Gastrointestinal distress, such as diarrhoea, cramping, vomiting, nausea and gastric pain are common among athletes during training and competition. The mechanisms that cause these symptoms are not fully understood. The stress of heat and oxidative damage during exercise causes disruption to intestinal epithelial cell tight junction proteins resulting in increased permeability to luminal endotoxins. The endotoxin moves into the blood stream leading to a systemic immune response. Tight junction integrity is altered by the phosphoylation state of the proteins occludin and claudins, and may be regulated by the type of exercise performed. Prolonged exercise and high-intensity exercise lead to an increase in key phosphorylation enzymes that ultimately cause tight junction dysfunction, but the mechanisms are different. The purpose of this review is to (1) explain the function and physiology of tight junction regulation, (2) discuss the effects of prolonged and high-intensity exercise on tight junction permeability leading to gastrointestinal distress and (3) review agents that may increase or decrease tight junction integrity during exercise.
- Stress response to exercise and adaptations of stress proteins to exercise training
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- Stress response to exercise and adaptations of stress proteins to exercise training
The intestine is the primary organ for absorption of fluids, nutrients and electrolytes. The mucosal layer of the intestinal tract is made up of epithelial cells, enterocytes, which are connected to one another by tight junctions (TJ) consisting of specialised proteins such as occludin, zona-occludens (ZO-1, ZO-2 and ZO-3) and claudins.1 ,2 Both TJ and the apical membrane of enterocytes constitute the intestinal barrier,3 which allows absorption of nutrients and water4 while also preventing the translocation of harmful substances from the gut to the bloodstream.1 The integrity of the intestinal barrier is regulated by the phosphorylation state of the TJ proteins where the type of kinase and binding site play a role.5 ,6
During prolonged exercise that increases core temperature, cardiovascular and thermoregulatory responses compromise intestinal blood flow. As core temperature approaches 39 s, intestinal temperature can be as high as 41 s7 leading to epithelial cell damage.8 In addition, high-intensity exercise redirects blood flow away from the splanchnic arteries and to the working muscle leading to an ischaemia reperfusion cycle where blood flow returns when exercise intensity is lowered,9 and may result in oxidative damage.10 ,11 Both heat and ischaemic/reperfusion stress can influence the phosphorylation state of TJ proteins resulting in increased permeability,12 ,13 endotoxin leakage and the provoking of a systemic inflammatory response.14 These mechanisms may contribute to the high prevalence of gastrointestinal distress reported among endurance athletes, where 60–90% report symptoms, including diarrhoea, nausea, stomach problems, bloating and intestinal cramps.15–17 Moseley and Gisolfi18 introduced the heat and oxidative pathway leading to gut permeability and endotoxin leakage, and this pathway was further developed by Lambert 20098 ,19–21 and additional contributions from Hall22 ,23 However, the underlying molecular mechanisms were not discussed. Therefore, we will build upon the Moseley/Gisolfi model by discussing the mechanisms that regulate the phosphorylation of TJ proteins dependent on the type of exercise (short high intensity vs long duration), and the protective effects of intracellular heat shock proteins (HSP). In addition, medications (NSAIDS), or dietary supplements that increase (quercetin) or decrease (glutamine, bovine colostrum) intestinal permeability will be discussed.
TJ function and protein components
Intestinal epithelial TJ are multiprotein complexes that connect adjacent cells on the apical and lateral membranes forming an extracellular border around the cell (figure 1).3 The TJs serve as a selective barrier, and regulate bidirectional paracellular movement of ions, water and other nutrients while providing protection against leakage of luminal toxins into the circulation.3 ,24 Activation of Na+/glucose transporters in response to feeding increases TJ permeability allowing nutrient absorption.25–27 An increase in intestinal volume that leads to pressure greater than 4 cm H2O enhances paracellular permeability and greater fluid absorption.28 Conditions of intense physical stress, such as exercise, cause TJ dysfunction leading to enhanced permeability allowing translocation of luminal toxins into the blood stream.29–32
TJ integrity is regulated by the assembly of the extracellular loops of the transmembrane proteins occludin and claudins, whereupon aggregation of these proteins at the site of the TJ increases barrier resistance.33 The intracellular plaque proteins zona occludens (ZO-1, ZO-2 and ZO-3) and PDZ link both occludin and claudins to the actin cytoskeleton,34 ,35 which is the transmembrane protein that upon activation shortens or ‘contracts’ the epithelial cell.36 Shortening of cytoskeleton is regulated by the state of phosphorylation through binding of myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP).36–39 Similar to vascular smooth muscle contraction, MLCK phosphorylates the myosin light chain of the epithelial actomyosin protein causing shortening and opening of the TJ, while MLCP dephosphorylates the actomyosin protein leading to closure of the TJ junction.37 A severe stimulus such as hyperthermia or ischaemia will disrupt the interaction between the TJ proteins zona occluden, occludin and claudins. The actin cytoskeleton is connected to the TJ proteins via the zona occludens (ZO-1, ZO-2 and ZO-3), and when disruption occurs the overall effect is reduced actin cytoskeleton regulation.
Regulation of TJ proteins
Occludin and claudins (claudin-1, claudin-2 and claudin-3) are tetraspanning membrane proteins with two extracellular loops, and cytoplasmic N-terminal and C-terminal domains (figure 2).40–42 The extracellular components form a barrier with adjacent epithelial cells, and regulate paracellular permeability. The C-terminal domain is the main site for interaction with the zona-occludens and PDZ proteins, and is required for the assembly at the TJ.5 ,43
Initially, occludin was thought to be the primary protein responsible in forming the TJ, as overexpression resulted in greater TJ resistance.44 ,45 However, occludin knock-out mice show normal TJ resistance, and barrier formation.46 This has led to the conclusion that occludin is not vital for TJ formation, but has a regulatory role in TJ assembly. Occludin is required in the ZO-1 and actomyosin cytoskeleton interaction, and through signalling molecules mediates the maintenance of intact TJ complexes, and barrier function.24 ,47 ,48 The claudin proteins (claudin-1, claudin-2 and claudin-3) are considered to be the primary seal forming protein, and have the ability to polymerise into linear fibrils, which is in contrast to occludin.39 ,49 ,50 Overexpression of claudin results in greater TJ resistance and claudin knock-out mice die within 1 day of birth.50
Both occludin and claudin formation at the TJ are regulated through phosphorylation by several proteins, including different isoenzyme forms of protein kinase C (PKC),51 protein kinase A (PKA),52 ,53 tyrosine kinase,54 ,55 MAPK6 and several more (figure 3).6 Occludin phosphorylation by conventional PKC (cPKC) and tyrosine kinases has been shown to decrease TJ assembly51 ,54 ,55 while phosphorylation by novel PKC (nPKC) improves TJ resistance (figure 3).47 ,51 The mechanism is through regulating the interaction of occludin with ZO-1, which is required for TJ formation. Claudin phosphorylation by nPKC promotes fibril formation and TJ assembly 56 while PKA has opposite effects (figure 3).52 Similar to occludin, the phosphorylation state regulates the claudin and ZO-1, ZO-2 and ZO-3 interaction. Research on the interactions between claudin and occludin is limited, but claudin-1 has been shown to be bound to occludin during TJ assembly.57 ,58
Heat and long-duration exercise effect on TJ proteins
Long-duration exercise, or exercise in a hot environment, often results in an increase in core temperature above 39 s.59 To defend core temperature blood flow is diverted from the splanchnic and renal arteries, to the cutaneous vascular bed to increase body heat loss. As core temperature rises intestinal wall temperature also rises, and may be slightly greater than core temperature.7 The reduction in blood flow may be the reason for heightened intestinal wall temperature as heat is not being removed due to vasoconstriction of the splanchnic arteries. Hyperthermia (>40o) has been shown to damage intestinal epithelial cells causing cell sloughing, shrinking of the villi, oedema and massive bleeding.60 ,61 Intestinal permeability is increased in runners and cultured human intestinal epithelial cells at temperatures above 39 s.29 ,62 The increase in TJ permeability leads to the translocation of lipopolysaccharide (LPS) into the blood circulation, where it attaches to lymphocyte TLR4, and CD14 receptors, triggering the release of pro-inflammatory cytokines such as tumour necrosis factor α (TNF-α), IFN-α, IL-1β or IL-6.63 TNF-α has been shown to damage the Na/K pump on the basolateral membrane of intestinal epithelial cells, resulting in reduced water absorption, fluid secretion from the vasculature into the lumen and diarrhoea.64 Inflammatory cytokines have been positively correlated with nausea, vomiting, diarrhoea and abdominal cramping among endurance athletes during competition.16
Heat stress may increase TJ permeability through activation of protein kinases resulting in phosphorylation of TJ proteins thus decreasing the interaction of occludin and claudin with the zona-occludins (figure 4).62 ,65 Heat activates phosphorylation enzymes tyrosine kinase and cPKC in epithelial cells, causing a decrease in TJ resistance.10 ,43 ,57 In summary, heat-induced intestinal permeability during exercise may be mediated by TJ phosphorylation by several key protein kinases.
Phosphorylation of TJ proteins, and their disassembly may be a result of endotoxin leakage and activation of proinflammatory cytokines during heat stress. Physical damage to the intestinal epithelial cells under hyperthermia conditions may cause the increase in intestinal permeability, endotoxin leakage and the cascade of immune responses (figure 5). IFN-γ and TNF-α synthesis have been shown to mediate actin cytoskeleton contraction, and TJ opening through the activation of MLCK, and phosphorylation of MLC.38 ,66 Injection of TNF-α into the intestines of rats activates cPKC resulting in TJ breakdown, inhibition of the Na/K exchanger and diarrhoea.64 Whether the phosphorylation of TJ proteins, and the loss of barrier integrity is a direct, or indirect result of heat stress is not known.
Ischaemic stress and high-intensity exercise effect on TJ proteins
Intestinal ischaemia can occur in as little as 10 min of high-intensity exercise as measured by gastric tonometry.9 ,67 Van Wijk et al9 showed splanchnic hypoperfusion 20 min into a 60 min bout of cycling at 70% VO2max, and complete reperfusion took place within the first 10 min of recovery. Intestinal hypoperfusion causes a rapid breakdown of ATP to AMP activating hypoxanthine, and during the reperfusion cycle hypoxanthine is reduced to xanthine by the calcium-activated enzyme xanthine oxidase.12 The increase in calcium may be a result of calcium pump dysfunction during ischaemia.12 The xanthine oxidase reaction then releases hydrogen peroxide, a potent free radical, which causes tissue breakdown and disruption of TJ proteins.12 ,68 Hydrogen peroxide levels increase in response to heavy aerobic exercise when measured indirectly through catalase levels and the ratio of glutathione to oxidised glutathione,69 where both catalyse the breakdown of hydrogen peroxide.
Intestinal permeability is elevated among athletes during high-intensity exercise and permeability correlates with markers of oxidant damage.9 In addition, intestinal permeability increases during exercise among patients with peripheral vascular disease, which is a result of ischaemia in peripheral tissue, such as the intestinal tract.70 The mechanism may be through hydrogen peroxide-induced tyrosine phosphorylation of occludin by the tyrosine kinase c-Src causing translocation of occludin into the intracellular membrane and reducing the ZO-1 interaction.55 ,71 In addition, tyrosine kinase inhibition restores TJ resistance.72 Hypoxic exposure to epithelial cells has been shown to activate an atypical isoezyme of PKC leading to occludin phosphorylation, and loss of TJ integrity, but claudin levels were not affected.73 In summary, high-intensity exercise causes intestinal ischaemia increasing the production of hydrogen peroxide, which activates protein kinases that phosphorylate TJ proteins leading to hyperpermeability.
Hydrogen peroxide production from ischaemic stress has also been shown to activate epithelial cell nuclear factor κB (NF-κB), which controls the transcription of proinflammatory cytokines (TNF-α, IL-6, IFN-γ, IL-1β).68 The release of TNF-α and IL-6 from the rat ileum increase in response to ischaemia/reperfusion injury, where the levels of cytokine release is related to the magnitude of the ischaemic insult.74 Incubation of intestinal cells with TNF-α, IFN-γ and IL-1β causes reorganisation of occludin, claudin and ZO-1.66 ,75 Ye et al66 explained the TNF-α molecular mechanism that leads to the decrease in TJ stability, where NF-κB mediates TNF-α synthesis leading to upregulation of MLCK promotor activity, and TJ permeability. It is believed that MLCK then phosphorylates the MLC of the actin cytoskeleton leading to the opening of the TJ.38
Therefore, high-intensity exercise can cause the production of hydrogen peroxide, which may contribute to the cause of ischaemia reperfusion injury. Hydrogen peroxide disorganises the TJ barrier by two mechanisms, that include (1) phosphorylation of TJ proteins through the activation of protein kinases, and (2) upregulation of NF-κB transcription of proinflammatory cytokines. It is important to mention that the ischaemic pathway leading to gastrointestinal distress has been challenged. Wright et al76 showed that splanchnic blood flow was compromised among athletes after a long-distance triathlon, but did not differ between those who suffered gastrointestinal symptoms, and those who did not. Measurements taken during the event would better support this argument as hypoperfusion occurs rapidly67 and has been associated with gastrointestinal (GI) damage.9
HSP protection against TJ phosphorylation
HSP are intracellular molecular chaperones that assist in protein synthesis and cell maintenance.77 Increased levels of intracellular HSP provide protection to the cell under stressful conditions. Intracellular HSP levels in peripheral blood mononuclear cells (PBMCs) of athletes are upregulated after a competitive endurance event,78 and trained athletes show a greater HSP response to exercise stress.79 In addition, HSP levels increase in response to heat acclimatisation, which provides greater thermotolerance.2 HSP also provide protection against gastrointestinal disease 80 where an increase in heat shock factor 1 (HSF-1), the cytosolic regulator of HSP synthesis, and HSP70 reduce the levels of gastric lesions and irritable bowel symptoms.81 ,82
In the gastrointestinal tract, increasing the levels of HSP70 increases the expression of actin fibres and prevents the breakdown of the TJ protein occludin.83 Furthermore, HSF-1 mediates the increase in occludin expression during heat stress.84 HSP70 has also been shown to protect the actin cytoskeleton of intestinal cells from hydrogen peroxide and hypoxia induced damage.85
HSP70 protects the intestinal epithelial cells under hyperthermic conditions by preventing the activation of cPKC, and reducing the phosphorylation of both MLC of the actin cytoskeleton, and the occludin protein.13 HSP27 reduces tyrosine kinase activation during ischaemia reperfusion injury resulting in a stronger occludin and ZO-1 interaction and stabilisation of the TJ.86 HSP also prevent NF-κb translocation into the nucleus of intestinal epithelial cells reducing the synthesis of proinflammatory cytokines such as TNF-α.83 Overexpression of HSP70 protects epithelial cells from TNF-α insult, and maintains TJ stability.87 This suggests that HSP may protect the intestinal barrier during both heat and ischaemic stress through the decrease in TJ protein phosporlyation and prevention of NF-κB activation.
Agents that protect the gut barrier
Recently, there has been a surge of research into identifying dietary supplements to upregulate HSPs and protect TJ proteins from stressors such as inflammatory bowel disease and exercise. Traditional methods for increasing HSP levels are through chronic stress exposures such as heat, or altitude acclimation, or exercise. However, this may not be feasible for some populations. Marchbank et al29 demonstrated upregulation of HSP70 in human intestinal cells in response to bovine colostrum supplementation to the cell culture media, along with a reduction in gut permeability in exercising subjects after 14-days of supplementation. Bovine colostrum has also been shown to protect the gut barrier during ischaemia reperfusion stress88 and hyperthermia89 in rats. Conversely, Buckley et al90 showed an increase in exercise-induced gut permeability in runners after 8 weeks of bovine colostrum supplementation. The explanation for the conflicting results between the Marchbank and Buckley studies is unclear, but because colostrum facilitates small molecule transport prior to gut closure in infants, colostrom may enhance the cellular transport of the lactulose and rhamnose sugar probes.91 This may have occurred among subjects in the Buckley study because the supplementation period was 8 weeks as compared with the 14-day trial in the Marchbank study.
Polaprezinc is an antiulcer drug containing zinc and several amino acids, which has been used primarily in Japan. It has been found to increase HSP levels in rat intestines while reducing permeability during hydrogen peroxide injury.92 In addition, zinc supplementation in humans prevented a rise in gut permeability after NSAID ingestion. In an in vivo follow-up study, zinc prevented rat intestinal cell villus shortening and oedema.93 It is thought that zinc is critical for TJ assembly,94 but whether or not it upregulates HSP levels is unknown.
Glutamine is the most abundant amino acid in the human body, and provides protection to many tissues in situations of stress.95–97 It has been used as treatment for patients suffering from irritable bowel and Crohn's disease.98 ,99 Oral glutamine supplementation in rats has been shown to increase HSP 70 expression in the gut in response to heat stress.100 These rats also demonstrated lower gut permeability 6 and 24 h postheat exposure. In addition, glutamine enhances HSP70 expression in vitro,101 and reduces proinflammatory cytokine release. The mechanism may be through glutamine-mediated increase in cytosolic HSF-1 translocation into the nucleus leading to HSP transcription.100
There is growing evidence that supports probiotic therapy for improving gut function and enhancing the integrity of the intestinal TJ.102 Probiotic supplementation has been shown to prevent phosphorylation of occludin, increasing the ZO-1103 and actin cytoskeleton interaction104 in a rat experimental colitis model. However, research in humans is limited, and if probiotics provide protection under conditions of exercise-induced heat and ischaemic stress is not known. The high temperature that probiotic bacteria are cultured in may allow it to withstand the rise in core temperature, and provide protection during prolonged exercise.
Agents that increase intestinal permeability
An agent that increases gut permeability in response to stress should increase the susceptibility of gastrointestinal distress. Inhibition of the HSP response to stress increases the breakdown of occludin, ZO-1, and claudin along with reducing barrier integrity. Quercetin, which is an antioxidant, has been shown to block the rise in HSP70 levels in response to heat stress.2 In addition, 7 days of quercetin supplementation prevented heat acclimation by decreasing thermoregulatory responses, which was mediated through the decrease in HSP levels.2 Quercetin is also commonly used as an HSP inhibitor in cell culture models,62 where it inactivates HSF-1.105 Recently, other antioxidant treatments were found to be ineffective against irritable bowel disease in rats.106 Conversely, a steady antioxidant infusion slowed gut mucosal damage during ischaemic injury in a porcine model.107 An antioxidant defence mechanism is to increase the levels of protective antioxidative enzymes, however, the capacity of these enzymes may be a limitation, and could be the reason why a constant infusion, but not a bolus, provides gut protection.
Non-steroidal anti-inflammatory drugs (NSAID) have been shown to increase gut permeability in humans during exercise,108 and induce damage in the intestines of rats.93 Overexpression of HSPs protect intestinal cells from NSAID damage, but the effect of NSAIDs on HSP levels in intestinal cells is not known. In myocardial and nerve tissues, NSAIDS increase HSP70 levels.109 ,110 If NSAIDs increase the HSP expression and also increase intestinal permeability then the effects on the gut may not be mediated through the HSP response, but through another pathway.
Nutrients, such as wheat, lactose and a bolus of a concentrated glucose solution, are shown to damage the intestinal barrier.111–113 Vigorous exercise combined with poor nutrition habits may enhance gut permeability during exercise.113 A food allergy, such as wheat intolerance,111 triggers an immune response causing the release of proinflammatory cytokines leading to TJ breakdown.66 ,114 ,115 HSP70 expression has been shown to protect the integrity of the TJ barrier in children suffering from celiac disease.116 There is some evidence that the celiac gene is located near the HSP gene cluster, which may cause the silencing of HSP expression.117 Therefore, food allergies, namely gluten, may cause TJ dysfunction by affecting HSP expression making the cell more susceptible to damage. Research into gut permeability during exercise combined with various nutrients is limited, but very important, and should be a future focus for exercise physiologists.
Many athletes suffer gastrointestinal problems during training and competition that can affect exercise tolerance, and sport performance. The regulation of TJ permeability may be the critical mechanism that causes GI distress. Exercise that changes local intestinal temperature, blood flow and oxidant damage could regulate the phosphorylation of the TJ proteins and determine the level of interaction between occludin, claudin, zona occludens and the cytoskeleton. HSP protect the intestine from both heat and ischaemic stress, and agents that increase the HSP response may provide benefit to athletes who are susceptible to GI distress. Therefore, a dietary substance that upregulates HSPs may reduce GI symptoms and improve overall athletic performance.
Summary of new findings
Exercise that increases core temperature (prolonged), or high-intensity exercise regulate the phosphorylation state of intestinal tight junction proteins leading to disruption and increased permeability.
The increase in permeability allows paracellular movement of endotoxin into the blood stream causing a cascade of immune and inflammatory responses that lead to reduced fluid absorption, fluid secretion and diarrhea.
Several supplements including, glutamine, bovine colostrum and prolaprezinc may increase the heat shock protein response during exercise leading to greater tight junction protein stability and lower permeability.
Contributors Each author contributed to the writing and editing of the manuscript.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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