We performed a cross-sectional study of 75 subjects: (a) T2D with increased body weight (obese and overweight, BMI ≥ 25 kg/m²) and hypertension (group A, n = 30), (b) T2D patients with increased body weight (obese and overweight, BMI ≥ 25 kg/m²) without hypertension (group B, n = 30) and nonobese (BMI ≥ 25 kg/m²) healthy controls (group C, n = 15). Exclusion criteria were BMI ≥ 35 kg/m², clinically significant renal or hepatic disease, anemia, diabetic retinopathy or symptomatic neuropathy, cardiac failure (New York Heart Association grades III and IV), angina pectoris, or recent myocardial infarction and severe uncontrolled hypertension. T2D was diagnosed in accordance with the criteria of World Health Organization: fasting plasma glucose ≥ 7 mmol or 2 h plasma glucose ≥ 11.1 mmol/L [16]. T2D patients were treated with oral antidiabetic agents, none of them were treated with insulin.

Hypertension was defined as (systolic/diastolic blood pressure (BP) (≥140/≥90 mmHg), according to Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure (JNC-7) criteria [17] or currently receiving antihypertensive agents. The patients without hypertension involved in this study were not on any medications lowering BP and were noted to have BP of less than 140/90.

The study was approved by the ethics review committee of Faculty of Medicine, University of Belgrade, Serbia and written informed consent was obtained from each participant. The study was conducted at the Clinic for Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Serbia, Faculty of Medicine, University of Belgrade.

At screening visit at the outpatients clinic subjects were interviewed about medical conditions, current medication and smoking habits. Antihyperglycemic, hypolipidemic and antihypertensive agents were stopped 24–48 h before the metabolic testing.

The presence of obesity was determined by using BMI which was calculated as weight/height² (kg/m²). Height was recorded to the nearest 0.5 cm, and weight was measured to the nearest 0.1 kg.

The BP was measured by using a standard mercury sphygmomanometer after the subjects had rested at least 10 min, in three separate measurements in sitting position, by the same doctor. SBP was recorded at the appearance of sounds, and DBP was recorded at the disappearance of sounds (V-phase Korotkov).

In each patient we performed the detection of: (a) insulin sensitivity, (b) plasma insulin (PI), (c) plasma glucose (c) IL-6 and TNF pro-inflammatory cytokines, (c) HbA1c and (d) lipid subfraction levels (total, HDL, LDL cholesterol and triglycerides).

Insulin sensitivity was evaluated by using the OGIS method [18]. Briefly, the plasma glucose and PI values measured from the samples during the standard OGTT were used to calculate the OGIS index according to Mari et al. [18]. The standard 75-g OGTT was performed after a 12-h overnight fast. Glucose solution was ingested within 2 min, and venous blood samples were collected for measurements of plasma glucose and insulin concentrations at 0, 30, 60, 90, and 120 min after glucose loading [16,18].

All analyses were carried out during the same day and blood samples drawn by the same study nurse after a 12 h overnight fast and were stored at −70 °C until assayed. PI levels were determined by radioimmunoassay (double antibody kits, INEP, Zemun, Serbia). Plasma glucose concentrations were measured using the glucose oxidase method using Beckman Glucose Analyzer (Beckman Instruments, Fullerton, CA, USA). Glycosylated hemoglobin (HbA1c) levels were determinate using turbidimetric immunoassay for HbA1c (Boehringer Mannheim, Mannheim, Germany). Total cholesterol, HDL cholesterol and triglyceride concentrations were determined with enzymatic methods (Boehringer Mannheim). LDL cholesterol concentrations were calculated using Friedewald formula. TNF-α and IL-6 were measured by ELISA system (ALPCO, Salem, NH, USA).

Data are expressed as means ± SD. Normality of distribution of the data was tested by the Kolmogorov-Smirnov Test, a p value greater than 0.05 indicated that the observed distribution of a variable is not statistically different from the normal distribution. Comparison of metabolic variables was done by Kruskal-Wallis Test when the data were not normally distributed. The continuous variables were analyzed with analysis of variance (ANOVA). Data with a p value less than or equal to 0.05 were considered statistically significant. The software package SPSS version 16.0 for Windows (Chicago, IL, USA) was used for all computations.

In this study we have found the highest IR (evaluated both by OGIS index and PI levels) in T2D patients with increased body weight (obese and overweight) and hypertension which is strongly associated with increases in the levels of pro-inflammatory cytokines, predominantly IL-6.

IR is thought to be an important pathogenic mechanism in the pathogenesis of essential hypertension [19,20,21,22]. A number of studies have been entirely consistent in showing that lower insulin sensitivity is associated with higher BP [19,20,21,22,23,24].

It was previously demonstrated that insulin sensitivity indices based on the OGTT significantly correlated with the glucose disposal rate (M-value) measured by the glucose clamp technique [18,25]. Previous studies have suggested that OGIS index, calculated from OGTT may provide a reliable estimate of insulin sensitivity especially in patients with T2D [18,25,26,27,28,29]. This was the reason why we used this index of insulin sensitivity based on the 75-g OGTT.

We found that insulin sensitivity is already substantially decreased in T2D patients with increased body weight. Moreover, the OGIS index as measurement of peripheral IR was significantly lower in the obese and overweight T2D patients with hypertension than in those with optimal BP. Our results imply that increases of IR might be an important factor influencing the development of hypertension in obese T2D patients.

Over the past decades many studies have suggested that low-grade inflammation related to obesity might be the key regulator in pathogenesis of T2D [30,31,32,33,34,35,36,37,38,39,40]. It has been confirmed that enlargement of adipose tissue is associated with increases of number of adipose tissue macrophages, which are responsible for increases in plasma concentration of pro-inflammatory cytokines, especially IL-6 and TNF-α expression [31]. IL-6 is released from macrophages of adipose tissue as well as from adipocytes and skeletal muscle [32,33]. In vitro and in vivo work has shown that IL-6 gene expression and circulating levels of IL-6 may be regulated by insulin and correlate well with central obesity [31,32,33]. TNF-α level is associated with IR, and this cytokine promotes serine phosphorylation of insulin receptor substrate 1 that impairs insulin signaling, resulting IR [33,34,35,36]. These pro-inflammatory cytokines appear in early stage of T2D and they are found to be capable to increase IR directly in adipocytes, muscle and hepatic cells leading to augmentation of the systemic IR [37,38,39].

Our results have confirmed these findings of increased levels of IL-6 and TNF-α in T2D patients with increased body weight (obese and overweight), but among them IL-6 and TNF-α were found to be significantly higher in the hypertensive patients. In addition, in our study the changes in IL-6 showed the same pattern as the increases in IR in the hypertensive obese and overweight T2D individuals.

However, we could not find the significant difference in the levels of TNF-α between hypertensive and normotensive T2D patients with increased body weight. The differences in the pattern of changes between IL-6 and TNF-α in our study might be caused by a different relationship between IR and each of the pro-inflammatory cytokines [5,34,35,36].

In past several decades different epidemiological studies showed the presence of a co-clustering of inflammation and hypertension in patients at high CVD risk [40,41,42,43]. Interestingly, these authors also observed that the association between the low-grade inflammation and the risk of becoming hypertensive remained statistically significant even after adjustment for features of the metabolic syndrome [40]. In addition, some recent epidemiological studies showed that the presence of a low-grade inflammation could anticipate the future development of hypertension [41,42]. This novel observation suggests that the increase in plasma levels of pro-inflammatory cytokines observed among hypertensive patients cannot be solely attributed to the vascular damage induced by high blood pressure [43].

Another factor that might be involved in the pathogenesis of hypertension in the settings of obesity-associated IR is increased sympathetic activity [22]. It has been recognized that obesity represents a condition of increased sympathetic activity, increase in norepinephrine (NE) concentrations and NE renal spillover, and this hyperactivity is associated with tissue IR. In pathogenesis of hypertension, some recent studies emphasize the role of arterial stiffening preceding the development of hypertension [44]. Interestingly, the impairments in pulse wave velocity, a measure of large vessels distension ability, was recently found to be associated with the increases in circulating levels of IL-6 and TNF- α [22,45,46] suggesting that low-grade inflammation may contribute to arterial stiffness.

New lines of research are now investigating the possibility of a direct pathogenic effect of pro-inflammatory mediators in altering mechanisms of vascular tone regulation leading to the onset of high blood pressure [47,48,49], which might clarify the mechanisms linking hypertension and low grade inflammation.

Lifestyle modification, physical activity and nutritional interventions [48,49], may reduce development of diabetes, but also the level of blood pressure and inflammation in patients with hypertension and T2D, which is important for the prevention of cardiovascular diseases [50]. Our results imply that this effect might be achieved by targeting IR and low-grade inflammation, predominantly IL-6 levels. The results of the National programme of early detection and prevention of T2D in Serbia is based on lifestyle modification aiming to reduce the risk not only for T2D but also to its complications and comorbidities, especially hypertension [51]. Our results imply that beneficial effect in that direction might be achieved primarily by targeting IR and low-grade inflammation, predominantly IL-6 levels.