Cell Biology International ISSN 1065-6995

doi: 10.1002/cbin.10711

SHORT COMMUNICATION

Nuclear co-repressor (NCoR) is required to maintain insulin

sensitivity in C2C12 myotubes
Abhijeet K. Choudhary and Chinmoy S. Dey*
Kusuma School of Biological Sciences (KSBS), Indian Institute of Technology (IIT)—Delhi, Hauz Khas, New Delhi 110016, India

Abstract
Nuclear co-repressor (NCoR) regulates peripheral insulin sensitivity; however, its role in modulating insulin sensitivity in
skeletal muscle remains elusive. Present study investigated protein expression and effect of NCoR on insulin sensitivity in
murine skeletal muscle cell line C2C12. Myotubes as compared to myoblasts of C2C12 cells were found to be more sensitive
in response to insulin as increase in insulin-stimulated phosphorylation of AKT at serine 473 residue (pAKTS473) was
significantly higher in myotubes. Incidentally, reduced protein level of NCoR coincided with differentiation of myoblasts into
myotubes of C2C12 cells. However, insulin stimulation per se failed to affect protein level of NCoR either in myoblasts or
myotubes of C2C12 cells. To assess the role of NCoR on insulin sensitivity, NCoR was transiently knocked down using siRNA
in myotubes of C2C12. In fact, transient silencing of NCoR led to significant reduction in insulin-stimulated pAKTS473 and
impaired glucose uptake. This observation is in contrast to published studies where NCoR has been reported to negatively
regulate insulin signaling cascade. Furthermore, transient silencing of NCoR failed to improve insulin sensitivity in chronic
hyperinsulinemia-induced insulin-resistant model of C2C12 cells. Importantly, inhibition of lysosomal protein degradation
pathway using ammonium chloride restored protein level of NCoR but failed to increase glucose uptake in serum-starved
C2C12 myotubes. Collectively, data from present study show differential protein level of NCoR under different cell state
(myoblast and myotubes) of C2C12 cells and NCoR proves to be vital for maintaining insulin sensitivity in C2C12 myotubes.

Keywords: AKT phosphorylation; ammonium chloride; glucose uptake; insulin resistance; lysosomal protein degradation
pathway; nuclear co-repressor (NCoR)

Introduction facilitating NCoR exit from nucleus for proteosomal

Nuclear co-repressor (NCoR) was discovered as receptor-
interacting factor in pursuit to delineate gene repressive
activity of nuclear receptors including Rev erb (Horlein et al.,
1995). NCoR is a large protein (molecular mass
270 kDa)
that primarily acts as a docking site for chromatin-modifying
enzymes including histone deacetylases 3 (HDAC3) to
facilitate transrepression of genes regulating circadian clock,
gluconeogenesis, and pro-inflammatory responses (Pascual
et al., 2005; Yin et al., 2007; Ghisletti et al., 2009).
Transrepression of genes by core nuclear complex involving
NCoR is intrinsically regulated via proteosomal protein
degradation pathway to ensure transient gene repression
(Mottis et al., 2013). In fact, ubiquitin ligase E3 including
Siah2 has been illustrated to maintain NCoR protein pool by
degradation (Frasor et al., 2005).
Other than modulating circadian and inflammatory
responses, NCoR has been reported to regulate differentia-
tion of different cell types including skeletal muscle cells.
Different studies have reported NCoR to modulate
transcription factors such as MyoD and MEF2 that regulate
myogenesis (Bailey et al., 1999; Yamamoto et al., 2011).
Interestingly, constitutive overexpression of NCoR has been
reported to ablate myogenesis in C2C12 cells (Bailey et al.,
1999). Conditional knock-down of NCoR in skeletal muscle
of mice promotes myogenesis via hyperacetylation and
activation of MEF2, an important transcription factor
regulating differentiation of muscle (Yamamoto et al., 2011).
Research over the last decade have identified non-
genomic role of NCoR in modulating phosphatidylinositol


Corresponding author: e-mail: csdey@bioschool.iitd.ac.in
Abbreviations: 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; NCoR, nuclear co-repressor; NH4Cl, ammonium chloride;
PKB/Akt, protein kinase B

Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology 1
NCoR maintains insulin sensitivity in C2C12
3-kinases (PI3K)-AKT signaling axis. A frameshift mutation
at C-terminal of thyroid hormone receptor beta (TRb)-1
(termed as PV) desensitized the receptor’s ability to bind to
thyroid hormone; however, they were found to avidly bind
to PI3K at C-terminal SH2 domain of its regulatory subunit
p85a resulting in sustained phosphorylation of AKT and
development of thyroid carcinoma (Furuya et al., 2006).
Subsequent studies by Furuya et al. (2007, 2009) identified
NCoR to compete with PV for binding to PI3K to negatively
regulate PI3K-AKT signaling axis. Incidentally, PI3K-AKT
signaling cascade plays a critical role in regulating insulin
signaling cascade too (Manning and Cantley, 2007). In fact, a
key physiological function of AKT is to facilitate glucose
uptake in response to insulin stimulation by activating
translocation of isoforms of GLUT to the plasma membrane
(Huang et al., 2002; Randhawa et al., 2008).
Recent studies employing tissue-specific gene knock-
down approach in murine models have highlighted
discrepant role of NCoR in insulin signaling. Tissue-specific
silencing of NCoR in skeletal muscle under in vivo condition
has been reported to improve oxidative function of skeletal
muscle by increasing the muscle mass and mitochondrial
activity (Yamamoto et al., 2011). However, no significant
improvement in insulin sensitivity was noted in this study
(Yamamoto et al., 2011). Whereas ablation of NCoR in
adipose tissue of mice has been shown to promote
adipogenesis and enhance peripheral insulin sensitivity by
constitutively activating peroxisome proliferator-activated
receptor-gamma (PPAR-g) (Li et al., 2011). Hence, the role
of NCoR in regulating PI3K-AKT signaling axis to modulate
insulin signaling at cellular level is poorly understood.
The present study was undertaken to determine the role of
NCoR on insulin signaling at cellular level using mouse
skeletal muscle cell line C2C12 cells which previously has
been used as a robust cell culture model to study insulin
sensitivity under insulin sensitive and chronic hyper-
insulinemia-induced insulin-resistant conditions (Arora
and Dey, 2014). The effect of in vitro differentiation of
myoblasts to myotubes on NCoR has been assessed too at
protein level as concurrent changes in transcription factors
mediating differentiation and NCoR has been reported
previously (Bailey et al., 1999).
Methodology
Chemicals and reagents
DMEM low glucose (5.5 mM) media, 2-(N-(7-nitrobenz-2-
oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), were
purchased from Life Technologies (USA). Fetal bovine serum
(FBS), horse serum, and Opti-MEM media were purchased
from Gibco BRL (USA). ECL Western blotting substrate and
primary antibody for NCoR were purchased from Thermo
2 Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology
A. K. Choudhary and C. S. Dey
Fisher Scientific (USA). Primary antibodies were bought for
pAKTS473, AKT (Cell Signalling Technology, USA), GAPDH,
myogenin, and FAK along with appropriate HRP conjugated
antibody (Santa Cruz Biotechnology, USA). Rest of the
chemicals and reagents were purchased from Sigma–Aldrich
(USA) unless otherwise stated in the text below.
Cell culture
Sub-culturing of C2C12
Murine skeletal muscle cell line C2C12 was sub-cultured every
48 h in DMEM low glucose (5.5 mM) media supplemented
with 10% FBS, 1% 1M Hepes buffer, and 1% antibiotics
(penicillin and streptomycin) along with 44 mM sodium
bicarbonate as reported previously (Kumar and Dey, 2002).
Differentiation of C2C12
C2C12 cells were differentiated into myotubes over a period
of 72 h in serum-free media containing equal mixture of
MCDB 201 and Ham’s F-12 medium (henceforth referred as
MF) along with supplements including 19 mM sodium
bicarbonate, 1% antibiotics, and 0.05% bovine serum
albumin (Kumar and Dey, 2002).
Comparative study on C2C12 myoblasts and myotubes
To study changes in expression of proteins between
myoblasts and myotubes, C2C12 cells were proliferated in
DMEM low glucose media for 48 h. Proliferated C2C12 cells
were either serum starved in DMEM media containing 0.5%
FBS for 2 h and used as myoblast samples or further
differentiated under MF condition for 72 h to get myotubes.
The experiments were terminated after 0.5 h of stimulation
with 100 nM insulin (henceforth referred as insulin
stimulation).
Serum shock treatment of C2C12 myotubes
Serum shock experiments were performed at end of 72 h
differentiation process by treating C2C12 myotubes with 40%
horse serum for 1 and 2 h prior to lysis.
Insulin-resistant model of C2C12
Chronic hyperinsulinemia-mediated insulin-resistant model
was generated by differentiating C2C12 myoblasts in MF
media supplemented with 100 nM insulin (henceforth
referred as MFI). Media change was performed after interval
of 12 h (Kumar and Dey, 2003).
Inhibition of protein degradation pathways
Chemical inhibitors MG132 and ammonium chloride
(NH4Cl) were used at 20 mM and 10 mM concentration,
respectively, to inhibit proteosomal and lysosomal protein
degradation pathways by pre-treating C2C12 cells for 12 h
during end of 72-h differentiation process with inhibitors.
A. K. Choudhary and C. S. Dey
siRNA transfection
Predesigned siRNA oligonucleotides (Qiagen, Germany)
against NCoR with targeted sequence 50 -CAGGAGAA-
TAATGAGAAGCAA-30 along with non-specific siRNA
(as control) were used to transfect C2C12 cells transiently at
200 nM concentration using lipofectamine 2000 as trans-
fecting reagent (Thermo Fischer Scientific, USA) in
reduced–serum Opti-MEM media. Briefly, 100,000 C2C12
cells plated for 48 h were treated with siRNA-transfecting
reagent complex for a period of 18 h prior to 72-h
differentiation process with MF media replacement
every 12 h.
Western blotting
Experiments were terminated by lysing cells in ice-cold lysis
buffer (50 mM HEPES, pH 7.5, containing 150 mM NaCl,
1.5 mM magnesium chloride, 10 mM sodium pyrophos-
phate, 10 mM sodium fluoride, 1 mM sodium orthovana-
date, 1 mM EGTA along with protease inhibitors including
1 mM PMSF, 10 mg/mL aprotinin, and 10 mg/mL leupeptin
and 1% (v/v) Triton X-100). Samples containing 30–40 mg
protein and protein standard were resolved on 10% SDS
polyacrylamide gels. The resulting fractionated samples were
transferred onto nitrocellulose membrane and were probed
with relevant primary antibodies followed by secondary
HRP conjugated antibody before being treated with ECL
Western blotting detection reagent to detect bands.
Densitometric quantification of immunoblots was per-
formed using Quantity One 1-D analysis software (Bio-
Rad Laboratories, USA) where relative values of the samples
were determined by giving an arbitrary value of 1.0 to the
control sample of each experiment with deduction of
background.
Glucose uptake assay
Glucose uptake was assayed as reported previously (Arora
and Dey, 2014) using fluorescently labeled analog of glucose
2-NBDG. Briefly, cells were incubated for 3 h at 37 C in
glucose starvation buffer (10 mM HEPES pH 7.4, 140 mM
NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgSO4, 1 mM
KH2PO4) at the end of differentiation of C2C12 cells. After
3 h of starvation, cells were stimulated with or without
100 nM insulin for 0.5 or 1 h. Cells were further incubated at
37 C with 50 mM of 2-NBDG for 1 h. Experiments were
terminated by lysing cells in lysis buffer (20 mM Tris–
HCl pH 7.4, 1% sodium deoxycholate, 1% Nonidet P-40,
40 mM KCl) for 10 min prior to being centrifuged at 16,000g
for 15 min at 4 C. Fluorescence of aliquots from super-
natants, containing equal amount of protein was measured
using Fluorescence Spectrometer LS55 (Perkin Elmer, USA)
Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology
NCoR maintains insulin sensitivity in C2C12
at excitation and emission wavelengths of 485 nm and
535 nm, respectively.
Statistical analysis
Data are expressed as mean  SEM computed from three
individual repeats of an experiment. Multiple treatment
groups were compared by one-way analysis of variance
ANOVA test followed by comparison between two groups
by two-tailed unpaired Student’s t-test with the signifi-
cance threshold set at P < 0.05 as measure of statistical
significance.
Results
Differential expression of NCoR in myoblasts and
myotubes of C2C12 cells
Serum starvation driven differentiation often results in
degradation of proteins that are not vital for cell survival. In
the present study, C2C12 myoblasts were subjected to serum
starvation for 72 h to acquire differentiated myotubes for
study of role of NCoR on insulin sensitivity.
A significant reduction of approximately 45% in protein
level of NCoR [Figures 1A Panel A, 1B: lane 1 (1.0  0) vs. 3
(0.55  0.03), P < 0.01] coincided with increase in protein
level of myogenin, (a differentiation marker), [Figures
1A Panel A and 1D: lane 1 (1.0  0) vs. 3 (1.5  0.01),
P < 0.0005] in C2C12 myotubes as compared to myoblasts
under basal (non-insulin stimulated) condition. Interest-
ingly, basal expression of GAPDH was reduced by
26% in
myotubes [Figures 1A Panel A, 1C: lane 1 (1.0  0) vs. 3
(0.7  0.01)] too. Previously, studies have reported differen-
tial expression of GAPDH during differentiation of primary
and L6 myoblasts (Pirkmajer and Chibalin, 2011). Hence,
FAK, which has been previously reported to remain
unchanged in myoblasts as well as myotubes (Clemente
et al., 2005), has been used for normalization of protein
expression in myoblasts and myotubes in the present study.
Cell lines are often subjected to shock treatment with high
concentrations of serum leading in increase in cellular
protein pool to study rhythmic changes in expression of
proteins specifically those involved in regulation of circadian
clock (Balsalobre et al., 1998; Wang et al., 2006; Yin et al.,
2006; Zhang et al., 2012; Peek et al., 2013). To further test the
influence of serum on protein expression of NCoR, C2C12
myotubes were supplemented with 40% horse serum for
1–2 h at the end of their 72-h differentiation. Significant
increase of
59% in protein expression of NCoR [Figures
1F and 1G: lane 1 (1.0  0) vs. 3 (1.6  0.11), P < 0.05] was
noted after 2-h incubation with horse serum. These data
collectively suggest serum availability to play a role in
regulating NCoR protein expression.
3
NCoR maintains insulin sensitivity in C2C12
Despite differential protein expression of NCoR and
GAPDH observed in C2C12 myoblasts and myotubes, no
notable change in their protein levels was observed under
basal and insulin-stimulated condition when selectively seen
Figure 1 Continued.
4 Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology
A. K. Choudhary and C. S. Dey
in myoblasts and myotubes [Figures 1A Panel A, 1B, and
1C). Nevertheless, insulin stimulation distinctly elevated
phosphorylation of AKT, one of the critical proteins of
insulin signal transduction, by
26% in myotubes as
compared to myoblasts of C2C12 cells [Figures 1A Panel
B, 1E: lane 2 (3.5  0.23) vs. 7.7 (1.6  0.11), P < 0.0005].
NCoR was found to be significantly reduced in a cell type,
that is, myotubes of C2C12 cells that was found to be more
sensitive to the effects of insulin, hence it was prudent to
determine the effect of NCoR on insulin signaling cascade in
C2C12 myotubes.
NCoR maintains insulin sensitivity in C2C12 myotubes
NCoR was transiently silenced using siRNA to validate the
hypothesis that reduction in cellular NCoR protein pool
might enhance insulin sensitivity in C2C12 myotubes.
NCoR-specific siRNA silenced NCoR by
50% under basal
condition [Figures 2A Panel A, 2B: lane 1 (1.0  0) vs. 4
(0.5  0.036), P < 0.0005]. Paradoxically, modest but
significant reduction by
22% in phosphorylation of AKT
was noted in NCoR silenced myotubes when subjected to
stimulation with insulin for 0.5 h [Figures 2A Panel B, 2C:
lane 2 (6.3  0.89) v 5 (4.9  0.025), P < 0.0005]. The
difference remained significant even when cells were
stimulated with insulin for 1 h [Figures 2A Panel B, 2C:
lane 3 (5.0  0.89) vs. 6 (3.9  0.11), P < 0.05].
Glucose uptake assay was performed to confirm that
decrease in pAKT upon insulin stimulation in NCoR
silenced myotubes eventually resulted in reduced uptake of
glucose by cells. Indeed, NCoR silencing led to a significant
reduction in insulin-mediated glucose uptake at 0.5 h
[Figure 2D: lane 2 (1.69  0.02) vs. 5 (1.46  0.03),
P < 0.01] and 1 h [Figure 2D: lane 3 (1.48  0.02) vs. 6
(0.95  0.06), P < 0.01].
3
Figure 1 Differential expression of proteins in C2C12 myoblasts and
myotubes and on serum shock in myotubes. (A) Representative
Western immunoblot images for protein expression of NCoR, myogenin,
GAPDH, FAK, pAKTS473, AKT in C2C12 myoblasts and myotubes in the
absence or presence of insulin stimulation at 100 nM concentration for
0.5 h. Relative densitometric values for (B) NCoR protein level normalized
to FAK protein, (C) GAPDH protein level normalized to FAK protein,
(D) Myogenin protein level normalized to FAK protein, (E) AKT
phosphorylation at serine 473 residue (pAKTS473) normalized to AKT
protein. (F) Representative Western immunoblot images for protein
expression of NCoR and GAPDH in C2C12 myotubes treated with 40%
horse serum to induce serum shock. Relative densitometric values for (G)
NCoR protein level normalized to GAPDH. Bar graphs represent relative
values as mean  SEM from three individual experiments. P-values
highlighted as  P < 0.05,  P < 0.01,  P < 0.005 are based on two-
tailed unpaired Student’s t-test between two groups.
A. K. Choudhary and C. S. Dey
Figure 2 Effect of transient knock-down of NCoR on insulin
sensitivity in C2C12 myotubes. (A) Representative Western immunoblot
images for protein expression of NCoR, GAPDH, pAKTS473, AKT in C2C12
myotubes transiently silenced for NCoR using specific oligonucleotides
(siNCoR) at 200 nM concentration in the absence or presence of
stimulation with 100 nM insulin for 0.5 or 1 h. Relative densitometric
values for (B) NCoR protein level normalized to GAPDH protein, (C) AKT
phosphorylation at serine 473 residue (pAKTS473) normalized to AKT
protein. Relative values for fluorescence intensity measured in cells
incubated for 1 h with fluorescent analog of glucose named (D) 2-NBDG
in C2C12 myotubes transiently silenced for NCoR in the absence or
presence of insulin stimulation for 0.5 or 1 h. Bar graphs represent relative
values as mean  SEM from three individual experiments. P-values
highlighted as  P < 0.05,  P < 0.01,  P < 0.005 are based on two-
tailed unpaired Student’s t-test between two groups.
Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology
NCoR maintains insulin sensitivity in C2C12
Interestingly, the decrease in glucose uptake as opposed
to AKT phosphorylation after 1 h insulin stimulation was
remarkable in NCoR-silenced C2C12 myotubes as glucose
uptake was comparable with baseline level. However, this
effect could be attributed to the fact that insulin
stimulation does not elicit identical degree of increase
in AKT phosphorylation and 2-NBDG uptake as is evident
by difference in relative value (y-axis) scale for the two
read-outs. Nevertheless, transient silencing of NCoR led to
distinct reduction in regulation of insulin signaling
cascade upon acute insulin stimulation. These observa-
tions were in contrast to the initial hypothesis that NCoR
negatively regulates insulin signaling but ascertain a role
of NCoR in maintaining insulin sensitivity in C2C12
myotubes.
Partial restoration of NCoR but not insulin sensitivity by
inhibition of lysosomal protein degradation pathway
Transient silencing of NCoR potentiated a modest insulin
desensitizing phenotype in C2C12 myotubes by reducing
phosphorylation of AKT (Figure 2C) and glucose uptake
(Figure 2D). Hence, it was prudent to assess whether
enhanced NCoR generates insulin sensitivity. Pharmaco-
logical inhibitors of protein degradation pathway, that is,
MG132 for inhibiting proteosomal and NH4Cl to inhibit
lysosomal degradation pathway at concentration reported
to inhibit these protein degradation pathways (Seglen
et al., 1979; Frasor et al., 2005) were employed to
transiently increase protein expression of NCoR. It was
observed that MG132 failed to elevate protein level of
NCoR in C2C12 myotubes at a concentration of 20 mM
(Figure 3A). Instead a lysosomotropic inhibitor, that is,
NH4Cl when used at 10 mM concentration during last
12 h of 72 h differentiation of C2C12 cells led to
approximately 30% increase in NCoR protein level
(Figure 3A).
NH4Cl was employed in transiently NCoR-silenced
C2C12 myotubes to assess whether transient increase in
NCoR improved insulin sensitivity. Expectedly, pretreat-
ment with NH4Cl resulted in
41% increase in protein
levels of NCoR in transiently NCoR-silenced C2C12
myotubes [Figures 3B Panel A, 3C: lane 5 (0.44 –0.03)
vs. 7 (0.75  0.04), P < 0.05]. This increase in NCoR
coincided with an increase in phosphorylation of AKT
when stimulated with insulin [Figures 3B Panel B, 3D: lane
6 (4.8  0.16) vs. 8 (5.6  0.03), P < 0.05]. However,
NH4Cl-mediated protein accumulation of NCoR along
with increase in insulin-mediated phosphorylation of AKT
in NCoR-silenced myotubes did not translate into increase
in glucose uptake (Figure 3E). Hence, these data suggest
that merely increasing cellular pool of NCoR protein may
unlikely improve insulin sensitivity.
5
NCoR maintains insulin sensitivity in C2C12
NCoR maintains insulin sensitivity in insulin-resistant
C2C12 myotubes
Furthermore, to understand the role of NCoR in insulin
resistance, C2C12 myotubes were transiently silenced for
Figure 3 Continued.
6 Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology
A. K. Choudhary and C. S. Dey
NCoR and subjected to chronic treatment with insulin (MFI
condition) for 72 h to achieve hyperinsulinemia-mediated
insulin resistance.
Hyperinsulinemia failed to have an effect on NCoR
protein level under basal condition [Figures 4A Panel A, 4B].
Importantly, insulin-stimulated phosphorylation of AKT in
NCoR-silenced C2C12 myotubes was distinctly reduced by
approximately 50% when stimulated with insulin under
hyperinsulinemic condition [Figures 4A Panel B, 4C: lane 6
(1.75  0.14) vs. 8 (0.9  0.23), P < 0.05]. However, this
exaggerated reduction in pAKTS473 failed to result in further
reduction in glucose uptake [Figure 4D: lane 6 (0.75  0.04)
vs. 8 (0.66  0.06), P > 0.05] as hyperinsulinemic condition
of its own [Figure 4D] was sufficient to reduce glucose
uptake to basal level.
Discussion
In the present study, the role of NCoR in maintaining insulin
sensitivity was being investigated. The data illustrate NCoR
to be dispensable under in vitro condition of differentiating
C2C12 myoblasts into myotubes. Serum availability and
lysosomal protein degradation pathway were noted to
distinctively regulate NCoR protein synthesis and degrada-
tion, respectively. Importantly, NCoR despite being lowly
expressed in C2C12 myotubes as compared to myoblasts is
required for maintenance of insulin sensitivity as transient
knock-down of NCoR modestly turned insulin resistant
phenotype.
Differential protein expression of NCoR observed in
C2C12 myotubes as compared to myoblasts concurs with
published studies where transcription downregulation of
3
Figure 3 Effect of pre-treatment with ammonium chloride
(NH4Cl), an inhibitor of lysosomal protein degradation pathway,
on insulin sensitivity in C2C12 myotubes transiently silenced for
NCoR. (A) Representative Western immunoblot images and relative
numbers for protein expression of NCoR normalized to GAPDH protein in
C2C12 myotubes transiently silenced for NCoR and treated with either
20 mM MG132 or 10 mM NH4Cl as single agent or in combination during
last 12 h of the 72 h of differentiation process under MF condition.
(B) Representative Western immunoblot images for protein expression of
NCoR, GAPDH, pAKTS473, AKT in C2C12 myotubes transiently silenced for
NCoR and pre-treated with 10 mM NH4Cl during last 12 h of the 72 h of
differentiation process prior to stimulation with 100 nM insulin for 0.5 h.
Relative densitometric values for (C) NCoR protein level normalized to
GAPDH protein, (D) AKT phosphorylation at serine 473 residue
(pAKTS473) normalized to AKT protein. Relative values for fluorescence
intensity measured in cells incubated for 1 h with fluorescent analog of
glucose named (E) 2-NBDG in C2C12 myotubes transiently silenced for
NCoR and pre-treated for 12 h with 10 mM NH4Cl prior to insulin
stimulation for 0.5 h. Bar graphs represent relative values as mean  SEM
from three individual experiments. P-values highlighted as  P < 0.05 is
based on two-tailed unpaired Student’s t-test between two groups.
A. K. Choudhary and C. S. Dey
NCoR was noted in C2C12 cells when cultured in low serum
differentiating media (Bailey et al., 1999). Regulation of
NCoR is found to be sensitive to nutrients too. Yamamoto
et al. (2011) have reported reduced expression of NCoR to be
associated with low glucose and high fat in mouse embryonic
fibroblasts. In fact, restoration of NCoR protein level in
C2C12 myotubes on being supplemented with horse serum in
Figure 4 Continued.
Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology
NCoR maintains insulin sensitivity in C2C12
the present study warrants further incisive research to
determine whether NCoR protein turnover in the cell can be
influenced by nutrients essentially amino acids that are
integral component of serum.
The present understanding of negative regulation of
PI3K-AKT signaling axis by NCoR is largely based on
studies performed in thyroid cells. NCoR is abundantly
expressed in thyroid cells and shown to compete with TRb1
to bind to PI3K and subsequently retard phosphorylation of
AKT (Furuya et al., 2007). Intriguingly, reduced expression
of NCoR (Bailey et al., 1999) and TRb (Milanesi et al., 2016)
is reported in C2C12 cells. Therefore, it is unlikely that NCoR
interaction with PI3K decisively regulates insulin sensitivity
in skeletal muscle. In fact, skeletal muscle-specific deletion of
NCoR failed to significantly improve glucose clearance (an
indicator of insulin sensitivity) as assessed by glucose
tolerance test under normal- and high-fat diet consumption
(Yamamoto et al., 2011). Furthermore, NCoR was observed
to positively regulate AKT phosphorylation and glucose
uptake in the present study, suggesting NCoR’s modulation
of insulin sensitivity to be dependent on cell type.
Previously, Li et al. (2011) showed selective abrogation of
NCoR in adipocytes to result into increased phosphorylation
of AKT in liver, adipocytes, and skeletal muscle of mice. The
contrasting data on the role of NCoR in modulating AKT
phosphorylation and insulin sensitivity between Li et al.
(2011) and present study are likely due to intracellular effect
of adiponectin. Adipocyte-specific deletion of NCoR in the
study by Li et al. (2011) led to metabolically favorable
remodeling of adipose tissue to promote adipogenesis as this
was evident by an increase in adiponectin despite weight
gain (Li et al., 2011). On the contrary, in the present in vitro
study, a modest reduction in AKT phosphorylation and
glucose uptake was solely dependent on transient knock-
down of NCoR. Furthermore, skeletal muscle-specific
deletion of NCoR failed to significantly improve insulin
3
Figure 4 Effect of transient knock-down of NCoR on insulin
sensitivity in C2C12 myotubes under insulin-resistant (MFI) condi-
tion. (A) Representative Western immunoblot images for protein
expression of NCoR, GAPDH, pAKTS473, AKT in C2C12 cells transiently
silenced for NCoR and differentiated under MF (insulin-sensitive) and MFI
(insulin-resistant) condition prior to stimulation with 100 nM insulin for
0.5 h. Relative densitometric values for (B) NCoR protein level normalized
to GAPDH protein, (C) AKT phosphorylation at serine 473 residue
(pAKTS473) normalized to AKT protein. Relative values for fluorescence
intensity measured in cells incubated for 1 h with fluorescent analog of
glucose named (D) 2-NBDG in C2C12 cells transiently silenced for NCoR
and differentiated under MF and MFI condition prior to insulin
stimulation for 0.5 h. Bar graphs represent relative values as mean  SEM
from three individual experiments. P-values highlighted as  P < 0.05,

P < 0.01,  P < 0.005 are based on two-tailed unpaired Student’s
t-test between two groups.
7
NCoR maintains insulin sensitivity in C2C12
sensitivity (Yamamoto et al., 2011). Together, these three
studies demonstrate different roles of NCoR at modulating
insulin sensitivity which plausibly is due to difference in
cellular and systemic effects arising due to variability in
models (in vivo vs. in vitro) and cell types (skeletal muscle vs.
adipocytes) that are being used for the study.
Interestingly, inhibition of lysosomal and not proteoso-
mal degradation pathway increased cellular pool of NCoR in
the present study as NH4Cl treatment led to significant
increase in AKT phosphorylation but not glucose uptake.
Hence, merely reinstating AKT phosphorylation does not
necessarily results in improved insulin sensitivity. However,
these data should be evaluated with caution as NH4Cl-
mediated neutralization of acidic pH may not be limited to
lysosomes alone and has a possibility to affect maturation of
recycling endosomes associated with GLUT translocation.
Previously, Siah2 has been reported to modulate NCoR
protein through proteosomal degradation in thryroid
hormone responsive breast cancer cell line (Frasor et al.,
2005). However, pre-treatment with MG132 failed to
increase protein level of NCoR in the present study. Hence,
a disparity in regulation of cellular protein pool of NCoR is
evident across different cell types too.
Under serum-starved condition, lysosomal protein degra-
dation pathway gets triggered to degrade non-functional
proteins to generate amino acids as nutrients for cell (Seglen
et al., 1979). This process is widely known as autophagy. In the
present study, autophagy may have been triggered as serum
deprivation was used as a tool to differentiate C2C12 myoblasts
into myotubes. It is evident from the fact that NH4Cl,
an inhibitor of lysosomal protein degradation pathway,
increased NCoR protein level in the present study. Recently,
two separate studies have reported onset of autophagy during
differentiation of C2C12 cells and mouse muscle satellite cell
(MSC)-derived myoblasts to promote mitochondrial
biogenesis (Fortini et al., 2016; Sin et al., 2016). In fact,
inhibition of autophagy distinctly interrupts with myogenesis
in these studies. Interestingly, two key proteins regulating
autophagy in skeletal muscle, that is, mTOR and FoxO3 lie
downstream of P13K-AKT pathway (Zhao et al., 2007).
Hence, it would be of interest to determine a role of NCoR in
modulating autophagy in skeletal muscle in future.
To conclude, our data display a modest but novel role of
NCoR in maintaining insulin sensitivity in murine skeletal
muscle cell culture. Transcriptional and post-translational
modification mechanisms governing the effects of NCoR on
insulin signaling are not completely understood and remains
of interest for further research.
Acknowledgements and funding
AKC is the recipient of a start-up research grant (SB/YS/LS-
150/2013) under Young Scientists scheme from the Science
8 Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology
A. K. Choudhary and C. S. Dey
and Engineering Research Board, Government of India, New
Delhi. This study was supported by a grant from the
Department of Science and Technology, Government of India,
New Delhi to CSD [SR/S2/JCB-24/2008(G) dated 30-12-2008].
Conflict of interest
None.
Author contribution
AKC and CSD conceived and designed the experiments.
AKC performed the experiments and analyzed the data.
AKC and CSD contributed to the writing, discussion, and
approval of the manuscript.
References
Arora A, Dey CS (2014) Sirt2 negatively regulates insulin resistance
in C2C12 skeletal muscle cells. Biochim Biophys Acta 1842:
1372–8.
Bailey P, Downes M, Lau P, Harris J, Chen SL, Hamamori Y,
Sartorelli V, Muscat GEO (1999) The nuclear receptor co-
repressor N-CoR regulates differentiation: N-CoR directly
regulates MyoD. Mol Endocrinol 13: 1155–68.
Balsalobre A, Damiola F, Schibler U (1998) A serum shock
induces circadian gene expression in mammalian tissue culture
cells. Cell 96: 929–37.
Clemente CFMZ, Corat MAF, Saad STO, Franchini KG (2005)
Differentiation of C2C12 myoblasts is critically regulated by
FAK signaling. Am J Physiol Regul Integr Comp Physiol 289:
R862–70.
Fortini P, Ferretti C, Iorio E, Cagnin M, Garribba L, Pietraforte D,
Falchi M, Pascucci B, Baccarini S, Morani F, Phadngam S, Luca
GD, Isidoro C, Dogliotti E (2016) The fine tuning of metabolism,
autophagy and differentiation during in vitro myogenesis. Cell
Death Dis 7: e2168.
Frasor J, Danes JM, Funk CC, Katzenellenbogen BS (2005)
Estrogen down-regulation of the corepressor N-CoR: mecha-
nism and implications for estrogen derepression of N-CoR-
regulated genes. Proc Natl Acad Sci USA 102: 13153–7.
Furuya F, Guigon CJ, Zhao L, Lu C, Hanover JA, Cheng S (2007)
Nuclear receptor corepressor is a novel regulator of phospha-
tidylinositol 3-kinase signaling. Mol Cell Biol 27: 6116–26.
Furuya F, Hanover JA, Cheng S (2006) Activation of phospha-
tidylinositol 3-kinase signaling by a mutant thyroid hormone b
receptor. Proc Natl Acad Sci USA 103: 1780–5.
Furuya F, Lu C, Guigon CJ, Cheng S (2009) Non genomic
activation of phosphatidylinositol 3-kinase signaling by thyroid
hormone receptors. Steroids 74: 628–34.
Ghisletti S, Huang W, Jepsen K, Benner C, Hardiman G,
Rosenfeld MG, Glass CK (2009) Cooperative NCoR/SMRT
interactions establish a corepressor-based strategy for integra-
tion of inflammatory and anti-inflammatory signaling path-
ways. Genes Dev 23: 681–93.
A. K. Choudhary and C. S. Dey
Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R,
Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG
(1995) Ligand-independent repression by the thyroid hormone
receptor mediated by a nuclear receptor co-repressor. Nature
377: 397–404.
Huang C, Somwar R, Patel N, Niu W, Torok D, Klip A (2002)
Sustained exposure of L6 myotubes to high glucose and insulin
decreases insulin-stimulated GLUT4 translocation but upre-
gulates GLUT4 activity. Diabetes 51: 2090–8.
Kumar N, Dey CS (2002) Metformin enhances insulin signalling
in insulin-dependent and independent pathways in insulin
resistant muscle cells. Br J Pharmacol 137: 329–36.
Kumar N, Dey CS (2003) Development of insulin resistance and
reversal by thiazolidinediones in C2C12 skeletal muscle cells.
Biochem Pharmacol 65: 249–57.
Li P, Fan W, Xu J, Lu M, Yamamoto H, Auwerx J, Sears DD,
Talukdar S, Oh DO, Chen A, Bandyopadhyay G, Scadeng M,
Ofrecio JM, Nalbandian S, Olefsky JM (2011) Adipocyte NCoR
knockout decreases PPAR phosphorylation and enhances
PPAR activity and insulin sensitivity. Cell 147: 815–26.
Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating
downstream. Cell 129: 1261–74.
Milanesi A, Lee J, Kim N, Liu Y, Yang A, Sedrakyan S, Kahng A,
Cervantes V, Tripuraneni N, Cheng S, Perin L, Brent GA
(2016) Thyroid hormone receptor a plays an essential role in
male skeletal muscle myoblast proliferation, differentiation,
and response to injury. Endocrinology 157: 4–15.
Mottis A, Mouchiroud L, Auwerx J (2013) Emerging roles of the
corepressors NCoR1 and SMRT in homeostasis. Genes Dev 27:
819–35.
Pascual G, Fong AL, Ogawa S, Gamliel A, Li AC, Perissi V, Rose
DW, Willson TM, Rosenfeld MG, Glass CK (2005)
A SUMOylation-dependent pathway mediates transrepression
of inflammatory response genes by PPAR-gamma. Nature 437:
759–63.
Peek CB, Affinati AH, Ramsey KM, Kuo H, Yu W, Sena LA, Ilkayeva
O, Marcheva B, Kobayashi Y, Omura C, Levine DC, Bacsik DJ,
Gius D, Newgard CB, Goetzman E, Chandel NS, Denu JM,
Mrksich M, Bass J (2013) Circadian clock NADþ cycle drives
mitochondrial oxidative metabolism in mice. Science 342: 1–8.
Cell Biol Int 9999 (2016) 1–9 © 2016 International Federation for Cell Biology
NCoR maintains insulin sensitivity in C2C12
Pirkmajer S, Chibalin AV (2011) Serum starvation: caveat
emptor. Am J Physiol Cell Physiol 301: C272–9.
Randhawa VK, Ishikura S, Talior-Volodarsky I, Cheng AWP,
Patel N, Hartwig JH, Klip A (2008) GLUT4 Vesicle recruitment
and fusion are differentially regulated by Rac, AS160, and
Rab8A in muscle cells. J Biol Chem 283: 27208–19.
Seglen PO, Grinde B, Solheim AE (1979) Inhibition of the
lysosomal pathway of protein degradation in isolated rat
hepatocytes by ammonia, methylamine, chloroquine and
leupeptin. Eur J Biochem 95: 215–25.
Sin J, Andres AM, Taylor DJR, Weston T, Hiraumi Y, Stotland A,
Kim BJ, Huang C, Doran KS, Gottlieb RA (2016) Mitophagy is
required for mitochondrial biogenesis and myogenic differen-
tiation of C2C12 myoblasts. Autophagy 12: 369–80.
Wang J, Yin L, Lazar MA (2006) The orphan nuclear receptor
Rev-erb regulates circadian expression of plasminogen activa-
tor inhibitor type 1. J Biol Chem 281: 33842–8.
Yamamoto H, Williams EG, Mouchiroud L, Canto C, Fan W,
Downes M, Heligon C, Barish GD, Desvergne B, Evans RM,
Schoonjans K, Auwerx J (2011) NCoR1 is a conserved
physiological modulator of muscle mass and oxidative
function. Cell 147: 827–39.
Yin L, Wang J, Klein PS, Lazar MA (2006) Nuclear receptor Rev-
erba is a critical lithium-sensitive component of the circadian
clock. Science 311: 1002–5.
Yin L, Wu N, Curtin JC, Qatanani M, Szwergold NR, Reid RA,
Waitt GM, Parks DJ, Pearce KH, Wisely GB, Lazar MA (2007)
Rev-erba, a heme sensor that coordinates metabolic and
circadian pathways. Science 318: 1786–9.
Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S,
Lecker SH, Goldberg AL (2007) FoxO3 coordinately activates
protein degradation by the autophagic/lysosomal and
proteasomal pathways in atrophying muscle cells. Cell Metab
6: 472–83.
Zhang X, Patel SP, McCarthy JJ, Rabchevsky AG, Goldhamer DJ,
Esser KA (2012) A non-canonical E-box within the MyoD core
enhancer is necessary for circadian expression in skeletal
muscle. Nucleic Acids Res 40: 3419–30.
Received 24 September 2016; accepted 26 November 2016.
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