The importance of appropriate functioning of the gut for the maintenance of health has been generally accepted. However, to what extent aging-induced functional decline in the small intestine and colon have a causal effect on the impaired health status of the increasing number of elderly in our modern Western society is still an unresolved issue. A large number of studies have already zoomed in on different features of the aging intestine separately, but in this study we have examined functional, structural and molecular effects of aging in the mouse small and large intestine. The adult life in C57BL/6J mice is characterized by different phases. The young adult phase starts at 3 and ends at 6 months of age and reflects a human age ranging from 20 to 30 years. Mice are considered to be in their old phase when they are between 18 and 24 months of age representing a human age ranging from 56 until 69 years [30, 31]. By comparing energy intake between C57BL/6J mice at the age of 4- and 21-months, we found no reduction in either the efficiency of energy absorption of the control 10E% fat diet or of the 45E% high-fat diet. The fact that energy intake is not altered in the mice implies that capacity to digest and absorb macronutrients by the gastrointestinal tract does not decline with age. In line with this observation, our microarray data discloses no significantly altered pathways in macronutrient metabolism in aging mice up to the age of 21-months. A previous study has analyzed gene expression regulation in the duodenum and colon of aging rats . Similar to our findings this study also observed a higher number of differentially expressed genes in the colon compared to the small intestine. Furthermore, in agreement with our results they found that in the colon, aging caused mainly increased gene expression, while in the small intestine most of the differentially expressed genes were decreased. However, while in our study the number of altered genes involved in metabolic processes was very low, Lee and colleagues reported altered expression of genes involved in nutrient digestion and absorption and energy metabolism, although this observation was not further supported by additional functional analyses . Differences between aging in rats and mice, different segments of the intestine analyzed or the use of scrapings versus whole intestine are factors that might contribute to the discrepancy between the obtained results.
Although we did not observe marked changes in expression of genes involved in macronutrient metabolism, changes in expression levels of genes playing a role in micronutrient metabolism including Cubn were detected. Cubn is of crucial importance for the absorption of vitamin B12 by mediating the transfer of the intrinsic factor-vitamin B12 complex over the apical membrane of enterocytes . Our microarray data show decreased expression of Cubn in the proximal and middle part of the small intestine as well as in the colon of aging mice. Vitamin B12 deficiency occurs frequently in elderly humans [34, 35] and has been shown to play a causal role in a variety of diseases like depression and impaired cognition [36, 37], cardiovascular diseases  and osteoporosis [39, 40]. Vitamin B12 deficiency in the elderly is mainly due to reduced food intake and, predominantly, malabsorption. It is believed that insufficient release of vitamin B12 from food and impaired coupling to intrinsic factor (IF) due to chronic atrophic gastritis  represent the underlying mechanisms of the malabsorption. However, our results imply that reduced expression of Cubn might contribute to the impaired absorption of vitamin B12 in the intestine and that this decreased expression puts the elderly at risk of becoming vitamin B12 deficient.
While aging-induced molecular responses occurred predominantly in the colon, in contrast, the major changes in gene expression induced by a high-fat dietary challenge were found in the small intestine. Furthermore, while IPA analysis of the differentially expressed genes showed that aging did not cause major changes in metabolic pathways, genes involved in a variety of metabolic processes dominated the molecular response induced by the high-fat diet, confirming the results of our previous study . Interestingly, exposure to a high-fat diet for two weeks resulted in increased villi length in the distal part of the small intestine in young and old mice. One could speculate that the capacity of the proximal and middle part of the small intestine is insufficient to clear the intestinal content of all lipids and/or fatty acids in case of a diet containing 45E% of fat. As a consequence, the final part of the small intestine is exposed to increased amounts of fat affecting the tissue structure in this part of the small intestine. The fact that the dietary composition affects tissue morphology of the intestine implies that identical nutritional conditions are of crucial importance to analyze aging-induced changes in tissue morphology of the intestine. In line with some other studies we did not observe aging-induced morphological changes in any of the 4 segments of the intestine while other studies reported changes in tissue morphology of the intestine as a consequence of aging. The inconsistency in the published data might be explained by differences in species used in the different studies or the different age groups that were compared. However, part of the diversity in effects might also be caused by artifacts induced by differences in the dietary conditions [13, 41–44].
The increased expression of genes involved in immune response and inflammation observed in the colon of the 21-month-old mice points to an affected immune system in this part of the intestine of aging mice. This observation is in agreement with the fact that changes in the immune system are one of the hallmarks of the aging body. Immunosenescence is the functional decline of the adaptive immune system brought on by natural aging whereby protection against infection by pathogens and the effectiveness of vaccination decline [45, 46]. The second aging-induced change in the immune system is called inflammaging which is characterized by a low-grade chronic inflammation process that contributes to the pathogenesis of many age-related diseases [47–49]. A large variety of cells with a defense function are present especially in the lamina propria and the submucosa of the intestine accomplishing immune protection via the innate as well as by the adaptive immune response. Interestingly, our microarray and Q-PCR data clearly show that activity of both branches of the immune system is enhanced in response to aging exclusively in the colon but not in the small intestine of old mice. Expression levels of well-established pro-inflammatory cytokines like IFNγ, TNFα, IL6 and IL1β turned out to be extremely low in the colon of both old and young mice and below the threshold of our microarray analysis. These low expression levels are probably due to the fact that these cytokines are predominantly produced by immune cells in the mucosa which is a rather low percentage of cells in relation to all cells present in the intestinal tissue. Q-PCR analysis confirmed the very low basal expression levels of these pro-inflammatory cytokines, yet a weak but significant induction of IFNγ TNFα and IL-1β in the colon of aging mice was observed. This result suggests that low-grade inflammation might be present in the colon of the aging mice in our study, although it should be noted that no altered expression of a number of established inflammation markers like Toll-like receptors (TLRs), C-type lectin receptors (CLRs) and retinoic acid-inducible receptors (RLRs)  was detectable.
Changes in the gut microbiota in terms of composition and functionality during the process of aging have previously been reported [19, 20, 51] and it has been postulated that these changes might contribute to the development of immunosenescence and inflammaging [18, 52]. To establish whether the enhanced expression of genes playing a role in the immune system are due to modifications in the microbiota we measured the total number of all bacteria and of the two most prominent phyla colonizing the colon, Bacteriodetes and Firmicutes, in the luminal content of the colon. We did not observe aging-related changes. More advanced techniques like pyrosequencing are required to determine whether total number of bacteria and changes in the composition of the microbiota might play a causal role in the observed changed expression of immune-related genes in the colon of our aging mice. Although it is difficult to assess the physiological consequences of the enhanced expression of genes involved in inflammation and immune response, it seems most likely that this effect is important for the health status of the aging colon.
Interestingly, also colorectal cancer, which occurs with increased frequency in the elderly, has been related to an altered immune status of the colon [53–55]. The fact that we only found increased expression of immune-related genes in the colon, but not in the small intestine is intriguing since tumor formation is a very rare event in the small intestine and molecular evidence explaining the extreme difference in incidence of tumor formation in the two segments of the intestine is currently still lacking. In contrast to what has previously been found in the colon of aging rats , none of the well-established genetic markers of colorectal cancer (i.e. APC, KRAS, p53 etc.) or genes involved in cell cycle arrest and apoptosis  showed marked differential expression in the colon of the old mice in our study. This observation might be related to the fact that colorectal cancer is only a rare spontaneous event in C57BL/6J mice and that specific predisposing mutations  or carcinogenic agents [58, 59] are required to induce colorectal cancer in this mouse strain. Apart from genetic events epigenetic modifications have become important markers of tumor development, in particular of colorectal tumors . Hypomethylation of repetitive sequences is frequently observed in tumor tissue, but is also recognized to be an established feature of normal aging tissue . Interestingly, our results show differential demethylation between the various segments of the intestine of normal old mice. Since no significant demethylation in the proximal part of the small intestine is observed, it can be speculated that this part of the intestine is less prone to tumor development. Further research is required to prove this hypothesis and to establish whether changes in DNA methylation are responsible for the altered levels of gene expression observed in the colon of our aging mice in line with what previously has been reported .