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Endocrinology, doi:10.1210/en.2006-0849
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Endocrinology Vol. 147, No. 11 5205-5216
Copyright © 2006 by The Endocrine Society

Arsenite Modulates Cardiac Substrate Preference by Translocation of GLUT4, But Not CD36, Independent of Mitogen-Activated Protein Kinase Signaling

Joost J. F. P. Luiken, Iman Momken, Daphna D. J. Habets, Mohammed El Hasnaoui, Will A. Coumans, Debby P. Y Koonen, Jan F. C. Glatz and Arend Bonen

Department of Molecular Genetics (J.J.F.P.L., D.D.J.H., M.E.H., W.A.C., D.P.Y.K., J.F.C.G), Cardiovascular Research Institute Maastricht, Maastricht University, NL-6200 MD Maastricht, The Netherlands; Department of Biochemical Physiology and Institute of Biomembranes (J.J.F.P.L.), Utrecht University, NL-3584 CH Utrecht, The Netherlands; and Department of Human Health and Nutritional Sciences (I.M., A.B.), University of Guelph, Guelph, Ontario, Canada N1G 2W1

Address all correspondence and requests for reprints to: Joost J. F. P. Luiken, Ph.D., Department of Molecular Genetics, Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands. E-mail: j.luiken{at}gen.unimaas.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The protein thiol-modifying agent arsenite, a potent activator of stress signaling, was used to examine the involvement of MAPKs in the regulation of cardiac substrate uptake. Arsenite strongly induced p38 MAPK phosphorylation in isolated rat cardiac myocytes but also moderately enhanced phosphorylation of p42/44 ERK and p70 S6K. At the level of cardiomyocytic substrate use, arsenite enhanced glucose uptake dose dependently up to 5.1-fold but failed to stimulate long-chain fatty acid uptake. At the substrate transporter level, arsenite stimulated the translocation of GLUT4 to the sarcolemma but failed to recruit CD36 or FABPpm. Because arsenite did not influence the intrinsic activity of glucose transporters, GLUT4 translocation is entirely responsible for the selective increase in glucose uptake by arsenite. Moreover, the nonadditivity of arsenite-induced glucose uptake and insulin-induced glucose uptake indicates that arsenite recruits GLUT4 from insulin-responsive intracellular stores. Inhibitor studies with SB203580/SB202190, PD98059, and rapamycin indicate that activation of p38 MAPK, p42/44 ERK, and p70 S6K, respectively, are not involved in arsenite-induced glucose uptake. In addition, all these kinases do not play a role in regulation of cardiac glucose and long-chain fatty acid uptake by insulin. Hence, arsenite’s selective stimulation of glucose uptake appears unrelated to its signaling actions, suggesting that arsenite acts via thiol modification of a putative intracellular protein target of arsenite within insulin-responsive GLUT4-containing stores. Because of arsenite’s selective stimulation of cardiac glucose uptake, identification of this putative target of arsenite within the GLUT4-storage compartment may indicate whether it is a target for future strategies in prevention of diabetic cardiomyopathy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE HEALTHY HEART, long-chain fatty acids (LCFAs) and glucose contribute 70 and 20%, respectively, to cardiac energy production (1). During development of cardiac hypertrophy due to pressure overload, there is a gradual decrease in fatty acid use, which is partly compensated for by an increase in glucose use (2). Conversely, during the development of diabetic cardiomyopathy, a reciprocal shift toward increased use of LCFAs has been observed (3). These shifts in substrate preference, toward either glucose use or fatty acid use, are accompanied by impaired cardiac functioning (2, 4). Hence, optimal cardiac performance appears dependent on maintaining the ratio between glucose use and LCFA use within a certain optimal range. Furthermore, modulation of cardiac substrate preference aimed at normalizing the balance between use of glucose and LCFAs might counteract the development and/or regress cardiac failure.

GLUT4 is the major cardiac glucose transporter, whereas the bulk of LCFA uptake is mediated by the concerted action of two structurally unrelated proteins, i.e. an 88-kDa heavily glycosylated fatty acid translocase, referred to as CD36 (5) and a 43-kDa plasma membrane fatty acid binding protein (FABPpm) (6). Both proteins are involved in LCFA uptake and are presumed to make up a functional transport system in which FABPpm is suggested to operate as a receptor for LCFA, and CD36 to operate as a flippase (7). In cardiac myocytes, uptake rates of glucose and LCFA are similarly regulated by inducing the translocation of GLUT4 and CD36, respectively, from intracellular stores to the sarcolemma. Insulin and contractions are two major physiological stimuli able to induce translocation of both transporters and hence enhance the uptake of both substrates (8). The similarity between regulation of glucose uptake and LCFA uptake also extends to the signaling pathways involved. Insulin-induced translocation of both GLUT4 and CD36 has been found to be dependent on activation of phosphatidylinositol-3 kinase (PI3K), whereas AMP-activated protein kinase (AMPK) activation is necessary for contraction-induced translocation of both transporters (8).

Whether GLUT4 and CD36 reside in the same intracellular storage compartment has not yet been determined. However, it appears to be possible to uncouple the simultaneous translocation of both transporters from each other. Notably, the cardiotonic agent dipyridamole induced the translocation of CD36 to the sarcolemma of cardiac myocytes without affecting subcellular localization of GLUT4, resulting in a selective increase in LCFA uptake (9). Dipyridamole exerts this effect via a yet-unidentified intracellular target, which is situated in the contraction signaling cascade downstream of AMPK. This finding may suggest that CD36 and GLUT4 are stored in separate intracellular pools or that, alternatively, both transporters reside within the same compartment but are recruited via distinct sorting mechanisms. In theory, dipyridamole, because of its ability to shift cardiac substrate use toward LCFAs, could be used to improve cardiac functioning during high pressure-induced cardiac hypertrophy. Conversely, a strategy to induce the translocation of GLUT4 without moving CD36 should selectively stimulate glucose uptake and thereby have antidiabetic potential.

Arsenite is recognized to stimulate glucose uptake in a number of mammalian cell types (10, 11). However, its effect on LCFA uptake is completely unknown. There is evidence, in adipocytes (11) and skeletal muscle cells (10), that the increase in glucose uptake by arsenite involves a stimulation of the intrinsic activity of glucose transporters, eventually in combination with a translocation of GLUT4 to the plasma membrane.

Besides activating glucose uptake, arsenite is also a potent activator of certain signaling kinases. It is known to activate p38 MAPK (12) and P70 S6 kinase (S6K) (13) in cardiac myocytes. The mechanism of action likely includes modification of vicinal sulfhydryl moieties in a number of limited arsenite-sensitive target proteins (14). The stimulatory effect of arsenite on P38 and on c-JUN NH2-terminal protein kinase, another related stress kinase, is probably due to modulating the activity of an unknown target protein (15), whereas arsenite’s effect on activation of P70 S6K is incompletely resolved (13). It has been speculated that P38 MAPK could be implicated in the stimulation of glucose uptake into adipocytes based on the partial inhibition of arsenite-induced glucose uptake by the specific MAPK inhibitor SB203580 (11, 16).

In the heart, the roles of p38 MAPK and related ERKs in substrate uptake have scarcely been explored. Interestingly, recent observations pinpoint p38 downstream of AMPK upon low flow- (17) or chemically induced ischemia (18) in the heart. Also p42/44 ERK has been positioned downstream of AMPK in contracting muscle preparations (19). Combined with the above-mentioned findings in adipocytes, a complex picture emerges in the way MAPKs are involved in cellular substrate use. Nevertheless, elucidation of the role of MAPKs in substrate use by the heart is important when considered that both p38 and ERK are involved in the regulation of cardiac contractility (20, 21).

In the present study, we used the ability of arsenite to stimulate MAPKs to assess the roles of these kinases in the regulation of substrate uptake by cardiac myocytes. In addition, we also examined the potential induction of MAPKs by insulin and the contraction-mimetic agent oligomycin. In contrast to many studies that have focused solely on the link between signaling and cardiac glucose uptake (e.g. Refs. 10, 11, 16), we investigated the uptake of both glucose and LCFAs. Notably, the comparison of the effects of arsenite on the regulation of GLUT4 translocation, compared with CD36 recycling, can shed new light on the role of MAPKs in cardiac substrate use. To couple signaling to glucose and LCFA uptake, we tested the ability of specific MAPK inhibitors to influence the uptake of both substrates into cardiac myocytes in the absence and presence of arsenite, insulin, and oligomycin. Collectively, the findings indicate that arsenite specifically stimulates glucose uptake by recruiting GLUT4 from the insulin responsive nonendosomal GLUT4-specific storage compartment. In addition, neither MAPKs nor S6Ks play a role in regulation of uptake of glucose and of LCFA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[1-14C]palmitic acid and [3H]deoxyglucose (fraction V) were obtained from Amersham Life Science Ltd. (Little Chalfont, UK). BSA, collagenase type VII, insulin, oligomycin, sodium arsenite, wortmannin, SB202190, SB203580, PD98059, and rapamycin were purchased from Sigma (St. Louis, MO). Collagenase type II was from Worthington (Freehold, NJ). Nonfat dry milk (Marvel) was obtained from Premier Brands (Moreton, UK). Western blot reagents were from Bio-Rad Laboratories (Hercules, CA) and the enhanced chemiluminescence kit was from Amersham Pharmacia Biotech (Buckingham, UK). CD36 was detected with a monoclonal antibody (MO25) directed against human CD36, kindly provided by Dr. N. Tandon (Otsuka Pharmaceuticals, Bethesda, MD). A rabbit polyclonal against rat hepatic membrane fatty acid binding protein was used to detect FABPpm (a gift from Dr. J Calles-Escandon, Section of Endocrinology and Metabolism, Wake Forest University School of Medicine and Baptist Medical Center, Winston-Salem, NC.). Antibodies directed against GLUT4 were obtained from Sanver Tech (Heerhugowaard, The Netherlands). Phospho-specific antibodies against signaling enzymes were from Cell Signaling (Danvers, MA). Rabbit antimouse immunoglobulin horseradish peroxidase and pork antirabbit immunoglobulin horseradish peroxidase were obtained from DAKO (Glostrup, Denmark).

Isolation of cardiac myocytes
Cardiac myocytes were isolated from male Lewis rats (200–250 g) using a Langendorff perfusion system and a Krebs-Henseleit bicarbonate medium supplemented with 11 mM glucose and equilibrated with a 95% O2-5% CO2 gas phase (medium A) at 37 C as previously described (22). After isolation, the cells were washed twice with medium A supplemented with 1.0 mM CaCl2 and 2% (wt/vol) BSA (medium B) and then suspended in 15 ml of medium B. The isolated cells were allowed to recover for approximately 2 h at room temperature. At the end of the recovery period, cells were washed and suspended in medium B. Only when more than 80% of the cells had a rod-shaped appearance and excluded trypan blue were they used for subsequent tracer uptake studies.

Substrate uptake by cardiac myocytes
Cells (2.0 ml; 5–8 mg wet mass per milliliter), suspended in medium B without glucose, were preincubated in capped 20-ml incubation vials for 15 min at 37 C under continuous shaking. To study palmitate uptake, 0.5 ml of the [1-14C]palmitate/BSA complex was added at the start of the incubations so that the final concentration of palmitate amounted to 100 µM with a corresponding palmitate/BSA ratio of 0.3. This palmitate/BSA complex was prepared as previously described (22). To study deoxyglucose uptake, [3H]deoxyglucose was added at the start of the incubations in 0.6 ml medium B without glucose to a final concentration of 100 µM. Cellular uptake of palmitate (3 min incubation) and deoxyglucose (3 min incubation) were determined upon washing the cells three times for 2 min at 100 x g in an ice-cold stop solution containing 0.2 mM phloretin as previously described (22). The washing procedure did not affect cellular integrity as evaluated by microscopical inspection.

Substrate uptake was stimulated by sodium arsenite (<0.3 mM), insulin (10 nM), and oligomycin (30 µM). These stimuli were added to the cell incubations 15 min before addition of radiolabeled substrate. A stock solution of oligomycin was prepared in dimethylsulfoxide, which never exceeded a final concentration of 0.5% in the cell suspensions. At this concentration, dimethylsulfoxide did not affect cellular substrate use.

Measurement of activation of signaling kinases
Cardiac myocytes (8–12 mg wet mass per milliliter) were incubated in medium B in the absence and presence of additions for 15 min. At the end of the incubation, cells were pulse centrifuged in an Eppendorf microfuge and then immediately dissolved in sample buffer containing 15 mM Tris-HCl (pH 6.8), 0.5 mM EDTA, 5 mM dithiothreitol, and 2% (wt/vol) sodium dodecyl sulfate and used for SDS-PAGE. Activation of p38 MAPK, p42/44 ERK, p70 S6K, Akt/protein kinase (PKB), AMPK, and acetyl-CoA carboxylase (ACC) was measured with phospho-specific monoclonal antibodies (Cell Signaling Technology). For this purpose, antibodies against phospho-p38 MAPK (Thr180/Tyr182), phospho-p42/44 ERK (Thr202/Tyr204), phospho-p70 S6K (Thr389), phospho-Akt (Ser473), phospho-AMPK{alpha} (Thr172), and phospho-ACC were used according to the manufacturer’s instructions.

To link activation of these kinases to their effects on metabolism, a variety of signaling inhibitors was used, i.e. the p38 MAPK inhibitors SB203580 and SB202190, the p42/44 ERK inhibitor PD98059, the p70 S6K inhibitor rapamycin, and the PI3K inhibitor wortmannin (200 nM). But before their use in substrate uptake studies, these inhibitors were tested whether they exerted their desired inhibitory effect on signaling in cardiac myocytes and whether this inhibitory effect was specific for one protein kinase.

All inhibitors were added at the lowest concentration at which they exerted their maximal effect. None of these agents, alone or in combination with pharmacological stimuli, was found to affect the percentage of cells that were rod shaped and excluded trypan blue, as parameters of cellular integrity.

At 10 µM, SB202190 largely inhibited arsenite-stimulated p38 MAPK phosphorylation but did not affect phenylephrine-induced p42/44 ERK phosphorylation, arsenite-induced p70 S6K phosphorylation, insulin-induced Akt/PKB phosphorylation, or oligomycin-induced ACC phosphorylation (Fig. 1Go). SB203580, at the same concentration used as SB202190, was similarly potent in blocking p38 MAPK phosphorylation (data not shown). PD98059 (10 µM) completely inhibited phenylephrine-induced p42/44 ERK phosphorylation but did not inhibit arsenite-induced p38 MAPK or p70 S6K phosphorylation, insulin-induced Akt/PKB phosphorylation, or oligomycin-induced ACC phosphorylation (Fig. 1Go). Rapamycin (20 nM) solely inhibited arsenite-induced S6K phosphorylation without altering phosphorylation of the other kinases on stimulation (Fig. 1Go). Finally, wortmannin (200 nM) completely blocked insulin-stimulated Akt/PKB- and arsenite-induced p70 S6K phosphorylation and left phenylephrine-induced p42/44 ERK- and oligomycin-induced ACC phosphorylation unaltered (Fig. 1Go). Because Akt/PKB and p70 S6K are recognized as protein kinases downstream of PI3K, this inhibitory action of wortmannin on both kinases is not surprising. However, when considered that p70 S6K can also be activated in a PI3K-independent manner, the observation that arsenite-stimulated S6K phosphorylation is wortmannin sensitive is a novel side observation. Taken together, at the concentrations used, all inhibitors exerted their claimed specific effects on signaling in cardiac myocyte incubations.


Figure 1
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  		FIG. 1. Verification of effectiveness and specificity of the applied signaling inhibitors on inhibition of protein kinase phosphorylation in cardiac myocytes. Cell suspensions were preincubated for 20 min with signaling inhibitors [10 µM SB202190 (SB); 10 µM PD98059 (PD); 20 nM rapamycin (Rp); or 200 nM wortmannin (Wrt)] and subsequently incubated for 20 min with metabolic stimuli (100 µM arsenite, 50 µM phenylephrine, 100 nM insulin, or 5 µM oligomycin). To stop the incubations, sample buffer was added and samples were used for gel electrophoresis and Western blotting for the detection of diphospho-p38 MAPK, diphospho-p42/44 ERK, phospho-p70 S6K, phospho-p60 Akt/PKB, or phospho-p280 ACC as detailed in Materials and Methods. A representative Western blot is shown of three experiments carried out with different cardiomyocyte preparations.

 
Subcellular fractionation of cardiac myocytes
Cardiac myocytes (2.25 ml; 20–25 mg wet mass per milliliter) were incubated for 15 min in medium B in the absence and presence of additions. At the end of the incubation, the total volume of cell incubations was quickly transferred to a tightly fitting 5-ml Potter-Elvejhem glass homogenizer on ice containing 1 ml cold H2O, after which NaN3 was added to a final concentration of 5 mM to stop ATP-dependent vesicular trafficking events. Immediately hereafter cell suspensions were homogenized with 10 strokes. Subsequently fractionation was carried out as described previously (23, 24). For determination of the GLUT4, CD36, and FABPpm content in the plasma membrane (PM) and low-density microsomes (LDMs), aliquots of the membrane fractions were separated with SDS-PAGE and Western blotting as previously described (7, 24, 25).

The purity of the fractions obtained by this fractionation procedure was previously checked (23). Specifically the PM fraction is 13.5-fold enriched with ouabain-sensitive p-nitrophenyl-phosphatase, whereas the specific activity of the sarcoplasmatic EGTA-sensitive Ca2+-ATPase was 3.6-fold decreased. In addition, no activity of p-nitrophenyl-phosphatase or of Ca2+-ATPase could be detected in the LDM fraction, indicating that this fraction was devoid of PM and sarcoplasmic reticulum. Furthermore, caveolin-3 was found to be 2.9-fold more abundant in the PM fraction than the LDM fraction (data not shown).

Isolation of giant vesicles
Giant vesicles were prepared from rat heart, as described previously (7). In general, tissues were cut into thin layers (~1–3 mm thick) and incubated in vesicle preparation medium [140 mM KCl per 10 mM 3[N-morpholino]propanesulfonic acid (MOPS) (pH 7.4)] supplemented with aprotinin (10 mg/ml), 0.8 mM CaCl2, and collagenase type II [0.3% (wt/vol)] for 1 h at 34 C in a shaking waterbath. After incubation, tissues were washed with KCl/MOPS and 10 mM EDTA. The supernatant was collected, and Percoll [final concentration of 16% (vol/vol)] and aprotinin (1.0 mg/ml) were added. The resulting suspension was placed at the bottom of a density gradient consisting of a 3-ml middle layer of 4% Nycodenz (wt/vol) and a 1-ml KCl/MOPS upper layer and centrifuged at 60 x g for 45 min at room temperature. Subsequently the vesicles were harvested from the interface of the upper and middle layer, diluted in KCl/MOPS, recentrifuged at 13,000 x g for 4 min, and finally resuspended in KCl/MOPS. For each assay, a protein concentration of 0.25–0.60 mg/ml was used. Vesicles were immediately used for glucose and LCFA transport experiments.

Substrate uptake by giant vesicles
Palmitate uptake was measured by addition of unlabeled and radiolabeled 0.3 µCi [9,10-3H]palmitate and 0.06 µCi [14C]mannitol in a 0.1% BSA KCl/MOPS solution to 40 µl of vesicles (~80 µg protein). The reaction was carried out at room temperature for precisely 15 sec. Palmitate uptake was terminated by addition of 1.4 ml of ice-cold KCl/MOPS-2.5 mM HgCl and 0.1% BSA. The sample was quickly recentrifuged at maximal speed in a microcentrifuge for 2 min. The supernatant was discarded, and radioactivity was measured in the tip of the tube. Nonspecific uptake was measured by adding the stop solution before the addition of the radiolabeled palmitate solution.

Glucose uptake studies were carried out by addition of 40 µl 0.1% BSA in KCl/MOPS containing 0.3 µCi [3H]D-glucose (200 µM) and 0.06 µCi [14C]mannitol. Incubations were carried out at room temperature for 1 min. The incubation was ended and radioactivity was determined as described above.

Arsenite and signaling inhibitors were added to giant vesicles 20 min before determination of palmitate or glucose uptake at the same concentrations as applied for cardiac myocyte studies (see above).

Other procedures
Cellular wet mass was obtained from cell samples taken during the incubation period and determined after centrifugation for 2–3 sec at maximal speed in a microcentrifuge and subsequent removal of the supernatant. Protein was quantified with the Bicinchichonic acid protein assay (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Statistics
All data are reported as mean ± SEM. Statistical difference between groups was tested with a Student’s t test. However, when the data failed to meet a test of normal distribution, we necessarily used the Mann-Whitney U test. P ≤ 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of arsenite on glucose and LCFA uptake into cardiac myocytes
Arsenite has been previously shown to stimulate glucose uptake into myocytes (10) and adipocytes (11), but it has never been used to study cellular LCFA uptake. Using cardiac myocytes, arsenite stimulated deoxyglucose uptake in a dose-dependent manner up to 5.1-fold at 100 µM, without a further increase at 300 µM (Fig. 2Go). In contrast, arsenite did not increase LCFA uptake, even at concentrations up to 300 µM (Fig. 2Go). Therefore, we chose a concentration of 100 µM to study arsenite’s effects on cardiac glucose and fatty acid use and signaling.


Figure 2
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  		FIG. 2. Dose-dependent effects of arsenite on glucose and LCFA uptake into cardiac myocytes. Cell suspensions were incubated for 20 min with various concentrations of arsenite and subsequently used for measurement of uptake of palmitate and 2-deoxy-D-glucose. Data are means ± SEM of three experiments carried out with different cardiomyocyte preparations. {square}, Deoxyglucose uptake; {diamondsuit}, palmitate uptake.

 
We previously demonstrated that contractile activity and insulin are two major physiological stimuli enhancing uptake of both glucose and LCFAs (24, 26). Oligomycin, an inhibitor of the mitochondrial proton pump, was able to mimic the contraction-induced increase in substrate use(26). In the present study, glucose uptake was increased on addition of insulin (10 nM) and oligomycin (5 µM) by 7.2- and 2.9-fold, respectively (Figs. 2Go and 3Go). More importantly, arsenite retained its stimulatory action on glucose uptake when added in combination with oligomycin, However, arsenite was ineffective on glucose uptake in insulin-stimulated cardiac myocytes (Fig. 3Go). Hence, the effect of arsenite on glucose uptake was additive to that of oligomycin but nonadditive to that of insulin. LCFA uptake was increased in the presence of insulin and oligomycin by 1.5- and 2.0-fold, respectively. In combination with insulin or oligomycin, arsenite did not significantly alter LCFA uptake (Fig. 3Go).


Figure 3
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  		FIG. 3. Effects of arsenite on glucose and LCFA uptake into cardiac myocytes: comparison with insulin and oligomycin. Cell suspensions were incubated in the absence of additions (basal) or presence of 100 µM arsenite, 100 nM insulin, 5 µM oligomycin (Oli), or combinations of these stimuli for 20 min before execution of 2-deoxy-D-glucose uptake studies (3 min; left panel) or palmitate uptake studies (3 min; right panel). Gray bars, Incubations in absence of arsenite; black bars, incubations in the presence of arsenite. Data are means ± SEM of four to six experiments carried out with different cardiomyocyte preparations. *, Significantly different from myocytes incubated without any additions (basal/none) (P < 0.05). **, Significantly different from corresponding myocytes incubated in the absence of arsenite (none) (P < 0.05).

 
Effects of blocking of signaling pathways on the uptake of glucose and LCFAs
To dissect the signaling mechanisms involved in the selective stimulatory effect of arsenite on glucose uptake, various signaling inhibitors were used at their optimal concentrations (see Materials and Methods), i.e. the p38 MAPK inhibitors SB203580 (10 µM) and SB202190 (10 µM), the p42/44 ERK inhibitor PD98059 (10 µM), the p70 S6K inhibitor rapamycin (100 nM), and the PI3K inhibitor wortmannin (200 nM). PD98059 and rapamycin had no effect on basal glucose uptake. Similarly, neither PD98059 nor rapamycin affected glucose uptake in the presence of any other stimuli (Fig. 4Go). In contrast, basal glucose uptake was, however, markedly inhibited by SB202190 (–45%) (Fig. 4Go). Similarly, SB202190 also inhibited glucose uptake to a comparable extent in the presence of either insulin (–33%) or arsenite (–42%). The inhibitor SB203580 also inhibited basal glucose uptake (–56%) and glucose uptake in the presence of insulin (–46%) or arsenite (–57%), effects that were similar to those of SB202190. It was only when the PI3K inhibitor wortmannin was used that the differing effects of insulin and arsenite on glucose uptake became evident. Whereas wortmannin did not inhibit basal glucose transport, insulin-stimulated glucose uptake was largely blocked by wortmannin. Remarkably wortmannin completely failed to inhibit the arsenite-stimulated glucose uptake.


Figure 4
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  		FIG. 4. Effects of signaling inhibitors on arsenite (Ars)- and insulin (Ins)-induced glucose and LCFA uptake. Cell suspensions were preincubated for 20 min with signaling inhibitors [10 µM SB202190 (SB); 10 µM PD98059 (PD); 20 nM rapamycin (Rapa); or 200 nM wortmannin (Wort)] and subsequently incubated for 20 min without (referred to as basal; the two upper panels) or with 100 nM insulin (the two middle panels) or 100 µM arsenite (the two lower panels). Immediately thereafter cells were subjected to 2-deoxy-D-glucose uptake studies (3 min; the three left panels) or palmitate uptake studies (3 min; the three right panels). The two inserts represent the effects of arsenite or insulin on glucose uptake (right inset) and LCFA uptake (left inset) in the absence of inhibitors. The data in the insets are from the same experiments as indicated under None. Data are means ± SEM of three to four experiments carried out with different cardiomyocyte preparations. *, Significantly different from myocytes without additions (none) (P < 0.05).

 
With respect to LCFA uptake, SB202190, PD98059 or rapamycin was not able to influence the uptake of this substrate into cardiac myocytes, whether in the absence or presence of insulin or arsenite (Fig. 4Go). However, wortmannin, although having no effect on basal LCFA uptake, completely blocked insulin-induced LCFA uptake, in agreement with our earlier observations (24, 25).

Effects of arsenite on intrinsic activity of transporters in giant vesicles
It is generally accepted the increase in glucose uptake into myocytes and adipocytes in the presence of physiological stimuli can largely be explained by a translocation of GLUT1 and/or GLUT4 from intracellular stores to the plasma membrane. An additional mechanism by which glucose uptake can be increased, includes an increase in the intrinsic activity of GLUT1 and/or GLUT4, as reported by Klip and coworkers (16, 27). To explore the possibility that arsenite stimulates glucose uptake through activation of transporters already located at the plasma membrane, giant vesicles were used for measurement of glucose uptake. These vesicles are formed by budding of the cardiomyocytic sarcolemma during incubation of cardiac tissue in KCl-containing buffers. During this budding process, cytoplasmic areas become sequestered within these giant vesicles. However, intracellular organelles such as the endosomal recycling compartment are absent (28) so that translocation of transporters cannot occur. Hence, during incubation of giant vesicles with selected stimuli, the number of transporters present at the vesicular membrane remains fixed, and therefore, alterations in transport rates can be due only to changes in the activities of transporters. Previously we found that a pharmacological stimulus (i.e. isobutyl-methylxanthine) is able to increase substrate transporter activity in giant vesicles (28). In the present study, arsenite did not influence either glucose uptake or LCFA uptake, thereby indicating that arsenite does not interact directly with GLUT4 or any LCFA transporters (Fig. 5Go).


Figure 5
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  		FIG. 5. Effects of arsenite (Ars) on intrinsic activity of substrate transporters in heart-derived giant vesicles. Giant vesicle suspensions were incubated with arsenite or signaling inhibitors (10 µM SB202190, 10 µM PD98059, 20 nM rapamycin, or 200 nM wortmannin) before execution of glucose uptake studies (2 min; upper panel) or palmitate uptake studies (15 sec; lower panel). Data are means ± SEM of three to four experiments carried out with different cardiomyocyte preparations. *, Significantly different from myocytes without additions (basal) (P < 0.05).

 
When dissecting the substrate uptake-related effects of the signaling inhibitors in giant vesicles, it was observed that none of these inhibitors affected LCFA uptake. Similarly, PD98059, rapamycin, and wortmannin also did not alter glucose uptake into giant vesicles. However, SB202190 (–27%) inhibited glucose uptake into giant vesicles (Fig. 5Go) to a similar extent as was observed in cardiac myocytes (–33%) (Fig. 4Go). Also, SB203580 inhibited glucose uptake into giant vesicles (–46%) to the same extent as in cardiac myocytes (–56%). Hence, these latter compounds directly inhibit glucose transporter activity at the plasma membrane.

Effects of arsenite on the translocation of GLUT4 and LCFA transporters
Because arsenite does not influence glucose transporter activity at the sarcolemma, an alternative explanation for the stimulation of glucose uptake by arsenite involves the translocation of GLUT4 to the sarcolemma. To investigate this notion, cardiac myocytes were incubated with arsenite for 20 min before subcellular fractionation. As a positive control for the fractionation procedure, parallel myocyte incubations were performed with insulin for 20 min. Indeed insulin decreased the contents of GLUT4 and CD36 in the LDM fraction by 41 and by 42%, respectively, and simultaneously increased the contents of both transporters in the PM fraction by 1.9- and 1.5-fold, respectively (Fig. 6Go). In contrast, insulin did not affect the distribution of FABPpm between the LDM and PM fraction. Because the insulin-induced translocation of GLUT4 is among the best-established intracellular trafficking events, our fractionation procedure is suitable for measuring stimulus-induced translocation processes. The insulin-induced translocation of CD36 and the inability of insulin to influence the subcellular recycling of FABPpm are in agreement with our previous findings (24, 29). In agreement with insulin’s effects on GLUT4 distribution, arsenite decreased the content of GLUT4 in the LDM fraction by 44%, and concomitantly increased its content in the PM fraction by 1.7-fold (Fig. 6Go). SB202190 did not affect this arsenite-induced GLUT4 translocation (Fig. 6Go, insert). With respect to LCFA transporter distribution, arsenite did not significantly influence the contents of CD36 and FABPpm in the LDM and PM fractions. In addition, the inclusion of SB202190 did not influence the subcellular distribution of both LCFA transporters (Fig. 6Go, inserts). When compared with insulin’s effects on transporter recycling, arsenite is almost as potent in inducing the translocation of GLUT4, but in contrast, arsenite completely fails to have any stimulatory effect on inducing the translocation of CD36.


Figure 6
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  		FIG. 6. Effects of arsenite on translocation of GLUT4, CD36, and FABPpm: comparison with effects of insulin. Cell suspensions were incubated for 20 min in the absence (basal) and presence of 100 nM insulin or 100 µM arsenite, after which NaN3 was added to stop ATP-demanding processes. Immediately thereafter cells were frozen in liquid nitrogen and, on thawing, subjected to subcellular fractionation. The collected fractions were analyzed on the relative contents of GLUT4 (45 kDa), CD36 (88 kDa), and FABPpm (43 kDa). Transporter content was expressed as multiple of control (basal; white bars) in the corresponding fraction. Data are means ± SEM of six experiments carried out with different cardiomyocyte preparations. A second set of experiments was performed to investigate the influence of SB202190 on the arsenite-induced changes in transporter recycling; 10 µM SB202190 was added to cell suspensions 20 min before addition of arsenite. Transporter content was expressed as multiple of values obtained in cell incubations with arsenite (black bars; see inserts). Data are means ± SEM of four experiments carried out with different cardiomyocyte preparations. SB, SB202190. Representative Western blots are shown. *, Significantly different from myocytes in the absence of additions (basal) (P < 0.05).

 
Effects of arsenite on cardiac signaling
Arsenite is considered a useful tool to investigate whether there is a relation between MAPK signaling and substrate use (11). The ability of arsenite to stimulate MAPK and other key signaling kinases was compared with that of insulin and oligomycin, two stimuli with profound effects on cardiac substrate uptake. Our focus was on: 1) the phosphorylation of MAPKs and S6K, known to be stimulated by arsenite (13); 2) phosphorylation of Akt/PKB, a hallmark for insulin signaling; and 3) phosphorylation of AMPK and ACC, key protein kinases in contraction signaling. As expected, incubation of cardiac myocytes with insulin during 20 min resulted in a large (>10-fold) increase in phosphorylation of Akt/PKB and a marked (2.8 ± 0.4-fold) increase in p70 S6K phosphorylation (Fig. 7Go). In addition, there was also a modest (1.8 ± 0.3-fold) increase in p42/44 ERK phosphorylation, which has also been previously reported in cardiac myocytes (30). Insulin, as expected, had no effect on the phosphorylation of AMPK and ACC. In contrast, incubation of cardiac myocytes with oligomycin for 20 min strongly induced the phosphorylation of AMPK and ACC (both > 10-fold). Concomitantly, there was also a markedly enhanced phosphorylation of p38 MAPK (5.7 ± 1.5-fold), but oligomycin did not increase the phosphorylation state of p42/44 ERK and Akt/PKB (Fig. 7Go).


Figure 7
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  		FIG. 7. Effects of arsenite on activation of signaling enzymes: comparison with insulin and oligomycin. Cell suspensions were incubated for 20 min with 100 nM insulin (Ins), 5 µM oligomycin (Oli), or 100 µM arsenite (Ars). At the stop of the incubations, sample buffer was added, and samples were used for gel electrophoresis and Western blotting for the detection of diphospho-p38 MAPK, diphospho-p42/44 ERK, phospho-p70 S6K, phospho-p60 Akt/PKB, or phospho-p280 ACC as detailed in Materials and Methods. A representative Western blot is shown of three to six experiments carried out with different cardiomyocyte preparations. C, Control.

 
The incubation of cardiac myocytes with arsenite for 20 min led to a potent induction of p38 MAPK phosphorylation (7.8 ± 1.8-fold; Fig. 7Go), a well-known effect of arsenite. However, p70 S6K phosphorylation was also appreciable (3.3 ± 0.7-fold). This effect had also been observed previously in cardiac myocytes (13). Finally, arsenite also induced mildly the phosphorylation of p42/44 ERK (2.3 ± 0.5-fold), whereas the phosphorylation of AMPK and ACC remained unaltered.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main purpose of this study was to assess the role of MAPKs in the regulation of cardiac glucose and LCFA uptake using arsenite as a tool to stimulate cardiac signaling. The following novel findings emerged from arsenite-treated cardiac myocytes in relation to signaling events and substrate uptake. First, arsenite stimulates glucose uptake into cardiac myocytes without affecting LCFA uptake. As a mechanism for this selective stimulation of glucose uptake, the present findings indicate that arsenite appears to recruit GLUT4 directly from the nonendosomal GLUT4-specific storage compartment. Second, the insulin-mimetic action of arsenite on glucose uptake appeared independent of its activation of p38 MAPK because insulin did not activate p38. Third, arsenite-induced signaling is likely not involved in arsenite’s selective stimulation of glucose uptake. Besides these main findings, other novel observations were made in the course of this study. Fourth, the specific p38 MAPK inhibitors SB203580 and SB202190 cannot be used to study the relation between MAPK signaling and glucose uptake due to direct interaction of these two p38 MAPK inhibitors with cell surface cardiac GLUTs. And finally, the signaling enzymes p38 MAPK, p42/44 ERK, and p70 S6K are involved in neither the short-term regulation of glucose uptake/GLUT4 translocation nor LCFA uptake/CD36 translocation by insulin.

Arsenite and subcellular recycling of cardiac substrate transporters
Arsenite appears the first compound characterized to be able to induce GLUT4 translocation without altering the subcellular localization of CD36. Previously we established in heart and skeletal muscle that many stimuli (e.g. insulin, electrical stimulation, oligomycin, and 5-amino-imidazole-4-carboxamide-1-ß-D-ribofuranoside) are able to induce GLUT4 translocation as well as increase CD36 translocation, leading to a general increase in substrate uptake in these tissues (8). Whereas these observations suggest that GLUT4 translocation and CD36 translocation appear to occur concomitantly, further studies, however, demonstrated that the coupling between GLUT4 translocation and CD36 translocation appears to be less coordinated. Recently selective translocation of CD36 was observed in the presence of the cardiotonic agent dipyridamole without alteration in the subcellular distribution of GLUT4, resulting in a selective increase in cardiac LCFA uptake (9). In the present study, selective GLUT4 translocation was observed in the presence of arsenite without alteration in the subcellular distribution of CD36. As a result, it is possible to modulate cardiac substrate preference via selective recruitment of substrate transporters.

Mechanism by which arsenite selectively stimulates glucose uptake
Arsenite has been previously shown to stimulate glucose uptake and GLUT4 translocation in adipocytes and skeletal muscle cell lines (10, 11). However, its selective action on glucose uptake but not on LCFA uptake, as observed in the present study, is a novel observation. Moreover, because arsenite did not alter the intrinsic activity of glucose transporters (Fig. 5Go), GLUT4 translocation (Fig. 6Go) must be fully responsible for arsenite’s selective stimulation of glucose uptake.

The arsenite-stimulated glucose uptake was completely additive to oligomycin-stimulated glucose uptake. This indicated that arsenite does not recruit GLUT4 from the intracellular contraction-responsive compartment(s). Because AMPK activation by electrical stimulation or oligomycin has also been found to induce CD36 translocation and LCFA uptake (26), activation of the contraction/AMPK-sensitive signaling pathway does not confer specific substrate selection for either glucose or LCFAs. As further support for this, we observed that, in contrast to the marked oligomycin-induced phosphorylation of AMPK and ACC, indeed, arsenite did not increase the phosphorylation of these two protein kinases, as was observed with oligomycin.

As opposed to arsenite’s fully retained potential to stimulate glucose uptake in oligomycin-treated cardiac myocytes, arsenite failed to stimulate glucose uptake further in insulin-stimulated cardiac myocytes. This suggests that arsenite mobilizes GLUT4 from the insulin-responsive stores and not from intracellular contraction-responsive compartments.

Signal transduction pathways involved in arsenite-stimulated glucose uptake
Because the kinetic evidence (i.e. nonadditivity of insulin-stimulated glucose uptake with arsenite-stimulated glucose uptake) indicates that arsenite interacts with the insulin-stimulated GLUT4 translocation process, we investigated the ability of arsenite to activate signaling enzymes that have been reported to be activated by insulin.

Akt/PKB.
The phosphorylation of Akt/PKB is a classical insulin response and is firmly established to be involved in GLUT4 translocation (31). Our evidence also implicates the PI3K-Akt/PKB axis to be responsible for CD36 translocation (8). Thus, this PI3K-Akt/PKB signaling pathway, just as the AMPK pathway, is not solely involved in the preferential uptake of glucose. Moreover, arsenite did not stimulate Akt/PKB phosphorylation, and inhibition of the PI3K-Akt/PKB axis by wortmannin (see Fig. 1Go) did not abrogate the arsenite-stimulated glucose uptake (Fig. 4Go). Altogether, these observations firmly exclude Akt/PKB to be involved in arsenite-stimulated GLUT4 translocation.

P38 MAPK.
Phosphorylation of 38 MAPK is the classical signaling response to arsenite exposure and has also been implicated in insulin-stimulated glucose uptake. Notably, Klip and coworkers (16) showed that insulin stimulated both Akt/PKB and p38 MAPK activation in adipocytes. The activation of both kinases was shown to occur by independent pathways, and they contributed independently to the large increase in insulin-stimulated glucose uptake in adipocytes (16, 27). Specifically Akt/PKB activation is causally related to GLUT4 translocation, and p38 MAPK activation is responsible for an increase in the intrinsic activity of GLUT4. However, whether this activation of GLUT4 involves a phosphorylation or another kind of protein modification has not yet been established. A large pillar in this hypothesis of dual activation of glucose transport was the ability of the widely used p38 MAPK inhibitor SB203580 to partly block insulin-stimulated glucose uptake. Because this compound does not affect insulin-induced GLUT4 translocation, it was, by default, expected to block GLUT4 activation by insulin (16, 27). However, the present study shows that insulin does not stimulate p38 MAPK phosphorylation in cardiac myocytes. This inability of insulin to activate cardiac p38 MAPK cannot be due to defective MAPK signaling because other stimuli such as arsenite and oligomycin potently stimulate phosphorylation of this serine/threonine kinase (Fig. 7Go). The discrepancy between insulin-induced p38 MAPK activation in adipocytes, as observed by Klip and coworkers (16) and also others (32), on one hand, and the lack of p38 MAPK phosphorylation on insulin exposure to cardiac myocytes, as observed in the present study, on the other hand, might be attributed to a tissue-specific action of insulin, although the ability of insulin to stimulate p38 MAPK in adipocytes has now also been disputed recently by others (33). With respect to cardiac myocytes, the lack of stimulation of p38 MAPK by insulin, as observed by us, is in agreement with results in earlier studies in primary cardiac myocyte cultures (34).

The inability of insulin to stimulate p38 MAPK in cardiac myocytes does not rule out the involvement of p38 MAPK in the insulin-mimetic action of arsenite on glucose uptake. In this scenario, arsenite-induced p38 MAPK activation could be part of a signaling pathway parallel to the PI3K/Akt/PKB axis, resulting in the activation of a downstream component that is also activated by Akt/PKB. Nonetheless, we were unable to link arsenite-induced p38 MAPK phosphorylation to arsenite-induced GLUT4 translocation. More specifically, the use of SB202190 and SB203580 indicates that p38 MAPK is not involved in arsenite-stimulated glucose uptake, notwithstanding the observation that both compounds partially inhibited arsenite-stimulated glucose uptake. Notably, both SBs partially inhibited glucose uptake in cardiac myocytes during arsenite stimulation and insulin stimulation as well as in unstimulated quiescent cardiac myocytes. However, in quiescent cardiac myocytes and during insulin stimulation, p38 MAPK is not activated (Fig. 7Go). Moreover, the inhibitory effect of both SBs on glucose uptake is completely retained in giant vesicles (Fig. 5Go), adding powerful evidence that these compounds directly interfere with the transport function of GLUTs that are located at the PM. Hence, because the magnitude of inhibition exerted by both SBs on glucose uptake is very similar between giant vesicles and arsenite-stimulated cardiac myocytes, the direct interaction of SBs with GLUT4 fully accounts for their inhibitory action on arsenite-stimulated glucose uptake. This also implies that the inhibition of p38 MAPK phosphorylation exerted by SB202190 (Fig. 1Go) and SB203580 (data not shown) does not play a role in inhibition of arsenite-stimulated glucose uptake by these SBs. Furthermore, the noteworthy conclusion can be drawn that both SBs cannot be used to discern the effects of p38 MAPK on glucose uptake. With respect to SB203580, there are recent reports that draw the same conclusion (35, 36), and a putative mechanism of action involves an endofacial interaction with GLUT4. This occupation of the endofacial substrate site of GLUT4 is then supposed to block the return of this substrate site to the extracellular leaflet, causing a drop in maximal velocity (33).

Because GLUT4 translocation is entirely responsible for the arsenite-induced increase in glucose uptake, conclusive evidence for the lack of involvement of p38 MAPK in arsenite-stimulated glucose uptake can be deduced from the inability of SB202190 to affect arsenite-induced GLUT4 translocation.

p42/44 ERK and p70 S6K.
Arsenite and insulin resemble each other in that they both moderately stimulated p42/44 ERK as well as p70 S6K by phosphorylation. On the other hand, oligomycin did not enhance phosphorylation of these kinases. The inhibitors PD98059 and rapamycin, under conditions at which they fully reverse agonist-induced ERK or S6K activation, respectively, have no direct inhibitory action on GLUT4 itself. They also did not affect arsenite- or insulin-stimulated glucose uptake. Hence, p42/44 ERK and p70 S6K are not involved in translocation of GLUT4 induced by each of these stimuli.

Taken altogether, our study suggests that despite the multiple (pleiotropic) effects of arsenite on selected signaling pathways, these signaling actions are not involved in arsenite’s selective stimulation of glucose uptake via GLUT4 translocation. It is well documented that intracellularly stored GLUT4 is divided over two compartments: the recycling endosomes and a nonendosomal GLUT4-specific storage compartment (8, 37, 38). This storage compartment harbors a relatively large pool of GLUT4 and is dedicated to mobilize GLUT4 specifically in response to insulin and not, for instance, in response to contractions (Fig. 8Go). As reviewed previously (8), it is unlikely that intracellular CD36 is present within the nonendosomal storage compartment. However, colocalization with Rab11 indicates that CD36 is present within the recycling endosomes (39), in which it can be mobilized by insulin and contractions. When insulin is added to cardiac myocytes, both the storage compartment and the recycling endosomes are activated to mobilize GLUT4 transporters through budding of small membrane vesicles. The GLUT4-containing vesicles mobilized from the nonendosomal storage compartment are not expected to migrate directly to the cell surface but first fuse with the recycling endosomes (38, 40, 41) (see Fig. 8Go). Simultaneously, transport vesicles will be assembled from the endosomal membranes for further transport of GLUT4 to the sarcolemma. As recently proposed (8), these endosomally derived vesicles would contain not only GLUT4 but also endosomally stored CD36. The specific action of arsenite on glucose uptake suggests that the intracellular target of arsenite must reside within the storage compartment rather than in the recycling endosomes (Fig. 8Go).


Figure 8
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  		FIG. 8. Hypothetical scheme of arsenite’s action on transporter recycling: comparison with those of insulin and contractions. Under basal conditions, the majority of GLUT4 and approximately 50% of CD36 is present in intracellular stores with the remaining portion of both transporters at the sarcolemma. Intracellularly GLUT4 is present in both the storage compartment and the recycling endosomes, whereas the localization of CD36 is restricted to the recycling endosomes. A, Insulin, through phosphorylation (P) of its receptor and downstream activation of PI3K and subsequently Akt/PKB, mobilizes GLUT4 from the storage compartment, and also both GLUT4 and CD36 from insulin-responsive recycling endosomes. GLUT4 mobilized from the storage compartment and on its way to the sarcolemma travels through the recycling endosomes, from which it shares its transport vesicle together with CD36. B, Arsenite does not affect the dynamics of the recycling endosomes but selectively recruits GLUT4 from the storage compartment through sulfhydryl modification of an unknown protein [intracellular target of arsenite (ITA)] within this compartment. Vesicles excised by thiol activation of ITA shortcut the endosomal route and migrate directly to the sarcolemma.

 
A hypothetical thiol modification of this target by arsenite would then result in excision of GLUT4-containing vesicles from the storage compartment. In contrast to insulin stimulation, the recycling endosomes are not activated by arsenite to mobilize transport vesicles, forcing the storage compartment-derived vesicles to bypass the endosomes and migrate directly to the sarcolemma. Although this proposed mechanism of arsenite action is speculative, it effectively explains arsenite’s selective effect on glucose uptake. It also explains the fact that arsenite does not behave as a contraction-mimetic agent such as oligomycin, and finally it explains that arsenite can behave as an insulin-mimetic agent with respect to glucose uptake, independent of the PI3K/Akt signaling system (Fig. 8Go).

Stimulation of LCFA uptake by insulin is not mediated via P38 MAPK, p42/44 ERK, and p70 S6K
Whereas PI3K has been firmly implicated in stimulation of LCFA uptake by insulin (31), virtually no information is available about the role of MAPKs and S6Ks in this insulin action. In the present study, arsenite does not increase LCFA uptake, despite activation of p38 MAPK, p42/44 ERK, and p70 S6K. On the contrary, blockade of arsenite signaling by the SBs, PD98059, or rapamycin had no effect on LCFA uptake by cardiac myocytes either under basal conditions or in the presence of insulin or arsenite. In contrast, wortmannin selectively blocks insulin-stimulated LCFA uptake, confirming earlier observations on the involvement of PI3K in insulin-induced LCFA uptake (24, 25). Hence, these observations firmly exclude a role of MAPKs and S6Ks in insulin-induced induction of CD36 translocation and stimulation of LCFA uptake in the heart.

Conclusion and future perspectives
The most important finding in this study is the arsenite-induced selective stimulation of glucose uptake into cardiac myocytes. We speculate that this selective stimulation of glucose uptake involves a thiol modification of an arsenite-sensitive protein. The insulin-mimetic action of arsenite on glucose uptake pinpoints this putative protein within the insulin-responsive storage compartment, harboring the majority of intracellular GLUT4. The important implication of the intrinsic ability of the heart to selectively up-regulate glucose uptake is that this would be favorable under conditions in which cardiac glucose uptake is repressed. Notably in the insulin-resistant and diabetic heart, there is a marked shift from glucose use to LCFA use (3) accompanied by a permanent relocation of CD36 to the sarcolemma (42). In the diabetic heart, there is also a decreased expression and/or activation of protein kinases (insulin receptor substrate-1, PI3K, and Akt/PKB) involved in insulin signaling (43, 44). The inability of GLUT4 to be recruited by insulin has been recognized to be responsible for the reduction in cardiac glucose uptake (45). Theoretically the hypothetical intracellular target of arsenite involved in stimulation of cardiac glucose uptake presents an ideal target to restore glucose uptake in the diabetic heart. In particular, a direct activation of this arsenite-sensitive target bypasses the defective insulin signaling cascade for recruitment of GLUT4 to the sarcolemma. Unfortunately, arsenite itself is not suitable as an antidiabetic therapeutic because of its atherogenic and carcinogenic actions. Interestingly, environmental exposure to arsenic from drinking water is associated with increased incidence of type 2 diabetes (46). An underlying mechanism for this arsenite-induced type 2 diabetes in humans could include ß-cell dysfunction, which is commonly observed on long-term arsenite exposure and is regarded an primary risk factor for this disease (14). Nevertheless, identification of the intracellular target of arsenite involved in selective stimulation of glucose uptake could provide novel information about regulation of recruitment of GLUT4 from its nonendosomal storage compartment. Moreover, it could be the basis of a novel antidiabetic strategy.


   Acknowledgments
 
Antibody MO25 was kindly provided by Dr. N. N. Tandon (Thrombosis Research Laboratory, Otsuka Maryland Medicinal Laboratories, Rockville, MD). FABPpm antisera were kindly provided by Dr. J. Calles-Escandon (Section of Endocrinology and Metabolism, Wake Forest University School of Medicine and Baptist Medical Center, Winston-Salem, NC). The authors thank J. Willems and W. J. Wijnen for their help with the illustrations.


   Footnotes
 
Present address for D.P.Y.K.: Department of Pediatrics, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada.

This work was supported by grants or fellowships from The Netherlands Organisation for Scientific Research (ZonMw VIDI Innovational Research Grant 016.036.305) (to J.J.F.P.L.), the European Community (Integrated Project LSHM-CT-2004-005272, Exgenesis), the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Heart and Stroke Foundation of Ontario, and the Canada Research Chair Program. J.F.C.G. is The Netherlands Heart Foundation Professor of Cardiac Metabolism. A.B. is the Canada Research Chair in Metabolism and Health.

Disclosure statement: none of the contributing authors has a potential conflict of interest.

First Published Online October 12, 2006

Abbreviations: ACC, Acetyl-coenzyme A carboxylase; AMPK, AMP-activated protein kinase: CD36, human homolog of rat fatty acid translocase; FABPpm, plasma membrane fatty acid-binding protein; LCFA, long-chain fatty acids; LDM, low-density microsomal; MOPS, 3[N-morpholino]propanesulfonic acid; PI3K, phosphatidylinositol-3 kinase; PKB, protein kinase B; PM, plasma membrane; S6K, S6 kinase.

Received June 22, 2006.

Accepted for publication July 25, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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R. W. Schwenk, J. J.F.P. Luiken, A. Bonen, and J. F.C. Glatz
Regulation of sarcolemmal glucose and fatty acid transporters in cardiac disease
Cardiovasc Res, July 15, 2008; 79(2): 249 - 258.
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