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In this study hind-limb perfusion was used to investigate the influence of thyroid hormones on some metabolic parameters in the skeletal muscle of the rat.Daily injection of 20 μg L-thyroxine (T4) per 100 g b. w. for a week caused a 25 % increase in oxygen consumption. Further enlargement of the T4dose had little additive effect. In the dose range 20–80 μg T4/100 g b. w., no important changes occurred in lactate production or glucose consumption. Only at the highest T4dose did the glucose consumption increase significantly. The most profound effect of T4was on lipolysis. A daily dose of 20 μg T4/100 g b. w. gave a doubling of glycerol production rate, the maximum occurring at a dose of 40 μg T4/100 g b. w Inactivation of the nervous system was without influence on the T4-induced increase in oxygen consumption. However, the T4-induced elevation of lipolysis disappeared after abolition of the nervous activity. This raises the possibility that the T4effect on lipolysis in skeletal muscle is a potentiation of catecholamine effects. The T4-induced oxygen consumption increase might be dependent not on the lipolytic process but rather on other energy-consuming cell processes.

Urinary 127I excretion, 24 h thyroid 131I uptake and serum values of thyrotrophin (TSH), triiodothyronine (T3) and protein-bound iodine (PBI) were measured in subjects from an area with severe (Segni) and moderate (Londjong) iodine deficiency. In Segni, 90 non-cretinous subjects and 40 cretins were studied. In both sub-groups from Segni non-compensated iodine deficiency was found. Although iodine excretion in these sub-groups was the same (mean: ± sd, 127I μg per g creatinine; non-cretins 16.9 ± 10.1 and cretins 15.2 ± 8.0) thyroid hormone serum levels were less in the cretins probably due to additional primary thyroid failure. In the subjects (non-cretins plus cretins) from Segni a positive relationship (r=0.39, P< 0.001) was found between urinary 127I excretion and serum PBI. Moreover in the same subjects a negative correlation was found between serum PBI and TSH (r=0.43; P< 0.001) while serum T3did not correlate with TSH. In the Londjong area (mean ± sd127I urinary excretion: 41.6 ± 18.6 μg per g creatinine) iodine deficiency appeared to be compensated in 52 subjects studied since mean serum levels of TSH, PBI and T3were within normal range. No correlation between PBI and serum TSH was found. PBI too did not correlate with iodine excretion despite the fact that 37 subjects excreted less than 48 μg 127I per g creatinine below which value iodine excretion varied in all but one of the studied subjects from Segni. It is suggested on the basis of a difference in the average iodine intake between the groups from Segni and Londjong, that lack of "iodine buffer capacity" of the thyroid gland in the Segni subjects leads to a situation where changes in iodine intake are readily reflected in T4production resulting in the correlation found between PBI and urinary excretion in this group.

In this study hind-limb perfusion was used to investigate conversion of T4to T3in skeletal muscle tissue. For this purpose the rats were depleted of thyroid hormones by thyroid ablation with 0.75 mCi 131I and were perfused 2 weeks later, when the skeletal muscle tissue consumed oxygen at a normal rate due to one subcutaneous dose of 10 μg T3/100 g b. w. 3 days before the perfusion experiments were started. T4*1) of high specific activity (> 2000 μCi/μg) was added to the perfusate. In the muscle (mixed type) a mean T4→ T3conversion of 2 % (range 0.5–3.9) was found after 120 min of perfusion. T3generation from T4in skeletal muscle did not correspond with T3muscle uptake. This observation makes a significant overestimation of T3by selective uptake of a small contamination of T3* in the T4* preparation highly improbable. In red muscle the T4and T3uptake was about 50 % higher than in white muscle. The observed Tetraccand T3cwere significantly higher in red than in white muscle. The uptake of thyroid hormones by both muscle types was not changed in hyperthyroid rats. The Tetrac and T3formation from T4, however, was increased in red muscles of hyperthyroid rats. The results show that thyroid hormone metabolism can vary markedly depending upon the type of muscle studied and they present a basis for a better understanding of clinical and biochemical evidence for a different susceptibility of red and white muscle fibers to thyroid hormones.

The influence of thyroid hormone depletion and experimental hyperthyroidism on red and white skeletal muscle of the rat during periods of 2, 4 and 8 weeks were studied. Body weight, muscle weight, mitochondrial protein content, and specific activities of the mitochondrial enzymes α-glycerophosphate dehydrogenase (EC 1.1.99.5) (α-GPD) and succinate dehydrogenase (EC 1.3.99.1) (SDH) were used as parameters. The largest differences in body weight gain and muscle weight gain (both red and white muscle) in the hypothyroid rats were seen after 8 weeks of T4treatment. In the hyperthyroid rats the weight of the red muscle and the ratio of the red muscle weight to the body weight increased, whereas the white muscle weight and the ratio of the white muscle weight to the body weight decreased relative to the control animals.In hypothyroid rats the mitochondrial protein content was lowered in both red and white muscle, the specific α-GPD activity only in the latter. No changes in specific SDH activity were observed in either type of muscle. The hyperthyroid rats showed an increase in the mitochondrial protein content and the specific α-GPD and SDH activity in the red muscle, whereas no significant changes were observed in the white muscle. The changes in the parameters under study show that the effect of the thyroid state differs in red and white muscle. An explanation for a possibly greater sensitivity of red than of white muscle to thyroid hormones is discussed.

The effect of thyroid hormone (L-tri-iodothyronine; T3) on the cytosolic free Ca2+ concentration ([Ca2+]i) in L6 myotubes was studied at rest and during activation to explore the possible mediating role of [Ca2+]i in the T3-induced net synthesis of fast-type sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The mean [Ca2+]i at rest was approx. 115 nM in myoblasts, control myotubes and T3-treated myotubes. Therefore it is unlikely that the T3-induced elevation of Ca(2+)-ATPase levels is mediated by [Ca2+]i changes. To investigate the influence of the 4-fold higher Ca(2+)-ATPase levels in T3-treated myotubes (compared with controls) on [Ca2+]i, interventions with caffeine (10 mM) and a high extracellular K+ concentration ([K+]o) (30 mM) were applied which initially mobilize Ca2+ predominantly from the SR. The results showed a lower (caffeine) or not significantly different (high [K+]o) increase in [Ca2+]i in T3-treated myotubes compared with controls. No rise in [Ca2+]i was found in myoblasts with caffeine or high [K+]o. The role of [Ca2+]i in the regulation of Ca(2+)-ATPase levels was investigated by varying [Ca2+]i through exposure of cells to different concentrations of extracellular Ca2+ (0.2-1.8 mM) and ionomycin (0.1-0.25 microM). At subnormal [Ca2+]i (55 nM) the T3-induced net synthesis of Ca(2+)-ATPase was virtually abolished, and at supranormal [Ca2+]i (195 nM) it was greatly depressed. Intermediate stimulation of net Ca(2+)-ATPase synthesis was found at [Ca2+]i of 95 and 165 nM, with an optimum at approx. 125 nM. Similar but less pronounced effects were found for the basal Ca(2+)-ATPase levels. In contracting primary rat myotubes, Ca(2+)-ATPase levels were significantly lower than in tetrodotoxin-arrested myotubes. The same results were obtained in the presence of T3. Since the mean [Ca2+]i in contracting cells is higher than in resting cells, these data agree with those obtained in the L6 cells with ionomycin. A major conclusion of this study is the existence of a [Ca2+]i optimum, near resting levels, for the expression of the fast-type Ca(2+)-ATPase in the L6 muscle cell line.

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.

Relaxation of skeletal muscle requires the re-uptake of Ca2+, which is mediated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Thyroid hormone (T3) stimulates the expression of the SERCA1 isoform, which is essential for fast skeletal muscle fiber phenotype. We have cloned and studied the first 962 base pairs of the 5'-flanking region of the rat SERCA1 gene. This sequence was tested for T3-regulated expression in transient transfection experiments using COS7 cells and for binding of thyroid hormone receptor (TR) alpha in mobility shift assays. A construct of the 5'-flanking region and a reporter gene was unresponsive to T3 in the absence of co-transfected thyroid hormone receptor. In the presence of TRalpha, a T3 induction ratio of almost 4.0 was found, and this induction ratio was doubled with co-transfection of an RXR expression plasmid. Analysis of progressive 5'-deletion fragments of the sequence indicated multiple regions involved in T3 responsiveness. Three regions, R1, R2, and R3, were identified that bound TR complexes in mobility shift assays and conferred T3 responsiveness to a heterologous promoter. The most potent of these thyroid hormone response elements, R3, increased the 2-fold background T3 stimulation of the thymidine kinase promoter to nearly 6-fold. Detailed analysis of this element showed that four TR-binding half-sites, comprising two independent thyroid hormone response elements, interact cooperatively to give the maximal T3 response. T3 regulation of SERCA1 expression is mediated by a complex thyroid hormone response element that may serve to provide a greater range of response in interaction with nuclear receptor partners or cell-specific transcription factors.

Hind-limb perfusion was used to study the effect of thyroidectomy on some metabolic parameters in the skeletal muscle of the rat. A week after thyroidectomy obtained by one dose of 3/4 mCi 131I, neither T4nor T3was detected in the blood. Lactate production and glycerol production were already decreased a week after the treatment and reached a base level at two weeks. At that time, the oxygen consumption was significantly lower (70 % of initial level) than in the control animals and decreased further in the third week to nearly 50 % of the control level.Glucose consumption and alanine release were decreased three weeks after thyroidectomy. One dose of T3(10 μg/100 g b. w.), administered to animals two weeks after the injection of 131I, restored the oxygen consumption, lactate production, and glycerol production to normal levels in 24 h. After 48 h, the glucose consumption was normal. Glycerol production was already significantly increased 6 h after T3injection in animals one week after thyroidectomy, and in another group of animals two weeks after thyroidectomy. Apparently the diminished oxygen consumption in the latter group does not retard the lipolytic response to T3. No direct relationship could be found between the activity of lipolytic process and the thyroid hormone controlled oxygen consumption.

The effect of sympathetic activity on T4and T3levels in cold-exposed rats was investigated. Administration of the highest dose of propranolol (2 mg/100 g b.w.) twice daily during 4 days decreased T4and T3concentrations in plasma of rats living at 23°C (T4from 46.4 ± 2.6 to 25.8 ± 5.3 nmol/l and T3from 1.08. ± 0.6 to 0.82 ± 0.12 nmol/l). No significant effect on T4and T3levels (49.0 ± 11.6 and 1.48 ± 0.16 n/mol, respectively) after the administration of the same dose regimen of propranolol was observed in rats exposed to cold for 4 weeks. T4and T3levels in rats exposed to cold for 4 weeks were not significantly altered 1 week after sympathectomy, while remaining in the cold. However, chemical sympathectomy before cold exposure delayed the cold induced T3elevation occurring during the first week of cold exposure (controls: from 1.16 ± 0.19 to 1.44 ± 0.29 nmol/l; sympathectomized rats: from 1.07 ± 0.12 to 1.17 ± 0.22 nmol/l). After 2 weeks of cold exposure the T3levels of controls and sympathectomized rats were not significantly different (controls: 1.45 ± 0.12 nmol/l, sympathectomized rats: 1.38 ± 0.15 nmol/l). No effect of sympathectomy was observed on T4levels.These experiments show that the role of sympathetic activity in increasing T3is not clear during cold exposure. They provide some evidence that sympathetic activity may play a role in the initiation of the process leading to increased T3plasma levels during cold exposure.

1. A modified method for the determination of thyroid hormone levels in muscle is described.2. The levels of T4and T3in skeletal muscle tissue of rats lie in the same range.3. The level of T4in plasma is about a hundred times higher than that of T3.4. To remove plasma hormones from the tissue in which hormone levels are to be determined, the animals should be perfused with saline before tissue samples are taken.

Rats were exposed to cold (4°C) for 1 and 4 weeks. The T4plasma concentrations initially declined (24.5 ± 7.7 nmol/l) after 1 week but returned to normal levels after 4 weeks (52.9 ± 14.2 nmol/l). The T3concentrations were elevated after both 1 and 4 weeks at 4°C (1.31 ± 0.21 and 1.38 ± 0.12 nmol/l, respectively). Control values (23°C) for T4were 42.6 ± 10.3 and for T31.11 ± 0.13 nmol/l. Addition of 0.015 g KI/l to the drinking water prevented the T4decrease in plasma after 1 week of cold exposure. No effect of iodide was observed at 23°C. The suppletion of KI did not change pattern of T3increase after cold exposure. After 4 weeks of cold exposure the T4levels of the iodide-supplemented animals did not differ from the non-supplemented group.No evidence was found that increased food intake is a contributory factor in the development leading to increased T3plasma levels during cold exposure.

The aim of this study was to investigate the mechanism(s) underlying the thyroid-hormone (L-tri-iodothyronine, T3)-induced elevation of fast-type sarcoplasmic-reticulum Ca(2+)-ATPase (SERCA1) levels in L6 myotubes and the potentiating effect of insulin-like growth factor-I (IGF-I) [Muller, van Hardeveld, Simonides and van Rijn (1991) Biochem. J. 275, 35-40]. T3 increased the SERCA1 protein level (per microgram of DNA) by 160%. The concomitant increase in the SERCA1 mRNA level was somewhat higher (240%). IGF-I also increased SERCA1 protein (110%) and mRNA levels (50%), whereas IGF-I + T3 increased SERCA1 protein and mRNA levels by 410% and 380% respectively. These SERCA1 mRNA analyses show that the more-than-additive action of T3 and IGF-I on SERCA1 expression is, at least in part, pre-translational in nature. Further studies showed that the half-life of SERCA1 protein in L6 cells (17.5 h) was not altered by T3. In contrast, IGF-I prolonged the half-life of SERCA1 protein 1.5-1.9-fold, which may contribute to the disproportional increase in SERCA1 protein content compared with mRNA by IGF-I. Measurements of SERCA1 mRNA half-life (as determined by actinomycin D chase) showed no difference from the control values (15.5 h) in the presence of T3 or IGF-I alone. When T3 and IGF-I were both present, the SERCA1 mRNA half-life was prolonged 2-fold. No significant effects of T3 and IGF-I were observed on the half-life of total protein (37.4 h) and total RNA (37.0 h). The absence of an effect of T3 on SERCA1 protein and mRNA stability, when it was present alone, suggested transcriptional regulation, which was confirmed by nuclear run-on experiments, showing a 3-fold increase in transcription frequency of the SERCA1 gene by T3. We conclude that the synergistic stimulating effects of T3 and IGF-I on SERCA1 expression are the result of both transcriptional and post-transcriptional regulation. T3 acts primarily at the transcriptional level by increasing the transcription frequency of the SERCA1 gene, whereas IGF-I seems to act predominantly at post-transcriptional levels by enhancing SERCA1 protein and mRNA stability, the latter, however, only in the presence of T3.

Net synthesis of the fast-type sarcoplasmic reticulum (SR) Ca2(+)-ATPase was studied in the muscle cell line L6AM using an immunochemical assay (e.l.i.s.a.). In addition, Ca2+ uptake by SR was monitored in muscle cell homogenates by a method employing the fluorescent Ca2+ indicator fura-2. Measurements were done both in differentiating myoblasts and in myotubes. Ca2(+)-ATPase levels were low (1 pmol/mg of protein) in undifferentiated myoblasts (controls) and only doubled over a period of 8 days in the absence of thyroid hormone (L-triiodothyronine; T3). This corresponded to a similar increase in Ca2+ uptake activity. Only half of the myoblasts fused under these conditions. Fusion was not increased in the presence of T3 (5 nM), but Ca2(+)-ATPase levels increased 4-fold and the Ca2+ uptake activity doubled compared with controls. In contrast, insulin-like growth factor-I (IGF-I) induced almost complete myotube formation (greater than 90% fusion), but only slightly stimulated (50%) net Ca2(+)-ATPase synthesis above control levels. However, the doubling of the Ca2+ uptake stimulation by IGF-I was comparable with that caused by T3. The effects of T3 plus IGF-I on Ca2(+)-ATPase levels and Ca2+ uptake activity were more than additive. Furthermore, the temporal relationship between the induction of Ca2(+)-ATPase net synthesis and Ca2+ uptake activity was identical with the two hormones. Qualitatively similar results were obtained when T3 and IGF-I were added to maximally fused cell cultures. The enhanced effect of T3 on Ca2(+)-ATPase net synthesis and Ca2+ uptake activity in the presence of IGF-I cannot therefore be explained by an increased myotube formation stimulated by the latter. In both differentiating myoblasts and myotubes the effect of T3 was more prominent on Ca2(+)-ATPase net synthesis than on Ca2+ uptake activity, whereas in myotubes the opposite was observed for IGF-I. This could imply complementary actions of the two agents in the development of a functional SR.

The effects of beta-adrenergic stimulation on the relaxation rate and the Ca2+-transport rate in sarcoplasmic reticulum of hypothyroid, euthyroid and hyperthyroid rat hearts were studied. Administration of isoproterenol (0.1 microM) to perfused, electrically stimulated hearts (5 Hz) caused a decrease in the half-time of relaxation (RT 1/2) the extent of which depended on the thyroid status, i.e. hypothyroid (-24%), euthyroid (-19%) or hyperthyroid (-8%). A similar decreasing effect was found for the stimulation of Ca2+ transport in isolated SR by cyclic AMP and protein kinase, i.e. hypothyroid (75%), euthyroid (37%) and hyperthyroid (20%). These alterations were not due to differences in endogenous protein kinase activity or cyclic AMP production. Estimations of Ca2+-ATPase and phospholamban (PL) content of the sarcoplasmic reticulum were obtained by measurement of the phosphorylated forms of Ca2+-ATPase (E-P) and phospholamban (PL-P) followed by electrophoresis and autoradiography. A 3-fold decrease of PL-P, accompanied by a 2-fold increase of E-P per mg of protein was observed in sarcoplasmic reticulum preparations in the direction hypothyroid----hyperthyroid. Consequently the E-P/PL-P ratio increased from 0.32 (hypothyroid), through 0.81 (euthyroid) to 1.69 (hyperthyroid). In spite of certain limitations inherent to quantification of Ca2+-ATPase and phospholamban by their phosphorylated products, these data provide strong evidence that during thyroid-hormone mediated cardiac hypertrophy, with concomitant proliferation of the sarcoplasmic reticulum, the relative amount of phospholamban decreases with respect to Ca2+-ATPase. This could provide an explanation for the observed gradual diminishment of the beta-adrenergic effect on the relaxation rate when cardiac tissue is exposed to increasing amounts of thyroid hormone.

Rats were made hyperthyroid by repeated sc injections of l-thyroxine (T4) in a two-week period. At several time-points after the last injection, the triiodothyronine (T3) and T4concentrations in plasma and red and white skeletal muscle were determined by a radioimmunoassay. This was done to investigate the relationship between the change in thyroid-hormone concentration in plasma and muscle and to see whether this change was similar in both muscle types. The results show that:1. Information about the T3and T4concentrations in the muscles of the hyperthyroid rats can only be obtained by direct measurement in the muscles and cannot be gathered from the plasma-T3concentration at any time-point or from below-normal plasma-T4concentrations.2. In hyperthyroid rats T4and T3are cleared more rapidly from the plasma than from the skeletal muscle.3. A proportionally higher T3concentration is present in the red compared with the white skeletal muscle of hyperthyroid rats.

This report describes the development of a histochemical method for the demonstration of sarcoplasmic reticulum Ca-ATPase activity in cross-sections of skeletal muscle. The demonstration of sarcoplasmic reticulum Ca-ATPase activity is complicated by the fact that capturing reagents for phosphate inhibit the enzyme. We present a minimal model for heavy-metal-phosphate precipitation reactions which gives a theoretical description of the effect of enzyme inhibition on the rate of phosphate precipitation in the section. The model indicates that the choice of capturing reagent is crucial: whether or not ATPase activity can be demonstrated depends mainly on the inhibition constant and the solubility product of the phosphate salt of the capturing reagent (but also on a fairly large number of other factors). All lanthanides tested can be used to demonstrate sarcoplasmic reticulum Ca-ATPase activity, but dysprosium results in the highest staining intensity. This suggests that dysprosium inhibits sarcoplasmic reticulum Ca-ATPase to a lesser degree than the other lanthanides and/or the solubility product of its phosphate salt is smaller. As an example, the method is used to investigate the effect of thyroid hormone on sarcoplasmic reticulum Ca-ATPase activity in individual fibres of the rat soleus muscle.

The effects of thyroid hormone depletion on skeletal muscle metabolism in relation to force development were studied. For this purpose, the triceps surae muscles were perfused and stimulated at 5 Hz. The basal oxygen consumption of the skeletal muscle preparation was 50% lower in hypothyroid rats as compared with euthyroid rats.The results show that:1. Active force development was the same in euthyroid and hypothyroid rats during 30 min of stimulation.2. The increase in oxygen consumption during contraction was twice as high in the euthyroid group compared with the hypothyroid group.3. Lactate release and glucose consumption were considerably higher in the euthyroid group than in the hypothyroid group during the last 15 min of stimulation. The data show that force development is not impaired in hypothyroid rats but, on the contrary, indicate that the contraction process proceeds more economically in hypothyroid rats than in euthyroid rats.

Basal plasma levels for adrenalin (A), noradrenalin (NA), l-triiodothyronine (T3), and l-thyroxine (T4) were determined in rats with a chronically inserted catheter. The experiments described in this report were started 3 days after the surgical procedure when T3and T4levels had returned to normal. Basal levels for the catecholamines were reached already 4 h after the operation.The T3/T4ratio in plasma was significantly increased after 3, 7, and 14 days in rats kept at 4°C and the same holds for the iodide in the 24-h urine after 7 and 14 days at 4°C. The venous NA plasma concentration was increased 6- to 12-fold during the same period of exposure to cold, whereas the A concentration remained at the basal level.During infusion of NA at 23°C the T3/T4ratio in plasma was significantly increased after 7 days compared to pair-fed controls, and the same holds for the iodide excretion in the 24-h urine. This paper presents further evidence for a role of the sympathetic nervous system on T4metabolism in rats at resting conditions.

T3and T4concentrations were determined in plasma and red and white skeletal muscle of the rat. Because of the small tissue samples (± 300 mg), the ultra-sensitive Wick radioimmunoassay (RIA) for serum was adapted for determination in ethanol extracts.The dilution curves of the plasma and tissue extracts showed excellent parallelism with the standard curves for both T3and T4.The mean T4level found in female rats (n = 6) was 22.6 ± 5.2 ng/ml in plasma and did not differ significantly between red (1.85 ± 0.28 ng/g) and white (1.90 ± 0.25 ng/g) skeletal muscle.The mean T3level in 11 normal female rats was 0.629 ± 0.098 ng/ml in the plasma and was significantly higher in the red muscle (2.07 ± 0.26 ng/g) than in the white muscle (1.65 ± 0.20 ng/g).The higher T3levels found in the red muscle as compared with the white muscle may help to elucidate the different responsiveness of these muscle types observed in altered thyroid states.