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6. Discussion

6.2. General discussion

6.2.2. Effect of exercise on gut leakage in CVD

When we started our study, there were limited reports whether an exercise intervention could affect circulating gut leakage markers. It had been shown that exercise increased the microbial diversity (137) and SCFA-producing taxa (188), both associated with a healthy, less leaky gut barrier. It had even been shown gut leakage markers LBP (152) and LPS and CD14 (153) were modified by exercise

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in diabetic subjects and obese adolescents, respectively. However, there were no reports on any effect of exercise on gut leakage markers in patients with CAD. We found no effect of 1 year of exercise intervention on any of the gut leakage markers (Paper I). This was somewhat surprising. As discussed in the paper, this could be due to the fact that the intervention group did in fact not improve their CRF, either because of the mode of training or because of relatively low exercise adherence. If so, an even stricter “per protocol” analysis or a different training programme focusing on increasing CRF could have given different results. However, analysis of individuals who improved their CRF did not show any effect either, although others have later found that increase in CRF improved the gut barrier integrity in patients with CAD assessed by I-FABP (189). Another possibility is of course that levels of gut leakage markers are not modifiable by exercise in comorbid patients with both T2DM and CAD. The conflicting results may be because our population might not readily be comparable to that of the other mentioned studies as our patients were older and with several comorbid complications accompanying their chronic disease. The diabetic subjects in the study by Motiani et.al (152) were newly diagnosed, and additionally patients with cardiac comorbidities were excluded. In the obese adolescent cohort (153), the intervention included a dietary regimen, making it impossible to detangle the exercise effect from any dietary effect.

Our combined CAD and T2DM patients were also heavily medicated with several medicines that might impose an effect on either the gut microbiota and the gut barrier itself, or that have anti-inflammatory effects with the potential of masking any subtle changes in gut related anti-inflammatory markers, as discussed in the Paper. It is therefore not possible to conclude on this matter on the basis of current investigations, and further research is needed to establish the role of exercise as a mode of treatment in the gut-heart axis.

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6.2.2.2. A single bout of strenuous exercise; short and long duration

It has been known for decades that strenuous exercise can induce endotoxemia, at least in athletes, and it has been reported that 2 hours duration seem to be the threshold for such exercise-induced gut leakage (147). We found in Paper II that even a short bout of strenuous exercise (median duration of 9 min 31 sec) increased both LPS, LBP and sCD14 significantly in all patients. Gut leakage induced by a significantly shorter exercise duration could be explained by our patient group who were older and untrained, whereas most research done on this subject previously has been conducted in healthy, endurance trained populations (147). Additionally, our subjects reached a close-to-maximal effort, and exercise intensity has proven to be the most important factor for inducing gut leakage (168).

I-FABP remained unchanged in the cohort of Paper II, probably due to an absence of intestinal ischemia and injury to enterocytes. We believe that the short duration of exercise, although intense, was not enough to elicit intestinal vasoconstriction large enough to cause ischemic damage. The observed leakage must therefore be either through paracellular or transcellular uptake. The transcellular uptake is most commonly associated with uptake of dietary fats, and as the patients were fasting, it is most likely that the leakage in this case was paracellular (71). It has been shown that exercise can influence the tight junctions regulating paracellular permeability (150). The

measurement of zonulin, a protein upregulated in states of increased paracellular permeability might have provided more insight into the mechanisms of leakage in this setting (190). Zonulin has been found to be elevated after 90 min of running in healthy individuals (191). However, zonulin was not available in our cohort. In contrast to in Paper II, we found I-FABP to be elevated after strenuous exercise of longer duration in Paper III. We believe this reflects exercise induced intestinal ischemia, as splanchnic hypoperfusion correlates with I-FABP concentrations (192). This is in line with what others have found on exercise of long duration and high intensity (193), although other modes of leakage cannot be ruled out and probably occur simultaneously.

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In Paper III, we also found a rise in sCD14 and LBP after strenuous exercise, further adding to the evidence of exercise induced gut leakage. This bout of exercise was considerably longer and exerted substantially more strain on the participants. However, they were endurance trained athletes, and thus not directly comparable to the cohort of patients with symptoms of CAD in Paper II. Either way, our results suggests that a single bout of exercise of a certain intensity is able to inflict gut leakage in both patients with symptoms of CAD and in healthy athletes. However, the mode of leakage may be different according to the duration and degree of intestinal ischemia.

Lastly, again in contrast to in Paper II, we found a significant fall in LPS the day after the race in Paper III, although the LPS-levels directly after the race were unaltered. As with the positive association between sCD14 and hours of weekly exercise discussed in the first section, we believe that this could be due to a high capacity for endotoxin clearance in healthy athletes that is not seen in the patients with symptoms of CAD. The athletes had high levels of LBP and sCD14 after the race, and even higher the day after the race, corresponding well to a high capacity for LPS sequestration. When additionally considering the probable higher levels of anti-LPS antibodies as a compensatory mechanism after repeated insults through frequent strenuous exercise in these athletes (145), as discussed in Paper III, we find it plausible that LPS translocation occurs in these athletes during strenuous exercise. We believe that our findings again highlight the different physiology of trained athletes compared to the average patient when it comes to exercise induced gut leakage.

In Paper III we also measured the gut leakage markers a week after the race in a subset of patients.

Both sCD14, LBP and I-FABP returned to baseline values. LPS, however, increased significantly from before to one week after the race. As discussed in the paper, we believe this to be due to the fall in LPS-binding molecules such as sCD14 and LBP, possibly combined with a change in diet in the individuals after the race that promote LPS uptake.

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6.2.2.3. Gut leakage and exercise as related to cardiac biomarkers and CAD In Paper II, we hypothesized that the observed exercise induced gut leakage would be more pronounced in patients who proved to have CAD, as increased levels of gut leakage markers have been associated independently with CAD (55, 60, 62), indicating a poorer gut barrier in these patients. This was not the case in this cohort. We found no difference in change or relative change between the three defined CAD groups. Neither was there any difference in the fasting baseline values of gut leakage markers prior to the EST in the groups of CAD, contrary to what others have found. As mentioned in section 6.1.4., the classification of the three groups of CAD may be

somewhat unnatural, and we therefore chose to compare patients with any degree of CAD to those without. However, this did not produce a different result. We believe that this is a strong signal that gut leakage induced by short-term strenuous exercise does not occur at different levels in those who have developed CAD and those who have not.

In Paper III we observed no relationship between exercise induced gut leakage and exercise induced increase in cTnT and NT-proBNP. We had hypothesised that the gut related inflammatory pathway might be in part responsible for the release of cardiac biomarkers during strenuous exercise, but our results indicate that these phenomena act independently of each other.

The explanation for both observations might again lie in the mode of leakage. As mentioned, leakage during exercise of short duration seems most likely to be paracellular due to disruption or alterations of the tight junctions. Gut leakage during longer, strenuous exercise may be both paracellular and due to injury to the single cell layer. In both cases LPS end up in the blood stream, allowing it to rapidly bind to LPS-binding molecules such as LBP, sCD14 and HDL, and to be transported via the portal system to the liver for detoxification. It has been shown that ~80% of infused LPS is cleared by the liver, and the HDL facilitates this clearance (74). Conversely, the elevated baseline levels of gut leakage markers observed in the larger cohorts mentioned above in relationship to cardiometabolic risk factors and chronic low-grade inflammation may be due to leakage associated to a high fat diet (194). In that case, LPS is translocated as part of chylomicrons, and transported via the lymph,

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thereby bypassing the first detoxification by the liver before it reaches the systemic circulation. LPS in the circulation is either cleared toward a detoxification process or toward inflammatory pathways depending on LPS clearance capacity. In other words, the distribution of LPS in the circulation seem to be crucial for their final pathway, and the subsequent handling of LPS may determine their immunostimulatory potential (194).

6.2.2.4. Associations with diet and n-3 PUFAs

As we have emphasised in this thesis, a healthy diet seems crucial to maintain a healthy gut microbial composition, and hence an intact gut barrier. In Paper IV, we therefore wanted to investigate the relationship between diet and gut leakage markers and corresponding genes expressed in SAT.

Surprisingly, we found no associations between diet and levels of gut leakage markers, neither circulating nor genetically expressed in SAT. As discussed in 6.1.5, a diet registration based on the patient’s memory may be biased, however, it is easy to access and reproducible. Especially intake of dietary fats seems essential in the question of chronic LPS-translocation across the gut barrier (71).

However, the SmartDiet form is actually evaluated to give a good estimate of dietary fat and fibre intake (164), and should therefore provide a good basis for evaluating the patients diets.

In patients taking n-3 PUFA supplements in Paper IV, we observed higher levels of circulating LPS. As n-3 PUFAs are thought to improve the gut microbiota and the intestinal barrier (65), we expected lower LPS-levels in these patients. However, although a growing body of literature supports a

positive effect of dietary n-3 PUFAs on gut microbiome and related LPS-translocation (195), studies in humans are scarce, and several important studies to date are performed in rodents (196). In healthy adults, some have found lower levels of LPS after intake of n-3 PUFAs (197), while others have found lower baseline levels, but elevated postprandial levels after n-3 PUFA consumption (198). Thus, the effects of dietary supplements of n-3 PUFAs on endotoxemia in humans remains unclear.

Lastly, when investigating gut-related inflammatory markers in SAT, we found TLR2 and CD14 to be downregulated in patients using n-3 PUFA supplements. n-3 PUFAs are thought to exert their effect

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mainly through the free fatty acid receptor 4, or through 3 PUFA-derived lipid mediators that are shown to ameliorate adipose tissue low-grade inflammation (199). It has, however, been shown that murine adipocytes downregulate their expression of TLR2, as well as production of inflammatory mediators, in response to n-3 PUFAs (200, 201), indicating the existence of other n-3 PUFA signalling pathways. It seems therefore likely that our observation reflects one of the anti-inflammatory effects of n-3 PUFAs (202).

6.2.3. Gut leakage in association to cardiometabolic disease states and risk