br Materials and methods br Results and discussion

Materials and methods
Results and discussion
Conflict of interest
Introduction In the presence of continuous and excessive levels of nutrients, adipocytes become hypertrophic and release several pro-inflammatory adipocytokines such as interleukin-6 (IL-6), monocyte chemoattractant protein-1/C-C motif chemokine ligand 2 (MCP-1/CCL2, hereafter CCL2), and tumor necrosis factor-alpha (TNF-α). These chemoattractants enhance the infiltration of macrophages and other immune cells into the adipose tissue. Moreover, in obesity, a high-fat diet influences the gut microbiota to enhance the baseline inflammatory response. This increases the circulating levels of inflammasomes such as lipopolysaccharide (LPS) [1], and thus toll-like receptors (TLRs) are activated in several cells, particularly adipocytes and macrophages [2]. Therefore, these receptors that respond to nutritional components might modulate adipocyte-macrophage interactions and contribute to the whole body metabolic status [2], [3]. It is now well documented that ω-3 polyunsaturated fatty EZLink NHS-Biotin Kit (ω-3 PUFA)–rich diets activate the G-protein coupled receptor 120 (GPR120), which inhibits TLR-mediated nuclear factor kappa B (NF-κB) activation. Through this TLR-NF-κB cascade, GPR120 activation eventually inhibits the pro-inflammatory activity in adipocytes [2]. However, although obesity is commonly associated with chronic low-grade inflammation [2], [3], GPR120 expression in adipose tissues is increased in obese subjects [4]. Therefore, the precise molecular mechanisms linking the nutritional sensing of GPR120 activation with inflammatory responses requires further clarification with respect to inflammatory responses in adipocytes. Ligand-bound GPR120 in adipocytes activates the Gαq/11 subunit of G proteins, which stimulates protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) [5]. Subsequently, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway is activated. This cascade facilitates cellular glucose uptake [5]. However, recent studies demonstrated that PKC-β inactivation in adipocytes enhanced fatty acid oxidation through the upregulation of mitochondrial peroxisome proliferator-activated receptor C coactivator 1alpha (PGC1α) and uncoupling protein-2 [6], [7]. In addition, PKC-δ activity in pancreatic acinar cells produced pro-inflammatory effects by activating the NF-κB pathway [8]. Taken together, the observations suggest that GPR120 activation may have differential effects on pro-inflammatory adipocytokines through the PKC and NF-κB pathways.
Materials and methods Please see the supplementary materials for the details of Materials and Methods.
Results
Discussion Obesity is associated with a systemic low-grade inflammatory milieu [2] including activated TLRs mediated by the high serum levels of LPS [1]. In the current study, we showed that agonizing GPR120 using GSK137647 abolished the LPS-induced phosphorylation of IκBα and the nuclear translocation of p65, which downregulated the transcriptional factors, IL-6 and CCL2 (Fig. 1). This observation is in agreement with the transcriptional property of p65 on inflammatory cytokines [11]. Of note, docosahexaenoic acid (DHA), a ω-3 PUFA reduced the TNFα induced phosphorylation of IKKβ in 3T3-L1 adipocytes [5], whereas GSK137647 had no effect through IKKβ (Fig. 1C and D). We assume that a non-GPR120 (i.e. peroxisome proliferator-activated receptor gamma) mediated activity of DHA is responsible for the effect through IKKβ [12]. Moreover, GPR120 mediated signaling in adipocytes via the Gαq/11 subunit activates the PKC-PI3K-Akt pathway [5]. In our experimental model, we found that agonizing GPR120 using GSK137647 did not remarkably influence the phosphorylation of Erk or Akt (Fig. 1E). Therefore, our findings suggest that the anti-inflammatory activities of GPR120 activation only involve the NF-κB pathway (Fig. 1E–G), and that the contribution of the PI3K/Akt and Erk cascade is negligible. This selective antagonism of NF-κB was also reported for DHA-mediated GPR120 activation in hypothalamic neurons [13].