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ARTICLE 5=-AMP-activated protein kinase increases glucose uptake independent of GLUT4 translocation in cardiac myocytes Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 05/26/14 For personal use only.

Christopher T. Lee, John R. Ussher, Askar Mohammad, Anna Lam, and Gary D. Lopaschuk

Abstract: Glucose uptake and glycolysis are increased in the heart during ischemia, and this metabolic alteration constitutes an important contributing factor towards ischemic injury. Therefore, it is important to understand glucose uptake regulation in the ischemic heart. There are primarily 2 glucose transporters controlling glucose uptake into cardiac myocytes: GLUT1 and GLUT4. In the non-ischemic heart, insulin stimulates GLUT4 translocation to the sarcolemmal membrane, while both GLUT1 and GLUT4 translocation can occur following AMPK stimulation. Using a newly developed technique involving [3H]2-deoxyglucose, we measured glucose uptake in H9c2 ventricular myoblasts, and demonstrated that while insulin has no detectable effect on glucose uptake, phenformin-induced AMPK activation increases glucose uptake 2.5-fold. Furthermore, insulin treatment produced no discernible effect on either Akt serine 473 phosphorylation or AMPK␣ threonine 172 phosphorylation, while treatment with phenformin results in an increase in AMPK␣ threonine 172 phosphorylation, and a decrease in Akt serine 473 phosphorylation. Visualization of a dsRed-GLUT4 fusion construct in H9c2 cells by laser confocal microscopy showed that unlike insulin, AMPK activation did not redistribute GLUT4 to the sarcolemmal membrane, suggesting that AMPK may regulate glucose uptake via another glucose transporter. These studies suggest that AMPK is a major regulator of glucose uptake in cardiac myocytes. Key words: AMPK, glucose uptake, GLUT4, glycolysis, metabolism, cardiac myocyte, 2-deoxyglucose. Résumé : La captation de glucose et la glycolyse sont accrues dans le cœur lors de l'ischémie, et cette modification métabolique constitue un facteur contributif important vers le dommage ischémique. En conséquence, il est important de comprendre la régulation de la captation du glucose dans le cœur ischémique. Il existe 2 transporteurs de glucose qui contrôlent la captation de glucose dans les myocytes cardiaques, GLUT1 et GLUT4. Dans le cœur non ischémique, l'insuline stimule la translocation de GLUT4 vers la membrane du sarcolemme, alors que la translocation de GLUT1 et GLUT4 peut survenir a` la suite de la stimulation de l'AMPK. Nous avons mesuré la captation de glucose dans les myoblastes ventriculaires H9c2 a` l'aide d'une nouvelle technique impliquant le [3H]2-déoxyglucose, et nous avons démontré que, alors que l'insuline n'exerce pas d'effet détectable sur la captation de glucose, l'activation de l'AMPK par la phenformine accroit la captation de glucose de 2.5 fois. De plus, le traitement a` l'insuline ne produisait par d'effet détectable sur la phosphorylation d'Akt sur la sérine 473 ou de l'AMPK␣ sur la thréonine 172, alors que le traitement a` la phenformine résultait en une augmentation de la phosphorylation de l'AMPK␣ sur la thréonine 172, et en une diminution de la phosphorylation d'Akt sur la sérine 473. L'observation en microscopie confocale au laser d'une construction impliquant une fusion dsRed-GLUT4 chez les cellules H92c a montré que contrairement a` l'insuline, l'activation de l'AMPK ne produit pas de redistribution de GLUT4 a` la membrane du sarcolemme, suggérant que l'AMPK peut réguler la captation de glucose par l'intermédiaire d'un autre transporteur de glucose. Ces études suggèrent que l'AMPK est un régulateur clé de la captation de glucose dans les myocytes cardiaques. [Traduit par la Rédaction] Mots-clés : AMPK, captation de glucose, GLUT4, glycolyse, métabolisme, myocytes cardiaques, 2-déoxyglucose.

Introduction 5=-AMP-activated protein kinase (AMPK) is a serine/threonine kinase that acts as a “cellular fuel gauge,” owing to its ability to increase energy producing pathways, and to inhibit energy consuming pathways during times of metabolic stress (Hardie and Carling 1997; Sambandam and Lopaschuk 2003; Dyck and Lopaschuk 2006). This role of AMPK is extremely relevant in the heart, the most metabolically demanding organ in the body. When faced with metabolic stress, such as ischemia, cardiac AMPK activation increases ATP production via stimulation of glucose uptake and glycolysis (Kudo et al. 1995; Ruderman et al. 2003; Russell et al. 2004; Dyck and Lopaschuk 2006; Fujii et al. 2006; Ussher and Lopaschuk 2006). Because the heart has minimal energy reserves,

AMPK's role as a fuel gauge is important for maintaining energy supply during times of metabolic stress (Kudo et al. 1995, 1996; Sambandam and Lopaschuk 2003; Dyck and Lopaschuk 2006). During ischemia, AMPK is rapidly activated (Kudo et al. 1995; Russell et al. 2004), where it acts to increase glucose uptake and glycolysis (Russell et al. 2004). This effect of AMPK on the ischemic heart has been proposed to be a protective mechanism, providing the heart with ATP anaerobically at a time when ATP levels are being rapidly depleted (Russell et al. 2004; Dyck and Lopaschuk 2006). Indeed, hearts from mice overexpressing a dominant negative isoform of the ␣2 catalytic subunit of AMPK fail to stimulate translocation of glucose transporter 4 (GLUT4) to the sarcolemmal membrane and subsequent glucose uptake (Russell et al. 2004).

Received 18 March 2013. Accepted 24 January 2014. C.T. Lee, A. Mohammad, A. Lam, and G.D. Lopaschuk.* Cardiovascular Research Centre, Mazankowski Alberta Heart Institute, University of Alberta, 8440 112 Street NW, Edmonton, AB T6G 2P4, Canada. J.R. Ussher. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Joseph and Wolf Lebovic Health Complex, 600 University Avenue, Toronto, ON M5G 1X5, Canada. Corresponding author: Gary D. Lopaschuk (e-mail: [email protected]). *Present address: 423 Heritage Medical Research Center, University of Alberta, Edmonton, AB T6G 2S2, Canada. Can. J. Physiol. Pharmacol. 92: 307–314 (2014) dx.doi.org/10.1139/cjpp-2013-0107

Published at www.nrcresearchpress.com/cjpp on 5 February 2014.

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Moreover, hearts from these animals have a poorer recovery during reperfusion following low-flow ischemia, and experience greater rates of apoptosis. In addition, it has been demonstrated that in isolated hearts from mice deficient for the ␣2 catalytic subunit of AMPK, reductions in exogenous glucose uptake and rates of glycolysis result in the rapid development of contracture during low flow ischemia (Zarrinpashneh et al. 2006). It is important to delineate the mechanisms by which AMPK activation in the heart increases glucose uptake and affords protection against ischemia. This has been investigated by many laboratories in recent years, and numerous studies have unequivocally shown that AMPK has a role in regulating glucose uptake in the heart, although the mechanism appears to differ from study to study (Li et al. 2004, 2005; Russell et al. 2004; Fujii et al. 2006; Yang and Holman 2006; Miller et al. 2008). One particular study from the laboratory of Holman and colleagues has shown that metformin-induced activation of AMPK increased glucose uptake in adult cardiac myocytes via prevention of endocytosis of GLUT4 transporters already at the sarcolemmal membrane (Yang and Holman 2006). Furthermore, Young and colleagues have shown in isolated papillary muscles that AMPK activation of endothelial nitric oxide synthase and p38 mitogen activated protein kinase both contribute to AMPK's ability to stimulate GLUT4 translocation and subsequent glucose uptake (Li et al. 2004, 2005). Although many of these studies support a role for AMPK in regulating glucose uptake in the heart, it is important to note that a major limitation with many studies investigating glucose uptake in the heart is the type of assay utilized. The majority of glucose uptake studies utilize a procedure where 2-deoxyglucose is either used at millimolar concentrations or in tracer amounts in the absence of glucose (Patel et al. 2001; Sajan et al. 2004, 2006; Carroll et al. 2005). Both of these methodologies are flawed, as 2-deoxyglucose used at millimolar concentrations can inhibit glycolysis (Hood et al. 1988), which itself can affect glucose uptake; whereas 2-deoxyglucose used in tracer amounts in the absence of glucose will have very different kinetics for the affinity of the glucose transporter compared with glucose at physiological levels (Doenst and Taegtmeyer 1998). Moreover, studies examining glucose uptake in isolated Langendorff or working hearts will often use measurements of glycolysis from exogenous glucose in the perfusate as an index of glucose uptake. Such measurements are also flawed, as they do not measure the radiolabel from exogenous glucose that is being incorporated into glycogen. Interestingly, looking at the radiolabel from 5-3H glucose being incorporated into glycogen and that being released from glycolysis, Jaswal et al. (2006) demonstrated that activation of AMPK by adenosine in hearts subjected to a transient ischemia did not actually increase glucose uptake. In this study, we developed an assay for measuring glucose uptake in H9c2 rat ventricular myoblasts, whereby tracer amounts of 3H 2-deoxyglucose were supplied to the medium in the presence of glucose. We also delivered fluorescently labeled GLUT4 to these H9c2 cells to directly assess GLUT4 translocation in these cells. Utilizing these assays, our aim was to determine whether AMPK activation truly increases glucose uptake in the heart, and to determine whether this effect of AMPK activation involves stimulation of GLUT4 translocation to the sarcolemmal membrane.

Materials and methods Cell culture H9c2 rat ventricular myoblasts (ATCC, Rockville, Maryland, USA) were grown to confluency in 60 mm (diameter) cell culture dishes in Dulbecco's modified Eagles' Medium (DMEM) containing 10% (v/v) fetal bovine serum, 1% (v/v) PenStrep, and 0.25 mmol·L–1 L-carnitine. Dishes were incubated in a water-jacketed CO2 incubator maintained at 37 °C with 95% O2 and 5% CO2 (v/v/v). Replen-

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ishment with fresh medium occurred every 48 h. Passages 10–25 were used for the experiments described in this study. Adenoviral vector generation and infections Adenoviruses containing either green fluorescent protein (GFP) or GLUT4 Discosoma spp. (reef coral) red fluorescence protein (dsRed) were subcloned into a pAdTrack-CMV shuttle vector, linearized, and inserted into an adenovirus using the pAdEasy-1 system for homologous recombination in Escherichia coli as previously described (He et al. 1998). At confluency, the medium for the H9c2 cells was switched to serum-free DMEM, and the cells were treated with adenoviruses containing either GFP or GLUT4-dsRed at a multiplicity of infection (MOI) of 100. Glucose uptake assay H9c2 cells were serum-starved for 24 h, then incubated with either 10 mmol·L–1 phenformin for 2 h or 100 nmol·L–1 insulin for 1 h. After incubation, the medium was removed and the cells were washed twice with 1× PBS with 10 mmol·L–1 HEPES. The cells were incubated with 1 mmol·L–1 glucose and (0.5 ␮Ci/mL) 3H 2-deoxyglucose for 10 min. The reaction was subsequently stopped with ice cold 20 ␮mol·L–1 phloretin. The phloretin reaction was then replaced with 0.1 N KOH (0.5 mL) and placed in a scintillation counter after the addition of 4 mL of scintillation fluid (EcoLite; ICN, California, USA). Non-specific glucose uptake was measured in the presence of 100 ␮mol·L–1 phloretin, and the values subtracted from the total uptake. AMPK activity assay Endogenous AMPK activity was measured as previously described (Kudo et al. 1995). Samples were diluted to a concentration of 1 mg/mL in resuspension buffer containing 50 mmol·L–1 Tris HCl (pH 8 at 4 °C), 1 mmol·L–1 EDTA, 10% glycerol (w/v), 0.02% Brij-35 (w/v), 1 mmol·L–1 dithiothreitol, protease, and phosphatase inhibitors (Sigma). Two microlitres of the above sample was then incubated with 200 ␮mol·L–1 synthetic AMARA (AMARAASAAALARRR) peptide, 200 ␮mol·L–1 [32P]ATP[␥-P], 0.8 mmol·L–1 dithiothreitol, 5 mmol·L–1 MgCl2, 200 ␮mol·L–1 AMP in buffer (pH 7.0) containing 40 mmol·L–1 HEPES– NaOH, 80 mmol·L–1 NaCl, and 8% (w/v) glycerol for 5 min at 30 °C (total volume 25 ␮L). This incubation results in incorporation of 32P into the AMARA peptide. At the end of 5 min, 15 ␮L of the incubation mixture was blotted onto 1 cm2 of phosphocellulose paper. From here, the phosphocellulose paper was washed 3 × 10 min in 150 mm phosphoric acid followed by a 5 min wash in acetone. The phosphocellulose papers were then dried and counted in 4 mL of scintillation fluid (EcoLite). AMPK activity was expressed as picomoles of 32P incorporated into AMARA peptide per minute per milligram of protein. Laser scanning confocal microscopy assessment of GLUT4 translocation H9c2 cells were grown to near confluency and plated onto sterile cover slips treated with fibronectin. They were subsequently infected with adenovirus containing the Glut4-dsRED expression construct at an MOI of 100. Cells were then serum-starved prior to treatment with insulin (100 nmol·L–1), or phenformin (10 mmol·L–1), or vehicle (control). Cells were then washed with 1× PBS and fixed with 2.5% paraformaldehyde for 5 min, and then mounted with Prolong Gold antifade reagent with DAPI (Molecular Probes P36935) prior to viewing under confocal microscopy. Immunoblot analysis Immunoblots were carried out to determine protein expression and phosphorylation as previously described (Ussher et al. 2012). Cells were lysed in buffer containing 50 mmol·L–1 Tris HCl (pH 8 at 4 °C), 1 mmol·L–1 EDTA, 10% glycerol (w/v), 0.02% Brij-35 (w/v), 1 mmol·L–1 dithiothreitol, and protease and phosphatase inhibitors. The cell lysate was collected into Eppendorf tubes and left on ice for 10 min before centrifugation at 800g for 20 min. The resulting Published by NRC Research Press

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Fig. 1. The effect of insulin and phenformin treatment on AMPK␣ and Akt phosphorylation in H9c2 cells. (A) AMPK␣ threonine 172 phosphorylation in H9c2 cells treated with either insulin (100 nmol·L–1 for 1 h) or phenformin (10 mmol·L–1 for 2 h). (B) AMPK activity was measured in H9c2 cells treated with either insulin (100 nmol·L–1 for 1 h) or phenformin (10 mmol·L–1 for 2 h) by measuring the incorporation of 32P into AMARA peptide. (C) AMPK␣ serine 485/491 phosphorylation in H9c2 cells treated with either insulin (100 nmol·L–1 for 1 h) or phenformin (10 mmol·L–1 for 2 h). (D) Akt serine 473 phosphorylation in H9c2 cells treated with either insulin (100 nmol·L–1 for 1 h) or phenformin (10 mmol·L–1 for 2 h). (E) LKB1 serine 428 phosphorylation in H9c2 cells treated with either insulin (100 nmol·L–1 for 1 h) or phenformin (10 mmol·L–1 for 2 h). (F) MO25␣ protein expression in H9c2 cells treated with either insulin (100 nmol·L–1 for 1 h) or phenformin (10 mmol·L–1 for 2 h). Values are the mean ± SE (n = 3–4 observations). Differences were determined using a one-way ANOVA followed by a Bonferroni post-hoc analysis. *, P < 0.05 compared with the control (vehicle treated) cells. control

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Results Treatment of H9c2 cells with phenformin results in activation of AMPK H9c2 cells were treated with the biguanide phenformin (10 mmol·L–1 for 2 h) to activate AMPK, or with insulin (100 nmol·L–1 for 1 h). Treatment of H9c2 cells with phenformin led to a marked increase in threonine 172 phosphorylation of the AMPK␣ subunit (Fig. 1A), indicative of AMPK activation. Moreover, utilizing a radioactive AMPK activity assay, we show that phenformin increases the ability of H9c2 cells to incorporate 32P into AMARA peptide (Fig. 1B). However, we did not observe an increase in ACC phosphorylation (an important downstream target of AMPK in regulating metabolism (data not shown)). Although previous studies have shown that insulin inhibits AMPK (Horman et al. 2006), we report here that insulin treatment of H9c2 cells has no effect on AMPK␣ threonine 172 phosphorylation or AMPK activity (Fig. 1A/B). In line with these findings, insulin treatment also had no effect on AMPK␣ serine 485/491 phosphorylation (Fig. 1C), which has previously been shown to be an AMPK inhibitory phosphorylation site (Horman et al. 2006; Soltys et al. 2006). To our surprise, phenformin treatment reduced AMPK␣ serine 485/ 491 phosphorylation in H9c2 cells (Fig. 1C), possibly due to a phenformin-mediated decrease in serine 473 phosphorylation of Akt (Fig. 1D), a key insulin signaling molecule that mediates insulin's ability to reduce AMPK activity via phosphorylating AMPK␣ at serine 485/491 (Horman et al. 2006; Soltys et al. 2006). Phenformin-induced AMPK activation in H9c2 cells was independent of LKB1, as we observed no effect on LKB1 expression or LKB1 serine 428 phosphorylation 2 h following treatment with 10 mmol·L–1 phenformin, and similar effects were observed following insulin treatment (Fig. 1E). Furthermore, expression of the LKB1 complex protein, mouse protein 25 (MO25␣), was also unaltered following treatment with either phenformin or insulin (Fig. 1F). Activation of AMPK in H9c2 cells results in an increase in glucose uptake We first confirmed the validity of our glucose uptake assay in neonatal cardiac myocytes by determining glucose uptake at increasing glucose concentrations, and demonstrate a concentrationdependent increase in glucose uptake that plateaued at 1 mmol·L–1 glucose (Fig. 2A). We therefore carried out all subsequent glucose uptake measurements in this study with 1 mmol·L–1 glucose. Next, a glucose uptake time-course in neonatal cardiac myocytes using 1 mmol·L–1 glucose and 3H-2-deoxyglucose (0.5 ␮Ci/mL) as a tracer demonstrated a linear increase (R2 = 0.987) in glucose uptake over 20 min (Fig. 2B). Utilizing this assay for measuring glucose uptake in H9c2 cells, we show that similar to previous studies (Li et al. 2004, 2005; Russell et al. 2004; Yang and Holman 2006; Miller et al. 2008), activation of AMPK with phenformin leads to a significant increase in glucose uptake (⬃2.5 fold) versus vehicle-treated cells (Fig. 3A). This phenformin-mediated effect was dependent on AMPK activation, as pre-treatment with the AMPK inhibitor com-

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Statistical analysis All values are the mean ± SE (n = observations). The significance of differences was determined by the use of a one-way analysis of variance (ANOVA), followed by a Bonferroni post-hoc analysis where appropriate. Differences were considered significant when P < 0.05.

Fig. 2. Validation of our glucose uptake assay. (A) Dose–response curve for glucose uptake in neonatal cardiac myocytes, demonstrating the plateau of glucose uptake at a glucose concentration of 1 mmol·L–1. (B) Glucose uptake time-course in neonatal cardiac myocytes incubated in medium containing 1 mmol·L–1 glucose and 3H-2-deoxyglucose (0.5 ␮Ci/mL); R2 = 0.987. Values are the mean ± SE (n = 4 observations).

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anti-phosphoSerine-473 Akt, anti-MO25␣ (Cell Signaling Technologies), peroxidase labeled streptavidin (KPL), or antiphosphoSerine-79 acetyl CoA carboxylase (ACC; Millipore), antibodies in 5% fatty acid-free bovine serum albumin. Immunoblots were visualized with the enhanced chemiluminescence Western blot detection kit (Perkin Elmer) and quantified with Quantity One (4.4.0; Bio-Rad).

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pound C (40 ␮mol·L–1), abolished the rise in glucose uptake (Fig. 3B). Interestingly, treatment of H9c2 cells with insulin did not increase glucose uptake (Fig. 3). Activation of AMPK and subsequent glucose uptake in H9c2 cells is not associated with GLUT4 translocation Upon reaching confluence, H9c2 cells were infected with adenoviruses expressing GFP or GLUT4-dsRed (Fig. 4A) at a MOI of 100. After a week of allowing the H9c2 cells to overexpress the proteins of interest, they were treated with either vehicle, insulin, or phenformin, and fixed in paraformaldehyde. Cells were then fixed onto cover slips and laser scanning confocal microscopy was used to determine the subcellular localization of GLUT4. Although insulin did not increase glucose uptake in H9c2 cells, it did cause GLUT4 translocation to the sarcolemmal membrane (Fig. 4C), in accordance with previous studies (Furtado et al. 2002). To our surprise, activation of AMPK in H9c2 cells did not cause GLUT4 translocation to the sarcolemmal membrane (Fig. 4D), indicating that AMPK activation in the heart may be mediating an increase in glucose uptake via some other mechanism. Published by NRC Research Press

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Fig. 3. AMPK activation increases glucose uptake in H9c2 cells. (A) Utilization of our modified glucose uptake assay reveals that activation of AMPK with phenformin (10 mmol·L–1 for 2 h) increases glucose uptake in cardiac myocytes. (B) This effect was dependent on AMPK activity, as pretreatment with the AMPK inhibitor compound C (40 ␮mol·L–1 for 30 min prior to phenformin treatment and throughout the 2 hr treatment), prevented the phenformininduced increase in glucose uptake. Values are the mean ± SE (n = 4 observations). Differences were determined using a one-way ANOVA followed by a Bonferroni post-hoc analysis; *, P < 0.05 compared with the control (vehicle treated) cells; #, P < 0.05 compared with the phenformin-treated cells.

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Discussion In this study we demonstrate that activation of AMPK in the heart increases glucose uptake independent of GLUT4 translocation to the sarcolemmal membrane, utilizing both measurements of glucose uptake and laser scanning confocal microscopy. Previous studies that have measured glucose uptake in-vitro in cultured cells have commonly used either 3H 2-deoxyglucose as a substrate (Patel et al. 2001; Kotani et al. 2004; Mahajan et al. 2004; Sajan et al. 2004, 2006; Carroll et al. 2005; Wijesekara et al. 2005) or, less frequently, 3H-3-O-methoxy glucose (Yu et al. 1999, 2000) and 18F-2-deoxyglucose (Botker et al. 1999). These glucose analogs are employed because of their structural similarity to glucose, and the fact that they cannot be further metabolized beyond the initial

phosphorylation step catalyzed by hexokinase once they enter the cell. Thus, accumulation of these molecules can be quantified if the substrates are radiolabeled as either 3H or 18F (Gjedde 1987). Currently, the vast majority of laboratories have employed the 3H-2-deoxyglucose method to measure glucose uptake (Patel et al. 2001; Kotani et al. 2004; Mahajan et al. 2004; Sajan et al. 2004, 2006; Carroll et al. 2005; Wijesekara et al. 2005). This method utilizes nonradioactive 2-deoxyglucose as a substrate in the culture medium, and 3H-2-deoxyglucose as a tracer. While this method will provide information on 2-deoxyglucose uptake, whether the result can be substituted for actual glucose uptake is debatable. This issue was first brought about by Sokoloff et al. (1977), while mapping various brain-region functions. More recently, Taegtmeyer and colleagues also raised similar concerns in the isolated working heart model (Hariharan et al. 1995; Doenst et al. 1999). Moreover, they also found that the increase in glucose uptake in the heart in response to different agonists depends on the substrate (analog) utilized in the uptake measurement (Doenst and Taegtmeyer 1998). This discrepancy in response to different agonists with different substrates was coined the “lump constant.” However, use of the lump constant is not ideal, as it requires numerous calculations that need to be performed before each and every study. Aside from these issues, 2-deoxyglucose is also an inhibitor of glycolysis (Hood et al. 1988). Thus, cells exposed to a substrate with the ability to inhibit glycolysis, which can itself influence glucose uptake, imposes a major limitation with the 2-deoxyglucose uptake assay. Despite these significant limitations, 2-deoxyglucose remains the preferred substrate choice for the majority of in-vitro glucose uptake assays, possibly due to the ease of carrying out the procedure and its general acceptance. To address these limitations with the 2-deoxyglucose uptake assay, we have developed an in-vitro glucose uptake assay that is simple to perform, and more reflective of true glucose uptake. The modified method utilizes glucose as a substrate in the culture medium and 3H-2-deoxyglucose only as a tracer. The advantage of this method is that the cell is exposed to its natural substrate, which allows us to measure accumulated radioactivity from 3H-2-deoxyglucose without any potential inhibition of glycolysis. Indeed, utilizing the classic 2-deoxyglucose uptake assay, we have shown that scenarios not causing an increase in glucose uptake may produce measurable increases with our modified procedure (A. Mohammad and G.D. Lopaschuk, unpublished data). Because AMPK is an important energy sensor that increases energy production during times of metabolic stress, it has received much attention with respect to the ischemic heart; a condition where enhanced energy production would be beneficial. It has been proposed that AMPK activation in the ischemic heart may be beneficial via increasing glucose uptake and glycolysis, although controversial results have been published in regards to AMPK activation being cardioprotective (Kudo et al. 1995; Dyck and Lopaschuk 2006; Ussher and Lopaschuk 2008, 2009; Folmes et al. 2009; Ussher et al. 2009), or whether it actually does increase glucose uptake (Jaswal et al. 2006). Utilizing our modified glucose uptake assay, we observed that AMPK activation in H9c2 cells did increase glucose uptake, consistent with previous studies (Li et al. 2004, 2005; Russell et al. 2004; Yang and Holman 2006). Interestingly, insulin had no effect on glucose uptake in H9c2 cells, but this may be due to the cancerous nature of the H9c2 cell line, as cancerous cells rely primarily on glycolysis for energy, and thus have significantly elevated basal glucose uptake rates (Ong et al. 2008; Ganapathy et al. 2009). Indeed, in our studies we chose to utilize H9c2 cells in the undifferentiated myoblastic state where they are able to proliferate at a relatively rapid rate. In contrast, if H9c2 cells are differentiated into myocytes with retinoic acid (Ussher et al. 2009), whereby they become devoid of the ability to proliferate, treatment with insulin results in a marked elevation of Akt phosphorylation (data not shown), suggesting that the cells Published by NRC Research Press

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Fig. 4. The stimulation of glucose uptake via AMPK activation in H9c2 cells is independent of GLUT4 translocation. (A) Schematic depicting the adenovirus containing the Glut4-dsRED expression construct. (B) Laser scanning confocal microscopy showing lack of GLUT4 translocation to the sarcolemmal membrane in H9c2 cells treated with vehicle control (yellow arrow indicates nucleus, whereas yellow arrowhead indicates GLUT4 throughout the cytosol). (C) Laser scanning confocal microscopy showing GLUT4 translocation to the sarcolemmal membrane in H9c2 cells treated with 100 nmol·L–1 insulin (yellow arrow indicates nucleus, whereas yellow arrowhead indicates GLUT4 at the sarcolemmal membrane). (D) Laser scanning confocal microscopy showing lack of GLUT4 translocation to the sarcolemmal membrane in H9c2 cells treated with 10 mmol·L–1 phenformin (yellow arrow indicates nucleus, whereas yellow arrowhead indicates GLUT4 throughout the cytosol).

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Fig. 5. The potential mechanisms by which AMPK activation mediates glucose uptake in cardiac myocytes. The activation of AMPK may mediate glucose uptake in cardiac myocytes by activating either p38 MAPK or eNOS, which subsequently increases GLUT4 translocation to the sarcolemmal membrane. In addition, the activation of AMPK may facilitate glucose uptake in cardiac myocytes by preventing endocytosis of GLUT4 transporters already present at the sarcolemmal membrane. Finally, the activation of AMPK may affect GLUT1 to enhance glucose uptake in cardiac myocytes.

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are now insulin sensitive. Similar findings have been reported by Chang et al., as treatment of differentiated H9c2 myocytes resulted in a marked increase in Akt phosphorylation and subsequent glucose utilization (Chang et al. 2013). AMPK has been proposed to stimulate GLUT4 translocation to the sarcolemmal membrane via its actions on both p38 MAPK or eNOS (Li et al. 2004, 2005) (Fig. 5). However, utilizing laser scanning confocal microscopy, we show that AMPK activation in H9c2 cells does not cause GLUT4 translocation to the sarcolemmal membrane, suggesting that AMPK activation in the heart increases glucose uptake via some other mechanism. However, it is important to note that our confocal experiments are only looking at translocation of GLUT4 that is expressed from the infected adenovirus. It may be possible that AMPK activation is causing translocation of endogenous GLUT4 transporters to the sarcolemmal membrane, but we are unable to detect it. In addition, AMPK activation has also been shown by Holman and colleagues to prevent endocytosis of GLUT4 transporters already at the sarcolemmal membrane (Fig. 5), which may represent the mechanism by which AMPK increases glucose uptake in the heart (Yang and Holman 2006). Other studies have also reported that AMPK may increase glucose uptake through a GLUT1 mechanism (Barnes et al. 2002; Kim et al. 2007). To address this possibility, we are in the process of generating a GLUT1-dsRed adenovirus. It has been proposed that AMPK may also act as an insulin sensitizer with its ability to enhance insulin-stimulated glucose uptake arising from inhibition of the mTOR–p70S6K pathway and over-activation of AS160 (Alkhateeb et al. 2009; Tanti and Jager 2009). In contrast, Ginion and colleagues observed that AMPK activation could enhance insulin-stimulated glucose uptake in both insulin sensitive and insulin resistant adult cardiac myocytes independent of AS160 and the mTOR–p70S6K pathway, suggesting that unidentified mechanisms are responsible for this effect (Ginion et al. 2011). It has also been speculated that AMPK activation enhances insulin signaling by converging on pathways activated via Akt, and potentiating the effect on glucose uptake, although this is unlikely to be involved in our study, as treatment with phenformin reduced Akt phosphorylation in H9c2 myoblasts. Furthermore, Chang et al. (2013) have also shown that berberinemediated AMPK activation can improve glucose uptake in insulinresistant cardiac myocytes without affecting Akt activity. It should also be noted in our study that our assessment of AMPK status and activity has focused solely on modifications to the ␣-subunit, as AMPK's catalytic activity resides in this particular subunit (Dyck and Lopaschuk 2006). However, recent findings have also suggested that myristoylation of the ␤-subunit of AMPK plays an important role in AMPK activity, as preventing AMPK ␤-subunit myristoylation results in marked increases in AMPK catalytic activity (Warden et al. 2001; Steinberg and Kemp 2009). We plan to explore whether alterations in the ␤-subunit of AMPK are responsible for some of AMPK's metabolic effects on glucose uptake in the heart during ischemia and heart failure in future studies. Overall, utilizing a modified method for measuring glucose uptake that is more reflective of true glucose uptake at more appropriate substrate levels, we confirm the results of previous studies indicating that AMPK activation increases glucose uptake in the heart. However, we show that this effect of AMPK occurs independently of GLUT4 translocation. Whether this effect of AMPK on glucose uptake is indeed beneficial to the ischemic heart remains to be determined.

Acknowledgements The authors would like to thank Dr. Amira Klip for kindly providing the GLUT4 construct used to generate our GLUT4-dsRed adenovirus. The authors would also like to thank Cory S. Wagg for his technical assistance, and Jagdip S. Jaswal for the insightful discussions with regards to AMPK and glucose uptake.

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This study was supported by a grant from the Canadian Institutes of Health Research (CIHR) to G.D.L., who is an Alberta Heritage Foundation for Medical Research (AHFMR) Scientist. C.T.L. is a fellow of the AHFMR. J.R.U. is a fellow of Alberta Innovates – Health Solutions as well as the CIHR. Disclosures: The authors have no conflicts of interest to disclose in connection with this study.

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5'-AMP-activated protein kinase increases glucose uptake independent of GLUT4 translocation in cardiac myocytes.

Glucose uptake and glycolysis are increased in the heart during ischemia, and this metabolic alteration constitutes an important contributing factor t...
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