enteroendocrine cells ͉ gastrointestinal chemosensation ͉ glucose sensor ͉ incretin G lucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretins, peptide hormones secreted from enteroendocrine L and K cells, respectively, that augment insulin secretion after oral intake of glucose (1). How carbohydrates in the gut lumen elicit the release of GLP-1 from L cells and GIP from K cells is unknown (2). Because i.v. glucose administration does not induce secretion of GLP-1 (3) it appears that glucose within the lumen of the gut acts on the luminal surface to stimulate secretion. Thus, we sought to determine what glucose-sensing mechanism in the gut lumen might underlie this L cell response.One mechanism for sensing glucose is by sweet taste receptors in taste receptor cells of the lingual epithelium (4). Sweet compounds bind to and activate specific G protein coupled receptors that couple through the G protein gustducin (5) to specific second messenger cascades (4, 6). Two type 1 taste G protein coupled receptors (T1Rs) heterodimerize to form the T1R2ϩT1R3 sweet taste receptor (7-11). Key elements of the taste transduction pathways are the ␣, , and ␥ subunits of gustducin (␣-gustducin, G 3 , and G␥ 13 ) (5, 12), phospholipase C2 (PLC2) (13), and transient receptor potential channel type M5 (14), a Ca 2ϩ -activated cation channel (15-17). ␣-Gustducin has been detected in brush cells of the stomach, duodenum, and pancreatic ducts in rat (18, 19), T1R2 and T1R3 are present in rodent gut and the enteroendocrine STC-1 cell line (20), and ␣-gustducin and GLP-1 are present in enteroendocrine cells of the human colon (21). However, the functional significance of expression of taste signaling elements in cells of the gastrointestinal (GI) tract had been unclear. Here, we present data that indicate that T1R3 and gustducin have a role in glucosemediated incretin release and may serve as the previously unknown gut lumen glucose sensor. ResultsWe examined L cells of the gut for the presence of taste receptors and elements of taste transduction pathways. In human duodenal biopsy sections ␣-gustducin was detected by immunofluorescence (
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...
An efficient and accurate method for controlled in vivo transgene modulation by site-directed recombination is described. Seven transgenic mouse founder lines were produced carrying the murine lens-specific aA-crystallin promoter and the simian virus 40 large tumor-antigen gene sequence, separated by a 1.3-kilobase-pair Stop sequence that contains elements preventing expression of the large tumorantigen gene and Cre recombinase recognition sites. Progeny from two of these lines were mated with transgenic mice expressing the Cre recombinase under control of either the murine aA-crystallin promoter or the human cytomegalovirus promoter. AU double-transgenic offspring developed lens tumors. Subsequent analysis confirmed that tumor formation resulted from large tumor-antigen activation via site-specific, Cre-mediated deletion of Stop sequences.A desired goal of transgene technology is efficient and accurate manipulation of DNA sequences after their integration in the germ line. DNA recombinases that mediate integration or excision of sequences at specific recognition sites in both prokaryotic (1-5) and eukaryotic (6-10) systems are well suited for this purpose. The bacteriophage P1 recombinase Cre catalyzes reciprocal recombination at a specific locus ofcrossing over (lox) (11-16). The lox sequence is composed of two 13-base-pair (bp) inverted repeats separated by an 8-bp spacer region. Upon binding to the inverted repeats, Cre synapses with a second lox site and then cleaves the DNA in the spacer region to initiate strand exchange with the synapsed lox partner. No additional factors are required in the recombination.In this study, we examine the potential of the cre/lox system to activate a dormant transgene in the mouse. The simian virus 40 (SV40) large tumor antigens (TAgs) directed to the lens by a murine aA-crystallin promoter (maA) cause malignant lens tumors (17). We inserted between maA and TAg a specially designed Stop sequence that prevents gene expression and is flanked by lox sequences. By crossing the dormant TAg transgenic mouse lines with Cre-expressing transgenic lines, we report here that the Cre protein recognizes the lox sites of the maA-Stop-TAg transgene and recombines the two lox sequences, thereby removing Stop and activating TAg. Our studies show that targeted transgene modification in the mouse can be performed efficiently and accurately with a prokaryotic recombinase.
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