0013-7227/90/1274-2025$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 4 Printed in U.S.A.

Vanadate Regulates Glucose Transporter (Glut-1) Expression in NIH3T3 Mouse Fibroblasts* KATHLEEN G. MOUNTJOY AND JEFFREY S. FLIER Charles A. Dana Research Institute, Harvard-Thorndike Laboratory, and the Department of Medicine, Beth Israel Hospital, and Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT. Vanadate, the major oxidized form of the essential trace element vanadium, has rapid effects on glucose transport in vitro and more delayed effects on glucose transport in vivo. We addressed the question that one potential mechanism for the delayed effects of vanadate on glucose homeostasis could be altered expression of one or more of the genes encoding glucose transporters. To do this we studied vanadate regulation of Glut-1 and Glut-4 in NIH3T3 mouse fibroblasts. Vanadate (5-40 MM) induced cells to proliferate to higher cell densities, and in addition, 40 nM vanadate caused the cells to exhibit a transformed morphology. Glut-1 mRNA was maximally induced 4- to 5-fold over the control value after 6-h exposure to 30 fiM vanadate. Unlike the response to serum and growth factors, the vanadate-induced increase in Glut-1 mRNA remained elevated over the control value in the presence of vanadate for 5 days. The vanadate effect was serum dependent and was fully revers-

V

ANADIUM is an essential trace element for higher animals (1, 2), but has not been identified with a specific physiological role. Almost a decade ago, however, vanadate, the oxidized form of vanadium, was observed to have insulin-like effects on glucose transport in vitro (3-7). Vanadate (3 mM) stimulates glucose transport within 30 min in isolated rat adipocytes (3). Evidence is consistent with this rapid effect being mediated by insulin receptor activation brought about by phosphotyrosine phosphorylation of insulin receptors via either direct phosphorylation or the inhibition of phosphotyrosine phosphatases by vanadate (7, 8) and subsequently increased translocation of glucose transporter proteins to the plasma membrane (9). In contrast to these rapid effects, more recent studies indicate that oral administration of vanadate (1 mM in the drinking water) normalizes blood glucose levels of streptozotocin-treated diabetic rats, but this effect occurs over a time course of several days (10). Although the Received November 29,1989. Address all correspondence and requests for reprints to: Dr. J. Flier, Diabetes Unit, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215. * This work was supported by a Juvenile Diabetes Foundation grant (to J.S.F.) and NIH Grant K-28082 (to J.S.F.).

ible when vanadate was removed from the medium. In the absence of vanadate, the half-life of Glut-1 mRNA was 0.5-1 h, whereas after treatment for 5 h with 30 nM vanadate the halflife was increased to 1.5-2 h. Thus, mRNA stabilization accounts for at least a part of the increase in glucose transporter mRNA levels after vanadate treatment. Glut-4 mRNA was not detected in these cells in either the absence or presence of vanadate. While the importance of this increased Glut-1 gene expression for the vanadate effect on normalization of blood glucose in vivo remains to be determined, an association between vanadateinduced cell proliferation and transformed phenotype, and vanadate-induced Glut-1 mRNA in vitro has been made. Possible potential therapeutic use of vanadate for treatment of diabetes must, therefore, be viewed with caution. (Endocrinology 127: 2025-2034,1990)

rapid effects on glucose uptake observed in vitro may be contributing to these more delayed effects of vanadate in vivo, it is also possible that the delayed effect of vanadate may have a distinct biochemical explanation. Several lines of evidence led us to believe that one potential mechanism would be altered expression of one or more of the genes encoding glucose transporters. First, vanadate has been shown to be a potent inhibitor of several enzymes that hydrolyze phosphate ester bonds, including ribonuclease (11), acid phosphatase, and alkaline phosphatase (12, 13). These effects of vanadate appear at least in part to result from vanadate binding to proteins, since its structure is analogous to that of phosphate (13). Tamura et al. (8) demonstrated in intact adipocytes that vanadate causes a net increase in the amount of phosphotyrosine incorporation into the /3-subunit of insulin receptors. Although the mechanism of vanadate action is generally attributed to its ability to specifically inhibit phosphotyrosine phosphatase (14, 15), in the case of insulin receptor phosphorylation, the elevated levels of phosphotyrosine may be partially due to a direct effect of vanadate to stimulate tyrosine kinase activity (7). Vanadate has also been shown to enhance phosphorylation of numerous other cellular proteins on tyrosine moieties (16-18), including the protooncogene

2025

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

2026

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA

c-src (19) and the oncogene v-src (20). Elevated levels of tyrosine phosphorylation resulting from tyrosine phosphorylation of those growth factor receptors that autophosphorylate and possess tyrosine kinase activity are associated with cellular growth and transformation (21, 22). It is, thus, not surpising that prolonged treatment with vanadate (1-3 days) in concentrations ranging from 5-50 /iM was found to stimulate DNA synthesis and mitogenesis in quiescent cultures of human fibroblasts (23) and Swiss mouse 3T3 and 3T6 cells (24) and to induce the morphological transformation of NRK-1 cells (16). Glucose transport is now known to be mediated by a family of structurally similar glucose transporters encoded by distinct genes (25-32). The encoded proteins are expressed in a tissue-specific manner and may differ functionally with respect to Km and ability to be activated by insulin. The first of these cDNA clones encoding nearly identical facilitative glucose transporters was isolated from a human hepatoma-derived cell line HepG2 (26) and rat brain (27). The expression of the mRNA encoding this transporter, referred to hereafter as the HepG2/brain glucose transporter or Glut-1, is induced by serum (33, 34); platelet-derived growth factor (35); fibroblast growth factor, insulin-like growth factor-I, and epidermal growth factor (33); insulin (36-38); and a tumor-promoting phorbol ester (33, 34, 39). The increased expression of the HepG2/brain glucose transporter mRNA by these factors is transient, peaking 4-8 h after exposure to the stimulus and returning to basal levels after 24 h. Furthermore, the HepG2/brain glucose transporter mRNA is elevated in cells transformed by certain oncogenes (39, 40), and recently the induction of this mRNA by the src oncoprotein was shown to be dependent upon the increased tyrosine kinase activity of the src oncoprotein (41). Given the known regulation of this gene by factors, including many tyrosine kinases, and the ability of vanadate to activate such receptors, we hypothesized the vanadate might be a potent regulater of this gene. In the present study we observed the effect of vanadate on HepG2/brain glucose transporter (Glut-1) gene expression in cultured NIH3T3 cells and attempted to correlate these changes with glucose transporter protein and glucose uptake.

Materials and Methods Materials Dulbecco's Modified Eagle's Medium (DMEM; no. 430,1600), a-D-glucose, Minimum Essential Medium vitamins solution, minimal essential amino acids, 2-deoxy-D-glucose (2DOG), phloretin 2-/3-D-glucoside, deoxyribonucleic acid type III, and Hoechst 33258 were purchased from Sigma Chemical

E n d o • 1990 Vol 127 • No 4

Co. (La Jolla, CA). Sodium orthovanadate was purchased from Fisher Scientific Co. (Springfield, NJ), calf serum from Whittaker Byproducts (Walkersville, MD), BSA (fatty acid-free fraction V) from ICN Immunologicals (Cleveland, OH), guanidine thiocyanate from Fluka Biochemika (Buchs, Switzerland), cesium chloride from Gallard Schlessenger Industries Inc. (New York, NY), and BCA protein assay kit from Pierce (Rockford, IL). 2-[G-3H]DOG, [«-32P]UTP, [a32P]dCTP, and [125I]protein-A were purchased from New England Nuclear (Boston, MA). A random primed DNA-labeling kit was purchased from Molecular Biology Boehringer Mannheim (Indianapolis, IN), and T7 RNA polymerase from Stratagene (La Jolla, CA). Cells

NIH3T3 mouse fibroblasts were obtained from Dr. T. Roberts, Dana Faber Institute (Boston, MA). NIH3T3 cells were grown in DMEM supplemented with 10% calf serum, aDglucose (4.5 g/liter, final concentration), penicillin (100 U/ml), and streptomycin (100 Mg/ml). The cells were passaged (split 1:100) weekly with 0.1% trypsin for 4-6 weeks, after which time early passage frozen stock were thawed. Cells were free of contamination by mycoplasma when tested using a mycoplasma T.C. detection kit from Gen-Probe (San Diego, CA). To prepare cultures for RNA extraction and cellular membrane fractionation, NIH3T3 cells were seeded (split 1:50) in 100 x 20-mm plastic culture dishes in 10 ml growth medium and fed on days 3 and 6. To prepare cultures for cell counts or 2-deoxy-D-glucose uptake, cells were seeded (split 1:50) in 35mm diameter, six-well plastic culture dishes in 2 ml growth medium and fed on days 3 and 6. On day 7 when NIH3T3 cells were confluent, medium was aspirated, and monolayers were exposed to vanadate (or not) in culture medium containing 10% calf serum. A fresh stock of vanadate (100 nM DMEM) was made for each experiment. Glucose levels, when monitored, were found to be not significantly decreased after 24-h exposure to vanadate. To study the effect of serum on the vanadate-induced alteration in gene activity, cells were grown to confluence and washed two times with 10 ml culture medium containing the indicated concentrations of serum or BSA over a period of 3 h; then the culture medium was aspirated, and the cells stimulated with medium with or without serum and with or without vanadate for 6 h. Cell growth was monitored by counting cells from triplicate 35-mm diameter wells. Cells were removed from culture plates using 0.1% trypsin (37 C; 5 min), dispersed in saline, and counted in a model ZF Coulter counter (Hialeah, FL), using a 100-jum aperture. Isolation and analysis of cellular RNA Confluent monolayers were washed twice with ice-cold PBS and then solubilized in 4 M guanidine thiocyanate. The RNA was then extracted by the method of Chirgwin et al, (42). The RNA (10-20 /xg) was size separated by electrophoresis on denaturing 1.2% agarose formaldehyde gels (43), then transferred to nylon filters, as described by Thomas (44). In some instances

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA RNA was further quantitated after applying 5 ng RNA to nitrocellulose filters using a Hybri-slot blot manifold (BRL, Bethesda, MD). The filters were hybridized at 65 C for 20 h with a 32P-labeled antisense RNA probe (2250 basepairs) generated by T7 RNA polymerase from a nearly full-length HepG2 glucose transporter cDNA (45). Northern blots were also probed with a random primed labeled 0-actin cDNA. Furthermore, some Northern blots and slot blots were also hybridized with a rat muscle/adipose glucose transporter (Glut-4) cDNA probe which is identical in sequence to the published rat muscle glucose transporter cDNA (30-32). Total cellular RNA (5 ^g) derived from rat heart was used as a positive control when probing for muscle glucose transporter, since this tissue expresses this insulin-sensitive glucose transporter in abundance (30). After hybridization and washing under stringent conditions appropriate to either cDNA or antisense RNA probes, filters were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -70 C and analyzed by scanning densitometry.

2027

2-DOG Confluent monolayers were washed three times at room temperature with PBS and then incubated with 0.98 ml 0.1% fatty acid-free BSA and glucose-free Minimum Essential Medium (MEM) at 37 C for 10 min. After this, 0.02 ml 2-DOG (2 X 105 cpm [3H]2-DOG (SA, 5 mCi/mmol) and 100 nM 2-DOG in glucose-free MEM were added, and the cells were incubated for a further 5 min at 37 C with mild shaking. Glucose uptake was terminated by the addition of 1 ml ice-cold PBS containing 0.3 mM phloretin. The plates were then thawed three times with cold PBS, the cells solubilized in 1 N NaOH (37 C; 1 h), and an aliquot from each plate was neutralized with concentrated HC1 and counted in 4.5 ml Liquiscint (National Diagnostics, Somerville, NJ) in a Searle (8-counter (Des Plaines, IL). A further aliquot from each plate was neutralized with 1 N HC1 and analyzed for DNA according to the method of Labarca and Paigen (47). Measurement of glucose transporter mRNA stability

Western blot analysis Confluent monolayers were washed twice with ice-cold PBS and then harvested in cold TESPI (20 mM Tris-HCl pH 7.6; 1 mM EDTA; 0.25 M sucrose; 5 mg/liter pepstatin; 5 mg/liter aprotonin; and 5 mg/liter leupeptin) by scraping with a rubber policeman. Cells were pelleted by centrifugation at 800 x g for 5 min at 4 C. The cell pellet was resuspended in 1 ml TESPI, and the cells ruptured with three 5-sec bursts using a Sonifier cell disrupter at a setting of 4. To prepare crude membranes, unbroken cells and nuclei were pelleted by spinning the cell homogenate at 800 X g for 3 min at 4 C. Crude membranes were prepared by spinning the resulting supernatant at 75,000 rpm for 15 min in a TL100.1 rotor in a Beckman TL100 benchtop centrifuge (Palo Alto, CA). The crude membrane pellets were resuspended in 100-200 /A TESPI and sonicated once for a 5-sec burst at a setting of 4. The protein concentrations were measured using a BCA protein assay kit. Cellular fractions (50 ng crude membrane) were electrophoresed on sodium dodecyl sulfate-10% polyacrylamide gels, and then the proteins were transferred to nitrocellulose, as described by Gershoni and Palade (46), for 16 h at 250 mmp at 4 C. The nitrocellulose filters were washed with distilled water then stained with Ponceau S (Sigma Chemical Co.) to check for even loading and transfer of proteins. The filters were washed for 20 min in TBS, 0.1% Tween 20, and then blocked with 5% nonfat milk, in 0.2% NP40 in TBS for 30 min at 37 C. After this the filters were exposed to an antibody (a gift from Dr. B. Thorens, Whitehead Institute, Cambridge, MA) raised against the 16 C-terminal amino acids of the HepG2 glucose transporter for 2 h at room temperature. The filters were then washed for 20 min each with 0.5% nonfat milk, 0.2% NP40 in TBS, and 0.2% NP40, TBS, and 0.1% Tween-20 in TBS and then blocked for 30 min at 37 C with 0.5% nonfat milk 0.2% NP40 in TBS. The three 20-min washes were repeated after a 1-h probing of the filters at 37 C with [125I] protein-A (0.09 /iCi/ml) in 0.5% nonfat milk-0.2% NP40 in TBS. The dried filters were exposed to Kodak XAR-5 film at —70 C and analyzed by scanning densitometry.

Transcription of glucose transporter mRNA was blocked by the addition of 5 /xg/ml actinomycin-D to confluent monolayers after 5-h stimulation with 30 nM vanadate. Cells (2 X 100-mm plates) were harvested for RNA at 0.15 and 30 min, and 1, 1.5, 2,3,4, and 6 h later. The RNA from these samples was analyzed by Northern blot and quantitated by slot blot hybridization. Nuclear runoff transcription assay Confluent monolayers (15-20 100-mm plates) were washed with 5 ml ice-cold PBS and harvested by scraping into cold PBS and centrifugation at 800 X g for 5 min. Cells were lysed by gently vortexing each pellet in 4 ml lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.5% NP-40, pH 7.4) and incubating on ice for 5 min. The nuclei were prepared for runoff transcription assay following the method of Hiraki et al. (33). For the nuclear runoff transcription assay, 195 ixl labeling mix [10/*l 1 M Tris-HCl, pH 8.0; 5 M 1 1 M MgCl2; 300 n\ 1 M KC1; 10 Ml 0.1 M ATP; 10 n\ 0.1 M CTP; 10 Ml 0.1 M GTP; 5 Ml 1 mM UTP; 5 MI 1 M dithiothreitol; 545 n\ H2O; and 100 n\ [32P] UTP (10 /xCi/fA)] were added to the frozen nuclei, and then the tubes were incubated for 30 min at 30 C. After this the nuclei were pelleted at 300 x g for 3 min and solubilized in 4 M guanidine thiocyanate, and the RNA transcripts were extracted by the method of Chirgwin et al. (42). Equal amounts of 32Plabeled RNA were hybridized in 1 ml hybridization buffer for 36 h at 65 C to nitrocellulose strips to which 10 ng of each of the following denatured plasmids or genomic DNA were bound: HepG2 glucose transporter plasmid, rat brain glucose transporter plasmid, pBR322 plasmid, mouse /3-actin plasmid, mouse c-fos plasmid, and 200 ng mouse genomic DNA.

Results Vanadate increases NIH3T3 cell density and alters cell morphology Vanadate (5-30 ^M) induced NIH3T3 cells to proliferate to higher cell densities than control cells (Figs. 1

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA

2028

Endo • 1990 Vol 127 • No 4

become refractile and exhibit a transformed morphology (Figs. 1 and 2). Vanadate induces HepG2/brain glucose transporter mRNA in NIH3T3 cells

FIG. 1. Increase in cell density and alteration in cell morphology by vanadate in NIH3T3 cells. Confluent NIH3T3 cells were fed DMEM with 10% calf serum (control; A), DMEM with 10% calf serum plus 30 ^M vanadate (B), or DMEM with 10% calf serum plus 40 nM vanadate (C) each day for 5 days. Cell density is increased after 5-day exposure to 30 MM vanadate compared with control values, and cell morphology is changed after 5-day exposure to 40 nM vanadate.

A dose-dependent induction of HepG2/brain glucose transporter mRNA by vanadate (5-40 juM) was observed after both 24 h and 5 days of exposure of cells to this ion (Fig. 3). Glucose transporter mRNA was maximally induced 4- to 5-fold by 20-40 ;uM vanadate, while 5 /iM vanadate had almost no effect. Actin mRNA was not consistently affected by the presence of vanadate in the culture medium, although in some experiments it appeared to decline modestly in a dose-independent manner. The time course for effects of 30 /LtM vanadate on glucose transporter mRNA was assessed, comparing the effects of control medium with 10% calf serum to those of medium with 10% calf serum and 30 /xM vanadate. The small transient increase in glucose transporter mRNA observed in the control cells fed culture medium containing only 10% calf serum (increased 30% at 6 h relative to zero time) reflects previously reported responses of this mRNA to serum (33, 34). The relatively small increase we detected in response to serum in this study, however, is a reflection that the cells were not serum deprived before stimulation. Expression of the HepG2/brain glucose transporter mRNA was induced 2GT Day 1

NO

'©

2-

B

1-

0

5 10 20 30 40

0

5 10 20 30 40

GT Day 5 ACTIN Day 5

o

10

20

30

40

Vanadate (uM)

FlG. 2. Increase in cell proliferation by vanadate in NIH3T3 cells. Confluent NIH3T3 cells were fed DMEM with 10% calf serum and the indicated concentrations of vanadate each day for 5 days. Cells were removed from culture plates using 0.1% tryspin (37 C; 5 min), dispersed in saline, and counted in a model ZF Coulter counter using a 100-//m aperture. *, P < 0.05; **, P < 0.001.

and 2). Vanadate at 40 JUM induced cells to proliferate to a higher cell density and, in addition, caused the cells to

VANADATE CONCENTRATION QiU)

FIG. 3. Induction of glucose transporter (GT) mRNA by increasing doses of vanadate in NIH3T3 cells. RNA (10 fig) samples prepared from confluent NIH3T3 cells that had been exposed to increasing doses of vanadate in the presence of 10% calf serum for 24 h (A) or 5 days (B) were analyzed by Northern blot analysis and probed with HepG2/ brain glucose transporter and /3-actin cDNAs. The data shown in A are from a single Northern blot (exposed for autoradiography for 24 h) and are representative of four independent experiments. The data shown in B are from a single Northern blot exposed for autoradiography for 4 h (glucose transporter) and 11 h (/3-actin).

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA to 5-fold over control values after 4-6 h of exposure of cells to 30 /LtM vanadate (Fig. 4). In the continuous presence of vanadate for 5 days absolute levels of this mRNA decreased between 6 and 24 h, but they remained 4-fold elevated over control values (Fig. 4B). The vanadate effect was fully reversible when vanadate was removed from the medium; glucose transporter mRNA levels returned to control levels within 2 days. The fiactin mRNA levels during this period of vanadate treatment were not different from control levels. Visualization of ethidium bromide staining of the ribosomal bands on each Northern blot was used to verify 1) even loading of RNA on the gel and 2) intactness of RNA. Levels of the mRNA encoding the insulin-sensitive glucose transporter cloned from rat muscle (Glut-4) were also assessed. This mRNA was not detectable in control or vanadate-treated cells. Increase in glucose transporter mRNA expression by vanadate involves mRNA stabilization To investigate the mechanism by which vanadate increases glucose transporter mRNA expression, the halflife of glucose transporter mRNA was determined in NIH3T3 cells. In the absence of vanadate, the half-life

2029

of glucose transporter mRNA was 0.5-1 h, whereas after treatment for 5 h with 30 /iM vanadate the half-life was increased to 1.5-2 h (Fig. 5). Thus, mRNA stabilization accounts for at least a part of the increase in glucose transporter mRNA levels after vanadate treatment. As assessed by nuclear runoff experiments, we could detect no increase in the rate of transcription of the HepG2/ brain glucose transporter gene after vanadate exposure for 15 min to 2 h (three experiments, data not shown). Since vanadate increases glucose transporter mRNA approximately 5-fold, and mRNA stability seems to account for more than half of this effect a 2-fold change in transcription rate might exceed the precision of this approach. We were able to confirm the findings of others, however, that serum increased transcription of rat brain glucose transporter, c-fos, and 0-actin genes after a 15min stimulation of cells (33, 48), indicating that our nuclear runoff methodology was capable of detecting changes in gene transcription in these cells. Serum is required for vanadate to increase glucose transporter mRNA Exposure of cells to several growth factors that are present in serum and bind to and activate specific tyro120 1

VANADATE CONTROL

m ft 0

• control m

1

•

• 2

• 4

6

m

• 8

vanadate

24

HOURS '•j

j -VANADATE VANADATE

CONTROL 6h

1

2 3 4 5 6 7 8

DAYS FIG. 4. Time course and reversibility of the vanadate induction of glucose transporter mRNA in NIH3T3 cells. Total cellular RNA was prepared from confluent cells that had been exposed to DMEM containing 10% calf serum (control) or DMEM containing 10% calf serum plus 30 fiM vanadate (vanadate) for 0-24 h (A) and 6 h to 5 days (B). Culture medium was changed daily, and on days 5, 6, and 7, the vanadate-treated cells were fed DMEM containing 10% calf serum without vanadate (B). Ten-microgram total RNA samples were analyzed by Northern blotting and probed with the HepG2/brain glucose transporter cDNA. The data in A are from a single Northern blot exposed for autoradiography for 17 h, and the data in B are from a single Northern blot exposed for autoradiography for 24 h. The vanadate effect was maximal by 6 h, persisted for the duration of exposure to the ion, and reversed when vanadate was removed.

Hours

FIG. 5. Analysis of glucose transporter mRNA stability in NIH3T3 cells with and without exposure to vanadate. Confluent NIH3T3 cells were exposed (or not) to 30 nM vanadate in DMEM containing 10% calf serum for 5 h. After this, actinomycin-D (5 fig/ml, final concentration) was added, and at the times indicated total cellular RNA was analyzed by Northern blot analysis and probed with the HepG2/brain glucose transporter to check the quality of the RNA. Total RNA (5 n%) was then analyzed by slot blot analysis, and the signals on the autoradiogram were quantitated by densitometry. The levels present at the time of actinomycin-D addition were arbitrarily made 100%. Subsequent levels of glucose transporter mRNA are shown as a percentage of this maximal level for both the untreated cells and the vanadatetreated cells. The data shown are representative of four independent experiments.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

2030

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA

sine kinase receptors has previously been shown to increase glucose transporter mRNA abundance (33-38). Vanadate inhibits phosphotyrosine phosphatases and, thus, increases the tyrosine phosphorylation state of many, if not all, of these receptors in vitro (8, 14). Thus, it is possible that the vanadate-enhanced tyrosine phosphorylation of one or more of these growth factor receptors is on the intracellular signalling pathway for vanadate-induced gene expression. We, therefore, wished to determine the dependence on serum of vanadates effect to increase glucose transporter mRNA. When NIH3T3 cells were washed free of serum for 3 h and then stimulated for 6 h with serum and vanadate, vanadate (30 JUM) had no effect on glucose transporter mRNA in the presence of 0.5% calf serum, but increased glucose transporter mRNA 3-, 4-, and 4-fold in the presence of 2%, 5%, and 10% calf serum, respectively (Fig. 6). In addition to the maximal response to vanadate increasing, the sensitivity of NIH3T3 cells to the vanadate effect on glucose transporter mRNA increased with increasing concentrations of serum, such that 10 juM vanadate increased glucose transporter mRNA 3-fold in the presence of 5% or 10% calf serum, but had only a minimal (40%) effect in the presence of 2% calf serum. Thus, serum increased the maximal effect and the sensitivity of the effect of vanadate to increase glucose transporter mRNA abundance. Vanadate increases glucose transporter protein NJH3T3 cells

in

The glucose transporter protein appears as a wide band with a mol wt of approximately 50K by Western analysis. This wide band is due to the heterogenous glycosylation of the HepG2 glucose transporter (49). Glucose transporter protein was induced by increasing doses of vanaFIG. 6. Vanadate induction of glucose transporter (GT) mRNA is serum dependent. Confluent NIH3T3 cells were washed twice with 10 ml culture medium containing 0.5-10% fetal calf serum over a period of 3 h. Total RNA was prepared from the cells after they were stimulated for 6 h with medium with or without serum and with and without the indicated vanadate concentrations. Ten-microgram total RNA samples were analyzed by Northern blotting and probed with the HepG2/brain glucose transporter cDNA. The data shown are from a single Northern blot (exposed for autoradiography for 10 h) and are representative of two independent experiments. A photo of the ethidium bromide staining of the ribosomal bands is also shown.

Endo • 1990 Vol 127 • No 4

date, and this 2- to 3-fold induction over control levels was maximal after 24-h exposure of the cells to 30 /xM vanadate (Fig. 7). The increase in glucose transporter protein peaked 4-8 h after exposure of cells to vanadate and, as was observed for vanadate-induced glucose transporter mRNA, remained elevated by approximately 2fold over control levels of glucose transporter protein at 24 h. The increase in glucose transporter protein was less than the increase in glucose transporter mRNA induced by vanadate, possibly reflecting translational and/or posttranslational control of glucose transporter protein abundance. Vanadate increases 2-DOG uptake in NIH3T3 cells Vanadate induced a dose-dependent (5-40 JUM) increase in the rate of 2-DOG uptake over control values after 24 h of exposure of cells to this ion (Fig. 8). The rate of 2-DOG uptake was maximally induced by approximately 40-80% over the control value by 30 fiM vanadate after 6 h and remained elevated in the presence of this dose of vanadate over 5 days (Fig. 9). This increase in 2DOG uptake is considerably less than the 5-fold increase in glucose transporter mRNA, and the 2- to 3-fold increase in glucose transporter protein induced by vanadate. The rate of 2-DOG uptake returned to control levels within 24 h of removal of vanadate from the culture medium, slightly more rapidly than the return of glucose transporter mRNA expression to control levels after vanadate withdrawal.

Discussion Vanadate has many effects on cells in vitro (50-53), including inhibition of phosphotyrosine phosphatases (12, 13), stimulation of DNA synthesis (23, 24), and induction of morphological transformation (16). We and

5%CS

10%CS 2.8kd

GT 0

10 30

0

10 30

0

10

30

Vanadate

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA

2031

160-,

50K 0

5 10 20 30 40

VANADATE CONCENTRATION (/IM) FIG. 7. Induction of glucose transporter protein by increasing doses of vanadate in NIH3T3 cells. Crude membranes were prepared from confluent NIH3T3 cells that had been exposed to increasing doses of vanadate for 24 h. Fifty micrograms of protein from each sample were analyzed by Western blot analysis using an antibody raised to the 16 C-terminal amino acids of the HepG2 glucose transporter. The HepG2 glucose transporter protein appears as a wide band with a mol wt of approximately 50K. The autoradiography was performed for 5 h. A 2to 3-fold induction of glucose transporter protein by 20-40 nM vanadate was quantitated by scanning densitometry.

2.00-

FIG. 9. Time course and reversibility of vanadate effect on 2-DOG uptake in NIH3T3 cells. Confluent monolayers of NIH3T3 cells were exposed to DMEM containing 10% calf serum (control) or DMEM containing 10% CS plus 30 /iM vanadate for 0-24 h (A) and 6 h to 5 days (B). Culture medium was changed daily, and on days 5, 6, and 7 the vanadate-treated cells were fed DMEM containing 10% calf serum without vanadate (B). 2-DOG uptake was measured as described in Materials and Methods and was analyzed as nanomoles of 2-DOG uptake per /ug DNA/5 min. Data at each time point are the means of triplicates and are expressed as a percentage of the control value. The arrow indicates the removal of vanadate and the reversal of the vanadate-induced increase in 2-DOG uptake.

1.50 "

1.00

Vanadate (uM) FIG. 8. Induction of 2-DOG uptake by increasing doses of vanadate in NIH3T3 cells. Confluent monolayers of NIH3T3 cells were exposed to DMEM containing 10% calf serum and increasing doses of vanadate for 24 h. 2-DOG uptake was measured as described in Materials and Methods and was analyzed as nanomoles of 2-DOG uptake per fig DNA/5 min. Each experiment was performed in triplicate, and the data expressed as the fold increase above basal values (no vanadate). The data shown are mean ± SEM of four independent experiments.

others have previously shown that certain cell membrane-associated tyrosine kinases are capable of regulating the expression of the HepG2/brain glucose transporter gene in cultured fibroblasts (33-38), and furthermore, that certain oncogene-transformed cells express elevated levels of this gene (39, 40). In this study we, therefore, investigated whether vanadate is capable of signalling HepG2 glucose transporter gene expression in NIH3T3 mouse fibroblasts. Klarlund (16) demonstrated that vanadate induced transformation of three different cell lines. In this study we have also demonstrated that vanadate (5-40

induces NIH3T3 cells to proliferate to higher cell densities than control cells, and 40 fxM vanadate induces the appearance of a transformed phenotype. Furthermore, we have demonstrated that vanadate can increase the expression of the HepG2/brain glucose transporter gene, a gene whose expression is linked to cellular growth and proliferation and the transformed phenotype (39, 40). This is the first demonstration of vanadate influencing the expression of a specific gene, an effect that appears to be at least in part due to an increase in glucose transporter mRNA stability. It is clear from this study that serum is required for vanadate to increase glucose transporter mRNA in NIH3T3 cells. This would be consistent with the proposed mechanism for vanadate regulating glucose transporter gene expression via enhancement of a serum growth factor-induced tyrosine phosphorylation state of one or more growth factor receptors. Alternatively, although less likely, vanadate could regulate gene expression via its known effect on stabilization of steroid re-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

2032

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA

ceptors. Vanadate, like molybdate, can stabilize steroid receptors and, thus, prevent them from being activated in the cell nucleus (54). Recently, dexamethasone was shown to decrease the expression of the HepG2/brain glucose transporter gene in cultured rat adipocytes (55). Thus, it is possible that vanadate increases glucose transporter gene expression by preventing a glucocorticoid inhibitory effect on this gene. The biochemical concomitants of the vanadate-induced increase in glucose transporter mRNA expression in fibroblasts was examined in NIH3T3 cells by analyzing glucose transporter protein abundance and basal 2DOG uptake after vanadate treatment. Both of these parameters were increased after vanadate exposure. However, the magnitude of changes in these two parameters after vanadate treatment was less than that for glucose transporter mRNA. Furthermore, the rate of 2DOG uptake returned to control levels within 24 h of removal of vanadate from the culture medium, and this appeared to be slightly more rapid than the return of glucose transporter mRNA expression to control levels after vanadate withdrawal. There are a number of possible explanations for this lack of quantitative concordance among glucose transporter mRNA, protein and transport, which we have previously noted (34, 41). First, in addition to vanadate inducing glucose transporter mRNA levels in NIH3T3 cells, this ion may also regulate translational or posttranslational mechanisms and thus prevent proportional expression of glucose transporter protein in these cells. Second, it has recently been established that there is a large family of glucose transporter genes, including the HepG2/brain, liver (28, 29), fetal muscle (25), muscle (30-32), and possibly others. Although the HepG2/brain glucose transporter is the only one that is currently known to be expressed in fibroblasts, it is possible that much of the basal 2-DOG uptake in these cells is mediated by an as yet undiscovered transporter species. Alternatively, hexose transport in human and/or Swiss 3T3 mouse fibroblasts can be acutely stimulated by several growth factors (56-58), indicating that at least one glucose transporter protein in these cells can be acutely activated by exogenous stimuli in the absence of changes in protein abundance or mRNA levels. Thus, the rate of 2-DOG uptake would not necessarily be expected to correlate with the level of glucose transporter mRNA or the total amount of glucose transporter protein. We have thus shown a vanadate-induced increase in gene expression of one member of the glucose transporter family which is expressed in a nearly ubiquitous manner. However, Glut-1 is not expressed in abundance in normal liver (45) and may be less abundant than other species of glucose transporter in tissues where glucose transport is acutely regulated by insulin, such as adipocytes (59)

Endo•1990 Vol 127 • No 4

and skeletal muscle (30,60). Since the role of this glucose transporter (Glut-1) in normal, diabetic, or insulin-resistant states is unknown, we can only speculate upon the significance of the vanadate-induced expression of this transporter for diabetes. If Glut-1 is a basal glucose transporter, as has previously been suggested (31), vanadate-induced increased expression of this protein may diminish blood glucose by increasing glucose uptake into many tissues, not just tissues where glucose transport is rapidly regulated by insulin. Vanadate increases glucose uptake into liver (10), a tissue where Glut-1 is absent but a specific liver glucose transporter is present. It is thus possible that in vivo vanadate also increases the expression of the liver glucose transporter protein, and this contributes to the normalization of blood glucose by vanadate in streptozocin diabetic rats. The insulin-sensitive glucose transporter Glut-4 is not expressed in fibroblasts (30-32), and furthermore, we did not detect any induction of this glucose transporter by vanadate in this cell type. Since insulin increases mRNA abundance of Glut-4 (61), and insulin and vanadate increase Glut-4 protein (62) in adipocytes of streptozotocin diabetic rats, it is likely that vanadate might also regulate the level of expression of this gene, which is thought to be important for both normal physiological regulation of glucose homeostasis and the dysregulated transport characteristics of diabetic states. In summary, vanadate induces a sustained increase in glucose transporter mRNA (Glut-1) expression in NIH3T3 cells, and this effect requires the presence of serum. While the relationship between this increased glucose transporter gene expression and the effect of vanadate to normalize blood glucose in vivo remains to be determined, an association between vanadate-induced transformed phenotype and vanadate-induced glucose transporter mRNA in vitro has been made. Possible potential therapeutic use of vanadate for treatment of diabetes must, therefore, be viewed with caution.

Acknowledgments The authors thank Dr. S. Tsuzaki for expert assistance with the preparation of the figures, Dr. B. Thorens for the glucose transporter antibody, Dr. D. Moller for the rat muscle glucose transporter cDNA probe, Dr. M. Birnbaum for the rat brain glucose transporter cDNA probe, Dr. M. Greenberg for the mouse c-fos probe, and Dr. B. Kahn for rat heart muscle RNA.

References 1. Underwood EJ 1977 Trace Elements in Human and Animal Nutrition, ed 4. Academic Press, New York, pp 416-424 2. Golden MHN, Golden BE 1981 Trace elements: potential importance in human nutrition with particular reference to zinc and vanadium. Br Med Bull 37:31 3. Dubyak GR, Kleinzeller A 1980 The insulin mimetic effects of vanadate in isolated rat adipocytes. J Biol Chem 255:5306 4. Schecter Y, Karlish SJD 1980 Insulin-like stimulation of glucose

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA oxidation in rat adipocytes by vanadyl (IV) ions. Nature 284:556 5. Hori C, Oka T 1980 Vanadate enhances the stimulatory action of insulin on DNA synthesis in cultured mouse mammary gland. Biochim Biophys Acta 612:235 6. Degani H, Gochin M, Karlish SJD, Schechter Y 1981 Electron paramagnetic resonance studies and insulin-like effects of vanadium in rat adipocytes. Biochemistry 20:5795 7. Tamura S, Brown TA, Whipple JH, Fujita-Yamaguchi Y, Dubler RE, Cheng K, Lamer J 1984 A novel mechanism for the insulinlike effect of vanadate on glycogen synthase in rat adipocytes. J Biol Chem 259:6650 8. Tamura S, Brown TA, Dubler RE, Lamer J 1983 Insulin-like effect of vanadate on adipocyte glycogen synthase and on phosphorylation of 95,000 dalton subunit of insulin receptor. Biochem Biophys Res Commun 113:80 9. Kono S, Robinson FW, Blevins TL, Ezaki O 1982 Evidence that translocation of the transport activity is the major mechanism of insulin action on glucose transport in fat cells. J Biol Chem 257:10942 10. Meyerovitch J, Farfel Z, Sack J, Schechter Y 1987 Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. J Biol Chem 262:6658 11. Lindquist RN, Lynn Jr JL, Lienhard GE 1973 Possible transitionstate analogs for ribonuclease. The complexes of uridine with oxovanadium (IV) and vanadium (V) ion. J Am Chem Soc 95:8762 12. Lopez V, Stevens T, Lindquist RN 1976 Vanadium ion inhibition of alkaline phosphatase-catalyzed phosphate ester hydrolysis. Arch Biochem Biophys 175:31 13. Van Etten RL, Waymack PP, Rehkop DM 1974 Transition metal ion inhibition of enzyme-catalyzed phosphate ester displacement reactions. J Am Chem Soc 96:6782 (Letter) 14. Swarup G, Cohen S, Garbers DL 1982 Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. Biochem Biophys Res Commun 107:1104 15. Nelson RL, Branton PE 1984 Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts. Mol Cell Biol 4:1003 16. Klarlund JK 1985 Transformation of cells by an inhibitor of phosphatases acting on phosphotyrosine in proteins. Cell 41:707 17. Earp HS, Rubin RA, Austin KS, Dy RC 1983 Vanadate stimulates tyrosine phosphorylation of two proteins in Raji human lymphoblastoid cell membranes. FEBS Lett 161:180 18. Nechay BR, Nanninga LB, Nechay PSE, Post RL, Grantham JJ, Macara IG, Kubena LF, Phillips RD, Nielsen FH 1986 Role of vanadium in biology. Fed Proc 45:123 19. Ryder JW, Gordon JA 1987 In vivo effect of sodium orthovanadate on pp60c-src kinase. Mol Cell Biol 7:1139 20. Brown DJ, Gordon JA 1984 The stimulation of pp60v-src kinase activity by vanadate in intact cells accompanies a new phosphorylation state of the enzyme. J Biol Chem 259:9580 21. Kaplan DR, Whitman M, Schaffhausen B, Pallas DC, White M, Cantley L, Roberts TM 1987 Common elements in growth factor stimulation and oncogenic transformation: 85kd phosphoprotein and phosphatidylinositol kinase activity. Cell 50:1021 22. Di Renzo MF, Ferracini R, Naldini L, Giordano S, Comoglia PM 1986 Immunological detection of proteins phosphorylated at tyrosine in cells stimulated by growth factors or transformed by retroviral-oncogene-coded tyrosine kinases. Eur J Biochem 158:383 23. Carpenter G 1981 Vanadate, epidermal growth factor and the stimulation of DNA synthesis. Biochem Biophys Res Commun 102:1115 24. Bingham Smith J 1983 Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells. Proc Natl Acad Sci USA 80:6162 25. Kayano T, Fukumoto H, Eddy R, Fan YS, Byers MG, Shows TB, Bell GI 1988 Evidence for a family of human glucose transporterlike proteins. J Biol Chem 263:15245 26. Mueckler M, Caruso C, Baldwin SA, Panico M, Blench I, Morris HR, Allard WJ, Lienhard GE, Lodish HF 1985 Sequence and structure of a human glucose transporter. Science 229:941 27. Birnbaum MJ, Haspel HC, Rosen OM 1986 Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein. Proc Natl Acad Sci USA 83:5784

2033

28. Thorens B, Sarkar H, Kaback HR, Lodish HF 1988 Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney and /3 pancreatic islet cells. Cell 55:281 29. Fukumoto H, Seino S, Imura H, Seino Y, Eddy RL, Fukushima Y, Byers MG, Shows TB, Bell GI 1988 Sequence, tissue distribution, and chromosomal localization of mRNA encoding a glucose transporter-like protein. Proc Natl Acad Sci USA 85:5434 30. James DE, Strube M, Mueckler M 1989 Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 338:83 31. Birnbaum MJ 1989 Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell 57:305 32. Charron MJ, Brosius III FC, Alper SL, Lodish HF 1989 A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci USA 86:2535 33. Hiraki Y, Rosen OM, Birnbaum MJ 1988 Growth factors rapidly induce expression of glucose transporter gene. J Biol Chem 263:13655 34. Mountjoy KG, Housey GM, Flier JS 1989 Overproduction of the betai form of protein kinase C enhances phorbol ester induction of glucose transporter mRNA. Mol Endocrinol 3:2018 35. Rollins BJ, Morrison ED, Usher P, Flier JS 1988 Platelet-derived growth factor regulates glucose transporter expression. J Biol Chem 263:16523 36. Kosaki A, Kuzuya H, Yoshimasa Y, Yamada K, Okamoto M, Nishimura H, Kakehi T, Takeda J, Seino Y, Imura H 1988 Regulation of glucose-transporter gene expression by insulin in cultured human fibroblasts. Diabetes 37:1583 37. Garcia de Herreros A, Birnbaum MJ 1989 The regulation by insulin of glucose transporter gene expression in 3T3 adipocytes. J Biol Chem 264:9885 38. Asano T, Shibasaki Y, Ohno S, Taira H, Jiann-Liang L, Kasuga M, Kanazawa Y, Akanuma Y, Takaku F, Oka Y 1989 Rabbit brain glucose transporter responds to insulin when expressed in insulinsensitive Chinese hamster ovary cells. J Biol Chem 264:3416 39. Flier JS, Mueckler MM, Usher P, Lodish HF 1987 Elevated levels of glucose transport and transporter messenger RNA are induced by ras and src oncogenes. Science 235:1492 40. Birnbaum MJ, Haspel HC, Rosen OM 1987 Transformation of rat fibroblasts by FSV rapidly increases glucose transporter gene transcription. Science 235:1495 41. Matsouka PT, Flier JS 1989 Relationship between c-src tyrosine kinase activity and the control of glucose transporter gene expression. Mol Endocrinol 3:1845 42. Chirgwin JM, Przybyla A, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294 43. Lehrach H, Diamond D, Wozney JM, Boedtker H 1977 RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical re-examination. Biochemistry 16:4743 44. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201 45. Flier JS, Mueckler M, McCall AL, Lodish HF 1987 Distribution of glucose transporter messenger RNA transcripts in tissues of rat and man. J Clin Invest 79:657 46. Gershoni JM, Palade GE 1983 Protein blotting: principles and applications. Anal Biochem 131:1 47. Labarca C, Paigen K 1980 A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344 48. Greenberg ME, Ziff EB 1984 Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 311:433 49. Wheeler TJ, Hinkle PC 1985 The glucose transporter of mammalian cells. Annu Rev Physiol 47:503 50. Cantley Jr LC, Josephson L, Warner R, Yanagisawa M, Lechene C, Guidotti G 1977 Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle. J Biol Chem 252:7421 51. Cantley Jr LC, Cantley LG, Josephson L 1978 A characterization of vanadate interactions with the (Na,K)-ATPase. J Biol Chem 240:1437

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.

2034

VANADATE REGULATES GLUCOSE TRANSPORTER mRNA

52. Torossian K, Freedman D, Fantus IG 1988 Vanadate down regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes. J Biol Chem 263:9353 53. Marshall S, Garvey WT, Monzon R 1987 Shunting of insulin from a retroendocytic pathway to a degradative pathway by sodium vanadate. J Biol Chem 262:12005 54. Pratt WB, Jolly DJ, Pratt DV, Hollenberg SM, Giguere V, Cadepond FM, Schweizer-Groyer G, Catelli MG, Evans RM, Baulieu E-E 1988 A region in the steroid binding domain determines formation of the non-DNA-binding, 9S glucocorticoid receptor complex. J Biol Chem 263:267 55. Garvey TW, Huecksteadt TP, Lima FB, Birnbaum MJ 1989 Expression of a glucose transporter gene cloned from brain in cellular models of insulin resistance: dexamethasone decreases transporter mRNA in primary cultured adipocytes. Mol Endocrinol 3:1132 56. Allard WJ, Gibbs EM, Witters LA, Lienhard GE 1987 The glucose transporter in human fibroblasts is phosphorylated in response to phorbol ester but not in response to growth factors. Biochim Biophys acta 929: 288

Endo • 1990 Vol 127 • No 4

57. Kitagawa K, Nishino H, Ogiso Y, Iwashima A 1987 Inhibition by pertusis toxin of fibroblast growth factor-stimulated hexone transport in Swiss 3T3 cells. Biochim Biophys Acta 931:110 58. Kitagawa K, Ogiso Y, Nishino H, Iwashima A 1987 Possible involvement of a Na + /H + antiporter in the stimulation of hexose transport in Swiss 3T3 cells by a phorbol ester and growth factors. Cell Struct Funct 12:115 59. Zorzano A, Wilkinson W, Kotliar N, Thoidis G, Wadzinkski BE, Ruoho AE, Pilch PF 1989 Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J Biol Chem 264:12358 60. Froehner SC, Davies A, Baldwin SA, Lienhard GE 1988 The bloodnerve barrier is rich in glucose transporter. J Neurocytol 17:173 61. Sivitz WI, De Sautel SL, Kayano T, Bell GJ, Pessin JE 1989 Regulation of glucose transporter messenger RNA in insulin-deficient states. Nature 340:72 62. Berger J, Biswas C, Pilch PF, Regulation of expression of the insulin-sensitive glucose transport protein in streptozotocin diabetic rats by insulin and vanadate. 49th Annual Meeting of the American Diabetes Association, Detroit, Michigan, 1989, p 63A (Abstract 250)

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 June 2016. at 06:49 For personal use only. No other uses without permission. . All rights reserved.