Nutrient sensing of gut luminal environment.

Sensing of nutrients by chemosensory cells in the gastrointestinal tract plays a key role in transmitting food-related signals, linking information about the composition of ingested foods to digestive processes. In recent years, a number of G protein-coupled receptors (GPCR) responsive to a range of nutrients have been identified. Many are localised to intestinal enteroendocrine (chemosensory) cells, promoting hormonal and neuronal signalling locally, centrally and to the periphery. The field of gut sensory systems is relatively new and still evolving. Despite huge interest in these nutrient-sensing GPCR, both as sensors for nutritional status and targets for preventing the development of metabolic diseases, major challenges remain to be resolved. However, the gut expressed sweet taste receptor, resident in L-enteroendocrine cells and responsive to dietary sweetener additives, has already been successfully explored and utilised as a therapeutic target, treating weaning-related disorders in young animals. In addition to sensing nutrients, many GPCR are targets for drugs used in clinical practice. As such these receptors, in particular those expressed in L-cells, are currently being assessed as potential new pathways for treating diabetes and obesity. Furthermore, growing recognition of gut chemosensing of microbial-produced SCFA acids has led further attention to the association between nutrition and development of chronic disorders focusing on the relationship between nutrients, gut microbiota and health. The central importance of gut nutrient sensing in the control of gastrointestinal physiology, health promotion and gut-brain communication offers promise that further therapeutic successes and nutritional recommendations will arise from research in this area.

Toll-like receptor 9 expressed in proximal intestinal enteroendocrine cells detects bacteria resulting in secretion of cholecystokinin.

Toll-like receptors (TLRs) play a key role in the recognition of microbes via detection of specific and conserved microbial molecular features. TLRs, mainly expressed in immune cells, interact with intestinal microbiome. Little is known about mechanism(s) of sensing of bacteria by the intestinal surface enteroendocrine cells (EECs). We show here that TLR9 is expressed by the EECs of proximal intestine in a range of species and is co-expressed with the satiety hormone cholecystokinin (CCK). CCK secreted in excess induces emesis (vomiting). Using an EEC model cell line, STC-1, we demonstrate that in response to the TLR9 agonist, DNA containing unmethylated CpG dinucleotide motifs, STC-1 cells secrete CCK and that this secretion is inhibited by specific inhibitors of TLR9. Exposure of STC-1 cells to heat-inactivated pathogenic bacteria, Escherichia coli O55/H7, Shigella flexneri 2457T, Salmonella typhimurium ST4/74, and non-pathogenic Lactobacillus amylovorus GRL1112, results to an increase in CCK secretion compared to untreated control. The magnitudes of CCK release are higher in response to pathogenic bacteria and lowest in response to the non-pathogenic L. amylovorus. The pathogenic strains not only have substantially bigger genomes than L. amylovorus, they also have significantly higher numbers/frequency of RR/CG/YY stimulatory CpG hexamers in their genomic DNA. Pathogen-induced excessive secretion of the gut hormone CCK, provoking emesis can serve as a protective mechanism against development of enteric infections.

Sweet taste receptor expression in ruminant intestine and its activation by artificial sweeteners to regulate glucose absorption.

Absorption of glucose from the lumen of the intestine into enterocytes is accomplished by sodium-glucose co-transporter 1 (SGLT1). In the majority of mammalian species, expression (this includes activity) of SGLT1 is upregulated in response to increased dietary monosaccharides. This regulatory pathway is initiated by sensing of luminal sugar by the gut-expressed sweet taste receptor. The objectives of our studies were to determine (1) if the ruminant intestine expresses the sweet taste receptor, which consists of two subunits [taste 1 receptor 2 (T1R2) and 3 (T1R3)], and other key signaling molecules required for SGLT1 upregulation in nonruminant intestines, and (2) whether T1R2-T1R3 sensing of artificial sweeteners induces release of glucagon-like peptide-2 (GLP-2) and enhances SGLT1 expression. We found that the small intestine of sheep and cattle express T1R2, T1R3, G-protein gustducin, and GLP-2 in enteroendocrine L-cells. Maintaining 110-d-old ruminating calves for 60d on a diet containing a starter concentrate and the artificial sweetener Sucram (consisting of saccharin and neohesperidin dihydrochalcone; Pancosma SA, Geneva, Switzerland) enhances (1) Na(+)-dependent d-glucose uptake by over 3-fold, (2) villus height and crypt depth by 1.4- and 1.2-fold, and (3) maltase- and alkaline phosphatase-specific activity by 1.5-fold compared to calves maintained on the same diet without Sucram. No statistically significant differences were observed for rates of intestinal glucose uptake, villus height, crypt depth, or enzyme activities between 50-d-old milk-fed calves and calves maintained on the same diet containing Sucram. When adult cows were kept on a diet containing 80:20 ryegrass hay-to-concentrate supplemented with Sucram, more than a 7-fold increase in SGLT1 protein abundance was noted. Collectively, the data indicate that inclusion of this artificial sweetener enhances SGLT1 expression and mucosal growth in ruminant animals. Exposure of ruminant sheep intestinal segments to saccharin or neohesperidin dihydrochalcone evokes secretion of GLP-2, the gut hormone known to enhance intestinal glucose absorption and mucosal growth. Artificial sweeteners, such as Sucram, at small concentrations are potent activators of T1R2-T1R3 (600-fold>glucose). This, combined with oral bioavailability of T1R2-T1R3 and the understanding that artificial sweetener-induced receptor activation evokes GLP-2 release (thus leading to increased SGLT1 expression and mucosal growth), make this receptor a suitable target for dietary manipulation.

Relevance of sodium/glucose cotransporter-1 (SGLT1) to diabetes mellitus and obesity in dogs.

Glucose transport across the enterocyte brush border membrane by sodium/glucose cotransporter-1 (SGLT1, coded by Slc5a1) is the rate-limiting step for intestinal glucose transport. The relevance of SGLT1 expression in predisposition to diabetes mellitus and to obesity was investigated in dogs. Cultured Caco-2/TC7 cells were shown to express SGLT1 in vitro. A 2-kbp fragment of the Slc5a1 5' flanking region was cloned from canine genomic DNA, ligated into reporter gene plasmids, and shown to drive reporter gene expression in these cells above control (P < 0.001). To determine the effect of the 3 known SNPs in this region on promoter function, new promoter/reporter constructs (all permutations of these 3 SNPs) were created by site-directed mutagenesis. No significant differences in promoter function were seen, suggesting that these SNPs do not have a significant effect on the constitutive transcription of SGLT1 mRNA in dogs. A search for novel SNPs in this region in dogs was made in 2 breeds predisposed to diabetes mellitus (Samoyed and cairn terrier), 2 breeds that rarely develop diabetes (boxer and German shepherd), and 2 breeds predisposed to obesity (Labrador retriever and cocker spaniel). The Slc5a1 5' flanking region was amplified from 10 healthy individuals of each of these breeds by high-fidelity PCR with the use of breed-labeled primers and sequenced by pyrosequencing. The sequence of the Slc5a1 5' flanking region in all individuals of all breeds tested was identical. On this evidence, variations in Slc5a1 promoter sequence between dogs do not influence the pathogenesis of diabetes mellitus or obesity in these breeds.

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.

Nonruminant Nutrition Symposium: intestinal glucose sensing and regulation of glucose absorption: implications for swine nutrition.

The Na(+/)glucose cotransporter (SGLT1) is the major route for the transport of dietary sugars from the lumen of the intestine into enterocytes. Regulation of this protein is essential for the provision of glucose to the body and avoidance of intestinal malabsorption. This has important nutritional implications in particular for young and growing animals. It has been demonstrated that dietary sugars and artificial sweeteners increase SGLT1 expression and the capacity of the gut to absorb monosaccharides. Furthermore, diets supplemented with artificial sweeteners have been shown to improve growth and performance of weaning piglets. In this review, after describing the organization of intestinal epithelium, the type of gut hormones released in response to dietary carbohydrates, the mechanism underlying the transcellular transport of glucose in the intestine is outlined. Next, a historical background to the work carried out in various laboratories aimed at identifying molecular mechanisms involved in regulation of intestinal glucose transporter, SGLT1, is described. Subsequently, the more recent data on the role of intestinal glucose, or sweet, sensor T1R2 + T1R3, a G protein-coupled receptor, required for upregulation of SGLT1 by dietary sugars and artificial sweeteners, are presented. The glucose sensor subunits, T1R2 + T1R3, are members of the taste receptor family 1, T1R, and are expressed in the gut enteroendocrine cells. Sensing of dietary sugars and artificial sweeteners by T1R2 + T1R3 activates a pathway in endocrine cells leading to secretion of gut hormones. Finally, after describing molecular mechanisms by which a specific gut hormone released by endocrine cells may regulate SGLT1 expression in the neighboring absorptive enterocytes, the application of these findings to enhancing intestinal capacity to absorb dietary sugars in weaning piglets is presented. A better understanding of the molecular events involved in regulation of SGLT1 will allow the identification of nutritional targets with attendant promise of avoiding nutrient malabsorption and enhancing growth and well-being of species.

Sodium/glucose cotransporter-1, sweet receptor, and disaccharidase expression in the intestine of the domestic dog and cat: two species of different dietary habit.

The domestic cat (Felis catus), a carnivore, naturally eats a very low carbohydrate diet. In contrast, the dog (Canis familiaris), a carno-omnivore, has a varied diet. This study was performed to determine the expression of the intestinal brush border membrane sodium/glucose cotransporter, SGLT1, sweet receptor, T1R2/T1R3, and disaccharidases in these species adapted to contrasting diets. The expression (this includes function) of SGLT1, sucrase, maltase and lactase were determined using purified brush border membrane vesicles and by quantitative immunohistochemistry of fixed tissues. The pattern of expression of subunits of the sweet receptor T1R2 and T1R3 was assessed using fluorescent immunohistochemistry. In proximal, middle, and distal small intestine, SGLT1 function in dogs was 1.9- to 2.3-fold higher than in cats (P = 0.037, P = 0.0011, P = 0.027, respectively), and SGLT1 protein abundance followed an identical pattern. Both cats and dogs express T1R3 in a subset of intestinal epithelial cells, and dogs, but not cats, express T1R2. In proximal and middle regions, there were 3.1- and 1.6-fold higher lactase (P = 0.006 and P = 0.019), 4.4- and 2.9-fold higher sucrase (both P < 0.0001), and 4.6- and 3.1-fold higher maltase activity (P = 0.0026 and P = 0.0005), respectively, in the intestine of dogs compared with cats. Dogs have a potential higher capacity to digest and absorb carbohydrates than cats. Cats may suffer from carbohydrate malabsorption following ingestion of high-carbohydrate meals. However, dogs have a digestive ability to cope with diets containing significant levels of carbohydrate.

Molecular insights into dietary induced colic in the horse.

Equine colic, a disorder manifested in abdominal pain, is the most frequent cause of emergency treatment and death in horses. Colic often requires intestinal surgery, subsequent hospitalisation and post operative care, with a strong risk of complications arising from surgery. Therefore strategies that explore approaches for preventing the condition are essential. To this end, a better understanding of the factors and mechanisms that lead to the development of colic and related intestinal diseases in the horse allows the design of preventive procedures. Colic is a multifactorial disorder that appears to be induced by environmental factors and possibly a genetic predisposition. One factor that seems to influence the risk of developing colic is the excessive consumption of diets containing high levels of carbohydrates. Therefore, major efforts have been made by various laboratories and institutions across the world to study the type and digestibility of various feed in order to formulate accurate and safe feed components and proportions. However, relatively little work has been carried out to characterise, in detail, the carbohydrate digestive and absorptive capacity and mechanisms underlying the potential adaptive response of equine gut epithelium to a changing diet. This review focuses on advances made towards understanding the molecular and cellular mechanisms involved in digestion and absorption of dietary carbohydrates in the equine gastrointestinal tract and the implication of these processes for the whole body physiology. It addresses the underlying mechanisms that may govern the adaptive response of equine small intestine to increased dietary hydrolysable carbohydrates. Furthermore, it describes changes that occur in the equine large intestinal microbiology and host tissue biology brought about by alterations in diet and in colic. It is hoped that a better understanding of the molecular and cellular processes that play important roles in the physiology and pathology of the equine gastrointestinal tract will assist the development of effective strategies to prevent equine colic.

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.

The importance of colonic butyrate transport to the regulation of genes associated with colonic tissue homoeostasis.

The transition from normality to malignancy in colorectal cancer is characterized by alterations in the expression of genes associated with the maintenance of tissue homoeostasis. Butyrate, a product of microbial fermentation of dietary fibre in the colon, is known to regulate a number of genes associated with the processes of proliferation, differentiation and apoptosis of colonic epithelial cells, and, hence, homoeostasis of colonic tissue. We have shown previously that the transport of butyrate into colonocytes is of fundamental importance to butyrate's regulatory ability, and therefore sought to assess the expression profile of butyrate-responsive genes in colon cancer tissue, where the expression of the colonic luminal-membrane butyrate transporter, MCT1 (monocarboxylate transporter 1), is significantly down-regulated. In the present paper, we first employed microarray analysis to assess global changes in butyrate-responsive genes using HT29 human colon carcinoma cells treated with butyrate. There was consistency in the butyrate response of selected genes in two other human colonic cell lines (HCT116 and AA/C1) using quantitative real-time PCR. Furthermore, we report that expression levels of selected butyrate-responsive genes involved in the processes of proliferation, differentiation and apoptosis, are deregulated in colon cancer tissue, correlating with decreased expression of MCT1. These findings support our hypothesis that a reduction in MCT1 expression, and hence butyrate transport, can lead to a reduction in the intracellular butyrate levels required to regulate gene expression. Collectively, our results highlight the important contribution of butyrate transport to the maintenance of tissue homoeostasis and disease prevention.

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.

The importance of butyrate transport to the regulation of gene expression in the colonic epithelium.

Butyrate is a naturally occurring monocarboxylate, produced in the lumen of the colon by microbial fermentation of complex carbohydrates that escape digestion in the small intestine. It serves as the principal metabolic fuel for colonic epithelial cells, and exerts a variety of effects important to intestinal health and function. This brief discussion focuses on the route, role and regulation of butyrate transport in the large intestine, with particular emphasis on the significance of butyrate transport to the ability of butyrate to modulate expression of genes important to the processes maintaining colonic tissue homoeostasis.

Molecular characterisation of fructose transport in equine small intestine.

REASONS FOR PERFORMING STUDY: Fructose can be a suitable carbohydrate supplement for horses before and/or during endurance exercise. In comparison to glucose, the ingestion of fructose results in a lower insulin peak and less marked fluctuations in blood glucose during exercise, potentially avoiding hypoglycaemia-induced exhaustion. OBJECTIVES: To assess the capacity of the equine small intestine to absorb fructose and to determine the mechanism, molecular structure and properties of equine intestinal fructose transport. METHODS: Using PCR-based strategies, RNA isolated from equine small intestine and primers designed to homologous regions of the fructose transporter, GLUT5, cDNA of other species, we cloned and sequenced equine GLUT5 (eGLUT5). Northern and western blot analyses, in conjunction with immunohistochemistry, utilising eGLUT5 cDNA and antibodies, assessed expression of eGLUT5 along the longitudinal and radial axes of the small intestine. Functional properties of fructose transport in intestinal brush-border membrane vesicles were measured using the rapid-filtration technique. RESULTS: eGLUT5 is expressed in the villus enterocytes with highest levels in duodenum>jejunum and lowest in the ileum. Kinetic studies indicate eGLUT5 is a low affinity, high capacity transporter. CONCLUSIONS: Equine small intestine has the capacity to absorb fructose. POTENTIAL RELEVANCE: The molecular probes produced in these studies can be used as diagnostic aids to determine equine intestinal monosaccharide malabsorption.

Mechanism of glucose sensing in the small intestine.

Sensing nutrients is a fundamental task for all living cells. For most eukaryotic cells glucose is a major source of energy, having significant and varied effects on cell function. Interest in identifying mechanisms by which cells sense and respond to variations in glucose concentration has increased recently. The epithelial cells lining the intestinal tract are exposed, from the luminal domain, to an environment with continuous and massive fluctuations in the levels of dietary monosaccharides. Enterocytes therefore have to sense and respond to the significant changes in the levels of luminal sugars, and regulate the expression of the intestinal glucose transporter (Na+/glucose co-transporter, SGLT1) accordingly. Our data, using a combination of in vivo and in vitro model systems, suggest that glucose in the lumen of the intestine is sensed by a glucose sensor residing on the external face of the enterocyte luminal membrane. Glucose binds to the sensor and generates an intracellular signal leading to enhancement in the expression of SGLT1. The generated signal is independent of glucose metabolism and is likely to operate via a G-protein-coupled receptor and cAMP/protein kinase A signalling cascade.

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.

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.

Expression of monosaccharide transporters in intestine of diabetic humans.

Noninsulin-dependent diabetes mellitus (NIDDM) is an increasingly common disease, which brings a number of life-threatening complications. In rats with experimentally induced diabetes, there is an increase in the capacity of the intestine to absorb monosaccharides. We have examined the activity and the expression of monosaccharide transporters in the intestine of patients suffering from NIDDM. Na(+)-dependent D-glucose transport was 3.3-fold higher in brush-border membrane (BBM) vesicles isolated from duodenal biopsies of NIDDM patients compared with healthy controls. Western analysis indicated that SGLT1 and GLUT5 protein levels were also 4.3- and 4.1-fold higher in diabetic patients. This was associated with threefold increases in SGLT1 and GLUT5 mRNA measured by Northern blotting. GLUT2 mRNA levels were also increased threefold in the intestine of diabetic patients. Analysis of other BBM proteins indicated that the activity and abundance of sucrase and lactase were increased by 1.5- to 2-fold and the level of the structural proteins villin and beta-actin was enhanced 2-fold in diabetic patients compared with controls. The increase in the capacity of the intestine to absorb monosaccharides in human NIDDM is due to a combination of intestinal structural change with a specific increase in the expression of the monosaccharide transporters SGLT1, GLUT5, and GLUT2.

Transcriptional regulation of the ovine intestinal Na+/glucose cotransporter SGLT1 gene. Role of HNF-1 in glucose activation of promoter function.

Dietary sugars D-glucose and D-galactose are transported across the intestinal brush-border membrane by the Na+/glucose cotransporter, SGLT1. In various species studied, it has been shown that the activity, and expression, of intestinal SGLT1 is regulated by dietary sugars. We report in this paper that regulation of the intestinal SGLT1 gene by lumenal sugar is due, in part, to an increase in transcription. Using deletion analyses of the -66/+21-bp fragment, we have identified the minimal region of the ovine SGLT1 promoter able to support transcription. Site-directed mutagenesis of the hepatic nuclear factor-1 (HNF-1) consensus motif within this domain eliminates basal promoter function. In addition, we show direct evidence for glucose-induced activation of the -66/+21-bp promoter region. There is a co-ordinated decline in the abundance of ovine intestinal HNF-1 and SGLT1 transcripts during transition from preruminant to adult ruminant. This decline is recovered after glucose infusion of adult sheep intestine. Similarly, as shown using DNA mobility-shift assays, the intensity of the HNF-1-binding complex to the target promoter sequence decreases during maturation of the animal; this is restored after intestinal sugar infusion. These data indicate that HNF-1 plays an important role in the glucose responsiveness of the ovine SGLT1 gene. This is the first report of in vitro glucose-induced activation of the intestinal SGLT1 promoter and identification of a glucose-responsive region of the ovine SGLT1 promoter.

Expression of the Na+/glucose co-transporter (SGLT1) in the intestine of domestic and wild ruminants.

The activity and abundance of the Na+/glucose co-transporter (SGLT1) was assessed in brush-border-membrane vesicles (BBMV) isolated from the intestine of grass- and roughage- (GR) consuming ruminants (sheep and dairy cattle), during the transition from the pre-ruminant to the mature ruminant state. The abundance of SGLT1 messenger ribonucleic acid (mRNA) was also compared in the intestinal tissue of the same animals. The dramatic developmental decline in the activity and expression of SGLT1 appears to be typical of GR-consuming ruminants and is coincident with the significant decline in the levels of lumenal monosaccharides. Expression of the ovine SGLT1 complementary deoxyribonucleic acid (cDNA) in Xenopus laevis oocytes confirmed that the isolated cDNA encodes for a functional Na+/glucose co-transporter. Determination of a bovine intestinal SGLT1 protein sequence (amino acids 347-658) indicated 99% similarity to the ovine SGLT1 protein with differences in the carboxyl terminus. In contrast to GR-consuming ruminants, the abundance of SGLT1 protein and SGLT1 mRNA remained significantly high in the intestine of ruminants in both the intermediate-mixed (IM) feeding goat and fallow deer and the concentrate-selecting (CS) moose and roe deer, dietary groups correlating with the availability of monosaccharides in the intestinal lumen.