Transformation of Skeletal Muscle from Fast- to Slow-Twitch during Acquisition of Cold Tolerance in the Chick
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内分泌学杂志 2005年第1期
Institute of Agriculture and Forestry (M.H., D.I., A.T., Yu.K.), University of Tsukuba, Tsukuba-shi, Ibaraki 305-8572, Japan; and Precursory Research for Embryonic Science and Technology (Ya.K.), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
Address all correspondence and requests for reprints to: M. Hirabayashi, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba-shi, Ibaraki 305-8572, Japan. E-mail: mhira@sakura.cc.tsukuba.ac.jp.
Abstract
Although birds lack brown adipose tissue, a thermogenic organ found in mammals, they possess other thermogenic mechanisms. In the current studies, we examined the molecular mechanisms of avian thermogenesis by studying how chicks acquire cold tolerance. We found that the acquisition of cold tolerance corresponded with an increase in the redness of the skeletal muscle, suggesting an increase in slow-twitch muscle fiber. This was confirmed by histological analysis. In addition, in chicks acquiring cold tolerance, there was an enhanced expression of the chicken homologue of peroxisome proliferator-activated receptor- coactivator-1, a protein involved in adaptive thermogenesis in mammalian brown adipose tissue and in slow-twitch fiber formation in mammalian skeletal muscle. Subtraction and differential display techniques further showed that, when chicks acquired cold tolerance, the expression of genes associated with slow-twitch fibers increased, whereas those associated with fast-twitch fibers decreased. There was also an enhanced expression of mitochondrial oxidative genes. Together, these results suggest that transformation of skeletal muscle fiber from fast-twitch to slow-twitch is involved in the acquisition of thermogenesis in chicks.
Introduction
BIRDS AND MAMMALS maintain their body temperature in a cold environment by thermogenesis. Shivering is an acute thermogenic response to protect organs, whereas nonshivering responses allow long-term adaptation to a cold environment. The brown adipose tissue (BAT) is a thermogenic organ, present in mammals, that mediates a nonshivering adaptive response. This organ is highly developed in small mammals, such as rodents, as well as in neonatal humans (1, 2, 3). BAT does not appear to be a major organ for thermogenesis in humans and other large mammals because it accounts for only 0.3% of the total body weight (4). Instead, skeletal muscle is thought to be involved in thermogenesis in humans and other large mammals because it is the largest organ in the human body, accounting for 40% of the total body weight (5, 6).
The molecular mechanism of BAT thermogenesis has been studied extensively in rodents. In the BAT of rodents, uncoupling protein (UCP)1 generates heat by uncoupling oxidative phosphorylation in electron transfer systems (1, 3, 7). Furthermore, exposure of rodents to cold induces expression of the transcriptional component, peroxisome proliferator-activated receptor- coactivator (PGC)-1, in the BAT and the skeletal muscle (8). PGC-1 activates the expression of many genes related to thermogenesis, including UCP1 and various mitochondrial genes (9, 10).
Although shivering is considered to be the primary mechanism for thermogenesis in birds, nonshivering responses may also play a role. Many studies have used cold-acclimated Muscovy ducklings to examine avian nonshivering thermogenesis (11, 12, 13, 14, 15). These investigations have found that, because birds have no BAT or related tissues, other organs must mediate thermogenesis. Due to its high capacity for oxygen consumption and energy expenditure (11, 12, 15, 16), skeletal muscle is considered to be the major organ for thermogenesis in birds as it is in large mammals. Thus, skeletal muscle appears to involve both shivering and nonshivering responses. Under conditions of severe cold, both shivering and nonshivering thermogenesis are thought to work together to ensure thermal homeostasis.
Although adult birds have a high tolerance for cold, very young birds cannot maintain their body temperature in cold environments until they acquire a capacity for thermogenesis and mature thermogenic organs (17, 18, 19, 20, 21). Very young chicks possess immature thermal organs, including feathers and fat, to maintain their temperature but are highly sensitivity to cold exposure. Although chicks are typically housed in temperature-controlled conditions (around 30 C) for breeding, studies on the ontogeny of thermogenesis in birds suggest that chicks acquire cold tolerance before 3–4 wk of age (17, 18, 19, 20, 21).
Many studies have investigated the molecular mechanism of thermogenesis in rodents by exposing them to 4 C for 24 h or 48 h (8, 22, 23, 24, 25). Therefore, in the current studies, we examined the effect of similar conditions on the development of chick thermogenesis. Unexpectedly, we observed gross morphological changes in the skeletal muscles associated with the acquisition of thermogenesis. Analysis of gene expression patterns combined with histological analysis showed that cold exposure caused a transformation of skeletal muscle fibers from fast-twitch to slow-twitch in chicks acquiring thermogenesis.
Materials and Methods
Animals and treatments
Fertilized eggs from genetically equalized Rhode Island Red chickens (Gallus gallus domesticus) were a gift of Ibaraki Prefectural Livestock Research Center (Ishioka, Ibaraki, Japan). Chicks were hatched and bred at the Agricultural and Forestry Research Center, University of Tsukuba. Breeding methods were according to the guidelines of the University of Tsukuba. Ninety-six chicks per batch were divided into two groups, and eight chicks/d from d 4–9 after hatching were exposed to a cold environment (4 C, 60% humidity, and 24 h continuous light). The same numbers of chicks for each day were kept in a thermo-regulated room (35 C, 60% humidity, and 24 h continuous light) as controls. The chicks had free access to food and water. To allow chicks continuous access to food, we kept the chicks under 24 h of light because birds are unable to see in the dark. Experiments were repeated five times and using five batches of chicks.
Blood samples were collected for metabolite analysis from chicks after decapitation under ethyl-ether anesthesia. Samples were obtained from 7-d-old control chicks or chicks exposed for 24 or 48 h to cold (eight chicks per group). Blood glucose, fatty acid, and triacylglycerol concentrations were determined by enzyme assays (SRL, Tokyo, Japan). After whole blood was taken, quadriceps and pectoralis muscles, white adipose tissues, livers, and brains were collected, frozen in liquid nitrogen, and stored at –80 C until use.
Histological analysis of skeletal muscle
Quadriceps muscles of 7-d-old control and 24-h cold-exposed chicks (three chicks per group) were examined. Samples were frozen in liquid nitrogen-cooled isopentane, and transverse serial sections were preincubated at pH 4.2 and stained by adenosine triphosphatase (ATPase) as described previously (26). The ratio of slow-twitch muscle fibers of skeletal muscle cells were determined by counting the number of cell stained by ATPase at pH 4.2 in three randomly selected cross-section from individual chicks.
RNA preparation
Total RNA was extracted from the tissues of 7-d-old control and 24- or 48-h cold-exposed chicks (eight chicks per group). RNA was purified using TRIzol reagent (Invitrogen, Tokyo, Japan). PolyA (+) mRNA of control and 24-h cold-exposed chicks was obtained by affinity chromatography on oligo deoxythymidine-cellulose using a polyA (+) tract mRNA isolation kit (Promega, Tokyo, Japan).
Suppression subtractive hybridization and screening of the subtracted cDNA
Suppression subtractive hybridization was performed using a PCR-select cDNA subtraction kit according to the manufacturer’s protocol (Clontech, Tokyo, Japan). Reverse transcription was performed using 2 μg polyA (+) mRNA from quadriceps muscles of control chicks (pooled mRNA from eight 7-d-old chicks) and 2 μg polyA (+) mRNA from the quadriceps muscles of chicks exposed to 4 C for 24 h (pooled mRNA from eight 7-d-old chicks). Two-directional (forward, cold-exposed sample minus control sample; and reverse, control sample minus cold-exposed sample) subtraction hybridizations were performed between the cold-exposed and control groups, and the subtractive hybridization products were amplified by suppression PCR. Products from the forward and reverse subtractions were cloned into the pT7Blue cloning vector (Novagen, Takara, Kyoto, Japan), and used to transform Escherichia coli DH5.
The PCR-select differential screening kit was used to identify differentially expressed products according to the manufacturer’s protocol (Clontech). Approximately 100 individual clones from each of the forward- or reverse-subtracted libraries were successfully sequenced with the T7 primer using an ABI Prism dye terminator cycle sequencer (Amersham Biosciences, Tokyo, Japan). Sequences were compared with the National Center for Biotechnology Information sequence database using the BLAST (basic local alignment search tool) program (http://www.ncbi.nlm.nih.gov/BLAST/).
Northern blot analysis
Total RNA (10 μg/lane) was separated by electrophoresis on a 1.2% formaldehyde-agarose gel and transferred to a nylon membrane. Hybridization, washing, and signal detection were performed as described previously (27). Hybridization to detect RNA from muscle and nonmuscle tissue was performed at 68 C and 42 C, respectively.
Statistical analysis
Results from control and cold-exposed chicks were compared using Student’s t test.
Results
Determination of the age at which chicks acquire the capacity for thermogenesis
We performed preliminary studies to determine the age at which chicks acquire thermogenesis. We monitored rectal temperature changes in 1- to 21-d-old chicks (eight chicks/d) exposed to 4 C for 24 h. Figure 1A shows the rectal temperature changes in 4- to 9-d-old chicks. The temperature in chicks less than 5 d old decreased when they were exposed to 4 C for 1 h, and they were unable to raise their temperature to 40 C. In chicks older than 5 d, there was an increase in the number of chicks with a temperature of 40 C during a 24-h cold exposure. All chicks older than 9 d were able to maintain a temperature of 40 C during a 24-h exposure to 4 C (data not shown). Furthermore, all chicks that were able to maintain a temperature of 40 C during a 24-h exposure to 4 C were also able to maintain the temperature for an additional 24 h. We therefore determined cold tolerance as the ability to maintain a temperature of 40 C during exposure to 4 C for 24 h. Figure 1B shows the rate of acquisition of cold tolerance in chicks from 4–9 d old. By 7 d of age, 80% of the chicks acquired cold tolerance. Thus, all experiments after this were performed using 7-d-old chicks.
FIG. 1. Acquisition of thermogenesis during chick development. A, Chicks from 4–9 d old were kept at 4 C for 24 h. The rectal temperature of eight chicks on each day was measured at 1, 3, 6, and 24 h. B, The graph shows the percentage of chicks with a body temperature of more than 40 C after 24-h cold exposure. The rectal temperature of eight chicks on each day was measured, and the measurements were repeated five times. Values correspond to the mean ± SE.
Blood metabolite levels in chicks with cold tolerance
To access the changes in fat and energy metabolism in response to the cold, we measured glucose, nonesterified fatty acid, and triacylglycerol concentrations in serum of control and cold-exposed (24 and 48 h) chicks (Table 1). The blood glucose level did not change between control and cold-exposed chicks. Nonesterified fatty acid concentrations in cold-exposed chicks were twice as high as those in control chicks. Finally, although the triacylglycerol concentration did not differ between control chicks and chicks exposed to cold for 24 h, the concentration of triacylglycerol decreased in the serum of chicks exposed to cold for 48 h.
TABLE 1. Serum concentration of glucose, nonesterified fatty acid, and triacylglycerol in chicks
Morphological and histological changes in skeletal muscle caused by cold exposure
To understand the changes that occur during the acquisition of cold tolerance, we examined the gross morphology of skeletal muscle in chicks exposed to cold for 24 or 48 h. We found that skeletal muscles of all chicks that could maintain their body temperature during cold exposure for 24 h became redder than those of control chicks (Fig. 2, A and B). Chicks exposed for 48 h to a cold environment also showed reddish muscle. We further performed histological analyses of quadriceps muscles after acid (pH 4.2) preincubation, which stains slow-twitch (red or type I) but not fast-twitch (white or type II) muscle fibers. We found that the number of slow-twitch fibers per area in the skeletal muscle was 2-fold higher in cold-exposed chicks than in control chicks (Fig. 2C). These results suggested that the skeletal muscle in chicks that acquired cold tolerance contained a higher content of slow-twitch fibers.
FIG. 2. Morphological and histological analysis of skeletal muscle in 7-d-old chicks. A, Side view of dissected chicks. The two chicks on the left are controls, and the two chicks on the right were kept at 4 C for 24 h. B, Dorsal view of the dissected chicks. Left, Control chick; right, cold-exposed chick. C, Light microscopy of ATPase (pH 4.2)-stained transverse sections of skeletal muscle specimens from control and cold-exposed chicks are shown on the left. Acid-stable ATPase-containing cells are stained black and correspond to slow-twitch fibers, and unstained cells correspond to fast-twitch fibers. The graph on the right shows the percent of acid-stable ATPase-stained myofibers to total myofibers. Values correspond to the mean ± SE from three chicks, wherein the value for each chick was the average of three randomly selected sections. **, P < 0.01 vs. control.
Expression of putative chick muscle transformation genes during the acquisition of cold tolerance
Because PGC-1 is involved in the conversion of mouse skeletal muscle fibers from fast-twitch to slow-twitch (28) and because the expression of PGC-1 is increased in the skeletal muscle exposed for 24 h to cold (8), we examined the expression of chicken PGC-1 in skeletal muscle of chicks with cold tolerance. Database searches using the BLAST program revealed that chicken-expressed sequence tag (EST) BM491854 is similar to the C-terminal portion of human and mouse PGC-1 (94% and 91% amino acid identity, respectively) and therefore probably corresponds to the C-terminal portion of chicken PGC-1 (Fig. 3A). For this reason, we used BM491854 as a probe to examine the changes in the expression of chicken PGC-1 during the acquisition of cold tolerance in chicks (Fig. 3B). Based on this, the level of chicken PGC-1 mRNA increased markedly after 24 h of cold exposure, and the level was maintained after 48 h of cold exposure.
FIG. 3. Northern blot analysis of chicken PGC-1, myoglobin, and UCP gene expressions in skeletal muscle. A, Sequence alignment of the primary amino acid sequences of human (hPGC), mouse (mPGC), and chicken PGC-1 (cPGC). The cPGC sequence was deduced from the EST (BM491854) sequence. Identical amino acids are indicated by an asterisk. The numbers show the positions of amino acids with the first methionine residue for human and mouse, and relative to the start of the chicken EST. B, The cPGC expression was assessed using its EST cDNA as a probe. The upper portion of the panel shows representative Northern blot data, and the lower portion shows the average densitometric ratio ± SE (n = 8) as a percentage of control. **, P < 0.01 (vs. control). C, Chicken myoglobin expression was examined using an EST (BU310745) as a probe. D, Chicken UCP expression was examined using a cDNA (AB088685) as a probe. *, P < 0.05 (vs. control). Each lane on the Northern blots represents a sample from an individual chick.
We further examined the expression of the myoglobin gene, which could contribute the red color to the skeletal muscle in the cold-tolerant chicks, because the skeletal muscles of myoglobin gene-defective mice are pale in color (29). Database searches using the BLAST program revealed that chicken EST BU310745 has high sequence similarity to the human and mouse myoglobin (77% and 75% amino acid identity, respectively; data not shown). Therefore, we examined the expression of chicken myoglobin BU310745 as a probe (Fig. 3C). We found that, although the expression of the myoglobin gene did not change after a 24-h exposure to cold, its expression was increased after exposure to cold for 48 h.
Finally, because UCP acts as a mitochondrial uncoupler and because it has been reported to increase after long-term cold exposure, in 1-wk-old ducklings (30) and in 3-wk-old chickens (31), we examined the expression of UCP during the acquisition of cold tolerance in chicks. For this analysis, we used a probe based on the chicken UCP (Gallus gallus UCP) AB088685 cDNA. However, chicken UCP expression decreased after 24 h of cold exposure, and the expression returned to normal after 48 h of cold exposure (Fig. 3D).
Changes in gene expression in the skeletal muscle of cold-exposed chicks
To estimate the involvement of thermogenesis and muscle fiber type conversion with the acquisition of cold tolerance, we investigated gene expression changes in the quadriceps muscles of cold-adapted chicks by subtraction and differential display analysis. We obtained 16 independent cold-induced genes, of which nine were known genes and the rest were ESTs. We also obtained 14 independent cold-repressed genes, of which five were known genes and the rest were ESTs. Expression of subtracted genes was confirmed by Northern blot analysis, and the relative expressions of known genes are shown in Fig. 4. The results show that, in skeletal muscle of cold-adapted chicks, there was an increase in the expression of genes related to the mitochondrial electron transfer system, such as reduced nicotinamide adenine dinucleotide-ubiquinone oxidoreductase, and cytochrome c oxidase (COX)-I, -II, and -IV. In addition, cold adaptation corresponded with an increase in the expression of slow-twitch muscle fiber-type lactate dehydrogenase and a decrease in the expression of the fast-twitch muscle fiber-type gene. Also, there was a decrease in the expression of the glycolysis pathway-related gene, glucose phosphomutase, the fast-twitch muscle fiber troponins T3 and T4, and myosin light chain 3f.
FIG. 4. Differential expression of skeletal muscle genes in control and cold-exposed chicks. Known genes obtained by subtraction hybridization and differential display analysis are listed, and confirmation of their expression levels by Northern blot analysis is shown on the right. The same RNA sample sets were blotted onto multiple membranes and hybridized with the indicated probes. Equal sample loading of each membrane was confirmed by ethidium bromide staining of 28S ribosomal RNA (not shown). Each lane on the Northern blots represents a sample from an individual chick. The average densitometric ratio and SE (in parentheses) compared with control is shown to the left of the autoradiogram (n = 8). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. control); UDP, uridine 5'-diphosphate; NADH, reduced nicotinamide adenine dinucleotide. .
We further examined the expression of the chicken PGC-1 gene and also a mitochondrial gene involved in oxidative phosphorylation in skeletal muscle. In pectolalis muscles, which consist mainly of fast-twitch fibers, the expression of the chicken PGC-1 gene and a representative gene engaged in mitochondrial electron transfer system, COX-II, was also increased in cold-adapted chicks (Fig. 5A). This corresponded with an increase in the redness of this muscle in the cold-exposed chicks, similar to the changes observed in quadriceps muscles (Fig. 2).
FIG. 5. Northern blot analysis of cPGC and COX-II gene expressions in pectoralis muscle (A), liver (B), white adipose tissue (C), and brain (D). Each lane on the Northern blots represents a sample from an individual chick. The average densitometric ratio and SE (in parentheses), compared with control, is shown below the autoradiogram (n = 3). *, P < 0.05 (vs. control).
Collectively, these results indicate that, in the skeletal muscle of cold-adapted chicks, there is an increase in the content of slow-twitch fibers, which contain many mitochondria and use oxidative phosphorylation to produce ATP. At the same time, there is a decrease in the content of fast-twitch fibers, which mainly perform glycolysis to produce ATP. This conclusion is consistent with our histological and gross morphological analyses.
Expressions of chicken PGC-1 and COX-II gene in nonmuscle tissues
We next examined whether the cold-adaptation responses seen in skeletal muscles, namely the increase of chicken PGC-1 and mitochondrial gene expression, were also induced in nonmuscle tissues, including liver, white adipose tissue, and brain (Fig. 5, B–D). In these tissues, the expressions of chicken PGC-1 and COX-II gene were less than those in leg muscles and were not different between control and cold-exposed chicks.
Discussion
In this study, we examined the mechanism by which chicks acquire the capacity for thermogenesis and tolerance to the cold. Morphological, histological, and gene expression analyses indicated that there is an increase in slow-twitch fibers in the skeletal muscle of chicks with cold tolerance. We also found the expression of chicken PGC-1 was enhanced in the skeletal muscle of chicks with cold tolerance. These results suggest that the acquisition of thermogenesis may be due to PGC-1-mediated transformation of skeletal myofibers from fast-twitch to slow-twitch. Our results are consistent with previous studies suggesting that skeletal muscle is the main tissue for both shivering and nonshivering thermogenesis in birds (11, 12, 15, 16, 19, 20, 21).
Many studies have examined the development of thermoregulation in birds (17, 18, 19, 20, 21). We used chicks for the current studies of the molecular mechanism of thermogenesis in birds because the largest body of genetic information on birds exists for domestic chickens. The chickens selected in this study were preserved as a pure line to maintain a common genetic background. Young chicks are more sensitive to cold exposure than adult chickens because they have immature thermal organs, including feather and fat tissue, and they have not acquired the capacity for thermogenesis. Therefore, we focused on the development of thermogenesis and cold tolerance in young chicks. Similar to studies in rodents (8, 22, 23, 24, 25), we studied the development of thermoregulation by exposing the chicks to 4 C for 24 h or more. Because most chicks older than 6 d tolerated cold for 24 h or more, we used 7-d-old chicks to investigate the molecular mechanism of muscle thermogenesis.
An initial gross morphological analysis revealed a remarkable color change of the skeletal muscle in cold-tolerant chicks. Histological and subtractive-differential expression analysis confirmed that this was due to an increase in the content of slow-twitch muscle fibers (red fibers). Using histological analysis, Duchamp et al. (14) also reported an increase in the level of slow-twitch fibers in the pectoralis muscle of ducklings after a long-term cold exposure. However, a morphological change has not been reported in studies of long-term cold exposure in birds (14, 15). The obvious morphological change of cold-exposed muscle observed in our study may be connected to the high plasticity of muscle structure in 7-d-old chicks (19, 21).
Mammalian PGC-1 expression is induced by cold exposure in BAT and skeletal muscle (8). PGC-1 improves adaptive thermogenesis in BAT by enhancing the expression of mitochondrial genes and inducing mitochondrial biogenesis (10). Furthermore, PGC-1 directs the transformation of mammalian skeletal muscle fibers from fast-twitch to slow-twitch (28). For these reasons, we examined the involvement of PGC-1 in the transformation of muscle fibers in chicks acquiring cold tolerance. We observed an increase in chicken PGC-1 expression in the skeletal muscle of chicks exposed to 4 C for 24 h. This suggests that chicken PGC-1 is involved in muscle fiber transformation in chicks during the acquisition of cold tolerance. Finally, the expression of the chicken PGC-1 gene was increased in chicks exposed to cold for 48 h, suggesting that a continuous high expression of this gene is necessary for muscle thermogenesis. Further studies are needed to determine whether the chicken PGC-1 directly drives muscle fiber transformation in chicks exposed to cold.
Because mouse PGC-1 induces the expression of myoglobin (28) and because myoglobin gives a red color to muscle (29), we examined the contribution of myoglobin to the reddish color in the skeletal muscle of chicks with cold tolerance. However, we found that the expression of myoglobin gene does not appear to be a main contributor to color change in the muscle of chicks with cold tolerance. Subtraction and differential display analysis found the up-regulation of characteristic genes of mitochondrial electron transfer system in muscle of chicks exposed to cold for 24 h, suggesting that, as in the BAT of rodents, an increase of the number of mitochondria may be responsible for the increased color.
In mammalian BAT-dependent thermogenesis, UCP1, but probably not UCP2 or UCP3, produces heat by uncoupling mitochondrial oxidative phosphorylation (1, 3, 7, 22, 32). UCP1 is expressed specifically in BAT, UCP2 is ubiquitously expressed (24), and UCP3 is expressed in BAT and skeletal muscle (23). Furthermore, avian UCP has been identified in skeletal muscle (30, 33) and has a higher homology with mammalian UCP2 and UCP3 than with UCP1 (55% identity vs. mouse UCP1 and 70% identity vs. mouse UCP2 and UCP3). Long-term cold exposure has been reported to increase avian UCP expression in 1-wk-old ducklings (30) and 3-wk-old chickens (31). However, in our studies, exposure of chicks to cold for 24 h decreased the expression of UCP in chicks with a high level of slow-twitch muscle fiber, and the expression of UCP returned to the control level after 48 h of cold exposure. Wang et al. (25) reported that the regulation of UCP3 expression in rats differs in various muscle types and, similar to our finding, that exposure of rats to cold for 24 h down-regulated UCP3 protein and mRNA in the soleus muscle (slow-twitch muscle). Therefore, although the function of avian UCP in thermogenesis is unclear, regulation of its expression may depend on the period of cold exposure and the type of muscle fiber.
We observed increased expression of chicken PGC-1 and COX-II gene not only in the leg muscles but also in the breast muscles. The white muscles of the chicken breast acquired a marked red color in cold-exposed chicks. Thus, transformation of the muscle seems to affect not only the muscles that already contain slow-twitch muscles but also the more glycolytic white flight muscles. Thus, other muscles in addition to the leg muscles may become thermogenic organs.
Collectively, our results suggest that enhancement of chicken PGC-1 expression may induce an increase in slow-twitch type fiber content, thereby allowing chicks to acquire thermogenesis. However, it is not certain that PGC-1 is the only protein that is involved in muscle fiber transformation and/or muscle thermogenesis. Indeed, many factors and molecules participate in muscle fiber transformation (34, 35, 36). In our study, subtraction and differential display analysis identified several ESTs that were either up- or down-regulated in the muscle of chicks with cold tolerance. Evaluating the functions and properties of these ESTs may provide an understanding of the molecular mechanism of avian thermogenesis and the role of muscle fiber transformation from fast-twitch to slow-twitch.
Acknowledgments
We sincerely appreciate the advice and expertise of Professor F. Kraemer, Stanford University, Palo Alto, CA). We thank A. Ushitani for secretarial assistance in the preparation of this manuscript, and T. Minematsu for technical assistance in the breeding of chicks.
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Address all correspondence and requests for reprints to: M. Hirabayashi, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba-shi, Ibaraki 305-8572, Japan. E-mail: mhira@sakura.cc.tsukuba.ac.jp.
Abstract
Although birds lack brown adipose tissue, a thermogenic organ found in mammals, they possess other thermogenic mechanisms. In the current studies, we examined the molecular mechanisms of avian thermogenesis by studying how chicks acquire cold tolerance. We found that the acquisition of cold tolerance corresponded with an increase in the redness of the skeletal muscle, suggesting an increase in slow-twitch muscle fiber. This was confirmed by histological analysis. In addition, in chicks acquiring cold tolerance, there was an enhanced expression of the chicken homologue of peroxisome proliferator-activated receptor- coactivator-1, a protein involved in adaptive thermogenesis in mammalian brown adipose tissue and in slow-twitch fiber formation in mammalian skeletal muscle. Subtraction and differential display techniques further showed that, when chicks acquired cold tolerance, the expression of genes associated with slow-twitch fibers increased, whereas those associated with fast-twitch fibers decreased. There was also an enhanced expression of mitochondrial oxidative genes. Together, these results suggest that transformation of skeletal muscle fiber from fast-twitch to slow-twitch is involved in the acquisition of thermogenesis in chicks.
Introduction
BIRDS AND MAMMALS maintain their body temperature in a cold environment by thermogenesis. Shivering is an acute thermogenic response to protect organs, whereas nonshivering responses allow long-term adaptation to a cold environment. The brown adipose tissue (BAT) is a thermogenic organ, present in mammals, that mediates a nonshivering adaptive response. This organ is highly developed in small mammals, such as rodents, as well as in neonatal humans (1, 2, 3). BAT does not appear to be a major organ for thermogenesis in humans and other large mammals because it accounts for only 0.3% of the total body weight (4). Instead, skeletal muscle is thought to be involved in thermogenesis in humans and other large mammals because it is the largest organ in the human body, accounting for 40% of the total body weight (5, 6).
The molecular mechanism of BAT thermogenesis has been studied extensively in rodents. In the BAT of rodents, uncoupling protein (UCP)1 generates heat by uncoupling oxidative phosphorylation in electron transfer systems (1, 3, 7). Furthermore, exposure of rodents to cold induces expression of the transcriptional component, peroxisome proliferator-activated receptor- coactivator (PGC)-1, in the BAT and the skeletal muscle (8). PGC-1 activates the expression of many genes related to thermogenesis, including UCP1 and various mitochondrial genes (9, 10).
Although shivering is considered to be the primary mechanism for thermogenesis in birds, nonshivering responses may also play a role. Many studies have used cold-acclimated Muscovy ducklings to examine avian nonshivering thermogenesis (11, 12, 13, 14, 15). These investigations have found that, because birds have no BAT or related tissues, other organs must mediate thermogenesis. Due to its high capacity for oxygen consumption and energy expenditure (11, 12, 15, 16), skeletal muscle is considered to be the major organ for thermogenesis in birds as it is in large mammals. Thus, skeletal muscle appears to involve both shivering and nonshivering responses. Under conditions of severe cold, both shivering and nonshivering thermogenesis are thought to work together to ensure thermal homeostasis.
Although adult birds have a high tolerance for cold, very young birds cannot maintain their body temperature in cold environments until they acquire a capacity for thermogenesis and mature thermogenic organs (17, 18, 19, 20, 21). Very young chicks possess immature thermal organs, including feathers and fat, to maintain their temperature but are highly sensitivity to cold exposure. Although chicks are typically housed in temperature-controlled conditions (around 30 C) for breeding, studies on the ontogeny of thermogenesis in birds suggest that chicks acquire cold tolerance before 3–4 wk of age (17, 18, 19, 20, 21).
Many studies have investigated the molecular mechanism of thermogenesis in rodents by exposing them to 4 C for 24 h or 48 h (8, 22, 23, 24, 25). Therefore, in the current studies, we examined the effect of similar conditions on the development of chick thermogenesis. Unexpectedly, we observed gross morphological changes in the skeletal muscles associated with the acquisition of thermogenesis. Analysis of gene expression patterns combined with histological analysis showed that cold exposure caused a transformation of skeletal muscle fibers from fast-twitch to slow-twitch in chicks acquiring thermogenesis.
Materials and Methods
Animals and treatments
Fertilized eggs from genetically equalized Rhode Island Red chickens (Gallus gallus domesticus) were a gift of Ibaraki Prefectural Livestock Research Center (Ishioka, Ibaraki, Japan). Chicks were hatched and bred at the Agricultural and Forestry Research Center, University of Tsukuba. Breeding methods were according to the guidelines of the University of Tsukuba. Ninety-six chicks per batch were divided into two groups, and eight chicks/d from d 4–9 after hatching were exposed to a cold environment (4 C, 60% humidity, and 24 h continuous light). The same numbers of chicks for each day were kept in a thermo-regulated room (35 C, 60% humidity, and 24 h continuous light) as controls. The chicks had free access to food and water. To allow chicks continuous access to food, we kept the chicks under 24 h of light because birds are unable to see in the dark. Experiments were repeated five times and using five batches of chicks.
Blood samples were collected for metabolite analysis from chicks after decapitation under ethyl-ether anesthesia. Samples were obtained from 7-d-old control chicks or chicks exposed for 24 or 48 h to cold (eight chicks per group). Blood glucose, fatty acid, and triacylglycerol concentrations were determined by enzyme assays (SRL, Tokyo, Japan). After whole blood was taken, quadriceps and pectoralis muscles, white adipose tissues, livers, and brains were collected, frozen in liquid nitrogen, and stored at –80 C until use.
Histological analysis of skeletal muscle
Quadriceps muscles of 7-d-old control and 24-h cold-exposed chicks (three chicks per group) were examined. Samples were frozen in liquid nitrogen-cooled isopentane, and transverse serial sections were preincubated at pH 4.2 and stained by adenosine triphosphatase (ATPase) as described previously (26). The ratio of slow-twitch muscle fibers of skeletal muscle cells were determined by counting the number of cell stained by ATPase at pH 4.2 in three randomly selected cross-section from individual chicks.
RNA preparation
Total RNA was extracted from the tissues of 7-d-old control and 24- or 48-h cold-exposed chicks (eight chicks per group). RNA was purified using TRIzol reagent (Invitrogen, Tokyo, Japan). PolyA (+) mRNA of control and 24-h cold-exposed chicks was obtained by affinity chromatography on oligo deoxythymidine-cellulose using a polyA (+) tract mRNA isolation kit (Promega, Tokyo, Japan).
Suppression subtractive hybridization and screening of the subtracted cDNA
Suppression subtractive hybridization was performed using a PCR-select cDNA subtraction kit according to the manufacturer’s protocol (Clontech, Tokyo, Japan). Reverse transcription was performed using 2 μg polyA (+) mRNA from quadriceps muscles of control chicks (pooled mRNA from eight 7-d-old chicks) and 2 μg polyA (+) mRNA from the quadriceps muscles of chicks exposed to 4 C for 24 h (pooled mRNA from eight 7-d-old chicks). Two-directional (forward, cold-exposed sample minus control sample; and reverse, control sample minus cold-exposed sample) subtraction hybridizations were performed between the cold-exposed and control groups, and the subtractive hybridization products were amplified by suppression PCR. Products from the forward and reverse subtractions were cloned into the pT7Blue cloning vector (Novagen, Takara, Kyoto, Japan), and used to transform Escherichia coli DH5.
The PCR-select differential screening kit was used to identify differentially expressed products according to the manufacturer’s protocol (Clontech). Approximately 100 individual clones from each of the forward- or reverse-subtracted libraries were successfully sequenced with the T7 primer using an ABI Prism dye terminator cycle sequencer (Amersham Biosciences, Tokyo, Japan). Sequences were compared with the National Center for Biotechnology Information sequence database using the BLAST (basic local alignment search tool) program (http://www.ncbi.nlm.nih.gov/BLAST/).
Northern blot analysis
Total RNA (10 μg/lane) was separated by electrophoresis on a 1.2% formaldehyde-agarose gel and transferred to a nylon membrane. Hybridization, washing, and signal detection were performed as described previously (27). Hybridization to detect RNA from muscle and nonmuscle tissue was performed at 68 C and 42 C, respectively.
Statistical analysis
Results from control and cold-exposed chicks were compared using Student’s t test.
Results
Determination of the age at which chicks acquire the capacity for thermogenesis
We performed preliminary studies to determine the age at which chicks acquire thermogenesis. We monitored rectal temperature changes in 1- to 21-d-old chicks (eight chicks/d) exposed to 4 C for 24 h. Figure 1A shows the rectal temperature changes in 4- to 9-d-old chicks. The temperature in chicks less than 5 d old decreased when they were exposed to 4 C for 1 h, and they were unable to raise their temperature to 40 C. In chicks older than 5 d, there was an increase in the number of chicks with a temperature of 40 C during a 24-h cold exposure. All chicks older than 9 d were able to maintain a temperature of 40 C during a 24-h exposure to 4 C (data not shown). Furthermore, all chicks that were able to maintain a temperature of 40 C during a 24-h exposure to 4 C were also able to maintain the temperature for an additional 24 h. We therefore determined cold tolerance as the ability to maintain a temperature of 40 C during exposure to 4 C for 24 h. Figure 1B shows the rate of acquisition of cold tolerance in chicks from 4–9 d old. By 7 d of age, 80% of the chicks acquired cold tolerance. Thus, all experiments after this were performed using 7-d-old chicks.
FIG. 1. Acquisition of thermogenesis during chick development. A, Chicks from 4–9 d old were kept at 4 C for 24 h. The rectal temperature of eight chicks on each day was measured at 1, 3, 6, and 24 h. B, The graph shows the percentage of chicks with a body temperature of more than 40 C after 24-h cold exposure. The rectal temperature of eight chicks on each day was measured, and the measurements were repeated five times. Values correspond to the mean ± SE.
Blood metabolite levels in chicks with cold tolerance
To access the changes in fat and energy metabolism in response to the cold, we measured glucose, nonesterified fatty acid, and triacylglycerol concentrations in serum of control and cold-exposed (24 and 48 h) chicks (Table 1). The blood glucose level did not change between control and cold-exposed chicks. Nonesterified fatty acid concentrations in cold-exposed chicks were twice as high as those in control chicks. Finally, although the triacylglycerol concentration did not differ between control chicks and chicks exposed to cold for 24 h, the concentration of triacylglycerol decreased in the serum of chicks exposed to cold for 48 h.
TABLE 1. Serum concentration of glucose, nonesterified fatty acid, and triacylglycerol in chicks
Morphological and histological changes in skeletal muscle caused by cold exposure
To understand the changes that occur during the acquisition of cold tolerance, we examined the gross morphology of skeletal muscle in chicks exposed to cold for 24 or 48 h. We found that skeletal muscles of all chicks that could maintain their body temperature during cold exposure for 24 h became redder than those of control chicks (Fig. 2, A and B). Chicks exposed for 48 h to a cold environment also showed reddish muscle. We further performed histological analyses of quadriceps muscles after acid (pH 4.2) preincubation, which stains slow-twitch (red or type I) but not fast-twitch (white or type II) muscle fibers. We found that the number of slow-twitch fibers per area in the skeletal muscle was 2-fold higher in cold-exposed chicks than in control chicks (Fig. 2C). These results suggested that the skeletal muscle in chicks that acquired cold tolerance contained a higher content of slow-twitch fibers.
FIG. 2. Morphological and histological analysis of skeletal muscle in 7-d-old chicks. A, Side view of dissected chicks. The two chicks on the left are controls, and the two chicks on the right were kept at 4 C for 24 h. B, Dorsal view of the dissected chicks. Left, Control chick; right, cold-exposed chick. C, Light microscopy of ATPase (pH 4.2)-stained transverse sections of skeletal muscle specimens from control and cold-exposed chicks are shown on the left. Acid-stable ATPase-containing cells are stained black and correspond to slow-twitch fibers, and unstained cells correspond to fast-twitch fibers. The graph on the right shows the percent of acid-stable ATPase-stained myofibers to total myofibers. Values correspond to the mean ± SE from three chicks, wherein the value for each chick was the average of three randomly selected sections. **, P < 0.01 vs. control.
Expression of putative chick muscle transformation genes during the acquisition of cold tolerance
Because PGC-1 is involved in the conversion of mouse skeletal muscle fibers from fast-twitch to slow-twitch (28) and because the expression of PGC-1 is increased in the skeletal muscle exposed for 24 h to cold (8), we examined the expression of chicken PGC-1 in skeletal muscle of chicks with cold tolerance. Database searches using the BLAST program revealed that chicken-expressed sequence tag (EST) BM491854 is similar to the C-terminal portion of human and mouse PGC-1 (94% and 91% amino acid identity, respectively) and therefore probably corresponds to the C-terminal portion of chicken PGC-1 (Fig. 3A). For this reason, we used BM491854 as a probe to examine the changes in the expression of chicken PGC-1 during the acquisition of cold tolerance in chicks (Fig. 3B). Based on this, the level of chicken PGC-1 mRNA increased markedly after 24 h of cold exposure, and the level was maintained after 48 h of cold exposure.
FIG. 3. Northern blot analysis of chicken PGC-1, myoglobin, and UCP gene expressions in skeletal muscle. A, Sequence alignment of the primary amino acid sequences of human (hPGC), mouse (mPGC), and chicken PGC-1 (cPGC). The cPGC sequence was deduced from the EST (BM491854) sequence. Identical amino acids are indicated by an asterisk. The numbers show the positions of amino acids with the first methionine residue for human and mouse, and relative to the start of the chicken EST. B, The cPGC expression was assessed using its EST cDNA as a probe. The upper portion of the panel shows representative Northern blot data, and the lower portion shows the average densitometric ratio ± SE (n = 8) as a percentage of control. **, P < 0.01 (vs. control). C, Chicken myoglobin expression was examined using an EST (BU310745) as a probe. D, Chicken UCP expression was examined using a cDNA (AB088685) as a probe. *, P < 0.05 (vs. control). Each lane on the Northern blots represents a sample from an individual chick.
We further examined the expression of the myoglobin gene, which could contribute the red color to the skeletal muscle in the cold-tolerant chicks, because the skeletal muscles of myoglobin gene-defective mice are pale in color (29). Database searches using the BLAST program revealed that chicken EST BU310745 has high sequence similarity to the human and mouse myoglobin (77% and 75% amino acid identity, respectively; data not shown). Therefore, we examined the expression of chicken myoglobin BU310745 as a probe (Fig. 3C). We found that, although the expression of the myoglobin gene did not change after a 24-h exposure to cold, its expression was increased after exposure to cold for 48 h.
Finally, because UCP acts as a mitochondrial uncoupler and because it has been reported to increase after long-term cold exposure, in 1-wk-old ducklings (30) and in 3-wk-old chickens (31), we examined the expression of UCP during the acquisition of cold tolerance in chicks. For this analysis, we used a probe based on the chicken UCP (Gallus gallus UCP) AB088685 cDNA. However, chicken UCP expression decreased after 24 h of cold exposure, and the expression returned to normal after 48 h of cold exposure (Fig. 3D).
Changes in gene expression in the skeletal muscle of cold-exposed chicks
To estimate the involvement of thermogenesis and muscle fiber type conversion with the acquisition of cold tolerance, we investigated gene expression changes in the quadriceps muscles of cold-adapted chicks by subtraction and differential display analysis. We obtained 16 independent cold-induced genes, of which nine were known genes and the rest were ESTs. We also obtained 14 independent cold-repressed genes, of which five were known genes and the rest were ESTs. Expression of subtracted genes was confirmed by Northern blot analysis, and the relative expressions of known genes are shown in Fig. 4. The results show that, in skeletal muscle of cold-adapted chicks, there was an increase in the expression of genes related to the mitochondrial electron transfer system, such as reduced nicotinamide adenine dinucleotide-ubiquinone oxidoreductase, and cytochrome c oxidase (COX)-I, -II, and -IV. In addition, cold adaptation corresponded with an increase in the expression of slow-twitch muscle fiber-type lactate dehydrogenase and a decrease in the expression of the fast-twitch muscle fiber-type gene. Also, there was a decrease in the expression of the glycolysis pathway-related gene, glucose phosphomutase, the fast-twitch muscle fiber troponins T3 and T4, and myosin light chain 3f.
FIG. 4. Differential expression of skeletal muscle genes in control and cold-exposed chicks. Known genes obtained by subtraction hybridization and differential display analysis are listed, and confirmation of their expression levels by Northern blot analysis is shown on the right. The same RNA sample sets were blotted onto multiple membranes and hybridized with the indicated probes. Equal sample loading of each membrane was confirmed by ethidium bromide staining of 28S ribosomal RNA (not shown). Each lane on the Northern blots represents a sample from an individual chick. The average densitometric ratio and SE (in parentheses) compared with control is shown to the left of the autoradiogram (n = 8). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. control); UDP, uridine 5'-diphosphate; NADH, reduced nicotinamide adenine dinucleotide. .
We further examined the expression of the chicken PGC-1 gene and also a mitochondrial gene involved in oxidative phosphorylation in skeletal muscle. In pectolalis muscles, which consist mainly of fast-twitch fibers, the expression of the chicken PGC-1 gene and a representative gene engaged in mitochondrial electron transfer system, COX-II, was also increased in cold-adapted chicks (Fig. 5A). This corresponded with an increase in the redness of this muscle in the cold-exposed chicks, similar to the changes observed in quadriceps muscles (Fig. 2).
FIG. 5. Northern blot analysis of cPGC and COX-II gene expressions in pectoralis muscle (A), liver (B), white adipose tissue (C), and brain (D). Each lane on the Northern blots represents a sample from an individual chick. The average densitometric ratio and SE (in parentheses), compared with control, is shown below the autoradiogram (n = 3). *, P < 0.05 (vs. control).
Collectively, these results indicate that, in the skeletal muscle of cold-adapted chicks, there is an increase in the content of slow-twitch fibers, which contain many mitochondria and use oxidative phosphorylation to produce ATP. At the same time, there is a decrease in the content of fast-twitch fibers, which mainly perform glycolysis to produce ATP. This conclusion is consistent with our histological and gross morphological analyses.
Expressions of chicken PGC-1 and COX-II gene in nonmuscle tissues
We next examined whether the cold-adaptation responses seen in skeletal muscles, namely the increase of chicken PGC-1 and mitochondrial gene expression, were also induced in nonmuscle tissues, including liver, white adipose tissue, and brain (Fig. 5, B–D). In these tissues, the expressions of chicken PGC-1 and COX-II gene were less than those in leg muscles and were not different between control and cold-exposed chicks.
Discussion
In this study, we examined the mechanism by which chicks acquire the capacity for thermogenesis and tolerance to the cold. Morphological, histological, and gene expression analyses indicated that there is an increase in slow-twitch fibers in the skeletal muscle of chicks with cold tolerance. We also found the expression of chicken PGC-1 was enhanced in the skeletal muscle of chicks with cold tolerance. These results suggest that the acquisition of thermogenesis may be due to PGC-1-mediated transformation of skeletal myofibers from fast-twitch to slow-twitch. Our results are consistent with previous studies suggesting that skeletal muscle is the main tissue for both shivering and nonshivering thermogenesis in birds (11, 12, 15, 16, 19, 20, 21).
Many studies have examined the development of thermoregulation in birds (17, 18, 19, 20, 21). We used chicks for the current studies of the molecular mechanism of thermogenesis in birds because the largest body of genetic information on birds exists for domestic chickens. The chickens selected in this study were preserved as a pure line to maintain a common genetic background. Young chicks are more sensitive to cold exposure than adult chickens because they have immature thermal organs, including feather and fat tissue, and they have not acquired the capacity for thermogenesis. Therefore, we focused on the development of thermogenesis and cold tolerance in young chicks. Similar to studies in rodents (8, 22, 23, 24, 25), we studied the development of thermoregulation by exposing the chicks to 4 C for 24 h or more. Because most chicks older than 6 d tolerated cold for 24 h or more, we used 7-d-old chicks to investigate the molecular mechanism of muscle thermogenesis.
An initial gross morphological analysis revealed a remarkable color change of the skeletal muscle in cold-tolerant chicks. Histological and subtractive-differential expression analysis confirmed that this was due to an increase in the content of slow-twitch muscle fibers (red fibers). Using histological analysis, Duchamp et al. (14) also reported an increase in the level of slow-twitch fibers in the pectoralis muscle of ducklings after a long-term cold exposure. However, a morphological change has not been reported in studies of long-term cold exposure in birds (14, 15). The obvious morphological change of cold-exposed muscle observed in our study may be connected to the high plasticity of muscle structure in 7-d-old chicks (19, 21).
Mammalian PGC-1 expression is induced by cold exposure in BAT and skeletal muscle (8). PGC-1 improves adaptive thermogenesis in BAT by enhancing the expression of mitochondrial genes and inducing mitochondrial biogenesis (10). Furthermore, PGC-1 directs the transformation of mammalian skeletal muscle fibers from fast-twitch to slow-twitch (28). For these reasons, we examined the involvement of PGC-1 in the transformation of muscle fibers in chicks acquiring cold tolerance. We observed an increase in chicken PGC-1 expression in the skeletal muscle of chicks exposed to 4 C for 24 h. This suggests that chicken PGC-1 is involved in muscle fiber transformation in chicks during the acquisition of cold tolerance. Finally, the expression of the chicken PGC-1 gene was increased in chicks exposed to cold for 48 h, suggesting that a continuous high expression of this gene is necessary for muscle thermogenesis. Further studies are needed to determine whether the chicken PGC-1 directly drives muscle fiber transformation in chicks exposed to cold.
Because mouse PGC-1 induces the expression of myoglobin (28) and because myoglobin gives a red color to muscle (29), we examined the contribution of myoglobin to the reddish color in the skeletal muscle of chicks with cold tolerance. However, we found that the expression of myoglobin gene does not appear to be a main contributor to color change in the muscle of chicks with cold tolerance. Subtraction and differential display analysis found the up-regulation of characteristic genes of mitochondrial electron transfer system in muscle of chicks exposed to cold for 24 h, suggesting that, as in the BAT of rodents, an increase of the number of mitochondria may be responsible for the increased color.
In mammalian BAT-dependent thermogenesis, UCP1, but probably not UCP2 or UCP3, produces heat by uncoupling mitochondrial oxidative phosphorylation (1, 3, 7, 22, 32). UCP1 is expressed specifically in BAT, UCP2 is ubiquitously expressed (24), and UCP3 is expressed in BAT and skeletal muscle (23). Furthermore, avian UCP has been identified in skeletal muscle (30, 33) and has a higher homology with mammalian UCP2 and UCP3 than with UCP1 (55% identity vs. mouse UCP1 and 70% identity vs. mouse UCP2 and UCP3). Long-term cold exposure has been reported to increase avian UCP expression in 1-wk-old ducklings (30) and 3-wk-old chickens (31). However, in our studies, exposure of chicks to cold for 24 h decreased the expression of UCP in chicks with a high level of slow-twitch muscle fiber, and the expression of UCP returned to the control level after 48 h of cold exposure. Wang et al. (25) reported that the regulation of UCP3 expression in rats differs in various muscle types and, similar to our finding, that exposure of rats to cold for 24 h down-regulated UCP3 protein and mRNA in the soleus muscle (slow-twitch muscle). Therefore, although the function of avian UCP in thermogenesis is unclear, regulation of its expression may depend on the period of cold exposure and the type of muscle fiber.
We observed increased expression of chicken PGC-1 and COX-II gene not only in the leg muscles but also in the breast muscles. The white muscles of the chicken breast acquired a marked red color in cold-exposed chicks. Thus, transformation of the muscle seems to affect not only the muscles that already contain slow-twitch muscles but also the more glycolytic white flight muscles. Thus, other muscles in addition to the leg muscles may become thermogenic organs.
Collectively, our results suggest that enhancement of chicken PGC-1 expression may induce an increase in slow-twitch type fiber content, thereby allowing chicks to acquire thermogenesis. However, it is not certain that PGC-1 is the only protein that is involved in muscle fiber transformation and/or muscle thermogenesis. Indeed, many factors and molecules participate in muscle fiber transformation (34, 35, 36). In our study, subtraction and differential display analysis identified several ESTs that were either up- or down-regulated in the muscle of chicks with cold tolerance. Evaluating the functions and properties of these ESTs may provide an understanding of the molecular mechanism of avian thermogenesis and the role of muscle fiber transformation from fast-twitch to slow-twitch.
Acknowledgments
We sincerely appreciate the advice and expertise of Professor F. Kraemer, Stanford University, Palo Alto, CA). We thank A. Ushitani for secretarial assistance in the preparation of this manuscript, and T. Minematsu for technical assistance in the breeding of chicks.
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