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Endocrinology Vol. 138, No. 3 1204-1214
Copyright © 1997 by The Endocrine Society

Articles

Effects of Thyroid Hormone on GLUT4 Glucose Transporter Gene Expression and NIDDM in Rats

Christopher J. Torrance, James E. deVente, Jared P. Jones and G. Lynis Dohm

Department of Biochemistry, East Carolina University School of Medicine, Greenville, North Carolina 27858

Address all correspondence and requests for reprints to: Christopher J. Torrance, Ph.D., Johns Hopkins University, Department of Oncology Research Labs, 424 North Bond Street, Baltimore, Maryland 21231.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that T3 coordinately stimulates GLUT4-glucose transporter messenger RNA (mRNA) and protein expression in mixed fiber-type skeletal muscle of the rat and produces a concomitant elevation in basal (noninsulin mediated) glucose uptake. The aim of the present study was to 1) determine the precise mechanism(s) for the T3-induced expression of GLUT4 in skeletal muscle, and 2) investigate the potential benefits of T3 on noninsulin dependent diabetes mellitus (NIDDM). Ten daily ip injections of T3 (100 µg/100 g BW) administered to hypothyroid male Sprague-Dawley rats, increased both GLUT4 mRNA and transcription approximately 70% (P < 0.05) in mixed fiber-type hindlimb skeletal muscle. Transcriptional induction was subsequently defined to be restricted to red (oxidative) muscle fibers (2.5-fold; P < 0.05), whereas GLUT4 protein was increased in both red and white (glycolytic) skeletal muscle. GLUT4 mRNA and protein expression were similarly inducible in the skeletal muscle of insulin-resistant Zucker rats. More importantly, T3 treatment totally ameliorated hyperinsulinemia in obese animals (P < 0.001), although their moderately elevated plasma glucose levels were not significantly altered. In conclusion, regulation of GLUT4 expression by T3 was shown to lie at the transcriptional level in red skeletal muscle, whereas in white muscle fiber types, it appears to operate via an alternative posttranscriptional mechanism. These data also support the potential of hormonally inducing glucose transporter expression in insulin-resistant muscle. However, high levels of T3 are associated with a number of adverse side-effects, in particular the stimulation of hepatic gluconeogenesis. Nevertheless, future studies may demonstrate, e.g. subthyrotoxic levels, to be similarly effective but without side effects, and thus perhaps find a clinical application in reducing both hyperinsulinemia and hyperglycemia in NIDDM.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The uptake of glucose into mammalian cells is mediated via a family of tissue-specific membrane transport proteins (GLUTs 1–4), the process of which represents the rate limiting step in glucose metabolism within most tissues (1, 2). The insulin-sensitive glucose transporter (GLUT4) is the predominant isoform expressed in skeletal muscle and functions largely to rapidly normalize circulating plasma glucose levels following a meal (3). Indeed, skeletal muscle accounts for greater than 85% of whole body insulin-stimulated glucose disposal (4), and defects in glucose transport within this tissue represent the primary cause of insulin resistance, hyperinsulinemia and hyperglycemia in noninsulin dependent diabetes mellitus (NIDDM) (3).

The molecular defect in glucose disposal leading to NIDDM has been suggested to reside within the insulin signaling pathway (5, 6, 7), although cause and effect have not been established in this disease. Nevertheless, GLUT4 protein levels in skeletal muscle are generally normal in type II diabetics (8). Indeed, elucidating positive regulators of GLUT4 gene expression, as well as their mode of action, are of considerable interest for the development of novel therapeutic agents to stimulate glucose disposal in type II diabetics. This hypothesis is exemplified by the development of transgenic mice, in which overexpression of either GLUT1 specifically within skeletal muscle of normal mice (9), or GLUT4 in the skeletal muscle and adipose tissue of diabetic mice (10), results in constitutively high levels of basal (noninsulin mediated) glucose disposal. Moreover, overexpression of GLUT4 in diabetic mice was shown to be entirely adequate for the restoration of efficient glycemic control in both the fed and fasted states (10).

Previous studies from our laboratory (11) and by Weinstein et al. (12, 13) have demonstrated that T3 directly stimulates basal, and to some extent, insulin-mediated glucose uptake in rat skeletal muscle. These studies also account for the numerous reports in the literature of elevated whole body (14, 15) and skeletal muscle (16, 17, 18, 19) glucose utilization in human and experimental models of hyperthyroidism. The mechanism for this induction was shown to be due primarily to an increase in GLUT4 protein expression (11); and subsequently, Weinstein et al. (13) demonstrated that GLUT4 protein and mRNA were coordinately regulated by T3 in rat skeletal muscle.

Thyroid hormone action on gene expression is mediated primarily at the level of gene transcription via a family of somewhat tissue specifically expressed nuclear receptor/transcription factors: c-erbA {alpha}1, ß1, and ß2 (20, 21, 22). However, T3 also regulates the splicing and/or mRNA stability of a number of gene products (23, 24, 25, 26, 27, 28), although the mechanisms for these processes are not understood. The initial aim of the present study was to define the precise mechanism(s) for the induction of GLUT4 messenger RNA (mRNA) by T3, and thus establish whether transcriptional induction by T3 represents a viable target to augment GLUT4 expression in insulin-resistant skeletal muscle. Indeed, GLUT4 expression was shown to be regulated at the transcriptional level by thyroid hormone. However, transcriptional induction was found to be restricted to red (oxidative) muscle fiber types.

The second aim of this study was to investigate the potential therapeutic application of T3 to stimulate GLUT4 expression in NIDDM; specifically, by determining its effects on skeletal GLUT4 expression, hyperinsulinemia, and hyperglycemia in obese/insulin resistant Zucker (fa/fa) rats. The Zucker rat is a well established and commonly used animal model of obesity and insulin resistance (29, 30). However, at variance with insulin resistant humans, obese Zucker rats do demonstrate lower levels of skeletal muscle GLUT4. Interestingly, this may be a consequence of their significantly lower levels of circulating T3. These differences notwithstanding, T3 was presently shown to be highly effective in stimulating GLUT4 gene expression in obese/insulin resistant Zucker rat skeletal muscle, and resulted in the total amelioration of their hyperinsulinemia. However, presumably due to the well established stimulatory effects of T3 on hepatic glucose production, the glycemic status of obese animals was unfortunately not improved. Nevertheless, the present study does serve to demonstrate the utility of hormonally targeting transcriptional induction of GLUT4 in insulin-resistant skeletal muscle. Moreover, further investigation may elucidate a means to avoid the side effects of high levels of T3, while maintaining the beneficial effects of T3 now apparent on glucose disposal, e.g. using subthyrotoxic doses and/or cotherapeutic regimens designed to inhibit hepatic gluconeogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
Male Sprague-Dawley rats (250–275 g) and male Zucker rats (lean, 304 ± 17 g; obese, 393 ± 9 g) were used in these studies (Harlan Sprague Dawley, Inc., Indianapolis, IN). In experiments using Sprague-Dawley rats, animals were pretreated with propylthiouracil administered in their drinking water (600 mg/liter) ad libitum to induce hypothyroidism before T3-treatment (confirmed by T3 assay). Animals received daily doses of T3 (100 µg/100 g BW) via ip injection for either 4 h, 3 days, or 10 days, with control animals receiving sham injections of the 25 mM NaOH carrier. In experimental groups spanning the entire time range, all animals (except controls) received T3 injections 4 h prior to death. Animals were stunned and killed by decapitation, and blood samples collected (where applicable) for the determination of total plasma T3, glucose, and insulin levels. The gastrocnemius and quadriceps muscle were excised, minced with scissors, pooled, and aliquots (~400 mg) quick-frozen for the isolation of RNA or protein for Northern and Western blot analyses, respectively. The remaining muscle (~8 g) was used to isolate nuclei for nuclear run-on experiments. In experiments requiring separated red and white muscle fibers, the red and white components of the gastrocnemius and quadriceps muscles were visually separated with scissors before pooling and mincing. Animal housing and protocols were approved by the Animal Use Committee of the School of Medicine, East Carolina University. All chemicals and reagents, unless otherwise stated, were purchased from Sigma (St. Louis, MO) or Fisher Scientific (Springfield, NJ).

Nuclei isolation
Nuclei were isolated from rat skeletal muscle by the method of Zahradka et al. (31) with certain modifications (32). Approximately 8 g of muscle tissue was homogenized in 500 ml of cold (4 C) lysis buffer [10 mM N-2-hydroxyethylpiperzine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 5 mM KCl, 10 mM MgCl, 5 mM ß-mercaptoethanol] containing 0.32 M sucrose using a polytron, and the homogenates kept on ice. Homogenates were filtered through four layers of cheese cloth and a 100-mesh stainless steel screen (Fischer Scientific) to remove cellular debris. A crude nuclear fraction was collected by low speed centrifugation (1000 x g) for 10 min in a Sorvall GSA rotor and resuspended thoroughly in 35 ml of cold lysis buffer (4 C) containing 2.2 M sucrose. This suspension was then subjected to high speed centrifugation at (27,000 rpm) for 90 min, 4 C, in a Beckman SW28 rotor. The resulting nuclear pellet was rinsed with cold (4 C) lysis buffer and resuspended in 2 ml of cold (4 C) storage buffer [75 mM HEPES, pH 7.5, 60 mM KCl, 15 mM NaCl, 0.5 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM EGTA, 40% glycerol]. This suspension was centrifuged for 10 min at 5000 x g, 4 C, and the pellet resuspended in 200 µl of cold storage buffer. Nuclei yields were determined spectrophotometrically by lysing 10 µl of the final suspension in 990 µl of 0.1% SDS, and calculating DNA concentrations from the absorbance values at 260 and 230 nm (33). Samples were then quick frozen in liquid nitrogen for storage at -80 C until required for analysis.

Nuclear run-on analysis
Nuclear run-on analysis was performed either by the modified method of Cornelius et al. (32), or by a modification of the method described by Srivastava et al. (34). In the first method, RNA was isolated using CsCl density gradient centrifugation, and in the second, a simplified method using TRIzol reagent was employed (GIBCO-BRL, Gaithersburg, MD). Before RNA isolation (i.e. the in vitro transcription reactions) all methods were as described previously by Neufer et al. (32). Briefly, suspensions containing 100–250 µg (180 µl) of nuclei from control and thyroid hormone treated groups were allowed to complete the synthesis of nascent RNA transcripts at 25 C for 30 min, in a reaction mixture containing final concentrations of 58.7 mM HEPES, pH 7.5, 80 mM KCl, 11.7 mM NaCl, 6.5 mM DTT, 5 mM MgCl, 78 mM EDTA, 78 mM EGTA, 0.6 mM ATP, 0.3 mM GTP, CTP, and 0.4 mM [{alpha}-32P]UTP (250 µCi/reaction) (Dupont-NEN, Boston, MA) with 40 U RNasin (Promega, Madison, WI) in a total volume of 230 µl. After 30 min, the samples were treated with 25 U RNase free DNase (Promega) for 5 min, followed either by the addition of 3 ml of 4 M guanidium thiocyanate and the subsequent CsCl isolation of RNA (method 1), or by 0.5 ml of TRIzol (GIBCO-BRL) (method 2) with the isolation of RNA as described in the following RNA isolation and Northern blot analysissection. Purified 32P RNA isolated by either method, was then resuspended in 0.5 ml hybridization solution (Hybrisol) (Oncor, Gaithersburg, MD), and the samples heated at 65 C for 10 min with intermittent vortexing to ensure complete RNA resuspension and denaturation. Two microliters of this suspension were removed to determine the yield of 32P RNA by scintillation counting. Equal counts per minute (cpm) per sample were then hybridized at 47 C with separate hybond filter strips containing 2 µg each of the following UV-immobilized cDNAs: the full length GLUT4 cDNA; plasmid (pGEM) serving as a negative background hybridization control; and 0.2 µg of either total rat genomic DNA, or a cDNA complimentary to chicken 18S ribosomal RNA, to control for variances in total 32P-RNA counts added in each hybridization bag. Following hybridization for 72 h, the nylon filter strips were then washed for 30 min at 50 C in 2 x standard saline citrate (SSC), 30 min at 37 C in 2 x SSC containing 10 µg/ml of RNase A, and finally for 30 min at 55 C in 0.1 x SSC, 0.1% SDS. After drying, filter strips were then subjected to either autoradiography for 7–14 days and quantitation by laser densitometry, or were visualized and quantified using a phosphoimager screen and scanning system (Molecular Dynamics, Sunnyvale, CA) after approximately 2 days of exposure.

RNA isolation and Northern blot analysis
Quick frozen aliquots of mixed, red, or white skeletal muscle obtained during nuclei preparation, were powdered in a cold steel mortar and pestle, and approximately 100 mg used for the isolation of RNA. Total RNA was isolated using TRIzol reagent (GIBCO-BRL) by a modified method of the manufacturer’s instructions. Briefly, samples were homogenized on ice using a polytron in 1 ml of TRIzol reagent, centrifuged at 12,000 x g, and the supernatant decanted from the cellular debris into a fresh microfuge tube. Two hundred microliters of chloroform were then added, and the samples incubated at room temperature for 5 min. The samples were then vortexed vigorously for 30 sec, incubated at room temperature for another 2–3 min, and centrifuged at 12,000 x g for 15 min at 4 C. The top aqueous phase was removed with a sterile pipette, placed into a fresh diethyl pyrocarbonate (DEPC)-treated microfuge tube, and the RNA precipitated by adding an equal volume of isopropanol for 10 min at room temperature and centrifugation in a microfuge at 12,000 x g for 15 min. The pellets were then washed with 0.5 ml of DEPC-treated 4 M LiCl, subjected to a second 5-min microfuge at 12,000 x g, and a final wash with 75% ethanol before air drying for approximately 10 min. Samples were then resuspended in 100 µl of DEPC-treated H2O, and the RNA concentrations calculated spectrophotometrically from the absorbance at 260 nm using a 1:100 diluted aliquot (10 µl) removed from each sample. Twenty micrograms of RNA per sample were then denatured and size fractionated on a 1.25% agarose, 2.0 M formaldehyde gel, and subjected to Northern blot analysis as described previously (32); except that the hybridization solution (Hybrisol) was obtained commercially from Oncor. All cDNA probes, i.e. full length GLUT4, 18S ribosomal RNA (pRibo), ß-actin, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were labeled with [{alpha}-32P]ATP by random priming using the method of Feinberg and Vogelstein (35). The resulting Northern blots were visualized in some cases by autoradiography and quantitated using laser densitometry (Figs. 1Go, 2Go, and 3Go), or were quantitated using phosphoimager analysis (Figs. 4Go and 5Go).



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  		Figure 1. Effect of 10 days T3 treatment on GLUT4 mRNA levels in mixed rat hindlimb skeletal muscle. Northern blot of 20 µg of total RNA isolated from the pooled quadriceps and gastrocnemius skeletal muscles of hypothyroid control (C), and 10 day T3-treated hyperthyroid rats (T). Fractionated total RNA was stained with ethidium bromide to verify 28 and 18S ribosomal RNA integrity (lower panel), transferred to a nylon membrane, and then probed sequentially for GLUT4, ß-actin, and 18S ribosomal RNA (pRibo). The results of densitometric scanning are expressed relative to hypothyroid controls (C) (±SEM), and normalized to 18S ribosomal RNA (pRibo) to account for variations in gel loading. (n) = five rats per group; *, P < 0.05, **, P < 0.01.

 


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  		Figure 2. Effect of 10 days T3 treatment on GLUT4 transcription in mixed rat hindlimb skeletal muscle. Representative results from a nuclear run-on experiment to determine GLUT4 transcription rates in hypothyroid control (C) (n = 6), and 10 day T3 treated hyperthyroid rats (T) (n = 7). Data from densitometric scanning of the GLUT4 signals are expressed relative to hypothyroid controls (±SEM), and normalized to genomic DNA to account for variations in total 32P-labeled RNA during hybridization. Plasmid = linearized pGEM plasmid and represents a negative control for nonspecific hybridization. *, P < 0.05.

 


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  		Figure 3. Effect of 3 days T3 treatment on GLUT4 mRNA expression in rat soleus skeletal muscle. Northern blot of 20 µg total RNA isolated from the soleus muscle of control hypothyroid (n = 6), and 3 day T3-treated hyperthyroid rats (T) (n = 6). Data from densitometric scanning are expressed relative to hypothyroid controls (±SEM), and normalized to pRibo (18S ribosomal RNA) for variations in gel loading. **, P < 0.01.

 


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  		Figure 4. Time course of T3 treatment on GLUT4 mRNA levels in red and white rat skeletal muscle. Northern blots of 20 µg total RNA isolated from the separated red and white fibers of the pooled quadriceps and gastrocnemius muscles of control hypothyroid (C), and T3-treated (T) hyperthyroid rats: A) Phosphoimager data for time points 4 h and 3 days (n = 4). B) Autoradiographic data for 10 day T3-treated rats (n = 6). All data are expressed relative to hypothyroid controls (±SEM) for each fiber type, and normalized to probe (18S ribosomal RNA) for variations in gel loading. *,P < 0.05. ADP < 0.01.

 


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  		Figure 5. Time course of T3 treatment on GLUT4 transcription in red and white rat skeletal muscle. Nuclear run-on experiments to determine GLUT4 transcription rates in the separated red and white fiber components of the pooled quadriceps and gastrocnemius muscles of control hypothyroid (C) and T3-treated (T) hyperthyroid rats: A, Autoradiographic data for time points 4 h and 3 days (n = 1; however, nuclei were pooled from two separate rats); B, phosphoimager data for 10 day T3-treated rats (n = 3). All data are expressed relative to hypothyroid controls (±SEM) for each fiber type, subtracted for hybridization to nonspecific plasmid DNA (pGEM), and normalized to either genomic DNA (left panel), or 18S Ribosomal RNA (pRibo; right panel) for variations in total 32P-labeled RNA during hybridization. Due to variability in genomic signals in this experiment, and for direct comparison of Northern and transcription data, pRibo was used to normalize these results. *, P < 0.05.

 
Simultaneous isolation of RNA and DNA
RNA and DNA were isolated simultaneously from rat skeletal muscle using TRIzol reagent according to the manufacturers’ instructions (GIBCO-BRL). Briefly, RNA was isolated as described above using 1 ml TRIzol and 100 mg tissue, except that centrifugation to remove cellular debris was omitted. In addition, after the removal of the aqueous phase, the remaining organic phase and interphase were retained in order to isolate cellular genomic DNA. DNA was isolated by adding 0.3 ml of 100% ethanol to the organic- and inter-phase, mixing by inversion several times, and then incubated at room temperature for 2–3 min. Samples were then centrifuged at less than 2000 x g in a microfuge for 5 min at 4 C, and the supernatant discarded. The pellet, which consists of mostly cellular debris (large and red colored due to contaminating dye from the organic phase), was washed extensively (4x) using 0.1 M sodium citrate/10% ethanol, incubating each time for 30 min, followed by centrifugation as before. DNA was then suspended in 1.5 ml 75% ethanol, incubated at room temperature for 30 min, and microfuged as before. The resulting DNA pellet (which may not be particularly visible due to the large cell debris pellet which still remains) was washed once again with 0.1 M sodium citrate/10% ethanol, pelleted, and then resuspended in 0.5 ml of 8 mM NaOH. Finally, the remaining particulate matter was removed by a high speed centrifugation for 10 min at 12,000 x g (4 C), and an aliquot of the supernatant removed for the spectrophotometric (A260) determination of the DNA yield (expected yields from skeletal muscle; DNA ~ 1.0–1.5 µg/mg tissue; RNA ~ 0.5–1.0 µg/mg tissue).

Western blotting
Total membrane preparations were isolated from frozen aliquots (50–100 mg) of powdered muscle by homogenizing in 2 ml of buffer [25 mM HEPES, pH 7.4, 25 mM benzamidine, 4 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM each of leupeptin, pepstatin, and aprotinin] using a polytron, followed by centrifugation at 150,000 x g for 1 h at 4 C. The resulting pellet was homogenized again in 200 µl buffer, Triton X-100 added to a final concentration of 1%, and the samples left to incubate at room temperature for 1.5 h. Solubilized total membrane proteins were then collected by a final spin at 150,000 x g for 1 h at 4 C, and the supernatant assayed for protein concentration using BCA reagent (Pierce, Rockford, IL). Samples were stored at -70 C until required for analysis. For the quantitation of GLUT4, 50 µg of total membrane protein per sample was subjected to SDS-PAGE on a 10% resolving gel by the method of Laemmli et al. (36). Western blot analysis was performed as described previously (32) using a C-terminal GLUT4 rabbit polyclonal antibody, and a horseradish peroxidase-conjugated donkey antirabbit immunoglobulin second antibody (Amersham, Arlington Heights, IL), with detection by the enhanced chemiluminescence method (Amersham) and quantitation by densitometric scanning of the autoradiographic GLUT4 signals.

Plasma T3 analysis
Total plasma T3 levels were determined in duplicate using a commercially available T3 microparticle enzyme immunoassay (MEIA) kit (Abbott Laboratories, Abbott Park, IL) performed on the automated IMx assay system (Abbott Laboratories) according to the manufacturer’s instructions. Plasma samples from T3-treated rats were diluted 1:10 before analysis.

Plasma insulin analysis
Plasma insulin concentrations (ng/ml) in experimental animals were determined using a commercially available RIA kit (Linco Research, St. Louis, MO.) according to the manufacturer’s instructions. Plasma samples from obese rats were diluted 1:10 before analysis.

Plasma glucose analysis
Plasma glucose levels (mg/dl) in experimental animals were determined using an enzymatic (glucose oxidase/O-dianisidine) colorimetric assay according to the manufacturer’s instructions (Sigma, St. Louis, MO). All samples were analyzed in duplicate and diluted 1:20 with H2O.

Statistics
Data were analyzed for statistical significance between experimental groups using two-way ANOVA, or Student’s t test, with significance set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thyroid hormone stimulates GLUT4 mRNA and transcription in mixed hindlimb rat skeletal muscle
To determine the mechanism(s) for the induction of GLUT4 mRNA by T3 in rat skeletal muscle, Northern blot, and nuclear run-on analyses were performed on total RNA and nuclei, respectively, isolated from the pooled gastrocnemius and quadriceps muscles of T3-treated male Sprague-Dawley rats (animals were made hypothyroid before T3-treatment to maximize the visualization of effects on GLUT4 expression). The Northern blot data (Fig. 1Go), demonstrated that 10 daily ip injections of T3 (100 µg/100 g BW; thyrotoxic doses) produced an approximate 70% (P < 0.05) increase in GLUT4 mRNA over hypothyroid carrier-injected controls. This value was somewhat lower than reported in a previous study (13) that demonstrated a 2.5-fold induction in GLUT4 mRNA in the mixed fiber type anterior tibialis muscle of 3 day T3-treated male Sprague-Dawley rats (100 µg/100 g BW). However, the previous data were normalized to ß-actin mRNA levels, which presently were shown to be down-regulated approximately 2-fold with 10 days of T3 treatment (Fig. 1Go). Therefore, an explanation for the greater stimulation of GLUT4 mRNA reported previously (13), would appear to be an artifact of using ß-actin as a loading control, and consequently, all Northern data in this report were normalized for variances in RNA loading using a complementary DNA (cDNA) probe complimentary to 18S ribosomal RNA (pRibo).

Nuclear run-on analysis using nuclei isolated from the same muscle preparations as above, demonstrated that 10 days of T3 treatment produced an identical 70% (P < 0.05) increase in GLUT4 transcription (Fig. 2Go). Therefore, these data indicate that induction of GLUT4 mRNA by T3 can be entirely accounted for by corresponding increase in GLUT4 gene transcription. Interestingly, however, the induction of GLUT4 mRNA in soleus muscle [a predominantly red (oxidative; type 1) muscle type] was found to be greater (approximately 2.5-fold) with 3 days T3 treatment (Fig. 3Go) than demonstrated by the mixed fiber type gastrocnemius and quadriceps muscles (Fig. 1Go). Due to this observation, as well as previous reports indicating fiber-type specificity in response to a number of other stimuli, e.g. exercise (32), a series of experiments were initiated to determine whether fiber type differences also exist for the induction of GLUT4 by T3.

Thyroid hormone stimulates GLUT4 mRNA and transcription specifically within red (oxidative) fibers in rat hindlimb skeletal muscle
To determine whether fiber type differences exist for the regulation of GLUT4 mRNA and transcription by T3, Northern blot and nuclear run-on analyses were performed as described above on the separated red (oxidative; insulin sensitive) and white (glycolytic; insulin resistant) components of the previously pooled gastrocnemius and quadriceps muscles. However, in addition to the standard 10 days of T3 treatment, hypothyroid rats were also treated for 4 h and 3 days to define a limited time course for the effects of T3 on GLUT4 gene transcription and mRNA.

Consistent with the previous data using soleus muscle (Fig. 3Go), GLUT4 mRNA induction by T3 was shown to be restricted exclusively to the red fibers of the gastrocnemius and quadriceps muscles (Fig. 4Go, A and B). In addition, a predictable result of this marked fiber type selectivity was the somewhat higher increase in GLUT4 mRNA observed with 10 days of T3 treatment (2.5-fold) (Fig. 4BGo) compared with the previous mixed muscle preparations (Fig. 1Go). This we presume was due to a dilution effect contributed by the unresponsive white muscle fiber components within the pooled gastrocnemius and quadriceps preparations. Moreover, nuclear run-on analysis demonstrated that induction of GLUT4 transcription was also limited to red muscle, with an identical 2.5-fold stimulation at 10 days (P < 0.05) (Fig. 5Go, A and B). In summary, these data corroborate with the previous mixed muscle experiments and demonstrate that the mechanism of GLUT4 mRNA induction by T3 lies solely at the transcriptional level; the effects of which can now be stated to be restricted to red skeletal muscle fiber types in the rat.

Thyroid hormone stimulates GLUT4 protein expression in both red and white skeletal muscle fiber types
Due to the novel observation of fiber type variances in GLUT4 mRNA induction by T3, a Western blot using total membrane protein isolated from the identical muscle preparations as used in Fig. 4BGo was performed to confirm whether the selective stimulation of GLUT4 in red muscle was retained at the protein level. Surprisingly, GLUT4 protein expression was increased approximately 5-fold (P < 0.01) in both red and white muscle fiber types with 10 days of T3 treatment (Fig. 6Go). This observation was also confirmed using a different set of control and T3-treated rats, in which an incremental stimulation of GLUT4 protein occurred in both red and white muscle over the entire 4-h, 3-day, and 10-day time course (data not shown). Therefore an additional posttranscriptional mechanism (presumably representing translational activation and/or a reduction in GLUT4 protein degradation) would appear to exist in skeletal muscle to stimulate GLUT4 protein expression. This conclusion is clearly apparent in white muscle fiber types because no specific increase in GLUT4 mRNA was previously demonstrated. However, this putative effect on GLUT4 protein expression would also appear to be manifest in red muscle due to the greater increase observed for GLUT4 protein over its effects on mRNA.



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  		Figure 6. Effect of 10 days T3 treatment on GLUT4 protein in red and white rat skeletal muscle. Representative Western blot of 50 µg total membrane protein to determine relative GLUT4 protein levels in red and white quadriceps and gastrocnemius muscle of control (C) (n = 6), and 10 day T3-treated hyperthyroid rats (T) (n = 6). The results of densitometric scanning were calculated using autoradiographic exposures within the linear range, and are expressed relative to hypothyroid controls (±SEM) for each fiber type. *, P < 0.01; **, P < 0.001.

 
T3 stimulates total RNA synthesis in rat skeletal muscle
Although a putative posttranscriptional mechanism would appear to be evident from the previous data, an observation made during the nuclear run-on experiments revealed perhaps an alternative, albeit nonspecific, effect which may explain the disparity between GLUT4 mRNA and protein induction by T3. Table 1Go illustrates the incorporation of 32P-UTP into nascent RNA during the in vitro transcription reactions of hypothyroid control and 10 day T3-treated muscle nuclei. These data, normalized for DNA content, demonstrate that nuclei derived from T3-treated animals synthesize approximately twice as much total RNA compared with the hypothyroid controls. Indeed, T3 has long been known to stimulate the synthesis of all classes of RNA in a number of other tissues (37, 38, 39), and we confirmed this effect by demonstrating an average 80% increase (P < 0.01) in the total cellular RNA/DNA ratio in both muscle types with T3 treatment (data not shown). Therefore, because ribosomal RNA (rRNA) comprises approximately 90% of total RNA, and that Northern blots were loaded per unit RNA and normalized to 18S ribosomal RNA, this nonspecific effect of T3 would lead to an approximate 2-fold underestimate of the specific effects on GLUT4 mRNA. While this may be sufficient to explain the additional increase in GLUT4 protein in red muscle, in the absence of specific increase in GLUT4 mRNA and/or transcription (Figs. 4Go and 5Go), this phenomenon alone is clearly inadequate to account for the 5-fold stimulation of GLUT4 protein observed in white muscle. This conclusion was further substantiated by loading RNA on a repeat Northern blot on a per unit DNA basis (data not shown).


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  		Table 1. Effect of 10 days T3 treatment on total transcription in whole rat hindlimb skeletal muscle

 
Effects of thyroid hormone on GLUT4 expression in obese Zucker rats
Due the large stimulatory effects of T3, particularly on GLUT4 protein expression, it was of great interest to investigate whether T3 has any beneficial properties on NIDDM. To this end, the effects of thyroid hormone treatment on GLUT4 gene expression in insulin-resistant skeletal muscle, hyperinsulinemia, and hyperglycemia were next studied in an animal model of insulin resistance; namely the obese (fa/fa) Zucker rat. Total plasma T3 levels in lean (Fa/fa), obese (fa/fa), and T3-treated lean and obese Zucker rats were first assayed to establish the thyroidal status of the experimental animals. Three days of T3 treatment (100 µg/100 g BW) clearly induced hyperthyroidism in both lean and obese animals, the levels of which were approximately 100-fold, and 200-fold, respectively, over the corresponding euthyroid (non-PTU treated) controls (P < 0.001) (Table 2Go). In addition, consistent with previous reports (40, 41), control obese Zucker rats exhibited significantly lower plasma T3 levels compared with their lean (euthyroid) control littermates (P < 0.05), although they remained within the euthyroid range.


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  		Table 2. Plasma T3 levels (±SEM) in control (euthyroid) lean and obese, and T3 treated lean and obese Zucker rats (n = 5 rats per group)

 
Northern blot and Western blot analyses were next performed on total RNA and total membrane protein, respectively, isolated from the pooled gastrocnemius and quadriceps muscles of control (euthyroid), and 3 day T3-treated (hyperthyroid) lean and obese Zucker rats. Northern blot analysis (Fig. 7Go) demonstrated that GLUT4 mRNA levels in lean euthyroid Zucker rat skeletal muscle were not significantly increased with 3 days of T3 treatment, although the trend was clearly present. In contrast, due to the initial trend toward reduced levels of GLUT4 mRNA expression in obese rat skeletal muscle (~25%), 3 days of T3 treatment resulted in a significant 2-fold (P < 0.05) increase in GLUT4 mRNA; and occurred without any significant changes in body weight (starting/final body weights: lean controls = 304 ± 17 g/304 ± 17 g; obese controls, 393 ± 19 g/404 ± 16 g; leans + T3, 285 ± 31 g/274 ± 28*; obese + T3 = 410 ± 16/385 ± 13; *, P < 0.05 vs. final weights of lean controls; no statistical differences were present between obese animal weights with T3 treatment). Moreover, in obese rats the extent of this induction was sufficient to elevate GLUT4 mRNA levels to equal that of their lean littermates. Western blot analysis (Fig. 8Go) demonstrated similar results; a significant (P < 0.05) 75% reduction in GLUT4 protein expression was observed in obese control rats, and an approximate 3.0-fold induction in obese rats with 3 days of T3 treatment (P < 0.05). In addition, a significant 2-fold (P < 0.05) increase in GLUT4 protein was demonstrated in lean T3-treated rats. In summary, these data demonstrated that administration of T3 was also highly effective in augmenting GLUT4 mRNA and protein expression in obese/insulin resistant rat skeletal muscle.



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  		Figure 7. Effect of 3 days T3 treatment on GLUT4 mRNA levels in lean and obese Zucker rat hindlimb skeletal muscle. Northern blot of 20 µg total RNA isolated from the pooled quadriceps and gastrocnemius skeletal muscles of control (euthyroid) lean (L), obese (O), and 3-day T3-treated (hyperthyroid) lean (L + T3) and obese (O + T3) Zucker rats. Fractionated total RNA was stained with ethidium bromide to verify 28 and 18S ribosomal RNA integrity (lower panel), transferred to a nylon membrane, and then sequentially for GLUT4 (upper panel), glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (middle panel), and 18S ribosomal RNA (data not shown). The results of densitometric scanning are expressed relative to lean controls (L) (±SEM), and normalized to G3PDH to account for variations in gel loading. n = 3 rats per group; +, P = <0.01 vs. obese controls (two-way ANOVA).

 


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  		Figure 8. Effect of 3 days T3 treatment on GLUT4 protein in lean and obese Zucker rat hindlimb skeletal muscle. Representative Western blot of 50 µg total membrane protein to determine relative GLUT4 protein levels in the pooled quadriceps and gastrocnemius muscles of euthyroid lean (L), obese (O), and 3 day T3-treated (hyperthyroid) lean (L + T3) and obese (O + T3) Zucker rats (n = 6 per group). The results of densitometric scanning were calculated using autoradiographic exposures within the linear range, and expressed relative to lean controls (±SEM). *, P < 0.01 vs. lean controls (top panel), +, P < 0.01 vs. obese controls. (All data analyzed using two-way ANOVA).

 
Thyroid hormone ameliorates hyperinsulinemia in obese Zucker rats
To determine whether T3, in addition to stimulating skeletal muscle GLUT4 expression, had any beneficial effects on hyperinsulinemia and/or glycemia in obese Zucker rats, plasma insulin, and glucose levels were assayed in the blood collected from the above control (euthyroid) and 3 day T3-treated (hyperthyroid) lean and obese Zucker rats. Plasma glucose levels were elevated in all groups compared with the lean controls (P < 0.05), and in fact a slight detrimental effect of T3 treatment was observed in lean animals (Fig. 9Go, top panel). Notwithstanding, a marked 80% reduction (P < 0.001) in plasma insulin levels in obese rats was demonstrated with 3 days of T3 treatment (Fig. 9Go, lower panel). Moreover, the extent of this reduction (final concentration = 5.4 ng/ml) was almost sufficient to restore insulin levels to within the normal range (0.5–2 ng/ml).



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  		Figure 9. Effect of 3 days T3 treatment on serum glucose and insulin levels in lean and obese Zucker rats. Plasma glucose concentrations (mg/dl) (top panel) and plasma insulin levels (ng/ml) (lower panel) in euthyroid lean (L), obese (O), and 3 day T3-treated (hyperthyroid) lean (L + T3) and obese (O + T3) Zucker rats (n = 6 per group) (statistical analysis via two-way ANOVA).

 
In conclusion, thyroid hormone treatment was shown to be highly effective in eliminating hyperinsulinemia in obese Zucker rats. However, presumably due to the previously described antagonistic effects of T3 on liver glucose production (42, 43), the moderate glycemia demonstrated by obese animals was unable to be lowered.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The initial aim of this study was to determine the relative effects of thyroid hormone on GLUT4 mRNA and transcription in skeletal muscle, and thus establish the mechanism(s) for the stimulatory effects of T3 on GLUT4 mRNA in this tissue. Consistent with a previous study suggesting the presence of a T3-responsive region within the GLUT4 promoter (44), the present experiments clearly demonstrated GLUT4 to be regulated at the transcriptional level within red fiber-types of rat skeletal muscle. The stimulation of GLUT4 mRNA was in fact restricted red muscle, and in concordance with this, the inductions of GLUT4 mRNA demonstrated in isolated red fiber types, or in soleus (which is comprised primarily of red muscle), were always higher than observed in mixed fiber-type muscle preparations. Nevertheless, even the maximal 2.5-fold increase in GLUT4 mRNA demonstrated presently with 10 days T3 treatment, only match the induction observed by Weinstein et al. (13) in mixed fiber-type mixed fiber type anterior tibialis muscle of 3 day T3-treated male Sprague-Dawley rats (rats were initially hypothyroid in both studies and received identical T3 doses; 100 µg/100 g BW). However, this variance probably derives from the fact that different controls were used in each study to normalize for differences in total RNA loaded onto Northern blot gels. Weinstein et al. (13) normalized their Northern data to ß-actin mRNA levels. However, we presently show ß-actin mRNA to be down-regulated approximately 2-fold in mixed fiber-type muscle with 10 days of T3 treatment (Fig. 1Go). Therefore, the mRNA data of Weinstein et al. are likely to be somewhat of an overestimate of the actual effects of T3 on GLUT4 expression.

Surprisingly, GLUT4 protein was found to be increased in both red and white skeletal muscle with T3 treatment. This increase in GLUT4 protein was greater than the stimulation of GLUT4 mRNA in red muscle, and in white muscle occurred without any perceptible increase in GLUT4 transcription or mRNA. These data were therefore suggestive of an additional posttranscriptional mechanism within both red and white muscle fiber types for the regulation of GLUT4 protein. However, T3 has been shown to nonspecifically increase the expression of all RNA classes in a number of tissues (37, 38, 39), and was presently shown to stimulate total cellular RNA synthesis in skeletal muscle. Therefore, because ribosomal RNA accounts for approximately 90% of cellular RNA, normalization of Northern and run-on data to 18S ribosomal RNA inevitably results in the under-estimation of the effects of T3 on a specific mRNA species, i.e. GLUT4. Hence, in red muscle the larger increase in GLUT4 protein over mRNA may be due primarily to a nonspecific stimulatory effect of T3 on total RNA synthesis. However, these data cannot discount the possibility that GLUT4 is also influenced to some degree at posttranscriptional level within red skeletal muscle.

An explanation for the selective stimulation of GLUT4 transcription and mRNA in red muscle was not readily apparent from these data. Conceivably, a qualitative and/or quantitative difference in thyroid hormone receptor expression in these two muscle fiber types may provide a plausible explanation for these results; however, preliminary studies from our laboratory would seem to discount this hypothesis (data not shown). Similarly, in white muscle a mechanism for the posttranscriptional regulation of gene expression by T3 is likewise unclear, although precedents do exist in the literature for the regulation of several genes at the posttranscriptional level by T3, e.g. hepatic triglyceride lipase (HTGL) (25), Na/K ATPase (24), malic enzyme (23, 26), and apolipoprotein B (27). Moreover, posttranscriptional regulation of GLUT4 has also been reported by a variety of other factors, i.e. muscle contraction/exercise (32), glucose (45), aging (46), and diabetes (47). It can be hypothesized, therefore, that a common mechanism may exist in this tissue to modulate GLUT4 protein expression in response to a diverse array of stimuli.

Interestingly, a recent observation may have shed some light on a potential posttranscriptional mechanism for the regulation of GLUT4 gene expression (48). In this study, the GLUT4 message in adipose and skeletal muscle was demonstrated to consist of two splice variants, with the longer mRNA species being less abundant but translated approximately five times more efficiently than the shorter spliced version (48). One can now perhaps visualize an explanation for a larger increase in GLUT4 protein over mRNA (especially within white skeletal muscle) due to the selective expression of the more translationally active splice variant. Indeed, consistent with this hypothesis, T3 has previously been shown to regulate the splicing of the apolipoprotein A-1 gene (27).

The second aim of this study was to determine whether T3 was able to stimulate GLUT4 gene expression in insulin resistant skeletal muscle, and thus perhaps demonstrate a beneficial effect on NIDDM. T3 was shown to be highly effective in stimulating GLUT4 mRNA and protein expression in obese/insulin-resistant Zucker rat skeletal muscle. More importantly, 3 days of T3 treatment resulted in the total amelioration of hyperinsulinemia in obese animals. Unfortunately, however, their plasma glucose levels were unable to be altered. Nevertheless, the extent of the reduction in hyperinsulinemia in obese animals was quite remarkable, especially in light of the fact that their moderately glycemic plasma glucose levels were not significantly reduced. Indeed, this observation may lead one to propose an intriguing hypothesis that a putative feedback loop may exist between skeletal muscle and pancreatic ß-cells; in this case to signal an adequate increase in basal glucose uptake (11, 12), and in nondiabetic individuals a sufficient degree of skeletal muscle insulin-sensitivity.

The lack of a desirable effect on glycemia, as well as a large number of undesirable side effects associated high levels of T3, especially in diabetic patients, e.g. tachycardia, would seem to negate any potential benefits of using T3 to stimulate glucose disposal in insulin-resistant skeletal muscle. However, the reason glycemia is not improved is also likely a consequence of using thyrotoxic doses of T3, i.e. high levels of T3 are known to stimulate hepatic gluconeogenesis (42, 43). Therefore, conceptually, it may still be possible to retain the beneficial effects of increasing GLUT4 expression, while avoiding stimulating hepatic glucose production and other side effects, by using, e.g. subthyrotoxic doses of T3. Interestingly, in a previous study which used a long term (6 weeks) regimen of powdered thyroid in obese Zucker rats (49), moderate reductions in both plasma insulin and glucose were demonstrated. However, in that study the effects of powdered thyroid (which is primarily composed of T4) could not be dissociated from the marked weight loss in the experimental animals. Indeed, promoting weight loss via stimulating basal metabolic rate would be another desirable effect of thyroid hormone because this has been established to have a marked positive impact on whole body insulin sensitivity (50, 51), and in many cases represents a real long-term solution for obese type II diabetics.

Notwithstanding, consistent with transgenic mice overexpressing GLUT4, the present study does serve at least to demonstrate the utility of hormonally inducing GLUT4 expression in insulin-resistant skeletal muscle. Moreover, because GLUT4 was also shown to be transcriptionally induced, at least in red muscle, and that preliminary data from our laboratory indicate that obese rat skeletal muscle possess the same pattern of {alpha}1-specific TR expression as normal skeletal muscle (52), a rationale is therefore suggested whereby skeletal muscle may be more effectively targeted for treatment in NIDDM using a future {alpha}1-isoform selective T3 analog. However, it needs to be determined whether T3 exerts its effects directly on GLUT4 expression via a thyroid hormone response element in the GLUT4 gene, the subject of which is investigated in the following paper.

In summary, this study established that GLUT4 gene expression in the rat is stimulated via transcriptional induction in red muscle, and a separate translational/posttranslational mechanism in white skeletal muscle. These studies also demonstrated the ability of T3 to stimulate GLUT4 gene expression in insulin-resistant skeletal muscle, and that T3 had a marked beneficial effect on hyperinsulinemia, but not glycemia in obese Zucker rats. Therefore, combined with the side-effects associated with high levels of T3 (in particular the counter-productive stimulation of hepatic gluconeogenesis), the clinical applications of this hormone in NIDDM would seem to be limited. However, the use of T3 in some form, e.g. subthyrotoxic doses, in combination with ß-blockers (53), or by a potentially muscle specific T3 analog, may be worthy of further investigation.

Received July 1, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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