Molecular effects of fermentation in the gut and its relevance for metabolism and satiety

Dietary fibres, the edible parts of plants that are resistant to digestion and absorption in the human small intestine, were shown to be important in the prevention of obesity and the metabolic syndrome. This association can partially be attributed to a fibre-induced increase in satiety. Dietary fibres can be fermented by bacteria, collectively referred to as the microbiota, in the large intestine (i.e. caecum and colon), resulting in the production of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate. Part of the effect of dietary fibres on satiety is thought to be mediated via the production of SCFAs. <strong>The objective of the research described in this thesis was to reveal the effects of fermentation in the large intestine using comprehensive approaches with focus on metabolism and satiety.</strong> The effect of 2-wk-consumption of resistant starch (RS), a dietary fibre highly fermentable by the gut microbiota, was studied in 2 pig experiments. In the first experiment, performed in adult female pigs, intestinal samples were collected from different areas of the gastrointestinal tract to measure luminal microbiota composition, luminal SCFA concentrations and the expression of host genes involved in SCFA uptake, SCFA signalling, and satiety regulation in mucosal tissue. In an additional study the effects of RS were investigated in young growing pigs fitted with a cannula in the proximal colon for repeated collection of tissue biopsies for whole-genome expression profiling and luminal content for measurement of SCFA concentrations and microbiota composition. To limit inter-individual variation, the RS diet was provided to the pigs in a 2 x 2 crossover design for 2 wk per diet. Furthermore, the behaviour of the pigs was monitored and the postprandial plasma response of satiety-related hormones and metabolites was measured at the end of each 2 wk period using repeated peripheral blood sampling via catheters. In order to determine the potential differences in post-prandial plasma protein profiles, minipigs were assigned to a control (C) diet or a diet containing the bulky fibre lignocellulose (LC), the viscous and fermentable fibre pectin (PEC) or RS for periods of 8 d in a 4 x 4 Latin square design. Portal and carotid blood samples were collected from catheters on d 8 of each treatment, both before and at several time points after an <em>ad libitum</em> morning meal. Male C57BL/6J mice were used to study the effect of background diet and SCFAs on colonic gene expression. Mice were fed a semi-synthetic low fat or high fat diet starting 2 wk before the treatment period. During treatment, mice received a rectal infusion of either an acetate, propionate, butyrate, or a control saline solution on 6 consecutive days, after which colon was collected to perform comprehensive gene expression profiling. RS enhanced satiety based on behavioural observations, as RS-fed pigs showed less feeder-directed and drinking behaviours than pigs fed a digestible starch (DS) diet. In both caecum and colon, differences in microbiota composition were observed between RS-fed pigs and DS-fed pigs. In the colon these included the induction of the healthy gut-associated butyrate-producing <em>Faecalibacterium prausnitzii</em>, whereas potentially pathogenic members of the <em>Gammaproteobacteria</em> were reduced in relative abundance in RS-fed pigs. Caecal and colonic SCFA concentrations were significantly higher in RS-fed pigs. Geneexpression profiling of the proximal colon revealed a shift upon RS consumption from the regulation of immune response towards lipid and energy metabolism. The nuclear receptor <em>PPARG</em> was identified as a potential key upstream regulator. At plasma level, SCFA concentrations were higher in RS-fed pigs throughout the day. Postprandial glucose, insulin and glucagon-like peptide 1 (GLP-1) responses were lower in RS-fed than in DS-fed pigs, whereas triglyceride levels were higher in RS-fed than in DS-fed pigs. In minipigs, plasma protein profiles were found to be most similar with C and LC consumption and with PEC and RS consumption, indicating that the consumption of diets with fermentable fibres results in a different plasma protein profile compared to a diet containing non-fermentable fibres or a diet without fibres. In mice we observed that dietary fat content had a major impact on colonic gene expression responses to SCFAs, especially after propionate treatment. Moreover, the diet- and SCFA-dependent gene expression changes pointed towards the modulation of several metabolic processes. Genes involved in oxidative phosphorylation, lipid catabolism, lipoprotein metabolism and cholesterol transport were suppressed by acetate and butyrate treatment, whereas propionate resulted in changes in fatty acid and sterol biosynthesis, and in amino acid and carbohydrate metabolism.Furthermore, SCFA infusion on the high fat diet background appeared to partially reverse the gene expression changes induced by high fat feeding without SCFA infusion. <strong>In conclusion, this thesis showed that the consumption of RS changed the microbiota composition in the colonic lumen, with a decrease in the abundance of potentially pathogenic bacteria</strong>and an increase in the abundance of SCFA-producing populations. Furthermore, <strong>colonic gene expression changes were observed after RS consumption and after colonic administration of SCFAs. With both treatments,</strong>among the <strong>changes in</strong>the transcriptional profile<strong>of the host were adaptations in</strong>metabolic processes, such as energy and lipid metabolism, and immune response.W<strong>e also showed that fat content in the background diet had a major impact on gene expression responses to SCFAs in colon</strong>. Overall, this thesis supports the implementation of fermentable dietary fibres into the human diet to improve colonic health and to reduce energy intake and body weight gain, which ultimately may prevent obesity and type 2 diabetes. Additional research is required to further elucidate the mechanisms via which fermentable dietary fibres can improve human health.