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Intracellular magnesium of obese and type 2 diabetes mellitus children

Introduction Magnesium is a critical cofactor in numerous enzymatic reactions. Diabetic patients and obese subjects are often reported to have intracellular magnesium ([Mg2+]i) deficiency. We studied the change of [Mg2+]i in obese children and children with type 2 diabetes mellitus (DM2) after educational intervention or treatment. Methods A total of 25 subjects were included: 13 with simple obesity (10 male, 3 female; mean age 16±8 years, intervention period 1.0±0.6 years), 12 with DM2 (8 male, 4 female; mean age 15±3 years, medication period 1.1±0.7 years), and 16 controls (8 male, 8 female; mean age 17±7 years). By using a fluorescent probe, mag-fura-2, we examined the basal and insulin-stimulated [Mg2+]i of platelets in the blood. Plasma leptin, ghrelin, adiponectin, and resistin levels were determined with the use of enzyme-linked immunosorbent assay (ELISA). Results Mean basal [Mg2+]i was lower in the obesity (160±65 μmol/L) and DM2 groups (140±30 μmol/L) compared with the control group (330±28 μmol/L). The elevated [Mg2+]i after insulin stimulation was also lower in these two groups (420±140 μmol/L, and 330±70 μmol/L, respectively) compared with the control group (690±270 μmol/L). In the DM2 group, the basal [Mg2+]i was significantly increased after treatment, while in the obesity group, stimulated [Mg2+]i was increased after intervention. Conclusion Platelet [Mg2+]i increased after intervention in children with obesity or DM2.


INTRODUCTION
Medical University. Written informed parental consent was obtained before recruitment.

Educational Intervention and Treatment
We educated the children and parents in a formal program of intensive lifestyle modification through anticipatory guidance about healthy dietary and activity habits. According to information from the American Academy of Pediatrics, 12 we prescribed and supported healthy eating habits such as avoiding the consumption of calorie-dense, nutrient-poor foods (for example, sweetened beverages, sports drinks, fruit drinks and juices, most "fast food" types, and caloriedense snacks). We prescribed eating timely, regular meals, particularly breakfast. We prescribed 30 minutes of daily moderate physical activity, such as walking briskly or riding a bicycle. We supported a decrease in time spent in sedentary activities, such as watching television, playing video games, or using computers for recreation.
Screen time was advised to be limited to 1-2 hours per day. No obesity subject was treated with any medication, including magnesium, and none showed any evidence of endocrine malfunction or recent use of drugs that might potentially alter electrolyte balance. The patients with DM2 were treated with metformin (250-750 mg orally twice daily), glybencramide 2.5 mg twice daily, and subcutaneous insulin 0.2-0.4 units/kg body weight twice daily, in combination with lifestyle modification.

Platelet Preparation
Blood samples were collected with citrate acid buffer and kept at 4°C. Platelets were isolated as previously described. 13 Approximately 5 mL of blood was taken and added to 3.8% (w/v) acid citrate buffer (10:1, v/v) then centrifuged at 200 g for 10 minutes at room temperature.
The association between magnesium deficiency and insulin resistance in children has been reported previously. 7

Subjects
These study groups consisted of children with simple obesity or DM2. Diabetes was defined using the criteria of the American Diabetes Association. 10 Consequently, if the diabetes criteria were met, the patient was considered to have DM2 regardless of body mass index (BMI). Height was measured to an accuracy of 0.1 cm and weight to 0.1 kg. Blood pressure was measured using a portable sphygmomanometer after the subject had rested in the sitting position for at least 5 minutes. A child was diagnosed as obese if his or her BMI was at least in the 95th percentile for age and sex or was over 25 kg/m 2 .
The percentage overweight definition was based on the percentage of a reference weight that is based on age, sex, and height. 11 The control group consisted of subjects with normal blood pressure and a negative family history of diabetes mellitus.
Samples from the control group were obtained once for the [Mg 2+ ] i measurements.

Ethical Considerations
This study was in compliance with the Declaration of Helsinki. The study protocol was approved by the ethics committee of the Kansai the methods of Ng et al. 16 Insulin was dissolved in deionized water. A total of 25 μL of insulin was added to 2.5 mL of platelet suspension. The final concentration of insulin was 0.72 nmol/L.

Enzyme-Linked Immunosorbent Assay
Plasma for enzyme-linked immunosorbent assay (ELISA) was separated immediately, stored at -80°C, and thawed only once before analysis.
Plasma glucose levels were measured by the glucose oxidase methods. Plasma leptin levels were determined with the use of a commercially available ELISA kit (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) with a detection limit of 195 ng/L (intra-assay and interassay coefficient of variations [CVs] of 6.9% and 7.7%, respectively). A plasma adiponectin assay was performed using a commercially available ELISA kit (R&D System Inc. Minneapolis, MN, USA) with a detection limit of 0.246 ng/mL (intra-assay and interassay CVs of 3.4% and 6.8%, respectively). Plasma resistin assay was performed using a commercially available ELISA kit (R&D System Inc. Minneapolis, MN, USA) with a detection limit of 0.026 ng/mL (intra-assay and interassay CVs of 5.3% and 8.2%, respectively). Plasma desacyl ghrelin concentrations were determined with the use of a commercially available ELISA kit (Mitsubishi Kagaku Iatrons, Inc., Tokyo, Japan) with a detection limit of 1.08 fmol/mL (intra-assay and interassay CVs of 5.3% and 9.8%, respectively). Platelets were studied within 4 hours after blood drawing.

Measurements of Intracellular [Mg 2+ ] i Concentrations
Ionic [Mg 2+ ] i concentrations were measured with a Hitachi F-2000 fluorescence spectrophotometer (Hitachi Instruments, Tokyo, Japan) by using an Mg-specific fura-2 probe as described by Raju et al. 14 A 2 μmol/L quantity of mag-fura-2/acetoxymethyl dye (fura dye) was added to the platelet suspension and incubated at 37°C for 30 minutes. After loading of the dyes, the platelets were washed twice with HBS, the fura dyes were removed by centrifugation, and the platelets were resuspended in HBS. The excitation wavelengths were set at 335/370 nm, and the emission wavelength was 510 nm. Each intracellular ionic concentration was calculated as described previously, 14,15 by using the dissociation constant K d =1500 μM. The maximum intensities were determined by disrupting the cells with 0.1% Triton in the presence of 30 mM MgCl 2 . The minimum intensities were the values determined in the presence of 60 mM EDTA. Manganese (II) chloride (0.05 mM) was used to quench the fluorescence from extracellular dye according to stated otherwise. Mag-fura-2/acetoxymethyl was purchased from Molecular Probes (Eugene, OR, USA).

Statistical Analysis
Data were expressed as the mean±SD.
Statistical significance was assessed using analysis of variance (ANOVA). Outcome variables were compared between the subgroups (obesity, DM2, and control) using t tests. A value of P<0.05 was considered significant. All statistical analyses were performed using StatView software (SAS Institute Inc., Cary, NC, USA).
Five patients were treated with metformin, one with glybencramide, and two with insulin in combination with lifestyle modification. The control group consisted of 16 subjects (8 male, 8 female; mean age 17±7 years).

Profile of Each Group
A profile of the study subjects is shown in Table 1. No statistical differences among the groups were observed for serum magnesium.
No significant difference in plasma leptin, adiponectin, ghrelin, resistin, and insulin levels existed between the obesity and DM2 groups (Table 2). Each group did not differ significantly in terms of medication or prescription. There was no difference of leptin values between genders (data not shown).

The Effect of Intervention or Treatment
In the obesity group, body weight, BMI, percentage overweight, and blood pressure were decreased after educational prescription.
Plasma leptin and resistin were decreased after intervention. Adiponectin and ghrelin were increased but not significantly. In the DM2 group, glycated hemoglobin (HbA 1c ), mean glucose, and blood pressure were