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(Circulation Research. 2000;86:326.)
© 2000 American Heart Association, Inc.

Cellular Biology

Parallel Stimulation of Glucose and Mg²⁺ Accumulation by Insulin in Rat Hearts and Cardiac Ventricular Myocytes

Andrea M. P. Romani, Veronica D. Matthews, Antonio Scarpa

From the Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio.

Correspondence to Andrea M.P. Romani, Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-4970. E-mail amr5{at}po.cwru.edu

	Abstract

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Abstract—The stimulation of ß-adrenoceptors in cardiac cells results in a rapid loss of cellular Mg²⁺. Because insulin physiologically counteracts several of the cellular effects mediated by the activation of ß-adrenoceptors and the elevation of cytosolic cAMP levels, we investigated whether insulin administration could prevent Mg²⁺ mobilization from rat hearts and ventricular myocytes. Rat hearts were perfused in a retrograde Langendorff system, and the changes in extracellular Mg²⁺ were measured by atomic absorbance spectrophotometry. Pretreatment of the hearts with 6 nmol/L insulin completely prevented the Mg²⁺ extrusion induced by the ß-adrenergic agonist isoproterenol. Furthermore, the administration of insulin per se induced an accumulation of Mg²⁺ by the heart. This accumulation was small but detectable in the presence of 25 to 35 µmol/L [Mg²⁺]_o and increased in proportion to [Mg²⁺]_o. Insulin-mediated Mg²⁺ accumulation was not observed in hearts perfused with a medium devoid of glucose or with a medium containing the inhibitors of glucose transport, cytochalasin B and phloretin. Insulin-stimulated [³H]2-deoxyglucose accumulation was measured in collagenase-dispersed cardiac ventricular myocytes in the presence of varying levels of [Mg²⁺]_o. Glucose transport was not observed below 25 µmol/L [Mg²⁺]_o, and it also increased in proportion to [Mg²⁺]_o. Taken together, these results indicate the presence of a major uptake of Mg²⁺ into cardiac cells that is stimulated by insulin and may require the insulin-induced operation of a glucose transporter. Hence, extracellular and/or intracellular Mg²⁺ may modulate glucose transport and/or utilization.

Key Words: Mg²⁺ • cardiac myocytes • hearts • insulin • glucose transport

	Introduction

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In recent years, a large number of reports have indicated that the selective stimulation of ß-adrenoceptors ^{1 2 3 4 5 6 7 8 9} results in a marked extrusion of cellular Mg²⁺ from cardiac myocytes,^{1 2 3} hepatocytes,^{4 5 6} and other cell types^{7 8 9} into the extracellular compartment. In addition, the infusion of isoproterenol or catecholamine results in a 15% to 20% increase in the total serum Mg²⁺ level in the anesthetized rat.^{10 11} At the cellular level, Mg²⁺ extrusion can be elicited by the administration of forskolin^{2 4} or cell-permeant cAMP analogues (eg, 8-bromo-cAMP)^{2 4 5 6 7 8 9} and be inhibited by the administration of Rp-cAMP,⁷ a cell-permeant blocking agent specific for protein kinase A. Taken together, these results support the idea that Mg²⁺ extrusion is mediated via a cAMP-dependent process, most likely the phosphorylation of a specific Mg²⁺ transporter.⁹

Experimental evidence suggests a role for insulin in regulating cellular or tissue Mg²⁺ content. Our laboratory has recently reported that because of its ability to prevent cAMP production¹² and accelerate cAMP catabolism via phosphodiesterase,¹³ insulin can effectively modulate the extrusion of Mg²⁺ induced by ß-adrenergic agonists in liver cells.⁶ In addition, evidence has been provided indicating that insulin increases cytosolic free [Mg²⁺] in beta pancreatic islets,¹⁴ 3T3 fibroblasts,¹⁵ and platelets¹⁶ by promoting an entry of Mg²⁺ across the plasma membrane and/or a release of Mg²⁺ from an intracellular organelle(s). Last, a marked decrease in cellular Mg²⁺ content has been observed in diabetes types I and II^{17 18 19} both in humans^{17 18} and animals,¹⁹ and this decrease has been suggested to be a possible cause of the long-term complications associated with diabetes.²⁰

In the present study, the ability of insulin to modulate cellular Mg²⁺ in cardiac myocytes was investigated. The results obtained indicate that insulin can modulate cellular Mg²⁺ content by limiting the amount of Mg²⁺ extruded from cardiac cells stimulated by ß-adrenergic agonists or by inducing a Mg²⁺ accumulation in the cells. Furthermore, the presence of a synergism between glucose transport and Mg²⁺ accumulation in cardiac cells suggests a key role of Mg²⁺ in controlling glucose utilization for energetic purposes within the cell.

	Materials and Methods

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Chemicals
Collagenase (CLS-1) was from Worthington. [³H]2-Deoxyglucose was from Amersham. All other chemicals were from Sigma Chemical Co. Whatman glass fiber filters were from Fisher.

Rat Heart Perfusion
Male Sprague-Dawley rats (250 g body weight) were anesthetized by intraperitoneal injection of sodium pentobarbital. The heart was removed and perfused in a Langendorff system at a flow rate of 7 mL/min with a medium containing (mmol/L) NaCl 120, KCl 3, KH₂PO₄ 1.2, CaCl₂ 1.2, MgCl₂ 1.2, glucose 20, HEPES 10, and NaHCO₃ 12, pH 7.2, equilibrated at 37°C with O₂/CO₂ (95:5 [vol/vol]).^{2 3} The [Mg²⁺]_o in the perfusion medium was varied from 0 to 1000 µmol/L. Where indicated in the figures, isoproterenol, 8-chloro-cAMP,² or insulin was added to the perfusion medium.

In the experiments performed in the absence of extracellular glucose, 5 mmol/L pyruvate and 5 mmol/L lactate were added to the perfusion medium. Alternatively, pyruvate and lactate were added to the perfusion medium in addition to glucose. Cytochalasin B or phloretin was dissolved in the perfusion medium 5 minutes before insulin administration.

Aliquots of the perfusate were collected every 30 seconds, and the Mg²⁺ content was measured by atomic absorbance spectrophotometry in a Perkin-Elmer 3100 spectrophotometer after proper dilution. Net Mg²⁺ accumulation was estimated as described previously.²¹

At the end of the experiment, the heart was homogenized in 10% HNO₃ and extracted overnight. The Mg²⁺ content in the acid supernatant was measured by atomic absorbance spectrophotometry as described previously.³

Isolation of Cardiac Ventricular Myocytes and Determination of Mg²⁺ Accumulation
Cardiac ventricular myocytes were isolated by collagenase digestion as described by De Young et al.²² An aliquot of cell suspension was washed at 600g for 1 minute and transferred into the incubation medium described previously, in the presence of varying concentrations of extracellular Mg²⁺ or glucose. Mg²⁺ accumulation into the cells was determined as reported previously.³

Determination of Glucose Transport
For the experiments in perfused hearts, 0.2 mCi/mL [³H]2-deoxyglucose was added to the perfusion medium. Half-milliliter aliquots of the perfusate were collected in duplicate and transferred in scintillation vials to measure the radioactivity by ß-scintillation counting in a Beckman LS7000 counter. At the end of the perfusion, the heart was homogenized in 10% HNO₃ and extracted overnight. The radioactivity accumulated into the tissue was measured by ß-scintillation counting in aliquots of the homogenate.

For the experiments in isolated myocytes, the cells were incubated as previously reported, in the presence of 0.2 mCi/mL [³H]2-deoxyglucose. After insulin administration, glucose accumulation was determined, as reported previously,²³ as the radioactivity retained onto glass fiber filters (NF Whatman, pore size 0.25 µm).

Protein was assessed by the procedure of Lowry et al²⁴ with bovine serum albumin used as a standard.

Statistical Analysis
The data were reported as mean±SE. Data were first analyzed by 1-way ANOVA. Multiple means were then compared by the Tukey multiple comparison test, which was performed with a q value established for significance at P<0.05.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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The administration of 1 or 10 µmol/L isoproterenol to rat hearts perfused in a Langendorff system resulted in a detectable increase in heart contractility (not shown) and in a marked extrusion of Mg²⁺ from the organ into the perfusate (Figure 1A). Mg²⁺ efflux became evident within 2 minutes after the addition of the ß-adrenergic agonist to the perfusion medium and persisted for an additional 5 minutes before returning toward basal levels. The time course of these changes was independent of the dose and persistence of the agonist in the perfusion medium, the rate of contractility elicited by the adrenergic agonist, and [Mg²⁺]_o, as already reported.^{2 3} Isoproterenol-induced Mg²⁺ extrusion was also observed when the concentration of Mg²⁺ in the perfusate was increased to 100 and 250 µmol/L. Despite the high noise-to-signal ratio observed under these experimental conditions, the net amount of Mg²⁺ extruded from the heart was similar (410.6±36.1 and 413.0±32.3 nmol Mg²⁺/g heart for 8 minutes at 100 and 250 µmol/L [Mg²⁺]_o, respectively, versus 528.4±23.6 nmol Mg²⁺/g heart for 8 minutes at 10 µmol/L [Mg²⁺]_o; n=5 for all the conditions).

View larger version (23K): [in this window] [in a new window]   Figure 1. Efflux of Mg2+ from perfused rat hearts stimulated by isoproterenol (iso or isoprot.) in the absence (A) or presence (B) of insulin. Mg2+ efflux from rat hearts perfused in a Langendorff retrograde manner was induced by administration of 1 or 10 µmol/L isoproterenol. Insulin (10 mU/mL=6 nmol/L) was administered 5 minutes before 10 µmol/L isoproterenol. Data were determined every 30 seconds but are represented at 90-second intervals for clarity. Data are mean±SE of 5 different hearts for all experimental conditions. *P<0.05 vs control. #P<0.05 vs 1 µmol/L isoproterenol.

Pretreatment of the heart with 10 mU/mL (6 nmol/L) insulin completely prevented the Mg²⁺ extrusion induced by 1 µmol/L (not shown) or 10 µmol/L isoproterenol (Figure 1B). A similar inhibition was also observed when 250 µmol/L 8-chloro-cAMP was used instead of isoproterenol² to mobilize Mg²⁺ (not shown). A similar inhibitory effect on isoproterenol-induced Mg²⁺ extrusion was also observed in hearts perfused in the presence of 15 µmol/L [Mg²⁺]_o when insulin was added 3 minutes after the ß-adrenergic agonist, ie, at a time point at which Mg²⁺ extrusion into the perfusate could be detected effectively, although it was not maximal (Figure 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Mg2+ extrusion from perfused hearts sequentially stimulated by isoproterenol and insulin in the presence of 15 µmol/L [Mg2+]o. Rat hearts, perfused in the presence of 15 µmol/L [Mg2+]o, were stimulated by 10 µmol/L isoproterenol and, 3 minutes later, by 10 mU/mL insulin. Data were determined every 30 seconds but are represented at 90-second intervals for clarity. Data are mean±SE of 4 hearts. *P<0.05 vs the average of the 5 time points preceding isoproterenol administration.

Because atomic absorbance spectrophotometry cannot measure unidirectional Mg²⁺ fluxes, in principle it is possible that the absence of Mg²⁺ extrusion is the result of a decreased Mg²⁺ efflux and/or an increased Mg²⁺ influx into cardiac cells. The latter possibility appears to be supported by the decline in the basal Mg²⁺ level below the initial value after insulin administration (Figure 2).

Hence, the possibility that insulin induced Mg²⁺ accumulation into cardiac cells was further investigated by increasing [Mg²⁺]_o in the perfusate from the contaminant concentration present in Figure 1. Insulin administration resulted in a small but detectable Mg²⁺ accumulation when the heart was perfused with 25 µmol/L [Mg²⁺]_o (not shown). The decrease in Mg²⁺ content in the perfusion medium, an indication of Mg²⁺ accumulation into cardiac cells, increased progressively in hearts perfused with 35 or 50 µmol/L [Mg²⁺]_o (Figure 3A) or higher levels of [Mg²⁺]_o (not shown). In Figure 3B, the net Mg²⁺ accumulation by the perfused heart during 8 minutes of insulin administration is reported as the total amount of Mg²⁺ disappearing from the perfusate. The net Mg²⁺ accumulation was accounted for by 500 to 600 nmol/g heart for [Mg²⁺]_o of 100 and 200 µmol/L and 700 to 800 nmol/g heart for [Mg²⁺]_o of 500 or 1000 µmol/L. The Mg²⁺ determination in acid extracts of the heart at the end of the experiment indicates an increase in total tissue Mg²⁺ content from 61.97±3.24 to 71.42±3.18 and to 77.21±4.50 nmol/mg protein^-1 in hearts perfused with 200 and 1000 µmol/L [Mg²⁺]_o, respectively (P<0.05, n=4 for all experimental conditions).

View larger version (19K): [in this window] [in a new window]   Figure 3. Mg2+ accumulation by perfused hearts stimulated by insulin in the presence of varying levels of [Mg2+]o. A, Rat hearts were perfused in the presence of 35 or 50 µmol/L [Mg2+]o and stimulated by insulin. Data were determined every 30 seconds but are represented at 90-second intervals for clarity. Data are mean±SE of 5 and 7 hearts for 35 and 50 µmol/L [Mg2+]o, respectively. *P<0.05 vs the average of the 5 time points preceding insulin administration. B, Net Mg2+ accumulation in perfused hearts stimulated by insulin in the presence of varying levels of [Mg2+]o is reported. Net Mg2+ accumulation during 8 minutes of stimulation by insulin was estimated as the disappearance of Mg2+ from the perfusate. Data are mean±SE of 7 hearts for 50 µmol/L [Mg2+]o and 5 hearts for all other levels of [Mg2+]o. *P<0.05 vs 10 µmol/L [Mg2+]o. #P<0.05 vs all other levels of [Mg2+]o. @P=NS vs 100 or 500 µmol/L.

A similar inhibitory effect of insulin on isoproterenol- or cAMP-induced Mg²⁺ extrusion has been observed in perfused liver.⁶ However, insulin per se did not induce any detectable Mg²⁺ uptake into liver cells, regardless of the [Mg²⁺]_o used.⁶ One notable difference between cardiac and liver cell metabolism is the different class of glucose transporter present in the plasma membrane, namely, Glut1 and Glut4 in cardiac cells²⁵ and Glut 2 in hepatocytes.²⁵ Glut4 transporters (and Glut1 to a lesser extent),²⁶ but not Glut2,²⁵ are recruited to the sarcolemma by insulin administration. Therefore, we next investigated the possibility that glucose transport is involved in mediating the accumulation of Mg²⁺ induced by insulin.

The requirement of glucose transport for Mg²⁺ accumulation is supported by the data reported in Figure 4. In the presence of 50 µmol/L [Mg²⁺]_o, the absence of glucose in the perfusate (replaced with lactate and pyruvate, Figure 4B) completely prevented the insulin-mediated Mg²⁺ accumulation (Figure 4A). To exclude the possibility that the lack of Mg²⁺ accumulation observed under these experimental conditions could be attributable to the sudden change in metabolic substrate, in a separate set of experiments, 5 mmol/L pyruvate and 5 mmol/L lactate were introduced into the perfusion medium at the start, in addition to glucose. Glucose was removed at the time of insulin administration, to be reintroduced after hormone removal, but pyruvate and lactate were maintained throughout the experimental protocol. Also, under these experimental conditions, insulin administration did not result in an accumulation of Mg²⁺ in the heart (total tissue Mg²⁺ content was 64.0±6.4 versus 61.7±5.4 nmol Mg²⁺/mg protein^-1 in control hearts versus insulin-treated hearts, n=4 for both experimental conditions, P>0.05). By contrast, when glucose was maintained throughout the experimental protocol in addition to pyruvate and lactate, the administration of insulin resulted in a disappearance of Mg²⁺ from the perfusate (Figure 4C) and an accumulation in the heart (total tissue Mg²⁺ content was 70.8±2.5 versus 60.8±3.4 nmol Mg²⁺/mg protein^-1 in the presence or in the absence of insulin, respectively).

View larger version (16K): [in this window] [in a new window]   Figure 4. Mg2+ accumulation in hearts perfused in the absence of extracellular glucose. A and B, Rat hearts were perfused in the presence of 50 µmol/L [Mg2+]o in the presence (A) or in the absence (B) of 15 mmol/L glucose (replaced with 5 mmol/L lactate and 5 mmol/L pyruvate) and stimulated by insulin for 8 minutes. C, Effect of insulin on rat hearts perfused in the presence of 5 mmol/L lactate and 5 mmol/L pyruvate plus 15 mmol/L glucose is shown. The infusion of glucose-free medium was initiated 5 minutes before insulin administration and limited to the time of insulin infusion. Data were determined every 30 seconds but are represented at 90-second intervals for clarity. Data are mean±SE of 5 different hearts for all experimental conditions. Values of P are as follows for panels A through C: A, *P<0.05 vs glucose-free medium. B, #P<0.05 vs glucose medium. C, *P<0.05 vs control and insulin minus glucose; #P<0.05 vs insulin minus glucose only.

In a separate set of experiments, cytochalasin B and phloretin were used as glucose transport inhibitors. Whereas cytochalasin B blocks the translocation of glucose transporters to the plasma membrane by disrupting cytoskeleton integrity, phloretin inhibits glucose transport operation at the plasma membrane by interacting at the extracellular site of the transporter.^{23 25} When 1 µmol/L cytochalasin B or 10 µmol/L phloretin was added to the perfusate in the presence of glucose, the insulin-induced Mg²⁺ accumulation in the heart was almost completely inhibited (Figure 5). Finally, when insulin-induced Mg²⁺ accumulation was measured at varying extracellular glucose concentrations, a minimal glucose concentration of 2 mmol/L appeared to be required for the Mg²⁺ accumulation to occur (Figure 6). Net Mg²⁺ accumulation accounted for 1.53±0.35 (n=4), 2.92±0.98 (n=4), and 17.80±2.32 nmol Mg²⁺/mg protein^-1 (n=5) for insulin-stimulated hearts perfused in the presence of 2, 5, and 10 mmol/L glucose, respectively. The last 3 time points under the curve of uptake with 10 mmol/L glucose are significantly different (P<0.05) compared with the corresponding time points reported in Figure 4A. Presently, we have no explanation for this discrepancy.

View larger version (28K): [in this window] [in a new window]   Figure 5. Effect of glucose transport inhibitors on insulin-induced Mg2+ accumulation in perfused hearts. Rat hearts were perfused in the presence of 50 µmol/L [Mg2+]o and 15 mmol/L glucose and stimulated by insulin. Where indicated, cytochalasin B (1 µmol/L) and phloretin (10 µmol/L) were added to the perfusion medium. Data were determined every 30 seconds but are represented at 90-second intervals for clarity. Data are mean±SE of 5 different hearts for all experimental conditions.

View larger version (32K): [in this window] [in a new window]   Figure 6. Mg2+ accumulation in perfused hearts stimulated by insulin in the presence of varying extracellular glucose concentrations. Rat hearts were perfused in the presence of 2, 5, and 10 mmol/L glucose and stimulated by insulin for 10 minutes. Data were determined every 30 seconds but are represented at 90-second intervals for simplicity. Data are mean±SE of 4 different hearts for all experimental conditions. *P<0.05 vs control. &P<0.05 vs 2 mmol/L glucose. #P<0.05 vs 5 mmol/L glucose. @P<0.05 vs all other conditions.

The presence of a synergism between glucose and Mg²⁺ accumulation is further corroborated by the results reported in Figure 7. Because nonphosphorylated glucose can cross the sarcolemma in either direction, [³H]2-deoxyglucose, which remains trapped in the cytosol after the phosphorylation by hexokinase,²⁵ was used to quantify the amount of glucose accumulated by cardiac ventricular myocytes after 5 minutes of stimulation by 10 mU/mL (6 nmol/L) insulin. Cardiac ventricular myocytes rather than perfused hearts were used to exclude possible artifacts related to perfusion flow rate and to cell heterogeneity. The data, reported in Figure 7A, indicate that [Mg²⁺]_o is required to observe an accumulation of glucose into cardiac cells. This accumulation accounted for 0.47±0.16 nmol glucose/10⁶ cells at 50 µmol/L [Mg²⁺]_o and increased to 1.07±0.20, 1.68±0.29, and 2.56±0.30 nmol/10⁶ cells when [Mg²⁺]_o was 100, 500, and 800 µmol/L, respectively. Under these experimental conditions, Mg²⁺ accumulation was 26.9±6.1 nmol/10⁶ cells for 5 minutes at 100 µmol/L [Mg²⁺]_o and 58.0±12.3 nmol/10⁶ cells for 5 minutes at 500 µmol/L [Mg²⁺]_o (Figure 7B). Based on the total cellular Mg²⁺ content of cardiac ventricular myocytes, these values account for increases of 10% and 22% in total Mg²⁺ content, respectively.

View larger version (16K): [in this window] [in a new window]   Figure 7. Net glucose (A) and Mg2+ (B) accumulation in collagenase-dispersed cardiac ventricular myocytes. Rat cardiac ventricular myocytes were incubated in the presence of 15 mmol/L glucose labeled with [3H]2-deoxyglucose (3H-2DOG) and varying levels of [Mg2+]o and stimulated by insulin for 5 minutes. See Materials and Methods for detail. Data are mean±SE of 5 different myocytes preparations, each of them performed in triplicate, for both glucose and Mg2+ determinations. Values of P are as follows for panels A and B: A, *P<0.05 vs 0 mmol/L [Mg2+]o. B, *P<0.05 vs respective control.

After it had been determined that the presence of extracellular glucose or the operation of glucose transporter is necessary to observe insulin-induced Mg²⁺ accumulation, rat hearts were perfused in the presence of pyruvate and lactate but in the absence of glucose and stimulated by 10 µmol/L isoproterenol and 10 mU/mL insulin to determine whether the effect of insulin on the ß-adrenoceptor–mediated Mg²⁺ extrusion observed in Figures 1 and 2 could be ascribed to an inhibitory effect on ß-adrenergic signaling^{12 13} and/or to a stimulated accumulation of Mg²⁺ into the heart. As Figure 8A shows, in the presence of 50 µmol/L [Mg²⁺]_o but in the absence of extracellular glucose, insulin was still able to block the extrusion of Mg²⁺ elicited by isoproterenol infusion. By contrast, in the presence of glucose, the administration of insulin before adrenergic agonist infusion resulted in an accumulation of Mg²⁺ that could not be reverted by the subsequent infusion of isoproterenol (Figure 8B).

View larger version (24K): [in this window] [in a new window]   Figure 8. Inhibitory effect of insulin (Insu) on isoproterenol (Iso)-induced Mg2+ extrusion in the absence (A) or presence (B) of extracellular glucose. Mg2+ efflux from rat hearts perfused in a Langendorff retrograde manner was elicited by administration of 10 µmol/L Iso. Insu (10 mU/mL=6 nmol/L) was administered 3 minutes before Iso infusion. Perfusion medium contained 50 µmol/L [Mg2+]o, 5 mmol/L lactate, 5 mmol/L pyruvate, and 15 mmol/L glucose. In panel A, glucose was removed from the incubation medium during Insu and Iso infusion. In panel B, extracellular glucose was maintained throughout the experimental protocol. Data were determined every 30 seconds but are represented at 90-second intervals for clarity. Data are mean±SE of 4 different hearts for all the experimental conditions. Values of P are as follows for panels A and B: A, *P<0.05 vs control and insulin-treated hearts. B, *P<0.05 vs control.

	Discussion

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The administration of ß-adrenergic agonists to cardiac cells elicits a marked extrusion of cellular Mg²⁺ in the extracellular compartment^{1 2 3} via an increase in cAMP and the activation of a specific Na⁺-Mg²⁺ exchanger.^{27 28} Recently, we have reported that insulin can modulate Mg²⁺ content in liver cells by preventing the ß-adrenoceptor–mediated Mg²⁺ mobilization from the cell.⁶

The present study, undertaken to investigate whether insulin has a similar modulatory role in cardiac cells, provides several novel observations. First, it provides evidence for a role of insulin in preserving Mg²⁺ content in cardiac cells by preventing the Mg²⁺ mobilization induced by ß-adrenoceptor stimulation. Second, it indicates that insulin induces an accumulation of Mg²⁺ into cardiac cells through a transport mechanism that is linked to the operation of glucose transporter in the cardiac sarcolemma. Third and most important, it suggests that Mg²⁺ is indispensable for the accumulation of glucose within cardiac myocytes. Although the physiological significance of the concomitant accumulation of glucose and Mg²⁺ needs further clarification, we can hypothesize that changes in cellular Mg²⁺ content are required for both proper glucose utilization and insulin signaling. These considerations may have particular importance in diabetes, a condition in which glucose transport and insulin signaling, as well as Mg²⁺ homeostasis, are markedly impaired.

Effect of Insulin on Cellular Mg²⁺ Homeostasis
The administration of insulin before isoproterenol (Figure 1) or cAMP addition (not shown) or after ß-agonist administration (Figure 2) can completely prevent the extrusion of Mg²⁺ elicited via activation of the ß-adrenergic signaling pathway. These effects of insulin can be explained by the ability of insulin to desensitize ß-adrenoceptors ^{12 29} at the cell membrane and stimulate calmodulin-dependent phosphodiesterase,¹³ thereby limiting the production and inducing a more rapid degradation of cellular cAMP. Overall, these results are consistent with the inhibitory effect observed previously in the perfused liver⁶ and would indicate a more general and physiological role of insulin at modulating ß-adrenoceptor–mediated Mg²⁺ extrusion in various tissues.

At variance with the observations involving liver cells,⁶ insulin induces an entry of Mg²⁺ into cardiac cells. This accumulation can be reliably detected when [Mg²⁺]_o is progressively increased from 25 to 1000 µmol/L Mg²⁺. Interestingly, under conditions in which [Mg²⁺]_o is >5 µmol/L, insulin-induced Mg²⁺ accumulation appears to prevail over isoproterenol-stimulated Mg²⁺ extrusion (Figures 2 and 8B). Consistent with the observations in liver cells⁶ and the modality of action on ß-adrenoceptors^{12 29} and phosphodiesterase,¹³ insulin still exerts a regulatory role on cellular Mg²⁺ homeostasis under conditions in which Mg²⁺ accumulation is prevented (ie, absence of extracellular glucose; Figure 8A). This may suggest that insulin and ß-adrenergic agonists regulate cellular Mg²⁺ homeostasis by activating distinct Mg²⁺ transport mechanisms and that insulin can inhibit the Mg²⁺ extrusion mechanism as well as activate the Mg²⁺ entry pathway. Because of the novelty of this observation, the physiological conditions that determine the modality of insulin action on cardiac Mg²⁺ homeostasis require further investigation.

Because insulin does not stimulate Mg²⁺ accumulation in the perfused liver⁶ regardless of [Mg²⁺]_o in the perfusion medium, cardiac but not liver cells must possess a specific entry mechanism activated by insulin. One of the main differences between cardiac and liver cells is the different class of glucose transporter present in the plasma membrane of these 2 cell types. In cardiac myocytes, insulin induces glucose accumulation by recruiting Glut4 (10- to 20-fold increase) and Glut1 transporters (2-fold increase) from a preconstituted intracellular pool to the sarcolemma.²⁶ The consequence of this recruitment is that glucose accumulation into cardiac myocytes increases severalfold and in a manner that is not simply proportional to the number of new transporters expressed in the sarcolemma.³⁰ By contrast, liver cells possess a distinct glucose transporter (Glut2) that is not affected in number and operation by insulin.²⁵

Involvement of Glucose Transport in Insulin-Mediated Mg²⁺ Accumulation
The results obtained in the absence of external glucose or in the presence of the inhibitors of glucose transport, cytochalasin B and phloretin, are consistent with the idea that insulin-induced Mg²⁺ accumulation requires operation and/or internalization of glucose transporters. Because insulin administration increases the expression of Glut4 to a great degree and Glut1 only marginally in the sarcolemma, it is conceivable that Glut4 is the main class of glucose transporters involved in Mg²⁺ accumulation. Support for this hypothesis is provided by the effect of glucose transport inhibitors. Whereas phloretin blocks both Glut1 and Glut4 in the sarcolemma by interacting at the extracellular site of the transporter, cytochalasin B (by disrupting cytoskeletal integrity) mainly affects Glut4, by preventing the recruitment of this transporter to the sarcolemma after insulin administration. In addition, Mg²⁺ accumulation requires a minimal extracellular glucose concentration of 2 mmol/L to occur, which falls well within the K_m of the Glut4 transporter.²⁵ However, because Mg²⁺ accumulation increases proportionally with the extracellular glucose concentration, the additional involvement of the Glut1 transporter (with a higher K_m²⁵ ) cannot be altogether excluded. Last, it is interesting to note that insulin and isoproterenol stimulate an accumulation and an extrusion of Mg²⁺, respectively, whereas they both induce a glucose accumulation in cardiac cells. Because insulin primarily activates Glut4 and isoproterenol activates Glut 1,²⁵ further indirect evidence is provided for the role of Glut4 in Mg²⁺ accumulation.

Our data do not clearly indicate whether Mg²⁺ is cotransported with glucose or whether the cation enters the cell through a transport pathway distinct from the glucose transporters. However, the inhibitory effect of cytochalasin B or phloretin suggests that the Mg²⁺ entry mechanism is activated by insulin indirectly via glucose transporter operation. Moreover, it appears that glucose reintroduction can induce Mg²⁺ accumulation even after insulin is removed from the system (Figure 4A). Most likely, this phenomenon is due to the persistence of an activated Glut4 transporter in the sarcolemma that is able to transport glucose after its reintroduction. In view of the fact that a glucose-triggered Mg²⁺ accumulation has been observed in pancreatic beta cells,³¹ the possibility that Mg²⁺ accumulation is generally associated with glucose transporter operation is a suggestive hypothesis that requires further investigation. Mg²⁺ uptake in cardiac myocytes appears to be 1 order of magnitude larger than glucose accumulation. Based on an estimated cell volume (Reference 3³ and references therein), the amount of Mg²⁺ accumulated into cardiac myocytes after insulin administration in the presence of physiological [Mg²⁺]_o would result in a potential severalfold increase in cytosolic free [Mg²⁺]. Yet, only minor changes in cytosolic free [Mg²⁺] were measured by fluorescent indicators in cells stimulated by insulin,^{15 16} suggesting that accumulated Mg²⁺ is rapidly redistributed among intracellular organelles. At the present time, the absence of Mg²⁺ accumulation under conditions in which glucose is replaced with pyruvate and lactate would reasonably exclude the possibility that Mg²⁺ accumulation is associated with, or dependent on, energy production.

Evidence for Role of Cellular Mg²⁺ in Mediating Effects of Insulin
A dependence of insulin-induced glucose transport in rat cardiac myocytes on intracellular Mg²⁺ has been reported by Eckel et al.³² The authors observed that insulin-stimulated glucose entry was completely abolished when EDTA buffer was used on A23187-treated myocytes³² and proposed an involvement of Mg²⁺ in insulin signaling. Whether this involvement is at the level of Mg²⁺-dependent hexokinase, cytoskeletal elements, or other intracellular enzymes involved in the translocation of glucose transporter to and from the sarcolemma is presently undefined. Evidence has been provided for a reduced autophosphorylation of insulin receptors and a reduced phosphorylation of insulin receptor–related kinases in Mg²⁺-deficient animals³³ and for significant alterations in glycemia and glucose utilization in rats after a long-term Mg²⁺-deficient diet.³⁴ Altogether, these observations strongly support a role of Mg²⁺ in modulating insulin response and cellular glucose utilization.

Conclusions
At the present time, we can only speculate about the physiological implication of Mg²⁺ accumulation in the heart. It is conceivable that Mg²⁺ plays 2 distinct though not mutually exclusive roles at the level of glucose entry and glucose utilization. As for glucose entry, Mg²⁺ may modulate the activity of cytoskeleton and kinases involved in the translocation of glucose transporters³⁵ or regulate allosterically glucose transport operation by changing the V_max of the transporter. A similar effect of extracellular Mg²⁺ on the inositol transporter in intestinal cells has been reported.³⁶ If corroborated by experimental evidence, either of these possibilities may explain why, after insulin stimulation, the rate of glucose transport increases by 20-fold versus the expected 10-fold increase calculated on the basis of the number of Glut4 recruited to the sarcolemma.³⁰ Alternatively, Mg²⁺ entry may be required to favor glucose utilization in cardiac cells, because many of the glycolytic enzymes, including hexokinase, are Mg²⁺ dependent to varying degrees. Our recent observation³⁷ that Mg²⁺ plays a regulatory role in the activity of several mitochondrial dehydrogenases supports the hypothesis that Mg²⁺ accumulated into the cell may be rapidly redistributed from the cytosol into the mitochondria or other organelles and regulate rates of respiration or concentrations of substrates necessary for specific metabolic pathways.

Together with previous evidence in the literature, the data reported in the present study indicate a close link between glucose transport and Mg²⁺ accumulation in cardiac ventricular myocytes. Although this link may be present in other cell types, it may be predominant in the heart, in which insulin modulates the operation of glucose transporters. Furthermore, indirect support for this link is provided by the observation that cellular Mg²⁺ levels and glucose utilization are markedly reduced in diabetic humans and animals. The relevance of this relation under both physiological and pathological conditions remains to be elucidated.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-18708 and R9-AA-11593A1 and by the Diabetes Association of Greater Cleveland (grant No. 397-A-97).

Received September 15, 1999; accepted November 22, 1999.

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