Metabolism-Induced Reactive Oxygen Species and Hif1α-Mediated Gene Regulation Control the Timing and Magnitude of Hematopoietic Stem Cell Induction

Abstract 1266

During development, the embryo is exposed to rapidly changing metabolic conditions and demands. The impact of elevated glucose levels on the developing hematopoietic system is not well characterized. Intriguingly, children born to mothers with gestational diabetes as well as those diagnosed with diabetes themselves have a higher risk of developing childhood leukemia, suggesting increased blood sugar concentrations may have lasting impact on hematopoiesis. To evaluate the consequences of elevated glucose levels on hematopoietic stem cell (HSC) development, zebrafish embryos were exposed to glucose and modifiers of glucose metabolism during embryonic blood cell specification from 12 to 36 hours post fertilization (hpf). HSCs in the Aorta-Gonad-Mesonephros (AGM) region were analyzed by in situ hybridization for the conserved markers runx1 and cmyb (n≥25–50 embryos/condition). D-glucose expanded HSCs in a dose-responsive manner, while the metabolically inactive enantiomer L-glucose did not affect HSCs. Quantitation by qPCR and FACS analysis of fluorescent HSC reporter embryos runx1:eGFP, cmyb:eGFP and CD41:GFP revealed a ∼3-fold increase in HSCs. Enhanced cellular proliferation (BrdU) was detected in the AGM in response to increased glucose levels. The effects of glucose on HSCs were sustained without any block in differentiation potential in larvae, and in adults after marrow injury. AGM time course analysis and ultrastructural assessment by electron microscopy (EM) revealed accelerated runx1 expression and hematopoietic cluster formation. EM and in situ hybridization for scl, gata1, and globin also demonstrated enhanced red cell numbers in response to elevated glucose levels. Genetic and chemical modulations of the metabolic hormone insulin did not alter the effects of glucose on HSC number. Activity of the glucose transporter glut1 was required to observe enhanced HSC induction, and glucose exposure increased glucose, ATP and lactate concentrations in the embryo. Additionally, incubation with inhibitors of glycolysis (lonidamine, ethyl-3-bromopyruvate) and oxidative phosphorylation (cyanide, oxaloacetate) reversed the beneficial effects of glucose, demonstrating that glucose affects HSCs specifically through energy metabolism. Enhanced oxidative phosphorylation produces excess reactive oxygen species (ROS), which can directly serve as hematopoietic signaling factors; treatment with the antioxidants N-acetylcysteine and MitoQ decreased HSC formation and blocked the effect of concomitant glucose exposure, while H₂O₂ expanded HSCs. Similarly, genetic enhancement of ROS levels by knockdown of peroxiredoxin 1 increased HSCs. ROS were visualized in vivo in erythrocytes and CD41+ cells using the fluorescent sensor peroxyfluorescein 2; increased glucose levels significantly enhanced ROS in the AGM as determined by FACS analysis of the lmo2:dsRed hematopoietic precursor and vascular reporter. ROS directly affects the stability of the cellular hypoxia sensor, hypoxia inducible factor 1α (hif1α), which can regulate expression of hematopoietic genes, such as vegf and epo. Stabilization of hif1α by cobalt chloride or morpholino knockdown of vhl enhanced HSCs and rescued the block in HSC induction following inhibition of energy metabolism or ROS production. Furthermore, by microarray and qPCR gene expression analysis, glucose exposure greatly impacted hematopoietic related transcripts, in addition to eliciting significant changes in the hif1α gene network: glucose increased expression of epo and epor during the primitive wave of hematopoiesis, confirming the effects on erythrocytes seen in vivo. In addition to vegf, nos and igf were induced during the definitive wave of hematopoiesis in a dose- and time-dependent fashion, providing signals to support HSC induction and expansion. These data indicate that fluctuations in glucose levels, and subsequent metabolic activity initially occurring in the dorsal aorta, are sensed by hif1α, which then functions to genetically enhance hematopoietic supply in advance of the anticipated demands of the growing organism during development. Our work provides direct evidence that the developing embryo responds dynamically to metabolic challenges by accelerating and expanding blood formation and demonstrates a connection between glucose metabolism and hif1α signaling in regulating HSC induction.