Hindawi Publishing Corporation
Journal of Nutrition and Metabolism
Volume 2012, Article ID 152902, 9 pages
Transient Silencing of a Type IV P-Type ATPase,
in Decreased Glucose Uptake in C2C12 Myotubes
S. E. Hurst,
S. C. Minkin,
and M. S. Dhar
Comparative and Experimental Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA
Department of Large Animal Clinical Sciences, College of Veterinar y Medicine, University of Tennessee, Knoxville, TN 37996, USA
Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996, USA
Correspondence should be addressed to S. E. Hurst, email@example.com
Received 15 August 2011; Revised 15 October 2011; Accepted 29 October 2011
Academic Editor: Duo Li
Copyright © 2012 S. E. Hurst et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of
ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-
speciﬁc siRNA. Glucose uptake assays revealed that insulin stimulation caused a signiﬁcant 2.54-fold decrease in 2-deoxyglucose
uptake in transfected cells coupled with a signiﬁcant upregulation of native mitogen-activated protein kinases (MAPKs), p38, and
p44/42. Additionally, glucose transporter-1 (GLUT1) was signiﬁcantly upregulated; no changes in glucose transporter-4 (GLUT4)
expression were observed. The involvement of MAPKs was conﬁrmed using the speciﬁc inhibitor SB203580, which downregulated
the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose
uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a
signiﬁcant role in the de velopment of insulin resistance and type 2 diabetes.
In humans, skeletal muscle accounts for nearly 40% of the
body’s mass and serves as the main tissue involved in glucose
uptake during insulin stimulation. Several researchers have
established that glucose consumption in skeletal muscle
decreases with typ e 2 diabetes mellitus. This reduced glu-
cose consumption is a result of impaired transduction of
insulin signals, such as insulin receptor substrate-1 phos-
phorylation, phosphatidylinositol 3-kinase (PI3K) activity,
mitogen-activated protein kinase (MAPK) activity, insulin-
responsive glucose transporters, namely, glucose tr ansporter-
4 (GLUT4), and/or other insulin-independent mechanisms
. Therefore, a detailed analysis of insulin signaling at the
cellular and molecular level is critical to understand the
pathogenesis of type 2 diabetes associated with obesity.
Heterozygous Atp10c mice present with the disease states
of insulin resistance and obesity, as well as a host of other
related disorders, including hyperlipidemia and hyperinsu-
linemia. Previous research using these mice indicates that
the Atp10c gene appears to be a strong candidate gene
for diet-induced obesity and type 2 diabetes mellitus .
Atp10c is a putative phospholipid translocase or “ﬂippase,”
which encodes for a type IV P-type ATPase. Atp10c maps
to the p-locus on mouse chromosome 7, to a region of a
quantitative trait locus associated with body weight, body
fat, and diabetic phenotypes. The human ortholog, ATP10C,
maps to the syntenic region on chromosome 15q12 and is
also associated with an elevated body mass index [3, 4].
Moreover, microarray gene proﬁling on Atp10c heterozygous
mice indicated signiﬁcant changes in the mRNA expres-
sion of factors involved in insulin-dependent and insulin-
independent glucose uptake .
Although ﬂippases, like Atp10c,havebeenstudiedfor
many years, their exact character and function remain
unclear. These proteins are believed to maintain the asymme-
try of the lipid bilayer by translocating speciﬁc phospholipids
from one leaﬂet to the other and vice versa , but they
may also participate in the formation of transport vesicles
. Moreover, deﬁciencies in these proteins have been
shown to cause defects in lipid metabolism and have been
implicated in the disease states of obesity, type 2 diabetes,
and nonalcoholic fatty liver disease . Not much is known
about the role of ATP10C in regulating insulin resistance in
skeletal muscle, if any, and its possible molecular and cellular
targets have not been investigated.
In view of the above literature, we hypothesized that
the type IV P-type ATPase, ATP10C, has an important role
in glucose metabolism. Since ATP10C is a transmembrane
protein it might exert its eﬀect via multiple signaling
pathways: (1) acting solely at the plasma membrane to
maintain the nonrandom distribution of phospholipids,
thus contributing to a proper membrane environment for
normal protein sequestration and function, (2) acting at
the plasma membrane aﬀecting the biogenesis of mem-
brane vesicles important for plasma membrane delivery
and/or retrieval of glucose transporter proteins in basal
and insulin-stimulated states, and (3) acting directly on
the expression, translocation, and/or function of glucose
transporter proteins themselves. Since the MAPK pathway
is known to be a key signaling cascade which mediates
glucose clearance/uptake by the skeletal muscle in presence
or absence of insulin, we tested whether the MAPKs, in
general, are the targets of ATP10C. To prove our hypothesis,
speciﬁc objectives were to (a) establish a tissue culture system
of mouse skeletal muscle wherein endogenous expression of
Atp10c could be monitored, (b) alter the endogenous level of
Atp10c expression by siRNA, and (c) measure glucose uptake
and assess changes in expression of MAPKs involved in this
2. Materials and Methods
2.1. Materials. MouseskeletalmusclecelllineC2C12,a
commercially available cell line, was kindly provided by Dr.
Seung B aek, College of Veterinary Medicine, the University
of Tennessee, Knoxville, TN, USA. Dulbecco’s modiﬁed
Eagle medium (DMEM) containing 4.5 mg/L glucose and
4.5 mM/L L-glutamine, antibiotics (100 IU/mL penicillin
and 100 μg/mL streptomycin), ABsolute Blue S YBR Green
ROX quantitative PCR mix, bovine calf serum (BCS), and
radioimmunoprecipitation assay (RIPA) buﬀer were from
Thermo Fisher Scientiﬁc (Waltham, MA). DMEM with 1%
antibiotics and 10% BCS is furthermore referred to as
the complete growth media. Horse serum, 2-deoxy [
glucose (2-DOG), protease inhibitor cocktail in DMSO
solution, MAPK inhibitor SB203580, and human insulin
solution (10 mg/mL in HEPES, pH 8.2) were from Sigma
Aldrich (St. Louis, MO). A bicinchoninic acid kit (BCA)
and an enhanced chemiluminescence (ECL) western blotting
Detection Kit were purchased from Pierce Biotech Inc.
(Rockford, IL) and used in protein experiments. Primary
antibodies (p38, phospho-p38, JNK, phospho-JNK, p44/42,
and phospho-p44/42) as well as the secondary antibody,
horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG,
were obtained from Cell Signaling Technology (Danvers,
MA). Caveolin-1 was used as an immunoblot control and
was purchased from Santa Cruz Biotechnologies (Santa
Cruz, CA). The secondary antibody, HRP-conjugated anti-
goat IgG, was also obtained from Santa Cruz Biotechnolo-
gies. HiPerfect tr ansfection reagent, Atp10c -speciﬁc siRNA
constructs, RNeasy Mini Kit, and QuantiTect primer assays
for MyoD and Atp10c were from Qiagen (Valencia, CA).
Quantitative PCR primers speciﬁc for mouse glyceraldehyde
3-phosphate dehydrogenase (Gapdh) were designed using
the Primer 3 program (http://primer3.sourceforge.net/)and
were commercially obtained from Operon (Huntsville, AL).
The iScript cDNA synthesis kit was acquired from Bio-Rad
Laboratories (Hercules, CA). Anti-GLUT1 and GLUT4 were
kindly provided by Dr. Samuel Cushman, National Institute
of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, MD. All immunoﬂuorescence
materials ( protein blocks [normal rabbit], negative control
[normal rabbit], and antibody diluent) were purchased
from BioGenex (San Ramon, CA). Millicell EZ slides from
Millipore (Billerica, MA) were used for immunoﬂuorescence
experiments. Secondary antibody speciﬁc for immunoﬂuo-
rescence application, Alexa Fluor 568 donkey anti-rabbit, as
well as Prolong Gold Antifade reagent was purchased from
Invitrogen (Carlsbad, CA).
2.2. Cell Culture and Treatments. C2C12 myoblasts were
cultured as described elsewhere [ 8, 9]. Roughly 2.0
were seeded in a 60 mm dish or a single well of a 6-well plate.
They were maintained at 37
C and 5% CO
growth media. Cells at 70% conﬂuency were diﬀerentiated in
the presence of 2% horse serum-enriched media for 3–5 days.
Completely diﬀerentiated myotubes (days 3–5) were either
subjected to various treatments described in the relevant
sections or harvested for subsequent experiments.
2.3. siRNA Transfection. Three diﬀerent siRNA oligonu-
cleotides against Atp10c were commercially obtained (Qia-
gen); one was generated from the sequence at the 3
of the Atp10c gene, SI00906220 (sense: r[CCU GGG UAU
UGA AAC CAA A]dTdT and antisense: r[UUU GGU UUC
AAU ACC CAG G]dTdG), and the second, SI00906213,
and third, SI00906206, were generated from the sequence
at the 5
end (sense: r[CGU CUU UGC UGC AAU GAA
A]dTdT and antisense: r[UUU CAU UGC AGC AAA GAC
G]dGdA). C2C12 myotubes were transiently transfected
with Atp10c-speciﬁc and scrambled siRNA using HiPerfect
transfection reagent (Qiagen) according to the manufac-
turer’s instructions. Myotubes transfected with siRNA were
either harvested or treated with the reagents indicated in the
relevant sections and/or used to measure glucose uptake.
In the ﬁrst experiment, the optimum concentration and
time of knockdown for each siRNA used were determined.
Brieﬂy, HiPerfect transfection regent and siRNAs were mixed
at various concentrations (0, 50, 100, and 200 nM) to
form a complex. The transfection complexes were then
applied to designated cells and incubated for 24, 48, or 72 h
before subsequent analysis. C2C12 myotubes demonstrating
eﬃcient Atp10c knockdown and, therefore, used in all further
experiments were designated as C210c/
(i.e., transfected with HiPerfect only) C2C12 myotubes
(C2wt) were used as corresponding controls.
2.4. RNA, cDNA Synthesis, and Quantitative PCR. The
following procedures were per formed as described elsewhere
[5, 10, 11]. Cells were washed with ice cold 1X PBS,
and total RNAs were isolated using RNeasy Mini RNA
kit (Qiagen), according to the manufacturer’s instructions.
Single-stranded cDNA was synthesized using the iScript
cDNA synthesis kit (Bio-Rad) and ampliﬁed using gene-
speciﬁc primers by quantitative PCR (qPCR by Quantitect
primer assays), with mouse Gapdh as the housekeeping
gene. All mRNA expressions were achieved by qPCR using
ABsolute SYBR Green ROX quantitative PCR mix on the
Strategene Mx3005P with MxPro analysis software under
the following PCR conditions: 1 cycle of 50
C for 2 min, followed by 40 cycles of 95
C for 1 min. The relative abundance
of target gene expression was calculated using the 2-ΔΔCT
and standard curve method, with ΔΔCT being the diﬀerence
between CT of the target gene normalized with respect to the
Gapdh CT [ 12 ].
2.5. Preparation of Cellular Extracts, Immunoblotting, and
Immunoﬂuorescence. Total cell lysates were isolated using
RIPA buﬀer according to standard methods [13, 14]. Brieﬂy,
cells were washed twice w ith 1X PBS and lysed in RIPA buﬀer
containing protease inhibitor cocktails at 4
C for 30 min.
Lysates were centrifuged at 16,000 g for 10 min at 4
Protein estimation was p erformed using the BCA kit (Pierce
Biotech), according to the manufacturer’s instructions.
Immunoblot analysis was carried out according to stan-
dard procedures [13, 14]. Equal concentrations (25
of proteins were resolved on 10% SDS-PAGE, using 5X
Laemmli sample buﬀer containing Tris-HCL (375 mM, pH
6.8), glycerol (48%), SDS (6%), beta-mercaptoethanol (6%),
and bromophenol (0.03%). Cell lysates were denatured
by heating before being applied to SDS-PAGE gel. After
electrophoresis, proteins were transferred to nitrocellulose
membranes, blocked for 1 h in blocking solution (1–
5% BSA in TBST buﬀer), and incubated with sp eciﬁc
primary antibodies overnight at 4
C. Primary antibodies
were detected with HRP-conjugated secondary antibodies,
and antibody-protein complexes were v isualized using ECL
(Pierce Biotech). Results are expressed as the ratio of target
protein expression to that of an internal loading control,
For i mmunoﬂuorescence, 2.0
cells were seeded
onto 4-well chamber slides (Millipore) and subjec ted to the
appropriate treatments as described in the relevant sections.
Immunoﬂuorescence assays were carried out according to
standard methods as described by others [8, 15]. Brieﬂy,
cells were ﬁxed with 1% paraformaldehyde in 0.1 M sodium
phosphate buﬀer, pH 7.3 for 10 min at room temperature.
Cells were washed in 1X PBS, permeabilized by incubating
with 0.01% Tween-20/PBS for 10 min, and then washed
again with 1X PBS. After the last wash, cells were blocked
using blocking buﬀer (1% BSA, 2% normal serum, 0.1%
Tween-20 in PBS) for 30 min. Blocking solution contained
normal rabbit serum, the animal in which the primary
antibody was generated. Once blocking was complete,
the cells were incubated with speciﬁc primary antibodies
overnight at 4
C. Bound antibody was visualized under
the microscope (Nikon Ti-E Eclipse, Nikon Instruments,
Melville, NY ) by incubating for 1 h with secondary antibody
labeled with Alexa Fluor 568 (TRITC). To visualize the
nucleus, cells were exposed for 5 min at room temperature
to a concentration of 300 nM DAPI (4
phenylindole, dilactate) in PBS. DAPI was prepared and
diluted based on manufacturer’s instructions (Invitrogen).
After washing, cells were mounted using ProLong Gold
Antifade reagent (Invitrogen). Slides were sealed and allowed
to dry overnight before imaging. Additionally, both positive
and negative controls were prepared and imaged alongside
the samples to correct for any background ﬂuorescence
and to serve as controls for quantitative analysis. Images
were captured using an epiﬂuorescence microscope (Nikon
Instruments) with a 60x objective lens (NA 1.49) and an
2.6. Glucose Uptake. Glucose uptake in myotubes was mea-
sured as previously described . Brieﬂy, myoblasts were
plated in 6-well cell culture plates at a density of 2
cells/well and allowed to diﬀerentiate under normal
conditions. After diﬀerentiation, cells were washed with IX
PBS and serum-starved in DMEM only for 3
stimulated with 100 nM insulin in DMEM for 30 min at
C. Each 6-well plate was setup so that wells 1, 2, and 3
did not contain insulin and wells 4, 5, and 6 did contain
insulin. Insulin induction was stopped by washing the
cells twice with 1 mL Krebs-Ringer HEPES (KRP) (121 mM
NaCl, 4.9 mM KCl, 1.2 mM MgSO
, 0.33 mM CaCl
HEPES) minus glucose at room temperature. Cytochalasin B
(5 μL of 1 mM stock/1 mL cocktail) was used to normalize for
nonspeciﬁc glucose uptake. Glucose uptake was determined
after the addition of
H-2-deoxyglucose (1 μL of 10 Ci/mmol
stock/1 mL cocktail) in KRP buﬀer at 37
C for 5 min.
Incorporation was terminated by washing the cells twice with
1 mL ice cold KRP plus 25 mM glucose. Cells were lysed
on ice for 30 min with RIPA buﬀer. Following incubation,
0.5 mL cell lysates were mixed with 10 mL scintillation ﬂuid
(Scintiverse) and subjected to liquid scintillation counting.
For protein quantitation (BCA method, Pierce Biotech),
250 μL of the lysate was used and processed according to the
manufacturer’s instructions. Glucose uptake was expressed
as disintegrations per minute per micro gram of total protein
(dpm/μg). Data is reported as the glucose uptake stimulation
expressed as the ratio of dpm/μg of total protein in presence
of insulin to that in the absence of insulin.
2.7. Densitometry Analysis. Relative densitometry analy-
ses of the immunoblots were determined using Scion
(http://rsb.info.nih.gov/ij/index.html) analysis software. By
giving an arbitrary value of 1.0 to the respective control
sample (caveolin-1) of each experiment, a ratio of relative
density was calculated for each protein of interest.
2.8. Immunoﬂuorescence Quant itation. Immunoﬂuorescence
images were analyzed using NIS-Elements software (AR v.
3.1) (Nikon Instruments). For each sample, 9 dual-channel
images were captured and stitched together to form a large
× 3). Mean ﬂuorescence intensity per cell was
calculated (MFI per cell
= total image ﬂuorescence/cell
count). Cell count was determined using a nuclear stain
(300 nM DAPI in PBS).
2.9. Statistical Analysis. Thedataareexpressedasmean
SE. For comparison of two groups, P values were calculated
using the two-tailed paired Student t test. In all cases P
0.05 was considered statistically signiﬁcant, and P ≤ 0.10 was
indicative of a trend.
3. Results and Discussion
To alleviate whole-animal complexities, C2C12, a mouse
skeletal muscle cell line, was selected as an in vitro model
to identify molecular and ce llular targets of ATP10C and
then assess its biological role, if any, in insulin signaling and
glucose metabolism [16, 17].
Under permissive conditions, C2C12 myoblasts undergo
diﬀerentiation to form myotubes. Diﬀerentiation of C2C12
myoblasts to myotubes was conﬁrmed by visual inspection
(see Supplemental Figure 1 in Supplementary Material
available online at doi.10.1155/2012/152902), as well as
by monitoring the expression of skeletal muscle-speciﬁc
mRNAs for MyoD and myogenin . Cells were collected
on days 1, 3, and 5 during the diﬀerentiation process.
Cells collected at day 1 represent the myoblasts, which are
then stimulated to diﬀerentiateintomyotubesbydays3–5.
The activation of cell diﬀerentiation is characterized by the
expression of myogenic regulatory factors including MyoD
and myogenin. After proliferation, cells express myogenin
which commits the myoblasts towards myogenic diﬀeren-
tiation. This is followed by the expression of additional
factors including but not limited to Myf-5 and MRF4 and
permanent exit from the normal growth and proliferation
cycles. As expected, the expression of myogenin increased as
myoblasts were stimulated to diﬀerentiate (30% to 52%),
and decreased when diﬀerentiation was complete (20%).
Similarly, qPCR showed that MyoD expression increased
dramatically on day 1 (172%), then steadily declined on days
3 and 5 (64% and 24%, resp.). Interestingly, Atp10c was
expressed in both cell types, and its expression increased
upon diﬀerentiation (42% to 64%) and steadily decreased
as myotubes were formed (47% on day 5). Changes in
Atp10c expression with diﬀerentiation were similar, but, not
as striking as that of myogenin and MyoD (Supplemental
Figure 2), ramiﬁcations of which are clearly beyond the scope
of this study. The data thus demonstrated the expression of
Atp10c mRNA in C2C12 cells, and gave us an opportunity
to modulate its expression in both myoblasts and myotubes,
Atp10c mRNA knockdown (%)
Figure 1: C2C12 cells were diﬀerentiated from myoblasts to
myotubes as described in Section 2.Myotubesweretransfected
at each concentration of s iRNA (SI00906220) (0, 50, 100, and
200 nM) collected at the above time points (24, 48, and 72 h).
Gapdh (housekeeping gene) and Atp10c gene mRNA expression was
analyzed using quantitative PCR. The percentage of knockdown
was calculated at each concentration and time point based on the
expression of the mock-transfected (0 nM) samples (
on Qiagen recommendations). Data represents three independent
experiments with each sample repeated in triplicate.
both of which can have signiﬁcant consequences in insulin
One of the limitations of global gene-targeting and
whole-animal approach is that adaptations over time might
occur, possibly producing secondary phenotypes that are not
directly linked to the mutation. In this case, the role of Atp10c
could be shown either by generating knockout mice or
using speciﬁc inhibitors against ATP10C. Since no inhibitors
are available, and to avoid whole-animal complexities in
transgenics, we modulated Atp10c expression in vitro in
C2C12 cells by t ransiently transfecting them with three inde-
pendent and commercially available Atp10c-speciﬁc siRNAs
(Qiagen). ATP10C is a putative transmembrane domain
protein, and as such a good antibody against ATP10C has
yet to be generated, making experiments to study ATP10C
challenging . Therefore, in this analysis, changes in Atp10c
expression were determined solely at the mRNA level by
quantitative PCR using QuantiTect primer assays (Qiagen).
Out of the three siRNAs tested, only one, SI00906220,
resulted in a signiﬁcant knockdown of
≥70% (data not
As shown in Figure 1, signiﬁcant knockdowns (70 to
100%) were observed in all samples with the exception of
200 nM, 72 h. Higher concentrations and longer time periods
resulted in cell death and poor quality of cells (as judged
visually). Taking these criteria into consideration as well as
the optimization of concentration and time of transfection,
50 nM of siRNA (SI00906220) and a time point of 24 h
were selected for all subsequent experiments. Gapdh was
used as the housekeeping gene, and no signiﬁcant mRNA
0 nM 24 h
50 nM 24 h
Glucose uptake stimulation (a.u.)
Figure 2: C2C12 cells were diﬀerentiated from myoblasts to
myotubes as described in Section 2.Myotubesweretransfected
at each concentration of siRNA (SI00906220) (0 and 50 nM) at
the designated time point (24 h). Cells were then stimulated with
insulin (100 nM, 30 min), and a 2-D OG uptake was performed
which is expressed as the ratio of dpm/μg of total protein in presence
of insulin to that in the absence of insulin.
changes in its corresponding expression in transfected cells
were observed. Results thus suggest that changes in Atp10c
expression after transfection with siRNA (SI00906220) were
not an artifact, and there was no deleterious eﬀect on
C2C12 myotubes due to Atp10c silencing. All further exper-
iments were carried out under these conditions in wild-
type C2C12 (C2wt) and Atp10c-silenced C2C12 myotubes
−) simultaneously. Since these transfections are
transient, Atp10c mRNA knockdown was conﬁrmed in each
and every experiment.
Previous experiments in our laboratory have shown that
on a high fat diet, there is a 35% decrease in glucose uptake
in soleus muscle in Atp10c heterozygotes . Based on
this ﬁnding, we next determined whether downregulation
of Atp10c expression had a similar eﬀect in vitro.Glucose
uptake was measured in C2wt and C210c/
− myotubes in
basal (without insulin) and stimulated (100 nM insulin,
30 min) states. Fold change representing the glucose uptake
stimulation/reduction between the C2wt and C210c/
compared. As shown in Figure 2, insulin stimulation caused
a signiﬁcant 2.54-fold decrease in 2-DOG uptake in C210c/
cells (P<0.05). Data thus complement the in vivo ﬁndings,
suggesting that ATP10C is necessary for insulin-stimulated
glucose uptake in skeletal muscle, and its knockdown renders
the myotubes insulin resistant.
Silencing Atp10c RNA decreases cellular glucose uptake,
which might be of consequence to impaired insulin signaling.
Insulin-induced glucose uptake into muscle and adipose
tissue involves a series of intracellular signaling cascades,
culminating in glucose disposal and metabolism [19–24].
The possible mechanisms include insulin-mediated activa-
tion of the insulin receptor and/or its downstream molecules,
ultimately eﬀecting GLUT4 expression and translocation.
Since these processes are a complex interplay of a variety of
proteins and because their changes have not been studied in
Atp10c silencing, we sought to identify changes in the key
proteins of one such signaling cascade, the MAPK pathway
in the absence of any insulin stimulation. Speciﬁcally, in the
present study, the eﬀect of Atp10c silencing was considered
on three essential MAPKs: p38 (MapK14), JNK, and p44/42,
(ERK1/2). Total proteins isolated from C2wt and C210c/
myotubes were subjected to a combination of immunoblot
and immunoﬂuorescence analysis (Figures 3(a)–3(c)).
For MAPK pathway analysis, results indicated signiﬁcant
upregulation of p38 (P
= 0.02) and p44/42 (P = 0.04)
and a signiﬁcant downregulation of JNK (P
and phospho-p44/42 (P
= 0.05) in C210c/− cells. While
not signiﬁcant, results indicated a trend for an increase in
= 0.1) (Figure 3(a)) and phospho-JNK
= 0.06) (Figure 3(c)). Beside these signal transduction
proteins, there was signiﬁcant upregulation of MyoD (P
0.01), Actin (P = 0.02) (Supplemental Figures 3(a) and
3(b)), and GLUT1 (P
= 0.03). Most importantly, there was
no signiﬁcant change in GLUT4 expression (Figure 4).
The exact mechanism by which the MAPKs mediate
that both in vitro and in vivo data are inconclusive. MAPKs,
speciﬁcally, p38 and p44/42 proteins, regulate g lucose uptake
via insulin-dependent and independent pathways [25–27].
Our results suggest that when myotubes become insulin
resistant by Atp10c silencing, as expected there is a signiﬁcant
increase in the expression of native MAPKs; however, they
are not activated into their phosphorylated forms at a
signiﬁcant level. Data demonstrates that p38 and p44/42 are
responsive to changes in Atp10c with increased expression,
whereas there is no similar eﬀectonJNK,suggestingthatitis
not a target at least in this pathway.
The importance of p38 in this process is further sup-
ported by the fact that there are signiﬁcant changes in glucose
transporter proteins as well. GLUT1 and GLUT4 have been
demonstrated to be the key players of glucose clearance
in peripheral tissues . GLUT1 is responsible for glucose
uptake in the basal state whereas GLUT4 is insulin responsive
. Defective uptake of glucose mediated by the GLUTs is
a central feature of obesity and type 2 diabetes. Interestingly,
microarray gene proﬁling of peripheral tissues obtained from
Atp10c heterozygote s which were fed a high fat diet for 4
weeks demonstrated upregulation of p38 in soleus muscle
Of relevance to our study, MAPK protein expression,
speciﬁcally p38, has been shown to aﬀect the expression of
GLUT1 and GLUT4, which can subsequently aﬀect glucose
uptake in peripheral tissues . The MAPK protein, p38,
reportedly upregulates the expression of GLUT1, thereby
altering glucose transport at the basal level, while also
involved in an insulin-induced enhancement of intrinsic
GLUT4 activity on the cell sur face. Since, we do not see
0 nM 24 h 50 nM 24 h
p38 and phospho-p38 fold change
0 nM 24 h 50 nM 24 h
p44/42 and phospho-p44/42 fold change
JNK and pJNK fold change
0 nM 24 h 50 nM 24 h
Figure 3: (a–c): C2C12 cells were diﬀerentiatedfrommyoblaststomyotubesasdescribedinSection 2. Myotubes were transfected at each
concentration of siRNA (SI00906220) (0 and 50 nM) and collected at the designated time point (24 h). Proteins were collected from these
samples and subjected to immunoblot analysis. Data shown are representative of multiple independent experiments (n
= 2 to 4), all analyzed
in triplicate (
any increase in the basal glucose uptake, we strongly feel
that the upregulation of GLUT1 is more of a compensatory
mechanism against insulin resistance in C210c/
−, since there
is no change in GLUT4; the precise underlying mechanism,
however, remains to be clariﬁed. This result may prove to
be the most crucial, as GLUT4 is the main player in insulin-
stimulated glucose metabolism. Reports additionally indicate
that the activation of p38 reduces insulin responsiveness ,
so the exact mechanism by which the MAPK pathway is
involved in glucose metabolism still remains controversial.
Up-regulation of MyoD and Actin in transfected cells
−) suggests a regulatory mechanism by which
the myotubes are tr ying to combat the stressful state of
insulin resistance. By aiding with GLUT4-containing vesicle
membrane movement and/or fusion, there is considerable
evidence that Actin is essential for insulin-regulated glucose
transport. Therefore, these cells may be overexpressing Actin
acutely in preparation for insulin-stimulated glucose uptake
To conﬁrm the involvement of p38, the p38-speciﬁc
inhibitor SB203580 was added to C210c/
− cells at a concen-
tration of 10 μmfor60min[30, 31]. Protein samples were
subjected to immunoblot analysis with p38 andphospho-p38
antibodies (Figure 5).
Our results indicate that C210c/
− cells treated with
10 nM SB203580 for 60 min eﬀected the expression of both
Mock 24 h 50 nM 24 h
Mean ﬂuorescence intensity/cell
Figure 4: C2C12 cells were seeded onto chamber slides and allowed
to diﬀerentiate from myoblasts to myotubes as described in the
Materials and Methods section. Myotubes were then transfected
at each concentration of siRNA (SI00906220) (0 and 50 nM) and
collected at the designated time point (24 h). After transfections,
cells underwent standard immunoﬂuorescence processing and were
imaged. Each sample was compared to negative and positive
controls, which were used to quantify the image results (
p38 (P = 0.1) and phospho-p38 (P = 0.08) (Figure 5).
While not signiﬁcant, it appears that the inhibitor was able
to partially restore the expression of all the proteins tested
suggesting its action on the MAPKs. This was further con-
ﬁrmed by the glucose uptake assay performed on C210c/
and MAPK-inhibited cells. Results from the assay showed
that glucose uptake remained unchanged, conﬁrming that
the inhibitors are acting directly on the MAPK proteins and
are not aﬀected by changes in Atp10c expression (Figure 6).
A phenomenon known as phospholipid randomization
aﬀects the structure and function of many channels, t rans-
porters, and signal transducing proteins and has been
implicated in several pathophysiology processes. Thus, main-
taining the organization and activ ity of the lipid bilayer is
essential for normal cell function. One class of proteins that
performs this action is the ﬂippases or type-IV ATPases .
In yeast studies, these proteins cause the translocation of
glycerophospholipids, and this movement is necessary for
intracellular membrane and protein traﬃcking . Atp10c
is one such phospholipid translocase which encodes for
0 nM control 50 nM control 0 nM
p38 and phospho-p38 fold change
Figure 5: C2C12 cells were diﬀerentiated from myoblasts to
myotubes as described in the Materials and Methods section.
Myotubes were transfected at each concentration of siRNA
(SI00906220) (0 and 50 nM) at the designated time point (24 h).
Cells were then treated with the inhibitor SB203580 (10 nM) and
collected after 60 min. Proteins were collected from these samples
and subjected to immunoblot analysis (
Glucose uptake stimulation
SB203580 + +
0 nM 24 h 50 nM 24 h
Figure 6: C2C12 cells were diﬀerentiated from myoblasts to
myotubes as described in the Materials and Methods section.
Myotubes were treated with the inhibitor SB203580 (10 nM) and
collected after 60 min. Cells were then stimulated with insulin
(100 nM, 30 min), and a 2-DOG uptake was performed (
a type IV P-type ATPase. Our laboratory has demonstrated
that Atp10c heterozygous mice are insulin resistant and
have an altered insulin-stimulated response in peripheral
tissues. Our obesity mouse model is dietinduced and shows
insulin resistance chara cterized by hyperinsulinemia, hyper-
glycemia, hyperlipidemia, and obesity in association with
glucose intolerance [2, 3, 11]. In fact, a recent publication
has sited ATP10C as a potential biomarker for obesity and
related metabolic disorders .
In the present study, we report, for the ﬁrst time, a
direct correlation between Atp10c mRNA expression levels
and glucose metabolism, at least in part via the MAPK
pathway in C2C12 skeletal muscle cells. Our results showed
no signiﬁcant change in the basal glucose uptake suggesting
that w hen Atp10c is silenced using Atp10c -speciﬁc siRNA,
an acute state of insulin resistance is observed. Conversely,
cells are insulin sensitive when normal levels of Atp10c
are maintained. The MAPK protein, speciﬁcally p38, is
potentially inﬂuencing this system by exerting an eﬀect
on the glucose transporter proteins. Results from these
experiments are interesting and lead to future experiments
that will involve other signaling cascades (PI3K), insulin
receptors, and substrates as well as other downstream factors,
including GLUT4 translocation.
The authors give special thanks to Erin Bartley. Her work
as our laboratory a ssistant is invaluable, and much of this
work would not have been possible without her help. They
would also like to especially thank the funding agencies: The
American Diabetes Association, The University of Tennessee
Center of Excellence in Livestock Diseases and Human
Health, and the University of Tennessee Obesity Research
Center. Additionally, they thank Misty Bailey for her editing
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