Alterations in Mitochondrial Function During Acute Hypoglycemia in Newborn Piglets • 1496

Abstract

Decreased substrate availability may lead to increased generation of oxygen free radicals in mitochondria due to partial reduction of oxygen. The present study tests the hypothesis that acute hypoglycemia alters mitochondrial function, resulting in oxygen free radical-induced mitochondrial damage. Studies were performed in newborn piglets exposed to normoglycemia (n=7) or 2 hr of hypoglycemia (n=6). Hypoglycemia (blood glucose 1 mmol/L) was induced with a bolus of regular insulin, 100 U/kg IV, followed by a continuous IV infusion of insulin, 2 U/kg/min. Mitochondria were isolated from cerebral cortex using a discontinuous Ficoll gradient and mitochondrial DNA was extracted. Cerebral oxidative phosphorylation was assessed biochemically by measuring tissue levels of ATP and phosphocreatine (PCr). Lipid peroxidation products (fluorescent compounds and conjugated dienes) were measured in mitochondrial membranes as an index of free radical-induced modification of membrane structure. Mitochondrial DNA analysis was performed using 1% agarose gel electrophoresis as an additional marker of mitochondrial damage. During hypoglycemia, blood glucose concentration decreased from the baseline value(mean ± SD) of 5.8 ± 1.2 mmol/l to 1.2 ± 0.4 mmol/L (p<0.005). ATP concentration (mmoles/g brain) was 4.7 ± 1.0 and 3.8± 0.7 in control and hypoglycemia respectively (p=ns); PCr (mmoles/g brain) decreased by 50% during hypoglycemia to 1.4 ± 0.8 from the control value of 2.7 ± 0.4 (p<0.05). Conjugated dienes were significantly increased in mitochondrial membranes during hypoglycemia to 4.14± 1.72 mmoles/g protein (p<0.05 compared to the reference value of 0 in controls). Fluorescent compounds (mg quinine sulfate/g protein) were also increased during hypoglycemia from the control value of 15.2 ± 9.5 to 62.2 ± 15.8 (p<0.001). Mitochondrial DNA from hypoglycemic brain showed marked fragmentation with a random distribution of fragment sizes ranging from 100-1500 bp, while control DNA showed no significant fragmentation. Thus we conclude that 2 hr of acute hypoglycemia in newborn piglets increased mitochondrial free radical generation as demonstrated by peroxidation of mitochondrial membranes. The observed pattern of mitochondrial DNA fragmentation could also be due to modification of DNA by oxygen free radicals. We speculate that free radical-induced changes in mitochondrial function during acute hypoglycemia may lead to progressive mitochondrial failure and subsequent cell death.