1 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
2 Department of Surgical Sciences, Karolinska Institutet, Stockholm, Sweden
3 Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, P.R. China
4 Department of Human Physiology, Copenhagen Muscle Research Centre, Institute of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark
5 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Uppsala, Sweden
6 Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala Biomedical Center, Uppsala, Sweden
Address correspondence and reprint requests to Juleen R. Zierath, PhD, Karolinska Institutet, Department of Surgical Sciences, Section of Integrative Physiology, von Eulers väg 4, 4th Floor, S-171 77 Stockholm, Sweden. E-mail: juleen.zierath{at}fyfa.ki.se
ACC, acetyl-CoA carboxylase; AMPK, 5'-AMPactivated protein kinase; CPT1b, carnitine palmitoyl transferase 1b; EDL, extensor digitorum longus; HAD, 3-hydroxyacylCoA dehydrogenase; HKII, hexokinase II; IMTG, intramuscular triglyceride; KHBB, Krebs-Henseleit bicarbonate buffer; LPL1, lipoprotein lipase 1
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ABSTRACT |
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5'-AMPactivated protein kinase (AMPK) is a cellular energy sensor that responds to alterations in the AMP-to-ATP ratio. Activation of AMPK in response to metabolic stress initiates several signaling cascades aimed at restoring energy balance, including stimulation of catabolic (ATP-generating) pathways, such as fatty acid oxidation (1), glucose uptake (2,3) and glycolysis, as well as inhibition of anabolic (ATP consuming) pathways, such as synthesis of fatty acids (4) and protein (5). Several physiological consequences of exercise, including muscle contraction, hypoxia, ischemia, heat shock, glycogen catabolism, and decreased pH, are associated with AMPK activation (6). However, multiple signal transduction cascades, including mitogen-activated protein kinases (7,8), calcineurin (9), hypoxia-inducible factor-1 (10), and calmodulin-dependent protein kinase (11) are engaged in response to exercise, thus the role of AMPK in specific exercise-induced responses in skeletal muscle is incompletely resolved.
Several lines of evidence reveal that AMPK is directly involved in glucose metabolism. Overexpression of a kinase-dead AMPK2 is associated with reduced skeletal muscle glycogen content and retarded after exercise glycogen resynthesis in mice (12,13). Moreover, a dominant missense mutation in the gene encoding AMPK
3 isoform enhances glycogen storage in glycolytic skeletal muscle in pigs (14) and transgenic mice (Tg-Prkag3225Q) harboring this mutation (15). Tg-Prkag3225Q mice have similar glycogen content immediately after exercise and elevated glycogen content after recovery when compared with wild-type mice (15). Conversely, AMPK
3 knockout mice (Prkag3/) have severely impaired glycogenesis after exercise (15). Furthermore, expression of other isoforms of AMPK subunits in Tg-Prkag3225Q and Prkag3/ mice is similar to the wild-type mice (15). Therefore, AMPK
3 appears to play a critical role in glycogen metabolism after exercise (16). However, because oxidative metabolism is important during endurance exercise, changes in lipid metabolism in response to AMPK activation may also affect glycogen metabolism in skeletal muscle.
Acute and chronic exercise promotes gene regulatory responses in skeletal muscle that may facilitate metabolic adaptations along pathways governing glycolytic and oxidative metabolism. Activation of AMPK by the adenosine analog 5'-amino-4-imidazolecarboxamide ribonucleoside increases transcription of metabolic genes in skeletal muscle that are also known to be regulated in response to exercise (17,18). A direct role for AMPK in promoting gene regulatory responses in skeletal muscle, including mitochondrial biogenesis in response to chronic energy deprivation, has been established using kinase-dead AMPK2 transgenic mice (19). Moreover, metabolic sensing in skeletal muscle also requires expression of the AMPK
3 subunit, because fasting-induced transcription of enzymes involved in lipid metabolism is retarded in Prkag3/ mice (20). Thus, AMPK plays an important role in the transcriptional regulation of multiple genes along divergent pathways controlling energy metabolism, cellular signaling, transcription, and translation (13).
Given that the metabolic requirement of fasting and long-term exercise promotes a shift from glycolytic to oxidative metabolism, we hypothesize that the AMPK3 isoform regulates glycogen resynthesis after exercise by altering the balance between glucose and lipid metabolism. Tg-Prkag3225Q and Prkag3/ mice were studied after a 2-h swimming bout or during recovery (2.5 h after swimming). Here, we provide evidence that the Prkag3225Q mutation, rather than the presence of a functional AMPK
3 subunit, directly promotes an enhanced reliance on lipid metabolism during endurance exercise, concomitant with a coordinated increase in expression of genes regulating lipid metabolism in skeletal muscle.
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RESEARCH DESIGN AND METHODS |
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Swimming protocol.
Overnight-fasted (16 h) wild-type, Tg-Prkag3225Q, or Prkag3/ mice were randomly assigned to either sedentary or swimming group. The swimming protocol has been previously described (21). Six mice swam together in plastic containers measuring 45 cm in diameter. Water temperature was maintained at 3233°C. Mice swam for four 30-min intervals separated by 5-min rest periods for a total swimming time of 2 h. After the last swim interval, mice were dried and either studied immediately or allowed to recover from the exercise bout for 2.5 h (recovery). At the onset of the recovery period, mice received an intraperitoneal glucose injection (0.5 mg/g body wt) and had free access to food and water.
Muscle incubations.
All incubation media were prepared from a stock solution of Krebs-Henseleit bicarbonate buffer (KHBB) containing 0.1% BSA (radioimmunoassay grade) (21). Media were continuously gassed with 95% O2/5% CO2. Muscles were preincubated (15 min at 30°C) in KHBB containing 5 mmol/l glucose and 15 mmol/l mannitol. Muscles were incubated in the absence or presence of insulin (12 nmol/l) for the duration of the incubation protocol.
Glucose uptake.
Muscles were transferred to KHBB containing 20 mmol/l mannitol and incubated for 10 min. Thereafter, muscles were transferred to KHBB containing 1 mmol/l 2-deoxy-[1,2-3H]glucose (2.5 µCi/ml) and 19 mmol/l [14C]mannitol (0.7 µCi/ml) and incubated for 20 min. Glucose transport activity is expressed as micromoles per milliliter of intracellular water per hour (22).
Glucose oxidation.
Muscles were incubated (30°C for 60 min) in the absence or presence of insulin (12 nmol/l) in preincubation media supplemented with [14C]glucose (0.2 mCi/ml). Thereafter, 0.2 ml Solvable (2% sodium hydroxide; Dupont, Hamburg, Germany) was injected into the center well of the incubation vial to collect liberated CO2, and 0.5 ml 15% perchloric acid was injected into the media to lyse the muscle. Glucose oxidation was assessed by collection of liberated CO2.
Oleate oxidation.
Muscles were harvested immediately after swim exercise and incubated (30°C for 60 min) in 1 ml KHBB media supplemented with 0.3 mmol/l [1-14C]oleate (0.4 µCi/ml). Thereafter, the media was acidified by 0.5 ml 15% pyrroline-5-carboxylic acid, and liberated CO2 was collected in center wells containing 0.2 ml Protosol (DuPont NEN Research Laboratories) for 60 min. Center wells were removed for scintillation counting. Results were expressed as nanomoles of oxidized oleate per gram of wet weight per hour.
Western blot analysis.
Phosphorylation of acetyl CoA-carboxylase (ACC) was determined by Western blot analysis. White gastrocnemius skeletal muscle was lysed in ice-cold buffer (23), and an aliquot of lysate (30 µg) was separated by SDSPAGE. Proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA) and probed with primary antibodies (described below) and secondary horseradish peroxidaseconjugated antibodies. Phosphorylation of ACC was determined using antiphospho-ACC (Ser227; Cell Signaling Technology, Beverly, MA) antibody. Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Biosciences, Uppsala, Sweden). Immunoreactive band intensity was quantified using the Image Gauge V3.01 software (Fujifilm, Tokyo, Japan).
Intramuscular triglyceride content.
Gastrocnemius muscles were removed from anesthetized mice, cleaned of fat and blood, and quickly frozen in liquid nitrogen. Triglycerides were analyzed using 1525 mg pulverized frozen skeletal muscle. Tissue was homogenized with 0.3 ml heptan-isopropanol-Tween mixture (3:2:0.01 by volume) and centrifuged (1,500 x g for 15 min at 4°C). Supernatants (upper phase containing extracted triglycerides) were collected and evaporated with vacuum centrifuge. Triglyceride content was measured with a triglyceride/glycerol blanked kit (Roche, Nutley, NJ). Seronorm lipid (SERO, Billingstad, Norway) was used as a standard. Samples were measured in duplicates.
RNA purification and cDNA synthesis.
Total RNA of white gastrocnemius muscle was purified using Trizol reagent (Sigma, St. Louis, MO), as specified by the manufacturer. Purified RNA was treated with DNase I using DNA-free kit (Ambion, Austin, TX), according to the manufacturers protocol. DNase-treated RNA served as a template for cDNA synthesis using oligo(dT) primers and SuperScript First Strand Synthesis System (Invitrogen, Carlsbad, CA).
Quantitative PCR.
Quantification of mRNA was performed using real-time PCR with the ABI PRISM 7700 Sequence Detector System (Applied Biosystems, Warrington, U.K.) and SYBR-green. The relative quantities of target transcripts were calculated from duplicate samples after normalization of the data against housekeeping gene 36B4 (acidic ribosomal phosphoprotein PO). Primers were selected by using PRIMER EXPRESS (Applied Biosystems). Transcript sequences obtained from ENSEMBL database were lipoprotein lipase 1 (LPL1; ENSMUST00000015715), carnitine palmitoyl transferase 1b (CPT1b; ENSMUST00000023287), 3-hydroxyacylCoA dehydrogenase (HAD; ENSMUST00000029610), uncoupling protein 3 (ENSMUST00000032958), GLUT4 (ENSMUST00000018710), and glycogen synthase (ENSMUST00000003964). Transcript sequences obtained from National Center for Biotechnology Information GenBank database were cytochrome c (NM007808), hexokinase II (HKII; Y11666), and 36B4 (BC003833).
Statistical analysis.
Differences between means were analyzed using Students t test or two-way ANOVA followed by Fishers least significant differences post hoc analysis. Significance was accepted at P < 0.05.
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RESULTS |
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Oleate oxidation.
EDL muscles were excised from mice directly after the swim bout and incubated for 2 h to determine the rate of oleate oxidation after exercise. Under these in vitro conditions, similar rates of oleate oxidation were observed among genotypes (data not shown). Therefore, despite enhanced IMTG utilization during exercise, Tg-Prkag3225Q mice maintained a similar rate of extracellular lipid oxidation in the state after exercise compared with wild type.
Quantitative PCR for metabolic genes.
We have previously determined mRNA expression of metabolic genes in white gastrocnemius muscles from wild-type, Tg-Prkag3225Q, and Prkag3/ mice under fed or fasted conditions (20). mRNA expression of genes involved in lipid metabolism through quantitative real-time PCR analysis in response to swim exercise or recovery was determined (Fig. 4). In Tg-Prkag3225Q mice, mRNA expression of LPL1 (P < 0.01), CPT1b (P < 0.05), HAD (P < 0.01), and cytochrome c (P < 0.01) was higher than the wild type after swimming exercise. In Prkag3/ mice, mRNA expression of CPT1b and cytochrome c was lower than the wild type (P < 0.01) after swimming. After recovery, mRNA expression of lipid metabolic genes was similar among Tg-Prkag3225Q, Prkag3/, and wild-type mice, with the exception of HAD, which was reduced in Prkag3/ mice (P < 0.05). When comparing mRNA expression levels after swimming and after 2.5 h of recovery in the same genotype, LPL1 was increased in wild-type (P < 0.05) and Prkag3/ (P < 0.01) mice. However, LPL1 (P < 0.01), CPT1b (P < 0.05), HAD (P < 0.01), and cytochrome c (P < 0.05) were decreased in Tg-Prkag3225Q mice.
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DISCUSSION |
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Glucose uptake, a rate limiting step for glycogen resynthesis, is unaltered among Tg-Prkag3225Q, Prkag3/, and wild-type mice either immediately after acute exercise or after 2.5 h of recovery. This was an unexpected observation because glycogen content is markedly elevated in Tg-Prkag3225Q and reduced in Prkag3/ mice after recovery when compared with the wild-type mice (15). However, a similar uncoupling between glucose transport and accelerated glycogen synthesis has been observed in mice overexpressing a constitutively active form of glycogen synthase in skeletal muscle (24,25), whereby the repartitioning of intracellular glucose intermediates toward glycogen synthesis after muscle contraction is enhanced (26). Glucose oxidation after intense anaerobic activity is markedly lower in Tg-Prkag3225Q and higher in Prkag3/ mice, respectively, (27), indicating a shift toward glucose incorporation into glycogen. However, in response to the 2-h endurance exercise bout, glucose oxidation was similar between Tg-Prkag3225Q and wild-type mice and reduced in Prkag3/ mice. Thus, any potential difference in glucose oxidation among the genotypes may be masked by an increased demand on lipid oxidation during steady-state endurance exercise. Collectively, these studies reveal muscle glycogen supercompensation can occur without excessive glucose transport, presumably through alterations in glucose handling between glucose oxidation and glycogenesis.
The contribution of fatty acid oxidation to total energy supply increases during long duration exercise. Thus, the metabolic shift toward utilization of fatty acids has a sparing effect on glucose utilization. Phosphorylation of ACC regulates the entry of fatty acids into the mitochondrial matrix. Immediately after swimming, ACC phosphorylation was increased in Tg-Prkag3225Q mice, suggestive of increased fatty acid availability for oxidation. Triglyceride content was reduced in skeletal muscle from Tg-Prkag3225Q mice directly after swimming. ACC phosphorylation and triglyceride content in skeletal muscle from Prkag3/ mice were similar to wild-type mice. Thus, the Prkag3225Q mutation, rather than the presence of a functional AMPK3 subunit, directly promotes a metabolic shift toward fatty acid utilization in response to exercise.
Exercise regulates transcriptional events in skeletal muscle partly through activation of AMPK. Chronic stimulation of AMPK increases protein expression of GLUT4, hexokinase (28), and the oxidative enzyme cytochrome c (19,28). We have previously determined the expression of several metabolic genes in skeletal muscle from wild-type, Tg-Prkag3225Q, and Prkag3/ mice under fed and fasted conditions, and we provide evidence AMPK plays a role in the coordinated expression of genes involved in lipid and glucose metabolism (20). Here, we observed a concerted upregulation of mRNA expression of genes involved in fatty acid availability (LPL1), transport into the mitochondria (CPT1b), and oxidation (cytochrome c and HAD) in Tg-Prkag3225Q mice compared with wild-type mice. The enhanced transcriptional response and phosphorylation of ACC in Prkag3225Q mice was associated with increased utilization of triglyceride, as was evident from the reduction in triglyceride content after swimming. In contrast, mRNA expression of genes involved in fatty acid metabolism in Prkag3/ mice, including CPT1b and cytochrome c, was diminished, with a tendency for reduced expression of LPL and HAD after swimming. Therefore, the AMPK3 subunit plays a role in modulating transcription of lipid metabolic genes and, importantly, lipid metabolism during endurance exercise. Nonetheless, expression of mRNA for lipid metabolic genes in Tg-Prkag3225Q and Prkag3/ mice was normalized to wild-type levels after recovery. Essentially, an enhanced response in transcription of lipid metabolic genes in Tg-Prkag3225Q mice and a diminished transcriptional response in Prkag3/ mice compared with wild-type mice was observed after swimming.
The transition between fed and fasted conditions promotes gene regulatory responses in skeletal muscle. We have previously observed a coordinated decrease in the mRNA expression of HKII and glycogen synthase in skeletal muscle from Prkag3225Q versus wild-type mice (20). Here, we report that mRNA expression of GLUT4, HKII, and glycogen synthase after exercise was higher in Tg-Prkag3225Q mice compared with wild-type mice. Thus, gene regulatory changes in Tg-Prkag3225Q mice are largely influenced by fasting and exercise. Change at the level of mRNA occurs in parallel with metabolic changes. An elevation in transcript levels of genes important for glycogen synthesis is consistent with the enhanced glycogen supercompensation of Prkag3225Q mice (27). The elevated expression of HKII, GLUT4, and glycogen synthase in Prkag3225Q mice during swimming was reduced after recovery, concomitant with the elevation in skeletal muscle glycogen content (15). After recovery from swimming, GLUT4 and HKII mRNA were increased in wild type when compared with the level immediately after swimming. In contrast, the transcriptional induction of GLUT4 and HKII during recovery is blunted in Prkag3/ mice. Thus, dysregulation of lipid and glucose metabolic gene expression in Prkag3/ mice provides evidence that the AMPK3 subunit plays an essential role in coordinating the transcription of lipid and glucose metabolic genes in response to metabolic challenges that include fasting, exercise, and recovery in skeletal muscle. Moreover, the Prkag3225Q mutation, rather than the presence of a functional AMPK
3 subunit enhances the transcriptional response to metabolic challenges in skeletal muscle.
In conclusion, AMPK activation achieved by overexpression of the Prkag3225Q mutation, rather than the presence of a functional AMPK 3 subunit, promotes fuel repartitioning and gene regulatory responses to facilitate lipid oxidation during endurance exercise and glycogen storage during recovery. Furthermore, the transcriptional and metabolic profile of the Prkag3/ mice diverges from that of the wild-type mice, suggesting that the AMPK
3 subunit plays a role in coordinating gene regulatory responses to exercise and recovery. Collectively, these results further support strategies aimed to activate AMPK in skeletal muscle as a means to improve impaired lipid and glucose homeostasis in metabolic disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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L.A. holds stock in Arexis (Gothenburg, Sweden).
Received for publication March 4, 2005 and accepted in revised form August 23, 2005
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REFERENCES |
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