Proinflammatory switch from Gαs to Gαi signaling by Glucagon-like peptide-1 receptor in murine splenic monocyte following burn injury
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.
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.
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.
KeywordsGlucagon-like peptide 1 G protein-coupled receptor Monocyte Inflammation Signal transduction Burn injury
This study was supported by National Natural Science Foundation (81272089), the National Basic Research Program of China (2012 CB518102), and Twelve-Five Plan for Military Scientific Foundation (BWS12J050).
QHZ: development of the concept, conduction of most experiments, generation of all the data and figures, drafting and final approval the paper. JWH, GLL, XJJ and XDY: conduct part of the experiments. YMY: providing part of the fund and the personnel.
Compliance with ethical standards
Conflict of interest
The authors report no conflict of interest.
- 9.Montrose-Rafizadeh C, Avdonin P, Garant MJ, Rodgers BD, Kole S, Yang H, et al. Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells. Endocrinology. 1999;140:1132–40.CrossRefPubMedGoogle Scholar
- 15.Watada S, Yu YM, Fischman AJ, Kurihara T, Shen CA, Tompkins RG, et al. Evaluation of intragastric vs intraperitoneal glucose tolerance tests in the evaluation of insulin resistance in a rodent model of burn injury and glucagon-like polypeptide-1 treatment. J Burn Care Res. 2014;35:e66–72.CrossRefGoogle Scholar
- 44.Solomon KR, Kurt-Jones EA, Saladino RA, Stack AM, Dunn IF, Ferretti M, et al. Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide. J Clin Investig. 1998;102:2019–27.CrossRefPubMedPubMedCentralGoogle Scholar