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Biochimica et Biophysica Acta, 1089 (1991) 27-32 © 1991 Elsevier Science Publishers B.V. 016%4781/91/$03.50 A D O N I S 016747819100124Q

BBAEXP 92237

Stimulation of glucose transporter (GLUT1) mRNA and protein expression by inhibitors of glycosylation F r a n c e s M a h e r * a n d L e o n a r d C. H a r r i s o n Burner Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research. Parkville. Victoria (Australia)

(Received 28 December 1990)

Key words: Glycosylation: Glucose transporter; mRNA

Glucose deprivation increases the steady-state levels of mRNA for the rat brain/HepG2-type glucose transporter (GLUTI) in L6 myncytes. Glucose deprivation also inhibits N-linked glycosylation. We therefore investigated a possible relationship between inhibition of glycosylation and GLUTI expression in cultured 1.6 myucytes by determining the effects on GLUTI expression of known inhibitors of glycosylation, namely tunicamycin, 2-deoxyglucose and glucosamine. All conditions prevented incorporation of [3H|mannose into TCA-preelpitable myucyte protein and resulted in a 2- to 5-fold increase in the level of GLUTI mRNA detected on Northern blots. Glucose deprivation and tunieamycin treatment caused an approx. 2-fold increase in GLUTI mRNA half-life. GLUTI protein, detected on immunoblots, accumulated 10- to 20-fuld in response to all glycosylation inhibitors, with apparent molecular masses of 40 kDa after glucose deprivation, 42 kDa after 2-dcoxyghicose and 38 kDa after glncosamine or tunicamyeln treatments, compared to 45-50 kDa in glucose-fed cells. However, glucose deprivation was the only condition in which the rate of 2-deoxy-[3H]glucose uptake increased (3- to 5-fold). These results demonstrate a direct correlation between inhibition of glycosylation and the induction of GLUTI mRNA and protein expression and suggest that the stability of GLUT! mRNA is controlled by a signal associated with glyeosylation.

Introduction

The effect of glucose deprivation to increase the rate of glucose uptake in cultured cells has been widely reported [1-10]. In some cells glucose deprivation has been shown to decrease the rate of glucose transporter degradation without altering translation or mRNA levels [9] and in others to enhance the rate of glucose transporter synthesis [10]. Recent studies in glucose deprived L6 myocytes and cultured glial cells have demonstrated increased expression of the rat brain/HepG2-type glucose transporter (GLUT1) mRNA [1,2,11] due to enhanced transcription [12] and mRNA stability [13]. Despite the recognition of transcriptional and translational control of GLUT1 by glucose deprivation, the biochemical mechanisms or signals underlying the effects of glucose deprivation remain unknown. Potential regulatory signals arising from glucose de-

Abbreviation: 3H-2DG, 2-deoxy-[3H]glucese. Correspondence and * present address: F. Maher, National Institutes of Health, Bldg 10, Rm 5N102, Bethesda. MD, 20892, U.S.A.

privation may include reduced metabolite concentrations or alterations in glycosylation pathways. Our previous studies in glucose deprived L6 myocytes [11] have shown that only fully metabolised hexoses (e.g., mannose) can substitute for glucose in the down-regulation of GLUTI mRNA and protein expression, but were unable to identify a metabolic intermediate which mimics the effects of glucose on GLUT1 expression. Metabolised hexoses, as well as substituting in glycolytic and oxidative pathways, are also utilised in glycosylation. In the present study we investigate the possible involvement of glycosylation as a regulator of GLUT1 expression. Glucose deprivation is known to inhibit glycoprotein synthesis by inhibiting the synthesis of lipid-linked oligosaccharides in the endoplasmic reticuhim, resulting in accumulation of short oligosaccharides (GIcNAc2 Mans) and, consequently, underglycosylated proteins [14-16]. The widely used antibiotic tunicamycin inhibits protein glycosylation by blocking the transfer of Nacetyiglucosamine to the dolichol carrier, the fh'st step in lipid-linked oligosaccharide synthesis in the endoplasmic reticulum [3]. In addition to antibiotics, sugar analogues such as 2-deoxyglucose and glucosamine are

28 well established inhibitors of glycosylation [3]. 2-Deoxyglucose is thought to act via the derivative GDP-2deoxyglucose, which substitutes for mannose in core glycoprotein synthesis, but these substituted oligosaccharides cannot be further elongated [3]. The site at which glucosamine inhibits glycosylation is not known but is thought to be at an early step in oligosaccharide synthesis [3]. We have investigated the effects of glucose deprivation, tunicamycin, 2-deoxyglucose and glucosamine on glycoprotein synthesis in L6 myocytes and compared this to their effects on expression of GLUTI mRNA and protein and glucose uptake activity to determine any relationship between glycosylation and GLUT1 gene expression. Materials and Methods

Cell culture L6 myocytes were grown to confluence in MEM (5 mM glucose) containing 10% fetal calf serum. For glucose deprivation the medium was changed to glucosefree DME containing 5% dialyzed fetal calf serum ( G medium). Control cells were incubated in the same medium containing 5 mM glucose (G ÷ medium).

Measurement of glycosvlation Myocytes in 6-well dishes were incubated in medium without and with glucose or inhibitors for 24 h. D-[23H(N)lMannose (New England Nuclear) was added at 2 5 / t C i / m l either for the final 8 h of the incubation or for the entire 24 h. Cells were washed with phosphatebuffered saline, lysed with 0.5 ml of 0.1 M NaOH and an aliquot taken for protein determination by the BioRad assay. Protein was precipitated from the remaining lysate by addition of 5 ml of 10% trichloroacetic acid (TCA), incubation for 30 min at 4 ° C and filtration through Whatman GF/C glass fibre filters. The filters were washed three times with 5 ml of 10% TCA, dried and the ~H-radioactivity counted in 3 ml scintillant. Statistical analysis was performed by Analysis of Variance, with P < 0.05 as the level of significance.

2-Deoxy-[3H]glucose (3H-2DG) uptake Cells were washed in glucose-free Krebs-Ringer bicarbonate buffer with Hanks' salts (HKRB) and incubated in 1 ml HKRB with 1.5 /~Ci 2-deoxy-D-[13H]glucose (17 Ci/mmol, Amersham International, Amersham, U.K.) for 10 rain at 37"C, washed with ice cold phosphate-buffered saline, solubilised in 1 ml of 1 M NaOH and counted in a liquid scintillation spectrometer. Non-specific uptake, measured in the presence of phloretin (500/~M), was < 10% of total uptake and was subtracted from total uptake. 3H-2DG uptake is expressed as pmol/mg cell protein per 10 rain. Statistical analysis was performed by Analysis of Variance, with P < 0.05 as the level of significance.

Northern blotting Total cytoplasmic RNA was extracted from L6 myocytes with SDS/urea as previously described [17,18]. RNA (25 #g) was electrophoresed in 1% agarose/2.2 M formaldehyde gels, transferred to nitrocellulose with 20 × SSC (3 M sodium chloride, 1.3 M sodium citrate). Filters were baked at 80"C in vacuo for 2 h, prehybridized for 4 - 6 h in 50% formamide, 5 x SSC, 5 x Denhardt's solution, 500 # g / m l salmon testis DNA and 1% SDS at 42"C, and hybridized for 18 h at 42°C in the same buffer containing 32P-labelled DNA probes. Filters were washed in 2 x SSC/0.1% SDS at room temperature for 1 h and 0.2 x SSC/0.1% SDS at 4 2 " C for 30 min, then autoradiographed on Hyperfilm MP (Amersham International, Amersham, U.K.). mRNA levels were quantitated by scanning the each lane on the autoradiographs two or three times with an LKB Ultroscan laser densitometer. GLUT1 mRNA was detected with an oligonucleotide probe to nucleotides 904-942 (within the putative cytoplasmic loop region) of the rat brain glucose transporter sequence [19!. The oligonucleotide was 5'-end labelled with [~,-32PIATP and T4 polynucleotide kinase (specific activity 10 ~ cpm/#g). A cDNA probe to glyceraldehydephosphate dehydrogenase (GAPDH) was labelled with [a-a2P]dATP by random priming (specific activity 109 cpm/#g).

lmmunoblotting Total cell membranes were isolated by hypotonic cell lysis and centrifugation as described previously [18]. Membrane protein was solubilized in 0.1 M Tris-HCI (pH 6.8), 0.1 M dithiothreitol, 0.1% SDS, 10% glycerol, 0.1% bromophenol blue, at 65°C for 5 rain, size fractionated in 10% SDS-polyacrylamide gels and transferred to nitrocellulose with an LKB Novabiot system (LKB, Turku, Finland). Filters were incubated for 2 h with phosphate-buffered saline (PBS) containing 0.05% Tween20 (PBS/Tween) and 5% milk powder then for 12 h at 4 ° C with P B S / T w e e n / l % BSA containing a 1/200 dilution of a rabbit antiserum raised against a synthetic C-terminal peptide (amino acids 480-492) of GLUT1 (antiserum 379, kindly provided by Dr. lan Simpson, NIDDK, National Institutes of Health, Bethesda, MD, U.S.A.). Filters were rinsed, incubated in P B S / T w e e n / l % BSA containing 0.2 p C i / m l J251-protein A for 1 h at room temperature, washed in PBS/0.2% Triton for 30 rain and autoradiographed. Results

Effects of glucose deprivation and inhibitors of glycosylation on [3H]mannose incorporation in L6 myocytes The effect of glucose deprivation ( G - ) on glycoprotein synthesis in L6 myocytes was determined by measuring the rate of [3H]mannose incorporation into

29 T C A - p r e c i p i t a b l e protein, a n d was c o m p a r e d to the effects o f glycosylation inhibitors t u n i c a m y c i n (20 / t g / m l ) , 2-deoxyglucose (20 m M ) a n d g l u c o s a m i n e (20 raM). [ ~ H ] m a n n o s e i n c o r p o r a t i o n was inhibited by 6 0 70% by glucose d e p r i v a t i o n , 4 5 - 5 5 % b y t u n i c a m y c i n , 3 5 - 4 0 % b y 2-deoxyglucose a n d 48% b y g l u c o s a m i n e w h e n expressed either as c p m / w e l l o r relative to the total p r o t e i n c o n t e n t p e r well (Table l). In c o n t r a s t , 3-O-methylglucose, a n o n - m e t a b o l i s a b l e glucose analogue, d i d n o t inhibit [ 3 H ] m a n n o s e i n c o r p o r a t i o n indic a t i n g that t h e effects o f 2-deoxyglucose a n d glucos a m i n e were n o t d u e to h e x o s e c o m p e t i t i o n with glucose. T h e c o m b i n a t i o n o f G - a n d g l u c o s a m i n e , 2-deo x y g l u c o s e o f 3 - O - m e t h y l g l u c o s e were n o t additive in the inhibition o f [ 3 H ] m a n n o s e i n c o r p o r a t i o n . Tunic a m y c i n a u g m e n t e d the G - effect, causing 80% inhibition o f [ 3 H ] m a n n o s e i n c o r p o r a t i o n .

A

a, lip

-.GT

"JGAPDH

B

Effects o f glycosylation mhibitors on G L U T I m R N A G l u c o s e d e p r i v a t i o n has previously been s h o w n to increase G L U T I m R N A levels in L6 m y o c y t e s by 2- to 4-fold [2,11,12]. Incubatit,~ o f anyocytes in G + m e d i u m c o n t a i n i n g t u n i c a m y c i n c a u s e d a 2- to 3-fold increase in G L U T 1 m R N A (Fig. la). E x p o s u r e t o , g l u c o s a m i n e o r 2-deoxyglucose also resulted in a n a p p r o x . 5-fold increase in G L U T I m R N A levels, w h e r e a s a d d i t i o n o f m a n n o s e , galactose, 3 - O - m e t h y l g l u c o s e a n d glucose-6-



~lmr 'mml~~

ell

ge -tGT

~ ~mr qglllPr

v

-,mGAPDH

TABLE !

Effect of glucose deprivation and glycos.vlation inhibt~or$on glycoprotein synthesis in 1,6 myocytes Myocytes in 6-well dishes were incubated for 24 h in G - medium or G ÷ medium with no addition or with 20/tg/ml tunicamycin. 20 mM 2-deoxyglucose, 3-O-methylglucose and ghicosamine. [3H]Mannose (25 ttCi/ml) was included for the final 8 h of the incubation. [3H]Mannose incorporation is expressed as both cpm in TCA-precipitable protein per well (cpm/well) and cpm in TCA-precipitable protein divided by the total amount of protein per well (cpm/mg protein). Protein content in glucose deprived or inhibitor-treated cells was typically reduced by 20~. The values are the mean_+S.D, of triplicate determinations. Comparable results were obtained in a second experiment in which [3H]mannose was included for the full 24 h incubation. * P < 0.05 compared to G+; ~IP < 0.05 compared to G-. [ 3H]Mannose in TCA-precipitable protein G+ G+ +tunicamycin

G + + 2-deoxyglucose G + + 3-O-methylglucose G + +glucosamine GG - + tunicamycin G - + 2-deoxyghicose G - + 3-O-methylglucose G - +glucosamine

cpm/well

cpm/mg protein

953_+58 437_+ 10 590_+ 19 1061_+56 507_+34 287_+33 132_+45 295 _+12 299_+ 13 319_+ 7

1466_+ 89 814± 19 * 967 4_- 32 * 1852z 98* 757_+ 50 * 566_+ 66 * 307_+ 104 ~s 750_+ 30 *s 640_+ 28 * 577_+ 13"

C 1 2 3 4

5 6 7 8

~GT

GAPUH

Fig. 1. Effects of glycosylation inhibitors on GLUT! mRNA. Myocytes were incubated for 24 h. (A) G + medium without or with 20/sg/ml tunicamycin. (B) G + medium containing additional l0 mM hexose; glucose, GLC; mannose, MAN; galactose. GAL; 2-deoxyglucose, 2DG: 3-O-methylglucose, 3 MG: glttcose-6-phosphate. G6P; 81ucosamine, GIcN. (C) G + (lanes 1-4) or G - (lanes 5-8) medium without other additions (lanes I and 5) or with l0 mM 2DG (lanes 2 and 6), 3MG (lanes 3 and 7) or GIcN (lanes 4 and 8). Total RNA (25/18/lane) was analyzed by Northern blot hybridization with an oligonucleotide to rat GLUTI sequence and a cDNA to GAPDH. Each blot is representative of at least three expetimems. p h o s p h a t e to G + m e d i u m d i d n o t alter G L U T 1 m R N A e x p r e s s i o n (Fig. lb). 2 - D e o x y g l u c o s e a n d glucosan,.ine were equally effective i n d u c e r s o f G L U T I m R N A ex-

30 Actinomycin

1

100

g

2

34

5 6 7 8

20097-

so i

68-

43!

10 0

i

6

8

12

Time(h) Fig. 2. Effects of glucose deprivation and tunicamycin on GLUTI mRNA half-life. Myocytes were incubated for 12 h in G* medium without (

Stimulation of glucose transporter (GLUT1) mRNA and protein expression by inhibitors of glycosylation.

Glucose deprivation increases the steady-state levels of mRNA for the rat brain/HepG2-type glucose transporter (GLUT1) in L6 myocytes. Glucose depriva...
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