Plasma and Muscle Myostatin in Relation to Type 2
Claus Brandt*, Anders R. Nielsen, Christian P. Fischer, Jakob Hansen, Bente K. Pedersen, Peter Plomgaard
Centre of Inflammation and Metabolism, Department of Infectious Diseases and CMRC, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen,
Objective: Myostatin is a secreted growth factor expressed in skeletal muscle tissue, which negatively regulates skeletal
muscle mass. Recent animal studies suggest a role for myostatin in insulin resistance. We evaluated the possible metabolic
role of myostatin in patients with type 2 diabetes and healthy controls.
Design: 76 patients with type 2 diabetes and 92 control subjects were included in the study. They were matched for age,
gender and BMI. Plasma samples and biopsies from the vastus lateralis muscle were obtained to assess plasma myostatin
and expression of myostatin in skeletal muscle.
Results: Patients with type 2 diabetes had higher fasting glucose (8.9 versus 5.1 mmol/L, P,0.001), plasma insulin (68.2
versus 47.2 pmol/L, P,0.002) and HOMA2-IR (1.6 versus 0.9, P,0.0001) when compared to controls. Patients with type 2
diabetes had 1.4 (P,0.01) higher levels of muscle myostatin mRNA content than the control subjects. Plasma myostatin
concentrations did not differ between patients with type 2 diabetes and controls. In healthy controls, muscle myostatin
mRNA correlated with HOMA2-IR (r=0.30, P,0.01), plasma IL-6 (r=0.34, P,0.05) and VO2 max (r=20.26, P,0.05),
however, no correlations were observed in patients with type 2 diabetes.
Conclusions: This study supports the idea that myostatin may have a negative effect on metabolism. However, the
metabolic effect of myostatin appears to be overruled by other factors in patients with type 2 diabetes.
Citation: Brandt C, Nielsen AR, Fischer CP, Hansen J, Pedersen BK, et al. (2012) Plasma and Muscle Myostatin in Relation to Type 2 Diabetes. PLoS ONE 7(5):
Editor: Jose A. L. Calbet, University of Las Palmas de Gran Canaria, Spain
Received September 16, 2011; Accepted April 18, 2012; Published May 16, 2012
Copyright: ? 2012 Brandt 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.
Funding: CIM is supported by a grant from the Danish National Research Foundation (# 02-512-55). This study was further supported by the Danish Council for
Independent Research – Medical Sciences and Natural Sciences and the Commission of the European Communities (Grant Agreement no. 223576 - MYOAGE). CIM
is part of the UNIK Project: Food, Fitness & Pharma for Health and Disease, supported by the Danish Ministry of Science, Technology, and Innovation. CIM is a
member of DD2 - the Danish Center for Strategic Research in Type 2 Diabetes (the Danish Council for Strategic Research, grant no. 09-067009 and 09-075724).
The Copenhagen Muscle Research Centre is supported by a grant from the Capital Region of Denmark. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Human myostatin was first cloned in 1998 . Myostatin, or
growth/differentiation factor 8 (GDF-8), belongs to the trans-
forming growth factor-b (TGF-b) superfamily and has been
identified as a major regulator of muscle mass . Myostatin is a
peptide hormone produced by skeletal muscle and secreted into
the circulation. Myostatin is a negative regulator of muscle mass
and is well preserved across species as judged from its expression in
fish, birds, cows and humans .
Interestingly, recent observations in animal models suggest that
myostatin is involved in the regulation of energy metabolism as
hypermuscular myostatin knock-out mice have reduced fat mass
and are protected from dietary-induced insulin resistance [4–6].
Furthermore, animal models have suggested a role for myostatin
in diabetic muscle atrophy as ob/ob diabetic mice have higher
levels of myostatin expression, and reduced muscle mass as well as
fiber cross-sectional area [7,8]. In vitro studies of myostatins effect
on glucose metabolism is contradictory as myostatin was shown to
inhibit glucose uptake in a placental cell line  however an
increased glucose uptake has also been demonstrated using human
placenta extracts . The finding that myostatin knock-out mice
are protected against obesity-induced insulin resistance as mea-
sured by a hyperinsulemeamic clamp  suggests an effect of
myostatin on insulin-mediated glucose uptake. Furthermore,
myostatin knock-out mice show an increased AMP-activated
protein kinase activity in skeletal muscle, which could explain the
increased insulin sensitivity . In contrast, myostatin has been
shown to increase AMP-activated protein kinase activity in C2C12
myotubes thereby improving glucose uptake . Taken together
in vitro and animal studies suggests that myostatin affects glucose
uptake, but the literature is not consistent. An inhibitory
association is supported by a gene expression study in which an
transcriptomic array revealed an increased myostatin expression in
skeletal muscles of patients with type 2 diabetes . Although loss
of muscle mass is a clear clinical feature of type 2 diabetes [14,15],
it is uncertain whether increased circulating myostatin plays a role
in the metabolic deterioration of skeletal muscle in individuals with
obesity and insulin resistance.
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To elucidate the associations between myostatin and insulin
resistance, lean body mass, fitness and low-grade inflammation, we
evaluated circulating levels of myostatin as well as skeletal muscle
expression of myostatin in patients with type 2 diabetes and in
controls, who were closely matched for gender and body mass
Materials and Methods
A cross-sectional design was employed. As previously described,
subjects (n=233) were recruited by advertising in a local
newspaper. They received oral and written information about
the experimental procedures before giving their written, informed
consent to participate. Assessment of the type 2 diabetes diagnosis
was based on information from each subject and confirmed by an
oral glucose tolerance test (OGTT). Thirty-four subjects were
excluded as they were classified to have an impaired glucose
tolerance (IGT) [16,17]. From 168 subjects (92 healthy controls
and 76 patients with type 2 diabetes) sufficient sample material was
available for analysis of myostatin. In brief, participants were
screened to isolate such metabolic conditions other than type 2
diabetes, which are known to influence body composition and the
immune system. Exclusion criteria were treatment with insulin,
recent or ongoing infection, a history of malignant disease and
known dementia. Participants reported to the laboratory between
8 and 10 am after an overnight fast. They did not take any
medication in the 24 h preceding the examination, and the type 2
diabetics did not take their oral anti-diabetic medication for 1
week preceding the examination. A general health examination
was performed. Blood samples were drawn from an antecubital
vein and a biopsy was obtained from the vastus lateralis muscle.
An oral glucose tolerance test (OGTT) was performed on the same
The study was approved by the Ethics Committee of the
Copenhagen and Frederiksberg Communities (KF 01-141/04).
Blood samples were drawn before and 1 and 2 h after the
participant had drunk 500 ml of water containing 75 g of
dissolved glucose. The WHO diagnostic criteria were applied.
Participants found to have IGT were excluded from the study.
Cardiorespiratory fitness was measured by the A˚strand-Rhym-
ing indirect test of maximal oxygen uptake .
Bone mass density (BMD), whole body fat and fat-free tissue
masses, trunk and extremities were measured using DXA scanning
(Lunar Prodigy Advance; GE Medical Systems Lunar, Milwaukee,
WI). DXA scanning does not distinguish between subcutaneous
and intraabdominal fat located in the trunk region. Software
(Prodigy, enCORE 2004, version 8.8, GE Lunar Corp., Madison,
WI) was used to estimate the mass of regional and total fat and fat-
Blood samples were drawn into glass tubes containing EDTA,
which were immediately spun at 3500 g for 15 min at 4uC. Plasma
was isolated and stored at 220uC until analysed.
Skeletal muscle biopsies were obtained from vastus lateralis
using a Bergstro ¨m biopsy needle . The biopsies were
immediately frozen in liquid nitrogen and stored at 280uC until
The plasma myostatin assay is a competitive immunoassay. The
standards and samples are pre-incubated with a polyclonal rabbit-
anti human recombinant myostatin (full length) antibody. During
this pre-incubation free myostatin is bound by the myostatin-
antibody. The pre-incubated samples and standards are then
transferred to a microtiterplate coated with human recombinant
myostatin (full length). The unbound antibodies bind to the
immobilized antigen on the microtiterplate. By use of a peroxidase
conjugated goat-anti-rabbit antibody the bound antibody is
detected. Tetramethylbenzidine (TMB) is used as a peroxidase
substrate. Finally, an acidic stop solution is added to terminate the
reaction, whereby the colour changes from blue to yellow. The
intensity of the yellow colour is inversely proportional to the
concentration of myostatin. A dose response curve of the
absorbance unit (optical density, OD at 450 nm) vs. concentration
is generated using the values obtained from the standard.
Myostatin in the samples is determined from this curve. Detection
limit: 0.273 ng/ml. Inter assay CV: ,15% Intra assay CV:
,10%. Immundiagnostik AG, Bensheim, Germany, conducted
the plasma myostatin measurements. Plasma concentrations of
TNF-a and IL-6 were measured by ELISA (R&D Systems,
Minneapolis, MN, USA). Samples were analysed in duplicate and
mean concentrations were calculated. In plasma, levels of
cholesterol (HDL and LDL), triglycerides, C-reactive protein
(CRP), glucose and insulin were measured using routine labora-
tory methods. Based on the fasting plasma concentrations of
glucose and insulin, the level of insulin resistance was calculated
using the homeostasis model assessment of insulin resistance,
version 2 (HOMA2-IR) of 1998 (software available at http://
RNA isolation, reverse transcription and real-time PCR
Total RNA was extracted from ,40 mg muscle tissue using
Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following the
manufacturer’s instructions. In summary, muscle tissue was
homogenized in 1 ml Trizol Reagent for 15 s using a Qiagen
Tissuelyser (Qiagen Nordic, Copenhagen, Denmark). Chloroform
was added and the phases were separated by centrifugation. The
aqueous phase with the RNA was transferred to a fresh tube and
the RNA precipitated by adding isopropanol and left at 220uC for
1 h. After another centrifugation, the RNA pellet was washed in
75% ethanol and finally dissolved in 50 ml diethylpyrocarbonate-
The RNA concentration was determined spectrophotometri-
cally and 2 mg total RNA was reversed-transcribed in a total
volume of 100 ml using the Taqman Reverse Transcription Kit
(Applied Biosystems, NJ, USA) and random hexamers as primers.
Real-time PCR was performed using an ABI 7900 Sequence
Detection System (Applied Biosystems). The mRNAs for myosta-
tin and the endogenous control, b-actin, were amplified using
predeveloped assays (Applied Biosystems). The PCR conditions
followed the procedure recommended by the manufacturer, with
10 ml reaction volume and each sample run in triplicate for 50
cycles. The mRNA content of both the target and the endogenous
control gene was calculated from the cycle threshold values by
using a standard curve constructed from a serial dilution of
aliquots of cDNA pooled from all the samples.
Myostatin and Diabetes
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Data are generally presented as means with confidence interval
of the mean. If the data were not normally distributed, a
logarithmic transformation was applied and the data were
presented as geometric means. Logarithmic transformation was
performed on all data except: age, BMI, BMD, lean body mass, fat
mass and LDL cholesterol. For comparisons between the groups
(control versus type 2 diabetes and low versus high myostatin) a t-
test was used for continuous variable whereas a x2test was used for
categorical variables. Analysis for correlations was performed
using Pearson’s approach. A multiple regression analysis was done
using a general linear model (PROC GLM). All analyses were
performed using SAS software version 9.1 (SAS institute, Cary,
NC, USA). P,0.05 was considered significant.
Characterization of control subjects and patients with
type 2 diabetes
Seventy-six patients with type 2 diabetes and 92 control subjects
were investigated in the study. The patients with diabetes were
slightly older than the control subjects, but gender distribution was
similar in the two groups, Table 1. BMI and fat mass as
determined by DXA scan were similar in both groups. Fasting
glucose, plasma insulin, and HOMA2-IR levels were higher,
whereas plasma total cholesterol, LDL cholesterol, HDL choles-
terol levels were lower in the patients with type 2 diabetes than in
the control subjects. Plasma triglycerides, TNF-a and IL-6 were
higher in the patients with diabetes than in the control subjects;
these differences remained significant after age and gender
adjustment, Table 1.
Myostatin levels are increased in patients with type 2
Skeletal muscle myostatin mRNA content was 1.4 fold (P,0.05)
higher in patients with type 2 diabetes when compared to the
control group, Figure 1A. This difference remained significant
after adjustment for age and gender (P,0.001). The plasma
myostatin concentration was slightly elevated in patients with type
2 diabetes 5.1 (4.6–5.7) mg/L compared to 4.5 (4.1–5.0) mg/L in
control subjects. However this difference was only significantly
different when correcting for age and gender (P=0.0261),
Figure 1B. When the data from the patients with diabetes and
the control subjects were combined, plasma and muscle myostatin
levels were similar in men and women (P=0.5 and P=0.2
Associations of muscle myostatin mRNA content and
plasma myostatin with clinical, glycaemic, lipid, and
To evaluate the association between clinical and biochemical
markers of insulin resistance Pearson’s correlations were per-
formed and appear from Table 2. The skeletal muscle content of
Table 1. Subject characteristics.
Type 2 diabetes
Age (years)53.2 (50.7–55.7) 58.2 (55.7–60.7)0.006 ----
Gender (M/F)64/28 57/190.43----
BMI (kg/m2) 30.0 (28.7–31.4)30.6 (29.2–31.9) 0.570.026
VO2 max(L/kg)28.7 (26.8–30.7) 23.3 (21.6–25.4)0.00010.0004
Bone mass density 3.0 (2.9–3.1)2.8 (2.7–2.9)0.03 0.002
Lean body mass 58.6 (55.9–61.3)57.9 (55.1–60.7)0.730.034
Fat mass30.5 (27.3–33.6) 29.3 (26.9–31.7) 0.550.033
Fasting glucose (mmol/L) 5.1 (5.0–5.2)8.9 (8.2–9.8)
Fasting insulin (pmol/L) 47.2 (40.8–54.5) 68.2 (56.7–82.1)0.002
HOMA2-IR 0.9 (0.8–1.0)1.6 (1.3–1.9)
Plasma cholesterol (mmol/L)5.3 (5.1–5.5)4.8 (4.5–5.1) 0.0090.0008
LDL cholesterol (mmol/L)3.5 (3.4–3.7) 2.9 (2.7–3.2)
HDL cholesterol (mmol/L) 1.5 (1.4–1.6)1.3 (1.2–1.4)0.006
Plasma triglycerides (mmol/L)1.2 (1.0–1.3)1.5 (1.3–1.8) 0.010.0011
CRP (mg/L) 2.4 (2.0–2.9) 3.0 (2.5–3.7)0.08 0.006
TNF-a (ng/L) 2.4 (2.3–2.5)2.7 (2.5–2.8) 0.0050.0045
IL-6 (ng/L)1.2 (1.1–1.5) 1.7 (1.4–2.0)0.020.0023
IL-18 (ng/L)224 (207–243)242 (221–264)0.200.0315
Body composition, glycaemic variables, plasma lipids, and inflammatory markers in healthy control subjects and type 2 diabetes patients. BMI; Body mass index, P
indicates a significant difference between the groups, P* is corrected for age and gender. P,0.05 is considered significant.
Myostatin and Diabetes
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myostatin mRNA correlated positively with fasting insulin,
HOMA2-IR, plasma IL-6, CRP, BMI and triglycerides, and
negatively with maximal oxygen uptake (VO2 max) when all
participants were analysed together. Only fasting blood glucose
correlated with plasma myostatin and only when the healthy
controls and patients with type 2 diabetes were analysed in
combination, Table 2. However when the groups were analysed
separately, muscle myostatin mRNA content only correlated
significantly in the control group. Plasma myostatin and muscle
myostatin mRNA content was positively correlated in the healthy
controls only. The healthy controls and patients with type 2
diabetes were divided into low (QL) and high (QH) muscle content
of myostatin mRNA and the fasting glucose, insulin, HOMA2-IR,
and plasma IL-6 levels were compared. In the patients with type 2
diabetes no difference was observed. However, in the healthy
controls with a high myostatin mRNA content in the vastus
muscle, a higher level of fasting insulin, HOMA2-IR and plasma
IL-6 could be demonstrated, Figure 2. No differences were found
when the same analysis was performed for circulating myostatin,
Figure 1. Skeletal muscle mRNA content (A) and plasma myostatin (B) in healthy control (n=92) and patients with type 2 diabetes
(n=76). Individual data are presented and the bar indicates the geometric mean. * indicates a significant difference between healthy controls and
patients with type 2 diabetes, P,0.05.
Figure 2. Muscle myostatin mRNA divided in low (QL) and high (QH) content in control subjects and patients with type 2 diabetes,
respectively. The bar represents geometric means for plasma (A), plasma insulin (B), HOMA2-IR (C) and plasma IL-6 (D). * indicates a difference
between low versus high muscle content of myostatin mRNA. P,0.05 is considered significant.
Myostatin and Diabetes
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To further investigate the relationship between the variables
found to correlate with muscle myostatin mRNA content, a
multivariate analysis was performed. Besides diabetes, age, and
gender, only predictors that correlated significantly were included.
As it appears from Table 3, age and plasma IL-6 remained
significant. Plasma myostatin was solely significantly correlated
with fasting glucose.
The present study demonstrates that skeletal muscle myostatin
mRNA is elevated in patients with type 2 diabetes when compared
to healthy control subjects. Furthermore we show that muscle
myostatin mRNA content is associated with impaired insulin
sensitivity, increased triglycerides, and low-grade chronic inflam-
mation as well as obesity and a poor fitness level. Interestingly,
clear associations were found in healthy controls, but were absent
in type 2 diabetes patients. Therefore, if a causal relationship exists
between myostatin and metabolism, it appears that the negative,
regulatory effects of myostatin on metabolism are overruled by
other factors in advanced type 2 diabetes. In accordance, a positive
association between plasma and muscle myostatin was only
observed in the healthy controls, which may suggest an alteration
in the regulatory mechanism with diabetes. It appears that plasma
myostatin, compared to muscle myostatin, was a less strong
marker of metabolism, as plasma myostatin was only associated
with fasting glucose.
Very few studies have assessed the plasma levels of circulating
myostatin in humans. Lakshman et al  applied an in house
developed ELISA to measure serum myostatin in 50 young and 48
old men and found serum concentrations at 8.0 and 7.0 mg/L,
respectively. In the present study, the average plasma level of
myostatin was 4.8 mg/L. The circulating levels are within the same
range; however the discrepancy could be due to the differences in
matrix (plasma versus serum) and differences in populations, as
well as to the large range of variation observed between
individuals. In the present study, no difference was detected
between young and old, which most likely was due to the low
number (n=7) of young participants (age,35 years). Even though
no differences were observed between young and old, a negative
association with age and muscle myostatin mRNA content was
observed, which remained significant when adjusting for insulin
resistance, inflammatory status and fitness. Lakshman et al did not
find an association between lean body mass and circulating
myostatin, which is in line with the present study, where no
correlation was found between lean body mass and neither plasma
myostatin, nor muscle myostatin mRNA content.
Myostatin KO mice demonstrate improved insulin sensitivity
[5,6], suggesting that myostatin is involved in glucose regulation.
However, these mice concomitantly had altered adiposity, but
interestingly treating ob/ob mice with anti-myostatin antibodies
resulted in an improved glucose clearance, without any changes in
fat mass . The effects of myostatin on glucose metabolism
could be due to effects on the muscle tissue itself, as only inhibition
of myostatin signaling in skeletal muscle and not adipose reveal an
improved insulin sensitivity . An alternative mechanism could
be via TNF-a , which is known to cause insulin resistance .
Interestingly, a positive association was observed between circu-
lating TNF-a and myostatin mRNA expression in the control
Figure 3. Plasma myostatin divided in low (QL) and high (QH) content in control subjects and patients with type 2 diabetes,
respectively. The bar represents geometric means for plasma (A), plasma insulin (B), HOMA2-IR (C) and plasma IL-6 (D). * indicates a difference
between low versus high muscle content of myostatin mRNA.
Myostatin and Diabetes
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subjects, supporting the observations made in mice. In the
multivariate analysis muscle myostatin mRNA content was
predicted by age and plasma IL-6, when adjusting for insulin
resistance, plasma triglycerides and obesity. It is noteworthy that
plasma IL-6 and fitness are inversely related [23,24]. A reduction
in myostatin mRNA with improved fitness is in line with a
reduction in muscle myostatin mRNA content after an acute bout
of exercise , however this inverse association in the present
data is not significant if adjusted for BMI. Very few studies have
evaluated the response of plasma myostatin in humans in relation
to exercise or training, whereas several have demonstrated a
reduction of muscle mRNA content [25–28]. One study reported
that after 10 weeks of resistance training, circulating levels of
myostatin have decreased by approximately 20% . The
present cross-sectional data suggest that plasma myostatin is a poor
marker of fitness, although this does not rule out the possibility that
individual changes in plasma myostatin could be a valuable
marker. Furthermore these human data reveal a positive
association between insulin resistance and myostatin mRNA
expression in the skeletal muscle in healthy subjects. Increased
myostatin mRNA expression might be a predisposing marker for
the development of insulin resistance in healthy subjects.
Table 2. Pearson’s correlations to plasma myostatin and myostatin mRNA expression in skeletal muscle tissue.
Control subjects (n=92)
Patients with type 2
diabetes (n=76)Combined (n=168)
Variable Plasma MusclePlasmaMuscle Plasma Muscle
VO2max (L O2/kg)0.09
Bone mass density0.07
Lean body mass0.080.160.090.150.08 0.14
Fasting glucose (mmol/L)0.02
Fasting insulin (pmol/L)
Plasma cholesterol (mmol/L)
LDL cholesterol (mmol/L)
HDL cholesterol (mmol/L)
Plasma triglycerides (mmol/L)0.090.24*0.09
IL-18 (ng/L) 0.070.130.001
Plasma myostatin (mg/L)0.21*
Muscle myostatin mRNA content0.21*
Pearson’s correlations coefficients r between plasma myostatin and muscle myostatin mRNA, respectively, and different clinical and biochemical variable.
Table 3. Multivariate analysis, including variables that were
found to correlate with muscle myostatin mRNA content.
Muscle myostatin mRNA.
Over all model: P=0.0011 n=168
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Interestingly, the fitness level correlated inversely with the
myostatin mRNA only in the group of healthy subjects, why it
could be speculated that an increased insulin resistance, which is
associated with increased myostatin can be counter acted by
Myostatin is involved in adipocyte differentiation  and
recently, Hittel et al  compared 6 lean (BMI,25) with 9
extremely obese (BMI.40) subjects using western blotting and
found an association with muscle and plasma myostatin to both
BMI and HOMA2-IR. In the present study a positive association
was also observed regarding muscle myostatin mRNA and both
BMI and insulin resistance as measured by HOMA in normal
controls subject. However, the present data contribute by allowing
adjustment for age, inflammation and fitness, which reveals that
the association with HOMA2-IR and BMI was no longer
In conclusion, high muscular expression of myostatin is
associated to impaired metabolism, systemic inflammation, obesity
and poor fitness level in healthy subjects. These associations are
disrupted in patients with type 2 diabetes, where no associations
are observed although myostatin mRNA levels are moderately
enhanced. The findings of the present study as well as data from
recent experimental reports make us suggest that muscle-produced
myostatin exerts direct and negative effects on glucose and lipid
metabolism. However, the metabolic effect of myostatin appears to
be overruled by other factors in full-blown type 2 diabetes.
The authors are grateful for the excellent assistance of the Centre of
Inflammation and Metabolism (CIM) technical staff.
Conceived and designed the experiments: CB ARN BKP PP JH CPF.
Performed the experiments: ARN CPF PP. Analyzed the data: CB ANR
JH PP. Wrote the paper: CB PP BKP.
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Myostatin and Diabetes
PLoS ONE | www.plosone.org7 May 2012 | Volume 7 | Issue 5 | e37236