This experiment has shown how three very different soluble fermentable dietary fibres, beta-glucan, FOS and pectin, all similarly decreased voluntary food intake, weight gain and adiposity in normal healthy young adult male rats over a 4-week period, and the associated increased plasma concentrations of PYY and GLP-1 were strongly indicative of increased satiety. While these data demonstrate an animal model of fibre-induced satiety, the dietary inclusion rate (10% w/w, or 6.6 mg/kJ) was approximately twice the US recommended daily fibre intake of 3.3 mg/kJ for men[33, 34] and further work is required to translate the findings to humans.
Whereas 10% w/w insoluble fibre cellulose had no effect on food intake compared with the control group, the same inclusion rate of the three soluble dietary fibres each had similar effects, decreasing cumulative intake by 10-19% over 4 weeks and daily intakes by ~12% after an initial week of adjustment. Although the immediate decreases in intake following introduction of the high fibre diets may have reflected some initial unfamiliarity and low palatability to the rats, the consistently lower daily intakes thereafter once they had become accustomed to the diets were more likely indicative of increased satiety since they were associated with increased circulating concentrations of two important satiety hormones. The three soluble dietary fibres specifically increased concentrations of total GLP-1 and PYY in plasma samples taken in the early part of the light phase at the end of the trial, indicative of raised tonic secretion of these hormones, and the magnitude of the increases were similar to those associated with increased satiety in other rat models[16, 35]. Predictably, changes in active GLP-1 were not detected since it only has a 1-2 minute half-life and samples were not taken immediately after feeding episodes; nonetheless, measurement of total GLP-1 includes both the intact hormone and its primary metabolite and thereby provides an accurate indication of overall GLP-1 secretion. Tonic plasma concentrations of ghrelin and CCK were unchanged. This was perhaps not surprising since ghrelin is secreted in the stomach and CCK is produced by I-cells that are located mainly in the duodenum and jejunum whereas the presence of indigestible dietary fibre in the gut is most likely to be detected nearer to where it is fermented in the large intestine. In line with this argument, PYY and GLP-1 are both secreted from L-cells situated mainly in the distal small intestine and proximal large intestine. Our data are consistent with findings in rats fed dietary resistant starch where total GLP-1 and PYY were up-regulated in a sustained day-long manner through fermentation. It is inferred that significant satiety-inducing effects of dietary fibre only manifest themselves after several days of consistently increased fibre intake and it is tempting to speculate that this reflects a minimum exposure time for the underlying chronic changes in the gut environment and in tonic gut satiety hormone secretion to develop.
The reductions in food intake on the three diets containing soluble fermentable fibre were associated with significant reductions in body weight gain and it is noteworthy that this was attributable to the arrested accumulation of fat with lean tissue growth maintained. Both body weight gain and fat mass gain were closely correlated with the cumulative food intake (Figure 2). These findings add important total body composition data to existing reports of dietary oat beta-glucan decreasing epididymal fat pad weights in growing rats and of dietary FOS decreasing fat pad mass in rats[10, 13] and lend support to the notion that fermentable dietary fibre has potential as a tool for body weight and body fat regulation through its effects on satiety. While differences in eating behaviour, physical activity and heat expenditure (from increased fermentation) may also have contributed to the observed differences between the soluble fibre-fed rats and controls, the overall outcome was a similar decrease in food intake, body weight and body fat in all three soluble fibre-fed groups.
Soluble dietary fibres may influence satiety by virtue of their physicochemical properties, for example those with high viscosity such as pectin and beta-glucan tend to slow the rate of gastric emptying and overall gut transit time, which can lead to reduced intakes[10, 14, 15]. However the present intake responses were also shown with FOS, which has negligible viscosity. The common feature of the three fibres studied herein was their fermentability in the large intestine, resulting in greatly increased total concentrations of fermentation products in the caecum and colon contents. The highest concentration was that of succinate, whereas dietary studies in the literature appear rarely to report succinate concentrations, concentrating rather on the main three SCFAs. Succinate is produced by intestinal bacterial species of the Bacteroides and Clostridium genera, numbers of Bacteroides have been shown to increase in rats given dietary inulin and FOS and increased caecal succinate concentrations of comparable magnitude to those reported herein have previously been reported in rats given 6% FOS or 5% pectin. It appears from these latter studies and from the present one that the production of large amounts of succinate is favoured when rats are given a rapidly soluble and fermentable fibre source in a purified diet and when the protein source is a highly digestible one such as casein; this is thought to be because a lack of nitrogen relative to the carbohydrate availability in the large intestine promotes high succinate concentrations. Moreover, succinate is absorbed by the intestinal mucosa more slowly than other SCFAs, which would lead to its relative accumulation. However, it is unlikely that the increased succinate per se elicited the satiety response in our model since succinate concentrations were lower in PECT than GLUC or FOS groups, yet the satiety response was similar. Furthermore, although increased luminal succinate was associated with increased PYY and GLP-1 secretion in the fermentable fibre-fed groups, there was no evidence for increased succinate signalling to the gut enteroendocrine cells via its receptor sucnr1. Indeed, the only increase in sucnr1 gene expression was unexpectedly seen in the CELL group, in the absence of elevated luminal succinate and without changes in PYY and GLP-1 secretion. It is unknown whether succinate activates the SCFA receptors, ffar2 and ffar3.
After succinate, in terms of relative concentrations, acetate was the dominant fermentation product in all three fermentable fibre groups, but whereas concentrations of propionate were greater than butyrate in PECT and GLUC groups, the reverse was observed in the FOS group. The differences in fermentation products between the soluble fibres may reflect different patterns of fermentation and/or different rates of fermentation and turnover, which the present measurements at a single time point would not have detected. Nonetheless, given the similar levels of satiety observed in all groups, it is hard to attribute relevance of these SCFA differences to signalling satiety.
It was perhaps surprising that there were no increases in concentrations of the main three SCFAs in caecum and colon contents of rats fed the fermentable fibres compared with controls, and indeed concentrations were unexpectedly lower for acetate in FOS and GLUC groups and for propionate in the FOS group. There may be a number of reasons for this, apart from the overwhelming dominance of the succinate: for example, measuring concentrations at a single time point would have given no indication of biologically significant changes in rates of turnover. Nonetheless, the volume of contents and hence the total pool of SCFAs would have been increased in the fermentable fibre groups because the large intestine was visibly enlarged. This observation was supported by measurements of increased caecum and colon weights in rats given 10% PECT in a separate experiment (Adam et al., unpublished results) and is in agreement with published data on rats fed the dietary fibre inulin. Although the caecum is the primary site for dietary fibre fermentation, SCFA concentrations and group differences were similar in colon and caecum contents. There was also visible leakage of caecal contents back into the distal ileum, indicating that enteroendocrine L-cells in both the distal ileum and proximal colon would have been exposed to SCFAs, from where satiety responses may have been elicited, and a recent report indicates that L-cell numbers are fairly evenly distributed along the rat jejunum, ileum and colon.
Whereas a recent review concludes overall that SCFAs may not play a role in appetite regulation, in vitro evidence shows that they can trigger GLP-1 release from cultured colonic L-cells via the SCFA receptors ffar2 and ffar3 and stimulate GLP-1 (proglucagon) mRNA expression in cultured enteroendocrine STC-1 cells. Some in vivo evidence indicates that oral administration of butyrate or propionate decreases food intake and butyrate stimulates GLP-1 and PYY secretion in mice, but these were ffar3-deficient animals indicating that ffar3 is not involved in SCFA stimulation of these gut satiety hormones. The present data are consistent with this since we found no evidence for increased ffar3 gene expression and indeed there was even a decrease in expression in the proximal colon of FOS and PECT groups (Table 4). Similarly we found little evidence for increased gene expression for ffar2, the one exception being an increase in the distal ileum of FOS-fed rats. The absence of increased gene expression found here for PYY and GLP-1 in distal ileum and proximal colon, despite the greatly increased plasma concentrations of these hormones, was another apparently anomalous finding and gene expression for both was even decreased in the PECT group. Since PYY and GLP-1 are secreted from intestinal L-cells, the increased plasma concentrations must reflect either increased secretory activity per cell or increased number of L-cells. The apparent anomalies are most likely attributable to an overall increase in L-cell number along the enlarged intestines, which was not detectable here since the real time PCR method measures expression per mg tissue. The same PECT diet given for 4 weeks to adult male rats in a separate experiment increased the weights and lengths of both colon and small intestine (Adam et al., unpublished results). This is not without precedent since a doubling of the L-cell population in the rat intestinal mucosa occurs with no change in L-cell density due to general gut hypertrophy both after Roux-en-Y gastric bypass and in the Zucker Diabetic Fatty rat model[43, 46]. These authors also report increased plasma GLP-1 concentrations in their models, associated with the increased L-cell number and overall increased proglucagon (GLP-1) gene expression (by in situ hybridisation) without any increase in gene expression per cell. Other reports of increased L-cell differentiation and number in the proximal colon of rats fed fermentable indigestible carbohydrate lend support to this argument[13, 23]. Altogether the present data are consistent with increased circulating PYY and GLP-1 concentrations arising from overall increased numbers of L cells along the ileum and colon exposed to increased amounts of fermentation products, but further mechanistic investigations need to acknowledge the potential influence of gut hypertrophy in this model.