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Department of Biomedical and Surgical Sciences, Section of Medicina Interna C, University of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 37134 Verona, Italy

(Requests for offprints should be addressed to P Delva; Email: pietro.delva{at}univr.it)

	Abstract

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Insulin is capable of increasing intracellular magnesium, although very little is known about the effect of insulin on the biologically active fraction of magnesium, i.e. the ionized quota (Mg_i²⁺), its interactions with glucose, and the cellular mechanisms involved in these processes. We studied the interactions of the effects of insulin and glucose on intracellular ionized magnesium in human lymphocytes. Mg_i²⁺ was measured using a fluorimetric method and the Mg²⁺-sensitive dye, furaptra. We found that insulin significantly increases the Mg_i²⁺(without insulin 227 ± 14 µM, with 10 µU/ml, insulin 301 ± 30 µM, P<0.0001, n = 12) in a dose-dependent manner in all three glucose concentrations tested (5, 7 and 15 mmol/l). The half-maximal effect of insulin was approximately 0.8 µU/ml. Glucose and insulin showed opposite effects in their ability to modify Mg_i²⁺ in lymphocytes. Inhibitors of the membrane Na⁺- Mg²⁺ transport system and of phosphatidylinositol (PI) 3-kinase abolish the insulin-mediated increase of Mg_i²⁺, thus suggesting that insulin is capable of increasing Mg_i²⁺ by modulating the activity of this transport system, possibly through the mediation of PI 3-kinase activation. Taking into account the relationship between insulin and glucose plasma levels and their opposing effects on Mg_i²⁺, this mechanism may represent the two limbs of a biphasic regulatory system of Mg_i²⁺ in both physiological and pathological conditions.

	Introduction

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A potential role of magnesium deficiency in the pathogenesis of type 2 diabetes mellitus has been recently proposed based on epidemiological, nutritional, and experimental data. In fact, large observational studies have described an association between low serum magnesium levels and type 2 diabetes (Ma et al. 1995). Accordingly, magnesium supplementation might be useful in the prevention and treatment of diabetes (Schnack et al. 1992). In vitro studies have shown an effect of magnesium on insulin secretion by the pancreas and on the mechanism of peripheral insulin resistance (Hwang et al. 1993). Our group has shown that intracellular ionized magnesium (Mg_i²⁺) concentrations are decreased in patients with insulin resistance syndrome (Delva et al. 1996, 1998a) and that peripheral insulin resistance is related to Mg_i²⁺ levels (Delva et al. 1998b). Furthermore, we have shown in vitro that high extracellular glucose at concentrations compatible with those found in diabetes can reduce the concentration of Mg_i²⁺ (Delva et al. 2002). Insulin has been shown to increase the Mg_i²⁺ concentration in human erythrocytes (Barbagallo et al. 1993, 1997), platelets (Hwang et al. 1993), and the total intracellular magnesium in rat ventricular myocytes (Romani et al. 2000), and prevents Mg²⁺ extrusion induced by isoproterenol (Romani et al. 2000). Nonetheless, very little is known about the effect of insulin on the biologically active fraction of magnesium, i.e. the ionized quota, in nucleated human-derived cells, its interactions with glucose, and the cellular mechanisms involved. We have studied the interactions between insulin and glucose on Mg_i²⁺ in human lymphocytes in order to shed light on the cellular mechanisms involved in these processes.

	Material and Methods

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Reagents

The Ficoll-Histopaque reagent was obtained from Sigma Chemical, as were the RPMI 1640, BSA free fatty acids, and all the sugars utilized. MgCl₂, lanthanum oxide (ultrapure grade), wortmannin, vandate and insulin were obtained from Sigma. Furaptra-acetoxymethyl ester and fura-2 acetoxy-methyl ester were provided by Molecular Probes Inc. (Eugene, OR, USA).

Preparations of human lymphocytes

All studies were performed on human lymphocytes obtained from the local blood bank from healthy blood donors. Consent was obtained from the subjects after the nature of the procedure was explained. Peripheral blood lymphocytes were isolated as follows: total blood was diluted with RPMI 1640 medium (HEPES modification, glucose-free and supplemented with MgSO₄ to reach the concentration of 0.8 mM) and layered carefully onto Histopaque 1077 and then centrifuged for 30 min at 400 g. The layer of lymphocytes was carefully aspirated and washed twice in RPMI 1640 for 10 min at 150 g. The cells thus obtained were allowed to sediment for 30 min in culture flasks. The supernatant was then transferred into tubes and centrifuged, and the lymphocytes thus obtained were resuspended in the same medium. The percentage of lymphocytes always exceeded 95% and the vitality assessed as Trypan Blue exclusion was always higher than 97%.

Measurement of free intralymphocyte magnesium

We used the method previously described by Delva et al.(1996). Lymphocytes were counted by means of a Coulter Counter (Coulter Electronics Ltd, Dunstable, Beds, UK). The cells were washed three times with RPMI 1640 and then three separate aliquots of lymphocytes (6 x 10⁶ cells each) were suspended in RPMI 1640 with insulin (or the relevant substance) and three aliquots (6 x 10⁶ cells each) without insulin (control cells). In some experiments, when the concentration of extracellular Mg²⁺ was varied, RPMI 1640 was substituted with Bulher solution containing (in mM) NaCl 145, KCl 5, Na₂HPO₄ 0.5, HEPES 10, CaCl₂ 1, MgSO₄ 0–3. All the aliquots had BSA 0.1% (v/v) and the cell permeant furaptra-acetoxymethyl ester 10 µM added to them for 1 h at 37 °C. After centrifugation, the cells were washed twice in the same medium to remove extracellular dye and resuspended in the same medium at room temperature for 45 min for complete deesterification of the dye. For the measurement of intracellular Mg²⁺, the buffer utilized contained (in mM) 140 NaCl, 5 KCl, 1.8 CaCl₂, 0.8 MgSO₄, and 15 HEPES with or without insulin for the insulin-incubated aliquots and without insulin for the insulin-free aliquots (pH 7.4 at 31 °C). After washing off the 1640 medium, the cells were added to the above pre-warmed medium (31 °C) just before the fluorimetric measurements were performed. Fluorescence emission at 510 nm (slit width 10 nm) was measured with alternate excitation at 335 and 370 nm (slit width 10 nm) within a thermostatically controlled cuvette holder (31 °C) in a Hitachi F-2000 fluorescence spectrophotometer. Autofluorescence contributed to less than 1% of the total fluorescence values. One centimeter quartz cuvettes were used for all experiments. After reading the initial fluorescence emission resulting from excitation at 335 and 370 nm, 5 mM EDTA and 5 mM EGTA were added to the cuvette. Since extracellular Mg²⁺ and Ca²⁺ were chelated in the medium, a rapid-step change in fluorescence at both wavelengths occurred due to dye leaking from inside the cells. The immediate (<10 s) change in fluorescence intensities at both wavelengths after the addition of EDTA and EGTA was considered in order to calculate the free resting intracellular Mg²⁺. Triton X-100 was then added to a final concentration of 0.1% (v/v) to lyse the cells, and since the cells were in a medium containing EDTA and EGTA, it was possible to determine the minimum fluorescence ratio, R_min. Subsequently, MgSO₄ (100 mM) was added to obtain the maximum fluorescence ratio, R_max. Intra-cellular-free Mg was measured in triplicate and was calculated according to (Raju et al. 1989). The K_d of intracellularly generated furaptra for Mg²⁺ in our experimental condition was calculated to be 2.1 mM as previously described (Delva et al. 2004).

Measurement of free intralymphocyte magnesium in the cytosol and intracellular organelles

We performed an indirect estimate of Mg_i²⁺ in the cytosol and intracellular organelles of lymphocytes, both in the presence of insulin and in its absence, by differential permeabilization of the cells (Delva et al. 2004). We used the procedure described above to measure lymphocyte steady-state Mg_i²⁺ further by adding, after the addition of EGTA/EDTA mixture, digitonin (25 mg/l) in order to permeabilize the cell plasma membrane. In this condition, the cytosolic free magnesium is expected to leave the cell following its outwardly directed gradient and all the fluorescence measured is derived from intracellular organelles. Then, we indirectly calculated the cytosolic free magnesium concentration by subtracting the magnesium-free concentration in the organelles from the lymphocyte steady-state Mg_i²⁺ concentration of the cell.

Measurement of free intralymphocyte calcium

The intracellular Ca²⁺ of lymphocytes was measured using fura-2 as previously described by Delva et al.(1996). Six aliquots of cells (6 x 10⁶ each) were incubated following the same strategy used for Mg_i²⁺ determinations (with or without insulin) with 5 µM fura-2-acetoxymethyl ester for 1 h at 37 °C, and then washed twice and left for 45 min at room temperature before any measurements were made. For fluorimetric measurements, the same buffer used for Mg_i²⁺ assay was utilized and the cells were added to a pre-warmed medium with a technique similar to that used for the Mg_i²⁺ assay. EGTA (10 mM) was added to chelate Ca²⁺ in the medium, thus producing a desaturation of fura-2, which leaked from inside the cells, and giving a rapid-step change in fluorescence emission at 510 nm with excitation set at both 340 and 380 nm wavelengths. This provided the ratio values for the intracellular Ca²⁺ resting calculation (Grynkiewicz et al. 1985) using a K_d value for 175 nM Ca²⁺/fura-2 complex as previously calculated in our experimental conditions (Delva et al. 2004).

The intra- and inter-assay variability (coefficient of variation) determinations were 5.8 and 3.9% for ionized Mg_i²⁺ and 4.6 and 12.7% for ionized Ca_i²⁺ determinations.

Measurement of total intralymphocyte magnesium

We utilized the method described by Elin and Hosseine (1985) with some modifications as previously described (Delva et al. 2004).

Measurement of intralymphocyte ATP

ATP content of human lymphocytes was measured as described by Nieminen et al.(1990) with minor variations (Delva et al. 2004).

Statistical analysis

Results are expressed as means ± S.D. if no other method is specified. Comparison between groups was performed using Student’s t-test and considered statistically significant when the probability of the null hypothesis was below at least 5%. Confidence limits for differences in means were also provided. For multiple comparisons, a one-way ANOVA test with Bonferroni’s correction for multiple comparisons was used.

	Results

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Effect of insulin on the intralymphocyte ionized magnesium concentration

The steady-state Mg_i²⁺ in the incubation media without insulin was 227 ± 14 µM (n = 12). Incubation of lymphocytes for 2.5 h with 10 µU/ml insulin significantly increased this value (301 ± 30 µM, P<0.0001, 95% confidence interval: 49–96 µM, n = 12) (Fig. 1C). Dose–response curves of Mg_i²⁺ to insulin at three extracellular glucose concentrations (5, 7, and 15 mmol/l) are shown in Fig. 1A. The effect of insulin on Mg_i²⁺ appears to be dose-dependent in all three extracellular glucose concentrations tested, with a half-maximal effect (EC₅₀) of approximately 0.8 µU/ml insulin. Three statistically different maximal Mg_i²⁺ values were found (glucose: 5 mmol/l, 277 ± 38; 7 mmol/l, 231 ± 40; 15 mmol/l, 204 ± 35; ANOVA: F = 3.5, P<0.01, n = 6). In Fig. 1B, the time course of the increase of Mg_i²⁺ by 10 µU/ml insulin is measurable after 30 min, reaching a maximal effect after 120 min. The in vitro effects of insulin comparisons to IGF-1 and insulin-mimetic stimuli, glucose analogs, inhibitors of insulin signaling and inhibitors of glucose metabolism are summarized in Table 1. The interrelationships between the in vitro effects of insulin and inhibitors or regulators of transmembrane Mg²⁺ fluxes in human lymphocytes are summarized in Table 2.

View larger version (17K): [in this window] [in a new window]   Figure 1 (A) Dose–response curves of Mgi2+ to insulin at three extracellular glucose concentrations (5, 7 and 15 mmol/l). The half-maximal effect (EC50) for the three curves is approximately equal (0.15 µU/ml insulin) and three statistically different maximal Mgi2+ values were found. One preparation of lymphocytes was exposed to one concentration of glucose and five different concentrations of insulin. The lymphocytes used for these experiments belong to a single donor. (B) Time course of the insulin-induced (10 µU/ml) Mgi2+ increase in human lymphocytes. (C) Bar graph showing the in vitro effect of 10 µU/ml insulin on Mgi2+ in human lymphocytes in the absence and presence of 5 mmol/l glucose. Each point is the mean ± S.D. obtained using six different preparations of lymphocytes.

In the absence of insulin, the lymphocyte whole-cell Mg_i²⁺, the digitonin-treated lymphocyte Mg_i²⁺ (compartmentalized Mg_i²⁺) and the lymphocyte cytosolic Mg_i²⁺ were 126 ± 23, 20 ± 7, 106 ± 23 µM, respectively (n = 5). In the presence of 10 µU/ml insulin, these values were 177 ± 26, 29 ± 4, and 147 ± 22 µM, ANOVA: F = 7.59, P = 0.002, respectively (n = 5). In the presence and absence of 10 µU/ml of insulin, the Mg_i²⁺ concentrations relating to lymphocyte whole-cell Mg_i²⁺, compartmentalized Mg_i²⁺ and cytosolic Mg_i²⁺, were all statistically significantly different (P = 0.01).

Under our experimental conditions, the incubation of lymphocytes for 2.5 h with 10 µU/ml of insulin did not produce any statistically significant modifications in the concentration of ATP_i, (control medium, 5.31 ± 0.73 mM; insulin-treated, 5.78 ± 0.85 mM, n = 6).

Total intracellular magnesium in lymphocytes incubated in the medium without insulin was 19 ± 2 mM (n = 12). Incubation of lymphocytes for 2.5 h with 10 µU/ml of insulin did not significantly modify this value (glucose-treated, 18.7 ± 1.5 mM, n = 12).

As far as ionized intracellular calcium is concerned, incubation of lymphocytes for 2.5 h with 100 µU/ml of insulin did not modify the concentration of Ca_i²⁺ in lymphocytes (without insulin, 60 ± 11 nM, with insulin 65 ± 12, n = 8).

	Discussion

Top Abstract Introduction Material and Methods Results Discussion References

 
Incubation of human lymphocytes with insulin at concentrations that mimic both physiological and pathological situations results in a significant increase in the Mg_i²⁺ concentration, confirming data obtained from anucleated human-derived cells such as erythrocytes (Barbagallo et al. 1993, 1997) and platelets (Hwang et al. 1993). However, in these previous studies, the interrelationships between intracellular magnesium, glucose and insulin were not investigated.

Paolisso et al.(1986) were the first to show in human erythrocytes that insulin in vitro increases erythrocyte magnesium, an effect that is entirely abolished by ouabain. The same authors then showed that the insulin-induced magnesium increase in erythrocytes is different in patients with type 2 diabetes (Paolisso et al. 1988) or obese subjects (Paolisso et al. 1990) compared with healthy subjects.

Dominguez et al.(1998) confirmed in human erythrocytes that insulin is capable of increasing intracellular free magnesium in a dose- and time-dependent manner (Barbagallo et al. 1993) and that IGF-1 has a similar effect.

Hwang et al.(1993) confirmed these results in a different cell model, namely the human platelet, and showed that insulin had a dose- and time-dependent effect on the increase of [Mg²⁺]_i and suggested that insulin can translocate Mg²⁺ from the extracellular space. Takaya et al.(1998) confirmed and extended the results on human platelets showing that IGF-1 produces the same increases in Mg_i²⁺ as insulin. The involvement of Na⁺–Mg²⁺ exchanger was judged as improbable and insulin/IGF-1 translocates Mg²⁺ from the extracellular space.

To our knowledge, only Hua et al.(1995) have studied the in vitro effects of insulin on human resting lymphocytes and confirmed the results obtained from erythrocytes and platelets by demonstrating that insulin in vitro can increase Mg_i²⁺ in human resting lymphocytes. The results obtained from the present work are in agreement with the above results obtained from a different cell model utilizing different methodological approaches. As far as we know, the only study performed in insulin-target cells was performed in rat hepatocytes by Keenan et al.(1996), who demonstrated that insulin is able to block the Mg²⁺ efflux from perfused rat livers stimulated by isoproterenol.

We have previously studied the in vitro effect of glucose alone on Mg_i²⁺ in human lymphocytes and found that in the absence of insulin, glucose is capable of decreasing the Mg_i²⁺ in a dose-dependent and stereospecific manner (Delva et al. 2002). The glucose-induced increase in intracellular ATP, binding a greater amount of Mg_i²⁺ and forming MgATP, seems to be the most likely cause for the glucose-induced decrease in ionized magnesium (Delva et al. 2002).

In the present paper, we studied the in vitro effects of insulin, both alone or in combination with glucose, on lymphocyte Mg_i²⁺. The latter situation, i.e. the interaction between insulin, glucose, and Mg_i²⁺, which obviously tries to mimic the physiological situation, is complex. As shown in Fig. 1A, insulin is capable of significantly increasing the Mg_i²⁺ in all glucose concentrations tested, which were intended to mimic conditions of overt hyperglycemia (15 mmol/l), hyperglycemia (10 mmol/l) and fasting (5 mmol/l). From a kinetic point of view, the three dose–response curves are sigmoidal with approximately the same EC₅₀. The time courses of the insulin-induced increase in Mg_i²⁺ (Fig. 1B) and glucose-induced decreases in Mg_i²⁺ (Delva et al. 2002) are superimposable as far as the timing of initial and maximal effect are concerned. The effect of both insulin and glucose on intralymphocyte-ionized magnesium does not begin immediately, but takes place after about 30 min.

Regarding the mechanisms involved in the insulin-mediated increase of Mg_i²⁺, the lack of effects of inhibitors of the membrane Na⁺-K⁺ pump (ouabain) and membrane Na⁺-K⁺-Cl^– cotransport (bumetanide) on the insulin-mediated increase of Mg_i²⁺ is at odds with the possibility that these membrane transport systems are involved. The substitution of extracellular sodium with choline, which blocks the membrane Na⁺-Mg²⁺ exchanger, abolishes the insulin-mediated increase of Mg_i²⁺. However, since the method for blocking the Na⁺-Mg²⁺ exchanger increases cytosolic Mg²⁺ to a very high level and may even be toxic, we used two well-known inhibitors of the Na⁺-Mg²⁺ exchanger: imipramine and quinidine (Feray and Garay 1986). In the presence of either inhibitor, no increase in Mg_i²⁺ was induced by insulin. This is in favor of the hypothesis that insulin is capable of increasing the Mg_i²⁺ by inhibiting this membrane transport.

In contrast with previous data (Romani et al. 2000), insulin is capable of increasing the Mg_i²⁺ even in the absence of extracellular glucose or magnesium. This latter fact would exclude that an Ca_o ²⁺-Mg_i²⁺ exchanger or an inwardly directed Mg²⁺ channel is involved in the process. By increasing the extracellular magnesium from 0 to 3 mmol/l, the Mg_i²⁺ increases progressively both in the absence and presence of insulin. In fact, the two curves are parallel, which, together with the lack of modification of total intracellular magnesium in the presence of insulin, suggests that the insulin-induced increase in Mg_i²⁺ is not due to increases in passive permeability. Data obtained with differential membrane permeabilization do not seem to localize the insulin-mediated increase of Mg_i²⁺ either in the cytosol or in cellular organelles.

In order to better understand the mechanism of the insulin effects on Mg_i²⁺, we evaluated molecules characterized by an insulin-mimicking action such as IGF-1 and vanadate. Dominguez et al.(1998) were the first to test in vitro the effects of IGF-1 on intracellular magnesium. IGF-1 is capable of increasing Mg_i²⁺ in erythrocytes from normotensive and hypertensive subjects. The results in which no effects of IGF-1 could be detected on lymphocyte Mg_i²⁺ are difficult to compare to those obtained by Dominguez et al.(1998) because different cell models and analytical methods were used. Barbagallo et al.(2001) have been the only investigators to assess the effect of vanadate on intracellular magnesium compared to the effects of insulin. In the experimental conditions utilized (in the erythrocyte measuring with nuclear magnetic resonance), vandate increased Mg_i²⁺ in a manner that was comparable to insulin at a concentration of 50 µmol/l, while at a concentration of 1 mmol/l, it had the opposite effect and produced a decrease. We tested two concentrations of vanadate on lymphocytes. At high concentrations (1 mmol/l), it decreased Mg_i²⁺, while at 50 µmol/l, vanadate did not show any effect on lymphocyte magnesium. The discrepancy of the effects of vanadate on Mg_i²⁺ in lymphocytes with respect to erythrocytes is difficult to reconcile with the current cellular model and the different methods of measurement. However, if this were the case, our results would also be different for insulin, which is in agreement with published studies. Our data suggests that in lymphocytes, vanadate does not mimic insulin action. The decrease in Mg_i²⁺ seen at high concentrations may be attributable to the toxicity of vanadate on various membrane transport systems (Domingo 2002).

PI 3-kinase is known to be a part of the signaling pathways for Na⁺ transporters. In adipose tissue, PI 3-kinase has been shown to be activated by insulin and to mediate the effect of insulin on glucose transport. To examine the role of PI 3-kinase on insulin-induced Mg_i²⁺ increase, we measured ionized magnesium in lymphocytes incubated with insulin in the presence and absence of wortmannin (WT), an established inhibitor of PI 3-kinase. Incubation of lymphocytes with 100 µU/ml insulin and 0.1 µmol/l WT completely blocked the insulin-stimulated Mg_i²⁺ increase, suggesting yet another role for the PI 3-kinase pathway in insulin-mediated cellular events. Ferreira et al.(2003) showed that insulin-stimulated Mg²⁺ efflux from Mg²⁺-loaded erythrocytes is blocked by WT. Despite this, an insulin-stimulated Mg_i²⁺ magnesium efflux does not explain the insulin-stimulated Mg_i²⁺ increase that we observed.

The use of 1,2-dioctanoyl-sn-glycerol, a cell-permeable diacylglycerol analog, mimics the effect of insulin on Mg_i²⁺, suggesting that insulin may act on intracellular Mg_i²⁺ at least in part by activation of protein kinase C (PKC).

Finally, we utilized iodoacetic acid to inhibit the hexose monophosphate shunt, and 2-deoxy-D-glucose to inhibit glycolysis. Both substances greatly increased Mg_i²⁺, as shown in Table 1, and we propose that it is likely to be due to a decrease in intracellular ATP and the consequent increase in free, unbound magnesium. The massive increase of ionized intracellular magnesium produced by both these substances prevents the measurement of any additional effects of insulin.

Even though the calcium determination was not the main aim of the study, intralymphocyte-free calcium was also measured, mainly to exclude substantial intracellular calcium increase that may theoretically affect magnesium determinations (Hurley et al. 1992). Insulin did not alter the Ca_i²⁺, thus confirming the results obtained in vitro in other cell models (Thomas et al. 1985).

In conclusion, we have shown for the first time in human nucleated cells the ability of insulin to modify in vitro the concentration of Mg_i²⁺ through a mechanism based on activation of a Na⁺–Mg²⁺ membrane transport system, possibly through the mediation of PI 3-kinase and PKC activation. Taking into account the relationship between insulin and glucose plasma levels and their opposite effects on Mg_i²⁺, this may represent the two limbs of a biphasic regulatory system of Mg_i²⁺ in both physiological and pathological conditions. An increase of insulin in response to eating, and therefore before an increase in plasma glucose levels, can be hypothesized, increasing Mg_i²⁺ and augmenting the activity of enzymes involved in glucose metabolism to bring about conditions that are more appropriate for metabolism and production of energy. Moreover, this effect would be produced as a final result of the increased enzymatic velocity and decrease in the extracellular concentration of glucose to reestablish glucose homeostasis. The decrease in extracellular glucose thus obtained would bring about a decrease in insulinemia that would precede a decrease in Mg_i²⁺, with reestablishment of cellular magnesium. In type 1 or 2 diabetes mellitus, the elevated plasma glucose concentrations in the absence of insulin or in situations of peripheral resistance to its actions would produce a prevailing effect, decreasing the inhibition of Mg_i²⁺. This would lead to a slowing of glucose degradation and would negatively influence magnesium-dependent enzymatic reactions. In addition, it would explain the frequent observation of reduced intracellular magnesium concentrations in patients with diabetes mellitus (Paolisso et al. 1988).

It is clear that this mechanism, which has been shown in lymphocytes, cannot easily be extrapolated to other cellular models that are much more important in the physiopathology of glucose metabolism, both in physiological conditions and in diabetes mellitus. Despite this, a direct correlation has been described between the concentration of magnesium in lymphocytes and that in striated muscular tissue (Dyckner and Wester 1985). It must also be remembered that some authors have shown that there is an alteration of pyruvate dehydrogenase (Curto et al. 1997) in the lymphocytes of patients with type 2 diabetes mellitus, which is in agreement with studies carried out in adipocytes (Mandarino et al. 1986) and in skeletal muscle (Kelley et al. 1992) of patients with type 2 diabetes mellitus. Finally, several investigators have described the presence of GLUT3 in human lymphocytes (Estrada et al. 1994), which is a transporter in some cell types that have been shown to be insulin sensitive (Bilan et al. 1992). Estrada et al.(1994) have also demonstrated that in contrast to monocytes, lymphocytes show a significant increase in the expression of GLUT3 in patients with insulin-dependent diabetes mellitus with respect to healthy control subjects.

	Funding

 
The present study was supported by grants from University of Verona (Fondi di Ateneo), Italy. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received in final form 15 April 2006
Accepted 28 April 2006
Made available online as an Accepted Preprint 11 May 2006

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