Alterations in GLUT1 and GLUT3 glucose transporter gene expression following unilateral hypoxia–ischemia in the immature rat brain

Abstract

The brain damage produced by unilateral cerebral hypoxia–ischemia in the immature rat results from major alterations in cerebral energy metabolism and glucose utilization which begin during the course of the insult and proceed into the recovery period. Consistent with a lack of pathology, the alterations in the hemisphere contralateral to the carotid artery ligation are transient and return to normal within 24 h of recovery, whereas the hemisphere ipsilateral to the ligation exhibits both early and late responses, and infarction. The facilitative glucose transporter proteins mediate glucose transport across the blood–brain barrier (55 kDa GLUT1), and into neurons and glia (GLUT3 and 45 kDa GLUT1), and demonstrate both early and late responses to perinatal hypoxia–ischemia. This study employed in situ hybridization histochemistry to investigate the temporal and regional patterns of GLUT1 and GLUT3 gene expression following a severe (2.5 h) hypoxic–ischemic insult in the 7-day old rat brain. Enhanced GLUT1 mRNA expression was apparent in cerebral microvessels of both hemispheres and remained elevated in the ipsilateral hemisphere through 24 h of recovery, consistent with our previous observation of increased microvascular 55 kDa GLUT1 protein. The expression of the neuronal isoform, GLUT3, was enhanced in penumbral regions, such as piriform cortex and amygdala, but was rapidly reduced in the affected areas of cortex, hippocampus and thalamus, reflecting necrosis. The late response, observed at 72 h of recovery, was characterized by extensive necrosis in the ipsilateral hemisphere, loss of GLUT3 expression, and a gliotic reaction including increased GLUT1 in GFAP-positive astrocytes. This study demonstrates that cerebral hypoxia–ischemia in the immature rat produces both immediate-early and long-term effects on the glucose transporter proteins at the level of gene expression.

Introduction

Glucose is the primary cerebral energy substrate for the adult, as well as the immature brain, and it is the only substrate able to sustain metabolism under anaerobic conditions. The delivery of glucose to the brain involves its passage across the endothelial cells of the blood–brain barrier (BBB) and the cell membranes of neurons and glia. These processes are mediated by the facilitative glucose transporter proteins: 55 kDa GLUT1 in the BBB, 45 kDa in choroid plexus and ependyma and in non-vascular brain, predominantly glia, and GLUT3 in neurons 8, 18, 37. The levels of these proteins are quite low in the immature rat brain and can be limiting to cerebral glucose utilization 34, 38. We have previously demonstrated that unilateral cerebral hypoxia–ischemia in the immature rat of sufficient duration to produce severe brain damage, results in significant alterations in both GLUT1 and GLUT3 proteins, in both ipsilateral (damaged) and contralateral (undamaged) hemispheres during the initial 24 h of recovery [39]. Major alterations in cerebral energy metabolism and glucose utilization, as well as several changes in gene expression also characterize this interval of recovery. The biochemical changes include altered levels of brain glucose, and brain-to-blood glucose ratios, increased lactate levels, and falling levels of ATP and phosphocreatine, reflective of the imbalance in the relationship between substrate supply and energy demand occurring during the evolution of the hypoxic–ischemic brain damage 26, 31, 33, 43. All of these studies involved hemispheric determinations of glucose transporter protein or metabolite levels, and thus do not provide any regional definition of selective alterations. More recent studies have demonstrated that additional early changes include induction of several immediate early and stress-response genes, such as c-fos and hsp70, observed throughout the affected hemisphere 1, 4, 22. The purpose of this study was to determine whether GLUT1 or GLUT3 gene expression is part of the early stress response in the immature brain and also to determine the temporal relationship between mRNA and protein expression. In addition, since reliable immunocytochemical localization of the glucose transporter proteins in brain is difficult, in situ hybridization analysis provides a regional definition of hypoxia/ischemia-induced alterations in GLUT1 and GLUT3 expression.