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      Gain-of-Function R225W Mutation in Human AMPKγ 3 Causing Increased Glycogen and Decreased Triglyceride in Skeletal Muscle

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          Abstract

          Background

          AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that is evolutionarily conserved from yeast to mammals and functions to maintain cellular and whole body energy homeostasis. Studies in experimental animals demonstrate that activation of AMPK in skeletal muscle protects against insulin resistance, type 2 diabetes and obesity. The regulatory γ 3 subunit of AMPK is expressed exclusively in skeletal muscle; however, its importance in controlling overall AMPK activity is unknown. While evidence is emerging that gamma subunit mutations interfere specifically with AMP activation, there remains some controversy regarding the impact of gamma subunit mutations [1][3]. Here we report the first gain-of-function mutation in the muscle-specific regulatory γ 3 subunit in humans.

          Methods and Findings

          We sequenced the exons and splice junctions of the AMPK γ 3 gene ( PRKAG3) in 761 obese and 759 lean individuals, identifying 87 sequence variants including a novel R225W mutation in subjects from two unrelated families. The γ 3 R225W mutation is homologous in location to the γ 2R302Q mutation in patients with Wolf-Parkinson-White syndrome and to the γ 3R225Q mutation originally linked to an increase in muscle glycogen content in purebred Hampshire Rendement Napole (RN -) pigs. We demonstrate in differentiated muscle satellite cells obtained from the vastus lateralis of R225W carriers that the mutation is associated with an approximate doubling of both basal and AMP-activated AMPK activities. Moreover, subjects bearing the R225W mutation exhibit a ∼90% increase of skeletal muscle glycogen content and a ∼30% decrease in intramuscular triglyceride (IMTG).

          Conclusions

          We have identified for the first time a mutation in the skeletal muscle-specific regulatory γ 3 subunit of AMPK in humans. The γ 3R225W mutation has significant functional effects as demonstrated by increases in basal and AMP-activated AMPK activities, increased muscle glycogen and decreased IMTG. Overall, these findings are consistent with an important regulatory role for AMPK γ 3 in human muscle energy metabolism.

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          Most cited references36

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          Human non-synonymous SNPs: server and survey.

          Human single nucleotide polymorphisms (SNPs) represent the most frequent type of human population DNA variation. One of the main goals of SNP research is to understand the genetics of the human phenotype variation and especially the genetic basis of human complex diseases. Non-synonymous coding SNPs (nsSNPs) comprise a group of SNPs that, together with SNPs in regulatory regions, are believed to have the highest impact on phenotype. Here we present a World Wide Web server to predict the effect of an nsSNP on protein structure and function. The prediction method enabled analysis of the publicly available SNP database HGVbase, which gave rise to a dataset of nsSNPs with predicted functionality. The dataset was further used to compare the effect of various structural and functional characteristics of amino acid substitutions responsible for phenotypic display of nsSNPs. We also studied the dependence of selective pressure on the structural and functional properties of proteins. We found that in our dataset the selection pressure against deleterious SNPs depends on the molecular function of the protein, although it is insensitive to several other protein features considered. The strongest selective pressure was detected for proteins involved in transcription regulation.
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            Accounting for human polymorphisms predicted to affect protein function.

            A major interest in human genetics is to determine whether a nonsynonymous single-base nucleotide polymorphism (nsSNP) in a gene affects its protein product and, consequently, impacts the carrier's health. We used the SIFT (Sorting Intolerant From Tolerant) program to predict that 25% of 3084 nsSNPs from dbSNP, a public SNP database, would affect protein function. Some of the nsSNPs predicted to affect function were variants known to be associated with disease. Others were artifacts of SNP discovery. Two reports have indicated that there are thousands of damaging nsSNPs in an individual's human genome; we find the number is likely to be much lower.
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              The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways.

              AMP-activated protein kinase (AMPK) is activated within the cell in response to multiple stresses that increase the intracellular AMP:ATP ratio. Here we show that incubation of muscle cells with the thiazolidinedione, rosiglitazone, leads to a dramatic increase in this ratio with the concomitant activation of AMPK. This finding raises the possibility that a number of the beneficial effects of the thiazolidinediones could be mediated via activation of AMPK. Furthermore, we show that in addition to the classical activation pathway, AMPK can also be stimulated without changing the levels of adenine nucleotides. In muscle cells, both hyperosmotic stress and the anti-diabetic agent, metformin, activate AMPK in the absence of any increase in the AMP:ATP ratio. However, although activation is no longer dependent on this ratio, it still involves increased phosphorylation of threonine 172 within the catalytic (alpha) subunit. AMPK stimulation in response to hyperosmotic stress does not appear to involve phosphatidylinositol 3-phosphate kinase, protein kinase C, mitogen-activated protein (MAP) kinase kinase, or p38 MAP kinase alpha or beta. Our results demonstrate that AMPK can be activated by at least two distinct signaling mechanisms and suggest that it may play a wider role in the cellular stress response than was previously understood.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS ONE
                plos
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2007
                19 September 2007
                : 2
                : 9
                Affiliations
                [1 ]Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
                [2 ]Division of Cardiology and Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Ottawa, Ontario, Canada
                [3 ]Ottawa Hospital Weight Management Clinic, Ottawa, Ontario, Canada
                [4 ]Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America
                [5 ]United States Department of Energy Joint Genome Institute, Walnut Creek, California, United States of America
                Lund University, Sweden
                Author notes
                * To whom correspondence should be addressed. E-mail: Maryellen.Harper@ 123456uottawa.ca

                Conceived and designed the experiments: LP RD RM MH. Performed the experiments: SC NK NA SC WS. Analyzed the data: LP RM MH SC NK NA SC WS. Contributed reagents/materials/analysis tools: LP RD RM MH. Wrote the paper: LP RD RM MH SC NK NA. Other: Designed the study: MH. Wrote the first draft of the paper: MH.

                Article
                07-PONE-RA-01766
                10.1371/journal.pone.0000903
                1964808
                17878938
                11942f84-100b-44dc-aaa2-43b9484212ec
                Costford et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 9
                Categories
                Research Article
                Diabetes and Endocrinology
                Diabetes and Endocrinology

                Uncategorized
                Uncategorized

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