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Endocrinology Vol. 144, No. 5 1703-1711
Copyright © 2003 by The Endocrine Society

Regulation of Hepatic GLUT8 Expression in Normal and Diabetic Models

Naira Gorovits, Lingguang Cui, Julia V. Busik, Mollie Ranalletta, Sylvie Hauguel de-Mouzon and Maureen J. Charron

Department of Biochemistry (N.G., L.C., M.R., M.J.C.), Albert Einstein College of Medicine, Bronx, New York 10461; Department of Physiology (J.V.B.), Michigan State University, East Lansing, Michigan 48824-1101; and Department of Genetics (S.H.d.-M.), Case Western Reserve University, Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: Maureen J. Charron, Department of Biochemistry Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: charron{at}aecom.yu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GLUT8 is a novel glucose transporter protein that is widely distributed in tissues including liver, a central organ of regulation of glucose homeostasis. The purpose of the current study was to investigate expression and regulation of hepatic GLUT8 mRNA and protein. Therefore, Northern and immunoblot analysis, semiquantitative RT-PCR, and immunofluorescence microscopy were performed using mouse livers at different stages of embryonic and postnatal development and in type 1 (streprozotocin treated) and type 2 (GLUT4 heterozygous) diabetes. GLUT8 mRNA and protein expression in embryonic liver was differentially regulated depending on the prenatal and postnatal developmental stage of the mice. Immunofluorescence microscopy of liver from wild-type mice demonstrated the highest levels of GLUT8 protein in perivenous hepatocytes pointing to its role in regulation of glycolytic flux. In diabetic scenarios, GLUT8 mRNA levels were correlated with circulating insulin; specifically, GLUT8 mRNA decreased in a type 1 diabetes model and increased in a type 2 diabetes model, suggesting a regulatory role for insulin in GLUT8 mRNA expression. While up-regulation of GLUT8 protein occurred in both models of diabetes, only in streptozotocin diabetic livers was GLUT8 zonation altered. These data demonstrate that GLUT8 mRNA and protein are differentially regulated in liver in response to physiologic and pathologic (diabetes) milieu and suggests that GLUT8 is intimately linked to glucose homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTRIGUING DATA OBTAINED from GLUT4 knockout mice (1, 2) led the efforts of several groups to clone a novel glucose transporter. In recent years, a novel 477-amino-acid glucose transporter-like protein, GLUT8, was discovered (3, 4, 5). GLUT8 is widely expressed in tissues including brain (cerebellum, brain stem, hippocampus, hypothalamus), adrenal gland, spleen, brown adipose tissue, white adipose tissue, muscle, heart, and liver (3, 4, 6). GLUT8 mRNA expression is greatest in testis, and its expression was linked to circulating gonadotropins (4, 7). In vitro studies demonstrated that GLUT8 transports glucose in a cytochalasin B-inhibitable manner (3, 4, 5). When transfected into HEK293T or COS-7 cells, GLUT8 protein migrates to 32 kDa and 60 kDa (3, 5). Several studies have shown GLUT8 is predominantly located in an intracellular compartment that is consistent with the presence of a dileucine motif in the amino terminus domain (3, 4, 5, 6, 8). On the other hand, GLUT8 promotes insulin-stimulated glucose transport in blastocysts (5). The presence of GLUT8 in the blastocyst is crucial for the embryonic development and survival (9). Further, GLUT8 gene expression is down-regulated by glucose deprivation and hypoxia in cultured differentiated 3T3-L1 adipocytes (7). These studies and the wide range of tissues that express GLUT8 suggest regulation of GLUT8 expression may play an important role in maintenance of glucose homeostasis.

Maintenance of normal circulating glucose in mammals is accomplished by hormone-regulated glucose uptake by peripheral tissues and glucose output by liver. The transport of glucose in liver is facilitated by a specific, high Michaelis constant, glucose transporter GLUT2 (10). GLUT2 is responsible for glucose transport into and out of liver under fed and fasted conditions, respectively. In the postprandial state, liver uses glucose and incorporates it into glycogen and fatty acids or oxidizes it into CO2. In the fasting state, liver mobilizes glycogen stores and releases glucose into the bloodstream to maintain normoglycemia. GLUT2 mRNA expression is regulated by glucose levels in different metabolic states and is expressed in every hepatocyte (11). GLUT1 is a glucose transporter expressed in perivenous hepatocytes and is regulated by nutrient flux (12). Interestingly, hepatocytes do not require GLUT2 for glucose release, suggesting an alternative pathway for transmembrane glucose transport (13, 14, 15, 16). Cellular localization and regulation of GLUT8 expression in the liver during development, in normal physiological states and in models of type I or type II diabetes is lacking. As liver plays a central role in regulation of glucose homeostasis, the present study was conducted to determine whether GLUT8 gene or protein expression is modulated in liver in normal or pathologic metabolic states.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Embryos and neonatal mice.
Fifteen-week-old female (CBA)mice (The Jackson Laboratory, Bar Harbor, ME) were housed at 24 C with light from 1000–2200 with free access to food and water. Mice were mated for 12 h, and the first day of pregnancy was verified by mucus plug appearance. Fourteen- and 17-d embryos and 1-, 4-, 6-, and 15-wk-old pups were killed and livers rapidly dissected, frozen in liquid nitrogen, and stored at -70 C until being processed for mRNA and protein extraction. A subset of livers were fixed in formalin and embedded in paraffin for immunofluorescence studies. All protocols were approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine in accordance with the Public Health Service Animal Welfare Policy.

Streptozotocin (STZ)-treated mice.
Ten- to 12-wk-old female (CBA) mice were housed at 24 C with light from 1000–2200 with free access to food and water. The mice were injected ip with 200 mg/kg of body weight STZ (Sigma, St. Louis, MO) dissolved in 0.1 M ice-cold sodium citrate. Three days after the injection, blood was taken from periorbital plexus, and diabetes was established when plasma glucose was 250–400 mg/dl with insulin levels 50% or less of controls. Glucose was measured using Medisense Precision QID glucose strips and glucometer (Abbott Laboratories, Abbott Park, IL). Mice were killed when the length of STZ-induced diabetes was 8 d. Livers were rapidly dissected, frozen in liquid nitrogen, and stored at -70 C until being processed for mRNA and protein extraction.

GLUT4 +/- knockout mice.
Male mice carrying a single disrupted allele of GLUT4 (GLUT4+/-) represent a model of progressive type 2 diabetes (17). Hyperglycemic/hyperinsulinemic (glucose > 250 mg/dl; insulin > 20 ng/ml) male GLUT4+/- mice (26–28 wk old) and age matched wild-type (WT) mice with normal fed glucose and insulin levels (glucose < 250 mg/dl; insulin < 20 ng/ml) were used for the study. Mice were killed after an overnight fast, liver rapidly dissected, frozen in liquid nitrogen, and stored at -70 C until being processed for mRNA and protein extraction. A subset of livers were fixed in formalin and embedded in paraffin for immunofluorescence studies.

Northern blot analysis
Total RNA was prepared using TriZol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Northern blot analysis was performed on approximately 25 µg of total liver RNA using Hybond-N+ nylon membrane optimized for nucleic acid transfer (Amersham Pharmacia Biotech, Piscataway, NJ) as described previously (18). An EcoRI-ApaI fragment from mouse GLUT8 cDNA was used as template for synthesizing random-primed 32P-labeled probes to mouse GLUT8. High stringency hybridization at 42 C was performed and membranes were washed twice for 15 min in 2x saline sodium citrate/0.1% sodium dodecyl sulfate at 42 C followed by two washes in 0.2x saline sodium citrate/0.1% sodium dodecyl sulfate at 42 C for 5 min. Membranes were exposed to x-ray film for 1–2 d. Results were quantified by scanning laser densitometry. Loading was normalized by hybridization with an end-labeled oligonucleotide for 18S rRNA (18).

RT-PCR analysis
Total RNA was extracted from 100 mg of mouse liver tissue using Tri-Reagent. For each reverse transcription reaction, 0.5 µg of RNA was used. Reverse transcription was carried out with Supercript II (Life Technologies, Inc., Gaithersburg, MD) as suggested by the manufacturer. Of the total reaction volume of 20 µl, 1 µl was used for the subsequent PCR. cDNA was amplified using both GLUT8 and ß-actin primers. The GLUT8 primers amplify a 351-bp fragment spanning bp 680–1030 of the mouse cDNA. The forward primer is a 21-oligomer primer, and the reverse primer is a 20-oligomer primer. The ß-actin primers amplify a 353-bp fragment. The PCR was prepared with Platinum Taq DNA Polymerase (Life Technologies, Inc.) and 32P {alpha}-deoxy-CTP as follows: initial annealing at 94 C for 2 min; then cycled at 94 C for 30 sec, 57 C for 30 sec, and 72 C for 30 sec for 30 cycles; then the final annealing step was 72 C for 7 min. Reactions for GLUT8 and ß-actin were carried out separately. Aliquots of each sample (25 µl) were run on an 8% polyacrylamide gel at 200 V for 1 h. Gels were dried under vacuum pressure at 80 C for 2 h, exposed for 2 h using a Phosphor screen and analyzed using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Values for GLUT8 were normalized to ß-actin.

GLUT8 antibody generation and immunoblot analysis
A polyclonal antibody to the carboxy-terminal 11 amino acids (LEQITAHFEGR) of the GLUT8 protein was generated as previously detailed (6, 19). The synthetic GLUT8 peptide (Research Genetics, Inc., Huntsville, AL) was linked to keyhole limpet hemacyanin (Pierce Chemical Co., Inc., Rockford, IL) and used to immunize rabbits (Covance Research, Inc., Berkeley, CA) (6). Purification of high affinity Igs to the GLUT8 peptide was performed using Sulfolink coupling gel (Pierce Chemical Co., Inc.) followed by elution according to the manufacturer’s instructions.

Livers were homogenized in a buffer containing 50 mM HEPES, 150 mM NaCl, 2 mM sodium pyrophosphate, 20 mM NaF, 4 mM EDTA, 20% glycerol, 10% Nonidet P-40, 4 mM phenylmethylsulfonyl fluoride, 20 nM leupeptin, and 20 nM aprotinin. Homogenates (75 µg protein) were separated by 10% SDS-PAGE and transferred to Hybond ECL nitrocellulose (Amersham Pharmacia Biotech). Rabbit polyclonal GLUT8 antibodies (1:500) were used to detect GLUT8 protein in total liver homogenates. For the competition study, as previously described, immunizing peptide was added to the immune serum at increasing concentrations (0.05 µg/ml–5 µg/ml) and incubated for 1 h at 4 C (6). The competed serum was used at a dilution of 1:500. Enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) detection was used in combination with scanning laser densitometry for quantitation.

Immunofluorescence detection of GLUT8
Paraffin-embedded sections (2 µm) were preheated at 37 C for 10 min. followed by incubation in xylene and stepwise rehydration in 100%, 95%, 70%, and 50% ethanol. After slides were washed in TBS the sections were blocked by addition of normal goat serum diluted in TBS for 1 h at room temperature. Affinity purified antibody to GLUT8 diluted in blocking solution was added at concentration of 2 µg/ml for 1 h at room temperature followed by three 3-min washes with TBS at RT. Cy3-conjugated antirabbit IgG was added to the sections for 1 h at room temperature followed by three 3-min washes in TBS at room temperature. Slides were mounted using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) and kept at 4 C until visualized.

Statistical analysis
Data are presented as mean ± SE of multiple determinations. Statistical significance was evaluated by two-tailed, unpaired, Student’s t test or by ANOVA using Fisher’s protected least significant difference test for post hoc analysis. Significance was accepted at P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GLUT8 regulation during fetal and postembryonic development
To determine the regulation of GLUT8 mRNA during different developmental stages, specimens were collected from embryos at d 14 (e14) and 17 (e17) of the development, neonates at d 1 (D1), 7 (D7), 14 (D14), 28 (D28), and adults at ages 6 wk (6W) and 15 wk (15W). Semiquantitative RT-PCR analysis of 5 µg of total liver RNA revealed differential expression of GLUT8 during development. High level expression of fetal liver GLUT8 mRNA at e14 was followed by a 6-fold decrease (P < 0.0001) at e17. GLUT8 expression gradually increased at D1, D7, and D28 of postnatal life (Fig. 1Go). At D28, GLUT8 mRNA expression increased about 5-fold compared with D7. At 6W, the GLUT8 mRNA expression returned to the levels established at e17.



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  		Figure 1. Developmental regulation of GLUT8 mRNA in liver. Quantitative of RT-PCR of GLUT8 mRNA in liver during fetal, neonatal, and adult development. RNA was isolated from WT embryos at e14 and e17 of development and neonatal days D1, D7, D28, 6-wk- (6W), and 15-wk-old (15W) adult WT mice. Two representative GLUT8 reactions are shown for each age (n = 3, results shown are means ± SEM; *, P < 0.05 and **, P < 0.0001 compared with 6W mice). GLUT8 mRNA was normalized using ß-actin primers.

 
A gradual decrease of the 32-kDa GLUT8 protein expression was demonstrated from e14 to 15W of age (Fig. 2Go). Interestingly, only negligible amounts of the 60 kDa GLUT8 immunoreactive species was detected at e14. The 60-kDa species was detected at D7 and its abundance increased gradually up to 15W. Both the 32-kDa and 60-kDa GLUT8 immunoreative bands were fully competed by addition of the GLUT8 immunizing peptide (Fig. 2BGo). Higher concentration of peptide was required to compete off the 32-kDa species than the 60-kDa species, indicating high specificity of the immune serum for the 32-kDa immunoreactive GLUT8 protein (Fig. 2BGo).



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  		Figure 2. Developmental regulation of GLUT8 protein in liver. A, Immunoblot analysis of GLUT8 in livers from WT embryos at d 14 (e14) and mice at 7 d (D7), 28 d (D28), and 15 wk. Immunoreactive GLUT8 antisera was used at 1:500 dilution. Representative immunoblot of 100 µg of total liver protein (upper panel) demonstrates highest expression of 32-kDa species of immunoreactive GLUT8 at e14 (n = 3–4 per group). Quantitation of the 32-kDa GLUT8 protein is shown on the lower panel. B, Sixty-kilodalton and 32-kDa immunoreactive species are efficiently competed off by addition 0.05 and 5 µg/ml of immunizing peptide to the immune sera. Representative immunoblot is shown from a total of eight studies.

 
Immunolocalization of GLUT8 protein in liver
Livers from 12- to 15- wk-old female mice were used for immunolocalization of GLUT8 using affinity purified antibodies. Intense staining of the perivenous hepatocytes and a gradual reduction in GLUT8 immunoreactivity with increasing distance from the central veins (Fig. 3Go, A and C) was detected. GLUT8 staining was negative in hepatocytes proximal to the portal triads (Fig. 3BGo).



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  		Figure 3. Immunolocalization of GLUT8 in WT normal liver. A, GLUT8 positive immunostaining in cells lining central vein (x10 magnification; arrows). B, Portal triad displays no GLUT8 positive cells (x10 magnification; arrow). C, x40 magnification of peri-central hepatocytes showing mainly cytoplasmic localization of GLUT8 (arrow). D, Negative control using rabbit IgG (2 µg/ml). E, Negative control using Cy3 secondary antibody only. F, Preabsorption of 2 µg/ml GLUT8 antibody with 60 µg/ml of immunizing peptide efficiently competes immunoreactivity in hepatocytes.

 
GLUT8 regulation in a model of short-term type 1 diabetes induced by STZ treatment
Semiquantitative RT-PCR analysis revealed that GLUT8 gene expression in the liver of short-term (8 d) STZ diabetic mice decreased slightly (10–12%) but significantly (P < 0.05), compared with nondiabetic mice (Fig. 4Go and Table 1Go). Alterations in GLUT8 mRNA seen in STZ diabetic liver were accompanied by opposite changes in abundance of the 32-kDa GLUT8 protein (Fig. 5Go and Table 1Go). STZ diabetes up-regulated expression of the 32-kDa GLUT8 protein 2-fold (P < 0.05). Conversely, levels of the 60-kDa GLUT8 immunoreactive protein were unaffected by STZ-induced diabetes (153 ± 6 and 159 ± 2.4, P = 0.5 for vehicle and STZ-treated groups, respectively (Fig. 5Go and Table 1Go). Immunofluorescence microscopy demonstrated a prominent increase in GLUT8 expression following STZ diabetes in agreement with the up-regulation of 32-kDa GLUT8 protein in immunoblot analysis. Importantly, GLUT8 was detected throughout the liver acinus including periportal hepatocytes (Fig. 6Go).



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  		Figure 4. Down-regulation of GLUT8 gene expression in insulinopenic liver of STZ-treated mice. Upper panel shows a representative scan of GLUT8 gene amplification product obtained by RT-PCR. GLUT8 gene expression is decreased 10–12% in STZ-treated mice. Data were normalized using ß-actin primers (data not shown). The lower panel shows quantitation of the normalized GLUT8 (n = 4, means ± SEM; *, P < 0.05).

 

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  		Table 1. Blood glucose and insulin levels and relative expression of hepatic GLUT8 mRNA and protein in STZ-induced and GLUT4+/- diabetic mice

 


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  		Figure 5. Alteration of GLUT8 protein in insulinopenic liver of STZ-treated mice. Immunoblot analysis of GLUT8 protein was performed as described in Fig. 2Go legend in liver of WT mice treated with vehicle (white bar) or STZ (black bar). Upper panel displays a representative immunoblot. One hundred micrograms of total liver protein were loaded per lane. Lower panel shows quantification of 32-kDa GLUT8 protein (n = 4 per group; results shown are means ± SEM; *, P < 0.05).

 


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  		Figure 6. Increased GLUT8 immunoreactivity in livers of insulinopenic STZ-treated mice. Immunostaining was performed using 2 µg/ml affinity purified GLUT8 antibody in combination with Cy3 secondary antibodies at dilution 1:400. Sections were visualized at magnification x10. GLUT8 immunofluorescence in hepatic central vein in vehicle (A) and STZ-treated (B) animals. Liver from vehicle-treated mice shows GLUT8 immunoreactivity specifically around the central vein. Livers from STZ-treated mice demonstrate perivenous staining and increased GLUT8 immunoreactivity in hepatocytes remote from the central vein.

 
GLUT8 regulation in a model of type 2 diabetes: GLUT4+/- mice
Hyperglycemic hyperinsulinemic male GLUT4+/- mice were used as a model of type 2 diabetes. GLUT8 mRNA was modestly (17%), though significantly (P < 0.01), increased in diabetic GLUT4+/- liver compared with WT controls (Fig. 7Go and Table 1Go). Similarly, immunoblot analysis detected a significant (P < 0.05) approximately 28% increase in the 32-kDa GLUT8 immunoreactive protein in the GLUT4+/- group compared with WT (Fig. 8Go and Table 1Go). Similar to that noted in STZ-treated mice, expression of the GLUT8 cross-reactive species of 60 kDa was unaltered in this model of type 2 diabetes (112.7 ± 2.8 vs. 115.3 ± 4.7, P = 0.7 for WT and GLUT4+/-, respectively; Fig. 8Go and Table 1Go). Moreover, in agreement with the regulation of 32-kDa GLUT8 protein, immunofluorescence microscopy demonstrated a notable elevation in GLUT8 signal detected in the perivenous hepatocytes of GLUT4+/- mice compared with the WT controls (Fig. 9Go). Importantly, no alteration of GLUT8 zonation occurred in the GLUT4+/- type 2 diabetic liver.



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  		Figure 7. Regulation of GLUT8 mRNA in liver of normal WT and diabetic GLUT4+/- mice. Northern blot analysis of 25 µg of liver RNA from normal WT control and diabetic GLUT4 +/- mice. 18S rRNA is shown to correct for loading differences between lanes. Quantitation of GLUT8 mRNA is shown below (n = 3–4). Results shown are means ± SEM; *, P < 0.05 between WT and GLUT4+/- animals.

 


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  		Figure 8. Regulation of GLUT8 protein in liver of normal WT and diabetic GLUT4+/- mice. Immunoblot analysis of GLUT8 protein from livers of WT and GLUT4+/-mice. Upper panel shows immunoblot analysis of WT and GLUT4+/- livers. Quantitation of the 32-kDa band is depicted in the lower panel (n = 4–6). Results shown are means ± SEM; *, P < 0.05 between WT and GLUT4+/- animals.

 


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  		Figure 9. Increased GLUT8 expression in liver of diabetic GLUT4+/- mice. GLUT8 immunolocalization in WT mice. Left panel displays a single layer of immunofluorescent cells around the central vein (arrow). Diabetic GLUT4+/- (right panel) liver displays increased GLUT8 immunofluorescence in hepatocytes surrounding central vein in GLUT4+/- liver (arrows).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Liver expresses three glucose transporters GLUT2, GLUT1, and the newly discovered GLUT8 (3, 4, 5, 10). Here we report the immunolocalization and regulation of GLUT8 expression in liver in several physiologic and pathologic paradigms in which hepatic glucose flux is modified in vivo. Specifically, we studied the developmental regulation of GLUT8 gene expression, effects of insulinopenic hyperglycemia, as well as hyperglycemia in combination with hyperinsulinemia on GLUT8 gene and protein expression and its cellular localization in the liver acinus. The results of this study demonstrated for the first time expression and regulation of hepatic GLUT8 mRNA and protein throughout different developmental stages of the mouse, insulin-dependent regulation of GLUT8 mRNA expression, and preferential expression of GLUT8 protein in perivenous hepatocytes.

GLUT8 gene expression was measured at e14 and e17 of fetal life and D1, D7, D28, 6W, and 15W of postnatal life. During embryonic development, liver receives a continuous supply of glucose from the maternal blood and therefore serves a mainly glycolytic role. In the present study, GLUT8 gene expression was shown to be six times higher (P < 0.0001) at e14 compared with e17. The fetal mouse liver is a hematopoetic organ throughout development with a peak activity at e11 through e16, followed by a dramatic decrease in hematopoetic activity by e19 (20). Hepatic GLUT8 gene and protein expression appeared to be up-regulated at the point in development when hematopoesis is predominant, suggesting that hematopoetic activity of the liver may play a regulatory role in GLUT8 expression. For instance, increased in energy demands during erythropoesis in the liver, concomitant with the up-regulation of GLUT8 at this stage might be an indicator of involvement of GLUT8 in transport of energy substrates into or within the hepatocytes. Alternatively, elevation of hepatic GLUT8 expression at this stage of the development could be a result of an increased property of hematopoetic cells. Moreover, the high density of glucose transporters on the membrane of red blood cells has been suggested to be important for generation of ATP and GTP (21). Thus, increased GLUT8 expression during hematopoesis may indicate that GLUT8 might be expressed in newly formed red blood cells. Regulation of GLUT8 expression throughout postnatal development was also assessed. GLUT8 expression in liver remained low until pups were 4 wk of age when GLUT8 expression peak significantly. This developmental stage is landmarked by weaning of pups onto a regular chow diet. During the suckling period, pups consume maternal milk that is low in carbohydrates, but rich in proteins and triacylglycerols. At weaning, mice are placed on a regular chow diet that is rich in carbohydrates and low in fat. It has been shown in rats that weaning onto a high carbohydrate diet leads to a 2-fold increase in plasma insulin and 3-fold increase of GLUT4 mRNA in muscle and fat tissue (22). Thus, GLUT8 gene expression might be regulated by the increased glycolytic flux due to the change in diet and increased circulating insulin. Indeed, immunolocalization of GLUT8 in murine blastocyst is significantly influenced by insulin (5).

Next, we examined GLUT8 protein expression in normal mouse livers. Studies by other investigators and us have identified 32-kDa species as GLUT8 protein in various tissues (3, 4, 6). It has been also reported that the overexpression of GLUT8 in various cell systems results in two molecular species detected by GLUT8 immune sera (3, 4). The appearance of 60-kDa species has been linked to the presence of reducing agents during sample preparation (3). We suggest that the origin of the 60-kDa band may be a result of dimerization of GLUT8 with itself or another protein. In the current study, the 32-kDa species demonstrated higher specificity to the immune sera as higher concentrations of competing peptide were needed to achieve complete attenuation of the signal. While it is not clear which molecular species of GLUT8 is biologically active, it is possible that both are involved in substrate handling. Our results demonstrate that the regulation of the 32-kDa species correlates with the behavior of GLUT8 expression measured by immunofluorescence microscopy. Thus, we focused on regulation of the 32-kDa species. Further studies, including transgenic overexpression and underexpression of GLUT8, will be needed to more completely understand the significance and function of each GLUT8 immunoreactive species.

Immunofluoresence studies demonstrated an intense level of GLUT8 expression in the perivenous hepatocytes and little or no immunoreactive GLUT8 in hepatocytes of portal triads. GLUT8 immunoreactivity was uniform throughout the cell suggesting mainly perinuclear localization (Fig. 3CGo). Nevertheless, some presence of GLUT8 at or near the plasma membrane could not be ruled out. According to the metabolic zonation model, hepatocytes lining central veins are mainly glycolytic as they express all the enzymes necessary for glycolysis and insulin receptor (23, 24, 25). These hepatocytes receive blood that is poorly oxygenated but rich in metabolic products. The hepatocytes proximal to portal triads are mainly gluconeogenic and liponeogenic and the blood supplied to them is highly oxygenated and rich in nutrients (26). It has been demonstrated that, whereas GLUT2 is present in all hepatocytes throughout the liver, GLUT1 is exclusively expressed in the perivenous area, and this localization of GLUT1 protein was linked to its ability to provide nutrient flow during fasting (12). The preferential immunolocalization of GLUT8 in the perivenous area and its negligible presence at the periportal area are in a good agreement with the metabolic zonation model and suggest GLUT8 involvement in substrate shuttling through the glycolytic pathway.

The regulation of hepatic GLUT8 expression was characterized in female mice treated with STZ that induces insulinopenia and severe hyperglycemia similar to type 1 diabetes (Table 1Go). Short-term STZ diabetes induced a significant decrease in GLUT8 mRNA that was accompanied by an increase in GLUT8 protein expression (Table 1Go). Interestingly, immunofluorescent detection demonstrated that STZ-induced diabetes led to up-regulation in GLUT8 protein expression in hepatocytes remote from central veins including periportal area. Thus, the effect of the metabolic milieu on translational and/or posttranslational processing of GLUT8 might be an important regulator of hepatic GLUT8 protein expression. It is also possible that the stability of GLUT8 protein is increased in the diabetic state. Previously, we reported such differential regulation of GLUT8 mRNA and protein in rat hippocampal neurons where STZ-induced diabetes led to a significant increase in GLUT8 mRNA expression, whereas GLUT8 protein levels remained normal (6). Interestingly, the magnitude of the increase of GLUT8 mRNA noted in rat hippocampus with STZ treatment was similar in magnitude to that demonstrated in liver in the present study, suggesting a very narrow range of transcriptional control of GLUT8 expression. This narrow window of GLUT8 mRNA and protein regulation is similar to the approximately 25% reduction in GLUT2 mRNA and unaltered amounts of GLUT2 protein following 7 d of STZ-induced diabetes in rat liver (11). It was suggested by Thorens et al. (11) that, despite the lack of profound alterations in GLUT2 mRNA and protein levels following STZ diabetes, its function may be altered on the level of the direction of glucose transport, namely, glucose uptake vs. glucose output by liver. Whereas GLUT2 functions at the plasma membrane, GLUT8 is predominately intracellular and, thus, its main action may involve intracellular trafficking of substrates. Our recent study has revealed one of the possible intracellular sites for GLUT8 function by demonstrating the redistribution of GLUT8 to the ER in response to a glucose challenge in rat hippocampal neurons (27). Future studies will determine whether the localization of GLUT8 within hepatocytes is modulated by nutrient availability and/or hormones such as insulin and glucagon.

To determine whether a defect in peripheral insulin sensitivity resulting from type 2 diabetes would alter GLUT8 mRNA or protein expression, livers from diabetic male GLUT4+/- mice were studied. Our previous studies demonstrated that the GLUT4+/- genetic lesion leads to severe insulin resistance in male but not female mice (17). Results of GLUT4 +/- mice showed an increase in GLUT8 mRNA in the diabetic hyperglycemic/hyperinsulinemic mice compared with WT (Table 1Go). This finding corroborates the STZ findings that suggested that GLUT8 gene expression is insulin sensitive. Moreover, GLUT8 protein levels were also increased in GLUT4+/- liver compared with WT mice (Table 1Go). Thus, chronic hyperglycemia and hyperinsulinemia is associated with the increased levels of GLUT8 protein. Immunofluorescence analysis of WT and GLUT4+/- livers demonstrated that the increase in GLUT8 protein expression involves hepatocytes surrounding central veins. Thus, this increase in the protein expression in GLUT4+/- livers is qualitatively different from STZ-induced hyperglycemia where GLUT8 protein expression increased in periportal hepatocytes. These findings may suggest that GLUT8 (immunodetection) expression in liver may vary depending upon the duration of hyperglycemia and/or changes in hormonal milieu (hyperinsulinemic vs. insulinopenic). Additionally, GLUT8 protein may be influenced by alterations in circulating fatty acids and triglycerides. STZ-induced diabetes results in significant dyslipidemia that is not seen in diabetic GLUT4+/- mice. Further studies are needed to determine the influence of lipids on GLUT8 expression and protein localization in normal and diabetic states. Importantly, it has been shown that GLUT8 expression is reduced in human testis following estrogen treatment and, thus, is regulated by gonadotropins (4). Because only female mice were subjected to STZ-induced diabetes in the current study, we cannot rule out that the regulation of GLUT8 in the diabetic models used in this study are not due to sexual dimorphisms and are independent of the different types of diabetes. Thus, gathering information on each sex independently will provide valuable information with regard to GLUT8 regulation.

In conclusion, we provide the first in vivo study that reveals metabolic regulation of the novel glucose transporter GLUT8 in liver, a central organ of glucose homeostasis. We demonstrate that GLUT8 expression in liver is regulated by physiologic and pathophysiologic changes in metabolism. GLUT8 gene expression is significantly affected when serum glucose and insulin levels are altered. Increase in circulating insulin is a common feature for the induction of hepatic GLUT8 gene, suggesting that its transcription is sensitive to insulin or its downstream effectors. It has been proposed previously that glucose flux into liver, mediated by insulin-activated glucokinase, alters expression of genes such as GLUT2, L-pyruvate kinase, Spot 14, and fatty acid synthase (28, 29, 30, 31, 32, 33). Also, glucose flux through gluconeogenesis in STZ diabetic and fasted rat livers or in cultured hepatocytes increases glucagon receptor gene expression (18, 32). Unlike the total amount of GLUT8 mRNA, which is correlated with circulating insulin levels, GLUT8 protein in the liver either follows GLUT8 gene regulation or is regulated in an opposite manner (Table 1Go). This finding suggests that, under hyperglycemic conditions, the posttranscriptional regulation of GLUT8, including an increase in its protein stability, may result in elevation of GLUT8 protein. Additionally, the influence of hypoxia on the preferential expression of GLUT8 in the perivenous hepatocytes cannot be ruled out. Hypoxia induced decrease mRNA expression of GLUT8 has been reported in a differentiated 3T3-L1 adipocytes but not fibroblasts in culture (7). For example, it has been established in rat hippocampus that one of the possible mechanisms for GLUT8 regulation in response to glucose challenge may involve its intracellular redistribution. Combined, the current study demonstrated that GLUT8 mRNA and protein levels are differentially regulated in liver in response to alterations in the milieu during normal development and in diabetes. We show that GLUT8 is localized primarily to the perivenous hepatocytes and propose that it may be playing a role in the regulation of the nutrient flow and glycolytic fluxes. Modulation of GLUT8 expression and/or localization in liver in various diabetic states provides an intriguing basis for studying its potential as a target for developing novel antidiabetic therapy.


   Acknowledgments
 
We thank Michele Causak and Xiu Quan Du for excellent technical assistance.


   Footnotes
 
This work was supported by grants from the NIH (DK-47425 and HL-58119), American Diabetes Association, Howard Hughes Medical Research Institute and Albert Einstein College of Medicine Cancer Center (5P30CA13330). M.J.C. is a recipient of an Irma T. Hirschl Career Scientist Award. This work was submitted in partial fulfillment of the requirements of the Ph.D. degree for the Albert Einstein College of Medicine (N.G.).

Abbreviations: e, Embryonic day; STZ, streptozotocin; WT, wild-type.

Received September 24, 2002.

Accepted for publication January 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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