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Characterization of human myotubes from type 2 diabetic and non-diabetic subjects using complementary quantitative mass spectrometric methods.

Thingholm, Tine LU ; Bak, Steffen; Beck-Nielsen, Henning; Jensen, Ole N and Gaster, Michael (2011) In Molecular & Cellular Proteomics 10(9).
Abstract
Skeletal muscle is a key tissue site of insulin resistance in type 2 diabetes. Human myotubes are primary skeletal muscle cells displaying both morphological and biochemical characteristics of mature skeletal muscle and the diabetic phenotype is conserved in myotubes derived from subjects with type 2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery strategy using iTRAQ and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring (SRM) to identify,... (More)
Skeletal muscle is a key tissue site of insulin resistance in type 2 diabetes. Human myotubes are primary skeletal muscle cells displaying both morphological and biochemical characteristics of mature skeletal muscle and the diabetic phenotype is conserved in myotubes derived from subjects with type 2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery strategy using iTRAQ and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring (SRM) to identify, quantify and validate changes in protein abundance between human myotubes obtained from non-diabetic lean, non-diabetic obese and type 2 diabetic subjects, respectively. A total of 2,832 unique proteins were identified and quantified using the iTRAQ strategy. Proteins from all major pathways known to be important in T2D were well-characterized in this study. This included pathways like the TCA cycle, lipid oxidation, oxidative phosphorylation, the glycolytic pathway and glycogen metabolism from which all but two enzymes were found in the present study. None of these enzymes were found to be regulated at the level of protein expression or degradation supporting the hypothesis that these pathways are regulated at the level of post-translational modification. Twelve proteins were, however, differentially expressed between the three different groups. Thirty-six proteins were chosen for further analysis and validation using SRM based on the regulation identified in the iTRAQ discovery study. The abundance of adenosine deaminase was considerably down-regulated in diabetic myotubes and as the protein binds DPP-IV, we speculate whether the reduced binding of adenosine deaminase to DPP-IV may contribute to the diabetic phenotype in vivo by leading to a higher level of free DPP-IV to bind and inactivate the anti-diabetic hormones, GLP-1 and GIP. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Molecular & Cellular Proteomics
volume
10
issue
9
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • wos:000294729200005
  • pmid:21697546
  • scopus:80052772366
ISSN
1535-9484
DOI
10.1074/mcp.M110.006650
language
English
LU publication?
yes
id
41ac8cd8-8db2-4e85-8c0e-1033a21a58fd (old id 2007833)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/21697546?dopt=Abstract
date added to LUP
2011-07-06 10:36:33
date last changed
2017-01-01 03:12:32
@article{41ac8cd8-8db2-4e85-8c0e-1033a21a58fd,
  abstract     = {Skeletal muscle is a key tissue site of insulin resistance in type 2 diabetes. Human myotubes are primary skeletal muscle cells displaying both morphological and biochemical characteristics of mature skeletal muscle and the diabetic phenotype is conserved in myotubes derived from subjects with type 2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery strategy using iTRAQ and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring (SRM) to identify, quantify and validate changes in protein abundance between human myotubes obtained from non-diabetic lean, non-diabetic obese and type 2 diabetic subjects, respectively. A total of 2,832 unique proteins were identified and quantified using the iTRAQ strategy. Proteins from all major pathways known to be important in T2D were well-characterized in this study. This included pathways like the TCA cycle, lipid oxidation, oxidative phosphorylation, the glycolytic pathway and glycogen metabolism from which all but two enzymes were found in the present study. None of these enzymes were found to be regulated at the level of protein expression or degradation supporting the hypothesis that these pathways are regulated at the level of post-translational modification. Twelve proteins were, however, differentially expressed between the three different groups. Thirty-six proteins were chosen for further analysis and validation using SRM based on the regulation identified in the iTRAQ discovery study. The abundance of adenosine deaminase was considerably down-regulated in diabetic myotubes and as the protein binds DPP-IV, we speculate whether the reduced binding of adenosine deaminase to DPP-IV may contribute to the diabetic phenotype in vivo by leading to a higher level of free DPP-IV to bind and inactivate the anti-diabetic hormones, GLP-1 and GIP.},
  author       = {Thingholm, Tine and Bak, Steffen and Beck-Nielsen, Henning and Jensen, Ole N and Gaster, Michael},
  issn         = {1535-9484},
  language     = {eng},
  number       = {9},
  publisher    = {American Society for Biochemistry and Molecular Biology},
  series       = {Molecular & Cellular Proteomics},
  title        = {Characterization of human myotubes from type 2 diabetic and non-diabetic subjects using complementary quantitative mass spectrometric methods.},
  url          = {http://dx.doi.org/10.1074/mcp.M110.006650},
  volume       = {10},
  year         = {2011},
}