PFKFB3 Inhibits Fructose Metabolism in Pulmonary Microvascular Endothelial Cells

Abstract

Pulmonary microvascular endothelial cells contribute to the integrity of the lung gas exchange interface, and they are highly glycolytic. Although glucose and fructose represent discrete substrates available for glycolysis, pulmonary microvascular endothelial cells prefer glucose over fructose, and the mechanisms involved in this selection are unknown. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is an important glycolytic enzyme that drives glycolytic flux against negative feedback and links glycolytic and fructolytic pathways. We hypothesized that PFKFB3 inhibits fructose metabolism in pulmonary microvascular endothelial cells. We found that PFKFB3 knockout cells survive better than wild-type cells in fructose-rich medium under hypoxia. Seahorse assays, lactate and glucose measurements, and stable isotope tracing showed that PFKFB3 inhibits fructose-hexokinase-mediated glycolysis and oxidative phosphorylation. Microarray analysis revealed that fructose upregulates PFKFB3, and PFKFB3 knockout cells increase fructose-specific GLUT5 (glucose transporter 5) expression. Using conditional endothelial-specific PFKFB3 knockout mice, we demonstrated that endothelial PFKFB3 knockout increases lung tissue lactate production after fructose gavage. Last, we showed that pneumonia increases fructose in BAL fluid in mechanically ventilated ICU patients. Thus, PFKFB3 knockout increases GLUT5 expression and the hexokinasemediated fructose use in pulmonary microvascular endothelial cells that promotes their survival. Our findings indicate that PFKFB3 is a molecular switch that controls glucose versus fructose use in glycolysis and help better understand lung endothelial cell metabolism during respiratory failure.

Carbonic anhydrase, its inhibitors and vascular function

“…It has been shown that CA IX expression is higher and declines with extracellular acidity, which is related to cell proliferation, in pulmonary microvascular endothelial cells (PMVEs) compared with pulmonary arterial endothelial cells (PAECs). Moreover, CA IX is essential for PMVECs angiogenesis during acidosis by controlling the extracellular and intracellular pH (Lee et al, 2018). CA II has been identified as an antigen which elicited humoral immune responses in melanoma patients and expressed in the endothelium of new growth vessels in cancer tissues, including melanoma, and lung, renal and esophageal cancer (Yoshiura et al, 2005).…”

“…It is likely that these differences in EC morphology and function, and interactions with other vascular cells in the vascular wall of the vessels in question relate to the effectiveness of CA inhibitors as vasodilators in different vascular beds. Still, although CA II and CA IV have been shown to be expressed in the corneal endothelium (Nguyen and Bonanno, 2011), only the membrane bound CA IV and CA IX isoforms have been shown to be present in normal vascular endothelial cells, and not the CA II (Lee et al, 2018;Annan et al, 2019). However, CA II present in other vascular cells may have indirect effects on vascular endothelial function, but the structural and functional heterogeneity of ECs is likely to determine to what extent and by which mechanism CA is involved in regulation of vascular function in the organ concerned.…”