Download Infusion of a biotinylated bis-glucose photolabel

Am J Physiol Endocrinol Metab 292: E1922–E1928, 2007.
First published March 6, 2007; doi:10.1152/ajpendo.00170.2006.
Innovative Methodology
Infusion of a biotinylated bis-glucose photolabel: a new method to quantify
cell surface GLUT4 in the intact mouse heart
Edward J. Miller,1 Ji Li,1 Kevin M. Sinusas,1 Geoffrey D. Holman,2 and Lawrence H. Young1
1
Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine,
New Haven, Connecticut; and 2Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom
Submitted 7 April 2006; accepted in final form 19 February 2007
glucose transport; perfused mouse heart; photolabeling; glucose transporter 4
ONE OR MORE OF THE 13 TRANSMEMBRANE glucose transporter
family of proteins (GLUTs) mediate glucose uptake in mammalian cells (24). Glucose transport in the heart is activated by
insulin (14), exercise (8, 16), hypoxia (44), and ischemia (51),
conditions under which glucose is an important cardiac substrate. Cardiac glucose uptake and glycolysis are protective in
the ischemic heart (33), and glucose transporter deficiency
decreases ischemic tolerance (46).
In the heart, as in all striated muscles, myocyte glucose
transport relies to a large extent on GLUT4, which facilitates
the entry of extracellular glucose when present in cell surface
membranes. GLUT4, typically considered the insulin-responsive glucose transporter, is highly expressed in the heart (23),
particularly in cardiomyocytes (42, 51). Heart GLUT4 is stored
Address for reprint requests and other correspondence: L. H. Young, Depts.
of Internal Medicine and Cellular and Molecular Physiology, Yale Univ.
School of Medicine, FMP 3, 333 Cedar St., New Haven, CT 06520 (e-mail:
[email protected]).
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in intracellular vesicles and translocates to cell surface membranes, including the sarcolemma and T-tubules following
insulin, exercise, and ischemia (8, 14, 39, 42, 51). GLUT4
is thought to be primarily responsible for the increase in
glucose uptake in response to insulin and ischemic stress
(39, 44, 46, 51).
GLUT1 is also expressed in the heart, both in cardiac
myocytes, where it has a role in basal glucose uptake (14, 41,
51), and in endothelial cells (9). GLUT1 undergoes modest
translocation to the sarcolemma with insulin and ischemia (14,
51), as it does in adipocytes (50). GLUT1 is also regulated at
the expression level during ischemia, in part through the action
of the transcription factor HIF1-␣ (21). In addition to GLUT1
and GLUT4, novel glucose transporters such as GLUT8 (6, 10)
and GLUT12 (37) are expressed in the heart, although their
physiological roles are uncertain.
The ability to quantify cell surface GLUT4 is critical to
understanding heart metabolism. In recent years, transgenic
mice have provided important insights into the molecular
mechanisms regulating the physiological response of the heart
to ischemia, pressure overload, and diabetes (1). Although
many techniques have been adapted to study mouse cardiac
physiology (15) and metabolism (4, 35), the quantification of
cell surface glucose transporters has proved challenging.
Mouse hearts typically weigh only 150 mg, limiting the application of conventional membrane fractionation techniques that
were developed for other species (13, 51). Immunohistochemical and immunofluorescence techniques (42, 51) require less
tissue, but the results are difficult to quantify. In addition,
glucose transport activity requires translocation, docking, and
fusion of GLUT4 vesicles with the surface membrane (7, 22),
and histological techniques often do not clearly discriminate
between GLUT4 vesicles associated with surface membranes
and active cell surface GLUT4.
Therefore, we sought to develop a new method to assess cell
surface glucose transporters directly in the isolated perfused
mouse heart. We used a cell surface impermeant biotinylated
bis-glucose photolabeling compound 4,4⬘-O-[2-[2-[2-[2-[2-[6(biotinylamino)hexanoyl]amino]ethoxy]ethoxy]ethoxy]-4-(1azi-2,2,2,-trifluoroethyl)benzoyl]amino-1,3-propanediyl]bis-Dglucose (bio-LC-ATB-BGPA) (19). Bis-mannose and bis-glucose
reagents have proven useful in assessing cell surface glucose
transporters in isolated cells (14, 26), as well as in excised
cardiac (27) and skeletal muscles (40). Thus, isolated mouse
hearts were retrogradely perfused for physiological study prior
to bio-LC-ATB-BGPA infusion through the aortic cannula.
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0193-1849/07 $8.00 Copyright © 2007 the American Physiological Society
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Miller EJ, Li J, Sinusas KM, Holman GD, Young LH. Infusion
of a biotinylated bis-glucose photolabel: a new method to quantify cell
surface GLUT4 in the intact mouse heart. Am J Physiol Endocrinol
Metab 292: E1922–E1928, 2007. First published March 6, 2007;
doi:10.1152/ajpendo.00170.2006.—Glucose uptake in the heart is
mediated by specific glucose transporters (GLUTs) present on cardiomyocyte cell surface membranes. Metabolic stress and insulin both
increase glucose transport by stimulating the translocation of glucose
transporters from intracellular storage vesicles to the cell surface.
Isolated perfused transgenic mouse hearts are commonly used to
investigate the molecular regulation of heart metabolism; however,
current methods to quantify cell surface glucose transporter content in
intact mouse hearts are limited. Therefore, we developed a novel
technique to directly assess the cell surface content of the cardiomyocyte glucose transporter GLUT4 in perfused mouse hearts, using
a cell surface impermeant biotinylated bis-glucose photolabeling
reagent (bio-LC-ATB-BGPA). Bio-LC-ATB-BGPA was infused
through the aorta and cross-linked to cell surface GLUTs. Bio-LCATB-BGPA-labeled GLUT4 was recovered from cardiac membranes
by streptavidin isolation and quantified by immunoblotting. Bio-LCATB-BGPA-labeling of GLUT4 was saturable and competitively
inhibited by D-glucose. Stimulation of glucose uptake by insulin in the
perfused heart was associated with parallel increases in bio-LC-ATBBGPA-labeling of cell surface GLUT4. Bio-LC-ATB-BGPA also
labeled cell surface GLUT1 in the perfused heart. Thus, photolabeling
provides a novel approach to assess cell surface glucose transporter
content in the isolated perfused mouse heart and may prove useful to
investigate the mechanisms through which insulin, ischemia, and
other stimuli regulate glucose metabolism in the heart and other
perfused organs.
Innovative Methodology
MOUSE HEART GLUCOSE TRANSPORTER PHOTOLABELING
The hearts underwent UV irradiation to cross-link the photoactivated diazirine group (19) to glucose transport proteins.
Photolabeled cell surface glucose transporters were isolated
from cell membranes by using streptavidin-agarose and then
were quantified by immunoblotting with specific GLUT antibodies.
MATERIALS AND METHODS
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Boiling longer than 30 min, or for a second time following storage at
⫺80°C, led to irreversible aggregation of GLUT4 in our samples and
was avoided.
GLUT1 and GLUT4 immunoprecipitation. In additional experiments, cardiac membranes (500 ␮g of protein) from photolabeled
hearts were initially immunoprecipitated with isoform-specific GLUT
antibodies bound to protein A/G beads (Amersham). Immunoprecipitation was performed overnight in buffer containing 125 mM Tris,
1 mM EDTA, 1 mM EGTA, 250 mM mannitol, 50 mM sodium
fluoride, 5 mM sodium pyrophosphate, 1 mM DTT, 1 mM benzamidine, 0.004% trypsin inhibitor, and 3 mM sodium azide, pH 7.5, at
4°C. Immunoprecipitated GLUT4 or GLUT1 was isolated by lowspeed centrifugation and extensively washed with buffer prior to
SDS-PAGE and immunoblotting, as described below with horseradish
peroxidase (HRP)-strepatavidin. The supernatants of the immunoprecipitates were also analyzed by immunoblotting to confirm complete
efficiency of the immunoprecipitation procedures.
Immunoblotting. Samples were diluted in SDS-containing sample
buffer and subjected to SDS-PAGE using 10% NuPAGE Bis-Tris gels
(Invitrogen, Carlsbad, CA) with MOPS buffer. Following transfer to
PVDF membranes, proteins were immunoblotted with either COOHterminal GLUT4 or GLUT1 antibodies (kind gifts of Dr. Samuel
Cushman, National Institutes of Health), Na⫹-K⫹-ATPase ␣1-subunit
antibody (Santa Cruz Biotechnology), or incubated with HRP-conjugated streptavidin (Pierce). Bands were detected with enhanced
chemiluminesence and quantified by densitometry after background
correction using ImageJ (version 1.33u).
Glucose uptake in perfused hearts. Rates of glucose uptake were
measured from the production of 3H2O from D-[2-3H]glucose, as
previously described (38). In brief, D-[2-3H]glucose (50 ␮Ci/l) was
added to the perfusate buffer, and the coronary effluent was sampled
every 5 min and subjected to ion exchange chromatography (Bio-Rad
AG1– 8X resin; Bio-Rad, Hercules, CA) to separate nonmetabolized
D-[2-3H]glucose from 3H2O, which was then subjected to scintillation
counting. The rate of glucose uptake was calculated by dividing the
rate of 3H2O production by the perfusate glucose specific activity (36).
Statistics. Results were compared using a two-sided Student’s t-test
or one-way ANOVA as appropriate and expressed as means ⫾ SE.
Differences were considered significant if P ⱕ 0.05.
RESULTS
Bio-LC-ATB-BGPA photolabeling dose titration. To determine the concentration of bio-LC-ATB-BGPA required to
optimally label cell surface GLUT4 in isolated perfused mouse
hearts, we varied bio-LC-ATB-BGPA concentrations (0 –500
␮M) in the buffer. Prior to labeling, these hearts were perfused
with a mixed-substrate buffer containing 100 ␮U/ml insulin for
30 min to increase the cell surface GLUT4 content. Cell
surface GLUT4 photolabeling was found to be maximal at a
concentration of ⬃400 ␮M (Fig. 1), which is consistent with
the Ki (⬃190 ␮M) for the inhibition of glucose transport by
bio-LC-ATB-BGPA and similar compounds in cardiomyocytes (14) and adipocytes (19). This concentration of bio-LCATB-BGPA (400 ␮M) was used for all subsequent photolabeling.
Efficiency and specificity of bio-LC-ATB-BGPA GLUT4
photolabeling and isolation. To confirm that retrograde infusion of bio-LC-ATB-BGPA into the aorta and the coronary
circulation delivered the photolabel uniformly throughout the
heart, we separated the LV free wall, RV, and intraventricular
septum. Similar labeling of cell surface GLUT4 was observed
in each of these myocardial regions (Fig. 2A).
To determine the efficiency of BGPA-labeled GLUT4 recovery, we compared the GLUT4 content in total cardiac
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Animals. Wild-type C57BL/6 male mice (age 10 –16 wk) were used
for all experiments. Animals were housed in accordance with guidelines from the American Association for Laboratory Animal Care and
fed a standard rodent chow diet with access to water ad libitum. All
procedures were approved by the Yale University Animal Care and
Use Committee.
Perfusion protocols. Hearts were isolated from anesthetized mice
after the intraperitoneal injection of heparin sulfate (100 U) and
pentobarbital sodium (100 mg/kg). Hearts were perfused in the Langendorff mode with Krebs-Henseleit buffer (KHB) containing (in
mM) 118 NaCl, 4.75 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 7
glucose, and 0.4 oleate bound to 1% BSA. Hearts were perfused with
a constant coronary flow of 4 ml/min to eliminate potentially confounding vasodilatory effects of insulin on the delivery of the photolabel to the myocardium. To assess the effects of insulin, the perfusion
protocol included an initial 30-min perfusion without insulin followed
by an additional 35 min with or without 100 ␮U/ml (0.7 nM) of
insulin.
Bio-LC-ATB-BGPA photolabeling. Following completion of heart
perfusion, the aortic cannula was flushed with 1 ml of ice-cold
glucose-free KHB followed by 1 ml of the same buffer containing
various concentrations of bio-LC-ATB-BGPA. The infused bio-LCATB-BGPA was allowed 15 min to bind in the heart at 4°C. To
improve exposure of the ventricular cavities to UV irradiation, the left
ventricle (LV) and right ventricle (RV) cavities were opened sagitally.
The bound bio-LC-ATB-BGPA was photochemically cross-linked to
cell surface GLUTs on ice using a Rayonet photochemical reactor
(340 nm, 3 min each on the epicardial and endocardial surfaces;
Southern New England Ultraviolet, Branford, CT). Hearts were
freeze-clamped and stored in liquid nitrogen until further analysis. In
experiments designed to study the specificity of bio-LC-ATB-BGPA
binding, D-glucose (0, 10, or 20 mM) was added to both the washout
and bio-LC-ATB-BGPA buffers. In experiments assessing the homogeneity of bio-LC-ATB-BGPA delivery to different regions of the
heart, the LV, RV, and intraventricular septum were dissected apart
after completion of cross-linking prior to freezing the tissues.
Bio-LC-ATB-BGPA-labeled GLUT isolation. Approximately 50 –
100 mg of myocardial tissue were homogenized in 400 ␮l of HEPESEDTA-sucrose (HES) buffer {20 mM HEPES, 5 mM Na-EDTA, 255
mM sucrose, 1 ␮g/l antipain, aprotinin, pepstatin, leupeptin, 100 ␮M
AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride],
pH 7.2}. Total cell membranes were isolated by ultracentrifugation
(227,000 g for 50 min at 4°C) and resuspended in phosphate-buffered
saline containing 2% Thesit. Insoluble cellular contents were removed
by centrifugation at 20,000 g for 30 min at 4°C. The supernatant
containing the membranes (“total membrane”) was saved and its
protein concentration measured by the Bradford method (Bio-Rad
protein assay; Bio-Rad, Hercules, CA). To isolate the photolabeled
GLUTs, 500 ␮g of total membrane protein were incubated with 100
␮l of streptavidin bound to 6% agarose beads (Pierce, Rockford, IL)
overnight at 4°C. The supernatant of the streptavidin-agarose isolation, which contained nonphotolabeled transporters, was saved as the
“unlabeled fraction.” The steptavidin-agarose isolated labeled fraction
of GLUTs was washed extensively with phosphate-buffered saline
containing decreasing concentrations of Thesit (1, 0.1, and 0%). The
labeled GLUTs were then dissociated from the streptavidin by boiling
in SDS-containing electrophoresis buffer for 30 min with the subsequent addition of 10% mercaptoethanol prior to to SDS-PAGE.
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MOUSE HEART GLUCOSE TRANSPORTER PHOTOLABELING
Fig. 2. Efficacy of heart bio-LC-ATB-BGPA GLUT4 photolabeling. A: regional homogeneity. Left ventricular (LV), right ventricular (RV), and intraventricular
septum myocardial tissues were processed separately after photolabeling hearts perfused without insulin. Streptavidin-isolated photolabeled GLUT4 was detected
by immunoblotting with GLUT4 antibody, quantified by densitometry, and expressed relative to total membrane GLUT4 content in each myocardial region.
B: recovery of GLUT4. Total cardiac membranes (“total membrane”) from photolabeled hearts were prepared by ultracentrifugation and isolated with
streptavidin-agarose to separate cell surface GLUTs (“labeled”) from non-cell surface GLUTs (“unlabeled”) that remained in the supernatant. Equal aliquots were
immunoblotted with GLUT4 antibody (top row) and quantified relative to total membrane GLUT4 in the bar graph (*P ⬍ 0.01, #P ⬍ 0.05 vs. total membrane
GLUT4). Photolabeled membrane protein was also detected directly with HRP-streptavidin (middle row) in the total membrane and streptavidin-isolated labeled
fractions, but not in the unlabeled fraction. Fractions were also immunoblotted with an anti-Na⫹-K⫹-ATPase ␣1-subunit antibody to exclude nonspecific recovery
of other membrane proteins (bottom row). C: inhibition of GLUT4 photolabeling by D-glucose. After heart perfusions, increasing concentrations of D-glucose
were added to the photolabel infusion buffer. Immunoblots of photolabeled GLUT4 (top) were quantified by densitometry (bottom; n ⫽ 3 each, *P ⫽ 0.05 vs.
0 mM glucose).
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Fig. 1. Dose dependence of heart bio-LC-ATB-BGPA GLUT4 photolabeling.
Varying concentrations of bio-LC-ATB-BGPA (0 –500 ␮M) where used to
label cell surface GLUT4 in hearts (n ⫽ 4) after 30-min perfusions with insulin
(100 ␮U/ml). Labeled GLUT4 was isolated from total heart membranes with
streptavidin-agarose and then detected after SDS-PAGE by immunoblotting
with GLUT4 antibody (top), quantified by densitometry, and expressed relative
to total membrane GLUT4 (bottom).
membranes prepared by ultracentrifugation with that in the
streptavidin-isolated “labeled” fraction and the post-streptavidin supernatant “unlabeled” fraction. Comparable aliquots
from each fraction were subjected to SDS-PAGE and quantified by densitometric analysis of GLUT4 immunoblots. Total
membrane GLUT4 was fully accounted for (99.6 ⫾ 3.6%) by
the sum of the labeled and unlabeled GLUT4 (Fig. 2B). To
further assess the efficiency of the isolation procedures, the
membrane proteins were also blotted with HRP-streptavidin.
These blots revealed a biotin-containing protein band at a
molecular mass of ⬃50 kDa, representing the bio-LC-ATBBGPA-glucose transporter conjugate, in both the total membrane and streptavidin-isolated fractions, but no residual
BGPA-GLUT4 in the streptavidin supernatant fraction (Fig. 2B).
Taken together, these results provide evidence that the recovery of bio-LC-ATB-BGPA-photolabeled GLUT4 was efficient.
In addition, to exclude the possibility that the streptavidin
isolation might nonspecifically recover other high-abundance
cell surface membrane transport proteins, we immunoblotted
the streptavidin isolates for Na⫹-K⫹-ATPase (␣1-subunit).
Na⫹-K⫹-ATPase was not detected in the streptavidin isolates,
providing evidence for the specificity of the bio-LC-ATBBGPA photolabeling and isolation methods (Fig. 2B).
Bio-LC-ATB-BGPA binding was generally performed without glucose in the labeling buffer in order to maximize the
labeling of GLUT4. However, when D-glucose (0 –20 mM)
was added to the photolabel-containing buffer, bio-LC-ATBBGPA binding to GLUT4 was inhibited in a dose-dependent
manner and was essentially abolished by a 50-fold higher
concentration of D-glucose (Fig. 2C). The competitive inhibi-
Innovative Methodology
MOUSE HEART GLUCOSE TRANSPORTER PHOTOLABELING
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DISCUSSION
New methods to quantify cell surface GLUT4 content in the
mouse heart are needed to advance our understanding of
myocardial glucose utilization in physiological and pathological states, such as ischemic preconditioning (32, 48), ischemiareperfusion (38), insulin-resistance (3), and heart failure (5).
Established methods are available in the isolated perfused mouse
heart to measure rates of glucose uptake and phosphorylation with
[2-3H]glucose, glycolysis with [5-3H]glucose, and glucose oxidation with [14C]glucose (4). Glucose uptake has been further
estimated by measuring the accumulation and phosphorylation
of deoxyglucose by tissue analysis (3, 5) and 31P NMR spectroscopy (46). However, these approaches do not address the
cellular mechanisms responsible for changes in glucose uptake
and also require that deoxyglucose and native glucose be
comparably transported and phosphorylated (18, 25, 31). The
photolabeling technique complements these methods and provides a direct and easily quantifiable measure of cell surface
GLUT4, which may prove useful in studying glucose transport
regulation in the isolated perfused mouse heart.
The isolated heart has advantages over excised heart muscles
(27) for the study of glucose transport regulation, because it is
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Fig. 3. Insulin stimulation of cell surface GLUT4 photolabeling and heart
glucose uptake. A: perfusion protocol. Following a 30-min baseline perfusion
without insulin, hearts underwent 35 min of additional perfusion with or
without insulin (100 ␮U/ml) before photolabeling with bio-LC-ATB-BGPA
(400 ␮M). B: insulin-stimulated bio-LC-ATB-BGPA cell surface GLUT4
photolabeling. Cell surface and total membrane GLUT4 from control and
insulin-stimulated photolabeled hearts, detected by immunoblotting (top) and
quantified with densitometry (bottom; n ⫽ 3, *P ⬍ 0.01 vs. control).
C: insulin-stimulated glucose uptake. Glucose uptake was measured during
perfusion with D-[2-3H]glucose in the perfusate (see MATERIALS AND METHODS)
(n ⫽ 3, *P ⱕ 0.0001 vs. without insulin).
an intact, contracting, and more physiological preparation. We
devised an infusion technique, which produced homogenous
photolabeling of cell surface GLUT4 throughout different
regions of the perfused heart. This might also prove important
in terms of studying pathological processes that involve specific regions of the heart. Successful implementation of this
approach required technical considerations that warrant emphasis. Effective perfusion of both the left and right coronary
arteries required that the aortic cannula be placed above the
aortic valve and the coronary artery ostia. This was facilitated
by cutting the aorta just prior to the innominate artery during
cardiac excision to allow an adequate aortic length for cannulation. Heparin administration to the mouse prior to heart
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tion of bio-LC-ATB-BGPA photolabeling of glucose transporters by D-glucose further supports the specificity of this
method in the intact heart.
Stimulation of GLUT translocation by insulin: quantification
of cell surface GLUT by bio-LC-ATB-BGPA GLUT4 photolabeling. Insulin is the prototypical stimulus for heart GLUT4
translocation to the cell surface (12, 14, 23, 39, 42, 49).
Therefore, we assessed whether bio-LC-ATB-BGPA photolabeling could effectively detect changes in cell surface GLUTs
after insulin stimulation in the isolated perfused mouse heart.
In these experiments, bio-LC-ATB-BGPA-labeled cell surface
GLUT4 was compared in control and insulin-stimulated hearts
(Fig. 3A). Hearts perfused with physiological concentrations of
insulin (100 ␮U/ml), had a three- to fourfold greater cell
surface GLUT4 compared with control hearts (P ⬍ 0.01;
Fig. 3B). During the same perfusions, glucose uptake measured by the production of 3H2O from D-[2-3H]glucose
increased comparably (3.5-fold, P ⬍ 0.001) in insulinstimulated hearts (Fig. 3C).
Heart GLUT1 cell surface labeling with bio-LC-ATB-BGPA.
GLUT1 is expressed in cardiomyocytes (13, 14, 51) and
endothelial cells (9). GLUT1 is generally considered to be
primarily involved in basal heart glucose transport but also
undergoes translocation to plasma membranes in response to
insulin (14, 51). We also analyzed the cell surface labeling of
GLUT1 in intact hearts after the infusion of bio-LC-ATBBGPA. GLUT1 was present on cell surface membranes in
control mouse hearts, and the content of labeled GLUT1
increased modestly (1.5-fold, P ⫽ 0.05) in response to insulin
(Fig. 4A). To examine the relative amounts of cell surface
GLUT4 and GLUT1, an additional strategy was developed in
which cell membranes from photolabeled hearts were immunoprecipitated with either GLUT4 or GLUT1 antibodies, submitted to SDS-PAGE, and then probed with HRP-streptavidin.
Assuming equal efficiency of GLUT1 and GLUT4 labeling
using this approach, GLUT4 accounted for 43 ⫾ 6% of total
labeled GLUTs after insulin stimulation (Fig. 4B).
Innovative Methodology
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MOUSE HEART GLUCOSE TRANSPORTER PHOTOLABELING
excision was needed to prevent coronary microthrombi that
might otherwise interfere with the uniform delivery of the
infused photolabel. Finally, meticulous attention was required
to avoid the introduction of air bubbles into the aortic cannula
when the heart was removed from the perfusion apparatus and
connected to the syringe containing the photolabel.
The concentrations of bio-LC-ATB-BGPA that saturated
GLUT4 labeling in our experiments (400 ␮M) are very similar
to those observed in isolated rat adipocytes (300 –500 ␮M)
(20). GLUT4 labeling was not only dose dependent but also
was competitively inhibited by the addition of glucose to the
photolabeling buffer. The specificity of the technique was
further supported by the absence of a high abundance cell
surface protein (Na⫹-K⫹-ATPase-␣1) in the streptavidin isolates. In addition, it would appear unlikely that there was
significant labeling of intracellular GLUT4 because of the
cell-impermeant nature of bio-LC-ATB-BGPA compound (19)
and the finding that only 15% of GLUT4 was present on the
cell surface in the absence of insulin, which is quite similar to
previous reports based on membrane fractionation in rat and
canine hearts (47, 51).
The biotin-containing compound bio-LC-ATB-BGPA has
significant technical advantages over tritiated photolabeling
reagents (14, 20) for assessing cell surface GLUT4 in the heart.
The use of the biotin compound avoids the radioactivity containment challenges that would be associated with the use of
tritiated compounds in intact hearts. It also allows for cell
surface GLUT4 quantification without the need to excise the
photolabeled glucose transporters from SDS-PAGE gels for
scintillation counting. The highly efficient recovery of cell
surface labeled GLUT4 with streptavidin-agarose can be attributed to both the high affinity of the biotin-streptavidin
interaction and the long side-chain design of bio-LC-ATBBGPA that makes the biotin moiety readily accessible (19).
Once cell surface labeled GLUT4 is separated from other
GLUT4 protein, immunoblotting techniques are intrinsically
highly specific for GLUT transporters. Therefore, the use of the
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biotinylated compound provides an effective measure of cell
surface GLUT4 in the perfused heart.
GLUT4 translocation to the cell surface is the principal
component of the cardiomyocyte response to insulin (12, 14,
39). Thus, as proof of principal, we assessed the ability of the
BGPA infusion method to measure changes in cell surface
GLUT4 content in response to a physiological concentration of
insulin (100 ␮U/ml). Insulin increased cell surface GLUT4
content three- to fourfold, which was consistent with the
increase in glucose uptake observed in this and previous
studies of perfused mouse hearts (3). The magnitude of insulin
stimulation in contracting hearts is less than that observed in
isolated noncontracting cardiac myocytes (3), highlighting the
importance of studying the heart under these more physiological conditions. One limitation of this method is that it does not
differentiate between cell surface GLUT4 in sarcolemma and
T-tubule membranes. However, given the size of bio-LC-ATBBGPA compound, it is unlikely to be excluded from the
T-tubule space, and tritiated-BMPA is known to label Ttubules in skeletal muscle (11).
The high efficiency of GLUT4 labeling observed in the
perfused hearts confirms that bio-LC-ATB-BGPA delivered
via aortic perfusion was readily able to reach cardiomyocytes,
since the heart contains little noncardiomyocyte GLUT4. Aortic smooth muscle cells express GLUT4 (25, 34), but GLUT4
does not appear to be present in heart blood vessels (9, 49).
Although infusion of photolabel leads to initial contact with
capillary endothelium, endothelial cells do not express GLUT4
transporters (9, 45).
The present results also indicate that the heart contains a
significant amount of cell surface GLUT1, consistent with
prior reports (13, 14, 51). GLUT1 is highly expressed in
endothelial cells (34, 36), and a recent immunogold electron
microscopy study demonstrated greater GLUT1 expression in
endothelial cells than in cardiomyocytes in the adult rat heart
(9). The photolabel experiments indicate that insulin modestly
increases cell surface GLUT1 in the heart but do not elucidate
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Fig. 4. Cell surface GLUT1 labeling with bio-LC-ATB-BGPA. A: cell surface and total membrane GLUT1 from control and insulin-stimulated photolabeled
hearts was detected by immunoblotting (top) and quantified with densitometry (bottom; n ⫽ 3 each, #P ⫽ 0.05 vs. control). Following a 30-min baseline perfusion
without insulin, hearts underwent 35 min of additional perfusion with or without 100 ␮U/ml insulin and then were photolabeled with bio-LC-ATB-BGPA (400
␮M). B: detection of BGPA-labeled GLUT1 and GLUT4 using HRP-streptavidin. Total heart GLUT1 or GLUT4 was immunoprecipitated (IP) from membranes
from control or insulin-stimulated hearts. Immunoprecipitated proteins were subjected to SDS-PAGE, transferred to PVDF, and the cell surface biotin-tagged
GLUTs were detected using HRP-streptavidin and quantified by densitometry. Unlabeled (UL) membrane proteins were prepared from perfused hearts that were
not photolabeled and served as negative controls (n ⫽ 3 each, *P ⫽ 0.01 GLUT1 vs. GLUT4 control, #P ⫽ 0.02 GLUT4 control vs. insulin).
Innovative Methodology
MOUSE HEART GLUCOSE TRANSPORTER PHOTOLABELING
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
GRANTS
18.
This work was supported in part by National Institutes of Health Grants
R01-HL-63811 and DK-59365 (Yale Mouse Metabolic Phenotyping Center)
and T32-HL-07950, and by the United Kingdom Medical Research Council
and the British Heart Foundation.
19.
REFERENCES
20.
1. Abel ED. Insulin signaling in heart muscle: lessons from genetically
engineered mouse models. Curr Hypertens Rep 6: 416 – 423, 2004.
2. Abel ED, Kaulbach HC, Tian R, Hopkins JC, Duffy J, Doetschman T,
Minnemann T, Boers ME, Hadro E, Oberste-Berghaus C, Quist W,
Lowell BB, Ingwall JS, Kahn BB. Cardiac hypertrophy with preserved
contractile function after selective deletion of GLUT4 from the heart.
J Clin Invest 104: 1703–1714, 1999.
3. Belke DD, Betuing S, Tuttle MJ, Graveleau C, Young ME, Pham M,
Zhang D, Cooksey RC, McClain DA, Litwin SE, Taegtmeyer H,
Severson D, Kahn CR, Abel ED. Insulin signaling coordinately regulates
cardiac size, metabolism, and contractile protein isoform expression.
J Clin Invest 109: 629 – 639, 2002.
4. Belke DD, Larsen TS, Lopaschuk GD, Severson DL. Glucose and fatty
acid metabolism in the isolated working mouse heart. Am J Physiol Regul
Integr Comp Physiol 277: R1210 –R1217, 1999.
5. Campbell FM, Kozak R, Wagner A, Altarejos JY, Dyck JR, Belke DD,
Severson DL, Kelly DP, Lopaschuk GD. A role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the control of cardiac
AJP-Endocrinol Metab • VOL
21.
22.
23.
24.
malonyl-CoA levels: reduced fatty acid oxidation rates and increased
glucose oxidation rates in the hearts of mice lacking PPARalpha are
associated with higher concentrations of malonyl-CoA and reduced expression of malonyl-CoA decarboxylase. J Biol Chem 277: 4098 – 4103,
2002.
Carayannopoulos MO, Chi MM, Cui Y, Pingsterhaus JM, McKnight
RA, Mueckler M, Devaskar SU, Moley KH. GLUT8 is a glucose
transporter responsible for insulin-stimulated glucose uptake in the blastocyst. Proc Natl Acad Sci USA 97: 7313–7318, 2000.
Chang L, Chiang SH, Saltiel AR. Insulin signaling and the regulation of
glucose transport (Review). Mol Med 10: 65–71, 2005.
Coven DL, Hu X, Cong L, Bergeron R, Shulman GI, Hardie DG,
Young LH. Physiological role of AMP-activated protein kinase in the
heart: graded activation during exercise. Am J Physiol Endocrinol Metab
285: E629 –E636, 2003.
Davey KA, Garlick PB, Warley A, Southworth R. An immunogold
labelling study of the distribution of GLUT1 and GLUT4 in cardiac tissue
following stimulation by insulin or ischemia. Am J Physiol Heart Circ
Physiol 292: H378 –H386, 2007.
Doege H, Schurmann A, Bahrenberg G, Brauers A, Joost HG.
GLUT8, a novel member of the sugar transport facilitator family with
glucose transport activity. J Biol Chem 275: 16275–16280, 2000.
Dudek RW, Dohm GL, Holman GD, Cushman SW, Wilson CM.
Glucose transporter localization in rat skeletal muscle. Autoradiographic
study using ATB-[2–3H]BMPA photolabel. FEBS Lett 339: 205–208,
1994.
Egert S, Nguyen N, Brosius FC, 3rd, Schwaiger M. Effects of wortmannin on insulin- and ischemia-induced stimulation of GLUT4 translocation and FDG uptake in perfused rat hearts. Cardiovasc Res 35:
283–293, 1997.
Egert S, Nguyen N, Schwaiger M. Myocardial glucose transporter
GLUT1: translocation induced by insulin and ischemia. J Mol Cell
Cardiol 31: 1337–1344, 1999.
Fischer Y, Thomas J, Sevilla L, Munoz P, Becker C, Holman G,
Kozka IJ, Palacin M, Testar X, Kammermeier H, Zorzano A. Insulininduced recruitment of glucose transporter 4 (GLUT4) and GLUT1 in
isolated rat cardiac myocytes. Evidence of the existence of different
intracellular GLUT4 vesicle populations. J Biol Chem 272: 7085–7092,
1997.
Georgakopoulos D, Kass D. Minimal force-frequency modulation of
inotropy and relaxation of in situ murine heart. J Physiol 534: 535–545,
2001.
Gertz EW, Wisneski JA, Stanley WC, Neese RA. Myocardial substrate
utilization during exercise in humans. Dual carbon-labeled carbohydrate
isotope experiments. J Clin Invest 82: 2017–2025, 1988.
Gosmanov AR, Stentz FB, Kitabchi AE. De novo emergence of insulinstimulated glucose uptake in human aortic endothelial cells incubated with
high glucose. Am J Physiol Endocrinol Metab 290: E516 –E522, 2006.
Hariharan R, Bray M, Ganim R, Doenst T, Goodwin G, Taegtmeyer
H. Fundamental limitations of [18F]2-deoxy-2-fluoro-D-glucose for assessing myocardial glucose uptake. Circulation 91: 2435–2444, 1995.
Hashimoto M, Hatanaka Y, Yang J, Dhesi J, Holman GD. Synthesis of
biotinylated bis (D-glucose) derivatives for glucose transporter photoaffinity labelling. Carbohydr Res 331: 119 –127, 2001.
Holman GD, Kozka IJ, Clark AE, Flower CJ, Saltis J, Habberfield
AD, Simpson IA, Cushman SW. Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol
ester. J Biol Chem 265: 18172–18179, 1990.
Huang Y, Hickey RP, Yeh JL, Liu D, Dadak A, Young LH, Johnson
RS, Giordano FJ. Cardiac myocyte-specific HIF-1alpha deletion alters
vascularization, energy availability, calcium flux, and contractility in the
normoxic heart. FASEB J 18: 1138 –1140, 2004.
Ishiki M, Klip A. Minireview: recent developments in the regulation of
glucose transporter-4 traffic: new signals, locations, and partners. Endocrinology 146: 5071–5078, 2005.
James DE, Strube M, Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 338: 83– 87,
1989.
Joost HG, Thorens B. The extended GLUT-family of sugar/polyol
transport facilitators: nomenclature, sequence characteristics, and potential
function of its novel members (Review). Mol Membr Biol 18: 247–256,
2001.
292 • JUNE 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 5, 2017
which cell type might be insulin responsive. Insulin is known
to increase cell surface GLUT1 in isolated cardiomyocytes
(14), but glucose uptake in endothelial cells is typically not
responsive to insulin at physiological concentrations except
when cultured in the presence of very high glucose concentrations (17). The observation that the degree of insulin stimulation of heart glucose uptake paralleled the increase in cell
surface GLUT4 content supports the contention that GLUT4 is
primarily responsible for the insulin effect, as suggested by
earlier studies in the cardiac-specific GLUT4 knockout mouse
(44). This observation is also consistent with the possibility
that a significant amount of GLUT1 in the heart functions as an
endothelial transporter (9) and that endothelial transport is not
rate limiting for insulin stimulation of total heart glucose
uptake. GLUT1 contributes to cardiomyocyte glucose transport
(14), and our results also do not exclude the possibility that
insulin activates GLUT1 activity in cardiomyocytes.
Additional glucose transporters, such as GLUT8 (10) and
GLUT12 (37), are also expressed in the heart, although their
roles in mediating glucose transport remain uncertain. The
photolabeling technique potentially could be used to assess the
cell surface content of these novel glucose transporters. Although we have been unable to detect these transporters on the
cell surface under control or insulin-stimulated conditions
using the bio-LC-ATB-BGPA approach (E. Miller, K. Moley,
S. Rogers, L. Young, unpublished data), it is possible that these
novel glucose transporters may undergo translocation to the
cell surface under pathological conditions or with other stimuli.
In conclusion, photolabeling with bio-LC-ATB-BGPA is a
novel, efficient, and quantifiable method that can be used to
assess cell surface GLUT4 in the isolated perfused mouse
heart. The infusion method may help to further elucidate the
molecular mechanisms controlling GLUT4 translocation and
the role of GLUT4 in the mouse heart under physiological and
pathophysiological conditions. Although we assessed cell surface GLUTs in the heart, the approach is potentially also more
broadly applicable to the investigation of additional cell surface transporters in the heart and other perfused organs.
E1927
Innovative Methodology
E1928
MOUSE HEART GLUCOSE TRANSPORTER PHOTOLABELING
AJP-Endocrinol Metab • VOL
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury. J Clin Invest 114: 495–503, 2004.
Russell RR 3rd, Yin R, Caplan MJ, Hu X, Ren J, Shulman GI, Sinusas
AJ, Young LH. Additive effects of hyperinsulinemia and ischemia on
myocardial GLUT1 and GLUT4 translocation in vivo. Circulation 98:
2180 –2186, 1998.
Ryder JW, Yang J, Galuska D, Rincon J, Bjornholm M, Krook A,
Lund S, Pedersen O, Wallberg-Henriksson H, Zierath JR, Holman
GD. Use of a novel impermeable biotinylated photolabeling reagent to
assess insulin- and hypoxia-stimulated cell surface GLUT4 content in
skeletal muscle from type 2 diabetic patients. Diabetes 49: 647– 654, 2000.
Sivitz WI, Lund DD, Yorek B, Grover-McKay M, Schmid PG. Pretranslational regulation of two cardiac glucose transporters in rats exposed to
hypobaric hypoxia. Am J Physiol Endocrinol Metab 263: E562–E569, 1992.
Slot JW, Geuze HJ, Gigengack S, James DE, Lienhard GE. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat.
Proc Natl Acad Sci USA 88: 7815–7819, 1991.
Slot JW, Moxley R, Geuze HJ, James DE. No evidence for expression
of the insulin-regulatable glucose transporter in endothelial cells. Nature
346: 369 –371, 1990.
Sun D, Nguyen N, DeGrado TR, Schwaiger M, Brosius FC 3rd.
Ischemia induces translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane of cardiac myocytes. Circulation
89: 793–798, 1994.
Thomas J, Linssen M, van der Vusse GJ, Hirsch B, Rosen P, Kammermeier H, Fischer Y. Acute stimulation of glucose transport by
histamine in cardiac microvascular endothelial cells. Biochim Biophys
Acta 1268: 88 –96, 1995.
Tian R, Abel ED. Responses of GLUT4-deficient hearts to ischemia
underscore the importance of glycolysis. Circulation 103: 2961–2966,
2001.
Tian R, Musi N, D’Agostino J, Hirshman MF, Goodyear LJ. Increased
adenosine monophosphate-activated protein kinase activity in rat hearts
with pressure-overload hypertrophy. Circulation 104: 1664 –1669, 2001.
Tong H, Chen W, London RE, Murphy E, Steenbergen C. Preconditioning enhanced glucose uptake is mediated by p38 MAP kinase not by
phosphatidylinositol 3-kinase. J Biol Chem 275: 11981–11986, 2000.
Watanabe T, Smith MM, Robinson FW, Kono T. Insulin action on
glucose transport in cardiac muscle. J Biol Chem 259: 13117–13122,
1984.
Yang J, Clark AE, Kozka IJ, Cushman SW, Holman GD. Development of an intracellular pool of glucose transporters in 3T3–L1 cells.
J Biol Chem 267: 10393–10399, 1992.
Young LH, Renfu Y, Russell R, Hu X, Caplan M, Ren J, Shulman GI,
Sinusas AJ. Low-flow ischemia leads to translocation of canine heart
GLUT-4 and GLUT-1 glucose transporters to the sarcolemma in vivo.
Circulation 95: 415– 422, 1997.
292 • JUNE 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 5, 2017
25. Kanda Y, Watanabe Y. Thrombin-induced glucose transport via Src-p38
MAPK pathway in vascular smooth muscle cells. Br J Pharmacol 146:
60 – 67, 2005.
26. Koumanov F, Yang J, Jones AE, Hatanaka Y, Holman GD. Cell
surface biotinylation of GLUT4 using bis-mannose photolabels. Biochem
J 330: 1209 –1215, 1998.
27. Li J, Hu X, Selvakumar P, Russell RR 3rd, Cushman SW, Holman
GD, Young LH. Role of the nitric oxide pathway in AMPK-mediated
glucose uptake and GLUT4 translocation in heart muscle. Am J Physiol
Endocrinol Metab 287: E834 –E841, 2004.
28. Liao R, Jain M, Cui L, D’Agostino J, Aiello F, Luptak I, Ngoy S,
Mortensen RM, Tian R. Cardiac-specific overexpression of GLUT1
prevents the development of heart failure attributable to pressure overload
in mice. Circulation 106: 2125–2131, 2002.
29. Lund S, Holman GD, Schmitz O, Pedersen O. Glut 4 content in the
plasma membrane of rat skeletal muscle: comparative studies of the
subcellular fractionation method and the exofacial photolabelling technique using ATB-BMPA. FEBS Lett 330: 312–318, 1993.
31. Ng C, Holden J, DeGrado T, Raffel D, Kornguth M, Gatley S.
Sensitivity of myocardial fluorodeoxyglucose lumped constant to glucose
and insulin. Am J Physiol Heart Circ Physiol 260: H593–H603, 1991.
32. Nishino Y, Miura T, Miki T, Sakamoto J, Nakamura Y, Ikeda Y,
Kobayashi H, Shimamoto K. Ischemic preconditioning activates AMPK
in a PKC-dependent manner and induces GLUT4 up-regulation in the late
phase of cardioprotection. Cardiovasc Res 61: 610 – 619, 2004.
33. Opie LH, Sack MN. Metabolic plasticity and the promotion of cardiac
protection in ischemia and ischemic preconditioning. J Mol Cell Cardiol
34: 1077–1089, 2002.
34. Park JL, Loberg RD, Duquaine D, Zhang H, Deo BK, Ardanaz N,
Coyle J, Atkins KB, Schin M, Charron MJ, Kumagai AK, Pagano PJ,
Brosius FC 3rd. GLUT4 facilitative glucose transporter specifically and
differentially contributes to agonist-induced vascular reactivity in mouse
aorta. Arterioscler Thromb Vasc Biol 25: 1596 –1602, 2005.
35. Park SY, Cho YR, Finck BN, Kim HJ, Higashimori T, Hong EG, Lee
MK, Danton C, Deshmukh S, Cline GW, Wu JJ, Bennett AM,
Rothermel B, Kalinowski A, Russell KS, Kim YB, Kelly DP, Kim JK.
Cardiac-specific overexpression of peroxisome proliferator-activated receptor-alpha causes insulin resistance in heart and liver. Diabetes 54:
2514 –2524, 2005.
36. Pekala P, Marlow M, Heuvelman D, Connolly D. Regulation of hexose
transport in aortic endothelial cells by vascular permeability factor and
tumor necrosis factor-alpha, but not by insulin. J Biol Chem 265: 18051–
18054, 1990.
37. Rogers S, Macheda ML, Docherty SE, Carty MD, Henderson MA,
Soeller WC, Gibbs EM, James DE, Best JD. Identification of a novel
glucose transporter-like protein-GLUT12. Am J Physiol Endocrinol Metab
282: E733–E738, 2002.
38. Russell RR 3rd, Li J, Coven DL, Pypaert M, Zechner C, Palmeri M,
Giordano FJ, Mu J, Birnbaum MJ, Young LH. AMP-activated protein