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Am J Physiol Endocrinol Metab 288: E1265–E1269, 2005. First published December 14, 2004; doi:10.1152/ajpendo.00533.2004. TRANSLATIONAL PHYSIOLOGY Hyperthyroidism and cation pumps in human skeletal muscle Anne Lene Dalkjær Riis,1 Jens Otto Lunde Jørgensen,1 Niels Møller,1,2 Jørgen Weeke,1 and Torben Clausen3 1 Medical Department M (Endocrinology and Diabetes) and 2Medical Research Laboratories, Aarhus University Hospital, and 3Institute of Physiology, University of Aarhus, Aarhus, Denmark Submitted 2 November 2004; accepted in final form 6 December 2004 Ca2⫹-activated adenosine triphosphatase; Na⫹-K⫹-activated adenosine triphosphatase; energy expenditure 6 h was fourfold larger than in soleus from euthyroid rats (11). Thus thyroid hormones markedly upregulate the capacity for Ca2⫹ uptake, allowing increased recycling of Ca2⫹ and ATP turnover. The stimulation of energy expenditure (EE) induced by thyroid hormones is for a significant part mediated by an increase in the capacity and activity of the Ca2⫹-ATPase in SR (4, 6, 25). The effects of thyroid hormones on the content and activity of Na⫹-K⫹-ATPase in skeletal muscle, however, accounts at most for only 15% of their thermogenic action (4). We have previously demonstrated that the content of Na⫹-K⫹ATPase in human skeletal muscle is closely correlated to plasma 3,5,3⬘-triiodothyronine (T3) and thyroxine (T4) (17), but the relations between thyroid status and the total content of Ca2⫹-ATPase in human skeletal muscle have not been characterized. In a controlled design, we examined the effect of hyperthyroidism on the contents of Ca2⫹-ATPase and Na⫹-K⫹-ATPase in human skeletal muscle in hyperthyroid patients with Graves’ disease before and after treatment and compared with healthy control subjects. We are testing the following hypotheses. 1) In hyperthyroid subjects, the contents of Ca2⫹-ATPase and Na⫹-K⫹-ATPase in skeletal muscle are increased and restored toward the normal level following standard treatment of hyperthyroidism. 2) The contents of Ca2⫹-ATPase and Na⫹-K⫹-ATPase in skeletal muscle are correlated to REE and plasma thyroid hormones. METHODS of resting energy expenditure (REE) in humans, and skeletal muscle constitutes the major target organ for the thermogenic action of thyroid hormones. Skeletal muscle is by far the most abundant tissue of the human body and accounts for more than 25% of the total resting O2 consumption (4, 23, 31). Thus even a modest change in heat production in skeletal muscles leads to a significant modification of total body heat production (6). Animal studies show that thyroid hormones upregulate the content of Ca2⫹ATPase in the sarcoplasmic reticulum (SR) and the maximum rate of Ca2⫹ uptake in SR from rat soleus increases over a fourfold range with thyroid status (14, 26). More recently, the amount of SR protein recovered from hyperthyroid rabbit muscle was found to be fourfold larger than that obtained from control animals (6). In intact soleus muscles prepared from hyperthyroid rats, the accumulation of 45Ca as measured over Participants. Eleven hyperthyroid patients (9 women and 2 men) with newly diagnosed Graves’ disease were studied before and after 3 mo of medical treatment with methimazole. The clinical diagnosis of diffuse toxic goiter was confirmed by measurement of thyroid-stimulating hormone (TSH) receptor antibodies ⬎2 IU/l. In two cases, muscle biopsies could not be obtained: in one female hyperthyroid patient (before treatment) and in one female euthyroid patient (after treatment). These two patients did not differ from the rest of the patients, and the data from those patients are included in the mean values and in the unpaired analysis comparing with healthy control subjects. In the paired comparisons before and after treatment, however, only nine patients could be included. An age-matched control group of eight healthy women using no medication was studied once. All participants gave their written informed consent after receiving oral and written information concerning the study according to the Declaration of Helsinki II. The Aarhus County Ethical Scientific Committee approved the study, approval number 1998/4372. Address for reprint requests and other correspondence: A. L. D. Riis, Medical Dept. M, Aarhus Univ. Hospital, 8000 Aarhus C, Denmark (E-Mail: [email protected]). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. LEAN BODY MASS IS A MAJOR DETERMINANT http://www.ajpendo.org 0193-1849/05 $8.00 Copyright © 2005 the American Physiological Society E1265 Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on June 15, 2017 Riis, Anne Lene Dalkjær, Jens Otto Lunde Jørgensen, Niels Møller, Jørgen Weeke, and Torben Clausen. Hyperthyroidism and cation pumps in human skeletal muscle. Am J Physiol Endocrinol Metab 288: E1265–E1269, 2005. First published December 14, 2004; doi:10.1152/ajpendo.00533.2004.—Skeletal muscle constitutes the major target organ for the thermogenic action of thyroid hormone. We examined the possible relation between energy expenditure (EE), thyroid status, and the contents of Ca2⫹-ATPase and Na⫹-K⫹-ATPase in human skeletal muscle. Eleven hyperthyroid patients with Graves’ disease were studied before and after medical treatment with methimazole and compared with eight healthy subjects. Muscle biopsies were taken from the vastus lateralis muscle, and EE was determined by indirect calorimetry. Before treatment, the patients had two- to fivefold elevated total plasma T3 and 41% elevated EE compared with when euthyroidism had been achieved. In hyperthyroidism, the content of Ca2⫹-ATPase was increased: (mean ⫾ SD) 6,555 ⫾ 604 vs. 5,212 ⫾ 1,580 pmol/g in euthyroidism (P ⫽ 0.04) and 4,523 ⫾ 1,311 pmol/g in healthy controls (P ⫽ 0.0005). The content of Na⫹-K⫹ATPase showed 89% increase in hyperthyroidism: 558 ⫾ 101 vs. 296 ⫾ 34 pmol/g (P ⫽ 0.0001) in euthyroidism and 278 ⫾ 52 pmol/g in healthy controls (P ⬍ 0.0001). In euthyroidism, the contents of both cation pumps did not differ from those of healthy controls. The Ca2⫹-ATPase content was significantly correlated to plasma T3 and resting EE. This provides the first evidence that, in human skeletal muscle, the capacity for Ca2⫹ recycling and active Na⫹-K⫹ transport are correlated to EE and thyroid status. E1266 CATION PUMPS IN HYPERTHYROIDISM Table 1. Clinical characteristics and thyroid hormones in 11 hyperthyroid patients before and after treatment and in 8 control subjects Patients Age, yr (range) BMI, kg/m2 EE, kcal/24 h Total T3, (1.1–2.6) nmol/l Total T4, (58–161) nmol/l Free T3, (3.7–9.5) pmol/l Free T4 (12–33) pmol/l TSH, mol/ml median (range) P Values Hyperthyroid Euthyroid P Values Ht. vs. Eut. 36⫾9 (26–49) 20.8⫾2.3 2091⫾384 7.7⫾2.5 253⫾49 43⫾18 140⫾51 ⬍0.002 22.4⫾2.6 1485⫾239 1.7⫾0.5 94⫾33 7.9⫾3.0 26⫾7 0.0019 (⬍0.002–0.07) 0.0007 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0004 0.0001 0.068 Control Subjects Cont. vs. Ht. Cont. vs. Eut. 40⫾8 (24–46) 24.5⫾4.1 1498⫾163 1.3⫾0.2 77⫾15 6.3⫾0.7 23⫾3 1.5 (0.02–2.58) 0.5 0.03 0.0009 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.2 0.9 0.02 0.2 0.2 0.3 0.0003 Procedures. REE was evaluated by indirect calorimetry (Deltatrac; Datex Instrumentarium, Helsinki, Finland). Under local anaesthesia, needle biopsies were taken from the vastus lateralis muscle with a Bergström needle and immediately frozen in liquid N2 for later measurements. Ca2⫹-activated adenosine triphosphatase (Ca2⫹-ATPase) and Na⫹K⫹-ATPase contents in the muscle biopsies were determined as described in detail elsewhere (10, 21). For the measurement of Ca2⫹-ATPase, ⬃30 mg of frozen muscle tissue were homogenized at 0°C in a HEPES-sucrose buffer (pH 7.4) with an Ultra-Turrax homogenizer. To obtain a smaller particle size, the suspension was further homogenized at 0°C using a glass homogenizer with a tightfitting Teflon pestle rotating at 1,000 rpm. Aliquots (0.2 ml) of the homogenate were reacted for 30 s at 0°C in 3 ml of a buffer containing (in mM): 100 imidazole, 100 KCl, 5 MgCl2, 0.5 EGTA, 0.05 ATP. [␥-32P]ATP (0.3 Ci/ml) was added as a tracer (pH 7.4). In a parallel assay, CaCl2 (0.55 mM) was added to the medium so as to establish a free Ca2⫹ content of ⬃50 M. The phosphorylation reaction was started by addition of the homogenate (0.2 to 2.8 ml of medium) and 30 s later quenched with trichloroacetic acid and centrifuged, the pellet was resuspended and recentrifuged, and the final pellet was prepared for counting of 32P bound to the protein. The increase in 32P uptake induced by the presence of Ca2⫹ was expressed in picomoles per gram tissue wet weight. Na⫹-K⫹-ATPase was measured as the [3H]ouabain binding capacity of muscle tissue specimens weighing ⬃5 mg (21). The tissue samples were equilibrated for 2 h at 37°C in a Tris-vanadate buffer containing [3H]ouabain (10⫺6 M and 0.6 Ci/ml) and henceforth washed for 4 ⫻ 30 min in ice-cold Trisvanadate buffer to remove the [3H]ouabain not bound to the tissue, blotted, weighed, and taken for counting. After correction for unspecific uptake, isotopic purity (95%), loss of 3H activity during the washout in the cold, and incomplete saturation, the [3H]ouabain bound was expressed in picomoles per gram tissue wet weight. The immunoblotting methods generally yield only relative values for tissue contents of cation ATPases. In the present study, therefore, the above-mentioned methods were preferred to obtain values that could be expressed in molar units per gram tissue wet weight. (5). Plasma thyroid hormones (total T3 and total T4) and TSH were measured by immunofluorescence methods (Immulite; DPC, Los Angeles, CA). Free T4 (fT4) and free T3 (fT3) were measured by RIA (28, 29). Statistical analysis. Statistical analyses were made using SPSS for Windows 10.0 (SPSS, Chicago, IL). All data (except TSH) are given as means ⫾ SD. Student’s two-tailed t-test for paired or unpaired data was used for comparison of data between groups as appropriate. Data corresponding to serum TSH are given as medians and ranges, and for comparisons Wilcoxon’s signed rank test or Mann-Whitney U-test were used. P values ⬍ 0.05 were considered statistically significant. In the two cases where muscle biopsies could not be obtained both AJP-Endocrinol Metab • VOL before and after treatment, the data are included in the mean values and in the unpaired comparison with healthy control subjects. The paired comparisons before and after treatment comprise nine patients as regards Ca2⫹-ATPase and eight patients as regards Na⫹-K⫹ATPase. Pearson’s product moment correlation with two-tailed probability values was used to measure the strength of association between the variables. RESULTS Thyroid status and cation pumps. In the hyperthyroid state, the patients had between two- and fivefold increased plasma total T3 and fT3 compared with posttreatment, when T3 decreased to normal levels (Table 1). Upon admission, the patients were clinically hyperthyroid with tachycardia and 41 ⫾ 18% increase in REE. Body weight increased 4.7 ⫾ 2.8 kg during treatment. As shown in Fig. 1, in hyperthyroid patients the content of Ca2⫹-ATPase in vastus lateralis was increased: 6,555 ⫾ 604 vs. 5,212 ⫾ 1,580 pmol/g after treatment (P ⫽ 0.04, paired comparison, n ⫽ 9) and 4,523 ⫾ 1,311 pmol/g in healthy controls (P ⫽ 0.0005). The mean relative increase in Ca2⫹-ATPase in hyperthyroidism makes up 26% of the values measured after euthyroidism had been restored and 45% of the Ca2⫹-ATPase content in healthy controls. As shown in Fig. 2, the content of Na⫹-K⫹-ATPase showed a 89% increase in Fig. 1. Skeletal muscle Ca2⫹-ATPase in 11 hyperthyroid patients before (n ⫽ 10) and after treatment (euthyroid, n ⫽ 10) and in 8 healthy controls. Mean values with bars showing SD. P values represent comparison between patients in the hyperthyroid and euthyroid states by use of paired t-test (n ⫽ 9) and comparisons of healthy controls with patients in the hyperthyroid or euthyroid state by use of unpaired t-test. NS, not significant. 288 • JUNE 2005 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on June 15, 2017 Data are presented as means ⫾ SD, except for thyroid-stimulating hormone (TSH) values, which are presented as medians and range. Normal ranges for thyroid hormones in our laboratory are stated in parentheses. Ht., hyperthyroid patients before treatment; Eut., hyperthyroid patients after treatment; Cont., controls; BMI, body mass index; EE, energy expenditure. T3, 3,5,3⬘-triiodothyronine; T4, thyroxine. P values represent comparisons between patients in the hyperthyroid and euthyroid states by paired t-test and comparisons of healthy controls with patients in the hyperthyroid or euthyroid state by unpaired t-test. CATION PUMPS IN HYPERTHYROIDISM E1267 0.54, P ⫽ 0.03 Na⫹, K⫹-ATPase: r ⫽ 0.63, P ⫽ 0.02. Na⫹, K⫹-ATPase correlations to total T3: r ⫽ 0.92, P ⬍ 0.0001, fT3: r ⫽ 0.91, P ⬍ 0.0001, fT4: r ⫽ 0.87, P ⬍ 0.0001, total T4: r ⫽ 0.84, P ⬍ 0.0001, EE/kg body wt: r ⫽ 0.79, P ⫽ 0.0003. DISCUSSION hyperthyroidism: 558 ⫾ 101 vs. 296 ⫾ 34 pmol/g in euthyroidism (P ⫽ 0.0001, paired comparison, n ⫽ 8) and 278 ⫾ 52 pmol/g in healthy controls (P ⬍ 0.0001). After restoration of euthyroidism, the contents of both cation pumps were not significantly different from those of healthy controls. In hyperthyroid patients and healthy controls, Ca2⫹-ATPase and Na⫹-K⫹-ATPase contents were positively correlated to circulating free and total thyroid hormone levels and to EE per kilogram of lean body mass, and the two cation pumps were intercorrelated (Fig. 3). Ca2⫹-ATPase correlations to total T3: r ⫽ 0.74, P ⫽ 0.001, fT3: r ⫽ 0.69, P ⫽ 0.006, fT4: r ⫽ 0.61, P ⫽ 0.022, total T4: r ⫽ 0.68, P ⫽ 0.004, EE/kg body wt: r ⫽ Fig. 3. Correlations in hyperthyroid patients and healthy controls between total 3,5,3⬘triiodothyronine (T3) and skeletal muscle Ca2⫹-ATPase and Na⫹-K⫹-ATPase (A) and between skeletal muscle Ca⫹-ATPase and Na⫹-K⫹-ATPase and energy expenditure (EE) per kg body wt (B). AJP-Endocrinol Metab • VOL 288 • JUNE 2005 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on June 15, 2017 Fig. 2. Skeletal muscle Na⫹-K⫹-ATPase in 9 hyperthyroid patients before and after treatment (euthyroid, n ⫽ 8) and in 8 healthy controls. Mean values with bars showing SD. P values represent the comparison between patients in the hyperthyroid and euthyroid states by use of paired t-test (n ⫽ 8) and comparisons of healthy controls with patients in the hyperthyroid or euthyroid state by use of unpaired t-test. Probably the most important new information gained from the present study is that in hyperthyroidism the content of Ca2⫹-ATPase in human skeletal muscle is increased. Moreover, this increase is normalized by achievement of euthyroidism by standard methimazole treatment and shows significant correlation to plasma thyroid hormone levels as well as to energy expenditure. Our values for the contents of Ca2⫹ATPase and Na⫹-K⫹-ATPase in skeletal muscle of healthy control subjects correspond closely to those previously published by our laboratory and others (5, 10). In all our patients, we confirm our previous finding of elevated Na⫹-K⫹-ATPase content in human skeletal muscle in hyperthyroidism and its normalization after treatment (17). Hyperthyroid patients suffer from muscle weakness, fatigue, and decreased contractility, which are normalized after treatment (22). The gross increase in skeletal muscle Na⫹-K⫹ATPase in hyperthyroidism is thus unlike the upregulation induced by physical training, not associated with increased muscle strength. This is probably due to a concomitant increase in the content of Na⫹ channels in muscle, as demonstrated in animal studies (15), allowing increased passive flux of Na⫹ into the muscle cells dissipating the membrane potential and reducing excitability and contractility (5). In the rat, the increase in the content of Na⫹ channels and passive Na⫹-K⫹ fluxes induced by thyroid hormone treatment precedes the E1268 CATION PUMPS IN HYPERTHYROIDISM AJP-Endocrinol Metab • VOL panied by normalization of REE. The increased content of Ca2⫹-ATPase in skeletal muscle can only in part explain the increase in REE in hyperthyroidism. Thyroid hormone affects energy balance by several mechanisms (24). In hyperthyroidism, spontaneous physical activity is increased (19). Thyroid hormone regulates gene expression of a large number of proteins known to regulate energy expenditure, for example uncoupling protein-3 (18), and increased protein synthesis also consumes energy (2). In summary, our data are consistent with the hypothesis that, in human subjects, the calorigenic actions of thyroid hormones are in part mediated by increased skeletal muscle content of Ca2⫹-ATPase, allowing for increased energy expenditure by both active Ca2⫹ transport and uncoupled ATPase activity. We confirm our previous finding of elevated Na⫹-K⫹-ATPase content in skeletal muscle in hyperthyroidism, which to a minor extent adds to the increased energy expenditure by increased active Na⫹-K⫹ transport. In a wider perspective, active Ca2⫹ transport in skeletal muscle may be a regulatory target for energy turnover and weight control. ACKNOWLEDGMENTS Tove Lindahl Andersen is thanked for excellent technical assistance. GRANTS The study was supported by Aarhus Universitets Forskningsfond and the Danish Biomembrane Research Center. Parts of the results were presented at the 85th Annual Scientific Meeting of the Endocrine Society 2003 in Philadelphia, PA. REFERENCES 1. Arai M, Otsu K, MacLennan DH, Alpert NR, and Periasamy M. Effect of thyroid hormone on the expression of mRNA encoding sarcoplasmic reticulum proteins. Circ Res 69: 266 –276, 1991. 2. Arruda AP, Da Silva WS, Carvalho DP, and de Meis L. Hyperthyroidism increases the uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2⫹-ATPase. Biochem J 375: 753–760, 2003. 3. Berchtold MW, Brinkmeier H, and Muntener M. Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. Physiol Rev 80: 1215–1265, 2000. 4. Clausen T, van Hardeveld C, and Everts ME. Significance of cation transport in control of energy metabolism and thermogenesis. Physiol Rev 71: 733–774, 1991. 5. Clausen T. 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Quantitative determination of Ca2⫹-dependent Mg2⫹-ATPase from sarcoplasmic reticulum in muscle biopsies. Biochem J 260: 443– 448, 1989. 11. Everts ME and Clausen T. Effects of thyroid hormones on calcium contents and 45Ca exchange in rat skeletal muscle. Am J Physiol Endocrinol Metab 251: E258 –E265, 1986. 12. Everts ME and Clausen T. Effects of thyroid hormone on Na⫹-K⫹ transport in resting and stimulated rat skeletal muscle. Am J Physiol Endocrinol Metab 255: E604 –E612, 1988. 288 • JUNE 2005 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on June 15, 2017 upregulation of Na⫹-K⫹-ATPase, indicating that the latter is a compensatory mechanism to maintain transmembrane Na⫹-K⫹ distribution and cell membrane potential (12, 15). The SR is a specialized Ca2⫹ storage membrane system in muscle cells and plays a central role in the contractile function of the muscle by virtue of its ability to regulate cytosolic Ca2⫹ concentration (3, 20). Ca2⫹ release from the SR initiates muscle contraction, whereas Ca2⫹ reuptake into the SR membrane vesicles lowers cytosolic Ca2⫹, causing muscle relaxation. The events that regulate Ca2⫹ release and removal can affect cytosolic Ca2⫹ levels and, hence, the rate and extent of muscle contraction and relaxation. The Ca2⫹ reuptake function of the SR is mediated by the Ca2⫹ transport pump, the sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA). Ca2⫹ release from the SR to trigger muscle contraction is mediated by Ca2⫹ channels, the ryanodine receptors. In animals, thyroid hormones increase the mRNA expression and protein of both the Ca2⫹-ATPase and the ryanodine receptor in skeletal muscle (1, 16). This dual upregulation explains that, in rat muscle, both contraction rates and relaxation rates are increased in hyperthyroidism and decreased in hypothyroidism (9, 13). In keeping with this, one of the classical clinical features of thyroid disorders is the shortening of the Achilles tendon reflex time in hyperthyroidism and its prolongation in hypothyroidism (30). The slow-to-fast transition in hyperthyroid red muscle is mainly due to increased SERCA1 isoform expression. SERCA1 seems to be the only SERCA isoform able to interconvert energy from ATP hydrolysis into heat without calcium transport, the uncoupled ATPase activity (7). Recently, an animal model described how the calorigenic action of thyroid hormone in hyperthyroidism might be due, at least in part, to the increased uncoupled ATPase activity in both red and white muscles (2). The observed increase in skeletal muscle Ca2⫹-ATPase in our study might in part be the result of a transition of muscle fiber types induced by hyperthyroidism from the type I muscle fibers with a low content of SR and Ca2⫹-ATPase to the type II muscle fibers with a high content of SR and Ca2⫹-ATPase (27). Moreover, because SERCA1 has a higher ATP turnover number (1,000 –1,100/min) than that of SERCA2 (450/min) (8), an increase in the ratio between type II and type I fibers further augments the capacity for Ca2⫹ recycling and the ATP turnover associated with this transport process. Therefore, the 45% increase in Ca2⫹-ATPase content demonstrated in the present study may be combined with an increased average ATP turnover number. This would allow for an even more pronounced relative rise in energy expenditure. We chose to reexamine the patients after 3 mo of treatment, and at that time the patients were clinically euthyroid, and circulating thyroid hormones and REE were normalized. We might have obtained a more complete normalization of the cation pumps after a longer period of euthyroidism, allowing for further regeneration of the muscles (22). Observations of correlations do not establish causality. 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