The present invention relates to a compound for use in the regulation of saccharide absorption via the gastrointestinal tract. The present invention further relates to a composition comprising the compound according to the present invention.

Absorption of monosaccharides in the small intestine is pivotal for caloric intake of mammalians and adjusted in accordance with food supply, food composition, and energy demand in diverse physiological and pathophysiological situations. In respect to caloric intake, d-glucose, d-galactose, and d-fructose are the most relevant monosaccharides. For absorption, monosaccharides must cross a layer of epithelial cells that are connected by tight junctions which do not allow permeation of monosaccharides in physiological concentrations. Because monosaccharides are hydrophilic, they cannot permeate cell membranes passively. Hence, for absorption of d-glucose, d-galactose, and d-fructose, transporters in the luminal brush border membrane (BBM) and basolateral membrane (BLM) of small intestinal epithelial cells (lECs) are required. The functions and membrane locations of transporters for d- glucose, d-galactose, and/or d-fructose expressed in the small intestine are well described in literature (see, for example: Koepsell H. Glucose transporters in the small intestine in health and disease. Pflugers Arch. 2020;472(9):1207-1248).

More than 650 million people worldwide have been diagnosed with obesity. Consumption of food high in sugars is one of the main factors contributing to obesity. Recent studies indicate that intestinal glucose absorption is significantly elevated in a cohort of obese patients, which elevated absorption is associated with increased expression of sodium-dependent glucose cotransporter 1 (SGLT1) found in the intestinal mucosa of the small intestine and glucose transport 2 (GLLIT2) found in the cellular membranes of the liver (see, for example: Hasan, Nesrin M., et al. "Intestinal stem cell-derived enteroids from morbidly obese patients preserve obesity-related phenotypes: Elevated glucose absorption and gluconeogenesis." Molecular metabolism 44 (2021): 101129). In addition, intestinal uptake of saccharides has also been shown to be increased in obese individuals compared to healthy control. Recent studies show that the intake of artificial sweeteners further increases the expression of glucose transporters, e.g. the expression of SGLT1 (see, for example: Xie, Ningning, et al. "Artificial sweeteners affect the glucose transport rate in the Caco- 2/NCI-H716 co-culture model." Journal of the Science of Food and Agriculture 100.13 (2020): 4887-4892 and Moran, Andrew W., et al. "Expression of Na+/glucose cotransporter 1 (SGLT1) is enhanced by supplementation of the diet of weaning piglets with artificial sweeteners." British Journal of Nutrition 104.5 (2010): 637-646).

Current strategies to induce weight loss include the decreasing digestion of saccharides, inducing satiety, modifying diet or performing exercise. However this is difficult to adhere to and especially due to the sedentary lifestyle, until 2025 the prevalence of people with obesity is expected to double.

Given the focus on downregulating saccharide caloric intake by mammalians suffering from obesity on the one hand, in sports, on the other hand, an increased saccharide caloric intake by mammalians is wanted in order to increase the exercise performance. Exercise performance (in particular exercise performance for endurance athletes) is currently increased by enhancing muscle function that can be achieved by increasing the oxidative capacity or substrate bioavailability. Current strategies of increasing the bioavailability of substrates like carbohydrates are focused on inducing the maximum uptake. Therefore, it is recommended to combine multiple monosaccharides glucose and fructose in a ratio of 2:1 (see, for example: Fuchs, Cas J., Javier T. Gonzalez, and Luc JC Van Loon. "Fructose co-ingestion to increase carbohydrate availability in athletes." The Journal of physiology 597.14 (2019): 3549- 3560). However, it is known that the muscle can handle more glucose than could be maximally taken up via the gastrointestinal system.

Given the above identified issues in relation to mammalians suffering from obesity and mammalian exercise performance, the present invention now provides for a compound regulating the saccharide absorption via the gastrointestinal tract. The compound of the present invention decreases or increases the maximal saccharide uptake by the gastrointestinal tract. By providing a compound that either downregulates (decreases) the saccharide absorption by the gastrointestinal tract or upregulates (increases) the saccharide absorption by the gastrointestinal tract, the present invention provides hereto a method for treating mammalians suffering from obesity or improving exercise performance, respectively. It was found that by providing the compound of the present invention, the expression of gastrointestinal saccharide transporters is influenced, i.e. downregulated or upregulated, as such and thus resulting in a prolonged time of modified maximal level of saccharide uptake by the mammalian. Therefore, by providing a compound that decreases the expression of monosaccharide transporters it is easier for the mammalian suffering from obesity to maintain a downregulated saccharide intake for a longer period of time than adhering to another lifestyle/(saccharide-low) diet regime.

In sports, i.e. exercise performance, by increasing the expression of monosaccharide transporters a more long-term effect on glucose bioavailability can be achieved than provided by the typical high-carbohydrate diet regime. In addition, it is noted that increasing exercise performance by increasing muscle oxidative capacity is a long and laborious process of multiple physical trainings and nutritional diet regimes. The present invention now provides for a relative convenient and comfortable way of improving exercise performance.

The present invention provides hereto a compound for use in the regulation of saccharide absorption via the gastrointestinal tract, wherein the compound is selected from the group consisting of hydroxycinnamic acids, curcuminoids and derivatives thereof. It was found that the group of hydroxycinnamic acids and derivatives thereof resulted in increasing saccharide absorption via the gastrointestinal tract, thus being of particular use in the improvement of exercise performance of a mammalian. It was further found that the group of curcuminoids and derivatives thereof resulted in decreasing saccharide absorption via the gastrointestinal tract, thus being of particular use in the treatment of mammalians suffering from obesity.

As used herein, the term ‘saccharide absorption’ or ‘saccharide uptake’ refers to the absorption of saccharides, such as d-glucose, d-galactose, d-fructose and ribose, via transporters in the gastrointestinal tract. Such transporters may be present in the luminal brush border membrane (BBM) and basolateral membrane (BLM) of small intestinal epithelial cells (lECs). Examples of such transporters may include (but are not limited to) transporters belonging to the SLC2 family with facilitative diffusion transporters (GLUTs) and the SLC5 family with Na ⁺ -d-glucose cotransporters (SGLTs). As used herein, the term ‘mammalian’ refers to an animal or human. Synonyms may include the terms ‘subject’ or ‘patient’.

The present invention relates to the use of a compound for use in the regulation of saccharide absorption via the gastrointestinal tract. The saccharide absorption preferably comprises the saccharide absorption via the small intestine. As used herein, the term ‘saccharide absorption’ may further refer to the absorption of monosaccharides, such as saccharides selected from the group consisting of d- glucose, d-glucose, d-galactose, d-fructose and pentoses, such as ribose, and combinations thereof.

In a first aspect, the present invention relates to the use of a compound for improving exercise performance, wherein the compound is selected from the group consisting of hydroxycinnamic acids and derivatives thereof.

The hydroxycinnamic acid is preferably selected from the group consisting of cinnamic acid, coumaric acid, ferulic acid, caffeic acid, chlorgenic acid and rosemarinic acid. In particular good results in increasing the saccharide absorption via the gastrointestinal tract are observed by using caffeic acid.

Where hydroxycinnamic acid is used as a compound for regulating saccharide absorption via the gastrointestinal tract, it was found that the amount and form of administration of hydroxycinnamic acid is preferably selected such that the bioaccessibility of hydroxycinnamic acid in the gastrointestinal tract, preferably the small intestine, is in the range of 100-400 pM hydroxycinnamic acid.

As used herein, the term ‘bioaccessibility of hydroxycinnamic acid’ refers to the amount of hydroxycinnamic acid released in the gastrointestinal tract. In other words, the amount of hydroxycinnamic acid available for absorption via the gastrointestinal tract. It is noted that the bioaccessibility depends on the amount administered and form of administration by the subject. A liquid formulation comprising a specific amount of hydroxycinnamic acid is expected to have a higher bioaccessibility than a solid formulation comprising the same amount of hydroxycinnamic acid.

In an embodiment of the present invention, the amount and form of administration of hydroxycinnamic acid is preferably selected such that the bioaccessibility of hydroxycinnamic acid in gastrointestinal tract, preferably the small intestine, is in the range of 125-350 pM, 150-300 pM or 175-250 pM hydroxycinnamic acid. In a preferred embodiment of the present invention, the amount and form of administration of hydroxycinnamic acid is preferably selected such that the bioaccessibility of hydroxycinnamic acid in gastrointestinal tract, preferably the small intestine is about 200 pM.

In a second aspect, the present invention relates to a compound for treating obesity, wherein the compound is selected from the group consisting of curcuminoids and derivatives thereof.

The curcuminoid is preferably selected from the group consisting of curcumin, demethoxycurcumin, dibenzoylmethane and bisdemethoxycurcumin. In particular good results in decreasing the saccharide absorption via the gastrointestinal tract are observed by using dibenzoylmethane, curcumin and bisdemethoxycurcumin.

Where curcuminoid is used as a compound for regulating saccharide absorption via the gastrointestinal tract, it was found that the amount and form of administration of curcuminoid is preferably selected such that the bioaccessibility of curcuminoid in the gastrointestinal tract, preferably the small intestine, is at least 100 pM curcuminoid.

As used herein, the term ‘bioaccessibility of curcuminoid’ refers to the amount of curcuminoid released in the gastrointestinal tract. In other words, the amount of curcuminoid available for absorption via the gastrointestinal tract. It is noted that the bioaccessibility depends on the amount administered and form of administration by the subject. A liquid formulation comprising a specific amount of curcuminoid is expected to have a higher bioaccessibility than a solid formulation comprising the same amount of curcuminoid.

In an embodiment of the present invention, the amount and form of administration of curcuminoid is preferably selected such that the bioaccessibility of curcuminoid in gastrointestinal tract, preferably the small intestine, is at least 125 pM, at least 150 pM, at least 175 pM, more preferably at least 200 pM or at least 400 pM curcuminoid.

The compound for use according to the present invention is preferably a nutraceutical. As used herein, the term ‘nutraceutical’ (or nutriceutical) refers to any food or food ingredient (or feed or feed ingredient for animals) considered to provide medical or health benefits, including the prevention and treatment of a disease. A nutraceutical may also be referred to as bioactive ingredient. It is further noted that a food product may comprise a food ingredient that has bioactive potential at a higher concentration than actually consumed (i.e. the concentration of the ingredient as consumed when consuming the food product). Therefore, the compound for use according to the present invention is preferably a food ingredient (or feed ingredient) having bioactive properties administered in an amount such that the food ingredient (or feed ingredient) exhibits the bioactive properties in the mammalian (human or animal) body.

In a third aspect, the present invention relates to a composition comprising the compound of the present invention. Preferably, the composition of the present invention is a food supplement.

Examples

Caco-2 cells

A differentiated Caco-2 cell line transwell model system was used to determine glucose and fructose transepithelial transport. To this extent, Caco-2 cells were differentiated in a transwell set-up and exposed to the compound of interest at different concentrations and exposure times. After a 3-day differentiation with butyrate and BSA to induce differentiation of Caco-2 cells to an intestinal epithelial phenotype in transwells, the cells were exposed to different doses (100 pM, 200 pM and/or 400 pM) of the compounds of interest for 4 hours. After that the cells are apically exposed to physiological glucose (1 mM) and fructose (0.5 mM) concentrations for 30 minutes. Subsequently, intestinal epithelial uptake of glucose and fructose was measured by determining the basolateral glucose and fructose spectrophotometrically by measuring absorbance (glucose amplex red assay) and fluorescence (NBD-labelled fructose).

The expression (fold change) of saccharide transporters, such as SGLT 1 and/or GLLIT5, was determined by QPCR.

In addition to the compounds of interest, the intestinal epithelial uptake of glucose and fructose as well as the expression of saccharide transporters SGLT 1 and GLLIT5 was measured for phlorizin and its aglucone phloretin, two known inhibitors of glucose transmembrane transport.

The results show that dibenzoylmethane (DBM) dose dependently decreases glucose transepithelial transport (Figure 1A), while fructose uptake is only significantly decreased at a concentration of 100 pM of dibenzoylmethane (Figure 1 B). These results are further confirmed by the glucose and fructose uptake results as shown in Figures 4A and 4B, i.e. the glucose uptake is significantly decreased at a concentration of 400 pM of dibenzoylmethane (Figure 4A), whereas the at the same concentration the fructose uptake is slightly increased (Figure 4B).

It is noted that at a concentration of 400 pM dibenzoylmethane further downregulates the SGLT1 expression (Figure 5) as compared to the slightly downregulation of the SGLT1 expression at a concentration of 100 pM dibenzoylmethane (Figure 3). On the other hand, the expression of the GLLIT5 is similar to the control (placebo) at a concentration of 400 pM of dibenzoylmethane (Figure 6). These findings are in line with the results observed regarding the regulation of the glucose (SGLT1) and fructose (GLLIT5) absorption.

Curcumin

Curcumin (200 pM) significantly increases fructose transepithelial transport (Figure 4B) and significantly decreases glucose transepithelial transport (Figure 4A). The decrease in glucose transepithelial transport is best explained by the significant downregulation of the SGLT1 expression (Figure 5). Similar to the results observed for dibenzoylmethane, curcumin does not significantly regulates the GLLIT5 expression (Figure 6). rcumin

Similar to dibenzoylmethane and curcumin, bisdemethoxycurcumin (200 pM) downregulates the glucose transepithelial transport (Figure 4A) and upregulates the fructose transepithelial transport (Figure 4B).

Caffeic acid

Caffeic acid (200 pM) increases glucose transepithelial transport (Figure 2A).

Caffeic acid has a small therapeutic window, since 100 pM and 400 pM already decreases glucose uptake (Figure 2A). An opposite pattern of fructose uptake has been seen as compared to glucose uptake for caffeic acid (Figure 2B).

The observed results are best explained by the significant upregulation of the SGLT1 expression (Figure 5).

Conclusion

The results show that the curcuminoids tested, i.e. dibenzoylmethane, curcumin and bisdemethoxycurcumin, decrease glucose transepithelial transport thus being a promising candidate for the downregulation of saccharide absorption via the gastrointestinal tract. Further, the results show that dibenzoylmethane and curcumin do have a significant downregulating effect on the SGLT1 expression. As SGLT1 overexpression is observed in obese patients (see, for example: Hasan, Nesrin M., et al. "Intestinal stem cell-derived enteroids from morbidly obese patients preserve obesity-related phenotypes: Elevated glucose absorption and gluconeogenesis." Molecular metabolism 44 (2021): 101129) it can be concluded that the curcuminoids are promising candidates for treating obesity.

The results further show that caffeic acid increases glucose transepithelial transport. It is further noted that caffeic acid significantly upregulates the expression of SGLT1 thus being a promising candidate for the upregulation of saccharide absorption via the gastrointestinal tract. As such, it can be concluded that caffeic acid is a promising candidate for improving exercise performance, in particular for improving exercise performance of endurance athletes.

Description of the figures

Figure 1A: glucose uptake results at different concentrations of dibenzoylmethane by measuring absorbance using a glucose amplex red assay;

Figure 1 B: fructose uptake results at different concentrations of dibenzoylmethane by measuring fluorescence using a NBD-labelled fructose;

Figure 2A: glucose uptake results at different concentrations of caffeic acid by measuring absorbance using a glucose amplex red assay;

Figure 2B: fructose uptake results at different concentrations of caffeic acid by measuring fluorescence using a NBD-labelled fructose; Figure 3: 100 pM DBM has been shown to decrease SGLT1 expression in the differentiated Caco-2 cells;

Figure 4A: glucose uptake results of phloretin (1 mM), phlorizin (500 pM), dibenzoylmethane (400 pM), curcumin (200 pM) and bisdemethoxycurcumin (200 pM) by measuring absorbance using a glucose amplex red assay;

Figure 4B: fructose uptake results of phloretin (1 mM), phlorizin (500 pM), dibenzoylmethane (400 pM), curcumin (200 pM) and bisdemethoxycurcumin (200 pM) by measuring fluorescence using a NBD-labelled fructose;

Figure 5: SGLT1 expression results in differentiated Caco-2 cells of phloretin (1 mM), phlorizin (500 pM), dibenzoylmethane (400 pM), curcumin (200 pM), bisdemethoxycurcumin (200 pM) and caffeic acid (200 pM); and

Figure 6: GLLIT5 expression results in differentiated Caco-2 cells of phloretin (1 mM), phlorizin (500 pM), dibenzoylmethane (400 pM), curcumin (200 pM) and bisdemethoxycurcumin (200 pM).