ORIGINAL
E n d o c r i n e
ARTICLE
R e s e a r c h
Slow-Twitch Fiber Proportion in Skeletal Muscle
Correlates With Insulin Responsiveness
Charles A. Stuart, Melanie P. McCurry, Anna Marino, Mark A. South,
Mary E. A. Howell, Andrew S. Layne, Michael W. Ramsey,
and Michael H. Stone
Department of Internal Medicine (C.A.S., M.P.M., A.M., M.E.A.H.), Quillen College of Medicine,
and the Department of Exercise and Sport Science (M.A.S., A.S.L., M.W.R., M.H.S.), Clemmer
College of Education, East Tennessee State University, Johnson City, Tennessee 37614
Context: The metabolic syndrome, characterized by central obesity with dyslipidemia, hypertension, and hyperglycemia, identifies people at high risk for type 2 diabetes.
Objective: Our objective was to determine how the insulin resistance of the metabolic syndrome
is related to muscle fiber composition.
Design: Thirty-nine sedentary men and women (including 22 with the metabolic syndrome) had
insulin responsiveness quantified using euglycemic clamps and underwent biopsies
of the vastus lateralis muscle. Expression of insulin receptors, insulin receptor substrate-1,
glucose transporter 4, and ATP synthase were quantified with immunoblots and
immunohistochemistry.
Participants and Setting: Participants were nondiabetic, metabolic syndrome volunteers and sedentary control subjects studied at an outpatient clinic.
Main Outcome Measures: Insulin responsiveness during an insulin clamp and the fiber composition
of a muscle biopsy specimen were evaluated.
Results: There were fewer type I fibers and more mixed (type IIa) fibers in metabolic syndrome
subjects. Insulin responsiveness and maximal oxygen uptake correlated with the proportion of type
I fibers. Insulin receptor, insulin receptor substrate-1, and glucose transporter 4 expression were not
different in whole muscle but all were significantly less in the type I fibers of metabolic syndrome
subjects when adjusted for fiber proportion and fiber size. Fat oxidation and muscle mitochondrial
expression were not different in the metabolic syndrome subjects.
Conclusion: Lower proportion of type I fibers in metabolic syndrome muscle correlated with the
severity of insulin resistance. Even though whole muscle content was normal, key elements of
insulin action were consistently less in type I muscle fibers, suggesting their distribution was
important in mediating insulin effects. (J Clin Endocrinol Metab 98: 2027–2036, 2013)
iabetes has been diagnosed in more than 10% of
adults in some regions of the United States. The
prevalence of obesity (body mass index [BMI] greater than
30 kg/m2) has more than doubled since 1980 (1). Because
of obesity-related illness, the average life expectancy in the
United States may soon decline for the first time (2). The
D
metabolic syndrome is a precursor to the development
of overt diabetes (3). Insulin resistance and hyperinsulinemia are key elements of the metabolic syndrome that
is characterized by visceral obesity, hypertension, hyperlipidemia, hyperglycemia, coronary heart disease,
and increased mortality (4). The severity of insulin re-
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
Received November 9, 2012. Accepted March 12, 2013.
First Published Online March 20, 2013
Abbreviations: BMI, body mass index; GIR, glucose infusion rate; GLUT4, glucose transporter 4; IRS-1, insulin receptor substrate-1; SSGIR, steady-state glucose infusion rate;
VO2max, maximal oxygen consumption.
doi: 10.1210/jc.2012-3876
J Clin Endocrinol Metab, May 2013, 98(5):2027–2036
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sistance correlates with risk for developing type 2
diabetes.
Because skeletal muscle accounts for 80% of insulinstimulated uptake of glucose (5) and represents 40% to
60% of body weight in normal women and men, muscle
is a principal tissue for evaluation of mechanisms of insulin
resistance. Key elements of insulin action have been quantified in the muscle of persons with type 2 diabetes. Insulin
receptors and the insulin-responsive glucose transporter 4
(GLUT4) have been normal or nearly normal in muscle
homogenates (6, 7), suggesting that their levels of expression could not explain the severe insulin resistance seen in
type 2 diabetes.
Previous studies have shown persons with type 2 diabetes had diminished type I fiber content in muscle biopsies (7, 8). Recently, the authors found nondiabetic subjects with the metabolic syndrome had muscle fiber
composition very similar to that seen in type 2 diabetes (9).
This study was performed to determine whether the muscle fiber composition of metabolic syndrome subjects correlated with measures of insulin resistance.
Materials and Methods
Materials
Monoclonal antibodies directed against slow-twitch myosin
heavy chain were purchased from Millipore (Billerica, Massachusetts). Fast-twitch myosin heavy chain antibodies (ab91506,
rabbit antihuman) and ATP synthase antibodies (ab110273)
were purchased from Abcam (Cambridge, Massachusetts). An
alkaline phosphatase-conjugated fast myosin antibody (Sigma
clone MY-32 alkaline phosphatase conjugate) was purchased
from Sigma-Aldrich (St Louis, Missouri). A peroxidase-conjugated rabbit antimouse IgG antibody (315-035-045) was purchased from Jackson ImmunoResearch Laboratories (West
Grove, Pennsylvania). GLUT4 antibodies (AB1049, goat antihuman) were purchased from Chemicon (Temecula, California).
Monoclonal antibodies directed at the human insulin receptor
␤-subunit (05-1104) were purchased from Millipore. Rabbit
polyclonal antibodies directed at the human insulin receptor
␤-subunit (07-724) were purchased from Millipore. Rabbit polyclonal antibodies directed against human insulin receptor substrate-1 (IRS-1) (2382) were purchased from Cell Signaling
(Danvers, Massachusetts).
Subject selection
Thirty-nine sedentary subjects were recruited. None of the
subjects had performed regular exercise for at least 1 year. The
research protocol and the consent documents were approved by
the East Tennessee State University Institutional Review Board.
Each subject provided written informed consent. Sedentary subjects were recruited into 2 groups: high risk for type 2 diabetes
(BMI ⱖ 30 kg/m2 and a family history of type 2 diabetes) and low
risk for type 2 diabetes (BMI ⬍ 30 kg/m2, no family history of
type 2 diabetes). The 22 subjects at high risk for diabetes qual-
J Clin Endocrinol Metab, May 2013, 98(5):2027–2036
ified for the designation metabolic syndrome, as set forward by
the International Diabetes Federation (4). All 22 of these subjects had BMI greater than 30 kg/m2, visceral obesity indicated
by waist circumference greater than 102 cm (40 in.), and insulin resistance with a 40 mU/m2/min euglycemic clamp
steady-state glucose infusion rate (SSGIR) less than 4.0 mg/
kg/min (⬎1 SD below the mean of the sedentary controls).
Seven of 22 had impaired fasting serum glucose concentrations (100 –125 mg/dL). Nineteen of 22 had dyslipidemia with
triglyceride concentration greater than 150 mg/dL and/or
high-density lipoprotein cholesterol less than 40 mg/dL ( 50
mg/dL in females). Four control subjects met these same criteria for dyslipidemia. Systolic blood pressure greater than
130 mm Hg was noted in 14 of 22 metabolic syndrome subjects and 3 of the sedentary controls.
The entry criteria for participation in these studies included
less than 150 min/wk of planned exercise for the previous year.
Each subject filled out a detailed Physical Activity Questionnaire
before beginning the study. The most common recreational
activity was walking that averaged less than 45 min/wk.
About half of the subjects engaged in some walking and/or
stair climbing at work amounting to less than 30 min/wk in
those participants. For the previous 12 months, none of the
metabolic syndrome subjects reported engaging in swimming,
hiking, aerobics, weight training, biking, jogging, martial
arts, basketball, rowing, or horseback riding. Nearly half of
the control subjects recorded participating in 1 or more of
these activities, but none more than once a month. All of the
volunteers were considered sedentary based on the activities
reported on this questionnaire.
Subject assessments
Body composition was measured by air displacement plethysmography (BodPod, Concord, California). Serum insulin
concentration was quantified after an overnight fast. Blood
pressures were the average of duplicates performed after sitting quietly for a minimum of 10 minutes. Glucose, insulin,
and cholesterol measurements were performed in a clinical
laboratory from serum obtained after an overnight fast. Endurance was measured using a SciFit cycle ergometer (M-F
Athletic, Cranston, Rhode Island). Maximal oxygen consumption (VO2max) and respiratory exchange ratio were
quantified using a TrueOne 2400 Metabolic Measurement
System (ParvoMedics, Sandy, Utah). Fat oxidation was calculated from the baseline respiratory exchange ratio by the
method of Frayn (10).
Muscle biopsies
Percutaneous needle biopsies of vastus lateralis were performed after an overnight fast and 2 hours of quiet recumbency
as previously described, using a Bergstrom-Stille 5-mm muscle
biopsy needle with suction (11). The sample was divided in 2,
with 1 piece frozen immediately in liquid nitrogen for later analysis, and the second piece for slide preparation was mounted on
cork and quickly frozen in a slurry of isopentane cooled by liquid
nitrogen.
Euglycemic-hyperinsulinemic clamp
After a 2-hour baseline period, a single infusion of regular
insulin was performed at 40 mU/m2/min for 2 hours to achieve
doi: 10.1210/jc.2012-3876
a physiological increment in insulin concentration of about 50
␮U/mL (350 pmol/mL) as previously described (12). Regular
human insulin was diluted to 0.3 U/mL in 100 mL normal saline
containing 1 mL of the subject’s own whole blood to provide
albumin at approximately 0.5 mg/mL. Infusion was done using
a single syringe pump (Thermo Fisher Scientific, Rockford, Illinois) set to deliver 40 mU/m2/min, usually about 15 mL/h
(range 11.9 –21.6 mL/h). At time 0, an iv bolus was given,
calculated based on body weight to increase the blood insulin
level by 50 ␮U/mL, generally 5% to 10% of the hourly amount
to be infused. Whole venous blood glucose concentration was
quantified using a Bayer Breeze 2 meter every 5 minutes. The
blood glucose concentrations were corroborated with serum
glucose values each hour done in the clinical laboratory. A
variable glucose infusion was adjusted frequently to maintain
the blood glucose at 85 ⫾ 5 mg/dL. The mean glucose infusion
rate during the last 30 minutes of the 120-minute insulin infusion was used to calculate the SSGIR to quantify insulin
responsiveness.
In earlier studies (9), insulin infusions were carried out for 3
hours with the last 30 minutes of glucose infusion used to calculate the SSGIR. We evaluated 48 consecutive 3-hour euglycemic clamp studies with insulin infused at 40 mU/m2/min and
determined that the infusion of 10% glucose was consistently
stable after about 90 minutes (Supplemental Figure 1, published
on The Endocrine Society’s Journals Online web site at http://
jcem.endojournals.org). The glucose infusion rate (GIR) calculated at 90 to 120 minutes (197 ⫾ 17 mL/h) was 94.7% of the
value calculated from the 150- to 180-minute interval (208 ⫾ 19
mL/h) with a correlation coefficient of 0.944. The subjects with
metabolic syndrome in these studies had GIR from 90 to 120 min
(169 ⫾ 15 mL/hr) that was 97.1% of that calculated from 150
to 180 min (174 ⫾ 17 mL/h). We have subsequently used a
2-hour protocol for the euglycemic clamps at 40 mU/m2/min
and compared these data with the 2-hour values from the
previous studies. Either 2- or 3-hour single-dose insulin infusions have been used by different groups (5, 13, 14), but
2-hour infusion periods are typical of multidose insulin clamp
studies (15–17).
Quantification of muscle fiber type composition
and fiber size
Fiber composition was determined using methods described by Behan et al (18). All sections were coded and then
quantified independently by 2 observers who were unaware of
which subject the image represented. Figure 1 displays a
bright-field image of a sample metabolic syndrome muscle
section stained using the Behan technique (18). All fiber size
data for the current study were calculated using the minimum
diameter measured for each fiber (19).
Immunoblots
Immunoblots to assess the content of the insulin receptor ␤-subunit, IRS-1, GLUT4, and ATP synthase were performed using muscle homogenates as previously described (9). Briefly, approximately
25 mg muscle was homogenized in 500 ␮L 0.25M sucrose, 20mM
HEPES (pH 7.4), containing Halt Protease Inhibitor Kit (Pierce,
Rockford, Illinois) using a hand-held Pellet Pestle Motor homogenizer (Kontes, Vineland, New Jersey) twice for 30 seconds. An aliquot of homogenate containing 10 ␮g protein was subjected to
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Figure 1. Metabolic syndrome subjects’ fiber composition of vastus
lateralis muscle is different from controls. A, Bright-field image of a
transverse section of vastus lateralis muscle from a subject with the
metabolic syndrome. Antibodies against slow-twitch myosin heavy
chain and fast-twitch myosin heavy chain are added sequentially as
described by Behan and coworkers (18). Type I fibers are darkest, type
IIx fibers are pink, and type IIa fibers are intermediate because they
contain both myosin types. Scale bar, 100 ␮m. B, Resulting fiber
composition data for sedentary controls and metabolic syndrome subjects.
The fiber composition data from subjects with the metabolic syndrome
demonstrated that there were fewer type I fibers (38% vs 48%) and more
type IIa fibers (27% vs 13%) than in muscle from sedentary controls. **,
Significant difference from the controls at P ⬍ .01.
SDS-PAGE with 10% polyacrylamide mini-gels (Pierce) or NuPAGE Novex TRIS-acetate 3% to 8% polyacrylamide gels for
IRS-1 (Invitrogen, Carlsbad, California). Protein was transferred
from gels to nitrocellulose membranes using a Mini Protein Transfer Unit from Bio-Rad (Hercules, California).
Immunohistochemistry
Immunohistochemical studies were performed as previously
described (20). Images were generated using a Leica confocal
microscope fluorescent system. Secondary antibodies conjugated with Alexa Fluors (Invitrogen) were used such that donkey
antigoat IgG (A11055) was fluorescent at 488 nm (green images), donkey antirabbit IgG (A31572) was 555 nm (red images),
and donkey antimouse (A31571) was 647 nm (image adjusted to
blue). Wavelength window settings for imaging on the confocal
scope were set (490 –530, 550 –590, and 660 – 690) and validated such that there was no overlap or bleedover between the 3
colors. Each specific labeled image was converted to a grayscale
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J Clin Endocrinol Metab, May 2013, 98(5):2027–2036
TIFF file for image signal intensity quantification of individual fibers using Quantity One image analysis software from
Bio-Rad. Thirty fibers were identified on the grayscale image
using side-by-side color images of the fibers labeled with either
slow-twitch or fast-twitch myosin heavy-chain–specific antibodies and mean pixel intensity quantified on each of at least
3 images. Each sample was coded such that the operator was
unaware of what subject or group corresponded to the image
being analyzed.
Muscle fiber composition of subjects with the
metabolic syndrome
Previous studies have found a lower proportion of type
I fibers in vastus lateralis muscle biopsies from type 2 diabetes (7, 8) and in metabolic syndrome subjects (9). With
this relatively large group of sedentary controls and metabolic syndrome subjects of both genders, the authors of
this study hoped to establish any difference in the proportion of type I, type IIa, and type IIx fibers in subjects with
the metabolic syndrome. Figure 1 summarizes the fiber
composition of the participants in this study. Metabolic
syndrome subjects had lower type I fiber content and
higher type IIa fiber content than the sedentary control
subjects. Average fiber diameters ranged from 51 to 78
␮m, with female control type IIa being the smallest and
male metabolic syndrome type IIx being the largest. Males
had consistently larger fiber diameters for all 3 types (7%–
33% larger). Only IIa fibers were significantly larger (66 ⫾
4 vs 58 ⫾ 3 ␮m, P ⫽ .05) in the metabolic syndrome group
including men and women.
Statistics
All data are displayed as mean ⫾ SEM, except as explicitly
indicated. Comparing data between the 2 groups was performed using the independent t test except as noted. Relationships between select variables were assessed using a Pearson correlation coefficient. Statistical procedures were
performed using SigmaPlot version 12.2 from Systat Software
(San Jose, California).
Results
Subject characteristics
The characteristics of the control and metabolic syndrome participants are displayed in Table 1. All of the
listed parameters were significantly different between the
2 groups, although comparisons of male with male or female with female were not always different.
Table 1.
Insulin responsiveness and muscle fiber
composition
The mean SSGIR achieved during euglycemic clamp
was 2.4 ⫾ 0.2 mg/kg/min for the metabolic syndrome
group and 6.1 ⫾ 0.5 mg/kg/min for the controls (P ⬎
Subject Characteristicsa
Controls
Number of
subjects
Age, y
BMI, kg/m2
Waist, cm
% fat
Systolic BP, mm
Hg
Diastolic BP, mm
Hg
Serum glucose,
mg/dL
Serum insulin,
pmol/L
Total
cholesterol,
mg/dL
TGs, mg/dL
HDL, mg/dL
LDL, mg/dL
Metabolic Syndrome
All
Females
Males
All
Females
Males
17
10
7
22
11
11
38 ⫾ 3
23.9 ⫾ 0.8
92 ⫾ 3
30 ⫾ 2
119 ⫾ 3
38 ⫾ 4
23.6 ⫾ 1.2
90 ⫾ 4
32 ⫾ 2
119 ⫾ 4
37 ⫾ 4
24.4 ⫾ 1.1
94 ⫾ 5
27 ⫾ 4
120 ⫾ 3
45 ⫾ 2b
34.5 ⫾ 0.7b
117 ⫾ 2b
43 ⫾ 1b
132 ⫾ 4b
44 ⫾ 3
33.6 ⫾ 0.7b
114 ⫾ 2b
46 ⫾ 1b
131 ⫾ 6
45 ⫾ 3
35.5 ⫾ 1.1b
120 ⫾ 3b
41 ⫾ 1b
132 ⫾ 4b
76 ⫾ 3
76 ⫾ 3
76 ⫾ 5
84 ⫾ 2b
84 ⫾ 3
85 ⫾ 3
91 ⫾ 3
88 ⫾ 3
96 ⫾ 6
103 ⫾ 3b
99 ⫾ 5
107 ⫾ 4
46 ⫾ 7
42 ⫾ 7
51 ⫾ 14
83 ⫾ 11b
111 ⫾ 21b
169 ⫾ 6
170 ⫾ 11
168 ⫾ 6
196 ⫾ 9b
198 ⫾ 14b
193 ⫾ 12b
127 ⫾ 22
50 ⫾ 3
94 ⫾ 6
108 ⫾ 20
57 ⫾ 2
91 ⫾ 8
154 ⫾ 44
39 ⫾ 4
98 ⫾ 10
181 ⫾ 29b
41 ⫾ 2b
119 ⫾ 8b
196 ⫾ 49b
45 ⫾ 2b
114 ⫾ 12b
159 ⫾ 26
37 ⫾ 3
124 ⫾ 10b
96 ⫾ 12b
Abbreviations: BP, blood pressure; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; TG, triglyceride.
a
Waist represents waist circumference at the umbilicus, percent fat represents body composition data by air plethysmography. All samples
(glucose, insulin, and lipids) were obtained after a 10-hour fast.
b
Significant difference from the corresponding control group (P ⬍ .05).
doi: 10.1210/jc.2012-3876
.001). The severity of insulin resistance is indicated by the
61% lower SSGIR and is also reflected by the more than
2-fold higher (⫹109%) fasting serum insulin shown in
Table 1. The insulin infusion (40 mU/m2/min) resulted in
an increment in serum insulin of 44.0 ⫾ 3.3 ␮U/mL
(13.8 ⫾ 1.7 to 57.7 ⫾ 4.4) in metabolic syndrome subjects
and an increment of 45.1 ⫾ 3.6 (6.5 ⫾ 1.0 to 51.6 ⫾ 3.8)
in the controls. Whole-blood glucose baseline was
102.9 ⫾ 2.9 and 91.4 ⫾ 3.2 mg/dL and in the last 30
minutes of the clamp study was 89.6 ⫾ 1.4 and 88.6 ⫾
2.2 mg/dL for the metabolic syndrome subjects and controls, respectively. The ratio of SSGIR to change in insulin concentration was 0.144 ⫾ 0.014 mg/kg/min per
␮U/mL for controls and 0.059 ⫾ 0.006 mg/kg/min per
␮U/mL (41 ⫾ 4% of control mean) for the metabolic
syndrome subjects.
The SSGIR correlated with several of the assessments
made in characterizing the participants. Fasting serum insulin inversely correlated strongly with SSGIR (R ⫽
Figure 2. Muscle type I fiber content predicts insulin responsiveness
and aerobic fitness. A, Graph of the SSGIR plotted against the type I
fiber percentage in the muscle biopsy specimens from 17 controls and
22 subjects with the metabolic syndrome. The open symbols represent
the controls, and the filled symbols represent the subjects with the
metabolic syndrome. R is the correlation coefficient and p is the
probability of the null hypothesis. B, VO2max plotted vs the percentage
of type I fibers in the muscle biopsies from the same controls and
metabolic syndrome subjects displayed in A.
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⫺0.527, P ⬍ .001). Figure 2 displays the correlation of
SSGIR with the type I fiber content. The content of type I
fibers in the muscle biopsy also correlated with VO2max
as shown in Figure 2B. Correlations between type I fiber
content and SSGIR and VO2max were no longer significant if the metabolic syndrome group or the control group
were analyzed separately.
There were other expected strong relationships that
were confirmed including BMI and percent body fat that
inversely correlated with SSGIR and VO2max. SSGIR and
VO2max strongly inversely correlated with BMI (r ⫽
⫺0.778, P ⬍ .001, and r ⫽ ⫺0.603, P ⬍ .001, respectively)
and with percent fat (r ⫽ ⫺0.792, P ⬍ .001, and r ⫽
⫺0.779, P ⬍ .001, respectively).
In the metabolic syndrome group, the proportion of
muscle fiber type I correlated with age (r ⫽ 0.522, P ⫽
.013). This relationship was not present in the control
group (r ⫽ 0.048, P ⫽ .856) or in the calculation with the
2 groups combined (r ⫽ 0.125, P ⫽ .447). The ages ranged
from 23 to 55 years in the metabolic syndrome subjects
and 24 to 54 years in the control group, but there were
more controls in their 20s. Dropping the youngest 6 control subjects from the data set allowed the mean age to rise
to 44 years, equivalent to the metabolic syndrome group.
Recalculating the relationships of the key data using this
smaller control group showed very similar correlations for
BMI and percent fat with SSGIR and VO2max. The correlation of the percentage of type I fibers was still present
(r ⫽ 0.351, P ⫽ .045) but with VO2max was no longer
significant (r ⫽ 0.290, P ⫽ .102).
Analysis of this muscle histology data set demonstrated other correlations of interest. The size of each
fiber was greater in males than females. The type IIx
tended to be larger than types I or IIa in the metabolic
syndrome subjects. Type IIa diameters tended to be
smaller than types I or IIx in both groups. Both type II
fiber types tended to be larger in metabolic syndrome
females compared with control females. Across all subjects, increasing fasting serum insulin concentrations
directly correlated with larger type IIa and IIx fiber diameters (r ⫽ 0.519, P ⬍ .001, and r ⫽ 0.426, P ⫽ .007).
Content of insulin receptor, IRS-1, and GLUT4 in
muscle from metabolic syndrome subjects
Muscle homogenate was subjected to polyacrylamide
electrophoresis with 10 ␮g protein per lane. After transfer
to membranes and incubation overnight with the primary
antibody, digital images were obtained and signal intensity was quantified. Figure 3 displays sample immunoblots, and the graph shows the summary of these studies.
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Figure 3. Muscle content of insulin receptor, IRS-1, and GLUT4.
Shown here are sample immunoblots for the 3 proteins in muscle
homogenates. In the blots shown, the first and third bands were from
control subject muscle. The second and fourth bands were from
metabolic syndrome muscle. None of expression of these proteins in
metabolic syndrome muscle was significantly different from that of the
control subjects. Each subject’s muscle sample was run in a minimum
of 3 separate analyses.
The metabolic syndrome subjects’ muscle expression of
insulin receptor, IRS-1, and GLUT4 was not different
from that of the controls.
Muscle fiber-specific content of insulin receptor,
IRS-1, GLUT4, and ATP synthase
Because the effectiveness of insulin to augment
whole-body glucose uptake in metabolic syndrome subjects was less than half that of the controls, it was expected there would be a dramatic defect in the muscle
pathways of insulin action. In muscle homogenates used
in immunoblots, insulin receptor and GLUT4 expression have shown little or no decrease in obesity or type
2 diabetes (21, 22). The following studies were to determine whether there were fiber-specific differences
(ie, type I fiber decreased insulin action pathway components) that would not be seen when homogenized
muscle was used.
Immunohistochemistry and analysis of images from
the Leica confocal microscope were performed on muscle sections from each subject in both groups of participants. Figure 4 displays examples of confocal images of
muscle specimens evaluated using specific rabbit polyclonal antibodies against human insulin receptor, human IRS-1, and human GLUT4, each used in concert
with mouse monoclonal antibodies against slow-twitch
J Clin Endocrinol Metab, May 2013, 98(5):2027–2036
myosin heavy chain. ATP synthase was assessed using a
specific monoclonal antibody with rabbit polyclonal
antifast myosin heavy chain antibodies to identify fiber
type. Evaluations of images similar to these were performed for each subject.
Figure 5, A–D, displays the means and SEs for the intensity
of the signal generated in each of the 3 muscle fiber types for
insulin receptor, IRS-1, GLUT4, and ATP synthase. Figure 5,
E–H, shows the same data adjusted for the fiber content and
size for each subject to reflect the total protein content in each
of the fiber types for the 2 subject groups.
Even though the metabolic syndrome muscle signal intensity was only slightly less in type I fibers, when calculated as the total relative amount contributed by the type
I subset of fibers, the decrease was significant. This calculated decrease is driven by the decreased portion of type
I fibers in the metabolic syndrome subject muscle.
The total insulin receptor in the muscle of metabolic
syndrome subjects determined by adding the amount in
each fiber type (Figure 5E) was 19% less than that of the
control subjects (P ⫽ .030). The metabolic syndrome muscle total IRS-1 (Figure 5F) and GLUT4 (Figure 5G) calculated this way were less than controls by 23% (P ⫽ .028)
and 38% (P ⫽ .048), respectively.
There were inverse relationships between fasting serum
insulin and the expression in type I fibers of the insulin
receptor (r ⫽ ⫺0.301, P ⫽ .070), IRS-1 (r ⫽ ⫺0.336, P ⫽
.052), and GLUT4 (r ⫽ ⫺0.315, P ⫽ .065), although none
of these achieved statistical significance. Paradoxically,
higher expression of the insulin receptor in type IIa fibers
corresponded with higher serum insulin concentrations
(r ⫽ 0.628, P ⫽ .038).
ATP synthase expression in type I, type IIa, and
type IIx muscle fibers
ATP synthase (also called complex V) is expressed in
mitochondria. Immunoblots of muscle homogenates
showed expression of ATP synthase was not different between control muscle and muscle from the metabolic syndrome subjects (100% ⫾ 20% vs 90% ⫾ 11% of baseline
control, respectively). Immunohistochemistry studies of
ATP synthase expression in the 3 types of muscle fibers
(sample slide images are shown in Figure 4, J–L) showed
that ATP synthase was expressed at 47% to 59% higher
levels in type IIx fibers relative to type I fibers in metabolic
syndrome subjects and controls. In contrast to the muscle
homogenate immunoblot data, control muscle expressed
ATP synthase at higher levels than metabolic syndrome
muscle in IIx and IIa fiber types (Figure 5D). When adjusted for the fiber diameter and proportion, the estimate
of total ATP synthase in type I and type IIx fibers was
lower in metabolic syndrome muscle but was greater in the
doi: 10.1210/jc.2012-3876
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(0.899% ⫾ 0.018% vs 0.873% ⫾
0.017% and 27.5% ⫾ 5.5% vs
35.7% ⫾ 5.6%, respectively, with P
values of .294 and .220).
Discussion
The subjects with the metabolic syndrome reported here have profound
insulin resistance reflected by fasting
hyperinsulinemia and euglycemic
clamp SSGIRs. The muscle of these
participants is different from that of
similarly sedentary control subjects
in important ways. Nondiabetic, insulin-resistant, metabolic syndrome
subject muscle had significantly
fewer type I muscle fibers. The fiber
composition of metabolic syndrome
Figure 4. Muscle fiber type-specific expression of insulin receptor, IRS-1, GLUT4, and ATP
synthase. Shown here are confocal microscopy images from 4 separate biopsy specimens labeled
subjects was essentially the same as
with antibodies against slow-twitch myosin heavy chain (stained blue) and antibodies specific to
that of patients with type 2 diabetes
the human insulin receptor ␤-subunit (green), IRS-1 (green), GLUT4 (red), or ATP synthase (blue).
(7). The insulin receptor, IRS-1, and
Panels A–C are from a single portion of a slide viewed on the confocal microscope. Panels D–F,
similarly, are one confocal view of another slide, as are panels G–I and J–L. A–C, Insulin receptor
GLUT4 were modestly decreased in
signal (A), slow-twitch myosin (B), and composite view of both antibodies (C). D–F, Antihuman
type I fibers and were slightly deIRS-1 (D), slow-twitch myosin (E), and composite image with IRS-1 and slow-twitch myosin signal
creased in whole muscle. The slow(F). G–I, Human GLUT4 signal (G), slow-twitch myosin (H), and composite image with GLUT4 and
the slow-twitch myosin heavy chain label (I). J–L, Signal from the ATP synthase-specific antibody
twitch, type I fibers represent a
(J), image of the same section probed with rabbit antifast twitch myosin heavy chain (K), and
smaller portion of the muscle in these
composite image of those of J and K (L). Note that in each of the 4 subjects displayed here, the
metabolic syndrome subjects and a
type IIa fibers can be identified in B, E, H, and K as the intermediate intensity stained fibers. Scale
smaller portion of key elements of
bar in J, 100 ␮m. All panels are at the same magnification.
the pathways of insulin action. That
the components of the insulin pathtype IIa fibers (Figure 5H). Not all subjects had more miway residing in type I fibers are especially important is
tochondria in type II fibers. Four controls and 3 metabolic
suggested by the correlation of insulin responsiveness (eusyndrome subjects had more ATP synthase expression in
glycemic clamp data of Figure 2A) with the portion of
type I fibers, but the means for the groups showed more in
muscle that is made up of type I fibers. This contrast in type
type II fibers.
I fiber content of insulin pathway components was driven
Immunohistochemical studies using cytochrome c anprimarily by the difference in the proportion of fiber types
tibodies confirmed the findings, although only a few subbecause the immunohistochemical signal intensity and the
jects had both ATP synthase and cytochrome c done. Samfiber sizes were similar. VO2max, a measure of aerobic
ple images are included in Supplemental Figure 2. The
confocal fluorescence window settings for the 488-, 555-, fitness, also correlated with the proportion of type I fibers
and 647-nm wavelength-generating probes were such that making up the vastus lateralis muscle.
Nyholm and coworkers (23) from the Copenhagen
there was no bleedover between the ATP synthase and fast
myosin heavy chain antibodies or the GLUT5 antibodies, Muscle Research Centre published results from a study of
eliminating a potential artifact. In addition, we confirmed similar design in 1997. Their subjects were 25 nonobese
in pilot studies of sequential sections that the fast- and relatives of patients with type 2 diabetes and 21 ageslow-twitch myosin heavy chain antibodies gave the same matched controls. Adjusting their euglycemic clamp data
fiber type identification as the pH 4.3 and 9.4 ATPase to body weight rather than lean body mass gave a control
GIR of 6.39 mg/kg/min and the relatives of diabetes pamethods, as reported by Behan and coworkers (18).
Neither the baseline respiratory exchange ratio nor the tients a GIR of 4.46 mg/kg/min, less insulin resistant than
fat oxidation percentage were different between the met- our obese metabolic syndrome subjects. Nyholm et al (23)
abolic syndrome and the sedentary control subjects found that the relatives of diabetes patients had a lower
2034
Stuart et al
Muscle Fiber Type and Insulin Resistance
Figure 5. Metabolic syndrome muscle type I fibers contain a lower
proportion of insulin receptor, IRS-1, GLUT4, and ATP synthase. The 4
panels on the left display the signal intensity in each of the 3 types of
fibers and compare controls and metabolic syndrome muscle. These
data were determined based on digital image analysis of
immunohistochemical studies, examples of which are shown in Figure
4. For each individual subject, an estimate of the total amount of each
of these 3 proteins associated with the specific fiber types was
calculated from the signal intensity, the percentage of each fiber type,
and the fiber size of each type. The means and SEM of these adjusted
data are shown in the panels on the right. *P value ⬍ .05 by t test. For
each of these proteins, less was present in the type I muscle fibers of
the metabolic syndrome subjects. More of IRS-1, GLUT4, and ATP
synthase was expressed in the IIa fibers. GLUT4 was significantly less in
both type I and type IIx fibers of metabolic syndrome subjects. The
mitochondrial marker data shown in D and H represent only the most
recently studied 18 subjects (7 controls and 11 metabolic syndrome
subjects), whereas the other panels represent the entire subject
groups. Unexpectedly, the IIx fibers expressed more mitochondrial
marker than the type I fibers in both groups. The difference between
type I and IIx ATP synthase signal intensity reached statistical
significance in the metabolic syndrome muscle only (P ⫽ .039, paired t
test, vs P ⫽ .101 for control muscle). As with the insulin receptor, IRS1, and GLUT4 data, the total ATP synthase expression in each fiber
type adjusted for fiber number and size was significantly different in
the metabolic syndrome muscle.
proportion of type I muscle fibers than the controls (41%
vs 44%, P ⫽ .37) and a lower proportion of type IIa fibers
(29% vs 34%, P ⫽ .20) and a higher proportion of type IIb
fibers (30% vs 21%, P ⬍ .05). That our type II fiber composition data were different with higher IIa and lower IIx
proportions is likely due to underestimation of the IIa con-
J Clin Endocrinol Metab, May 2013, 98(5):2027–2036
tent by the ATPase pH methods used in the earlier study.
Nyholm and coworkers (23) found a correlation similar
to what we found relating fiber type with insulin responsiveness and VO2max. Glucose rate of disposal,
Rd, correlated with the percent type I fibers (r ⫽ 0.39,
P ⬍ .01). The correlation of VO2max with type I was
also significant in their study (r ⫽ 0.47, P ⬍ .01). The
type I fiber content, GIR, and VO2max data from nonobese relatives of patients with diabetes are similar to the
data we present for metabolic syndrome subjects, and
the correlations of percent type I muscle fibers with GIR
(Rd in their study) and VO2max show the same relationship in both studies.
In the study reported here, the sedentary controls were
somewhat younger than the metabolic syndrome subjects.
However, because the proportion of type I fibers tended to
increase with age in the metabolic syndrome group, higher
age might be expected to lessen the difference from
controls.
What is unique about type I fibers that would make their
proportion reflect whole-body insulin responsiveness? Type
I fibers classically contain more mitochondria that in turn
mediate fat oxidation. Unexpectedly, both metabolic syndrome and control subjects had more mitochondrial marker
enzymes in type II fibers. Why this occurs is puzzling, but the
authors speculate that it may be related to the very sedentary
lifestyle of all of our subjects before the study. Compared
with metabolic syndrome subjects, the control subjects
tended to have more mitochondria in muscle homogenate,
more mitochondria in each muscle fiber type, and higher
whole-body fat oxidation.
When and how the fiber proportion is determined and
why long-term training in man does not result in significant change are important questions that are not resolved.
Normal human skeletal muscle is made up of nearly equal
numbers of slow-twitch (type I, endurance, fatigue resistant) and fast-twitch (type II, strength) fibers that are arranged in a roughly checkerboard pattern (19). Fiber composition in humans is largely fixed and not amenable to
training-related changes other than minor shifts between
IIx and IIa (24). Humans, in contrast to mice, appear to
have the skeletal muscle fiber composition determined before birth (25, 26). This may be entirely genetically determined, or there could be an epigenetic modification of the
intrinsic genes or posttranslational modification of key
development regulatory factors by maternal nutrition and
activity (27, 28).
The reason that a higher proportion of type I fibers
leads to better insulin responsiveness or higher utilization
of oxygen during exercise is elusive. There are modestly
more insulin receptors, IRS-1, and GLUT4 in control subject type I fibers, but the overall muscle content of these
doi: 10.1210/jc.2012-3876
factors is not different from that present in the muscle of
metabolic syndrome subjects. Mitochondria appear to be
expressed more in type II fibers, and the higher amount of
ATP synthase in control muscle fibers is modest. The percentage of fat oxidation was not different between the 2
groups. It is possible that several modest differences that
were observed add up to a large difference in insulin responsiveness, but in situations such as these, additional
mechanisms should still be sought.
The data presented here document hyperinsulinemia
and severe insulin resistance in men and women with
central obesity and a family history of type 2 diabetes.
These subjects all met the criteria for the designation
metabolic syndrome (4). Biopsy specimens from the vastus lateralis muscle show 21% fewer type I muscle fibers
but a more than 2-fold increase in the mixed type IIa
fibers in these subjects. The metabolic syndrome type I
muscle fiber contributed 19% less insulin receptor,
23% less IRS-1, and 38% less GLUT4, compared with
control type I fiber. The decrease in whole-body insulin
responsiveness (60%) is out of proportion to the diminished insulin receptor, IRS-1, and GLUT4, suggesting
that there is a functional road block in another element
of the pathway of insulin action. Thus, the full explanation of the mechanism of insulin resistance in metabolic syndrome subjects remains elusive, but the search
may be narrowed now to include the phosphorylation
status of IRS-1 and expression and activation of downstream targets such as subunits of phosphatidylinositol-3 kinase, Akt, and protein kinase C.
Acknowledgments
We thank the subjects who volunteered for these studies, without whose participation these data would not be available.
The friendly cooperation of the ETSU Physician and Associates clinic staff in performing the muscle biopsies and insulin
infusions was very valuable to the research team. Research
nurses Susie Cooper Whitaker and Mary Ward were invaluable in this effort.
Address all correspondence and requests for reprints to:
Charles A. Stuart, MD, East Tennessee State University, Quillen
College of Medicine, P.O. Box 70622, Johnson City, Tennessee
37614-0622. E-mail: [email protected].
These studies were funded by a grant from the National Institutes of Health (DK080488) to C.A.S., M.H.S., and M.W.R.
C.A.S., M.W.R., and M.H.S. designed the study; C.A.S.,
A.M., M.A.S., A.S.L., M.W.R., and M.H.S. performed clinical
testing; C.A.S., M.P.M., and M.E.A.H. performed bench studies;
C.A.S., M.P.M., M.A.S., M.E.A.H., and A.S.L. did data analysis;
C.A.S. prepared the manuscript; and all authors reviewed, edited, and approved the final manuscript.
jcem.endojournals.org
2035
Disclosure Summary: The authors have no conflicts of interest to disclose.
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