AMP-activated protein kinase (AMPK) consists of three subunits: α, β, and γ. Two isoforms exist for the α-subunit (α1 and α2), two for the β-subunit (β1 and β2), and three for the γ-subunit (γ1, γ2, and γ3). Although the specific roles of the β- and γ-subunits are not well understood, the α-subunit isoforms contain the catalytic site and also the phosphorylation/activation site for the upstream kinase. This study was designed to determine the role of thyroid hormones in controlling expression levels of these AMPK subunits and of one downstream target, acetyl-CoA carboxylase (ACC), in muscle. AMPK subunit and ACC levels were determined by Western blots in control rats, in rats given 0.01% propylthiouracil (PTU) in drinking water for 3 wk, and in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 1 or 3 wk. In gastrocnemius muscle, all isoforms of AMPK subunits were significantly increased in rats given thyroid hormones for 3 wk vs. those treated with PTU. Similar patterns were seen in individual muscle types. Expression of muscle ACC was also significantly increased in response to 3 wk of treatment with excess thyroid hormones. Muscle content of malonyl-CoA was elevated in PTU-treated rats and depressed in thyroid hormone-treated rats. These data provide evidence that skeletal muscle AMPK subunit and ACC expression is partially under the control of thyroid hormones.
amp-activated protein kinase (AMPK) is an enzyme consisting of three subunits: α, β, and γ (12, 13, 42, 45). Two isoforms exist for the α-subunit (α1 and α2), two for the β-subunit (β1 and β2), and three for the γ-subunit (γ1, γ2, and γ3). Although the specific roles of the β- and γ-subunits are not well understood, the α-subunit isoforms contain the catalytic site and also the phosphorylation/activation site for the upstream kinase (AMPKK). In humans, the genes for these isoforms are located on chromosomes 1, 2, 5, 7, and 12 (seehttp://us.expasy.org/cgi-bin/niceprot;http://bioinfo.weizmann. ac.il/cards-bin/carddisp and Refs.2 and 32). The locus for the gene for the α1-subunit (5p12) is different from that for the α2-subunit (1p31). The gene for the β1-subunit is located at 12q24.1 and the gene for the β2-subunit at 1q12-q21. The positions for the γ1-, γ2-, and γ3-subunit isoforms are 12q13.1, 7q36, and 2q35, respectively.
Exceptionally large changes (200-fold) can occur in the activity of AMPK, inasmuch as it is regulated by covalent modification and allosteric mechanisms (6, 12, 13). It is phosphorylated (threonine 172 of the α-subunit) and, thereby, activated by an upstream kinase, AMPKK, which is activated allosterically by 5′-AMP (14, 33). The phosphorylated AMPK is also allosterically activated by increases in 5′-AMP and inhibited by high concentrations of ATP and phosphocreatine (12, 13, 28, 42, 45). Any perturbation that will increase 5′-AMP or decrease phosphocreatine or ATP will result in activation of the kinase. The most common natural conditions when this occurs include muscle contraction and hypoxia (12, 13, 34, 39, 42, 45). Thus the enzyme can be thought of as a sensitive energy monitor in the muscle fiber (12, 13, 40,42, 45).
The actions of AMPK appear to be designed to prevent ATP depletion in the muscle fiber. Thus AMPK phosphorylates and inactivates acetyl-CoA carboxylase (ACC), resulting in a decrease in malonyl-CoA, relieving inhibition of carnitine palmitoyltransferase (CPT) and allowing fatty acid oxidation to proceed (21, 42, 44-46). Accumulating evidence indicates that AMPK stimulates glucose transport into the muscle by causing translocation of GLUT-4 from microvesicles into the muscle sarcolemma and T tubules (21, 42). Sensitivity of muscle to insulin has recently been discovered to be influenced by prior AMPK activation (8). With chronic activation of AMPK over a period of several days, there is an increase in expression of GLUT-4 and hexokinase, apparently due to an enhancement of transcription (24, 42, 52). Recently, insulin receptor substrate-1 has been shown to be a target for AMPK (15). Mitochondrial enzymes of the citric acid cycle and electron transport chain also increase in response to chronic activation of muscle AMPK by injection of 5-aminoimidazole-4-carboxamide-riboside into rats (42).
Despite its apparent central role in regulation of fatty acid oxidation, glucose uptake, insulin sensitivity, and GLUT-4 expression in muscle, little is known regarding the control of expression of subunit isoforms of AMPK in muscle. In a recent study on the effect of endurance training on AMPK subunit expression, it appears that only γ3-subunit expression in fast-twitch high-oxidative muscle (type IIa fibers) is increased (3-fold) by endurance training (7). Previous studies have clearly demonstrated that mitochondrial enzymes, GLUT-4, and hexokinase levels in muscle increase with chronic treatment of rats with excess thyroid hormones (43,47). The mechanisms of these effects are not completely understood. Likewise, many of the effects of thyroid hormones on fat metabolism and body composition are not entirely explained with available data. It seemed reasonable that AMPK, which also appears to be involved in control of expression of several muscle proteins and of carbohydrate and fat metabolism, may be governed in part by the thyroid state. This study was designed to test the hypothesis that thyroid hormones influence AMPK expression in muscle.
MATERIALS AND METHODS
All procedures were approved by the Institutional Animal Care and Use Committee of Brigham Young University. Male Sprague-Dawley rats (Sasco, Wilmington, MA) were housed in individual cages in a room lighted from 6 AM to 6 PM. Rats were fed powdered Harlan Teklad rodent diet (Madison, WI). Rats weighed an average of 205 ± 2 g at the beginning of treatments. Rats were divided into four treatment groups. The first group (controls) received powdered food and water ad libitum. The second group received 0.01% propylthiouracil (PTU) in drinking water for 3 wk for the purpose of inhibiting thyroid hormone synthesis. This dose of PTU results in marked hypertrophy of the thyroid (54.3 ± 9.1 vs. 14.2 ± 0.5 mg in controls). The third group was provided with powdered Harlan Teklad rodent diet containing 3 mg of thyroxine and 1 mg of 3,5,3′-triiodothyronine per kilogram for 3 wk (T21d). The fourth group was given the same amount of thyroid hormones in their food but only for 1 wk (T7d). This dose has previously been shown to increase mitochondrial enzymes in liver and muscle and to increase heart weight (43, 47).
On the afternoon before the rats were killed, they were given 16 g of rat chow. After the respective treatments, rats were anesthetized by injection of pentobarbital sodium (35–50 mg/kg ip). The soleus muscle, the gastrocnemius muscle, the red and white regions of the vastus lateralis muscle, and the liver were quickly removed and immediately frozen using stainless steel block clamps at liquid nitrogen temperature. Tissues were kept frozen at −80°C until analyses. The epididymal and retroperitoneal fat pads were weighed as an indicator of adiposity.
Anti-α1, anti-β1, anti-β2, and anti-phosphorylated ACC antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-α2, anti-γ1, anti-γ2, and anti-γ3antibodies were prepared as described previously (7).
Muscles for determination of ACC and AMPK activity were ground to powder under liquid nitrogen. The powder was weighed and homogenized (1 g muscle/9 ml buffer) in a buffer containing 100 mM mannitol, 50 mM NaF, 10 mM Tris, 1 mM EDTA, 10 mM β-mercaptoethanol, pH 7.5, and proteolytic enzyme inhibitors (8 trypsin-inactivating units aprotinin/l, 10 mg/l leupeptin, and 10 mg/l antitrypsin). The homogenate was centrifuged at 48,000 g for 30 min. ACC was precipitated from the supernatant by addition of 144 mg ammonium sulfate/ml followed by stirring for 30 min on ice. The precipitate was collected by centrifugation (48,000 g, 30 min). The pellet was dissolved in 10% of the original volume of the homogenate buffer, and this solution was centrifuged again to remove insoluble protein. ACC activity was determined on the supernatant at citrate/magnesium concentrations of 0–20 mM by measuring the rate of incorporation of [14C]bicarbonate into malonyl-CoA at 37°C for 10 min, as described previously (46). The ACC activity data were fitted to the Hill equation using the Grafit program (Sigma, St. Louis, MO). This program allows determination of the activation constant for citrate (K a) and the maximal velocity as a function of citrate concentration (V max). AMPK activity was determined (46) using the same resuspended ammonium sulfate precipitate used to determine ACC activity. Neutralized perchloric acid extracts of the gastrocnemius muscle were used for the determination of malonyl-CoA (20). Glycogen content of the powdered muscle was determined by the method of Passonneau and Lowry (26).
Immunoblotting for AMPK subunits and ACC.
Muscle was ground to powder under liquid nitrogen and homogenized (1 g muscle/9 ml buffer) in a buffer containing 100 mM mannitol, 50 mM NaF, 10 mM Tris, 1 mM EDTA, 10 mM β-mercaptoethanol, pH 7.5, and proteolytic enzyme inhibitors (8 trypsin-inactivating units aprotinin/l, 10 mg/l leupeptin, and 2 mg/l antitrypsin). The homogenate was centrifuged at 700 g at 4°C for 10 min. The supernatant was diluted [1:2:1 (vol/vol/vol) homogenate-water-Laemmli buffer], and 40 μl of this solution were loaded onto the gel. Blots for ACC and phosphorylated ACC were run using 5% Tris · HCl Ready Gels (Bio-Rad, Hercules, CA). The AMPK subunit blots were done using 7.5 or 10% Tris · HCl Ready Gels. After SDS-PAGE (200 V for 40 min), proteins were transferred from gel to nitrocellulose membrane (100 V for 50 min). Membranes were blocked in 5% nonfat dry milk (Bio-Rad) in PBST (139 mM NaCl, 2.7 mM KH2PO4, 9.9 mM Na2HPO4, and 0.1% Tween 20) for 1 h and then incubated overnight with the specific antibodies to the AMPK subunits or phosphorylated ACC in 5% nonfat dry milk in PBST. After the membranes were washed twice with PBST and twice with PBS, antibodies bound to the nitrocellulose membrane were detected with anti-rabbit immunoglobulin-horseradish peroxidase (HRP)-linked antibody (Amersham Pharmacia Biotech, Piscataway, NJ) or with donkey anti-sheep HRP (in the case of the α2- and γ3-subunits). The membrane designated for ACC quantitation was exposed to streptavidin-HRP conjugate (Amersham Pharmacia). After they were washed twice in PBST and twice in PBS (139 mM NaCl, 2.7 mM KH2PO4, and 9.9 mM Na2HPO4), membranes were incubated with enhanced chemiluminescence detection reagent and visualized on enhanced chemiluminescence hyperfilm (Amersham Pharmacia Biotech). Relative amounts of each protein were then quantified using a Hewlett-Packard Scan Jet 5370C and SigmaGel software (SPSS, Chicago, IL). Extracts from each treatment group were run on the same gel and blot. All data are expressed relative to muscle extracts from the control group.
Values are means ± SE. Results of the four different treatment groups were compared using analysis of variance followed by Fisher's least significant differences test. Differences were considered to be statistically significant when P < 0.05.
Body weight, fat pad weight, food intake, malonyl-CoA, and glycogen.
The final body weights were 298 ± 11, 255 ± 2, 289 ± 10, and 282 ± 7 g for control, PTU, T21d, and T7d rats, respectively. The mean of the PTU-treated group was significantly different from all others (P < 0.05). Food intake for the penultimate 2 days before the animals were killed was 26 ± 1, 17 ± 1, 35 ± 1, and 24 ± 1 g, respectively. The means for the PTU and T21d rats were significantly different from all others (P < 0.05). The combined mass of the epididymal and retroperitoneal fat pads expressed as percent body weight were 0.59 ± 0.03, 0.53 ± 0.02, 0.21 ± 0.04, 0.39 ± 0.06 for control, PTU, T21d, and T7d rats, respectively. Both thyroid hormone-treated groups had significantly reduced fat pad weight compared with control and PTU-treated rats (P < 0.05).
Malonyl-CoA content of the gastrocnemius muscle was significantly increased in PTU-treated rats and significantly reduced in thyroid hormone-treated rats compared with controls (Fig.1). Glycogen content of gastrocnemius muscle and glucose and lactate concentrations of the blood were not markedly influenced by the thyroid hormone treatments (data not shown).
AMPK subunit isoform Western blots in skeletal muscle.
Figure 2 shows the Western blot data for subunit isoforms of AMPK in gastrocnemius muscle. Subunit isoforms migrated in the approximate positions as expected on the basis of standards consisting of known molecular mass proteins (α-subunit at 63 kDa, β1-subunit at 32 kDa, β2-subunit at 30 kDa, γ1-subunit at 35 kDa, γ2-subunit at 63 kDa, and γ3-subunit at 55 kDa). All subunit isoforms were present in significantly greater amounts in T21d than in PTU rats. The direction of change appeared to be the same for all subunit isoforms. Table 1 demonstrates that the pattern of expression in the red and white regions of the quadriceps (composed of type IIa and IIb fibers, respectively) was similar to that of the gastrocnemius (composed predominantly of type IIa and IIb fibers). Although much greater variation occurred in the soleus, it appeared that larger changes occurred in this muscle than in the type IIa or IIb fast-twitch muscle fibers.
ACC activity, AMPK activity, ACC, and phosphorylated ACC Western blots.
ACC activities as a function of citrate concentration are shown in Fig.3. The V max values were 26.4 ± 1.2, 20.0 ± 1.8, 39.9 ± 0.6, and 26.6 ± 2.5 nmol · g−1 · min−1for control, PTU, T21d, and T7d rats, respectively (n = 5–6). The V max values for PTU and T21d rats were significantly different from all other groups (P< 0.05). The K a values were 5.6 ± 1.1, 5.6 ± 1.2, 4.6 ± 0.6, and 5.8 ± 1.5, respectively. These values were not significantly different from one another. The liver ACC activity was also determined for comparative purposes. TheV max values were 317 ± 48, 100 ± 15, and 495 ± 87 nmol · g−1 · min−1for control, PTU, and T21d rats, respectively (n = 5–6). The values were significantly different from each other (P < 0.05). The ACC activity appears to vary as a function of thyroid state in both tissues, although the range ofV max activity was fivefold in liver and twofold in muscle when PTU rats were compared with T21d rats. ACC activity data from individual muscle types showed the same pattern as in the gastrocnemius (Table 2).
Total gastrocnemius AMPK activity was 248 ± 54, 155 ± 33, 332 ± 54, and 371 ± 95 nmol · g−1 · min−1for control, PTU, T21d, and T7d rats, respectively. Although trends were apparent between the PTU- and thyroid hormone-treated rats, these values were not significantly different from one another.
Data from Western blots for ACC and phosphorylated ACC for gastrocnemius muscle are shown in Fig. 4. As with the ACC activity measurements, the amount of ACC was increased in response to thyroid hormones. In addition, the amount of phosphorylated ACC increased as a function of thyroid hormones. A summary of these two measurements in the different muscle types is shown in Table 3. Again, the pattern of adaptation in the individual muscles types in response to varying thyroid hormone treatment was similar to that of the mixed-fiber gastrocnemius muscle.
Previous studies have clearly demonstrated marked fiber type-specific adaptations of skeletal muscle in response to thyroid hormone treatment. Oxidative enzymes of the citric acid cycle, electron transport chain, ketone oxidation enzymes, hexokinase, and GLUT-4 increase in response to thyrotoxicosis (36, 43, 47). Contractile proteins and sarcoplasmic reticulum proteins, as well as contractile properties (soleus), change with thyroid state (5, 9,10). Because AMPK is very likely involved in control of fatty acid oxidation, glucose uptake, and specific gene expression in skeletal muscle, it seemed reasonable that expression of AMPK subunit isoforms might also be regulated by thyroid state. This indeed proved to be the case. It appears that there is coordinated regulation of all subunits. For the most part, all subunits appear to be increasing in response to exposure to increasing levels of thyroid hormones. It will be interesting to determine whether all genes involved have thyroid response elements in their promoter regions. These studies do not provide information on the mechanism of the increase in total amounts of each subunit in response to thyroid hormones. This could be due to thyroid hormone-induced increased rate of transcription of the respective genes or to other mechanisms such as reduction in rate of mRNA or protein degradation.
One previous study reported a small downregulation of mRNA for an AMPK subunit in brain and thyroid of a hyperthyroid goat (27). It is unclear whether this is a tissue-specific adaptation or possibly a species difference.
Previous studies demonstrated that expression of citrate synthase and other mitochondrial oxidative enzymes in response to thyroid hormones is fiber type dependent (47). The soleus muscle (composed predominantly of the slow-twitch, type I fibers) has been shown to respond with much larger increases in mitochondrial oxidative enzymes than did the fast-twitch high-oxidative (type IIa) and the fast-twitch low-oxidative (type IIb) fibers (47). In the present study, the change in expression of the AMPK subunits in response to PTU and thyroid hormones in the red (type IIa fibers) and white (type IIb fibers) regions of the quadriceps was similar to that observed in the gastrocnemius, which consists predominantly of type IIa and IIb fibers. Much larger changes were observed in the soleus, particularly in the α1-subunit (23-fold difference between the PTU and the T21d group) and in the γ3-subunit (10-fold difference between the PTU and the T21d group). Although the specific roles of different combinations of the different subunits have not been ascertained, it is possible that intracellular targeting may be one function of the β- and/or γ-subunit isoforms.
The stimulatory effect of thyroid hormones on liver proteins involved in lipogenesis was described many years ago. Fatty acid synthetase, ACC, malic enzyme, and spot 14 protein show dramatic increases in liver in response to treatment of rats with thyroid hormones (4, 16,30, 35, 37, 51). Muscle is not considered to be a lipogenic tissue, yet it contains significant amounts of a unique isoform of ACC (3, 11, 38, 41, 50). This isoform is thought to play a role in regulating rates of muscle fatty acid oxidation by controlling levels of malonyl-CoA, an inhibitor of CPT I (18, 19, 31). Muscle ACC is phosphorylated and inactivated by AMPK (activated in response to contraction) (25, 42, 45, 46, 49). The consequent decline in malonyl-CoA relieves inhibition of CPT I, allowing fatty acid oxidation to proceed. Little is known regarding control of expression of the muscle isoform of ACC. In a recent study, expression of ACC in rat skeletal muscle was not influenced by a rigorous program of training by treadmill running (7). Fasting and refeeding have marked effects on expression of liver lipogenic enzymes but not on ACC in the muscle (48). In the present study, the magnitude of differences in activity of ACC between PTU- and thyroid hormone-treated rats is much greater in liver than in skeletal muscle, but the direction of change is the same.
In vitro and in vivo studies have demonstrated that phosphorylation of muscle ACC by AMPK results in a shift of the citrate activation curve to the right (with consequent increase in K a) and a decrease in V max (21, 22, 25, 46,49). This results in a decrease in ACC activity, particularly at physiological concentrations of citrate. Although the amount of ACC phosphorylated in the site equivalent to serine 79 of the liver isoform (serine 221 in the human muscle isoform) was increased in the present study in response to the increase in thyroid hormones, the kinetic properties of ACC determined in enzyme activity measurements were not altered. This provides evidence that the same proportion of the total ACC was phosphorylated. We would have expected an increase in the measured K a if a greater proportion of the total ACC were phosphorylated by AMPK in the rats given excess thyroid hormones. It appears that the increase in absolute amount of phosphorylated ACC was proportional to the increase in total ACC protein.
It is conceivable that AMPK activation in response to thyroid hormone could mediate some of the effects on metabolism, stimulating an increase in glucose uptake and an increase in fatty acid oxidation to support the elevated rate of energy consumption. AMPK activity, although tending to be higher in the thyroid hormone-treated rats, was not significantly increased. It should be pointed out that, with intraperitoneal injection of pentobarbital for anesthesia, some variability occurs in the time to surgical level of anesthesia. This may account for some of the variability in muscle AMPK response. It is unlikely that thyroid hormones would directly activate AMPK, since most thyroid hormone effects are thought to be mediated by regulation of gene transcription, but it is possible that AMPK could be activated secondarily to the increase in metabolic rate. High-energy phosphates do not appear to be markedly influenced (in soleus muscle) at this level of hyperthyroidism, however (43).
ACC activity and ACC protein (quantified by Western blot) were significantly higher in muscle of thyroid hormone-treated rats than in muscle of control or PTU-treated rats. The malonyl-CoA content, however, was highest in the PTU-treated rats and lowest in the thyroid hormone-treated rats. This implies that the malonyl-CoA content of the muscle of rats in these treatment groups is determined by mechanisms other than long-term changes in muscle content of ACC. Possible means of regulation include cytoplasmic citrate concentration (allosteric activator of ACC), long-chain acyl-CoA (allosteric inhibitor of ACC) concentration, and phosphorylation state. Although a greater absolute amount of ACC was phosphorylated in the muscle of thyroid hormone-treated rats, the shape of the citrate activation curve was not significantly changed, implying that substrate supply or short-term allosteric mechanisms were more important in determining malonyl-CoA content of the muscle in rats of different thyroid hormone status. Whatever the mechanism, the lower muscle malonyl-CoA in thyroid hormone-treated rats would be expected to result in an increase in rate of long-chain fatty acid oxidation. With the hypermetabolic state extending to 3 wk, a 64% reduction in epididymal fat pad weight was observed in the face of a 35% increase in food intake. This implies a marked increase in fat oxidation in response to excess thyroid hormones. A knockout mouse lacking muscle ACC exhibited increased rate of muscle fat oxidation and reduced body fat content (1). In human subjects, experimentally induced mild hyperthyroidism results in a significant reduction in total fat stores within 6–9 wk (17).
A previous study from our laboratories indicated an association between the training-induced increase in γ3-subunit expression and increased muscle glycogen content (7). The fact that no difference in muscle glycogen content was observed between PTU- and thyroid hormone-treated rats, despite a significant difference in γ3-subunit expression, indicates that there may be no cause-and-effect relationship. It is recognized that numerous factors may influence muscle glycogen content.
In summary, expression of AMPK subunits and ACC in skeletal muscle is greater in rats treated with excess thyroid hormone than in rats treated with PTU to inhibit thyroid activity. In general, the same pattern is seen in slow-twitch red (type I), fast-twitch red (type IIa), and fast-twitch white (type IIb) fibers. To our knowledge, this is the first indication that thyroid state influences expression of AMPK and ACC in skeletal muscle.
This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-41438 (to W. W. Winder) and by Project Grant RD99/0001901 from Diabetes UK (to D. G. Hardie). S. R. Paulsen and S. R. Gammon were supported in part by an undergraduate student mentoring grant from the College of Biological and Agricultural Sciences, Brigham Young University. K. J. Mustard was supported by a studentship from the Biotechnology and Biological Sciences Research Council (UK) and by a grant from Novo-Nordisk.
Address for reprint requests and other correspondence: W. W. Winder, 545 WIDB, Dept. of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602 (E-mail:).
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- Copyright © 2002 the American Physiological Society