Sensing of prebiotic carbohyddrates in the gut via taste receptors: role in the control of food intake.

The obesity rates continue to rise worldwide and are associated with adverse health problems, including increased risk of type 2 diabetes. Excessive weight gain is often considered to be the result of excessive food intake and/or insufficient physical activity. In addition, the food landscape has shifted dramatically over the past several decades and the increased consumption of soft drinks and other sugar-sweetened beverages is considered as a major contributor to the obesity epidemic. Not surprisingly, non-caloric sweeteners have increased in popularity over the years as a mean to facilitate weight loss by reducing the sugar content of meals without affecting its taste. Next to these non-caloric sweeteners, low-caloric prebiotic sweeteners (oligofructose; OFS) have been proposed as functional food ingredients that could improve lipid metabolism and body weight through beneficial effects ascribed to their fermentation products, the short-chain fatty acids (SCFAs). When dietary changes are insufficient, pharmacotherapy can be added, although the risks and modest nature of weight loss that can be achieved with these anti-obesity drugs highlights the need for new treatment strategies. The gastrointestinal tract is an obvious target for new anti-obesity treatment strategies as it coordinates the release of gut hormones, such as the ‘hunger hormone’ ghrelin and satiety hormones GLP-1 and PYY, to regulate energy uptake and utilization. Restoring postprandial gut hormone levels (ghrelin, GLP-1, PYY), known to be dysregulated in obesity, may play a role in the metabolic improvements after bariatric surgery such as Roux-en-Y gastric bypass (RYGB) surgery. RYGB surgery is still an invasive technique but mimicking the sustained and enhanced release of GLP-1 and PYY through combination therapy might be a valuable, non-invasive alternative for bariatric surgery. The magnitude of postprandial gut hormone release depends on the meal composition. Several taste receptors (sweet, umami, bitter, fatty acid) and the taste receptor coupled G-protein, α-gustducin, are not only present on taste buds of the tongue but also on entero-endocrine cells (EECs) and may tune gut hormone release according to the macronutrient composition of the meal. The physiological role of taste receptors on EECs has not been fully elucidated yet.

In this thesis, we aimed to unravel whether α-gustducin coupled sweet taste receptors (TAS1R2-TAS1R3) play a role in the sensing of carbohydrates and sweeteners by the ghrelin cell. In addition, we elucidated whether intragastric supplementation of artificial sweeteners (sucralose) or prebiotic sweeteners (OFS) can prevent the deleterious effects of a high-fat diet in mice by altering gut hormone release through gustducin-mediated taste receptor activation. As a last aim, we investigated the effect of nutrient rerouting during RYGB surgery on the gustducin-mediated signaling pathways that contribute to the metabolic improvements and physiological adaptations along the gut after RYGB surgery.

In the first part of this thesis we investigated if α-gustducin mediated sweet taste receptor signaling is involved in the sensing of sweeteners by the ghrelin cell in three different experimental models (a ghrelinoma cell line, ex vivo intestinal segments, in vivo experiments).

The carbohydrate D-glucose and prebiotic sweetener OFS decreased ghrelin release from a gastric ghrelinoma cell line at concentrations physiological to the postprandial luminal fluid. In contrast, the artificial sweetener sucralose increased ghrelin release in vitro at a supraphysiological (200mM) concentration. Furthermore, by using pharmacological inhibitors we showed that neither sweet taste receptor activation, nor glucose transport (SGLT-1, GLUT family) played a role in the effect of D-glucose, OFS or sucralose on ghrelin release from the ghrelinoma cell line. Ghrelin release from gastric (only containing the TAS1R3 subunit) and jejunal (containing the TAS1R2 and TAS1R3 subunit) segments from WT and α-gust-/-mice, mimicked the in vitro effects of the sweeteners in the ghrelinoma cell line to a similar extent in both genotypes. These findings indicate that the effect of D-glucose, OFS and sucralose on ghrelin release is neither α-gustducin nor region-dependent and thus does not involve the α-gustducin coupled TAS1R2-TAS1R3 heterodimer. Intragastric, but not intravenous administration of D-glucose decreased plasma octanoyl ghrelin levels in WT and α-gust-/- mice. These results indicate that the sensing of D-glucose is polarized and occurs via the luminal side of the X/A cell. In contrast, neither OFS nor sucralose at “equisweet” concentrations affected octanoyl ghrelin release after an intragastric administration in WT or α-gust-/- mice. In conclusion, our findings indicate that α-gustducin-mediated sweet taste receptor signaling does not play a functional role in the effect of sweeteners on ghrelin release. In contrast to the in vitro findings, only acute intragastric administration of D-glucose but not OFS or sucralose affected ghrelin release.

In the second part of this thesis we studied whether daily intragastric administration of equisweet concentrations of an artificial sweetener (sucralose) or a prebiotic sweetener (OFS) for 8 weeks can prevent high-fat diet induced body weight gain, glucose intolerance and impairment of gut permeability, via activation of taste receptors coupled to α-gustducin, using WT and α-gust-/- mice.

Sucralose administration did not modulate gut hormone release nor did it prevent body weight gain or glucose intolerance. Instead we provided evidence that OFS (300 mg/day) administration decreased HFD-induced body weight gain with about 20% without improving glucose homeostasis. This effect was not accompanied by a reduced food intake. Furthermore, OFS induced a similar but delayed decrease in body weight gain in α-gust-/- mice, indicating that the α-gustducin mediated signaling pathway did not play a major role in this effect. OFS administration did not affect plasma levels of ‘the hunger hormone’ ghrelin and ‘satiety hormone’ PYY, but decreased plasma levels of ‘the satiety hormone’ GLP-1 in WT mice. These changes in gut hormone levels cannot explain the beneficial effects on body weight. Neither OFS, nor sucralose administration altered the mRNA expression levels of the TAS1R2 or TAS1R3 subunit of the sweet taste receptor in the gastro-intestinal tract. However, OFS supplementation decreased cecal acetate and butyrate levels, downregulated colonic short chain fatty acid receptor (FFAR2/3) mRNA levels and upregulated FFAR2 in peripheral adipose tissue. These findings suggest that, not sweet taste receptor activation, but enhanced uptake of SCFAs produced by the fermentation of OFS interacting with FFAR2 in peripheral adipose tissue may reduce adipogenesis and lead to the decrease (60%) in fat mass. Moreover, OFS improved the increased colonic permeability which results in metabolic complications in obesity, independent from taste receptors coupled to α-gustducin. In conclusion, this study provided evidence that despite the controversy in the field, artificial sweeteners are metabolically inert. Furthermore, neither OFS nor sucralose affected TAS1R2 or TAS1R3 mRNA levels, while OFS supplementation altered FFAR2/3 expression levels in the gastrointestinal tract and on adipose tissue. Therefore, not sucralose but OFS and especially the produced SCFAs, are interesting metabolites that could beneficially affect body weight gain.

In the third part we studied the role of gustducin-mediated signaling in the metabolic improvements and intestinal adaptations along the gut after RYGB surgery in obese WT and α-gust-/- mice.

We showed that RYGB surgery decreased body weight in WT and a-gust-/- mice. Furthermore, pair-feeding to the RYGB group induced similar blood glucose and plasma insulin profiles during an oral glucose tolerance test compared to RYGB surgery, indicating that the reduced food intake after RYGB surgery was responsible for the improved glucose homeostasis. Moreover, a-gust-/- mice were partially protected from the diabetogenic properties of a western style diet, highlighting the importance of the gustatory signaling pathway in glucose homeostasis. After RYGB surgery plasma GLP1 levels were increased in both genotypes, plasma PYY levels were increased in α-gust-/-mice and plasma octanoyl ghrelin levels were not affected. The mechanism behind the postsurgical changes in gut hormone levels seemed to differ between WT and a-gust-/- mice.

In WT mice, nutrients act via α-gustducin to increase L-cell differentiation (in the Roux limb which comes in contact with more undigested nutrients) and L-cell number (Roux limb and colon) after RYGB surgery, in a region-dependent manner. However, this nutrient rerouting did not alter the mRNA expression levels of nutrient sensors in the Roux Limb, nor did it alter bacterial fermentation in the caecum of WT mice. In contrast, a-gust-/- mice did not display an altered L-cell number or L-cell differentiation in the Roux limb or colon. However, a-gust-/- mice did show increased mRNA expression levels of the glucose transporters (SGLT1 and GLUT2) and the protein sensor (LPAR5) in the Roux limb. Furthermore, RYGB surgery changed bacterial fermentation in the caecum of a-gust-/- mice, which showed increased butyrate and propionate levels compared to WT mice. This resulted in decreased colonic FFAR2/3 mRNA levels in a-gust-/- mice. These results suggest that a changed L-cell number and differentiation will be responsible for the increased plasma GLP-1 levels in WT mice. In contrast, alterations in nutrient signaling in the foregut, and altered bacterial fermentation and short-chain fatty acid sensing in the distal gut of a-gust-/- mice could explain the increased plasma GLP-1 and PYY levels in this genotype.

Finally, signaling via α-gustducin plays a role in the increased ion transport of the foregut but not in the improvement in colonic barrier function.

To summarize, our findings do not indicate a major contribution of gustducin-mediated signaling in the metabolic effects of RYGB. Nevertheless, RYGB activated several regulatory systems in which the gustducin mediated signaling pathway plays a role. This study highlights that nutrients cannot only serve as fuel but may regulate a number of physiological processes after RYGB surgery such as tuning of gut hormone release which is the result of multifaceted intestinal adaptations along the gut. Importantly, these gut hormones could contribute to the observed metabolic improvements after RYGB surgery.

In conclusion our studies suggest that the canonical sweet taste receptor signaling pathway does not seem to play a major role in the sensing of carbohydrates or sweeteners by the ghrelin cell. Furthermore, in vivo targeting of sweet taste receptors on entero-endocrine cells by supplementing artificial sweeteners directly into the stomach for several weeks does not help to prevent the development of obesity nor type 2 diabetes. In contrast, prebiotic sweeteners seem to be more promising since their fermentation products target SCFA receptors. On a long term basis their effect on short-chain fatty acid induced gut hormone release does not seem to play a major role in the development of obesity but their capacity to alter expression levels of SCFA receptors on adipose tissue might be relevant. Finally, gustducin-mediated nutrient sensing does not play a role in the effect of RYGB surgery on the energy-and glucose homeostasis, but altered gut hormone levels might.