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

Abstract

Omega-3 fatty acids (omega-3 FAs), DHA and EPA, exert anti-inflammatory effects, but the mechanisms are poorly understood. Here, we show that the G protein-coupled receptor 120 (GPR120) functions as an omega-3 FA receptor/sensor. Stimulation of GPR120 with omega-3 FAs or a chemical agonist causes broad anti-inflammatory effects in monocytic RAW 264.7 cells and in primary intraperitoneal macrophages. All of these effects are abrogated by GPR120 knockdown. Since chronic macrophage-mediated tissue inflammation is a key mechanism for insulin resistance in obesity, we fed obese WT and GPR120 knockout mice a high-fat diet with or without omega-3 FA supplementation. The omega-3 FA treatment inhibited inflammation and enhanced systemic insulin sensitivity in WT mice, but was without effect in GPR120 knockout mice. In conclusion, GPR120 is a functional omega-3 FA receptor/sensor and mediates potent insulin sensitizing and antidiabetic effects in vivo by repressing macrophage-induced tissue inflammation.

FIG. 1. Expression level of GPR120 and GPR120-mediated anti-inflammatory response in RAW 264.7 cells

(A) The mRNA expression pattern of various lipid sensing GPCRs is shown in adipose tissue, (B) CD11c⁺ bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMDMs), IPMacs, 3T3-L1 preadipocytes, differentiated 3T3-L1 adipocytes, RAW 264.7 cells and L6 myocytes. Ribosomal protein S3 (RPS3) was used as internal control. (C) Expression of GPR120 in SVF, adipocytes and hepatic Kupffer cells from chow (NC)- or HFD-fed mice was examined by q-PCR. Data are expressed as the mean±SEM of at least three independent experiments in triplicate. *, p<0.05 versus NC. (D) RAW 264.7 cells, transfected with scrambled (Scr) or GPR120 #2 siRNA (GPR120 KD), were treated with 100 μM of GW9508 for 1 hr prior to LPS (100 ng/ml) treatment for 10 min and then subjected to western blotting. Left panel is a representative image from three independent experiments, and the scanned bar graph (right panel) shows fold induction over basal conditions. Knockdown efficiency of GPR120 siRNA is shown in Fig. S1. (E) Cytokine secretion level was measured in RAW 264.7 cells by ELISA. Data are expressed as the mean±SEM of three independent experiments. *, p<0.05 versus LPS treatment in scrambled siRNA transfected cells. See also Figs. S1 and S2.

FIG. 2. Omega-3 FA stimulates GPR120 and mediates anti-inflammatory effects

(A) GPR120-mediated SRE-luc activity after treatment with various FAs. Results are fold activities over basal. Each data point represents mean±SEM of three independent experiments performed in triplicate. Black lines indicate SRE-luc activities without GPR120 transfection or with non-stimulating FAs. DHA inhibits LPS-induced inflammatory signaling (B), cytokine secretion (C) and inflammatory gene mRNA expression level (D) in RAW 264.7 cells, but not in GPR120 knockdown cells. (E and F) GPR120 stimulation inhibits LPS-induced inflammatory response in WT primary macrophage. Data are expressed as the mean±SEM of three independent experiments. *, p<0.05 versus LPS treatment in scrambled siRNA transfected cells or WT IPMacs. See also Fig. S2.

FIG. 3. GPR120 internalization with β-arrestin2 mediates anti-inflammatory effects

(A) RAW 264.7 cells were transfected with siRNA as indicated and stimulated with or without 100 μM of DHA 1 hr prior to LPS (100 ng/ml) treatment for 10 min and then subjected to western blotting. (B) TNF-α secretion was measured in RAW 264.7 cell cultured media with or without RNA interference as indicated. (C) Phosphorylation of TAK1 and MKK4 in RAW 264.7 cells with or without siRNA transfection as indicated. (D) HEK 293 cells were co-transfected with HA-GPR120 and β-arrestin2·GFP to analyze GPR120 internalization after DHA stimulation for the indicated times. GPR120 (red) and β-arrestin2 (green) were localized by confocal microscopy. (E) Co-immunoprecipitation between GPR120 and β-arrestin2 with DHA stimulation for 30 min in RAW 264.7 cells and, (F) HEK 293 cells (HA-GPR120 and β-arrestin2·GFP), respectively. Lysate indicates 1/10 input in each experiment. Interaction between TAB1 and β-arrestin2 (G), and interaction between TAB1 and TAK1 (H) were detected by co-immunoprecipitation and the scanned bar graph quantitates the association in RAW 264.7 cells. (I) Schematic diagram of the β-arrestin2 and GPR120-mediated anti-inflammatory mechanism. Red colored letters and arrows indicate the DHA-mediated anti-inflammatory effect, and black colored letters and arrows indicate the LPS- and TNF-α-induced inflammatory pathway. See also Fig. S2.

FIG. 4. GPR120 activation enhances GLUT4 translocation and glucose uptake

(A) 3T3-L1 adipocytes were transfected with a dually tagged HA-GLUT4-GFP construct. Total GLUT4 expression was determined by GFP fluorescence, and GLUT4 translocation to the cell surface after 100 ng/ml insulin or 100 μM DHA stimulation for 30 min was determined by indirect immunofluorescence of the HA-conjugated with Alexa 594 in fixed cells. Translocation following insulin stimulation was expressed as a percentage of the maximum response. Bar graph represents the mean±SEM data from four independent experiments. *, p<0.05 vs. vehicle treatment. (B) Glucose uptake was measured in WT and GPR120 KO mouse primary adipose tissue and in (C–F) 3T3-L1 adipocytes±siRNA with the indicated treatment. Data are expressed as mean±SEM of three independent experiments in triplicate. *, p<0.05 vs. basal activity. The indicated siRNA knockdown efficiency was validated by western blotting. See also Fig. S3.

FIG. 5. In vivo metabolic studies in GPR120 KO mice

(A) GTT in NC-fed WT and GPR120 KO mice. n=7 per group. (B) Insulin concentration were measured at the indicated time points and (C) area-under-curve analysis of the insulin data shows a significant difference between WT and GPR120 KO mice on NC. (D) Hyperinsulinemic/euglycemic clamp studies in chow-fed WT and GPR120 KO mice on. (E) Clamp studies in HFD, ω-3 FA supplemented (+ω3), and Rosiglitazone treated HFD mice (+Rosi). n=8 per group, *, p<0.05 compared to HFD-fed WT group. (F) mean±SEM plasma concentration (mole (%)) of DHA and EPA for each diet in WT and GPR120 KO mice. n= 7 per each group. *, p<0.05, compared to NC, and **, p<0.05 compared to HFD. Data are represented as mean±SEM. See also Figs. S4, S5, S6 and supplemental Table 2.

FIG. 6. Omega-3 FA enriched diet decreases inflammatory macrophage infiltration in adipose tissue

(A) Confocal merged images from epididymal fat pads from HFD and ω-3 FA enriched HFD (HFD+ω3)-fed WT and GPR120 KO mice, co-stained with anti-F4/80 (green) and anti-Caveolin1 (blue) antibodies, left 4 panels, or anti-MGL1 (green) and anti-Caveolin1 (red) antibodies, right 4 panels. The image is representative of similar results from 3–4 independent experiments. Scale bar represents 100 μm. (B) Dot plot representation of CD11b versus CD11c expression for FACS data obtained from adipose tissue SVF of NC, HFD or HFD+ω3-fed WT and GPR120 KO. Scattergram is representative from three independent mice from each group. (C and D) Migratory capacity of IPMacs from WT and GPR120 KO mice as measured using an in vitro transwell chemotaxis assay as described under supplemental experimental procedures. Data are expressed as mean±SEM of three independent experiments in triplicate. *, p<0.05 vs. CM treatment.

FIG. 7. M1 and M2 inflammatory gene expression levels in adipose tissue from WT vs. GPR120 KO mice

Relative mRNA levels for M1 pro-inflammatory genes (A) and M2 anti-inflammatory genes (B) in NC, HFD or HFD+ω3 (+ω3)-fed WT and GPR120 KO mice, as measured by q-PCR. Data are expressed as mean±SEM of three independent experiments in triplicate. n=7 per group, *, p<0.05 compared to the HFD-fed WT group. **, p<0.05 compared to the WT vs. GPR120 KO on NC. See also Fig. S7. Primer sequences are shown in supplemental Table 1.