The ancient wisdom of Hippocrates who believed that ‘all disease begins in the gut’ is being validated by modern science, with poor gastrointestinal (GIT) health linked to an increasing number of diseases. Zonulin is a protein that has been the subject of much research in recent years, particularly in relation to its role in dysfunctional GIT functionality and disease pathogenesis.
Zonulin is a pre-haptoglobulin (HP2) protein that regulates small intestinal and gastro-duodenal permeability through its effects on tight junctions (TJ), which are located between epithelial cells.[1-3]
TJs are comprised of transmembrane and peripheral proteins joined to a cytoskeleton. Zonulin modulates intestinal permeability by binding to the epidermal growth factor and protease activated receptors in the epithelium, resulting in phosphorylation of TJ proteins and subsequent modification of both cytoskeletal and TJ structure.[1-4]
Encoded from the HP2 gene, the uncleaved form of zonulin in human serum is a product of both intestinal and extra-intestinal tissue secretion, including the liver, adipose tissue, brain, heart, immune cells, lungs, kidney and skin.[1,2,5]
In individuals genetically predisposed to coeliac disease, zonulin release in the acute disease phase follows intercommunication between gliadin and the CXCR3 chemokine receptor, which is over-expressed in coeliac intestinal tissue. This stimulates adaptive immune-regulated intestinal damage and increased permeability characteristic of the disease.
In type 1 diabetes, impaired intestinal permeability has been observed to develop before the onset of the disease, and increased zonulin is associated with antibodies of the wheat storage protein Glo-3a and islet autoimmunity in genetically predisposed children.[1,6,9] About 50% of people with type 1 diabetes have been observed to have elevated serum zonulin levels, which correlate with increased intestinal permeability.
In a study on individuals with concomitant coeliac disease and type 1 diabetes, it was observed that, compared with healthy controls, diseased subjects had higher serum zonulin levels that also correlated with both enterovirus density and severe atrophic small intestinal damage. They also had an increased expression of FOXP3 mRNA, a mediator of regulatory T cells.
Other autoimmune diseases that have been found to have upregulated zonulin include ankylosing spondylitis, systemic lupus erythematous (SLE) and rheumatoid arthritis.
In a recent study investigating the role of ileal bacteria in ankylosing spondylitis, high zonulin levels were associated with an impaired gut vascular barrier and elevated serum levels of lipopolysaccharide (LPS) and LPS-binding protein, while adherent and invasive bacteria was significantly correlated with gut inflammation. An in vitro analysis in the same study also observed that zonulin release was stimulated by ileal bacteria, which subsequently adversely affected endothelial TJs.
Elevated zonulin levels and altered intestinal permeability have also been observed in non-autoimmune diseases including type 2 diabetes, obesity, cardiovascular disease, polycystic ovarian syndrome (PCOS), non-alcoholic fatty liver disease (NAFLD), autism spectrum disorder, some cancers and nervous system diseases.[2,4,6,7,11-14]
In overweight and obese subjects, zonulin is secreted from intestinal, adipose and liver tissues, and the concomitant presence of impaired glucose metabolism, inflammation and dysbiosis in this population are thought to play a role in its release.
In glucose-intolerant obese males, serum zonulin levels were significantly increased in obese versus non-obese subjects, and were also associated with higher body mass index (BMI) levels, waist to hip ratio (WHR), fasting insulin and triglycerides, uric acid and interleukin (IL)-6.
In a subsequent study on individuals with type 2 diabetes, subjects were found to have significantly higher serum zonulin levels compared to impaired or normal glucose intolerant subjects. High serum zonulin levels were also correlated with BMI, WHR, fasting and two-hour plasma glucose, HbA1c, HOMA-IR, triglyceride, total and HDL-cholesterol, TNF-alpha and IL-6.
Similar results were observed in a recent population-based prospective cohort study with elevated zonulin levels associated with higher waist circumference and fasting plasma glucose, overweight, obesity and hyperlipidaemia.
In obese children with NAFLD, zonulin levels were found to be significantly higher compared to those without the disease, and were also positively associated with whole body insulin sensitivity index, two-hour insulin and hepatic fat levels.
This was also observed in a separate study on adults with NAFLD, with higher serum zonulin levels in the diseased group versus healthy controls, as well as positive correlations between zonulin and BMI, fasting insulin and HOMA-IR, triglycerides, alanine transaminase and IL-6. Zonulin was also found to be of diagnostic value in the occurrence of non-alcoholic steatohepatitis, with the authors suggesting a potential role of zonulin in NAFLD pathogenesis.
High serum zonulin levels have also been seen in women with PCOS, along with positive associations between zonulin, HOMA-IR and insulin sensitivity index, and correlations between zonulin and PCOS severity.
Serum zonulin levels have been found to be elevated in patients with coronary artery disease (CAD) compared with non-CAD subjects. An in vitro analysis in the same study also observed that an enteric-derived bacteria Pseudomonas uorescens increased the expression and secretion of zonulin from caco-2 cells and subsequently caused increased intestinal permeability.
While all of these studies vary in subject characteristics and the pathologies investigated, they demonstrate the concomitant interaction between abnormal metabolic parameters such as inflammation, glucose and lipid metabolism and impaired GIT function as underlying factors in disease aetiology. What needs to be further elucidated is the specific relevance of zonulin as a disease biomarker or antecedent in the context of this endogenous milieu.
The role of exogenous nutritional influences on zonulin is a topic of ongoing research, however some recent studies have investigated the effect of dietary and nutritional compounds on zonulin levels.[5,8,17-20]
For 12 weeks, obese and normal-weight subjects with type 2 diabetes followed an Okinawan-based diet for 12 weeks, with low carbohydrate and high fat, fibre and protein content. At the end of the treatment period, zonulin levels (serum and faeces) increased and there were positive correlations between elevated serum zonulin and the percentage of energy from protein, and inverse correlations with the percentage of energy from carbohydrates. There was also a correlation between calprotectin, a marker of gut inflammation, and zonulin after 12 weeks.
In a separate study on obese subjects, plasma zonulin levels was found to be proportional to daily energy intake from higher fat consumption, and inversely proportional to percentage protein intake in obese and normal weight subjects. Positive correlations between elevated zonulin levels and total faecal bacterial count were also found.
In overweight pregnant women, lower zonulin levels were associated with higher intakes of omega-3 fatty acids and fibre. The low zonulin group also had greater bacterial richness compared with the high zonulin group, with lower levels of bacteroidaceae and bacteroides and higher levels of faecalibacterium compared with the high zonulin group.
While these studies indicate that macronutrients differentially influence zonulin secretion, further investigations are necessary to confirm the specific effects of acute and chronic nutritional exposure on zonulin levels in a range of population groups.
It has been established that pathogenic bacteria are involved zonulin secretion,[1,2] and the effect of probiotics in modulating endogenous zonulin has been the subject of a few recent studies.[19,20]
In overweight and obese subjects supplemented with Bifidobacterium animalis ssp. lactis (10 billion CFU daily) with fibre for 6 months, circulating levels of zonulin were lower compared with placebo. Zonulin levels were also associated with reductions in trunk fat mass and high-sensitive C-reactive protein.
In healthy exercise-trained men, faecal levels of zonulin and serum TNF-alpha were significantly lower following supplementation with multi-species probiotics (B. bifidum, B. lactis, Enterococcus faecium, Lactobacillus acidophilus, L. brevis, Lactococcus lactis) at a dose of 10 billion CFU daily for 14 weeks compared to placebo levels.
In an investigation of subjects with colorectal liver metastases given either probiotics (2.6 x 1014 CFU daily of three unspecified strains) or placebo pre-operatively for 6 days and post-operatively for 10 days, probiotics reduced serum zonulin concentrations and plasma endotoxin levels. In a subsequent sub-analysis, subjects with normal intestinal permeability in the control group had significantly elevated zonulin levels compared with the probiotic group, which had similar zonulin levels before and after treatment.
Colostrum may also have a beneficial effect on zonulin metabolism. In a double-blind, placebo controlled study, supplementation of athletes during a peak training period for 20 days with 500mg of colostrum resulted in a decrease in intestinal zonulin levels and improved intestinal permeability.
Along with the potential role of probiotics in modulating zonulin levels, further research is required on the specific role of zonulin in intestinal and systemic physiology, if it is a biomarker or precursor of abnormal metabolic parameters and its specific involvement in the pathogenesis of disease.
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- Ohlsson B, Orho-Melander M, Nilsson PM. Higher levels of serum zonulin may rather be associated with increased risk of obesity and hyperlipidemia, than with gastrointestinal symptoms or disease manifestations. Int J Mol Sci 2017;8:82. [Full text]
- Vanuytsel T, Vermeire S, Cleynen I. The role of haptoglobin and its related protein zonulin, in inflammatory bowel disease. Tiss Barr 2013;1:5:e27321. [Full text]
- Li C, Gao M, Zhang W, et al. Zonulin regulates intestinal permeability and facilitates enteric bacteria permeation in coronary artery disease. Sci Rep 2016;6:29142. [Full text]
- Zak-Golab Z, Pocelak P, Aptekorz M, et al. Gut microbiotia, microinflammation, metabolic profile and zonulin concentration in obese and normal weight subjects. Int J Endocrin 2013;2013:674106. [Full text]
- Fasano A. Intestinal permeability and its regulation by zonulin: diagnostic and therapeutic implications. Clin Gastroenterol Hepatol 2012;10(10):1096-1100. [Full text]
- Zhang D, Zhang L, Yue F, et al. Serum zonulin is elevated in women with polycystic ovarian syndrome and correlates with insulin resistance and severity of anovulation. Eur J Endocrinol 2015;172:29-36. [Full text]
- Liu Z, Li C, Huang M, et al. Positive regulatory effects of perioperative probiotic treatment on postoperative liver complications after colorectal liver metastases surgery: a double-centre and double-blind randomized clinical trial. BMC Gastroenterol 2015;15:34. [Full text]
- Taplin CE, Mojibian M, Simpson M, et al. Antibodies to the wheat storage globulin Glo-3A in children before and at diagnosis of celiac disease. J Pediatr Gastroenterol Nutr 2011;52(2):21-25. [Full text]
- Vorobjova T, Raikkerus H, Kadaja L, et al. Circulating zonulin correlates with density of enteroviruses and tolerogenic dendritic cells in the small bowel mucosa of celiac disease patients. Dig Dis Sci 2017;62(2):358-371. [Abstract]
- Ciccia F, Guggino G, Rizzo A, et al. Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis. Ann Rheum Dis 2017;76(6):1123-1132. [Abstract]
- Pacifico L, Bonci E, Marandola L, et al. Increased circulating zonulin in children with biopsy-proven non-alcoholic fatty liver disease. World J Gastroenterol 2014;20(45):17107-17114. [Full text]
- Hendy OM, Elsabaawy MM, Aref MM, et al. Evaluation of circulating zonulin as a potential marker in the pathogeneiss of NAFLD. APMIS 2017 Apr 21. [Epub ahead of print] [Abstract]
- Esnafoglu E, Cirrik S, Ayyildiz SN, et al. Increased serum zonulin levels as an intestinal permeability marker in autistic subjects. J Pediatr 2017 May 11. [Epub ahead of print] [Abstract]
- Moreno-Navarrete M, Sabater M, Ortega F, et al. Circulating zonulin, a marker of intestinal permeability, is increased in association with obesity-associated insulin resistance. PLoS One 2012;7(5):e37160 [Full text]
- Zhang D, Zhang L, Zheng Y, et al. Circulating zonulin levels in newly diagnosed Chinese type 2 diabetes patients. Diabetes Res Clin Pract 2014;106(2):312-318. [Abstract]
- Mokkala K, Roytio H, Munukka E, et al. Gut microbiota richness and composition and dietary intake of overweight pregnant women are related to serum zonulin concentration, a marker for intestinal permeability. J Nutr 2016;146(9):1694-1700. [Abstract]
- Ohlsson B, Roth B, Larsson E, et al. Calprotectin in serum and zonulin and faeces are elevated after introduction of a diet with lower carbohydrate content and higher fibre, fat and protein content. Biomed Rep 2017;6(4):411-422. [Full text]
- Stenman LK, Lehtinen MJ, Meland N, et al. Probiotic with or without fibre controls body fat mass, associated with serum zonulin, in overweight and obese adults: randomized controlled trial. EBioMed icine 2016;3:190-200. [Full text]
- Lamprecht M, Bogner S, Shippinger G, et al. Probiotic supplementation affects markers of intestinal barrier, oxidation, and inflammation in trained men: a randomized, double-blinded, placebo-controlled trial. J Int Soc Sports Nutr 2012;9:45. [Full text]
- Halasa M, Maciejewska D, Baskiewicz-Halasa M, et al. Oral supplementation with bovine colostrum decreases intestinal permeability and stool concentrations of zonulin in athletes. Nutrients 2017;9:370. [Full text]