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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]). E1922 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. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 0193-1849/07 $8.00 Copyright © 2007 the American Physiological Society http://www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 5, 2017 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 AJP-Endocrinol Metab • VOL 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 292 • JUNE 2007 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 5, 2017 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. E1923 Innovative Methodology E1924 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). AJP-Endocrinol Metab • VOL 292 • JUNE 2007 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 5, 2017 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 E1925 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 AJP-Endocrinol Metab • VOL 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 292 • JUNE 2007 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 5, 2017 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 E1926 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 AJP-Endocrinol Metab • VOL 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 292 • JUNE 2007 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 5, 2017 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