Glucose sensing by gut endocrine cells and activation of the vagal afferent pathway is impaired in a rodent model of type 2 diabetes mellitus

Glucose in the gut lumen activates gut endocrine cells to release 5-HT, glucagon-like peptide 1/2 (GLP-1/2), and glucose-dependent insulinotropic polypeptide (GIP), which act to change gastrointestinal function and regulate postprandial plasma glucose. There is evidence that both release and action of incretin hormones is reduced in type 2 diabetes (T2D). We measured cellular activation of enteroendocrine and enterochromaffin cells, enteric neurons, and vagal afferent neurons in response to intestinal glucose in a model of type 2 diabetes mellitus, the UCD-T2DM rat. Prediabetic (PD), recent-diabetic (RD, 2 wk postonset), and 3-mo diabetic (3MD) fasted UCD-T2DM rats were given an orogastric gavage of vehicle (water, 0.5 ml /100 g body wt) or glucose (330 μmol/100 g body wt); after 6 min tissue was removed and cellular activation was determined by immunohistochemistry for phosphorylated calcium calmodulin-dependent kinase II (pCaMKII). In PD rats, pCaMKII immunoreactivity was increased in duodenal 5-HT (P < 0.001), K (P < 0.01) and L (P < 0.01) cells in response to glucose; glucose-induced activation of all three cell types was significantly reduced in RD and 3MD compared with PD rats. Immunoreactivity for GLP-1, but not GIP, was significantly reduced in RD and 3MD compared with PD rats (P < 0.01). Administration of glucose significantly increased pCaMKII in enteric and vagal afferent neurons in PD rats; glucose-induced pCaMKII immunoreactivity was attenuated in enteric and vagal afferent neurons (P < 0.01, P < 0.001, respectively) in RD and 3MD. These data suggest that glucose sensing in enteroendocrine and enterochromaffin cells and activation of neural pathways is markedly impaired in UCD-T2DM rats.     

Gut bitter taste receptor signalling induces ABCB1 through a mechanism involving CCK

T2Rs (bitter taste-sensing type 2 receptors) are expressed in the oral cavity to prevent ingestion of dietary toxins through taste avoidance. They are also expressed in other cell types, including gut enteroendocrine cells, where their physiological role is enigmatic. Previously, we proposed that T2R-dependent CCK (cholecystokinin) secretion from enteroendocrine cells limits absorption of dietary toxins, but an active mechanism was lacking. In the present study we show that T2R signalling activates ABCB1 (ATP-binding cassette B1) in intestinal cells through a CCK signalling mechanism. PTC (phenylthiocarbamide), an agonist for the T2R38 bitter receptor, increased ABCB1 expression in both intestinal cells and mouse intestine. PTC induction of ABCB1 was decreased by either T2R38 siRNA (small interfering RNA) or treatment with YM022, a gastrin receptor antagonist. Thus gut ABCB1 is regulated through signalling by CCK/gastrin released in response to PTC stimulation of T2R38 on enteroendocrine cells. We also show that PTC increases the efflux activity of ABCB1, suggesting that T2R signalling limits the absorption of bitter tasting/toxic substances through modulation of gut efflux membrane transporters.     

Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle

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

Effect of GLP-2 on mucosal morphology and SGLT1 expression in tissue-engineered neointestine

Using tissue-engineering techniques, we have developed a neointestine that regenerates the structural and dynamic features of native small intestine. In this study, we tested neointestinal responsiveness to glucagon-like peptide 2 (GLP-2). Neointestinal cysts were engineered by seeding biodegradable polymers with neonatal rat intestinal organoid units. The cysts were matured and anastomosed to the native jejunum of syngeneic adult recipients. Animals were treated with GLP-2 [Gly2] (twice daily, 1 microg/g body wt) or vehicle alone (control) for 10 days. Rats were then killed, and tissues were harvested for analysis. Na+-glucose cotransporter (SGLT1) mRNA expression was assessed with Northern blotting and in situ hybridization. SGLT1 protein was localized by using immunofluorescence. GLP-2 administration resulted in 1.8- and 1.7-fold increases (P < 0.05) in neointestinal villus height and crypt depth, respectively. GLP-2 administration also resulted in a 2.4-fold increase (P < 0.01) in neomucosal SGLT1 mRNA expression. SGLT1 mRNA expression was localized to enterocytes throughout the villi, and SGLT1 protein was localized to the brush border of enterocytes along the entire length of villi from the neointestine of GLP-2-treated animals. The response of tissue-engineered neointestine to exogenous GLP-2 includes mucosal growth and enhanced SGLT1 expression. Therefore, tissue-engineering principles may help in dissecting the regulatory mechanisms mediating complex processes in the intestinal epithelium.     

Transformation of postingestive glucose responses after deletion of sweet taste receptor subunits or gastric bypass surgery

The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) + type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K(+) (K(ATP)) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3(-/-) mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3(+/+), but not T1R3(-/-), ileum explants; this secretion was not mimicked by the K(ATP) channel blocker glibenclamide. T1R2(-/-) mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was K(ATP) channel-dependent and glucose-specific emerged in the large intestine of T1R3(-/-) mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB.     

Influence of glucagon-like peptide 2 on energy homeostasis

Glucagon like peptide-2 (GLP-2) is a gastrointestinal hormone released from enteroendocrine L-type cells together with glucagon like peptide-1 in response to dietary nutrients. GLP-2 acts through a specific receptor, the GLP-2 receptor, mainly located in the gut and in the brain. Classically, GLP-2 is considered a trophic hormone involved in the maintenance of intestinal epithelial morphology and function. This role has been targeted for therapies promoting repair and adaptive growth of the intestinal mucosa. Recently, GLP-2 has been shown to exert beneficial effects on glucose metabolism specially in conditions related to increased uptake of energy, such as obesity. Several actions of GLP-2 are related to a positive energy balance: GLP-2 increases not only the absorptive surface, but also expression and activity of epithelial brush-border nutrient transporters and digestive enzymes, intestinal blood flow, postprandial chylomicron secretion and it inhibits gastrointestinal motility, providing the opportunity to increase absorption of nutrients. Other actions, including anorexigenic effects, appear in opposition to the energy intake. In this review, we discuss the GLP-2 functions related to energy homeostasis. GLP-2 could be considered an hormone causing positive energy balance, which, however has the role to mitigate the metabolic dysfunctions associated with hyper-adiposity.     

Differential rAAV2 transduction efficiencies and insulin secretion profiles in pure and co-culture models of human enteroendocrine L-cells and enterocytes

BACKGROUND: Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than insulin injections. Previously, we developed an engineered human enteroendocrine L-cell model for regulated insulin release via recombinant adeno-associated virus serotype 2, or rAAV2, transduction. The aim of this study was to evaluate the efficiency and selectivity of rAAV2-mediated insulin gene delivery to enteroendocrine L-cells in co-culture with a prevailing number of enterocytes, which are the predominant cell type in intestinal epithelium. METHODS: We tested rAAV2 transduction in pure and co-culture models of human cell lines of enterocytes (Caco-2 and T84 cell lines) and enteroendocrine L-cells (NCI-H716 cell line). Non-viral, chemical-mediated transfection was used as a control. Transduced and transfected co-cultures were subjected to insulin secretion studies. RESULTS: In pure cultures, rAAV2 exhibited a low transduction efficiency towards both Caco-2 and T84 enterocytes, as opposed to a strong reporter expression in permissive NCI-H716 L-cells. In co-cultures of NCI-H716 L-cells and Caco-2 or T84 enterocytes, rAAV2 exhibited differential transduction efficiency with a strong preference towards NCI-H716 L-cells. The rAAV2-transduced co-culture achieved regulated insulin release against stimulation, whereas the chemically transfected co-culture failed to respond. CONCLUSIONS: This study demonstrated that rAAV2-mediated insulin gene transfer can differentiate human intestinal cell types in vitro, in particular enterocyte and enteroendocrine L-cell lines. We consider the AAV2 vector a useful tool in developing enteroendocrine L-cell-specific insulin gene delivery for IDD treatment, in terms of AAV2 avoiding enterocytes and targeting selectively L-cells.     

The Emerging Role Of Adjunctive Noninsulin Antihyperglycemic Therapy In The Management Of Type 1 Diabetes

Objective: Review available data on adjunctive therapies for type 1 diabetes (T1D), with a special focus on newer antihyperglycemic agents.Methods: Published data on hypoglycemia, obesity, mortality, and goal attainment in T1D were reviewed to determine unmet therapeutic needs. PubMed databases and abstracts from recent diabetes meetings were searched using the term “type 1 diabetes” and the available and investigational sodium-glucose cotransporter (SGLT) inhibitors, glucagon-like peptide 1 (GLP-1) receptor agonists, dipeptidyl peptidase 4 inhibitors, and metformin.Results: The majority of patients with T1D do not meet glycated hemoglobin (A1C) goals established by major diabetes organizations. Hypoglycemia risks and a rising incidence of obesity and metabolic syndrome featured in the T1D population limit optimal use of intensive insulin therapy. Noninsulin antihyperglycemic agents may enable T1D patients to achieve target A1C levels using lower insulin doses, which may reduce the risk of hypoglycemia. In pilot studies, the SGLT2 inhibitor dapagliflozin and the GLP-1 receptor agonist liraglutide reduced blood glucose, weight, and insulin dose in patients with T1D. Phase 2 studies with the SGLT2 inhibitor empagliflozin and the dual SGLT1 and SGLT2 inhibitor sotagliflozin, which acts in the gut and the kidney, have demonstrated reductions in A1C, weight, and glucose variability without an increased incidence of hypoglycemia.Conclusion: Newer antihyperglycemic agents, particularly GLP-1 agonists, SGLT2 inhibitors, and dual SGLT1 and SGLT2 inhibitors, show promise as adjunctive treatment for T1D that may help patients achieve better glucose control without weight gain or increased hypoglycemia.Abbreviations:A1C = glycated hemoglobinBMI = body mass indexCI = confidence intervalDKA = diabetic ketoacidosisDPP-4 = dipeptidyl peptidase 4GLP-1 = glucagonlike peptide 1PYY = polypeptide tyrosine tyrosineSGLT = sodium-glucose cotransporterSGLT1 = sodium-glucose cotransporter 1SGLT2 = sodium-glucose cotransporter 2T1D = type 1 diabetesT2D = type 2 diabetesTDD = total daily dosage     

Frontiers in glucagon-like peptide-2: multiple actions, multiple mediators

Glucagon-like peptide-2 (GLP-2) is a pleiotropic hormone that affects multiple facets of intestinal physiology, including growth, barrier function, digestion, absorption, motility, and blood flow. The mechanisms through which GLP-2 produces these actions are complex, involving unique signaling mechanisms and multiple indirect mediators. As clinical trials have begun for the use of GLP-2 in a variety of intestinal disorders, the elucidation of such mechanisms is vital. The GLP-2 receptor (GLP-2R) is a G protein-coupled receptor, signaling through multiple G proteins to affect the cAMP and mitogen-activated protein kinase pathways, leading to both proliferative and antiapoptotic cellular responses. The GLP-2R also demonstrates unique mechanisms for receptor trafficking. Expression of the GLP-2R in discrete sets of intestinal cells, including endocrine cells, subepithelial myofibroblasts, and enteric neurons, has led to the hypothesis that GLP-2 acts indirectly through multiple mediators to produce its biological effects. Indeed, several studies have now provided important mechanistic data illustrating several of the indirect pathways of GLP-2 action. Thus, insulin-like growth factor I has been demonstrated to be required for GLP-2-induced crypt cell proliferation, likely involving activation of beta-catenin signaling. Furthermore, vasoactive intestinal polypeptide modulates the actions of GLP-2 in models of intestinal inflammation, while keratinocyte growth factor is required for GLP-2-induced colonic mucosal growth and mucin expression. Finally, enteric neural GLP-2R signaling affects intestinal blood flow through a nitric oxide-dependent mechanism. Determining how GLP-2 produces its full range of biological effects, which mediators are involved, and how these mediators interact is a continuing area of active research.     

Sweet-taste-suppressing compounds: current knowledge and perspectives of application

Sweet-tasting compounds are recognized by a heterodimeric receptor composed of the taste receptor, type 1, members 2 (T1R2) and 3 (T1R3) located in the mouth. This receptor is also expressed in the gut where it is involved in intestinal absorption, metabolic regulation, and glucose homeostasis. These metabolic functions make the sweet taste receptor a potential novel therapeutic target for the treatment of obesity and related metabolic dysfunctions such as diabetes. Existing sweet taste inhibitors or blockers that are still in development would constitute promising therapeutic agents. In this review, we will summarize the current knowledge of sweet taste inhibitors, including a sweet-taste-suppressing protein named gurmarin, which is only active on rodent sweet taste receptors but not on that of humans. In addition, their potential applications as therapeutic tools are discussed.     

Molecules Implicated in Glucose Homeostasis are Differentially Expressed in the Trachea of Lean and Obese Zucker Rats

Recent studies indicate that the processes mediated by the (T1R2/T1R3) glucose/sugar receptor of gustatory cells in the tongue, and hormones like leptin and ghrelin contribute to the regulation of glucose homeostasis. Altered plasma levels of leptin and ghrelin are associated with obesity both in humans and rodents. In the present study, we evaluated the ultrastructure of the mucosa, and the expression of molecules implicated in the regulation of glucose homeostasis (GLUT2, SGLT1, T1R3, ghrelin and its receptor) in the trachea of an animal model of obesity (Zucker rats). We found that the tracheal epithelium of obese animals was characterized by the presence of poorly differentiated cells. Ciliated and secretory cells were the cell lineages with greatest loss of differentiation. Severe epithelial alterations were associated with marked deposit of extracellular matrix in the lamina propria. The expression pattern of GLUT2 and SGLT1 glucose transporters was similar in the trachea of both the Zucker rat genotypes, whereas that of T1R3 was reduced in ciliated cells of obese rats. A different immunolocalization for ghrelin was also found in the trachea of obese rats. In conclusion, the tracheal morphological alterations in obese animals seem to compromise the expression of molecules involved in the homeostasis of glucose.Key words: Obesity, ultrastructure, ghrelin, ghrelin receptor, sweet receptor, immunohistochemistry     

Sugar sensing by enterocytes combines polarity, membrane bound detectors and sugar metabolism

Sugar consumption and subsequent sugar metabolism are known to regulate the expression of genes involved in intestinal sugar absorption and delivery. Here we investigate the hypothesis that sugar-sensing detectors in membranes facing the intestinal lumen or the bloodstream can also modulate intestinal sugar absorption. We used wild-type and GLUT2-null mice, to show that dietary sugars stimulate the expression of sucrase-isomaltase (SI) and L-pyruvate kinase (L-PK) by GLUT2-dependent mechanisms, whereas the expression of GLUT5 and SGLT1, did not rely on the presence of GLUT2. By providing sugar metabolites, sugar transporters, including GLUT2, fuelled a sensing pathway. In Caco2/TC7 enterocytes, we could disconnect the sensing triggered by detector from that produced by metabolism, and found that GLUT2 generated a metabolism-independent pathway to stimulate the expression of SI and L-PK. In cultured enterocytes, both apical and basolateral fructose could increase the expression of GLUT5, conversely, basolateral sugar administration could stimulate the expression of GLUT2. Finally, we located the sweet-taste receptors T1R3 and T1R2 in plasma membranes, and we measured their cognate G alpha Gustducin mRNA levels. Furthermore, we showed that a T1R3 inhibitor altered the fructose-induced expression of SGLT1, GLUT5, and L-PK. Intestinal gene expression is thus controlled by a combination of at least three sugar-signaling pathways triggered by sugar metabolites and membrane sugar receptors that, according to membrane location, determine sugar-sensing polarity. This provides a rationale for how intestine adapts sugar delivery to blood and dietary sugar provision.     

Density distribution of free fatty acid receptor 2 (FFA2)-expressing and GLP-1-producing enteroendocrine L cells in human and rat lower intestine,and increased cell numbers after ingestion of fructo-oligosaccharide

Glucagon-like peptide 1 (GLP-1) is a multifunctional hormone in glucose metabolism and intestinal function released by enteroendocrine L-cells. The plasma concentration of GLP-1 is increased by indigestible carbohydrates and luminal infusion of short-chain fatty acids (SCFAs). However, the triggers and modulators of the GLP-1 release remain unclear. We hypothesized that SCFAs produced by bacterial fermentation are involved in enteroendocrine cell proliferation and hormone release through free fatty acid receptor 2 (FFA2, also known as FFAR2 or GPR43) in the large intestine. Fructo-oligosaccharide (Fructo-OS), fermentable indigestible carbohydrate, was used as a source of SCFAs. Rats were fed an indigestible-carbohydrate-free diet (control) or a 5% Fructo-OS-containing diet for 28 days. FFA2-, GLP-1-, and 5-hydroxytryptamine (5-HT)-positive enteroendocrine cells were quantified immunohistochemically in the colon, cecum, and terminal ileum. The same analysis was performed in surgical specimens from human lower intestine. The coexpression of FFA2 with GLP-1 was investigated both in rats and humans. Fructo-OS supplementation in rats increased the densities of FFA2-positive enteroendocrine cells in rat proximal colon, by over two-fold, relative to control, in parallel with GLP-1-containing L-cells. The segmental distributions of these cells in human were similar to rats fed the control diet. The FFA2-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HT-containing EC cells, in both human and rat colon and terminal ileum. Fermentable indigestible carbohydrate increases the number of FFA2-positive L-cells in the proximal colon. FFA2 activation by SCFAs might be an important trigger for produce and release GLP-1 by enteroendocrine L-cells in the lower intestine.     

Maternal dexamethasone and GLP-2 have early effects on intestinal sugar transport in their suckling rat offspring

Both glucagon-like peptide 2 (GLP-2) and glucocorticosteroids enhance intestinal uptake in mature animals. Maternal stimuli may cause intestinal adaptation in the offspring. We hypothesized that administering GLP-2, dexamethasone (DEX) or a combination of GLP-2+DEX to rat dams during pregnancy and lactation would enhance intestinal sugar uptake in their offspring. Rat dams were treated with GLP-2 (0.1 microg/g/day), DEX (0.128 microg/g/day), a combination of GLP-2+DEX or placebo. Glucose and fructose uptake was assessed in their suckling offspring using an in vitro intestinal ring uptake technique. The protein abundance of SGLT1, GLUT5, GLUT2, Na(+)K(+)-ATPase and selected signals was determined by immunohistochemistry; GLP-2 caused hypertrophy of the jejunal enterocytes and increased ileal villous height. Jejunal fructose uptake was reduced by GLP-2, DEX and GLP-2+DEX. V(max) for jejunal glucose uptake was reduced with DEX and GLP-2+DEX. These declines were not explained by alterations in transporter abundance. Decreases in Akt and mTOR abundance were associated with declines in transporter activity. We speculate that the intrinsic activity of the sugar transporters was modified via the P13K pathway. In conclusion, maternal GLP-2 and DEX reduced intestinal sugar uptake in their offspring. This may have nutritional implications for the offspring of mothers treated with GLP-2 or steroids.     

Spatiotemporal Modeling of Triggering and Amplifying Pathways in GLP-1 Secreting Intestinal L Cells

Glucagon-like peptide 1 (GLP-1) is secreted by intestinal L-cells, and augments glucose-induced insulin secretion, thus playing an important role in glucose control. The stimulus-secretion pathway in L-cells is still incompletely understood and a topic of debate. It is known that GLP-1 secreting cells can sense glucose to promote electrical activity either by the electrogenic sodium-glucose cotransporter SGLT1, or by closure of ATP-sensitive potassium channels after glucose metabolism. Glucose also has an effect on GLP-1 secretion downstream of electrical activity. An important aspect to take into account is the spatial organization of the cell. Indeed, the glucose transporter GLUT2 is located at the basolateral, vascular side, while SGLT1 is exposed to luminal glucose at the apical side of the cell, suggesting that the two types of transporters play different roles in glucose sensing. Here, we extend our recent model of electrical activity in primary L-cells to include spatiotemporal glucose and Ca²⁺ dynamics, and GLP-1 secretion. The model confirmed that glucose transportation into the cell through SGLT1 cotransporters can induce Ca²⁺ influx and release of GLP-1 as a result of electrical activity, while glucose metabolism alone is insufficient to depolarize the cell and evoke GLP-1 secretion in the model, suggesting a crucial role for SGLT1 in triggering GLP-1 release in agreement with experimental studies. We suggest a secondary, but participating, role of GLUT2 and glucose metabolism for GLP-1 secretion via an amplifying pathway that increases the secretion rate at a given Ca²⁺ level.