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Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. K. Sreekumaran Nair, Mayo Clinic, 200 First Street SW, Joseph 5-194, Rochester, Minnesota 55905. E-mail: nair.sree{at}mayo.edu.
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Abstract |
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Objective: The objective of the study was to determine whether treatment of hyperthyroidism causes changes in amino acid fluxes, synthesis rates of muscle proteins, and expression of muscle myosin heavy chain (MHC) that may impact skeletal muscle function and metabolic rate.
Methods: Eight hyperthyroid patients were studied (TSH 0.008 ± 0.001 mU/liter) before treatment and at least 9 months after correction of hyperthyroidism (TSH 2.3 ± 0.4) (P < 0.03). Fluxes of leucine and phenylalanine as well as muscle protein synthesis rates were measured using L[1,2 13C] leucine and L(15N) phenylalanine as tracers. mRNA levels of selected genes were measured in muscle biopsy samples.
Results: Treatment decreased resting metabolic rate that paralleled changes in fluxes of leucine and phenylalanine accompanied by improved muscle strength and mass. Synthesis rates of mixed muscle proteins (P = 0.01), sarcoplasmic (P = 0.04), and mitochondrial (P = 0.08) proteins decreased, whereas MHC synthesis was unchanged. Selective increases in mRNA abundance of muscle MHC1 isoform (P = 0.04) and decrease of MHCIIA (P = 0.007) and MHCIIx (P = 024) were observed. Muscle mitochondrial oxidative enzymes and mRNA levels of mitochondrial proteins were unchanged, but uncoupling protein2 and uncoupling protein3 mRNA levels (P = 0.02) decreased.
Conclusion: Increased amino acid flux, mixed muscle protein synthesis, and synthesis of sarcoplasmic proteins are consistent with the hypermetabolic state in hyperthyroidism. After treatment, MHC synthesis rates were unchanged, but mRNA levels of isoforms of MHC found in slow-twitch and fast-twitch fibers increased and decreased, respectively. These results offer a mechanistic explanation for posttreatment improvement in muscle functions in hyperthyroidism.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The cause of muscle wasting and weakness is poorly understood, although recent studies in rats (4) and humans (5) have shown that, despite increased energy flux in this condition, it appears likely that uncoupling of oxidative phosphorylation in skeletal muscle also occurs. Thyroid hormones also play a pivotal role in regulating body metabolism at different levels (6). There is very limited information, however, regarding the impact of treatment on muscle protein metabolism at the biochemical and molecular levels. Changes in muscle protein synthesis are likely to have major impact on muscle function. Previous studies demonstrated that the hyperthyroid state is associated with a net increase in efflux of branched chain amino acids, phenylalanine, and tyrosine from the muscle bed (7, 8, 9), indicating net muscle protein catabolism. Furthermore, tracer dilution studies have shown that hyperthyroidism is associated with increased muscle protein breakdown (7, 8), whereas a forearm study showed no effect of muscle protein synthesis in this condition (7). Direct measurement of the incorporation of amino acids in to muscle proteins has not been reported in human hyperthyroid state.
Studies in rats have demonstrated that hyperthyroidism is associated with enhanced metabolic activities in muscles with high oxidative capacity such as the soleus as opposed to less oxidative muscles such as the plantaris (4). This metabolic action is specifically noted in muscle mitochondria with enhancement of oxidative phosphorylation and increased expression of uncoupling proteins (UCPs). Moreover, feeding rodents with T3 has been shown to effect the expression of isoforms of myosin heavy chain (MHC) (10), which is a major contractile protein involved in ATPase actions in skeletal muscle. This effect seems to be muscle specific, and whether these effects are present in humans with mostly mixed muscle fiber remains to be determined. Nor has it been determined in humans whether the hyperthyroid state is associated with changes in muscle MHC isoform expression that alters muscle oxidative capacity. The current studies were undertaken to further characterize the metabolic changes that occur in human skeletal muscle after treatment of hyperthyroidism and better understand the underlying mechanisms that might mediate them.
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Patients and Methods |
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shows subject characteristics. The diagnosis of hyperthyroidism was confirmed by clinical examination and laboratory tests that included suppressed levels of serum TSH in the presence of elevated levels of serum free T4 (Table 2). None of the patients had concomitant musculoskeletal conditions or were taking medications known to influence skeletal muscle metabolism. The initial study was performed before the initiation of ß-blocker therapy and radioactive iodine (131I) administration. The follow-up study was performed after an interval of at least 9 months after the restoration of a euthyroid state. Treatment of hyperthyroidism resulted in hypothyroidism diagnosed clinically and confirmed by a posttreatment rise in serum TSH. Restoration of a euthyroid state was achieved by the daily oral levothyroxine treatment and confirmed by normalization serum TSH and free T4.
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Body composition was measured using dual x-ray absorptiometry (DEXA-DPX-IA; Lunar Corp., Madison, WI) (11), and thigh muscle area was measured using computed tomography scan (12) before and after treatment. Leg muscle strength was assessed by dynamometry. Knee flexor and extensor isometric muscle strength at 60 °/sec and 180 °/sec were assessed before and after treatment.
The studies were performed at the Mayo General Clinical Research Center. Patients were placed on an isocaloric diet appropriate to their resting energy expenditure for 3 d before the study day. They were studied after an overnight fast and received a priming dose of L[1,213C] leucine (1 mg/kg), L[15N] phenylalanine (0.6 mg/kg), and [13C] NaBicarbonate (0.2 mg/kg) at 0400 h with a continuous infusion of [1,2 13C] leucine (1.0 mg/kg·h) and L[15N] phenylalanine (0.6 mg/kg·h) for a 7-h period. L-[1,2 13C] leucine was purchased from Isotec Inc. (Miamisburg, OH) and Mass Trace (Woburn, MA). L[15N] phenylalanine and [13C] NaBicarbonate were purchased from Cambridge Isotope Labs (Andover, MA). Blood samples were collected at 0 time and 30-min intervals during the last 5 h. Needle biopsies of each vastus lateralis muscle were performed at 3 and 7 h, respectively, after isotope infusion.
Hormones and glucose measurements
Plasma insulin was measured by chemoluminescent sandwich assay (Sanofi Diagnostics, Chaska, MN). IGF free, IGF total, IGF-II, IGF binding protein (IGFBP)1, IGFBP2, and IGFBP3 were measured using a two-site immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX). Blood glucose was measured by the glucose oxidase method using glucose autoanalyzer (Beckman Instruments, Fullerton, CA).
Plasma isotope enrichment of phenylalanine, leucine, and ketoisocaproate (KIC)
Plasma and tissue fluid enrichment levels of [15N] phenylalanine and [1,2-13C] leucine and plasma levels of [1,2-13C] KIC were determined by gas chromatography/mass spectrometry. The amino acids from plasma were isolated by ion exchange chromatography and the tertiary butyldimethylsilyl ether derivative was prepared using N-methyl-N(t-butyldimethylsilyl)-trifluoroacetamide with 1% t-butyl-dimethylchlorosilane in acetonitrile (Regis Technologies Inc., Morton Grove, IL) by reaction at room temperature overnight. The amino acids were separated on a 30 m x 0.25 mm x 0.25 µm DB5MS column (J&W Chromatography, Folsom, CA). Under electron ionization, selected ions were monitored at m/z 302 and 304 for leucine and [1,2-13C]leucine, respectively, and m/z 336 and 337 for phenylalanine and its 15N analog, respectively, using a Hewlett Packard MS engine (Hewlett Packard, Avondale, CA) to determine the isotopic enrichment. [1,2-13C]KIC was also isolated by ion exchange chromatography and its quinoxalinol-trimethylsilyl ether derivative prepared using o-phenylenediamine followed by Bis(trimethylsilyl) trifluoracetamide (Regis Technologies). Ions at m/z 232 and 234 for KIC and [1,2-13C]KIC, respectively, were monitored using a Finnigan Voyager gas chromatography/mass spectrometry (Thermo Electron Corp., Waltham, MA) under electron ionization conditions (13). Muscle tissue fluid was obtained by homogenizing the muscle biopsy tissue in 1 M perchloric acid. After centrifugation, the supernatant containing the free pool of amino acids was removed and purified by a cation exchange column. The acidified samples were applied to the column, and the amino acids were eluted with 2 ml of 4 N NH4OH. The detailed description of this process has been reported previously (13). The processing of muscle samples for mixed muscle protein (13) mitochondrial and MHC (14, 15), sarcoplasmic proteins (16), mitochondrial proteins (17), and measurements of isotopic enrichment by gas chromatograph to combustion to isotope ratio mass spectrometry were performed as previously described (18).
Fractional synthesis rates of mixed muscle proteins
We calculated fractional synthesis rates of muscle proteins using plasma and tissue fluid leucine, plasma KIC, and plasma and tissue fluid phenylalanine enrichment as precursor pools as previously described (13, 19). Plasma KIC enrichment is theoretically a better representation of intracellular leucine isotopic enrichment than plasma leucine enrichment, and muscle tissue fluid leucine and phenylalanine enrichment values are closer to amino acyl tRNA, the obligatory precursor of protein synthesis (13, 20). KIC is the obligatory precursor of leucine oxidation and therefore used as precursor for calculation of leucine oxidation.
RNA isolation and cDNA synthesis
RNA isolation and cDNA synthesis were completed as described (21). Briefly, skeletal muscle biopsy samples were immediately frozen in liquid nitrogen after collection and kept at –80 C until analysis. Total RNA was extracted for approximately 50 mg skeletal muscle by the guanidinium method (TriReagent; Molecular Research Center, Cincinnati, OH). Total RNA (1 µg) was treated with DNase (Life Technologies, Gaithersburg, MD) and reverse transcribed using a TaqMan reverse transcription kit (PE Biosystems, Foster City, CA) according to the manufacturer’s instruction.
Quantitative PCR
Primer and probe sequences and the quantitative PCR procedure were as previously described (21). Briefly, primers and probes for 28S rRNA and MHC isoforms were selected using the Primer Express v.1.0 software (PE Biosystems) and screened for mispriming of isoforms using the Oligo Primer analysis software v.5.0 (National Biosciences, Plymouth, MN). The probes were labeled at the 5' end with the reporter dye 6'-carboxyfluorescein and at the 3' end with the quencher dye 6'-carboxytetramethylrhodamine and were phosphate blocked at the 3' end to prevent extension. The TaqDNA polymerase allows for the separation of the reporter from the quencher. The resulting fluorescence was measured at each cycle of amplification by the ABI sequence detection system (ABI Prism 7700). All samples were quantitated by normalizing the MHC signal with the 28S rRNA signal. The final quantitation was achieved with a relative standard curve.
Statistics
Differences between the hyperthyroid and treated (euthyroid) conditions were compared using a paired t test. Two-tailed t test was used in all cases except when hypotheses being tested are clearly one sided (muscle strength). P < 0.05 was considered significant. Regression analysis was performed to determine whether amino acid fluxes are related to energy expenditure.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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)Treatment of hyperthyroidism and rendering the patients euthyroid status resulted in significant increase in body weight, body mass index, fat mass, fat-free mass, and thigh muscle area.
Hormones and substrates (Table 2)
Free T4 and TSH levels confirmed hyperthyroidism at the time of the initial study and restoration of a euthyroid state at the time of the follow-up study. Whereas total IGF-I and IGF-II did not change, a significant reduction in IGFBP1 and IGFBP2 with a nonsignificant reduction of IGFBP3 was noted. Both nonesterified fatty acid (NEFA) and glycerol levels also decreased significantly, whereas glucose levels did not change.
Amino acids (Table 3)
A significant reduction in glutamine, histidine, thionine, valine, leucine, and total amino acid concentrations occurred after treatment.
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Muscle strength showed significant improvement or near significant improvement in all cases, as expected. When muscle strength was normalized for fat-free mass of leg, the trend to improve muscle strength per leg fat-free mass persisted, but only some of the parameters showed statistically significant changes.
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Fig. 1A shows a significant reduction in leucine flux (based on both plasma leucine and KIC isotopic enrichment), and phenylalanine flux was noted after treatment. Fig. 1B shows significant reduction in CO2 production, O2 consumption, respiratory quotient, and energy expenditure. The energy expenditure showed a significant correlation to leucine flux (hyperthyroid: r2 = 0.898, P < 0.01; euthyroid: r2 = 0.638, P < 0.051) and phenylalanine flux (hyperthyroid: r2 = 0.752, P < 0.05; euthyroid: r2 = 0.252, P > 0.1).
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FSR calculations were performed using plasma and tissue fluid leucine, plasma KIC, and plasma and tissue fluid phenylalanine enrichment as precursor pools. It demonstrated that mixed muscle protein FSR (Fig. 2A) and sarcoplasmic protein FSR significantly decreased after treatment, irrespective of the precursor pool and tracer used. Analysis could be performed in only a limited number of samples (n = 5) in case of mitochondrial proteins FSR, which showed a consistent (P < 0.08) trend to decrease with treatment. MHC FSR also showed a trend to increase, but no statistical significance was observed. Because of the limited amount of MHC and mitochondrial proteins that could be purified from the needle muscle samples, we could not get a significant amount of muscle phenylalanine levels to measure its isotopic enrichment in gas chromatograph/combustion isotope ratio mass spectrometer, and therefore, the measurements were based on leucine as a tracer.
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)After the restoration of a euthyroid state, there was a 60% increase in mRNA for MHC I (P < 0.04) accompanied by 22 and 35% declines in mRNA for MHCIIa (P < 0.007) and -IIx (P < 0.02), respectively.
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) did not change with treatment of hyperthyroidism. No changes in mRNA levels (Fig. 4) of NADH subunit 4 (ND4), cytochrome c oxidase subunit 3 (COX3), or cytochrome c oxidase subunit 4 (COX4) occurred upon restoration of a euthyroid state. However, mRNA levels of UPC2 and UPC3 decreased upon restoration of a euthyroid state.
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Discussion |
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The study demonstrated that treatment of overt hyperthyroidism results in reduction of whole-body fluxes of leucine and phenylalanine, indicating a reduction in proteins turnover at the whole-body level. These results are consistent with the previous reports at the whole-body level and across muscle beds showing higher protein breakdown in hyperthyroid patients (7, 8). A novel and important finding of the current study is that treatment of hyperthyroidism in human subjects reduces the average synthesis rates of muscle proteins as demonstrated by mixed muscle protein synthesis rate measurements. However, these changes in muscle protein synthesis rates are not similar for all components of muscle proteins. Although the synthesis rate of sarcoplasmic proteins also decreased after treatment, synthesis rates of MHC protein, although trending upward, did not reach a statistical significance. Moreover, the changes in mRNA levels of MHC isoforms suggest that hyperthyroidism has differential effects on individual muscle proteins with a clear tendency to promote fast-twitch muscle action. The changes in protein metabolism and muscle mRNA levels occurred in association with substantial changes in both muscle performance and energy metabolism. The biochemical and molecular level changes helped to further our understanding of the mechanism of improvement in muscle performance and decrease in energy expenditure in response to treatment of hyperthyroidism.
The current study did not measure muscle protein breakdown, which involves arteriovenous catheterization in addition to muscle needle biopsies that are required for the measurement muscle protein synthesis. However, the whole-body amino-acid flux in combination with previous reports (7) is consistent with an increased muscle protein and whole-body breakdown during the hyperthyroid state. The results suggest that many of the muscle proteins that show increased synthesis rates are sarcoplasmic. Sarcoplasmic proteins reflect enzyme systems intimately involved in multiple metabolic functions and anaerobic ATP production. The synthesis rates of mitochondrial proteins also showed a decrease with treatment, although the changes are of borderline significance. In addition, we found no changes in the activities of citrate synthase and cytochrome c oxidase, the two key mitochondrial oxidative enzymes. We also did not find any significant changes in mRNA levels of genes encoding mitochondrial proteins involved in electron chain transport. It is possible that these changes occur rather acutely and may not be seen in chronic situations. We noted, however, a lowering of mRNA levels of UCP2 and UCP3 after treatment. These findings are consistent with our previously reported results in rodent studies that showed increased mRNA levels of UCP2 and UCP3 (22) and uncoupling of oxidative phosphorylation in hyperthyroid human subjects (5). In rodents, we also noted increased muscle mitochondrial ATP production and oxidative enzyme activities in the hyperthyroid state (22). It is also possible that differences in the duration and/or severity of hyperthyroidism between rodents and humans may explain the observed differences, or alternatively they reflect species differences.
MHC isoform mRNA levels changed significantly after treatment. A significant increase in mRNA levels of MHC1 accompanied by a decrease in MHCIIa and MHCIIx were noted. MHC1 proteins are expressed in slow-twitch muscle fibers, whereas MHCII isoforms are expressed in fast-twitch muscle fibers. The higher expression of MHCIIa mRNA levels is consistent with the reports in rat models, especially in certain muscle groups (23). Because MHCIIx expression predominates in humans, direct comparison with rodents is not possible. Both MHCIIa and MHCIIx proteins are predominant in glycolytic fibers. It is therefore likely that a phenotypic change in muscle fiber type occurs in response to hyperthyroidism that favors fast-twitch, glycolytic fibers. These fibers produce ATP primarily through anaerobic pathways that are promoted by a number of sarcoplasmic proteins. The increased synthesis rates of sarcoplasmic proteins combined with the greater expression of genes favoring type II fibers in hyperthyroidism are consistent with enhanced fast-twitch muscle action (24) that primarily depends on anaerobic respiration. Of note, MHC synthesis rates, which reflect average synthesis rates of all isoforms, were not significantly altered by treatment. The mRNA data indicate that MHC1 and MHCII isoforms changed in opposite directions. Therefore, if a similar change in MHC synthesis rates in each direction occurs, then the average of both MHCI and MHCII may be expected to be little changed.
The results demonstrated an improvement in fat and muscle mass after treatment. The changes in energy expenditure show substantial decrease in energy expenditure with treatment that could account for increased energy store as fat. There was also a decline in NEFAs and glycerol concentrations consistent with reduced lipolysis. Of interest, there were substantial decreases in IGFBP1 and IGFBP2 with a small nonsignificant trend in decreasing IGFBP3 as well with treatment. These changes in binding proteins, despite any unaltered IGF total, were expected to increase free IGF levels. However, based on the assay we used, no significant change in IGF free levels was noted. This may reflect the difficulty to measure free IGF-I.
The current results, in combination with the reports from previous studies (25), offer some insight to the underlying mechanism of improved muscle functions after treatment of hyperthyroidism. The net protein anabolic effect (net reduction of muscle protein turnover) of treatment of hyperthyroidism is likely to have caused the increase in muscle mass, which in turn contributed to increased muscle strength. We observed that some parameters of muscle strength improved even after normalizing for muscle mass, indicating an improvement of efficiency or quality of muscle.
In summary, the current study demonstrated that treatment of hyperthyroidism is followed by a substantial net protein anabolic effect. Treatment of hyperthyroid patients not only decreased overall metabolic rate but also reduced protein turnover. Hyperthyroid patients have a net overall increase in muscle protein synthesis. Based on previous reports of increased muscle protein breakdown in the hyperthyroid state, it appears that there is an overall increase in muscle protein turnover, and net muscle loss is likely to result from a greater increase in muscle protein breakdown than on synthesis rate. The improvement in muscle strength seems to result by not only increasing muscle mass but also improving the quality (or efficiency) of the muscle.
Changes in skeletal muscle metabolism and MHC isoform composition in response to even mild degrees of hyperthyroidism may have particular relevance in certain patient populations including elderly people who may be already at a disadvantage due to age-related sarcopenia.
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Footnotes |
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Abbreviations: FSR, Fractional synthesis rate; IGFBP, IGF binding protein; KIC, ketoisocaproate; MHC, myosin heavy chain; NEFA, nonesterified fatty acid; UCP, uncoupling protein.
Received May 17, 2006.
Accepted August 23, 2006.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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