AMP-activated Protein Kinase α2 Activity Is Not Essential for Contraction- and Hyperosmolarity-induced Glucose Transport in Skeletal Muscle
2005; Elsevier BV; Volume: 280; Issue: 47 Linguagem: Inglês
10.1074/jbc.m504208200
ISSN1083-351X
AutoresNobuharu Fujii, Michael F. Hirshman, Erin M. Kane, Richard C. Ho, Lauren Peter, Matthew Seifert, Laurie J. Goodyear,
Tópico(s)Autophagy in Disease and Therapy
ResumoTo examine the role of AMP-activated protein kinase (AMPK) in muscle glucose transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive α2 (α2i TG) and α1 (α1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In α2i TG mice basal α2 activity was barely detectable, whereas basal α1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK α2 activity in α2i TG mice, whereas α1 activation was attenuated by only 30–50%. Glucose transport was measured in vitro using isolated EDL muscles from α2i TG mice. AICAR- and rotenone-stimulated glucose transport was fully inhibited in α2i TG mice; however, the lack of AMPK α2 activity had no effect on contraction- or sorbitol-induced glucose transport. Similar to these observations in vitro, contraction-stimulated glucose transport, assessed in vivo by 2-deoxy-d-[3H]glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in α2i TG mice. Thus, AMPK α2 activation is essential for some, but not all, insulin-independent glucose transport. Muscle contraction- and hyperosmolarity-induced glucose transport may be regulated by a redundant mechanism in which AMPK α2 is one of multiple signaling pathways. To examine the role of AMP-activated protein kinase (AMPK) in muscle glucose transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive α2 (α2i TG) and α1 (α1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In α2i TG mice basal α2 activity was barely detectable, whereas basal α1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK α2 activity in α2i TG mice, whereas α1 activation was attenuated by only 30–50%. Glucose transport was measured in vitro using isolated EDL muscles from α2i TG mice. AICAR- and rotenone-stimulated glucose transport was fully inhibited in α2i TG mice; however, the lack of AMPK α2 activity had no effect on contraction- or sorbitol-induced glucose transport. Similar to these observations in vitro, contraction-stimulated glucose transport, assessed in vivo by 2-deoxy-d-[3H]glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in α2i TG mice. Thus, AMPK α2 activation is essential for some, but not all, insulin-independent glucose transport. Muscle contraction- and hyperosmolarity-induced glucose transport may be regulated by a redundant mechanism in which AMPK α2 is one of multiple signaling pathways. Recent reports suggest that AMP-activated protein kinase (AMPK), 2The abbreviations used are:AMPKAMP-activated protein kinaseα2i TG micetransgenic mice expressing an inactive form of α2 in skeletal muscleα1i TG micetransgenic mice expressing an inactive form of α1 in skeletal muscleα1/2i TG micetransgenic mice expressing an inactive form of both α1 and α2 in skeletal muscleEDLextensor digitorum longusAICAR5-aminoimidazole-4-carboxamide-1-β-4-ribofuranosideGLUT1glucose transporter 1GLUT4glucose transporter 4IRSinsulin receptor substrateKRBKrebs-Ringer bicarbonateDNPdinitrophenolCLAMSComprehensive Lab Animal Monitoring SystemZMP5-aminoimidazole-4-carboxamide ribonucleotide 2The abbreviations used are:AMPKAMP-activated protein kinaseα2i TG micetransgenic mice expressing an inactive form of α2 in skeletal muscleα1i TG micetransgenic mice expressing an inactive form of α1 in skeletal muscleα1/2i TG micetransgenic mice expressing an inactive form of both α1 and α2 in skeletal muscleEDLextensor digitorum longusAICAR5-aminoimidazole-4-carboxamide-1-β-4-ribofuranosideGLUT1glucose transporter 1GLUT4glucose transporter 4IRSinsulin receptor substrateKRBKrebs-Ringer bicarbonateDNPdinitrophenolCLAMSComprehensive Lab Animal Monitoring SystemZMP5-aminoimidazole-4-carboxamide ribonucleotide a member of a metabolite-sensing protein kinase family, controls blood glucose homeostasis by regulating glucose transport in skeletal muscle and glucose production in the liver (1Rutter G.A. 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AMP-activated protein kinase transgenic mice expressing an inactive form of α2 in skeletal muscle transgenic mice expressing an inactive form of α1 in skeletal muscle transgenic mice expressing an inactive form of both α1 and α2 in skeletal muscle extensor digitorum longus 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside glucose transporter 1 glucose transporter 4 insulin receptor substrate Krebs-Ringer bicarbonate dinitrophenol Comprehensive Lab Animal Monitoring System 5-aminoimidazole-4-carboxamide ribonucleotide AMP-activated protein kinase transgenic mice expressing an inactive form of α2 in skeletal muscle transgenic mice expressing an inactive form of α1 in skeletal muscle transgenic mice expressing an inactive form of both α1 and α2 in skeletal muscle extensor digitorum longus 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside glucose transporter 1 glucose transporter 4 insulin receptor substrate Krebs-Ringer bicarbonate dinitrophenol Comprehensive Lab Animal Monitoring System 5-aminoimidazole-4-carboxamide ribonucleotide AMPK is a serine/threonine kinase consisting of a catalytic α subunit and regulatory β and γ subunits (15Hardie D.G. 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Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 17Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). It has long been believed that there are two major signaling mechanisms leading to glucose transport stimulation in skeletal muscle. One mechanism is insulin-activated signaling through the insulin receptor, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase. The other is insulin-independent signaling for stimuli such as exercise, hyperosmolarity, mitochondrial uncoupling, and hypoxia (23Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar), which recent reports suggest to be regulated by AMPK. Initial evidence in support of a role for AMPK in muscle glucose transport came from studies using 5-aminoimidazole-4-carboxamide riboside (AICAR), a compound that is taken up into skeletal muscle and metabolized to ZMP, an analog of AMP. It was first shown that AICAR infusion enhances insulin-stimulated glucose transport in perfused rat hindlimb skeletal muscles (3Merrill G.F. Kurth E.J. Hardie D.G. Winder W.W. Am. J. Physiol. 1997; 273: E1107-E1112Crossref PubMed Google Scholar). After this report, our group (4Hayashi T. Hirshman M.F. Kurth E.J. Winder W.W. Goodyear L.J. Diabetes. 1998; 47: 1369-1373Crossref PubMed Scopus (702) Google Scholar) and Bergeron et al. (5Bergeron R. Russell Spaceiiiqq R.R. Young L.H. Ren J.M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar) showed that AICAR directly stimulates glucose transport in the absence of insulin in isolated rat muscle. Similar to contraction-induced glucose transport, AICAR-stimulated glucose transport was not affected by inhibition of phosphatidylinositol 3-kinase (4Hayashi T. Hirshman M.F. Kurth E.J. Winder W.W. Goodyear L.J. Diabetes. 1998; 47: 1369-1373Crossref PubMed Scopus (702) Google Scholar, 5Bergeron R. Russell Spaceiiiqq R.R. Young L.H. Ren J.M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar). In addition to studies of muscle contraction, AMPK activation closely correlates with increased glucose transport in isolated rat muscle in response to other insulin-independent stimuli such as hypoxia, inhibition of mitochondrial respiration, and hyperosmolarity (24Hayashi T. Hirshman M.F. Fujii N. Habinowski S.A. Witters L.A. Goodyear L.J. Diabetes. 2000; 49: 527-531Crossref PubMed Scopus (378) Google Scholar). Similar to contraction, these stimuli also reduce cellular ATP, phosphocreatine, and glycogen levels (4Hayashi T. Hirshman M.F. Kurth E.J. Winder W.W. Goodyear L.J. Diabetes. 1998; 47: 1369-1373Crossref PubMed Scopus (702) Google Scholar). A potential role for AMPK in glucose transport induced by hyperosmolarity (6Fryer L.G. Foufelle F. Barnes K. Baldwin S.A. Woods A. Carling D. Biochem. J. 2002; 363: 167-174Crossref PubMed Scopus (156) Google Scholar, 25Fryer L.G. Hajduch E. Rencurel F. Salt I.P. Hundal H.S. Hardie D.G. Carling D. Diabetes. 2000; 49: 1978-1985Crossref PubMed Scopus (158) Google Scholar, 26Patel N. Khayat Z.A. Ruderman N.B. Klip A. Biochem. Biophys. Res. Commun. 2001; 285: 1066-1070Crossref PubMed Scopus (47) Google Scholar) and by reduction of ATP generation by mitochondria (25Fryer L.G. Hajduch E. Rencurel F. Salt I.P. Hundal H.S. Hardie D.G. Carling D. Diabetes. 2000; 49: 1978-1985Crossref PubMed Scopus (158) Google Scholar, 27Chen H.C. Bandyopadhyay G. Sajan M.P. Kanoh Y. Standaert M. Farese Jr., R.V. Farese R.V. J. Biol. Chem. 2002; 277: 23554-23562Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) has also been suggested based on findings in cultured muscle cells. Therefore, AMPK may be a key molecule that is responsible for insulin-independent glucose transport caused by cellular stress in skeletal muscle. Consistent with the hypothesis that AMPK regulates glucose transport in rat skeletal muscle, transgenic mice that express a muscle-specific inactive mutant of AMPKα that reportedly has a dominant inhibitory effect have full inhibition of AICAR- and hypoxia-stimulated glucose transport, whereas contraction-stimulated glucose transport was reduced by 30–40% (28Mu J. Brozinick Jr., J.T. Valladares O. Bucan M. Birnbaum M.J. Mol. Cell. 2001; 7: 1085-1094Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar). In contrast, Jorgensen et al. (29Jorgensen S.B. Viollet B. Andreelli F. Frosig C. Birk J.B. Schjerling P. Vaulont S. Richter E.A. Wojtaszewski J.F. J. Biol. Chem. 2004; 279: 1070-1079Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar) showed that contraction-induced glucose transport in isolated muscles was not altered in either α1 or α2 whole body AMPK knock-out mice. These reports have led us to reconsider the role of the two catalytic subunits of AMPK in glucose transport regulation by muscle contraction and other non-insulin stimuli. For this purpose, we generated three types of muscle-specific transgenics; they are 1) mice expressing an inactive form of the α1 catalytic subunit isoform (α1i TG mice), 2) mice expressing an inactive form of α2 (α2i TG mice), and 3) a cross of α1i TG and α2i TG (α1/2i TG mice). We show that although AMPK activity is necessary for some insulin-independent glucose transport (AICAR and mitochondrial respiratory Complex I inhibition), glucose transport induced by contraction and hyperosmolarity does not require active α2 protein. Therefore, we conclude that at least three distinct or overlapping intracellular signaling pathways (i.e. an insulin-dependent, an AMPK-dependent, and an insulin/AMPK-independent pathway) are present in murine skeletal muscle for the regulation of glucose transport. Generation of Transgenic Mice—To render the catalytic subunit inactive, the aspartic acid at amino acid residue 157 of rat AMPK α2 subunit was substituted to alanine by a PCR-based site direct mutagenesis as described by Stein et al. (30Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (479) Google Scholar). Complementary primers spanning the residue to be mutated (30Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (479) Google Scholar) were synthesized and used for the mutagenesis. Mice expressing the inactive α2 tagged at the amino terminus with a hemagglutinin epitope (α2i TG mice) were generated by injecting the recombinant DNA driven by a muscle creatine kinase promoter (a gift from Drs. Kohjiro Ueki and C. Ronald Kahn, Joslin Diabetes Center, Boston, MA) into fertilized FVB mouse oocytes at the Brigham and Women's Hospital Transgenic Mouse Facility (Boston, MA). Three α2i TG lines were confirmed, and the founders were bred to FVB mice. A similar strategy was used with the goal of generating mice that express an inactive α1 in skeletal muscle (α1i TG mice). Rat AMPK α1 cDNA, in which the lysine at amino acid residue 45 was changed to arginine to render the catalytic subunit inactive (31Dyck J.R.B. Gao G. Widmer J. Stapleton D. Fernandez S.C. Kemps B.E. Witters L.A. J. Biol. Chem. 1996; 271: 17798-17803Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), was tagged at the amino terminus with the Myc epitope. Six α1i TG lines were confirmed, and the founders were bred to FVB mice. Transgenic mouse founders were identified by polymerase chain reaction-based methods, and transgene product expression was confirmed by immunoblots with an antibody for each tagged epitope. Mice expressing both transgenes were generated by breeding an α2i TG line to an α1i TG line (α1/2i TG mice). Ten-to-sixteen-week-old mice from F2 and F3 generations were used for experiments. Comparisons of the transgenic mice were made against littermate wild type mice. All procedures used were approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center. Muscle Incubation and Contraction in Vitro—Mice were sacrificed, and the extensor digitorum longus (EDL) muscles were rapidly removed and treated as previously described (4Hayashi T. Hirshman M.F. Kurth E.J. Winder W.W. Goodyear L.J. Diabetes. 1998; 47: 1369-1373Crossref PubMed Scopus (702) Google Scholar, 24Hayashi T. Hirshman M.F. Fujii N. Habinowski S.A. Witters L.A. Goodyear L.J. Diabetes. 2000; 49: 527-531Crossref PubMed Scopus (378) Google Scholar). Both ends of each EDL muscle were tied with suture (silk 4–0) and mounted on an incubation apparatus. Muscles were preincubated in 6 ml of Krebs-Ringer bicarbonate (KRB) buffer (117 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 24.6 mm NaHCO3, pH 7.5) containing 2 mm pyruvate for 20 min. The muscles were then incubated in KRB buffer in the absence or presence of 2 mm AICAR (20 min), 500 μm dinitrophenol (DNP; 20 min), 3 μm rotenone (40 min), or 120 mm sorbitol (30 min). We have previously reported that muscle treatment with DNP, rotenone, and sorbitol decreases ATP, phosphocreatine, and glycogen in skeletal muscle (24Hayashi T. Hirshman M.F. Fujii N. Habinowski S.A. Witters L.A. Goodyear L.J. Diabetes. 2000; 49: 527-531Crossref PubMed Scopus (378) Google Scholar). For the wortmannin studies muscles were preincubated in KRB buffer containing 2 mm pyruvate for 30 min in the presence or absence of wortmannin (100 nm). The maximal concentration of vehicle (Me2SO) was 0.1%, which did not affect any assay. The buffers were kept at 37 °C throughout the experiment and gassed continuously with 95% O2 and 5% CO2. For muscle contraction, muscles were transferred to a tissue support with stimulating electrodes (Harvard Apparatus, Holliston, MA), and resting tension was set to 0.5 g. Muscles were stimulated for 10 min with the following parameters: train rate = 1/min; train duration = 10 s; pulse rate = 100 pulse/s; duration = 0.1 ms; volts = 100 V. Force production during contraction was monitored with an isometric force transducer (Kent Scientific, Litchfield, CT), and the converted digital signal was captured by a data acquisition system (iWorx114, CB Sciences, Dover, NH). Muscle force generation was determined with data analysis software (LabScribe, CB Sciences, Dover, NH) and represented by g × 10 s. In the second series of experiments muscles from wild type mice were stimulated 2–3 times before the 10-min contraction period to find an optimum voltage to match muscle force production with the α2i TG mice. Prior contractions were very short (train rate = 0.1/min; train duration = 180 ms), and we determined that these short contractions did not affect force generation during the subsequent 10 min of contraction. The standard 10-min contraction protocol was then carried out with the determined optimum voltage (8.4–21 V). Measurement of Glucose Transport in Vitro—Immediately after muscle incubation or contraction, the muscles were transferred to KRB buffer containing 1 mmol/liter 2-deoxy-d-glucose (1.5 μCi/ml) and 7 mmol/liter d-[14C]mannitol (0.3 μCi/ml) (PerkinElmer Life Sciences) at 30 °C and incubated for 10 min. The same concentration of AICAR, DNP, rotenone, sorbitol, or insulin was included in each buffer if present during the previous incubation period. Glucose transport was terminated by dipping muscles in KRB buffer containing 80 μmol/liter cytochalasin B at 4 °C, and muscles were frozen in liquid nitrogen. Muscles were weighed and digested by incubating in 250 μl of 1 m NaOH at 80 °C for 10 min. Digests were neutralized with 250 μl of 1 m HCl, and particulates were precipitated by centrifugation at 13,000 × g for 5 min. Radioactivity in aliquots of the digested muscles was determined by liquid scintillation counting for dual labels, and the extracellular and intracellular spaces were calculated as previously described (4Hayashi T. Hirshman M.F. Kurth E.J. Winder W.W. Goodyear L.J. Diabetes. 1998; 47: 1369-1373Crossref PubMed Scopus (702) Google Scholar). In Situ Muscle Contraction—Mice that had been fasted for 12 h were anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg). The sciatic nerves were bilaterally isolated, and subminiature electrodes were placed around each nerve and interfaced with a Grass model S88 electrical stimulation unit as previously described (32Goodyear L.J. Giorgino F. Balon T.W. Condorelli G. Smith R.J. Am. J. Physiol. 1995; 268: E987-E995Crossref PubMed Google Scholar, 33Ruderman N.B. Houghton C.R.S. Hems R. Biochem. J. 1971; 124: 639-651Crossref PubMed Scopus (319) Google Scholar). Hindlimb muscles on one side were stimulated to induce contractions for 15 min (1 train/s, 500-ms train duration, 100 Hz, 0.1-ms duration, 1–5 V), whereas the contralateral side remained unstimulated and served as a sham-treated control. Measurement of Glucose Uptake in Vivo—Muscle glucose uptake in vivo was measured as described previously (34Ho R.C. Alcazar O. Fujii N. Hirshman M.F. Goodyear L.J. Am. J. Physiol. 2004; 286: R342-R349Crossref PubMed Scopus (83) Google Scholar). Briefly, base-line blood samples were collected from the tails of mice, the jugular vein was catheterized, and an intravenous bolus of 2-deoxy-d-[3H]glucose (10 μCi/mouse) was administered. After injection of the tracer, mice were subjected to the 15 min in situ muscle contraction protocol described above. Blood samples were obtained at 5, 10, 15, 25, 35, and 45 min for the determination of blood glucose concentrations and 2-deoxy-d-[3H]glucose specific activities. After collection of the last blood sample, animals were sacrificed, and the EDL, the tibialis anterior, and gastrocnemius muscles were removed and snap-frozen in liquid nitrogen. Muscles were digested by incubating in 400 μlof1 m NaOH at 80 °C for 10 min and neutralized with 400 μl of 1 m HCl. Aliquots of this neutralized solution were added to either perchloric acid or Ba(OH)2/ZnSO4 and centrifuged at 13,000 × g for 5 min. The radioactivity of an aliquot of each of these two supernatants was determined by liquid scintillation counting. Phosphorylated 2-deoxy-d-glucose in each tissue was then calculated as the difference between the radioactivity in the perchloric acid and Ba(OH)2/ZnSO4 supernatants and used to calculate rates of uptake (35Ferre P. Leturque A. Burnol A.-F. Penicaud L. Girard J. Biochem. J. 1985; 228: 103-110Crossref PubMed Scopus (261) Google Scholar). Measurement of Isoform-specific AMPK Activity—AMPK activity was measured as previously described (4Hayashi T. Hirshman M.F. Kurth E.J. Winder W.W. Goodyear L.J. Diabetes. 1998; 47: 1369-1373Crossref PubMed Scopus (702) Google Scholar). Briefly, muscle lysates (150 μg protein) were immunoprecipitated with specific antibodies against the α1 or α2 catalytic subunits (36Fujii N. Hayashi T. Hirshman M.F. Smith J.T. Habinowski S.A. Kaijser L. Mu J. Ljungqvist O. Birnbaum M.J. Witters L.A. Thorell A. Goodyear L.J. Biochem. Biophys. Res. Commun. 2000; 273: 1150-1155Crossref PubMed Scopus (283) Google Scholar) and protein A beads. The kinase reaction was carried out in 40 mm Hepes, pH 7.0, 0.1 mm synthetic SAMS peptide, 0.2 mm AMP, 80 mm NaCl, 0.8 mm dithiothreitol, 5 mm MgCl2, and 0.2 mm ATP (2 μCi of [γ-32P]ATP) for 20 min at 30 °C. Reaction products were spotted on Whatman P81 filter paper, the papers were extensively washed in 1% phosphoric acid, and radioactivity was assessed with a scintillation counter. Kinase activity was assessed by incorporated ATP (pmol) per immunoprecipitated protein (mg) per min as previously described (37Winder W.W. Hardie D.G. Am. J. Physiol. 1996; 270: E299-E304Crossref PubMed Google Scholar). AICAR Tolerance Test—AICAR (0.25 g/kg) was injected intraperitoneally to wild type and α2i TG mice. Blood was collected from the tail before (0 min) and after AICAR injection (20, 40, and 60 min). Blood glucose was determined using a glucometer (ONE TOUCH Ultra, LifeScan, PA). Noninvasive Physiological and Behavioral Characterization—Fourteen-week-old wild type and α2i TG mice were subjected to noninvasive physiological and behavioral characterization using the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus) at the Physiology Core Laboratory of the Joslin Diabetes Center. The mice were monitored for 24 h to assess oxygen consumption (ml/kg/h), carbon dioxide generation (ml/kg/h), heat generation calculated from the gas exchange data (kcal/h), food consumption (g/24 h), water consumption (ml/24 h), and locomotive activity, evaluated by three-dimensional fixed point observation (counts/h). Monitoring started at 10:00 h, and CLAMS assessment was made during both the light cycle (07:00 to 19:00 h) and dark cycle (19:00 to 7:00 h). Immunoblotting—Immunoblotting was done using standard procedures as previously described (38Fujii N. Boppart M.D. Dufresne S.D. Crowley P.F. Jozsi A.C. Sakamoto K. Yu H. Aschenbach W.G. Kim S. Miyazaki H. Rui L. White M.F. Hirshman M.F. Goodyear L.J. Am. J. Physiol. 2004; 287: C200-C208Crossref PubMed Scopus (41) Google Scholar). Antibodies to the α1 and α2 catalytic subunit isoforms were generated by standard methods (36Fujii N. Hayashi T. Hirshman M.F. Smith J.T. Habinowski S.A. Kaijser L. Mu J. Ljungqvist O. Birnbaum M.J. Witters L.A. Thorell A. Goodyear L.J. Biochem. Biophys. Res. Commun. 2000; 273: 1150-1155Crossref PubMed Scopus (283) Google Scholar). Other antibodies were from commercial sources including hemagglutinin (Roche Diagnostics), IRS-1 and Akt (Upstate Biotechnology, Inc.), and glucose transporter 1 and 4 (GLUT1, GLUT4) (Chemicon International, Inc., Temecula, CA). Statistics—Statistical evaluation was performed by two-way analysis of variance or Student's two-tailed t test. When analysis of variance revealed significant differences, the Bonferroni t test was used as a post hoc test for multiple comparisons. AMPK Activity and Subunit Expression in α2i TG Mice—Immunoblot analysis using an isoform-specific AMPK α2 antibody showed that the α2 protein in the α2i TG mice possessed a slower mobility (Fig. 1A, upper arrow) compared with wild type mice (lower arrow), likely due to the hemagglutinin tag. In α2i TG mice, endogenous α2 was only detected with very long exposure times, suggesting that almost all endogenous α2 protein was replaced by the mutated α2 in α2i TG mice. Band densities revealed that expression of the α2 mutant protein was 1.6-fold higher than the endogenous α2 expressed in wild type mice (Fig. 1A, left). To characterize the effects of α2 transgene expression on AMPK activity, EDL muscles were isolated from α2i TG mice and their wild type littermate controls. Muscles were incubated in vitro in the absence or presence of AICAR. Both basal and AICAR-stimulated α2 activities were severely reduced in α2i TG mice compared with wild type mice (Fig. 1B, left). On the other hand, basal levels of α1 activity were only slightly decreased in the muscle of the α2i TG mice (Fig. 1B, right). AICAR-stimulated α1 activity was reduced by 50% in α2i TG mice, but this still represented a significant 2-fold increase above basal. We also characterized AMPK activation in muscles of α2i TG mice using DNP, a chemical mitochondrial uncoupler which reduces cellular ATP levels and activates AMPK. Similar to AICAR, DNP-induced α2 activation was abolished, and α1 activation was partially inhibited in α2i TG mice (Fig. 1C). Forced expression of an inactive α subunit can act as a dominant-negative by preventing the native α subunit from binding to the regulatory subunits (39Woods A. Azzout-Marniche D. Foretz M. Stei
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