Free radical production is increased in subjects with

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1 Effects of Vitamin E and Glutathione on Glucose Metabolism Role of Magnesium Mario Barbagallo, Ligia J. Dominguez, Maria Rosaria Tagliamonte, Lawrence M. Resnick, Giuseppe Paolisso Abstract Vitamin E is an antioxidant that has been demonstrated to improve insulin action. Glutathione, another natural antioxidant, may also be important in blood pressure and glucose homeostasis, consistent with the involvement of free radicals in both essential hypertension and diabetes mellitus. Our group has recently suggested that the effects of reduced glutathione on glucose metabolism may be mediated, at least in part, by intracellular magnesium levels (Mg [i] ). Recent evidence suggests that vitamin E enhances glutathione levels and may play a protective role in magnesium deficiency induced cardiac lesions. To directly investigate the effects of vitamin E supplementation on insulin sensitivity in hypertension, in relation to the effects on circulating levels of reduced (GSH) and oxidized (GSSG) glutathione and on Mg [i], we performed a 4-week, double-blind, randomized study of vitamin E administration (600 mg/d) versus placebo in 24 hypertensive patients and measured whole-body glucose disposal (WBGD) by euglycemic glucose clamp, GSH/GSSG ratios, and Mg [i] before and after intervention. The relationships among WBGD, GSH/GSSG, and Mg [i] in both groups were evaluated. In hypertensive subjects, vitamin E administration significantly increased WBGD ( to mol/kg of fat-free mass per minute; P 0.01), GSH/GSSG ratio ( to ; P 0.01), and Mg [i] ( to mmol/l; P 0.01). In basal conditions, WBGD was significantly related to both GSH/GSSG ratios (r 0.58, P 0.047) and Mg [i] (r 0.78, P 0.003). These data show a clinical link between vitamin E administration, cellular magnesium, GSH/GSSG ratio, and tissue glucose metabolism. Further studies are needed to explore the cellular mechanism(s) of this association. (Hypertension. 1999;34[part 2]: ) Key Words: glutathione magnesium hypertension glucose insulin resistance antioxidants Free radical production is increased in subjects with non insulin-dependent diabetes mellitus (NIDDM) 1,2 and those with essential hypertension, 3,4 but the mechanism responsible for the linkage among increased oxidative stress, impaired glucose metabolism, and blood pressure is still debated. Vitamin E improves the free radical defense system potential and may have a beneficial effect in improving glucose transport and insulin sensitivity. 5 7 Glutathione in the reduced state (GSH) is present in human plasma; intracellularly, it has antioxidant properties to inhibit free radical formation and functions more generally as a redox buffer. 1,8,9 Recent evidence suggests that GSH may also be important in blood pressure and glucose homeostasis, consistent with the involvement of free radicals in both essential hypertension and diabetes mellitus Changes in the reduced/oxidized glutathione (GSH/GSSG) ratio affected the -cell response to glucose and improved insulin action. 10 Additionally, glutathione infusions both lowered blood pressure 11 and directly potentiated insulin secretion in subjects with insulin resistance and impaired glucose tolerance. 9 However, despite the above evidence, the mechanisms underlying the contribution of vitamin E and GSH to vascular tone and carbohydrate metabolism remain undefined. While investigating the ionic aspects of insulin resistance in diabetes and hypertension, we suggested that the depletion of intracellular free magnesium common to both conditions may help to explain their frequent clinical association, especially because all kinases and other ATP-related enzymes and channels regulating insulin action and vascular tone 19,20 are magnesium dependent. Magnesium deficiency is also associated with increased free radical dependent oxidative tissue damage, 21,22 and magnesium supplementation may lower blood pressure 23,24 and improve circulating glucose levels and tissue glucose oxidation in subjects with NIDDM. 25 We previously showed that antioxidants improve insulin sensitivity in diabetics and in aged subjects, 5,6,9,10 and Received May 9, 1999; first decision June 1, 1999; revision accepted July 8, From the Institute of Internal Medicine and Geriatrics University of Palermo, Palermo, Italy (M.B., L.J.D.); Division of Endocrinology, Metabolism and Hypertension, Wayne State University, Detroit, Mich (L.J.D., L.M.R.); and Department of Geriatric Medicine and Metabolic Diseases II University of Naples, Naples, Italy (R.T., G.P.). Correspondence to Mario Barbagallo, MD, PhD, Viale F. Scaduto 6/c, Palermo, Italy. mabar@unipa.it 1999 American Heart Association, Inc. Hypertension is available at

2 Barbagallo et al Vitamin E, Glutathione, and Glucose Metabolism 1003 we recently suggested that an increase in intracellular magnesium may mediate, at least in part, the favorable relation of GSH to glucose metabolism. 26 In light of such experimental evidence, the purpose of the present study was to investigate whether vitamin E, a natural antioxidant, has similar effects in vivo in another state associated with increased oxidative stress, such as essential hypertension. We report here the effects of vitamin E supplementation on insulin action in hypertensive patients in relation to the effects on intracellular magnesium (Mg [i] ) and circulating levels of reduced and oxidized glutathione. Methods Twenty-four newly diagnosed hypertensive patients were recruited and monitored from the outpatient clinic of the Department of Geriatric Medicine and Metabolic Diseases of II University of Naples. The study protocol was approved by the Ethics Committee of the institution; the study was conducted according to the guidelines of the Helsinki declaration, and informed consent was obtained from each subject after the nature of the procedures had been explained. Essential hypertension was diagnosed on the basis of outpatient blood pressure 140/90 mm Hg on 3 occasions and the absence of any history, physical examination, or laboratory evidence of secondary forms of hypertension. None of the subjects had a family history of diabetes. At the time of enrollment, all patients underwent a 75-g oral glucose tolerance test. Patients with diabetes mellitus or glucose intolerance were excluded from the study. After a 4-week run-in period, all patients were assigned to 1 of the 2 research groups in a random, double-blind manner, as explained below. All hypertensive subjects from both groups had not taken medications for 4 weeks and were given diuretic therapy (furosemide 25 mg/d) at baseline for ethical reasons. No patient had significant renal dysfunction, as assessed by serum creatinine levels. After an overnight fast, all subjects underwent measurement of whole-body glucose disposal (WBGD) by the euglycemic glucose clamp technique, as described below. Blood samples for Mg [i] and plasma glutathione concentration measurements were always taken at the beginning of the euglycemic clamp. After the euglycemic glucose clamp test, the 2 study groups were respectively given vitamin E (600 mg/d PO) or placebo treatment for 4 weeks. At the end of the intervention period, all subjects repeated the euglycemic glucose clamp. Total red blood cell intracellular magnesium (Mg [i] ) levels and plasma glutathione levels were also obtained at this stage. The relationships among WBGD, GSH/GSSG levels, and Mg [i] in both groups were evaluated. Eight nondiabetic normotensive subjects matched for age, sex, and body mass index with the study groups also underwent a euglycemic glucose clamp before and after 4 weeks supplementation with vitamin E (600 mg/d). Anthropometric and Fat-Free Mass Measurements Weight and height were measured by standard techniques. Fat-free mass (FFM) was measured with a 4-terminal bioimpedance analyzer (BIA) (RJL Spectrum Bioelectrical Impedance, BIA 101/SC Akern). Measurements were performed with subjects in the supine position, after they had fasted overnight and emptied their bladders. Prediction of FFM by BIA was done with equations validated for subjects with a wide age range. Body mass index was calculated as body weight divided by height squared. Euglycemic Glucose Clamp Euglycemic glucose clamp was performed according to the method of De Fronzo et al. 27 Specifically, with a fixed insulin infusion rate (1 mu kg 1 min 1 Humulin, Eli Lilly), the pump delivered a variable amount of glucose (as 20%) solution supplemented with 0.26 mmol/l KCl to maintain euglycemia and basal plasma potassium levels throughout the experiment. During the glucose clamp, blood samples were drawn for measurements of glucose and insulin at 20, 5, and 0 minutes and then every 20 minutes until the end of the test. WBGD was calculated during the final 60 minutes of the clamp according to the following formula: WBGD glucose infusion rate pool correction where the pool correction takes into account the change in the whole body glucose pool, as estimated from the change in plasma glucose concentration. This correction was always 5% of the glucose infusion rate during the glucose clamp. This calculation is valid when no entry into plasma of glucose from the liver occurs. 24 Furthermore, preliminary studies in our group have demonstrated that an infusion rate of 1 mu kg 1 min 1 fully suppresses hepatic glucose output in both control subjects and hypertensive patients. Analytical Methods Glutathione Measurements Fasting blood was obtained for analysis of plasma total glutathione, GSH, GSSG, and Mg [i]. Samples for plasma total glutathione determinations were collected according to the techniques described by Beutler and Gelbart. 8 Plasma levels of total glutathione, GSH, and GSSG levels were determined by use of an enzymatic assay 28 that allows a recovery of GSH 90% and has no appreciable interference with other thiols present in the plasma or in the reactive mixture. Intracellular Magnesium Measurement by Atomic Absorption Spectroscopy Blood samples for Mg [i] measurements were collected into tubes containing heparin. We used a method previously described in detail elsewhere. 18 Briefly, erythrocytes were isolated by centrifugation (5000 rpm for 15 minutes), and the precipitate was washed 3 times with an isotonic saline solution (150 mmol/l NaCl). Subsequently, cells were incubated for 90 minutes in a Krebs-Ringer buffer with the following composition: 2.5 mmol/l NaCl 2, 1.2 mmol/l MgCl 2, and 20 mmol/l NaHCO 3. Solutions were continuously gassed with a mixture of 95% O 2 and 5% CO at ph 7.4, and the temperature was kept at 37 C. Cells were counted to normalize samples, then were lysed osmotically by the addition of deionized water, with the solution allowed to stand for 30 minutes. Then the solution was centrifuged and the supernatant kept at 20 C until magnesium determinations were made by atomic absorption spectrophotometry with a Perkin-Elmer apparatus. All assays were performed in duplicate. Plasma Metabolites Plasma glucose was determined by the glucose-oxidase method (Beckman Auto-Analyzer), and serum immunoreactive insulin was measured by standard radioimmunoassay techniques. Statistical Analysis Data are expressed as mean SD. Differences between hypertensive patients and control subjects were assessed by unpaired t tests. Pearson correlation coefficients were used to analyze the linear correlations between variables. Differences were considered to be statistically significant for probability values Results Clinical and laboratory characteristics of the study subjects are reported in the Table. As shown, baseline fasting plasma total glutathione concentrations, Mg [i], vitamin E, and WBGD were not different between the 2 groups. Four weeks of vitamin E treatment significantly elevated vitamin E (from to mol/l; P 0.01; Table), WBGD ( to mol/kg of FFM per minute; P 0.01; Figure 1A), and glutathione levels (GSH/GSSG ratio increased from to ; P 0.01; Figure 1B). Mg [i] was also significantly increased in the vitamin E supplemented group ( to mmol/l; P 0.01; Figure 1C).

3 1004 Hypertension October 1999 Part II Clinical Characteristic of Study Subjects Placebo (n 12)* Vitamin E (n 12)* Baseline Follow-Up Baseline Follow-Up Age, y Gender, M/F 5/7 6/6 BMI, kg/m BF, % FPG, mmol/l FPI, pmol/l SBP, mm Hg (P 0.001) (P 0.001) DBP, mm Hg (P 0.005) (P 0.005) HR, bpm Vitamin E, mol/l (P 0.001) BMI indicates body mass index; BF, body fat; FPG, fasting plasma glucose; FPI, fasting plasma insulin; SBP, systolic blood pressure; DBP, diastolic blood pressure; and HR, heart rate. *All patients received furosemide 25 mg/d. All results are mean SD. P vs placebo. Vitamin E concentrations, glutathione levels, WBGD, and Mg [i] were not altered in the placebo group (Table and Figure 1). In nondiabetic normotensive subjects, vitamin E supplementation did not alter WBGD ( to mol/kg of FFM per minute; P NS). When all hypertensive subjects were taken into consideration, WBGD at baseline was significantly related to Mg [i] (r 0.826, P 0.001). In the vitamin E supplemented group, WBGD at the end of treatment was significantly related to GSH/GSSG ratio (r 0.58, P 0.047) (Figure 2A) and to Mg [i] (r 0.78, P 0.003) (Figure 2B), and the increase in WBGD ( WBGD) was also significantly related to the increase in GSH/GSSG ratio ( GSH/GSSG) (r 0.660, P 0.019). Discussion Increased free radical activity and alterations of antioxidant status have been reported previously in essential hypertension, 3,4,29 in women with pregnancy-induced hypertension, 30 and in other cardiovascular diseases. 29 Current interest in the protective role of endogenous antioxidants led our group to examine the effects of vitamin E and glutathione on insulin sensitivity, and the positive effects of these compounds were Figure 1. WBGD (A), GSH/GSSG ratio (B), and Mg [i] (C) at baseline and after 4 weeks of treatment with vitamin E (Vit. E) or placebo. *P Figure 2. Relations of WBGD to GSH/GSSG ratio (A) and to total red blood cell intracellular magnesium (B) in vitamin E treated patients.

4 Barbagallo et al Vitamin E, Glutathione, and Glucose Metabolism 1005 shown in diabetic and in aged subjects. 5,6,9,10 Recently, we 26 reported that GSH has a direct effect in vivo and in vitro to increase Mg [i] levels, which suggests that this effect may mediate, at least in part, the positive effects of GSH on glucose metabolism. We wondered whether vitamin E has similar effects in vivo in another state associated with increased oxidative stress, such as essential hypertension, and to what extent the previously reported effects of vitamin E on peripheral insulin action and blood pressure may be explained by interactions with glutathione and Mg [i] content. The present study demonstrated the following: (1) an increase of insulin sensitivity in hypertensive subjects treated with vitamin E; (2) a rise of GSH/GSSG ratio in the vitamin E treated group; (3) a concurrent increase in Mg [i] in the vitamin E treated subjects; and (4) a significant direct relationship between glucose disposal and endogenous circulating GSH/GSSG ratio and between glucose disposal and Mg [i] levels. It is therefore reasonable to suggest that the effects of vitamin E supplementation on glucose and insulin metabolism may derive at least in part from its effects on GSH/GSSG ratio and cellular magnesium concentrations. Our results are consistent with previous observations in the literature. Pharmacological doses of vitamin E enhance red blood cell levels of reduced glutathione 31 and plasma GSH/ GSSG ratio in humans. 32 Increased oxygen free radical production, which may contribute to several human diseases, 33 is associated with both low plasma GSH/GSSG ratios 10 and low Mg [i] concentrations. 24 Vitamin E has been demonstrated to protect against magnesium deficiency induced myocardial injury 21,34,35 and magnesium deficiency associated cerebral vascular damage. 36 Conversely, prior magnesium depletion renders cells more sensitive to oxidative damage. 21 Furthermore, magnesium may itself possess antioxidant properties, scavenging oxygen radicals, possibly by affecting the rate of spontaneous dismutation of the superoxide ion. 37 Chronic hypomagnesemia results in excessive production of oxygen-derived free radicals, 34 which supports a role for magnesium in altering the threshold antioxidant capacity. Hypertension, as well as type II diabetes and aging, is associated with increased production of oxygen free radicals, a rise in plasma fasting free radicals, hyperinsulinemia, and insulin resistance. 3,4,12,17,29,33 However, the mechanisms underlying the relationship between elevated plasma free radical concentration and poor insulin-mediated glucose uptake are still unclear, 2,8 10 and whether oxidative stress precedes or follows hyperinsulinemia/insulin resistance is still an open question. Indeed, chronic slight hyperinsulinemia is a prooxidant factor. In intact human fat cells, exposure to insulin leads to accumulation of hydrogen peroxidase in the suspension medium. 38 Conversely, in vitro data indicate that oxidative stress might be responsible for a decline in insulin action. 39,40 In particular, it has been demonstrated that oxidative stress may be associated with a reduced exposition of glucotransporter 4 39 and/or an impairment of insulin signaling. 40 Thus, a vicious circle among hyperinsulinemia and plasma free radical concentrations might occur in these subjects. Recent evidence suggests that antioxidants may also improve impaired insulin-mediated glucose uptake in vascular smooth muscle cells. 41 Preliminary data reported here suggest that vitamin E may have no effect on insulin sensitivity in subjects without oxidative stress (nondiabetic, normotensive subjects). However, the action of antioxidants in subjects without oxidative stress in a preventive perspective should be addressed in future studies. We did not observe in the present study any additive effect of vitamin E on blood pressure with respect to furosemide treatment (25 mg/d), which was administered to all hypertensive patients for ethical reasons. However, the diuretic administration may have masked any effect of vitamin E on blood pressure. Our present data suggest that the relation between vitamin E and glucose metabolism may be mediated, at least in part, by the ability of vitamin E to stimulate glutathione and Mg [i]. We have previously formulated an ionic hypothesis in which altered intracellular steady-state concentrations of calcium and magnesium ions act as a final common pathway to regulate cellular metabolism in general and in particular, cellular glucose homeostasis, insulin sensitivity, peripheral vascular tone, and blood pressure. 12 Previous studies indicated a role for magnesium in insulin action ,16 18,26 The insulin-induced changes in magnesium are directly proportional to the initial Mg [i] level; the depletion of magnesium from normal cells renders them insulin resistant, 42 and dietary-induced magnesium deficiency is also associated with a decrease in insulin action. 43 These observations emphasize the potential contribution of altered cellular magnesium as an independent determinant of insulin action. In conclusion, our data demonstrate that in patients with essential hypertension, chronic consumption of pharmacological doses of vitamin E improves insulin-mediated glucose disposal and enhances GSH/GSSG ratio and Mg [i] content, and they suggest a role for glutathione and Mg [i] in mediating the effects of vitamin E on insulin action. Further studies are needed to explore the cellular mechanism(s) of this association. References 1. Hwang C, Sinskey AJ, Lodish HF. Oxidized redox state of glutathione in the endoplasmic reticulum. Science. 1992;257: Collier A, Wilson R, Bradley H, Thomson JA, Small M. Free radical activity in type II diabetes. Diabet Med. 1990;7: Sagar S, Kallo IJ, Kaul N, Ganguly NK, Sharma BK. Oxygen free radicals in essential hypertension. Mol Cell Biochem. 1992;111: Moran JP, Cohen L, Greene JM, Xu G, Feldman EB, Hames CG, Feldman DS. Plasma ascorbic acid concentrations relate inversely to blood pressure in human subjects. 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