Co-localization of GLUT1 and GLUT4 in the blood–brain barrier of the rat ventromedial hypothalamus

Brain Research 900 (2001) 1–8 www.elsevier.com / locate / bres

Research report

Co-localization of GLUT1 and GLUT4 in the blood–brain barrier of the rat ventromedial hypothalamus a

a

Chardpraorn Ngarmukos , Elisabeth L. Baur , Arno K. Kumagai a

a,b,c ,

*

Department of Internal Medicine, 5570 MSRB-2, Box 0678, University of Michigan Medical School, Ann Arbor, MI 48109, USA b Michigan Diabetes Research and Training Center, University of Michigan Medical School, Ann Arbor, MI 48109, USA c JDRF Center for Complications in Diabetes, University of Michigan Medical School, Ann Arbor, MI 48109, USA Accepted 23 January 2001

Abstract The ventromedial hypothalamus (VMH) has been proposed to be a glucose sensor within the brain and appears to play a critical role in initiating the counterregulatory response to hypoglycemia. Transport of glucose across the brain capillaries and into neurons in this region is mediated by different isoforms of the sodium-independent glucose transporter gene family. The objective of the present study was to identify the specific glucose transporter isoforms present, as well as their cellular localization, within the VMH. Immunohistochemistry was performed for GLUT1, GLUT2 and GLUT4 in frozen sections of hypothalami from normal rats. GLUT1 was present on the endothelial cells of the blood–brain barrier (BBB) of the VMH. GLUT2 immunoreactivity was seen in the ependymal cells of the third ventricle and in scattered cells in the arcuate and periventricular nuclei. There was no GLUT2 expression in the VMH. The insulin-sensitive GLUT4 isoform was localized to vascular structures within the VMH. Double-labeled immunohistochemistry demonstrated co-localization of GLUT4 with GLUT1 and with the tight junction protein ZO-1 in the VMH and suggested that VMH GLUT4 expression was restricted to the BBB. The role of GLUT4 in the brain and within the VMH is unknown, but given its location on the BBB, it may participate in brain sensing of blood glucose concentrations.  2001 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Blood–brain barrier Keywords: Blood–brain barrier; Hypothalamus; GLUT1; GLUT2; GLUT4; Ventromedial hypothalamus

1. Introduction The brain has an absolute dependence on glucose for normal metabolism. Because glycogen stores in the brain are negligible in comparison to metabolic demand, the brain requires constant transport of glucose from the peripheral circulation [32]. In states of fasting or hypoglycemia, glucose availability for transport into brain is preserved through counterregulation, a hierarchical series of hormonal responses involving first, the inhibition of

Abbreviations: BBB, Blood–brain barrier; IDDM, Insulin-dependent diabetes mellitus; VMH, Ventromedial hypothalamus *Corresponding author. Tel.: 11-734-936-5035; fax: 11-734-9366684. E-mail address: [email protected] (A.K. Kumagai).

endogenous insulin secretion, followed sequentially by the secretion of glucagon, epinephrine, norepinephrine, cortisol and growth hormone. These compensatory responses serve to raise blood glucose concentrations and ensure adequate delivery of this essential substrate to the brain [11]. Glucose transport into the brain involves first, transport across the endothelial cells of the brain microvessels, and subsequently, transport across the neuronal cell membranes into the cytosol of the neuronal cells, where glucose becomes available for critical cellular processes. The microvasculature of the brain — the first interface across which glucose is transported — is structurally distinct from other microvascular beds. Unlike those of other organs, the microvessels of the brain are characterized by the presence of non-fenestrated, ‘continuous’ endothelia that are connected by tight junctions (zonulae occludens) which prevent the passive intercellular or transendothelial diffusion

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02184-9

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of polar compounds from the blood into the brain parenchyma [43]. This so-called ‘blood–brain barrier’ (BBB) requires that all non-lipophilic compounds be transported via selective transport processes. Because the surface area of the neuronal elements of the brain far exceeds that of the BBB, glucose transport into the brain is limited at the interface of the brain microvessel endothelial cells [32,43]. Furthermore, under normal physiological conditions, glucose transport exceeds hexokinase-mediated phosphorylation of glucose; however, in certain pathological states, such as insulin-induced hypoglycemia, hexokinase activity declines such that glucose transport becomes the limiting step in brain glucose metabolism [32,43]. Glucose crosses the BBB and gains entry into the brain via a facilitative transport process mediated by members of the sodium-independent glucose transporter gene family [39]. Within the brain, GLUT1 and GLUT3 appear to be predominant and are expressed on the BBB and in the plasma membranes of the neural elements, respectively [41,44]. Several groups, however, have reported the expression in brain of other glucose transporter isoforms as well [34], including GLUT2 [23,30], GLUT4 [2,9,15, 25,29,37,49,53], and GLUT5 [35,47]. Various lines of evidence suggest that the brain, and in particular, the ventromedial hypothalamus (VMH) plays a major role in the sensing of blood glucose concentrations and activation of the counterregulatory response to hypoglycemia. In dogs, cerebral perfusion of glucose abolishes the counterregulatory response in the presence of peripheral hypoglycemia [4]. In rats, selective destruction of the VMH by injection of ibotenic acid or localized perfusion of concentrated glucose solutions into the ventromedial nuclei abolishes the counterregulatory response [5,6]. In contrast, selective glycopenia in the cells within the VMH by perfusion of 2-deoxyglucose activates counterregulation, even in the presence of normal circulating blood glucose concentrations [7]. Given the regional heterogeneity of both glucose transport and metabolism in the brain [19,31], changes in glucose transport in the VMH may allow sensing of declining blood glucose concentrations and the activation of the counterregulatory response during periods of hypoglycemia. In order to begin to investigate the changes, if any, that occur in glucose transport and glucose transporter expression in the VMH during hypoglycemia, it is essential first to identify and localize the different isoforms of glucose transporters present in this putative brain glucose sensor. The objective of the present study was to investigate the expression of GLUT2 and the insulin-sensitive glucose transporter GLUT4 in the VMH of the healthy adult rat using immunohistochemical techniques. We report that GLUT4, and not GLUT2, is expressed selectively on the capillaries of the VMH and co-localizes with the BBB markers GLUT1 and ZO-1 in this area proposed to be the brain’s glucose sensor.

2. Materials and methods

2.1. Animals All experiments were performed with the approval of the University of Michigan Committee on Use and Care of Animals. Healthy adult male Sprague–Dawley rats (Harlan, Indianapolis, IN), weighing 300–400 g, were kept in cages with a 12-h light / dark cycle and given ad libitum access to chow and tap water. On the day of the experiment, the rats were given a lethal dose of halothane and decapitated. Brains were quickly isolated and rinsed in cold phosphate-buffered saline (PBS, pH 7.4) and after removal of the meninges, the brains were transferred to a brain block (Harvard Apparatus, So. Natick, MA). The brains were cut into three coronal sections of 3–5 mm in width, and the sections were coated with M1 embedding matrix (Shandon Lipshaw, Pittsburgh, PA) and rapidly frozen in powdered dry ice. The sections containing the hypothalamus were transferred to a pre-chilled cryostat, cut into 10-mm cross sections and thaw-mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA). The sections containing the VMH were identified by anatomical landmarks according to a rat brain atlas [46]. All slides were air-dried and stored at 2808C until immunohistochemical staining was performed. Samples of rat pancreas and soleus muscle also were collected and prepared in an identical manner to be used as positive controls for GLUT2 and GLUT4, respectively.

2.2. Immunohistochemistry 2.2.1. Antisera The anti-GLUT1 antiserum (a gift of C. Carter-Su, University of Michigan) used in these experiments was a rabbit polyclonal antibody raised against purified erythrocyte glucose transporter and was used at a working concentration of 1:1,000. The anti-GLUT2 antibody (a gift of W.M. Pardridge, UCLA School of Medicine) was a rabbit polyclonal antibody raised against a synthetic peptide corresponding to the carboxyl terminus of rat GLUT2 and used at a concentration of 1:250. The anti-GLUT4 antibody (Santa Cruz Biochemicals, Santa Cruz, CA) was a goat polyclonal antiserum directed against a synthetic peptide corresponding to the carboxyl terminus of rat GLUT4 and used at 1:200. An additional GLUT4 antibody, called in the present study GT4 (a gift of S. Cushman, National Institutes of Health), was a rabbit polyclonal antibody against the rat GLUT4 carboxyl terminus and used at 1:1,000. The anti-ZO-1 antiserum (Zymed Inc., So. San Francisco, CA) was a rabbit polyclonal antibody directed against the amino terminus of rat ZO-1. The anti-GLUT1, anti-GLUT2 and anti-GLUT4 antisera have been characterized previously [10,24,51]. Unless otherwise specified, all experiments were per-

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formed at room temperature. Specimen-mounted slides were thawed at room temperature for 10 min, fixed in ice-cold acetone for 10 min and rehydrated by incubation in PBS twice for 5 min. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide (Sigma, St Louis, MO) in PBS for 10 min. Slides were washed for 5 min three times in PBS. One hundred microliters of 3% serum were applied on each specimen to block nonspecific binding: 3% normal goat serum for blocking anti-GLUT1, anti-GLUT2, GT4 and anti-ZO-1 antisera; 3% normal rabbit serum for blocking goat polyclonal anti-GLUT4. After a 30-min incubation, excess blocking solution was drained from the slides, and 100 ml of each primary antiserum was applied. The slides were incubated at room temperature for 90 min or at 48C overnight. The specimens were washed in PBS for 5 min three times and incubated with biotinylated secondary antibodies for 30 min at room temperature. The working concentrations of the secondary antibodies were: a 1:1,000 dilution of biotinylated goat anti-rabbit IgG (Vector Labs, Burlingame, CA) for the anti-GLUT1 antibody; a 1:500 dilution of biotinylated goat anti-rabbit IgG (Vector) for the antiGLUT2 antibody; and a 1:500 dilution of biotinylated rabbit anti-goat IgG (Vector) for the goat polyclonal antiGLUT4 antibody. The slides were washed with PBS and followed by a 30-min incubation in an avidin–biotin– peroxidase complex (Vectastain ABC Elite kit, Vector). Following three washes in PBS, the peroxidase reaction was carried out in the presence of 3-amino-9-ethylcarbazyl (AEC, 0.18 mg / ml, Sigma) and 0.2% hydrogen peroxide at 378C. The slides were washed in running tap water to terminate the reaction, counterstained with Meyers Hematoxylin (Sigma) and air-dried at room temperature. All slides were mounted with coverslips for light microscopic examination. Negative controls for each of the antisera were as follows: normal rabbit serum for the anti-GLUT1, anti ZO-1 and the GT4 antisera; and pre-absorption with the corresponding synthetic peptide for the anti-GLUT2 and the goat polyclonal anti-GLUT4 antibodies. For preabsorption, the calculated, undiluted volume of primary antibodies were allowed to mix with the synthetic peptides at a 103 concentration of the primary antibody on an end-over-end rocker for 2 h at room temperature. PBS was then added to correct to the working dilution of the primary antibodies before incubation. For double immunofluorescent staining, the specimens were stained as described above except that fluorescent secondary antibodies were used FITC-conjugated donkey anti-rabbit IgG (Jackson Laboratories, West Grove, PA) at a concentration of 10 mg / ml for the anti-GLUT1 and antiZO-1 and their controls, and Alexa 594-conjugated donkey anti-goat IgG (Molecular Probes, Eugene, OR) at a concentration 20 mg / ml for the anti-GLUT4 antibody. Two primary antibodies (anti-GLUT1 and anti-GLUT4 or antiGLUT4 and anti-ZO-1) were combined at the appropriate

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concentrations and applied as described above. After washing, the specimens were incubated in secondary antisera for 90 min, and then were washed in PBS for 5 min three times. Excess buffer was carefully removed from the slides, approximately 25 ml of Vectashield (Vector labs) was added, and the specimens were covered with coverslips. The specimens were examined under using a Zeiss Axiophot fluorescent microscope (Carl Zeiss Inc, Thornwood, NY). All immunohistochemistry experiments were performed in specimens from at least three different rats.

3. Results GLUT1 staining of rat brain specimens (Fig. 1A, left) revealed positive staining of capillaries throughout the brain sections, except for non-blood–brain barrier areas, such as the median eminence [56]. The apical surface of the ependymal cells lining the third ventricle also stained for GLUT1, as has been reported previously by Farrell and coworkers [16]. The specificity of the staining was confirmed with an absence of staining in specimens where normal rabbit serum was used in place of the primary antibody (Fig. 1A, right). The sensitivity and specificity of the anti-GLUT2 antiserum used in these studies were confirmed by positive staining of pancreatic islets and no staining in the sections in which pre-absorbed antiserum was employed (data not shown). As has been previously demonstrated [30], the ependymal cells lining the third ventricle demonstrated immunoreactivity for GLUT2 (Fig. 1B, left panel). In these cells, GLUT2 expression was localized to the apical membrane, as was observed for GLUT1 (data for GLUT1 not shown). In addition, GLUT2 immunoreactivity of scattered cells adjacent to the third ventricle was observed (Fig. 1B, left, arrowheads). These findings were confirmed on immunofluorescent staining sections (Fig. 1C), which demonstrated staining of ependymal cells and stellateshaped cells in the paraventricular and arcuate nuclei, as has been previously described [23,30]. Careful examination of these cells suggested that they were not endothelial cells. Notably, there was an absence of GLUT2 staining in the ventromedial hypothalamus. Immunoreactive GLUT4 was detected in soleus muscle sections, specifically on the sarcolemma and, to a lesser extent, in the cytoplasm (Fig. 1D, upper panel). There was no immunoreactivity for GLUT4 in the specimens stained with the pre-absorbed anti-GLUT4 antiserum (Fig. 1D, lower panel). In the hypothalamus, GLUT4 expression was restricted to vascular structures in ventromedial hypothalamus (Fig. 1E, left panel). No significant GLUT4 immunoreactivity was seen in the hypothalamus outside of the VMH. The specificity of GLUT4 immunoreactivity in the VMH was confirmed by additional experiments in

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which immunohistochemistry for GLUT4 was performed using a different anti-GLUT4 antiserum, GT4. In these experiments, GLUT4 immunoreactivity was localized to vascular structures within the VMH (Fig. 1F). In order to confirm the presence of GLUT4 on the blood–brain barrier of the VMH, we performed double-label immunohistochemistry for GLUT1 and GLUT4 in rat hypothalamic sections. As is seen in Figs. 1G and H, co-localization of GLUT1 and GLUT4 was detected on the BBB of the VMH. Because GLUT1 has also been reported to be expressed in nonvascular structures of the brain, and in particular, glial cells [34,38,57], we used another blood– brain barrier marker, the tight junction protein ZO-1 [54], to verify the localization of GLUT4 to the BBB of the VMH. As can be seen in Figs. 1I and J, GLUT4 and ZO-1 were clearly co-localized to the same vascular structures within the VMH.

4. Discussion The present study demonstrates immunoreactive GLUT1 expression in the microvessels comprising the BBB of the ventromedial hypothalamus (Fig. 1A) and confirms a similar pattern localization for GLUT1 in whole brain [44] and in the hypothalamus [56]. The present study also confirms observations of GLUT2 immunoreactivity in the ependymal cells lining the third ventricle [30] and reports the co-localization of GLUT1 and GLUT2 in the apical membranes of these cells. In support of previous studies [23,30], isolated cells within the arcuate and periventricular nuclei of the rat hypothalamus were shown to express GLUT2; however, unlike the findings of Jetton and coworkers, GLUT2 staining within the hypothalamus in the present study did not appear to be associated with vascular structures (Fig. 1B). The staining of scattered

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stellate-shaped cells suggested cells of glial origin. These findings are in agreement with those of Leloup et al., who have demonstrated GLUT2 immunoreactivity restricted to astrocytes in the lateral hypothalamus and arcuate and periventricular nuclei [30]. In the present study, immunoreactive GLUT2 was not detected in the VMH, a finding that complements reverse transcription polymerase chain reaction (RT–PCR) experiments that suggest the absence of GLUT2 transcript in this region of the hypothalamus [30]. Previous experiments by Rayner and coworkers could detect neither GLUT2 protein or mRNA in the hypothalamus by Western blotting and Northern blotting, respectively [49]; however, given that GLUT2 expression is restricted to a small number of cells within the hypothalamus, it is likely that GLUT2 could not be detected using the relatively insensitive techniques in Rayner’s study. The present study also demonstrates colocalization of GLUT1 and GLUT4 in the BBB of the rat ventromedial hypothalamus by double immunohistochemical staining (Figs. 1G and H). These observations are similar to those of McCall and colleagues, who demonstrated the presence of GLUT4 on the BBB of the frontal cortex [37] and support studies documenting the presence of GLUT4 mRNA and protein in the rat hypothalamus [9,29]. The present study differs from those of Leloup and coworkers, who reported neuronal-selective expression of GLUT4 protein in the perirhinal cortex and GLUT4 in the periventricular nucleus of the rat hypothalamus [29]. In Leloup’s study, GLUT4 mRNA was detected by RT–PCR in the rat VMH; however, immunoreactive GLUT4 was not [29]. Recent work by El Messari et al. and Apelt et al. report similar neuronal expression of GLUT4 in the basal forebrain, cerebral cortex, hippocampus, cerebellum and spinal cord of the rat [2,15] and colocalization of immunoreactive GLUT4 and GLUT3 mRNA in cerebral cortical, hip-

Fig. 1. Immunohistochemical staining of rat brain sections containing the ventromedial hypothalamus (VMH). (A, left panel) GLUT1 immunoreactivity in a cross-section of rat hypothalamus. Positive immunoreactivity for GLUT1 is detected by the presence of a reddish-brown reaction product. Anti-GLUT1 antiserum at 1:1,000. (A, right panel) Control slide stained with normal rabbit serum at 1:1,000. Magnification53400. (B, left panel) GLUT2 immunoreactivity in cross-section of rat hypothalamus and third ventricle. Anti-GLUT2 antiserum at 1:250. Positive immunoreactivity is seen in the ependymal cells lining the third ventricle (V) and scattered cells in the areas of the periventricular and arcuate nuclei (arrowheads). (B, right panel) Similar section of hypothalamus after staining with anti-GLUT2 antibody previously pre-absorbed with corresponding synthetic peptide. Magnification53200. (C) Fluorescent microscopy of GLUT2 staining of rat hypothalamus. Immunopositive cells (green) are seen lining the third ventricle, with scattered, stellate-shaped GLUT2-positive cells in the arcuate and periventricular nuclei. Magnification53200. (D, upper panel) Cross-section of soleus muscle stained with a 1:200 dilution of anti-GLUT4 antiserum. Positive immunoreactivity for GLUT4 is seen predominantly outlining the sarcolemma. (D, lower panel) Soleus muscle cross-section stained with anti-GLUT4 antibody previously pre-absorbed with its corresponding synthetic peptide. Magnification53 200. (E, left panel) GLUT4 immunoreactivity in rat VMH. Positive reaction product (reddish-brown) is present in vascular structures, seen here en face and in longitudinal section (arrowheads). (E, right panel) Similar cross-section of VMH stained with anti-GLUT4 antibody previously pre-absorbed with corresponding synthetic peptide. Magnification53400. (F) Longitudinal section of vascular structure stained with another anti-GLUT4 antiserum, GT4, at 1:500 dilution. Secondary antibody is Alexa-594-conjugated goat anti-rabbit IgG at a concentration of 20 mg / ml. Magnification531,000. (G, H) Double fluorescent immunostaining of rat VMH for GLUT1 (G) and GLUT4 (H). (G) GLUT1-positive immunoreactivity (green) in vascular structures after reaction with anti-GLUT1 antiserum at 1:1,000 and FITC-conjugated secondary antibody at a concentration of 10 mg / ml. (H) GLUT4-positive immunoreactivity (red) in same structures after reaction with anti-GLUT4 antiserum at 1:200 and Alexa 594-conjugated secondary antibody at a concentration of 20 mg / ml. Magnification53400. (I, J) Double fluorescent immunohistochemistry for ZO-1 (I) and GLUT4 (J). (I) Z0-1 immunoreactivity (green) outlining endothelial cells of vascular structures. Z0-1 antiserum at a 1:500 dilution with a FITC-conjugated secondary antibody at a concentration of 10 mg / ml. (J) GLUT4 immunoreactivity (red) of same vascular structures after reaction with anti-GLUT4 antiserum at 1:200, followed by an Alexa 594-conjugated secondary antibody at a concentration of 20 mg / ml. Magnification531,000.

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pocampal and cerebellar neurons [2]. In contrast, the present study documents selective expression of GLUT4 on the endothelial cells of the VHM BBB. Expression of GLUT4 in the hypothalamus was not addressed in Apelt’s study [2], and no immunoreactive GLUT4 was detected in the VMH in El Messari’s experiments [15]. Discrepancies in GLUT4 localization in the various studies may possibly be attributed to differences in anti-GLUT4 antisera. The current observations of selective localization of GLUT4 on the BBB within the VMH are supported by three major lines of evidence. First, the specificity of the anti-GLUT4 antiserum used in these studies was confirmed by the characteristic, specific labeling of skeletal muscle and by complete absence of staining after pre-absorption with synthetic peptide (Fig. 1D); second, specific labeling of vascular structures within the VMH was seen after staining with a second anti-GLUT4 antiserum (Fig. 1F); and third, colocalization of GLUT4 with GLUT1 and ZO-1, two markers of the BBB, was observed (Figs. 1G–I). The current studies therefore suggest that, unlike in other brain regions, GLUT4 within the VMH is selectively expressed on the microvessels of the BBB. The role of the insulin-sensitive GLUT4 in the brain, and specifically in the VMH, is unclear. Insulin receptors are present on the BBB [45], and as detailed above, GLUT4 is expressed in several different regions in the brain; however, despite a report to the contrary [20], the majority of studies have found that glucose uptake in the brain is independent of insulin [3,18,21]. In this regard, cytochalasin-B binding studies and quantitative Western blotting analysis of GLUT1 on isolated brain capillary preparations suggest that glucose transport across the BBB is predominantly mediated by GLUT1 [14]. Glucose transport mediated by GLUT1, unlike that mediated by GLUT4, is known to be insulin-insensitive [40]. Nonetheless, given the restricted expression of hypothalamic GLUT4 to the VMH BBB found in the present study, it is possible that studies of insulin effects on glucose transport in whole brain may not detect changes in transport in response to insulin in this small brain region. Furthermore, although not a direct measurement of glucose transport per se, an autoradiographic study by Lucignani and coworkers using labeled 2-deoxyglucose provides evidence that glucose metabolism within the ventral hypothalamus is insulin-sensitive [31]. Glucose-sensitive neurons have been reported in the hypothalamus — specifically, the VMH and lateral hypothalamus — and in the nucleus of the solitary tract in the brain stem [42]. In experiments using isolated hypothalamic slices, these neurons modulate their rate of electrical discharge in response to changes in the extracellular concentrations of glucose [42]; however, before glucose reaches these glucose-sensitive neurons in the VMH, it must cross the BBB, and since the surface area of the capillaries is but a fraction of that of the neuronal elements, both in whole brain [32] and within the VMH

[50], availability of glucose of neurons in the VMH, as in whole brain, is limited by its transport across the BBB. It is currently unknown which specific glucose transporter isoform, if any, plays a critical role in glucose sensing by the brain in response to hypoglycemia. As described above, while both immunoreactive GLUT1 and GLUT4 are present in the VMH, GLUT2 is notably absent. The reported presence of GLUT2 in conjunction with glucokinase in the ventrolateral hypothalamus is intriguing [23], since GLUT2 and glucokinase participate in glucose sensing in the beta cells of the pancreas [17]. Furthermore, GLUT2 and glucokinase expression have also been reported in the brainstem [33], another area proposed to act as a central nervous system glucose sensor. The present study, however, was unable to document the presence of GLUT2 in the VMH, which is the area of the brain most closely associated with modulation of the counterregulatory response to hypoglycemia [5–7]. Recently, two novel glucose transporters, GLUTX1 and GLUT8, have been identified and have identical amino acid sequences [13,22]. GLUTX1 / 8 have been reported to be expressed in high abundance in testis, in insulin-sensitive tissues and in brain. In the brain GLUTX1 mRNA has been reported in several regions, including the cerebellum, brainstem, hippocampus and hypothalamus [22]. The cellular localization of GLUTX1 / 8 within the hypothalamus is at present unclear; however, given its presence in this area of the brain, it is possible that GLUTX1 may participate in brain glucose sensing. Several studies have suggested that changes in brain glucose transport and glucose transporter expression play a role in the physiological adaptation to hypoglycemia. In animal models of sustained hypoglycemia, transport of glucose across the BBB is increased [28,36,48], a phenomenon that occurs via an upregulation of GLUT1 in the brain [26], and specifically on the brain microvasculature comprising the BBB [28]. In similar animal models, neuronal GLUT3 is also upregulated [52]. These changes in brain glucose transport have a possible clinical correlation in the management of type 1 or insulin-dependent diabetes mellitus (IDDM). Individuals with IDDM who are attempting to achieve tight metabolic control often develop severe deficits in the counterregulatory response to hypoglycemia and a blunting of awareness of declining blood glucose levels [1,11]. These impairments underlie the significantly increased risk of severe hypoglycemia of individuals under intensive insulin therapy [12] and have been associated with a relative increase in whole brain BBB glucose transport [8]. The application of the abovecited studies towards understanding the impairment in the counterregulatory response in type 1 diabetes, however, must be done with caution. First, the hypoglycemia that is typically seen in the setting of intensive insulin therapy in type 1 diabetes is intermittent rather than sustained [55], and second, nothing is known about changes, if any, in glucose transport and glucose transporter expression in

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specific areas of the brain that are suspected to act in the sensing of blood glucose concentrations. Nonetheless, given the regional heterogeneity of both glucose transport and metabolism in the brain [19,31], changes in regional glucose transport within the glucose-sensing areas of the brain in the setting of intermittent hypoglycemia may underlie the impairments in counterregulatory responses seen in the treatment of type 1 diabetes mellitus [27]. In summary, the present study demonstrates the localization of GLUT4 to the microvessels comprising the blood– brain barrier of the rat ventromedial hypothalamus and co-expression of GLUT4 with both GLUT1 and ZO-1 on the endothelial cells of these capillaries. The questions of whether the presence of GLUT4 on the BBB of the VMH confers insulin sensitivity to this area, and whether changes in the expression of hypothalamic GLUT1, GLUT2, GLUT4 or a novel glucose transporter play a role in brain glucose sensing and regulation of the counterregulatory response to hypoglycemia, remain to be explored.

Acknowledgements The authors would like to thank Drs Christin Carter-Su, William M. Pardridge, and Samuel W. Cushman for their generous gifts of the glucose transporter antisera and Drs Lisa Larkin and Frank C. Brosius for invaluable discussions. This work was supported by grants from the American Diabetes Association and the Michigan Diabetes Research and Training Center to AKK. AKK is supported by the Juvenile Diabetes Foundation Center for Complications in Diabetes, and is supported in part by National Institutes of Health Grant RPO60DK-20572, which supports the Michigan Diabetes Research and Training Center.

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