GLUT-4 and GLUT-1 Glucose Transporter Expression Is Differentially ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 20, Issue of July 15, pp. 14998-15003,1993 Printed in U.S.A.

GLUT-4 and GLUT-1 Glucose TransporterExpression Is Differentially Regulated by ContractileActivity in Skeletal Muscle* (Received for publication, February 8, 1993, and in revised form, March 19,1993)

Anna Castello$§, Joan CadefauQV,Roser CussoV, Xavier TestarS,John E. HeskethII , Manuel Palacin$, and Antonio Zorzano$** From the SDepartament de Bioquimica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda.Diagonal 645, 08028 Barcelona, Spain, the YDepartament de Ciencies Fisiohgiques Humanes i de la Nutricio, Facultat de Medicina, Universitat de Barcelona, 08028 Barcelona, Spain, and the IIDivision of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, United Kingdom

Mammalian skeletal muscle expresses GLUT-4 and GLUT-4 and GLUT-1 glucose transporters (Klip and MarGLUT-1 glucose transporters. Here, we have investi- ette, 1992; Marette et al., 1992; Handberg et al., 1992), and gated whetherGLUT-1 and GLUT-4 expressionis reg- the molar ratio of GLUT-1 toGLUT-4 in red skeletal muscle ulatedin muscle by contractileactivity.GLUT-1 membranes from the adult rat is about 1:20 (Klip and Marette, mRNA levels were high in skeletal muscle at days 16 1992).Furthermore, plasma membranes of red muscle contain and 17 of fetal life and decreased markedly by days 19 80% more GLUT-4 transporters and 280%more GLUT-1 and 21. In contrast, GLUT-4 mRNA levels were clearly transporters than plasma membranes of white muscle (Mardetectable at day 2 1 of fetal life, and they increased ette et al., 1992). progressively during postnatal life. The timing data for GLUT-4 inductionand GLUT-1 repression suggest Subcellular fractionation data indicate thatGLUT-1 is that these processes are related to skeletal muscle in- located mainly at the cell surface, is nottranslocated in response to insulin (Douen et al., 1990a, 1990b;Marette et al., nervation.GLUT-4 mRNA decreasedmarkedlyin adult rat and rabbit tibialis anterior muscle after sev- 19921, and may have a major role catalyzing basal glucose erage of peroneal nerve. In contrast, GLUT-1 mRNA uptake by the muscle cell. On the other hand, immunocytolevels showed a 9-fold increase in rat muscle 3 days chemical and biochemical studies reveal that under basal after denervation. Direct stimulationof rabbit tibialis conditions GLUT-4 is mainly intracellular (Friedman et al., 1991; Bornemann et al., 1992; Rodnick et al., 1992) and that anterior muscle with extracellular electrodes protected GLUT-4 mRNA levels against the effect of denerva- insulin and exercise cause the translocation of GLUT-4tion. This indicates that the repression of GLUT-4 containing vesicles from an intracellular siteto thecell surface mRNA associated with denervation is due, at least in (Douen et al., 1990a, 1990b; Klip et al., 1990; Hirschman et part, to electrical activity. Increased contractile actival., 1990; Friedman et al., 1991; Bornemann et al., 1992; ity induced for 24 h by indirect electrical stimulation Rodnick et al., 1992). at low frequency caused a marked and specific increase Expression of various glucose transporter isoforms appears in GLUT-1 mRNA levels in rabbit tibialis anterior to be regulated at both pretranslational and post-translational muscle. steps. In brown and white adipose tissue, diabetes and fasting Our results indicate that(a)innervation-dependent basal contractile activity regulates in an inverseman- drastically decrease GLUT-4 protein andmRNA levels (Berner theexpression of GLUT-1 and GLUT-4 in skeletal ger et al., 1989; Sivitz et al., 1989; Kahn et al., 1989; Garvey et al., 1989,1991; Sinha et al., 1991; Camps et al., 1992).These muscle, and ( b ) enhanced contractile activity stimulates GLUT-1 expression in the absence of modifica- data suggest that a decrease in plasma insulin blocks GLUTtions to GLUT-4 expression. This suggests the exist- 4 expression in adipose tissue due to impairment at a pretranence of different factors which depend on contractile slational step. In skeletal muscle, it has been reported that activity and which control GLUT-1 and GLUT-4 glu- diabetes causes a decrease in GLUT-4 expression (Garvey et al., 1989; Strout et al., 1990; Bourey et al., 1990). However, cose transporter expression in skeletalmuscle. insulinopenic conditions only cause moderate alterations in GLUT-4 protein and mRNA levels in skeletal muscle, suggesting that insulin does not exert the pivotal role in the Skeletal muscle is the main tissue responsible for insulin- maintenance of GLUT-4 expression already substantiated in induced glucose utilization in humans and in rodents (Deadipose tissue (Richardson et al., 1991; Kahn et al., 1991; Fronzo et al., 1981; James et al., 1985), and glucose transport Camps et al., 1992). in this tissueis rate-limiting for glucose disposal under most It has recently been reported that denervation, a situation conditions (Furler et al., 1991). Skeletal muscle expresses both characterized by impairment of muscle insulin action (Burant et al., 1984; Smith and Lawrence, 1984), causes a profound * This work was supported inpart by Research Grants PB89/0331 (to A. Z.) and DEP89/0581 (to R. C.) from the Direccion General de modification of glucose transporter expression in skeletal Investigaci6n Cientifica y TBcnica, Spain. The costs of publication of muscle. Thus, GLUT-4 decreased markedly, and GLUT-1was this article were defrayed in part by the payment of page charges. enhanced in muscles after resection of sciatic nerves (Block This article must therefore be hereby marked “advertisement” in et al., 1991; Henriksen et al., 1991; Coderre et al., 1992).These accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. observations raise the important question of whether muscle § Recipients of predoctoral fellowships from the Ministerio de glucose transporter isoforms are regulated by nervous stimuli Educaci6n y Ciencia, Spain. or by contractile activity. In this study, we have investigated ** To whom correspondence and reprint requests should be ad( a ) the possible regulation of muscle GLUT-1 and GLUT-4 dressed. 14998

Regulation of Glucose Transporter Expression in Muscle glucose transporter expression by electrical activity and ( b ) the effect of increased contractile activity on glucose transporter expression in skeletalmuscle. EXPERIMENTAL PROCEDURES

Materials-Hybond N was purchased from Amersham and a random priming DNA labeling kit from Boehringer. Electrophoresis reagents were from Sigma and Boehringer Mannheim. Molecular weight markers were obtained from Bethesda Research Laboratories. Most commonly used chemicals were from Sigma. Animals and Tissue Sampling-Female Wistar rats (150-200 g) from our own colony were mated, and gestation was timed from the appearance of spermatozoids in vaginal smears. The rats were fed with Purina Laboratory chow ad libitum and housed in animal quarters maintained at 22 "C with a 12-hlight, 12-h darkcycle. Atdifferent gestational times (16-21 days), mothers were anesthesized with sodium pentobarbital (5-7mg/100 g body weight). Fetuses were removed, and theirhindlimbs were rapidly collected and frozen in liquid nitrogen. When the postnatal period was studied, pups remained with their mothers after delivery and were anesthesized with sodium pentobarbital at different times, before tissue sampling. After collection, tissues were rapidly frozen and kept a t -80 "C until analysis. For denervation studies, the peroneal nerve of anesthesized female New Zealand rabbits, ormale rats (ketamine, 20 mg/kg body weight), was unilaterally severed. The contralateral control legwas shamoperated. At different times after denervation, tibialis anterior muscles were dissected and frozen in liquid nitrogen. In a different set of experiments, stimulation of rabbit tibialis anterior muscle was performed immediately after bilateral section of the peroneal nerve. One week before denervation, electrodes were implanted at both distal (anteriorly) and proximal (posteriorly) ends of tibialis anterior muscle from one hindlimb. The muscle was stimulated chronically for 2 days in 100-Hz trains, 2.5-s duration, applied once every 30 min. The trains of pulses were 18 mA for 0.15 ms. The contralateral unstimulated muscle served as a control. In another set of experiments, rabbit muscles were subjected to low frequency stimulation. To thatend, 1week before the experiment, electrodes were implanted laterally to the peroneal nerve to induce low frequency stimulation as described elsewhere (Schwarz et al., 1983). This entailed the continuous stimulationin10-Hz trains (trains of pulses of 18 mA for 0.15 ms) for 24 h/day. After a 24-h stimulation period, tibialis anterior muscles were removed and rapidly frozen in liquid nitrogen. The unstimulated contralateral muscles were also collected. Hooded Lister rats of the Rowett strain (30 days old) were used for experiments in which compensatory hypertrophy was induced by tenotomy. Tenotomy was performed as previously described (Whitelaw and Hesketh, 1992). Left legs were sham-operated. An additional six animals were designated as unoperated controls. At various times after the operation, the animals were killed, and plantaris muscles were removed and rapidly frozen. RNA Isolation and NorthernBlot Analysis-Total RNA from muscle was extracted using the acid guanidinium isothiocyanate/phenol/ chloroform method as described by Chomczynski and Sacchi (1987). All samples had a 260/280 absorbance ratio above 1.7. After quantification, total RNA (15-30 pg) was denatured at 65 "C in the presence of formamide, formaldehyde, and ethidium bromide (Rosen and Villa-Komaroff, 1990) to allow the visualization of RNA. RNA was separated on a 1.2% agarose/formaldehyde gel and blotted on Hybond N filters. The RNA in gels and filters was visualized with ethidium bromide and photographed by UV transillumination to ensure the integrity of RNA, to check the loading of equivalent amounts of total RNA, and to confirm proper transfer. RNA was transferred in 10 X standard saline citrate (SSC; 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). Blots were initially prehybridized for 4 h a t 45 "C in 50% formamide, 5 X Denhardt's (1 X Denhardt's solution is 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 0.5% SDS, 5 X SSPE (1X SSPE is 0.15 M NaCI, 1 mM EDTA, 10 mM NaH,PO,, pH 7.4), and 200 pg/ml denatured salmon sperm DNA. The blots were then hybridized to the corresponding probes overnight at 42 "C in 50% formamide, 5 X Denhardt's, 0.5% SDS,5 x SSPE, 10% dextran sulfate, and 200 pg/ml denatured salmon sperm DNA. The human cDNA probe for GLUT-1 was a 1,346-base pair EcoRI fragment, the rat cDNA probe for GLUT-1 was a 2,521-base pair fragment, the rabbit cDNA probe for GLUT-1 was a 2,119-base pair Hind111 fragment, the human cDNA probe for GLUT-4 was a 2,007-

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base pair Sal1 fragment, and the ratcDNA probe for GLUT-4 was a 2,470-base pair EcoRI fragment. Human cDNA probes were obtained from Dr. Graeme I. Bell (University of Chicago). The rat cDNA probes for GLUT-1 and GLUT-4 were obtained from Dr. Morris Birnbaum (University of Harvard). The rabbit cDNA probe for GLUT-1 was obtained from Dr. Yoshitomo Oka (University of Tokyo). The cDNA probes were labeled with [32P]dCTPby random oligonucleotide priming. The probes were included at 2 X lo6 cpm/ ml. Filters from GLUT-1 and GLUT-4 assays werewashed for 15 min in 2 X SSC at room temperature, then for 20 min in 0.4 X SSC, 0.1% SDS a t 55 "C, and finally for 25 min in 0.1 x SSC, 0.1% SDS at 55 "C. The abundance of specific glucose transporter message was quantified by scanning densitometry (Ultrascan X L enhancer laser densitometer, LKB) of autoradiograms as described above, and data were expressed as a percentage of control values. RESULTS AND DISCUSSION

GLUT-I and GLUT-4 mRNALevels Are Differentially Modified duringLate Muscle Development-In rodents, critical processes related to muscledevelopmentoccur during late fetal life. Thus, primary myotubes appear in ratcalf muscles by embryonic day 16 (Harris et al., 1989). Thereafter, nervemuscle connections are established, and, by embryonic day 18, secondary myotubes are already developed (Ontell and Kozeka, 1984; Ontell et al., 1988). To determinewhether glucose transporter levels are regulated by innervation, we analyzed the expression of their transcripts during late muscle development (16- to 21-day fetuses). For these studies, total RNA was isolated from hindlimbmuscle of 16-21-day fetuses and neonates, and GLUT-4 and GLUT-1 mRNA species were determined by Northern blot. A greater yield of RNA was obtained in skeletal muscle during fetal (3.9 4 0.2 mg/g of tissue) or early neonatal life (2.1 0.2 mg/g of tissue) than in the adult state(0.93 f 0.02 mg/g of tissue). The presenceof GLUT-1 mRNAwas determined by Northern hybridization using a rat cDNA probe under high stringency conditions. GLUT-1 mRNAlevels were high in skeletal muscle in 16-17-day fetuses (Fig. l),and a substantial drop was observed in 19-21-day fetuses (GLUT-1 mRNAlevels at 19- and 21-day fetuses accounted for 42 and 29% of levels on day 16) (Fig. 1). A further decrease was noted in postnatal levels in life; thus, at day 5 afterbirth,GLUT-1mRNA skeletal muscle accounted for 7% of 16-day fetal levels (Fig. 1).No difference in GLUT-1 mRNA electrophoretic migration was detected throughoutdevelopment. The abundanceof GLUT-4 mRNAwas also determined by Northern blot using rat and human cDNA probes (Birnbaum, 1989) under high stringency conditions. GLUT-4 mRNAwas barelydetectableinfetuses at days 16,17, and19 which precluded their precise quantification. However, GLUT-4 mRNA levels were already clearly detectable in21-day fetuses (5.7% of adult levels) (Fig. 2). The transcripts for GLUT-4 increased progressively afterbirth, so by day 5 postnatal GLUT-4 mRNA accountedfor 25% of adult levels. On day 9 postnatal, GLUT-4 mRNA levels accounted for 68% of adult levels (Fig. 2). On day 21 postnatal, GLUT-4 mRNA levels were still lower than in adult skeletal muscle (79% of adult levels). As with GLUT-1 mRNA, nodifference in electrophoretic migration of GLUT-4 mRNA was detected during development. Our data indicate that the transcriptscoding for GLUT-1 and GLUT-4glucose transporters are regulated in an inverse manner during development in skeletal muscle. Thus, fetal life is associated with high expression of GLUT-1 and low expression of GLUT-4 in cardiac muscle and brown adipose tissue (Santalucia et al., 1992). Here, we have substantiated that ( a ) GLUT-1 mRNA levels decrease markedly between days 17 and 19 of fetal life in skeletal muscle, and (21) the

Regulation of Glucose Transporter Expression in Muscle

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FIG.1. Expression of GLUT-1 mRNA in rat skeletalmuscle during development. Total RNA was purified from pooled skeletal muscle obtained from rats (from day 16 of fetal life through day 18 postnatal and adult rats). 30 pgof total RNA from the different groups was applied on gels. GLUT-1 mRNA was detected after hybridization with a rat GLUT-1 cDNA probe, as described under “Experimental Procedures” (top panel). Autoradiograms were subjected to scanning densitometry. The results (mean f S.E.) of three separate experiments are shown and expressed as a percentage of fetal (day 16) values (bottom p a n e l ) . F = fetuses; N = neonates.

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et al., 1990), our data strongly suggest a possible involvement of muscle innervationin GLUT-4 induction and GLUT-1 repression during the perinatal period. GLUT-4 and GLUT-1 Transcripts AreDifferentially Altered by Denervation;Direct Electrical Stimulation Up-regulates GLUT-4 mRNA after Denervation-In order to assess the effects of innervationon the expression of GLUT-1 and GLUT-4 glucose transporter, we measured their levels after denervation. In contrastto previous studiesin which the sciatic nerve was severed (Henriksen et al., 1991; Block et al., 1991; Coderre et al., 1992), we performed a localized denervation by resection of peroneal nerve, which affects the innervation of tibialis anterior and extensor digitorum longus but which allows the maintenance of the normal hindlimb posture. RNA was obtained from control and denervated tibialis anterior muscle from rats 3 days after denervation. Under conditions in which no alteration in RNA yield was detected (0.93 k 0.02 mg/g of tissue and 1.02 k 0.01 mg/g of tissue in control and denervated muscle, respectively), a 69% reductionin GLUT-4 mRNA levels was substantiated in tibialis anterior muscle after denervation (Fig. 3). GLUT-1 mRNA increased markedly (950%) in tibialisanterior muscles from 3-day denervated rats (Fig. 3). RNA was also obtained from denervated and control tibialis anterior muscles from rabbits after different periods following denervation. Denervation did not affect the yield of RNA (0.40 f 0.02 mg/g of tissue and 0.38 f 0.05 mg/g of tissue in control and denervated muscle, respectively). GLUT-4 mRNA GLUT-4 mRNA

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FIG.2. Expression of GLUT-4 mRNA in rat skeletalmuscle during development. Total RNA was purified from pooled skeletal muscle obtained from rats (from day 16 of fetal life through day 21 postnatal and adult rats). 30 pg of total RNA from the different groups was applied on gels. GLUT-4 mRNA was detected after hybridization with a rat GLUT-4 cDNA probe, as described under “Experimental Procedures” (top panel). Autoradiograms were subjected to scanning densitometry. The results of two to threeseparate experiments areshown and expressed as a percentage of adult values (bottom panel).F = fetuses; N = neonates; A = adult rats.

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FIG.3. Expression of GLUT-4 and GLUT-1 mRNA in rat tibialis anterior muscle after denervation. Total RNA was purified from 3-day denervated (Dn)and contralateral rat tibialis anterior muscles (C). RNA wasquantitated spectrophotometrically, and onset of GLUT-4 mRNA induction occurs a t late fetal life as the integrity and relative amounts of RNA in each sample used were by ethidium bromide staining on the same gel. 30 pg of total observed recently and independently by Wallace et al. (1992). checked RNA from the different groups was applied on gels. GLUT-4 (top Based on previous observations regarding the timing of muscle panel) and GLUT-1 (bottom panel) mRNA were detected after hyinnervation and appearance of secondary myotubes in rodent bridization with rat cDNA probes, as described under “Experimental muscle (Ontell and Kozeka, 1984; Ontell et al., 1988; Condon Procedures.” Representative autoradiograms are shown.

Regulation of Glucose Transporter Expression

in Muscle

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GLUT-4 mRNA E t Br levels remained unaltered 1 day after denervation (Fig. 4). However, there was a marked reduction in GLUT-4 mRNA content (38%of control) 2 days after denervation (Fig. 4). A further reduction was noted at day 4 after denervation in tibialis anterior muscle (14% of control) (Fig. 4). This pattern was detected in the absence of alterations in the amount of rRNA species (Fig. 4). GLUT-1mRNA levels were undetectable in control rabbit muscles when Northern blot assayswere performed using a rabbit GLUT-1 cDNA probe. However, GLUT-1 mRNA was detectable, although a t a low level, in muscle from 2-day denervated rabbits (data notshown). This low expression level of GLUT-1 mRNA precluded it from Dn S Dn S further study. Innervation may regulate muscle gene expression by trophic FIG. 5. Electrical activity up-regulates the expression of factors that arereleased by the nerve and/or by the electrical GLUT-4mRNA in denervated rabbit muscle.Total RNA was activity resulting from muscle depolarization during neuropurified from denervated/stimulated ( S ) and contralateral denervamuscles (Dn).RNAwas muscular transmission. To testwhether electrical activity per ted/unstimulatedrabbittibialisanterior quantitatedspectrophotometrically,andtheintegrityand relative se stimulates GLUT-4 expression, electrodes were implanted in tibialis anterior muscles, and, immediately after peroneal amounts of RNA in each sample usedwerechecked by ethidium bromide staining on the same gel (right panel). 30 pg of total RNA nerve resection, muscles were stimulated for 2days (see from the different groups was applied on gels. GLUT-4 mRNA was “Experimental Procedures”).After stimulation, RNA was ob- detectedafterhybridizationwithratandhumanGLUT-4 cDNA tained from denervated/stimulated muscles and from dener- probes, as described under “Experimental Procedures” and in the vated muscles (contralateral leg) of each rabbit. Under these legend to Fig. 2. A representativeautoradiogram,obtainedafter conditions, the RNA yield was greater in the denervated/ hybridization with a human cDNA probe, is shown (left panel). stimulated group than in thedenervated one (0.88 It 0.22 mg/ g of tissue and 0.50 f 0.02 mg/g of tissue in denervated/ al., 1990). In contrast, GLUT-1 is expressed in skeletalmuscle stimulated and denervated groups, respectively). Direct stim- in different cell types (Handberg et al., 1992; Marette et al., ulation of tibialis anterior muscle with extracellular electrodes 1992), which raises the question of whether the alterationsin suppressed the reduction of GLUT-4 mRNA induced by de- the levels of GLUT-1 mRNA are a consequence of enhanced nervation (Fig. 5). The GLUT-4 mRNA levels in the dener- expression in the muscle fiber itself. In this regard, in situ vated/stimulated muscles were on 250% of those found in the hybridization analysisindicates thatGLUT-1 mRNA incontralateral denervated/unstimulated muscles. These exper- creases in muscle myofiber after denervation of rat peroneal iments demonstrate that muscle electrical activityper se can nerve.’ Whether GLUT-1expression is also affected in other selectively suppress, at least in part, thedecrease of GLUT-4 cell types in skeletalmuscle remains to be determined. transcripts caused by denervation. Taken together, our data reveal an inverse pattern of alterGLUT-4 expression is restricted to muscle fibers (Slot et ations in glucose transporter expression in response to denervation and late muscle development. This supports the view GLUT-4 mRNA that innervationtightlycontrols glucose transporter gene expression in skeletal muscle, maintaining high levels of GLUT-4 transcripts and very low levels of GLUT-1. Our data also indicate that neurotransmitter-dependent electrical activity, rather thantrophic agentsreleased from nerve endings, 2.8Kb- ’ is the critical factor by which innervation regulates glucose transporter expression in muscle. Regarding the possible mechanisms involved in the reguCC Jn -Dn C o n C-Dn lation of glucose transporter expression by innervation-deDays 0 1 2 4 pendent electricalactivity, it has been reported that the Et Br transcripts for the myogenic helix-loop-helix proteins of the MyoD family, myoD, myogenin, mrf-4, and myf-5 also undergo major changes after denervation and muscle development (Eftimie et al., 1991; Buonanno et al., 1992). Thus, denervation and development lead to changes in transcripts for myoD, myogenin, and myf-5 very similar to those described for GLUT-l mRNA (Eftimie et al., 1991; Buonanno C O n C O n C On C Dn et al., 1992). On the otherhand, mrf-4 mRNA shows a pattern Days 0 1 2 4 of changes with development similar to that described here FIG. 4. Expression of GLUT-4 mRNA in rabbit tibialis an- for GLUT-4 (Bober et al., 1991; Buonanno et al., 1992; Hinterberger et al., 1992). Furthermore, muscle electrical activity terior muscle after denervation. Total RNA was purified from denervated (On)and contralateral rabbit tibialis anterior muscles per se, in the absence of nerve, represses the increases of (C). RNA was quantitated spectrophotometrically, and the integrity mRNA levels for myoD, myogenin, and myf-5 associated with and relative amounts of RNA in each sample used were checked by muscle denervation (Eftimie et al., 1991; Buonanno et al., ethidium bromide staining on the same gel (bottom panel). 15 pg of total RNA from the different groups was applied on gels. GLUT-4 1992). It should also be mentioned that mRNA levels of muscle mRNA was detected after hybridization with rat and human GLUT4 cDNA probes, as described under “Experimental Procedures” and nicotinic acetylcholine receptor a-subunit rise after denervain thelegend to Fig. 2. Similar results were obtained with human and A. CastellB, J. Cadefau, R. Cuss6, X. Testar, E. Soriano, M. rat cDNA probes.A representative autoradiogram, obtained after Palacin, and A. Zorzano, unpublished observations. hybridization with a human cDNA probe, is shown (toppanel).

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Regulation of Glucose Transporter Expression in Muscle

GLUT-1 mRNA tion and diminish during muscle development (Evans et al., 1987; Tsay and Schmidt,1989; Merlie and Kornhauser, 1989), = and thisgene has been found tobe transactivated by proteins of the MyoD family (Piette et al., 1990). Infact, MyoD, myogenin, and myf-5 have alsobeen reported to activate other muscle-specific genes such as the creatine kinase gene and 2.8 kbthe myosin light chain gene (Lassar etal., 1989; Chakraborty et al., 1991; Wentworth et al., 1991). Thus, we postulate that the proteins of the MyoD family might have an important role in the control of GLUT-1 and GLUT-4 expression in skeletal muscle during late muscle development and after muscle denervation. c s c s GLUT-1 mRNA Selectively Accumulates afterIncreased Contractile Activity-It is well established that the tissue levels of proteins involved in energy metabolism are adjusted, GLUT-4 mRNA in skeletal muscle, to the functional demands imposed. Thus, increased contractile activity induces changes in the content of mitochondrial proteins and enzymes involved in energy generation, whichin many cases is due to increased gene expression (Williams etal., 1987; Hood et al., 1989). Based on the above observations, which indicate a regulatory role of contractile activity in muscle glucose transporter expression, 2.8 kb we next investigated the earlyeffect of increased contractile activity. Rabbit tibialis anterior muscles were continuously stimulated a t low frequency (10 Hz) for 24 h. This protocol increases muscle glucose metabolism and induces many fastto-slow transitions in phenotypic properties of the muscle c s c s (Pette and Diisterhoft,1992). After 24 h of stimulation, RNA FIG. 6. Effect of low frequency stimulation of rabbit tibialis was obtained from stimulated muscle and from contralateral anterior muscle on GLUT-4 and GLUT-1 mRNA expression. unstimulated muscle of each rabbit. Under these conditions, Total RNA was purified from1-day low frequency (10-Hz)stimulated no difference in RNA yield was observed (0.54 f 0.01 mg/g ( S )and contralateral unstimulated tibialis anterior muscle (C). RNA of tissue and 0.61 & 0.01 mg/g of tissue in unstimulated and was quantitated spectrophotometrically,and the integrity and relative stimulated groups, respectively). Indirect low frequency stim- amounts of RNA in each sample used were checked by ethidium ulation of tibialis anterior muscle for 24 h did not modify bromide staining on the same gel. 30 pgof total RNA from the groupswas applied on gels. GLUT-4 (bottom panel) and tissue GLUT-4 mRNA levels (Fig. 6) (values from the stim- different GLUT-1 (top panel)mRNA species were detected after hybridization ulated group were 86 f 14% of control unstimulated group). with the human and rat GLUT-4 and GLUT-1cDNA probes, as However, under these conditions, GLUT-1 mRNA was very described under “Experimental Procedures.” Representative autoramarkedly enhancedinthestimulated group, so whereas diograms, obtained after hybridization with the human GLUT-4 and GLUT-1 mRNAlevels were undetectable in the unstimulated GLUT-I cDNA probes, are shown. muscle, a very intense label was observed in the stimulated TABLE I group (Fig. 6). Effect of tenotomy on GLUT-4 mRNA expression in rat plantaris We also investigated the effect of compensatory workload muscle afterseverance of thetendon to a synergistic muscle on Total RNA was purified from plantaris muscles from sham-operglucose transporter expression. The early responses to a hy- ated and tenotomized legs. 25pgof total RNA from the different pertrophic stimulus of compensatory work overload were in- groups was applied on gels. GLUT-4 mRNA was detected as described vestigated 6, 9, 12, and 48 h after tenotomy. Under the same under “Experimental Procedures”and in the legend to Fig. 2. Results conditions, a very rapid increase in c-myc mRNA levels after are mean S.E. of 3 to 5 observations per group. Data wereexpressed initiation of the work overload has recently been reported as a Dercentage of sham-oDerated ~ O U D S . GLUT-4mRNA (WhitelawandHesketh, 1992). GLUT-4mRNA levels in (arbitrary units) plantaris muscle were similar in 6-h and 9-h tenotomized Sham-operated 100 groups andsham-operatedcontrols(TableI). A marginal decrease was detected in GLUT-4 mRNA levels 12 h after Tenotomized 6h 85.8 f 9.5 ( n = 3) tenotomy, which disappeared 48 h after surgery (Table I). 82.4 f: 11.4 ( n = 3) 9h Similar results were observed when data were expressed per 12 h 64.3 f: 13.0” ( n = 5) pg of RNA or per unit of 18 S rRNA (data not shown). In 76.1 zk 27.6 ( n = 4) 48 h both sham-operated and tenotomized groups, the GLUT-4 Significant difference from sham-operated group, at p < 0.05. mRNA levels 6 h, 9 h, and 12h after tenotomywere markedly greater (aproximately 2-fold) than in the control unoperated All theseresultsindicate that increased neuromuscular group (data not shown). This increase in GLUT-4 mRNA activity induced by 24 h of low frequency stimulation causes levels in sham-operated and tenotomized groups compared to a rapid increase in GLUT-1 expression, in the absence of the unoperatedgroup disappeared 48h after surgery (data not alterations in GLUT-4 mRNA content, observed by low freshown). The transient increase in muscle GLUT-4 mRNA quency stimulation and compensatory workload. Based on the levels detected after surgery might be a consequence of en- factthatchronicincreasedcontractileactivitystimulates hanced plasmaglucocorticoid levels. In keeping with this view, glucose uptake by muscle (Augert etal., 1985) and that under greater muscle GLUT-4 mRNA levels have been detected in our experimental conditions enhanced concentrationsof gludexamethasone-treated rats (Haber and Weinstein, 1992). cose and hexose phosphates were also detected (Green etal.,

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Regulation of Glucose Transporter Expression 1990),2these data suggest that the alterations found after enhanced contractile activity occur within myofibers. In summary, our data demonstrate that GLUT-1 and GLUT-4 are regulated by innervation-dependent electrical activity in an inverse manner in skeletal muscle. Thus, as a consequence of signals dependent on muscle electrical activity, GLUT-1is expressed only at a very low level, and GLUT4 expression is high. The nature of the electrical activityinduced intracellular signals that control glucose transporter expression remains unknown. In addition, increased chronic contractile activity must generate another signal which derepresses GLUT-1 gene expression. Therefore, we propose the existence of different intracellular signals, which depend on the degree of muscle contractile activity, and which impose different types of regulatory control on GLUT-1 and GLUT4 gene expression. Acknowledgments-We thank Robin Rycroft for his editorial help and Philippa Whitelaw for preparation of RNA from samples in the tenotomy experiments. We are indebted to Dr. Dirk Pette for introducing us to the use of the electrostimulation model. REFERENCES Augert, G., Van de Werve, G. & Le Marchand-Brustel, Y. (1985) Diabetologia 2 --,~-295-2171 -. - - - Berger, J., Biswas, C., Vicario, P. P., Strout, H. V., Saperstein, R. & Pilch, P. F. (1989) Nature 3 4 0 , 70-72 Birnbaum, M. J. (1989) Cell 57,305-315 Block, N. E., Menick, D. R., Robinson, K. A. & Buse, M. G. (1991) J. Clin. Inuest. 88, 1546-1552 Bober, E., Lyons, G. E., Braun, T., Cossu, G., Buckingham, M. & Arnold, H. H. (1991) J. CellBiol. 113,1255-1264 Bornemann, A., Ploug, T. & Schmalbruch, H. (1992) Diabetes 4 1 , 215-221 Bourey, R. E., Koranyi, L., James, D. E., Mueckler, M. & Permutt, M.A. (1990) J. Clin. Invest. 86, 542-547 Buonanno, A., Apone, L., Morasso, M. I., Beers, R., Brenner, H. R. & Eftimie, R. (1992) Nucleic Acids Res. 20,539-544 Burant, C. F., Lemmon S. K., Treutelaar, M. K. & Buse, M. G. (1984) Am. J. Physiol. 2 4 7 , E657-E666 Camps, M., Castell6, A,, Mudoz, P., Monfar, M., Testar, X., Palacin, M. & Zorzano, A. (1992) Biochem. J . 2 8 2 , 765-772 Chakraborty, T., Brennan, T. J., Li, L., Edmonson, D. & Olson, E. N. (1991) Mol, Cell. Btol. _ 11.~ 3633-3641 . Chomczynski, P . & Sacchi, N. (1987) Anal. Biochem. 1 6 2 , 156-159 Coderre, L., Monfar, M.M., Chen, K. S., Heydrick S. J., Kurowski T. G., Ruderman, N. B. & Pilch, P. F. (1992) Endoerinobiy 131,1821-182'5 Condon, K., Silberstein, L., Blau, H. M. & Thompson, W. J. (1990) Deu. BioL 138.256-274 ""

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