Impact of plasma omentin levels on glucose metabolic disorders and early stage of endothelial dysfunction in elderly patients

Background: Diabetes-induced endothelial dysfunction is a critical and initiating factor in the genesis of diabetic vascular complications. Omentin exerts direct influence on glucose metabolic disorders and vascular dysfunction. But the association between omentin and cardiovascular events reported by recent studies are paradoxical. Thus, we intend to investigate the relationship between omentin and vascular function in patients with different glucose metabolism states. Methods: A total of 162 elderly patients were stratified into three groups by the status of glucose tolerance test [normal glucose tolerance (NGT), impaired glucose regulation (IGR), type 2 diabetes mellitus (T2DM)]. All participants underwent complete clinical work-up. Fasting blood samples were collected for biochemical measurement. Circulating omentin, adiponectin, interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) levels were detected by enzyme-linked immunosorbent assays. Endothelial and vascular smooth muscle function were evaluated by flow-mediated dilatation (FMD) and nitrate-mediated dilation test (NMD) respectively. Results: Circulating omentin levels were lower in IGR group compared with NGT and were lowest in individuals with T2DM group (P=0.004). FMD has a significant and negative association with systolic blood pressure (SBP), glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG), fasting insulin and interleukin-6 (IL-6), while have a positive relationship with log (omentin) by bivariate correlation analysis in all subjects. After adjusting for traditional cardiovascular factors, log (omentin) was independent related factor that influenced FMD (β=-0.346P=0.01) in IGR subjects, but not in the subgroups NGT and T2DM. Conclusions: A decreased omentin levels had been suggested to be a biomarker of glucose metabolic disorders and potentially cardiovascular risk. Higher circulating omentin (2) Diabetic patients show depressed vascular function, mainly refers to endothelial dysfunction, even in the prediabetes status (IGR). (3) Plasma omentin levels were positively associated with FMD, while in subjects with IGR, omentin showed a significantly and negatively relationship with FMD after adjustment for traditional cardiovascular risk factors. Which may suggest higher omentin concentrations in individuals at risk of atherosclerosis reflecting a compensatory mechanism in early stage of diabetes. Our findings may lay a foundation for studying the pathogenesis of early vascular endothelial damage in prediabetic individuals and new therapies to retard the progression of diabetic cardiovascular complications.

level are independent determining factors for endothelial dysfunction in impaired glucose regulation (IGR) subjects suggesting and may be a compensatory mechanism for early diabetic vascular injury. which suggest omentin may act as potential markers for assessing endothelial dysfunction and identifying high risk of cardiovascular complications among asymptomatic prediabetic individuals.

Background
Type 2 diabetes mellitus (T2DM) represents a major risk factor for the development of cardiovascular diseases and ischemic stroke. A collaborative meta-analysis of 102 prospective studies indicated that diabetes confers about a two-fold excess risk for coronary heart disease, major stroke subtypes, and deaths attributed to other vascular causes, independently from other conventional risk factors. Besides, patients with diabetes are more likely to develop fatal myocardial infarction than those with normal blood glucose 1 . Although we have made several efforts in reducing cardiovascular morbidity and mortality with novel hypoglycemic drugs and regular management, convincing improvements in outcomes have not been achieved yet 2 .The morbidity and mortality rates of diabetes remain high. According to the latest data from the International Diabetes Federation (IDF), approximately 12% to 31.7% older people with type 1 and type 2 diabetes have coronary artery disease, which accounted for about 25% of vascular deaths in populations in developed countries 3 .
Endothelial and vascular smooth dysfunction are considered early abnormalities in the development of atherosclerotic process in cardiovascular diseases, with a significant prognostic role in high-risk population 4 . Impaired endothelial function has been consistently demonstrated in the macro-and microcirculation of individuals with type 2 diabetes, moreover, it appears long before symptoms. Unfortunately, a majority of older people with diabetes are unaware of having diabetes complications, thus, physicians always deal with diabetic patients with various complications rather than the early stage of the diseases. However, most complications can be detected in their early stages by screening programmes. Decreased bioavailability of nitric oxide is considered to be an important feature of vascular endothelial dysfunction, which can be quantified by the noninvasive technique of flow-mediated dilation (FMD) 5 . Nitrate-mediated dilation of brachial artery (NMD) using the exogenous nitrate administration to relax vascular smooth muscle (VSM), which is in an endothelium-independent manner, thus reflecting VSM function 6 . The noninvasive nature and repeatable characteristic of the technique allow its widespread usage in predicting cardiovascular outcomes in patients at high risk of developing cardiovascular diseases. However, there are also technical and interpretive limitations of this technique, which limit its widely clinical application.
Normalization of glycemia does not reduce macrovascular events suggesting other mediators may participate to increase the residual cardiovascular risk in diabetic patients 7 . Accumulating evidence indicated that adipose tissue secretes several bioactive mediators termed adipokines, which link metabolic disorders and atherosclerotic cardiovascular diseases 8 . Omentin is among the anti-inflammatory and anti-atherogenic adipokine that has potentially beneficial effects on glucose metabolism and cardiovascular disorders 9 10 . However, recent studies indicate a paradoxical association between omentin and cardiovascular events 11 12 13 14 . In this study, we investigated the association of circulating omentin levels and endothelial function in the elderly subjects with different glucose metabolism conditions, at the aim of finding potential biomarkers recommended for the clinical use in the prevention or treatment of diabetic patients who at high risk of developing cardiovascular diseases.

Population
We recruited a total of 162 elderly patients with different blood glucose levels at Geriatric Department of Ruijin hospital affiliated to Shanghai Jiao Tong University School of Medicine between December 2017 and May 2019. According to the plasma glucose criteria, patients were divided into three groups: 48 subjects with normal glucose tolerance (NGT) ,56 subjects with impaired glucose regulation (IGR, also known as prediabetes), including 28 subjects with impaired glucose tolerance (IGT) and 28 subjects with impaired fasting glucose (IFG), 58 subjects with type 2 diabetes mellitus (T2DM).
Diagnoses were made according to the American Diabetes Association Criteria 15 . A 75 g oral glucose tolerance test were performed in all subjects. Subjects with fasting plasma glucose(FPG) value <5.6 mmol/l and 2-h plasma glucose (2-h PG) value during a 75-g oral glucose tolerance test (OGTT) <7.8 mmol/l are considered as normal glucose tolerance (NGT); IFG is defined as FPG levels between 100 and 125 mg/dL (between 5.6 and 6.9 mmol/L) and IGT as 2-h post-load plasma glucose between 140 and 199 mg/dL (between 7.8 and 11.0mmol/L). Patients with IGR are defined by the presence of IFG and/or IGT and/or HbA1C 5.7-6.4%. The fasting plasma glucose (FPG)≥126 mg/dL (7.0 mmol/L) or the 2-h plasma glucose (2-h PG) value ≥200mg/dL (11.1mmol/L), or HbA1C criteria ≥6.5% are diagnosed as type 2 diabetes mellitus (T2DM). Patients with type 1 diabetes or other types of diabetes were not included in this study. The clinical characteristics of the three groups are shown in Table 1.
Exclusion criteria: Those with acute cardiovascular and cerebrovascular diseases, acute infectious diseases, chronic heart failure, liver and kidney dysfunction, valvular diseases, arrhythmias, autoimmune diseases, malignancies and hyperthyroidism were excluded in this study.

This study was approved by the Clinical Research Ethics Committee of Ruijin Hospital
Shanghai Jiao Tong University School of Medicine. Informed consent was obtained from each participant after the purpose of this study was explained.

Anthropometric and biochemical measurements
The subjects' age, gender, previous history and medication history were recorded by special personnel. Body weight and height were measured in all participators using standard protocols, BMI was calculated according to the following formula: BMI=weight/height squared (kg/m 2 ). Blood pressure was measured on the right arm after a 10-minute rest for three times, values used in the analysis are the average of three readings taken at 5-minute intervals. Those who has a history of hypertension or whose systolic blood pressure (SBP)≥140mmHg and/or diastolic blood pressure (DBP)≥90mmHg were defined as hypertension.
Fasting elbow venous blood samples were collected in the morning after 12-h fasting period for biochemical test. Fasting plasma glucose was evaluated by the automated glucose oxidase method. HbA1c was measured by high-performance liquid chromatography. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) and serum triglyceride (TG) concentrations were measured by using enzymatic assays. Insulin resistance was calculated by the homeostasis model assessment of insulin resistance (HOMA-IR), which was automatically calculated by the following formula: HOMA-IR=fasting insulin (μU/ml) x fasting glucose (mmol/L)/22.5.
Serum concentrations of omentin, adiponectin, TNF-α, IL-6 were determined by an enzyme-linked immunosorbent assay (ELISA) kit (provided by Shanghai Senxiong Bio-Tech CO. Ltd). All standards are strictly handled according to the instructions of the kit.

The assessment of macrovascular function
All patients underwent assessment of arterial dilator function, following the International Brachial Artery Reactivity Task Force guidelines 5 . Before testing, all subjects were in a fasting state for at least 6 hours, vasoactive drugs (such as angiotensin converting enzyme inhibitors, Calcium antagonist, beta receptor blockers), alcohol, caffeine, tea, cigarettes and nicotine were withheld for more than 12 hours. Subjects rested in the supine position for at least 10 minutes in a quiet and warm (temperature of 22-24℃) room, A 10-MHz multifrequency linear array probe attached to a high-resolution ultrasound machine (Toshiba Artida) was used to acquire images of the right brachial artery. The lumen diameter of the artery was defined as the distance from the echo front edge of the near wall-lumen interface to the echo front edge of the far wall-lumen interface. In brief, the basal diameter of the brachial artery was obtained in the cubital region before inducing forearm ischemia, recorded as D0 (mm). Subsequently, a forearm cuff, positioned 1 cm above the antecubital fossa, was inflated to 50 mmHg above systolic blood pressure for 5min and then released to elicit forearm reactive hyperemia. The diameter at the same point of the artery was recorded as D1 (the maximum dilatation after deflation). Flow mediated dilation of brachial artery (FMD), described as the maximum percent change of the brachial artery diameter during forearm hyperemia compared to the basal diameter, was calculated by the following formula: FMD (%) = (D1-D0)/D0*100. After 15 minutes, the brachial artery returned to the baseline. Brachial artery diameter was monitored continuously until achievement of maximal dilatation after administration of 0.5mg of sublingual nitroglycerin, which was measured as D2, and Nitrate-mediated dilation of brachial artery (NMD) was expressed as the percent maximal increase of the artery diameter after nitroglycerin compared to baseline [NMD(%)=(D2-D0)/D0*100]. We use flow mediated dilatation (FMD) to quantify vascular endothelial function, while apply nitrate-mediated dilation (NMD) to assess endothelium-independent dilator function. The value of FMD 10% indicates impaired endothelial function, and the lower the FMD value is, the worse the endothelial function is. The variability for this ultrasound determination of FMD and NMD showed a coefficient of variation of 1.2%-4.2% and 3.97±0.24% respectively, suggesting our method has good reproducibility 16 .
Throughout the measurement of each individual, the probe was maintained in a fixed position. All operations were performed by the same experienced sonographer who was blind to the subjects' clinical and biochemical characteristics.

Statistical analysis
Date are expressed as the mean and standard deviations (SDs), or median (interquartile range), or as percentage according to the variables were normal distributing or not, which were examined by the Kolmogorov-Smirnov equality of distributions test. Skewed parameters, such as triglycerides levels, HDL-C, fasting glucose, fasting insulin, HOMA-IR TNF-α, adiponectin and omentin levels were logarithmically transformed to normalize their distributions before regression analysis. Differences among the three groups were tested using ANOVA for normally variables while Kruskal-Wallis H test was used for non-normally distributed variables. Pearson's correlation coefficient was used for calculation of associations between serum omentin levels and clinical factors, so as the FMD. While multiple stepwise linear regression analysis was used to determine significant confounding factors for circulating omentin levels and FMD separately. Clinically and statistically significant variables were added in the regression model. Data were analyzed using SPSS 22.0 software. We regarded P value of less than 0.05 as statistically meaningful.

Anthropometric, clinical and biochemical characteristics
A total of 162 elderly subjects were recruited in our study. The mean age of participators was 78.64 ± 8.96 and 84% were male. Baseline characteristics of subjects stratified by the status of glucose tolerance test (NGT, IGR and T2DM) are presented separately in Table 1. BMI, blood pressure, TG, TC, LDL-C, HDL-C were similar among the three groups. The levels of FPG, FINS, 2 h PG, HbA1c and HOMA-IR were higher in T2DM group compared with NGT and IGR groups. However, no significant differences in terms of FPG, FINS, 2 h-PG, HOMA-IR and HbA1c were detected between NGT and IGR groups. Besides, Subjects with T2DM were more likely to be older, showing higher levels of TNF-α, IL-6 when compared with NGT groups, but no significant differences were detected within them.  The results were show in Table 2.
When omentin levels were analyzed according to vascular endothelial function status, we found plasma omentin levels were significantly lower in endothelial dysfunction (ED) than normal endothelial function (NEF) in all subjects (20.12 ± 13.32 ng/ml verse 14.44 ± 8.19 ng/ml, P = 0.007) Table 3. Similar result was indicated in participators with NGT, data wasn't showed. Then we conducted an analysis in each subgroup respectively found the plasma omentin levels in IGR subjects with endothelial dysfunction were higher than those of normal endothelial function (18.511 ± 9.35 ng/ml verse 13.46 ± 5.16 ng/ml, P = 0.014).
To further study the relationship between plasma omentin levels and FMD, multiple regression analyses were performed separately in the subgroups. Even after adjusting for age, BMI, SBP, log (FPG), log (INS), log (HOMA-IR), log (TG), log (HDL-C), log (APN), log (TNF-α), and IL-6, log (omentin) was independent related factor that influenced FMD (β=-0.346,P = 0.01) in IGR subjects, but not in the subgroups NGT and T2DM. In T2DM group, The effect of glycosylated hemoglobin on vascular dysfunction is greater than that of lower omentin levels (β=-0.525, P = 0.009). Data was show in Table 5.  Endothelial dysfunction is implicated in a variety of cardiovascular diseases characterized by a reduced production and availability of nitric oxide (NO). The measurement of flowmediated dilation (FMD) of the brachial artery after forearm ischemia is supposed to be a non-invasive method to assess endothelial production and release of NO, which has been widely used to assess vascular function. At present, it is generally accepted that the value of FMD less than 10% indicates impaired endothelial function, and the lower the FMD value is, the worse the endothelial function is 5 . Besides, FMD has demonstrated to be a good predictor of future cardiovascular events. A current meta-analysis found that a 1 SD increase or decrease of FMD was associated with 50% lower risk or doubled risk of cardiovascular events 24 25 . In our study, FMD was decreased in subjects with T2DM and IGR compared with NGT group, no difference was found in terms of NMD, which shows that Vitro studies indicated omentin might play a preventive role in the pathogenesis of obesity and diabetes-related vascular complications 12 32 . In agreement with previous cross-sectional studies reported that circulating omentin concentrations were lower in people with coronary artery disease or ischemic stroke compared with individuals without cardiovascular disease 33 34 , our study demonstrated that plasma omentin levels decreased in subjects with the early stage of atherosclerosis, i.e. endothelial dysfunction, especially in patients with diabetes. After adjustment for other factors, no significant association was found between log(omentin) and FMD in all subjects and T2DM group.
However, in IGR group, the higher circulating omentin levels are independent determining factors for endothelial dysfunction. Although the detailed mechanisms are not yet completely understood. Omentin could be upregulated in response to metabolic and inflammatory stimuli that contribute to vascular endothelial dysfunction in the early stage of diabetes. One potential explanation for the adverse association between omentin and endothelial dysfunction in IGR subjects is higher omentin levels in individual at risk of atherosclerosis reflecting a compensatory mechanism, just like there exist insulin resistance in prediabetes, but this self-regulating mechanism is insufficient to protect against the onset of endothelial dysfunction when hyperglycemia persists or reaches the diagnostic criteria for diabetes, then lower omentin levels in diabetic patients appears. If this were true, associations could be explained by reverse causation because higher omentin levels would represent early symptoms of prediabetic endovascular injury.

Limitations
Our study both has strengths and limitations. It is the first study to demonstrates that circulating omentin levels strongly predicts endothelial function in elderly patients with impaired glucose regulation independently from traditional cardiovascular risk factors and from adiponectin level. Additionally, our study mainly focused on patients with impaired glucose regulation (IFG and IGT), which was considered as pre-diabetes. The main limitation of our study was its cross-sectional design, we are not yet sure of the causal relationship between the plasma omentin concentration and diabetic vascular dysfunction.
In addition, our sample size was too small to make the case, besides, the elderly mainly centered in Shanghai may have regional differences and are not representative. Finally, we did not take the duration of diabetes and medication histories into account when we included the study subjects, which may affect the experimental results.