Role of glucose in modulating Mg2+ homeostasis in liver cells from starved rats

Lisa M. Torres, Jonathan Youngner, and Andrea Romani

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

Submitted 19 November 2003 ; accepted in final form 7 September 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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{alpha}1- and {beta}-Adrenoceptor stimulation elicits Mg2+ extrusion from liver cells in conjunction with hepatic glucose output (T. Fagan and A. Romani. Am J Physiol Gastrointest Liver Physiol 279: G943–G950, 2000.). To characterize the role of intrahepatic glucose on Mg2+ transport, male Sprague-Dawley rats were starved overnight before being anesthetized and used as organ donors. Perfused livers or collagenase-dispersed hepatocytes were stimulated by {alpha}1 (phenylephrine)- or {beta} (isoproterenol)-adrenergic agonists. Mg2+ extrusion was assessed by atomic absorbance spectrophotometry. In both experimental models, the administration of pharmacological doses of adrenergic agonists did not elicit Mg2+ extrusion. The determination of cellular Mg2+ indicated an ~9% decrease in total hepatic Mg2+ content in liver cells after overnight fasting, whereas the ATP level was unchanged. Hepatocytes from starved rats accumulated approximately four times more Mg2+ than liver cells from fed animals. This enlarged Mg2+ accumulation depended in part on extracellular glucose, since it was markedly reduced in the absence of extracellular glucose or in the presence of the glucose transport inhibitor phloretin. The residual Mg2+ accumulation observed in the absence of extracellular glucose was completely abolished by imipramine or removal of extracellular Na+. Taken together, these data indicate 1) that hepatic glucose mobilization is essential for Mg2+ extrusion by adrenergic agonist and 2) that starved hepatocytes accumulate Mg2+ via two distinct pathways, one of which is associated with glucose transport, whereas the second can be tentatively identified as an imipramine-inhibited Na+-dependent pathway.

magnesium; adrenergic stimulation; amiloride; magnesium transport


IN THE PAST TWO DECADES, the modality of Mg2+ transport in and out of the cell and its role on cellular function has been investigated extensively. In most mammalian cells, a total cellular Mg2+ concentration of 16–20 mM and a cytosolic free Mg2+ concentration of 0.5–1 mM have been measured (1416, 40). Several laboratories, including ours (19, 21, 22, 29, 35, 3739, 44, 46), have provided compelling evidence for the extrusion of cellular Mg2+ after the administration of adrenergic agonists, such as phenylephrine ({alpha}1-adrenoceptor agonist; see Refs. 5 and 6), isoproterenol ({beta}-adrenoceptor agonist; see Refs. 25 and 37), and epinephrine (mix {alpha}1- and {beta}-adrenoceptor agonists; see Refs. 5 and 25), in various cell types. These fluxes are quite large, accounting for 5–10% of total cellular Mg2+, and occur within a few minutes from the administration of the stimulus to the cell (5, 6, 21, 35, 3739). Mg2+ extrusion occurs via a putative cAMP-phosphorylated Na+/Mg2+ exchanger (9, 18, 19) and an Na+-independent pathway (16, 37). Whereas the former transporter is strictly Na+-dependent and able to operate in either direction (2, 17), at least in purified plasma membrane vesicles (2), the second pathway would exchange Mg2+ for extracellular Ca2+, Mn2+ (8, 16, 20, 35, 36), or other cations (16, 40) or would cotransport Mg2+ together with HCO3 or Cl (16, 40). Comparable amounts of Mg2+ can also be accumulated in the cell within the same period of time after the administration of hormones, like insulin (37) and vasopressin (35), or protein kinase C-activating agents, like diacylglycerol analogs or phorbol myristate derivates (35), in a Na+-dependent (35), Ca2+-independent (7) manner.

Among the various metabolic functions within the cell, Mg2+ plays an essential role in regulating glycolysis (10). In Ehrlich ascites tumor cells, extracellular Mg2+ may regulate the key glycolytic enzyme phosphofructokinase (45). In human erythrocytes, glucose metabolism is dependent on the intracellular concentration of Mg2+ (27). Furthermore, removing Mg2+ from cells inhibits glycolysis (24) and glucose accumulation (37). In fact, lowering Mg2+ levels in red blood cells below an optimal concentration inhibits the hexokinase-phosphofructokinase control system, de facto decreasing the glycolytic rate (11) and limiting the amount of glucose transported in the cell.

Endogenous catecholamine, together with glucagon and other proglycemic hormones, is released in the circulation in response to stress stimuli to maintain glycemia. One of catecholamine's main targets is the liver, which responds to the hormone by mobilizing glucose in the blood stream. Three-day starvation of rats has been shown to cause increased sensitivity to insulin in rats (26) as a result of glucose/glycogen depletion. The investigation of hormonal-stimulated Mg2+ transport in cardiac (37) and liver cells (5) in our laboratory has evidenced a connection between Mg2+ and glucose transport. The administration of catecholamine or glucagon resulted in the parallel mobilization of glucose and Mg2+ from liver cells into the extracellular compartment, whereas the presence of glucose transport inhibitors decreased Mg2+ extrusion (5). In cardiac cells, instead, the stimulation by insulin resulted in the parallel accumulation of glucose and Mg2+ in the myocytes (37). Also in this model, the presence of glucose transport inhibitors or the absence of extracellular glucose impaired Mg2+ transport (37).

The present study was undertaken to investigate the role of intra- and extrahepatic glucose on Mg2+ homeostasis and transport in and out liver cells. Overnight-starved rats were used, since this procedure results in the depletion of hepatic glycogen/glucose. The data reported here indicate that starvation resulted in the spontaneous loss of ~9–10% of total cellular Mg2+, rendering liver cells unresponsive to the subsequent administration of catecholamine. Hepatocytes from starved rats, however, accumulated approximately fourfold more Mg2+ than liver cells from fed animals via a glucose-dependent mechanism and an imipramine-inhibited (Na+-dependent) pathway. Taken together, these data support an essential role of intra- and extrahepatic glucose in modulating Mg2+ homeostasis and transport in liver cells.


    MATERIALS AND METHODS
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Chemicals. Collagenase was from Worthington (Lakewood, NJ). Vasopressin, oleyl-arachidonoil-glycerol (OAG), phorbol myristate acetate (PMA), and all other agents were from Sigma (St. Louis, MO). [2-3H]deoxyglucose and 125I-labeled RIA assay for cAMP were from Amersham (Piscataway, NJ). Scintillation cocktail was from Fisher (Pittsburgh, PA).

Perfused livers. Fed or overnight-starved male Sprague-Dawley rats (250–300 g body wt) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). Liver was perfused without recirculation (open system) via the portal vein with a medium containing (in mM): 120 NaCl, 3 KCl, 1.2 KH2PO4, 10 glucose, 12 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, and 10 HEPES, pH 7.2 at 37°C, equilibrated with O2-CO2 (95:5 vol/vol) gas mixture. After removal of the liver from the abdomen and placement on a platform, the perfusion medium was switched to one having similar composition but devoid of Mg2+. Mg2+ contaminant was measured by atomic absorbance spectrophotometry (AAS) in a Perkin-Elmer 3100 AA spectrophotometer and found to be ~5–7 µM. The liver was perfused for 15 min at a rate of 4 ml·g wet wt–1·min–1. Perfusate was collected at 30-s intervals, and Mg2+ content was measured by AAS. Phenylephrine (5 µM), epinephrine (5 µM), or isoproterenol (10 µM) was dissolved in the perfusion medium and administered for the time indicated in Figs. 19. Pharmacological doses of these agonists were used to exclude that a reduced Mg2+ extrusion could be ascribed to reduced receptor responsiveness. The absence of liver damage was assessed enzymatically as lactate dehydrogenase (LDH) activity in aliquots of the perfusate throughout the perfusion protocol. Release of cellular K+ in the perfusate was also assessed by AAS (5, 6).



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Fig. 1. A: Mg2+ extrusion in perfused livers from fed (A) and overnight-starved (B) rats. Livers from fed (A) and starved (B) rats were perfused as reported under MATERIALS AND METHODS. At the time reported, 5 µM phenylephrine (Phe), 10 µM isoproterenol (Iso), or 5 µM epinephrine (Epi) was infused for 8 min. Data are means ± SE of 8 fed and 5 starved livers for each experimental condition. All the points under the curve of extrusion in A were statistically significant vs. the corresponding time points in control livers reported in A and vs. the corresponding time points during agonist infusion in B. Labeling was omitted for simplicity.

 


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Fig. 9. Net Mg2+ accumulation in hepatocytes from fed and starved rats incubated in the absence of extracellular Na+ and glucose. Hepatocytes from fed or starved rats were incubated in the absence of both extracellular Na+ and glucose. Accumulation of Mg2+ was induced by addition of 20 nM OAG, 100 µM PDBU, or 20 nM AVP. Data are means ± SE of 3 preparations for each experimental condition, each performed in duplicate. All data are statistically significant vs. corresponding values in hepatocytes incubated in the presence of extracellular Na+ and glucose. Labeling was omitted for simplicity.

 
In a separate set of experiments, portions of hepatic lobules were removed from the organ during the perfusion protocol immediately before isoproterenol or epinephrine administration, 5 min into the agonist infusion, and 15 min after the removal of the agonist from the perfusion medium to measure tissue cAMP level by 125I-labeled RIA assay, as previously reported (42). The lobule portions were weighted, homogenized (20% wt/vol) in the assay medium provided with the RIA kit, boiled for 5 min, and stored at –20°C until used (42).

Estimation of total Mg2+ extrusion. The Mg2+ content of the perfusate at the last five points before adrenergic agonist addition were averaged and subtracted from the subsequent time points under the curve of efflux. The perfusion rate and collection intervals were taken into account to estimate the total amount of Mg2+ extruded (expressed as µmol; see Refs. 5 and 6). At the end of the perfusion protocol, the liver was blotted gently on absorbing paper and weighted. One hepatic lobe was homogenized (10% wt/vol) in 10% HNO3. After overnight digestion, the acid extract was sedimented (5,000 g for 5 min), and the Mg2+, Na+, K+, and Ca2+ content was measured by AAS (32, 33). Another lobe was homogenized in 5% perchloric acid and extracted in ice for 5 min. The mixture was neutralized by addition of 2 vol of 1 M KHCO3 and sedimented at 2,000 g for 10 min in a refrigerated J6B Beckman centrifuge. The supernatant was removed and stored at –20°C until used for ATP determination (42). To exclude possible miscalculations resulting from an increased hepatic protein catabolism in starved animals, tissue cation, ATP, and cAMP content levels were normalized based on RNA content.

Hepatocyte isolation. Hepatocytes were isolated by collagenase digestion and resuspended (1 x 106 cells/ml) in a medium containing (in mM): 120 NaCl, 3 KCl, 1.2 KH2PO4, 12 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, 10 glucose, and 10 HEPES, pH 7.2, at room temperature, under O2-CO2 (95:5 vol/vol), as previously reported (5). Cell viability (88 ± 3, n = 12) was assessed by trypan blue exclusion test and did not change significantly throughout the duration of the experiment (87 ± 4, n = 12). No appreciable differences in viability were observed in hepatocytes from starved vs. fed animals.

Mg2+ determination in cell suspensions. Aliquots of cell suspension (1 ml) were transferred in a microfuge tube and sedimented at 500 g for 30 s. The cells were washed with 1 ml of the medium reported above but devoid of Mg2+ (incubation medium) and incubated therein in the presence of contaminant (0.03 mM), 0.5 mM or 1.2 mM extracellular Mg2+, at 37°C, under O2-CO2 flow. After 3 min of equilibration, vasopressin (20 nM), OAG (20 nM), or PMA (100 µM) was added to the cell suspension to stimulate Mg2+ accumulation. Aliquots of the incubation mixture (700 µl) were withdrawn in duplicate before or at 2-min intervals after agonist addition and sedimented in microfuge tubes (3,500 g for 45 s), and the cells were sedimented through an oil layer (dibutyl phthalate-dioctyl phthalate, 2:1 vol/vol; see Refs. 35 and 36) to minimize Mg2+ carryover. The supernatant and oil layer were removed by vacuum suction, and the cell pellets were digested overnight in 500 µl 10% HNO3 (also see Refs. 35 and 36). Mg2+ and Na+ content of the acid extract was determined by AAS after sedimentation of denaturated protein (5,000 g for 5 min). The cation content at the two time points before agonist addition were averaged and subtracted from the subsequent time points to calculate net change in cellular Mg2+ or Na+ content. To estimate cellular Mg2+ partitioning, hepatocytes were incubated in the presence of contaminant extracellular Mg2+. After 3 min of equilibration, digitonin (80 µg/ml), 4-(trifluoromethoxy)-phenylhydrazone [FCCP (2 µg/ml)], and A-23187 (2 µg/ml) were added to the incubation mixture at 5-min intervals. The ionophore A-23187 has been routinely used to mobilize Ca2+ or Mg2+ from cellular pools in that it promotes countertransport of 2 H+ for 1 Ca2+ or Mg2+ (3) down a concentration gradient. Aliquots of the cell suspension were withdrawn in quadruplicate and sedimented in microfuge tubes (10,000 g for 2 min). The supernatant was removed, and Mg2+ content was determined by AAS. The Mg2+ content of the pellet was also measured by AAS after overnight digestion in 10% HNO3 and sedimentation of denaturated protein, as indicated previously. Cellular Na+ content could not be measured under these conditions because of the large contamination exerted by the physiological extracellular Na+ concentration present in the buffer.

To assess the role of extracellular Na+ on Mg2+ accumulation, hepatocytes from fed and starved rats were incubated in a medium in which Na+ was replaced with choline chloride (in mM): 120 choline chloride, 3 KCl, 1.2 KH2PO4, 12 KHCO3, 1.2 CaCl2, 1.2 MgCl2, 10 glucose, and 10 HEPES, pH 7.2 with KOH, under O2-CO2 (95:5 vol/vol), as previously reported (35, 36).

For the experiments in cells, Mg2+ content, glucose transport, and cAMP content were normalized per 106 cells to minimize possible miscalculated erroneous determinations of cellular Mg2+ content resulting from an increased hepatic protein catabolism in starved animals. As an additional control, the above parameters were normalized based on cellular nucleic acid content, with qualitative similar results. For simplicity, all cellular results are reported as nanomoles Mg2+ per 106 cells.

Glucose accumulation. Glucose accumulation was determined in aliquots of cell suspensions incubated in the presence of 5, 10, or 20 mM glucose labeled with 1 µCi/ml [2-3H]deoxyglucose.

Aliquots of the labeled incubation mixture were withdrawn in duplicate before, or 6 min after, agonist administration and diluted 10-fold in ice-cold 250 mM sucrose containing 20 µM phloretin (37). The mixture was then filtered on glass fiber filters (Whatman C) under vacuum. The radioactivity retained on the filters was measured by {beta}-scintillation counting (Beckman 2100).

Other procedures. Protein was measured according to the procedure of Lowry et al. (28). ATP was measured by the luciferin-luciferase assay (Sigma kit), using a Berthold LB 9501 Luminometer (42). Tissue and cellular cAMP level was determined in perfused livers and suspension of isolated hepatocytes by 125I-labeled RIA (Biotrak; Amersham), as reported previously by us (42). Total tissue or cell nucleic acid content was measured as reported by Quamme and Rabkin (32).

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


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Mg2+ extrusion from perfused livers. Consistent with previous reports, the infusion of the adrenergic agonist epinephrine, phenylephrine, or isoproterenol into rat livers resulted in a marked extrusion of cellular Mg2+ in the perfusate (Fig. 1A). Mg2+ mobilization reached the maximum at time 8 min after agonist administration and declined afterward toward basal levels despite the persistence of the agonist in the medium. Under these conditions, no release of LDH in the perfusate was detected (data not shown; see Refs. 5 and 6). By contrast, unstimulated livers did not release a detectable amount of Mg2+ in the perfusate over the same period of time. Phenylephrine, isoproterenol, and epinephrine mobilized ~1.45, 1.13, and 2.45 µmol Mg2+ over 8 min, respectively (n = 9 for each experimental condition), which accounted for ~3.5, ~2.8, and ~6.5% of the total hepatic Mg2+ content. The administration of epinephrine (a mix of {alpha}1- and {beta}-adrenoceptor agonists) caused the largest Mg2+ extrusion, equivalent to the sum of the amounts mobilized by phenylephrine and isoproterenol alone (Fig. 1A). As previously reported (4), glucose extrusion accompanied Mg2+ extrusion as a result of adrenergic stimulation in fed rat livers. The administration of phenylephrine, isoproterenol, or epinephrine resulted in the mobilization of 28.4 ± 2.2, 21.3 ± 1.9, and 24.3 ± 2.0 µmol glucose/ml, respectively, over 8 min of agonist infusion (n = 9 for each experimental condition). When similar experiments were performed on livers of overnight-starved rats, no detectable extrusion of Mg2+ (Fig. 1B) or glucose (data not shown) was observed.

To determine whether the lack of Mg2+ extrusion in starved livers depended on a defective Mg2+ transport or an extrusion during the overnight starvation, total Mg2+ content in livers of fed and starved animals was determined. Table 1 shows an ~9% decrease in total Mg2+ content in starved rat livers compared with their fed counterparts. The decrease in Mg2+ content was associated with a marked increase (15%, Table 1) in Na+ content and a modicum change in K+ content (–7%, Table 1) within the tissue, whereas Ca2+ content did not show appreciable differences (data not shown). Hepatic ATP content and tissue cAMP level were also unchanged (Table 1), excluding the possibility that Mg2+ loss depended on a decreased buffering capacity of this moiety, as observed under alcoholic experiments (42), or on a defective activation of the cAMP-signaling pathway. Cellular cAMP level was also measured in suspension of isolated hepatocytes from starved and fed animals upon in vitro stimulation with isoproterenol or catecholamine, with qualitatively similar results (data not shown).


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Table 1. Cation, ATP, and cAMP content in liver from fed and overnight-starved rats

 
A quantitatively similar decrease in cellular Mg2+ content (–15%) was observed in isolated hepatocytes. As indicated in Fig. 2, the decrease appeared to predominantly affect the cytoplasm (digitonin-accessible pool), and mitochondria (FCCP-accessible pool), whereas the postmitochondrial pools (or A-23187-accessible pools) were essentially unaffected. Experiments of adrenergic-induced Mg2+ extrusion were also performed in a suspension of isolated hepatocytes, with results qualitatively similar to those obtained in perfused livers (data not shown).



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Fig. 2. Total cellular and intracellular Mg2+ distribution in hepatocytes from fed and starved rats. Hepatocytes from fed and starved animals were isolated and incubated as reported under MATERIALS AND METHODS. After 3 min of equilibration, aliquots of the incubation mixture were withdrawn in quadruplicate (time 0 min) and sedimented in microfuge tubes to establish a baseline before the addition of digitonin, 4-(trifluoromethoxy)-phenylhydrazone, and A-23187 at 5-min intervals, as described under MATERIALS AND METHODS. After the addition of each of these agents, aliquots of the incubation mixture were withdrawn in quadruplicate and sedimented in microfuge tubes. Cellular Mg2+ content was determined by atomic absorbance spectrophotometry (AAS) after acid digestion of the cell pellet with 10% HNO3. Data are means ± SE of 6 different preparations, each performed in triplicate. *Statistically significant vs. "fed" sample.

 
Mg2+ accumulation in isolated hepatocytes. We have previously reported that stimulation by carbachol, vasopressin, diacylglycerol analogs, or PMA derivates causes an accumulation of Mg2+ from the extracellular environment in the hepatocyte (35, 39). To evaluate whether hepatocytes from starved animals retained the ability to accumulate Mg2+ and the process was actually enhanced to replenish cellular Mg2+ content, liver cells from fed and starved rats were incubated in a medium containing contaminant (i.e., ~0.03 mM) or 0.5 or 1.2 mM extracellular Mg2+. Figure 3A shows a typical incubation experiment in which nonstimulated cells did not release or accumulate significant amounts of Mg2+ over 8 min of incubation. In agreement with the data reported in Table 1 and Fig. 2, hepatocytes from starved rats presented a basal cellular Mg2+ content lower than hepatocytes from fed rats. The addition of OAG, PMA, phorbol 12,13-dibutyrate (PDBU), or vasopressin resulted in a time-dependent accumulation of Mg2+ in both hepatocytes from fed and starved rats, which reached the maximum within 6 min from the agonist addition. For simplicity, net Mg2+ accumulation at this time point was calculated as indicated under MATERIALS AND METHODS and is reported for the following experiments. Hepatocytes from fed rats, stimulated with the indicated agonists, accumulated ~5 nmol Mg2+·106 cells–1·6 min–1 irrespective of the extracellular Mg2+ concentration (also see Refs. 35 and 36). Hence, only the net Mg2+ accumulation in the presence of 1.2 mM external Mg2+ is reported in Fig. 3B for simplicity. In contrast, hepatocytes from starved rats accumulated larger amounts of Mg2+ than the hepatocytes from fed rats. The amount of Mg2+ accumulated increased proportionally to the Mg2+ concentration present in the medium, reaching ~20 nmol·106 cells–1·6 min–1 for cells incubated in the presence of 1.2 mM external Mg2+ (Fig. 3B). Concentrations of Mg2+ >1.2 mM were not tested at this time.



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Fig. 3. Typical incubation experiment (A) and net Mg2+ accumulation (B) in isolated hepatocytes from fed and starved rats. Hepatocytes were isolated and incubated in the presence of 20 mM extracellular glucose and varying extracellular Mg2+ concentrations as described under MATERIALS AND METHODS. A typical experiment out of 6 for cells incubated in the presence of 1.2 mM extracellular Mg2+ is reported in A. Net Mg2+ accumulation at time 6 min after the addition of 20 nM oleyl-arachidonoil-glycerol (OAG), 100 µM phorbol-12,13 dibutyrate (PDBU), or 20 nM arginine vasopressin (AVP) is reported in B. CTL, control; [Mg2+]o, extracellular Mg2+ concentration. Data are means ± SE of 6 different preparations, each performed in duplicate. *Statistically significant vs. corresponding value in fed hepatocytes.

 
In cardiac cells, Mg2+ accumulation is a glucose-dependent process (37). Hence, we tested the hypothesis that the Mg2+ accumulation observed in starved hepatocytes was also associated with the transport of glucose in the hepatocytes to replenish the hepatic glycogen store. To this purpose, hepatocytes from starved animals were incubated in the presence of 1.2 mM extracellular Mg2+, either in the absence of external glucose or in the presence of 20 mM glucose and 20 µM phloretin as an inhibitor of hepatic glucose transport (Fig. 4A). Under these experimental conditions, the Mg2+ accumulation elicited by OAG, PDBU (an analog of PMA that also induces Mg2+ accumulation; see Ref. 35), or vasopressin stimulation was largely attenuated (only ~9 nmol Mg2+·106 cells–1·6 min–1 were accumulated under either experimental condition). In contrast, fed hepatocytes stimulated by OAG, PDBU, or vasopressin accumulated quantitatively similar amounts of Mg2+ (~4–4.5 nmol Mg2+·106 cells–1·6 min–1) irrespective of the availability or nonavailability of glucose in the extracellular milieu (see Figs. 4B and 3B for comparison). To assess whether extracellular glucose concentration modulated the amplitude of Mg2+ accumulation in a dose-dependent manner, hepatocytes from fed and starved rats were incubated in the presence of 1.2 mM Mg2+ and varying extracellular glucose concentrations (5, 10, or 20 mM) and stimulated by addition of OAG or PMA (Fig. 5). As seen in Fig. 5, in "starved" hepatocytes Mg2+ accumulation increased proportionally to the external glucose concentration. When similar experiments were performed on hepatocytes from fed rats, total Mg2+ accumulation varied little (~10–15%) according to the extracellular glucose concentration (e.g., 4.38 ± 0.78 nmol Mg2+·106 cells–1·6 min–1 after OAG stimulation in the presence of 5 mM extracellular glucose, n = 7, vs. 5.25 ± 0.94 nmol Mg2+·106 cells–1·6 min–1 for cells incubated in the presence of 20 mM glucose, n = 7, Fig. 5).



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Fig. 4. Effect of extracellular glucose removal or glucose transport inhibitor on net Mg2+ accumulation in hepatocytes from starved and fed rats. Hepatocytes from starved (A) or fed (B) animals were incubated in the presence of 1.2 mM extracellular Mg2+, either in the absence of extracellular glucose or in the presence of 20 mM extracellular glucose ± 20 µM phloretin as glucose transport inhibitor. Accumulation of Mg2+ was induced by addition of 20 nM OAG, 20 nM AVP, or 100 µM PDBU. Data are means ± SE of 6 different preparations, each performed in duplicate. *Statistically significant vs. corresponding value in hepatocytes incubated in the presence of external glucose without phloretin.

 


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Fig. 5. Effect of varying extracellular glucose concentrations on Mg2+ accumulation in hepatocytes from fed or starved animals. Hepatocytes from fed (open bars; F) or starved (filled bars; S) rats were incubated in the presence of varying extracellular glucose concentrations ([glucose]o) and 1.2 mM extracellular Mg2+. Mg2+ accumulation was induced by addition of 20 nM OAG or 100 µM PMA. Data are means ± SE of 4 different incubations, each performed in duplicate. *Statistically significant vs. corresponding values in fed hepatocytes. #Statistically significant vs. values in "starved" hepatocytes incubated in the presence of 5 mM extracellular glucose.

 
Conversely, varying the concentration of extracellular Mg2+ affected glucose uptake in starved hepatocytes. In the presence of contaminant Mg2+ level (0.03 mM), no significant increase in glucose accumulation was observed after the addition of vasopressin or OAG (Fig. 6, A and B). The increase of external Mg2+ concentration to 1.2 mM enhanced OAG- and vasopressin-stimulated glucose accumulation in both fed and starved hepatocytes vs. the corresponding values obtained in the presence of contaminant extracellular Mg2+, at both 5 (Fig. 6A) and 20 (Fig. 6B) mM external glucose. As shown in Fig. 6, glucose accumulation increased by approximately two- and threefold in "fed" hepatocytes incubated in the presence of 5 and 20 mM extracellular glucose, respectively. In contrast, glucose accumulation in starved hepatocytes increased from approximately twofold in the presence of 5 mM external glucose to approximately eightfold in the presence of 20 mM extracellular glucose. This represents a net two- to threefold increase in glucose uptake in starved vs. fed hepatocytes.



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Fig. 6. Glucose accumulation in liver cells from fed and starved rats. Hepatocytes were incubated in the presence of 5 (A) or 20 (B) mM glucose labeled with 1 µCi/ml [2-3H]deoxyglucose, in the presence of contaminant (~0.030 mM) or 1.2 mM extracellular Mg2+. Glucose accumulation was assessed as described under MATERIALS AND METHODS. The amount of glucose accumulated after OAG or AVP stimulation is expressed as nmol·106 cells–1·6 min–1. Data are means ± SE of 3 different preparations for both fed and starved cells. PMA, phorbol myristate acetate; [Mg2+]o, extracellular Mg2+ concentration. *Statistically significant vs. corresponding values in fed hepatocytes.

 
Previous data from our laboratory indicate that Mg2+ accumulation in liver cells is an Na+-dependent process (35) in that it does not occur in the absence of a physiological concentration of extracellular Na+. These results were confirmed in hepatocytes from starved animals as well. Figure 7 shows that liver cells isolated from fed animals accumulated negligible amounts of Mg2+ in the absence of extracellular Na+. Under similar experimental conditions, hepatocytes from starved rats retained a partial Mg2+ accumulation that accounted for ~50–60% of the accumulation observed in the presence of a physiological concentration of extracellular Na+. To confirm these data, and ascertain that the residual Mg2+ accumulation observed in starved hepatocytes incubated in the absence of glucose and the basal Mg2+ accumulation observed in fed hepatocytes occurs via the Na+-dependent pathway, hepatocytes from fed and starved rats were incubated in a medium containing 1.2 mM, varying extracellular glucose concentrations, and 500 µM imipramine, a nonselective inhibitor of the Na+/Mg2+ exchanger. We have previously reported that this agent inhibits the Na+-dependent Mg2+ accumulation mechanism present in the basolateral domain of the hepatocyte (2). As shown in Fig. 8A, in the presence of 20 mM extracellular glucose, the addition of imipramine resulted in minimal variations in cellular Mg2+ content in the absence of any stimulatory agent in both fed and starved hepatocytes. After the stimulation by OAG, PMA, or vasopressin, no Mg2+ accumulation was observed in hepatocytes isolated from fed rats, whereas hepatocytes from starved animals accumulated ~10–12 nmol Mg2+·106 cells–1·6 min–1 (Fig. 8A). When similar experiments were repeated in the absence of extracellular glucose (0 mM), the already reduced Mg2+ accumulation observed in starved hepatocytes (~9 nmol Mg2+·106 cells–1·6 min–1 vs. ~22 nmol Mg2+·106 cells–1·6 min–1 in the absence and the presence of extracellular glucose, respectively) was completely abolished by the presence of imipramine (Fig. 8B). In fed hepatocytes, instead, the Mg2+ accumulation elicited by OAG, PMA, or vasopressin was marginally reduced (~15%) by the removal of extracellular glucose (Fig. 8B) compared with the accumulation occurring in the presence of 20 mM glucose (Fig. 8A) but was completely abolished by the addition of imipramine (Fig. 8B). Glucose accumulation was decreased by ~30% in both fed and starved hepatocytes by the presence of imipramine (data not shown).



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Fig. 7. Effect of extracellular Na+ removal on net Mg2+ accumulation in hepatocytes from fed and starved rats. Hepatocytes from fed or starved rats were incubated in the absence or in the presence of extracellular Na+ and stimulated for Mg2+ accumulation in the presence of 20 mM extracellular glucose and 1.2 mM external Mg2+. Accumulation of Mg2+ was induced by addition of 20 nM OAG, 100 µM PMA, or 20 nM AVP. [Na+]o, extracellular Na+ concentration. Data are means ± SE of 4 preparations for each experimental condition, each performed in duplicate. *Statistically significant vs. corresponding values in the presence of extracellular Na+.

 


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Fig. 8. Effect of imipramine on net Mg2+ accumulation in hepatocytes from fed and starved rats. Hepatocytes from fed or starved rats were stimulated for Mg2+ accumulation in the presence of 20 mM (A) or 0 mM (B) extracellular glucose concentrations, 1.2 mM external Mg2+, and 500 µM imipramine as an inhibitor of Na+-dependent Mg2+ transport. Accumulation of Mg2+ was induced by addition of 20 nM OAG, 100 µM PMA, or 20 nM AVP. Data are means ± SE of 4 preparations for each experimental condition, each performed in duplicate. *Statistically significant vs. corresponding values in the absence of imipramine.

 
Consistent with the data reported above, when hepatocytes from starved and fed animals were incubated in the absence of extracellular Na+ (replaced with choline chloride) and extracellular glucose, no significant Mg2+ accumulation was observed after stimulation by PMA or vasopressin (Fig. 9).


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In the last two decades, several aspects of Mg2+ transport and regulation in mammalian cells have been uncovered. Several laboratories, including ours, have indicated that catecholamine, selective {alpha}1- and {beta}-adrenoceptor agonists, insulin, or vasopressin all play a significant role in modulating Mg2+ transport in or out of mammalian cells (see Refs. 9, 21, 25, and 40 as a review). Although no functional transporter has been cloned to date in mammalian cells, the main route for cellular Mg2+ transport appears to be an Na+-dependent Mg2+ transport mechanism that, in the absence of more detailed information, has been tentatively identified as an Na+/Mg2+ exchanger (9). A little more uncertainty exists about the pathway used for Mg2+ accumulation. Also, this process appears to require a physiological concentration of Na+ (35) but not Ca2+ (7) in the extracellular milieu. Yet, the intrinsic nature of the transport mechanism remains undefined. Experimental evidence obtained in purified liver plasma membrane vesicles suggests the operation in reverse of the Na+/Mg2+ exchanger present in the basolateral domain of the hepatocyte cell membrane (2). Yet, the operation of such a mechanism in the intact cells is dubious in that the cellular Na+ gradient is inwardly directed and therefore not ideal to support Mg2+ accumulation. Hence, in the intact cell, the operation of an alternative Mg2+ entry mechanism has to be postulated. Consistent with this possibility, data obtained in cardiac cells have suggested the operation of a channel (32). More recently, increasing evidence has been provided by several laboratories that transient receptor potential (TRP) mammalian type (TRPM)6 and TRPM7, two members of the TRP channel family, can transport Mg2+, at least under well-defined conditions (30, 43). It is presently undefined whether the operation of these channels is coupled to an Na+ transmembrane electrochemical gradient.

A second point of uncertainty is the physiological significance of Mg2+ transport. At the hepatic level, little insight is currently available. Gaussin et al. (12) have reported that an increase in cytosolic free Mg2+ in hepatocytes is associated with activation of glycogen phosphorylase and glycolysis. Garner and Rosett (11) investigated the effect of Mg2+ on glycolytic enzymes and found that hexokinase, phosphofructokinase, and pyruvate kinase, among other enzymes, are activated by changes in Mg2+ concentrations. Recently, our laboratory has reported that inhibition of glucose transport by phloretin decreases Mg2+ extrusion by ~50%, whereas conditions that hamper Mg2+ transport negatively affect glucose transport as well (5).

The present study was undertaken to further elucidate the role played by glucose on Mg2+ homeostasis and transport in and out of the hepatocyte.

Role of intrahepatic glucose on Mg2+ extrusion. Adrenergic stimulation of liver cells results in the activation of glycogenolysis and in a marked extrusion of glucose in the bloodstream to maintain euglycemia under stress, starvation, or under conditions in which energy consumption increases. Especially during starvation, catecholamine and other proglycemic hormones, like glucagons and cortisol, are released to mobilize glucose reserves from liver and skeletal muscle. Stimulation by catecholamine or glucagon also results in an increased extrusion of Mg2+ from liver cells (5). Based on the reports of Gaussin et al. (12), Garner and Rosett (11), and Garfinkel and Garfinkel (10), it is evident that Mg2+ plays an essential role in regulating glycogenosynthetic, glycogenolytic, and glycolytic enzymes, and possibly the switching from one metabolic state to the other. The observation that, after overnight starvation, liver cells have already lost ~9–10% of their cellular Mg2+ content (i.e., an amount that closely resembles that mobilized by adrenoceptor stimulation; Fig. 1 and Refs. 35 and 39) and become unresponsive to the in vitro stimulation by pharmacological doses of adrenergic agonists indicates that Mg2+ extrusion already occurred during the mobilization of hepatic glucose to maintain glycemia. The concomitant increase in hepatic Na+ content also suggests that Mg2+ extrusion occurred via an exchange for extracellular Na+. Because the Na+/Mg2+ exchange process operates electrogenically in cells (9) and in plasma membrane vesicles (2) as well, it is conceivable that the decrease in total tissue K+ content (Table 1) occurs for charge compensation purposes. In this respect, it is worth noting that, in percent terms, the increase in Na+ content (~15%) matches the combined decrease in Mg2+ and K+ contents (~9% and ~7%, respectively).

The inability of liver cells to extrude additional Mg2+ when stimulated by adrenergic agonists also indicates that the hepatocyte possesses a finite pool of Mg2+ that can be mobilized after adrenergic stimulation. Within the cell, Mg2+ is lost essentially at the level of cytoplasm and mitochondria. The larger decrease in Mg2+ content observed in isolated cells compared with the whole tissue is likely to depend on the procedure used to determine the cellular Mg2+ partitioning. The agents used, in fact, induce changes in Mg2+ equilibrium and distribution among the various compartments but also affect to a lesser extent other cations (e.g., Ca2+) that may, in turn, affect Mg2+ redistribution (5, 6). In addition, it cannot be excluded that the Mg2+ determination in the whole tissue is underestimated because of the presence of other cell types (e.g., Kuppfer, stellar, and endothelial cells) that do not actively participate in Mg2+ homeostasis under fed-fasted conditions.

Mg2+ accumulation in starved rat livers. We have previously reported that the administration of vasopressin, diacylglycerol analogs, or phorbol myristate acetate derivates (PMA and PDBU) to hepatocytes from fed rats induces a marked Mg2+ accumulation that does not significantly vary in response to variations in extracellular Mg2+ (35, 36) or glucose concentration (Fig. 5).

In contrast, hepatocytes from starved rats accumulate approximately four times more Mg2+ than cells from fed rats, and the process is directly proportional to the extracellular Mg2+ (Fig. 3B) or glucose (Fig. 4) concentration. Although the increased Mg2+ accumulation can be interpreted as an attempt to restore the intracellular Mg2+ pool depleted after overnight starvation, not a clear explanation is at hand for the glucose dependence of Mg2+ accumulation. The process is not new, in that a similar association between glucose and Mg2+ accumulation has been observed in cardiac myocytes stimulated by insulin (37) and in {beta}-cells (23). Yet, the reason(s) behind the process remain(s) unclear. Physiologically, several glycolytic enzymes, including hexokinase, phosphofructokinase, phosphoglycerate mutase, phosphoglycerate kinase, enolase, and pyruvate kinase, are active at low and inhibited at high Mg2+ concentrations (31). The Mg2+ concentrations at which activation and inhibition occur (0.5–1 mM) are well within the fluctuations in free Mg2+ content measured in the hepatocyte (10, 31). Considering the essential role that Mg2+ plays in modulating hexokinase activity (11), a reasonable explanation is that the increased Mg2+ accumulation is essential to support the high phosphorylation rate of the kinase (10) to generate glucose 6-phosphate and maintain an inwardly oriented gradient for glucose accumulation. This hypothesis would explain the increased glucose accumulation observed in hepatocytes from starved rats vs. liver cells from fed animals stimulated by vasopressin, as well as the lack of a clear stoichiometry ratio between the amounts of glucose and Mg2+ accumulated. In addition, by inhibiting phosphofructokinase activity (10, 31), the increase in cellular Mg2+ concentration will prevent the formation of fructose 1,6-diphosphate and progression of glycolysis, favoring at the same time the utilization of glucose in the glycogenosynthetic process. Overall, the increased Mg2+ accumulation would support glucose accumulation and storage as hepatic glycogen. Alternatively, it has to be postulated that Mg2+ modulates the glucose transport rate by changing the affinity of the GLUT2 transporter in a manner similar to what has been described for the intestinal myoinositol transporter (13). Irrespective of the physiological significance, it is presently unclear which pathway Mg2+ uses to enter the hepatocyte. A major difference can be noticed in hepatocytes from fed vs. starved rats. Consistent with previous reports (7, 35), fed hepatocytes accumulate Mg2+ almost exclusively via what appears to be an Na+-dependent mechanism, as indicated by the Na+ removal and imipramine data, whereas variations in extracellular glucose enlarge the amplitude of Mg2+ accumulation by 10–15% at the most. Yet, as mentioned previously, the Na+ electrochemical gradient across the cell membrane makes unlikely that Mg2+ entry occurs via the reverse operation of the Na+/Mg2+ exchanger, as observed in plasma membrane vesicles isolated from the basolateral domain of the hepatocyte (2). On the other hand, imipramine is a nonspecific inhibitor in that it affects all Na+ transport mechanisms and K+ channels. Therefore, it is conceivable that this drug can affect Mg2+ transport by altering the membrane potential. In line with this possibility, Mg2+ enters the cell via a pathway [e.g., the channel proposed by Quamme and Rabkin (32) or Nadler and collaborators (30)], possibly coupled to the cell membrane potential. In "fasted" hepatocytes, instead, Mg2+ is accumulated to a larger extent via two distinct mechanisms. One mechanism, responsible for ~50% of total Mg2+ accumulation, is the same mechanism present in fed hepatocytes operating in the starved cells at about two times the rate observed in their fed counterpart (Fig. 8B). The remaining 50% of Mg2+ accumulation occurs via the glucose-linked mechanism that in fasted hepatocytes is approximately seven to eight times more active than in fed cells (Fig. 8A), most likely as a result of intracellular glucose depletion. The operation of two distinct Mg2+ accumulation pathways is also supported by the observation that the amount of Mg2+ accumulated in starved hepatocytes incubated in the presence of glucose and 1.2 mM Mg2+ (Fig. 3B) is quantitatively equivalent to the combined amounts of Mg2+ accumulated under conditions in which extracellular glucose (Fig. 4) or Na+ (Figs. 7 and 8) is not available to support Mg2+ accumulation. At the present time, we have no information about the nature of the second transport pathway. A couple of possibilities, however, can be excluded. First, no experimental evidence is there to support an entry via the GLUT2 transporter. Second, Mg2+ does not enter the cell via an increased operation of the transport mechanism operating under basal conditions in fed hepatocytes. If this was the case, Mg2+ accumulation should be completely inhibited by Na+ removal or imipramine addition, as observed in fed hepatocytes (Figs. 7 and 8). Furthermore, data obtained in purified liver plasma membranes (1) indicate that changes in extravesicular glucose concentration do not affect the amplitude of Mg2+ movement via the basolateral or apical transport mechanisms.

In conclusion, the data reported here indicate that the mobilization of hepatic glycogen/glucose after endogenous catecholamine release is associated with the mobilization of a well-defined pool of cellular Mg2+. In addition, our data indicate that hepatocytes depleted of Mg2+ after overnight starvation accumulate larger amounts of Mg2+ than hepatocytes from fed rats in a manner that is directly proportional to the extracellular Mg2+ and glucose concentration. This Mg2+ accumulation appears to occur via two distinct pathways modulated by glucose and Na+. Further studies are required to elucidate the nature of these mechanisms and the significance of an (enlarged) Mg2+ accumulation in starved hepatocytes.


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This work was supported by National Institutes of Health Grants R9AA-11593 and HL-18708.


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Address for reprint requests and other correspondence: A. Romani, Dept. of Physiology and Biophysics, School of Medicine, Case Western Reserve Univ., 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.


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