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.

Bothropsenvenomation is a public health problem in Brazil. Despite the advances in the knowledge of the pathogenesis of systemic and local effects induced byBothropsvenom, the target tissues to this venom are not completely characterised. As preadipocytes are important cells of the adipose tissue and synthesize inflammatory mediators, we investigated the ability ofB.moojenisnake venom (Bmv) to stimulate an inflammatory response in 3T3-L1 preadipocytesin vitro, focusing on (1) the release of PGE₂, IL-6, TNF-α, MCP-1, KC, leptin and adiponectin; (2) the mechanisms involved in PGE₂release and (3) differentiation of these cells. Cytotoxicity of Bmv was determined by MTT assay. The concentrations of PGE₂, cytokines and adipokines were quantified by EIA. Participation of the COX-1 and COX-2 enzymes, NF-κB and PGE₂receptors (EP1-4) was assessed using a pharmacological approach, and protein expression of the COX enzymes and P-NF-κB was analysed by western blotting. Preadipocyte differentiation was quantified by Oil Red O staining. Bmv (1 μg/mL) induced release of PGE₂, IL-6 and KC and increased expression of COX-2 in preadipocytes. Basal levels of TNF-α, MCP-1, leptin and adiponectin were not modified. Treatment of cells with SC560 (COX-1 inhibitor) and NS398 (COX-2 inhibitor) inhibited Bmv-induced PGE₂release. Bmv induced phosphorylation of NF-κB, and treatment of the cells with TPCK and SN50, which inhibit distinct NF-κB domains, significantly reduced Bmv-induced PGE₂release, as did the treatment with an antagonist of PGE₂receptor EP1, unlike treatment with antagonists of EP2, EP3 or EP4. Bmv also induced lipid accumulation in differentiating cells. These results demonstrate that Bmv can activate an inflammatory response in preadipocytes by inducing the release of inflammatory mediators; that PGE₂production is mediated by the COX-1, COX-2 and NF-κB pathways; and that engagement of EP1 potentiates PGE₂synthesis via a positive feedback mechanism. Our findings highlight the role of the adipose tissue as another target for Bmv and suggest that it contributes toBothropsenvenomation by producing inflammatory mediators.

Snakebites are a worldwide public health problem with high-cost treatment in many countries. Crotalus genus snakes, commonly known as rattlesnakes, cause the most lethal snakebites in Brazil. Crotalus ssp envenomation is characterised by systemic neurotoxicity, myotoxicity, renal failure and minor local effects. Despite the knowledge of envenomation’s pathogenesis, the impact of Crotalus venom on adipose tissue (AT) cells is unknown. AT is an endocrine organ capable of releasing diverse immunomodulatory molecules, including prostaglandin E₂ (PGE₂). Herein, we investigated the effects of Crotalus durissus terrificus venom (CdtV) on preadipocytes in vitro, focussing on the release of PGE₂ and mechanisms involved. CdtV (5 and 10 μg/mL) induced a marked release of PGE₂ by preadipocytes (3–24 h) compared to controls. Pre-treatment of cells with SC-560 or NS-398, selective inhibitors for cyclooxygenase (COX)-1 and COX-2 enzymes, respectively, decreased CdtV-induced PGE₂ release after 24 h. CdtV (1 and 10 μg/mL) did not change COX-1 protein expression by preadipocytes, but induced COX-2 protein expression at all time intervals evaluated. Pre-treating preadipocytes with AH6809, an inhibitor of the PGE₂ EP2 receptor, significantly increased the CdtV-induced PGE₂ release. EP1, EP3, or EP4 receptor antagonists did not change the CdtV-induced PGE₂ release. Additionally, CdtV did not alter the EP2 receptor protein expression in preadipocytes. These findings demonstrate that CdtV activates preadipocytes to produce PGE₂ via COX-1 activation and COX-2 protein expression and PGE₂ triggers a negative feedback loop via EP2 for its own production. These results highlight AT as another target for CdtV that may contribute to envenomation.