An increased proportion of mitochondrial common DNA deletions is associated with mitochondria-related problems such as an overall decrease in energy supply and lactic acidosis. As such, it is an important factor correlated with the severity of mitochondrial disease symptoms (29, 22). It has been suggested that among the deletions observed in humans, mtDNA4977may be involved in the age-dependent decline of cell or tissue function (19, 18). In addition, mtDNA4977, which is considered a pathogenic mutation, may disrupt bioenergetic function by affecting the respiratory chain as previously reported (13, 14). It is believed that if the ratio of deletions to wild-type mtDNA exceeds a threshold for a cell or tissue, this leads to a decrease in mitochondrial ATP production and, as a result, a decrease in the ATP/ADP ratio of associated cells. In addition, experimental studies demonstrate that intestinal permeability is increased by depletion of ATP, suggesting that mitochondrial energy metabolism is vital for maintaining the intestinal barrier (30). Considering that mitochondrial dysfunction is implicated in an extensive list of various aging pathologies including intestinal barrier dysfunction (7, 31), this deletion may alter the metabolic properties of the intestines during aging and cause age-related changes in intestinal permeability.
Although a layer of epithelial cells forms the primary physical barrier between the lumen and mucosal tissues, intestinal organization formed by intestinal cell populations such as smooth muscle cell layers, enteric nervous system and blood vessels, as well intestinal epithelium, is essential for intestinal functions, including intestinal permeability regulation (32). Therefore, we performed mtDNA analyses of tissue homogenates to reflect the general status of intestinal cell populations. In this study, the abundance of mtDNA4834 deletion did not correlate with the rat age of sampled intestinal tissue. There were no correlations between the mutation load in intestinal tissue and intestinal permeability markers.
We also assessed the bioenergetic consequences of accumulating mtDNA4834 deletion levels by measuring the MMP and ATP synthesis rate in intestinal tissue mitochondria. Isolation of mitochondria through tissue homogenization and differential centrifugation is ideal for studying mitochondrial bioenergetics free from the influence of other cellular factors (e.g., cytoskeleton and endoplasmic reticulum) (33). In addition, use of isolated mitochondria enhances the ability to measure a fluorescence-based method for measuring mitochondrial membrane potential, because the isolation is allowed the mitochondria to diffuse in a small volume. Therefore, we also prefer to determine mitochondrial membrane potential in isolated mitochondria rather than whole cells. Our results showed that, in compared with young rats, the mitochondria of the intestinal tissue of old rats did not have a decrease in mitochondrial function. Several studies have speculated that the common deletion during human life may be due to tissue properties and that post-mitotic tissues accumulate mitochondrial damage faster than mitotically active tissues (3, 24). Therefore, a high turnover of the intestinal epithelium may not allow an age-dependent accumulation of this deletion. Furthermore, in an mtDNA-polymerase γ (PolgD257A) mutated mouse model, it has been shown that mtDNA mutations expand clonally, causing a mosaic pattern of the oxidative phosphorylation complex defects in aging tissues, including the small intestine. These defects have been shown to have cellular effects such as a decrease in cell proliferation, an ATP-consuming process, and an increase in the frequency of apoptosis in small intestine cells (34). Although the functional consequences of these effects have not yet been fully elucidated, Fox et al. (34) suggest that the transition from little functional impact to mitochondrial respiratory chain dysfunction may be slightly different in various cell types of intestinal tissue as well as slightly different in various tissues. Therefore, with our experimental design, we cannot be sure whether clonally expanded mitochondrial common deletion or other mtDNA mutations in rat intestine tissue may be responsible for direct or indirect functional outcomes in the context of age-related increased intestinal permeability.
This study compared mtDNA copy number in younger and older rats, as an increased mtDNA copy number has been suggested as a possible compensatory mechanism for aging-related increases in energy demand (35). However, we found no significant differences between the two groups, suggesting that mtDNA copy number status in aging intestinal tissue might not play a compensatory role for ATP tissue demands. On the other hand, glycolysis, which is the first step of the glucose oxidation process that occurs in the cytosol and produces only two ATP molecules and leads to pyruvate, is mainly involved in a wide range of biological processes. Experimental evidence demonstrates that the glycolytic pathway is much more important than the oxidative phosphorylation respiratory pathway in producing energy in intestinal epithelial cells (20, 21). Recently, researchers have shown that glycolytic genes in various tissues are down-regulated with aging, which in turn causes a reduction of glycolysis and glucose metabolism (36). Thus intestinal permeability with age may be affected by impaired glycolysis. To the best of our knowledge, this association is unclear. Therefore, we also examined whether increased intestinal permeability could be, at least in part, the result of age-related inactivation of glycolysis. Since it is pointed out that monitoring the lactate levels and L/P ratio in cells helps to detect imbalances in age-related energy metabolism between aerobic glycolysis and mitochondrial oxidation (37, 38), we also examined this parameter in intestinal cell homogenates. We detected a marked decrease in lactate levels and, accordingly, L/P ratio in the intestinal tissue samples of aged rats and found significant negative correlations between intestinal permeability parameters and tissue L/P ratios. As total ATP levels in the intestinal cell homogenates do not change in the aged tissue, our data imply the preferential use of aerobic glycolysis to generate ATP instead of mitochondrial oxidative phosphorylation in the intestinal tissue of young rats. Therefore, an imbalance in the age-related alteration of glycolytic and oxidative capacities of intestinal tissue contribute to increased permeability. At present, the basis for the change in the L/P ratio in the intestinal tissue of aged rats is unknown. However, in contrast to our results, Fleming et al., (20) found that glycolytic flux was not different for aged vs. young rats as it related to the proximal small intestine.
Numerous studies have shown the beneficial effects of microbiota-derived lactate as a significant factor in inducing enterocyte hyperproliferation and intestinal stem-cell-mediated epithelial development (39, 40). Although various factors such as changes in diet, medication, and lifestyles lead to changes in the intestinal microbiome, its diversity generally decreases with age (41, 42). This knowledge suggests that lactate levels measured in rat intestinal tissue are affected by bacteria-derived lactate, independent of the altered glycolytic and oxidative capacities of the intestinal tissue of aged animals. With our experimental design and the literature published to date, it is not possible to provide a sufficient explanation as to what causes the decrease in lactate levels of the intestinal tissue in aged rats and whether this decrease affects intestinal permeability. Therefore, further studies are required to understand the factors affecting intestinal permeability in association with aging.
Our study has several limitations. First, we only used tissue samples from Wistar rats having the same genetic background born and raised in the same facility under the same dietary pattern and environmental conditions. As known, lifestyle and environmental factors may influence the mutation rate in tissues and intestinal permeability. Thus, intestinal tissue from the rat model may not reflect the characteristics of human tissue. Second, the assessment of intestinal permeability was made through blood biomarkers as an indirect measurement of epithelial barrier integrity. Another limitation was that we did not separately analyze the different parts of the intestinal tract that have different functions. Therefore, these issues could have contributed to the results obtained.
In conclusion, our study indicates that the intestinal tissues of aged rats are not subject to the accumulation of mtDNA common deletion. The findings also demonstrated that the deletion load in the tissue did not correlate with the increased intestinal permeability of naturally aging rats. Considering a decrease in glycolytic flux in the intestinal tissue of aging rats in our study, it seems to be more likely that altered glycolytic capacity could be a link to increased intestinal permeability with age. This observation strengthens assertions that the balance between glycolysis and mitochondrial metabolism may play a critical role in intestinal barrier functions.