189
Molecular and Cellular Endocrinology, 75 (1991) 189-196 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50
MOLCEL
02435
Regulation of glucose transporter synthesis in cultured human skin fibroblasts Atsushi Kosaki, Hideshi Kuzuya, Haruo Nishimura, Gen Inoue, Motozumi Takako Kakehi, Mikiko Okamoto, Shigeo Kono, Ikuko Maeda, Masashi and Hiroo Imura
Okamoto, Kubota
Second Division, Department of Medicine, Kyoto University School of Medicine, Kyofo, Japan (Received
Key wore?:
Transporter
glucose:
Glucose:
Insulin;
31 August
1990; accepted
Fibroblasts,
S November
1990)
human
Summary expression and The present study was designed to see the effects of glucose on glucose transporter glucose transport activity using cultured human skin fibroblasts. When the cells were incubated with various concentrations of glucose (11.1-44.4 mM), no differences were found in the HepG2 glucose transporter mRNA, protein levels and basal and insulin-stimulated 2-deoxyglucose uptake. Glucose deprivation, however, resulted in appro~mately 4-fold increases in the mRNA and 3-fold increases in the protein and the basal 2-deoxyglucose uptake. Chronic exposure to insulin increased the glucose transporter protein levels to similar degrees in the cells incubated with 11.1, 22.2 and 44.4 mM glucose accompanied by increases in the glucose transport activity. Effects of insulin on the glucose transporter mRNA and protein levels, however, were not evident in the glucose-deprived cells. It is concluded that glucose transport activity correlates closely with HepG2 glucose transporter expression in cultured human fibroblasts and that glucose (11.1-44.4 mM) does not affect the glucose transporter expression and glucose transport activity.
Introduction Glucose transport into cells is mediated by integral membrane protein glucose transporter. Recently several species of glucose transporters have been identified by cDNA cloning. The cDNA of human erythrocyte glucose transporter (GLUT1) was first cloned from the human hepatoma cell
Address for correspondence: Atsushi Kosaki, M.D., Second Division, Department of Medicine, Kyoto University School of Medicine, Shogoin Kawaharacho 54, Sakyo-ku, Kyoto 606, Japan.
line HepG2 by Mueckler et al. in 1985 (Mueckler et al., 1985). The protein, termed HepG2 glucose transporter, is expressed in most tissues. More recently a new species of glucose transporter was identified from adipocytes, skeletal muscles and heart (James et al., 1988, 1989; Birnbaum, 1989; Fukumoto et al., 1989; Kaestner et al., 1989). This adipocyte/ muscle type glucose transporter (GLUT-41 may be the major species in these insulin-target cells and account for most of insulinstimulated glucose uptake. It has been reported that insulin stimulates glucose uptake by both promoting the translocation of the glucose transporter from an intracellular pool to the plasma
190
membrane (Cushman and Wardzala, 1980; Kono et al., 1982; James et al., 1988; Gould et al., 1989) and enhancing the intrinsic activity of the molecule (Kahn and Cushman, 1987). Cellular glucose transport activity could change in a variety of physiological and pathological conditions, and thus be involved in altered glucose metabolism. However, factors which are responsible for the long-term regulation of glucose transport activity have not been fully elucidated and could vary depending on the species of glucose transporter. Obviously studies on the regulation of glucose transport will help to understand the pathophysiology of abnormal glucose metabolism in diabetes mellitus. We previously reported that insulin increases the HepG2 glucose transporter gene expression using cultured human skin fibroblasts (Kosaki et al., 1988). The present study was designed to further examine if glucose can regulate cellular glucose transport activity and if it modulates the insulin effect on glucose transporter synthesis in cultured human skin fibroblasts. Materials and methods Materials Tissue culture medium, serum, and reagents were obtained from Flow (Irvine, CA, U.S.A.) and NBM Laboratory (Kyoto, Japan). 2-Deoxy[ 3H]glucose and [ cu-32P]dCTP were purchased from New England Nuclear (Boston, MA, U.S.A.) and Amersham (Arlington Heights, IL, U.S.A.), respectively. The HepG2 glucose transporter cDNA was a generous gift from Dr. G.I. Bell (University of Chicago). Human p-actin pseudogene was obtained from Wako Chemical Industries (Osaka, Japan). Cell culture Human diploid fibroblast cultures were established from forearm skin of normal volunteers. The cells were grown as monolayers in a-minimal essential medium (aMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 50 units/ml penicillin, 50 pg/ml streptomycin and 250 pg/ml fungizone in a humidified atmosphere of 95% air5% CO, at 37°C. Stock cultures were maintained in 75 cm2 flasks, subcultured in 100 mm dishes and grown to confluence. The culture medium was
changed every 3 or 4 days. After 24-48 h when cells approached confluency, the medium was aspirated and replaced with serum-free (YMEM containing varying concentrations of glucose and 0.1% (w/v) bovine serum albumin (BSA). After 36 h at 37°C a small aliquot of phosphate-buffered saline (PBS) with or without 100 nM insulin was added. The cells were incubated at 37°C for the indicated hours and then used for the experiments. Isolation of cellular RNA Total cellular RNA was isolated as previously described (Glisin et al., 1974). Briefly, the cells, scraped off the dishes with a rubber policeman and harvested quickly, were lysed with GIT buffer (4 M guanidine isothiocyanate, 2.5 mM sodium acetate, 120 mM 2-mercaptoethanol, pH 6.9) and then centrifuged through a 3 ml CsCl cushion (5.7 M CsCl, 2.5 mM sodium acetate, pH 6.0) at 160,000 x g for 20 h at 15°C. The pellets were resuspended in 0.3 M sodium acetate, pH 6.0, and RNA was precipitated with 2.5 volumes of 100% ethanol. Dot blot analysis RNA (5520 pg) obtained as described above was denatured with 7% formaldehyde and immobilized on a nitrocellulose filter (BA-85, Schleicher & Schuell) with a microfiltration manifold. The blot was prehybridized and hybridized to the nick-translated [ CY32P]dCTP-labeled HepG2 glucose transporter cDNA fragment (phGT2-1, approximate 2.0 kilobase pairs) that included the region downstream from the internal EcoRI site (Mueckler et al., 1985) or the 443 base pair HinfI fragment of human &actin pseudogene, that was used for control marker (Nakajima-Iijima et al., 1985). Prehybridization was carried out in 50% deionized formamide, 5 X SSC (1 X SSC = 0.15 M NaCI, 15 mM sodium citrate, pH 7.0) 5 X Denhardt’s (1 x Denhardt’s = 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.2% BSA), 50 mM sodium phosphate, pH 6.5, 0.25% sodium dodecyl sulfate (SDS), and 0.3 mg/ml denatured sheared salmon sperm DNA at 42°C for 16 h. Hybridization with 32P-labeled probe (> 10’ cpm/pg DNA) was performed in 50% deionized formamide, 5 X SSC, 2 X Denhardt’s, 20 mM sodium phosphate, pH
191
6.5, 0.25% SDS, 0.1 mg/ml denatured sheared salmon sperm DNA, and 10% dextran sulfate at 42°C for 24 h. The blot was washed twice for 5 min at room temperature and 3 times for 20 min at 55°C with 0.1 x SSC, and 0.1% SDS, and then exposed to Konika X-ray film with an intensifying The dots on the blot were screen at -70°C. quantified by a liquid scintillation counter to determine the relative amounts of HepG2 glucose transporter mRNA. Peptide synthesis and production of polyclonal antibodies The two peptides corresponding to the carboxyl-terminal portion of HepG2 glucose transporter (residues 477-492) and of adipocyte/ muscle type glucose transporter (residues 498-509) were synthesized by the solid-phase method (Merrifield, 1963) using a peptide synthesizer. Each peptide was coupled to keyhole limpet hemocyanin (KLH) using maleimidobenzol-N-hydroxysuccinimide or glutaraldehyde, respectively, as previously described (Walter et al., 1980; Gentry et al., 1983). The peptide-KLH conjugate was emulsified with complete Freund’s adjuvant and injected intradermally to a female Japanese White rabbit at multiple sites in the back. An additional two injections of the conjugate in incomplete Freund’s adjuvant were given with a 4-week interval, and then the rabbit was bled 2 weeks after the last immunization. Antisera were heated at 56’C for 30 min to inactivate complement. Antibody to the peptide was affinity purified using the peptide-coupled Sepharose 4B column. Western blot analysis The cells were solubilized in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% Triton X-100 containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.4 mg/ml aprotinin at 4°C for 60 min and then sonication was performed twice for 20 s. Insoluble material was removed by centrifugation at 200,000 x g for 60 min at 4°C. 80 pg protein of the supernatant was separated by 9% SDS-polyacrylamide gel electrophoresis (PAGE) by the Laemmli method (Laemmli, 1970) and electrophoretically transferred to nitrocellulose filter (BA-85) at 100 V for 16 h. The filters were blocked with 5% BSA in TBS buffer (20 mM Tris-HCl, pH
7.5, 500 mM NaCl), incubated with rabbit polyclonal anti-glucose transporter antibody in 1% BSA-TBS buffer and sequentially with goat 1251labeled anti-rabbit IgG antibody in 1% BSA-TBS buffer. The filters were then washed 3 times with TBS buffer containing 0.05% Tween-20 and twice with TBS buffer for 10 min at room temperature, dried and subjected to autoradiography. The bands on the autoradiograms were quantified using a scanning densitometer to determine the relative amounts of HepG2 glucose transporter protein. Glucose transport assay 2-Deoxy-D-glucose transport studies were performed on the cells maintained in 60 X 15 mm tissue culture dishes as previously described with slight modifications (Howard et al., 1979). The medium was removed and replaced with KRP buffer (140 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl,, 1.47 mM KH,PO,, 8.06 mM Na,HPO,, 0.49 mM MgCl,, 0.1% BSA, pH 7.4). Then 1 PM insulin (acute insulin stimulation) or KRP buffer alone (basal uptake) was added. The cells were further incubated for 30 min at 37°C. The rate of glucose uptake was then determined by adding 2-deoxy[ 3H]glucose (50 PM, 5 pCi/nmol) at 37°C for 5 min. Glucose uptake was terminated by aspiration of the buffer, followed by three rapid washes with ice-cold PBS containing 0.1 mM phloretin. The cells were solubilized in 0.1% SDS and subjected to scintillation counting. The specific uptake was normalized for the protein content (per dish) determined by the method of Bradford (Bradford, 1976). Statistical analysis Data were presented as mean + SEM and differences among groups were tested by two-way layout analysis of variance and Scheffe type multiple comparison (Morrison, 1976) or by Student’s paired t-test. Results Effects of glucose and insulin treatment on glucose transporter gene expression Fig. 1A demonstrates the HepG2 glucose transporter mRNA levels determined in six strains of fibroblasts obtained from six healthy subjects. The
192
lns"l~nflOOnMi
Glucose (mM\
-
+ 111
~
+ 222
Fig. 1. Effects of glucose and insulin on HepG2 glucose transporter mRNA in cultured human fibroblasts. Cells were preincubated with the indicated glucose concentration for 36 h and further incubated with or without 100 nM insulin for 3 h. HepG2 glucose transporter mRNA was determined by dot blot analysis. A: HepG2 glucose transporter mRNA level. B: Ratio of HepG2 glucose transporter mRNA to P-actin mRNA. Each point represents the mean k SEM of six strains. * * p < 0.01, * p < 0.05 vs. cells incubated without insulin. ‘p < 0.05 vs. cells incubated with glucose (11.1 or 22.2 mM) and insulin.
cells were preincubated with varying concentrations of glucose for 36 h. Insulin (100 nM) or buffer alone was then added and the cells were further incubated for 3 h. As indicated in the figure there were no differences in the mRNA levels among the cells incubated with 11.1, 22.2 and 44.4 mM glucose. As we previously reported, insulin increased the mRNA levels. The magnitude of the insulin-induced increase in mRNA was comparable in the cells treated with 11.1 and 22.2 mM glucose, but a little smaller in the cells treated with 44.4 mM glucose. Similar findings were obtained when the ratio of HepG2 glucose transporter to P-actin mRNA was calculated, suggesting that the effect of insulin is specific (Fig. 1B). Using the same protocol we found that glucose deprivation (36 h) resulted in approximately 4-fold increases in the HepG2 glucose transporter mRNA levels compared with glucose (22.2 mM)-treated cells (Fig. 2A). Since P-actin mRNA levels were decreased, the ratio of mRNA of HepG2 glucose transporter to /3-actin was increased by 6-fold in the glucose-deprived cells (Fig. 2B). Insulin stimu-
Fig. 2. Effects of glucose deprivation on HepG2 glucose transporter mRNA in cultured human fibroblasts. Cells were preincubated without glucose or with glucose (22.2 mM) for 36 h and further incubated in the absence or presence of 100 nM insulin for 3 h. HepG2 glucose transporter mRNA was determined by dot blot analysis. A: HepG2 glucose transporter mRNA level. B: Ratio of HepG2 glucose transporter mRNA to p-actin mRNA. Each point represents the mean+SEM of five strains. * p < 0.01 vs. cells incubated with 22.2 mM glucose. * * p < 0.01, * p < 0.05 vs. cells incubated with 22.2 mM glucose without insulin.
193
123456
180 5 z i
140
;
100
.
0
4
8
Incubation
16 Time
(h)
Fig. 3. Time course of changes in HepG2 glucose transporter protein in cultured human fibroblasts stimulated with insulin. Cells were preincubated with serum-free (rMEM (11.1 mM glucose) for 36 h and then 100 nM insulin was added. After further incubation for the indicated hours, HepG2 glucose transporter protein in Triton X-100 soluble fraction was determined by Western blot analysis and quantified using a scanning densitometer. Each point is expressed as a percentage of control cells incubated for the indicated hours without insulin.
lation of the mRNA, however, in the glucose-deprived cells.
was not significant
Effects of glucose and insulin treatment on cellular glucose transporter protein levels We determined total cellular HepG2 glucose transporter levels in fibroblasts by the Western blotting using antibody against the carboxylterminal portion of HepG2 glucose transporter. HepG2 glucose transporter was detected as the 55 kDa band as previously described (Haspel et al., 1985; Homer et al., 1987). Insulin (100 nM) increased HepG2 glucose transporter in a time-dependent manner (Fig. 3). The levels of HepG2 glucose transporter reached the plateau at 4 h after the addition of insulin. Next we studied effects of glucose on basal and insulin-stimulated HepG2 glucose transporter protein levels. The cells were preincubated with varying concentrations of glucose for 36 h. Insulin (100 nM) or buffer alone was then added and the cells were further incubated for 6 h. There were no differences in the basal HepG2 glucose transporter level (without insulin) among the cells treated with 11.1, 22.2 and 44.4 mM glucose (Fig. 4, Table 1). Insulin increased the glucose transporter levels to similar extents in the cells treated with 11.1, 22.2 and 44.4 mM glucose (Fig. 4, Table 1). Again, glucose-deprived cells exhibited a higher level of glucose transporter than the cells incubated with glucose
Insulin Glucose
,-
+,
,-
11 .l
+,
,-
22.2
+ 44.4
(mM)
Fig. 4. Effects of glucose and insulin on HepG2 glucose transporter protein in cultured human fibroblasts. Cells were preincubated with the indicated glucose concentrations for 36 h and further incubated in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of 100 nM insulin for 6 h. HepG2 glucose transporter in T&on X-100 soluble fraction of the cells was determined by Western blot analysis.
(Fig. 5, Table 1). The effect of insulin was not evident in the glucose-deprived cells. In a different series of experiments we examined if adipocyte/ muscle type glucose transporter is expressed in the cultured fibroblasts by the Western blotting using antibody against the carboxyl-terminal portion of the glucose transporter. Adipocyte/ muscle type
TABLE 1 EFFECTS OF GLUCOSE AND INSULIN ON HepG2 GLUCOSE TRANSPORTER CONTENT IN CULTURED HUMAN FIBROBLASTS The data are expressed as percent of glucose transporter con tent in the cells incubated with 11.1 mM glucose without insulin. Values are the means* SEM of three or five experiments performed independently. Glucose
Insulin (nM)
(mM)
0
100
336.9 k 53.2 a 100 89.5k 9.7 101.6+11.8
335.0 + 50.1 125.3 k 6.1 138.4k 7.6 141.3k11.8
0
11.1 22.2 44.4
a b ’ b
a Difference from cells incubated with 11.1 mM glucose without insulin at p < 0.05. b Difference from cells incubated without insulin at p < 0.05.
194
12345678
123456
Insulin Glucose
Insulin
-
+,
,-
+,
,-
+
Glucose
-
-
+
Xylose
+
-
-
Fig. 5. Effects of glucose deprivation on HepG2 glucose transporter protein in cultured human fibroblasts. Cells were preincubated without glucose (lanes 7, 2, 3, and 4) or with glucose (22.2 mM: lanes 5 and 6) for 36 h and further incubated in the absence (lanes 1. 3, and 5) or presence (lanes 2, 4. and 6) of 100 nM insulin for 6 h. HepG2 glucose transporter in Triton X-100 soluble fraction of the cells was then determined by Western blot analysis. Xylose (22.2 mM) was added to adjust the osmotic pressure in lanes 1 and 2.
,-
+, ,0
9
+, I-
11.1
+, (-
22.2
+,
Adipo.
44.4 (mM)
Fig. 6. Absence of adipocyte/muscle type glucose transporter protein in cultured human fibroblasts. Cells were preincubated with the indicated glucose concentrations for 36 h and further incubated in the absence (lanes 1, 3, 5, and 7) or presence (lanes 2, 4, 6, and 8) of 100 nM insulin for 6 h. Adipocyte/ muscle type glucose transporter was determined in the Triton X-100 soluble fraction by Western blot analysis. No band was detected under these experimental conditions. Adipocyte/ muscle type glucose transporter in total crude membrane fraction of rat adipocytes (lanes 9) was shown as positive control. This is a representative result from experiments performed 3 times.
glucose transporter, however, could not be detected in the fibroblasts under any experimental conditions examined (Fig. 6). Effects of glucose and insulin treatment on 2-deoxyglucose uptake Effects of glucose and insulin on 2-deoxyglucase uptake by cultured fibroblasts were studied to see if the changes found in the glucose transporter expression are reflected in the cellular glucose transport activity. After the preincubation with glucose (O-44.4 mM) for 36 h, basal and maximal insulin (1 PM, 30 min)-stimulated 2-deoxyglucose uptake were determined (Fig. 7, columns B and I, respectively). In parallel experiments, to see the long-term effect of insulin, insulin (100 nM) was added to the preincubation medium for the last 6 h and then maximal insulin (1 PM, 30 min)-stimulated 2-deoxyglucose uptake was determined (Fig. 7, columns II). In the cells preincubated without glucose, the basal uptake was increased compared with those pretreated with
11 1
Glucose
Concentration
22 2
44 4
(mMI
Fig. 7. Effects of glucose and insulin on 2-deoxyglucose uptake in cultured human fibroblasts. Cells were preincubated with the indicated glucose concentrations for 36 h and then the basal (B) and acute (1 pM, 30 min) effects of insulin (I) on 2-deoxyglucose uptake were determined. To see long-term effects of insulin (II), insulin (100 nM) was added to the preincubation medium for the last 6 h and then insulin (1 ,uM, 30 min)-stimulated 2-deoxyglucose uptake was determined. These data represents the mean+SEM of six experiments using four strains. ‘p < 0.01 vs. B of the same group. * * p < 0.01 vs. I and B of the same group. t p < 0.01 vs. cells incubated with glucose (11.1, 22.2 and 44.4 mM).
195
glucose. However, acute and long-term effects of insulin were not evident. On the other hand, there were no differences in the basal uptake among the cells preincubated with 11.1, 22.2 and 44.4 mM glucose. Insulin stimulated 2-deoxyglucose uptake to the same degrees (Fig. 7, columns I). Moreover, long-term treatment with insulin enhanced insulin-stimulated 2-deoxyglucose uptake to the same degree irrespective of the glucose concentrations in the preincubation medium (Fig. 7, columns II). Thus the changes in cellular glucose transport activity reflected those of HepG2 glucose transporter mRNA and protein levels in cultured human fibroblasts. Discussion In the present study we examined the effects of insulin and glucose on the glucose transport system in cultured human skin fibroblasts. Although it has been well known that insulin stimulates glucose uptake in cultured fibroblasts (Howard et al., 1979), we did not detect the adipocyte/muscle type glucose transporter in fibroblasts by the Western blotting using antibody against the carboxyl-terminal portion of the adipocyte/ muscle type glucose transporter. Thus, it seems likely that the HepG2 glucose transporter is the major species involved in insulin-stimulated glucose uptake in cultured fibroblasts. Of course, at present, we cannot rule out a possibility of contribution of other species of glucose transporter thus far not identified (Kayano et al., 1988; Mesmer et al., 1990). We found that long-term treatment of the cells with insulin resulted in parallel increases in the cellular levels of HepG2 glucose transporter mRNA and protein accompanied by increases in insulin-stimulated glucose uptake. There was a good correlation between the increase in the mRNA levels and that in the glucose transport activity. These findings are in agreement with those previously reported in 3T3-Ll adipocytes (Tordjman et al., 1989) and in rat L6 skeletal muscle cell lines (Walker et al., 1989). In contrast, an elevation of glucose concentrations in the medium from 11.1 to 44.4 mM was without effect on the HepG2 glucose transporter mRNA and protein levels. Both basal and
insulin-induced (acute) 2-deoxyglucose uptake were not altered. Furthermore, although incubation with the highest concentration of glucose (44.4 mM) slightly diminished the extent of insulin-induced increase of the glucose transporter mRNA levels, glucose (11.1-22.2 mM) did not affect insulin’s long-term effects on the glucose transporter mRNA and protein levels and the glucose transport activity. Thus from the present study it seems unlikely that high concentrations of glucose modulate the insulin effects to increase the HepG2 glucose transporter synthesis and glucose transport activity in cultured human fibroblasts. It should be noted, however, that the present findings may not be extrapolated to other tissues and other species of glucose transporters. Garvey et al. (1987) for example, have recently shown that glucose and insulin pretreatment can synergistically impair insulin-induced translocation of glucose transporters to the cell surface in primary cultured rat adipocytes, thus resulting in a decrease in insulin responsiveness. They found, however, no changes in the total numbers of D-glucase-inhibitable cytochalasin B binding sites among the cells preincubated for 24 h as control cells and in the presence of 100 ng/ml insulin with and without 15 mM D-&COSe. Although there were no major differences in the glucose transporter expression and glucose transport activity among the cells treated with glucose (11 .l-44.4 mM), glucose deprivation, from the medium for 36 h resulted in marked increases in these parameters. The acute and long-term effects of insulin were not observed in the glucosedeprived cells. Several previous studies have also reported that glucose deprivation induces an increase in hexose transport. For example, Haspel et al. (1986) have shown that 3T3-C2 murine fibroblasts deprived of glucose exhibit increases in hexose transport and glucose transporter. Walker et al. (1988) have shown similar results in rat brain glial cells. We confirmed these findings in cultured human skin fibroblasts and further clarified that enhanced HepG2 glucose transporter expression mainly contributes to the increase in cellular glucose transporter levels. Similar conclusions were reached in rat L6 skeletal muscle cell lines (Walker et al., 1989). The reasons for the lack of both acute and chronic effects of insulin in the glucose-de-
196
prived cells remain to be elucidated. Recently it has been shown that HepG2 glucose transporter can translocate from an intracellular membrane compartment to the cell surface in response to insulin in 3T3-Ll adipocytes (Gould et al., 1989) and Chinese hamster ovary cells (Asano et al., 1989). If this is the case in cultured fibroblasts, insulin-induced translocation of glucose transporters might be hampered by some unknown mechanisms, for example energy depletion, in the cells deprived of glucose. Alternatively newly synthesized glucose transporters may be distributed mainly to the plasma membrane in glucosedeprived cells (Walker et al., 1990). Determinations of glucose transporter in intracellular and plasma membrane compartments will be necessary in future. Regarding the lack of chronic effects of insulin on the glucose transport system, glucose deprivation might maximally stimulate the rate of glucose transporter synthesis. Acknowledgments We are grateful to Dr. G.I. Bell (Howard Hughes Medical Institute, University of Chicago) for providing the HepG2 glucose transporter cDNA and to Dr. T. Mitani (Sanwa Kagaku Co. Ltd.) for synthetic C-terminal peptide of glucose transporter. We thank Kayoko Furukawa for secretarial assistance. References Asano, T., Shibasaki, Y., Ohno, S., Taira, H., Lin, J.L., Kasuga, M., Kanazawa, Y., Akanuma, Y., Takaku, F. and Oka, Y. (1989) J. Biol. Chem. 264, 3416-3420. Bimbaum, M.J. (1989) Cell 57, 305-315. Bradford, M.M. (1976) Anal. B&hem. 72, 248-254. Cushman, S.W. and Wardzala, L.J. (1980) J. Biol. Chem. 255, 4758-4762. Fukumoto, H., Kayano, T., Buse, J.B., Edwards, Y., Pilch, P.F., Bell, G.I. and Seino, S. (1989) J. Biol. Chem. 264, 7776-7779. Garvey, W.T., Olefsky, J.M., Matthaei, S. and Marshall, S. (1987) J. Biol. Chem. 262, 189-197. Gentry, L.E., Rohrschneider, L.R., Casnellie, J.E. and Krebs, E.G. (1983) J. Biol. Chem. 258, 11219-11228.
Glisin, V., Crkvenjakuv, R. and Byns, C. (1974) Biochemistry 13, 2633-2637. Gould, G.W., Derechin, V., James, D.E., Tordjman, K., Ahem, S., Gibbs, E.M., Lienhard, G.E. and Mueckler, M. (1989) J. Biol. Chem. 264, 2180-2184. Haspel, H.C., Bimbaum, M.J., Wilk, E.W. and Rosen, O.M. (1985) J. Biol. Chem. 260, 7219-7225. Haspel, H.C., Wilk, E.W., Bimbaum, M.J., Cushman, S.W. and Rosen, O.M. (1986) J. Biol. Chem. 261, 6778-6789. Homer, H.C., Munck, A. and Lienhard, G.E. (1987) J. Biol. Chem. 262, 17696-17702. Howard, B.V., Mott, D.M., Fields, R.M. and Bennett, P.H. (1979) J. Cell. Physiol. 101, 129-138. James, D.E., Brown, R., Navarro, J. and Pilch, P.F. (1988) Nature 333, 183-185. James, D.E., Strube, M. and Mueckler, M. (1989) Nature 338, 83-87. Kaestner, K.H., Christy, R.J., McLenithan, J.C., Braiterman, L.T., Cornelius, P., Pekala, P.H. and Lane, M.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3150-3154. Kahn, B.B. and Cushman, S.W. (1987) J. Biol. Chem. 262, 5118-5124. Kayano, T., Fukumoto, H., Eddy, R.L., Fan, Y.S., Byers, M.G., Shows, T.B. and Bell, G.I. (1988) J. Biol. Chem. 263, 15245-15248. Kono, T., Robinson, F.W., Blevins, T.L. and Ezaki, 0. (1982) J. Biol. Chem. 257, 10942-10947. Kosaki, A., Kuzuya, H., Yoshimasa, Y., Yamada, K., Okamoto, M., Nishimura, H., Kakehi, T., Takeda, J., Seino, Y. and Imura, H. (1988) Diabetes 37, 1583-1586. Laemmli, U.K. (1970) Nature 227, 680-685. Merrifield, R.B. (1963) J. Am. Chem. Sot. 85, 2149-2154. Mesmer, O.T., Gordon, B.A., Pupar, C.A. and Lo, T.C.Y. (1990) B&hem. J. 265, 823-829. Morrison, D.F. (1976) Multivariate Statistical Methods, 2nd edn., McGraw Hills, New York. Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W.J., Lienhard, G.E. and Lodish, H.F. (1985) Science 229, 941-945. Nakajima-Iijima, S., Hamada, H., Reddy, P. and Kakunaga, T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6133-6137. Tordjman, K.M., Leingang, K.A., James, D.E. and Mueckler, M.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7761-7765. Walker, P.S., Donovan, J.A., Van Ness, B.C., Fellows, R.E. and Pessin, J.E. (1988) J. Biol. Chem. 263, 15594-15601. Walker, P.S., Ramlal, T., Donovan, J.A., Doering, T.P., Sandra, A., Klip, A. and Pessin, J.E. (1989) J. Biol. Chem. 264, 6587-6595. Walker, P.S., Ramlal, T., Sarabia, V., Koivisto, U.M., Bilan, P.J., Pessin, J.E. and Khp, A. (1990) J. Biol. Chem. 265, 1516-1523. Walter, G., Scheidtmann, K.H., Carbone, A., Laudano, A.P. and Doolittle, R.F. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5197-5200.
Molecular and Cellular Endocrinology, 75 (1991) 189-196 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50
MOLCEL
02435
Regulation of glucose transporter synthesis in cultured human skin fibroblasts Atsushi Kosaki, Hideshi Kuzuya, Haruo Nishimura, Gen Inoue, Motozumi Takako Kakehi, Mikiko Okamoto, Shigeo Kono, Ikuko Maeda, Masashi and Hiroo Imura
Okamoto, Kubota
Second Division, Department of Medicine, Kyoto University School of Medicine, Kyofo, Japan (Received
Key wore?:
Transporter
glucose:
Glucose:
Insulin;
31 August
1990; accepted
Fibroblasts,
S November
1990)
human
Summary expression and The present study was designed to see the effects of glucose on glucose transporter glucose transport activity using cultured human skin fibroblasts. When the cells were incubated with various concentrations of glucose (11.1-44.4 mM), no differences were found in the HepG2 glucose transporter mRNA, protein levels and basal and insulin-stimulated 2-deoxyglucose uptake. Glucose deprivation, however, resulted in appro~mately 4-fold increases in the mRNA and 3-fold increases in the protein and the basal 2-deoxyglucose uptake. Chronic exposure to insulin increased the glucose transporter protein levels to similar degrees in the cells incubated with 11.1, 22.2 and 44.4 mM glucose accompanied by increases in the glucose transport activity. Effects of insulin on the glucose transporter mRNA and protein levels, however, were not evident in the glucose-deprived cells. It is concluded that glucose transport activity correlates closely with HepG2 glucose transporter expression in cultured human fibroblasts and that glucose (11.1-44.4 mM) does not affect the glucose transporter expression and glucose transport activity.
Introduction Glucose transport into cells is mediated by integral membrane protein glucose transporter. Recently several species of glucose transporters have been identified by cDNA cloning. The cDNA of human erythrocyte glucose transporter (GLUT1) was first cloned from the human hepatoma cell
Address for correspondence: Atsushi Kosaki, M.D., Second Division, Department of Medicine, Kyoto University School of Medicine, Shogoin Kawaharacho 54, Sakyo-ku, Kyoto 606, Japan.
line HepG2 by Mueckler et al. in 1985 (Mueckler et al., 1985). The protein, termed HepG2 glucose transporter, is expressed in most tissues. More recently a new species of glucose transporter was identified from adipocytes, skeletal muscles and heart (James et al., 1988, 1989; Birnbaum, 1989; Fukumoto et al., 1989; Kaestner et al., 1989). This adipocyte/ muscle type glucose transporter (GLUT-41 may be the major species in these insulin-target cells and account for most of insulinstimulated glucose uptake. It has been reported that insulin stimulates glucose uptake by both promoting the translocation of the glucose transporter from an intracellular pool to the plasma
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membrane (Cushman and Wardzala, 1980; Kono et al., 1982; James et al., 1988; Gould et al., 1989) and enhancing the intrinsic activity of the molecule (Kahn and Cushman, 1987). Cellular glucose transport activity could change in a variety of physiological and pathological conditions, and thus be involved in altered glucose metabolism. However, factors which are responsible for the long-term regulation of glucose transport activity have not been fully elucidated and could vary depending on the species of glucose transporter. Obviously studies on the regulation of glucose transport will help to understand the pathophysiology of abnormal glucose metabolism in diabetes mellitus. We previously reported that insulin increases the HepG2 glucose transporter gene expression using cultured human skin fibroblasts (Kosaki et al., 1988). The present study was designed to further examine if glucose can regulate cellular glucose transport activity and if it modulates the insulin effect on glucose transporter synthesis in cultured human skin fibroblasts. Materials and methods Materials Tissue culture medium, serum, and reagents were obtained from Flow (Irvine, CA, U.S.A.) and NBM Laboratory (Kyoto, Japan). 2-Deoxy[ 3H]glucose and [ cu-32P]dCTP were purchased from New England Nuclear (Boston, MA, U.S.A.) and Amersham (Arlington Heights, IL, U.S.A.), respectively. The HepG2 glucose transporter cDNA was a generous gift from Dr. G.I. Bell (University of Chicago). Human p-actin pseudogene was obtained from Wako Chemical Industries (Osaka, Japan). Cell culture Human diploid fibroblast cultures were established from forearm skin of normal volunteers. The cells were grown as monolayers in a-minimal essential medium (aMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 50 units/ml penicillin, 50 pg/ml streptomycin and 250 pg/ml fungizone in a humidified atmosphere of 95% air5% CO, at 37°C. Stock cultures were maintained in 75 cm2 flasks, subcultured in 100 mm dishes and grown to confluence. The culture medium was
changed every 3 or 4 days. After 24-48 h when cells approached confluency, the medium was aspirated and replaced with serum-free (YMEM containing varying concentrations of glucose and 0.1% (w/v) bovine serum albumin (BSA). After 36 h at 37°C a small aliquot of phosphate-buffered saline (PBS) with or without 100 nM insulin was added. The cells were incubated at 37°C for the indicated hours and then used for the experiments. Isolation of cellular RNA Total cellular RNA was isolated as previously described (Glisin et al., 1974). Briefly, the cells, scraped off the dishes with a rubber policeman and harvested quickly, were lysed with GIT buffer (4 M guanidine isothiocyanate, 2.5 mM sodium acetate, 120 mM 2-mercaptoethanol, pH 6.9) and then centrifuged through a 3 ml CsCl cushion (5.7 M CsCl, 2.5 mM sodium acetate, pH 6.0) at 160,000 x g for 20 h at 15°C. The pellets were resuspended in 0.3 M sodium acetate, pH 6.0, and RNA was precipitated with 2.5 volumes of 100% ethanol. Dot blot analysis RNA (5520 pg) obtained as described above was denatured with 7% formaldehyde and immobilized on a nitrocellulose filter (BA-85, Schleicher & Schuell) with a microfiltration manifold. The blot was prehybridized and hybridized to the nick-translated [ CY32P]dCTP-labeled HepG2 glucose transporter cDNA fragment (phGT2-1, approximate 2.0 kilobase pairs) that included the region downstream from the internal EcoRI site (Mueckler et al., 1985) or the 443 base pair HinfI fragment of human &actin pseudogene, that was used for control marker (Nakajima-Iijima et al., 1985). Prehybridization was carried out in 50% deionized formamide, 5 X SSC (1 X SSC = 0.15 M NaCI, 15 mM sodium citrate, pH 7.0) 5 X Denhardt’s (1 x Denhardt’s = 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.2% BSA), 50 mM sodium phosphate, pH 6.5, 0.25% sodium dodecyl sulfate (SDS), and 0.3 mg/ml denatured sheared salmon sperm DNA at 42°C for 16 h. Hybridization with 32P-labeled probe (> 10’ cpm/pg DNA) was performed in 50% deionized formamide, 5 X SSC, 2 X Denhardt’s, 20 mM sodium phosphate, pH
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6.5, 0.25% SDS, 0.1 mg/ml denatured sheared salmon sperm DNA, and 10% dextran sulfate at 42°C for 24 h. The blot was washed twice for 5 min at room temperature and 3 times for 20 min at 55°C with 0.1 x SSC, and 0.1% SDS, and then exposed to Konika X-ray film with an intensifying The dots on the blot were screen at -70°C. quantified by a liquid scintillation counter to determine the relative amounts of HepG2 glucose transporter mRNA. Peptide synthesis and production of polyclonal antibodies The two peptides corresponding to the carboxyl-terminal portion of HepG2 glucose transporter (residues 477-492) and of adipocyte/ muscle type glucose transporter (residues 498-509) were synthesized by the solid-phase method (Merrifield, 1963) using a peptide synthesizer. Each peptide was coupled to keyhole limpet hemocyanin (KLH) using maleimidobenzol-N-hydroxysuccinimide or glutaraldehyde, respectively, as previously described (Walter et al., 1980; Gentry et al., 1983). The peptide-KLH conjugate was emulsified with complete Freund’s adjuvant and injected intradermally to a female Japanese White rabbit at multiple sites in the back. An additional two injections of the conjugate in incomplete Freund’s adjuvant were given with a 4-week interval, and then the rabbit was bled 2 weeks after the last immunization. Antisera were heated at 56’C for 30 min to inactivate complement. Antibody to the peptide was affinity purified using the peptide-coupled Sepharose 4B column. Western blot analysis The cells were solubilized in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% Triton X-100 containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.4 mg/ml aprotinin at 4°C for 60 min and then sonication was performed twice for 20 s. Insoluble material was removed by centrifugation at 200,000 x g for 60 min at 4°C. 80 pg protein of the supernatant was separated by 9% SDS-polyacrylamide gel electrophoresis (PAGE) by the Laemmli method (Laemmli, 1970) and electrophoretically transferred to nitrocellulose filter (BA-85) at 100 V for 16 h. The filters were blocked with 5% BSA in TBS buffer (20 mM Tris-HCl, pH
7.5, 500 mM NaCl), incubated with rabbit polyclonal anti-glucose transporter antibody in 1% BSA-TBS buffer and sequentially with goat 1251labeled anti-rabbit IgG antibody in 1% BSA-TBS buffer. The filters were then washed 3 times with TBS buffer containing 0.05% Tween-20 and twice with TBS buffer for 10 min at room temperature, dried and subjected to autoradiography. The bands on the autoradiograms were quantified using a scanning densitometer to determine the relative amounts of HepG2 glucose transporter protein. Glucose transport assay 2-Deoxy-D-glucose transport studies were performed on the cells maintained in 60 X 15 mm tissue culture dishes as previously described with slight modifications (Howard et al., 1979). The medium was removed and replaced with KRP buffer (140 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl,, 1.47 mM KH,PO,, 8.06 mM Na,HPO,, 0.49 mM MgCl,, 0.1% BSA, pH 7.4). Then 1 PM insulin (acute insulin stimulation) or KRP buffer alone (basal uptake) was added. The cells were further incubated for 30 min at 37°C. The rate of glucose uptake was then determined by adding 2-deoxy[ 3H]glucose (50 PM, 5 pCi/nmol) at 37°C for 5 min. Glucose uptake was terminated by aspiration of the buffer, followed by three rapid washes with ice-cold PBS containing 0.1 mM phloretin. The cells were solubilized in 0.1% SDS and subjected to scintillation counting. The specific uptake was normalized for the protein content (per dish) determined by the method of Bradford (Bradford, 1976). Statistical analysis Data were presented as mean + SEM and differences among groups were tested by two-way layout analysis of variance and Scheffe type multiple comparison (Morrison, 1976) or by Student’s paired t-test. Results Effects of glucose and insulin treatment on glucose transporter gene expression Fig. 1A demonstrates the HepG2 glucose transporter mRNA levels determined in six strains of fibroblasts obtained from six healthy subjects. The
192
lns"l~nflOOnMi
Glucose (mM\
-
+ 111
~
+ 222
Fig. 1. Effects of glucose and insulin on HepG2 glucose transporter mRNA in cultured human fibroblasts. Cells were preincubated with the indicated glucose concentration for 36 h and further incubated with or without 100 nM insulin for 3 h. HepG2 glucose transporter mRNA was determined by dot blot analysis. A: HepG2 glucose transporter mRNA level. B: Ratio of HepG2 glucose transporter mRNA to P-actin mRNA. Each point represents the mean k SEM of six strains. * * p < 0.01, * p < 0.05 vs. cells incubated without insulin. ‘p < 0.05 vs. cells incubated with glucose (11.1 or 22.2 mM) and insulin.
cells were preincubated with varying concentrations of glucose for 36 h. Insulin (100 nM) or buffer alone was then added and the cells were further incubated for 3 h. As indicated in the figure there were no differences in the mRNA levels among the cells incubated with 11.1, 22.2 and 44.4 mM glucose. As we previously reported, insulin increased the mRNA levels. The magnitude of the insulin-induced increase in mRNA was comparable in the cells treated with 11.1 and 22.2 mM glucose, but a little smaller in the cells treated with 44.4 mM glucose. Similar findings were obtained when the ratio of HepG2 glucose transporter to P-actin mRNA was calculated, suggesting that the effect of insulin is specific (Fig. 1B). Using the same protocol we found that glucose deprivation (36 h) resulted in approximately 4-fold increases in the HepG2 glucose transporter mRNA levels compared with glucose (22.2 mM)-treated cells (Fig. 2A). Since P-actin mRNA levels were decreased, the ratio of mRNA of HepG2 glucose transporter to /3-actin was increased by 6-fold in the glucose-deprived cells (Fig. 2B). Insulin stimu-
Fig. 2. Effects of glucose deprivation on HepG2 glucose transporter mRNA in cultured human fibroblasts. Cells were preincubated without glucose or with glucose (22.2 mM) for 36 h and further incubated in the absence or presence of 100 nM insulin for 3 h. HepG2 glucose transporter mRNA was determined by dot blot analysis. A: HepG2 glucose transporter mRNA level. B: Ratio of HepG2 glucose transporter mRNA to p-actin mRNA. Each point represents the mean+SEM of five strains. * p < 0.01 vs. cells incubated with 22.2 mM glucose. * * p < 0.01, * p < 0.05 vs. cells incubated with 22.2 mM glucose without insulin.
193
123456
180 5 z i
140
;
100
.
0
4
8
Incubation
16 Time
(h)
Fig. 3. Time course of changes in HepG2 glucose transporter protein in cultured human fibroblasts stimulated with insulin. Cells were preincubated with serum-free (rMEM (11.1 mM glucose) for 36 h and then 100 nM insulin was added. After further incubation for the indicated hours, HepG2 glucose transporter protein in Triton X-100 soluble fraction was determined by Western blot analysis and quantified using a scanning densitometer. Each point is expressed as a percentage of control cells incubated for the indicated hours without insulin.
lation of the mRNA, however, in the glucose-deprived cells.
was not significant
Effects of glucose and insulin treatment on cellular glucose transporter protein levels We determined total cellular HepG2 glucose transporter levels in fibroblasts by the Western blotting using antibody against the carboxylterminal portion of HepG2 glucose transporter. HepG2 glucose transporter was detected as the 55 kDa band as previously described (Haspel et al., 1985; Homer et al., 1987). Insulin (100 nM) increased HepG2 glucose transporter in a time-dependent manner (Fig. 3). The levels of HepG2 glucose transporter reached the plateau at 4 h after the addition of insulin. Next we studied effects of glucose on basal and insulin-stimulated HepG2 glucose transporter protein levels. The cells were preincubated with varying concentrations of glucose for 36 h. Insulin (100 nM) or buffer alone was then added and the cells were further incubated for 6 h. There were no differences in the basal HepG2 glucose transporter level (without insulin) among the cells treated with 11.1, 22.2 and 44.4 mM glucose (Fig. 4, Table 1). Insulin increased the glucose transporter levels to similar extents in the cells treated with 11.1, 22.2 and 44.4 mM glucose (Fig. 4, Table 1). Again, glucose-deprived cells exhibited a higher level of glucose transporter than the cells incubated with glucose
Insulin Glucose
,-
+,
,-
11 .l
+,
,-
22.2
+ 44.4
(mM)
Fig. 4. Effects of glucose and insulin on HepG2 glucose transporter protein in cultured human fibroblasts. Cells were preincubated with the indicated glucose concentrations for 36 h and further incubated in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of 100 nM insulin for 6 h. HepG2 glucose transporter in T&on X-100 soluble fraction of the cells was determined by Western blot analysis.
(Fig. 5, Table 1). The effect of insulin was not evident in the glucose-deprived cells. In a different series of experiments we examined if adipocyte/ muscle type glucose transporter is expressed in the cultured fibroblasts by the Western blotting using antibody against the carboxyl-terminal portion of the glucose transporter. Adipocyte/ muscle type
TABLE 1 EFFECTS OF GLUCOSE AND INSULIN ON HepG2 GLUCOSE TRANSPORTER CONTENT IN CULTURED HUMAN FIBROBLASTS The data are expressed as percent of glucose transporter con tent in the cells incubated with 11.1 mM glucose without insulin. Values are the means* SEM of three or five experiments performed independently. Glucose
Insulin (nM)
(mM)
0
100
336.9 k 53.2 a 100 89.5k 9.7 101.6+11.8
335.0 + 50.1 125.3 k 6.1 138.4k 7.6 141.3k11.8
0
11.1 22.2 44.4
a b ’ b
a Difference from cells incubated with 11.1 mM glucose without insulin at p < 0.05. b Difference from cells incubated without insulin at p < 0.05.
194
12345678
123456
Insulin Glucose
Insulin
-
+,
,-
+,
,-
+
Glucose
-
-
+
Xylose
+
-
-
Fig. 5. Effects of glucose deprivation on HepG2 glucose transporter protein in cultured human fibroblasts. Cells were preincubated without glucose (lanes 7, 2, 3, and 4) or with glucose (22.2 mM: lanes 5 and 6) for 36 h and further incubated in the absence (lanes 1. 3, and 5) or presence (lanes 2, 4. and 6) of 100 nM insulin for 6 h. HepG2 glucose transporter in Triton X-100 soluble fraction of the cells was then determined by Western blot analysis. Xylose (22.2 mM) was added to adjust the osmotic pressure in lanes 1 and 2.
,-
+, ,0
9
+, I-
11.1
+, (-
22.2
+,
Adipo.
44.4 (mM)
Fig. 6. Absence of adipocyte/muscle type glucose transporter protein in cultured human fibroblasts. Cells were preincubated with the indicated glucose concentrations for 36 h and further incubated in the absence (lanes 1, 3, 5, and 7) or presence (lanes 2, 4, 6, and 8) of 100 nM insulin for 6 h. Adipocyte/ muscle type glucose transporter was determined in the Triton X-100 soluble fraction by Western blot analysis. No band was detected under these experimental conditions. Adipocyte/ muscle type glucose transporter in total crude membrane fraction of rat adipocytes (lanes 9) was shown as positive control. This is a representative result from experiments performed 3 times.
glucose transporter, however, could not be detected in the fibroblasts under any experimental conditions examined (Fig. 6). Effects of glucose and insulin treatment on 2-deoxyglucose uptake Effects of glucose and insulin on 2-deoxyglucase uptake by cultured fibroblasts were studied to see if the changes found in the glucose transporter expression are reflected in the cellular glucose transport activity. After the preincubation with glucose (O-44.4 mM) for 36 h, basal and maximal insulin (1 PM, 30 min)-stimulated 2-deoxyglucose uptake were determined (Fig. 7, columns B and I, respectively). In parallel experiments, to see the long-term effect of insulin, insulin (100 nM) was added to the preincubation medium for the last 6 h and then maximal insulin (1 PM, 30 min)-stimulated 2-deoxyglucose uptake was determined (Fig. 7, columns II). In the cells preincubated without glucose, the basal uptake was increased compared with those pretreated with
11 1
Glucose
Concentration
22 2
44 4
(mMI
Fig. 7. Effects of glucose and insulin on 2-deoxyglucose uptake in cultured human fibroblasts. Cells were preincubated with the indicated glucose concentrations for 36 h and then the basal (B) and acute (1 pM, 30 min) effects of insulin (I) on 2-deoxyglucose uptake were determined. To see long-term effects of insulin (II), insulin (100 nM) was added to the preincubation medium for the last 6 h and then insulin (1 ,uM, 30 min)-stimulated 2-deoxyglucose uptake was determined. These data represents the mean+SEM of six experiments using four strains. ‘p < 0.01 vs. B of the same group. * * p < 0.01 vs. I and B of the same group. t p < 0.01 vs. cells incubated with glucose (11.1, 22.2 and 44.4 mM).
195
glucose. However, acute and long-term effects of insulin were not evident. On the other hand, there were no differences in the basal uptake among the cells preincubated with 11.1, 22.2 and 44.4 mM glucose. Insulin stimulated 2-deoxyglucose uptake to the same degrees (Fig. 7, columns I). Moreover, long-term treatment with insulin enhanced insulin-stimulated 2-deoxyglucose uptake to the same degree irrespective of the glucose concentrations in the preincubation medium (Fig. 7, columns II). Thus the changes in cellular glucose transport activity reflected those of HepG2 glucose transporter mRNA and protein levels in cultured human fibroblasts. Discussion In the present study we examined the effects of insulin and glucose on the glucose transport system in cultured human skin fibroblasts. Although it has been well known that insulin stimulates glucose uptake in cultured fibroblasts (Howard et al., 1979), we did not detect the adipocyte/muscle type glucose transporter in fibroblasts by the Western blotting using antibody against the carboxyl-terminal portion of the adipocyte/ muscle type glucose transporter. Thus, it seems likely that the HepG2 glucose transporter is the major species involved in insulin-stimulated glucose uptake in cultured fibroblasts. Of course, at present, we cannot rule out a possibility of contribution of other species of glucose transporter thus far not identified (Kayano et al., 1988; Mesmer et al., 1990). We found that long-term treatment of the cells with insulin resulted in parallel increases in the cellular levels of HepG2 glucose transporter mRNA and protein accompanied by increases in insulin-stimulated glucose uptake. There was a good correlation between the increase in the mRNA levels and that in the glucose transport activity. These findings are in agreement with those previously reported in 3T3-Ll adipocytes (Tordjman et al., 1989) and in rat L6 skeletal muscle cell lines (Walker et al., 1989). In contrast, an elevation of glucose concentrations in the medium from 11.1 to 44.4 mM was without effect on the HepG2 glucose transporter mRNA and protein levels. Both basal and
insulin-induced (acute) 2-deoxyglucose uptake were not altered. Furthermore, although incubation with the highest concentration of glucose (44.4 mM) slightly diminished the extent of insulin-induced increase of the glucose transporter mRNA levels, glucose (11.1-22.2 mM) did not affect insulin’s long-term effects on the glucose transporter mRNA and protein levels and the glucose transport activity. Thus from the present study it seems unlikely that high concentrations of glucose modulate the insulin effects to increase the HepG2 glucose transporter synthesis and glucose transport activity in cultured human fibroblasts. It should be noted, however, that the present findings may not be extrapolated to other tissues and other species of glucose transporters. Garvey et al. (1987) for example, have recently shown that glucose and insulin pretreatment can synergistically impair insulin-induced translocation of glucose transporters to the cell surface in primary cultured rat adipocytes, thus resulting in a decrease in insulin responsiveness. They found, however, no changes in the total numbers of D-glucase-inhibitable cytochalasin B binding sites among the cells preincubated for 24 h as control cells and in the presence of 100 ng/ml insulin with and without 15 mM D-&COSe. Although there were no major differences in the glucose transporter expression and glucose transport activity among the cells treated with glucose (11 .l-44.4 mM), glucose deprivation, from the medium for 36 h resulted in marked increases in these parameters. The acute and long-term effects of insulin were not observed in the glucosedeprived cells. Several previous studies have also reported that glucose deprivation induces an increase in hexose transport. For example, Haspel et al. (1986) have shown that 3T3-C2 murine fibroblasts deprived of glucose exhibit increases in hexose transport and glucose transporter. Walker et al. (1988) have shown similar results in rat brain glial cells. We confirmed these findings in cultured human skin fibroblasts and further clarified that enhanced HepG2 glucose transporter expression mainly contributes to the increase in cellular glucose transporter levels. Similar conclusions were reached in rat L6 skeletal muscle cell lines (Walker et al., 1989). The reasons for the lack of both acute and chronic effects of insulin in the glucose-de-
196
prived cells remain to be elucidated. Recently it has been shown that HepG2 glucose transporter can translocate from an intracellular membrane compartment to the cell surface in response to insulin in 3T3-Ll adipocytes (Gould et al., 1989) and Chinese hamster ovary cells (Asano et al., 1989). If this is the case in cultured fibroblasts, insulin-induced translocation of glucose transporters might be hampered by some unknown mechanisms, for example energy depletion, in the cells deprived of glucose. Alternatively newly synthesized glucose transporters may be distributed mainly to the plasma membrane in glucosedeprived cells (Walker et al., 1990). Determinations of glucose transporter in intracellular and plasma membrane compartments will be necessary in future. Regarding the lack of chronic effects of insulin on the glucose transport system, glucose deprivation might maximally stimulate the rate of glucose transporter synthesis. Acknowledgments We are grateful to Dr. G.I. Bell (Howard Hughes Medical Institute, University of Chicago) for providing the HepG2 glucose transporter cDNA and to Dr. T. Mitani (Sanwa Kagaku Co. Ltd.) for synthetic C-terminal peptide of glucose transporter. We thank Kayoko Furukawa for secretarial assistance. References Asano, T., Shibasaki, Y., Ohno, S., Taira, H., Lin, J.L., Kasuga, M., Kanazawa, Y., Akanuma, Y., Takaku, F. and Oka, Y. (1989) J. Biol. Chem. 264, 3416-3420. Bimbaum, M.J. (1989) Cell 57, 305-315. Bradford, M.M. (1976) Anal. B&hem. 72, 248-254. Cushman, S.W. and Wardzala, L.J. (1980) J. Biol. Chem. 255, 4758-4762. Fukumoto, H., Kayano, T., Buse, J.B., Edwards, Y., Pilch, P.F., Bell, G.I. and Seino, S. (1989) J. Biol. Chem. 264, 7776-7779. Garvey, W.T., Olefsky, J.M., Matthaei, S. and Marshall, S. (1987) J. Biol. Chem. 262, 189-197. Gentry, L.E., Rohrschneider, L.R., Casnellie, J.E. and Krebs, E.G. (1983) J. Biol. Chem. 258, 11219-11228.
Glisin, V., Crkvenjakuv, R. and Byns, C. (1974) Biochemistry 13, 2633-2637. Gould, G.W., Derechin, V., James, D.E., Tordjman, K., Ahem, S., Gibbs, E.M., Lienhard, G.E. and Mueckler, M. (1989) J. Biol. Chem. 264, 2180-2184. Haspel, H.C., Bimbaum, M.J., Wilk, E.W. and Rosen, O.M. (1985) J. Biol. Chem. 260, 7219-7225. Haspel, H.C., Wilk, E.W., Bimbaum, M.J., Cushman, S.W. and Rosen, O.M. (1986) J. Biol. Chem. 261, 6778-6789. Homer, H.C., Munck, A. and Lienhard, G.E. (1987) J. Biol. Chem. 262, 17696-17702. Howard, B.V., Mott, D.M., Fields, R.M. and Bennett, P.H. (1979) J. Cell. Physiol. 101, 129-138. James, D.E., Brown, R., Navarro, J. and Pilch, P.F. (1988) Nature 333, 183-185. James, D.E., Strube, M. and Mueckler, M. (1989) Nature 338, 83-87. Kaestner, K.H., Christy, R.J., McLenithan, J.C., Braiterman, L.T., Cornelius, P., Pekala, P.H. and Lane, M.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3150-3154. Kahn, B.B. and Cushman, S.W. (1987) J. Biol. Chem. 262, 5118-5124. Kayano, T., Fukumoto, H., Eddy, R.L., Fan, Y.S., Byers, M.G., Shows, T.B. and Bell, G.I. (1988) J. Biol. Chem. 263, 15245-15248. Kono, T., Robinson, F.W., Blevins, T.L. and Ezaki, 0. (1982) J. Biol. Chem. 257, 10942-10947. Kosaki, A., Kuzuya, H., Yoshimasa, Y., Yamada, K., Okamoto, M., Nishimura, H., Kakehi, T., Takeda, J., Seino, Y. and Imura, H. (1988) Diabetes 37, 1583-1586. Laemmli, U.K. (1970) Nature 227, 680-685. Merrifield, R.B. (1963) J. Am. Chem. Sot. 85, 2149-2154. Mesmer, O.T., Gordon, B.A., Pupar, C.A. and Lo, T.C.Y. (1990) B&hem. J. 265, 823-829. Morrison, D.F. (1976) Multivariate Statistical Methods, 2nd edn., McGraw Hills, New York. Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W.J., Lienhard, G.E. and Lodish, H.F. (1985) Science 229, 941-945. Nakajima-Iijima, S., Hamada, H., Reddy, P. and Kakunaga, T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6133-6137. Tordjman, K.M., Leingang, K.A., James, D.E. and Mueckler, M.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7761-7765. Walker, P.S., Donovan, J.A., Van Ness, B.C., Fellows, R.E. and Pessin, J.E. (1988) J. Biol. Chem. 263, 15594-15601. Walker, P.S., Ramlal, T., Donovan, J.A., Doering, T.P., Sandra, A., Klip, A. and Pessin, J.E. (1989) J. Biol. Chem. 264, 6587-6595. Walker, P.S., Ramlal, T., Sarabia, V., Koivisto, U.M., Bilan, P.J., Pessin, J.E. and Khp, A. (1990) J. Biol. Chem. 265, 1516-1523. Walter, G., Scheidtmann, K.H., Carbone, A., Laudano, A.P. and Doolittle, R.F. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5197-5200.
