Good, Bad, or Risky? : intestinal permeability to protein: human and in vitro studies

The gut plays a pivotal role in human wellbeing by forming the body’s largest barrier between the external and internal environment. It has a major function in the digestion and absorption of nutrients and at the same time protects the body from the uptake of potentially harmful compounds. An increase in intestinal permeability could affect the uptake of larger fragments of food components such as partly undigested dietary proteins. Endurance exercise is a widely experienced physiological challenge known to increase intestinal permeability, but the subsequent effects on protein uptake are less studied. Furthermore, several underlying mechanisms for exercise-induced permeability have been hypothesized, but have not been substantiated. The aim of this thesis was to examine the effect of exercise intestinal permeability to dietary protein and peptides. Several in vivo proof-of-concept studies have been performed to study the effect of exercise on intestinal permeability towards small inert sugars versus small (casein-derived betacasomorphin-7) and large (peanut-derived allergen Ara h 6) dietary peptides. An in vitro model was developed to study one of the possible underlying mechanisms for an increase in intestinal permeability. Chapter 1 provides background on intestinal integrity and permeability and their role in digestion and absorption of dietary protein. Furthermore, the in vivo and in vitro models for intestinal permeability as applied in the next chapters are introduced. Additionally, the research aim and the outline of this thesis are presented. Chapters 2, 3, 4, and 5 describe in vivo findings on intestinal permeability. Chapters 2 and 3 show findings of the Protégé study; this study consisted of 12 well-trained healthy young men who consumed a casein protein solution in rest or directly after completing a strenuous dual exercise protocol. Chapter 2 shows the effect of the strenuous exercise on urinary peptide excretion and markers of intestinal function and recovery, and inflammation. The exercise protocol resulted in increased urinary excretion of the casein-derived peptide betacasomorphin-7, even though intestinal permeability as measured with inert sugars was less pronounced. The exercise protocol also resulted in major changes in post-prandial amino acid profiles in plasma. It was concluded that strenuous exercise could have an effect on the amount of food-derived (bioactive) peptides crossing the epithelial barrier. In chapter 3 the test-retest repeatability of the outcomes of this strenuous exercise intervention is studied, since the complete intervention was performed twice in two separate weeks. It was shown that the metabolic effect parameters, such as glucose, lactate, and energy expenditure showed low test-retest variation, while stress response parameters to the exercise, such as creatine kinase, fibroblast growth factor 21 and intestinal permeability as measured with a lactulose/rhamnose challenge test showed high test-retest variation. It was concluded that even in well-trained young men an adapted response can be seen in exercise-induced stress after only a single repetition of the exercise intervention. This finding has implications for the design of human studies aiming at evaluating physiological responses to exercise. In chapters 4 and 5, we focused on peanut protein instead of casein and compared to the Protégé study less trained individuals were included. These individuals reflect the general population level of training. Chapter 4 describes the PEANUTS Pilot study, which was performed to develop an ELISA method to detect the major peanut allergen Ara h 6 in serum after peanut consumption. This protein could not be detected in four out of ten tested individuals. It was shown with spiking experiments that high levels of Ara h 6-specific immunoglobulin G in the blood hampered the detection of this allergen. This may be a broader phenomenon in studies on the uptake of food allergens in the circulation, and may explain why variable levels of food allergen in serum have been reported in literature. In chapter 5 the developed ELISA detection method from chapter 4 is used in the PEANUTS Study. Potential participants were first screened for the ability to detect Ara h 6 in their serum by a spiking experiment. The PEANUTS study consisted of 10 untrained healthy young men and women who consumed a lactulose/rhamnose test solution and 100 grams of mildly-roasted unsalted peanuts. The first week consumption of the peanuts was followed by rest, the second week each of the participants had to cycle for 60 minutes at 70% of their maximal output directly after the consumption of the sugar solution and the peanuts. The endurance exercise significantly increased intestinal permeability (lactulose/rhamnose ratio) as well as the uptake of Ara h 6. The lactulose/rhamnose ratio and the levels of Ara h6 were strongly correlated. From this it was hypothesized that endurance exercise after food consumption leads to increased paracellular intestinal uptake of food proteins. However, increased permeability due to increased transcytosis or decreased cell integrity could also add to the observed increase in the protein uptake. Chapter 6 shows findings on the newly developed in vitro model for intestinal permeability. This chapter describes the development of an in vitro cell model mimicking to a higher extent the in vivo situation with regard to metabolic phenotype. The Caco-2 monolayers grown in galactose medium instead of glucose medium showed a more oxidative phenotype dependent on mitochondrial ATP production. Obstruction of this mitochondrial energy production resulted in decreased cellular ATP levels coinciding with increased monolayer permeability. Gene expression analysis of tight junction proteins was inconclusive, but pointed towards a defence mechanism of the Caco-2 cells during energy stress. It was concluded that mitochondrial functioning may be essential for maintaining a gut barrier function with high integrity. In chapter 7 the main findings of this thesis are discussed. Furthermore, several methodological considerations are made for future design of in vivo studies on intestinal permeability including an exercise challenge and in vitro studies on intestinal permeability with Caco-2 cells. Next, future implications of my research is placed in the context of the broader aim of the IPOP Customized Nutrition research program with regard to the health effects of novel proteins. Lastly, some general conclusions of my thesis research are drawn

Mitochondrial ATP Depletion Disrupts Caco-2 Monolayer Integrity and Internalizes Claudin 7

Objective:In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation.Methods: Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed.Results: Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins (TJP) 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation.Conclusions: Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the distribution of claudin 7

Endurance Exercise Increases Intestinal Uptake of the Peanut Allergen Ara h 6 after Peanut Consumption in Humans

Controlled studies on the effect of exercise on intestinal uptake of protein are scarce and underlying mechanisms largely unclear. We studied the uptake of the major allergen Ara h 6 following peanut consumption in an exercise model and compared this with changes in markers of intestinal permeability and integrity. Ten overnight-fasted healthy non-allergic men (n = 4) and women (n = 6) (23 ± 4 years) ingested 100 g of peanuts together with a lactulose/rhamnose (L/R) solution, followed by rest or by 60 min cycling at 70% of their maximal workload. Significantly higher, though variable, levels of Ara h 6 in serum were found during exercise compared to rest (Peak p = 0.03; area under the curve p = 0.006), with individual fold changes ranging from no increase to an increase of over 150-fold in the uptake of Ara h 6. Similarly, uptake of lactulose (2–18 fold change, p = 0.0009) and L/R ratios (0.4–7.9 fold change, p = 0.04) were significantly increased which indicates an increase in intestinal permeability. Intestinal permeability and uptake of Ara h 6 were strongly correlated (r = 0.77, p < 0.0001 for lactulose and Ara h 6). Endurance exercise after consumption may lead to increased paracellular intestinal uptake of food proteins

The effect of endurance exercise on intestinal integrity in well-trained healthy men

Exercise is one of the external factors associated with impairment of intestinal integrity, possibly leading to increased permeability and altered absorption. Here, we aimed to examine to what extent endurance exercise in the glycogen-depleted state can affect intestinal permeability toward small molecules and protein-derived peptides in relation to markers of intestinal function. Eleven well-trained male volunteers (27 ± 4 years) ingested 40 g of casein protein and a lactulose/rhamnose (L/R) solution after an overnight fast in resting conditions (control) and after completing a dual – glycogen depletion and endurance – exercise protocol (first protocol execution). The entire procedure was repeated 1 week later (second protocol execution). Intestinal permeability was measured as L/R ratio in 5 h urine and 1 h plasma. Five-hour urine excretion of betacasomorphin-7 (BCM7), postprandial plasma amino acid levels, plasma fatty acid binding protein 2 (FABP-2), serum pre-haptoglobin 2 (preHP2), plasma glucagon-like peptide 2 (GLP2), serum calprotectin, and dipeptidylpeptidase-4 (DPP4) activity were studied as markers for excretion, intestinal functioning and recovery, inflammation, and BCM7 breakdown activity, respectively. BCM7 levels in urine were increased following the dual exercise protocol, in the first as well as the second protocol execution, whereas 1 h-plasma L/R ratio was increased only following the first exercise protocol execution. FABP2, preHP2, and GLP2 were not changed after exercise, whereas calprotectin increased. Plasma citrulline levels following casein ingestion (iAUC) did not increase after exercise, as opposed to resting conditions. Endurance exercise in the glycogen depleted state resulted in a clear increase of BCM7 accumulation in urine, independent of DPP4 activity and intestinal permeability. Therefore, strenuous exercise could have an effect on the amount of food-derived bioactive peptides crossing the epithelial barrier. The health consequence of increased passage needs more in depth studies

Adaptation of exercise-induced stress in well-trained healthy young men

Strenuous exercise induces different stress-related physiological changes, potentially including changes in intestinal barrier function. In the Protégé Study (ISRCTN14236739; www.isrctn.com) we determined the test-retest repeatability in responses to exercise in well-trained individuals. Eleven well-trained males (27 ± 4 years old) completed an exercise protocol that consisted of intensive cycling intervals, followed by an overnight fast and an additional 90 min cycling phase at 50% Wmax the next morning. The day before (rest), and immediately after the exercise protocol (exercise) a lactulose/rhamnose solution was ingested. Markers of energy metabolism, lactulose/rhamnose ratio, several cytokines and potential stress-related markers were measured at rest and during exercise. In addition, untargeted urine metabolite profiles were obtained. The complete procedure (Test) was repeated one week later (Retest) to assess repeatability. Metabolic effect parameters with regard to energy metabolism and urine metabolomics were similar for both the Test and Retest period, underlining comparable exercise load. Following exercise, intestinal permeability (one hour plasma lactulose/rhamnose ratio), serum interleukin-6, interleukin-10, fibroblast growth factor-21, and muscle creatine kinase levels were only significantly increased compared to rest during the first test and not when the test was repeated. Responses to strenuous exercise in well-trained young men, as indicated by intestinal markers and myokines, show adaptation in Test-Retest outcome. This might be due to a carry-over effect of the defense mechanisms triggered during the Test. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise

Adaptation of exercise-induced stress in well-trained healthy young men

Strenuous exercise induces different stress-related physiological changes, potentially including changes in intestinal barrier function. In the Protégé Study (ISRCTN14236739; www.isrctn.com) we determined the test-retest repeatability in responses to exercise in well-trained individuals. Eleven well-trained males (27 ± 4 years old) completed an exercise protocol that consisted of intensive cycling intervals, followed by an overnight fast and an additional 90 min cycling phase at 50% Wmax the next morning. The day before (rest), and immediately after the exercise protocol (exercise) a lactulose/rhamnose solution was ingested. Markers of energy metabolism, lactulose/rhamnose ratio, several cytokines and potential stress-related markers were measured at rest and during exercise. In addition, untargeted urine metabolite profiles were obtained. The complete procedure (Test) was repeated one week later (Retest) to assess repeatability. Metabolic effect parameters with regard to energy metabolism and urine metabolomics were similar for both the Test and Retest period, underlining comparable exercise load. Following exercise, intestinal permeability (one hour plasma lactulose/rhamnose ratio), serum interleukin-6, interleukin-10, fibroblast growth factor-21, and muscle creatine kinase levels were only significantly increased compared to rest during the first test and not when the test was repeated. Responses to strenuous exercise in well-trained young men, as indicated by intestinal markers and myokines, show adaptation in Test-Retest outcome. This might be due to a carry-over effect of the defense mechanisms triggered during the Test. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise