Association Between Visceral Adipose Tissue and Estradiol with Bone Health among Obese Women with Metabolic Syndrome

INTRODUCTION

Literatures revealed that about 50% of adults will be classified as obese by 2030¹. Recently, factors that typically affect older adults have dramatically increased, for example, the rates of disease spreading and unfavorable environmental conditions. The last three decades have witnessed an elevated prevalence of obesity among all age groups, in addition to doubling in the number of obese older adults^2,3. Factors which contribute to obesity epidemic include: A disturbed balance between energy intake and expenditure, age-related hormonal changes, genetic, environmental and social factors. Although, obesity has many direct consequences of on individual’s health, yet it also increases the risks of numerous chronic diseases which deteriorate the quality of life and may contribute to premature death^2,3.

Metabolic Syndrome (MS) is typically defined as a cluster of at least three out of five clinical risk factors which include visceral obesity, elevated blood pressure, higher TG, lower HDL and insulin resistance “IR”⁴. MS causes major problem to health services and deteriorates health economies as well. However, visceral obesity or ectopic fat accumulation around the viscera is directly related to the development of both insulin resistance “IR”¹ and enlarged and dysfunctional adipose cells ‘sick fat’⁵.

Vitamin D has many important functions. It is plays a major role to skeletal tissues, as it affects bone mineralization (BMD), bone turnover rate, occurrence of fractures, osteoporosis⁶. Moreover, accumulation of vitamin D in adipose tissue is important for its subsequent release when needed as for example during winter when the fat storage decreases⁷. It also plays a key role in bone metabolism as it increases the plasma levels of both Ca and phosphorus, which are essential for bone mineralization⁶. However, it’s prolonged deficiency (reduced serum Vit. D) results in bone loss, osteopenia and finally osteoporosis⁸.

The mechanism explaining the direct effect of menopause on vitamin D levels remains uncertain. However, low levels of E2 could be an important factor for vitamin D metabolism in post-menopausal women. This can be attributed to the fact that, E2 not only has a critical role in both reproductive and sexual functioning, but also affects bone metabolism⁹.

During the process of bone formation, type I collagen which is the main protein of bone matrix, cleaves into 2 propeptides, namely N-terminal (P1NP) and C-terminal (P1CP). Serum procollagen type I N propeptides (P1NP) is responsible for bone formation, while, serum C-terminal propeptides of type I collagen (P1CP) is responsible bone resorption¹⁰.

So, this study aimed to investigate the impact of visceral fat and serum E₂levels on bone health markers (vitamin D, C-terminal peptide, Ca and BMD) in obese women with and without MS.

MATERIALS AND METHODS

This cross-sectional study involved 64 Egyptian obese women with age range 25-65 years and mean age 48.85±9.88 years. Their BMI was >30 kg m^–² and their Waist Circumference (WC) >94 cm. They were all recruited and randomly chosen from employees and workers, from all categories at the “Medical Excellence Research Center (MERC)” which is part of the “National Research Centre”. This research paper was derived from a cross-sectional survey of a project funded by National Research Centre (NRC) Egypt, 2016-2019 entitled ‘‘Bone mass among Overweight and Obese Women: Mechanism and Intervention.” (11th Research Plan of the NRC), with an approval obtained from Ethics Committee of NRC (Registration Number is 11010110). A written informed consent was obtained from all participants after being informed about the purpose of the study.

For each participant women, the following parameters were taken: blood pressure, anthropometric measurements, visceral fat, DEXA and laboratory investigations.

Blood pressure measurement: Blood pressure was measured using the standardized mercury sphygmomanometer with a suitable cuff size. It was measured on the right arm, while the participant was sitting relaxed for 5 min. Two readings were obtained and the average was recorded. Systolic Blood Pressure (SBP) was determined by the onset of the “tapping” korotkoff sounds (K1), while the fifth korotkoff sound (K5) or the disappearance of korotkoff sounds as the definition of Diastolic Blood Pressure (DBP).

Anthropometric measurements: Anthropometric assessments included body weight, height and WC following the recommendations of the International Biological Program¹¹. Body Weight (Wt) was determined to the nearest 0.01 kg by using a Seca Scale Balance, with the woman wearing minimal clothing and with no shoes. Body Height (Ht) was measured to the nearest 0.1 cm using a Holtain portable anthropometer. The WC was measured using non-stretchable tape and approximated to the nearest 0.1 cm. It was measured, at the umbilicus level with the participant in an upright standing position, relaxed shoulders, face directed forward and breathing normally. The BMI was calculated using this Eq:

Visceral fat: Visceral fat (ratio) was assessed by using Tanita Body Composition Analyzer [bioelectrical impedance apparatus (BIA)]. Visceral fat was measured based on the woman’s age, body weight and height approximated to the nearest units. Women with visceral fat values >13 are regarded as having visceral obesity, according to the machine instructions manual (Tanita Body Composition Analyzer-MC-780 MA III).

DEXA measurements: Both Bone Mineral Density (BMD) (g cm^–²) and BMD t-score at both the femoral neck and lumbar spines sites were measured using dual-energy DEXA (DEXA Norland XR-46 version 3.9.6/2.3.1, USA). Full body DEXA scan based on the subject’s age, weight and height was performed with the participant keeping the precise distance between her arms and legs according to the machine instructions manual. A well-qualified operator executed and evaluated all analyses using the same protocol for all assessments.

Laboratory investigations: After overnight 8 h fasting, participant’s venous blood samples were obtained by venipuncture in the morning to assessthe following parameters: Fasting Blood Sugar (FBS), serum calcium (Ca), serum Vit D, C-terminal peptide and E2. Another venous blood samples were obtained after 12-4 h fasting to measure lipid profile (TG, Tchol and HDL). The blood samples were left to clot were then centrifuged at 5000 rpm for 10 min to separate sera and were then stored at -80ºC to be assayed later on.

Fasting Blood Sugar (FBS), serum Ca, Tchol, HDL-C and TG levels were measured by using the automated clinical chemistry analyzer Olympus AU 400 analyzer. LDL-C was calculated by using the formula developed by Friedewald et al.¹² as follows:

Serum Vit D and C-terminal peptide were measured using ELISA kit, Cataloge number SL1831 HU., SL2109 HU., Sun long Biotech. Co. LTD and E2 Cataloge number ES180S. Dal Biotech, respectively. The assessment of these parameters were done in the laboratory of “Medical Excellence Research Center (MERC), which is part of “National Research Centre (NRC), Egypt.

Statistical analyses: Data were analyzed using the Statistical Package for Social Sciences (SPSS/Windows Version 16, SPSS Inc., Chicago, IL, USA). Normality of data were tested using the Kolmogorov-Smirnov test. The p<0.05 was regarded as statistically significant for all tests. Blood pressure, FBS, Vit.D, C-terminal peptides and E2 were normally distributed, on the contrary most of the variables such as; the data of DEXA, weight, BMI, VAT, lipid profile and Ca were not normally distributed.

Criteria for MS were defined according to the International Diabetes Federation¹³ for example, central obesity (WC >94 cm and/or BMI >30 kg m^–²) plus any two of the following: Elevated TG (>1.7 mmol L^–¹), reduced HDL (<1.03 mmol L^–¹), elevated blood pressure (systolic >130 mm Hg^–¹ or diastolic >85 mm Hg^–¹), elevated FBS (>5.6 mmol L^–¹) or drug treatment for the individual features.

The study sample was classified into 2 groups as follows: A group with MS (n = 25) and another without MS (n = 39). The parametric data were expressed as mean±SD, whereas, the qualitative ones (frequency distribution) were expressed as number and percentage (%). Various variables of the 2 groups were analyzed and compared using Mann-Whitney test for independent groups.

Spearmen’s correlation coefficients, associations between anthropometric parameters, body composition and bone data, were all used to examine the correlation between both VAT and E2 and between the other variables in the present study.

RESULTS

Characteristics of the current sample: Elevated level visceral fat (>13) among both women without MS (38.5%) and women with MS (40%) has revealed insignificant differences. All participant women in the current study were suffering from central obesity (WC >94 cm and/or BMI >30 kg m^–²). The other criteria for MS have shown highly significant differences between the two groups. Among women without MS, 51.3% had no criteria and 48.7% had only one criterion in addition to central obesity. Among women with MS in addition to central obesity, 68% had 2 criteria, 24% had 3 criteria and 8% had 4 criteria (Table 1). The most common criteria for MS among women of current study were decreased HDL (39.1%), increased SBP (34.4%), increased fasting blood glucose (26.6%), increased DBP (21.9%) and increased triglycerides (17.2%), respectively.

Table 1:	Characteristics of the current sample

**p-value<0.01: Highly significant differences

Table 2:	Comparison between the different criteria for women with and without Ms (Mann-Whitney test)

p-value<0.05: Significant differences, Z: Difference between groups, *p-value<0.01: Highly significant differences, P: Significance (2-tailed)

Comparative results: Comparing women with and without MS (Table 2) revealed highly significant differences in blood pressure (both systolic and diastolic) and TG, where women with MS had the higher values. Women with MS had significantly lower value of HDL than those without MS. In contrast, insignificant differences were shown in age, anthropometric measurements (weight, height, BMI and WC), visceral fat, BMD and its T-score at lumbar spines and femoral neck and the other laboratory findings including FSB, Tchol, LDL, Ca, Vit. D, C-terminal peptide and E2.

Correlation results: Spearman’s correlations revealed insignificant association between visceral fat and BMD and its T-score at lumbar spines and femoral neck and the related laboratory findings as Ca, Vit. D and C-terminal peptide, in both the total sample and the 2 groups.

Visceral fat showed significant positive correlations with: (a) Body weight and BMI (in the total sample and in the 2 groups), (b) WC (in the total sample and in the group with MS group only) and (c) Age and TG (in the total sample and for the group without MS group). On the contrary, visceral fat showed significant negative correlations with body height and E2 in the group without MS (Table 3).

Spearman’s correlation between E2 and DEXA findings revealed highly significant positive correlations between E2 and BMD and its T-score at lumbar spines and femoral neck, for the total sample and for women with MS.

Table 3:	Spearman’s correlation between visceral fat ratio and the other variables

p-value<0.05: Significant differences, *p-value<0.01: Highly significant differences

In addition, there were significant positive correlations between E2 and body weight, BMI and FBS for women with MS. Among women without MS, significant negative correlations were found between E2 and systolic blood pressure, visceral fat and Ca. Among total sample, E2 had significant positive correlations with body weight and height, while it had significant negative correlation with HDL (Table 4).

DISCUSSION

The last decades witnessed a rapid increase in the prevalence of both obesity and osteoporosis¹⁴. The relation between bone and fat, commonly known as bone-fat relationship is a complex one, since the published findings are still unclear. The prevalence Metabolic Syndrome (MS), one of the major public health problems has increased dramatically during the recent decades. Moreover, obesity and MS are associated with low-grade inflammation¹⁵. Many researches has focused on the role of VAT on bone, since Visceral Adipose Tissue (VAT) produces inflammatory cytokines that are harmful to bone¹⁶.

The current study aimed to investigate the association between visceral fat levels, E2 and MS, as well as its components, in obese women with and without MS. The study also aimed to investigate the impact of visceral fat and serum levels of E2 on bone markers (Vit. D, C-terminal peptide, Ca and BMD) in obese women with and without MS.

In the present study, the prevalence (percentage) of MS was 39.1% in obese subjects and 60.9% in the non-MS ones. Visceral fat, which is recently recognized as a common health problem is found to be related to several chronic diseases including osteoporosis, diabetes and CVD¹ and it is to be a cause for increasing the risk of vitamin D insufficiency and deficiency in Chinese adults¹⁷.

The current results revealed that MS has no effect on the bone health and the other related laboratory investigations (Ca, Vit. D, C-terminal peptide and estradiol).

Table 4:	Spearman’s correlation between estradiol and the other variables

p-value<0.05: Significant differences, *p-value<0.01: Highly significant differences

However, visceral fat showed insignificant correlations with BMD and its T-score at lumbar spines and femoral neck sites, as well as with the related laboratory investigations (Ca, Vit. D and C-terminal peptide). On the contrary, visceral fat showed significant positive correlations with BMI and WC among the total sample and women with MS group, while, it had weak significant negative correlations with estradiol among the women without MS.

Among women with MS, estradiol showed significant positive correlations with both bone health and BMI, while it has insignificant correlations with visceral fat. Moreover, estradiol showed significant negative correlations calcium, among women without MS.

Cohen¹⁸ stated that large VAT depot is associated with the elevated levels of E2, which acts as a bone protective substance. In contrast, our results revealed that E2 level had significant negative correlation with visceral fat, while it showed significant positive correlation with BMD. Khosla et al.¹⁹ in Rochester found no association between bone density and sex steroid levels. Choi et al.²⁰ in Korea, also found that VAT was negatively correlated with BMD.

In concordance with the present study, Maggio et al.²¹ investigated adults with and without MS and found higher E2 levels in subjects with MS. Pedersen et al.²² demonstrated that E2, only through the estrogen receptor α, inhibits adrenaline-stimulated lipolysis in human subcutaneous fat cells by increasing the amount of α2-adrenergic antilipolytic receptors. This may explain how E2 is related to typical female subcutaneous adipose tissue distribution, since this inhibition is not observed in visceral fat depots. In women, E2 may shift accumulation of fat from visceral into subcutaneous depots⁴.

As, it was concluded that fat distribution is affected by E2 level, it was expected to find a positive association between E2 levels and fat volume, due to increased aromatase activity¹⁸. The study showed significant correlation between E2 and VAT in women without MS.

Current analysis revealed that VAT was insignificantly associated with BMD, although, it was associated with E2 level. In Denmark, it is concluded that^23,24 when including metabolic markers in an analysis, E2 is still the only marker to be significantly associated with BMD.

The findings indicated that visceral fat is significantly associated with BMI, WC and FBS, which represented the principal causative factors of the development of MS. Thus, the findings are in agreement with those of Damiri et al.²⁵ in Palestine. Park et al.²⁶ have concluded that, higher BMI is associated with lower levels of serum 25(OH)D. However, some studies could not reveal the association between Vit. D deficiency the occurrence of MS²⁷. These are in agreement with observations of the current research, where insignificant negative correlations were found between Vit. D and both E2 and VAT.

The findings of the present research showed that the level of C-terminal peptide was insignificantly higher in obese women with MS than in those without MS. Several studies suggested that C-terminal peptide is a reliable factor for early declines in BMD and can be regarded as a risk factor for rapid bone loss in Iraq²⁸ and in China²⁹. In spite of the insignificant low level of Vit. D in women with MS, observed in this research as well as in other studies, we recommend further investigations to confirm the effect of using Vit. D, as prophylaxis for improving bone health and to preventing occurrence of osteoporosis.

CONCLUSION

The prevalence of MS increases significantly with increased obesity and older age. More attention, through health care providers should be given to adult population who are at risk. The high VAT, BMI, age and low E₂ level have a magnitude implying clinical importance for predicting future fracture risk in obese women suffering from MS.

SIGNIFICANCE STATEMENT

This study discover the clinical significance of the interaction between old age, increased visceral adiposity and BMI and low estradiol level that can be beneficial for prediction of bone health risk among obese women suffering from MS. This study will help the researcher to uncover the critical areas of bone health among obese women that many researchers were not able to explore, for early protection from osteoporosis. Thus, a new theory on these interactions may be arrived at.

ACKNOWLEDGMENTS

We would like to acknowledge our institute "National Research Centre', Egypt", without its fund this study could not be done. Also, we would like to acknowledge all individuals who participated in this study, the employers of our institute, the technicians who helped in the laboratory analysis and the doctors who participated in collection of the data'. Without their help, this study couldn’t have been completed.