T3 increases mitochondrial ATP production in oxidative muscle despite increased expression of UCP2 and -3

Kevin R. Short, Jonas Nygren, Rocco Barazzoni, James Levine, and K. Sreekumaran Nair

Endocrinology Research Unit, Mayo Clinic, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Triiodothyronine (T3) increases O2 and nutrient flux through mitochondria (Mito) of many tissues, but it is unclear whether ATP synthesis is increased, particularly in different types of skeletal muscle, because variable changes in uncoupling proteins (UCP) and enzymes have been reported. Thus Mito ATP production was measured in oxidative and glycolytic muscles, as well as in liver and heart, in rats administered T3 for 14 days. Relative to saline-treated controls, T3 rats had 80, 168, and 62% higher ATP production in soleus muscle, liver, and heart, respectively, as well as higher activities of citrate synthase (CS; 63, 90, 25%) and cytochrome c oxidase (COX; 119, 225, 52%) in the same tissues (all P < 0.01). In plantaris muscle of T3 rats, CS was only slightly higher (17%, P < 0.05) than in controls, and ATP production and COX were unaffected. mRNA levels of COX I and III were 33 and 47% higher in soleus of T3 rats (P < 0.01), but there were no differences in plantaris. In contrast, UCP2 and -3 mRNAs were 2.5- to 14-fold higher, and protein levels were 3- to 10-fold higher in both plantaris and soleus of the T3 group. We conclude that T3 increases oxidative enzymes and Mito ATP production and Mito-encoded transcripts in oxidative but not glycolytic rodent tissues. Despite large increases in UCP expression, ATP production was enhanced in oxidative tissues and maintained in glycolytic muscle of hyperthyroid rats.

triiodothyronine; adenosine 5'-triphosphate; citrate synthase; cytochrome c oxidase; uncoupling proteins; metabolic rate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE REGULATION OF OXIDATIVE METABOLISM by thyroid hormone has been of considerable interest for many years. In hyperthyroid rodents, a failure to maintain normal growth of body mass (41, 50) despite increased food consumption (50, 51) has been reported. This is likely the consequence of higher energy expenditure, as shown by increased rates of whole body oxygen consumption (VO2) (20, 40, 42). Thyroid hormones have been recognized as major regulators of oxidative energy metabolism at the level of the mitochondrion. Mitochondria isolated from hyperthyroid animals display increased VO2 rates (30, 40, 42) due to accelerated import and oxidation of fuel substrates (30, 31, 34). This increased metabolic flux is also facilitated by higher activities of enzymes in the oxidative pathway (7, 33, 42, 49, 51).

An important aspect of mitochondrial metabolism that has yet to be fully defined in the hyperthyroid state is ATP production. The increases in substrate oxidation (30, 40, 42) and enzymatic capacity (7, 33, 42, 49, 51) in various tissues suggest that ATP production capacity may be enhanced by thyroid hormone. Conversely, much of the energy from fuel oxidation may be lost as heat rather than being used for ATP production, because there can be increased mitochondrial proton leak, as shown in liver preparations from hyperthyroid rodents (3, 15-17). Potential mechanisms regulating the proton leak include changes in the size and phospholipid composition of the inner mitochondrial membrane (5) and activity of membrane carrier proteins like the adenine nucleotide transporter (ANT) (39). Considerable attention has also been given to the recently discovered uncoupling protein (UCP) homologs UCP2, expressed in many tissues, and UCP3, found mainly in skeletal muscle and heart (35). Both UCP2 and UCP3 have been shown to stimulate proton leak in vitro (19, 35). Additionally, UCP3 transgenic mice have increased metabolic rate and resistance to weight gain (8), whereas UCP3 knockout mice have reduced rates of VO2 in muscle mitochondria (14, 44), thus strongly supporting the metabolic importance of UCPs in muscle. In response to in vivo administration of thyroid hormones, transcript levels of both UCP2 and UCP3 rapidly increase in several rodent tissues including highly oxidative tissues like heart, liver, and muscle (13, 20-23, 26). These observations support the possibility that increased UCP expression in hyperthyroid animals may limit the ability of mitochondria to generate ATP.

Several studies have examined the effect of hyperthyroidism on oxidative phosphorylation in isolated mitochondria, but these have typically been confined to a single tissue, either liver (11, 18) or heart (28, 30, 40), although Tata et al. (42) included both liver and mixed skeletal muscle in their study. Collectively, these findings demonstrated that mitochondrial VO2 is increased in hyperthyroid heart (30, 40), liver (18, 42), and mixed skeletal muscle (42) and that the ratio of ADP to molecular oxygen consumed (ADP:O, an index of coupling) is not significantly altered. These data indirectly suggest that mitochondrial ATP production capacity is enhanced by hyperthyroidism. However, in some studies the calculated ATP production was unchanged by thyroid hormones, either because mitochondrial VO2 and ADP:O remained unaffected (11) or because there was a decline in ADP:O that offset the increase in VO2 (28).

It is unknown whether hyperthyroidism affects mitochondrial ATP production to the same extent in different tissues, particularly in skeletal muscles with different metabolic fiber types. Tata et al. used mixed-composition hindlimb muscle in their study. Previous studies had shown that mitochondrial enzyme activities increase after thyroid hormone administration more rapidly and to a greater extent in oxidative than in glycolytic muscles (7, 49, 51). Furthermore, a recent study found that UCP2 and -3 mRNA levels were unchanged in the oxidative soleus but were increased in the glycolytic gastrocnemius muscle of hyperthyroid mice (20). Together, these findings suggest that hyperthyroidism leads to a greater increase in the ATP production capacity of oxidative than of glycolytic muscles, but this has not been previously demonstrated.

In the present study, we have measured the ATP production rate in mitochondria from soleus and plantaris muscles of hyperthyroid rats in conjunction with enzyme activities and mRNA levels of UCP2, UCP3, and the mitochondrially encoded cytochrome c oxidase (COX) subunits I and III to better understand the differential responses of these muscles to thyroid hormone. In addition, the mitochondrial ATP production capacity of liver and heart from the same animals was measured so that the magnitude of the effect of thyroid hormone could be compared among tissues with different oxidative capacities.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals and treatment. Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing 325-350 g were randomly allocated to thyroid hormone-treated or control groups (n = 9 animals per group). Triiodothyronine (T3, Spectrum Quality Products, Gardena, CA) was administered for 14 days via a subcutaneous osmotic pump (Alzet, Alza Scientific Products, Palo Alto, CA) implanted above the shoulders. The pump delivered 200 µg/day of T3, which was dissolved in saline vehicle containing 20 mM NaOH, 50 mM Na2CO3, and 5% (wt/vol) bovine serum albumin. Control animals were implanted with vehicle-only pumps for 14 days. Animals were housed in individual Plexiglas cages in a controlled environment (12:12-h light-dark cycle, 20-22°C, 50-60% relative humidity). Food consumption and body mass were recorded on alternate days throughout the treatment period. Standard laboratory rat chow (Lab Diet 5001, PMI International, Brentwood, MO) and water were provided ad libitum up to 2 h before the animals were killed. The institutional committee for animal research approved all animal care and experimental procedures.

VO2. Whole body VO2 was measured in six hyperthyroid and six control animals by use of a customized indirect calorimetry chamber (Oxymax, Columbus Instruments, Columbus, OH). Each rat was studied on the day before the osmotic pump was implanted and again from the morning of day 13 until day 14 of the intervention period, which permitted paired analysis of results. Calibration of the gas analyzers and volume flowmeter with standard gases and volumes was performed before all trials. Animals were allowed to become acclimated for 120 min before data collection was initiated, which was then continued for 24 h. Data recordings were made every 60 s and were averaged over the entire collection period.

Tissue collection and mitochondrial analysis. Animals were anesthetized by intraperitoneal injection of pentobarbital sodium. Tissues were then quickly removed and cleaned of visible blood and connective tissue. Samples (40-70 mg) from the soleus and plantaris muscles, liver, and heart (apex of left ventricle) were kept on ice in saline-soaked gauze for mitochondrial studies. The remaining portion of each tissue was immediately frozen in liquid nitrogen and used for quantifying enzyme activities and mRNA abundance. Blood samples, obtained via cardiac puncture, were used for determination of circulating thyroid hormone levels. Total thyroxine (T4) and T3 were measured by competitive chemiluminescence immunoassay with the ACS-180 automated immunoassay system (Bayer Diagnostics, Tarrytown, NY).

Mitochondrial separation procedures and buffer solutions were similar to those described by Wibom et al. (47). Samples were homogenized in buffer A (in mM: 100 KCl, 50 Tris, 5 MgCl2, 1.8 ATP, 1 EDTA) and spun at 720 g in an Eppendorf 5417C centrifuge at 4°C. The supernatant was spun at 10,000 g, and the resulting pellet was washed in buffer A, recentrifuged at either 3,000 (liver) or 9,000 g (muscles and heart), and finally suspended in buffer B (in mM: 180 sucrose, 35 KH2PO4, 10 Mg acetate, 5 EDTA) and kept on ice. Aliquots of the suspension were used for measuring mitochondrial ATP production rate, enzyme activities, and protein concentration (DC Protein Assay, Bio-Rad, Hercules, CA).

ATP production was determined using a bioluminescence technique (46, 47). Mitochondrial suspensions diluted in ATP-monitoring reagent (AMR, formula SL; BioThema AB, Dalarö, Finland; reconstituted according to supplier's recommendation in 1.2× concentration of buffer B) were added to cuvettes containing AMR, substrate, and ADP. Substrates added (in mM final concentration) were either 1) 1 pyruvate + 1 malate or 2) 1 pyruvate + 0.05 palmitoyl-L-carnitine + 1 malate + 10 alpha -ketoglutarate, with additional blank tubes used for measuring background. For each animal, all ATP production reactions for the four tissue preparations were analyzed in duplicate simultaneously. ATP production was monitored at 25°C with an automated routine in a BioOrbit 1251 luminometer (BioOrbit Oy, Turku, Finland). Internal calibration of each reaction cuvette was achieved by the addition of an ATP standard. The time required to prepare the mitochondrial suspensions (~90 min) and perform the ATP production assay (35 min) was standard for all animals studied. The variation between duplicate measurements of ATP production from a given preparation was consistently <6%. The reproducibility of the method was established before the study by dividing rat muscles or livers into 4-5 pieces and performing separate parallel analyses. The coefficients of variation for these preparations (9% for liver, 11% for muscle) were similar to those reported for human muscle studies (46).

The integrity of each fresh mitochondrial preparation was monitored by measuring the activity of CS before and after membrane disruption by freeze-thawing and the addition of Triton X-100 (36). According to this procedure, mitochondria were 94 ± 1% intact in soleus and plantaris, 96 ± 3% in liver, and 92 ± 1% in heart, with no differences between treatment groups. Frozen tissue samples from the same animals were used to determine the whole tissue activities of CS (36) and COX (12). CS activities in mitochondria and tissue homogenates were used to calculate the ATP production in the whole tissue as previously described (46).

Northern blot analysis. Soleus and plantaris muscles were used for the measurement of mRNA levels of COX subunits and UCPs. RNA analysis for the mitochondrially encoded COX polypeptides, COX I and COX III, was performed as previously described (2). The same samples and procedures were used to analyze the UCP transcripts, as follows. cDNA probes for UCP2 and UCP3 as well as for 28S rRNA transcripts were generated by RT-PCR amplification from rat skeletal muscle total RNA. Primers for the UCP2 probe corresponded to nt 6251-6274 (forward) and 6615-6635 (reverse, PCR product of 381 bp) of the rat UCP2 sequence (GenBank accession no. X14848). Primers for the UCP3 probe corresponded to nt 6251-6274 (forward) and 6615-6635 (reverse, PCR product of 381 bp) of the rat UCP3 sequence (GenBank accession no. X14848). Primers for the 28S rRNA probe corresponded to nt 4203-4222 (forward) and 4370-4389 (reverse, PCR product 186 bp) of the rat ribosomal RNA genome (GenBank accession no. V01270). Amplification products were cloned into the TA-plasmid vector (TA Cloning KIT, Invitrogen, Carlsbad, CA) used to transfect competent bacteria (TA Cloning KIT, Invitrogen) isolated (Endo Free Plasmid Maxi Kit, Qiagen, Valencia, CA) and sequenced before use. Total RNA was isolated from 30-50 mg of plantaris and soleus muscles by the guanidinium method (TRI reagent, Molecular Research Center). For each tissue, 10 µg of total RNA from each animal were separated on one 1.5% agarose-2.2 M formaldehyde gel and then transferred overnight to nylon membranes (HyBond N+, Amersham, Arlington Heights, IL) (38). Probes were radiolabeled with [32P]CTP (Decaprime KIT, Ambion, Austin, TX), and membranes were sequentially hybridized with the UCP3, UCP2, and 28S rRNA probes. For hybridization, all membranes were prehybridized at 68°C for 30 min (ExpressHyb Hybridization Solution, Clontech, Palo Alto, CA), hybridized to the radioactive probe for 1 h at 68°C and then washed at room temperature three times for 10 min in 2× standard sodium citrate (SSC)-0.05% SDS and at 50°C two times for 20 min in 0.1× SSC-0.1% SDS. Membranes were then exposed to film at -80°C for 3-16 h (Kodak Biomax MR, Kodak, Rochester, NY). The resulting images were quantified by laser densitometry (Ultroscan, Amersham Pharmacia, Uppsala, Sweden), UCP bands were normalized to the corresponding 28S rRNA band, and individual results were expressed as a relative proportion of the average value for control animals.

Western blot analysis. Mitochondrial proteins isolated from soleus and plantaris muscles of three control and three T3-treated rats were used for detection of UCP2 and -3 proteins. Mitochondrial proteins (50 mg/sample) were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immun-Blot, Bio-Rad). Membranes were blocked overnight in buffer containing 1× Tris-buffered saline (TBS), 0.1% Tween-20, and 5% (wt/vol) powdered milk. Membranes were then incubated with affinity-purified rabbit anti-human antiserum either for UCP3 (Chemicon, Temecula, CA) for 2 h at room temperature, or for UCP2 (Alpha Diagnostic International, San Antonio, TX), overnight at 4°C. The membranes were washed in TBS-Tween-20 (TBS-T) and then incubated with goat-anti-rabbit secondary antibody conjugated with horseradish peroxidase (Amersham Pharmacia) for 1 h at room temperature. After being washed again in TBS-T, the membranes were exposed with enhanced chemiluminescence (Amersham Pharmacia) on Kodak BioMax film. Protein bands were quantified by densitometry and expressed as a relative proportion of the average value for control animals.

Statistical analysis. Analysis of variance for repeated measures was used to analyze treatment effects on body mass, food consumption, and VO2. Where appropriate, post hoc comparisons were performed using Tukey's test. The remaining data were tested for treatment effects with Student's unpaired t-test. Associations between UCP levels and VO2 rates were assessed using Pearson's correlation test. Significance for all analyses was accepted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Whole body variables. Clear evidence of the treatment effect on circulating thyroid hormone was present. Serum concentrations of total T3 were higher (P < 0.001) in the group receiving thyroid hormone (755 ± 57 ng/dl) than in the control animals (57 ± 1.3). In contrast, total T4 in serum was 1.8 ± 0.1 µg/dl in control rats but was suppressed below the detectability level of the assay (<20 ng/dl) in the T3-treated animals.

At baseline, there were no between-group differences in body mass, food consumption, or VO2 (Table 1). After the 14-day experiment, body mass increased 13% in controls (pre- to postdifference, P < 0.001) but was unchanged in T3-treated rats. The daily food intake of control animals was not significantly changed during the study, whereas T3 animals increased (P < 0.01) their food intake by ~60% above their baseline as well as the intake levels of controls. Likewise, there was not a significant change in VO2 in the control group during the study, but in T3 rats, there was a 53% increase in VO2 relative to baseline (P < 0.01), which also was higher than control values (P < 0.01).

                              
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Table 1.   Body mass, food intake, and oxygen consumption at baseline and after 14 days of vehicle (control) or T3 (hyperthyroid) administration

Mitochondrial ATP production rate. In previous studies of human muscle, the substrate combination of pyruvate + palmitoyl-L-carnitine + alpha -ketoglutarate + malate was found to give higher rates of ATP production than pyruvate + malate (46). However, in the current study of rat mitochondria, there was generally close agreement within each tissue in the ATP production rates obtained with the two different substrate combinations (Figs. 1 and 2). The ATP production capacity varied considerably among the four tissues examined (Fig. 1), as expected, but there were differences in their response to hyperthyroidism. T3-treated animals had higher (P < 0.01) ATP production per unit of tissue weight (Fig. 1) in heart (62 and 60% higher than posttreatment control values with pyruvate + malate and pyruvate + palmitoyl-L-carnitine alpha -ketoglutarate + malate, respectively), liver (166 and 130%), and soleus (80 and 76%). In plantaris, there were no significant differences in ATP production between groups, although there was a trend for higher rates in T3 animals. When expressed relative to mitochondrial protein (Fig. 2), ATP production rates were higher (P < 0.03) in mitochondria from hyperthyroid heart (59 and 56% higher with pyruvate + malate and pyruvate + palmitoyl-L-carnitine alpha -ketoglutarate + malate, respectively), liver (86 and 62%), and soleus muscle (57 and 55%) but not in plantaris.


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Fig. 1.   Mitochondrial ATP production rate per gram of tissue in control (open bars, n = 9) and hyperthyroid (filled bars, n = 6) rats. Substrate combinations are pyruvate + malate (A) and pyruvate + palmitoyl-L-carnitine + alpha -ketoglutarate + malate (B). Sol., soleus; Plan., plantaris. Data are shown as means ± SE. Differences between groups are denoted as *P < 0.01, **P < 0.001.



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Fig. 2.   ATP production rate per milligram of mitochondrial protein from control (open bars) and hyperthyroid (filled bars) rats. Substrates used in A and B and number of animals used are the same as in Fig. 1. Data are shown as means ± SE. *Higher than control values, P < 0.03.

Mitochondrial enzyme activities. Relative to the controls, CS values (Fig. 3A) in T3 rats were higher by 25% in heart (P < 0.02), 90% in liver (P < 0.001), 63% in soleus (P < 0.001), and 17% in plantaris (P < 0.05). CS activity in the isolated mitochondria (Fig. 3B) was also higher in hyperthyroid animals by 23% in heart (P < 0.05), 27% in liver (P < 0.04), and 47% in soleus (P < 0.001), but in the plantaris muscle there was not a significant difference between groups. COX activities (Fig. 4A) in tissue homogenates were also higher in T3 rat heart (76% higher than control values, P < 0.04), liver (225%, P < 0.001), and soleus muscle (119%, P < 0.001) but not in the plantaris muscle. Likewise, COX activities measured in the mitochondrial suspensions were higher (P < 0.001) in T3 heart (24% higher than control values), liver (74%), and soleus muscle (91%), but not in plantaris muscle.


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Fig. 3.   Citrate synthase activity in control (open bars, n = 9) and hyperthyroid (filled bars, n = 6) rats. A: activity in tissue homogenates expressed per gram of wet tissue. B: activity in isolated mitochondria expressed per milligram of mitochondrial protein. Data are shown as means ± SE. Differences between groups are denoted as *P < 0.05, **P < 0.001.



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Fig. 4.   Cytochrome c oxidase (COX) activity in control (open bars, n = 9) and hyperthyroid (filled bars, n = 6) rats. A: activity in tissue homogenates expressed per gram of wet tissue. B: activity in isolated mitochondria expressed per milligram of mitochondrial protein. Data are shown as means ± SE. Differences between groups are denoted as *P < 0.01, **P < 0.001.

COX and UCP transcripts. COX I and COX III mRNA levels in soleus were 33 and 47% higher (P < 0.01), respectively, in hyperthyroid animals compared with corresponding values in controls (Fig. 5). In the plantaris, there were no significant differences between groups in COX transcripts. In contrast, UCP expression was higher (P < 0.001) in T3 animals in both soleus and plantaris (Fig. 6). Compared with control muscles, UCP2 levels were 2.5- and 3.0-fold higher in T3 soleus and plantaris, respectively, whereas UCP3 mRNA levels were 14-fold higher in soleus and 6-fold higher in plantaris. When data from both control and thyroid-treated animals were included, UCP2 and UCP3 mRNA levels in the soleus were positively correlated (P < 0.01) with whole body VO2 (r = 0.91 and 0.89, respectively). UCP2 and UCP3 mRNA levels in the plantaris were also positively correlated (P < 0.05) with VO2 (r = 0.53 and 0.78, respectively).


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Fig. 5.   COX mRNA expression in skeletal muscle. Mitochondrially encoded subunits I (A) and III (B) of COX were measured using Northern blot and were normalized for 28S rRNA. Mean ± SE data are presented as the relative mRNA abundance in hyperthyroid rats (filled bars, n = 9) compared with control rats (open bars, n = 9) after assigning a value of 1 to the control average. *Higher than control values, P < 0.01.



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Fig. 6.   Uncoupling protein (UCP) mRNA expression in skeletal muscle. UCP2 (A) and UCP3 transcripts (B) were measured using Northern blot and normalized for 28S rRNA. Mean ± SE data are presented as the relative mRNA abundance in hyperthyroid rats (filled bars, n = 9) compared with control rats (open bars, n = 9) after assigning a value of 1 to the control average. *Higher than control values, P < 0.001.

UCP Western blots. The UCP2 protein level in soleus was significantly higher in T3-treated rats than in the control animals (3.3 ± 0.4 vs. 1.0 ± 0.1 relative units, P < 0.04). In the plantaris, however, UCP2 protein was not detectable in either control or T3 animals. In contrast, the level of UCP3 protein in plantaris was evident in both treatment groups, with significantly higher levels in T3 animals than in controls (9.8 ± 1.0 vs. 1.0 ± 0.2, P < 0.02). UCP3 was not detectable in soleus muscle from control rats but was present in T3 rats. As a point of reference, the UCP3 protein level in T3 soleus was 4.5-fold higher than the UCP3 levels in control plantaris analyzed on the same blot.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The current study demonstrates that experimental hyperthyroidism in rodents results in increased mitochondrial ATP production, enzyme activities, and gene expression of mitochondrial-encoded transcripts in oxidative, but not glycolytic, skeletal muscle. In contrast, both muscle types had increased expression of the nuclear-encoded UCP2 and UCP3 at the protein and/or mRNA level. These data demonstrate that T3 regulation of mitochondrial function and protein expression is highly complex. Like the soleus muscle, the heart and liver, two other highly oxidative tissues, also responded to hyperthyroidism with large increases in mitochondrial ATP production and enzyme activities. Together, the current findings illustrate that the effects of T3 on mitochondrial metabolism are regulated in a tissue-specific manner, which may be related to the mitochondrial content of each tissue.

The tissue-specific effect of T3 on mitochondrial ATP production was shown in this study by characterizing the ATP production rate in skeletal muscles with different fiber types. The production of ATP by mitochondria is a complex, multifactorial process with the potential for regulation at many sites. The finding that ATP production was enhanced by T3 in the soleus, liver, and heart, but not in the plantaris, suggests that T3 alters the quality and/or composition of mitochondria. This could be accomplished by T3 having a selective regulatory effect on the content or function of proteins in the oxidative pathway, such as the oxidative enzymes CS and COX. The relatively greater change in enzyme activities in soleus than in plantaris is in agreement with previous comparisons of red and white muscles from hyperthyroid rodents (7, 49, 51).

In this study, we were particularly interested in the regulation of COX in muscle, because it has been implicated as a key regulatory point of oxidative phosphorylation (45) and because COX activity was ~100% higher in soleus but unchanged in plantaris of hyperthyroid animals. COX is also unique because it is encoded by a combination of mitochondrial and nuclear genes that must be coordinately expressed. We found that the transcript abundance of the mitochondrially encoded subunits I and III were increased in the soleus but not in the plantaris of hyperthyroid rodents. This differential response in mRNA levels is one potential mechanism that could regulate the change in COX activity in these two muscles. It is probable that the increase in COX transcripts in soleus arises from increased transcription, because thyroid hormone receptors have been identified that can bind directly to promoter regions of mtDNA, leading to increased transcription of mitochondrial genes, independent of nuclear factors (10, 52). There may also be changes in mRNA stability, although we are unaware of any reports that have examined the effects of thyroid hormone on transcripts for mitochondrial genes in skeletal muscles.

It should also be emphasized that the increase in COX activity in soleus was disproportionately higher than the change in COX I and III transcript levels, indicating that other regulatory mechanisms are involved. One likely mechanism is allosteric enhancement of COX activity by cardiolipin, a mitochondrial membrane lipid that is increased by T3 administration (5, 29). Another study has proposed that T3 enhances COX activity by increasing the affinity of ADP binding to regulatory subunits (1), although this requires further confirmation. Transcription and translation of the nuclear-encoded subunits of COX probably also have some effect on COX protein levels, although it is not yet clear how the complex coordination of the mitochondrial and nuclear genomes is controlled by thyroid hormone in different tissues. Wiesner et al. (48) reported that, after a single dose of T3 in hypothyroid rats, mitochondrially encoded COX transcripts increased more rapidly than nuclear subunits in gastrocnemius muscle, whereas the opposite pattern was observed in the liver. Discordant effects of T3 on mitochondrial-encoded (i.e., COX I, COX II, cytochrome b) vs. nuclear-encoded (i.e., COX IV, COX Va, cytochrome c1, ANT2) mRNA levels have also been shown in hepatoma cells (43) and rat liver (25). The extent to which this variation in transcript availability affects translation and function of mitochondrial proteins among tissues remains a question of considerable interest.

In addition to oxidative enzymes, other mitochondrial proteins that are under T3 control may also enhance ATP production. Transport of substrates needed for oxidative phosphorylation, such as pyruvate, fatty acids, and phosphate, are all increased by thyroid hormones (30-32, 34). Like COX, the proteins that perform these functions are allosterically controlled by cardiolipin content (30, 31, 34). The activity of ANT, which exchanges ADP and ATP across the mitochondrial membrane, is also increased by thyroid hormone administration (9, 27, 37). Some authors have suggested that T3 and thyroxine can bind directly to ANT, causing it to adopt a conformation highly favorable to inward ADP flux (27, 37). This could cause a rapid change in ANT activity and, potentially, ATP production, although this has not been confirmed. T3 has also been shown to increase ANT gene expression and protein accumulation in liver and heart (9). Because many mitochondrial proteins in the oxidative pathway appear to be allosterically controlled by T3, either directly or indirectly, even small increases in their concentration could exert disproportionately large effects on oxidative phosphorylation. This may potentially explain why ATP production per unit of mitochondrial protein was increased in heart, liver, and soleus (Fig. 2). However, still unknown is how any of these changes could be regulated in a tissue-specific manner so that mitochondrial function in plantaris would remain relatively unaffected by T3.

The regulation of UCP2 and -3 expression and their function in the oxidative phosphorylation process is a complex process that is also not yet fully understood. In this study we showed that T3 administration was associated with higher levels of UCP2 and -3 transcripts and, with the exception of UCP2 in the plantaris, higher levels of UCP protein in both soleus and plantaris. The disproportionate increases in mRNA and protein levels of these UCPs, particularly UCP2 in plantaris, implies that posttranscriptional mechanisms play an important role in UCP expression. The lack of detectable levels of UCP2 protein in plantaris is in agreement with a recent study in which UCP2 was undetectable in hindlimb mixed muscle of rats (6). Previous work has shown that T3 administration increases the mRNA levels of UCP2 in heart (20), liver (20), and soleus (26) and of UCP3 in mixed leg muscle (21, 23). Gong et al. (13) also showed that, in rat gastrocnemius, UCP3 transcripts increased within 18 h after acute exposure to T3, but no change in UCP2 was detected for up to 4 days of treatment. In contrast, in mice treated with T3 for 6 days, both UCP2 and UCP3 transcripts were elevated in the gastrocnemius, whereas no changes in UCPs were detected in soleus (20). It is unclear whether this lack of T3 effect on UCPs in mouse soleus is related to species differences or to other methodological reasons.

Like others previously (20, 21), we found that UCP2 and -3 mRNA levels in soleus and plantaris were positively correlated with whole body VO2 (r values between 0.53 and 0.91). These findings cannot be interpreted to imply that a direct relationship exists, however, because our results indicate that the degrees of change in UCP mRNAs and protein were disproportionate. Nevertheless, the fact that the protein levels of one or both of these UCPs were increased in muscles of hyperthyroid rats is consistent with the increase in metabolic rate. Recent data from in vitro systems (19, 35) and UCP3-knockout and -transgenic mice (8, 14, 44) are strongly in favor of UCP2 and -3 actually uncoupling oxidative phosphorylation by proton translocation. There is also ample evidence that mitochondrial protein leak is increased in hyperthyroid rats (3, 15-17). However, we found that mitochondrial ATP production was unchanged (plantaris) or increased (soleus) in muscles that experienced up to 13-fold increases in UCP2 and -3 transcripts and protein levels. It is possible that increases in proton leak prevented further increases in ATP production that could have otherwise occurred. We also cannot exclude the possibility that the mitochondrial isolation process somehow disrupts the normal in vivo functions of UCPs. Another possibility that must be considered is that UCP-mediated proton leak during fully active oxidative phosphorylation, as measured in the current study, may be qualitatively less important to the total energy flux, and perhaps undetectable, compared with the resting state, as suggested by studies in liver mitochondria (4, 15). There also may be other, as yet unknown, factors involved in mediating proton leak by UCP2 and -3 in muscle. Cadenas et al. (6) have shown that 24-h starvation results in four- to fivefold increases in UCP2 and -3 mRNA and twofold increases in UCP3 protein levels in rat hindlimb muscle, but they did not detect a significant change in membrane proton leak. Although those data suggest that caution is needed in interpreting the results of UCP expression data, the short duration of their study (24 h) does not rule out the possibility that UCP-mediated proton conductance is slower to respond than protein production.

Increased metabolic rate and hyperactivity are well documented in people with hyperthyroidism (24). Muscle weakness occurs in a large number of hyperthyroid patients, the cause of which may be multifactorial. Although proton leak may increase, our study shows that mitochondrial ATP production is increased in oxidative muscle of thyrotoxic rodents. The relevance of these findings to humans should be interpreted cautiously, because human skeletal muscle in general contains a more mixed fiber type, and fiber type can vary considerably among subjects of different age or exercise training backgrounds. Further human studies are needed before the findings of the current investigation can be translated to human hyperthyroidism. The large increase in serum T3 concentration in our rodent model may exceed that of human hyperthyroidism, but humans also tend to have elevated T4 levels, whereas in rats, T4 was suppressed (24).

In conclusion, we have shown that thyroid hormone enhances mitochondrial ATP production rate and enzyme activities in highly oxidative tissues like the soleus muscle, liver, and heart but not in the glycolytic plantaris muscle. Increases in mitochondrial enzyme activities, such as citrate synthase and cytochrome c oxidase, likely contribute to the ATP production capacity by permitting increased energy flux through the oxidative pathway. The muscle-specific increase in cytochrome c oxidase activity may be mediated in part by increased transcript levels of mitochondrially encoded transcripts, which are increased in oxidative, but not in glycolytic, muscle. UCP2 and -3 expression was enhanced at the protein and/or transcript level in both glycolytic and oxidative skeletal muscles of hyperthyroid animals, but ATP production was either maintained or increased in these muscles, indicating that the role of these UCPs has yet to be fully defined. The mechanisms underlying patterned gene expression in different tissues, particularly muscles with different fiber types, remains unknown.


    ACKNOWLEDGEMENTS

The authors thank Dr. Aizhong Fu, Dawn Morse, Rebecca Miller, Jane Kahl, and Sara Kuecker for their excellent assistance in data collection and analysis. We also thank Dr. Rolf Wibom for helpful discussions regarding the ATP production assay.


    FOOTNOTES

This project was supported by Public Health Service Grant RO1-DK-41973, the Mayo Foundation, and the David Murdock Dole Professorship (K. S. Nair), National Research Service Award T32-DK-07352 (K. R. Short), and by the Swedish Society of Medicine, The Medical Research Council, the Henning and Johan Throne-Holsts Foundation, and the Wenner-Gren Center Foundation (J. Nygren).

Address for reprint requests and other correspondence: K. S. Nair, Endocrine Research Unit, 5-194 Joseph, Mayo Clinic and Foundation, Rochester, MN 55905 (E-mail: nair.sree{at}mayo.edu).

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.

Received 23 August 2000; accepted in final form 26 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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