Cerebral glucose utilization and glucose transporter expression: response to water deprivation and restoration.

The relationship between local rates of cerebral glucose utilization (ICMRglc) and glucose transporter expression was examined during physiologic activation of the hypothalamoneurohypophysial system. Three days of water deprivation, which is known to activate the hypothalamoneurohypophysial system, resulted in increased ICMRglc and increased concentrations of GLUT1 and GLUT3 in the neurohypophysis; mRNA levels of GLUT1 and GLUT3 were decreased and increased, respectively. Water deprivation also increased ICMRglc in the hypothalamic supraoptic and paraventricular nuclei; mRNA levels of GLUT1 and GLUT3 appeared to increase in these nuclei, but the changes did not achieve statistical significance. Restoration of water for 3 to 7 days reversed all observed changes in GLUT expression (protein and mRNA): restoration of water also reversed changes in ICMRglc in both the neurohypophysis and the hypothalamic nuclei. These results indicate that under conditions of neural activation and recovery, changes in ICMRglc and the levels of GLUT1 and GLUT3 are temporally correlated in the neurohypophysis and raise the possibility that GLUT1 and GLUT3 transporter expression may be regulated by chronic changes in functional activity. In addition, increases in the expression of GLUT5 mRNA in the neurohypophysis after dehydration provide evidence for involvement of microglial activation.

Effects of barbiturates on facilitative glucose transporters are pharmacologically specific and isoform selective.

Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [3H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC50s of 0.2-2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all 10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms.

Blood-brain barrier glucose transporter: effects of hypo- and hyperglycemia revisited.

The transport of glucose across the blood-brain barrier (BBB) is mediated by the high molecular mass (55-kDa) isoform of the GLUT1 glucose transporter protein. In this study we have utilized the tritiated, impermeant photolabel 2-N-[4-(1 -azi-2,2,2-trifluoroethyl)[2-3H]propyl]-1,3-bis(D-mannose-4-ylo xy)-2-propylamine to develop a technique to specifically measure the concentration of GLUT1 glucose transporters on the luminal surface of the endothelial cells of the BBB. We have combined this methodology with measurements of BBB glucose transport and immunoblot analysis of isolated brain microvessels for labeled luminal GLUT1 and total GLUT1 to reevaluate the effects of chronic hypoglycemia and diabetic hyperglycemia on transendothelial glucose transport in the rat. Hypoglycemia was induced with continuous-release insulin pellets (6 U/day) for a 12- to 14-day duration; diabetes was induced by streptozotocin (65 mg/kg i.p.) for a 14- to 21-day duration. Hypoglycemia resulted in 25-45% increases in regional BBB permeability-surface area (PA) values for D-[14C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarly, there was a 23+/-4% increase in total GLUT1/mg of microvessel protein and a 52+/-13% increase in luminal GLUT1 in hypoglycemic animals, suggesting that both increased GLUT1 synthesis and a redistribution to favor luminal transporters account for the enhanced uptake. A corresponding (twofold) increase in cortical GLUT1 mRNA was observed by in situ hybridization. In contrast, no significant changes were observed in regional brain glucose uptake PA, total microvessel 55-kDa GLUT1, or luminal GLUT1 concentrations in hyperglycemic rats. There was, however, a 30-40% increase in total cortical GLUT1 mRNA expression, with a 96% increase in the microvessels. Neither condition altered the levels of GLUT3 mRNA or protein expression. These results show that hypoglycemia, but not hyperglycemia, alters glucose transport activity at the BBB and that these changes in transport activity result from both an overall increase in total BBB GLUT1 and an increased transporter concentration at the luminal surface.

Glucose transporter expression in brain: relationship to cerebral glucose utilization.

Glucose is the principle energy source for mammalian brain. Delivery of glucose from the blood to the brain requires its transport across the endothelial cells of the blood-brain barrier and across the plasma membranes of neurons and glia, which is mediated by the facilitative glucose transporter proteins. The two primary glucose transporter isoforms which function in cerebral glucose metabolism are GLUT1 and GLUT3. GLUT1 is the primary transporter in the blood-brain barrier, choroid plexus, ependyma, and glia; GLUT3 is the neuronal glucose transporter. The levels of expression of both transporters are regulated in concert with metabolic demand and regional rates of cerebral glucose utilization. We present several experimental paradigms in which alterations in energetic demand and/or substrate supply affect glucose transporter expression. These include normal cerebral development in the rat, Alzheimer's disease, neuronal differentiation in vitro, and dehydration in the rat.

Monocarboxylate transporter expression in mouse brain.

Although glucose is the major metabolic fuel needed for normal brain function, monocarboxylic acids, i.e., lactate, pyruvate, and ketone bodies, can also be utilized by the brain as alternative energy substrates. In most mammalian cells, these substrates are transported either into or out of the cell by a family of monocarboxylate transporters (MCTs), first cloned and sequenced in the hamster. We have recently cloned two MCT isoforms (MCT1 and MCT2) from a mouse kidney cDNA library. Northern blot analysis revealed that MCT1 mRNA is ubiquitous and can be detected in most tissues at a relatively constant level. MCT2 expression is more limited, with high levels of expression confined to testes, kidney, stomach, and liver and lower levels in lung, brain, and epididymal fat. Both MCT1 mRNA and MCT2 mRNA are detected in mouse brain using antisense riboprobes and in situ hybridization. MCT1 mRNA is found throughout the cortex, with higher levels of hybridization in hippocampus and cerebellum. MCT2 mRNA was detected in the same areas, but the pattern of expression was more specific. In addition, MCT1 mRNA, but not MCT2, is localized to the choroid plexus, ependyma, microvessels, and white matter structures such as the corpus callosum. These results suggest a differential expression of the two MCTs at the cellular level.

GLUT4 glucose transporter expression in rodent brain: effect of diabetes.

This study describes the regional and cellular expression of the insulin-sensitive glucose transporter, GLUT4, in rodent brain. A combination of in situ hybridization, immunohistochemistry and immunoblot techniques was employed to localize GLUT4 mRNA and protein to the granule cells of the olfactory bulb, dentate gyrus of the hippocampus and the cerebellum, with the greatest level of expression being in the cerebellum. Estimates of the concentration of GLUT4 in cerebellar membranes indicate that this transporter isoform is present in significant amounts, relative to the other isoforms, GLUT1 and GLUT3. Cerebellar GLUT4 expression was increased in the genetically diabetic, hyperinsulinemic, db/db mouse relative to the non-diabetic control, and even higher levels were observed in db/db female than db/db male mice. Levels of expression of GLUT4 protein in cerebellum appear to respond to the level of circulating insulin, and are reduced in the hypoinsulinemic streptozotocin-diabetic rat. Exercise training also results in reduced insulin levels and comparably reduced levels of GLUT4 in the cerebellum. These studies demonstrate a chronic insulin-sensitive regulation of GLUT4 in rodent brain and raise the possibility of acute modulations of glucose uptake in these GLUT4 expressing cells.

Effects of central injection of kyotorphin and L-arginine on oxytocin and vasopressin release and blood pressure in conscious rats.

Intracerebroventricular (I.C.V.) administration of an inhibitor of nitric oxide synthase (NOS) increases oxytocin but not vasopressin secretion, in dehydrated rats [38]. Surprisingly, central injection of L-arginine, the substrate for NOS, caused a similar effect. Kyotorphin (L-tyrosyl-L-arginine), a dipeptide formed from L-arginine by kyotorphin synthetase in the brain may mediate this magnocellular response. Therefore, the dose and time responses of hormone release were compared following I.C.V. injection of kyotorphin and L-arginine to conscious rats that were normally hydrated or deprived of water for 24 h. In water-sated rats, both L-arginine and kyotorphin increased blood pressure and plasma glucose levels coincident with elevating circulating levels of oxytocin, but not vasopressin. In dehydrated animals, both L-arginine and kyotorphin increased plasma oxytocin levels with a similar time course but only kyotorphin decreased vasopressin release. D-arginine, like L-arginine, stimulated secretion of oxytocin, indicating a nonstereospecific effect. A kyotorphin receptor antagonist (L-leucyl-L-arginine) given I.C.V. to dehydrated animals elevated plasma oxytocin and prevented the decrease in vasopressin levels after kyotorphin. Thus, kyotorphin, but not L-arginine, appears to attenuate release of vasopressin either directly from magnocellular neurons or indirectly via modulating compensatory reflexes activated by the pressor response. On the other hand, an excess of L-arginine and kyotorphin within the CNS may mimic the stress response by augmenting release of oxytocin and activating the sympathetic nervous system to increase blood pressure and plasma glucose levels.

Effects of chronic central infusion of oxytocin on the response of the magnocellular neuroendocrine system to osmotic stimulation.

The effect of continuous intracerebroventricular (i.c.v.) infusion of oxytocin (OT) on the release of OT and vasopressin (VP) following osmotic stimulation was studied in ovariectomized rats treated peripherally with gonadal steroids to simulate late gestation/lactation. Artificial cerebrospinal fluid (CSF) with or without OT (2 ng/microg) was infused (0.5 microl/h) i.c.v. continuously for 7 days along with sequential peripheral administration of progesterone (2 mg/kg i.m.) for 4 days, then 17-beta-estradiol (200 microg/kg i.m.) for 2 days. Following 7 days of OT infusion, isotonic (0.15 mol/l NaCl) or hypertonic (1.5 mol/l NaCl) saline was injected (15 ml/kg s.c.); the animals were decapitated 1 h later. Animals infused centrally with OT had higher basal levels of OT in plasma (p < 0.01 vs. CSF). While osmotic stimulation increased plasma levels of both OT and VP (0.15 mol/l NaCl < 1.5 mol/l NaCl; p < 0.01), only circulating VP was enhanced further (p < 0.01) in animals infused with OT compared with those receiving CSF. These changes in hormone levels could not be explained by differences in neural lobe stores of OT or VP or by alterations in daily water intake during the infusion period. Thus, chronic i.c.v. infusion of OT stimulates basal release of OT and increases the response of the VP system to osmotic stimulation.

Thrombin-induced translocation of GLUT3 glucose transporters in human platelets.

Platelets derive most of their energy from anaerobic glycolysis; during activation this requirement rises approx. 3-fold. To accommodate the high glucose flux, platelets express extremely high concentrations (155+/-18 pmol/mg of membrane protein) of the most active glucose transporter isoform, GLUT3. Thrombin, a potent platelet activator, was found to stimulate 2-deoxyglucose transport activity 3-5-fold within 10 min at 25 degrees C, with a half-time of 1-2 min. To determine the mechanism underlying the increase in glucose transport activity, an impermeant photolabel, [2-3H]2N-4-(1-azi-2,2,2-trifluoethyl)benzoyl-1,3, -bis-(d-mannose-4-ylozy)-2-propylamine, was used to covalently bind glucose transporters accessible to the extracellular milieu. In response to thrombin, the level of transporter labelling increased 2.7-fold with a half-time of 1-2 min. This suggests a translocation of GLUT3 transporters from an intracellular site to the plasma membrane in a manner analogous to that seen for the translocation of GLUT4 in insulin-stimulated rat adipose cells. To investigate whether a similar signalling pathway was involved in both systems, platelets and adipose cells were exposed to staurosporin and wortmannin, two inhibitors of GLUT4 translocation in adipose cells. Thrombin stimulation of glucose transport activity in platelets was more sensitive to staurosporin inhibition than was insulin-stimulated transport activity in adipose cells, but it was totally insensitive to wortmannin. This indicates that the GLUT3 translocation in platelets is mediated by a protein kinase C not by a phosphatidylinositol 3-kinase mechanism. In support of this contention, the phorbol ester PMA, which specifically activates protein kinase C, fully stimulated glucose transport activity in platelets and was equally sensitive to inhibition by staurosporin. This study provides a cellular mechanism by which platelets enhance their capacity to import glucose to fulfil the increased energy demands associated with activation.