Soraya P. Shirazi-Beechey

T1R3 and gustducin in gut sense sugars to regulate expression of Na-glucose cotransporter 1

Dietary sugars are transported from the intestinal lumen into absorptive enterocytes by the sodium-dependent glucose transporter isoform 1 (SGLT1). Regulation of this protein is important for the provision of glucose to the body and avoidance of intestinal malabsorption. Although expression of SGLT1 is regulated by luminal monosaccharides, the luminal glucose sensor mediating this process was unknown. Here, we show that the sweet taste receptor subunit T1R3 and the taste G protein gustducin, expressed in enteroendocrine cells, underlie intestinal sugar sensing and regulation of SGLT1 mRNA and protein. Dietary sugar and artificial sweeteners increased SGLT1 mRNA and protein expression, and glucose absorptive capacity in wild-type mice, but not in knockout mice lacking T1R3 or α-gustducin. Artificial sweeteners, acting on sweet taste receptors expressed on enteroendocrine GLUTag cells, stimulated secretion of gut hormones implicated in SGLT1 up-regulation. Gut-expressed taste signaling elements involved in regulating SGLT1 expression could provide novel therapeutic targets for modulating the guts capacity to absorb sugars, with implications for the prevention and/or treatment of malabsorption syndromes and diet-related disorders including diabetes and obesity.

Expression of sweet taste receptors of the T1R family in the intestinal tract and enteroendocrine cells

The composition of the intestinal luminal content varies considerably with diet. It is important therefore that the intestinal epithelium senses and responds to these significant changes and regulates its functions accordingly. Although it is becoming evident that the gut epithelium senses and responds to luminal nutrients, little is known about the nature of the nutrient sensing molecule and the downstream cellular events. A prototype example is the modulation in the capacity of the gut to absorb monosaccharides via the intestinal luminal membrane Na(+)/glucose cotransporter, SGLT1. The experimental evidence suggests that luminal sugar is sensed by a glucose sensor residing on the luminal membrane of the gut epithelium and linked to a G-protein-coupled receptor, cAMP/PKA (protein kinase A) pathway, resulting ultimately in modulation of intestinal monosaccharide absorption. Here we report the expression, at mRNA and protein levels, of members of the T1R sweet taste receptors, and the alpha-subunit of the G-protein gustducin, in the small intestine and the enteroendocrine cell line, STC-1. In the small intestine, there is a highly coordinated expression of sweet taste receptors and gustducin, a G-protein implicated in intracellular taste signal transduction, throughout the gut. The potential involvement of these receptors in sugar sensing in the intestine will facilitate our understanding of intestinal nutrient sensing, with implications for better nutrition and health maintenance.

Molecular characterisation of carbohydrate digestion and absorption in equine small intestine.

Dietary carbohydrates, when digested and absorbed in the small intestine of the horse, provide a substantial fraction of metabolisable energy. However, if levels in diets exceed the capacity of the equine small intestine to digest and absorb them, they reach the hindgut, cause alterations in microbial populations and the metabolite products and predispose the horse to gastrointestinal diseases. We set out to determine, at the molecular level, the mechanisms, properties and the site of expression of carbohydrate digestive and absorptive functions of the equine small intestinal brush-border membrane. We have demonstrated that the disaccharidases sucrase, lactase and maltase are expressed diversely along the length of the intestine and D-glucose is transported across the equine intestinal brush-border membrane by a high affinity, low capacity, Na+/glucose cotransporter type 1 isoform (SGLT1). The highest rate of transport is in duodenum > jejunum > ileum. We have cloned and sequenced the cDNA encoding equine SGLT1 and alignment with SGLT1 of other species indicates 85-89% homology at the nucleotide and 84-87% identity at the amino acid levels. We have shown that there is a good correlation between levels of functional SGLT1 protein and SGLT1 mRNA abundance along the length of the small intestine. This indicates that the major site of glucose absorption in horses maintained on conventional grass-based diets is in the proximal intestine, and the expression of equine intestinal SGLT1 along the proximal to distal axis of the intestine is regulated at the level of mRNA abundance. The data presented in this paper are the first to provide information on the capacity of the equine intestine to digest and absorb soluble carbohydrates and has implications for a better feed management, pharmaceutical intervention and for dietary supplementation in horses following intestinal resection.

Ontogenic development of lamb intestinal sodium-glucose co-transporter is regulated by diet.

1. The ontogenic development of the intestinal Na(+)‐glucose co‐transporter was measured in lambs as a function of diet. Transport activity was assayed in brush‐border membrane vesicles and the expression of transport protein in the brush‐border membrane determined by Western analysis. 2. Na(+)‐dependent D‐glucose transport increased to a maximum (300‐700 pmol mg‐1 s‐1) within the first 2 weeks of birth and then declined to negligible amounts (less than 10 pmol mg‐1 s‐1) over the next 8 weeks. There was no further change over the next 2‐3 years. Early changes were associated with modifications in both the maximum velocity Vmax for transport and expression of carrier protein in the brush‐border plasma membrane. 3. Maintaining lambs on a milk replacer diet beyond the normal weaning period prevented the normal decline in the expression of Na(+)‐glucose co‐transport. At 5 weeks the transport rate was 433 +/‐ 150 pmol mg‐1 s‐1 in lambs maintained on milk replacer, but only 79 +/‐ 40 pmol mg‐1 s‐1 in normally reared control lambs. 4. Infusing the proximal intestine of 2‐ to 3‐year‐old sheep with 30 mM‐D‐glucose for four days increased the rate of transport 40‐ to 80‐fold above that found in control animals perfused with mannitol. A similar but smaller increase was observed in one animal perfused with the non‐metabolizable sugar alpha‐methyl‐D‐glucopyranoside. The induced increase in glucose transport was correlated with the expression of the co‐transporter protein in the brush‐border plasma membrane. 5. It is concluded that the age‐related decline in Na(+)‐glucose co‐transport in the sheep intestine is directly due to the decrease in D‐glucose (and D‐galactose) reaching the small intestine after development of the rumen. These results further suggest that luminal sugar substrates for the co‐transporter promote both the maintenance and the up‐regulation of the brush‐border transport protein and it is the intact sugar itself which controls gene expression during enterocyte maturation.

Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1

Butyrate is the principal source of energy for colonic epithelial cells, and has profound effects on their proliferation, differentiation and apoptosis. Transport of butyrate across the colonocyte luminal membrane is mediated by the monocarboxylate transporter 1 (MCT1). We have examined the regulation of expression of human colonic MCT1 by butyrate, in cultured colonic epithelial cells (AA/C1). Treatment with sodium butyrate (NaBut) resulted in a concentration‐ and time‐dependent upregulation of both MCT1 mRNA and protein. At 2 mm butyrate, the magnitude of induction of mRNA (5.7‐fold) entirely accounted for the 5.2‐fold increase in protein abundance, and was mediated by both activation of transcription and enhanced mRNA stability. The other monocarboxylates found naturally in the colon, acetate and propionate, had no effect. The properties of butyrate uptake by AA/C1 cells were characteristic of MCT1. Induction of the MCT1 protein resulted in a corresponding increase in the maximal rate of butyrate transport. The Vmax for uptake of [U‐14C]butyrate was increased 5‐fold following pre‐incubation with 2 mm NaBut, with no significant change in the apparent Km. In conclusion, this study is the first to show substrate‐induced regulation of human colonic MCT1. The basis of this regulation is a butyrate‐induced increase in MCT1 mRNA abundance, resulting from the dual control of MCT1 gene transcription and stability of the MCT1 transcript. We suggest that butyrate‐induced increases in the expression and resulting activity of MCT1 serve as a mechanism to maximise intracellular availability of butyrate, to act both as a source of energy and to influence processes maintaining cellular homeostasis in the colonic epithelium.

Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport l‐lactate as well as butyrate

1 Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription‐polymerase chain reaction (RT‐PCR). The sequence of the RT‐PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT‐PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well‐characterized human and pig colonic lumenal membrane vesicles (LMV). 2 Properties of the colonic lumenal membrane l‐lactate transporter were studied by the uptake of L‐[U‐14C]lactate into human and pig colonic LMV. l‐lactate uptake was stimulated in the presence of an outward‐directed anion gradient at an extravesicular pH of 5.5. Transport of l‐lactate into anion‐loaded colonic LMV appeared to be via a proton‐activated, anion exchange mechanism. 3 l‐lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl− and SO42−. The uptake of l‐lactate was inhibited by phloretin, mercurials and α‐cyano‐4‐hydroxycinnamic acid (4‐CHC), but not by the stilbene anion exchange inhibitors, 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS) and 4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulphonic acid (SITS). 4 The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of l‐lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.

Molecular changes in the expression of human colonic nutrient transporters during the transition from normality to malignancy.

Healthy colonocytes derive 60–70% of their energy supply from short-chain fatty acids, particularly butyrate. Butyrate has profound effects on differentiation, proliferation and apoptosis of colonic epithelial cells by regulating expression of various genes associated with these processes. We have previously shown that butyrate is transported across the luminal membrane of the colonic epithelium via a monocarboxylate transporter, MCT1. In this paper, using immunohistochemistry and in situ hybridisation histochemistry, we have determined the profile of MCT1 protein and mRNA expression along the crypt to surface axis of healthy human colonic tissue. There is a gradient of MCT1 protein expression in the apical membrane of the cells along the crypt-surface axis rising to a peak in the surface epithelial cells. MCT1 mRNA is expressed along the crypt-surface axis and is most abundant in cells lining the crypt. Analysis of healthy colonic tissues and carcinomas using immunohistochemistry and Western blotting revealed a significant decline in the expression of MCT1 protein during transition from normality to malignancy. This was reflected in a corresponding reduction in MCT1 mRNA expression, as measured by Northern analysis. Carcinoma samples displaying reduced levels of MCT1 were found to express the high affinity glucose transporter, GLUT1, suggesting that there is a switch from butyrate to glucose as an energy source in colonic epithelia during transition to malignancy. The expression levels of MCT1 in association with GLUT1 could potentially be used as determinants of the malignant state of colonic tissue.

Glucose sensing and signalling; regulation of intestinal glucose transport

Epithelial cells lining the inner surface of the intestinal epithelium are in direct contact with a lumenal environment that varies dramatically with diet. It has long been suggested that the intestinal epithelium can sense the nutrient composition of lumenal contents. It is only recently that the nature of intestinal nutrient-sensing molecules and underlying mechanisms have been elucidated. There are a number of nutrient sensors expressed on the luminal membrane of endocrine cells that are activated by various dietary nutrients. We showed that the intestinal glucose sensor, T1R2+T1R3 and the G-protein, gustducin are expressed in endocrine cells. Eliminating sweet transduction in mice in vivo by deletion of either gustducin or T1R3 prevented dietary monosaccharide- and artificial sweetener-induced up-regulation of the Na+/glucose cotransporter, SGLT1 observed in wild-type mice. Transgenic mice, lacking gustducin or T1R3 had deficiencies in secretion of glucagon-like peptide 1 (GLP-1) and, glucose-dependent insulinotrophic peptide (GIP). Furthermore, they had an abnormal insulin profile and prolonged elevation of postprandial blood glucose in response to orally ingested carbohydrates. GIP and GLP-1 increase insulin secretion, while glucagon-like peptide 2 (GLP-2) modulates intestinal growth, blood flow and expression of SGLT1. The receptor for GLP-2 resides in enteric neurons and not in any surface epithelial cells, suggesting the involvement of the enteric nervous system in SGLT1 up-regulation. The accessibility of the glucose sensor and the important role that it plays in regulation of intestinal glucose absorption and glucose homeostasis makes it an attractive nutritional and therapeutic target for manipulation.

Intestinal glucose sensing and regulation of intestinal glucose absorption

SGLT1 (Na(+)/glucose co-transporter 1) transports the dietary sugars, D-glucose and D-galactose, from the lumen of the intestine into enterocytes. SGLT1 regulation has important consequences for the provision of glucose to the respiring tissues and is therefore essential for maintaining glucose homoeostasis. SGLT1 expression is directly regulated in response to changes in the sugar content of the diet. To monitor these variations, there is a requirement for a glucose-sensing system located on the luminal membrane of gut cells. This short review focuses on recent findings on intestinal sugar sensing and the downstream mechanisms responsible for enhancement in SGLT1 expression.

Sensing of amino acids by the gut-expressed taste receptor T1R1-T1R3 stimulates CCK secretion

CCK is secreted by endocrine cells of the proximal intestine in response to dietary components, including amino acids. CCK plays a variety of roles in digestive processes, including inhibition of food intake, consistent with a role in satiety. In the lingual epithelium, the sensing of a broad spectrum of L-amino acids is accomplished by the heteromeric amino acid (umami) taste receptor (T1R1-T1R3). T1R1 and T1R3 subunits are also expressed in the intestine. A defining characteristic of umami sensing by T1R1-T1R3 is its potentiation by IMP or GMP. Furthermore, T1R1-T1R3 is not activated by Trp. We show here that, in response to L-amino acids (Phe, Leu, Glu, and Trp), but not D-amino acids, STC-1 enteroendocrine cells and mouse proximal small intestinal tissue explants secrete CCK and that IMP enhances Phe-, Leu-, and Glu-induced, but not Trp-induced, CCK secretion. Furthermore, small interfering RNA inhibition of T1R1 expression in STC-1 cells results in significant diminution of Phe-, Leu-, and Glu-stimulated, but not Trp-stimulated, CCK release. In STC-1 cells and mouse intestine, gurmarin inhibits Phe-, Leu-, and Glu-induced, but not Trp-stimulated, CCK secretion. In contrast, the Ca(2+)-sensing receptor antagonist NPS2143 inhibits Phe-stimulated CCK release partially and Trp-induced CCK secretion totally in mouse intestine. However, NPS2143 has no effect on Leu- or Glu-induced CCK secretion. Collectively, our data demonstrate that functional characteristics and cellular location of the gut-expressed T1R1-T1R3 support its role as a luminal sensor for Phe-, Leu-, and Glu-induced CCK secretion.