Whole body and forearm substrate metabolism in hyperthyroidism: evidence of increased basal muscle protein breakdown
Anne Lene Dalkjær Riis,1
Jens Otto Lunde Jørgensen,1
Signe Gjedde,2
Helene Nørrelund,1
Anne Grethe Jurik,3
K. S. Nair,4
Per Ivarsen,5
Jørgen Weeke,1 and
Niels Møller5
1Medical Department M (Endocrinology and Diabetes), 2Medical Department C and 3Department of Radiology R, Aarhus University Hospital, Aarhus; 5Medical Research Laboratories, University of Aarhus, Aarhus, Denmark; and 4Endocrine Research Unit, Mayo Clinic, Rochester Minnesota
Submitted 14 June 2004
; accepted in final form 7 January 2005
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ABSTRACT
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Thyroid hormones have significant metabolic effects, and muscle wasting and weakness are prominent clinical features of chronic hyperthyroidism. To assess the underlying mechanisms, we examined seven hyperthyroid women with Graves' disease before (Ht) and after (Eut) medical treatment and seven control subjects (Ctr). All subjects underwent a 3-h study in the postabsorptive state. After regional catheterization, protein dynamics of the whole body and of the forearm muscles were measured by amino acid tracer dilution technique using [15N]phenylalanine and [2H4]tyrosine. Before treatment, triiodothyronine was elevated (6.6 nmol/l) and whole body protein breakdown was icreased 40%. The net forearm release of phenylalanine was increased in hyperthyroidism (µg·100 ml1·min1): 7.0 ± 1.2 Ht vs. 3.8 ± 0.8 Eut (P = 0.04), 4.2 ± 0.3 Ctr (P = 0.048). Muscle protein breakdown, assessed by phenylalanine rate of appearance, was increased (µg·100 ml1·min1): 15.5 ± 2.0 Ht vs. 9.6 ± 1.4 Eut (P = 0.03), 9.9 ± 0.6 Ctr (P = 0.02). Muscle protein synthesis rate did not differ significantly. Muscle mass and muscle function were decreased 1020% before treatment. All abnormalities were normalized after therapy. In conclusion, our results show that hyperthyroidism is associated with increased muscle amino acid release resulting from increased muscle protein breakdown. These abnormalities can explain the clinical manifestations of sarcopenia and myopathy.
hyperthyroidism; skeletal muscle; amino acids; stable isotopes; tracers; protein synthesis; protein breakdown; energy metabolism
THYROID HORMONES HAVE PROFOUND metabolic effects, and chronic hyperthyroidism is characterized by increased energy expenditure (EE) with increased oxidation of protein, glucose, and lipids (19, 28). Loss of muscle mass and subsequent sarcopenia are prominent clinical features of hyperthyroidism (27), and recovery of muscle mass and function is prolonged, lasting several months (24). Accelerated whole body protein catabolism has been demonstrated in experimental hyperthyroidism (16), but studies of whole body leucine kinetics in clinical and experimental hyperthyroidism have yielded inconsistent results. Studies of protein metabolism in hyperthyroid patients before and after treatment have suggested that the net protein catabolism is mainly because of depressed rates of whole body protein synthesis (7, 20) with low or normal rates of proteolysis. In experimental hyperthyroidism, increased rates of proteolysis with no change in protein synthesis rates have been reported (6, 16), whereas Tauveron et al. (35) found both increased proteolysis and synthesis. Thyroid hormones have both anabolic and catabolic effects; therefore, the net effect on protein metabolism may vary, and the above inconsistencies may relate to heterogeneity both of the hyperthyroid subjects, in terms of severity and duration of hyperthyroidism, and of the methods employed.
The metabolism of muscle protein in hyperthyroid subjects has previously been described measuring urinary excretion or arteriovenous differences of 3-methylhistidine to estimate myofibrillar degradation, giving conflicting results. Some report normal values (6, 7, 20) and others report increased values (1, 30, 42) that were normalized after treatment. In addition, increased net muscle release of certain amino acids has been reported in hyperthyroid subjects. To our knowledge, no studies have hitherto addressed the issue of whether muscle loss and sarcopenia in hyperthyroidism is caused by defective muscle protein synthesis or breakdown employing tracer dilution techniques regionally across a muscle bed.
The current protocol was specifically designed to define the local mechanisms leading to loss of striated muscle mass and function in hyperthyroid patients. We examined seven patients before and after therapy and seven healthy control subjects, and we used infusion of phenylalanine and tyrosine tracers combined with catheterization across the forearm vascular bed to quantify muscle protein breakdown and synthesis.
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MATERIALS AND METHODS
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Subjects.
Seven hyperthyroid women, aged 2649 yr, with newly diagnosed diffuse toxic goitre [Graves' disease, thyrotropin (TSH) receptor antibodies >2 IU/l] were consecutively recruited and studied before treatment and reexamined after 3 mo of medical treatment with methimazole. An age-matched control group of seven healthy women using no medication were 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.
Materials.
We used L-[15N]phenylalanine, L-[15N]tyrosine, and L-[2H4]tyrosine from Cambridge Isotope Laboratories. The chemical, isotopic, and optical purity of the isotopes were tested before use. Solutions were prepared under sterile conditions and were shown to be free of bacteria and pyrogens before use.
Methods and study design.
The participants were admitted to the Clinical Research Center the evening before the day of the examinations. The investigations were carried out in the postabsorptive state the morning after an overnight fast (1012 h) without any caffeine consumption or cigarette smoking; only ingestion of tap water was allowed, and the participants were placed in the supine position under thermoneutral conditions. One intravenous catheter (Viggo, Helsingborg, Sweden) was placed in an antecubital vein for infusions, another in the contralateral antecubital vein for deep venous samples, and a third in a superficial vein draining the ipsilateral hand, which was heated in a box with an air temperature of 65°C to provide arterialized blood (17). Preceding every deep venous sampling, forearm blood flow was determined by venous occlusion plethysmography.
After priming the amino acid pool with bolus injections of [15N]phenylalanine (0.7 mg/kg), [15N]tyrosine (0.3 mg/kg), and [2H4]tyrosine (0.5 mg/kg), continuous infusions of [15N]phenylalanine (0.7 mg·kg1·h1) and [2H4]tyrosine (0.5 mg·kg1·h1) were maintained for 3 h. After 150 min with continuous infusions, steady state is accomplished, and blood samples were taken in triplicate during the last 30 min of each study. Enrichments of [15N]phenylalanine, [15N]tyrosine, and [2H4]tyrosine were measured by mass spectrometry as their t-butyldimethylsilyl ether derivates under electron ionization conditions, and concentrations of phenylalanine and tyrosine were measured using L-[2H8]phenylalanine and L-[13C6]tyrosine as internal standards (22). Whole blood concentrations of amino acids were determined by the HPLC technique (Biotek Kontron Instruments autosampler 465, System 265, and fluorescence detector SFM 25) with precolumn O-phthaldehyde derivatization, after deproteinizing the blood samples with 10% 5-sulfosalicylic acid (12). Thyroid hormones [total triiodothyronine (T3) and total thyroxine (T4)] and TSH were measured by immunofluorecent methods (Immulite; DPC, Los Angeles, CA). Free thyroid hormones T4 and T3 were measured by ultrafiltration and RIA (39, 40). The clinical diagnosis of diffuse toxic goiter (Graves' disease) was confirmed by measurements of thyrotropin receptor antibodies (Lumitest TRAK human; Brahms Diagnostica). We used a two-site immunoassay ELISA (2) to measure serum insulin. A double monoclonal immunoflourometric assay (Delfia, Wallac, Finland) was used to measure serum growth hormone (GH), while plasma glucagon (26) and serum C-peptide (Immunoclear, Stillwater, MN) were measured by RIAs. Serum IGF-I was measured with an in-house time-resolved fluoroimmunoassay and urea with a commercially available kit (COBAS, INTEGRA; Roche, Hvidovre, Denmark). Serum free fatty acids (FFA) were determined by a colorimetric method employing a commercial kit (Wako Chemicals, Neuss, Germany). Blood samples were deproteinized with Perchloric acid for determination of glycerol, 3-hydroxybutyrate and lactate by an automated fluorometric method (14).
Respiratory exchange rates (RQ) and total EE were measured by indirect calorimetry (Deltatrac; Datex Instrumentarium, Helsinki, Finland), and anthropometrical measurements and whole body DEXA scanning (Hologic QDR 1000/2000/W scanner) were performed to evaluate changes in body composition before and after treatment. Cross-sectional computed tomography (CT) scanning of the femur was performed to assess muscle area, as described previously (24). The maximal voluntary isometric strength of the left quadriceps muscle and the right biceps muscle was assessed by means of an electronic dynamometer (Metitur), and the values reported are the highest of five attempts.
Calculations of phenylalanine kinetics.
The equations of Thompson et al. (37) were used for measurements of whole body phenylalanine kinetics. Phenylalanine flux (QPhe) and tyrosine flux (QTyr) were calculated as follows:
in which i is the rate of tracer infusion (µmol·kg1·h1) and Ei and Ep are enrichment of the tracer infused and plasma enrichment of the tracer at isotopic plateau, respectively. The rate of phenylalanine conversion by hydroxylation to tyrosine (Ipt) was calculated as follows:
where [15N]Tyrei and [15N]Pheei are the isotopic enrichments of the respective tracers in plasma and IPhe is the infusion rate of [15N]phenylalanine (µmol·kg1·h1).
Phenylalanine incorporation into protein is calculated by subtracting Ipt from QPhe, since phenylalanine is irreversibly lost from the bloodstream either by its hydroxylation into tyrosine or by incorporation into protein.
In the forearm study, phenylalanine balance (PheBal) was calculated as follows using Fick's principle:
in which Phea and Phev are arterial and deep venous phenylalanine concentrations and F is blood flow in the forearm. Regional phenylalanine kinetics were calculated, using the equations described by Nair et al. (22). The forearm protein breakdown represented by phenylalanine rate of appearance (Ra Phe) was calculated as follows (4):
in which PheEa and PheEv represent phenylalanine isotopic enrichment in arteries and veins. The local rate of disappearance, which represents the muscle protein synthesis rate, was calculated as:
where Rd Phe is phenylalanine synthesis rate.
Statistics.
All the data were tested for normal distribution using SPSS for Windows 10.0 (SPSS, Chicago, IL), and Student's paired t-test or Student's unpaired t-test were employed for comparisons. Results are expressed as means ± SE. P values <0.05 were considered significant.
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RESULTS
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Clinical characteristics: Thyroid hormones, body composition, muscle strength, and indirect calorimetry.
In the hyperthyroid state, the patients had a two- to fivefold elevation of total and free T3, compared with after treatment, when T3 decreased to normal levels (Table 1). Patients and control subjects were of comparable age and height, and, after treatment of the patients, their body weight was not significantly different from the control subjects. All patients were treated with methimazole, which inhibits thyroid hormone synthesis in the thyroid gland. Methimazole has no known metabolic effects. The patients gained an average of 4 kg body wt during treatment, and DEXA scans showed proportional increments in fat and lean body mass. The cross-sectional area of muscle at the midfemoral level increased with treatment, whereas the fat area remained unchanged, indicating that the increase of body fat mass with treatment was mainly in the upper body. Before treatment, the maximum isometric contraction of both the upper or lower limbs were decreased compared with after treatment, and the quadriceps contractility was decreased by 11 ± 4% (P = 0.04) in hyperthyroidism compared with the euthyroid state. In the hyperthyroid state, total EE was increased (1,989 ± 79 vs. 1,552 ± 87 kcal/24 h after treatment) and RQ was decreased, indicating increased lipid oxidation (Table 2).
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Table 1. Clinical characteristics and thyroid hormones in hyperthyroid patients before and after treatment and in control subjects
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Table 2. Fasting circulating hormones and metabolites in hyperthyroid patients before and after treatment and in control subjects
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Circulating metabolites and hormones.
In hyperthyroidism, fasting levels of glucose (Table 2) tended to be higher, glucose arteriovenous difference was lower, whereas circulating levels of insulin, C-peptide, glucagon, lactate, urea, GH, and IGF-I did not differ with thyroid state in our study. As for the metabolites of lipid metabolism, glycerol levels were elevated during hyperthyroidism, and the concentrations of FFA and 3-hydroxybutyrate tended to be elevated.
Whole body amino acid kinetics.
Isotopic enrichments reached a plateau at the end of the study period (Fig. 1). This was assessed based on the observation that, when isotopic enrichment values for phenylalanine and tyrosine were plotted against time, the ensuing slopes were not different from zero (P values between 0.20 and 0.29). In the hyperthyroid state, whole body phenylalanine and tyrosine fluxes were increased (Table 3) compared with the euthyroid state. Phenylalanine conversion to tyrosine (reflecting amino acid degradation) was increased, and protein synthesis (phenylalanine disposal not accounted for by phenylalanine conversion to tyrosine) was increased.
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Table 3. Fasting amino acid fluxes and forearm blood flow in hyperthyroid patients before and after treatment and in control subjects
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Amino acid levels.
Whole blood concentrations of amino acids are shown in Table 4. The total amino acid concentrations did not differ between the study groups. Concentrations of phenylalanine, tyrosine, and the branched-chain amino acids (leucine, isoleucine, and valine) were elevated in hyperthyroidism. By contrast, the gluconeogenic amino acids (alanine, serine, and glycine) were decreased, although not significantly (P = 0.06).
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Table 4. Whole blood concentrations of amino acids in hyperthyroid patients before and after treatment and in control subjects
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Regional muscle amino acid kinetics.
We saw a net forearm release of phenylalanine in all study groups, the release being increased in hyperthyroidism (Fig. 2, P = 0.04 and 0.007 vs. euthyroid patients and controls, respectively). Muscle protein breakdown, assessed by phenylalanine rate of appearance, was increased (P = 0.03) in hyperthyroid patients and became normalized with treatment. Muscle protein synthesis rate did not differ (P = 0.3). Forearm blood flow was increased 35% in hyperthyroidism and was normalized after treatment.

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Fig. 2. Forearm protein turnover in hyperthyroid patients before and after treatment and in healthy controls. Data are presented as means ± SE. A: phenylalanine arteriovenous balance across the forearm. B: rate of appearance of phenylalanine (Ra Phe; muscle protein breakdown). C: rate of disappearance of phenylalanine (Rd Phe; muscle protein synthesis).
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DISCUSSION
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The present study, which was designed to define the effects of thyroid hormone excess on muscle protein metabolism and the mechanisms leading to sarcopenia and myopathy in hyperthyroidism, shows a number of abnormalities in muscle protein metabolism. Sarcopenia was confirmed by DEXA and CT scans, and we found decreased contractile force. A previous study has shown muscle fiber atrophy in muscle biopsies from hyperthyroid patients (41), supporting the concept that hyperthyroidism leads to decreased muscle protein and fiber content. We observed an increased net release of amino acids across the forearm muscular bed, and application of phenylalanine tracer dilution technique revealed that the excessive loss of muscle protein was the result of accelerated muscle protein breakdown rather than defective protein synthesis.
The hyperthyroid patients had increased whole body fluxes of both phenylalanine and tyrosine, reflecting a high turnover state with concomitant increases of protein breakdown and synthesis. This is in keeping with the notion that thyroid hormones have both anabolic and catabolic actions, as suggested by the reports of Rochon et al. (29) and Tauveron et al. (35) using a leucine tracer method. At the whole body level, we observed a 40% increase in protein synthesis rate (calculated as total phenylalanine flux phenylalanine-to-tyrosine degradation), and in the forearm we saw a similar nonsignificant increase in regional muscle protein synthesis. The fact that the increase in muscle protein synthesis was nonsignificant could represent a type 2 error, and the findings, in all probability, reflect that nonmuscle and muscle protein synthesis is increased to a similar degree by thyroid hormones.
The results of our forearm studies showed that the changes in whole body fluxes, to a large extent, were because of increased muscle protein breakdown, since the forearm rate of appearance for phenylalanine was elevated by 60% in hyperthyroidism, whereas whole body protein breakdown (phenylalanine flux) was only elevated by 40%. This is supported by a previous observation of increased release of branched-chain amino acids by the forearm (31) and by Morrison et al. (20), who reported a sixfold increase in net tyrosine release across the leg in patients with untreated hyperthyroidism. The latter study, however, failed to observe any increase in 3-methylhistidine efflux from the leg, possibly because the analytical performance of 3-methylhistidine measurements is not sufficient to detect changes in muscle metabolism. Compared with other amino acids, the concentrations of 3-methylhistidine in the blood are very low, and arteriovenous gradients of 3-methylhistidine are >1,000-fold below that of certain other amino acids, making it very difficult to detect differences in regional muscle 3-methylhistidine efflux.
Branched-chain amino acids are known to stimulate protein synthesis and inhibit protein breakdown (15, 43), and the increase of blood concentrations of branched amino acids in hyperthyroidism may thus be a compensatory mechanism, whereby protein is preserved. The pattern of increased branched-chain amino acid concentrations and decreased gluconeogenic amino acid concentrations in hyperthyroidism corresponds to what others have reported (38), and probably reflects increased splanchnic utilization of gluconeogenic amino acids and increased peripheral mobilization of branched-chain amino acids (8, 9).
It is noteworthy that protein catabolism prevails in hyperthyroidism despite increased concentrations of a number of anabolic agents. Circulating levels of FFA, glycerol, and ketone bodies are, in general, elevated, and these lipid intermediates have been shown to stimulate protein anabolism (36); this may represent a protein-sparing mechanism in hyperthyroidism (23, 28). Furthermore, basal insulin levels, if anything, tend to be elevated in hyperthyroid patients. Insulin inhibits whole body and muscle protein breakdown (11, 13) and may stimulate protein synthesis in the presence of high amino acid concentrations. Previous studies have shown that GH exerts stimulation of protein synthesis and inhibition of breakdown (25), and, even though we could not detect any difference in single GH measurements in the present study, it has been shown earlier that the 24-h production rate of GH in thyrotoxicosis is increased nearly fourfold (10). It is possible that these anabolic fuels counteracted the increased catabolic drive in hyperthyroidism.
In the present study, measurements of protein turnover were performed using phenylalanine and tyrosine tracers. The essential amino acid phenylalanine is an attractive tracer because it is not synthesized endogenously and has only two fates in the body: incorporation into protein or irreversible hydroxylation into tyrosine, which occurs in the liver and the kidney (18). We therefore assume that the flux of phenylalanine measured across the forearm area represents skeletal muscle protein turnover (22). As with other tracer methods, the major assumptions of the method are that phenylalanine truly reflects the turnover of protein in the body, that there is a single homogenous pool of free amino acids, that the infused tracer is uniformly mixed in the entire pool, and that the tracer is in steady state, i.e., all fluxes are constant. In our study, enrichment levels in arterialized and venous plasma were constant during the 30-min sampling period, indicating steady-state conditions. Determination of blood flow represents another possible source of error; if, for instance, blood flow determinations are spuriously high, this would give rise to calculation of elevated values for net forearm amino acid release and muscle protein breakdown and protein synthesis. In the present study, we recorded a 35% increase in forearm blood flow. Previous studies have reported 50280% increases in basal resting forearm blood flow (5, 19) and >100% increases in leg blood flow in hyperthyroid subjects (20). The validity of our data is further supported by the fact that only muscle protein breakdown was significantly increased in hyperthyroidism (P values between 1.8 and 3.1%), whereas protein synthesis was not significantly affected (P values between 25 and 29%). It is possible that the increased blood flow may contribute to increased protein breakdown and relatively decreased synthesis via mass action by maintaining a lower amino acid concentration in the blood nourishing the muscle cells. Our data do not provide any answers to the issue of how hyperthyroidism affects the kinetics of specific muscle proteins, such as, for instance, myosin heavy chain. This question awaits answers from studies using a combination of tracer dilution and muscle biopsy techniques. Three previous studies (1, 30, 42) have, however, shown that urinary excretion of 3-methylhistidine, a marker of muscle myofibrillar protein degradation, is increased in hyperthyroidism. This strongly supports the view that myofibrillar degradation is increased in hyperthyroidism. The findings of normal 3-methylhistidine release across a muscle bed (20) and normal urinary 3-methylhistidine excretion (7) in other studies may relate to methodological insensitivity of the arteriovenous technique in the former and possibly intake of food items containing 3-methylhistidine in the latter. Our observations of 1020% decrements in both muscle strength and area also support that the myofibrillar content of muscle is decreased in hyperthyroidism.
We found that both protein breakdown and synthesis at the whole body level are increased in hyperthyroidism, supporting the concept that thyroid hormones are both anabolic and catabolic and indicating that some of the excessive EE in hyperthyroidism, apart from glucose and FFA, is due to futile substrate cycling of protein and amino acids (21, 32, 33). Other energy-consuming processes could be increased activity of Na+-K+-ATPase and Ca2+-ATPase (3). Spontaneous physical activity is increased in hyperthyroidism, and nonexercise activity thermogenesis (34) may contribute.
In summary, we find that hyperthyroidism is characterized by elevated basal whole body protein breakdown and synthesis. The mechanisms maintaining sarcopenia and myopathy in hyperthyroid subjects include an inappropriately high rate of net muscle amino acid release due to increased muscle protein breakdown.
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GRANTS
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This study was supported by The Aarhus University Research Fund and Musikforlæggerne Agnes og Knut Mørks fund.
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ACKNOWLEDGMENTS
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Lone Svendsen and Iben Christensen are thanked for excellent technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: A. L. Riis, Medical Dept. M, Aarhus Univ. Hospital, DK-8000 Aarhus C, Denmark (E-mail: anne.lene.riis{at}ki.au.dk)
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.
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