Upregulation of Blood-Brain Barrier GLUT1 Glucose Transporter Protein and mRNA in Experimental Chronic Hypoglycemia Arno K. Kumagai, Young-Sook Kang, Ruben J. Boado, and William M. Pardridge An in vivo model of chronic hypoglycemia was used to investigate changes in blood-brain barrier (BBB) glucose transport activity and changes in the expression of GLUT1 mRNA and protein in brain microvasculature occurring as an adaptive response to low circulating glucose levels. Chronic hypoglycemia was induced in rats by constant infusion of insulin via osmotic minipumps; control animals received infusions of saline. The criterion for chronic hypoglycemia was an average blood glucose concentration of <2.3 mmol/1 (42 mg/dl) after 5 days. The average blood glucose concentration at the end of the experimental period in the rats selected for study was 2.0 ± 0.1 mmol/1 (36 ± 1 mg/dl) vs. 4.9 ± 0.1 mmol/1 (88 ± 1 mg/dl) in the controls. Internal carotid artery perfusion studies demonstrated an increase in the BBB permeabil- ity-surface area (PS) product of 40% (i» < 0.0005) in the chronically hypoglycemic animals as compared with con- trols. Western blotting of solubilized isolated brain cap- illaries demonstrated a 51% increase (i> < 0.05) in immunoreactive BBB GLUT1 in the chronically hypogly- cemic rats, and Northern blotting of whole-brain poly(A+) mRNA revealed a 50% increase in the GLUT1- to-actin ratio in the insulin-treated group (P < 0.05). Northern blotting analysis of microvessel-depleted total brain poly(A-H) showed that the increase in GLUT1 mRNA in the chronically hypoglycemic rats was restricted to the BBB. The present study demonstrates increased expression of GLUT1 mRNA and protein at the BBB in chronic hypoglycemia and suggests that this increase is responsible for the compensatory increase in BBB glu- cose transport activity that occurs with chronically low circulating blood glucose levels. Diabetes 44:1399-1404, 1995 I n the brain, glucose represents the principal metabolic substrate required for normal neuronal function (1). Glucose gains entry into cerebral neurons from the blood by transport first across the brain capillaries comprising the blood-brain barrier (BBB) and second across neuronal cell membranes. Cytochalasin B binding assays From the Department of Medicine (A.K.K., Y.-S.K., R.J.B., W.M.P.) and Brain Research Institute (R.J.B., W.M.P.)> University of California at Los Angeles School of Medicine, Los Angeles, California. Address correspondence and reprint requests to Dr. Arno K. Kumagai, Division of Endocrinology, Department of Medicine, University of California at Los Angeles, Campus Mail: 168217, Los Angeles, CA 90095-1682. Received for publication 12 December 1994 and accepted in revised form 10 August 1995. ADU, arbitrary densitometric unit; BBB, blood-brain barrier; PS, permeability- surface area; SDS, sodium dodecyl sulfate. have indicated that the concentration of glucose transporters in brain capillaries is 5-10 times that found in brain cell membranes (2,3). However, the amount of capillary protein per gram of brain tissue is <0.2% of that of brain parenchyma (4), owing to the fact that the surface area of the cerebral neuronal and glial cell membranes far exceeds that of the cerebral microvasculature (5). Consequently, the concentra- tion of glucose transporters associated with brain microves- sels represents only a minute fraction of that of the total glucose transporters in the brain, an observation that sug- gests that the rate-limiting step in glucose transport from blood to brain is at the brain microvasculature or BBB. Glucose transport across the BBB and across neuronal cell membranes is mediated by GLUT1 (6-10) and GLUT3 (8,11), respectively, isoforms of the sodium-independent glucose transporter gene family (12,13). Under normal physiological conditions, cerebral glucose metabolism is limited by the rate of glucose phosphorylation within the cytoplasm of the neuronal cells, which exceeds that of glucose transport by approximately twofold (14). However, in the setting of a low systemic blood glucose concentration, as occurs in hypoglycemia, glucose transport becomes the limiting step of cerebral glucose utilization (15,16). To protect the brain against the deleterious effects of hypoglycemia, compensatory mechanisms at the BBB serve to maintain BBB glucose transport in the face of decreased peripheral glucose concentrations. Clinical evidence of this compensation in chronic hypoglycemia may be seen in patients with insulinoma who experience hypoglycemia- induced adrenergic and neuroglycopenic symptoms at lower blood glucose levels than nonchronically hypoglycemic indi- viduals (17). Elucidation of the physiological basis for such compensation has come in the form of in vivo studies in chronically hypoglycemic rats, which have demonstrated increased brain extraction of glucose in the chronically hypoglycemic animals as compared with euglycemic con- trols (18). Recent in vivo studies have demonstrated in- creased abundance of GLUT1 protein and mRNA in whole- brain specimens in chronically hypoglycemic rats (19) and mice (20). Glucose deprivation studies of bovine brain endo- thelial cell cultures in vitro has demonstrated an increase in the abundance of GLUT1 mRNA (21,22) and protein (23), an increase which is mediated through posttranscriptional reg- ulation (22). It has been assumed from data concerning GLUT1 expression in whole brain that GLUT1 expression at the BBB—the location of the rate-limiting step in glucose metabolism in hypoglycemia—is similarly altered in chronic hypoglycemic states (24). To date, however, no in vivo studies have specifically addressed this issue. Therefore, the DIABETES, VOL. 44, DECEMBER 1995 1399 Downloaded from http://diabetesjournals.org/diabetes/article-pdf/44/12/1399/361184/44-12-1399.pdf by guest on 27 January 2023