Metabolic Endocrine Regulators of Hematopoietic Stem Cell Formation and Function.

Abstract 1496

Poster Board I-519

Obesity and subsequent diabetes have emerged as major health problems in the U.S. While the consequences of elevated blood glucose levels on the cardiovascular system and other organs are well known, the direct effects on the hematopoietic system are more elusive. Similarly, the impact of gestational diabetes on embryonic hematopoiesis has not been examined in detail. The zebrafish has emerged as an important model system to study conserved regulators of organ development and homeostasis. In order to evaluate the role of elevated glucose levels on hematopoietic stem cell (HSC) production, zebrafish embryos were exposed to increasing doses of D-glucose from 5 somites to 36 hours post fertilization (hpf); HSC number, as indicated by in situ hybridization for the conserved markers runx1 and cmyb in the Aorta-Gonad-Mesonephros (AGM) region, was increased at 0.5, 1% and 2% glucose; results were confirmed by in analysis of CD41 expression. Quantification using FACS analysis of fluorescent HSC reporter embryos and qPCR revealed a 2-3-fold enhancement following 1% glucose treatment. Other mono, di-, and trisaccharide sugars had similar effects, causing increased numbers of HSCs, however, L-glucose had no impact. BrdU incorporation in the AGM region was elevated after 1% glucose treatment, while acridine orange staining revealed an inhibitory effect on apoptosis. To evaluate potential mediators of these glucose-responsive effects, embryos were injected with antisense morpholino oligonucleotides (MO) against both the insulin (insr), and insulin-like growth factor receptors (igfr); insr and igfr receptors can each bind insulin, released following elevations in blood sugar levels. MO knockdown of insra or igfrb, but not igfra, influenceded runx1+ HSCs substantially, indicating an important role of these endocrine regulatory signaling pathways in HSC formation. However, D-glucose completely reversed these effects, implying either functional redundancy, or a multi-step, multi-effector process of HSC regulation by endocrine factors. To further clarify when insr- and/or igfr-mediated activity was influencing HSC formation and to correlate that effect with elevated glucose exposure, embryos were treated for defined periods with either 1% glucose, insulin, or IGF; exposure from 10 somites to 24 hpf influences the formation and arterial/venous specification of dorsal aorta, the conserved site of initial definitive HSC production, while exposure from 24 to 36 hpf regulates HSC induction. IGF exerted a positive effect on HSCs only after the establishment of the hematopoietic niche (>24hpf). Glucose treatment, however, positively influenced HSC formation at all time points examined, suggesting it works not only in the HSC niche to induce HSCs, but also prior to HSC formation. MO knockdown of the glucose transporter glut1 resulted in diminished HSC production, confirming a direct role of glucose in this process. To determine whether the effect of glucose elevation was mediated by changes in cellular energy production, embryos were exposed to chemical inhibitors of oxidative phosphorylation. Cyanide and oxaloacetate reversed the beneficial effects of D-glucose, indicating that energy production can modulate HSC formation. Investigation into the functional redundancy and cross-regulation of insulin and IGF on HSC self-renewal and the evolutionary conservation of the effects of energy metabolism on HSC production are ongoing; further studies will be needed to determine if glucose maintains an influential role on HSC homeostasis or bone marrow recovery following injury. These results could have an impact on methods for HSC modulation for therapeutic purposes, and may further unveil specific risks of obesity and diabetes for hematopoiesis and HSC homeostasis during gestation and in the adult.