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Heterotrimeric G-protein subunit G?i2 contributes to agonist-sensitive apoptosis and degranulation in murine platelets.

ABSTRACT: G?i2 , a heterotrimeric G-protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet-expressed G?i2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether G?i2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen-related peptide (CoRP), which are further known to enhance degranulation and activation of ?IIb ?3-integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking G?i2 and their wild-type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 ?g/mL or 5 ?g/mL) significantly upregulated PS-exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P-selectin, and ?IIb ?3-integrin activation. These molecular alterations were significantly less pronounced in G?i2 -deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of G?i2 signaling in governing platelet activation and apoptosis.

SUBMITTER: Cao H

PROVIDER: S-EPMC6125243 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Heterotrimeric G-protein subunit Gαi2 contributes to agonist-sensitive apoptosis and degranulation in murine platelets.

Cao Hang H Qadri Syed M SM Lang Elisabeth E Pelzl Lisann L Umbach Anja T AT Leiss Veronika V Birnbaumer Lutz L Nürnberg Bernd B Pieske Burkert B Voelkl Jakob J Gawaz Meinrad M Bissinger Rosi R Lang Florian F

Physiological reports 20180901 17

Gαi2 , a heterotrimeric G-protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet-expressed Gαi2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gαi2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (P ...[more]

PMID: 30187671

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Project description:ObjectivesTypically, obesity results from an inappropriate balance between energy uptake from nutrient consumption and burning of calories, which leads to a pathological increase in fat mass. Obesity is a major cause of insulin resistance and diabetes. Inhibitory G proteins (Gαi) form a subfamily that is involved in the regulation of adipose tissue function. Among the three Gαi members, i.e. Gαi1, Gαi2, Gαi3, the Gαi2, protein is predominantly expressed in adipose tissue. However, the functions of the Gαi2 isoform in adipose tissue and its impact on the development of obesity are poorly understood.MethodsBy using AdipoqCreERT2 mice, we generated adipocyte-specific Gnai2-deficient mice to study Gαi2 function, specifically in white and brown adipocytes. These mice were fed either a control diet (CD) or a high fat diet (HFD). Mice were examined for obesity development, insulin resistance and glucose intolerance. We examined adipocyte morphology and the development of inflammation in the white adipose tissue. Finally, intracellular cAMP levels as an indicator of Gαi signaling and glycerol release as an indicator of lipolysis rates were measured to verify the impact of Gαi2 on the signaling pathway in brown and white adipocytes.ResultsAn adipocyte-specific deficiency of Gαi2 significantly reduced diet-induced obesity, leading to decreased fat masses, smaller adipocytes and decreased inflammation in the white adipose tissue relative to littermate controls. Concurrently, oxygen consumption of brown adipocytes and in vivo measured energy expenditure were significantly enhanced. In addition, glucose tolerance and insulin sensitivity of HFD-fed adipocyte-specific Gnai2-deficient mice were improved compared to the respective controls. In the absence of Gαi2, adrenergic stimulation of intracellular adipocyte cAMP levels was increased, which correlated with increased lipolysis and energy expenditure.ConclusionWe conclude that adipocyte Gαi2 is a major regulator of adipocyte lipid content in diet-induced obesity by inhibiting adipocyte lipolysis in a cAMP-dependent manner resulting in increased energy expenditure.

Project description:Background: Relaxins are small peptide hormones, which are novel candidate molecules that play important roles in cardiometablic syndrome. Relaxins are structurally related to the insulin hormone superfamily, which provide vasodilatory effects by activation of G-protein-coupled relaxin receptors (RXFPs) and stimulation of endogenous nitric oxide (NO) generation. Recently, relaxin could be demonstrated to activate Gi proteins and phosphoinositide 3-kinase (PI3K) pathways in cultured endothelial cells in vitro. However, the contribution of the Gi-PI3K pathway and their individual components in relaxin-dependent relaxation of intact arteries remains elusive. Methods: We used Gαi2- (Gnai2-/-) and Gαi3-deficient (Gnai3-/-) mice, pharmacological tools and wire myography to study G-protein-coupled signaling pathways involved in relaxation of mouse isolated mesenteric arteries by relaxins. Human relaxin-1, relaxin-2, and relaxin-3 were tested. Results: Relaxin-2 (∼50% relaxation at 10-11 M) was the most potent vasodilatory relaxin in mouse mesenteric arteries, compared to relaxin-1 and relaxin-3. The vasodilatory effects of relaxin-2 were inhibited by removal of the endothelium or treatment of the vessels with N (G)-nitro-L-arginine methyl ester (L-NAME, endothelial nitric oxide synthase (eNOS) inhibitor) or simazine (RXFP1 inhibitor). The vasodilatory effects of relaxin-2 were absent in arteries of mice treated with pertussis toxin (PTX). They were also absent in arteries isolated from Gnai2-/- mice, but not from Gnai3-/- mice. The effects were not affected by FR900359 (Gαq protein inhibitor) or PI-103 (PI3Kα inhibitor), but inhibited by TGX-221 (PI3Kβ inhibitor) or AS-252424 (PI3Kγ inhibitor). Simazine did not influence the anti-contractile effect of perivascular adipose tissue. Conclusion: Our data indicate that relaxin-2 produces endothelium- and NO-dependent relaxation of mouse mesenteric arteries by activation of RXFP1 coupled to Gi2-PI3K-eNOS pathway. Targeting vasodilatory Gi-protein-coupled RXFP1 pathways may provide promising opportunities for drug discovery in endothelial dysfunction and cardiometabolic disease.

Dataset Information

Heterotrimeric G-protein subunit G?i2 contributes to agonist-sensitive apoptosis and degranulation in murine platelets.

Publications

Heterotrimeric G-protein subunit Gα<sub>i2</sub> contributes to agonist-sensitive apoptosis and degranulation in murine platelets.

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