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.