Glucose Catabolism with Resultant Formation of Reactive Oxygen Species and Local Hypoxia Modulates the Induction and Expansion of Hematopoietic Stem Cells

Abstract 408

Metabolic disorders, including obesity, diabetes, and their related complications have become the leading cause of preventable death in the U.S. While effects of increased blood sugar on the cardiovascular system and other organs are well established, consequences for the hematopoietic system are less well characterized. Specifically, the effects of gestational diabetes on embryonic blood formation have not been elucidated in detail. To determine the impact of elevated glucose concentrations on hematopoietic stem cell (HSC) induction and expansion, we treated zebrafish embryos with D-glucose from 12 to 36 hours post fertilization (hpf). HSCs in the Aorta-Gonad-Mesonephros (AGM) region were expanded in a dose-responsive manner, as assessed by in situ hybridization for the conserved HSC markers, runx1 and cmyb; this effect was verified by analysis of CD41 expression (n≥25–50 embryos/condition). A significant 3-fold enhancement in HSC number was observed after 1% glucose treatment by qPCR for runx1 and FACS analysis of fluorescent HSC reporter embryos, Tg(cmyb:eGFP) and Tg(-6.0itga2b:eGFP)la2 (CD41:GFP). Other mono-, di-, and tri-saccharides exerted similar effects, expanding the number of HSCs. However, L-glucose, the inactive enantiomer, did not affect runx1 expression, implying that the effect of the metabolizable saccharides is independent of osmotic dynamics. Furthermore, treatment with glucose from 12 to 120 hpf resulted in a sustained increase in HSC number throughout the duration of treatment. Increased cellular proliferation was observed in the AGM region following 1% glucose treatment, as assessed by elevated BrdU incorporation and cyclin D1 expression. Additionally, acridine orange staining revealed a decrease in apoptotic cell number. HSC formation was impaired by morpholino knockdown of the glucose transporter glut1, indicating the requirement of glucose uptake for HSC formation. Exposure to chemical inhibitors of glycolysis (ethyl-3-bromopyruvate) and of oxidative phosphorylation (cyanide and oxaloacetate) reversed the beneficial effects of glucose on HSCs. In contrast, pharmacological and genetic modulations of the metabolic endocrine hormones IGF and insulin did not alter the effects of glucose treatment. Treatment with the glycolytic intermediate pyruvate expanded HSCs, while exposure to glucosamine, the first component of the hexosamine biosynthetic pathway, had no effect on blood stem cells. These results suggest that glucose catabolism specifically is responsible for HSC expansion. A potential consequence of heightened glucose metabolism is increased generation of reactive oxygen species (ROS), which may serve as important signaling factors mediating the observed effects on HSCs. In support of this hypothesis, treatment with the antioxidant N-acetylcysteine, which alone decreased HSC formation, could not be rescued by concomitant glucose exposure, while the oxidant, 1-phenyl-2-thiourea, elicited an expansion in HSC number. Using the fluorescent ROS sensor, dihydroethidium, we observed ROS in circulating erythrocytes, a subpopulation of CD41+ cells and in the adjacent somitic muscle. ROS are known inducers of the hypoxia sensor, hif1α, which regulates important hematopoietic genes including vegf and epo, as well as the glut1 glucose transporter, indicating a potential feedback loop controlling HSC induction. Chemical induction of hypoxia by cobalt chloride similarly enhanced HSC formation. Intriguingly, excess glucose can overcome the reduction of HSC number normally observed in silent heart mutants lacking blood circulation, possibly by ROS/hif1α-mediated induction of vegf and NO in the hematopoietic niche. The beneficial effect of glucose on hematopoiesis was conserved in adult zebrafish: FACS analysis of kidney marrow following sublethal irradiation revealed a more rapid recovery of the progenitor population after glucose exposure. Our data suggest that, in conjunction with the cardiovascular system, energy metabolism plays a key role in regulating hematopoetic induction and homeostasis. These results could lead to novel therapeutic approaches for HSC modulation, and may unveil specific risks of obesity and diabetes for hematopoiesis during gestation and in the adult. (equal contribution: WG, TEN).