Advanced Search
Figures

Figure 1

Alignment of ovine taste 1 receptor 2 (T1R2) AA sequence for (A) 174 and (B) 115 AA fragments deduced from the cloned mRNA nucleotide sequence, with the corresponding regions of cow, pig, human, and mouse T1R2 (numbers in parentheses relate to initiating Met residue). Color version available in the online PDF.

Figure 2

Alignment of ovine taste 1 receptor 3 (T1R3) AA sequence (321 AA fragments deduced from the cloned mRNA nucleotide sequence) with the corresponding region of cow, pig, human, and mouse T1R3 (numbers in parentheses relate to initiating Met residue). Color version available in the online PDF.

Figure 3

A representative figure showing immunohistochemical localization of taste 1 receptor 2 (T1R2), taste 1 receptor 3 (T1R3), and α-gustducin in the ovine small intestine. Sections of ovine jejuna were probed with fluorescently labeled antibodies to T1R2 (A and B), T1R3 (C and D), and α-gustducin (E and F). In figures B, D, and F, nuclei are stained with 4′,6-diaminido-2-phenylindole (darker gray;/blue); images shown at 1,000× magnification; scale bar = 10 µm. Color version available in the online PDF.

Figure 4

A representative image demonstrating that bovine L-enteroendocrine cell expresses taste 1 receptor 2 (T1R2), taste 1 receptor 3 (T1R3), and α-gustducin. (A) Sections of bovine duodenal tissues were probed with antibodies to T1R2 (a), T1R3 (d), α-gustducin (Gust; g), and chromogranin A (ChA; b, e, and h). Merged images (c, f, and i) demonstrate co-expression. (B) Co-localization (l) of T1R2 (j) and glucagon-like peptide-2 (GLP-2; k) shows that T1R2 is present in l-enteroendocrine cells expressing GLP-2. A similar co-expression pattern was obtained for T1R3 and GLP-2. Images shown at 1,000× magnification; scale bar = 10 µm.

Figure 5

Expression of sodium-glucose co-transporter 1 (SGLT1) mRNA and protein function in bovine intestine in response to supplementation of feed with the artificial sweetener Sucram (saccharin and neohesperidin dihydrochalcone). Brush border membrane vesicles (BBMV) and RNA were isolated from duodenal tissues of calves maintained for 50 d on a milk-replacer diet (Con 1, n = 4, group 1) or supplemented with Sucram (SUC 1, n = 4, group 2), with two other groups switched for a further 60 d to a starter concentrate (Con 2, n = 4, group 3) or the same dietary regimen but supplemented with Sucram (SUC 2, n = 4, group 4). (A) Steady-state levels of SGLT1 mRNA abundance normalized to RNA polymerase II (RP2). (B) Initial rates of Na+-dependent [U14C]-d-glucose uptake into BBMV. Data were generated in triplicate. Results are shown as mean ± SEM; statistically significant results were determined using a one-way ANOVA and Dunnett’s multiple comparison post-test, where an asterisk (*) indicates P < 0.05.

Figure 6

Expression of sodium-glucose co-transporter 1 (SGLT1) protein in the bovine intestine in response to feed supplementation with the artificial sweetener Sucram (saccharin and neohesperidin dihydrochalcone). Two groups of cows in the dry period, 45 to 60 d before parturition, were fed either a control (Con, n = 2) diet or the same diet supplemented with Sucram (SUC, n = 4). (A) Expression of SGLT1 and β-actin proteins in brush border membrane vesicles isolated from duodenal biopsies assessed by Western blotting. (B) Densitometric analysis of Western blots normalized SGLT1 protein abundance to β-actin. Results are expressed as mean ± SEM; statistically significant results were determined using a one-way ANOVA and Dunnett’s multiple comparison post-test, where two asterisks (**) indicates P < 0.01.

Figure 7

Villus height and crypt depth increase in the duodenum of ruminating calves in response to supplementation with the artificial sweetener Sucram (saccharin and neohesperidin dihydrochalcone). (A) Representative light micrographs of duodenal tissues of 50-d-old milk-fed calves (Con 1), 50-d-old milk-fed calves with Sucram supplementation (SUC 1), 110-d-old ruminating calves (Con 2), and 110-d-old ruminating calves with Sucram supplementation (SUC 2). Images were obtained at 40× magnification. (B) Morphometric analyses of villus height and crypt depths are shown as histograms (µm ± SEM): Con 1 (white bar), SUC 1 (gray bar), Con 2 (dotted bar), SUC 2 (checked bar); n = 3 to 4 animals. Statistically significant results were determined using one-way ANOVA with Dunnett’s post-test. An asterisk (*) indicates P < 0.05; 2 asterisks (**) indicates P < 0.01; 3 asterisks (***) indicates P < 0.001. Color version available in the online PDF.

Figure 8

Changes in specific activities of maltase and alkaline phosphatase in the bovine intestine in response to supplementation with the artificial sweetener Sucram (saccharin and neohesperidin dihydrochalcone). Brush border membrane vesicles (BBMV) were isolated from duodenal tissues of calves maintained for 50 d on a milk-replacer diet (Con 1, n = 4, group 1) or supplemented with Sucram (SUC 1, n = 4, group 2), with 2 other groups switched to a starter concentrate for further 60 d (Con 2, n = 4, group 3) or the same dietary regimen but supplemented with Sucram (SUC 2, n = 4, group 4). Maltase- (A) and alkaline phosphatase-specific (B) activities were measured in BBMV. Data were generated in triplicate. Results are shown as mean specific activity (nmol/min per milligram of protein) ± SEM; statistically significant results were determined using a one-way ANOVA and Dunnett’s multiple comparison post-test. An asterisk (*) indicates P < 0.05.

Figure 9

Effects of glucose, mannitol, sucralose, saccharin, and neohesperidin dihydrochalcone (NHDC) on glucagon-like peptide-2 (GLP-2) secretion from the sheep intestine. Sheep jejunal tissues were cut into ~2-cm sheets with the serosa removed, as previously described ( Daly et al., 2012 ), and incubated in 500 µL of media consisting of low-glucose (5.55 mM) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum, 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 20 µL/mL of dipeptidyl peptidase-4 inhibitor, gassed with carbogen (5% CO2, 95% O2), and supplemented with either 300 mM glucose, 300 mM mannitol, or 5 mM sucralose, saccharin, or NHDC for 1 h at 37°C, 5% CO2. The GLP-2 content of media was measured as described (n = 4). Results are expressed as mean + SEM, where P-values are based on one-way ANOVA with Bonferroni’s multiple comparison post-test; 2 asterisks (**) indicates P < 0.01 and 3 asterisks (***) indicates P < 0.001.

Abstract

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 60 d 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.

Related Articles

Searching for related articles..