Effects of fasting and insulin-induced hypoglycemia on brain cell membrane function and energy metabolism during hypoxia–ischemia in newborn piglets

Abstract

This study was done to determine the effects of 12 h fasting-induced mild hypoglycemia (blood glucose 60 mg/dl) and insulin-induced moderate hypoglycemia (blood glucose 35 mg/dl) on brain cell membrane function and energy metabolism during hypoxia–ischemia in newborn piglets. Sixty-three ventilated piglets were divided into six groups; normoglycemic control (NC, n=8), fasting-induced mildly hypoglycemic control (FC, n=10), insulin-induced moderately hypoglycemic control (IC, n=10), normoglycemic/hypoxic–ischemic (NH, n=11), fasting-induced mildly hypoglycemic/hypoxic–ischemic (FH, n=12) and insulin-induced moderately hypoglycemic/hypoxic–ischemic (IH, n=12) group. Cerebral hypoxia–ischemia was induced by occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min. The brain lactate level was elevated in NH group and this change was attenuated in FH and IH groups. The extent of cerebral lactic acidosis during hypoxic–ischemic insult showed significant positive correlation with blood glucose level (r=0.55, p<0.001). Cerebral Na⁺, K⁺-ATPase activity and concentrations of high-energy phosphate compounds were reduced in NH group and these changes were not ameliorated in FH or IH group. Cortical levels of conjugated dienes, measured as an index of lipid peroxidation of brain cell membrane, were significantly elevated in NH, FH and IH groups compared with NC, FC and IC groups and these increases were more profound in FH and IH with respect to NH. Blood glucose concentration showed significant inverse correlation with levels of conjugated dienes (r=−0.35, p<0.05). These findings suggest that, unlike in adults, mild or moderate hypoglycemia, regardless of methods of induction such as fasting or insulin-induced, during cerebral hypoxia–ischemia is not beneficial and may even be harmful in neonates.

Introduction

It is well known that, in the adults, hyperglycemia aggravates 2, 28and hypoglycemia mitigates 20, 29, 38hypoxic ischemic brain injury. The mechanism might be attributed to the differences in the extent of cerebral lactic acidosis during hypoxia–ischemia 22, 30, 32, 34, 45

However, in neonates, the available data on the role of glucose in modulating hypoxic–ischemic brain injury is still controversial. There are some recent studies that report the benefits of glucose supplement during hypoxia–ischemia to brain injury 25, 33, 41, while on the other hand, there are reports that showed that it is as deleterious in neonates as it is in adults 4, 17. Further studies will be necessary to clarify whether this age-specific paradoxical role of glucose in cerebral hypoxia–ischemia really exists or not.

In our previous study [4], we demonstrated that hyperglycemia during hypoxia–ischemia in the newborn piglet was not beneficial and might be even harmful, as evidenced by increased lipid peroxidation products and accentuating cerebral lactic acidosis. Thus, the next step was to carry out a study in neonates as in the adults to find the effects of hypoglycemia on hypoxic–ischemic brain injury and to determine whether hypoglycemia in neonates could ameliorate hypoxic–ischemic brain injury, as in the adults. LeBlanc et al. [17]demonstrated pathologic aggravation and worse neurologic examination scores in the hyperglycemic/hypoxic–ischemic newborn piglets compared to the insulin-induced hypoglycemic/hypoxic–ischemic piglets. In another study of a newborn piglet model [18], they reported that insulin-induced hypoglycemia during hypoxia–ischemia reduced the cerebral lactate level significantly compared to normoglycemic animals, however, neuropathologic outcomes were not compared.

In adults, fasting 14, 21or insulin-induced mild, but not moderate, hypoglycemia 12, 20ameliorated hypoxic–ischemic brain injury. Besides by reducing peripheral blood glucose levels, insulin has also been known to be neuroprotective by directly reacting with brain tissue 1, 43. However, this neuroprotective effect of insulin has not been demonstrated in neonates and even higher mortality than in control animals have been reported 39, 40, 44. In addition, since neonatal brain can utilize ketone bodies more efficiently than the adults, adequate fasting to produce hypoglycemia and ketonemia in the immature animals have been postulated to be more neuroprotective during hypoxia–ischemia than in insulin-induced hypoglycemia [46].

This study was done to determine the effects of hypoglycemia on the function of brain cell membranes and energy metabolism during cerebral hypoxia–ischemia in newborn piglets. We tested the following hypotheses: (1) Mild or moderate hypoglycemia in newborn piglets mitigates hypoxic–ischemic brain injury via the mechanisms of decreased brain lactic acidosis, which is dependent on blood glucose levels. (2) This modulating effect of hypoglycemia on hypoxia–ischemia induced cerebral dysfunction is different according to the methods of inducing hypoglycemia such as fasting or insulin-induced. We used the same hypoxic–ischemic model of newborn piglets previously described [4]. Mild hypoglycemia (blood glucose level in 60 mg/dl) was induced by overnight (12 h) fasting without any other intervention. Moderate hypoglycemia (blood glucose level in 35 mg/dl) was induced by modified glucose clamp technique with insulin infusion. Changes in brain cell membrane structure, function and energy metabolism were determined by measuring lipid peroxidation products (conjugated dienes), Na⁺, K⁺-ATPase activity and concentration of high-energy phosphate compounds in the cerebral cortex.