EP2197433A2 - Regulating glp-1 and sglt-1 in gastrointestinal cells - Google Patents
Regulating glp-1 and sglt-1 in gastrointestinal cellsInfo
- Publication number
- EP2197433A2 EP2197433A2 EP08798295A EP08798295A EP2197433A2 EP 2197433 A2 EP2197433 A2 EP 2197433A2 EP 08798295 A EP08798295 A EP 08798295A EP 08798295 A EP08798295 A EP 08798295A EP 2197433 A2 EP2197433 A2 EP 2197433A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- sweet taste
- mammal
- potentiator
- patient
- sweet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
Definitions
- the present invention relates to the regulation of GLP-I and SGLT-I in gastrointestinal cells.
- GLP-I glucose-dependent insulinotropic polypeptide
- GIP glucose-dependent insulinotropic polypeptide
- GPCRs G- protein coupled receptors
- TlRs Two type 1 taste GPCRs
- ⁇ -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-I cell line (20), and ⁇ -gustducin and GLP-I are present in enteroendocrine cells of the human colon (21).
- GI gastrointestinal
- T1R2+T1R3 a heterodimer of type 1 taste receptor subunits (9, 10).
- the taste receptor cells of the anterior tongue that express T1R2+T1R3 typically also express gustducin, a transducin-like heterotrimeric G protein (5).
- Gustducin's ⁇ -subunit (G ⁇ t ) has been detected in brush cells of the rat stomach, duodenum, and pancreatic ducts (46).
- 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-I (47).
- a first aspect of the present invention relates to a method of decreasing or inhibiting carbohydrate absorption by a mammal. This method involves administering an effective amount of a sweet taste inhibitor to the gastrointestinal tract of the mammal under conditions effective to decrease expression and/or inhibit upregulation of SGLT-I in the mammal, thereby decreasing or inhibiting carbohydrate adsorption by the mammal.
- a second aspect of the present invention relates to a method of promoting insulin secretion in a mammal.
- a third aspect of the present invention relates to a method of treating obesity in a patient. This method involves selecting a patient that is obese, and administering an effective amount of a sweet taste inhibitor to the gastrointestinal tract of the patient under conditions effective to decrease expression and/or inhibit upregulation of SGLT-I in the patient, thereby decreasing or inhibiting carbohydrate adsorption by the patient and treating obesity in the patient.
- a fourth aspect of the present invention relates to a method of promoting weight loss in a mammal.
- This method involves administering an effective amount of a sweet taste inhibitor to the gastrointestinal tract of the mammal under conditions effective to decrease expression and/or inhibit upregulation of SGLT-I in the mammal, thereby decreasing or inhibiting carbohydrate adsorption by the mammal and promoting weight loss in the mammal.
- a fifth aspect of the present invention relates to a method of treating diabetes in a patient.
- This method involves selecting a patient that has diabetes, and administering an effective amount of a sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the patient under conditions effective to increase the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby promoting insulin secretion by the patient and treating diabetes in the patient.
- a sixth aspect of the present invention relates to a method of treating obesity in a patient.
- This method involves selecting a patient that is obese, and administering an effective amount of a sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the patient under conditions effective to increase the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby inducing satiety in the patient and treating obesity in the patient.
- a seventh aspect of the present invention relates to a method of promoting weight loss in a mammal.
- This method involves administering an effective amount of a sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the mammal under conditions effective to increase the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby inducing satiety in the mammal and promoting weight loss in the mammal.
- An eighth aspect of the present invention relates to a method of regulating blood sugar levels in a mammal. This method involves administering an effective amount of a sweet taste inhibitor or sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the mammal under conditions effective to increase or decrease the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby regulating insulin secretion by the mammal and regulating blood sugar levels in the mammal.
- a ninth aspect of the present invention relates to a method of increasing or inducing carbohydrate absorption by a mammal.
- a tenth aspect of the present invention relates to a method of treating a disorder associated with insufficient carbohydrate absorption in a patient.
- This method involves selecting a patient that has a disorder associated with insufficient carbohydrate absorption, and administering an effective amount of a sweet taste potentiator to the gastrointestinal tract of the patient under conditions effective to increase expression and/or induce upregulation of SGLT-I in the patient, thereby increasing or inducing carbohydrate adsorption by the patient and treating the disorder in the patient.
- An eleventh aspect of the present invention relates to a method of promoting weight gain in a mammal.
- This method involves administering an effective amount of a sweet taste potentiator to the gastrointestinal tract of the mammal under conditions effective to increase expression and/or induce upregulation of SGLT-I in the mammal, thereby increasing or inducing carbohydrate adsorption by the mammal and promoting weight gain in the mammal.
- Figures IA-F relate to the presence of taste signaling elements in L cells of the human duodenum.
- Figure IA Indirect immunofluorescent imaging showing co- expression of taste signaling elements (left column) with GLP-I (center column). Nuclei in the merged images (right column) are stained blue. Scale bars, 15 ⁇ m.
- Figure IB top row, cells showing ⁇ -gustducin cytosolic expression and dense apical immunostaining (arrows) projecting into the gut lumen. Scale bars, 5 ⁇ m.
- Middle row low-magnification fields showing immunostaining of ⁇ -gustducin, GLP-I and GIP.
- FIG. 1C Co-expression of T1R2 sweet taste receptor subunit with ⁇ -gustducin ( ⁇ -gust), GLP-I and T1R3 in duodenal enteroendocrine cells. Scale bars, 15 ⁇ m.
- Figure ID Triple staining showing expression of both GLP-I and GIP in an ⁇ -gustducin expressing cell (upper row, arrow). The same image, taken at a different depth, shows a cell that expresses GLP-I and ⁇ -gustducin but not GIP (lower row, arrowhead).
- Figure IE Quantitation of cells expressing ⁇ -gustducin, GLP- 1, or GIP, statistically significant results determined by Student's t-test, values are means ⁇ s.e.m.
- Figure IF RT-PCR amplification of ⁇ -gustducin mRNA in the indicated sub- populations of laser-captured cells.
- Figures 2A-E relate to altered secretion of GLP-I, GIP, and insulin in response to gavage-administered glucose in ⁇ -gustducin null (a-gusf " ) mice.
- Figure 2A Plasma GLP-I (top panel), GIP (middle panel) and insulin (lower panel) levels after glucose gavage (5g/Kg body weight).
- Figure 2B Plasma glucose after glucose gavage (2g/Kg body weight).
- Figure 2C Plasma glucose after post- fasting feeding on chow.
- Figure 2D Plasma GLP-I responses from surgically isolated duodenum in vivo: the duodenum was ligated away from the stomach and rest of the intestines, and circulatory contact maintained.
- Figures 3A-D relate to secretion of GLP-I in response to glucose, sucrose, and sucralose in NCI-H716 cells.
- Figures 3A-B Glucose- sucrose- and sucralose-mediated GLP-I secretion from NCI-H716 cells.
- the sweet receptor inhibitor lactisole inhibited sucralose-mediated GLP-I secretion.
- Figure 3C siRNA-mediated diminution of both ⁇ - gustducin protein levels (by immunoblotting) and glucose-induced (but not basal (BSL)) GLP-I secretion from NCI-H716 cells.
- Figure 3D Immunoblotting of ERK and pERK phosphorylated in from NCI-H716 cells in response to increasing concentrations of glucose and sucralose.
- BSL basal. Experiments were carried out in triplicate and replicated at least twice. Statistical significance determined by ANOVA, values are means ⁇ s.e.m; *p ⁇ 0.05, ***p ⁇ 0.001.
- Figure 4 relates to the coupling of taste receptors to G-protein ⁇ -subunits in
- NCI-H716 cells NCI-H716 cells. Membranes from NCI-H716 cells were preincubated with the indicated concentrations of glucose and sucrose for 10 min at 25°C in the presence of 5-10 ⁇ Ci [ 32 P] GTP-22 azidoanilide, then irradiated to cross link the GTP analog to G-proteins. G-protein specific immune complexes (anti-G ⁇ -gustducin, anti-G ⁇ , anti-G ⁇ s , and control IgG) were separated by SDS-PAGE, transferred to membranes and autoradiographically imaged. [0023] Figures 5A-E are indirect immuno fluorescent images of various gastrointestinal tissues.
- Figure 5 A There is no fluorescent staining of enteroendocrine cells in human duodenum sections when the primary antibody was omitted. Scale bars, top row, 50 ⁇ m: bottom row, 15 ⁇ m.
- Figure 5B Indirect immunofluorescence of duodenum showing co-expression of ⁇ -gustducin ( ⁇ -gust) with GLP-I and GLP-2. Intrinsic fluorescence of green fluorescent protein (GFP) showing co-expression of GLP-2 in the ⁇ -gustducin-expressing cells of ⁇ -gustducin-GFP mice.
- Figure 5C Duodenal sections from ⁇ -gustducin-GFP transgenic mice show colocalization of GFP with ⁇ -gustducin ( ⁇ -gust), GLP-I, and GLP-2.
- FIG. 5D-E Indirect immunofluorescence of jejunum and ileum, respectively, showing co-expression of ⁇ -gustducin with GLP-I, GLP-2, and PYY. Scale bars, 15 ⁇ m.
- Figures 6A-C show that treatment of wild type (a-gust +/+ ) and homozygous null (a-gusf " ) mice with exendin-4, a GLP-I receptor agonist, and systemic glucose (in order to bypass the bowel) elicited similar insulin secretory responses in both sets of mice.
- Figure 6A Exendin-4 (0.4 nM/Kg) was given intravenously, blood was taken at the times indicated and plasma insulin assayed by ELISA.
- FIG. 6B Intraperitoneal injection of glucose (lg/kg) in wild type (a-gust +/+ ) and homozygous null (a-gust ⁇ A ) mice elicited similar insulin secretory responses; blood was taken at the times indicated and plasma insulin assayed.
- Figure 6C GLP-I secretory responses to 5% glucose from isolated duodenal villi.
- Figure 7 shows that age-matched wild type (a-gust +/+ ) and homozygous null
- FIG. 8 is a stained image of isolated villi of mice.
- FIGS 9A-B show that NCI-H716 cells express taste signaling components.
- Figure 9A Indirect immunofluorescence showing presence in NCI-H716 cells of multiple taste elements, GLP-I (nucleus stained red), GLP-I along with PYY. Scale bars, 30 ⁇ m.
- Figure 9B RT-PCR products showing that NCI-H716 cells express multiple taste elements, a fatty acid receptor (Gpr40) and the bile acid receptor (Tgr5). Numbers are product sizes. CHO cells and tongue are negative and positive ⁇ -gustducin controls.
- Figure 10 relates to GLP-I secretion from stimulated NCI-H716 cells.
- Figure 1OA Glucose-potentiated receptor-operated Ca 2+ entry, assessed by depleting Ca 2+ stores with thapsigargin (TG, lO ⁇ M) in the absence of exogenous Ca 2+ , then replenishing with 2mM Ca 2+ .
- Thapsigargin was added to both cells at the first arrow (labeled TG).
- Glucose was added to one set of cells (closed triangles) at the point indicated by the arrow. The experiment was initiated in cells maintained in buffer without calcium. Calcium (2 mM final) was restored to the buffer at the time indicated.
- Figure 1OB Representative traces show changes in [Ca 2+ J 1 after the addition (indicated by arrow) of glucose or 2-deoxyglucose (non-metabolizable glucose).
- Figure 1OC Representative images of [Ca 2+ J 1 levels are depicted on a pseudo-color scale, acquired prior to, (i), and after, (ii) and (iii), exposure to glucose.
- Figure 10D Glucose-stimulated Ca 2+ mobilization in NCI-H716 cells depends upon PLC.
- FIG. 12 relates to G protein activation by ligand, positive control.
- Treatment with GLP-I led to a dose-dependent increase in cross-linked G ⁇ s (GTP-azidoanilide cross- linked subunit) in a GLP-I receptor-expressing cell line, CHO-GLP-IR cells, due to coupling of the activated receptor to G ⁇ s .
- Figures 13A-C relate to increased SGLTl expression in response to dietary carbohydrate in wild-type, but not in G ⁇ gust or T1R3 knockout, mice.
- Wild-type (WT), G ⁇ t , and T1R3 knockout mice were given low (L) or high (H) carbohydrate diets for two weeks.
- Figure 13 A Steady-state levels of SGLTl mRNA determined by QPCR were normalized to ⁇ -actin mRNA.
- Figure 13B SGLTl protein from brush-border membrane vesicles (BBMV) isolated from mid small intestine was detected in Western blots ⁇ Left).
- BBMV brush-border membrane vesicles
- Figures 14A-D relate to increased SGLTl expression in response to dietary supplementation with artificial sweeteners in wild-type, but not in or T1R3 knockout, mice.
- Wild-type (WT), Gcigust, and T1R3 knockout mice were given a low-carbohydrate diet without (L), or with 2 mM sucralose (L+suc) for 2 weeks.
- Figures 14A-C Steady-state levels of SGLTl mRNA ( Figure 14A), SGLTl protein ( Figure 14B), and SGLTl -mediated glucose transport rates (Figure 14C), were measured (see Figures 13 A-C).
- Figures 15A-I relate to detection and localization of TlR receptors and G ⁇ gust along the crypt-villus axis of small intestine.
- Figure 15 A In situ hybridization with antisense riboprobes to T1R2, T1R3, and G ⁇ gust . Complementary sense probes to all targets did not hybridize to any transcripts within the tissue sections (see Figure 18).
- Figures 15B-C Immuno fluorescent detection of the T1R3 taste receptor subunit (red) and G ⁇ guSt (green) in serial wax sections of mouse duodenum.
- Figure 15D Immuno fluorescent detection of the T1R3 (red) and T1R2 (green) taste receptor subunits in a single wax section of human duodenum.
- Figures 15E-G Immunofluorescent detection of the T1R2 and T1R3 taste receptor subunits (green), and G ⁇ t (red) in a single wax section of human duodenum.
- Figure 15H Chromogenic detection of SGLTl in wax sections of mouse small intestine.
- Figure 151 Chromogenic detection of G ⁇ gus t and chromogranin in serial wax sections of mouse proximal intestine. The boxed cell expresses both G ⁇ gust and chromogranin.
- Figures 16A-B show that sucralose stimulation of endogenously expressed sweet taste receptors in GLUTag cells leads to GLP-I and GIP release.
- Figures 17A-C show that intestinal structure is not affected by diet or gene deletion.
- Figures 17A-B Mean villus height (Figure 17A) and crypt depth (Figure 17B) of intestinal tissues from wild-type mice maintained on low- (L) or high- (H) carbohydrate diets or on the low-carbohydrate diet supplemented with drinking water containing 20 mM saccharin (sac), 1 mM aspartame (asp), or 10 mM acesulfame K (ace-K).
- sac saccharin
- asp 1 mM aspartame
- ace-K 10 mM acesulfame K
- FIG. 17C Western blots of villin and ⁇ -actin in wild-type (WT), and G ⁇ gust and T1R3 knockout, mice on both low- (L) and high- (H) carbohydrate diets show no significant differences in levels of these proteins between wild-type and knockout mice and no change in the level of either protein as a function of dietary carbohydrate.
- Villin protein gives a good indication of brush-border membrane recovery and purity, both of which are unaffected by diet or gene deletion.
- the weaker signal for low-abundance villin vs. high-abundance ⁇ -actin is due to adjusting the exposure time so that both signals are measured within the linear range for densitometric analysis.
- Statistical significance determined by ANOVA data are expressed as means ⁇ SD.
- Figure 18 relates to in situ hybridization controls. In situ hybridization histochemistry with complementary sense probes to T1R2, T1R3, G ⁇ gus t, and SGLTl did not nonspecifically hybridize with targets within the mouse small intestine.
- Figure 19 relates to immunohistochemistry controls. Omission of the primary antibodies for gustducin and T1R3 with mouse (FITC Mouse and Cy3 Mouse, respectively) and human (FITC Human and Cy3 Human, respectively) tissues showed no nonspecific immunoreactivity with targets within the mouse small intestine.
- Figure 20 relates to detection of taste signaling elements in enteroendocrine cell lines.
- Figures 21A-B show that gurmarin inhibits mouse T1R2+T1R3 sweet taste receptor activity.
- HEK 293 cells were transfected with plasmids encoding mouse T1R2, mouse T1R3, and Gal6/gust44 loaded with the fluorescent calcium indicator dye Fluo-4 and then exposed to sweeteners with or without the mouse sweet taste inhibitor gurmarin.
- FIG. 1A Fluo-4 Fluorescence, in arbitrary units (a.u.), measures calcium mobilization in response to sweet taste receptor activation by the addition of 10 mM sucralose (Sue) (open circles). Preincubation (15 min ) with gurmarin (1 mg) abolishes the response (filled circles).
- Figure 2 IB DeltaF/F measures the peak amplitude of calcium mobilization in response to addition of sweeteners: 10 mM sucralose (Sue), 10 mM saccharin (Sac) and 10 mM acesulfame-K (AceK) and 20 mM cyclamate (Cyc).
- the expressed mouse T1R2+T1R3 receptor does not respond to the human-specific sweetener cyclamate (see 61).
- Preincubation (15 min) with gurmarin (1 mg) abolishes the responses to sucralose, saccharin, and acesulfame-K.
- Figure 22 shows that gurmarin inhibits calcium mobilization in sucralose- stimulated GLUTag cells. Intracellular free Ca + in GLUTag cells transfected, to maximally enhance detection of calcium mobilization, with Gal6/gust44, YC3.60, and REEP-EI was monitored continuously by fluorescence confocal microscopy.
- a first aspect of the present invention relates to a method of decreasing or inhibiting carbohydrate absorption by a mammal.
- This method involves administering an effective amount of a sweet taste inhibitor to the gastrointestinal tract of the mammal under conditions effective to decrease expression and/or inhibit upregulation of SGLT-I in the mammal, thereby decreasing or inhibiting carbohydrate adsorption by the mammal.
- the sweet taste inhibitor inhibits activation of one or more taste signaling molecules contained within a gastrointestinal endocrine cell in the mammal.
- Exemplary taste signaling molecules include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ i 3 , G ⁇ 3 , G ⁇ i, T1R2, T1R3, T1R2+T1R3 dimeric receptor, Trpm5, Trpm4, PLC ⁇ 2, IP 3 receptor type 3, Type I PDEs, and PDE-IA.
- Suitable sweet taste inhibitors include lactisole, gurmarin, gymnemic acid, substituted sulfamates, and substituted cyclamate sulfamates. [0043] In some embodiments, the adsorption of glucose, fructose, and/or other dietary monosaccharides is decreased or inhibited.
- the sweet taste inhibitor is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste inhibitor.
- the sweet taste inhibitor is administered primarily to the stomach, small intestine, and/or large intestine of the mammal.
- the sweet taste inhibitor is encapsulated in a pharmaceutical carrier that releases the sweet taste inhibitor into the stomach, small intestine, and/or large intestine of the mammal.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste inhibitor to taste cells in the mouth of the mammal.
- a second aspect of the present invention relates to a method of promoting insulin secretion in a mammal.
- This method involves administering an effective amount of a sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the mammal under conditions effective to increase the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby promoting insulin secretion by the mammal.
- the sweet taste potentiator activates one or more taste signaling proteins in the gastrointestinal endocrine cell of the mammal. Suitable taste signaling proteins include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3, G ⁇ 1?
- Suitable sweet taste potentiators include sweet tasting compounds selected from the group of sugars, peptides, proteins, natural non-sugar sweeteners, glycyrrhizic acid, stevioside, and artificial non-sugar sweeteners.
- the sweet taste potentiator is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste potentiator.
- the sweet taste potentiator is administered primarily to the stomach, small intestine, and/or large intestine of the mammal.
- the sweet taste potentiator is encapsulated in a pharmaceutical carrier that releases the sweet taste potentiator into the stomach, small intestine, and/or large intestine of the mammal.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste potentiator to taste cells in the mouth of the mammal.
- a third aspect of the present invention relates to a method of treating obesity in a patient.
- This method involves selecting a patient that is obese, and administering an effective amount of a sweet taste inhibitor to the gastrointestinal tract of the patient under conditions effective to decrease expression and/or inhibit upregulation of SGLT-I in the patient, thereby decreasing or inhibiting carbohydrate adsorption by the patient and treating obesity in the patient.
- the patient is selected from the group of a human, a dog, and a non-ruminant livestock.
- the sweet taste inhibitor inhibits activation of one or more taste signaling molecules contained within a gastrointestinal endocrine cell in the patient.
- taste signaling molecules include heterotrimeric gustducin, ⁇ -gustducin,
- Suitable sweet taste inhibitors include lactisole, gurmarin, gymnemic acid, substituted sulfamates, and substituted cyclamate sulfamates.
- the adsorption of glucose, fructose, and/or other dietary monosaccharides is decreased or inhibited.
- the sweet taste inhibitor is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste inhibitor.
- the sweet taste inhibitor is administered primarily to the stomach, small intestine, and/or large intestine of the patient.
- the sweet taste inhibitor is encapsulated in a pharmaceutical carrier that releases the sweet taste inhibitor into the stomach, small intestine, and/or large intestine of the patient.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste inhibitor to taste cells in the mouth of the patient.
- a fourth aspect of the present invention relates to a method of promoting weight loss in a mammal.
- This method involves administering an effective amount of a sweet taste inhibitor to the gastrointestinal tract of the mammal under conditions effective to decrease expression and/or inhibit upregulation of SGLT-I in the mammal, thereby decreasing or inhibiting carbohydrate adsorption by the mammal and promoting weight loss in the mammal.
- the mammal is selected from the group of a human, a dog, and a non-ruminant livestock.
- the sweet taste inhibitor inhibits activation of one or more taste signaling molecules contained within a gastrointestinal endocrine cell in the mammal.
- Exemplary taste signaling molecules include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ i 3 , G ⁇ 3 , G ⁇ i, T1R2, T1R3, T1R2+T1R3 dimeric receptor, Trpm5, Trpm4, PLC ⁇ 2, IP 3 receptor type 3, Type I PDEs, and PDE-IA.
- Suitable sweet taste inhibitors include lactisole, gurmarin, gymnemic acid, substituted sulfamates, and substituted cyclamate sulfamates. [0059] In some embodiments, the adsorption of glucose, fructose, and/or other dietary monosaccharides is decreased or inhibited.
- the sweet taste inhibitor is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste inhibitor.
- the sweet taste inhibitor is administered primarily to the stomach, small intestine, and/or large intestine of the mammal.
- the sweet taste inhibitor is encapsulated in a pharmaceutical carrier that releases the sweet taste inhibitor into the stomach, small intestine, and/or large intestine of the mammal.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste inhibitor to taste cells in the mouth of the mammal.
- a fifth aspect of the present invention relates to a method of treating diabetes in a patient.
- This method involves selecting a patient that has diabetes, and administering an effective amount of a sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the patient under conditions effective to increase the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby promoting insulin secretion by the patient and treating diabetes in the patient.
- the patient is selected from the group of a human, a dog, and a non-ruminant livestock.
- the sweet taste potentiator activates one or more taste signaling proteins in the gastrointestinal endocrine cell of the patient.
- Suitable taste signaling proteins include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3 , G ⁇ ls T1R2, T1R3, T1R2+T1R3 dimeric receptor, Trpm5, Trpm4, PLC ⁇ 2, IP 3 receptor type 3, Type I PDEs, and PDE-IA.
- Suitable sweet taste potentiators include sweet tasting compounds selected from the group of sugars, peptides, proteins, natural non-sugar sweeteners, glycyrrhizic acid, stevioside, and artificial non- sugar sweeteners.
- the sweet taste potentiator is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste potentiator.
- the sweet taste potentiator is administered primarily to the stomach, small intestine, and/or large intestine of the patient.
- the sweet taste potentiator is encapsulated in a pharmaceutical carrier that releases the sweet taste potentiator into the stomach, small intestine, and/or large intestine of the patient.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste potentiator to taste cells in the mouth of the patient.
- a sixth aspect of the present invention relates to a method of treating obesity in a patient.
- This method involves selecting a patient that is obese, and administering an effective amount of a sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the patient under conditions effective to increase the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby inducing satiety in the patient and treating obesity in the patient.
- the patient is selected from the group of a human, a dog, and a non-ruminant livestock.
- the sweet taste potentiator activates one or more taste signaling proteins in the gastrointestinal endocrine cell of the patient.
- Suitable taste signaling proteins include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3 , G ⁇ 1? T1R2, T1R3,
- T1R2+T1R3 dimeric receptor Trpm5, Trpm4, PLC ⁇ 2, IP3 receptor type 3, Type I PDEs, and
- Suitable sweet taste potentiators include sweet tasting compounds selected from the group of sugars, peptides, proteins, natural non-sugar sweeteners, glycyrrhizic acid, stevioside, and artificial non- sugar sweeteners.
- the sweet taste potentiator is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste potentiator.
- the sweet taste potentiator is administered primarily to the stomach, small intestine, and/or large intestine of the patient.
- the sweet taste potentiator is encapsulated in a pharmaceutical carrier that releases the sweet taste potentiator into the stomach, small intestine, and/or large intestine of the patient.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste potentiator to taste cells in the mouth of the patient.
- This method involves administering an effective amount of a sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the mammal under conditions effective to increase the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby inducing satiety the mammal and promoting weight loss in the mammal.
- the mammal is selected from the group of a human, a dog, and a non-ruminant livestock.
- the sweet taste potentiator activates one or more taste signaling proteins in the gastrointestinal endocrine cell of the mammal.
- Suitable taste signaling proteins include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3 , G ⁇ 1? T1R2, T1R3, T1R2+T1R3 dimeric receptor, Trpm5, Trpm4, PLC ⁇ 2, IP 3 receptor type 3, Type I PDEs, and PDE-IA.
- Suitable sweet taste potentiators include sweet tasting compounds selected from the group of sugars, peptides, proteins, natural non-sugar sweeteners, glycyrrhizic acid, stevioside, and artificial non-sugar sweeteners.
- the sweet taste potentiator is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste potentiator.
- the sweet taste potentiator is administered primarily to the stomach, small intestine, and/or large intestine of the mammal.
- the sweet taste potentiator is encapsulated in a pharmaceutical carrier that releases the sweet taste potentiator into the stomach, small intestine, and/or large intestine of the mammal.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste potentiator to taste cells in the mouth of the mammal.
- An eighth aspect of the present invention relates to a method of regulating blood sugar levels in a mammal.
- This method involves administering an effective amount of a sweet taste inhibitor or sweet taste potentiator to a gastrointestinal endocrine cell in the gastrointestinal tract of the mammal under conditions effective to increase or decrease the synthesis and/or secretion of GLP-I by the gastrointestinal endocrine cell, thereby regulating insulin secretion by the mammal and regulating blood sugar levels in the mammal.
- the mammal is selected from the group of a human, a dog, and a non-ruminant livestock.
- the sweet taste inhibitor or sweet taste potentiator is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste inhibitor or sweet taste potentiator.
- the sweet taste inhibitor or sweet taste potentiator is administered primarily to the stomach, small intestine, and/or large intestine of the mammal.
- the sweet taste inhibitor or sweet taste potentiator is encapsulated in a pharmaceutical carrier that releases the sweet taste inhibitor or sweet taste potentiator into the stomach, small intestine, and/or large intestine of the mammal.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste inhibitor or sweet taste potentiator to taste cells in the mouth of the mammal.
- the sweet taste potentiator when a sweet taste potentiator is administered, activates one or more taste signaling molecules contained within a gastrointestinal endocrine cell in the mammal.
- taste signaling molecules include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3, G ⁇ 1? T1R2, T1R3, T1R2+T1R3 dimeric receptor, Trpm5, Trpm4, PLC ⁇ 2, IP 3 receptor type 3, Type I PDEs, and PDE-IA.
- Suitable sweet taste potentiators include sugars, peptides, proteins, natural non-sugar sweeteners, glycyrrhizic acid, stevioside, and artificial non-sugar sweeteners.
- a sweet taste potentiator when administered, insulin secretion in the mammal is downregulated, thereby increasing blood sugar levels in the mammal.
- the sweet taste inhibitor inhibits activation of one or more taste signaling molecules contained within a gastrointestinal endocrine cell in the mammal.
- Exemplary taste signaling molecules include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3 , G ⁇ ls T1R2, T1R3, T1R2+T1R3 dimeric receptor, Trpm5, Trpm4, PLC ⁇ 2, IP 3 receptor type 3, Type I PDEs, and PDE-IA.
- Suitable sweet taste inhibitors include lactisole, gurmarin, gymnemic acid, substituted sulfamates, and substituted cyclamate sulfamates.
- a sweet taste inhibitor when administered, insulin secretion in the mammal is upregulated, thereby decreasing blood sugar levels in the mammal.
- the sweet taste inhibitor or sweet taste activator is administered under conditions effective to maintain healthy blood sugar levels in the mammal.
- a ninth aspect of the present invention relates to a method of increasing or inducing carbohydrate absorption by a mammal. This method involves administering an effective amount of a sweet taste potentiator to the gastrointestinal tract of the mammal under conditions effective to increase expression and/or induce upregulation of SGLT-I in the mammal, thereby increasing or inducing carbohydrate adsorption by the mammal.
- the sweet taste potentiator activates one or more taste signaling proteins in the gastrointestinal endocrine cell of the mammal.
- Suitable taste signaling proteins include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3, G ⁇ 1? T1R2,
- Suitable sweet taste potentiators include sweet tasting compounds selected from the group of sugars, peptides, proteins, natural non-sugar sweeteners, glycyrrhizic acid, stevioside, and artificial non-sugar sweeteners.
- the sweet taste potentiator is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste potentiator.
- the sweet taste potentiator is administered primarily to the stomach, small intestine, and/or large intestine of the mammal.
- the sweet taste potentiator is encapsulated in a pharmaceutical carrier that releases the sweet taste potentiator into the stomach, small intestine, and/or large intestine of the mammal.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste potentiator to taste cells in the mouth of the mammal.
- a tenth aspect of the present invention relates to a method of treating a disorder associated with insufficient carbohydrate absorption in a patient.
- This method involves selecting a patient that has a disorder associated with insufficient carbohydrate absorption, and administering an effective amount of a sweet taste potentiator to the gastrointestinal tract of the patient under conditions effective to increase expression and/or induce upregulation of SGLT-I in the patient, thereby increasing or inducing carbohydrate adsorption by the patient and treating the disorder in the patient.
- the patient is selected from the group of a human, a dog, and a non-ruminant livestock.
- the sweet taste potentiator activates one or more taste signaling proteins in the gastrointestinal endocrine cell of the patient.
- Suitable taste signaling proteins include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3, G ⁇ ls T1R2, T1R3,
- T1R2+T1R3 dimeric receptor Trpm5, Trpm4, PLC ⁇ 2, IP 3 receptor type 3, Type I PDEs, and
- Suitable sweet taste potentiators include sweet tasting compounds selected from the group of sugars, peptides, proteins, natural non-sugar sweeteners, glycyrrhizic acid, stevioside, and artificial non- sugar sweeteners.
- the sweet taste potentiator is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste potentiator.
- the sweet taste potentiator is administered primarily to the stomach, small intestine, and/or large intestine of the patient.
- the sweet taste potentiator is encapsulated in a pharmaceutical carrier that releases the sweet taste potentiator into the stomach, small intestine, and/or large intestine of the patient.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste potentiator to taste cells in the mouth of the patient.
- Disorders that can be treated according to this aspect of the present invention include anorexia, bulimia, intestinal malabsorption syndromes, and celiac disease.
- An eleventh aspect of the present invention relates to a method of promoting weight gain in a mammal.
- This method involves administering an effective amount of a sweet taste potentiator to the gastrointestinal tract of the mammal under conditions effective to increase expression and/or induce upregulation of SGLT-I in the mammal, thereby increasing or inducing carbohydrate adsorption by the mammal and promoting weight gain in the mammal.
- the mammal is selected from the group of a human, a dog, and a non-ruminant livestock.
- the sweet taste potentiator activates one or more taste signaling proteins in the gastrointestinal endocrine cell of the mammal.
- Suitable taste signaling proteins include heterotrimeric gustducin, ⁇ -gustducin, G ⁇ 13 , G ⁇ 3, G ⁇ 1? T1R2,
- Suitable sweet taste potentiators include sweet tasting compounds selected from the group of sugars, peptides, proteins, natural non-sugar sweeteners, glycyrrhizic acid, stevioside, and artificial non-sugar sweeteners.
- the sweet taste potentiator is administered in a pharmaceutical formulation.
- the pharmaceutical formulation consists essentially of the sweet taste potentiator.
- the sweet taste potentiator is administered primarily to the stomach, small intestine, and/or large intestine of the mammal.
- the sweet taste potentiator is encapsulated in a pharmaceutical carrier that releases the sweet taste potentiator into the stomach, small intestine, and/or large intestine of the mammal.
- the pharmaceutical carrier can be formulated to substantially prevent administration of the sweet taste potentiator to taste cells in the mouth of the mammal.
- Suitable pharmaceutical formulations for use in the methods of the present invention include the sweet taste inhibitor/potentiator and any pharmaceutically acceptable adjuvants, carriers, excipients, and/or stabilizers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.
- the percentage of the sweet taste inhibitor/potentiator in these compositions may, of course, be varied and may conveniently be from about 0.01% to about 99% by weight, preferably between about 0.1% to about 99% percent, more preferably from about 2% to about 60%, of the weight of the unit together with the adjuvants, carriers and/or excipients.
- suitable amounts of the sweet taste inhibitor/potentiator include about 10%, about 20%, about 30%, about 40%, about 50%, about 70%, about 80%, and about 90% of the weight of the unit.
- the amount of the sweet taste inhibitor/potentiator in such therapeutically useful compositions is such that a suitable dosage will be obtained.
- the sweet taste inhibitor/potentiator may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
- the tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, or alginic acid; and a lubricant such as magnesium stearate.
- a liquid carrier such as a fatty oil.
- Solutions or suspensions of the sweet taste inhibitor/potentiator can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.
- oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
- water, saline, aqueous dextrose and related sugar solutions are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
- Administration of the sweet taste inhibitor/potentiator can be accomplished either via systemic administration to the subject or via targeted administration to affected tissues, organs, and/or cells.
- the therapeutic agent i.e., a sweet taste inhibitor/potentiator
- the therapeutic agent will be administered to a patient in a vehicle that delivers the therapeutic agent(s) to the target cell, tissue, or organ.
- the therapeutic agent will be administered as a pharmaceutical formulation, such as those described above.
- Exemplary routes of administration include, without limitation, orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intraventricularly, and intralesionally; by intratracheal inoculation, aspiration, airway instillation, aerosolization, nebulization, intranasal instillation, oral or nasogastric instillation, intraperitoneal injection, intravascular injection, intravenous injection, intra-arterial injection (such as via the pulmonary artery), intramuscular injection, and intrapleural instillation; by application to mucous membranes (such as that of the nose, throat, bronchial tubes, genitals, and/or anus); and by implantation of a sustained release vehicle.
- intratracheal inoculation aspiration, airway instillation, aerosolization, nebulization, intranasal instillation, oral or nasogastric instillation, intraperitoneal injection, intravascular
- a peptide of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
- suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
- the peptides of the present invention also may be administered in a non-pressurized form.
- Exemplary delivery devices include, without limitation, nebulizers, atomizers, liposomes (including both active and passive drug delivery techniques) (63-70), transdermal patches, implants, implantable or injectable sweet taste inhibitor/potentiator depot compositions, and syringes.
- Other delivery systems which are known to those of skill in the art can also be employed to achieve the desired delivery of the sweet taste inhibitor/potentiator to the desired organ, tissue, or cells.
- Administration can be carried out as frequently as required and for a duration that is suitable to provide the desired effect.
- administering can be carried out once or multiple times, and can be carried out with a single sustained-release dosage formulation or with multiple (e.g., daily) doses.
- the amount to be administered will, of course, vary depending upon the particular conditions and treatment regimen.
- the amount/dose required to obtain the desired effect may vary depending on the agent, formulation, disease or condition, the duration for which treatment is desired, and the individual to whom the agent is administered.
- Effective amounts can be determined empirically by those of skill in the art.
- determination of effective amounts for in vivo administration may involve in vitro assays in which varying doses of the sweet taste inhibitor/potentiator is administered to cells in culture and the concentration effective for achieving the desired result is determined in order to calculate the concentration required in vivo.
- Effective amounts may also be based on in vivo animal studies.
- Example 1 Gut-expressed Gustducin and Taste Receptors Regulate Secretion of Glucagon-like Peptide-1.
- GLP-I Glucagon-like peptide-1
- gut endocrine L cells released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-I secretion is unknown.
- human duodenal L cells express sweet taste receptors, the taste G-protein gustducin, and several other taste transduction elements.
- Mouse intestinal L cells also express ⁇ -gustducin. Ingestion of glucose by ⁇ -gustducin null mice revealed deficiencies in secretion of GLP- 1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from ⁇ - gustducin null mice showed markedly defective GLP-I secretion in response to glucose.
- the human L cell line NCI-H716 expresses ⁇ -gustducin, taste receptors and several other taste signaling elements.
- GLP-I release from NCI-H716 cells was promoted by sugars and the non-caloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for ⁇ -gustducin.
- lactisole or siRNA lactisole or siRNA for ⁇ -gustducin.
- L cells of the gut "taste” glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-I secretion in gut "taste cells” may provide an important treatment for obesity, diabetes and abnormal gut motility.
- T1R3 and gustducin have a role in glucose-mediated incretin release and may serve as the previously unknown gut lumen glucose sensor.
- a-gusf f ⁇ mice maintained their GIP concentrations at a near constant level.
- the wild-type mice showed a higher and more transient rise in GIP concentrations, peaking at 20 min after glucose administration.
- Glucose homeostasis was also altered in the a-gusf f ⁇ mice: plasma glucose concentrations after gavage-administration of glucose (Figure 2B) or after eating lab chow following an 18h fast ( Figure 2C) were higher in the a-gusf ' mice than in their wild-type littermates, and were maintained at this elevated level for more than 2hrs.
- Gavage-administration of glucose might stimulate ⁇ -gustducin-expressing brush cells of the stomach, depending upon what receptors are expressed in these cells. Conceivably, these brush cells might contribute to duodenal release of GLP-I.
- GLP-I secretion in a-gusf f ⁇ and a-gust +/+ mice in which glucose was infused directly into the duodenum that had been isolated from the stomach and the rest of the small intestine, but remained in circulatory contact.
- plasma concentrations of GLP-I peaked 10 min after duodenal infusion of 10% glucose, and then returned to baseline within 20 min (Figure 2D).
- That glucose stimulated GLP-I release from the tissue and villi of a-gusf A mice indicates that one or more gustducin-independent mechanisms also contribute to this effect: possibilities include effects on L cell channels (e.g. closure of K ATP channels or transporter associated electrogenic currents), enhanced L cell metabolism, or sweet receptor coupling to other L cell-expressed G proteins.
- L cell channels e.g. closure of K ATP channels or transporter associated electrogenic currents
- enhanced L cell metabolism e.g. closure of K ATP channels or transporter associated electrogenic currents
- sweet receptor coupling to other L cell-expressed G proteins.
- PYY in NCI-H716 cells a human enteroendocrine L cell line, and detected the presence in this cell line of several taste signaling elements: ⁇ -gustducin, G ⁇ 3 , G ⁇ 13 , PLC ⁇ 2, TRPM5, TlRl, T1R2, T1R3, IP 3 type III receptor (IP 3 R-3), numerous phosphodiesterases (Pdes) including Pdela, which has been described in taste cells (4), the fatty acid receptor Gpr40, and the bile acid receptor Tgr5 (25) ( Figures 9A-B).
- Example 2 Immunofluorescence and Confocal Microscopy.
- the human paraffin-embedded duodenal sections were from anonymous post-mortem samples. Tissue processing and immunofluorescence were performed as described previously (22). For double immuno fluorescent staining sections were incubated overnight at 4°C with both primary antibodies (Table 1), washed and incubated with secondary antibodies (Alexa 568 donkey anti-goat antibody for GLP-I, Alexa 488 donkey anti-rabbit antibody for others and Cy3 -conjugated goat anti-rabbit antibody for GLP-I and GLP-2 in the gustducin- GFP mice) for Ih, incubated with TOPRO ® -3 (Molecular Probe) for nuclear staining, washed and mounted with fluorescence mounting medium, Vectashield ® (Vector Laboratories).
- Example 3 Laser capture microdissection of single cells and PCR.
- mice [0129] The design and production of ⁇ -gustducin null (a-gusf " ) mice (6) and the mice expressing GFP from the 7.6-kb promoter region upstream from the ⁇ -gustducin gene (gustducin-GFP mice) have been described (12, 23, 24). The genetic background of all mice is C57BL/6. For comparisons of wild type and ⁇ -gustducin null mice a-gust +/+ , a-gust +/ ⁇ and a-gusf f ⁇ littermate mice were generated from a-gust +/ ⁇ x a-gust +/ ⁇ matings.
- Example 5 Gavage-administration of Glucose Load in Wild Type and Transgenic Mice.
- DPPIV dipeptidyl peptidase IV
- Example 6 Tissue Measurements of Incretin Levels.
- Example 7 Blood Glucose in Wild Type and Transgenic Mice After Alimentation.
- the blood glucose concentration was measured from tail vein by Freestyle glucometer (Therasense): measurements were taken immediately before feeding (time 0) and 15, 30, 45, 60, 90, and 120 min after feeding.
- Example 8 Secretion of GLP-I from Intact Duodenum in Vivo.
- duodenum for mincing median laparotomy was done as above, then an incision was made in the antimesenteric side of the duodenum near the gastroduodenal junction, and the duodenum was flushed with 20ml of HBSS (calcium and magnesium free). The proximal 5cm of duodenum were dissected out, the serosal layer stripped off, then minced pieces of tissue ( ⁇ l-2 mm 2 ) were placed in culture medium (40) (DMEM with or without 10% glucose with 10% fetal bovine serum, 100U/ml penicillin, lOOmg/ml streptomycin and 20 ⁇ l/ml DPPIV inhibitor).
- culture medium 40
- Tissues were incubated in 5% CO 2 for Ih at 37°C, the media was collected and GLP-I levels assayed to determine unstimulated (baseline control) levels as well as that released into media.
- cell viability was confirmed by exclusion of trypan blue.
- Duodenal villi were obtained from the proximal duodenum by scraping with mild pressure from the short edge of a glass slide. The isolated tissue was allowed to settle at the bottom of a tube on ice and than was washed three times with HBSS. After the final wash purified villi were resuspended in DMEM as above for minced tissue, aliquoted and incubated as above but with 5% glucose to determine GLP-I release.
- Example 11 RNA Isolation and RT-PCR of NCI-H716 Cells.
- Qiagen OneStep RT-PCR kit
- Example 12 siRNA preparation and NCI-H716 Cell Transfection.
- siRNA sequence targeting human ⁇ -gustducin (GenBank Accession No.
- XM 294370 was from position 177-195 relative to the start codon. This ⁇ -gustducin sequence was reversed and used as unspecific siRNA control. 21-nt RNAs were purchased from Dharmacon in deprotected and desalted form, and the formation of siRNA duplex (annealing) was performed according to the manufacturer's instructions (Dharmacon). Subconfluent differentiated NCI-H716 cells were transiently transfected with siRNAs using Lipofectamine 2000TM according to the manufacturer's protocol (Life Technologies). The entire mixture was then added to the cells in one dish resulting in a final concentration of 30OnM for the siRNAs. Cells were usually studied 48h after transfection.
- Example 13 Calcium Imaging.
- Example 15 Membrane Preparation, Labeling and Immunoprecipitation of Membrane-associated G Proteins.
- GTP-azidoanilide was from ALT.
- the clarified supernatants 160 ⁇ l were transferred to tubes containing 5-1 O ⁇ l polyclonal rabbit antisera raised against G ⁇ -gustducin, G ⁇ s (Santa Cruz) and G ⁇ ,2 (Affinity BioReagents). Immunoprecipitation was performed as described previously (42). An aliquot (20 ⁇ l) of the samples was subjected to SDS-PAGE. [0141] Gels were transferred to membrane, and band intensity was quantified by electronic autoradiography using a Storm (Molecular Dynamics). The protein concentration was determined by the Bradford method.
- GLP-I data represents means ⁇ s.e.m. Differences between mean values for variables within individual experiments were compared statistically by ANOVA and followed by post hoc testing with Scheffe's test. Comparisons were performed using Graphpad Prism (GraphPad Software, Inc. San Diego, CA). P ⁇ 0.05 was viewed as significant.
- ⁇ -gustducin null mice are defective for secretion of GLP-I in response to lumenal glucose.
- isolated duodenum and duodenal villi from ⁇ -gustducin null mice likewise show deficient release of GLP-I in response to glucose.
- NCI-H716 enteroendocrine L cells we observed GPCR-mediated activation of ⁇ -gustducin by glucose and sucrose. Decreasing ⁇ -gustducin expression in NCI-H716 cells by siRNA resulted in decreased glucose-mediated GLP-I secretion.
- GLP-I receptor agonists and GLP-I analogs are under intense investigation as treatments for type 2 diabetes and obesity.
- Exendin-4 an agonist of the GLP-I receptor, has recently been approved for human use in type 2 diabetes because of its insulinotropic and weight-reducing properties.
- An alternative mode of modulating signaling through GLP-I receptors would be to develop secretagogues to increase plasma levels of endogenous ligands, analogous to the use of sulfonylureas in increasing insulin secretion for the treatment of type 2 diabetes.
- Example 17 T1R3 and Gustducin in Gut Sense Sugars to Regulate Expression of Na + -Glucose Cotransporter 1
- SGLTl sodium-dependent glucose transporter isoform 1
- Dietary sugars are transported from the intestinal lumen into absorptive enterocytes by the sodium-dependent glucose transporter isoform 1 (SGLTl). Regulation of this protein is important for the provision of glucose to the body and avoidance of intestinal malabsorption. Although expression of SGLTl is regulated by luminal monosaccharides, the luminal glucose sensor mediating this process was unknown. Examples 17-29 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 SGLTl mRNA and protein.
- TlR taste receptors and G ⁇ gus t are expressed in the mouse small intestinal epithelium and proposed that they function as luminal sugar sensors to control SGLTl expression in response to dietary sugar (20).
- dietary sugars and artificial sweeteners increase SGLTl mRNA and protein expression and glucose-absorptive capacity in wildtype mice, but not in T1R3 or G ⁇ gust knockout mice.
- TlR taste receptors and G ⁇ gust are expressed in human and mouse enteroendocrine cells.
- T1R2+T1R3 sweet receptor is involved in intestinal sugar sensing, then artificial sweeteners that activate this receptor in taste cells of the tongue might increase intestinal expression of SGLTl .
- Example 20 Expression of TlRs and G ⁇ gust in Enteroendocrine Cells.
- T 1R3 -expressing cells In mouse villi, most T 1R3 -expressing cells expressed G ⁇ gus t ( Figure 15B) or were in close proximity to such cells (Figure 15C). In human duodenum, virtually all T 1R3 -expressing cells also expressed G ⁇ t ( Figures 15F-G). Omission of the primary antibodies for gustducin or T1R3 showed no nonspecific immunoreactivity in small intestine ( Figure 19). The cells that contain TlRs and appear to be either triangular or flask-like in shape, suggesting they are of enteroendocrine type (Figure 15G), and immunohistochemistry of serial sections of mouse intestine showed that the enteroendocrine cell marker chromogranin was indeed coexpressed with G ⁇ guSt ( Figure 151).
- Example 21 Sucralose Activates Sweet Taste Receptors on Enteroendocrine Cells to Elicit Hormone Secretion.
- GLUTag and STC-I cells two mouse enteroendocrine cell lines, have been shown to secrete hormones, including glucagon- like peptide- 1 (GLP-I) and glucose-dependent insulinotrophic peptide (GIP), in response to glucose (50, 51). GLUTag cells also respond to fructose and nonmetabolizable glucose analogues (35, 51). STC-I cells have been shown to express TlRs, T2Rs, and taste G proteins (20, 47). We have examined GLUTag cells and found that they express TlRs and G ⁇ g ust (Figure 20).
- GLUTag cells Tonically release GLP-I and GIP into the culture medium; however, the addition of sucralose elicited increased release of GLP-I ( Figure 16A) and GIP ( Figure 16B) and an elevation of intracellular calcium ( Figure 22).
- Example 22 Mice, Diets, and Tissue Collection.
- mice 8-week-old C57BL/6 G ⁇ gus t ' and T1R3 ⁇ ⁇ mice and their wild-type littermates of the same origin were used. Animals were placed individually in standard tub cages in a room with automatically controlled temperature, humidity, and 12-h light/12-h dark cycle. Mice were divided into three groups, with equal number of wild-type, and T1R3 ⁇ ⁇ animals of both genders. Group one was fed a high-carbohydrate diet (High-carbohydrate Diet (70%), Testdiet #5810; Purina Mills, Richmond, IN), group two was fed a low-carbohydrate diet (Low-carbohydrate Diet (1.9%), Testdiet #590N; Purina Mills).
- high-carbohydrate diet High-carbohydrate Diet (70%), Testdiet #5810; Purina Mills, Richmond, IN
- group two was fed a low-carbohydrate diet (Low-carbohydrate Diet (1.9%), Test
- the carbohydrate content of both diets consisted of sucrose.
- the two diets are equicaloric, being 3.73 and 3.86 (Kcal/g) (42) for the high- and low-carbohydrate diets, respectively.
- Group three was maintained on the low-carbohydrate diet, and 2 mM sucralose solution was supplied instead of water.
- a 2 mM concentration of sucralose was used because C57BL/6 wild-type mice displayed markedly increased behavioral responses to this concentration (11). The sucralose solution was changed every second day. All diets were provided ad libitum.
- mice were fed the low-carbohydrate diet and given the following artificial sweetener solutions instead of water: 10 mM acesulfame K, 20 mM saccharin, and 1 mM aspartame. Saccharin and acesulfame K at these concentrations taste sweet to mice and activate expressed mouse T1R2+T1R3 (4, 9-11). Aspartame tastes sweet to humans but not to rodents; neither 1 mM nor 10 mM concentrations of aspartame activate the mouse sweet receptor (5, 9, 10). After 2 weeks, animals were killed by cervical dislocation, and intestines were immediately excised, flushed with ice-cold saline, immersed in liquid nitrogen, and stored at -80 0 C until use.
- RNA isolated from intestinal tissue by using the RNeasy Mini Kit with on-column DNase 1 digestion (Qiagen, Crawley, U.K.) was used for cDNA synthesis using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo(dT)i2-i8 primers.
- cDNA was cleaned up by using the Machery-Nagel Nucleospin extract kit (AB Gene, Epsom, U.K.), and 50 ng of cDNA was used per reaction.
- PCR primers and probes for the amplification of TlRl, T1R2, T1R3, G ⁇ gust , and the NaVglucose cotransporter, SGLTl, (F AM/T AMRA), along with ⁇ -actin (JOE/T AMRA- labeled) were designed by using Primer Express (Applied Biosystems, Warrington, U.K.), and purchased from Eurogentec (Seraing, Belgium) (Table 3). For real-time PCRs, the enzyme was activated by heating at 95°C for 2 min.
- a two-step PCR procedure was used, 15 s at 95°C and 60 s at 60 0 C for 45 cycles in a PCR mix containing 5 ml of cDNA template, lxJumpstart qPCR master mix (Sigma- Aldrich, Poole, U.K.), 900 nM concentrations of each primer, and 250 nM probe in a total volume of 25 ml.
- the ⁇ -actin primers were primer-limiting and used at 600 nM. All reactions were performed in a Rotor-Gene 3000 (Corbett Research). Relative amounts of mRNA were normalized to ⁇ -actin mRNA within each sample.
- Assays on Demand Taqman primer/probe mixes to human SGLTl (FAM/MGB) and ⁇ -actin (VIC/MGB) were purchased from Applied Biosystems.
- Example 24 Western Blotting and Glucose Transport.
- BBMV were isolated from mouse small intestine by the method described (59) in the presence of a mixture of protease inhibitors (Roche Diagnostics, Indianapolis, IN). Western blot analysis was performed as described (44) with antisera to SGLTl (44, 45), villin (clone 1D2C3; Abeam, Cambridge, U.K.), and ⁇ -actin (clone AC-15; Sigma- Aldrich). Immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibodies (DAKO, Carpenteria, CA) and enhanced chemiluminescence (Amersham Biosciences).
- 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).
- 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 (44, 59).
- 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). The filter was washed five times with 1 ml of stop buffer, and the radioactivity retained on the filter was measured by liquid scintillation counting. All uptakes were measured in triplicate.
- Morphometry was performed on 5- ⁇ m sagittal sections prepared from paraffin-embedded intestinal samples and stained with hematoxylin and eosin. Digital images were captured with an Eclipse microscope and DXM 1200 digital camera (Nikon, East Rutherford, NJ) and analyzed by using Image J software (National Institutes of Health, Bethesda, MD). Crypt depth was measured as the distance from crypt base to crypt-villus junction on an average of 17 well oriented crypts from 10 different sagittal sections of intestinal tissues from mice maintained on different diets and sweetener supplements. Villus height was measured as villus base to villus tip from an average of 18 well oriented villi.
- Tissue sections (fixed for 4 h in 4% paraformaldehyde in PBS) were paraffin wax embedded and sectioned at 5- to 7-mm thickness. After dewaxing and hydration, sections were permeabilized by a 20-min incubation in 0.2 M HCl, followed by two 3-min washes in 2xSSC, 1 h of incubation at 37°C in 50 mM Tris ⁇ Cl (pH 7.4) containing 2 ⁇ g/ml proteinase K (Sigma), and two 3-min washes in 0.2% glycine/PBS.
- Sections were prehybridized in hybridization buffer [50% deionized formamide/300 mM NaCl/20 mM Tris ⁇ Cl (pH 8.0)/5 mM EDTA/lxDenhardt's/lxRNA Protect (Sigma)/100 mg/ml dextran sulfate] for 1 h at 60 0 C and hybridized overnight at 25°C in hybridization buffer containing 100 ⁇ g/ml tRNA and 100 ng/ml DIG-labeled probe (Table 4).
- slides were washed for 1 h in 2xSSC, followed by 4 h at 25°C in 300 mM NaCl, 200 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50% formamide, lxDenhardt's and then 30 min in 2xSSC and 30 min in 0.2xSSC.
- Bound DIG-labeled probes were detected by 5 -min equilibration in digoxigenin-alkaline phosphatase (DIG-AP) buffer [100 mM Tris-HCl (pH 7.5)/150 mM NaCl], followed by 30- min block in DIG-AP buffer containing 1% (wt/vol) DIG blocking reagent (Roche), overnight incubation in DIG-AP buffer containing 1% (wt/vol) DIG blocking reagent and anti-DIG AP-conjugated antibody diluted 1 :1,000, 5-min wash in DIG-AP buffer, 5-min equilibration in NBT/BCIP buffer [100 mM Tris-HCl (pH 9.5)/100 mM NaCl/50 mM MgCl 2 ], 1-h incubation in the dark in NBT/BCIP buffer containing 10% polyvinyl alcohol and NBT/ BCIP mixture (Roche) diluted 1 :50, and 5-min wash in 10 m
- Sections of paraffin-embedded tissue (6- ⁇ m thick) were dewaxed in xylene and rehydrated; antigen retrieval was performed by incubation of tissue sections with 0.05% pronase (Roche) solution for 15 min at 37°C in a humidified chamber. Sections were washed in PBS and blocked for 30 min in 3% BSA, 0.3% Triton X-100, 2% goat serum, and 0.1% Na azide.
- Serial sections were incubated with primary antibody against G ⁇ gust (Santa Cruz Biotechnology, Santa Cruz, CA), 1 :1,000; T1R2 (Santa Cruz Biotechnology), 1 :500; or T1R3 (11) 1 :1 ,000, washed in PBS three times for 5 min, and incubated with Cy3 -conjugated goat anti-rabbit secondary antibody, (Jackson ImmunoResearch, West Grove, PA), and FITC- conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR). Images from serial sections were merged by using Volocity imaging software for confocal microscope (Impro vision).
- the Zenon Alexa Fluor Rabbit IgG Labeling kit (Molecular Probes) was used: in brief, antibodies were preincubated with Alexa Fluor 488-labeled fab fragments (green) or Alexa Fluor 594-labeled fab fragments (red) for 10 min after 10-min incubation with blocking reagent (nonspecific IgG) to absorb excess labeling reagent. Directly labeled rabbit anti- G ⁇ gust /T1R2/T1R3 antibodies were applied for 1 h. The sections were washed in PBS three times for 5 min after final incubation and mounted.
- Negative controls omitting the primary antibodies for gustducin and T1R3 were done with mouse and human tissues and were uniformly negative.
- antigen retrieval was performed by autoclaving in 10 mM Tris buffer (pH 10) for 22 min, and slides were pretreated for peroxidase blocking by incubation in 3% H2O2/PBS for 15 min. Sections were blocked for 1 h at room temperature in 5% B S A/PBS.
- the slides were then incubated at room temperature overnight with primary antibodies to SGLTl or chromogranin A+B (Abeam), diluted 1 : 100 in 1% BSA/TBS, washed, and incubated with horseradish peroxidase-conjugated swine anti- rabbit secondary antibody (DAKO) diluted 1 :200 in 1% BSA/TBS for 2 h at room temperature.
- DAKO horseradish peroxidase-conjugated swine anti- rabbit secondary antibody
- Another three 5-min washes were performed, and then the slides were developed in 0.05% DAB/0.03% H 2 O 2 /50 mM Tris ⁇ Cl (pH 7.6) for 10 min at room temperature in the dark.
- the slides were then counterstained in 1% chloroformextracted methyl green and mounted by using DPX (Raymond Lamb, Eastbourne, Eastshire, U.K.).
- Example 28 GLUTag Cells, GLP-I, GIP, and Ca 2+ Imaging Assays.
- GLUTag cells 60 were incubated in PBS with or without gurmarin (3 mg/ml) for 15 min, followed by incubation (1 h) with sucralose (50 mM final concentration).
- the concentrations of GLP-I and GIP (total) (active form) in the culture medium were determined by using GLP-I and GIP ELISA kits (LincoResearch).
- GLUTag cells were transiently transfected by using Lipofectamine 2000 (Invitrogen) with the following plasmids: Gal6/gust44 [a plasmid encoding a Gal 6- G ⁇ gus t chimeric G protein ⁇ -subunit that couples to sweet taste receptors (61)], YC3.60 (a ratiometric fluorescent indicator of free Ca 2+ ) (62), and REEP-EI (a plasmid encoding a receptor-enhancing protein that promotes function of olfactory (62) and gustatory receptors.
- Gal6/gust44 a plasmid encoding a Gal 6- G ⁇ gus t chimeric G protein ⁇ -subunit that couples to sweet taste receptors (61)
- YC3.60 a ratiometric fluorescent indicator of free Ca 2+
- REEP-EI a plasmid encoding a receptor-enhancing protein that promotes function of olfactory (62) and gustatory receptors.
- Results are expressed as means ⁇ SD or means ⁇ SEM, as indicated. Data were analyzed, as appropriate for the data set, by ANOVA with Dunnett's post test, or by unpaired two-tailed Student's t test (GraphPad Prism). P ⁇ 0.05 was considered significant. [0165] We thank Drs. A. Ellis (Consultant Gastroenterologist, Royal Liverpool and
- the Na /glucose cotransporter SGLTl 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, thus, is important for maintenance of glucose homeostasis.
- Examples 17-29 show that T1R3 and G ⁇ gus t are expressed in enteroendocrine cells and are required for the enhanced expression of SGLTl shown by enterocytes in vivo in response to luminal sugars or sweeteners.
- G ⁇ gust and TlRs are expressed in enteroendocrine cells, whereas SGLTl is expressed in enterocytes, implies that a chemical signaling event takes place between the chemosensory enteroendocrine cells and absorptive enterocytes.
- Enteroendocrine cells in response to luminal nutrients, secrete endocrine hormones including cholecystokinin (CCK), peptide tyrosine tyrosine (PYY), neurotensin, GLP-I, GLP-2, and GIP (35, 51, 54-56).
- GLP-I and GIP known as incretins, are secreted in response to dietary sugars, and influence glucose transport, metabolism, and homeostasis (57).
- Luminal glucose above a threshold level may activate in enteroendocrine sensor cells a signaling pathway involving T1R2+T1R3, G ⁇ gust , and other taste signaling elements, resulting in the secretion of GLP-I, GIP, and/or other endocrine products.
- enteroendocrine sensor cells a signaling pathway involving T1R2+T1R3, G ⁇ gust , and other taste signaling elements, resulting in the secretion of GLP-I, GIP, and/or other endocrine products.
- T1R2+T1R3, G ⁇ gust , and other taste signaling elements resulting in the secretion of GLP-I, GIP, and/or other endocrine products.
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CANI PATRICE D ET AL: "Dietary non-digestible carbohydrates promote L-cell differentiation in the proximal colon of rats" BRITISH JOURNAL OF NUTRITION, vol. 98, no. 1, July 2007 (2007-07), pages 32-37, XP002593206 ISSN: 0007-1145 * |
CANI PATRICE D ET AL: "Improvement of glucose tolerance and hepatic insulin sensitivity by oligofructose requires a functional glucagon-like peptide 1 receptor" DIABETES, vol. 55, no. 5, May 2006 (2006-05), pages 1484-1490, XP002593207 ISSN: 0012-1797 * |
See also references of WO2009026389A2 * |
SEIFARTH C ET AL: "Prolonged and enhanced secretion of glucagon-like peptide 1 (7-36 amide) after oral sucrose due to .alpha.-glucosidase inhibition (acarbose) in Type 2 diabetic patients" DIABETIC MEDICINE, JOHN WILEY & SONS, LTD, GB, vol. 15, no. 6, 1 June 1998 (1998-06-01), pages 485-491, XP002576743 ISSN: 0742-3071 * |
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