Streptozotocin-induced diabetes impairs Mg2+ homeostasis and uptake in rat liver cells

Theresa E. Fagan, Christie Cefaratti, and Andrea Romani

Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

Submitted 2 May 2003 ; accepted in final form 6 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Male Sprague-Dawley rats rendered diabetic by streptozotocin injection presented 10 and 20% decreases in total hepatic Mg2+ content at 4 and 8 wk, respectively, following diabetes onset. This decrease was associated with a parallel decrease in K+ and ATP content and an increase in Na+ level. In diabetic liver cells, the Mg2+ extrusion elicited by {alpha}1-adrenoceptor stimulation was markedly reduced compared with nondiabetic livers, whereas that induced by {beta}-adrenoceptor stimulation was unaffected. In addition, diabetic hepatocytes did not accumulate Mg2+ following stimulation of protein kinase C pathway by vasopressin, diacylglycerol analogs, or phorbol 12-myristate 13-acetate derivates despite the reduced basal content in cellular Mg2+. Experiments performed in purified plasma membrane from diabetic livers located the defect at the level of the bidirectional Na+/Mg2+ exchanger operating in the basolateral domain of the hepatocyte cell membrane, which could extrude but not accumulate Mg2+ in exchange for Na+. The impairment of Mg2+ uptake mechanism, in addition to the decrease in cellular ATP level, can contribute to explaining the decrease in liver Mg2+ content observed under diabetic conditions.

magnesium; adrenergic signaling; protein kinase c; adenosine 5'-triphosphate; plasma membrane


MAMMALIAN CELLS regulate Mg2+ homeostasis and transport across the cell membrane via complex mechanisms that are under tight hormonal control (14, 32). Cardiac myocytes (39, 31), hepatocytes (9, 27), erythrocytes (16, 20), and lymphocytes (41) all extrude a sizable amount of cellular Mg2+ as a result of the increase in cAMP that follows the activation of {beta}-adrenergic (27, 31, 39), glucagon (9), or progesterone receptors (41) or the administration of cell-permeant cAMP analogs (i.e., 8-Cl-cAMP or dibutyryl-cAMP) (27, 31, 39) or forskolin (27, 31), an agent that activates adenylyl cyclase. Further evidence for a role of cAMP in eliciting Mg2+ extrusion is provided by the effect of the Rp-cAMP isomer, which stably blocks adenylyl cyclase and prevents cAMP-mediated Mg2+ extrusion (41). Liver cells can also extrude Mg2+ following activation of {alpha}1-adrenoceptor (9, 10). Under all these conditions, Mg2+ is transported across the cell membrane via a putative Na+/Mg2+ exchanger (reviewed in Ref. 13). Although the transporter has not been cloned to date, the Na+ dependence of Mg2+ extrusion is supported by results in various experimental models, which indicate that Mg2+ extrusion is largely inhibited in the absence of extracellular Na+ (29, 30) or in the presence of the Na+ transport inhibitors amiloride (15) and imipramine (12).

Mammalian cells can also accumulate Mg2+ as a result of protein kinase C activation or a decrease in cAMP level. Agents such as carbachol or insulin that decrease cellular cAMP level, in fact, induce a marked accumulation of Mg2+ within liver cells (28), cardiac myocytes (28, 31), platelets (36), or fibroblasts (17). Activation of protein kinase C by cell-permeant analogs of diacylglycerol (25), derivates of phorbol 12-myristate 13-acetate (PMA) (28), or hormones like vasopressin (27) also results in an increase in cellular Mg2+ content. It is presently undefined whether Mg2+ entry is mediated by the reverse operation of the Na+/Mg2+ exchanger, as suggested by data obtained in purified liver plasma membranes (5, 6), or a distinct pathway, possibly a channel, as indicated by results in cardiac myocytes (25) or kidney cells (24).

Because of the relative recent interest in Mg2+ homeostasis, scarce data are available about defects in Mg2+ transport and regulation under pathological conditions. Experimental and clinical evidence, however, indicates that a loss of tissue and plasma Mg2+ is observed under both type 1 and type 2 diabetes (26, 40). The modality by which this decrease in tissue and plasma Mg2+ occurs is largely unknown, as it is unclear to what extent the decrease in Mg2+ content is responsible for the onset of short- and long-term complications of diabetes.

The present study is the first to investigate the changes in hepatic Mg2+ homeostasis and transport under type 1 diabetic conditions. The results reported here indicate that the lack of insulin results in a time-dependent loss of Mg2+ from liver cells and possibly other tissues as well. In the hepatocyte, this loss is consequent to a decrease in cellular ATP content and the inability of the cell to accumulate Mg2+ following specific hormonal stimuli and restore cellular Mg2+ homeostasis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of diabetes. Male Sprague-Dawley rats (200–220 g body wt) were rendered diabetic by a single intraperitoneal injection of 65 mg/kg body wt of streptozotocin in citrate buffer (pH 4.0). Urinary levels of glucose were measured by glucose strips (Fisher, Pittsburgh, PA) twice a week to monitor onset and progression of diabetes. The insurgence of diabetes was determined as the appearance of glucose in the urine, which occurred within 36 h of streptozotocin administration. During the period of this study, no insulin supplement was provided. The animals were maintained on a 12:12-h light-dark cycle and had free access to food and water. Fewer than 5% of the animals injected with streptozotocin did not become diabetic. These animals were used as additional controls to exclude the possibility that the observed effects were due to streptozotocin metabolism.

Determination of tissue ion content. At 4 and 8 wk after diabetes induction, diabetic and age-matched nondiabetic rats were anesthetized by intraperitoneal injection of a saturated pentobarbital sodium solution (50 mg/kg body wt). The abdomen was opened and the liver removed, rinsed in 250 mM sucrose, blotted on absorbing paper, weighed, and homogenized in 10% HNO3. After overnight digestion, aliquots of the acid extracts were transferred into microfuge tubes, and the denatured protein was sedimented at 8,000 g for 5 min. The acid supernatants were removed and assessed for Mg2+, Ca2+, Na+, and K+ content by atomic absorbance spectrophotometry (AAS) in a Perkin-Elmer 3100 after proper dilution. Cation content was normalized for protein content, measured according to the procedure of Lowry et al. (19) with bovine serum albumin as a standard.

Liver perfusion. Four- and eight-week diabetic and age-matched nondiabetic rats were anesthetized as indicated above. The abdomen was opened and the portal vein cannulated. The liver was perfused with a medium containing (in mM) 120 NaCl, 3 KCl, 1 CaCl2, 0.8 MgCl2, 1.2 K2HPO4, 12 NaHCO3, 15 glucose, 10 HEPES, pH 7.2, at 37°C, equilibrated with an O2-CO2 (95:5 vol/vol) gas mixture. The liver was quickly removed from the abdominal cavity, placed on a platform, and perfused at a flow rate of 3.5–4 ml·g–1·min–1. After a few minutes of equilibration, the perfusion medium was switched to one having a similar composition but devoid of Mg2+ (Mg2+-free medium). The contaminant Mg2+ present in the medium was measured by AAS and found to range between 7 and 10 µM. Samples of the perfusate were collected at 30-s intervals, and the Mg2+ content in the perfusate was measured by AAS. The first 10 min provided a baseline for the subsequent addition of adrenergic agonist. Phenylephrine (5 µM) or isoproterenol (10 µM) was dissolved directly into the perfusion medium and administered for the time reported in the figures. Pharmacological doses of the agonists were used to exclude reduced adrenoceptor responsiveness. To estimate the total amount of Mg2+ extruded from the organ, the Mg2+ content in the perfusate of the last six points before the adrenergic agonist addition was averaged and subtracted from each of the time points under the curve of efflux. The net amount of Mg2+ mobilized into the perfusate (nmol/ml) was then calculated, taking into account the perfusion rate (3.5–4 ml·g–1·min–1) and the time of collection (30 s), and expressed as micromoles. The residual Mg2+ content of perfused livers was also calculated in tissue homogenate, as described previously. The absence of cell damage was assessed by enzymatically measuring lactate dehydrogenase (LDH) activity in aliquots of the perfusate at 1-min intervals throughout the experimental procedure. The release of K+ from potentially damaged cells was also measured by AAS in aliquots of the perfusate (9, 31).

Hepatocyte isolation. Hepatocytes were isolated by collagenase digestion according to the procedure of Seglen (34). After isolation, the hepatocytes were resuspended in a medium having a composition similar to that reported in the previous section, containing 0.8 mM MgCl2, and kept at room temperature, under constant flow of O2-CO2 (95:5) until used. Cell viability, assessed by Trypan blue exclusion test, was found to be 90 ± 3, n = 9, and did not change significantly over the course of 4 h (88 ± 4, n = 8).

To determine Mg2+ transport, 1 ml of cell suspension was transferred to a microfuge tube, and the cells were rapidly sedimented at 600 g for 30 s and washed with 1 ml of the Mg2+-free medium described above. After the washing, the cells were transferred to 8 ml of Mg2+-free incubation medium, prewarmed at 37°C, and incubated under continuous O2-CO2 flow and stirring. After 2 min of equilibration, agonists for {alpha}1- or {beta}-adrenergic receptor or for protein kinase C signaling were added to the incubation system. At the time reported in the figures, 0.7 ml of incubation mixture was withdrawn in duplicate, and the cells were sedimented in microfuge tubes. The supernatants were removed, and their Mg2+ content was determined by AAS. The cell pellets were digested overnight in 10% HNO3, and the Mg2+ content of the acid extract was measured by AAS after sedimentation of the denatured protein in microfuge tube at 8,000 g for 5 min.

In a separate set of experiments, hepatocytes were incubated in the presence of 0.5 or 0.8 mM extracellular Mg2+. At selected times, aliquots of the incubation mixture were withdrawn in duplicate as reported above and sedimented in microfuge tubes through an oil layer containing dibutyl phthalate-dioctyl phthalate (2:1 vol/vol) to remove excess extracellular Mg2+. The supernatant and oil layer were removed by vacuum suction, and the cell pellet was digested overnight in 0.5 ml of 10% HNO3. The acid mixture was sonicated for 20 min, and the denaturated protein was sedimented at 8,000 g for 5 min. The Mg2+ content of the acid supernatant was measured by AAS.

Cellular Mg2+ distribution. To estimate the total cellular Mg2+ content and its distribution among cytoplasm, mitochondria, and other cellular organelles (mainly endoplasmic reticulum), hepatocytes isolated from diabetic and nondiabetic animals were sedimented, washed, and incubated in Mg2+-free medium as described above. Digitonin (50 µg/ml final concentration), FCCP (2 µg/ml), and A-23187 (2 µg/ml) were sequentially added to the incubation system at 5-min intervals, and aliquots of the incubation mixture were withdrawn and sedimented at 10,000 g for 2 min. The Mg2+ content of the supernatant was measured by AAS. The residual Mg2+ content in the cell pellets was also measured by AAS after acid digestion performed as reported in the previous section. The Mg2+ content present in the cell pellet or in the extracellular space before the addition of any stimulatory agent was calculated and subtracted from the following time point of incubation to determine the net amount of Mg2+ retained within the cell or released into the incubation medium.

Additional determinations. Aliquots of the perfusate were collected every minute, and glucose content was determined by enzymatic kit (Sigma) as the variation in hexokinase-coupled NADH+ content at 340 nm. LDH activity in the perfusate or in the extracellular medium was measured with an enzymatic kit (Sigma) sensitive enough to detect changes in the microunit per milliliter range and expressed as units per liter or as a percentage of the total amount of LDH releasable from digitonin-permeabilized hepatocytes. cAMP level was determined in aliquots of cell extract by 125I-labeled RIA (Amersham) as reported previously (38). Cellular ATP content was also measured in isolated hepatocytes by luciferin-luciferase assay (38), and by HPLC technique for comparison (7).

Plasma membrane purification. Total liver plasma membrane (tLPM) vesicles from diabetic and nondiabetic rats were isolated and stored as described in detail elsewhere (5). Plasma membrane purity was assessed by using 5'-nucleotidase, cytochrome c oxidase, and glucose-6-phosphatase activities as markers for plasma membrane, mitochondria, and endoplasmic reticulum, respectively (5). Negligible levels of cytochrome c oxidase and glucose-6-phosphatase activities were detected in both diabetic and nondiabetic tLPM compared with total liver homogenate. A fourfold enrichment in 5'-nucleotidase activity compared with total homogenate was observed in both preparations. Total LPM orientation, determined by measuring Na+-K+-ATPase and 5'-nucleotidase activities (5), indicated that >=90% of diabetic and nondiabetic tLPM loaded with Mg2+ were in the "inside-in" configuration. Na+-K+-ATPAse activity in diabetic tLPM was ~20% of that measured in nondiabetic tLPM (0.034 ± 0.001 vs. 0.18 ± 0.02 µmol Pi·mg protein–1·min–1, respectively; n = 7, P < 0.05).

Loading of LPM. Aliquots of tLPM were loaded with 20 mM Mg2+ or Na+, according to our published protocols (1). The Mg2+-loaded vesicles were resuspended in 5 ml of 250 mM sucrose and 25 mM K-HEPES, pH 7.4, in the absence of added Mg2+ (Mg2+-free medium) and stored in ice until used. Loading efficiency was assessed by treating the vesicles with ionophore (A-23187) or detergent (Triton X-100) and measuring the amount of Mg2+ extruded in the extravesicular space or retained within the vesicle pellet by AAS (1).

Measurement of Mg2+ fluxes. Mg2+-loaded tLPM were incubated in the Mg2+-free medium mentioned above, at 37°C under continuous stirring, at the final concentration of ~300 µg protein/ml. After 2 min of equilibration, aliquots of the incubation mixture were withdrawn in duplicate (t = 0 min), and the vesicles were sedimented in microfuge tubes at 7,000 g for 45 s. Total Mg2+ content in the supernatants was measured by AAS. The residual Mg2+ content within the vesicles was also measured by AAS after overnight digestion of the vesicle pellets in 500 µl of 10% HNO3 and sedimentation of denaturated proteins at 7,000 g for 5 min in microfuge tubes. The first time point after the equilibration period (t = 0) was used to assess basal vesicular and extravesicular Mg2+ level. After the withdrawal of the sample, Mg2+ transport was stimulated by addition of 25 mM Na+ to the incubation mixture. The incubation was continued for 6 additional minutes, and samples were withdrawn in duplicate at 2-min intervals. Because Mg2+ content in the supernatant could vary considerably among preparations as a result of the loading procedure and carry-over, the data are reported as the net variation in extravesicular (or vesicular) Mg2+ content, normalized per milligram of protein for simplicity. To determine net Mg2+ extrusion, Mg2+ content in the supernatant at t = 0 min was calculated and subtracted from the values of the subsequent time points of incubation.

Similar experimental procedures were used for Na+-loaded, Mg2+-stimulated vesicles.

Statistical analysis. The data are reported as means ± SE. Data were first analyzed by one-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for statistical significance of P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatic cation content. Rats rendered diabetic by streptozotocin injection presented an ~30% decrease in body weight compared with age-paired controls (256 ± 12 vs. 377 ± 18 g, respectively; n = 10 for each group) after 4 wk from the induction of diabetes. The decrease in body weight progressed with time and became more marked (~40%) at 8 wk from diabetes onset. Four-week diabetic animals presented a four- to fivefold increase in glycemia compared with age-matched controls (435 ± 25 vs. 85 ± 5 mg/100 ml, respectively). As shown in Table 1, total Mg2+ content decreased by ~10% in liver of rats diabetic for 4 wk. Potassium content also decreased to a comparable extent, whereas Na+ content increased. Calcium content also appeared to increase, although the amplitude of the changes was smaller compared with that of Na+, Mg2+, or K+ (not shown) and did not reach statistical significance. The changes in tissue cation content progressed over time. At 8 wk after diabetes induction, liver Mg2+ and K+ content were decreased by ~20% compared with age-matched nondiabetic rats, whereas tissue Na+ content increased proportionally (Table 1). Liver Mg2+ and K+ content were similarly lower compared with the levels determined in livers of weight-matched nondiabetic rats (not shown), thus excluding that the observed changes depended on difference in development. Streptozotocin-injected animals that did not develop diabetes presented hepatic cation content similar to that of nondiabetic animals (not shown).


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Table 1. Total liver cation content in 4- and 8-wk diabetic and age-matched nondiabetic animals

 

Mg2+ mobilization. In the past decade, our laboratory (9, 10, 2731) has steadily investigated the hormonal mechanisms that control cellular Mg2+ homeostasis in liver cells. Hence, we used perfused livers and collagenase-dispersed hepatocytes to assess whether 1) alteration in transmembrane Mg2+ transport could be responsible for the depletion in hepatic Mg2+content, and 2) an accumulation of Mg2+ into liver cells could be induced to restore cellular cation homeostasis.

Perfused livers of 4-wk diabetic and age-paired nondiabetic (normal) rats were stimulated by 10 µM isoproterenol (Fig. 1A) or 5 µM phenylephrine (Fig. 1B). As Fig. 1A shows, in the absence of any stimulating agent (basal conditions), no Mg2+ is extruded from nondiabetic or diabetic livers into the perfusate during the time course of our protocol. Under similar experimental conditions, the infusion of isoproterenol resulted in a marked extrusion of Mg2+ into the perfusate, which was quantitatively similar in diabetic and nondiabetic livers (1.7 vs. 1.5 µmol, respectively, as total net Mg2+ extrusion). Similar results were also obtained in livers stimulated by 250 µM 8-Cl-cAMP (a cell-permeant cAMP analog) instead of isoproterenol (not shown). The stimulation of {alpha}1-adrenoceptor by phenylephrine instead elicited a sizable extrusion of Mg2+ from nondiabetic livers but not from livers of diabetic rats, in which it was ~70% reduced (~3.75 vs. ~1.1 µmol, respectively; Figs. 1B and 2A). The estimate of the total amount of Mg2+ mobilized from the liver into the perfusate, calculated as reported in MATERIALS AND METHODS, confirmed a reduced responsiveness of {alpha}1-adrenoceptor signaling in diabetic livers, while indicating that phenylephrine mobilizes a larger amount of Mg2+ than isoproterenol in nondiabetic livers (~3.75 and ~1.50 µmol, respectively; Fig. 2A). Similar results were obtained in livers stimulated by methoxamine as {alpha}1-adrenoceptor agonist (data not shown). As expected, a negligible amount of glucose (<5 µmol/ml) was mobilized from diabetic livers by {alpha}1- or {beta}-adrenergic receptor stimulation compared with ~30 µmol/ml glucose extruded by either agonist in nondiabetic livers (Fig. 2B). The administration of isoproterenol and phenylephrine elicited similar results in livers from 8-wk diabetic rats (Figs. 3, A and B, respectively). The profile of Mg2+ extrusion induced by the mixed adrenergic agonist epinephrine was also markedly different in diabetic compared with nondiabetic livers (Fig. 3C). In both nondiabetic and diabetic livers, epinephrine elicited a Mg2+ extrusion that appeared to be quantitatively equivalent to the amounts of Mg2+ mobilized separately by phenylephrine and isoproterenol, as reported previously (9). In diabetic livers, however, epinephrine infusion resulted in a smaller and transient increase in Mg2+ extrusion, which returned toward basal level within 4 min from the agonist administration. At later time points following agonist removal, Mg2+ baseline appeared to be slightly elevated compared with nonstimulated livers and with nondiabetic livers treated with epinephrine (Fig. 3C); yet, the difference did not achieve statistical significance, nor was it accompanied by release of cellular LDH or K+. The Mg2+ extruded at these later time points was not taken into account in estimating total Mg2+ extrusion under these conditions, and the phenomenon was not further investigated at the present time. Glucose output from livers of 8-wk diabetic animals remained considerably lower than the values measured in age-matched nondiabetic rats (not shown).



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Fig. 1. Mg2+ extrusion from perfused livers stimulated by {beta}- and {alpha}1-adrenergic agonist. Livers from 4-wk diabetic and age-matched nondiabetic rats were perfused as described in MATERIALS AND METHODS. After a few minutes of equilibration, 10 µM isoproterenol (A) or 5 µM phenylephrine (B) were dissolved directly into the perfusion medium and administered for 10 min. Data are means ± SE of 6 different preparations for each experimental condition. *All data points under the curve of efflux in B were statistically significant vs. corresponding values in nondiabetic animals. Labeling omitted for simplicity.

 


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Fig. 2. Net Mg2+ and glucose extrusion from 4-wk-perfused rat livers. The net amount of Mg2+ (A) and glucose (B) extruded from rat livers reported in Fig. 1 were estimated as described in MATERIALS AND METHODS. Data are means ± SE of 6 different preparations for each experimental condition. *Statistically significant vs. value in nondiabetic animals.

 


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Fig. 3. Mg2+ extrusion from perfused livers stimulated by {beta}- and {alpha}1-adrenergic agonist. Livers from 8-wk diabetic and age-matched nondiabetic rats were perfused as described in MATERIALS AND METHODS. After a few minutes of equilibration, 10 µM isoproterenol (A), 5 µM phenylephrine (B), or 5 µM epinephrine (C) were dissolved directly into the perfusion medium and administered for 10 min. Data are means ± SE of 6 different preparations for each experimental condition. *All data points under the curve of efflux in B and C were statistically significant vs. corresponding values in nondiabetic animals. Labeling omitted for simplicity.

 

To better quantitate the amplitude of Mg2+ extrusion and to determine whether the loss of cellular Mg2+ resulted in a particular redistribution of Mg2+ within the cell, collagenase-dispersed hepatocytes were used under in vitro conditions. Hepatocytes isolated from 4-wk diabetic rats presented a 25% decrease in total Mg2+ content compared with age-paired controls (26.0 ± 0.6 vs. 36.1 ± 0.8 nmol/mg protein, respectively) and an ~20% decrease compared with liver cells from weight-matched nondiabetic animals (34.7 ± 0.5 nmol/mg protein; n = 4). When determined in hepatocytes from 8-wk diabetic rats, total Mg2+ content was further reduced vs. age-matched controls (24.2 ± 0.5 vs. 36.9 ± 0.4 nmol/mg protein, or minus ~35%). The decrease in cellular Mg2+ content was associated with a 17% decline in total ATP content at 4 wk after diabetes onset (~3.2 vs. 4.1 nmol ATP/mg protein in diabetic vs. nondiabetic hepatocytes, respectively). This decline was essentially similar to that determined in total liver homogenate of 4-wk diabetic rats compared with agematched nondiabetic animals (11.5 ± 0.1 vs. 14.1 ± 0.3 nmol ATP/mg protein, respectively; n = 9). A similar decline was also observed in liver cells from 8-wk diabetic rats vs. their nondiabetic controls (3.0 ± 0.2 vs. 4.1 ± 0.3 nmol ATP/mg protein, respectively). The decline in cellular Mg2+ and ATP content directly correlated at both 4 wk (r = 0.999, P < 0.0001) and 8 wk (r = 0.998, P < 0.01). Similar correlation values were also obtained when the declines in ATP and Mg2+ were correlated as percent changes. The decrease in Mg2+ content appeared to affect to a varying extent all cellular compartments (Fig. 4). The sequential administration of digitonin, mitochondria uncoupler FCCP, and ionophore A-23187 indicated a 20% decrease in cytosolic Mg2+ (digitonin), a 35% decrease in mitochondrial Mg2+ (FCCP), and a ~50% decrease in postmitochondrial Mg2+ (A-23187) in hepatocytes from 4-wk diabetic rats compared with age-matched nondiabetic animals. Figure 5A illustrates the incubation protocol utilized to assess Mg2+ transport in suspensions of hepatocytes from 4-wk diabetic and nondiabetic rats. In the absence of any stimulatory agent, hepatocytes in suspension did not release a detectable amount of Mg2+ into the extracellular space. After the addition of adrenergic agonist, the cells extruded a significant amount of Mg2+ into the external compartment. The net amount of Mg2+ mobilized by phenylephrine or isoproterenol from nondiabetic hepatocytes accounted for 1.6 ± 0.4 and 1.2 ± 0.3 nmol Mg2+/mg protein, respectively, over 4 min of stimulation (Fig. 5B). Under similar experimental conditions, diabetic hepatocytes mobilized 0.2 ± 0.04 and 1.1 ± 0.2 nmol Mg2+/mg protein, respectively (Fig. 5B). Also in this experimental model, epinephrine elicited a Mg2+ extrusion that was quantitatively equivalent to the amounts mobilized separately by isoproterenol and phenylephrine, although it remained significantly smaller in diabetic compared with nondiabetic hepatocytes (1.8 ± 0.3 vs. 3.5 ± 0.5 nmol Mg2+·mg protein–1·6 min–1, respectively; n = 8, P < 0.05). The inability of phenylephrine (or methoxamine, not shown) to mobilize Mg2+ from diabetic hepatocytes cannot not be ascribed merely to a defect in receptor responsiveness, as a similar lack of extrusion was observed in hepatocytes treated with thapsigargin, an agent that mimics {alpha}1-adrenoceptor-mediated Mg2+ extrusion in liver cells bypassing the receptor (10). The addition of 2 µM thapsigargin, in fact, elicited the extrusion of 0.4 ± 0.1 nmol Mg2+·mg protein–1·6 min–1 from diabetic hepatocytes compared with 1.7 ± 0.2 nmol Mg2+·mg protein–1·6 min–1 from nondiabetic cells (n = 8, P < 0.05). Cellular Mg2+ partitioning in nondiabetic hepatocytes undergoing stimulation by adrenergic agonists indicated a decrease at the cytoplasmic and mitochondrial levels following stimulation by isoproterenol (–7 and –37%, respectively, vs. basal values) and a decrease in the postmitochondrial pool (–21% vs. basal value) after phenylephrine stimulation (Fig. 6). In hepatocytes from diabetic rats instead, isoproterenol stimulation resulted in a further decrease in cytoplasm and mitochondria pools compared with the level before agonist addition (–6 and –18%, respectively), whereas phenylephrine addition did not elicit an appreciable decrease in the postmitochondrial pool (Fig. 6). When correlated with the basal Mg2+ content present in the different cellular compartments, the amount of Mg2+ extruded by isoproterenol or phenylephrine stimulation in nondiabetic hepatocytes resulted in the values r = 0.177, P < 0.01 and r = 0.133, P < 0.02, respectively, for the cytosol; r = –0.948, P < 0.01 and r = 1.55e–15, P < 0.001, respectively, for mitochondria, and r = 0.755, P < 0.1 and r = –0.07, P < 0.1, respectively, for the postmitochondrial pools. In diabetic hepatocytes, similar correlation analysis resulted in values of r = 0.42, P < 0.06 and r = –0.73, P < 0.03, respectively, for the cytosol; r = 0.24, P < 0.01 and r = –0.91, P < 0.01, respectively, for mitochondria, and r = –0.53, P < 0.03 and r = 0.98, P < 0.01, respectively, for the postmitochondrial compartments. Comparable values were obtained when basal Mg2+ content was correlated with percent magnesium extrusion following {beta}- and {alpha}1-adrenoceptor agonist stimulation. The correlation between the Mg2+ content present within the various cellular compartments before agonist stimulation and the Mg2+ content remaining therein after adrenergic stimulation resulted in the values r = 0.994, P < 0.002 and r = 0.998, P < 0.001 for isoproterenol- and phenylephrine-stimulated nondiabetic hepatocytes, respectively, and r = 0.993, P < 0.002 and r = 0.998, P < 0.001, respectively, in diabetic hepatocytes. Finally, the correlation between the amount of Mg2+ lost from the different compartments and the net amount of Mg2+ mobilized into the extracellular compartment resulted in the values r = 0.30, P < 0.02 and r = 0.23, P < 0.01 for isoproterenol- and phenylephrine-stimulated nondiabetic cells, respectively. In diabetic hepatocytes, inverse correlations were measured for both isoproterenol (r = –0.59, P < 0.01) and phenylephrine stimulation (r = –0.10, P < 0.01).



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Fig. 4. Net Mg2+ extrusion from various intracellular compartments. Hepatocytes from 4-wk diabetic and age-matched nondiabetic rats were isolated and incubated as indicated in MATERIALS AND METHODS. After a few minutes of equilibration, digitonin (50 µg/ml), the mitochondria uncoupler FCCP (52 µg/ml), and the ionophore A-23187 (2 µg/ml) were added sequentially at 5-min intervals. Net Mg2+ extrusion was calculated as described in MATERIALS AND METHODS. Data are means ± SE of 6 different preparations, each performed in quadruplicate. *Statistically significant vs. nondiabetic hepatocytes.

 


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Fig. 5. Mg2+ extrusion from isolated hepatocytes. Hepatocytes from 4-wk diabetic and age-matched nondiabetic rats were incubated in a medium devoid of Mg2+ (see MATERIALS AND METHODS). After a few minutes of equilibration, cells were stimulated by addition of isoproterenol (ISO, 10 µM) or phenylephrine (PHE, 5 µM). Ctrl, control. A: typical incubation profile. Net Mg2+ extrusion for hepatocytes stimulated by adrenergic agonists (B) was determined as described in MATERIALS AND METHODS. Data are means ± SE of 6 different experiments for all experimental conditions, each performed in quadruplicate. B: *statistically significant vs. nondiabetic hepatocytes.

 


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Fig. 6. Net Mg2+ extrusion from various intracellular compartments. Hepatocytes from 4-wk diabetic and age-matched nondiabetic rats were isolated and incubated as indicated in MATERIALS AND METHODS. After a few minutes of equilibration, cells were stimulated with 10 µM isoproterenol or 5 µM phenylephrine for 6 min. Digitonin (50 µg/ml), FCCP (52 µg/ml), and A-23187 (2 µg/ml) were then added sequentially at 5-min intervals. Net Mg2+ extrusion was calculated as described in MATERIALS AND METHODS using the values at 6 min as basal level. Data are means ± SE of 6 different preparations, each performed in quadruplicate. All values for diabetic hepatocytes are statistically significant vs. nondiabetic hepatocytes. Labeling omitted for simplicity.

 

The experimental protocol reported in Fig. 5A was also used to investigate whether hepatocytes from 4-wk diabetic animals could accumulate Mg2+ following stimulation of the protein kinase C-signaling pathway. After 3 min of equilibration, various agents that stimulate Mg2+ accumulation in liver cells were added to the incubation mixture. As Fig. 7A indicates, the administration of vasopressin, insulin, oleoyl-arachidonoyl glycerol (OAG, a cell-permeant diacylglycerol analog), or phorbol 12,13-dibutyrate (PDBu, an analog of PMA), all induced a net accumulation of ~1–1.5 nmol Mg2+·mg protein–1·4 min–1 in hepatocytes from nondiabetic animals. In contrast, hepatocytes from diabetic animals were unable to accumulate Mg2+ irrespective of the agonist (Fig. 7A), the dose of the agent (not shown), or the extracellular Mg2+ concentration (Fig. 7B) utilized. In nondiabetic hepatocytes, the amplitude of Mg2+ accumulation elicited by the various protein kinase C agonists was unaffected by changes in extracellular Mg2+ concentration (not shown; see Ref. 29). Qualitatively similar results in terms of Mg2+ accumulation and extrusion were also observed in hepatocytes isolated from 8-wk diabetic animals (not shown).



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Fig. 7. Mg2+ accumulation in isolated hepatocytes. Hepatocytes from 4-wk diabetic and age-matched nondiabetic rats were incubated in a medium containing trace contaminant (A) or 1 mM external Mg2+ (B). After a few minutes of equilibration, the hepatocytes were stimulated by addition of vasopressin (AVP, 20 nM), insulin (10 nM), oleoyl-arachidonoyl glycerol (OAG, 20 nM), and phorbol 12,13-dibutyrate (PDBu, 20 µM). After 4 min of stimulation, total Mg2+ content in the cell pellet was determined as described in MATERIALS AND METHODS. Data are means ± SE of 6 different experiments for all experimental conditions, each performed in quadruplicate. A: *statistically significant vs. nondiabetic hepatocytes. B: all data points statistically significant vs. nondiabetic hepatocytes values reported in A. Labeling omitted for simplicity.

 

We (5) have evidenced the operation of bidirectional Na+-dependent Mg2+ transport mechanisms in purified tLPM vesicles. To determine whether the lack of Mg2+ accumulation depended on the defective operation of the Mg2+ entry mechanisms, tLPM vesicles were purified from 4-wk diabetic and age-matched nondiabetic rats and loaded with 20 mM Mg2+ or 20 mM Na+. As Fig. 8A indicates, Mg2+-loaded diabetic tLPM vesicles extruded about three times more Mg2+ than tLPM from nondiabetic livers following addition of 25 mM Na+ in the extravesicular space. In contrast, when the cation gradient was reversed, diabetic tLPM did not accumulate Mg2+ in exchange for vesicular entrapped Na+ (Fig. 8B). Under similar experimental conditions, nondiabetic tLPM accumulate ~200 nmol Mg2+/mg protein within 2 min from the addition of 20 mM extravesicular Mg2+ (Fig. 8B).



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Fig. 8. Mg2+ transport in total liver plasma membranes (tLPM). tLPM from 4-wk diabetic and nondiabetic rats were isolated and loaded with 20 mM Mg2+ (A) or 20 mM Na+ (B). Mg2+ extrusion was elicited by extravesicular addition of 25 mM Na+ (A). Magnesium accumulation was elicited by addition of 20 mM Mg2+ in the extravesicular space (B). *Statistically significant vs. corresponding data point in nondiabetic tLPM.

 

Streptozotocin-injected animals that did not develop diabetes were used as additional controls throughout the study. Liver cells and tLPM from these animals extruded and accumulated Mg2+ in a manner similar to nondiabetic animals (not shown), excluding therefore that the observed effects in perfused livers, isolated cells, or tLPM were due to drug metabolism.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The past decade has registered an increasing interest in understanding how Mg2+ is transported and regulated in mammalian cells as well as the implications that changes in cellular Mg2+ content have for the overall cell functioning under physiological and pathological conditions.

As indicated in the introductory section, experimental and clinical evidence indicates that plasma and tissue Mg2+ contents are markedly decreased in both type 1 and type 2 diabetes (26, 40). Although a decreased phosphorylation of insulin receptor and downstream signaling effectors has been observed in Mg2+-depleted tissues of animals fed a Mg2+-deficient diet (35), no organic study has been carried out to date to elucidate the modality by which Mg2+ loss occurs in diabetic animals or patients and the short- and long-term implications of Mg2+ deficiency for the proper functioning of cellular metabolic processes under this pathological condition.

The present study was undertaken to address some of these questions. In particular, we wanted to investigate whether the decrease in Mg2+ content could be ascribed to an altered transport across the cell membrane of the hepatocyte.

Mg2+ homeostasis within the hepatocyte. The onset of type 1 diabetes results in a time-dependent loss of hepatic Mg2+, which is associated with a comparable loss of K+ and ATP and an increase in Na+ content. The increase in tissue Na+ content (and Ca2+; not shown) excludes the possibility that Mg2+ and K+ loss is a nonspecific process. Although the decreased activity of Na+-K+-ATPase observed in tLPM and homogenate under diabetic conditions can explain the modifications in Na+ and K+ content, other mechanisms have to be invoked to explain the decrease in hepatic Mg2+ content.

The larger loss in Mg2+ content detected in hepatocytes compared with total liver extract is probably due to the removal of collagen and tissue components other than the hepatocytes, which introduces some degree of inaccuracy in total tissue determination. Within the cell, Mg2+ loss appears to affect all organelles and compartments, including cytoplasm. In fact, the depletion in cytosolic Mg2+ (~20%; Fig. 4) directly correlates with and is almost superimposable on the 17% decrease in ATP content, further supporting the notion that ATP represents the main regulatory component of cytosolic Mg2+ content (33). Based on a total ATP content of 4 mM, a 17% decrease corresponds to ~700 µM. As ATP is degraded to ADP and AMP, the dissociation constant of these moieties for Mg2+ decreases from ~80 µM (Mg-ATP) to ~8.13 mM (Mg-AMP), resulting in the dissociation of a significant amount of Mg2+ that ultimately is extruded from the cell. The discrepancy between the total amount of cellular Mg2+ (10 nmol/mg protein) vs. ATP content (~1 nmol/mg protein) lost from the diabetic hepatocytes can be explained by the fact that Mg2+ is lost from all cellular organelles and compartments (in particular the postmitochondrial compartments) and not merely from the cytoplasm and mitochondria, in which ATP is predominantly located. Although phosphocreatine was not measured in the present study, a decrease in ATP content of the amplitude observed here (~17%) would imply a decrease in this ATP-regenerating moiety. Because phosphocreatine binds cytoplasmic Mg2+ as well (30), its decrease is likely to contribute to the decrease in cytoplasmic Mg2+ observed in diabetic hepatocytes. A similar decrease in Mg2+ and ATP content has been observed in hepatocytes treated acutely (38) or chronically (43) with EtOH. This result suggests that the cell possesses a specific but still-unidentified mechanism that senses variations in cytosolic Mg2+ and determines the extrusion of excess Mg2+ across the plasma membrane. As for the changes in other cellular compartments, they can be either secondary to the changes in bound/free Mg2+ in the cytoplasm or consequent to a direct modification of the organelle functioning under diabetic conditions. It has been reported that Mg2+ plays a key role in regulating various organelle enzymes, including mitochondrial dehydrogenases (23) and reticular ATPases (8). Hence, it can be speculated that a loss of Mg2+ within organelles can contribute to some of the metabolic changes observed under diabetic conditions. This speculation is supported by the correlation between Mg2+ content within cellular organelles and the amplitude of Mg2+ extrusion following {beta}- and {alpha}1-adrenergic stimulation in nondiabetic and diabetic hepatocytes.

Mg2+ entry across the cell membrane. The most important information provided by this study is that liver cells from diabetic animals are unable to accumulate Mg2+ to restore the cation cellular homeostasis. This defect persists irrespective of the extracellular Mg2+ concentration (contaminant or physiological 1 mM) and is consistent with the hypothesis that Mg2+ entry does not depend on the Mg2+ gradient across the cell membrane but is a tightly regulated process (14, 32). Previous reports indicate that Mg2+ accumulation in liver cell is regulated via protein kinase C activation (28) and inhibited under conditions in which protein kinase C is downregulated (28) or intracellular Ca2+ is elevated (30). Under diabetic conditions, defects in protein kinase C signaling (37) and an altered Ca2+ cycling between cytoplasm and sarcoendoplasmic reticulum (18) have both been reported and attributed to changes in the plasma membrane phospholipid environment (22), an altered cross talk among signaling molecules (11), or an abnormal operation of the reticular Ca2+-sequestering/releasing mechanisms (18). The inability of diabetic tLPM to accumulate Mg2+ in exchange for intravesicular Na+ (Fig. 8B) via the putative bidirectional Na+/Mg2+ exchanger (5) indicates that functional (e.g., faulty signaling or phosphorylation) and/or structural modifications (e.g., glycation at the extracellular side or alteration in the phospholipid environment) affect directly or indirectly the Mg2+ transport mechanism. Further studies are necessary to discriminate among these possibilities. The possibility that Mg2+ entry is an energy-dependent process (e.g., a pump) that becomes defective in diabetic hepatocytes due to the reduced cellular ATP level appears to be unlikely, as results obtained in nondiabetic tLPM indicate that Mg2+ enters the vesicle in exchange for entrapped Na+ in the absence of intravesicular ATP (Fig. 8B; see also Ref. 5). Yet, a reduced phosphorylation/activation of the Mg2+ transporter as a consequence of the reduced cellular level of ATP cannot be excluded altogether.

Mg2+ extrusion across the cell membrane. Our results also indicate that a difference exists between {alpha}1- and {beta}-adrenoceptor-mediated Mg2+ extrusion processes. Although the latter process appears to operate normally irrespective of the time elapsed since diabetes onset, the {alpha}1-adrenergic receptor-mediated process is markedly reduced (~50% or more). As both classes of adrenoceptor activate the same Mg2+ extrusion mechanism [i.e., the Na+/Mg2+ exchanger (9, 10)] that appears to be operative in cells stimulated by {beta}-adrenoceptor agonist and tLPM (Fig. 7A), we have to hypothesize that the defect is at the level of 1) the {alpha}1-adrenoceptor, 2) the associated signaling pathway activating the transporter, or 3) the cellular compartment from which Mg2+ should be mobilized. Although the first two possibilities are not excluded, the inability of thapsigargin to restore Mg2+ extrusion bypassing the {alpha}1-adrenoceptor (10) suggests that the depletion of the cellular Mg2+ compartment targeted by {alpha}1-adrenoceptor stimulation (most likely the endoplasmic reticulum) is one of the main reasons for the lack of Mg2+ extrusion under these conditions. Further support for this possibility is provided by the estimate of cellular Mg2+ compartmentalization (Fig. 4), which indicates a ~50% depletion of postmitochondrial pools under diabetic conditions, which does not change significantly following phenylephrine stimulation (Fig. 6). In contrast, administration of isoproterenol to diabetic hepatocytes results in a further decrease in cytoplasmic and mitochondrial Mg2+ content compared with basal level (Fig. 6). These results, however, do not necessarily exclude the occurrence of Mg2+ redistribution among cellular compartments to an extent that may vary in diabetic compared with nondiabetic cells. Additional, albeit indirect, evidence for the mobilization of Mg2+ from distinct cellular pools following {alpha}1- and {beta}-adrenoceptor stimulation (9) is provided by the normal response to {beta}-adrenoceptor agonist (isoproterenol) or its second messenger cAMP and by the partial responsiveness of diabetic liver cells to the mixed adrenoceptor agonist epinephrine. As mitochondrial and cytosolic Mg2+ pools appear to be less depleted than postmitochondrial (ionophore-sensitive) pools, it can be speculated that they constitute the intracellular pool(s) involved in {beta}-adrenergic-mediated Mg2+ extrusion. This hypothesis will be consistent with an early report from this laboratory (27) about the occurrence of a cAMP-mediated Mg2+ extusion from permeabilized hepatocytes or isolated liver mitochondria. It must be noted that the normal Mg2+ extrusion elicited via {beta}-adrenergic receptor activation in liver cells appears to contrast with data available in the literature indicating a defective responsiveness of this signaling pathway in the heart of diabetic animals (21). The reason for this differing response is not apparent, although the different {beta}-adrenoceptor subtypes expressed in these tissues [{beta}1 in cardiac cells and {beta}2 in liver cells (4)] and differences in postreceptor signaling between {beta}1- and {beta}2-adrenoceptor, as reported by various laboratories (42), can contribute to explain it.

Overall, the comparison between the percent and absolute decrease in cellular and intraorganelle Mg2+ content (–35 to –45%, according to the compartment considered), and the decreased amplitude of Mg2+ extrusion by phenylephrine or mixed adrenergic agonist (~50%) or uptake following OAG or PMA stimulation observed in diabetic hepatocytes suggests a reduced steady state in cellular Mg2+ as a result of a reduced buffering capacity and intracellular storing. This would be consistent with a general property of ion homeostasis, by which cellular steady-state level regulates cellular responsiveness to extracellular and intracellular mobilizing stimuli (1, 2) However, caution must be exercised in interpreting our data, in that the high concentration of glucose utilized under our experimental conditions, which is two- to threefold larger than the concentration utilized in other studies on liver cells and resembles extracellular diabetic environment, may per se alter intracellular ion concentration (1, 3).

In conclusion, the results of our study indicate that liver cells from type 1 diabetic rats present a considerable loss of Mg2+. This Mg2+ loss depends on a decrease in cellular ATP and the inability of the hepatocyte to accumulate Mg2+ from the extracellular compartment and restore cellular Mg2+ homeostasis. The defect is localized at the level of the Mg2+ entry mechanism in the plasma membrane and possibly on its activating signaling. Overall, these results provide a basic understanding of the defect(s) responsible for the decrease in cellular Mg2+ content under diabetic conditions as well as providing an important framework for further studies aimed at determining the role this Mg2+ loss plays for the short- and long-term complications of diabetes.


    ACKNOWLEDGMENTS
 
GRANTS

This study was supported by National Institute of Alcohol Abuse & Alcoholism Grant R9A11593 and National Heart, Lung, and Blood Institute Grant HL-18708.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Romani, Dept. of Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106–4970 (E-mail: amr5{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barbagallo M, Dominguez LJ, Bardicef O, and Resnick LM. Altered cellular magnesium responsiveness to hyperglycemia in hypertensive subjects. Hypertension 38: 612–615, 2001.[Abstract/Free Full Text]
  2. Barbagallo M, Gupta RK, Bardicef O, Bardicef M, and Resnick LM. Altered ionic effects of insulin in hypertension: Role of basal ion levels in determining cellular responsiveness. J Clin Endocrinol Metab 82: 1761–1765, 1997.[Abstract/Free Full Text]
  3. Barbagallo M, Shan J, Pang PK, and Resnick LM. Glucose-induced alterations of cytosolic free calcium in cultured rat tail artery vascular smooth muscle cells. J Clin Invest 95: 763–767, 1995.[ISI][Medline]
  4. Barnes PJ. Beta-adrenergic receptors and their regulation. Am J Respir Crit Care Med 152: 838–860, 1995.[ISI][Medline]
  5. Cefaratti C, Romani A, and Scarpa A. Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver. Am J Physiol Cell Physiol 275: C959–C1008, 1998.
  6. Cefaratti C, Romani A, and Scarpa A. Differential localization and operation of distinct Mg2+ transporters in apical and basolateral sides of rat liver plasma membrane. J Biol Chem 275: 3772–3880, 2000.[Abstract/Free Full Text]
  7. De Young MB, Giannattasio B, and Scarpa A. Isolation of calcium-tolerant atrial and ventricular myocytes from adult rat heart. Methods Enzymol 173: 662–676, 1989.[ISI][Medline]
  8. Duggleby RC, East M, and Lee AG. Luminal dissociation of Ca2+ from the phosphorylated Ca2+-ATPase is sequential and gated by Mg2+. Biochem J 339: 351–357, 1999.[ISI][Medline]
  9. Fagan TE and Romani A. Activation of Na+- and Ca2+-dependent Mg2+ extrusion by {alpha}1- and {beta}-adrenergic agonists in rat liver cells. Am J Physiol Gastrointest Liver Physiol 279: G943–G950, 2000.[Abstract/Free Full Text]
  10. Fagan TE and Romani A. {alpha}1-adrenoceptor-induced Mg2+ extrusion from rat hepatocytes occurs via Na+-dependent transport mechanism. Am J Physiol Gastrointest Liver Physiol 280: G1145–G1156, 2001.[Abstract/Free Full Text]
  11. Farese RV. Insulin-sensitive phospholipid signaling systems and glucose transport. Update II. Exp Biol Med 226: 283–295, 2001.[Abstract/Free Full Text]
  12. Feray J-C and Garay R. Demonstration of a Na+:Mg2+ exchange in human red cells by its sensitivity to tricyclic antidepressant drugs. Naunyn-Schmied Arch Pharmacol 338: 332–337, 1988.[ISI][Medline]
  13. Flatman PW. Mechanisms of magnesium transport. Annu Rev Physiol 53: 259–272, 1991.[ISI][Medline]
  14. Gunther T. Functional comparmentation of intracellular magnesium. Magnesium 5: 53–59, 1986.[ISI][Medline]
  15. Gunther T and Vormann J. Mg2+ efflux is accomplished by an amiloride-sensitive Na+/Mg2+ antiport. Biochem Biophys Res Commun 130: 540–545, 1985.[ISI][Medline]
  16. Gunther T and Vormann J. Characterization of Na+-independent Mg2+ efflux from erythrocytes. FEBS Lett 271: 149–151 1990.[ISI][Medline]
  17. Ishijima S and Tatibana M. Rapid mobilization of intracellular Mg2+ by bombesin in Swiss 3T3 cells. Mobilization through external Ca2+ and tyrosine kinase-dependent mechanism. J Biochem (Tokyo) 115: 730–737, 1994.[Abstract]
  18. Levy J. Abnormal cell calcium homeostasis in type 2 diabetes mellitus: a new look on old disease. Endocrine 10: 1–6, 1999.[ISI][Medline]
  19. Lowry OH, Rosebrough NJ, Farr Al, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  20. Matsuura T, Kanayama Y, Inoue T, Takeda T, and Morishima I. cAMP-induced changes of intra-cellular Mg2+ levels in human erythrocytes. Biochim Biophys Acta 1220: 31–36, 1993.[ISI][Medline]
  21. Molinoff PB. Alpha- and beta-adrenergic receptors subtypes, properties, distribution and regulation. Drugs 28: 1–14, 1984.[ISI][Medline]
  22. Nassar K, Cheng S, and Levy D. The effect of diabetes on hepatocyte plasma membrane fluidity and concanavalin A-induced agglutination. Exp Cell Res 132: 99–104, 1981.[ISI][Medline]
  23. Panov A and Scarpa A. Independent modulation of the activity of {alpha}-ketoglutarate dehydrogenase complex by Ca2+ and Mg2+. Biochemistry 35: 427–432, 1996.[ISI][Medline]
  24. Quamme GA and Dai LJ. Presence of a novel influx pathway for Mg2+ in MDCK cells. Am J Physiol Cell Physiol 259: C521–C525, 1990.[Abstract/Free Full Text]
  25. Quamme GA and Rabkin SW. Cytosolic free magnesium in cardiac myocytes: identification of a Mg2+ influx pathway. Biochem Biophys Res Commun 167: 1406–1412, 1990.[ISI][Medline]
  26. Resnick LM, Altura BT, Gupta RK, Laragh JH, Alderman MH, and Altura BM. Intracellular and extracellular magnesium depletion in type w (noninsulin-dependent) diabetes mellitus. Diabetologia 36: 767–770, 1993.[ISI][Medline]
  27. Romani A, Dowell E, and Scarpa A. Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria. J Biol Chem 266: 24376–24384, 1991.[Abstract/Free Full Text]
  28. Romani A, Marfella C, and Scarpa A. Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C. FEBS Lett 296: 135–140, 1992.[ISI][Medline]
  29. Romani A, Marfella C, and Scarpa A. Regulation of magnesium uptake and release in the heart and in isolated ventricular myocytes. Circ Res 72: 1139–1148, 1993.[Abstract]
  30. Romani A, Marfella C, and Scarpa A. Hormonal regulation of Mg2+ uptake in hepatocytes. J Biol Chem 268: 15489–15495, 1993.[Abstract/Free Full Text]
  31. Romani A and Scarpa A. Hormonal control of Mg2+ in the heart. Nature 346: 841–844, 1990.[ISI][Medline]
  32. Romani A and Scarpa A. Regulation of cell magnesium. Arch Biochem Biophys 298: 1–12, 1992.[ISI][Medline]
  33. Scarpa A and Brinley FJ. In situ measurements of free cytosolic magnesium ions. Fed Proc 40: 2646–52, 1981.[ISI][Medline]
  34. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 13: 29–83, 1976.[Medline]
  35. Suarez A, Pulido N, Casla A, Casanova B, Arrieta FJ, and Rovira A. Impaired tyrosine-kinase activity of muscle insulin receptors from hypomagnesaemic rats. Diabetologia 38: 1262–1270, 1995.[ISI][Medline]
  36. Takaya J, Higashino H, Miyazaki R, and Kobayashi Y. Effects of insulin and insulin-like growth factor-1 on intracellular magnesium of platelets. Exp Mol Pathol 65: 104–109, 1998.[ISI][Medline]
  37. Tang EY, Parker PJ, Beattie J, and Houslay MD. Diabetes induces selective alterations in the expression of protein kinase C isoforms in hepatocytes. FEBS Lett 326: 117–123, 1993.[ISI][Medline]
  38. Tessman PA and Romani A. Acute effect of EtOH on Mg2+ homeostasis in liver cell: evidence for the activation of an Na+/Mg2+ exchanger. Am J Physiol Gastrointest Liver Physiol 275: G1115–G1116, 1998.
  39. Vormann J and Gunther T. Amiloride sensitive net Mg2+ efflux from isolated perfused rat hearts. Magnesium 6: 220–224, 1987.[ISI][Medline]
  40. Wallach S and Verch R. Tissue magnesium content in diabetic rats. Magnesium 6: 302–306, 1987.[ISI][Medline]
  41. Wolf FI, Di Francesco A, Covacci V, and Cittadini A. Regulation of magnesium efflux from rat spleen lymphocytes. Arch Biochem Biophys 344: 397–403, 1997.[ISI][Medline]
  42. Xiao RP, Hohl C, Altschuld R, Jones L, Livingston B, Ziman B, Tantini B, and Lakatta EG. Beta 2-adrenergic receptor-stimulated increase in cAMP in rat heart cells is not coupled to changes in Ca2+ dynamics, contractility, or phospholamban phosphorylation. J Biol Chem 269: 19151–19156, 1994.[Abstract/Free Full Text]
  43. Young A, Cefaratti C, and Romani A. Chronic EtOH administration alters liver Mg2+ homeostasis. Am J Physiol Gastrointest Liver Physiol 284: G57–G67, 2003.[Abstract/Free Full Text]




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