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Endocrinology 2010 151:4665-4677 originally published online Aug 4, 2010; , doi: 10.1210/en.2010-0116
Klip and S. Lavandero
A. E. Contreras-Ferrat, B. Toro, R. Bravo, V. Parra, C. Vásquez, C. Ibarra, D. Mears, M. Chiong, E. Jaimovich, A.
Cardiomyocytes
Insulin-Stimulated Glucose Transporter 4 Translocation and Glucose Uptake in
Receptor Pathway Is Required for3)-IP3An Inositol 1,4,5-Triphosphate (IP
Society please go to: http://endo.endojournals.org//subscriptions/
or any of the other journals published by The EndocrineEndocrinologyTo subscribe to
Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online
An Inositol 1,4,5-Triphosphate (IP
3
)-IP
3
Receptor
Pathway Is Required for Insulin-Stimulated Glucose
Transporter 4 Translocation and Glucose Uptake in
Cardiomyocytes
A. E. Contreras-Ferrat, B. Toro, R. Bravo, V. Parra, C. Va´ squez, C. Ibarra,
D. Mears, M. Chiong, E. Jaimovich, A. Klip, and S. Lavandero
Centro Estudios Moleculares de la Ce´ lula, Facultad de Medicina (A.E.C.-F., B.T., R.B., V.P., C.V., C.I.,
D.M., M.C., E.J., S.L.) and Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias
Químicas y Farmace´ uticas (A.E.C.-F., B.T., R.B., V.P., C.V., C.I., M.C., S.L.), Universidad de Chile,
Santiago 838-0492, Chile; University of Texas Southwestern Medical Center (S.L.), Dallas, Texas 75235;
and Cell Biology Program (A.K.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8
Intracellular calcium levels ([Ca
2⫹
]
i
) and glucose uptake are central to cardiomyocyte physiology,
yet connections between them have not been studied. We investigated whether insulin regulates
[Ca
2⫹
]
i
in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose
uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4).
Primary neonatal rat cardiomyocytes were preloaded with the Ca
2⫹
fluorescent dye fluo3-ace-
toxymethyl ester compound (AM) and visualized by confocal microscopy. Ca
2⫹
transport pathways
were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using
[
3
H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyo-
cytes transfected with GLUT4-myc-enhanced green fluorescent protein.
Insulin elicited a fast, two-component, transient increase in [Ca
2⫹
]
i
. Nifedipine and ryanodine pre-
vented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP
3
)-
receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP
3
receptor
knockdown via small interfering RNA or by transfected G
␥
peptidic inhibitor

ARKct. Insulin-stimu-
lated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetra-acetic acid-AM,
2-amino-ethoxydiphenylborate, and

ARK-ct but not by nifedipine or ryanodine. Similarly, insulin-
dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by
bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetra-aceticacid-AM and xestospongin C but not by nifedipine.
Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca
2⫹
release and
GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase
␥
inhibited the
latter.
In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca
2⫹
release via IP
3
receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unre-
alized contribution of IP
3
-sensitive Ca
2⫹
stores to insulin action. This pathway may influence cardiac
metabolism in conditions yet to be explored in adult myocardium. (Endocrinology 151: 4665–4677,
2010)
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2010 by The Endocrine Society
doi: 10.1210/en.2010-0116 Received January 29, 2010. Accepted June 25, 2010.
First Published Online August 4, 2010
Abbreviations: 2-APB, 2-Amino-ethoxydiphenylborate; 2,4-DNP, 2,4-dinitrophenol; Ad, ade-
novirus; AM, acetoxymethyl ester compound; BAPTA, bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-
tetra-acetic acid; [Ca
2⫹
]
i
, intracellular calcium; eGFP, enhanced green fluorescent protein; GFP,
green fluorescent protein; GLUT, glucose transporter; IP
3
, inositol-1,4,5-trisphosphate; IP
3
R,
inositol-1,4,5-trisphosphate receptor; KD, kinase dead; LTCC, L-type calcium channel; myr,
myristoylated; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PTX, pertussis toxin;
RyR, ryanodine receptor; SER, sarcoendoplasmic reticulum; si, small interfering; TeTx, tetanus
toxin; WT, wild type.
DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL
Endocrinology, October 2010, 151(10):4665–4677 endo.endojournals.org 4665
Cardiac muscle is an energy-consuming tissue that re-
quires constant supply of oxygen and metabolic fuels
to maintain contractile function. Under physiological con-
ditions, fatty acids are major metabolic substrates for the
heart. However, up to 30% of myocardial ATP is gener-
ated by glucose and lactate, with lesser contribution from
ketones and amino acids. Although glucose is not the ma-
jor metabolic substrate in the beating heart, it is para-
mount in several pathological conditions (1).
Insulin is an important regulator of cardiac size and
metabolism, and the absence of insulin receptors impedes
the switch of cardiac substrate utilization from glucose to
fatty acids (2). Like skeletal muscle cells and adipocytes,
most insulin actions in cardiomyocytes have been linked to
activation of two canonical signaling pathways: the phos-
phatidylinositol 3-kinase (PI3K)-protein kinase Akt path-
way and the Ras-MAPK pathway (3). In addition, a Cbl-
CAP-TC10 pathway has also been described in cardiac
muscle (4). Interestingly, the insulin receptor has also been
reported to activate heterotrimeric G proteins in adipo-
cytes and fibroblasts (5, 6).
Glucose enters cardiomyocytes via the SLC2 family of
facilitative glucose transporters (GLUTs) (7). The most
abundant GLUT in the heart is GLUT4, essential for in-
sulin-dependent glucose uptake in this tissue (8). Insulin-
dependent gains in GLUT4 at the cell surface have been
detected in cardiac tissue by immunocytochemistry, sub-
cellular fractionation, and surface photolabeling (9–12).
In the heart, there is also significant expression of GLUT1,
which under certain circumstances is responsible for a sig-
nificant component of cardiac glucose uptake (13).
In skeletal muscle, insulin-dependent signals regulating
GLUT4 translocation include IRS-1, class IA PI3K, Akt,
AS160, and atypical protein kinase C (14). In contrast,
beyond PI3K, the insulin signals regulating GLUT4 trans-
location and glucose uptake in cardiac cells remain to be
defined (15). In this context, it is interesting to inquire
whether intracellular calcium levels ([Ca
2⫹
]
i
) contribute
to regulating glucose uptake. In adipocytes, interfering
with [Ca
2⫹
]
i
decreased insulin-stimulated glucose uptake
and Akt phosphorylation (16), and, in skeletal muscle fi-
bers, Ca
2⫹
influx was found important for full stimulation
of glucose uptake (17, 18). Surprisingly, despite the prom-
inent regulation of Ca
2⫹
in cardiomyocyte function (19),
its participation in the metabolic actions of insulin in this
tissue is unknown. In isolated adult cardiomyocytes, in-
sulin stimulates voltage-activated L-type Ca
2⫹
channels
(LTCCs) (20) and Na
⫹
-Ca
2⫹
exchange (21), but whether
this regulates glucose uptake has not been explored.
Cultured neonatal cardiomyocytes are an interesting in
vitro model to study regulation of glucose uptake by in-
sulin because, at approximately the perinatal period, the
predominant metabolism of the heart shifts from non-ox-
idative glucose utilization to fatty acids oxidation (22).
This shift is associated with changes in expression of a
number of regulatory proteins of glucose and fatty acids
metabolism (23, 24). Importantly, GLUT4 is already ex-
pressed in neonatal cardiomyocytes (12, 24) and repre-
sents up to 40% of the adult content (25). In these cells, we
have observed a contribution of both IGF-1 and testos-
terone to calcium homeostasis (26, 27).
Here we show that, at physiological doses, insulin in-
duces a transient [Ca
2⫹
]
i
increase into neonatal rat cardio-
myocytes, revealed by a fluorescent Ca
2⫹
indicator. The
signal has two components: one dependent on extracel-
lular Ca
2⫹
influx and a second one dependent on Ca
2⫹
release from sarcoendoplasmic reticulum (SER) stores.
Notably, the latter contributes to the insulin-dependent
increase in glucose uptake through internal GLUT4 vesicle
translocation and fusion with the plasma membrane. Fi-
nally, we established the participation of a G protein,
PI3K
␥
and 1,4,5-inositol-triphosphate (IP
3
) receptor
(IP
3
R) axis in the mechanism leading to the Ca
2⫹
signal
and GLUT4-mediated increase in glucose uptake.
Materials and Methods
Animals
Rats were bred in the Animal Breeding Facility, Faculty of
Chemical and Pharmaceutical Sciences, University of Chile (San-
tiago, Chile). Studies were approved by the Institutional Bioethi-
cal Committee, Faculty of Chemical and Pharmaceutical Sci-
ences, University of Chile, in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals (28).
Primary culture of neonatal rat cardiomyocytes
Cardiomyocytes were prepared from hearts of 1- to 3-d-old
Sprague Dawley rats as described previously (29). Cultured car-
diomyocytes were more than 95% pure based on

-myosin
heavy chain expression. Serum was removed 24 h before insulin
(Novo Nordisk Pharma, Hellerup, Denmark) stimulation. Un-
der these conditions, cardiomyocytes do not show spontaneous
Ca
2⫹
oscillations.
Recombinant adenoviruses
Adenoviruses (Ad) for

ARK-ct (Ad

ARK-ct), green fluores-
cent protein (GFP) (AdGFP), and an empty construct (AdEmpty)
were a gift from Dr. W. J. Koch (Duke University, Durham, NC).
Ad

ARK-ct leads to expression of the C-terminal portion of

-adrenergic receptor kinase that binds G
␥
subunits and be-
haves as dominant-negative inhibitor of G
␥
signaling (30).
Transduction efficiency was more than 95% monitored with
AdGFP.
Intracellular Ca
2ⴙ
levels
[Ca
2⫹
]
i
was determined in cardiomyocytes preloaded with
fluo3-acetoxymethyl ester compound (AM) as described previ-
4666 Contreras-Ferrat et al. IP
3
-Mediated Ca
2⫹
Release in GLUT4 Translocation Endocrinology, October 2010, 151(10):4665– 4677
ously (26). Insulin was added directly to the microscope cham-
ber, and fluorescence images were collected every 0.985 s and
then analyzed with NIH ImageJ software.
Glucose uptake
Cardiomyocytes were rinsed twice with HEPES-buffered saline
containing either 2 mMCaCl
2
or2mMEGTA as indicated and
maintained in this condition for 1 h. Inhibitors were added during
the last 30 min and insulin during the last 10 min. Ad transduction
was performed 24 h before insulin stimulation. Glucose uptake was
measured using 10
M[
3
H]2-deoxyglucose (31).
Transfections and immunofluorescence microscopy
Cardiomyocytes were transfected with 2
g GLUT4-myc-
enhanced GFP (eGFP) (32), wild-type (WT) PI3K
␥
, kinase-dead
(KD) PI3K
␥
, myristoylated (myr) PI3K
␥
, Akt-myr (donated by
Dr. T. R. Jackson, University of Newcastle, Newcastle, UK), or
tetanus toxin (TeTx) light chain (33) using 2
l/ml Lipo-
fectamine 2000 (Invitrogen, Carlsbad, CA). Transfection effi-
ciency was 7%. Type 2 IP
3
R mRNA was knocked down using
100 nMof a mix of four different small interfering (si) IP
3
R2
(Thermo Fisher Scientific, Waltham, MA) with 1
l/ml Dhar-
maFECT (Thermo Fisher Scientific). siRNA to the unrelated pro-
tein emerin (Sigma-Proligo) served as control. Plasmids or
siRNA-treated cells were incubated 24 or 72 h before experi-
ments, respectively. Cell surface GLUT4-myc-eGFP was deter-
mined as described previously (32), from the ratio of surface myc
signal to total cellular eGFP fluorescence in each cell.
Membrane potential determinations
Membrane potential was recorded from single, cultured car-
diomyocytes using the perforated patch configuration of the
whole-cell recording technique. Patch electrodes were formed
from thin-walled borosilicate capillary glass using a horizontal
puller and filled with the following: 120 mMK-gluconic acid, 20
mMKCl, 8 mMNaCl, 1 mMMgCl
2
,10mMHEPES, and 240
mg/ml amphotericin B (pH 7.4), tip resistance of approximately
2.5 mW. Experiments were performed with the cells bathed in an
extracellular solution containing the following (in mM): 145
NaCl, 5 KCl, 1 MgCl
2
, 2.5 CaCl
2
, 10 HEPES, and 5.6 glucose
(pH 7.4). The electrode was sealed onto the membrane of a car-
diomyocyte with gentle suction, and membrane potential record-
ings began when the amphotericin B in the pipette had perforated
the membrane under the patch to an access resistance less than
40 mW (usually 5–15 min). Membrane potential was recorded in
current-clamp mode using an EPC-7 patch-clamp amplifier
(HEKA, Lambrecht/Pfalz, Germany) and a Digidata 1200B data
acquisition system (Molecular Devices, Sunnyvale, CA), at an
acquisition rate of 200 samples/s. Insulin was added to the bath
solution from a stock solution in H
2
O, after recording basal
membrane potential for at least 3 min. In some experiments, the
amplifier was switched to voltage-clamp mode to record voltage-
dependent membrane currents in response to families of depo-
larizing voltage pulses, before and after addition of insulin.
Statistical analysis
Results are expressed as mean ⫾SD of the number of inde-
pendent experiments indicated (n) and subjected to ANOVA,
and comparisons between groups were performed using a pro-
tected Tukey’s ttest assigning P⬍0.05 as limit of statistical
significance. Experiments illustrated are representative of assays
performed on at least three separate occasions.
Results
Insulin induces a biphasic [Ca
2ⴙ
]
i
increase in
cultured cardiomyocytes
Changes in [Ca
2⫹
]
i
were determined in cardiomyocytes
preloaded with fluo3-AM. Quiescent cardiomyocytes,
maintained in Ca
2⫹
-containing media, exhibited basal in-
tracellular Ca
2⫹
levels with an average peak fluorescence
of 24 ⫾15 [⌬F/F] ⫻100 (n ⫽10) relative to the first 10 s
mean basal reading (Supplemental Fig. 1, A and C, pub-
lished on The Endocrine Society’s Journals Online web site
at http://endo.endojournals.org), whereas in Ca
2⫹
-free
media, the basal peak fluorescence was 8 ⫾1[⌬F/F] ⫻100
(n ⫽10) (Supplemental Fig. 1, B and C). These values
correspond to 5 min of recording.
In cardiomyocytes maintained in Ca
2⫹
-containing me-
dia, insulin (10 and 100 nM) induced a transient [Ca
2⫹
]
i
increase that exhibited two kinetically distinguishable
phases (Fig. 1A). The first phase was a fast, almost instan-
taneous [Ca
2⫹
]
i
increase reaching a maximal mean fluo-
rescence intensity of 224 ⫾23 [⌬F/F] ⫻100 (n ⫽4) within
the first second of stimulation. The second phase was re-
corded as a plateau in [Ca
2⫹
]
i
levels right after the first
phase, with maximal mean fluorescence intensity of 144 ⫾
10 [⌬F/F] ⫻100 (n ⫽4) that lasted 26 ⫾5 s before
returning to basal fluorescence. In cardiomyocytes main-
tained in Ca
2⫹
-free media, insulin (10 and 100 nM) in-
duced a single-phase [Ca
2⫹
]
i
increase that lasted 75 ⫾6s,
with a maximum fluorescent intensity of 219 ⫾38 [⌬F/F] ⫻
100 (n ⫽4), at 7.8 ⫾1.4 s after stimulation (Fig. 1B). To
establish the concentration-response relationship, cardi-
omyocytes were stimulated with different concentrations
of insulin in Ca
2⫹
-containing and Ca
2⫹
-free resting media
(Supplemental Fig. 1D). Insulin at 0.01 nMdid not increase
[Ca
2⫹
]
i
, whereas 0.1–1 nMinsulin induced the maximal
increase of [Ca
2⫹
]
i
in approximately 80% of cardiomyo-
cytes analyzed. Insulin at 1
Minduced an increase of
[Ca
2⫹
]
i
in practically all the cells analyzed but showed
different Ca
2⫹
kinetics profile, possibly related with a
nonspecific activation of other receptors (data not shown).
We chose 10 nMinsulin for all the following experiments
because it is near physiological concentration that induced
a consistent, large [Ca
2⫹
]
i
response, ensuring specificity.
In either case, the magnitude of these changes in [Ca
2⫹
]
i
is
comparatively smaller than those occurring during nor-
mal heart beat and hence would not be expected to modify
cardiac function.
To confirm that insulin also caused release of Ca
2⫹
from intracellular stores, we first depleted SER Ca
2⫹
using
Endocrinology, October 2010, 151(10):4665– 4677 endo.endojournals.org 4667
thapsigargin (a SER Ca
2⫹
-ATPase inhibitor). In the ab-
sence of extracellular Ca
2⫹
, thapsigargin induced a
rapid increase in intracellular fluorescence. Subsequent
addition of caffeine or insulin did not evoke any increase
in [Ca
2⫹
]
i
levels (Supplemental Fig. 1E). These results
indicate that the insulin-induced [Ca
2⫹
]
i
gain in cardio-
myocytes maintained in Ca
2⫹
-free media is mainly at-
tributable to Ca
2⫹
release from thapsigargin-sensitive
internal stores. To test whether the effect of 10 nMin-
sulin was through its receptors, cardiomyocytes were
preincubated with genistein (tyrosine kinase receptor
inhibitor). Genistein blocked the insulin-dependent in-
crease in [Ca
2⫹
]
i
in both the presence or absence of
extracellular Ca
2⫹
(Supplemental Fig. 1F). These data
suggest that the increase in [Ca
2⫹
]
i
induced by insulin
depend on the activation of insulin receptor.
The first phase of [Ca
2ⴙ
]
i
rise
involves extracellular Ca
2ⴙ
influx
through L-type Ca
2ⴙ
channels and
Ca
2ⴙ
release through ryanodine
receptors
To begin mapping the pathways of
Ca
2⫹
influx into the cytosol, we used
well-defined molecular inhibitors or ac-
tivators of distinct channels. Nifedipine
(1
M), an exclusive inhibitor of LTCCs
at low concentrations, virtually blocked
the first phase of insulin-stimulated
[Ca
2⫹
]
i
transient in cardiomyocytes incu-
bated in Ca
2⫹
-containing media (Fig.
1C). Conversely, the more delayed insu-
lin-induced [Ca
2⫹
]
i
increase observed
was insensitive to nifedipine (Fig. 1D),
confirming distinct participation of intra-
cellular Ca
2⫹
stores in this process.
To determine the possible role of
plasma membrane depolarization in the
insulin-induced intracellular fast Ca
2⫹
rise observed in cardiomyocytes, we
measured membrane potential from in-
dividual cultured cardiomyocytes using
the perforated patch recording method.
Under basal conditions, the majority of
cardiomyocytes fired simple or com-
pound action potentials of variable fre-
quency and duration from a hyperpo-
larized resting potential (Supplemental
Fig. 2A, left and middle panels). The
remaining cells displayed a stable, hy-
perpolarized membrane potential and
fired very few action potentials (Supple-
mental Fig. 2A, right panel). Insulin in-
duced a transient depolarization and
volley of action potentials, after which the membrane po-
tential returned to its baseline level (Supplemental Fig.
2B). Another type of response observed was a gradual but
sustained depolarization of the membrane, reaching levels
between ⫺40 and ⫺20 mV (data not shown). Cardiomy-
ocytes displaying this response ceased firing action poten-
tials, probably as a result of inactivation of voltage-de-
pendent sodium channels at the depolarized membrane
potentials. These responses were not artifacts caused by
mechanical disruption of the preparation during the ad-
dition of solution to the static bath, because such re-
sponses were not seen when vehicle alone was added to
the bath solution (n ⫽6). These results support the idea
that the nifedipine-sensitive, fast Ca
2⫹
component in-
duced by insulin is attributable to calcium influx
FIG. 1. Insulin increases [Ca
2⫹
]
i
in neonatal rat cardiomyocytes by a Ca
2⫹
influx, followed by
aCa
2⫹
release from intracellular stores. Changes in [Ca
2⫹
]
i
were investigated in individual,
fluo3-AM-loaded cells maintained in medium with or without Ca
2⫹
.A,B,[Ca
2⫹
]
i
changes
evoked by 0.01 nM(‚), 10 nM(䡺), and 100 nM(f) insulin. C, D, Cardiomyocytes exposed to
1
Mnifedipine 5 min before and during insulin stimulation. E, F, Cardiomyocytes were
preincubated with ryanodine (50
M) for 1 h, and fluo3-AM was added in the final 30 min.
A, C, E, Fluorescence of cells in Ca
2⫹
-containing medium. B, D, F, Fluorescence of cells in
Ca
2⫹
-free medium. Results are expressed as relative total fluorescence [ratio of fluorescence
difference (F ⫺F
o
), to basal value (F
o
)] ⫻100 as a function of time and are representative of
five independent experiments in which at least 60 cells were analyzed in each case. Response
of cardiomyocytes preincubated with fluo3-AM (5.4
M) for 30 min and throughout insulin
(10 nM) stimulation.
4668 Contreras-Ferrat et al. IP
3
-Mediated Ca
2⫹
Release in GLUT4 Translocation Endocrinology, October 2010, 151(10):4665– 4677
through LTCCs after plasma membrane depolarization
of cardiomyocytes.
There are two major routes of Ca
2⫹
efflux from SER,
one mediated by a channel inhibitable by ryanodine [chan-
nel hence termed ryanodine receptor (RyR)], the other by
a channel activated by IP
3
(IP
3
R). The RyR is tightly cou-
pled to activation by surface LTCC for excitation-con-
traction coupling (34). In cells preincubated with ryano-
dine (50
M) and then placed in Ca
2⫹
-containing media,
the peak of first-phase insulin-induced [Ca
2⫹
]
i
transient
was both reduced and delayed (Fig. 1E). These results sug-
gest that the first phase of the insulin-induced [Ca
2⫹
]
i
tran-
sient requires both nifedipine-sensitive entry of external
Ca
2⫹
(presumably through LTCCs) and
coupled ryanodine-sensitive Ca
2⫹
-in-
duced Ca
2⫹
release. Importantly, ryano-
dine did not affect the [Ca
2⫹
]
i
transient
evoked by insulin in cardiomyocytes in
Ca
2⫹
-free media (Fig. 1F).
The second phase of insulin-
dependent [Ca
2ⴙ
]
i
rise is mediated
by Ca
2ⴙ
efflux through IP
3
R
From Fig. 1, it emerges that the sec-
ond phase of insulin-dependent [Ca
2⫹
]
i
transient occurs through Ca
2⫹
release
into the cytosol from intracellular stores
but is not mediated by RyR. To evaluate
Ca
2⫹
release through IP
3
R channels, we
used two well-described inhibitors,
2-amino-ethoxydiphenylborate (2-APB)
and xestospongin C (26), as well as
knockdown of the type 2 IP
3
R via siRNA
(for knockdown efficiency, see Supple-
mental Fig. 3). Remarkably, all three ma-
neuvers completely inhibited the insulin-
induced [Ca
2⫹
]
i
increase (Fig. 2, A–C).
These results suggest that the second
phase of insulin-dependent [Ca
2⫹
]
i
in-
crease is mediated by type 2 IP
3
R-gated
Ca
2⫹
channels. The intracellular Ca
2⫹
-
chelator bis(2-aminophenoxy)ethane-
N,N,N⬘,N⬘-tetra-acetic acid (BAPTA)-
AM (Fig. 2D) completely suppressed the
insulin-induced [Ca
2⫹
] increase, con-
firming that the signal arises from
changes in the levels of this cation.
The second phase of insulin-
dependent [Ca
2ⴙ
]
i
rise is signaled
via trimeric G proteins,
phospholipase C, and PI3K
The canonical pathways that acti-
vate IP
3
R are initiated by membrane-bound receptors that
engage trimeric G proteins, whose
␥
subunits in turn
activate downstream effectors, including phospholipase C
(PLC). This latter enzyme generates IP
3
, the agonist that
opens the IP
3
-ligated Ca
2⫹
channels (i.e. IP
3
R) from the
endoplasmic reticulum. To explore whether a similar
pathway operates in cardiomyocytes stimulated with in-
sulin, we tested the effect of diverse strategies that inhibit
␥
and PLC on the insulin-dependent second phase of the
[Ca
2⫹
]
i
transient.
Overexpression of

ARK-ct (inhibitor of
␥
subunits)
effectively abolished the insulin-induced [Ca
2⫹
]
i
transient
FIG. 2. The second phase of insulin-stimulated Ca
2⫹
increase involves Ca
2⫹
release through the
IP
3
R, trimeric G protein, PLC, and PI3K. Cardiomyocytes were analyzed in Ca
2⫹
-free media with
the respective inhibitors and then stimulated with 10 nMinsulin. Fluo3-AM and corresponding
inhibitors were added during preincubation and during stimulation. Fluorescence images were
acquired, and relative fluorescence was calculated as described in Figure 1. A, 2-APB (20
M); B,
xestospongin C (100
M); C, siRNA to rat type 2 IP
3
R (100 nM); D, BAPTA-AM (50
M); E,
Ad-

ARKct (multiplicity of infection of 300) for 24 h; F, PTX (1
g/ml, preincubated by 1 h); G,
U-73122 (50
M); H, LY-294002 (50
M). The results are representative of five independent
experiments, and at least 60 cells were analyzed.
Endocrinology, October 2010, 151(10):4665– 4677 endo.endojournals.org 4669
in Ca
2⫹
-free media (Fig. 2E). However, pertussis toxin
(PTX) (an inhibitor of G
i/o
) only partially inhibited the
insulin-induced [Ca
2⫹
]
i
increase (Fig. 2F). These observa-
tions suggest that
␥
subunits (dissociated from a hetero-
trimeric G protein) play a critical role in insulin-induced
[Ca
2⫹
]
i
increase, but such heterotrimeric G protein is un-
likely to be exclusively G
i/o
.
To determine whether insulin-stimulated [Ca
2⫹
]
i
tran-
sient involves activation of PLC, we preincubated
fluo3-AM preloaded cardiomyocytes with U-73122
[1-[6[[(17

)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]
hexyl]-1H-pyrrole-2,5-dione] (PLC inhibitor, 10
M) and
then stimulated with insulin. Under these conditions, the
insulin-dependent second-phase [Ca
2⫹
]
i
transient was to-
tally eliminated (Fig. 2G), supporting the participation of
PLC in this response.
The results in Fig. 3 reveal a novel pathway from the
insulin receptor to eventual activation of a putative G pro-
tein whose
␥
subunits and PLC contribute to opening
IP
3
R. Given that insulin classically activates PI3K, we
asked whether this enzyme participates in the insulin-de-
pendent Ca
2⫹
influx into the cytosol. The second-phase
[Ca
2⫹
]
i
transient was obliterated by LY-294002 [2-(4-
morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one] (Fig.
2H), raising the possibility that such PI3K may be PI3K
␥
,
a known upstream activator of PLC (35) (see below).
The above experiments were performed in Ca
2⫹
-free
medium for easier visualization of the second phase, hav-
ing eliminated the first phase of Ca
2⫹
influx. Parallel ex-
periments in Ca
2⫹
-containing media rendered compara-
ble results (Supplemental Fig. 4). To investigate whether
the parallel, nonrelated signaling pathways are still func-
tional in the presence of inhibitors, we tested the mem-
brane depolarization and excitation-contraction coupling
induced by KCl in cardiomyocytes maintained in Ca
2⫹
-
containing media. Cells preincubated for 30 min with LY-
294002, U-73122, or xestospongin C were stimulated
with 50 mMKCl. In all conditions, KCl induced a fast
increase of [Ca
2⫹
]
i
and showed Ca
2⫹
oscillations related
with a Ca
2⫹
-induced Ca
2⫹
-release response (Supplemen-
tal Fig. 5, A–C). These results suggest that unrelated sig-
naling pathways are perfectly operative when the insulin-
dependent Ca
2⫹
signals are blocked.
IP
3
-dependent Ca
2ⴙ
release is required for glucose
uptake and GLUT4 translocation in cardiomyocytes
Although there is evidence in both adipocytes and skel-
etal muscle that Ca
2⫹
may play a role in insulin stimula-
tion of glucose uptake (16, 17), the underlying mecha-
nisms are unresolved, and moreover it is not known
whether this occurs in cardiomyocytes. Uptake of [
3
H]2-
deoxyglucose was significantly elevated by insulin in cells
incubated in Ca
2⫹
-containing or Ca
2⫹
-free media. Glu-
cose uptake increased during the first minutes of insulin
stimulation, reaching values 3.2 ⫾0.2 and 3.5 ⫾0.3 times
higher than the control, in the presence and absence of
extracellular Ca
2⫹
, respectively (Supplemental Fig. 6A).
Cytochalasin B markedly reduced insulin-stimulated glu-
cose uptake in both extracellular Ca
2⫹
conditions (Sup-
plemental Fig. 6A), confirming that glucose uptake oc-
curred through GLUT-mediated transporters. Indinavir
(100
M), a blocker of glucose influx through GLUT4
(36), inhibited insulin-stimulated glucose uptake to reach
basal values, suggesting that GLUT4 is the preeminent
pathway for glucose uptake in insulin-stimulated cardio-
myocytes (Supplemental Fig. 6A).
Neither nifedipine nor ryanodine affected the insulin-
dependent stimulation glucose uptake, whether in Ca
2⫹
-
containing or Ca
2⫹
-free media, suggesting that stimula-
tion does not require extracellular Ca
2⫹
influx or Ca
2⫹
release through RyR (Supplemental Fig. 6B). Notably,
however, insulin-stimulated glucose uptake was com-
FIG. 3. Participation of G
␥
, PI3K, PLC, and IP
3
R in insulin-induced
glucose uptake. Glucose uptake was measured in cardiomyocytes as
described in Materials and Methods, in the absence of external Ca
2⫹
.A,
Cells were transduced with Ad

ARK-ct (multiplicity of infection of 300)
for 24 h or preincubated for 1 h with PTX (1
g/ml), and increases in
glucose uptake induced by insulin (10 nM) were markedly inhibited, as
well as the preincubation with BAPTA-AM (50
M), U-73122 (10
M), or
2-APB (20
M) (B). Knockdown of type 2 IP
3
R with a specific siRNA
(siIP
3
R2) decreased significantly the insulin response vs. control
(nonrelated siRNA, siNR) (C), LY-294002 (50
M), and Akt
i
1/2 (10
M)
given 30 min before insulin (10 nM) abolished the response of glucose
uptake (D). Values represent the mean ⫾SD of four different
experiments. *, P⬎0.05 and **, P⬍0.01 vs. control; †, P⬍0.05;
and ††, P⬍0.01 vs. insulin.
4670 Contreras-Ferrat et al. IP
3
-Mediated Ca
2⫹
Release in GLUT4 Translocation Endocrinology, October 2010, 151(10):4665– 4677
pletely inhibited by BAPTA-AM, Ad

ARK-ct, U-73122,
and 2-APB and was also partially inhibited by siRNA to
type 2 IP
3
R (Fig. 3, A–C). These results strongly suggest
that insulin-dependent stimulation of glucose uptake re-
quires the second-phase Ca
2⫹
transient, which was simi-
larly sensitive to these selective inhibitors of the G
␥
-PLC-
IP
3
R axis. Interestingly, the insulin-dependent stimulation
of glucose uptake was not inhibited by PTX (Fig. 3A). The
results illustrated refer to cells in Ca
2⫹
-free media, and
similar results were obtained in the presence of external
Ca
2⫹
(data not shown). These findings suggest that glu-
cose uptake depends on a signaling pathway involving
G
␥
, PLC, type 2 IP
3
R, and Ca
2⫹
.
Hence, a Ca
2⫹
-dependent signal is fundamental for the
insulin-dependent stimulation of glucose uptake into car-
diomyocytes. This response requires PI3K and its down-
stream target Akt in adipose and skeletal muscle cells, and
we confirmed its role in cardiomyocytes (Fig. 3D). The
observation that both G
␥
-PLC-IP
3
R-Ca
2⫹
and PI3K-
Akt inputs are required raised the question whether Ca
2⫹
might influence Akt activation. Indeed, although insulin
increased Akt phosphorylation in cardiomyocytes main-
tained in the presence or absence of extracellular Ca
2⫹
,
U-73122 and BAPTA-AM decreased the insulin-induced
Akt phosphorylation in Ca
2⫹
-free media (Fig. 4A). More-
over, the IP
3
R inhibitor 2-APB precluded insulin-depen-
dent Akt phosphorylation (Fig. 4B). These data reveal that
the second phase of the [Ca
2⫹
]
i
transient induced by in-
sulin is required to allow Akt activation, which also re- quires classical input from PI
3
K (given its inhibition by
LY-294002).
Ultimately, mechanistic information on the stimulation of
glucose uptake requires demonstration of regulation of
GLUT4 translocation. To evaluate the participation of cy-
tosolic Ca
2⫹
in the process of GLUT4 exposure at the plasma
membrane, cardiomyocytes were transiently transfected
with a cDNA encoding a GLUT4-myc-eGFP chimera. Insu-
lin caused a notable and consistent 2-fold to 3-fold gain in
surface GLUT4-myc at the cell surface. To demonstrate that
insulin-dependent exofacial exposure of myc epitope relies
on fusion of GLUT4-containing vesicles, the involvement of
the vesicle soluble N-ethylmaleimide sensitive factor attach-
ment protein receptor vesicle-associated membrane protein 2
(VAMP-2) was tested. Transfection of TeTx light chain
(which proteolyses VAMP-2) completely abrogated the
GLUT4 exposition (Fig. 5).
In nonpermeabilized cells maintained in Ca
2⫹
-free me-
dium, insulin induced a significant increase in exofacial ex-
posure of the myc epitope. Nifedipine did not alter this re-
sponse, and ryanodine only slightly decreased the insulin
effect from 0.95 ⫾0.04 to 0.70 ⫾0.07 (normalized cyanine
3/eGFP ratio) (Fig. 6, A and B). In contrast, major reductions
in insulin-induced surface gain in GLUT4 were provoked by
BAPTA-AM, Akt
i
1/2, or the IP
3
R blocker xestospongin C.
FIG. 5. VAMP-2 is required for insulin-dependent exofacial exposure
of myc epitope in cultured cardiomyocytes. Cells were transiently
cotransfected with GLUT4-myc-eGFP and TeTx construct for 24 h (A, B)
and stimulated with 10 nMinsulin for 10 min in Ca
2⫹
-free medium.
The effect of light chain of TeTx in insulin-dependent exofacial
exposure of myc epitope is showed in single cells (A) and quantified
(B). Values represent the average ⫾SD of four different experiments.
**, P⬍0.01 vs. basal condition. ††, P⬍0.01 vs. maximal response.
FIG. 4. Activation of Akt induced by insulin is dependent on the
intracellular Ca
2⫹
rise. Cell lysates were separated by 10% SDS-PAGE
and electrotransferred to nitrocellulose, blocked with 5% BSA, Tris-
buffered saline (pH 7.6), and 0.1% Tween 20, then incubated with
phospho-Akt, total-Akt, type 2 IP
3
R, or

-actin antibodies (1:1000 in
blocking buffer), and revealed with horseradish peroxidase-linked
secondary antibody (1:5000). Bands were detected using ECL and
quantified by densitometry. Akt phosphorylation at Ser473 induced by
10 nMinsulin for 10 min was independent of extracellular Ca
2⫹
and
was reduced by U-73122 (10
M) or BAPTA-AM (50
M) (A) and by
2-APB (20
M) or LY-294002 (50
M) (B). Results shown are
representative of three independent experiments.
Endocrinology, October 2010, 151(10):4665– 4677 endo.endojournals.org 4671
Moreover, specific knockdown of type 2 IP
3
R by siRNA also
markedly decreased the exofacial exposure of myc epitope
induced by insulin (Fig. 6C). These observations strongly
support a role of Ca
2⫹
release through type 2 IP
3
R in GLUT4
translocation and insertion into the cardiomyocyte plasma
membrane. To determine whether this mechanism is specific
to insulin pathways, we studied the effect of IGF-1 and 2,4-
dinitrophenol (2,4-DNP) on the exofacial exposure of myc
epitope. IGF-1 (10 nM) induced a small increase in exofacial
exposure of GLUT4-myc, and exposure
was completely inhibited by xestospon-
gin C (Fig. 6D). Conversely, 2,4-DNP
increased exofacial exposure of GLUT4-
myc that was not inhibited by xestospon-
gin C in cardiomyocytes maintained in
Ca
2⫹
-free resting media (Fig. 6E). These
results suggest that, although free cytoso-
lic Ca
2⫹
by itself triggers translocation/
fusion of GLUT4 with the plasma
membrane, the IP
3
R-dependent re-
lease participate in specific pathways
aimed to that purpose.
Finally, the involvement of
␥
sub-
units of a heterotrimeric G protein in
GLUT4 translocation raised the possible
participation of the
␥
isoform of PI
3
Kin
this process. To test this hypothesis, car-
diomyocytes were cotransfected with
GLUT4-myc-eGFP and WT-PI3K
␥
, KD-
PI3K
␥
, or myr-PI3K
␥
. Remarkably, the
non-active form of PI3K
␥
decreased in-
sulin-dependent exofacial exposure of
myc epitope, whereas WT-PI3K
␥
had no
effect (Fig. 7, A and B). Moreover, myr-
PI3K
␥
caused a small, if not statistically
significant, elevation of the basal level of
surface GLUT4-myc and also failed to
inhibit insulin-mediated GLUT4-myc
translocation (Fig. 7C). As positive con-
trol, myr-Akt was shown to produce the
expected gain in surface GLUT4-myc in
the absence of insulin (Fig. 7D). To-
gether, these results support the partici-
pation of PI3K
␥
in the mechanism of
GLUT4 membrane translocation and in-
sertion induced by insulin.
Discussion
Here we show a novel, insulin-induced
transient increase in [Ca
2⫹
]
i
levels in
neonatal rat cardiomyocytes. This
Ca
2⫹
transient consists of a first phase of extracellular
Ca
2⫹
influx and a second phase of SER Ca
2⫹
release into
the cytosol. The latter appears to be a physiologically rel-
evant signal and can be studied in isolation in the absence
of external Ca
2⫹
. This second phase of insulin-induced
[Ca
2⫹
]
i
increase involves
␥
subunit of a heterotrimeric G
protein, PI3K
␥
, PLC, and type 2 IP
3
R channels. We spec-
ulate that these signals act as a linear sequence of events,
FIG. 6. Insulin-induced exofacial exposure of GLUT4-myc-eGFP depends on [Ca
2⫹
]
i
, Akt, and
IP
3
R type 2 activation. Cells were transiently transfected with GLUT4-myc-eGFP cDNA for 24 h (A,
B) or cotransfected with GLUT4-myc-eGFP and siRNA type 2 IP
3
R (siIP
3
R2) or nonrelated siRNA
(siNR) for 72 h (C, D) and stimulated with 10 nMinsulin for 10 min in Ca
2⫹
-free medium.
Extracellular exposure of the myc epitope was detected by immunofluorescence in
nonpermeabilized cells as described in Materials and Methods. Insulin induced an increase in
cyanine 3/eGFP ratio independently of extracellular Ca
2⫹
presence. Cardiomyocytes were
preincubated with nifedipine (1
M) for 5 min, with ryanodine (50
M) for 1 h, or with BAPTA-AM
(50
M), Akt
i
1/2 (10
M), or xestospongin C (100
M) for 30 min, before insulin stimulation (A).
The quantification was calculated as described in Materials and Methods (B). The effect of
knockdown of type 2 IP
3
R in insulin-dependent exofacial exposure of myc epitope is showed in
single cells (C) and quantified (D). Values represent the average ⫾SD of four different
experiments. **, P⬍0.01 vs. control basal conditions. †, P⬍0.05 and ††, P⬍0.01 vs. insulin-
stimulated control condition (siNR). D, IGF-1 (10 nM) for 10 min induced a modest increase in
exofacial exposure of myc epitope that was inhibited by xestospongin C (100
M). E, 2,4-DNP (0.5
mM) for 10 min induced an increase in exofacial exposure of myc epitope that was insensitive to
xestospongin C (100
M) pretreatment. Values represent the average ⫾SD of four different
experiments. *, P⬍0.05 and **, P⬍0.01 vs. basal conditions. †, P⬍0.05 and ††, P⬍0.01 vs.
insulin or IGF-1-stimulated condition.
‡‡
,P⬍0.01 vs. insulin plus 2,4-DNP stimulated
cardiomyocytes.
4672 Contreras-Ferrat et al. IP
3
-Mediated Ca
2⫹
Release in GLUT4 Translocation Endocrinology, October 2010, 151(10):4665– 4677
as summarized in Fig. 8. We further show that each of the
above elements is required for insulin-dependent GLUT4
translocation, revealing an unrealized connection be-
tween IP
3
R-mediated Ca
2⫹
release and insulin action. The
intersection between Ca
2⫹
and the canonical PI3K-Akt
insulin pathway may occur at several levels, including
Ca
2⫹
-dependent facilitation of Akt activation, and poten-
tially vesicle traffic and/or fusion with the membrane (Fig.
8). These conclusions are expanded below.
Ca
2ⴙ
entry pathways into the cytosol
The [Ca
2⫹
]
i
debate in insulin action has centered
around the difficulty in detecting changes in the levels of
the cation in response to the hormone. Whereas ratiomet-
ric dyes sampling the entire cytosol have failed to detect
any rise in [Ca
2⫹
]
i
in response to insulin in 3T3-L1 adi-
pocytes (37), L6 myotubes (38), and isolated skeletal mus-
cle (39), the hormone activated the signal of a membrane-
associated Ca
2⫹
sensor (FIP18) in the latter system (39).
Here we show that fluo3, a dye with dynamic range higher
than the ratiometric dye indo-1, clearly detected [Ca
2⫹
]
i
transients in the cytosol of neonatal cardiomyocytes. This
suggests that, in these cells, the change in [Ca
2⫹
]
i
may be
more pronounced than in the other cell types and/or that
fluo3 can reveal [Ca
2⫹
]
i
transients more effectively than
ratiometric dyes.
The first phase of insulin-dependent [Ca
2⫹
]
i
increase in
cultured rat cardiomyocytes involves Ca
2⫹
influx through
LTCCs, because this rapid effect was totally prevented by
both Ca
2⫹
-free/EGTA-containing resting solution and ni-
fedipine. The inhibition by ryanodine further suggests in-
volvement of a Ca
2⫹
-induced Ca
2⫹
-release mechanism,
i.e. Ca
2⫹
influx through the LTCC activates the RyR
which in turn release Ca
2⫹
from the SER. This mechanism
participates in normal cardiac muscle contraction, albeit
producing comparatively larger elevations in [Ca
2⫹
]
i
. The
changes in [Ca
2⫹
]
i
produced by insulin are clearly insuf-
ficient to drive contraction.
In the heart, IP
3
is generated through the action of dis-
tinct PLCs on phosphatidylinositol 4,5-bisphosphate (40,
41). Some neurohumoral agonists (e.g. acetylcholine, en-
dothelin, catecholamines, prostaglandins) activate a G
q
-
dependent PLC

(42, 43). In contrast, purines or angio-
tensin II stimulate a tyrosine kinase-dependent PLC
␥
(40,
44). IP
3
induces slow release of Ca
2⫹
from vesicular prep-
arations and activates contraction in skinned ventricular
rat muscle and chick heart preparations (45). In the adult
rat, ventricular (46) and atrial (47) myocytes express mainly
type 2 IP
3
Rs, and all isoforms of IP
3
R have been identified in
neonatal rat cardiomyocytes, with individual intracellular
localization (26). The [Ca
2⫹
]
i
increase induced by insulin
reported here was inhibited by the expression of

ARK-ct,
LY-294002, and type 2 IP
3
R knockdown, suggesting par-
ticipation of G
␥
subunits from heterotrimeric G protein as
well as the PI
3
K and type 2 IP
3
R.
Participation of G
␥
and PI3K
␥
in insulin action in
cardiomyocytes
According to the canonical insulin signaling pathway,
insulin binding to its receptor activates class I PI3K (cat-
alytic p110
␣
and

subunits) through binding of the reg-
ulatory p85 subunit to insulin receptor substrate proteins,
a mechanism that is independent of G protein action.
However,
␥
subunits of heterotrimeric G proteins can
activate PI
3
K
␥
by direct interaction with two domains of
the catalytic p110
␥
subunit (48). During pressure load
hypertrophy, PI3K is activated by a G
␥
-dependent
process in mice (49). PI3K
␥
is crucial for the purinergic
regulation of spontaneous Ca
2⫹
spiking in rat cardio-
myocytes, in which LY-294002 prevented membrane
translocation of PLC
␥
and IP
3
generation (50). By anal-
ogy, we hypothesize that insulin might activate PI
3
K
(p110
␥
) through
␥
subunits of a heterotrimeric G pro-
tein. In support of this hypothesis, p110
␥
physically as-
sociates with and phosphorylates G
␣
q/11
in fibroblasts
overexpressing insulin receptors (5). This may explain the
inhibition of insulin-induced [Ca
2⫹
]
i
increase and GLUT4
translocation caused by the PI3K inhibitor LY-294002,
as well as the inhibition of GLUT4 translocation by
KD-PI3K
␥
.
FIG. 7. Insulin-dependent exofacial exposure of GLUT4-myc requires
the PI3K
␥
isoform. Cells were transiently cotransfected with GLUT4-
myc-eGFP and WT-PI3K
␥
(A) or KD-PI3K
␥
(B), or myr-PI3K
␥
(C) or myr-
Akt (D) constructs for 24 h and stimulated with 10 nMinsulin for 10
min in Ca
2⫹
-free medium. KD-PI3K
␥
decreased significantly the insulin-
dependent exofacial exposure of myc epitope as showed in single cells.
Values represent the mean ⫾SD of four different experiments. *, P⬍
0.05 and **, P⬍0.01 vs. control; †, P⬍0.05 vs. basal conditions;
¶
,P⬍0.05 vs. maximal response.
Endocrinology, October 2010, 151(10):4665– 4677 endo.endojournals.org 4673
Participation of Ca
2ⴙ
released via IP
3
Rin
insulin-dependent glucose uptake and GLUT4
translocation
Here we show that insulin induces an increase in glu-
cose uptake in cardiomyocytes maintained in both Ca
2⫹
-
containing and Ca
2⫹
-free resting media, suggesting that
the effect of insulin is independent of extracellular Ca
2⫹
.
Extracellular Ca
2⫹
also does not regulate glucose uptake
in isolated working heart (51). Although that study spec-
ulated that cytoplasmic Ca
2⫹
may not contribute to the
regulation of glucose uptake in working conditions, our
results clearly show that [Ca
2⫹
]
i
is required for insulin-
induced glucose uptake in cardiomyocytes, and this effect
seems to be independent of excitation-contraction [Ca
2⫹
]
i
signals.
The participation of [Ca
2⫹
]
i
in insulin-dependent stim-
ulation of glucose uptake has been debated in studies using
primary and cultured adipocytes, L6 myotubes, and iso-
lated skeletal muscle. Early studies in rat adipocytes
showed that, by changing extracellular Ca
2⫹
, a window of
[Ca
2⫹
]
i
ensued that was permissive for insulin-stimulated
glucose uptake (52), and that beyond a narrow value of
140–270 nM, [Ca
2⫹
]
i
instead correlated with insulin re-
sistance (53). In 3T3-L1 adipocytes, as shown here for
cardiomyocytes, BAPTA-AM reduced the stimulation of
Akt and the gain in surface GLUT4 (16,
54). Although neither study determine
whether the hormone increases the lev-
els of [Ca
2⫹
]
i
or merely requires the
resting [Ca
2⫹
]
i
levels, they both showed
inhibition of Akt phosphorylation, a
finding we have corroborated in neona-
tal cardiomyocytes. Worrall and Olef-
sky (54) proposed that [Ca
2⫹
]
i
has neg-
ative input in insulin signals proximal
to the receptor and a positive input on
more distal ones, including Akt. White-
head et al. (16) further proposed two
possible positive inputs for the cation
based on the use of BAPTA-AM and
Ca
2⫹
ionophores: one allowing the
transporter to reach the plasma mem-
brane and another one allowing its fu-
sion with the membrane. This was as-
certained from comparisons of the
levels of GLUT4 associated with mem-
brane lawns and the extent of hemag-
glutinin-GLUT4 epitope exposure to
the outer medium. The authors pre-
sented a lucid discussion of potential
molecules affected, and to date these re-
main attractive Ca
2⫹
targets. Finally,
that study found that BAPTA-AM di-
rectly inhibited glucose uptake in adipocytes, consistent
with the high susceptibility of GLUT4 to inhibition by very
diverse chemical agents, which however does not invalidate
the effects of BAPTA-AM on GLUT4 traffic. In contrast to
these findings, a less effective chelator of intracellular
[Ca
2⫹
]
i
, Quin2-AM (2-[(2-bis[carboxymethyl] amino-5-
methylphenoxy]-6-methoxy-8-bis[carboxymethyl]-amin-
oquinolinetetrakis acetoxymethyl ester), did not reduce glu-
cose uptake in L6 myotubes or 3T3-L1 adipocytes (37, 38),
and dantrolene, an inhibitor of Ca
2⫹
release from the SER
through RyRs, did not prevent GLUT4 translocation in L6
myotubes expressing GLUT4-myc (55). Finally, in rat skel-
etal muscle, GLUT4 coprecipitates with the TRPC3 chan-
nel, and TRPC3 knockdown decreased in insulin-mediated
glucose uptake (18), suggesting that, in this tissue the chan-
nel, and possibly ions flowing through it, are required for
insulin-stimulated glucose uptake. All these findings
beg for analysis of cell-specific responses of Ca
2⫹
to
insulin and for a more in depth exploration of the
source of the regulatory Ca
2⫹
. In the present study, we
find that this cation is likely released from endomem-
branes through IP
3
R and propose a signaling pathway
leading to such release. This mechanism may be unique
to cardiomyocytes or may be more amply operative,
FIG. 8. Proposed model. Insulin binding to its receptor activates a biphasic [Ca
2⫹
]
i
response.
The first phase involves the LTCC activation, extracellular Ca
2⫹
influx, activation of RyR Ca
2⫹
channel, and Ca
2⫹
release from SER. The second phase involves a non-inhibitory G protein-
coupled receptor, the G
␥
subunits that activates the lipid-kinase PI3K
␥
to generate
phosphatidylinositol-3,4,5-trisphosphate (PIP
3
) from phosphatidylinositol 4,5-bisphosphate
(PIP
2
). This is a strong signal for recruitment of the pleckstrin homology domain of PLC,
leading to its binding to the inner side of the cell surface. PLC then converts PIP
3
to IP
3
and
diacylglycerol. IP
3
activates the IP
3
R, leading to calcium release from SER. This Ca
2⫹
is
involved in GLUT4 vesicle traffic and/or fusion with the cell surface, finally producing an
increase in glucose uptake. CCLT, L-type Ca
2⫹
channels; SERCA, sarcoendoplasmic reticulum
Ca
2⫹
-ATPase; SR, sarcoendoplasmic reticulum.
4674 Contreras-Ferrat et al. IP
3
-Mediated Ca
2⫹
Release in GLUT4 Translocation Endocrinology, October 2010, 151(10):4665– 4677
and future work in each of the insulin-sensitive tissues
should provide answers to these possibilities.
We also show that either IGF-1 or 2,4-DNP can induce
an increase in plasma membrane GLUT4 levels indepen-
dent of the presence of extracellular Ca
2⫹
. In these con-
ditions, the pretreatment of cardiomyocytes with xesto-
spongin C inhibited only the effect of IGF-1. We have
shown previously that IGF-1 also increases Ca
2⫹
in car-
diomyocytes but with clear difference in the amplitude,
frequency, duration, or subcellular localization of the
Ca
2⫹
signals elicited by insulin (26). These last findings
show that a link exists between IP
3
R-dependent Ca
2⫹
re-
lease and GLUT4 translocation, but this mechanism is
particular for certain stimuli.
In particular, the link between IP
3
R-dependent Ca
2⫹
release and mobilization of GLUT4 is a novel contribution
of this study. Moreover, our study provides an explana-
tion for the documented contribution of endogenous
G
␣
q/11
in insulin-stimulated GLUT4 translocation in
3T3-L1 adipocytes (5) and the emulation of this process by
a constitutively active form of G
␣
q
(Q209L-G
␣
q
) acting
via a PI3K-dependent mechanism (6).
The observations made in adipocytes, skeletal muscle,
and ours in cardiomyocytes beg the question of what is the
precise role of [Ca
2⫹
]
i
that impacts on GLUT4 transloca-
tion. The regulation of Akt shown both here and by James
et al. (16) may be an important point of action. Addition-
ally, Ca
2⫹
may regulate GLUT4 vesicle fusion, potentially
through interaction with molecules functionally akin to
synaptotagmin. Furthermore, Ca
2⫹
ions may participate
in the recently described calmodulin kinase II-dependent
regulation of GLUT4 traffic (16, 33). Future studies
should reveal the molecular action of [Ca
2⫹
]
i
and whether
the IP
3
R implicated in this study is directly present on
GLUT4 vesicles, thereby providing local levels of elevated
Ca
2⫹
in the vicinity of the membrane.
In summary, insulin induces [Ca
2⫹
]
i
transients in neo-
natal cardiomyocytes, and Ca
2⫹
release from IP
3
-sensitive
stores is an important physiological regulator of the insu-
lin-dependent stimulation of glucose uptake via GLUT4.
These novel observations suggest a link between intracel-
lular Ca
2⫹
homeostasis and metabolism and raise the con-
cept that alterations in Ca
2⫹
regulation, so common in
cardiomyopathies, may impinge on the ability of cardio-
myocytes to regulate nutrient availability. Because cardiac
glucose uptake is paramount for energy procurement in
conditions of ischemia and hypoxia, the concomitant im-
pairment in Ca
2⫹
fluxes may be fundamental for energy
availability in the failing heart. Such a scenario would be
important in cardiomyopathy accompanying insulin re-
sistance and diabetes.
Acknowledgments
We thank the Graduated Student’s Exchange Program from the
Canadian International Education Board, International Com-
merce Exterior Relationship Department, The Hospital for Sick
Children (Research Institute, Cell Biology Department), and
University of Toronto for the travel fellowship to A.E.C.-F. We
also thank Fidel Albornoz and Ruth Marquez for their excellent
technical assistance and Dr. Philip J. Bilan for valuable advice.
Address all correspondence and requests for reprints to: Dr.
Sergio Lavandero, Facultad de Ciencias Químicas y Farmace´u-
ticas, Universidad de Chile, Olivos 1007, Santiago 838-0492,
Chile, E-mail: slavander@uchile.cl; or Dr. Amira Klip, The Hos-
pital for Sick Children, McMaster Building, Room 5004B, To-
ronto, Ontario, Canada M5G 1X8, E-mail: amira@sickkids.ca.
This work was supported by Fondo Nacional de Desarrollo
Científico y Tecnolo´ gico Grant 1080436 (to S.L.), Fondo Na-
cional de Desarrollo Científico y Tecnolo´ gico-Centros de Exce-
lencia en Investigacio´ n Avanzada Grant 15010006 (to S.L. and
E.J.), Mejoramiento de la Calidad de la Educacio´ n Superior
Grant UCHO802 (to S.L. and E.J.), and Canadian Institutes of
Health Research Grant MT 7307 (to A.K.). A.E.C.-F., B.T., R.B.,
V.P., and C.I. hold Ph.D. fellowships from Comisio´ n Nacional
de Investigacio´ n Científica y Tecnolo´ gica, Chile. A.E.C.-F. also
hold a travel fellowship from Graduated Student’s Exchange
Program, Canadian International Education Board, Interna-
tional Commerce Exterior Relationship Department and from
the University of Toronto.
S.L. is on sabbatical leave at the University of Texas South-
western Medical Center (Dallas, TX).
Disclosure Summary: The authors have nothing to disclose.
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