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Hypertension. 2001;38:612-615
doi: 10.1161/hy09t1.095764
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(Hypertension. 2001;38:612.)
© 2001 American Heart Association, Inc.

Cardiovascular Biology

Altered Cellular Magnesium Responsiveness to Hyperglycemia in Hypertensive Subjects

Mario Barbagallo; Ligia J. Dominguez; Orit Bardicef; Lawrence M. Resnick

From the Institute of Internal Medicine and Geriatrics, University of Palermo (M.B., L.J.D.), Italy; and Hypertension Center, New York Presbyterian Hospital/Weill Medical College of Cornell University (O.B., L.M.R.).

Correspondence to Mario Barbagallo, MD, Professor of Medicine, Head of Geriatric Medicine, University of Palermo, Via F. Scaduto 6/c, 90144 Palermo Italy. E-mail mabar{at}unipa.it

Abstract

Abstract—— Previous studies by our group have identified ionic aspects of insulin resistance in hypertension, in which cellular responses to insulin were influenced by the basal intracellular ionic environment—the lower the cytosolic free magnesium (Mgi), the less Mgi increased following insulin stimulation. To investigate whether this ionic insulin resistance represents a more general abnormality of cellular responsiveness in hypertension, we studied Mgi responses to nonhormonal signals such as hyperglycemia (15 mmol/L) and used 31P-nuclear magnetic resonance (NMR) spectroscopy to measure Mgi in erythrocytes from normal (NL, n=14) and hypertensive (HTN, n=12) subjects before and 30, 60, 120, and 180 minutes after in vitro glucose incubations. Basal Mgi levels were significantly lower in HTN subjects than in NL subjects (169±10 versus 205±8 µmol·L-1, P<0.01). In NL cells, hyperglycemia significantly lowered Mgi, from 205±8 µmol·L-1 (basal, T=0) to 181±8, 162±6, 152±7, and 175±9 µmol·L-1 (T=30, 60, 120, and 180, respectively; P<0.005 versus T=0 at all times). In HTN cells, maximal Mgi responses to hyperglycemia were blunted, from 169±10 µmol·L-1 (basal, T=0) to 170±11, 179±12, 181±14, and 173±15 µmol·L-1 (T=30, 60, 120, and 180, respectively; P=NS versus T=0 at all times). For all subjects, Mgi responses to hyperglycemia were closely related to basal Mgi levels: the higher the Mgi, the greater the response (n=26, r=0.620, P<0.001). Thus, (1) erythrocytes from hypertensive vis-à-vis normotensive subjects are resistant to the ionic effects of extracellular hyperglycemia on Mgi levels, and (2) cellular ionic responses to glucose depend on the basal Mgi environment. Altogether, these data support a role for altered extracellular glucose levels in regulating cellular magnesium metabolism and also suggest the importance of ionic factors in determining cellular responsiveness to nonhormonal as well as hormonal signals.


Key Words: magnesium • hyperglycemia • glucose • diabetes • magnetic resonance spectroscopy

Clinical conditions such as type 2 diabetes mellitus, obesity, and hypertension frequently coexist1–5; each of these conditions has a cellular resistance to insulin action and also a common underlying abnormality of cellular ion content, viz., higher basal cytosolic free calcium (Cai), and/or reciprocally lower cytosolic free magnesium (Mgi) levels.6–8 Although usually defined on the basis of impaired insulin-mediated glucose utilization, insulin resistance is itself an ionic phenomenon,9,10 and in erythrocytes, where glucose uptake is independent of insulin, the lower the intracellular free magnesium, or the higher the free calcium, the less the ionic effects of insulin.10 On the basis of these and other observations, an ionic hypothesis was formulated, in which the insulin resistance of both hypertension and type 2 diabetes results, at least in part, from a cellular Mgi deficiency. Because basal cellular free ion levels, in addition to influencing cellular insulin responses,10 also predict blood pressure6–8 and the extent of cardiac hypertrophy,11 we suggested that insulin resistance in hypertension, rather than representing a unique, disease-specific property, might rather be only 1 example of altered cell responses to a variety of stimuli that depend on the cellular ionic environment.

If this hypothesis is correct, that the intracellular ionic milieu determines physiological cellular responsiveness, then altered cellular responses or resistance to other, nonhormonal stimuli should also be demonstrable in hypertension and other states with similarly altered basal ion content. One such stimulus is hyperglycemia, which lowers Mgi levels in normal erythrocytes.12 We therefore used 31P-nuclear magnetic resonance (NMR) spectroscopy to noninvasively measure Mgi responses to hyperglycemia in erythrocytes from normal and essential hypertensive individuals. Our results support the above hypothesis and demonstrate that Mgi responses to in vitro hyperglycemia are blunted in hypertensive subjects and that for all subjects these responses are closely linked to basal Mgi levels.

Methods

Subjects
Twenty milliliters of venous blood were drawn from normotensive controls (n=14) and unmedicated essential hypertensive outpatients (n=12) between 9:00 AM and noon after an overnight fast. Hypertensives and controls were randomly selected from patients and staff at the hypertension clinic by one of the authors (LMR). Patients were off any therapy for at least 3 weeks and had not received diuretics for at least 3 months before the study. Essential hypertension was previously diagnosed on the basis of at least 3 blood pressure readings >150/90 mm Hg in the absence of signs or symptoms of secondary forms of hypertension. A history of myocardial infarction, angina pectoris, or stroke in the 6 months before the study, as well as renal or hepatic failure, excluded the subject from consideration.

Glucose was added to cells from both normotensive and hypertensive subjects to achieve a final tube concentration of 15 mmol/L. This glucose concentration was based on previously published dose-response relationships for glucose and magnesium, in which the maximal ionic effect was observed at this concentration under the same experimental conditions.12 Intracellular free magnesium concentrations were measured serially before and 30, 60, 120, and 180 minutes after the addition of glucose.

31P-NMR Analysis of Free Mgi
Erythrocyte Mgi levels were measured according to methods previously described.8 Briefly, blood samples were spun at 2000 rpm for 10 minutes, and the plasma was discarded. The remaining packed erythrocyte fraction was decanted into 12-mm NMR tubes, and 31P-NMR spectra were recorded at 81 MHz and at 37°C with a GE 300-MHz NMR spectrometer in the Fourier transform mode and with wide-band proton noise decoupling. Under these conditions, measured Mgi levels remain stable for 8 to 12 hours. Intracellular free magnesium was calculated as Mgi=KdMgATP({Phi}-1-1), where {Phi}=(ATP)free/(ATP)total, the fraction of uncomplexed ATP derived from the 31P-NMR spectrum, and KdMgATP is the dissociation constant for the reaction MgATP=Mg2++ATP, which is 38 µmol·L-1 at 37°C and pH 7.2.

Statistical Analyses
Serial Mgi responses to hyperglycemia were analyzed with repeated-measures ANOVA with subsequent post-hoc Friedman testing for statistical significance. The statistical significance of differences in Mgi responses to glucose among HTN versus NL subjects was estimated by 1-way ANOVA, using an appropriate post-hoc t test for multiple comparisons. The relations between measured variables were assessed by linear regression analysis and Pearson correlation coefficients. Statistical tests were performed with CRUNCH software on an IBM compatible computer. All data are reported as mean±SEM.

Results

Basal Mgi levels were significantly lower in HTN subjects than in NL subjects (169±10 versus 205±8 µmol·L-1, P<0.01). In NL, hyperglycemia significantly suppressed Mgi from 205±8 µmol·L-1 (basal, T=0) to 181±8, 162±6, 152±7, and 175±9 µmol·L-1 (T=30, 60, 120, and 180, respectively; P<0.01 versus basal at all times). In contrast, in HTN Mgi responses to hyperglycemia were blunted, with 15 mmol/L glucose not significantly altering Mgi levels from 169±10 µmol·L-1 (basal, T=0) to 170±11, 179±12, 181±14, and 173±15 µmol·L-1 (T=30, 60, 120, and 180, respectively; P=NS versus basal at all times) (Figure 1). Furthermore, the changes in Mgi relative to basal Mgi levels at any time of incubation also differed significantly in cells from hypertensive and normotensive subjects (Table).



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  		Figure 1. Mgi levels before (basal T=0) and 30, 60, 120, and 180 minutes after the addition of glucose (15 mmol/L) to erythrocytes from normotensive (A) and essential hypertensive (B) subjects. C, Glucose-induced changes in Mgi at the listed times compared with that at 0 minutes in hypertensive vs normotensive subjects. *P<0.01 vs basal Mgi levels.


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  		Table 1. Glucose-Induced Changes in Mgi Levels From Baseline Values in Erythrocytes From NT and HTN Subjects

For all subjects, regardless of diagnostic clinical blood pressure category, the cellular Mgi responses to hyperglycemia were closely linked to basal Mgi levels—the higher the basal Mgi, the greater the response to glucose (n=26, r=0.620, P<0.001) (Figure 2).



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  		Figure 2. Ion dependence of cellular glucose responses: relationship between basal Mgi values and the maximal magnesium responses to glucose hypertensive and normotensive subjects (n=26).

Discussion

Magnesium, the second most abundant intracellular cation, is involved in a number of important biochemical reactions, including all ATP transfer reactions. Possibly because of its relevance to all protein kinases, magnesium appears to modulate hormonal and biochemical aspects of cellular glucose utilization.13 The intracellular magnesium deficiency that has been demonstrated in insulin-resistant states such as hypertension and type 2 diabetes may thus contribute to suppressed glucose metabolism and insulin action. Indeed, in essential hypertensive and/or diabetic subjects, both the level of the blood pressure and the hyperinsulinemic response to glucose loading are closely related to steady-state fasting levels of intracellular free magnesium—the lower the magnesium, the higher the blood pressure and the more hyperinsulinemic the response to oral glucose loading.6–8 Similarly, decreasing intracellular free magnesium in cells from normal individuals rendered them resistant to the ionic effects of insulin, an effect indistinguishable from the insulin resistance observed in cells from hypertensive subjects.10 One question arising from these observations was whether this cellular ionic resistance to insulin reflected a unique abnormality of cellular insulin action or whether it represented a more general property of cells, in which cellular magnesium deficiency necessarily also alters cellular ionic responses to other nonhormonal stimuli as well.

We approached this problem by comparing the effect of hyperglycemia on Mgi content in erythrocytes from normal versus hypertensive subjects. We have previously reported the primary ionic actions of glucose in vitro and in vivo to directly suppress Mgi early after oral glucose loading in erythrocytes and in skeletal muscle.12,14–17 These ionic effects of glucose may be clinically relevant, because hyperglycemia per se significantly contributes to insulin resistance in diabetes mellitus and because reducing Mgi promotes vasoconstriction.18–20 This MgI-lowering effect of glucose is not inconsistent with the rise in cellular total and free magnesium levels following glucose loading in vivo, which occurs over a longer time course and is accompanied by increased circulating insulin levels, insulin elevating Mgi.9,21

In the present study, we observed that (1) hyperglycemia reduced Mgi levels in cells from NL but not HTN subjects, and (2) independently of their designation as normotensive or hypertensive, glucose-induced changes in Mgi levels were closely related to basal Mgi levels—the lower the basal Mgi, the less responsive was the cell to glucose action (Figure 2). These results suggest that Mgi levels may be regulated by ambient glucose levels, that this effect is blunted in hypertension, and, more broadly, that the basal Mgi environment in turn influences the cellular ionic responses to glucose. These data also provide at least 1 mechanism—the lowering of intracellular free magnesium, by which hyperglycemia itself contributes to insulin resistance. Lastly, because the magnesium-dependence of this nonhormonal ionic glucose stimulus so closely corresponds to the similar magnesium-dependence of insulin-induced ionic responses,10 we hypothesize a central role for Mgi depletion, present in both hypertension and diabetes mellitus,6–8 in mediating the variety of altered cellular responses characteristic of these conditions. One example of this is the in vitro stimulation of erythrocyte Mgi response by captopril, in which the same behavior is observed—the greater the basal Mg, the more Mgi responded.22

Advantages and disadvantages of NMR spectroscopy-based measurements of erythrocyte Mgi, which provide values that are considerably lower than those of other techniques,23,24 have been discussed in the literature.25 Nevertheless, consistently reproducible results have been obtained using this technique in the present study, in our previous work, and in the work of other groups as well, despite remaining uncertainty about the absolute Mgi levels measured.

Our present findings support the overall hypothesis that the intracellular ionic milieu is at least 1 determinant of physiological cellular responsiveness to extracellular stimuli. Thus, following an oral glucose tolerance test, the rate of glucose disappearance from the circulation was inversely related to the basal skeletal muscle free magnesium content in hypertensives, and magnesium uptake was blunted in type 2 diabetes, both in the same manner—the lower the Mgi, the slower the decrease in extracellular glucose and the more blunted the magnesium uptake. This is consistent with an a priori cellular magnesium deficiency as the cause of, rather than merely the result of, peripheral insulin resistance.17,21 The primacy of Mgi levels in determining insulin action, rather than vice versa, was also suggested by the induction of cellular insulin resistance in normal cells following intracellular magnesium depletion to levels observed in hypertensive or diabetic cells.10 Hence, regardless of the primary origins of glucose intolerance and insulin resistance in syndromes such as hypertension or diabetes, our observations emphasize the potential contribution of altered cellular Mgi as an independent determinant of glucose and insulin action. In support of this, substances such as IGF-I that increase Mgi in normotensive and hypertensive cells independently of basal Mgi levels26 improve insulin sensitivity clinically, and we have suggested that this effect results at least in part from reversing the intracellular magnesium deficiency accompanying insulin-resistant states. Furthermore, because dietary magnesium loading contributes significantly to the blood pressure effects of "healthy diets,"27 it is possible that the overlap of cellular abnormalities in hypertension and diabetes may, at least in part, have a dietary nutritional basis. Future studies are needed to determine whether dietary magnesium loading can alter basal Mgi levels in hypertension, and/or the altered cell responses observed in the present study.

Received March 29, 2001; first decision June 5, 2001; accepted July 4, 2001.

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