Bases de presentación y consolidación de las cuentas

Compared with the transcriptional profiles induced by SCFA, profiles induced by dietary fibers were different to SCFA infusions (mainly with respect to metabolic, catabolic gene sets) (chapter 2, 3). Several factors might play a role in this discrepancy. For example, it has been shown that individual SCFA can have opposite effects in metabolic pathways [20], [21]. Hence, the studied individual SCFA might act differently when applied in combination, as it is the case in more physiological conditions. Another point that can be taken into account for explaining differences between response to SCFA infusion and dietary fibers are fluxes of SCFA vs. snapshot concentrations. By measuring SCFA concentrations, production and uptake rates of SCFA remain unknown. It has been shown that actual concentrations of SCFA do less correlate with parameters of metabolic syndrome after feeding guar gum to mice than do flux measurements of SCFA [22]. Hence, different fibers can have similar SCFA concentrations but different uptake rates, which might lead to different effects.

Role of concentration dependent effect of butyrate

Butyrate in different concentrations induced different sets of genes (chapter 5; 1mM vs. 8mM) which points to other mechanism being involved at different concentrations. This is further supported by the fact that butyrate regulated also many genes Pparγ independently (243 genes regulated in both Pparγ WT and KO mice, chapter 6). It was shown that butyrate induced different mechanisms for concentrations > 5mM where HDAC inhibition takes place which is different at lower concentrations [23]. It is suggested that the different concentrations may play a role for cell turnover along the lumen-to-crypt axis of the colon [24]. At lower concentrations, which can be found at the crypt cells, butyrate induces proliferation of cells via usage as energy substrate and histone acetylation via acetyl-CoA. Whereas at the luminal site butyrate concentrations are higher, exceeding metabolic capacity of the cells and hence inducing apoptosis of cells via HDAC (histone deacetylase) inhibition. Hence, detailed mechanism about the different concentrations not only along the intestine, in small or large intestine, proximal or distal, but also along the crypt-villus axis should be studied in more detail. Experiments on the crypt or surface cells can reveal more evidence for the expected role of butyrate and also PPAR along the crypt-villus axis. Transcriptome analysis of crypt and surface cells isolated by laser capture microdissection can be used to dissect the molecular mechanism on the single cell level along crypt-villus axis [25].

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might explain differences in colonic gene expression response.

Intestinal metabolites

In general, intestinal metabolites modulated or produced by microbiota include bile acid derivatives, indole, choline metabolites, phenolic, benzoyl, phenyl derivatives, vitamins, polyamines, lipids [26]. During fermentation of carbohydrates mainly SCFA are formed, but also formate, hydrogen sulphide, ethanol, succinate and lactate. In chapter 6, besides SCFA, another metabolite (succinate) was found to be important for driving the intestinal metabolome after feeding mice inulin. Succinate is a di-carboxylic acid and an intermediate product of fermentation which is converted into propionate by luminal bacteria [27]. Metabolites such as succinate are rather used in the metabolic network of the gut bacteria to finally produce SCFA, propionate [28]. Succinate can be taken up by large intestinal mucosa [29]. Little is known about effects on gene expression in intestinal mucosa by succinate. Succinate can be metabolized in the TCA cycle. Succinate was further shown to be a signaling molecule in inflammation [30], [31] and linking TCA cycle dysfunction to oncogenesis [32]. In vitro studies showed that succinate can inhibit growth and proliferation of colon cancer cells and that some polyphenols can increase the cecal succinate levels in rats fed HFD [33]. Other products derived from carbohydrate degradation are less well studied with respect to host metabolism.

Alternatives

What would be alternatives to study impact of SCFA in dietary fiber mediated effects? Germ free animal models can be applied to study the direct effects of dietary fibers on colonic mucosa without influences of SCFA, which are not produced in germ free mice. This model, however, has several marked disadvantages such as large differences between germ free and conventional mice with respect to immune cells, epithelial cell turnover, mucosal gene expression and metabolic functions [34] making it difficult to compare to physiological conditions. Also short-term antibiotics use to diminish the number of bacteria could be applied to study effects of dietary fiber without SCFA production [35]. A shift in the balance of microbial ecosystem [36], however, might influence the response to dietary fibers in the colonic epithelium. Furthermore, to test the causal effect of SCFA in dietary fiber induced gene expression profile one could use SCFA transporter knockout models. SCFA are taken up via transport proteins (monocarboxylic acid transporters), mainly by MCT1-4. Also sodium-coupled transporter SMCT1 has been found to transport SCFA. Fluxes of SCFA correlate with mRNA level of these transporters [22] implicating the importance of these transporters in uptake and subsequent metabolism of SCFA. Hence, blocking uptake of SCFA via knock out of these transporters might help to better understand the impact of intracellular SCFA metabolism. Next to SCFA, the monocarbox- ylate transporter also transport other metabolites (lactate, pyruvate, nicotinate and ketone

bodies) [37]. Knock down of MCT1 and MCT4 by siRNA has been seen to lower both lactate and butyrate uptake into rat intestinal cells [35]. From these metabolites, lactate and pyruvate are formed as intermediary products by bacteria during carbohydrate fermentation, which are utilized by other bacteria to form SCFA. Hence, uptake of these metabolites by these transporters might play only a minor role for host response to fermentation. Besides uptake via transporters, however, diffusion of the protonated form of SCFA has also been suggested [38] and the primary transporter for butyrate on the apical site has not been unequivocally identified yet. Nevertheless, for answering the question in as how much SCFA are mediating the colonic response to fermentation of dietary fiber, Smct1 knock out might be most appealing since in chapter 6 we showed that mRNA level were significantly increased by dietary fibers in Pparγ dependent manner and might hence be important for mediating effects of dietary fiber.