Overweight-Related Hypertension in Middle-Aged Men: Pathophysiological Impact of Serum Leptin, TNF-α, IL-6, Cholesterol and Testosterone

1. Introduction

The age and sex specific data of the worldwide prevalence of hypertension (HT) shows that it is an important health challenge and requires to be screened, prevented and treated with high priority [1]. One of the major causes of HT is the transition of normal weight status to overweight (OW) status and obesity in a population [2] that leads to cardiovascular disease (CVD) and other disorders. It was revealed that there is a significant association between body weight (BW) and HT [3]. It was found that high levels of body mass index (BMI) (in either OW or obese subjects) is one of the main factors leading to several chronic disorders e.g., CVD, diabetes mellitus (DM) and HT [4]. Furthermore, both normal weight (NW) and OW subjects showed an association between blood pressure (BP) and BMI [5]. The OW status increases the risk of the development of high BP. It is well studied in the Framingham Study that high BP or HT [6] and OW [7] are independent risk factors for CVD [8]. It is documented that the occurrence of HT could increase the BMI [9]. The data of all age groups show that the obese subjects with HT ranged 60%-77% with increasing BMI and that was significantly higher as compared to 34% in NW subjects [10].

The tendency in public of having gradual increase in BP with increase in BW or BMI was also noted [11] and over the life course including late in life [12]. Although the various BP range levels have been classified into three grades of HT based on systolic blood pressure (SBP)/ diastolic blood pressure (DBP) in mmHg as: grade 1: 140-159/ 90-99, grade 2: 160-179/ 100-109, and grade 3: ≥ 180/ ≥ 110, the management with proper lifestyle and medication is essentially required regardless of any grade [13].

The BP is found higher in OW/obese people than in non-obese people and the lifestyle changes decrease the BP in OW and obese men [14]. In view of the interactive effects and association of BW (OW/obesity) and HT, it is highly recommended to have the combined management for both risk factors, even in non-hypertensive (NHT) subjects [15]. Furthermore, the patients with HP and OW/obesity require proper management especially including the change in lifestyle specially reducing the BW that decreases the risk of HT for those who were even never OW or obese [16].

There are various studies documenting the association of serum leptin (Lep) with other variables including serum tumor necrosis factor-alpha (TNF-α), serum testosterone (ST) and interleukin-6 (IL-6) in HT [17,18,19,20] and OW-related HT [21,22,23]. However, there are reports that contradict these findings [24,25]. No clearly known interactive role of these factors are yet known, that requires further studies to be conducted for exploring the role of serum Lep, TNF-α, IL-6, ST, total cholesterol (TC), fasting blood glucose (FBG), and hepcidin (Hp) in NC, HT and OHT subjects. Therefore, the purpose of present research work was to study and investigate the involvement of the mentioned factors in hypertension and overweight-related hypertension.

2. Materials and Methods

2.1. Study Design, Participants and Data Collection

The design and detailed proposal for the present study was prepared and submitted to the Ethical Approval Committee of the Faculty of Medicine, Umm Al-Qura University. With the issuance of the ethical approval (Approval No: HAPO-02-K-012-2021-02-124), the research work was commenced. The participants in the present study were informed during their first visit that the data of subjective information and the blood samples would be collected, and other tests will be carried out considering their personal consent whether they decide or not to take part in the present study. After the selection of each subject for the present study, the detailed interview and assessment were conducted. For this purpose, proper diagnostic procedures were employed and subjects/ patients were characterized for the current study.

Present study comprised the participants classified into three groups (each with age range of 51-60 years) as: normal control (NC, n: 98), hypertensive (HT, n: 97) and overweight-related hypertensive (OHT, n: 97) subjects. The NC were normotensive (NT) male subjects with NW at BMI 23-24.9 kg/m². The HT were hypertensive male subjects with NW at BMI 23-24.9 kg/m². Whereas the OHT were hypertensive male subjects with OW status at BMI 28-29.9 kg/m².

The NC and HT were those subjects having high normal weight (HNW) BMI, and OHT were considered the subjects with high overweight (HOW) BMI. Subjects having lower and higher levels of BMI other than the mentioned for NC, HT and OHT were not included in the present study.

Furthermore, the NT subjects had SBP/ DBP as <120/<80 mmHg, and the HT subjects in the range of 140-159/ 90-99 (grade 1-HT) [13]. The subjects with lower and higher values of SBP/DBP for HT and OHT groups were not included in the current study. Moreover, subjects having grade 2 hypertension (SBP/DBP: 160-179/ 100-109) and grade 3 hypertension (SBP/DBP: ≥ 180/ ≥ 110) [13]. were also not entertained for the present work.

Sample size calculator was used for assessing the required number of samples for the present study. General features of the subjects and history of patients were obtained and recorded in the Questionnaire. The present study was conducted in the Umm Al-Qura University affiliated hospitals in Makkah, Saudi Arabia from June 2021 till Dec 2022. Considering the previous report [26], it was planned to collect the data for serum Lep, TNF-α, testosterone and inflammatory status.

2.2. Measurements and Methods

Routine kit method and enzymatic colorimetric method was employed for the determination of TC. The BMI levels were assessed by dividing the BW (kilograms) with the square of body height (meters) (kg/m²) [27,28]. The already published levels of HNW related BMI and HOW related BMI for the Saudi population were followed [29]. However, the subjects having low normal weight (LNW) and medium normal weight (MNW) related BMI and low overweight (LOW), and medium overweight (MOW) related BMI were not included in the present study.

The BP (SBP and DBP) levels determined by routine methods [30] in the NT subjects were in normal range and in HT subjects were in the HT range [31,32]. However, the subjects only with grade-1 HT in the range of 140-159/ 90-99 [13] were included. The measurement of BP was carried out using the mercury sphygmomanometer, (MS-S1500-Mercury Sphygmomanometer, Medical Sources Co., Limited, Nanjing, Jiangsu). The blood samples obtained from NC, HT & OHT groups were taken, serum separated, and analyzed using ELISA (enzyme-linked immunosorbent assay) kits for serum Lep, TNF-α, ST, TC and IL-6 and other variables.

2.3. Data Analysis

The data analysis was performed using the Statistical Package for Social Sciences (version 24.0) for Windows (SPSS Inc., Chicago, IL, USA). The GraphPad Prism (version 6.0) software, San Diego, CA, USA, was used. The values of the analyzed data were obtained as mean ± standard deviation (SD). Distribution of the quantitative characteristics of present data corresponded to the normal one. The two tailed P-value was obtained by applying the Student t-test (unpaired two samples t–test). The one-way analysis of variance (ANOVA) was applied for comparing the variables of three subject groups. The results obtained by Student t-test were further confirmed by the Tukey’s Kramer post hoc test. For the determination of positive or negative correlations, linear regression lines were plotted. The values of coefficient of determination (R²) were obtained that showed the level of significance. The value of significance (P) was obtained considering the biostatistical applications [33]. The significance level was considered as P ≤ 0.05.

3. Results

Characteristic features and related parameters in subjects with hypertension and overweight-related hypertensive middle-aged men were compared (Table 1). Age of NC, HT and OHT subject groups respectively was 55.37±2.86, 55.35±2.82 and 55.36±2.78 having no significant difference. The high normal weight BMI (kg/m²) and high overweight BMI (kg/m²) in range and mean±SD of these subject groups are given in Table 1.

3.1. Two Samples Comparisons and Analysis of Variance for Serum Leptin, TNF-α, IL-6, Cholesterol, Testosterone and Other Variables in Hypertensive and Overweight-Related Hypertensive Men

Comparisons for Lep, TNF-α, and TC showed significant results for group comparisons (Table 1). The ST indicated significant difference for OHT vs. NC but did not show significant variation for other comparisons. The IL-6 gave significant difference for OHT vs. NC and. OHT vs. HT, but not for. HT vs. NC. Comparison for FBG and Hp did not present significant variations for HT vs. NC, OHT vs. NC, and OHT vs. HT.

One-way ANOVA presented significant change among subject groups for the levels of Lep, TNF-α, IL-6, TC and ST (Table 2). However, no significant variation was found among groups for the remaining variables.

3.2. Association of Serum Leptin with Other Variables in Hypertensive and Overweight-Related Hypertensive Men

The results for association of serum Lep with other variables in NC, HT and OHT men are given in Table 3. Serum Lep plotted against TNF-α presented highly significant positive linear correlation for the subject groups. Similar results were obtained for association of Lep with ST, though the correlation of Lep with ST was negative linear. Serum Lep associated significantly with IL-6 and TC showing positive linear correlation for subject groups. Other variables of Hp and FBG did not present significant correlation with Lep (Table 3).

3.3. Association of Serum TNF-alpha with Other Variables in Hypertensive and Overweight-Related Hypertensive Men

Results for the association of serum TNF-alpha with other variables in subject groups are shown in Table 3. Correlation of serum TNF-alpha with ST was found to be negatively linear. Its association with Lep, IL-6 and TC was significant with positive linear in subject groups.

3.4. Association of Serum Testosterone with Other Variables in Hypertensive and Overweight-Related Hypertensive Men

The association of serum testosterone (ST) with other variables in subject groups are shown in Table 3. The ST gave negative linear and significant correlation with IL-6. Negative linear but non-significant correlation of ST with TC was noted in subject groups except in NC group that showed significant negative linear correlation. Lep and TNF-α both associated with ST showing significant negative correlation (Table 3).

3.5. Association of Serum Interleukin-6 with Other Variables in Hypertensive and Overweight-Related Hypertensive Men

The association of serum IL-6 with each of TC, Lep, and TNF-α in subject groups (Table 3) showed positive and significant linear correlations, whereas its association with ST was negative and significant linear. Other variables did not show significant correlation with IL-6.

3.6. Association of Total Cholesterol with Other Variables in Hypertensive and Overweight-related Hypertensive Men

Association of serum cholesterol with each of Lep, TNF-α and IL-6 (Table 3) presented significant positive linear correlations. However, non-significant correlations in HT and OHT subject groups were obtained for TC when plotted against ST. Serum cholesterol did not show significant correlations with the remaining variables.

4. Discussion

It was attempted to assess and explore the pathophysiological role of the variations and associations of serum Lep, TNF-α, ST, IL-6, cholesterol, fasting blood glucose (FBG), and hepcidin (Hp) in NC, HT and OHT or OW-related HT subjects. Study in NW and OW male subjects [26] provided information of the significant variation and positive linear correlation among serum Lep, BMI and BP, and various other cytokines and inflammatory factors though that study was conducted in 18-20 years old healthy male university students and was quite descriptive involving various low, medium and high BMI levels in NW and OW subjects.

Considering the aim of the present study, we were more interested to explore the changes or associations at a specific level of BMI and BP. We selected the subjects at a specific BMI level of 23-24.9 kg/m² (high normal weight BMI) for NC and HT and 28-29.9 kg/m² (high overweight BMI) for OHT. We could not study the low normal weight BMI and middle normal weight BMI for NC and HT, and low overweight BMI and middle overweight BMI for OHT subjects. Furthermore, only grade-1 HT subjects were included in the present study. Hence, the present study is well controlled for gender, age, BMI, and BP (normotensive & grade-1 hypertensive subjects).

The values were compared for each two groups and among groups, and associations were found for various variables with each other rather than determining their associations with BMI or SBP/ DBP. We could not manage though it was more interesting to obtain data of additional subjects at low normal weight BMI and middle normal weight BMI for NC and HT, and low overweight BMI and middle overweight BMI for OHT subjects as well. It could not be accomplished owing to insufficient funding.

We found significant difference of serum Lep for both HT and OHT subject groups against NC subject group. A positive correlation was found between serum Lep with OHT and it was revealed that Lep has an important role in the pathophysiology of OW-related HT [34]. It was investigated that it is the Lep having influencing role in the OW-related HT; and the HT and endothelial dysfunction partly relate to serum levels of Lep, and other adipokines [35]. Inflammation caused by the cytokines causes inflammation dependent aortic stiffening that may lead to ventricular stiffness [36]. These observations identify the impact of Lep via inflammatory processes in the progressive development of HT. Furthermore, OW and obesity can lead to dysfunction of perivascular adipose tissue (PVAT) that releases Lep, and other cytokines/ chemokines to the vascular wall [37,38] and causes endothelial dysfunction and inflammation [37]. These reports support the changes found in the present study for Lep interacted with IL-6 and TNF-α in HT and OHT subjects compared to NC subjects.

The OW and obesity status increases the production of contractile factors and incorporates increased arterial vasoconstriction and greater vessel tone, producing different effects on the control of vascular tone [37]. The PVAT derived contractile factors e.g., Lep causes vasoconstriction. It was found that anticontractile activity is decreased in patients with HT that is an indirect association of lep in HT [38].

Arterial stiffness via promoting vascular smooth muscle cell proliferation as well as migration associated with HP has been suggested to be due to Lep receptors located in vessels especially in aorta [39], tunica media, and adventitia in arteries and in atherosclerotic plaques [40]. Furthermore, the function of Lep in promoting the angiogenesis, activating the immune system, increasing the platelet aggregation, producing radical oxygen species (ROS) [41], inducing endothelial oxidative stress and ROS in experimental human cell models [42] interactively present a mechanism whereby risk of the development of HT increases. Adiponectin having protective effects on arteries associates negatively with Lep, and hence hypoadiponectinemia associates with increased Lep levels [43].

Another mechanism explaining our present results is that an increased activity of sympathetic nervous system in OW subjects has been revealed in several studies [44,45,46], and it was further found that increased BW associated with HT stimulates the sympathetic stimulation causing increased development and progression of HT with increase in Lep levels in association with increase in pro-inflammatory cytokines, and hence further increased HT condition [44,45,46].

The second important factor that we investigated in the present work is the increased level of TNF-α in OW and HT subjects. This investigation can be explained with the help of several related reports. The cross-link between the concept of immune system–adiposity–inflammation–blood pressure¸ makes a set of vicious events [47] having a major role in HT [48]. The TNF-α is an important and effective pro-inflammatory adipocytokine that is involved in causing the low-grade inflammation and influencing the CVD involving OW/ obesity, atherosclerosis and other disorders [49]. The OW and inflammation in HT men showed significant role of TNF-α [23].

The low-grade inflammation related to the adipose tissue and excess fats [17] that relates to increased level of serum cholesterol in HT and OHT subjects in the present study associates with the immune complications leading to HT and other disorders [17,18]. There are a variety of immune factors involved in regulating the immune processes in vascular pathologies caused by adipose tissue derived and a most notorious pro-inflammatory cytokine TNF-α and other adipokines [50]. Probable immune-related inflammatory changes in newly diagnosed HT with higher BMI showed higher levels of TNF-α, and other factors better characterizing the CV risk in HT patients [51].

The immune mechanisms related to immune system activation explains our observations in the current clinical study. Inflammatory processes are involved in the pathogenesis and progression of CVD complications [52,53], and the inflammatory markers may serve as emergent therapeutic targets [52]. Increased levels of IL-6 and TNF-α in HT and OHT groups in the present study fit nicely in the proposed mechanism of inflammatory processes leading to CVD complications. The inflammatory status facilitated by the CV risk factors [54] is then regulated by the mechanisms related to immune system activation [51,55].

Decreased levels of ST in HT and OHT men in the present investigation, and especially significant decreased ST levels in OHT (OHT vs. NC) can be described with the help of other reports. The mechanism whereby testosterone decreases in response to HT in OW status has been reported and it was found that serum levels of ST associate with the incidence of the morbidity and mortality of CVD diseases in men [56]. Furthermore, the effect of ST on CVD processes was revealed [57,58,59].

It has been shown in male patients with coronary heart disease (CHD) that acute intracoronary administration of ST brings coronary artery dilation and hence, increases the blood flow and improved coronary activities [57]. The long-term administration of normal ST doses decreases the symptoms of angina and cardiac ischemia in men [58,59]. The ST is low in men with OW and obese status than in the NW people [60]. On contrary, the lifestyle changes bring the ST to increased level in men with OW and obesity [61]. The low levels of ST in men in obesity/ OW increases BP that leads to CVD complications, though it is not known, how does ST bring this change. The OW and obesity are associated with the decrease in ST [62]. High increase in BW accompanies elevated levels of aromatase from fat that decreases the hypothalamic-pituitary-testis pathway and hence causes decrease in testosterone production by Leydig cells in testes [63].

Testosterone deficiency occurs in OW/ obesity leading to changes in blood vessels and vice versa [20,22,64,65]. However, it has not been studied thoroughly to have clear idea of the role of testosterone in OW subjects. Therefore, an association between BW change and ST variations is not simple [20]. Furthermore, there are reports showing the occurrence of hypogonadism due to decreased levels of ST associated with BW changes [22,64,65]. Hence, it seems reasonable to think that the manipulations for decreasing the BW can bring increase in ST [22].

The subjects with increased levels of BMI associated with the occurrence of vascular dysfunctions, decreased ST and increased inflammation [19]. This investigation is quite supportive for our observation of decreased ST and increased inflammation (IL-6 and TNF-α). Based on these observations, it was suggested for manipulating the procedures leading to weight loss and involving testosterone and anti-inflammatory agents that may prevent from vascular dysfunction [19]. The ST was found decreased in subjects with high BMI compared to that in low BMI [19].

On the other hand, decreased levels of ST stimulates the fat formation in viscera leading to chain of events causing more decrease in the levels of ST [66]. The Lep levels increase in OW and obesity and have negative effect on luteinizing hormone (LH) and the production of human chorionic gonadotropin (hCG)-stimulated testicular androgen [67]. The adipose tissue, fat formation, endocrine events of testosterone homeostasis, and OW/ obesity are associated with the inflammatory processes through release of pro-inflammatory and anti-inflammatory factors [68], and decreased blood supply to adipocytes that causes hypoxia [69]. It then becomes a cascade causing further inflammation by releasing TNF-α and IL-6, [70,71] decreasing nitric oxide (NO) in blood vessels and vascular dysfunction [21]. This interactive set of investigations is a landmark in our present study.

It is not only the testosterone, but other sex hormones including estradiol have also shown cardioprotective actions in association with the vascular physiology [57,59], though we could not measure the other sex hormones. Suppressing the activity of androgens was found to increase the BP in male subjects [72] that verifies the effect of testosterone decreasing BP in men i.e., ST and BP are negatively associated. This indicates that any manipulation to increase the ST could bring decrease in BP in OW and obese men [61]. The low levels of ST associates with the morbidity and mortality of cardiovascular disorders in men [61]. These reports verify our present results.

There are other studies that did not find change in BP in response to lifestyle changes for decreasing body weight [24,25]. They found little change in body weight. The present study comprises the NW subjects with HT and OW subjects with HT and hence changes occurred accordingly. Therefore, we could find progressive increase in various variables with increased BMI in OHT compared to that in HT and NC.

Cardiovascular events in HT increase the morbidity and mortality. The pharmacological interventions, exercise and diet plans including various forms of therapeutic patient education (TPE) help in reducing the morbidity and mortality due to cardiovascular involvements. Such aspects have not frequently been studied considering with the well-organized and personalized approaches.

The data presented in the Results section clearly shows the significance level between each two groups (Table 1) for Lep, TNF-α, IL-6, TC and ST, and among all three groups (Table 2) for various variables. Based on that, we interpreted the results with the help of already published reports. However, the results for various correlations given in Table 3 show significant associations not only for OHT and HT groups but also for NC group. Such sort of results might have been due to the reason that we collected the data of high normal weight BMI in normal controls (NCs) and hypertensives (HTs), and high overweight BMI in overweight hypertensives (OHTs). For further evidence, it is required to carry out as studies including low and medium weight BMI in normal controls (NCs) and hypertensives (HTs), and low and medium overweight BMI in overweight hypertensives (OHTs) as well. Hopefully, such management can clarify the associations of various variables with each other and with the mentioned levels of BMI.

We followed the already published data for the levels of high normal weight (HNW) related BMI and high overweight (HOW) related BMI for the Saudi population [29]. Therefore, we suggest for carrying out wide range BMI studies, since such studies have not been performed yet in Saudi Arabia. These suggested studies may clarify whether the correlations obtained in the present study appear in similar or different pattern in subjects with low, medium and high normal weight BMIs and low, medium and high overweight BMIs.

In case, only the upper or high levels of BMIs (high normal weight BMI in NCs and HTs; and high overweight BMI in OHTs) show similar correlations with various variables after performing wide range BMI studies, then it would be necessary to suggest to the related Health Department for planning to carry out comprehensive mega-studies for confirming the existing categorization/ classification of BMI in Saudi population since BMI and BMI categories are region/ country-based and public health/ lifestyle based specifications. It could be that the upper or high level of the BMI in normal individuals is actually the low level of overweight individuals, and upper level of the overweight BMI actually the low level of obese individuals for Saudi population.

Additionally, we suggest that the cytokines are not only the inflammatory or anti-inflammatory factors/ biomarkers, but also are the physiological regulatory factors/ substances as are the hormones that also alter in physiological and pathophysiological conditions. The Lep, TNF-α and IL-6-the cytokines, and testosterone-a hormone in our present study are the physiological regulatory factors as well. This viewpoint could partly explain the existence of significant associations/ correlations between each two variables to certain extent even in the NC subject group. The comparisons in NC, HT and OHT subject groups given in Table 1 & 2 provide the significance levels of variations for impressive physiological/ pathophysiological interpretations.

Conclusively, the current report provides pathophysiological evidence of the interactive role of serum leptin, TNF-α, testosterone, total cholesterol, and IL-6 in normotensive, hypertensive and overweight-related hypertensive middle-aged men. The physiological approach of assessing the biochemical parameters including those determined in the present study, and a variety of other factors may further help understanding the cardiovascular events and the influencing factors in hypertension and overweight related hypertension.

5. Limitations

We found wide variations in our pilot studies for the age and gender-controlled levels of serum Lep, TNF-α, IL-6, TC, ST and other variables in NC, HT and OHT men. Hence, we carried out the present study in men of a specified age-51-60 years. However, to understand the clear evidence of the general role of Lep, ST, TNF-α, inflammatory status and lipid profile in NC, HT and OHT, it seems necessary to have the data of wide age range in both men and women. Hopefully the existing and future studies would clarify further the impact of wide range of age in men and women.

We measured the levels of ST but could not determine the levels of other gonadal/ steroid hormones and gonadotrophic hormones for understanding the interplay of endocrine changes in hypertension and overweight-related hypertension.

There is a need to assess the interactive role of leptin with other adipokines especially adiponectin for investigating the overweight-related hypertension. We could not include the determinations of adiponectin and other related adipokines mainly due to limited funding available for the proposal related to current report.

Indeed, other groups of subjects comprising obese, and obese hypertensives are the highly important group of subjects that could not be investigated in the present study, since our proposal was not related to obesity related hypertension. However, we wish to overcome this limitation by carrying out further studies in future for other proposals for studying the obese subjects and obesity related hypertension.

The data of several diseases including mainly the CHD, DM, renal ischemia/ failure and other kidney disorders, and cerebral ischemia/ stroke were excluded in the present study though these and other diseases are prevalent in overweight/ obese subjects with/ without hypertension.