data for: Cystathionine y-lyase and hydrogen sulfide modulates glucose transporter Glut1 expression via NF-κB and PI3k/Akt in macrophages during inflammation
Macrophages play a crucial role in inflammation, a defense mechanism of the innate immune system. Metabolic function powered by glucose transporter isoform 1 (Glut1) is necessary for macrophage activity during inflammation. The present study investigated the roles of cystathionine-γ-lyase (CSE) and its byproduct, hydrogen sulfide (H2S), in macrophage glucose metabolism to explore the mechanism by which H2S acts as an inflammatory regulator in lipopolysaccharide- (LPS) induced macrophages. Our results demonstrated that LPS-treated macrophages increased Glut1 expression. LPS-induced Glut1 expression is regulated via nuclear factor (NF)-κB activation and is associated with phosphatidylinositol-3-kinase PI3k activation. Small interfering (si) RNA-mediated silencing of CSE decreased the LPS-induced NF-κB activation and Glut1 expression, suggesting a role for H2S in metabolic function in macrophages during pro-inflammatory response. Confoundingly, treatment with GYY4137, an H2S-donor molecule, also displayed inhibitory effects upon LPS-induced NF-κB activation and Glut1 expression. Moreover, GYY4137 treatment increased Akt activation, suggesting a role in promoting resolution of inflammation. Our study provides evidence that the source of H2S, either endogenous (via CSE) or exogenous (via GYY4137), supports or inhibits the LPS-induced NF-κB activity and Glut1 expression, respectively. Therefore, H2S may influence metabolic programming in immune cells to alter glucose substrate availability that impacts the immune response.
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Protein extraction and Western blot Proteins were detected using enhanced chemiluminescence detection kit (Bio-Rad). The band density was quantified by Image J 1.8.0172 software (National Institutes of Health) and the representative data were experiment normalized to nontreated control. RNA extraction and RT-qPCR PowerUp SYBR Green Mix (Applied Biosystems, Waltham, MA) was used according to the manufacturer’s instructions in a 384-well format. Ct values were acquired on QTower (JenaAnalytik). To compare the mRNA levels between different samples, the 2-ΔCt method was used; and data were normalized to GAPDH. Experiments were run in triplicate; each sample represents three technical repeats. NF-κB activity The commercial kit NF-κB phospho-p65 InstantOne ELISA (eBioscience, 85-86083-11) was used to detect phosphorylated NF-κB in whole cell lysates following the manufacturer’s instructions. Akt activity The commercial kit Akt (phosphor) pSer473 InstantOne ELISA (eBioscience, 85-86042-11) was used to detect activated (phosphorylated) Akt in whole cell lysates. Flow cytometry to measure Glut1 surface levels For immunofluorescence surface staining of macrophages primary anti-Glut1 and secondary-PE conjugated antibodies were used, and cells were read by flow cytometry (Guava MUSE cell analyzer). Data files were analyzed for mean fluorescence intensity by floreada.io software. Glutathione level assay GSH level in macrophages was determined using a one-step fluorometric kit (Fluorometric-Green, ab138881, Abcam)