Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
Submitted 2 May 2003 ; accepted in final form 6 October 2003
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ABSTRACT |
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magnesium; adrenergic signaling; protein kinase c; adenosine 5'-triphosphate; plasma membrane
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.
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MATERIALS AND METHODS |
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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 3.54 ml·g1·min1. 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 (3.54 ml·g1·min1) 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 1- or
-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 protein1·min1, 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.
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RESULTS |
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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 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
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
1-adrenoceptor agonist (data not shown). As expected, a negligible amount of glucose (<5 µmol/ml) was mobilized from diabetic livers by
1- or
-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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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 protein1·6 min1, 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
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 protein1·6 min1 from diabetic hepatocytes compared with 1.7 ± 0.2 nmol Mg2+·mg protein1·6 min1 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 = 1.55e15, 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
- and
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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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 11.5 nmol Mg2+·mg protein1·4 min1 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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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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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.
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DISCUSSION |
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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
- and
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 1- and
-adrenoceptor-mediated Mg2+ extrusion processes. Although the latter process appears to operate normally irrespective of the time elapsed since diabetes onset, the
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
-adrenoceptor agonist and tLPM (Fig. 7A), we have to hypothesize that the defect is at the level of 1) the
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
1-adrenoceptor (10) suggests that the depletion of the cellular Mg2+ compartment targeted by
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
1- and
-adrenoceptor stimulation (9) is provided by the normal response to
-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
-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
-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
-adrenoceptor subtypes expressed in these tissues [
1 in cardiac cells and
2 in liver cells (4)] and differences in postreceptor signaling between
1- and
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.
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ACKNOWLEDGMENTS |
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This study was supported by National Institute of Alcohol Abuse & Alcoholism Grant R9A11593 and National Heart, Lung, and Blood Institute Grant HL-18708.
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FOOTNOTES |
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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.
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REFERENCES |
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