A Representative GIIA Phospholipase A2 Activates Preadipocytes to Produce Inflammatory Mediators Implicated in Obesity Development

Abstract

Adipose tissue secretes proinflammatory mediators which promote systemic and adipose tissue inflammation seen in obesity. Group IIA (GIIA)-secreted phospholipase A₂ (sPLA₂) enzymes are found to be elevated in plasma and adipose tissue from obese patients and are active during inflammation, generating proinflammatory mediators, including prostaglandin E₂ (PGE₂). PGE₂ exerts anti-lipolytic actions and increases triacylglycerol levels in adipose tissue. However, the inflammatory actions of GIIA sPLA₂s in adipose tissue cells and mechanisms leading to increased PGE₂ levels in these cells are unclear. This study investigates the ability of a representative GIIA sPLA₂, MT-III, to activate proinflammatory responses in preadipocytes, focusing on the biosynthesis of prostaglandins, adipocytokines and mechanisms involved in these effects. Our results showed that MT-III induced biosynthesis of PGE₂, PGI₂, MCP-1, IL-6 and gene expression of leptin and adiponectin in preadipocytes. The MT-III-induced PGE₂ biosynthesis was dependent on cytosolic PLA₂ (cPLA₂)-α, cyclooxygenases (COX)-1 and COX-2 pathways and regulated by a positive loop via the EP₄ receptor. Moreover, MT-III upregulated COX-2 and microsomal prostaglandin synthase (mPGES)-1 protein expression. MCP-1 biosynthesis induced by MT-III was dependent on the EP4 receptor, while IL-6 biosynthesis was dependent on EP3 receptor engagement by PGE₂. These data highlight preadipocytes as targets for GIIA sPLA₂s and provide insight into the roles played by this group of sPLA₂s in obesity.

Keywords: EP receptors; adipokines; cytokines; phospholipase A2; preadipocytes; prostaglandins.

Figure 1

MT-III induces production of PGE₂, PGI₂, TXA₂ and LTB₄ by 3T3-L1 preadipocytes. Cells were incubated with MT-III (0.4 µM) or DMEM (control) for 1 to 48 h. Bar graphs show the MT-III-induced release of PGE₂ (A), PGI₂ (B), TXA₂ (C) and LTB₄ (D) by preadipocytes. Concentrations were quantified in culture supernatants by EIA commercial kit. Results are expressed as mean ± SEM from 3 independent experiments. * p < 0.05 as compared with control group (two-way ANOVA and Bonferroni posttest).

Figure 2

MT-III activates COX-1 and COX-2 pathways for release of PGE₂ by 3T3-L1 preadipocytes. (A) Cells were incubated with either valerylsalicylate (VSA) (10 µM), or NS-398 (10 µM), or both for 1 h, followed by incubation with MT-III (0.4 µM) for 6 h. PGE₂ concentrations were quantified in culture supernatants by EIA commercial kit. (B–E) 3T3-L1 preadipocytes were incubated with MT-III (0.4 µM) or DMEM (control) for 1 up to 48 h. (B) Western blotting of COX-1 and β-actin (loading control) showing immunoreactive bands. (D) Western blotting of COX-2 and β-actin (loading control) showing immunoreactive bands. Densitometric analysis of immunoreactive (C) COX-1 and (E) COX-2 bands. Density data (in arbitrary units) were normalized with those of β-actin. Results are expressed as mean ± SEM from 3 independent experiments. * p < 0.05 as compared with control group and # p < 0.05 as compared with MT-III group (two-way ANOVA and Bonferroni posttest).

Figure 3

MT-III upregulates protein expression of mPGES-1 in 3T3-L1 preadipocyte. Cells were incubated with MT-III (0.4 µM) or DMEM (control) for 1 up to 12 h. (A) Western blotting of mPGES-1 and β-actin (loading control) showing immunoreactive bands. (B) Densitometric analysis of immunoreactive mPGES-1 bands. Density data (in arbitrary units) were normalized with those of β-actin. Results are expressed as mean ± SEM from 3 experiments. * p < 0.05 as compared with the control group (two-way ANOVA and Bonferroni posttest).

Figure 4

MT-III-induced PGE₂ release is dependent on cPLA2-α in 3T3-L1 preadipocytes. Cells were incubated with Pyr-2 (1 μM) for 1 h followed by incubation with MT-III (0.4 µM) for 3 h. PGE₂ concentrations were quantified in culture supernatants by EIA commercial kit. * p < 0.05 as compared with control group and # p < 0.05 as compared with MT-III group (two-way ANOVA and Bonferroni posttest).

Figure 5

EP4 receptor participates in MT-III-induced PGE₂ biosynthesis in 3T3-L1 preadipocytes. (A) Preadipocytes were incubated with SC-19220 (10 μM), AH6809 (10 μM), L-798106 (1 μM) or AH23848 (10 μM) for 1 h followed by incubation with MT-III (0.4 µM) for 6 h. PGE₂ concentrations were quantified in culture supernatants by EIA commercial kit. (B–I) 3T3-L1 cells were incubated with MT-III (0.4 µM) or DMEM (control) for 1 up to 48 h. (B,D,F,H) Western blotting of EP1, EP2, EP3 and EP4 receptors, respectively, and β-actin (loading control), showing immunoreactive bands. (C,E,G,I) Densitometric analysis of immunoreactive bands for EP1, EP2, EP3 and EP4 receptors, respectively. Results are expressed as mean ± SEM from 3 independent experiments. * p < 0.05 as compared with control group and # p < 0.05 as compared with MT-III group (one-way ANOVA and Bonferroni posttest in (A) and two-way ANOVA and Bonferroni posttest in (C,E,G,I)).

Figure 5

EP4 receptor participates in MT-III-induced PGE₂ biosynthesis in 3T3-L1 preadipocytes. (A) Preadipocytes were incubated with SC-19220 (10 μM), AH6809 (10 μM), L-798106 (1 μM) or AH23848 (10 μM) for 1 h followed by incubation with MT-III (0.4 µM) for 6 h. PGE₂ concentrations were quantified in culture supernatants by EIA commercial kit. (B–I) 3T3-L1 cells were incubated with MT-III (0.4 µM) or DMEM (control) for 1 up to 48 h. (B,D,F,H) Western blotting of EP1, EP2, EP3 and EP4 receptors, respectively, and β-actin (loading control), showing immunoreactive bands. (C,E,G,I) Densitometric analysis of immunoreactive bands for EP1, EP2, EP3 and EP4 receptors, respectively. Results are expressed as mean ± SEM from 3 independent experiments. * p < 0.05 as compared with control group and # p < 0.05 as compared with MT-III group (one-way ANOVA and Bonferroni posttest in (A) and two-way ANOVA and Bonferroni posttest in (C,E,G,I)).

Figure 6

MT-III induces MCP-1 and IL-6 production by 3T3-L1 preadipocytes. Cells were incubated with MT-III (0.4 μM), or DMEM (control) for ½ up to 48 h. Bar graphs show concentrations of (A) MCP-1 and (B) IL-6 released by cells incubated with MT-III. Cytokines concentrations were quantified in culture supernatants by Cytometric Bead Array (CBA). Results are expressed as mean ± SEM from 5 experiments. * p < 0.05 as compared with control group (two-way ANOVA and Bonferroni posttest).

Figure 7

EP3 and EP4 receptors participate in the MT-III-induced release of IL-6 and MCP-1, respectively, by 3T3-L1 preadipocytes. Cells were incubated with AH23848 (10 μM) or L-798106 (1 μM) or vehicle for 1 h followed by incubation with MT-III (0,4 μM) for 12 or 24 h. Graphs show participation of EP4 receptor in the MT-III-induced release of MCP-1 (A) and participation of the EP3 receptor in the MT-III-induced release of IL-6 (B). Concentration of cytokines were quantified from culture supernatants by Cytometric Bead Array (CBA). Results are expressed as mean ± SEM from 5 experiments. * p < 0.05 as compared with control group and # p < 0.05 as compared with MT-III group (one-way ANOVA and Bonferroni posttest).

Figure 8

MT-III upregulates gene expression of leptin and adiponectin by 3T3-L1 preadipocytes. Cells were incubated with MT-III (0.4 μM) or DMEM (control) for 1, 3 or 6 h. Graphs show gene expression of leptin (A), adiponectin (B) and resistin (C) in the presence of MT-III. Concentrations of adipokines were quantified in cell lysates by qPCR. Results are expressed as mean ± SEM from 5 experiments. * p < 0.05 as compared with the control group (two-way ANOVA and Bonferroni posttest).

Scheme 1

Proinflammatory pathways activated by MT-III, a GIIA snake venom sPLA₂, in 3T3-L1 preadipocytes. (1) MT-III stimulates preadipocytes to release PGE₂ and (2) PGI₂; (3) PGE₂ release induced by MT-III is dependent on cPLA₂-α, (4) COX-1, COX-2, mPGES-1 and (5) EP4 receptor, which triggers a positive loop for PGE₂ production; (6) MT-III up-regulates COX-2 and mPGES-1, key enzymes involved in PGE biosynthesis. Moreover, MT-III induces the release of (7) MCP-1, dependent on the EP4 receptor, and (8) IL-6, dependent on the EP3 receptor. Furthermore, (9) MT-III up-regulates leptin and adiponectin gene expression