The free fatty acid receptor G protein-coupled receptor 40 (GPR40) protects from bone loss through inhibition of osteoclast differentiation

Abstract

The mechanisms linking fat intake to bone loss remain unclear. By demonstrating the expression of the free fatty acid receptor G-coupled protein receptor 40 (GPR40) in bone cells, we hypothesized that this receptor may play a role in mediating the effects of fatty acids on bone remodeling. Using micro-CT analysis, we showed that GPR40(-/-) mice exhibit osteoporotic features suggesting a positive role of GPR40 on bone density. In primary cultures of bone marrow, we showed that GW9508, a GRP40 agonist, abolished bone-resorbing cell differentiation. This alteration of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation occurred via the inhibition of the nuclear factor κB (NF-κB) signaling pathway as demonstrated by decrease in gene reporter activity, inhibitor of κB kinase (IKKα/β) activation, inhibitor of κB (IkBα) phosphorylation, and nuclear factor of activated T cells 1 (NFATc1) expression. The GPR40-dependent effect of GW9508 was confirmed using shRNA interference in osteoclast precursors and GPR40(-/-) primary cell cultures. In addition, in vivo administration of GW9508 counteracted ovariectomy-induced bone loss in wild-type but not GPR40(-/-) mice, enlightening the obligatory role of the GPR40 receptor. Then, in a context of growing prevalence of metabolic and age-related bone disorders, our results demonstrate for the first time in translational approaches that GPR40 is a relevant target for the design of new nutritional and therapeutic strategies to counter bone complications.

FIGURE 1.

GPR40^−/− mice exhibit an osteoporotic phenotype. A, micro-CT analysis with corresponding images of left femurs from wild-type (WT) and GRP40^−/− mice (males; 16 weeks old mice; n = 11/group; BV/TV, bone volume/total volume; Tb.N, trabecular number; Tb.Sp, trabecular spaces; Tb.Th, trabecular thickness). B, bone mineral density. C, Western blot analysis of GPR40 expression in bone (tibia). Error bars represent standard deviation (±S.D.); *, p < 0.05.

FIGURE 2.

GW9508 inhibits RANKL-induced osteoclast differentiation in primary cell cultures obtained from mouse bone marrow tissues. A and B, TRACP staining: wild-type (WT) versus GPR40^−/− mice (KO). Magnification, ×10 (Zeiss). Osteoclast differentiation was induced by murine recombinant RANKL (50 ng/ml). DMSO was used as a vehicle. GPR40 agonist GW9508 was used at either 10 or 50 μm (GW10 or GW50). C, osteoclast marker expression analyzed by real-time RT-PCR. Error bars represent standard deviation (±S.D.); *, p < 0.05.

FIGURE 3.

GW9508 inhibits RANKL-induced osteoclastogenesis in RAW264.7 cell cultures. A, RT-PCR and Western blot analysis. Cells were invalidated for GPR40 expression (Sh) or transfected with a nontargeting vector (SCR). B, TRACP enzymatic activity. Osteoclast differentiation was induced by murine recombinant RANKL (50 ng/ml). DMSO was used as a vehicle. GPR40 agonist GW9508 was used at either 10 or 50 μm (GW10 or GW50). C, osteoclast marker expression analyzed by real-time RT-PCR. GPR40 agonist GW9508 was used at either 10 or 50 μm. *, p < 0.05. D, XTT cell viability assay. *, p < 0.05 versus DMSO control; error bars represent standard deviation (±S.D.).

FIGURE 4.

GW9508 inhibits RANKL-induced signaling pathways in RAW264.7 cell cultures. A, Western blot analysis of IkBα and IKKα/β phosphorylation. B, NF-κB-dependent luciferase assay. DMSO was used as a vehicle. GPR40 agonist GW9508 was used at 50 μm (GW50). Relative light units were measured at 6 and 24 h after incubation and reported to total protein content. C, NFATc1 expression analyzed by real-time RT-PCR in cells invalidated for GPR40 expression (Sh) or transfected with a nontargeting vector (SCR). *, p < 0.05. DMSO was used as a vehicle. GPR40 agonist GW9508 was used at either 10 or 50 μm (GW10 or GW50). Error bars represent standard deviation (±S.D.).

FIGURE 5.

GPR40 agonist as a protective agent against in vivo OVX-induced bone loss. A, bone microarchitecture (females; OVX, ovariectomy; Sh, Sham-operated; 14-week-old mice; n = 7 per group; BV/TV, bone volume/total volume; Tb.N, trabecular number; Tb.Sp, trabecular spaces; Tb.Th, trabecular thickness). Mice received either DMSO as vehicle or GPR40 agonist GW9508 (8 mg/kg of body weight: GW). B, representative bone tissue marker expression analyzed by TLDA on whole tibiae (Applied Biosystems 7900HT real-time PCR system). Tissues were collected from OVX or Sham-operated mice that received either DMSO as vehicle or GPR40 agonist GW9508 (8 mg/kg of body weight: GW). Different letters are attributed to significantly different groups (p < 0.05); error bars represent standard deviation (±S.D.).

FIGURE 6.

GW9508 protective effect on bone is dependent on GPR40 expression. A, food intake. Consumption was analyzed weekly throughout the protocol. B, mouse body weight. Mice were weighed every week throughout the experimental period. C and D, EchoMRI body composition analysis. Mice received either DMSO as vehicle or GPR40 agonist GW9508 (8 mg/kg of body weight: GW). E, bone mineral density (females; OVX, ovariectomy; SH, Sham-operated; 14-week-old mice; n = 7/group; wild-type (WT) versus GPR40^−/− mice). Different letters are attributed to significantly different groups (p < 0.05); error bars represent standard deviation (±S.D.).