Summary of study ST000431

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000334. The data can be accessed directly via it's Project DOI: 10.21228/M8D02C This work is supported by NIH grant, U2C- DK119886.

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Study IDST000431
Study TitleC2C12 stretch cessation models muscle atrophy and anaplerotic changes in metabolism
Study TypeGC-MS non-targeted metabolomic profiling
Study SummaryStudies of skeletal muscle disuse either in patients on bed rest or experimentally in animals(immobilization) have demonstrated that decreased protein synthesis is common, with transient parallel increases in protein degradation. Muscle disuse atrophy involves a process of transition from slow to fast myosin fiber types 6 . A shift toward glycolysis, decreased capacity for fat oxidation, and substrate accumulation in atrophied muscles have been reported as has accommodation of the liver with an increased gluconeogenic capacity. Recent studies have modeled skeletal muscle disuse by using cyclic stretch of differentiated myotubes (C2C12), which mimics the loading pattern of mature skeletal muscle, followed by cessation of stretch.We utilized this model to determine the metabolic changes using non-targeted metabolomics analysis of the media. We identified increases in amino acids resulting from protein degradation (largely sarcomere) that occurs with muscle atrophy that are involved in feeding the Kreb’s cycle through anaplerosis. Specifically, we identified increased alanine/proline metabolism (significantly elevated proline, alanine, glutamine, and asparagine) and increased -ketoglutaric acid, the proposed Kreb’s cycle intermediate being fed by the alanine/proline metabolic anaplerotic mechanism. Additionally, several unique pathways not clearly delineated in previous studies of muscle unloading were seen, including: 1) elevated ethanolamine and elevated keto-acids (e.g. 2-ketoleucine and 2-keovaline) represent intermediates in the Ehlrich amino acid degradation pathway, which feeds into a metabolic pathway supplying acetyl-CoA and 2-hydroxybutyrate (also significantly increased); and 2) elevated guanine, an intermediate of purine metabolism, was seen at 12 hours unloading. Given the interest in targeting different aspects of the ubiquitin proteasome system to inhibit protein degradation, this C2C12 system may allow the identification of direct and indirect alterations in metabolism due to anaplerosis or through other yet to be identified mechanisms using a non-targeted metabolomics approach.
Institute
University of North Carolina at Chapel Hill
DepartmentMcAllister Heart Institute
LaboratoryMutliple Centers
Last NameIlaiwy;WIllis
First NameAmro;Monte
Address111 Mason Farm road, Chapel Hill, North Carolina, 27599-7126, USA
Emailamroilaiwy@gmail.com, monte_willis@med.unc.edu
Phone919-3607599
Submit Date2016-05-28
Analysis Type DetailGC-MS
Release Date2016-09-23
Release Version1
Amro Ilaiwy Amro Ilaiwy
Monte WIllis Monte WIllis
https://dx.doi.org/10.21228/M8D02C
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000334
Project DOI:doi: 10.21228/M8D02C
Project Title:C2C12 stretch cessation models muscle atrophy and anaplerotic changes in metabolism
Project Type:GC-MS non targeted qualitative analysis
Project Summary:Studies of skeletal muscle disuse either in patients on bed rest or experimentally in animals(immobilization) have demonstrated that decreased protein synthesis is common, with transient parallel increases in protein degradation. Muscle disuse atrophy involves a process of transition from slow to fast myosin fiber types 6 . A shift toward glycolysis, decreased capacity for fat oxidation, and substrate accumulation in atrophied muscles have been reported as has accommodation of the liver with an increased gluconeogenic capacity. Recent studies have modeled skeletal muscle disuse by using cyclic stretch of differentiated myotubes (C2C12), which mimics the loading pattern of mature skeletal muscle, followed by cessation of stretch.We utilized this model to determine the metabolic changes using non-targeted metabolomics analysis of the media. We identified increases in amino acids resulting from protein degradation (largely sarcomere) that occurs with muscle atrophy that are involved in feeding the Kreb’s cycle through anaplerosis. Specifically, we identified increased alanine/proline metabolism (significantly elevated proline, alanine, glutamine, and asparagine) and increased -ketoglutaric acid, the proposed Kreb’s cycle intermediate being fed by the alanine/proline metabolic anaplerotic mechanism. Additionally, several unique pathways not clearly delineated in previous studies of muscle unloading were seen, including: 1) elevated ethanolamine and elevated keto-acids (e.g. 2-ketoleucine and 2-keovaline) represent intermediates in the Ehlrich amino acid degradation pathway, which feeds into a metabolic pathway supplying acetyl-CoA and 2-hydroxybutyrate (also significantly increased); and 2) elevated guanine, an intermediate of purine metabolism, was seen at 12 hours unloading. Given the interest in targeting different aspects of the ubiquitin proteasome system to inhibit protein degradation, this C2C12 system may allow the identification of direct and indirect alterations in metabolism due to anaplerosis or through other yet to be identified mechanisms using a non-targeted metabolomics approach.
Institute:University of North Carolina at Chapel Hill
Department:McAllister heart Institute, Department of Internal medicine
Laboratory:Multiple Centers
Last Name:Ilaiwy; Willis
First Name:Amro; Monte
Address:111 Mason Farm road, Chapel Hill, North Carolina, 27599-7126, USA
Email:amroilaiwy@gmail.com, monte_willis@med.unc.edu
Phone:210-596-0171
Funding Source:NIH, Fondation Leducq

Subject:

Subject ID:SU000452
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Species Group:Mammal

Factors:

Subject type: Animal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Group
SA021671p12 #212 hour reversal
SA021672p12 #112 hour reversal
SA021673p12 #312 hour reversal
SA021668p1 #31 hour reversal
SA021669p1 #11 hour reversal
SA021670p1 #21 hour reversal
SA021674p3 #33 hour reversal
SA021675p3 #13 hour reversal
SA021676p3 #23 hour reversal
SA021677p6 #36 hour reversal
SA021678p6 #26 hour reversal
SA021679p6 #16 hour reversal
SA021680p0 #16 hour stretch
SA021681p0 #26 hour stretch
SA021682p0 #36 hour stretch
SA021683p6 no stretch #1no stretch-serum free
SA021684p1 no stretch #1no stretch-serum free
SA021685p3 no stretch #1no stretch-serum free
SA021686p0 no stretch #1no stretch-serum free
SA021687serum 6hr no stretch #3serum-no stretch
SA021688serum 6hr no stretch #2serum-no stretch
SA021689serum 6hr no stretch #1serum-no stretch
Showing results 1 to 22 of 22

Collection:

Collection ID:CO000446
Collection Summary:Media was collected from all different 6 wells, and stored in -80 C
Sample Type:Muscle

Treatment:

Treatment ID:TR000466
Treatment Summary:N/A

Sample Preparation:

Sampleprep ID:SP000459
Sampleprep Summary:N/A

Combined analysis:

Analysis ID AN000681
Analysis type MS
Chromatography type GC
Chromatography system Agilent 6890N
Column Agilent DB5-MS (15m × 0.25mm, 0.25um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5975
Ion Mode POSITIVE
Units Peak values (Log transformed)

Chromatography:

Chromatography ID:CH000493
Chromatography Summary:GC/MS methods follow previous studies using a 6890 N GC connected to a 5975 Inert single quadrupole MS (Agilent Technologies, Santa Clara, CA) (Bonikos et al. 1975; Fiehn 2008; Kind et al. 2009). The two wall-coated, open-tubular GC columns connected in series are both from J&W/Agilent (part 122–5512), DB5-MS, 15 meters in length, 0.25 mm in diameter, with an 0.25-l m luminal film. Positive ions generated with conventional electron-ionization at 70 eV are scanned broadly from 600 to 50 m/z in the detector throughout the 45 min cycle time.
Instrument Name:Agilent 6890N
Column Name:Agilent DB5-MS (15m × 0.25mm, 0.25um)
Chromatography Type:GC

MS:

MS ID:MS000607
Analysis ID:AN000681
Instrument Name:Agilent 5975
Instrument Type:Single quadrupole
MS Type:EI
Ion Mode:POSITIVE
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