thyroid hormone increases basal and insulin-stimulated glucose

Thyroid Hormone Increases Basal andInsulin-Stimulated Glucose Transport inSkeletal MuscleThe Role of GLUT4 Glucose Transporter ExpressionSteven P. Weinstein, Elizabeth O'Boyle, and Richard S. Haber

In skeletal muscle, the main site of insulin-mediatedglucose disposal, the major muscle glucose transporterGLUT4 is induced by thyroid hormone. To test the hy-pothesis that thyroid hormone alters muscle glucosetransport, we examined the effect of L-triiodothyronine(T3) on glucose transport and GLUT4 protein content inisolated rat skeletal muscles. Euthyroid rats were treatedwith or without T3 for 3 days, and [3H]2-deoxy-D-glucose(2-DG) uptake in soleus and extensor digitorum longus(EDL) muscles was measured under conditions in whichtransport was rate limiting for uptake in the absence orpresence of 10 nmol/l insulin. In control animals, insulinstimulated 2-DG uptake sevenfold in soleus and fivefoldin EDL. T3 treatment increased basal 2-DG uptake insoleus and EDL by 115 ± 29% and 136 ± 23%, respec-tively, and increased insulin-stimulated 2-DG uptake insoleus and EDL by 55 ± 9 and 42 ± 12%, respectively.Immunoblot analysis revealed that T3 treatment in-creased GLUT4 protein content in soleus by 43 ± 6% andin EDL by 56 ± 13%. These data demonstrate that thyroidhormone increases basal and insulin-stimulated glucosetransport in skeletal muscle. The percentage increase ininsulin-stimulated transport in T3-treated muscles is sim-ilar to the increase in GLTJT4 protein content, whereasthe percentage change in basal transport greatly exceedsthe change in GLUT4. Thus, increased insulin-stimulatedglucose transport in T3-treated muscle can be accountedfor by the induction of GLUT4 protein. However, in-creased basal glucose transport in T3-treated musclemust reflect additional mechanisms, such as increasedsubcellular partitioning of GLUT4 to plasma membrane.Diabetes 43:1185-1189, 1994

Consistent with its stimulatory action on metabolicrate, thyroid hormone increases glucose utiliza-tion in both experimental animals (1,2) and hu-mans (3-5). Both basal and insulin-stimulated

whole-body glucose utilization are increased by thyroid

From the Department of Medicine, Mount Sinai School of Medicine, New York,New York.

Address correspondence and reprint requests to Dr. Steven P. Weinstein, Box1055, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY10029.

Received for publication 29 March 1994 and accepted in revised form 2 June1994.

T3, L-triiodothyronine; T4, L-thyroxine; EDL, extensor digitorum longus; 2-DG,2-deoxy-D-glucose; BSA, bovine serum albumin; 2-DG-6-P, [3H]2-deoxyglucose-6-phosphate; TCA, trichloroacetic acid.

hormone excess (1-5). As the main site of insulin-mediatedglucose uptake (6), skeletal muscle probably plays a domi-nant role in this enhancement of glucose utilization. Indeed,expression of the major skeletal muscle glucose transportprotein GLUT4 and its mRNA is increased by thyroid hor-mone in rats and decreased in hypothyroidism (7,8). Thisobservation suggested that muscle glucose transport mightbe regulated by thyroid hormone because GLUT4 contentmay be a determinant of maximally stimulated glucosetransport in muscle (9). However, whether thyroid hormonedoes increase insulin-stimulated glucose transport in skeletalmuscle is unclear. Dimitriadis et al. (10) reported thatL-triiodothyronine (T3) treatment for 2-10 days increasedinsulin-stimulated glycolysis, glucose oxidation, and hexosephosphorylation in isolated rat soleus muscles, suggesting anincrease in glucose transport. In contrast, administration ofL-thyroxine (T4) to rats for 10 or 30 days was found toincrease basal glucose uptake in a perfused hindlimb prep-aration, but insulin-stimulated glucose uptake was reportedto be unchanged, despite increased GLUT4 protein in gas-trocnemius extracts (8). Sugden et al. (11) reported thatadministration of T3 for 3 days increased a basal glucoseutilization index in several rat skeletal muscles, but respon-siveness in these muscles was not assessed.

The goals of this study were 7) to determine whetherthyroid hormone treatment increases basal and insulin-stimulated glucose transport measured in isolated rat mus-cles, and 2} to determine whether concomitant changes inGLUT4 protein content induced by T3 in these musclesquantitatively explain changes in transport. Because regula-tion of glucose transport may vary in muscles of differentfiber-type composition (12), we chose to study both soleus(predominantly type I/slow-twitch oxidative fibers) and ex-tensor digitorum longus (EDL) (mixed type Ila/fast-twitchoxidative-glycolytic and type Ilb/fast-twitch glycolytic fibers)muscles (13). We report that T3 treatment increases basaland insulin-stimulated glucose transport in both muscles.Insul in-stimulated glucose transport is increased by T3 inproportion to increased GLUT4 protein content, but thepercentage increase in basal glucose transport exceeds thatof GLUT4. These data suggest that thyroid hormone in-creases maximal insulin-stimulated glucose transport inmuscle by increasing GLUT4 gene expression, but an addi-tional mechanism must underlie the increase in basal glu-cose transport.

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T, INCREASES GLUCOSE TRANSPORT IN MUSCLE

RESEARCH DESIGN AND METHODSRabbit antiserum against the COOH-terminal 12-amino acid peptide ofrat GLUT4 (14) was obtained from East Acres Biologicals (Southbridge,MA). 2-Deoxy-D-glucose (2-DG), sodium salt (T3), bovine insulin, radio-immunoassay-grade bovine serum albumin (BSA), 0.3 N zinc sulfate, and0.3 N barium hydroxide were from Sigma (St. Louis, MO). 2-[3H(G)]deoxy-D-glucose (8 Ci/mmol), 2-[l,2-3H(N)]deoxy-D-glucose (26 Ci/mmol), D-[l-14C]mannitol (49 mCi/mmol), and [125I]protein A (9 |xCi/jag)were purchased from Du Pont-NEN (Boston, MA).Treatment of animals. Male Sprague-Dawley rats (37-45 g) (CharlesRiver, Wilmington, MA) were fed standard diet. Small rats were chosento minimize muscle size (diffusion distance) in hexose uptake studies(15). T3 was administered in the drinking water at a concentration of 12mg/1; control rats received tap water alone. Based on an observedaverage water intake of 15 ml/day, treated rats received a daily dose of360 |xg T3/100 g body wt, sufficient to saturate T3 receptors (16). After 3days of treatment, rats were killed by CO2 inhalation, after which soleusand EDL muscles were rapidly excised for hexose uptake measurementsor preparation of detergent extracts. This short treatment interval waschosen to avoid marked muscle atrophy that occurred at longer inter-vals.

Hexose uptake measurements in isolated skeletal muscles. 2-DGuptake was measured by a modification (17) of the method of Henriksenet al. (9). Longitudinally split muscles (muscle halves still joined at thedistal tendon) were preincubated for 60 min and then incubated for 30min in the presence or absence of 10 nmol/1 insulin, a maximallyeffective concentration. After washing and additional incubation for 10min in fresh glucose-free buffer (with or without insulin as appropriate),uptakes were started by adding 2-[3H(G)]2-deoxy-D-glucose (1.5 jxCi/ml),D-[l-MC]mannitol (0.3 |xCi/ml), and 1 mmol/1 unlabeled 2-DG (in someexperiments the latter was omitted). Because insulin-stimulated uptakesin soleus and EDL muscles were found to be linear for at least 30 min,uptakes were routinely terminated after 30 min. Muscles were extractedin 10% trichloroacetic acid (TCA), and 3H and 14C radioactivity in theextracts and in the uptake medium was determined. The extracellularspace was calculated from the uptake of [14C]mannitol, and intracellular[3H]2-DG uptake was calculated by subtracting the amount of [3H]2-DGin the extracellular space from the total [3H]2-DG uptake.Determination of unphosphorylated intracellular [3H]2-DG con-centration. [3H]2-Deoxyglucose-6-phosphate (2-DG-6-P) in muscle ex-tracts was precipitated by using a modification of the method of Kipnisand Cori (18), which is based on the Somogyi procedure (19). Afteruptake of 2-DG, frozen muscles were extracted in 1 ml of ice-cold 10%TCA overnight, and radioactivity in 0.4 ml of the extract was determined.To precipitate phosphorylated 2-DG, a second 0.4-ml aliquot of theextract was brought to 0.8 ml with water and mixed first with 1.6 ml 0.3N zinc sulfate and then with 2.0 ml 0.3 N barium hydroxide. The mixturewas incubated for 20 min at room temperature and then centrifuged at1,200 g for 10 min. A 1-ml supernatant aliquot was subjected todual-channel liquid scintillation counting. Total supernatant volume wascalculated by dividing the total [14C]mannitol disintegrations per minuteadded to each tube by the concentration of [l4C]mannitol disintegrationsper minute in the supernatant. This volume was used to determine thetotal amount of unphosphorylated [3H]2-DG in each supernatant. Theamount of [3H]2-DG in the supernatant contributed by uptake into theextracellular space was then subtracted, yielding the amount of intra-cellular unphosphorylated [3H]2-DG. In these experiments, 2-[l,2-3H(N)]deoxy-D-glucose was used for uptakes to avoid contamination ofsupernatants with 3H2O, which could potentially be released fromgenerally labeled [3H]2-DG on phosphorylation at carbon 6.

To assess the efficiency of precipitation of phosphorylated 2-DG,2-[l,2-3H(N)]deoxy-D-glucose was quantitatively phosphorylated withhexokinase and MgATP, and the reaction product was precipitated in10% TCA by using the procedure described above. When equal volumesof the zinc sulfate and barium hydroxide reagents were used asoriginally described by Somogyi (19), the precipitation procedure left upto 10% of the [3H]2-DG-6-P in the supernatant. The ratio of bariumhydroxide to zinc sulfate proved critical for efficient precipitation: 1.6 mlof zinc sulfate reagent and 2.0 ml of barium hydroxide reagent wasoptimal, and precipitated >98% of the 3H radioactivity, a percentageindistinguishable from the purity of the starting material. We alsoconfirmed that neither [3H]2-DG nor [14C]mannitol was precipitated bythis procedure.Preparation of detergent extracts from skeletal muscle. Pairedsoleus or EDL muscles were excised from each animal, frozen in liquidN2, weighed, and suspended in 30 vol of ice-cold extraction buffer (0.25

TABLE 1Effect of T3 treatment on basal and insulin-stimulated [3H]2-DGuptake in rat soleus and EDL muscles

[3H]2-DG uptake (ujnol

Soleus

Control T3 treated

era wet wt

Control

M O m

EDL

T3 treated

Basal 0.27 ±0.03 0.58 ± 0.06* 0.22 ± 0.01 0.52 ± 0.05*n 15 15 9 9

Insulin 2.0 ± 0.1 3.1 ± 0.2* 1.2 ± 0.1 1.7 ± 0.1*n 24 24 12 12

Rats were treated with T3 for 3 days, after which [3H]2-DG uptakewas measured in isolated soleus and EDL muscles in the presenceor absence of 10 nmol/1 insulin, as described in METHODS. Theexternal 2-DG concentration was 1 mmol/1. The data shown arepooled from eight independent experiments; n, number of muscles.Because T3 treatment did not alter the intracellular water volume(0.48 ml/g wet wt, see Ref. 35 for method of calculation), the effectof T3 on 2-DG uptake is equivalent when uptakes are expressed ona per gram or per milliliter intracellular water basis. *P < 0.01 vs.control.

mol/1 sucrose, 50 mmol/1 Tris-HCl [pH 7.5], 0.5% sodium deoxycholate,0.5% Triton X-100, 1.25 mmol/1 EGTA, 1 mmol/1 phenylmethylsulfonylfluoride, and 1:1,000 [vol:vol] aprotinin solution [Sigma A-6279]). Themuscles were then homogenized and extracted as previously described(17). Virtually all of the immunoreactive GLUT4 protein in the muscleswas found in the extract supernatant (17). Protein concentration in theextracts was determined on 1:10 dilutions in water by the method ofLowry et al. (20) with BSA as a standard.Immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis of detergent extracts (20-40 |xg protein per lane) and quanti-tative immunoblotting with antiserum against GLUT4 protein with theuse of l25I-labeled protein A to detect bound antibody were performed aspreviously described (7). Sections of the blots corresponding to the45-kDa GLUT4 band on autoradiograms were excised, and boundradioactivity was determined with a 7-counter, with correction forbackground radioactivity on the blot. The GLUT4 signal was shown to belinear with the amount of protein loaded per lane.Statistical analysis. Data are expressed as means ± 1 SE. P valueswere calculated with the use of the Student's /, test (two-tailed).

RESULTSEffect of T3 treatment on body and muscle weight. Asexpected, induction of hyperthyroidism by T3 treatmentdecreased the rate of gain of body weight. Animals in bothcontrol (n = 50) and T3-treated (n - 56) groups initiallyweighed 44 ± 0.6 g; by the end of the 3-day treatment, controlanimals weighed 57 ± 1.1 g, whereas T3-treated animalsweighed only 50 ± 1.1 g. Soleus muscles from control andT3-treated animals weighed 22 ± 0.4 mg (n = 74) and 19 ±0.3 mg (n = 74), respectively, and EDL muscles weighed 26± 0.6 mg (n = 41) and 23 ± 0.6 mg (n = 41), respectively.Thus, T3 treatment for 3 days caused only a small differencein muscle size.Effect of T3 treatment on basal and insulin-stimulated[3H]2-DG uptake in skeletal muscle. In control animals,insulin at a maximally effective concentration (10 nmol/1)stimulated [3H]2-DG uptake 7.4-fold in soleus muscles and5.5-fold in EDL muscles (Table 1). The absolute rate ofinsulin-stimulated [3H]2-DG uptake in soleus was substan-tially greater than that in EDL as previously reported (9),which is consistent with the observation that insulin respon-siveness is greater in red versus white skeletal muscle (21).T3 treatment increased basal [3H]2-DG uptake in soleus andEDL by 115 ± 29 and 136 ± 23%, respectively, and increased

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TABLE 2Effect of T3 treatment and external 2-DG concentration on intracellular unphosphorylated 2-DG concentration in insulin-stimulatedsoleus muscles

Control soleus T3-treated soleus

External [2-DG]Intracellular [2-DG]Intracellular [2-DG] as percentage

of external [2-DG]

53nmol/l-3.6 ± 1.3 nmol/1

-6.8 ± 2.4

1 mmol/10.017 ± 0.027 mmol/1

1.7 ± 2.9

53 nmol/10.44 ±2.7 nmol/1

0.8 ± 5.1

1 mmol/10.18 ±0.07 mmol/1

18 ± 7.1

Rats were treated with T3 for 3 days, after which [3H]2-DG uptake was measured in isolated soleus muscles in the presence 10 nmol/1 insulin,as described in METHODS. The external 2-DG concentration was 53 nmol/1 or 1 mmol/1. Intracellular unphosphorylated 2-DG concentrationmeasured as described in METHODS is presented in molar units and as a percentage of the external 2-DG concentration. The data shown arepooled from 3 independent experiments; n, 9 muscles per group. The negative value shown for intracellular [2-DG] (in control soleus at 53nmol/1 external 2-DG) is close to zero and reflects the difficulty in accurately determining intracellular unphosphorylated 2-DGconcentrations when they are very low relative to the external concentration.

insulin-stimulated [3H]2-DG uptake in soleus and EDL by 55± 9 and 42 ± 12%, respectively.

Because at higher rates of transport (e.g., with insulinstimulation and at higher hexose concentrations) intracellu-lar phosphorylation rather than membrane transport maybecome partly rate limiting for 2-DG uptake, two approacheswere used to confirm that thyroid hormone stimulates mem-brane transport. First, the magnitude of the effect of T3 oninsulin-stimulated 2-DG uptake in soleus was measured attracer (53 nmol/1) instead of 1 mmol/1 external 2-DG, theconcentration used in the above-described experiments.Insulin-stimulated 2-DG uptake at tracer external 2-DG con-centration was 130 ± 8 pmol • g wet wt"1 • 30 min"1 (n = 12muscles) and 200 ± 18 pmol • g wet wt"1 • 30 min"1 (n = 12muscles) in control and T3-treated soleus, respectively (datapooled from four experiments; P < 0.01). Thus, at low ratesof uptake, when membrane transport is rate limiting, theeffect of thyroid hormone on insulin-stimulated 2-DG uptakeis fully preserved (54% at tracer concentrations of 2-DG vs.55% at 1 mmol/1 2-DG).

For the second approach, intracellular unphosphorylated2-DG concentration was measured after uptake of 2-DG(Table 2). During conditions in which membrane transportrather than phosphorylation by hexokinase is rate limitingfor 2-DG uptake, the intracellular unphosphorylated 2-DGconcentration will remain low relative to the extracellularconcentration. After uptakes at tracer external 2-DG (53nmol/1), intracellular unphosphorylated 2-DG concentrationwas <5% of extracellular levels in insulin-stimulated soleus(both control and T3-treated), as well as after uptakes at 1mmol/1 external 2-DG in control insulin-stimulated soleus.Only in T3-treated insulin-stimulated soleus after uptakes at 1mmol/1 external 2-DG did a significant accumulation ofintracellular unphosphorylated 2-DG occur (18% of the ex-ternal 2-DG concentration). These data indicate that, duringthe uptake conditions used here, accumulation of intracellu-lar unphosphorylated 2-DG is minimal, confirming that ouruptake measurements primarily reflect membrane transport.Effect of T3 treatment on GLUT4 protein content inskeletal muscle. Detergent-extractable protein yields incontrol soleus and EDL muscles were 52 ± 2.1 mg/g wet wt(n = 10 muscle pairs) and 57 ± 1.4 mg/g wet wt (n = 10muscle pairs), respectively, and were unaffected by treat-ment with thyroid hormone (data not shown). Extracts wereanalyzed by immunoblotting, with the use of an antiserumagainst rat GLUT4. The antiserum recognized a major proteinband in the 45,000- to 50,000-ATr range, which is the GLUT4protein, and a minor protein band at a lower relative molec-

ular weight that is more prominent in extracts of EDL (Fig.1). GLUT4 protein content in EDL was 70% of that in soleus(Table 3), as previously reported (9). T3 treatment increasedGLUT4 protein content in soleus by 43 ± 6% and in EDL by56 ± 13% (Table 3).

Figure 2 compares the effects of T3 treatment on 2-DGuptake and GLUT4 protein content in both soleus and EDLmuscles. The percentage increments in insulin-stimulated2-DG uptake induced by T3 treatment in soleus and EDLwere similar to the percentage increments in GLUT4 proteincontent, whereas the increase in basal 2-DG uptake greatlyexceeded the increase in GLUT4 protein content.

DISCUSSIONThe first goal of this study was to determine whether thyroidhormone increases insulin-stimulated and basal glucosetransport in skeletal muscle. We chose to measure [3H]2-DGuptake in soleus and EDL by using an in vitro incubationmethod, which permits control of the extracellular environ-ment and which eliminates the potential confounding vari-able of altered regional capillary flow inherent in in vivomethods. T3 administration for 3 days increased both basaland insulin-stimulated [3H]2-DG uptake in both soleus andEDL (Table 1) under conditions in which uptake is a mea-surement of membrane transport (see RESULTS). Moreover,the percentage changes in [3H]2-DG uptake induced by T3

treatment were similar in the two muscles, demonstratingthat thyroid hormone stimulates muscle glucose uptake inmuscles having markedly different fiber type composition.These results directly demonstrate that thyroid hormoneincreases both basal and insulin-stimulated glucose trans-port in mammalian skeletal muscle. Because skeletal muscleis the major site of insulin-mediated glucose disposal andbecause membrane transport is normally rate limiting forglucose uptake in this tissue (22), this finding may explainprevious observations that thyroid hormone excess in-creases insulin-mediated whole-body glucose utilization (2-5). Our results are consistent with a previous report ofincreased insulin-stimulated 2-DG uptake in isolated ratsoleus from thyrotoxic rats, in which the relative role oftransport versus phosphorylation was not directly addressed(10). In a second previous study (8), basal glucose uptake ina perfused hindlimb preparation was found to be increasedin thyrotoxic rats, as in this study, but thyroid hormone didnot affect insulin-stimulated glucose uptake, in contrast tothis study. Although the explanation for this discrepancy isunclear, it should be noted that the hindlimb perfusion


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T, INCREASES GLUCOSE TRANSPORT IN MUSCLE

Soleus EDL

- 3 3

FIG. 1. Autoradiogram of a GLUT4 immunoblotof detergent extracts of soleus and EDL musclesfrom control and T3-treated rats. GLUT4 proteinin muscle extracts (40 \ig protein/lane) wasdetected with an antiserum against theCOOH-terminal region of GLUT4, as described inMETHODS. GLUT4 protein in soleus and EDLextracts from two control and two T3-treatedanimals is shown. Positions of molecular weightmarkers (in kilodaltons x 10~3) are shown onright.

- - + +method does not measure glucose uptake rates in individualmuscles.

Because of the clinical association of hyperthyroidismwith glucose intolerance in some patients (23), the observa-tion that thyroid hormone increases insulin-mediated glu-cose uptake might appear paradoxical. However, enhancedhepatic glucose output with incomplete suppression byinsulin may account for hyperglycemia associated with thy-rotoxicosis (3).

The second goal of this study was to determine whetherincreased expression of the major muscle glucose trans-porter GLUT4 could quantitatively explain the effects ofthyroid hormone on muscle glucose transport. T3 treatmentincreased GLUT4 protein expression in both soleus and EDL(Table 3), and the percentage increases in GLUT4 proteinconcentration were similar to the increases in insulin-stimu-lated 2-DG uptake (transport) in these muscles (Fig. 2). Thus,increased insulin-stimulated glucose transport in T3-treatedskeletal muscle can be accounted for by the induction ofGLUT4 protein, probably reflecting a direct effect of T3 onGLUT4 gene transcription (24). In contrast, the percentageincreases in basal [3H]2-DG uptake induced by T3 treatmentin both soleus and EDL were substantially greater than thepercentage increases in insulin-stimulated uptake (Table 1)and exceeded the percentage increases in GLUT4 concentra-

TABLE3Effect of T3 treatment on GLUT4 protein content in rat soleus andEDL muscles

Control

100(n-

±3= 10)

GLUT4 protein

Soleus

content

T3 treated

143:(n =

±5*10)

(arbitrary units/g wet wt)

Control

70(w =

±5= 10)

EDL

T3 treated

109(n =

± 7*= 10)

Rats were treated with T3 for 3 days, and GLUT4 protein indetergent extracts of soleus and EDL muscles was quantitated byimmunoblotting, as described in METHODS. The data shown arepooled from three independent experiments and are expressed as apercentage of the mean value in control soleus muscles, n, numberof muscle pairs. * P < 0.001 vs. control value.

tion (Fig. 2). Increased basal glucose transport thus cannotbe fully accounted for by GLUT4 induction alone. Thisincrease in basal transport is unlikely to reflect changes inmuscle GLUT1 content, because we have previously shownthat the low levels of GLUT1 in rat skeletal muscle are notaltered by T3 treatment (7).

The effects of thyroid hormone on both basal and insulin-stimulated glucose transport in muscle reported here may beexplained in terms of the current understanding of thesubcellular trafficking of GLUT4. Abundant evidence indi-cates that GLUT4 protein in both adipocytes and muscle ispartitioned between intracellular vesicles and the plasmamembrane (25) and cycles between these two pools (26,27).In the basal state, most GLUT4 resides intracellularly, butinsulin rapidly induces an increase in the fractional partition-ing of GLUT4 to the surface (plasma membrane) pool,resulting in increased membrane transport of glucose(28,29). Because insulin-stimulated 2-DG uptake increased inproportion to GLUT4 concentration in muscles from T3-

+ 1 6 0 I

I I Basal 2-DG uptake

Insulin-stimulated2-DG uptake

GLUT4 proteincontent

Soleus EDL

FIG. 2. Percentage increases in 2-DG uptake and GLUT4 protein contentinduced by T3 treatment in soleus and EDL. The percentage changeinduced by T., treatment is plotted for basal and insulin-stimulated 2-DGuptake and for GLUT4 protein content (data from Tables 1 and 3).Percentage change for individual data points in T3-treated animals wascalculated based on mean control values in each experiment. Barsindicate 1 SE.

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treated animals, the increase in maximal insulin-stimulated2-DG uptake in these muscles may be attributed to anincrease in the total GLUT4 pool, with little or no effect of T3

on the partitioning of GLUT4 between plasma membrane andintracellular sites in the insulin-stimulated state. In contrast,the observation that the percentage effect of T3 on basal2-DG uptake exceeded the percentage increase in the totalGLUT4 pool implies that thyroid hormone increased thefractional partitioning of GLUT4 to the plasma membrane inthe basal state.

We conclude that the increase in maximal insulin-stimu-lated 2-DG uptake in muscles from T3-treated rats is, in largepart, caused by the increase in GLUT4 protein content. Thisconclusion is supported by recent reports that overexpres-sion of GLUT4 mRNA and protein in transgenic mice resultsin increased insulin action and glycemic control (30,31). Inaddition, chronic exercise training may increase insulin-stimulated glucose transport in muscle by increasing GLUT4protein expression (32,33). Although skeletal muscle insulinresistance is generally not associated with reductions inmuscle GLUT4 content (34), the experimental evidence thatupregulation of GLUT4 gene expression in muscle mayincrease insulin-stimulated glucose transport suggests a the-oretical rationale for attempting to manipulate muscleGLUT4 expression as one potential therapeutic approach toinsulin resistance in skeletal muscle.

ACKNOWLEDGMENTSThis work was supported by National Institutes of HealthResearch Grants DK-41674 and DK-02057, a grant from BootsPharmaceuticals, and an Irma T. Hirschl Career ScientistAward (to R.S.H.).

The results were presented in part at the 67th annualmeeting of the American Thyroid Association, Tampa, FL, 13November 1993 (Thyroid 3 [Suppl.]:T-94, 1993).

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