Preparation and properties of brush-border membrane vesicles from human small intestine

Shirazi-Beechey, Soraya P.; Davies, Antony G.; Tebbutt, K.; Dyer, Jane; Ellis, Angela E.; Taylor, C J; Fairclough, P. D.; Beechey, R. B.

doi:10.1016/0016-5085(90)90288-c

Cited by 95 publications

(104 citation statements)

References 24 publications

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“…Western Blotting and Glucose Transport. BBMV were isolated from mouse small intestine by the method described (27) in the presence of a mixture of protease inhibitors (Roche Diagnostics, Indianapolis, IN). Western blot analysis was performed as described (5) with antisera to SGLT1 (5, 6), villin (clone 1D2C3; Abcam, Cambridge, U.K.), and ␤-actin (clone AC-15; SigmaAldrich).…”

Section: Methodsmentioning

confidence: 99%

“…Relative levels of SGLT1 protein were normalized to ␤-actin levels within each sample. Glucose transport assays were performed as described (5,27). D-glucose uptake was initiated by the addition of 100 l of incubation medium containing 100 mM NaSCN (or KSCN), 100 mM mannitol, 20 mM Hepes/Tris (pH 7.4), 0.1 mM MgSO 4, 0.02% (wt/vol) NaN 3 , and 0.1 mM D-[U 14 C]glucose to BBMV (100 g of protein).…”

Section: Methodsmentioning

confidence: 99%

“…D-glucose uptake was initiated by the addition of 100 l of incubation medium containing 100 mM NaSCN (or KSCN), 100 mM mannitol, 20 mM Hepes/Tris (pH 7.4), 0.1 mM MgSO 4, 0.02% (wt/vol) NaN 3 , and 0.1 mM D-[U 14 C]glucose to BBMV (100 g of protein). The reaction was stopped after 3 sec by addition of 1 ml of ice-cold stop buffer, containing 150 mM KSCN, 20 mM Hepes/Tris (pH 7.4), 0.1 mM MgSO 4 , 0.02% (wt/vol) NaN 3 , and 0.1 mM phlorizin (5,27). A 0.9-ml portion of the reaction mixture was removed and filtered under vacuum through a 0.22-m pore cellulose acetate/nitrate filter (GSTF02500; Millipore, Bedford, MA).…”

Section: Methodsmentioning

confidence: 99%

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

Margolskee

Dyer

Kokrashvili

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

774

843

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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 ␣-gustducin. Artificial sweeteners, acting on sweet taste receptors expressed on enteroendocrine GLUTag cells, stimulated secretion of gut hormones implicated in SGLT1 upregulation. Gut-expressed taste signaling elements involved in regulating SGLT1 expression could provide novel therapeutic targets for modulating the gut's capacity to absorb sugars, with implications for the prevention and/or treatment of malabsorption syndromes and diet-related disorders including diabetes and obesity.carbohydrate absorption ͉ gastrointestinal chemosensation ͉ glucose sensor ͉ glucose uptake T o date, the only identified sugar sensors in the mammalian gastrointestinal tract are those involved in taste transduction (1). Although the gut epithelium senses luminal sugars and modulates its glucose absorptive capacity accordingly, the nature of the sugar-sensing molecule(s) and downstream events remain unknown. Several studies have shown that in many species expression of the intestinal sodium-dependent glucose transporter 1 (SGLT1) is directly regulated by monosaccharides in the lumen of the gut independently of metabolism and appears to involve a G protein-linked second messenger pathway (2-6). Furthermore, the addition of membrane-impermeable glucose analogues to the lumen of the intestine stimulates SGLT1 expression, implying that a glucose sensor on luminal membranes is involved (6).In taste cells, the detection of sugars and sweeteners depends on T1R2ϩT1R3, a heterodimer of type 1 taste receptor subunits (T1Rs) (7,8). The taste receptor cells of the anterior tongue that express T1R2ϩT1R3 typically also express gustducin, a transducin-like heterotrimeric G protein (9). Gustducin's ␣-subunit (G␣ gust ) has been detected in brush cells of the rat stomach, duodenum, and pancreatic ducts (10). G␣ gust and bitter-responsive type 2 taste receptors (T2Rs) are expressed in mouse intestinal endocrine cells and in the murine enteroendocrine cell line STC-1 (11).We reported previously that T1R taste receptors and G␣ gust are expressed in the mouse small intestinal epithelium and proposed that they function as luminal sugar sensors to control SGLT1 expression in response to dietary sugar (12). Here, we provide three lines of evidence in favor of T1R taste receptors an...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Margolskee

Dyer

Kokrashvili

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

774

843

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“…Brush-border membrane vesicles (BBMV) were prepared from equine small intestinal mucosal scrapings using a method based on that of Shirazi-Beechey et al (1988, 1990. Briefly, mucosal scrapings were thawed in a solution (100 mmol/l mannitol, 2 mmol/l HEPES/Tris, pH 7.1), containing a cocktail of protease inhibitors (Complete protease inhibitor cocktail tablets; D-glucose U V test Kit) 1 (Rowell et al 1997).…”

Section: Isolation Of Brush-border Membrane Vesiclesmentioning

confidence: 99%

Molecular characterisation of carbohydrate digestion and absorption in equine small intestine

Dyer

Merediz

Salmon

et al. 2002

Equine Veterinary Journal

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SummaryD i e t a ry 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 micro b i a l populations and the metabolite products and predispose the horse to gastrointestinal diseases. We set out to determine, at the m o l e c u l a r level, the mechanisms, pro p e rt i e s and the site of e x p ression 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 brushb o r d e r membrane by a high aff i n i t y, low capacity, Na + / g l u c o s e c o t r a n s p o rt e r type 1 isoform (SGLT1). The highest rate of t r a n s p o rt 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 corre l a t i o n 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 p roximal to distal axis of the intestine is regulated at the level of m R N A abundance. The data presented in this paper a re 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 d i e t a ry supplementation in horses following intestinal re s e c t i o n .

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“…These membranes differ in structure, function and surface charges (6) ; properties that have facilitated their isolation in pure form as brush-border or basolateral membrane vesicles. Brush-border membrane vesicles have been used frequently for measurements of digestive enzyme activities and nutrient transport functions expressed on the brush-border membrane (7) . Enteroendocrine cells, dispersed among the cells lining the intestinal epithelium, represent about 1 % of the entire gut epithelial population ( Fig.…”

mentioning

confidence: 99%

Glucose sensing and signalling; regulation of intestinal glucose transport

et al. 2011

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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.Intestine: Glucose sensor: Nutrient: Neuroendocrine: SGLT1The intestinal epithelium is lined with a single layer of epithelial cells that undergoes rapid and continuous renewal. The stem cell located near the base of the crypt of Lieberkühn gives rise to absorptive enterocytes, and three types of secretory cells; mucus producing goblet, Paneth and enteroendocrine. Paneth cells, which secrete antimicrobial peptides, digestive enzymes and growth factors, complete their differentiation at the crypt base. The other three epithelial lineages differentiate during a highly organised upward migration from the crypt to the villus tip where they are extruded into the intestinal lumen (1,2) . This process takes about 2-3 d in most species (3)(4)(5) . Enterocytes constitute the majority (90 %) of cells lining the villus (Fig. 1). They are involved in vectorial transport of nutrients from the lumen of the intestine to the systemic system. These polarised cells possess two distinct membrane domains, the apical (brush border) and basolateral. These membranes differ in structure, function and surface charges (6) ; properties that have facilitated their isolation in pure form as brush-border or basolateral membrane vesicles. Brush-border membrane vesicles have been used frequently for measurements of digestive enzyme...

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Preparation and properties of brush-border membrane vesicles from human small intestine

Cited by 95 publications

References 24 publications

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

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

Molecular characterisation of carbohydrate digestion and absorption in equine small intestine

Glucose sensing and signalling; regulation of intestinal glucose transport

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Preparation and properties of brush-border membrane vesicles from human small intestine

Cited by 95 publications

References 24 publications

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

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

Molecular characterisation of carbohydrate digestion and absorption in equine small intestine

Glucose sensing and signalling; regulation of intestinal glucose transport

Contact Info

Product

Resources

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

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