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Increased secretion and expression of myostatin in skeletal muscle from extremely obese women.

Hittel DS, Berggren JR, Shearer J, Boyle K, Houmard JA - Diabetes (2008)

Bottom Line: To test this hypothesis, we used a "bottom-up" experimental design using stable isotope labeling by amino acids in culture (SILAC) and liquid chromatography/mass spectometry/mass spectometry (LC-MS/MS) to both identify and quantify proteins secreted from cultured myotubes derived from extremely obese compared with healthy nonobese women.Myostatin was subsequently shown to increase in skeletal muscle (23%, P < 0.05) and plasma (35%, P < 0.05) and to correlate (r(2) = 0.6, P < 0.05) with the severity of insulin resistance.This may contribute to systemic metabolic deterioration of skeletal muscle with the progression of insulin resistance to type 2 diabetes.

View Article: PubMed Central - PubMed

Affiliation: Human Performance Laboratory, Faculty of Kinesiology, Roger Jackson Center for Health and Wellness, University of Calgary, Calgary, Alberta, Canada. dhittel@kin.ucalgary.ca

ABSTRACT

Objective: Obesity is associated with endocrine abnormalities that predict the progression of insulin resistance to type 2 diabetes. Because skeletal muscle has been shown to secrete proteins that could be used as biomarkers, we characterized the secreted protein profile of muscle cells derived from extremely obese (BMI 48.8 +/- 14.8 kg/m(2); homeostasis model assessment [HOMA] 3.6 +/- 1.0) relative to lean healthy subjects (BMI 25.7 +/- 3.2 kg/m(2); HOMA 0.8 +/- 0.2).

Research design and methods: We hypothesized that skeletal muscle would secrete proteins that predict the severity of obesity. To test this hypothesis, we used a "bottom-up" experimental design using stable isotope labeling by amino acids in culture (SILAC) and liquid chromatography/mass spectometry/mass spectometry (LC-MS/MS) to both identify and quantify proteins secreted from cultured myotubes derived from extremely obese compared with healthy nonobese women.

Results: Using SILAC, we discovered a 2.9-fold increase in the secretion of myostatin from extremely obese human myotubes. The increased secretion and biological activity of myostatin were validated by immunoblot (3.16 +/- 0.18, P < 0.01) and a myoblast proliferation assay using conditioned growth medium. Myostatin was subsequently shown to increase in skeletal muscle (23%, P < 0.05) and plasma (35%, P < 0.05) and to correlate (r(2) = 0.6, P < 0.05) with the severity of insulin resistance.

Conclusions: Myostatin is a potent antianabolic regulator of muscle mass that may also play a role in energy metabolism. These findings show that increased expression of myostatin in skeletal muscle with obesity and insulin resistance results in elevated circulating myostatin. This may contribute to systemic metabolic deterioration of skeletal muscle with the progression of insulin resistance to type 2 diabetes.

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Related in: MedlinePlus

LC-MS/MS identification and quantification of secreted myostatin protein. A: The top spectrum shows a peptide doublet at m/z 571.8 and 574.8 corresponding to unlabeled and 13C6-Lys peptides from human myostatin. The labeled and unlabeled peptides are 3 Da apart in a 3:1 ratio and agree with one 13C6-Lys residue in the doubly charged peptide. The peptide sequence shown on top of the spectrum was obtained by MS/MS analysis of the fragmented peptide. B: Shown in bold an underlined are the relative positions of the other myostatin peptides identified in the primary sequence of human myostatin.
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f2: LC-MS/MS identification and quantification of secreted myostatin protein. A: The top spectrum shows a peptide doublet at m/z 571.8 and 574.8 corresponding to unlabeled and 13C6-Lys peptides from human myostatin. The labeled and unlabeled peptides are 3 Da apart in a 3:1 ratio and agree with one 13C6-Lys residue in the doubly charged peptide. The peptide sequence shown on top of the spectrum was obtained by MS/MS analysis of the fragmented peptide. B: Shown in bold an underlined are the relative positions of the other myostatin peptides identified in the primary sequence of human myostatin.

Mentions: The resulting peptide solutions were analyzed by LC-MS/MS at the southern Alberta mass spectrometry center at the University of Calgary (Calgary, AB, Canada). Chromatographic separations of peptides were performed with a C18 analytical column using an Agilent 1100 nanoLC system (Agilent Technologies, Santa Clara, CA). Peptides were loaded onto an enrichment and eluted with 0.2% formic acid and 10% water in acetonitrile over 50 min. The analytical column was connected online to a Qstar XL Hybrid quadrupole time-of-flight (TOF) mass spectrometer fitted with a nanospray ion source (Applied Biosystems, Foster City, CA). TOF MS experiments were performed in positive ion mode over an m/z range of 400–1,500. Automated tandem MS analyses were carried out using a standard data-dependent configuration, in which the three most intense peptides (2+ or 3+ charge states) in an MS scan were automatically selected for sequencing. Protein searches were performed against the human NCBInr and SwissProt databases using the Mascot distiller (Matrix Science, London) using the Mascot search algorithm. The mass tolerance for the precursor peptide ion was set at 200 parts per million, and the mass tolerance for the MS/MS fragment ions was set to 0.5 Da. Quantitation was performed manually according to the manufacturer's instructions (Invitrogen). Briefly, we used the Mascot peptide summary list generated for each LC-MS/MS run and Analyst QS 1.1 Software (Applied Biosystems) to analyze selected ion chromatograph data from the raw WIFF file generated by the LC-MS/MS analysis. The retention time and protein identification for each peptide were then confirmed by the y ions from each MS/MS and the relative abundance of selected peptide pairs (Figs. 1 and 2) calculated as previously described (8). The search parameters allowed for variable modifications, including amidomethylation of cysteine, oxidation of methionine, and presence of 13C6-Lys. Secreted proteins had to have a minimum of one 13C6-Lys peptide identified with high confidence and had to be verified by manual inspection for having a consecutive series of y ions (8).


Increased secretion and expression of myostatin in skeletal muscle from extremely obese women.

Hittel DS, Berggren JR, Shearer J, Boyle K, Houmard JA - Diabetes (2008)

LC-MS/MS identification and quantification of secreted myostatin protein. A: The top spectrum shows a peptide doublet at m/z 571.8 and 574.8 corresponding to unlabeled and 13C6-Lys peptides from human myostatin. The labeled and unlabeled peptides are 3 Da apart in a 3:1 ratio and agree with one 13C6-Lys residue in the doubly charged peptide. The peptide sequence shown on top of the spectrum was obtained by MS/MS analysis of the fragmented peptide. B: Shown in bold an underlined are the relative positions of the other myostatin peptides identified in the primary sequence of human myostatin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2606890&req=5

f2: LC-MS/MS identification and quantification of secreted myostatin protein. A: The top spectrum shows a peptide doublet at m/z 571.8 and 574.8 corresponding to unlabeled and 13C6-Lys peptides from human myostatin. The labeled and unlabeled peptides are 3 Da apart in a 3:1 ratio and agree with one 13C6-Lys residue in the doubly charged peptide. The peptide sequence shown on top of the spectrum was obtained by MS/MS analysis of the fragmented peptide. B: Shown in bold an underlined are the relative positions of the other myostatin peptides identified in the primary sequence of human myostatin.
Mentions: The resulting peptide solutions were analyzed by LC-MS/MS at the southern Alberta mass spectrometry center at the University of Calgary (Calgary, AB, Canada). Chromatographic separations of peptides were performed with a C18 analytical column using an Agilent 1100 nanoLC system (Agilent Technologies, Santa Clara, CA). Peptides were loaded onto an enrichment and eluted with 0.2% formic acid and 10% water in acetonitrile over 50 min. The analytical column was connected online to a Qstar XL Hybrid quadrupole time-of-flight (TOF) mass spectrometer fitted with a nanospray ion source (Applied Biosystems, Foster City, CA). TOF MS experiments were performed in positive ion mode over an m/z range of 400–1,500. Automated tandem MS analyses were carried out using a standard data-dependent configuration, in which the three most intense peptides (2+ or 3+ charge states) in an MS scan were automatically selected for sequencing. Protein searches were performed against the human NCBInr and SwissProt databases using the Mascot distiller (Matrix Science, London) using the Mascot search algorithm. The mass tolerance for the precursor peptide ion was set at 200 parts per million, and the mass tolerance for the MS/MS fragment ions was set to 0.5 Da. Quantitation was performed manually according to the manufacturer's instructions (Invitrogen). Briefly, we used the Mascot peptide summary list generated for each LC-MS/MS run and Analyst QS 1.1 Software (Applied Biosystems) to analyze selected ion chromatograph data from the raw WIFF file generated by the LC-MS/MS analysis. The retention time and protein identification for each peptide were then confirmed by the y ions from each MS/MS and the relative abundance of selected peptide pairs (Figs. 1 and 2) calculated as previously described (8). The search parameters allowed for variable modifications, including amidomethylation of cysteine, oxidation of methionine, and presence of 13C6-Lys. Secreted proteins had to have a minimum of one 13C6-Lys peptide identified with high confidence and had to be verified by manual inspection for having a consecutive series of y ions (8).

Bottom Line: To test this hypothesis, we used a "bottom-up" experimental design using stable isotope labeling by amino acids in culture (SILAC) and liquid chromatography/mass spectometry/mass spectometry (LC-MS/MS) to both identify and quantify proteins secreted from cultured myotubes derived from extremely obese compared with healthy nonobese women.Myostatin was subsequently shown to increase in skeletal muscle (23%, P < 0.05) and plasma (35%, P < 0.05) and to correlate (r(2) = 0.6, P < 0.05) with the severity of insulin resistance.This may contribute to systemic metabolic deterioration of skeletal muscle with the progression of insulin resistance to type 2 diabetes.

View Article: PubMed Central - PubMed

Affiliation: Human Performance Laboratory, Faculty of Kinesiology, Roger Jackson Center for Health and Wellness, University of Calgary, Calgary, Alberta, Canada. dhittel@kin.ucalgary.ca

ABSTRACT

Objective: Obesity is associated with endocrine abnormalities that predict the progression of insulin resistance to type 2 diabetes. Because skeletal muscle has been shown to secrete proteins that could be used as biomarkers, we characterized the secreted protein profile of muscle cells derived from extremely obese (BMI 48.8 +/- 14.8 kg/m(2); homeostasis model assessment [HOMA] 3.6 +/- 1.0) relative to lean healthy subjects (BMI 25.7 +/- 3.2 kg/m(2); HOMA 0.8 +/- 0.2).

Research design and methods: We hypothesized that skeletal muscle would secrete proteins that predict the severity of obesity. To test this hypothesis, we used a "bottom-up" experimental design using stable isotope labeling by amino acids in culture (SILAC) and liquid chromatography/mass spectometry/mass spectometry (LC-MS/MS) to both identify and quantify proteins secreted from cultured myotubes derived from extremely obese compared with healthy nonobese women.

Results: Using SILAC, we discovered a 2.9-fold increase in the secretion of myostatin from extremely obese human myotubes. The increased secretion and biological activity of myostatin were validated by immunoblot (3.16 +/- 0.18, P < 0.01) and a myoblast proliferation assay using conditioned growth medium. Myostatin was subsequently shown to increase in skeletal muscle (23%, P < 0.05) and plasma (35%, P < 0.05) and to correlate (r(2) = 0.6, P < 0.05) with the severity of insulin resistance.

Conclusions: Myostatin is a potent antianabolic regulator of muscle mass that may also play a role in energy metabolism. These findings show that increased expression of myostatin in skeletal muscle with obesity and insulin resistance results in elevated circulating myostatin. This may contribute to systemic metabolic deterioration of skeletal muscle with the progression of insulin resistance to type 2 diabetes.

Show MeSH
Related in: MedlinePlus