Conceived and designed the experiments: JMM-N WR JMF-R. Performed the experiments: JMM-N MS FO. Analyzed the data: JMM-N MS. Contributed reagents/materials/analysis tools: MS FO. Wrote the paper: JMM-N JMF-R.
The authors have declared that no competing interests exist.
Zonulin is the only physiological mediator known to regulate intestinal permeability reversibly by modulating intercellular tight junctions. To investigate the relationship between intestinal permeability and obesity-associated metabolic disturbances in humans, we aimed to study circulating zonulin according to obesity and insulin resistance. Circulating zonulin (ELISA) was measured in 123 caucasian men in association with inflammatory and metabolic parameters (including minimal model-measured insulin sensitivity). Circulating zonulin increased with body mass index (BMI), waist to hip ratio (WHR), fasting insulin, fasting triglycerides, uric acid and IL-6, and negatively correlated with HDL-cholesterol and insulin sensitivity. In multiple regression analysis, insulin sensitivity (p = 0.002) contributed independently to circulating zonulin variance, after controlling for the effects of BMI, fasting triglycerides and age. When circulating IL-6 was added to this model, only BMI (p = 0.01) contributed independently to circulating zonulin variance. In conclusion, the relationship between insulin sensitivity and circulating zonulin might be mediated through the obesity-related circulating IL-6 increase.
Obesity has been associated with increased intestinal permeability and absorption
Intestinal permeability regulates molecular trafficking between the intestinal lumen and the submucosa, leading to either tolerance or immunity to non–self-antigens
Zonulin is the only physiological mediator known to regulate intestinal permeability reversibly by modulating intercellular TJs
To gain insight in the relationship between intestinal permeability and obesity-associated metabolic disturbances in humans, we hypothesized a possible association between circulating zonulin, obesity and insulin sensitivity according to glucose tolerance.
NGT | GI | P | |
|
82 | 41 | |
|
48.29±11.7 | 55.9±10.3 |
|
|
26.6±3.2 | 28.1±3.8 |
|
|
0.929±0.064 | 0.952±0.069 | 0.06 |
|
92.9±7.3 | 101.15±10.47 |
|
|
8.1±3.6 | 11.1±6.5 | 0.01 |
|
4.78±0.32 | 4.9±0.42 | 0.1 |
|
0.58±0.2 | 0.43±0.16 |
|
|
52.8±12.9 | 52.2±10.1 | 0.8 |
|
83 (58.5–120.5) | 100 (69.5–129) | 0.5 |
|
5.63±1.3 | 6.12±1.4 | 0.05 |
|
1.22±1.1 | 1.37±0.9 | 0.4 |
|
9.1±4.5 | 10.9±4.8 |
|
NGT, participants with normal glucose tolerance; GI, participants with glucose intolerance; WHR, waist to hip ratio; HbA1c, Glycosylated haemoglobin; IS, insulin sensitivity.
One hundred twenty three Caucasian men were recruited and studied in ongoing study dealing on non-classical cardiovascular risk factors in Northern Spain. Subjects were randomly localized from a census and they were invited to participate. The participation rate was 71%. A 75 g oral glucose tolerance test according to the American Diabetes Association Criteria was performed in all subjects. All subjects with normal glucose tolerance (n = 82) had fasting plasma glucose <7.0 mM and two-hour post-load plasma glucose <7.8 mM after a 75 g oral glucose tolerance test. Glucose intolerance was diagnosed in 41 subjects according to the American Diabetes Association Criteria (post-load glucose between 7.8 and 11.1 mmol/l). Inclusion criteria included the following: 1) BMI <40 kg/m2, 2) absence of systemic disease, and 3) absence of infection within the previous month. None of the control subjects were under medication or had evidence of metabolic disease other than obesity. Alcohol and caffeine were withheld within 12 h of performing the insulin sensitivity test. Liver disease and thyroid dysfunction were specifically excluded by biochemical work-up. All subjects gave written informed consent after the purpose of the study was explained to them. The ethical committee of the Hospital Universitari Dr. Josep Trueta (Comitè d’Ètica d’Investigació Clínica, CEIC) approved the protocol.
Subjects were studied in the post-absorptive state. BMI was calculated as weight (in kilograms) divided by height (in meters) squared. Subjects’ waists were measured with a soft tape midway between the lowest rib and the iliac crest; hip circumference was measured at the widest part of the gluteal region; and waist-to-hip ratio (WHR) was accordingly calculated.
All participants(N = 123) | NGT(N = 82) | GI(N = 41) | ||||
r | p | r | p | r | p | |
|
0.18 | 0.05 | 0.22 |
|
−0.03 | 0.8 |
|
0.28 |
|
0.16 | 0.1 | 0.42 |
|
|
0.2 |
|
0.18 | 0.1 | 0.17 | 0.2 |
|
−0.01 | 0.9 | −0.03 | 0.7 | −0.11 | 0.4 |
|
0.37 |
|
0.18 | 0.1 | 0.57 |
|
|
0.14 | 0.1 | 0.24 |
|
−0.04 | 0.8 |
|
−0.28 |
|
−0.22 |
|
−0.36 |
|
|
−0.21 |
|
−0.27 |
|
−0.04 | 0.8 |
|
0.21 |
|
0.22 |
|
0.15 | 0.35 |
|
0.2 |
|
0.24 |
|
0.01 | 0.9 |
|
0.29 |
|
0.31 |
|
0.22 | 0.2 |
NGT, participants with normal glucose tolerance; GI, participants with glucose intolerance; WHR, waist to hip ratio; HbA1c, Glycosylated haemoglobin; IS, insulin sensitivity.
Insulin sensitivity was measured using the frequently sampled intravenous glucose tolerance test (FSIVGTT). In brief, basal blood samples were drawn at –15 and –5 min, after which glucose (300 mg/kg body wt) was injected over 1 min starting at time 0. At 20 min, regular insulin (Actrapid, Novo, Denmark; 0.03 U/kg) was injected as a bolus. Additional samples were obtained from a contralateral antecubital vein at times 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 20, 22, 23, 24, 25, 27, 30, 40, 50, 60,70, 80, 90, 100, 120, 140, 160, and 180 min. Samples were rapidly collected via a three-way stopcock connected to the butterfly needle. Data from the FSIVGTT were submitted to computer programs that calculate the characteristic metabolic parameters by fitting glucose and insulin to the minimal model that describes the times course of glucose and insulin concentrations. The glucose disappearance model, by accounting for the effect of insulin and glucose on glucose disappearance, provides the parameters SI (10−4) per minute per microunit per milliliter) or the insulin sensitivity index, a measure of the effect of insulin concentrations above the basal level to enhance glucose disappearance. The estimation of model parameters was performed according to the MINMOD computer program
Model 1 | Beta (Standardized coefficients) | T | p |
|
0.157 | 1.77 | 0.08 |
|
0.066 | 0.59 | 0.5 |
|
−0.263 | −2.95 |
|
|
0.132 | 1.45 | 0.15 |
|
|||
|
0.134 | 1.16 | 0.25 |
|
0.159 | 1.34 | 0.18 |
|
−0.150 | −1.29 | 0.20 |
|
0.067 | 0.579 | 0.50 |
|
0.23 | 2.03 | 0.04 |
Serum glucose concentrations were measured in duplicate by the glucose oxidase method using a Beckman glucose analyzer II (Beckman Instruments, Brea, California). Glycosylated haemoglobin
Statistical analyses were performed using SPSS 12.0 software. Unless otherwise stated, descriptive results of continuous variables are expressed as mean and SD for Gaussian variables. Parameters that did not fulfill normal distribution were mathematically transformed to improve symmetry for subsequent analyses. The relation between variables was analyzed by simple correlation (Pearson’s test) and multiple regression analyses. Unpaired t tests were used to compare subjects with NGT and AGT subjects. Levels of statistical significance were set at
In all subjects, as a whole circulating zonulin was significantly increased in obese (n = 33) versus non-obese (n = 90) subjects (12.5±4.6 vs. 9.3±5.1, p = 0.007) and in subjects with glucose intolerance (10.9±4.8 vs. 9.1±4.5, p = 0.03) (
The associations of circulating zonulin with metabolic variables according to glucose tolerance are described in
In multiple regression analysis, insulin sensitivity (p = 0.002) contributed independently to circulating zonulin variance, after controlling for the effects of BMI, fasting triglycerides and age (
To the best of our knowledge, this is the first study that associates circulating zonulin concentration (a marker and modulator of intestinal permeability
Recent evidence suggests a possible role of gut in obesity. Recent findings emphasize the important symbiotic contributions (in diversity and function) to the human metabolism made by the collection of microbial genomes known as the
In subjects with glucose intolerance, circulating zonulin was strongly associated with insulin resistance and obesity. Otherwise, in subjects with normal glucose tolerance (NGT), zonulin concentrations were also associated with high levels of uric acid, HbA1c, circulating IL-6 and low HDL-Cholesterol. In conclusion, circulating zonulin might help us to know the contribution of small intestine permeability on glucose intolerance and insulin resistance. Functional ex-vivo studies with recombinant zonulin in mouse intestinal segments have shown that zonulin increased significantly intestinal permeability
We acknowledge the technical assistance of Oscar Rovira and the administrative help of Roser Rodriguez.