocytes, TUG-891 increased the [Ca2+]i and phosphorylation level of ERK1/2. Pretreatment with inhibitors of either ERK1/2 (U0126) or [Ca2+]i (BAPTA-AM) notably attenuated the GPR120-mediated adipogenesis. These results show that GPR120 promotes adipogenesis by increasing PPARγ expression via [Ca2+]i and ERK1/2 signal pathway in 3T3-L1 adipocytes.

Introduction

White adipose tissue (WAT), which is considered as the main reservoir to store energy, plays an important role as an endocrine organ in glucose metabolism and immune functions by secreting a vast range of regulatory factors (Kershaw and Flier, 2004). Given the vital role of WAT in the health implications and energy homeostasis, it is essential to investigate the molecular mechanisms of adipogenesis. Until now, a variety of transcription factors, including CCAAT enhancer binding protein α (C/EBPα), C/EBPβ, C/EBPδ and peroxisome proliferator activated receptor γ (PPARγ), have been demonstrated to be involved in this process (Otto and Lane, 2005, Tang and Lane, 2012). Among them, PPARγ is considered as the master regulator of the complex transcriptional cascade in adipogenic differentiation and the terminal differentiation does not occur without PPARγ (Cristancho and Lazar, 2011, Rosen et al., 1999).

Recently, the novel fatty acid receptor, GPR120 (also called FFAR4), has been shown to be implicated in diverse physiological homeostasis, such as insulin sensitization, anti-inflammation and regulation of appetite (Hirasawa et al., 2005, Oh et al., 2010). Notably, it is abundantly expressed in WAT and mature adipocytes, whereas it is undetectable in preadipocytes (Gotoh et al., 2007, Ichimura et al., 2012, Miyauchi et al., 2009, Oh et al., 2010). GPR120 knockdown reduces the expression of PPARγ and fatty acid binding protein 4 (FABP4, also known as aP2) in 3T3-L1 adipocytes (Gotoh et al., 2007). Consistently, suppression of these adipogenic marker genes was detected in the mouse-embryonic fibroblast (MEF) derived adipocytes isolated from the GPR120-deficient mouse (Ichimura et al., 2012). These evidences indicate that GPR120 contributes to the process of adipogenesis. However, its precise molecular function in the regulation of adipogenic processes remains unclear.

As a G protein-coupled receptor (GPCR), GPR120 is located in the cell surface and induces a wide range of cellular responses when responding to appropriate ligand binding. Several experimental observations showed that GPR120 stimulated by fatty acids or synthetic agonists increases intracellular calcium concentration ([Ca2+]i) and elevates the phosphorylation level of extracellular signal-regulated kinase1/2 (ERK1/2) cascade (Hirasawa et al., 2005, Hudson et al., 2013, Ichimura et al., 2012, Katsuma et al., 2005). ERK1/2 facilitates the early stage of adipogenic differentiation but needs to be turned off at the adipocyte maturation phase, suggesting a dual role of ERK1/2 in adipogenesis (Bost et al., 2005b, Prusty et al., 2002). On the other hand, [Ca2+]i also plays a time-dependent role in adipogenesis. Improving intracellular calcium concentration inhibits the early stage but accelerates the maturation stage of adipogenic differentiation in both murine and human adipocyte (Neal and Clipstone, 2002, Shi et al., 2000). Hence, it would be interesting to understand the roles of [Ca2+]i and ERK1/2 signaling in GPR120-induced adipogenesis. In this study, we determined the expression of GPR120 during the differentiation progress of 3T3-L1 adipocytes and found that GPR120 facilitates 3T3-L1 adipogenesis. Moreover, we for the first time showed that [Ca2+]i and ERK1/2 signaling contributes to GPR120-induced adipogenesis.

Section snippets

Materials

Oil Red O, paraformaldehyde, insulin, dexamethasone, isobutylmethylxanthine, collagenase I, BAPTA-AM, U0126, Docosahexoenoic acid (DHA, 22:6n-3), α-linolenic acid (ALA, 18:3n-3), linoleic acid (LA, 18:2n-6), Dimethylsulfoxidewere (DMSO) were purchased from Sigma Aldrich. TUG-891 was obtained from R&D systems. Rabbit anti-PPARγ and anti-p-ERK1/2 (T202/Y204) were purchased from Affinity biosciences Inc. DHA, ALA and LA were dissolved in DMSO. The stock solutions (500 mM) were aliquoted and stored

GPR120 knockdown decreased adipogenesis in 3T3-L1 cells

To test whether the fatty acid receptor GPR120 plays a role in adipogenesis, the mRNA expression profile of GPR120 was detected using qPCR at the indicated time points (0, 0.5, 1, 2, 4, 5, 6 day) during the adipogenesis of the 3T3-L1 cells. As shown in Fig. 1A, the results indicated that GPR120 was weakly expressed at the beginning of adipogenic differentiation. Upon the induction of differentiation, the expression of GPR120 and the adipogenic marker genes (PPARγ2 and aP2) was dramatically

Discussion

Until now, there have been increasing evidences demonstrating that the adipose tissue has profound effects on glucose and energy homeostasis (Rosen and Spiegelman, 2014). The adipogenesis process is regulated by a large body of factors ranging from the extracellular space to the nuclear depot and involves a complex and orchestrated program of adiposity-related gene expression (Otto and Lane, 2005, Tang and Lane, 2012). Several studies have demonstrated that GPR120 may function as a novel fatty

Conflict of interest

The corresponding author states that there is no conflict of interest.

Acknowledgment

This work was supported by National Program on Key Basic Research Project of China (No. 2013CB127305); The National Natural Science Foundation of China (No. 314020785); Hubei Provincial Creative Team Project of Agricultural Science and Technology (No. 2007-620); Hubei Provincial Natural Science Foundation (No. 2013CFA010); The Key Technology Research and Development Program of Hubei Province (No. 2014ABB014, No. 2014ABC012).

References (43)

  • G. Murali et al.

    Differential effects of eicosapentaenoic acid and docosahexaenoic acid in promoting the differentiation of 3T3-L1 preadipocytes

    Prostagl. Leukot. Essent. Fat. Acids

    (2014)
  • J.W. Neal et al.

    Calcineurin mediates the calcium-dependent inhibition of adipocyte differentiation in 3T3-L1 cells

    J. Biol. Chem.

    (2002)
  • J.M. Ntambi et al.

    Role of Ca2+ in the early stages of murine adipocyte differentiation as evidenced by calcium mobilizing agents

    Differentiation

    (1996)
  • D.Y. Oh et al.

    GPR120 is an Omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects

    Cell

    (2010)
  • D. Prusty et al.

    Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor gamma (PPAR gamma) and C/EBP alpha gene expression during the differentiation of 3T3-L1 preadipocytes

    J. Biol. Chem.

    (2002)
  • E.D. Rosen et al.

    PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro

    Mol. Cell

    (1999)
  • E.D. Rosen et al.

    What we talk about when we talk about fat

    Cell

    (2014)
  • J. Wang et al.

    Linoelaidic acid enhances adipogenic differentiation in adipose tissue-derived stromal cells through suppression of Wnt/β-catenin signaling pathway in vitro. Prostaglandins

    Leukot. Essent. Fat. Acids (PLEFA)

    (2016)
  • F. Bost et al.

    The extracellular signal-regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis

    Diabetes

    (2005)
  • C.P. Briscoe et al.

    Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules

    Br. J. Pharmacol.

    (2006)
  • E. Burgermeister et al.

    MAPK kinases as nucleo-cytoplasmic shuttles for PPAR gamma

    Cell Cycle

    (2007)
  • Cited by (0)

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