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The 5′-AMP-activated Protein Kinase γ3 Isoform Has a Key Role in Carbohydrate and Lipid Metabolism in Glycolytic Skeletal Muscle

2004; Elsevier BV; Volume: 279; Issue: 37 Linguagem: Inglês

10.1074/jbc.m405533200

ISSN

1083-351X

Autores

Brian R. Barnes, Stefan Marklund, Tatiana L. Steiler, Mark Walter, Göran Hjälm, Valérie Amarger, Margit Mahlapuu, Ying Leng, Carina Johansson, Dana Galuska, Kerstin Lindgren, Magnus Åbrink, David Stapleton, Juleen R. Zierath, Leif Andersson,

Tópico(s)

Adipose Tissue and Metabolism

Resumo

5′-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific γ3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK γ3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse γ3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK γ3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK γ3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK γ3 isoform as a therapeutic target for prevention and treatment of insulin resistance. 5′-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific γ3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK γ3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse γ3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK γ3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK γ3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK γ3 isoform as a therapeutic target for prevention and treatment of insulin resistance. AMPK 1The abbreviations used are: AMPK, 5′-AMP-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside; RN, Rendement Napole; KHB, Krebs-Henseleit bicarbonate buffer; EDL, extensor digitorum longus; ACC, acetyl-CoA carboxylase.1The abbreviations used are: AMPK, 5′-AMP-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside; RN, Rendement Napole; KHB, Krebs-Henseleit bicarbonate buffer; EDL, extensor digitorum longus; ACC, acetyl-CoA carboxylase. is a heterotrimeric serine/threonine protein kinase composed of a catalytic α subunit and non-catalytic β and γ subunits (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1273) Google Scholar, 2Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). The mammalian genome contains seven AMPK genes encoding two α, two β, and three γ isoforms. AMPK signaling is elicited by cellular stresses that deplete ATP (and consequently elevate AMP) by either inhibiting ATP production (e.g. hypoxia) or accelerating ATP consumption (e.g. muscle contraction). AMPK is activated allosterically by AMP and through phosphorylation of Thr172 in the α subunit by an upstream AMPK kinase, the tumor-suppressor protein kinase LKB1 (3Hong S.P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (477) Google Scholar, 4Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; (September 24, jbiol.com/content/2/4/28)PubMed Google Scholar). AMPK is likely to be important for diverse functions in many cell types, but particular interest has been focused on elucidating the role of AMPK in the regulation of lipid and carbohydrate metabolism in skeletal muscle (5Hayashi T. Hirshman M.F. Kurth E.J. Winder W.W. Goodyear L.J. Diabetes. 1998; 47: 1369-1373Crossref PubMed Scopus (707) Google Scholar, 6Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1671) Google Scholar, 7Zong H. Ren J.M. Young L.H. Pypaert M. Mu J. Birnbaum M.J. Shulman G.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15983-15987Crossref PubMed Scopus (830) Google Scholar, 8Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3440) Google Scholar, 9Koistinen H.A. Galuska D. Chibalin A.V. Yang J. Zierath J.R. Holman G.D. Wallberg-Henriksson H. Diabetes. 2003; 52: 1066-1072Crossref PubMed Scopus (203) Google Scholar, 10Bergeron R. Russell R.R.I. Young L.H. Ren J.M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar). AMPK activity has been correlated with an increase in glucose uptake and fatty acid oxidation and an inhibition of glycogen synthase activity and fatty acid synthesis. Exercise, as well as skeletal muscle contractions in vitro, leads to AMPK activation. Pharmacological activation of AMPK also can be achieved using 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Once taken up by the cell, AICAR is phosphorylated to 5-aminoimidazole-4-carboxamide riboside monophosphate (ZMP) and mimics effects of AMP on AMPK (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1273) Google Scholar, 2Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). AMPK function is closely related to glycogen storage. AMPK phosphorylates glycogen synthase in vitro (11Carling D. Hardie D.G. Biochim. Biophys. Acta. 1989; 1012: 81-86Crossref PubMed Scopus (255) Google Scholar) and co-immunoprecipitates with glycogen synthase and glycogen phosphorylase from skeletal muscle (12Chen Z. Heierhorst J. Mann R.J. Mitchelhill K.I. Michell B.J. Witters L.A. Lynch G.S. Kemp B.E. Stapleton D. FEBS Lett. 1999; 460: 343-348Crossref PubMed Scopus (114) Google Scholar). Mutations of the γ3or γ2 subunit, respectively, affect glycogen storage in pigs (13Milan D. Jeon J.T. Looft C. Amarger V. Thelander M. Robic A. Rogel-Gaillard C. Paul S. Iannuccelli N. Rask L. Ronne H. Lundström K. Reinsch N. Gellin J. Kalm E. Le Roy P. Chardon P. Andersson L. Science. 2000; 288: 1248-1251Crossref PubMed Scopus (610) Google Scholar, 14Ciobanu D. Bastiaansen J. Malek M. Helm J. Woollard J. Plastow G. Rothschild M. Genetics. 2001; 159: 1151-1162Crossref PubMed Google Scholar) or glycogen storage associated with cardiac abnormalities in humans (15Arad M. Benson D.W. Perez-Atayde A.R. McKenna W.J. Sparks E.A. Kanter R.J. McGarry K. Seidman J.G. Seidman C.E. J. Clin. Investig. 2002; 109: 357-362Crossref PubMed Scopus (460) Google Scholar). The recent identification of a glycogen-binding domain in the AMPK β1 subunit provides a molecular relationship between AMPK and glycogen (16Hudson E. Pan D. James J. Lucocq J. Hawley S. Green K. Baba O. Terashima T. Hardie D.G. Curr. Biol. 2003; 13: 861-866Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 17Polekhina G. Gupta A. Michell B.J. van Denderen B. Murthy S. Feil S.C. Jennings I.G. Campbell D.J. Witters L.A. Parker M.W. Kemp B.E. Stapleton D. Curr. Biol. 2003; 13: 867-871Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). The formation of heterotrimers appears to be rather promiscuous, and the different subunits (α1, α2, β1, β2, γ1, γ2, and γ3) can form a maximum of 12 different AMPK heterotrimers. The functional diversification of the different isoforms is largely unknown. The dominant Rendement Napole (RN) phenotype identified in Hampshire pigs is associated with a single missense mutation (R225Q) in PRKAG3, encoding the muscle-specific AMPK γ3 isoform (13Milan D. Jeon J.T. Looft C. Amarger V. Thelander M. Robic A. Rogel-Gaillard C. Paul S. Iannuccelli N. Rask L. Ronne H. Lundström K. Reinsch N. Gellin J. Kalm E. Le Roy P. Chardon P. Andersson L. Science. 2000; 288: 1248-1251Crossref PubMed Scopus (610) Google Scholar). RN pigs have a ∼70% increase in glycogen content in glycolytic skeletal muscle, whereas liver and heart glycogen content is unchanged (18Estrade M. Vignon X. Rock E. Monin G. Comp. Biochem. Physiol. 1993; 104B: 321-326Google Scholar, 19.Monin, G., Brard, C., Vernin, P. & Naveau, J. (1992) in 38th International Congress on Meat Science and Technology, Vol. 3, pp. 391–394, Clermont-Ferrand, FranceGoogle Scholar). The mutation has a large impact on meat characteristics and leads to a low pH because of the anaerobic glycogen degradation occurring postmortem. A second mutation (V224I) identified in pigs at the neighboring amino acid residue is associated with an opposite effect, low glycogen and high pH, compared with the RN allele (14Ciobanu D. Bastiaansen J. Malek M. Helm J. Woollard J. Plastow G. Rothschild M. Genetics. 2001; 159: 1151-1162Crossref PubMed Google Scholar). We have found that γ3 is the predominant AMPK γ isoform in glycolytic (white, fast-twitch type II) muscle, whereas it is expressed at very low levels in oxidative (red, slow-twitch type I) muscle and is undetectable in brain, liver, or white adipose tissue (20Mahlapuu M. Johansson C. Lindgren K. Hjälm G. Barnes B. Krook A. Zierath J. Andersson L. Marklund S. Am. J. Physiol. Endocrinol. Metab. 2004; 286: E194-E200Crossref PubMed Scopus (150) Google Scholar). Furthermore, γ3 primarily forms heterotrimers with α2 and β2 isoforms in glycolytic skeletal muscle. Here we report the characterization of the metabolic consequences of genetic modification of AMPK γ3 expression in skeletal muscle. Animal Care—Mice were maintained in a temperature- and light-controlled environment and were cared for in accordance with regulations for the protection of laboratory animals. The regional animal ethical committee approved all experimental procedures. Animals had free access to water and standard rodent chow. In some experiments, female mice were placed on either a standard chow or a high fat diet (21Zierath J.R. Houseknecht K. Gnudi L. Kahn B.B. Diabetes. 1997; 46: 215-223Crossref PubMed Google Scholar) from 4 to 16 weeks of age. Generation of Transgenic Mice—The complete coding sequence of mouse Prkag3 was amplified by reverse transcriptase-PCR using skeletal muscle mRNA (Clontech). The forward (5′-CACCATGGAGCCCGAGCTGGAGCA) and reverse (5′-GTCTCAGGCGCTGAGGGCATC) primers included the translation start and stop codons (in bold), respectively. The forward primer also included a Kozak element (CACC, underlined above) (22Kozak M. Mol. Cell. Biol. 1989; 9: 5073-5080Crossref PubMed Scopus (374) Google Scholar) in front of the start codon to facilitate initiation of translation. The reverse transcriptase-PCR product (∼1.5 kb) was ligated into the pCRII TA TOPO cloning vector (Invitrogen). A clone with this consensus sequence (100% identity to Prkag3) was used for the transgene constructs. The R225Q mutation (13Milan D. Jeon J.T. Looft C. Amarger V. Thelander M. Robic A. Rogel-Gaillard C. Paul S. Iannuccelli N. Rask L. Ronne H. Lundström K. Reinsch N. Gellin J. Kalm E. Le Roy P. Chardon P. Andersson L. Science. 2000; 288: 1248-1251Crossref PubMed Scopus (610) Google Scholar) was introduced by in vitro mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene) changing codon 225 from arginine to glutamine. EcoRI fragments containing the wild type or the mutant (225Q) form were ligated into the pMLC vector (23Gros L. Riu E. Montoliu L. Ontiveros M. Lebrigand L. Bosch F. Hum. Gene Ther. 1999; 10: 1207-1217Crossref PubMed Scopus (36) Google Scholar) along with flanking sequences for the myosin light chain 1 (MLC1) promoter and enhancer and the SV40 3′-untranslated region (Fig. 1a). The constructs were cut from the plasmid and microinjected into mouse oocytes (CBA × C57Bl/6J). The founder mice were tested for transgenesis by PCR analysis using a forward primer in Prkag3 exon 12 (5′-GCTGCCCAGCAAACCTACAAC) and a reverse primer in SV40 3′-untranslated region. This amplicon spans the SV40 3′-untranslated region intron (66 bp) and thereby allowed for confirmation of the transgene both in genomic DNA (amplicon size = 453 bp) and in cDNA (amplicon size = 387 bp). Genomic DNA prepared from mouse tails was used for PCR. Generation of Knock-out Mice—Prkag3 knock-out mice were generated through traditional gene-targeting techniques. Briefly, exons 1–4 and exons 11–13 of Prkag3 were cloned into the pKOV923 selection plasmid (Stratagene) with a neomycin resistance (neor) gene inserted. The predicted result was a Prkag3 transcript with exons 1–4 joined with exons 11–13, including a frameshift after residue 211 and a premature stop codon at residue 235, skipping most of the 489 amino acids encoded by the wild type transcript (Fig. 1b). The construct was linearized using NotI and used for electroporation of embryonic stem cells. Knock-out recombinant embryonic stem cells were injected into blastocysts. Screening for knock-out recombinant embryonic stem cells was performed using Southern analysis, with SpeI digestions and a 1034-bp cDNA probe, representing the mitochondrial vitamin D (3Hong S.P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (477) Google Scholar) 25-hydroxylase (Cyp27) gene (Fig. 1b). The probe was amplified by reverse transcriptase-PCR from mouse skeletal muscle mRNA. Long range PCR (MasterAmp high fidelity long PCR kit, Epicenter Technologies) was used to screen for knock-out recombinant embryonic stem cells and identification of heterozygous (Prkag3+/–) and homozygous (Prkag3–/–) carriers of the knock-out recombinant allele, respectively. Founder mice were back-crossed to C57Bl/6J mice for three generations. In all experiments, knock-out homozygote mice were compared with homozygous wild type littermates. Relative Quantification of mRNA—Quantification of mRNA representing different isoforms of AMPK γ subunits from adult mouse tissues was performed using reverse transcription and real time PCR. Relative quantities of mRNA were calculated for duplicate tissue samples from 1–2 mice and normalized for Actb (β-actin). Cell Culture and Transfections—Briefly, cDNA encoding the γ3 subunit of AMPK was inserted into cloning vector pDONR201 included in the Gateway cloning system (Invitrogen) per the manufacturer's instructions. Site-directed mutagenesis was used to create γ3 V224I and γ3 R225Q cDNA constructs, which were cloned into Gateway cloning system pDEST26 (Invitrogen) for subsequent expression in mammalian cell culture. Cultured COS7 cells were transiently transfected with cDNA encoding α2, β2, and γ3 wild type, γ3 V224I, or γ3 R225Q. Post-transfection, cells were lysed, insoluble material was removed, and lysates were exposed to protein G-Sepharose-bound monoclonal antibody. After incubation, α2 containing immune complexes were harvested, washed, and halved for subsequent activity assays and immunoblotting. AMPK Activity Assay—AMPK activity was measured by phosphate incorporation of the ADR1 (222–234)Pro229 peptide substrate, LKKLTLRPSFSAQ (24Michell B.J. Stapleton D. Michelhill K.I. House C.M. Katsis F. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 28445-28450Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). AMPK immunocomplexes were assayed for 10 min, reactions were spotted on phosphocellulose paper (P81), and radioactivity was assessed by liquid scintillation analyzer. AMPK activities were calculated as pmol of phosphate incorporated into the ADR1 peptide/min in the presence of equal amounts of the heterotrimer. Western Blot Analysis—Quantitative analysis of the expression of different AMPK subunits was performed as described previously (20Mahlapuu M. Johansson C. Lindgren K. Hjälm G. Barnes B. Krook A. Zierath J. Andersson L. Marklund S. Am. J. Physiol. Endocrinol. Metab. 2004; 286: E194-E200Crossref PubMed Scopus (150) Google Scholar). Skeletal muscle protein lysate was separated by SDS-PAGE, transferred to Immobilon-P membranes (Millipore), and probed with primary AMPK isoform- or phospho-specific antibodies and secondary horseradish peroxidase-conjugated antibodies. Glycogen and Triglyceride Analyses—Mice were studied under fed or fasted conditions or after swim exercise, as described previously (25Ryder J.W. Kawano Y. Galuska D. Fahlman R. Wallberg-Henriksson H. Charron M.J. Zierath J.R. FASEB J. 1999; 13: 2246-2256Crossref PubMed Scopus (60) Google Scholar). Fasted mice swam for four 30-min intervals separated by 5-min rest periods. After the last swim interval, mice were studied immediately or dried and returned to cages for 2.5 h (recovery). At the onset of the recovery period, mice received an intraperitoneal glucose injection (0.5 mg/g of body mass) and were subsequently given free access to chow and water. Gastrocnemius muscles were removed from anesthetized mice (Avertin; 2,2,2-tribromoethanol 99+% and tertiary amyl alcohol, 15 μl/g of body mass), cleaned of fat and blood, and quickly frozen in liquid nitrogen. Glycogen content was determined fluorometrically on HCl extracts as described previously (26Wallberg-Henriksson H. Zetan N. Henriksson J. J. Biol. Chem. 1987; 262: 7665-7671Abstract Full Text PDF PubMed Google Scholar). Triglyceride content was determined with a triglycerides/glycerol blanked kit (Roche Applied Science) using Seronorm™ lipid (SERO) as a standard. Glucose Tolerance Test—Glucose (2 g/kg of body mass) was administered to fasted mice by intraperitoneal injection. Blood samples were obtained via the tail vein prior to and 15, 30, 60, and 120 min following glucose injection for measurement of glucose concentration (One Touch Basic glucose meter; Lifescan). Skeletal Muscle Incubation Procedure—Incubation medium was prepared from a stock solution of Krebs-Henseleit bicarbonate buffer (KHB) supplemented with 5 mmol/liter HEPES and 0.1% bovine serum albumin (RIA grade) and continuously gassed with 95% O2, 5% CO2. Mice were anesthetized, and extensor digitorum longus (EDL) muscles were isolated and preincubated at 30 °C for 30 (glucose uptake) or 60 (for AMPK and acetyl-CoA carboxylase (ACC) phosphorylation) min in KHB containing 5 mmol/liter glucose and 15 mmol/liter mannitol in the absence or presence of insulin (12 nmol/liter) or AICAR (2 mmol/liter). Some muscles were subjected to 10 min of in vitro electrical stimulation, as described previously (27Ryder J.W. Fahlman R. Wallberg-Henriksson H. Alessi D.R. Krook A. Zierath J.R. J. Biol. Chem. 2000; 275: 1457-1462Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), before (glucose uptake) or during the final 10 min (for AMPK and ACC phosphorylation) of the incubation. Metabolic Assays—2-Deoxyglucose uptake was assessed in EDL muscles as described previously (26Wallberg-Henriksson H. Zetan N. Henriksson J. J. Biol. Chem. 1987; 262: 7665-7671Abstract Full Text PDF PubMed Google Scholar). Results are expressed as μmol/ml of intracellular water/h (26Wallberg-Henriksson H. Zetan N. Henriksson J. J. Biol. Chem. 1987; 262: 7665-7671Abstract Full Text PDF PubMed Google Scholar). Oleate oxidation was assessed in EDL muscles as described by Young et al. (28Young D.A. Ho R.S. Bell P.A. Cohen D.K. McIntosh R.H. Nadelson J. Foley J.E. Diabetes. 1990; 39: 1408-1413Crossref PubMed Google Scholar) with minor modifications. Muscles were preincubated in the presence of insulin (60 nmol/liter) without or with AICAR (2 mmol/liter) for 20 min in KHB containing 5 mmol/liter HEPES, 3.5% fatty acid-free bovine serum albumin, and 10 mmol/liter glucose. Thereafter, muscles were incubated in 1 ml of identical medium containing 0.3 mmol/liter [1-14C]oleate (0.4 μCi/ml) for 60 min. The medium was acidified by 0.5 ml of 15% Δ′-pyrroline-5-carboxylic acid, and liberated CO2 was collected in center wells containing 0.2 ml of Protosol (PerkinElmer Life Sciences) for 60 min. Center wells were removed for scintillation counting. Results were expressed as nmol of oxidized oleate/g of wet mass/h. Statistical Analyses—Differences between two groups were determined by an analysis of variance with multiple comparisons. Differences between more than two groups were determined by one-way analysis of variation followed by Fisher's least significant difference post hoc analysis. Significance was accepted at p < 0.05. Three novel mouse models were used to genetically dissect the functional role of the AMPK γ3 isoform in skeletal muscle. AMPK γ3-transgenic (Tg-Prkag3wt and Tg-Prkag3225Q) and Prkag3–/– mice had normal growth rates (data not shown). The expression of Prkag3 wild type (Tg-Prkag3wt) or an R225Q mutant form (Tg-Prkag3225Q) transgene was restricted to skeletal muscles containing a high proportion of glycolytic fibers, consistent with the expression profile of the endogenous Prkag3 transcript (Table I). We found a marked overexpression of the wild type transgene in EDL, gastrocnemius, and quadriceps muscle (∼16.5-, 6.6-, and 1.7-fold, respectively), but no or only a moderate overexpression of the mutant transgene (∼0.7-, 1.0-, and 1.8-fold, respectively). Positional effects or number of integrated copies most likely explain the difference in expression level between the two transgenic models. Levels of endogenous Prkag3 transcript in Tg-Prkag3225Q mice tended to be decreased.Table IRelative mRNA expression levels from endogenous Prkag1 and Prkag3 and transgenic Prkag3 in mice as measured by real time PCRTissueMouse linePrkag1 endogenousPrkag3EndogenousTransgenicLiverWTaWT, wild type.2.4 ± 0.90.0AMPK γ3wt1.70.00.0AMPK γ3225Q2.7 ± 1.30.00.0HeartWT22.9 ± 9.31.2 ± 0.4AMPK γ3wt12.50.10.0AMPK γ3225Q12.9 ± 10.90.3 ± 0.10.0Extensor digitorum longusbMuscle containing primarily glycolytic muscle fibers.WT19.9 ± 14.924.2 ± 3.0AMPK γ3wt23.3 ± 3.518.3 ± 4.21646 ± 492AMPK γ3225Q18.714.173.2GastrocnemiusbMuscle containing primarily glycolytic muscle fibers.WT30.9 ± 0.8100 ± 25.3AMPK γ3wt26.3 ± 8.562.1 ± 13.2659 ± 222AMPK γ3225Q19.4 ± 8.011.1 ± 2.499.5 ± 28.3AMPK γ3-/-9.53.8QuadricepsbMuscle containing primarily glycolytic muscle fibers.WT45.7 ± 3.141.3 ± 3.2AMPK γ3wt27.526.8167.2AMPK γ3225Q35.012.9182.8SoleuscMuscle containing primarily oxidative muscle fibers.WT33.4 ± 8.73.0 ± 0.8AMPK γ3wt30.8 ± 0.62.6 ± 0.811.1 ± 6.9AMPK γ3225Q28.7 ± 5.22.7 ± 0.56.5 ± 4.5DiaphragmcMuscle containing primarily oxidative muscle fibers.WT7.0 ± 4.01.3 ± 0.6AMPK γ3wt12.82.55.0AMPK γ3225Q10.3 ± 8.01.3 ± 0.80.9 ± 0.9BrainWT1.5 ± 0.00.0AMPK γ3wt2.2 ± 0.30.00.0AMPK γ3225Q1.70.00.0White adipose tissueWT1.3 ± 0.40.0AMPK γ3wt0.90.00.0AMPK γ3225Q1.20.00.0a WT, wild type.b Muscle containing primarily glycolytic muscle fibers.c Muscle containing primarily oxidative muscle fibers. Open table in a new tab The amount of expressed γ3 protein, as well as α, β, or the other γ subunits, was unchanged in Tg-Prkag3225Q mice (Fig. 2). Thus, AMPK expression in Tg-Prkag3225Q mice resembles the expression pattern in wild type mice, both in regard to tissue distribution and protein expression. The mutant form (225Q) presumably replaced endogenous γ3, based on the relative mRNA expression. However, endogenous and exogenous forms, as assessed by Western blot, were indistinguishable because they differed by a single amino acid substitution. An absolute quantification of the relative expression of the mutant and wild type protein is not crucial for the interpretation of our results because the R225Q mutation is fully dominant. Moreover, there is no significant difference in glycogen content between pigs expressing 50 or 100% of the mutant form. 2L. Andersson, unpublished observation. Overexpression of the wild type transgene led to an increase in the amount of γ3 protein and a concomitant increase in α1, α2, and β2 subunits (Fig. 2). Thus, the total amount of AMPK heterotrimers in glycolytic muscles was increased in Tg-Prkag3wt mice. Southern and Western blot analysis confirmed the successful disruption of Prkag3 and concomitant complete absence of γ3 expression in skeletal muscle in Prkag3 knock-out (Prkag3–/–) mice (Fig. 1, c and d). The homozygous knock-out animals were fully viable, and a standard pathological examination revealed no obvious phenotypic consequences of the γ3 disruption. Real time PCR analysis of mRNA from skeletal muscle expectedly revealed a low abundance of Prkag3 transcripts, as the PCR primers were designed against a part of the 3′-region that was not deleted by the gene-targeting event (Fig. 1b and Table I). The low abundance of this aberrant transcript likely reflects degradation by the nonsense-mediated mRNA decay pathway (29Culbertson M.R. Leeds P.F. Curr. Opin. Genet. Dev. 2003; 13: 207-214Crossref PubMed Scopus (111) Google Scholar). Western blot analysis did not reveal any compensatory increase in γ1 and γ2 isoform expression in skeletal muscle (Fig. 2), indicating that these isoforms do not compete with γ3 for the same pool of α-β chains or do not form AMPK heterotrimers in the same cell or cellular compartment. Glycogen content in the glycolytic portion of the gastrocnemius muscle was 2-fold higher in Tg-Prkag3225Q mice compared with wild type mice under both fed and fasted conditions, whereas glycogen content was unaltered in Tg-Prkag3wt or Prkag3–/– mice (Fig. 3a). The results provide definitive evidence that R225Q is the causative mutation for the RN phenotype in pigs (13Milan D. Jeon J.T. Looft C. Amarger V. Thelander M. Robic A. Rogel-Gaillard C. Paul S. Iannuccelli N. Rask L. Ronne H. Lundström K. Reinsch N. Gellin J. Kalm E. Le Roy P. Chardon P. Andersson L. Science. 2000; 288: 1248-1251Crossref PubMed Scopus (610) Google Scholar) because the phenotype is replicated in mice by introducing this single missense mutation. Furthermore, this mutation alters the biochemical regulation of AMPK, as the increase in AMPK expression in the Tg-Prkag3wt mice failed to cause a glycogen phenotype. Glycogen content after swimming exercise was similar between wild type and all genetically modified mice (Fig. 3a). Thus, the R225Q mutation does not impair glycogen utilization during exercise. Because postexercise glycogen levels were appropriately depleted in Prkag3–/– mice, our data also suggests that AMPK γ3 is not required for glycogen degradation. Glycogen content 2.5 h after exercise was significantly higher in Tg-Prkag3225Q mice compared with wild type mice (Fig. 3a). A similar tendency (N. S) for increased glycogen content after exercise was also noted in Tg-Prkag3wt mice. In contrast, glycogen content was significantly lower in Prkag3–/–versus wild type mice 2.5 h after exercise, demonstrating that γ3 is important for glycogen resynthesis. Fasted insulin and glucose levels (data not shown) and glucose tolerance (Fig. 3b) were normal in transgenic and Prkag3–/– mice. Thus, despite a very distinct phenotype for skeletal muscle glycogen content, blood glucose homeostasis was normal in Tg-Prkag3225Q mice, consistent with the phenotype noted in pigs carrying the R225Q mutation. 3B. Essén-Gustavsson, M. Jensen-Waern, R. Jonasson, and L. Andersson, unpublished observation. AMPK phosphorylation under basal conditions or after activation with AICAR or contraction was similar between genotypes (Fig. 3c). Phosphorylation of the AMPK downstream target ACC was elevated under basal conditions and after AICAR stimulation in TgPrkag3225Q mice (Fig. 3d). This was unexpected because our transfection experiments (see below) revealed that the R225Q mutant γ3 isoform is AMP-independent and thus would be predicted to be AICAR-insensitive. However, the elevated ACC phosphorylation may be an indirect effect of the R225Q mutant γ3 isoform caused by an altered metabolic state of the cell. In fact, the γ3 isoform may not mediate ACC phosphorylation, consistent with unaltered ACC phosphorylation in Prkag3–/– mice (Fig. 3d). Glucose transport in isolated EDL muscle from fasted mice was determined in response to insulin, in response to AICAR, or after electrically stimulated contractions (Fig. 3e). Basal glucose transport was similar between genotypes. Insulin-stimulated glucose transport was normal in Tg-Prkag3wt and Prkag3–/– mice but was significantly reduced in Tg-Prkag3225Q mice. The reduction in insulin-stimulated glucose transport was not observed in fed mice (data not shown). AICAR-induced glucose transport was normal in Tg-Prkag3wt, but significantly reduced (∼50%) in Tg-Prkag3225Q, compared with wild type fasted (Fig. 3e) and fed (data not shown) mice. Thus, the mutation in γ3 may occur at a site that is directly involved with the interaction with both AMP and AICAR, rendering a mutant form that is partially resistant to AICAR. This interpretation is supported by evidence that this region of the γ subunit directly binds AMP and that the presence of this mutation at the corresponding site in the γ1 (R70Q) or γ2 (R302Q) subunit impairs AMP and ATP binding (30Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Investig. 2004; 113: 274-284Crossref PubMed Scopus (605) Google Scholar, 31Adams J. Chen Z.P. Van Denderen B.J. Morton C.J. Parker M.W. Witters L.A. Stapleton D. Kemp B.E. Protein Sci. 2004; 13: 155-165Crossref PubMed Scopus (130) Google Scholar). However, we cannot exclude a partial inhibition because of excessive glycogen content (32Wojtaszewski J.F. Jorgensen S.B. Hellsten Y. Hardie D.G. Richter E.A. Diabetes. 2002; 51: 284-292Crossref PubMed Scopus (232) Google Scholar, 33Wojtaszewski J.F. MacDonald C. Nielsen J.N. Hellsten Y. Hardie D.G. Kemp B.E. Kiens B. Richter E.A. Am. J. Physiol. Endocrinol. Metab. 2002; 284: E813-E822Crossref PubMed Scopus (273) Google Scholar). Interestingly, the AICAR effect on glucose uptake in EDL muscle was completely abolished in Prkag3–/– mice (Fig. 3e). Thus, AMPK complexes containing the γ3 subunit are required for AICAR-induced glucose transport in skeletal muscle, and other γ isoforms fail to compensate for the loss of γ3 function. This result is consistent with the reduced glycogen resynthesis in vivo after exercise in Prkag3–/– mice (Fig. 3a). In contrast to the results for AICAR, in vitro contraction of isolated EDL muscle led to a similar increase in glucose uptake in all genotypes in fasted (Fig. 3e) or fed mice (data not shown). Similarly, AICAR- but not contraction-induced glucose uptake was abolished in AMPK α2, but not in α1 knock-out mice (34Jorgensen S.B. Viollet B. Andreeli 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 (467) Google Scholar). Because the γ3 subunit primarily forms heterotrimers with α2 (20Mahlapuu M. Johansson C. Lindgren K. Hjälm G. Barnes B. Krook A. Zierath J. Andersson L. Marklund S. Am. J. Physiol. Endocrinol. Metab. 2004; 286: E194-E200Crossref PubMed Scopus (150) Google Scholar), disruption of either α2 or γ3 should confer a similar glucose transport defect in skeletal muscle. AICAR-induced glucose uptake was also abolished in kinase-dead AMPK α2-transgenic mice (35Mu J. Brozinick J.T.J. Valladares O. Bucan M. Birnbaum M.J. Mol. Cell. 2001; 7: 1085-1094Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). In contrast to the α2 AMPK knock-out and the Prkag3–/– mice, contraction-mediated glucose transport was significantly blunted (30%) in kinase-dead AMPK α2-transgenic mice, possibly because of contraction-induced hypoxia, as these mice have an impaired hypoxia response. Collectively, these results challenge the hypothesis that contraction increases glucose transport through an AMPK-mediated mechanism. In contrast, activation of AMPK is directly linked to AICAR-stimulated glucose transport. Although AICAR and contraction both increase AMPK activity, the AMPK response to in vitro contraction may be inconsequential for activation of glucose transport. Although γ3-containing AMPK complexes are required for AICAR-mediated glucose uptake, they appear to be dispensable for AICAR-mediated fatty acid oxidation in chow-fed mice. AICAR-mediated fatty acid oxidation was similar between genotypes (Fig. 3f), consistent with the observed normal level of ACC phosphorylation in the Prkag3–/– mice (Fig. 3d). AMPK has been identified as a molecular target for pharmacological intervention to treat insulin resistance and type II diabetes mellitus. However, genetic validation of this target is lacking. We challenged wild type, Tg-Prkag3225Q, and Prkag3–/– mice with a high fat diet for 12 weeks and evaluated metabolic responses. Muscle glycogen content was unaffected by the high fat diet (data not shown). However, triglyceride content was increased (Fig. 4b), and insulin action on glucose transport was impaired in wild type and Prkag3–/– mice (Fig. 4, c and d). In contrast, Tg-Prkag3225Q mice were protected against triglyceride accumulation (Fig. 4b) and insulin resistance (Fig. 4c), presumably because of increased fat oxidation (Fig. 4a) in skeletal muscle. This phenotype closely resembles the original phenotype described for mutant pigs (13Milan D. Jeon J.T. Looft C. Amarger V. Thelander M. Robic A. Rogel-Gaillard C. Paul S. Iannuccelli N. Rask L. Ronne H. Lundström K. Reinsch N. Gellin J. Kalm E. Le Roy P. Chardon P. Andersson L. Science. 2000; 288: 1248-1251Crossref PubMed Scopus (610) Google Scholar). The high frequency of the RN– mutation (PRKAG3225Q) in Hampshire pigs was likely caused by the strong selection for lean meat content in commercial pig populations, as pigs carrying this mutation are leaner (more muscle, less fat) than wild type pigs. Mutant pigs also have a higher oxidative capacity, as measured by an increase in activity of citrate synthase and β-hydroxyacyl-coenzyme A dehydrogenase (36Estrade M. Ayoub S. Talmant A. Monin G. Comp. Biochem. Physiol. 1994; 108: 295-301Google Scholar, 37Lebret B. Le Roy P. Monin G. Lefaucheur L. Caritez J.C. Talmant A. Elsen J. Sellier P. J. Anim. Sci. 1999; 77: 1482-1489Crossref PubMed Scopus (89) Google Scholar). The relationship between the R225Q mutation and increased oxidative capacity in both mutant pigs and transgenic mice prompted us to investigate whether this was associated with an altered muscle fiber type composition. Real time PCR analysis of Atp1b1 and Atpb2 (20Mahlapuu M. Johansson C. Lindgren K. Hjälm G. Barnes B. Krook A. Zierath J. Andersson L. Marklund S. Am. J. Physiol. Endocrinol. Metab. 2004; 286: E194-E200Crossref PubMed Scopus (150) Google Scholar), markers of glycolytic and oxidative skeletal muscle fibers (38Hundal H.S. Marette A. Ramlal T. Liu Z. Klip A. FEBS Lett. 1993; 328: 253-258Crossref PubMed Scopus (68) Google Scholar), revealed a similar ratio of Atp1b1 and Atpb2 expression in gastrocnemius and EDL muscle from Tg-Prkag3225Q and Prkag3–/– mice (data not shown). Furthermore, myoglobin protein expression in Tg-Prkag3225Q and Prkag3–/– did not differ from wild type mice (data not shown). Thus, R225Q alters skeletal muscle oxidative capacity without altering fiber type composition. AMPK activity and phosphorylation of Thr172 were determined in COS cells transfected with AMPK trimers containing α2, β2, and either wild type γ3 or mutant γ3 (R225Q or V224I). AMPK activity and phosphorylation on Thr172 in the absence of AMP were elevated in cells transfected with α2-β2-γ3 R225Q and unchanged in cells transfected with α2-β2-γ3 V224I. Both mutations resulted in diminished AMP dependence on AMPK (Fig. 5). The ranking of basal AMPK activity in the three genotypes is consistent with the in vivo effects of the corresponding pig mutations, as the R225Q and V224I mutants are associated with increased and decreased muscle glycogen content, respectively (13Milan D. Jeon J.T. Looft C. Amarger V. Thelander M. Robic A. Rogel-Gaillard C. Paul S. Iannuccelli N. Rask L. Ronne H. Lundström K. Reinsch N. Gellin J. Kalm E. Le Roy P. Chardon P. Andersson L. Science. 2000; 288: 1248-1251Crossref PubMed Scopus (610) Google Scholar, 14Ciobanu D. Bastiaansen J. Malek M. Helm J. Woollard J. Plastow G. Rothschild M. Genetics. 2001; 159: 1151-1162Crossref PubMed Google Scholar). We provide definitive evidence that PRKAG3 R225Q is the causative mutation for the elevated glycogen content in skeletal muscle from RN pigs. Tg-Prkag3225Q mice have elevated glycogen levels and have increased glycogen resynthesis after exercise. Our in vitro studies reveal that AMPK complexes containing the R225Q γ3 subunit have a higher basal AMPK activity and lack AMP dependence. Thus, R225Q can be considered a loss-of-function mutation that abolishes allosteric regulation by AMP/ATP, causing increased basal AMPK activity.

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