Objective: Glucagon-like peptide-1 (GLP-1)-based therapy via G protein-coupled receptor (GPCR) GLP-1R, to attenuate hyperglycemia in critical care has attracted great attention. However, the exaggerated inflammation by GLP-1R agonist, Exendin-4, in a mouse model of burn injury was quite unexpected. Recent studies found that GPCR might elicit proinflammatory effects by switching from Gαs to Gαi signaling in the immune system. Thus, we aimed to investigate the possible Gαs to Gαi switch in GLP-1R signaling in monocyte following burn injury.
Materials and methods: Splenic monocytes from sham and burn mice 24 h following burn injury were treated with consecutive doses of Exendin-4 alone or in combination with an inhibitor of Gαi signaling (pertussis toxin, PTX), or a blocker of protein kinase A (H89). Cell viability was assessed by CCK-8, and the supernatant was collected for cytokine measurement by ELISA. Intracellular cAMP level, phosphorylated PKA activity, and nuclear NF-κB p65 were determined by ELISA, ERK1/2 activation was analyzed by Western blot. The expression of GLP-1R downstream molecules, Gαs, Gαi and G-protein coupled receptor kinase 2 (GRK2) were examined by immunofluorescence staining and Western blot.
Results: Exendin-4 could inhibit the viability of monocyte from sham rather than burn mice. Unexpectedly, it could also reduce TNF-α secretion from sham monocyte while increase it from burn monocyte. The increased secretion of TNF-α by Exendin-4 from burn monocyte could be reversed by pretreatment of PTX or H89. Accordingly, Exendin-4 could stimulates cAMP production dose dependently from sham instead of burn monocyte. However, the blunt cAMP production from burn monocyte was further suppressed by pretreatment of PTX or H89 after 6-h incubation. Nevertheless, phosphorylated PKA activity was significantly increased by low dose of Exendin-4 in sham monocyte, by contrast, it was enhanced by high dose of Exendin-4 in burn monocyte after 1-h incubation. Following Exendin-4 treatment for 2 h ex vivo, total nuclear NF-κB and phosphorylated NF-κB activity, as well as cytoplasmic pERK1/2 expressions were reduced in sham monocyte, however, only pERK1/2 was increased by Exendin-4 in burn monocytes. Moreover, reduced expressions of GLP-1R, GRK-2 and Gαs in contrast with increased expression of Gαi were identified in burn monocyte relative to sham monocyte.
Conclusions: This study presents an unexpected proinflammatory switch from Gαs to Gαi signaling in burn monocyte, which promotes ERK1/2 and NF-κB activation and the downstream TNF-α secretion. This phenomenon is most probably responsible for proinflammatory response evoked by Gαs agonist Exendin-4 following burn injury.
Keywords: Burn injury; G protein-coupled receptor; Glucagon-like peptide 1; Inflammation; Monocyte; Signal transduction.