Myotubes from lean and severely obese subjects

Am J Physiol Cell Physiol 308: C548 –C556, 2015.
First published January 22, 2015; doi:10.1152/ajpcell.00314.2014.
Myotubes from lean and severely obese subjects with and without type 2
diabetes respond differently to an in vitro model of exercise
Yuan Z. Feng,1 Nataša Nikolić,1 Siril S. Bakke,1 Eili T. Kase,1 Kari Guderud,1 Jøran Hjelmesæth,2,3
Vigdis Aas,4 Arild C. Rustan,1 and G. Hege Thoresen1,5
1
Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway; 2The Morbid Obesity
Center, Vestfold Hospital Trust, Tønsberg, Norway; 3Department of Endocrinology, Morbid Obesity and Preventive Medicine,
Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 4Faculty of Health, Oslo and Akershus University College of
Applied Sciences, Oslo, Norway; and 5Department of Pharmacology, Institute of Clinical Medicine, Faculty of Medicine,
University of Oslo and Oslo University Hospital, Oslo, Norway
Submitted 17 September 2014; accepted in final form 20 January 2015
in vitro exercise model; insulin sensitivity; myotubes from severely
obese subjects; oleic acid and glucose metabolism; PPAR␦ activation
PHYSICAL ACTIVITY plays an important role in both prevention
and treatment of obesity and type 2 diabetes (T2D). Skeletal
muscle is the largest insulin-sensitive organ in humans, accounting for ⬎80% of insulin-stimulated glucose disposal (15).
Physical training leads to extensive adaptations in skeletal
muscles (14, 46), but the molecular mechanisms underlying
these adaptations are still poorly understood, and there is
increasing evidence that some individuals may be inherently
less responsive to exercise (20, 53). Regular physical activity
Address for reprint requests and other correspondence: Y. Z. Feng, School
of Pharmacy, P.O. Box 1068 Blindern, 0316 Oslo Norway (e-mail:
[email protected]).
C548
not only improves oxidative capacity, such as increased lipid
and glucose oxidation (39), but is also known to increase
insulin-stimulated glucose uptake (45). The effect of exercise
on substrate oxidation is less clear in obese and type 2 diabetic
subjects than in lean subjects (37). Impairments have been
noted in skeletal muscle glucose and fatty acid oxidation in
obesity and T2D in the resting state (6, 29, 30). It is therefore
important to determine whether interventions, either exercise
or pharmacologically, can effectively reverse this impairment.
Plasticity of skeletal muscle in response to regular exercise
extends beyond the metabolic changes, and peroxisome proliferator-activated receptor-␦ (PPAR␦) activation is one of many
pathways proposed to be involved in exercise adaptations
(15, 35). PPAR␦ is an important regulator of skeletal muscle
metabolism, in particular lipid oxidation. It is well known that
endurance exercise increases mitochondrial content (46, 54)
and that myosin heavy chain type I, slow-twitch oxidative
(MHCI) muscle fibers are associated with higher mitochondrial
content compared with MHCII (type II, fast-twitch glycolytic)
fibers (32). On the other hand, it is debated whether conversion
of muscle type II to type I phenotype actually happens in adult
humans (22).
Moreover, skeletal muscle has been identified as a secretory
organ that releases a diversity of biologically active proteins
classified as myokines that can have autocrine, paracrine, or
endocrine functions. Muscle contraction during exercise is a
major stimulus of these endocrine functions, and the myokines
are thought to mediate beneficial effects of exercise and may
have a role in the protection against conditions associated with
low-grade inflammation, such as T2D and metabolic syndrome
(38, 42, 56). The myokines, interleukin-6 (IL-6) and IL-8, are
markedly produced in contracting muscles and, at least for
IL-6, released into plasma during the postexercise period (2,
51) when the insulin sensitivity is enhanced (23). It seems that
IL-6 works as an energy sensor and preserves fuel availability
during exercise (40) by enhancing insulin-stimulated glucose
disposal and fatty acid metabolism (10, 55). Interestingly,
while IL-6 released from skeletal muscle may promote insulin
sensitivity (21), IL-6 secreted from adipose tissue may induce
insulin resistance in muscle (41). Thus, while it has become
evident that contracting skeletal muscle releases myokines that
may influence metabolism and function of muscle tissue and
other tissues and organs, the secretion of myokines from obese
and/or diabetic muscle is yet to be fully clarified.
Previously, we have established an in vitro model of regular
exercise of cultured human skeletal muscle cells (myotubes) by
0363-6143/15 Copyright © 2015 the American Physiological Society
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Feng YZ, Nikolić N, Bakke SS, Kase ET, Guderud K, Hjelmesæth J, Aas V, Rustan AC, Thoresen GH. Myotubes from lean and
severely obese subjects with and without type 2 diabetes respond
differently to an in vitro model of exercise. Am J Physiol Cell Physiol
308: C548 –C556, 2015. First published January 22, 2015;
doi:10.1152/ajpcell.00314.2014.—Exercise improves insulin sensitivity and oxidative capacity in skeletal muscles. However, the effect of
exercise on substrate oxidation is less clear in obese and type 2
diabetic subjects than in lean subjects. We investigated glucose and
lipid metabolism and gene expression after 48 h with low-frequency
electrical pulse stimulation (EPS), as an in vitro model of exercise, in
cultured myotubes established from lean nondiabetic subjects and
severely obese subjects (BMI ⱖ 40 kg/m2) with and without type 2
diabetes. EPS induced an increase in insulin sensitivity but did not
improve lipid oxidation in myotubes from severely obese subjects.
Thus, EPS-induced increases in insulin sensitivity and lipid oxidation
were positively and negatively correlated to BMI of the subjects,
respectively. EPS enhanced oxidative capacity of glucose in myotubes
from all subjects. Furthermore, EPS reduced mRNA expression of
slow fiber-type marker (MYH7) in myotubes from diabetic subjects;
however, the protein expression of this marker was not significantly
affected by EPS in either of the donor groups. On the contrary, mRNA
levels of interleukin-6 (IL-6) and IL-8 were unaffected by EPS in
myotubes from diabetic subjects, while IL-6 mRNA expression was
increased in myotubes from nondiabetic subjects. EPS-stimulated
mRNA expression levels of MYH7, IL-6, and IL-8 correlated negatively with subjects’ HbA1c and/or fasting plasma glucose, suggesting
an effect linked to the diabetic phenotype. Taken together, these data
show that myotubes from different donor groups respond differently
to EPS, suggesting that this effect may reflect the in vivo characteristics of the donor groups.
MYOTUBES FROM DIFFERENT DONORS RESPOND DIFFERENTLY TO EPS
MATERIALS AND METHODS
Human myotubes. Human myotubes grown from satellite cells
were isolated as previously described (18) from the musculus obliquus
internus abdominis from lean or severely obese subjects. The lean
nondiabetic donor biopsies were obtained from subjects donating a
kidney at Oslo University Hospital, Norway and the severely obese
donor biopsies were obtained from subjects undergoing bariatric
surgery at The Morbid Obesity Center, Vestfold Hospital Trust,
Norway. The isolation of satellite cells from all biopsies was performed at the same location and by the same researchers. The biopsies
were obtained with written informed consent and were approved by
the National Committee for Research Ethics, Oslo, Norway (S-04133
and S-09078d).
Classification of the severely obese donors into the two groups,
severely obese nondiabetic subjects (SO-nD) and severely obese
subjects with T2D (SO-T2D), was based on fasting plasma glucose
values (ⱖ7.0 mmol/l), HbA1c (glycosylated hemoglobin, ⱖ6.5%),
and/or use of one or more antidiabetic drugs. The cells were cultured
in DMEM-Glutamax (5.5 mmol/l glucose) with supplements during
proliferation and differentiation as previously described (25). Briefly,
at ⬃80% confluence, growth medium was replaced by differentiation
medium to induce fusion of myoblasts into multinucleated myotubes.
Experiments were performed on cells from passage 2 to 5 and after 7
days of differentiation. There was no difference in mRNA expression
levels of muscle fiber type I marker between the myotubes from lean
nondiabetic and severely obese subjects or between the myotubes
from severely obese nondiabetic and diabetic subjects (data not
shown).
Electrical pulse stimulation of human myotubes. Human myotubes
grown on Corning CellBIND six-well plates (Corning Life-Sciences,
Schiphol-Rijk, The Netherlands) were stimulated via carbon electrodes by applying chronic, low-frequency EPS (single, bipolar pulses
of 2 ms, 30 V, 1 Hz) continuously for the last 48 h of the differentiation period, as previously described (36). Culture media were
changed every 12 or 24 h during EPS. Electrical pulses were generated by a muscle stimulator built at the Electronics Lab, Institute of
Chemistry, University of Oslo, Norway. Neither applying EPS for 48
h to the myotubes nor changing the media every 12 or 24 h showed
differences in floating cells evaluated by microscopic inspection or by
measurement of cell protein content in the three donor groups (data
not shown).
Immunoblotting. After EPS, cells were harvested in Laemmli
buffer (0.5 mol/l Tris·HCL, 10% SDS, 20% glycerol 10% ␤-mercaptoethanol, and 5% Bromophenol blue). Total cell lysates were electrophoretically separated on 4 –20% Mini-Protean TGX gels (BioRad, Copenhagen, Denmark) with Tris/glycine buffer (pH 8.3) followed by blotting to nitrocellulose membrane and incubation with
antibodies against total Akt kinase, Akt phosphorylated at Ser473,
␤-actin, ␣-tubulin (all from Cell Signaling Technology, Beverly,
MA), MHCI (Millipore, Billerica, MA), and an OXPHOS antibody
cocktail recognizing Complex I subunit NDUFB8, Complex II subunit, Complex III subunit core 2, Complex IV subunit II and ATP
synthase subunit ␣ (Abcam, Cambridge, UK). Immunoreactive bands
were visualized with enhanced chemiluminescence (Chemidoc XRS,
Bio-Rad) and quantified with Image Lab software (version 4.0).
␤-Actin or ␣-tubulin was used to normalize the protein-antibody
signals versus protein loading.
Glucose metabolism. After EPS, myotubes were exposed to serumfree DMEM supplemented with [14C(U)]glucose (0.5 ␮Ci/ml, 1
mmol/l, PerkinElmer, Boston, MA) and 1 mmol/l pyruvate in the
presence or absence of 100 nmol/l insulin for 3 h to study basal and
insulin-stimulated glycogen synthesis as previously described (16).
Glucose oxidation was measured after 3 h of incubation, 900 ␮l cell
medium was transferred to airtight flasks, and 300 ␮l of phenyl
ethylamine:methanol (1:1, vol/vol) was added to a center well containing a folded filter paper. Subsequently, 200 ␮l of 1 mol/l perchloric acid was added to the cell medium through the stopper tops using
a syringe, as previously described (36). The flasks were placed at
room temperature to trap labeled CO2 for 2 h, and radioactivity was
counted by liquid scintillation. All data were related to protein
content.
Fatty acid oxidation. Myotubes were treated with 0.1% DMSO
(vehicle) or 10 nmol/l GW501516 for 96 h, and EPS was applied to
the cells for the last 48 h of the treatment period. After EPS, the
myotubes were exposed to 1 ml of DPBS supplemented with HEPES
(10 mmol/l), NaHCO3 (44 mmol/l), [1-14C]oleic acid (1 ␮Ci/ml, 100
␮mol/l, PerkinElmer), 40 ␮mol/l BSA, and 1 mmol/l L-carnitine in a
5% CO2 incubator at 37°C. After 2 h, CO2 and cell-associated
radioactivity were measured as previously described (36). Briefly, 500
␮l of cell medium was transferred to airtight flasks, and 300 ␮l of
phenyl ethylamine:methanol (1:1, vol/vol) was added to a center well
containing a folded filter paper. Subsequently, 100 ␮l of 1 mol/l
perchloric acid was added to the cell medium through the stopper tops
using a syringe. The flasks were placed at room temperature to trap
labeled CO2 for 2 h. The cells were placed on ice and washed twice
with PBS, lysed with 0.1 mol/l NaOH, and cell-associated radioactivity was counted by liquid scintillation to determine uptake of oleic
acid. All data were related to protein content.
Live imaging. After EPS, the myotubes were incubated at 37°C and
5% CO2 with MitoTracker Red FM (100 nmol/l, Invitrogen, Carlsbad,
CA) for 15 min to stain mitochondria, Bodipy 493/503 (2 ␮g/ml,
Invitrogen) for 15 min to stain neutral lipids and Hoechst 33258 (2.5
␮g/ml, Invitrogen) for 15 min to stain nuclei and washed with PBS in
between. Live imaging was carried out as previously described (25).
Images were randomly taken in 25–36 positions per well with a ScanR̂
platform (Olympus IX81 inverted fluorescence microscope) equipped
with a temperature incubator for long-term live imaging. After the
aggregates and dead cells were gated out, each parameter was determined from ⬃240 images per donor group (average of 38 ⫾ 4 nuclei
per image). MitoTracker Red FM and Bodipy 493/503 intensity per
image was related to number of nuclei per image since for multinucleated myotubes the absolute number of cells cannot be determined.
This is an accepted method to account for differences in cell numbers
(13, 28, 34, 36). For the myotubes from lean nondiabetic subjects
(L-nD), some of the cells were also treated with GW501516 for 96 h
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applying chronic, low-frequency electrical pulse stimulation
(EPS) (36), thereby enabling the study of exercise-induced
cellular mechanisms under controlled conditions. Human myotubes are considered a valid model for studying metabolic
disorders as obesity and T2D because perturbances evident in
vivo, such as depressed lipid oxidation and insulin resistance,
are retained in myotubes in culture (1, 7, 19). After EPS,
myotubes established from lean nondiabetic subjects showed
an increase in glucose and lipid metabolism as well as indications of fast-twitch glycolytic muscle fiber transformation into
slow-type oxidative fibers (36) and enhanced expression and
release of IL-6 and IL-8 (31, 44, 48). In the present study, we
wanted to explore whether there are differences in EPS response on insulin sensitivity, glucose and lipid metabolism,
and gene expression in myotubes from three groups of donors—lean nondiabetic, severely obese nondiabetic, and severely obese subjects with established T2D—and whether
these differences reflect the in vivo characteristics of the donor
groups. Moreover, PPAR␦ agonists and exercise training are
shown to act synergistically to increase the content of oxidative
muscle fibers, and PPAR␦ agonists have been proposed as
exercise mimetics (35). Therefore, we wanted to explore the
combined effect of EPS together with the PPAR␦ agonist,
GW501516, on lipid metabolism as well.
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MYOTUBES FROM DIFFERENT DONORS RESPOND DIFFERENTLY TO EPS
RESULTS
Clinical characteristics of the biopsy donors. Clinical characteristics of the donors are presented in Table 1. Fasting
plasma glucose and HbA1c were significantly higher in severely obese subjects with T2D (SO-T2D) than in nondiabetic
subjects with similar body mass index (SO-nD). Fasting
plasma levels of insulin, triacylglycerol, and high-density lipoprotein were not different between the two obese groups. As
expected, clinical characteristics of lean nondiabetic subjects
(L-nD) were significantly different from severely obese subjects. The age was not significantly different between the three
donor groups.
Effect of EPS on insulin sensitivity in myotubes. Myotubes
from severely obese subjects with T2D maintained their diaTable 1. Clinical characteristics of biopsy donors
Age, yr
BMI, kg/m2
Fasting glucose,
mmol/l
HbA1c, %
Insulin, pmol/l
TAG, mmol/l
HDL, mmol/l
L-nD (n ⫽ 14)
SO-nD (n ⫽ 9)
SO-T2D (n ⫽ 9)
49 ⫾ 4
24.3 ⫾ 0.9
41 ⫾ 2
42.0 ⫾ 1.7*
48 ⫾ 3
44.6 ⫾ 2.2*
5.6 ⫾ 0.3
4.8 ⫾ 0.1
5.5 ⫾ 0.1
89.4 ⫾ 16
1.6 ⫾ 0.2*
1.1 ⫾ 0.1*
8.2 ⫾ 0.5†
7.2 ⫾ 0.6 ‡
62.4 ⫾ 18
2.0 ⫾ 0.3*
1.0 ⫾ 0.1*
1.0 ⫾ 0.1
1.6 ⫾ 0.1
Values are means ⫾ SE. Glycosylated hemoglobin (HbA1c) and plasma
insulin were not measured in the lean nondiabetic cohort (L-nD). *Significantly different from the lean group; †significantly different from both of the
nondiabetic groups; ‡significantly different from severely obese nondiabetic
(SO-nD) (P ⬍ 0.05). SO-T2D, severely obese with type 2 diabetes; BMI, body
mass index; TAG, triacylglycerol; HDL, high-density lipoprotein.
betic phenotype in culture shown as absence of insulin-stimulated glucose uptake in myotubes from SO-T2D, and furthermore, phosphorylation of Akt tended to be lower in myotubes
from SO-T2D compared with SO-nD (4). To examine whether
EPS could increase insulin sensitivity, insulin-stimulated phosphorylation of Akt (Ser473) and glycogen synthesis were
measured in myotubes established from L-nD, SO-nD, and
SO-T2D after EPS stimulation. In absence of insulin, phosphorylation of Akt was not affected by EPS (Fig. 1A), whereas
insulin-stimulated phosphorylation of Akt was significantly
increased by EPS in myotubes from SO-T2D (Fig. 1B). In the
absence of insulin, glycogen synthesis was not affected by EPS
(Fig. 1C), whereas insulin-stimulated glycogen synthesis after
EPS was increased in myotubes from both SO-nD and SO-T2D
subjects (Fig. 1D). Furthermore, EPS-induced effect on insulin-stimulated phosphorylation of Akt and glycogen synthesis
in the myotubes correlated positively with BMI of the subjects
(Fig. 1, E and F), i.e., the EPS effect is increased with increasing
BMI of the donors.
Effect of EPS on glucose and oleic acid metabolism. We
wanted to explore whether there were differences in the EPSinduced response on oxidative capacity in myotubes established from lean, nondiabetic, and severely obese subjects with
and without T2D. Glucose oxidation after EPS was increased
in myotubes from both lean and severely obese subjects (Fig. 2A),
whereas EPS had an effect on oleic acid oxidation in myotubes
from lean subjects only (Fig. 2B). Furthermore, oleic acid
oxidation after EPS was significantly different in myotubes
from lean subjects compared with myotubes from severely
obese subjects (Fig. 2B), and the EPS-induced effect on oleic
acid oxidation correlated also negatively with the BMI of the
subjects irrespective of donor group (Fig. 2C). Mitochondrial
content shown as MitoTracker intensity related to nuclei was
significantly increased by EPS in myotubes from L-nD subjects
(Fig. 2D), but not in myotubes from severely obese subjects
(SO-nD, P ⫽ 0.076; SO-T2D, P ⫽ 0.32). Also the overall
expression levels of proteins involved in mitochondrial oxidative phosphorylation (OXPHOS proteins) were significantly
increased by EPS in myotubes from L-nD subjects (Fig. 2, E
and F). Without EPS, lipid droplet number (Fig. 2G) and
neutral lipid content (data not shown) measured with Bodipy
493/503 were not different between the myotubes from lean
nondiabetic and severely obese subjects, or between myotubes
from severely obese nondiabetic and diabetic subjects. EPS
revealed no further differences in lipid droplet number between
the groups (Fig. 2G). In line with this, cellular uptake of oleic
acid, assessed as the sum of cell-associated and CO2-trapped
radioactivity, was not affected by EPS in either of the donor
groups (Fig. 2H).
Effect of EPS on oleic acid metabolism in the presence of
PPAR␦ activation. To examine whether the PPAR␦ agonist
GW501516 and exercise could act synergistically in our cell
model, myotubes were exposed to both EPS and GW501516.
PPAR␦ activation increased oleic acid oxidation in myotubes
from both lean and severely obese subjects, whereas no further
increase with GW501516 and EPS in combination compared
with GW501516 alone (Fig. 3A) was seen. Cellular uptake of
oleic acid was increased after GW501516 treatment in myotubes
from all three donor groups, whereas there was no further increase
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and EPS was applied to the cells for the last 48 h of the treatment
period.
Gene expression analysis by qPCR. After EPS, total RNA was
isolated from the myotubes, and qPCR was performed as previously
described (36). Briefly, total RNA from cells was isolated by Agilent
Total RNA isolation kit according to the supplier’s protocol. RNA
was reverse-transcribed with oligo primers using a PerkinElmer Thermal Cycler 9600, and qPCR was performed using an ABI PRISMT
7000 Detection System (Applied Biosystems, Warrington, UK). Transcription levels were normalized to the housekeeping genes acidic
ribosomal phosphoprotein P0 (36B4) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). The following forward and reverse primers
(Molecular Probes, Invitrogen, Carlsbad, CA) were used at a concentration of 30 ␮mol/l: 36B4 (accession no. M17885); GAPDH (accession no. NM_002046); IL-6 (accession no. NM_000600); IL-8 (accession no. NM_000584.2); and MYH7 (accession no. NM_
000257.2).
Data presentation and statistics. Data are presented as means ⫾
SE. Statistical analyses were performed using GraphPad Prism 5.0 for
Windows (GraphPad Software, San Diego, CA). The value n represents the number of different donors used, and each with at least
duplicate observations. Two-tailed paired Student’s t-tests were performed to determine the effects of treatments, and unpaired Student’s
t-tests (independent samples) were performed to determine the difference in effects of EPS between the donor groups. For correlation
studies, Spearman correlation analysis was performed. Linear mixed
model analysis (LMM, SPSS 20.0.0.1, IBM SPSS, Chicago, IL) was
used to determine the effect of EPS in OXPHOS experiments. The
linear mixed model includes all observations (complex I–V) in the
statistical analyses and at the same time takes into account that not all
observations are independent. P ⬍ 0.05 was considered statistically
significant.
MYOTUBES FROM DIFFERENT DONORS RESPOND DIFFERENTLY TO EPS
C551
with GW501516 and EPS in combination (Fig. 3B). Although
only studied in myotubes established from lean nondiabetic subjects, mitochondrial content measured as MitoTracker intensity
was not further increased in cells treated with both GW501516
and EPS compared with EPS alone (Fig. 3C).
Effect of EPS on slow-oxidative (MHCI) fiber type. We
wanted to investigate the effect of EPS on expression of genes
and proteins for type I fibers (MYH7, the gene that regulates
protein expression of MHCI). After EPS, MYH7 gene expression was reduced in myotubes from SO-T2D subjects (Fig. 4A).
Furthermore, the EPS effect on gene expression of MYH7 in
myotubes correlated negatively with fasting plasma glucose
(Fig. 4B) and HbA1c (Fig. 4C) levels of the subjects. However,
results obtained for protein expression of MHCI (Fig. 4D)
showed no significant differences between the donor groups.
Effect of EPS on gene expression of IL-6 and IL-8. To
investigate whether EPS-induced gene regulation of myokines
seen in myotubes from lean nondiabetic subjects (48) also
occurred in myotubes originating from severely obese subjects,
qPCR on selected genes was performed. Gene expression of
IL-6 was increased after EPS in myotubes established from
L-nD and SO-nD subjects (Fig. 5A). Furthermore, EPS-induced effect on IL-6 gene expression in myotubes correlated
negatively with fasting plasma glucose (Fig. 5B) as well as
HbA1c (r ⫽ ⫺0.60, P ⬍ 0.05) levels of the subjects. Similar
results were also obtained for IL-8; although the EPS-increased
IL-8 expression was not significant (Fig. 5C). In line with this,
effect of EPS on gene expression of IL-8 also correlated
negatively with fasting plasma glucose levels of the subjects
(Fig. 5D).
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Fig. 1. Electrical pulse stimulation (EPS) improved insulin sensitivity in myotubes derived
from severely obese subjects. EPS was applied
to cultured myotubes for the last 48 h of the
differentiation period. A and B: after termination
of EPS, myotubes were incubated for 15 min in
the presence or absence of 100 nmol/l insulin,
and immunoblot analysis with antibodies against
phosphorylated (p)Akt (Ser473) and total Akt
was performed (n ⫽ 4 –5). A: pAkt/total Akt
without insulin. B: insulin-stimulated pAkt/total
Akt. C and D: after termination of EPS, glycogen
synthesis was measured as [14C]glucose (1
mmol/l) incorporation into glycogen in the presence or absence of 100 nmol/l insulin for 3 h (n ⫽
6 – 8). C: glycogen synthesis without insulin. D:
insulin-stimulated glycogen synthesis. E and F:
EPS-induced effect on insulin-stimulated pAkt/
total Akt (E, n ⫽ 13) and glycogen synthesis (F,
n ⫽ 21) in the myotubes correlated positively
with BMI of the donor subjects. L-nD, lean
nondiabetic; SO-nD, severely obese, nondiabetic;
SO-T2D, severely obese, type 2 diabetes. *P ⬍
0.05 vs. control.
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MYOTUBES FROM DIFFERENT DONORS RESPOND DIFFERENTLY TO EPS
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Fig. 2. EPS improved oxidative capacity of human
myotubes. Myotubes were electrically stimulated for
48 h. Glucose and oleic acid oxidation, mitochondrial
staining, OXPHOS, and lipid droplets were measured
after termination of EPS. A: glucose oxidation.
Myotubes were exposed to [14C]glucose (1 mmol/l)
for 3 h before CO2 assessment (n ⫽ 6 –7). B: oleic
acid oxidation. Myotubes were exposed to [14C]oleic acid (100 ␮mol/l) for 2 h before CO2 assessment (n ⫽ 7– 8). C: EPS-induced effect on oleic acid
oxidation correlated negatively with BMI of the
subjects (n ⫽ 21). D: myotubes were stained for
mitochondrial content with MitoTracker Red FM,
and nuclei with Hoechst 33258. Data are shown as
MitoTracker intensity related to nuclei (n ⫽ 5– 6).
E: expression of proteins involved in mitochondrial
oxidative phosphorylation, OXPHOS (Complex I
subunit NDUFB8, Complex II subunit, Complex III
subunit core 2, Complex IV subunit II, and ATP
synthase subunit ␣). Expression levels were normalized to ␣-tubulin (n ⫽ 4 – 6). Linear mixed model
was performed for OXPHOS experiments. F: representative immunoblots from one experiment (LnD). Ctr, control. G: myotubes were stained for lipid
droplets with Bodipy 493/503. Data are shown as
lipid droplets related to nuclei. H: cellular uptake of
oleic acid was assessed as the sum of cell-associated
and CO2-trapped radioactivity (n ⫽ 7– 8). *P ⬍
0.05 vs. control. #P ⬍ 0.05 vs. L-nD.
DISCUSSION
In this study, we show that insulin sensitivity was increased
in myotubes established from severely obese subjects after
EPS. Furthermore, EPS enhanced oxidative capacity of glucose
in myotubes from all subjects, while oleic acid oxidation and
mitochondrial content were improved only in myotubes from
lean subjects. Oleic acid oxidation and uptake were also
increased after GW501516 treatment in myotubes from all
subjects, whereas combination of GW501516 treatment and
EPS showed no additional effect on oleic acid oxidation or
uptake. Of particular interest, the impact of EPS on gene
expression of IL-6 and IL-8 was different in myotubes originating from lean and severely obese nondiabetic subjects
compared with myotubes from severely obese diabetic sub-
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MYOTUBES FROM DIFFERENT DONORS RESPOND DIFFERENTLY TO EPS
C553
jects. EPS reduced mRNA expression of slow fiber-type
marker (MYH7) in myotubes from diabetic subjects, while
protein expression of this marker was not affected by EPS in
neither of the donor groups.
Our findings suggest that human myotubes display the same
phenotype as intact muscle in vivo, in accordance with other
studies (17, 19, 24). The precise mechanisms by which myotubes are able to retain the in vivo characteristics are not
known. However, a combination of genetic and epigenetic
mechanisms is probably involved, and this has been reviewed
in a recent paper (1). For example, epigenetic regulation of
skeletal muscle stem cells and skeletal muscle differentiation,
exercise, diet, and a family history of T2D have all been
described to influence DNA methylation in human skeletal
muscle, and these are traits that might follow the isolated
satellite cells into their corresponding cultured myotubes (1).
Exercise increases insulin sensitivity in vivo (23, 46), and in
line with this, we have shown here that EPS can increase
Fig. 4. Effect of EPS on gene and protein expression
of myosin heavy chain type I (MHCI). The myotubes were electrically stimulated for 48 h. A: after
termination of EPS, expression of MYH7, the gene
that regulates protein expression of MHCI, was
measured by qPCR and compared relative to the
housekeeping genes 36B4 and GAPDH (n ⫽ 5– 6).
B and C: EPS effect on gene expression of MYH7
correlated negatively with fasting plasma glucose
(B) and HbA1c (C) of the subjects (n ⫽ 16). D:
immunoblot analysis with antibodies against MHCI,
related to ␤-actin, presented relative to unstimulated
control. *P ⬍ 0.05 vs. control.
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Fig. 3. Effect of EPS combined with peroxisome proliferator-activated receptor-␦ (PPAR␦) activation on oleic acid metabolism. Myotubes were treated with 10
nmol/l GW501516 for 96 h and EPS was applied to the cells for the last 48 h of the treatment period. A and B: after termination of EPS, myotubes were exposed
to [14C]oleic acid (100 ␮mol/l) for 2 h. A: oleic acid oxidation (n ⫽ 5–10). B: cellular uptake of oleic acid, assessed as the sum of cell-associated and CO2-trapped
radioactivity (n ⫽ 5–7). C: after termination of EPS, myotubes were stained for mitochondrial content with MitoTracker Red FM, and nuclei with Hoechst 33258.
Data are shown as MitoTracker intensity related to nuclei and normalized to unstimulated control cells (n ⫽ 5– 6). *P ⬍ 0.05 vs. control.
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MYOTUBES FROM DIFFERENT DONORS RESPOND DIFFERENTLY TO EPS
insulin sensitivity in insulin-resistant myotubes from severely
obese diabetic subjects. The observed effect on insulin sensitivity was more evident in myotubes from severely obese
subjects, which is consistent with other studies reporting
greater exercise-induced changes in insulin sensitivity in subjects who were more insulin resistant at baseline (5, 20), and
more evident in obese men compared with lean men (20). This
suggests that interventions to increase physical activity may be
particularly effective at improving insulin sensitivity in population groups who are more insulin resistant or have an increased predisposition to insulin resistance. Lack of EPSinduced increase in insulin sensitivity in myotubes from lean
nondiabetic subjects could also be explained by maximal
responsiveness to insulin in those cells per se and limited
capacity to respond further. In agreement with other studies
reporting enhanced carbohydrate oxidation after exercise in
obese subjects (9) and in subjects with T2D (50), we showed
an increase in glucose oxidation after EPS in myotubes from all
three donor groups. In contrast, we have previously (36) and in
the present study shown that mitochondrial content was increased after EPS in myotubes from lean nondiabetic subjects,
and this was followed by increased rate of oleic acid oxidation.
In this study, however, we showed that this is not the case in
myotubes from both groups of severely obese subjects. These
data are in contrast to studies reporting an increase in muscle
ex vivo palmitate oxidation in obese subjects after in vivo
exercise (33). The discrepancy may to some extent be explained by differences between in vivo and in vitro exercise
models. Without EPS, there was no difference in basal lipid
content between myotubes from the three donor groups, ex-
cluding the possibility that the absence of EPS effect on oleic
acid oxidation was due to label dilution when using a labeled
fatty acid as a measurement of fatty acid oxidation. At resting
state, the current consensus is that fatty acid oxidation is
reduced in obese versus lean muscle (6, 26). However, some
studies have also reported similar fatty acid oxidation in
myotubes from lean and obese diabetic subjects (49) and even
higher oxidation in obese than lean muscle (3). The reason
behind this discrepancy is unclear but may be a result of
different subgroups of obese populations studied. It is an
ongoing debate whether the impairment in oxidative capacity
in obese and insulin-resistant subjects is due to lower mitochondrial content or intrinsic mitochondrial dysfunction (54).
Although it has been shown that exercise improves both
mitochondrial content and intrinsic mitochondrial function in
obese and insulin-resistant subjects (43), it has also been
reported that there were no changes in key regulators of
mitochondrial biogenesis in skeletal muscles in diabetic men
after in vivo exercise (12). Consistent with this, in our exercise
model, myotubes from subjects with higher BMI were less
responsive to exercise-induced effects on fatty acid oxidation
than myotubes from lean subjects. It has been suggested that
insulin resistance in obesity and T2D is associated with intracellular accumulation of lipid in skeletal muscles (29). Consequently, much focus has been on the possibility of increasing
lipid utilization by exercise to avoid lipid accumulation. However, evidence has emerged that increased lipid oxidation
impairs glucose uptake by metabolic feedback, as suggested by
the Randle cycle (reviewed in ref. 29). In line with this, we
have shown that EPS was able to improve insulin sensitivity
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Fig. 5. Effect of EPS on gene expression of IL-6 and
IL-8. The myotubes were electrically stimulated for
48 h and harvested after termination of EPS. Expression of IL-6 (A) and IL-8 (C) was measured by
qPCR and studied relative to the housekeeping
genes 36B4 and GAPDH. Fasting plasma glucose
levels were inversely related to the effect of EPS on
gene expression of IL-6 (B, n ⫽ 22) and IL-8 (D, n ⫽
19). *P ⬍ 0.05 vs. control.
MYOTUBES FROM DIFFERENT DONORS RESPOND DIFFERENTLY TO EPS
ACKNOWLEDGMENTS
The authors thank Terje Grønås (Electronics Lab, Institute of Chemistry,
University of Oslo) for support and technical assistance. We are also thankful
to Gerbrand Koster and Oddmund Bakke from the NORMIC-UiO imaging
platform, Department of Molecular Biosciences, University of Oslo, for
support, use of equipment, and excellent technical assistance.
GRANTS
This work was funded by the University of Oslo, Diabetesforbundets
forskningsfond, and Anders Jahres fond til vitenskapens fremme.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Y.Z.F., N.N., S.S.B., and G.H.T. conception and design of research; Y.Z.F.,
N.N., S.S.B., E.T.K., and K.G. performed experiments; Y.Z.F. and S.S.B.
analyzed data; Y.Z.F., A.C.R., and G.H.T. interpreted results of experiments;
Y.Z.F. prepared figures; Y.Z.F. drafted manuscript; Y.Z.F., N.N., S.S.B.,
E.T.K., K.G., J.H., V.A., A.C.R., and G.H.T. edited and revised manuscript;
Y.Z.F., N.N., S.S.B., E.T.K., K.G., J.H., V.A., A.C.R., and G.H.T. approved
final version of manuscript.
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