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Cited by 324Muscle Physiology and DisordersAdipokines, Inflammation, and Metabolic DiseasesDiabetes, Cardiovascular Risks, and Lipoproteins

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Increased Secretion and Expression of Myostatin in Skeletal Muscle From Extremely Obese Women

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Hittel, Dustin S., Berggren, Jason R., Shearer, Jane, Boyle, Kristen, Houmard, Joseph A.·2009·Diabetes
DOI 10.2337/db08-0943PMC PMC2606890
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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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FIG. 1. LC-MS/MS identification and quantification of 13C6-Lys and unlabeled secreted proteins from lean and extremely obese primary human myotubes. A: Proteins from 18-h conditioned, serum-free media from unlabeled (lean) and 13C6-Lys (extremely obese) myotubes were combined 1:1 and resolved by 4–16% SDS-PAGE. B: The base peak chromatograph showing overall peak intensities and retention time of all the peptides extracted from a gel slice between 37 and 15 kDa. C: A peptide eluting at 31.8 min as a doublet at m/z 706.4 and 709.4, corresponding to the unlabeled and 13C6-Lys peptide from human GAPDH. The peptide sequence shown on top of the spectrum was obtained by MS/MS analysis. The labeled and unlabeled peptides are 3 Da apart in a 1:1 ratio and agree with one 13C6-Lys residue in the doubly charged peptide. (Please see http://dx.doi.org/10.2337/db08-0943 for a high-quality digital representation of this figure.)
FIG. 1. LC-MS/MS identification and quantification of 13C6-Lys and unlabeled secreted proteins from lean and extremely obese primary human myotubes. A: Proteins from 18-h conditioned, serum-free media from unlabeled (lean) and 13C6-Lys (extremely obese) myotubes were combined 1:1 and resolved by 4–16% SDS-PAGE. B: The base peak chromatograph showing overall peak intensities and retention time of all the peptides extracted from a gel slice between 37 and 15 kDa. C: A peptide eluting at 31.8 min as a doublet at m/z 706.4 and 709.4, corresponding to the unlabeled and 13C6-Lys peptide from human GAPDH. The peptide sequence shown on top of the spectrum was obtained by MS/MS analysis. The labeled and unlabeled peptides are 3 Da apart in a 1:1 ratio and agree with one 13C6-Lys residue in the doubly charged peptide. (Please see http://dx.doi.org/10.2337/db08-0943 for a high-quality digital representation of this figure.)CC BY-NC-ND · PubMed Central
FIG. 2. 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.
FIG. 2. 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.CC BY-NC-ND · PubMed Central
FIG. 3. Myostatin secretion is increased from extremely obese primary human myotubes. Equal amounts of protein from 24-h–conditioned, serum-free media resolved by 15% SDS-PAGE. Myostatin (A) and GAPDH (B) protein were detected with polyclonal goat anti-myostatin and monoclonal mouse anti-GAPDH antibodies, respectively. The mature form of myostatin is indicated in a representative blot, and the molecular masses of the detected bands are indicated. Number of replicated are indicated for each sample, and the asterisk indicates significant differences (P < 0.05). Data are normalized to average lean expression level ± SE. ExOb, exobese. (Please see http://dx.doi.org/10.2337/db08-0943 for a high-quality digital representation of this figure.)
FIG. 3. Myostatin secretion is increased from extremely obese primary human myotubes. Equal amounts of protein from 24-h–conditioned, serum-free media resolved by 15% SDS-PAGE. Myostatin (A) and GAPDH (B) protein were detected with polyclonal goat anti-myostatin and monoclonal mouse anti-GAPDH antibodies, respectively. The mature form of myostatin is indicated in a representative blot, and the molecular masses of the detected bands are indicated. Number of replicated are indicated for each sample, and the asterisk indicates significant differences (P < 0.05). Data are normalized to average lean expression level ± SE. ExOb, exobese. (Please see http://dx.doi.org/10.2337/db08-0943 for a high-quality digital representation of this figure.)CC BY-NC-ND · PubMed Central
FIG. 4. Myostatin protein is increased in extremely obese primary human myotubes. Equal amounts of total protein from primary human myotubes were resolved by 15% SDS-PAGE. A: Myostatin protein was detected with goat polyclonal anti-myostatin antibodies. Precursor, mature, and LAP forms of myostatin are indicated in a representative blot. Molecular masses of the detected bands are indicated. B: Only the 52-kDa precursor form of myostatin was quantified; however, the LAP appeared to follow a similar expression profile. Small amounts of the 26-kDa mature myostatin were detected only in extremely obese myotubes. Number of replicated are indicated for each sample, and the asterisk indicates significant differences (P < 0.05). Data are normalized to average lean expression level ± SE. (Please see http://dx.doi.org/10.2337/db08-0943 for a high-quality digital representation of this figure.)
FIG. 4. Myostatin protein is increased in extremely obese primary human myotubes. Equal amounts of total protein from primary human myotubes were resolved by 15% SDS-PAGE. A: Myostatin protein was detected with goat polyclonal anti-myostatin antibodies. Precursor, mature, and LAP forms of myostatin are indicated in a representative blot. Molecular masses of the detected bands are indicated. B: Only the 52-kDa precursor form of myostatin was quantified; however, the LAP appeared to follow a similar expression profile. Small amounts of the 26-kDa mature myostatin were detected only in extremely obese myotubes. Number of replicated are indicated for each sample, and the asterisk indicates significant differences (P < 0.05). Data are normalized to average lean expression level ± SE. (Please see http://dx.doi.org/10.2337/db08-0943 for a high-quality digital representation of this figure.)CC BY-NC-ND · PubMed Central
FIG. 5. Myostatin protein is increased in skeletal muscle and plasma from extremely obese subjects. Equal amounts of total protein from human skeletal muscle and plasma were resolved by 15% SDS-PAGE. A: Myostatin protein was detected with goat polyclonal anti-myostatin antibodies. Precursor (52 kDa) and mature dimer (26 kDa) forms of myostatin are indicated in representative blots. B: Only the 52-kDa precursor form of myostatin was quantified in muscle and the 26-kDa mature form in plasma. Number of replicated are indicated for each sample, and the asterisk indicates significant differences (P < 0.05). Data are normalized to average lean expression level ± SE. (Please see http://dx.doi.org/10.2337/db08-0943 for a high-quality digital representation of this figure.)
FIG. 5. Myostatin protein is increased in skeletal muscle and plasma from extremely obese subjects. Equal amounts of total protein from human skeletal muscle and plasma were resolved by 15% SDS-PAGE. A: Myostatin protein was detected with goat polyclonal anti-myostatin antibodies. Precursor (52 kDa) and mature dimer (26 kDa) forms of myostatin are indicated in representative blots. B: Only the 52-kDa precursor form of myostatin was quantified in muscle and the 26-kDa mature form in plasma. Number of replicated are indicated for each sample, and the asterisk indicates significant differences (P < 0.05). Data are normalized to average lean expression level ± SE. (Please see http://dx.doi.org/10.2337/db08-0943 for a high-quality digital representation of this figure.)CC BY-NC-ND · PubMed Central
FIG. 6. Linear regression analysis of myostain expression in muscle. Linear regression analyses were conducted using the least squares method to compare muscle myostatin expression levels to HOMA (A) and BMI (B). Rsqr, r2.
FIG. 6. Linear regression analysis of myostain expression in muscle. Linear regression analyses were conducted using the least squares method to compare muscle myostatin expression levels to HOMA (A) and BMI (B). Rsqr, r2.CC BY-NC-ND · PubMed Central
FIG. 7. Proliferation rate of C2C12 mouse myoblasts grown in human myotube-conditioned growth medium. Equal numbers of cells were grown in lean or extremely obese human myotube conditioned growth medium, which was preincubated with or without goat polyclonal anti-myostatin antibodies to neutralize myostatin activity. Plotted values represent the average of three experiments ± SD. Asterisks indicate significant differences (P < 0.05). Control cells were grown in unconditioned growth medium.
FIG. 7. Proliferation rate of C2C12 mouse myoblasts grown in human myotube-conditioned growth medium. Equal numbers of cells were grown in lean or extremely obese human myotube conditioned growth medium, which was preincubated with or without goat polyclonal anti-myostatin antibodies to neutralize myostatin activity. Plotted values represent the average of three experiments ± SD. Asterisks indicate significant differences (P < 0.05). Control cells were grown in unconditioned growth medium.CC BY-NC-ND · PubMed Central

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Research works printed but not linked: 1

  1. Allen DL, Cleary AS, Speaker KJ, Lindsay SF, Uyenishi J, Reed JM, Madden MC, Mehan RS: Myostatin, activin receptor IIb, and follistatin-like-3 gene expression are altered in adipose tissue and skeletal muscle of obese mice. Am J Physiol Endocrinol Metab 294:E918–E927,2008 · DOI · Paper page

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Metadata

Authors
Hittel, D. S., Berggren, J. R., Shearer, J., Boyle, K., & Houmard, J. A.
Affiliations
  • University of Calgary (CA)
  • East Carolina University (US)
ORCID
Venue
Diabetes
Publisher
American Diabetes Association
Published
2009-01-01
Volume
58
Issue
1
Pages
30-38
ISSN
  • 0012-1797 (ISSN-L)
  • 1939-327X
DOI
10.2337/db08-0943
PMC
PMC2606890
OpenAlex
W2132813206
Cited by
324
References
47
Citations by year
2026: 10 · 2025: 19 · 2024: 12 · 2023: 18 · 2022: 19 · 2021: 21 · 2020: 20 · 2019: 14 · 2018: 26 · 2017: 13 · 2016: 11 · 2015: 25 · 2014: 31 · 2013: 24 · 2012: 22
FWCI
5.35
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Diabetes (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • The Scholarship East Carolina University's Institutional Repository (East Carolina University) (submittedVersion)
Funders
  • Heart and Stroke Foundation of Canada
  • Canadian Diabetes Association
  • Fondation pour la Recherche Médicale
  • National Institutes of Health
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK056112)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adipokines, Inflammation, and Metabolic Diseases
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Diabetes, Cardiovascular Risks, and Lipoproteins
Keywords
myostatin, obesity, insulin resistance, skeletal muscle, myotubes, secreted proteins, type 2 diabetes, myoblast proliferation, energy metabolism, women
MeSH terms
Adult, Chromatography, Liquid, Female, Humans, Insulin Resistance, Middle Aged, Obesity, Immunoblotting, Muscle, Skeletal, Muscle Fibers, Skeletal, Tandem Mass Spectrometry, Myostatin

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