ArticlePDF Available

Abstract and Figures

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 development of insulin resistance and type 2 diabetes.
Content may be subject to copyright.
Hindawi Publishing Corporation
Journal of Nutrition and Metabolism
Volume 2012, Article ID 152902, 9 pages
doi:10.1155/2012/152902
Research Article
Transient Silencing of a Type IV P-Type ATPase,
Atp10c
, Results
in Decreased Glucose Uptake in C2C12 Myotubes
S. E. Hurst,
1, 2
S. C. Minkin,
3
J. Biggerstaff,
3
and M. S. Dhar
2
1
Comparative and Experimental Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA
2
Department of Large Animal Clinical Sciences, College of Veterinar y Medicine, University of Tennessee, Knoxville, TN 37996, USA
3
Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996, USA
Correspondence should be addressed to S. E. Hurst, shurst6@utk.edu
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.
1. Introduction
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
[1]. 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 [2].
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 [5].
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 [6], but they
may also participate in the formation of transport vesicles
[7]. 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,
2 JournalofNutritionandMetabolism
and nonalcoholic fatty liver disease [2]. 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 eect 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 aecting 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
process.
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. Dulbeccos 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) buer 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 [
3
H]
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
× 10
5
cells
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
2
in complete
growth media. Cells at 70% confluency were dierentiated in
the presence of 2% horse serum-enriched media for 3–5 days.
Completely dierentiated 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 dierent siRNA oligonu-
cleotides against Atp10c were commercially obtained (Qia-
gen); one was generated from the sequence at the 3
end
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
ecient Atp10c knockdown and, therefore, used in all further
experiments were designated as C210c/
. Mock-transfected
JournalofNutritionandMetabolism 3
(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
Cfor15min
and 95
C for 2 min, followed by 40 cycles of 95
Cfor25s,
52
Cfor25s,and72
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 dierence
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 buer according to standard methods [13, 14]. Briefly,
cells were washed twice w ith 1X PBS and lysed in RIPA buer
containing protease inhibitor cocktails at 4
C for 30 min.
Lysates were centrifuged at 16,000 g for 10 min at 4
C.
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
100 μgs)
of proteins were resolved on 10% SDS-PAGE, using 5X
Laemmli sample buer 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 buer), 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,
caveolin-1.
For i mmunofluorescence, 2.0
× 10
4
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 buer, 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 buer (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
, 6-diamidino-2-
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
automated stage.
2.6. Glucose Uptake. Glucose uptake in myotubes was mea-
sured as previously described [11]. Briefly, myoblasts were
plated in 6-well cell culture plates at a density of 2
×
10
5
cells/well and allowed to dierentiate under normal
conditions. After dierentiation, cells were washed with IX
PBS and serum-starved in DMEM only for 3
5h.Cellswere
stimulated with 100 nM insulin in DMEM for 30 min at
37
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
4
, 0.33 mM CaCl
2
,12mM
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
3
H-2-deoxyglucose (1 μL of 10 Ci/mmol
stock/1 mL cocktail) in KRP buer 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 buer. 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://scion-image.software.informer.com/)andImageJ
(http://rsb.info.nih.gov/ij/index.html) analysis software. By
giving an arbitrary value of 1.0 to the respective control
4 JournalofNutritionandMetabolism
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
image (3
× 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
dierentiation to form myotubes. Dierentiation 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 [18]. Cells were collected
on days 1, 3, and 5 during the dierentiation process.
Cells collected at day 1 represent the myoblasts, which are
then stimulated to dierentiateintomyotubesbydays35.
The activation of cell dierentiation is characterized by the
expression of myogenic regulatory factors including MyoD
and myogenin. After proliferation, cells express myogenin
which commits the myoblasts towards myogenic dieren-
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 dierentiate (30% to 52%),
and decreased when dierentiation 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 dierentiation (42% to 64%) and steadily decreased
as myotubes were formed (47% on day 5). Changes in
Atp10c expression with dierentiation 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,
20
40
60
80
100
120
0
Atp10c mRNA knockdown (%)
100 nM
200 nM
50 nM
100 nM
200 nM
50 nM
100 nM
200 nM
50 nM
48 h
72 h
24 h
Figure 1: C2C12 cells were dierentiated 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 (
P<0.7based
on Qiagen recommendations). Data represents three independent
experiments with each sample repeated in triplicate.
both of which can have significant consequences in insulin
signaling pathways.
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
shown).
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
JournalofNutritionandMetabolism 5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 nM 24 h
50 nM 24 h
Glucose uptake stimulation (a.u.)
Figure 2: C2C12 cells were dierentiated 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
(
P<0.05).Dataisreportedastheglucoseuptakestimulation
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 eect 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
(C210c/
) 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 [5]. Based on
this finding, we next determined whether downregulation
of Atp10c expression had a similar eect 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/
was
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 [1924].
The possible mechanisms include insulin-mediated activa-
tion of the insulin receptor and/or its downstream molecules,
ultimately eecting 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 eect 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
= 0.001)
and phospho-p44/42 (P
= 0.05) in C210c/ cells. While
not significant, results indicated a trend for an increase in
phospho-p38 (P
= 0.1) (Figure 3(a)) and phospho-JNK
(P
= 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
glucoseuptakeisdebatable.Thisissuchacomplexprocess
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 [2527].
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 eectonJNK,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 [5]. GLUT1 is responsible for glucose
uptake in the basal state whereas GLUT4 is insulin responsive
[28]. 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
[5].
Of relevance to our study, MAPK protein expression,
specifically p38, has been shown to aect the expression of
GLUT1 and GLUT4, which can subsequently aect glucose
uptake in peripheral tissues [28]. 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
6 JournalofNutritionandMetabolism
0
1
2
3
4
5
6
7
8
9
10
0 nM 24 h 50 nM 24 h
p38
pp38
p38 and phospho-p38 fold change
#
(a.u.)
(a)
0
5
10
15
20
25
30
0 nM 24 h 50 nM 24 h
p44/42
pp44/42
p44/42 and phospho-p44/42 fold change
(a.u.)
(b)
0
1
2
3
4
5
6
7
JNK
pJNK
JNK and pJNK fold change
#
0 nM 24 h 50 nM 24 h
(a.u.)
(c)
Figure 3: (a–c): C2C12 cells were dierentiatedfrommyoblaststomyotubesasdescribedinSection 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 (
P<0.05,
#
P<0.1).
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 [25],
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
(C210c/
) 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
[29].
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 eected the expression of both
JournalofNutritionandMetabolism 7
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Glut1
Glut4
Mock 24 h 50 nM 24 h
Mean fluorescence intensity/cell
(a.u.)
Figure 4: C2C12 cells were seeded onto chamber slides and allowed
to dierentiate 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 (
P<0.05).
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 aected by changes in Atp10c expression (Figure 6).
4. Conclusions
A phenomenon known as phospholipid randomization
aects 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 [6].
In yeast studies, these proteins cause the translocation of
glycerophospholipids, and this movement is necessary for
intracellular membrane and protein tracking [6]. Atp10c
is one such phospholipid translocase which encodes for
0
0.5
1
1.5
2
2.5
3
0 nM control 50 nM control 0 nM
SB203580
50 nM
SB203580
p38
pp38
p38 and phospho-p38 fold change
#
#
#
siRNA
SB203580
+
−−
+
+
+
(a.u.)
Figure 5: C2C12 cells were dierentiated 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 (
P<0.05,
#
P<0.1).
0
0.5
1
1.5
2
Glucose uptake stimulation
SB203580 + +
0 nM 24 h 50 nM 24 h
(a.u.)
Figure 6: C2C12 cells were dierentiated 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 (
P<0.05,
#
P<0.1).
8 JournalofNutritionandMetabolism
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 [32].
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 eect
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.
Acknowledgments
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
assistance.
References
[1] K. Hayata, K. Sakano, and S. Nishinaka, “Establishment of new
highly insulin-sensitive cell lines and screening of compounds
to facilitate glucose consumption, Journal of Pharmacological
Sciences, vol. 108, no. 3, pp. 348–354, 2008.
[2] M. S. Dhar, C. S. Sommardahl, T. Kirkland et al., “Mice
heterozygous for atp10c, a putative amphipath, represent a
novel model of obesity and type 2 diabetes, The Journal of
Nutrition, vol. 134, no. 4, pp. 799–805, 2004.
[3] M.Dhar,L.S.Webb,L.Smith,L.Hauser,D.Johnson,andD.B.
West, A novel ATPase on mouse chromosome 7 is a candidate
gene for increased body fat, Physiol Genomics, vol. 4, no. 1,
pp. 93–100, 2000.
[4] M. Meguro, A. Kashiwagi, K. Mitsuya et al., A novel mater-
nally expressed gene, ATP10C, encodes a putative aminophos-
pholipid translocase associated with Angelman syndrome,
Nature Genetics, vol. 28, no. 1, pp. 19–20, 2001.
[5]M.S.Dhar,J.S.Yuan,S.B.Elliott,andC.Sommardahl,
A type IV P-type ATPase aects insulin-mediated glucose
uptake in adipose tissue and skeletal muscle in mice, Journal
of Nutritional Biochemist ry, vol. 17, no. 12, pp. 811–820, 2006.
[6] C. C. Paulusma and R. P. J. Oude Elferink, “Diseases of
intramembranous lipid transport, FEBS Letters, vol. 580, no.
23, pp. 5500–5509, 2006.
[7] G. Lenoir and J. C. Holthuis, “The elusive flippases, Current
Biology, vol. 14, no. 21, pp. R912–R913, 2004.
[8] N. Kumar and C. S. Dey, “Development of insulin resistance
and reversal by thiazolidinediones in C2C12 skeletal muscle
cells, Biochemical Pharmacology, vol. 65, no. 2, pp. 249–257,
2003.
[9] B. Bisht, H. L. Goel, and C. S. Dey, “Focal adhesion kinase
regulates insulin resistance in skeletal muscle, Diabetologia,
vol. 50, no. 5, pp. 1058–1069, 2007.
[10] S. Roshwalb, S. Gorman, S. Hurst et al., “mRNA expression of
canine ATP10C, a P4-type ATPase, is positively associated w ith
body condition score, The Veterinary Journal, vol. 190, no. 1,
pp. 174–175, 2011.
[11] A. L. Peretich, M. Cekanova, S. Hurst, S. J. Baek, and M. S.
Dhar, “PPAR agonists down regulate the expression of atp10c
mRNA during adipogenesis, The O pen Obesit y Journal, vol. 1,
pp. 41–54, 2009.
[12] M. W. Pfa, A new mathematical model for relative quantifi-
cation in real-time RT-PCR, Nucleic Acids Research, vol. 29,
no. 9, p. e45, 2001.
[13] M. Hance, M. S. Dhar, and H. K. Plummer III, Tobacco
carcinogens stimulate dierent signaling pathways in breast
cancer, Breast Cancer, vol. 1, pp. 25–34, 2008.
[14] M. S. Dhar and H. K. Plummer III, “Protein expression of
G-protein inwardly rectifying potassium channels (GIRK) in
breast cancer cells, BMC Physiology, vol. 6, article no. 8, 2006.
[15] B. Bisht and C. S. Dey, “Focal Adhesion Kinase contributes to
insulin-induced actin reorganization into a mesh harboring
glucose transporter-4 in insulin resistant skeletal muscle cells,
BMC Cell Biology, vol. 9, article no. 48, 2008.
[16] T. N. Lui, C. W. Tsao, S. Y. Huang, C. H. Chang, and J. T.
Cheng, Activation of imidazoline I2B receptors is linked with
AMP kinase pathway to increase glucose uptake in cultured
C2C12 cells, Neuroscience Letters, vol. 474, no. 3, pp. 144–147,
2010.
[17] T. Nedachi and M. Kanzaki, “Regulation of glucose trans-
porters by insulin and extracellular glucose in C2C12
myotubes, American Journal of Physiology, vol. 291, no. 4, pp.
E817–E828, 2006.
[18] T. Shimokawa, M. Kato, O. Ezaki, and S. Hashimoto,
“Transcriptional regulation of muscle-specific genes dur-
ing myoblast dierentiation, Biochemical and Biophysical
Research Communications, vol. 246, no. 1, pp. 287–292, 1998.
[19] E. B. Katz, R . Burcelin, T. S. Tsao, A. E. Stenbit, and M.
J. Charron, “The metabolic consequences of altered glucose
transporter expression in transgenic mice, Journal of Molecu-
lar Medicine, vol. 74, no. 11, pp. 639–652, 1996.
[20] R. T. Watson, M . Kanzaki, and J. E. Pessin, “Regulated mem-
brane tracking of the insulin-responsive glucose transporter
4 in adipocytes, Endocrine Review s , vol. 25, no. 2, pp. 177–204,
2004.
[21] G. I. Shulman, “Cellular mechanisms of insulin resistance,
Journal of Clinical Investigation, vol. 106, no. 2, pp. 171–176,
2000.
[22] R. T. Watson and J. E. Pessin, “Intracellular organization of
insulin signaling and GLUT4 translocation, Recent Progress in
Hormone Research, vol. 56, pp. 175–193, 2001.
[23] P. R. Shepherd and B. B. Kahn, “Glucose transporters and
insulin action: implications for insulin resistance and diabetes
mellitus, New England Journal of Medicine, vol. 341, no. 4, pp.
248–257, 1999.
JournalofNutritionandMetabolism 9
[24] H. Wallberg-Henriksson and J. R. Zierath, “GLUT4: a key
player regulating glucose homeostasis? Insights from trans-
genic and knockout mice, Molecular Membrane Biology, vol.
18, no. 3, pp. 205–211, 2001.
[25] M. Fujishiro, Y. Gotob, H. Katagiri et al., “MKK6/3 and p38
MAPK pathway activation is not necessary for insulin-induced
glucose uptake but regulates glucose transporter expression,
Journal of Biological Chemistry, vol. 276, no. 23, pp. 19800–
19806, 2001.
[26] N. Kumar and C. S. Dey, “Gliclazide increases insulin receptor
tyrosine phosphorylation but not p38 phosphorylation in
insulin-resistant skeletal muscle cells, Journal of Experimental
Biology, vol. 205, no. 23, pp. 3739–3746, 2002.
[27] N. Wijesekara, F. S. L. Thong, C. N. Antonescu, and A. Klip,
“Diverse signals regulate glucose uptake into skeletal muscle,
Canadian Journal of Diabetes, vol. 30, no. 1, pp. 80–88, 2006.
[28] M. Ariga, T. Nedachi, H. Katagiri, and M. Kanzaki, “Func-
tional role of sortilin in myogenesis and development
of insulin-responsive glucose transport system in C2C12
myocytes, Journal of Biological Chemistry, vol. 283, no. 15, pp.
10208–10220, 2008.
[29] A. M. McCarthy, K . O. Spisak, J. T. Brozinick, and J. S. Elmen-
dorf, “Loss of cortical actin filaments in insulin-resistant
skeletal muscle cells impairs GLUT4 vesicle tracking and
glucose transport, American Journal of Physiology, vol. 291,
no. 5, pp. C860–C868, 2006.
[30] M. Koistinaho and J. Koistinaho, “Role of p38 and p44/42
mitogen-activated protein kinases in microglia, Glia, vol. 40,
no. 2, pp. 175–183, 2002.
[31] H. Wang, Q. Xu, F. Xiao, Y. Jiang, and Z. Wu, “Involvement of
the p38 mitogen-activated protein kinase α, β,andγ isoforms
in myogenic dierentiation, Molecular Biology of the Cell, vol.
19, no. 4, pp. 1519–1528, 2008.
[32] F. I. Milagro, J. Campi
´
on, P. Cordero et al., A dual epigenomic
approach for the search of obesity biomarkers: DNA methy-
lation in relation to diet-induced weight loss, The FASEB
Journal, vol. 25, no. 4, pp. 1378–1389, 2011.
... [93] In order to facilitate the understanding of ATP10C in insulin signalling and glucose metabolism, one study explored the MAPK signalling cascade in C2C12 myotubes. [94] In C2C12 cells, Atp10c mRNA is expressed in both myoblasts and myotubes. Compared to wild-type C2C12 cells, it was reported that glucose uptake was diminished by 2.5-fold in Atp10c-silenced C2C12 myotubes when stimulated by 100 nM insulin, suggesting that the knockdown of Atp10c rendered insulin resistance and reduced insulin-dependent glucose uptake. ...
... Glucose uptake was diminished by 2.5-fold in Atp10c-silenced C2C12 myotubes when stimulated by 100 nM insulin, therefore the knockdown of Atp10c rendered insulin resistance and reduced insulindependent glucose uptake [94] NYGGF4 genes NYGGF4 gene is involved in insulin signalling pathway, and its silencing could be a potential target to augment glucose uptake for treatment of insulin resistance [98] Thrombospondin 1 In the presence of insulin at 10 nM and 100 nM, thrombospondin 1 treatment attenuated Akt phosphorylation in C2C12 myotubes by 16% and 26%, respectively. Therefore, therapeutic agents that can suppress thrombospondin 1 would be a potential treatment option for insulin resistance [106] implication on insulin signalling pathway can affect GLUT-4 expression ultimately. ...
... As Atp10c-silenced C2C12 myotubes did not alter glucose uptake significantly at the basal level, it was suggested that insulin resistance occurred in cells. [94] NYGGF4 genes ...
Article
Objectives The myoblast cell line, C2C12, has been utilised extensively in vitro as an examination model in understanding metabolic disease progression. Although it is indispensable in both preclinical and pharmaceutical research, a comprehensive review of its use in the investigation of insulin resistance progression and pharmaceutical development is not available. Key findings C2C12 is a well‐documented model, which can facilitate our understanding in glucose metabolism, insulin signalling mechanism, insulin resistance, oxidative stress, reactive oxygen species and glucose transporters at cellular and molecular levels. With the aid of the C2C12 model, recent studies revealed that insulin resistance has close relationship with various metabolic diseases in terms of disease progression, pathogenesis and therapeutic management. A holistic, safe and effective disease management is highly of interest. Therefore, significant efforts have been paid to explore novel drug compounds and natural herbs that can elicit therapeutic effects in the targeted sites at both cellular (e.g. mitochondria, glucose transporter) and molecular level (e.g. genes, signalling pathway). Summary The use of C2C12 myoblast cell line is meaningful in pharmaceutical and biomedical research due to their expression of GLUT‐4 and other features that are representative to human skeletal muscle cells. With the use of the C2C12 cell model, the impact of drug delivery systems (nanoparticles and quantum dots) on skeletal muscle, as well as the relationship between exercise, pancreatic β‐cells and endothelial cells, was discovered.
... Heterozygous deletions of Atp10a in mice lead to diet-induced obesity, insulin resistance, and nonalcoholic fatty liver disease [45]. Atp10a is also implicated in regulation of insulin-stimulated glucose uptake via regulation of the MAPK signaling pathway in skeletal muscle and adipose tissue [46,47]. Nevertheless, our insulin tolerance tests revealed that the peripheral insulin sensitivity seemed not to be affected by the QTL containing the Atp10a gene (Fig. 4). ...
Article
Full-text available
Background A genome-wide mapping study using male F2zinc transporter 7-knockout mice (znt7-KO) and their wild type littermates in a mixed 129P1/ReJ (129P1) and C57BL/6J (B6) background identified a quantitative trait locus (QTL) on chromosome 7, which had a synergistic effect on body weight gain and fat deposit with the znt7-null mutation. Results The genetic segment for body weight on mouse chromosome 7 was investigated by newly created subcongenic znt7-KO mouse strains carrying different lengths of genomic segments of chromosome 7 from the 129P1 donor strain in the B6 background. We mapped the sub-QTL for body weight in the proximal region of the previously mapped QTL, ranging from 47.4 to 64.4 megabases (Mb) on chromosome 7. The 129P1 donor allele conferred lower body weight gain and better glucose handling during intraperitoneal glucose challenge than the B6 allele control. We identified four candidate genes, including Htatip2, E030018B13Rik, Nipa1, and Atp10a, in this sub-QTL using quantitative RT-PCR and cSNP detection (single nucleotide polymorphisms in the protein coding region). Conclusions This study dissected the genetic determinates of body weight and glucose metabolism in znt7-KO mice. The study demonstrated that a 17-Mb long 129P1 genomic region on mouse chromosome 7 conferred weight reduction and improved glucose tolerance in znt7-KO male mice. Among the four candidate genes identified, Htatip2 is the most likely candidate gene involved in the control of body weight based on its function in regulation of lipid metabolism. The candidate genes discovered in this study lay a foundation for future studies of their roles in development of metabolic diseases, such as obesity and type 2 diabetes. Electronic supplementary material The online version of this article (10.1186/s12863-019-0715-2) contains supplementary material, which is available to authorized users.
... Our findings are in good agreement with reports demonstrating the role of ATP10A (formerly ATP10C), another class 5 P4-type ATPase, in diet-induced obesity, T2D and insulinstimulated glucose uptake [38][39][40]. Like ATP10D, ATP10A depends upon interaction with TMEM30A/CDC50A to translocate from the endoplasmatic reticulum to the plasma membrane. ...
Article
Full-text available
Background Sequence variants near the human gene for P4-type ATPase, class V, type 10D (ATP10D) were shown to significantly associate with circulating hexosylceramide d18:1/16:0 and d18:1/24:1 levels, obesity, insulin resistance, plasma high density lipoprotein (HDL), coronary stenotic index and intracranial atherosclerotic index. In mice Atp10d is associated with HDL modulation and C57BL/6 mice expressing a truncated, non-functional form of ATP10D easily develop obesity and insulin resistance on high-fat diet. Results We analyzed metabolic differences of ATP10D deficient C57BL/6J wild type and ATP10D transgenic C57BL/6J BAC129 mice. ATP10D transgenic mice gain 25% less weight on high-fat diet concomitant with a reduced increase in fat cell mass but independent of adipocyte size change. ATP10D transgenic mice also had 26% lower triacylglycerol levels with approximately 76% bound to very low density lipoprotein while in ATP10D deficient wild type mice 57% are bound to low density lipoprotein. Furthermore increased oxygen consumption and CO2 production, 38% lower glucose and 69% lower insulin levels and better insulin sensitivity were observed in ATP10D transgenic mice. Besides decreased hexosylceramide species levels were detected. Part of these effects may be due to reduced hepatic stearoyl-CoA desaturase 1 (SCD1) expression in ATP10D transgenic mice, which was reflected by altered fatty acid and lipid species patterns. There was a significant decrease in the hepatic 18:1 to 18:0 free fatty acid ratio in transgenic mice. The ratio of 16:1 to 16:0 was not significantly different. Interestingly both ratios were significantly reduced in plasma total fatty acids. Summary In summary we found that ATP10D reduces high-fat diet induced obesity and improves insulin sensitivity. ATP10D transgenic mice showed altered hepatic expression of lipid-metabolism associated genes, including Scd1, along with changes in hepatic and plasma lipid species and plasma lipoprotein pattern.
... Heterozygous deletion of ATP10A in mice causes diet-induced obesity, type 2 diabetes, and nonalcoholic fatty liver disease, implicating ATP10A in obesityrelated metabolic abnormalities (26). ATP10A is 3 also implicated in regulation of insulin-stimulated glucose uptake (27,28). However, the flippase activity and substrate specificities of ATP10A remain to be determined. ...
Article
Full-text available
We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine [PS] and phosphatidylethanolamine [PE]), and ATP8B1 and ATP8B2 have flippase activity toward phosphatidylcholine (PC). Here, we show that localization of class 5 P4-ATPases to the plasma membrane (ATP10A, ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant, ATP10A(E203Q), dramatically increased PC flipping, but not flipping of PS or PE. Depletion of CDC50A made ATP10A retained at the ER instead of delivered to the plasma membrane, and abrogated the increased PC-flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A, and specifically flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC-flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... Mice heterozygous for the P4-ATPase Atp10a (also named Atp10c) develop insulin resistance, hyperlipidemia and are hyperinsulinemic and provide a model for type-2 diabetes mellitus and diet-induced obesity [116,117]. ATP10A has been implicated in the regulation of insulin-stimulated glucose uptake by plasma membrane mobilization of GLUT4-containing vesicles [116,117,129]. Moreover, mRNA expression levels of a canine ATP10A orthologue in visceral adipose tissue were found to be five times higher in obese dogs in comparison with lean dogs, suggesting an involvement of ATP10A in response to diet-induced obesity [130]. ...
Article
Full-text available
P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed "lipid flipping". Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse) of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role.
... The substrate specificities of ATP10A, ATP10B and ATP10D have not yet been defined. In mice, ATP10A and ATP10D are associated with obesity and diabetes [86][87][88][89][90]. There are also reports that link imprinting mutations and deletions in the region of ATP10A with Angelman syndrome, a form of autism-spectrum disorder [91,92]. ...
Article
Full-text available
Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle tranfficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis among others. P(4)-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membranes proteins that actively transport phospholipids across cellular membranes. P(4)-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P(4)-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
Article
P4-ATPases, a subfamily of P-type ATPases, were initially identified as aminophospholipid translocases in eukaryotic membranes. These proteins generate and maintain membrane lipid asymmetry by translocating aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the exoplasmic/lumenal leaflet to the cytoplasmic leaflet. The human genome encodes 14 P4-ATPases, and the cellular localizations, substrate specificities, and cellular roles of these proteins were recently revealed. Numerous P4-ATPases, including ATP8A1, ATP8A2, ATP11A, ATP11B, and ATP11C, transport phosphatidylserine. By contrast, ATP8B1, ATP8B2, and ATP10A transport phosphatidylcholine but not aminophospholipids, although there is a discrepancy regarding the substrate of ATP8B1 in the literature. Some yeast and plant P4-ATPases can also translocate phosphatidylcholine. At least 2 P4-ATPases (ATP8A2 and ATP8B1) are associated with severe human diseases, and other P4-ATPases are implicated in various pathophysiologic conditions in mouse models. Here, we discuss the cellular functions of phosphatidylcholine flippases and suggest a model for the phenotype of progressive familial intrahepatic cholestasis 1 caused by a defect in ATP8B1.-Shin, H.-W., Takatsu, H. Substrates of P4-ATPases: beyond aminophospholipids (phosphatidylserine and phosphatidylethanolamine).
Article
Full-text available
Previous data from our laboratory have indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in breast cancer cell lines and that these pathways are involved in growth regulation of these cells. To determine functionality, MDA-MB-453 breast cancer cells were stimulated with ethanol, known to open GIRK channels. Decreased GIRK1 protein levels were seen after treatment with 0.12% ethanol. In addition, serum-free media completely inhibited GIRK1 protein expression. This data indicates that there are functional GIRK channels in breast cancer cells and that these channels are involved in cellular signaling. In the present research, to further define the signaling pathways involved, we performed RNA interference (siRNA) studies. Three stealth siRNA constructs were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence. These constructs were transfected into MDA-MB-453 cells, and both RNA and protein were isolated. GIRK1, β(2)-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, β(2) mRNA levels were unchanged by the GIRK1 knockdown. GIRK1 protein levels were also reduced by the knockdown, and this knockdown led to decreases in beta-adrenergic, MAP kinase and Akt signaling.
Article
Full-text available
Epigenetics could help to explain individual differences in weight loss after an energy-restriction intervention. Here, we identify novel potential epigenetic biomarkers of weight loss, comparing DNA methylation patterns of high and low responders to a hypocaloric diet. Twenty-five overweight or obese men participated in an 8-wk caloric restriction intervention. DNA was isolated from peripheral blood mononuclear cells and treated with bisulfite. The basal and endpoint epigenetic differences between high and low responders were analyzed by methylation microarray, which was also useful in comparing epigenetic changes due to the nutrition intervention. Subsequently, MALDI-TOF mass spectrometry was used to validate several relevant CpGs and the surrounding regions. DNA methylation levels in several CpGs located in the ATP10A and CD44 genes showed statistical baseline differences depending on the weight-loss outcome. At the treatment endpoint, DNA methylation levels of several CpGs on the WT1 promoter were statistically more methylated in the high than in the low responders. Finally, different CpG sites from WT1 and ATP10A were significantly modified as a result of the intervention. In summary, hypocaloric-diet-induced weight loss in humans could alter DNA methylation status of specific genes. Moreover, baseline DNA methylation patterns may be used as epigenetic markers that could help to predict weight loss.
Article
Full-text available
We have shown that Atp10c, a type 4 P-type ATPase, is a strong candidate affecting glucose and lipid metabolism in humans and mice. Atp10c is a putative phospholipid translocase associated with cell signaling and intracellular protein trafficking. In order to examine the biological role of Atp10c, semiquantitative reverse transcriptase – polymerase chain reaction was carried out. Atp10c mRNA is expressed in 3T3-L1 cells and in primary preadipocytes and adipocytes generated from mice. Atp10c mRNA is regulated during fat cell differentiation and modulated by PPAR agonists and antagonists as well as by hormonal factors (insulin and dexamethasone). Atp10c expression is regulated by both the process of adipocyte differentiation and by effectors of fat and glucose metabolism. Taken together these data along with the published phenotype of the Atp10c heterozygote mice suggest that ATP10C is a newly identified protein with a possible biological role in the development of obesity and obesity-related metabolic disorders.
Article
Full-text available
We have shown that Atp10c, a type 4 P-type ATPase, is a strong candidate affecting glucose and lipid metabolism in humans and mice. Atp10c is a putative phospholipid translocase associated with cell signaling and intracellular protein trafficking. In order to examine the biological role of Atp10c, semiquantitative reverse transcriptase - polymerase chain reaction was carried out. Atp10c mRNA is expressed in 3T3-L1 cells and in primary preadipocytes and adipocytes generated from mice. Atp10c mRNA is regulated during fat cell differentiation and modulated by PPARγ agonists and antagonists as well as by hormonal factors (insulin and dexamethasone). Atp10c expression is regulated by both the process of adipocyte differentiation and by effectors of fat and glucose metabolism. Taken together these data along with the published phenotype of the Atp10c heterozygote mice suggest that ATP10C is a newly identified protein with a possible biological role in the development of obesity and obesity-related metabolic disorders.
Article
Full-text available
To obtain compounds that promote glucose uptake in muscle cells, the novel cell lines A31-IS derived from Balb/c 3T3 A31 and C2C12-IS from mouse myoblast C2C12 were established. In both cell lines, glucose consumption was induced by insulin and suppressed by the addition of Akt-activating kinase inhibitor. The A31-IS cells highly express the insulin receptor beta chains, Glut4, and uncoupling protein-3, as compared to the parent Balb/c 3T3 A31 cells, and C2C12-IS cells highly express the insulin receptor beta chain as compared to its parent cell line. Using A31-IS cells, we screened our library compounds and obtained three compounds, DF-4394, DF-4451, and DG-5451. These compounds dose-dependently promoted glucose consumption in A31-IS cells and facilitated [3H]-2-deoxyglucose uptake in differentiated C2C12-IS cells. The compounds that we obtained from the library screening will be good candidates for improving insulin resistance in muscle cells.
Article
Studies in which GLUT4 has been overexpressed in transgenic mice provide definitive evidence that glucose transport is rate limiting for muscle glucose disposal. Transgenic overexpression of GLUT4 selectively in skeletal muscle results in increased whole body glucose uptake and improves glucose homeostasis. These studies strengthen the hypothesis that the level of muscle GLUT4 affects the rate of whole body glucose disposal, and underscore the importance of GLUT4 in skeletal muscle for maintaining whole body glucose homeostasis. Studies in which GLUT4 has been ablated or 'knocked-out' provide proof that GLUT4 is the primary effector for mediating glucose transport in skeletal muscle and adipose tissue. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle and adipose tissue. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycaemia, understanding the molecular regulation of glucose transport in ske...
Article
RÉSUMÉ Skeletal muscle glucose uptake is mediated by glucose trans- porter 4, the major isoform that is responsive to hormones such as insulin, and by energy-demanding conditions such as exercise and hypoxia. While participation of the phosphati- dylinositol 3-kinase (PI3K) pathway in insulin-stimulated glucose uptake is well established, the signals involved in mediating glucose uptake in response to exercise and hypox- ia (collectively termed "alternative pathway" since it is PI3K- independent) are largely unknown. 5'-AMP-activated kinase (AMPK) and Ca 2+ have been implicated in these insulin-
Article
Mouse and human Atp10c genes are strong candidates for changes in bodyweight and glucose homeostasis. Using comparative genomic analysis, a novel canine P4-type ATPase, ATP10C, was identified. Expression of ATP10C was compared between sex-matched lean (body condition score, BCS<8; n=7) and obese (BCS⩾8, n=8) client-owned dogs of comparable ages. Canine ATP10C is highly expressed in visceral and subcutaneous fat at approximately 3-fold levels compared to the omental adipose depot. There was a 5-fold significant increase (P<0.0001) in mRNA expression of ATP10C in dogs with a BCS⩾8.
Article
Guanidine, the active ingredient extracted from Galega officinalis, is introduced as a ligand for imidazoline I2 receptor (I2R) because guanidine decreased plasma glucose via an activation of I2BR to increase glucose uptake into skeletal muscle isolated from Wistar rats. However, the signals for this action of guanidine remained obscure. In the present study, we used the cultured skeletal muscle fibroblast named C2C12 cell line to investigate this point. We found that guanidine increased the phosphorylation of AMP-activated protein kinase (AMPK) in addition to the higher of glucose transporter GLUT4 expression and glucose uptake. These effects of guanidine were blocked by the pretreatment with I2R antagonist BU224 but not by the blockade of I2AR amiloride. Moreover, compound C at concentrations sufficient to inhibit AMPK blocked the guanidine-induced glucose uptake and GLUT4 protein level. These results suggested that guanidine increases glucose uptake via an activation of I2BR through AMPK activation in skeletal muscle cell; this view has not been mentioned before.