The role of cystathionine y-lyase and hydrogen sulfide in glucose transporter Glut1 expression in macrophages

Abstract

Hydrogen sulfide (H₂S) is an endogenous gasotransmitter that regulates immune function and energy metabolism in macrophages. While it is known to impair mitochondrial respiration at high levels, recent studies have shown that it promotes aerobic glycolysis, highlighting its potential role in regulating macrophage inflammatory response. The investigation of the link between H₂S and glucose metabolism in macrophages is of particular significance due to the role of glycolysis in driving macrophage innate immune functions. Inflammation stimulates macrophages to increase glycolysis by upregulating the expression of Glut1, the primary glucose transporter in these cells. Nuclear factor (NF)-κB is a transcription factor that elicits inflammation by regulating the expression of pro-inflammatory cytokines and chemokines in response to stimuli such as lipopolysaccharide (LPS), a bacterial toxin. However, NF-κB controls Glut1 expression in macrophages during immune responses. The phosphoinositide 3-kinase (PI3k)/ protein kinase B (Akt) signaling also plays a role in regulating NF-κB activity and glucose metabolism. The precise impact of H₂S on the PI3k/Akt and NF-κB signaling pathways, as well as Glut1 expression in macrophages, remains poorly understood. The goal of my thesis research is to test for a link between endogenously produced H₂S and Glut1 expression during the pro-nflammatory response in macrophages. Small interfering (si)RNAs were used to knockout cystathionine g-lyase (CSE) gene expression and block endogenous H₂S production in LPS-stimulated macrophages and the effect on LPS-induced Glut1 protein and mRNA expression was measured. Silencing CSE in LPS-stimulated macrophages reduced Glut1 mRNA levels suggesting that H₂S regulates Glut1 levels in inflammatory macrophages. Pre-treatment of macrophages with drug inhibitors targeting NF-κB or PI3k/Akt prevented LPS-induced Glut1 expression, implicating the signaling activity of these proteins as regulators of inflammation induced Glut1 expression. Silencing CSE decreased NF-κB activation in LPS-stimulated macrophages, suggesting that endogenous H₂S acts via NF-κB to supports Glut1 expression. To determine whether H₂S supports the LPS/NF-κB/Glut1 response, we treated LPS induced macrophages with GYY4137, a slow releasing H₂S-donor molecule. Low levels of exogenous H₂S did not change Glut1 expression in LPS treated cells. However, high levels of H₂S inhibited NF-κB activation and Glut1 expression and increased Akt activation, suggesting an antiinflammatory role of elevated H₂S levels. The anti-inflammatory effect of elevated levels of H₂S on LPS-induced NF-κB activation and Glut1 expression shows a marked difference from the pro-inflammatory impact of enzymatically produced levels, emphasizing the importance of distinguishing between the source and concentration of H₂S. These findings suggest that H₂S plays a role in inflammatory Glut1 expression through modulating NF-κB and Akt activity in macrophages.