Hypoglycemia-induced effects on cell proliferation, cell death, and glucose metabolism in organogenesis-stage embryonic mouse heart

Hypoglycemia is a common clinical condition and is believed to cause cardiac malformations in offspring born to diabetic mothers. This study investigates the in vivo and in vitro effects of hypoglycemia on glucose metabolism, cell death and cell proliferation in the organogenesis-stage embryonic mouse heart. This study also offers a possible way to prevent excessive cell death due to hypoglycemia by over expressing Hsp70. Effects on glucose metabolism were evaluated by 13C-NMR spectroscopy to study glucose utilization and lactate production in embryonic day (E) 10.5 embryonic hearts exposed to hypoglycemia (40 mg/dl glucose) compared to normoglycemia (150 mg/dl glucose). Cell death was evaluated by TUNEL, LysotrackerRTM, active caspase-3 protein expression in vivo and in vitro, and by flow cytometry using TUNEL and myosin heavy chain in vivo. Cell proliferation was evaluated by p53, and PCNA immunohistochemistry and Western analysis. Hsp70 over-expression was achieved in isolated heart cultures and the hearts post-culture were stained with LysotrackerRTM or used for Western analysis to analyze Hsp70 over-expression and active caspase-3 expression. 13C-NMR spectroscopy revealed increased lactate production in hearts exposed to hypoglycemia as compared to normoglycemia indicative of increased glucose metabolism by glycolysis. TUNEL, LysotrackerRTM, and active caspase-3 assays indicate increased cell death induced by hypoglycemia in E9.5 and E10.5 embryonic hearts but not in E8.5 embryonic hearts. Flow cytometry results are similar to TUNEL and LysotrackerRTM results. p53 and PCNA analysis demonstrates decreased cell proliferation in response to hypoglycemia during organogenesis. Over-expressing Hsp70 helped prevent excessive cell death induced by hypoglycemia in embryonic mouse hearts. Over-expressing Hsp70 significantly reduced active capase-3 expression as demonstrated by Western analysis. The results indicate that 24 hr hypoglycemic exposure in vivo and in vitro significantly alters glucose metabolism, cell death and cell proliferation in the organogenesis-stage embryonic mouse heart. These effects may eventually contribute to lethality in the embryo or congenital cardiac defects observed in diabetic offspring.