Insulin resistance, oxidative stress markers and the blood antioxidant system in overweight elderly men

Abstract

The purpose of the study was to examine in the blood of overweight men aged from 62 to 83 years, the relationships between age and insulin resistance, selected parameters of the oxidative stress, and the antioxidant defense system. The population studied was divided into two groups: the group ‘young-old’ consisted of men aged 62 to 74 years old, and the group ‘old-old’– of men aged between 75 and 83 years. The total antioxidative status (TAS) and concentrations of thiobarbituric acid reactive substances (TBARS) were measured in the blood plasma. In the serum samples, the levels of antibodies against oxidized LDL (oLAB), glucose, total cholesterol, HDL-cholesterol, triglycerides and insulin were measured. Homeostasis Model Assessment of Insulin Resistance (HOMA_IR) was calculated. Concentration of reduced glutathione (GSH) and glutathione peroxidase activity (GPx) were determined in the red blood cells hemolysate. The results of the study did not show significant differences between groups investigated with respect to concentrations of TBARS, TAS, GSH and GPx. However, significantly higher concentrations of glucose and antibodies against oxLDL (p < 0.05) were observed in the group of men over 74 years old in comparison to the group of ‘young-old’ men. It was indicated that the increased insulin resistance and hyperglycemia in elderly men are related to body mass and that they cause intensified oxidative modifications of LDL.

Keywords:

• HOMA_IR
• oxidative stress
• antioxidant defence system
• elderly men

Introduction

Insulin resistance is defined as a lower than normal insulin activity with respect to stimulation of glucose transport and its metabolism in target tissues. According to data published by the European Group for the Study of Insulin Resistance, it is correlated to age [1]. Metabolic consequence of the insulin resistance is hyperglycemia – being an effect of decreased insulin transport predominantly to the muscle tissue [2],[3]. Hyperglycemia has been acknowledged as one of the main factors increasing the level of oxygen free radicals as a result of autooxidation of glucose, protein glycation or formation of final products of progressive glycation [4]. In addition, the increase of the glucose level in the blood above reference values intensifies its increased transport to the endothelial cells, and causes in them increased catabolism in the polyol pathway. The reduction of glucose into sorbitol decreases accessibility of NADPH for regeneration of intracellular antioxidants and causes prooxidative effects [5]. Moreover, the enlarged fat deposition of the body is not only associated to the glucose intolerance and insulin resistance, but to the oxidative stress as well [6],[7].

The metabolic disturbances mentioned above have inspired us to investigate relationships between age and the insulin resistance, oxidative stress markers and the antioxidant defense system in the blood of overweight men, aged between the seventh and ninth decades of life.

Methods

The study was conducted on 118 men aged 62–83 years – members of ‘Senior's Club’ in Poznań. All declared good health status. Data concerning detailed health condition, dietary habits, alcohol consumption, smoking status and medication were obtained with a questionnaire. Patients with inflammatory disorders, recent infections, renal or hepatic insufficiency, active coronary artery disease, diabetes, hypertension (≥160/100 mm Hg), heart failure, obvious nutritional deficiencies or using corticosteroids were not included in the study.

The subjects under the study were divided on the basis of Spirduso [8] criteria, identifying elderly men from their calendar age. The group of ‘young-old’ consisted of men between 62 and 74 years, the group of ‘old-old’ consisted of men between 75 and 83 years. Before starting the study, all participants were delivered an explanation of methods and purposes of the investigations. After basic anthropometrical measurements (height and body mass) the body mass index (BMI) was calculated and subsequently the body composition was assessed with the method of electric bioimpedance analysis (BIA), using 101/S analyzer produced by Akern (Florence, Italy).

In order to determine biochemical indices, the venous blood was taken from the antecubital vein, after an overnight fast, between 7 and 8 a.m. In the heparinized blood plasma the total antioxidant status (TAS) was measured with the commercially available assay (Randox Laboratories Ltd, Crumlin, Co. Antrim, UK) and the concentration of thiobarbituric acid reactive substances (TBARS) were assessed using a modified spectrophotometric method with chromogen extraction with n-butanol, as described by Buege and Aust [9].

In the serum samples, levels of antibodies to oxidized LDL (oLAB) were measured with the commercially available enzyme-linked immunoassay (Biomedica GmbH, Wien, Austria), while the concentrations of glucose, total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides (TG) were measured by tests made by Cormay (Warsaw, Poland). The insulin level was assessed using a commercially available radioimmunoassay (INS-IRMA, BioSource SA, Nivelles, Belgium). The LDL-cholesterol concentration was calculated with the formula proposed by Friedewald [10]. Insulin sensitivity index HOMA_IR (Homeostasis Model Assessment of Insulin Resistance) was calculated according to the formula of Matthews et al. [11]: HOMA_IR = C_INS (μU/mL) × C_GLUC (mmol/L)/22.5.

In the red blood cells haemolysate the amount of reduced glutathione (GSH) was measured with BIOXYTECH® GSH-400™ assay (Oxis International Inc., Portland, USA) and the activity of glutathione peroxidase (GPx) was estimated using BIOXYTECH® GPx-340™ (Oxis International Inc., USA). The concentrations of haemoglobin in the red blood cells haemolysate were measured with HemoCue Blood Hemoglobin analyzer (HemoCue, Angelholm, Sweden), and afterwards levels of GSH and activity of GPx per 1 gram of haemoglobin were calculated.

The technicians performing biochemical analyses were not aware of patients' clinical data. The results of the study were presented as mean values±standard deviations (SD). The statistical analysis was performed with the nonparametric Mann-Whitney test and the Spearman's rank correlation test. The level of significance was set at p = 0.05.

Informed, written consent was obtained from each subject prior to the enrolment in the study. The study protocol was accepted by the local Ethics Committee for Research in Humans.

Results

Tables 1–3 present comparative analysis between mean values of parameters measured in two groups of men under the study –‘young-old’ (<74 years) and ‘old-old’ (>74 years). The significant differences were found with respect to the mean body mass, body mass index and body fat mass (p < 0.05; Table I). The mean BMI in the group under 74 years amounted to 29.15±3.57 kg/m², while in the group above 74 years to 27.3±3.06 kg/m², indicating overweight in both groups of men.

Table I.  Basic characteristics of the population under the study. Mean values ± SD are presented.

	Men<74 years old (n = 83)	Men>74 years old (n = 26)	Mann-Whitney test
Age (years)	66.0±4.09	77.0±2.77	p < 0.001
Body mass (kg)	83.5±13.86	77.8±11.46	p < 0.05
Body height (m)	1.7±0.56	1.69±0.74	0.428
BMI (kg/m^2)	29.15±3.57	27.3±3.06	p < 0.05
Body fat mass (kg)	27.91±6.92	24.66±4.57	p < 0.05

BMI = body mass index.

Table II.  Biochemical parameters and comparisons between the ‘young-old’ and the ‘old-old’ groups of subjects. Mean values ± SD are presented.

	Men <74 years old (n = 83)	Men >74 years old (n = 26)	Mann-Whitney test
Total cholesterol (mmol/L)	4.92±0.62	4.79±0.61	0.333
HDL (mmol/L)	1.41±0.27	1.36±0.26	0.419
LDL (mmol/L)	3.02±0.62	2.87±0.57	0.273
Triglycerides (mmol/L)	1.1±0.38	1.03±0.31	0.531
Glucose (mmol/L)	5.56±1.22	6.08±0.99	p < 0.05
Insulin (μU/mL)	9.45±3.42	8.48±2.67	0.147
HOMA IR	2.51±1.24	2.35±0.94	0.669

HDL = high-density lipoproteins; LDL = low-density lipoproteins; HOMA_IR = Homeostasis Model Assessment of Insulin Resistance.

Table III.  Comparison of selected markers of the oxidative stress and the antioxidant system between the ‘young-old’ and the ‘old-old’ groups of elderly men. Mean values ± SD are presented.

	Men <74 years old (n = 83)	Men >74 years old (n = 26)	Mann-Whitney test
oLAB (mU/mL)	363.81±275.87	471.45±274.07	p < 0.05
GSH (μmol/gHb)	2.9±0.87	2.79±0.64	0.604
GPx (U/gHb)	29.24±10.34	28.76±7.6	0.344
TAS (mmol/L)	0.97±0.26	0.87±0.33	0.892
TBARS (μmol/L)	3.65±0.85	3.61±0.9	0.513

GSH = reduced glutathione; GPx = glutathione peroxidase; oLAB = antibodies to oxidized low-density lipoproteins; TAS = plasma total antioxidant status; TBARS = thiobarbituric acid reactive substances.

Parameters of carbohydrate and lipid metabolism are compared inTable II. No significant differences in the blood lipid profile were observed, but the concentration of glucose measured in older men was significantly higher than in the younger group (p < 0.05).

The selected markers of the oxidative stress (oLAB and TBARS), the total antioxidant status in the plasma and selected antioxidant indices in the red blood cells, i.e. the concentration of reduced glutathione (GSH) and the activity of glutathione peroxidase (GPx) are compared in Table III. The significant differences between the ‘young-old’ and the ‘old-old groups of elderly men were noted only with respect to concentrations of oLAB antibodies (p < 0.05). In the case of men under 74 years the mean value of this parameter amounted to 363.81±275.87 mU/ml, while in the older group the mean value amounted to 471.45±274.07 mU/ml.

The Spearman's rank correlation test in the ‘young-old’ group revealed the positive correlation between BMI and the HOMA _IR index (r = 0.41; p < 0.01), between BMI and the insulin concentration (r = 0.42; p < 0.01), as well as between BMI and the TBARS concentration (r = 0.41; p < 0.05). The negative correlation was found between TAS and age of subjects under the study (r = −0.25; p < 0.01). In the ‘old-old’ group, the positive correlation was revealed between TAS and the GSH concentration (r = 0.29; p < 0.01) and between BMI and the concentration of insulin (r = 0.43; p < 0.05), while the negative concentration was found between TAS and age of subjects under the study (r = −0.28; p < 0.01).

Discussion

Physiological and pathological changes occurring in the organism during the ageing process are more intensive in people with impaired long-term dietary habits (leading to overweight and obesity), limited motor activity [12],[13] as well as hormonal changes coinciding with menopause in women and andropause in men [14], [15]. Effects of the positive energetic balance and of the low level of physical activity are metabolic processes that stimulate development of the atheromatosis and insulin resistance [7],[16].

In this study, to describe the insulin resistance in elderly men between 63 and 74, and between 75 and 83 years, we have used HOMA_IR index by Matthews et al. [11]. The results have shown that in the ‘young-old’ group of men, the lower glucose concentration accompanies higher insulin concentration and higher HOMA_IR index, whereas in the ‘old-old’ group, the higher glucose concentration is associated with the lower insulin concentration and the lower HOMA_IR index (Table II). Such an image confirms the hypothesis, proposed by Rowe et al. [17], that the increased secretion of insulin, reducing the glucose concentration in the blood to the physiological level, is impaired in elderly men. The mean values of the HOMA_IR index established in our study in groups of ‘young-old’ and ‘old-old’ elderly overweight men (BMI = 29.15±3.57 kg/m², BMI = 27.3±3.06 kg/m², respectively ) are considerably higher than those described by Matthews et al. [11] who have reported that the insulin resistance in healthy subjects with regular BMI is on the normal level when the HOMA_IR index is comprised in the range 1.25-1.41. However, other authors have established higher HOMA_IR indices: 2.1 [18], 2.7 [19] and 3.8 [20]. Such variability indicates that values of this index are highly correlated with age and BMI. This has been confirmed in our study by high correlation coefficients calculated with respect to BMI and the HOMA_IR index in the ‘young-old’ group (r = 0.41; p < 0.01) and significant, positive correlations between BMI and the insulin concentration in both groups investigated (r = 0.42 and r = 0.43; p < 0.01, in the ‘young-old’ and ‘old-old’ groups, respectively).

No significant differences between groups have been revealed with respect to the mean parameters of the lipid profile in the blood, whereas the concentration of glucose in the group of older men has been higher by the average of 0.52 mmol/L (p < 0.05; Table II), in comparison to the younger group. Studies of Sakai et al. [21] and Laight et al. [22] have shown that the excessive diffusion of glucose to pancreatic beta cells may lead to the suppression of the first phase of insulin secretion (which is induced by this diffusion) and in consequence may contribute to development of the insulin resistance. According to Ceriello et al. [4], the hyperglycaemia and insulin resistance influence generation of reactive forms of oxygen. This has been previously confirmed by results of our studies in overweight and obese men at the age of 62-83 years [16], as well as by investigations of Trevisan et al. [23], conducted on New York citizens between 35 and 79 years old.

The level of the oxidative stress has been assessed by measurements of concentrations of thiobarbituric acid reactive substances (TBARS). Despite no significant differences between mean values of this parameter between the groups investigated, lower mean values of the antioxidant defense system have been found in the older men. This points to the higher consumption of antioxidants with age (Table III), which seems to be confirmed by the negative correlation between the parameters measured (r = −0.25 and r = −0.28; p < 0.01, for the groups of men under and over 74 years, respectively). One should also notice that mean TAS levels in both groups investigated (0.97±0.29 mmol/L and 0.87±0.33 mmol/L, respectively) have appeared to be lower than reference values of the test's producer. The total antioxidative status of the plasma is commonly regarded as the index of the current prooxidative-antioxidative balance in the blood [24]. Similar observations have been made by Aguirre et al. [25] in elderly men with the type II diabetes. These authors have also reported that the decreased TAS is correlated to the increased level of oxidative-modified low-density lipoproteins.

Mean values of TBARS measured in two groups under the study (3.65±0.85 and 3.61±0.9 mmol/L, for the groups of men under and over 74 years, respectively) are comparable to those previously described for men of 67–80 years old for various levels of physical activity [26]. Moreover, low values of the lipid peroxidation products have been noted by Kłapcińska et al. [27] in people over one hundred years old, as well as Cals et al. [28] in the population of old people. On the other hand, high TBARS concentration has been observed in old subjects by Cangy et al. [13]. In the latter case, however, subjects investigated have also suffered from numerous diseases and haven't presented an active lifestyle. This allows us to suggest that the TBARS concentration is connected rather to the akinesia than age.

Our results also point to a significant interrelationship between the body mass index and the concentration of antibodies against oxidized LDL (oLAB) (r = 0.43; p < 0.05). These antibodies are acknowledged as one of the most important markers of prooxidative processes [29] and have appreciated diagnostic value in assessment of atheromatosis in the circulatory system [30]. The reference values of this parameter, measured in the blood serum with a commonly used immunological test by Biomedica GmgH (Wien, Austria), are comprised in the range from 195 to 600 mU/mL. Despite the fact that the mean values of oLAB measured in both groups have been comprised below these reference values, higher concentrations have been noticed in the group of older men (>74 years) – in which also lower levels of the antioxidant defense system parameters have been established. However, in the group of younger men (<74 years; BMI = 29.15±3.57 kg/m²), the significant correlation has been found between BMI and the concentrations of oLAB (r = 0.43; p < 0.05) and TBARS (r = 0.41; p < 0.05). The above correlation coefficients in the group of ‘young-old’ group of men (but with higher overweight) seem to confirm that the body mass, and not age, is the factor determining development of disadvantageous metabolic modifications leading to the insulin resistance and oxidative modifications of the LDL cholesterol.

The comparative analysis of the mean values of parameters of the antioxidant defense system in the red blood cells, as the concentration of reduced glutathione (GSH) and the activity of glutathione peroxidase (GPx), have not indicated significant variability (Table III). Comparable levels of GSH have been previously described by Lang et al. [31] in the study performed in American women between 60 and 103 years old. The authors haven't noticed a relationship between age of women and the GSH concentration and have shown that this concentration is similar to values measured in women between 20 and 39 years old. They have also suggested that high GSH concentration in the blood of elderly people might be connected to their good physical and mental conditions. Our previous investigations [26],[32] have confirmed that elderly men with higher levels of physical activity have higher GSH concentrations in the red blood cells in comparison to men at the same age, but low physical activity. Similar results have been obtained by Kłapcińska et al. [27] in people one hundred years old.

Conclusions

The results of the study confirm the following:

• The increased insulin resistance and hyperglycemia in elderly men are associated particularly to the body mass and lead to the intensified oxidative modifications of low-density lipoproteins.

• Age of the subjects does not influence oxidative stress markers and the antioxidant defensive system of red blood cells. This lets us hypothesize that the endogenous antioxidant defense system in elderly men depends rather on the current physical and mental health status than on age.