Certain nutrients stimulate glucagon-like peptide-1 (

-) secretion from the intestinal enteroendocrine L-cells, but due to rapid degradation by the DPP-4 enzyme, >90% is converted to inactive metabolite before reaching the target organs via circulation. Plants are a source of potent bioactive compounds that promote endogenous secretion of - from L-cells. To search for the effective bioactive compound from a vast library of natural compounds, a reliable and low-cost assay is required considering the high cost of commercial assays. We developed a low-cost sandwich enzyme-linked immunosorbent assays (s-‍ELISAs) for detecting ‘total’, ‘sensitive active’, and ‘wide-range active’ -. The s-ELISAs exhibited high sensitivity with measurement ranges between 0.94~240, 0.98~62.5, and 4.8~4,480 pmol/L, respectively. High precision was observed; i.e., CVs within 5% and 20% for intra- and inter-assay variations, respectively, and excellent recovery of exogenous - from assay buffer. The developed s-ELISAs had the same performance as the commercial kits and approximately 80% cheaper cost. For their application, cinnamtannin A2-induced - secretion was confirmed in STC-1 cells consistent with our previous findings. The s-ELISAs were further validated by measuring plasma - level in mice after oral administration of black soy bean seed coat extract containing cinnamtannin A2.

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Energy metabolism and circadian rhythms are closely related together, i.e., the timing of nutrient intake affects metabolism under the regulation of circadian rhythms. Previously, we have reported that cacao liquor procyanidin (CLPr) promotes energy metabolism, resulting in preventing obesity and hyperglycemia. However, it is not unclear whether CLPr regulates clock gene expression. In this study, we investigated whether the administration timing of CLPr affected clock gene expression and found that CLPr regulated the circadian clock gene expression through the glucagon-like peptide-1 (

-) signaling pathway. CLPr administration at Zeitgeber time 3 increased the expression level of Per family and Dbp in the liver. At the same administration timing, CLPr increased - and insulin concentration in the plasma and phosphorylation of AMPK in the liver. It was noteworthy that an antagonist for - receptor Exendin (9-39) canceled CLPr-increased expression of Per family and Dbp and phosphorylation of AMPK in the liver, in addition to insulin secretion. These results strongly suggest that CLPr-induced - regulates the changes in clock gene expression in the liver through increased insulin. Thus, CLPr is a possible functional food material for prevention and/or amelioration of metabolic disorders through preventing circadian disruption through - and AMPK pathways.

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Introduction : -, a cAMP mobilizing agonist, is an insulinotropic peptide released from the intestinal L cell in response to a meal. However, the underlying mechanisms of the stimulatory effect of - on insulin secretion remain fully elucidated. Aim: The present study was conducted to examine whether - can activate PKCalpah and PKCepsilon in INS-1 cells at a substimulatory concentration of glucose. Methods: We employed either GFP or DsRed-tagged proteins related to PKC signaling pathway using epifluorescence microscopy and total internal reflection fluorescence microscopy. Results: We first showed that - translocated endogenous PKCalpah and PKCepsilon from the cytosol to the plasma membrane. Then we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), as a maker of PKC activation. - translocated GFP-tagged MARCKS from the plasma membrane to the cytosol and the --evoked translocation of MARCKS-GFP was blocked by PKC inhibitors. The above observations were verified in three different ways using live cell imaging technique.In addition to these results, PKC inhibitors reduced forskolin-induced insulin secretion in INS-1 cells and rat islet beta cells. Conclusion: - can activate PKCalpah and PKCepsilon, and the --activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose. [J Physiol Sci. 2006;56 Suppl:S67]

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Glucagon-like peptide1 (-), a cAMP mobilizing agonist, is an insulinotropic peptide. However, the underlying mechanisms of the stimulatory effect of - on insulin secretion remain fully elucidated. One of the mechanisms by which - potentiates glucose-induced insulin secretion is to modulate Ca²⁺signaling. Recently, we have demonstrated that depolarization-evoked Ca²⁺ influx alone can activate conventional protein kinase C (PKC) and novel PKC in INS-1 cells.The present study was conducted to examine whether - can activate PKCα and PKCε, which are predominantly expressed in INS-1 cells at a substimulatory concentration of glucose. We first showed that - translocated endogenous PKCα and PKCε from the cytosol to the plasma membrane. Then we assessed the phosphorylation state of the PKC substrate, MARCKS, as a maker of PKC activation. - translocated GFP-tagged MARCKS from the plasma membrane to the cytosol and the --evoked translocation of MARCKS-GFP was blocked by a PKC inhibitor. The above observations were verified in three different ways by demonstrating: 1) Br-cAMP induced translocation of GFP-tagged C1 domain of PKCγ, 2) Br-cAMP induced translocation of GFP-tagged pleckstrin homology domain of phospholipase Cδ1, 3) forskolin-induced translocation of PKCα and PKCε, as makers of PKC activation.Taken together, these results demonstrate that - can activate PKCα and PKCε through a Ca²⁺-dependent phospholipase C activation. [Jpn J Physiol 55 Suppl:S34 (2005)]

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Insulin secretion from pancreatic β cells is modulated by numerous extracellular signaling molecules. Glucagon-like peptide 1 (-) among them is a powerful insulinotropic peptide. Upon binding of its receptor, GLP-I increases cyclic adenosine monophosphate (cAMP) levels via a G-protein-coupled activation of adenylate cyclase (AC), leading to activation of protein kinase A (PKA). - through cAMP-PKA signaling pathway modulates Ca²⁺ signals. Our recent work has demonstrated that depolarization-evoked Ca²⁺ influx via voltage dependent Ca²⁺ channels in insulin secreting cells (INS-1 cells) induces activation of conventional PKC (cPKC) as well as structurally Ca²⁺-independent novel PKC (nPKC). We investigated whether - as a Ca²⁺ modulator can activate PKCα and PKCε as representatives of cPKC and nPKC in INS-1 cells. - and 8-Br-cAMP (Br) evoked-Ca²⁺ signal induced GFP-tagged PKCα and PKCε. This result indicated that --evoked Ca²⁺ signals can activate PKCs. We verified this by showing in three deferent ways: firstly, --induced translocation of GFP-tagged myristoylated alanine-rich C kinase substrate, secondly, --induced translocation of GFP-tagged C1 domain of PKCγ as a diacylglycerol (DAG) sensor, thirdly --induced translocation of GFP-tagged pleckstrin homology domain as a marker of phospholipase C (PLC) activation. Thus, we concluded from these observations that - -evoked Ca²⁺ signal generates DAG through PLC activation thereby activating both PKCα and PKCε. [Jpn J Physiol 54 Suppl:S74 (2004)]

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The beneficial effect of dipeptidyl peptidase-4 inhibition on diet-induced extra-pancreatic effects, especially on liver tissue remains poorly understood. Thus, we made the experimental designs as follows; five-week-old male ob/ob mice, which develop type 2 diabetic mellitus and nonalcoholic fatty liver disease by taking a high-carbohydrate diet (HCD), were divided into a group in which a HCD was given for 8 weeks as control, and another in which a HCD added with 0.0018% sitagliptin was given for 8 weeks. Hepatic steatosis was seen in all mice, but the mean grade of steatosis in the sitagliptin-administrated mice was significantly decreased. The acetyl-CoA concentrations were lower in sitagliptin-administrated mice, although the differences were not significant. However, the malonyl-CoA concentrations were significantly lower in sitagliptin-administrated mice. The expression of acetyl-CoA carboxylase 1 was inhibited in sitagliptin-administrated mice, irrespective of expressions of carbohydrate responsive element-binding protein (ChREBP) or sterol regulatory element-binding protein (SREBP)-1c. In conclusion, sitagliptin may affect the development of nonalcoholic fatty liver disease by inhibiting the production of malonyl-CoA and thus synthesis of fatty acids in the liver.

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Glucagon-like peptide 1 (

-) is an enteroendocrine hormone which is released from L-cells in the intestine and stimulates insulin secretion and inhibits glucagon secretion. It also shows appetite suppressing effects by mainly central action. - is also a neuropeptide synthesized by neurons in the nucleus tractus solitarius (NTS). These neurons project to hypothalamic nuclei and release - which inhibit food intake. - receptors are also expressed in the NTS which is the first gate region receiving sensory vagal inputs from peripheral organs including gastrointestinal organs. However, the physiological roles of the - receptors in the NTS were not examined precisely. So, we examined the effects of - receptor activation on excitatory synaptic transmission in the NTS second order neurons.

Liraglutide (1 μM:

- receptor agonist) did not changed the frequency and amplitude of spontaneous EPSCs (sEPSCs) but increased the amplitude of evoked EPSCs (eEPSCs). Paired pulse ratio of eEPSCs was decreased by liraglutide which suggested presynaptic action. Another - receptor agonist exendin-4 (1 μM) also showed similar effects with liraglutide on both sEPSCs and eEPSCs.

These results suggest that the activation of

- receptors in the NTS facilitates evoked excitatory synaptic transmission although no effects on spontaneous synaptic transmission. These effects may partly contribute to the weight reducing effects of - agonist.

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The anorexigenic gut hormones, cholecystokinin (CCK), glucagon-like peptide ()- and peptide tyrosine-tyrosine (PYY), are released in response to food intake from the intestines. Dietary nutrients have been shown to stimulate these hormones. Some non-nutrients such as polyphenols show anorexigenic effects on humans. In the present study, we examined whether dietary polyphenols can stimulate secretion of these gut hormones. Caco-2 cells expressed mRNA of the gut hormones, CCK, PC1 (prohormone convertase 1), GCG (glucagon) and PYY. CCK, - and PYY were secreted from Caco-2 cells after adding sugars, amino acids or fatty acids. Using Caco-2 cells, epigallocatechin-3-gallate (EGCG), chlorogenic acid and ferulic acid induced secretion of anorexigenic gut hormones. Particularly, EGCG induced secretion of all three hormones. In an ex vivo assay using murine intestines, EGCG also released CCK from the duodenum, and - from the ileum. These results suggest that EGCG may affect appetite via gut hormones.

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Abnormal G-protein-coupled receptor kinase 2 (GRK2) accumulation has a crucial role in the development of insulin resistance and diabetes. Previous report showed that impaired insulin-induced relaxation in diabetes is improved by suppressing the GRK2 levels. Glucagon-like peptide-1 (

-) is a gut hormone that promote insulin secretion. However, it is unknown whether - directly affects diabetic endothelial dysfunction, especially GRK2 signaling. In this study, we investigated the relationship between - and GRK2 under insulin stimulation for endothelial dysfunction. - increased the impaired insulin-induced NO-dependent relaxation responses in diabetes. However, these responses are disappeared by treatment of Exendin-9, a - receptor antagonist. Furthermore, phosphorylation levels of Akt and eNOS were higher in diabetes under -/insulin stimulation than non-stimulation. Additionally, in diabetes GRK2 activity was inhibited under -/insulin stimulation compared with non-stimulation, although GRK2 expression was not altered between the two groups. Those results suggest that in diabetes, - improves the endothelial dysfunction by suppressing of GRK2 activity, which might provide a therapeutic target for diabetic vascular complications.

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Palatinose (isomaltulose), a slowly digested disaccharide, is used as a non-cariogenic sugar and as a sucrose substitute in several foods. Because of its ability to lower postprandial glycemia, palatinose may be beneficial as a treatment for impaired glucose metabolism. Glucagon-like peptide-1 (-) improves glycemia via enhancing pancreatic beta-cell functions. The secretion of - is stimulated by sugars, including glucose and artificial sweeteners. In this study, we examined whether palatinose induced - secretion in vivo and in vitro. Firstly, portal - and glucose were measured after oral administration of palatinose or sucrose in conscious rats. Secondly, portal - and glucose were measured after jejunal or ileal administration of each sugar in anesthetized rats. Finally, GLUTag, a murine --producing cell line, was exposed to several sugars, including palatinose and sucrose, to observe the direct effect of these sugars on - secretion. Compared with sucrose, palatinose enhanced portal - level when administered orally in conscious rats. Both palatinose and sucrose induced a significant increase in portal - after jejunal or ileal administration of each sugar in anesthetized rats. Ileal administration triggered a greater response than did jejunal administration. Glycemic responses were higher in sucrose-treated rats than in palatinose-treated rats in every experiment. In GLUTag cells, glucose induced a significant increase in - secretion, but neither sucrose nor palatinose had an effect. These data demonstrate that luminal palatinose induces - secretion in rats. However, it is likely that - secretion is triggered mainly by glucose released in the lumen rather than by palatinose itself.

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The gut hormone glucagon-like peptide-1 (-) is proposed for treatment of Type II diabetes mellitus. However, the short half life of - in vivo is a major limitation for its application due to the frequent invasive administrations. To provide a optimal formulation to overcome this limitation, we developed a - entrapped microspheres to achieve sustained release - for 4-week. - was stabilized by --zinc complexation with zinc carbonate and encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA) with S/O/O solvent extraction to obtain - loaded PLGA microspheres (MS). The characteristics of MS were evaluated as follows: The surface morphyology was assessed by scanning electron microscopy (SEM); The drug encapsulation efficiency and - controlled release profile was tested by HPLC; The sustained release of - MS in vivo and pharmacological efficacy were studied in normal mice and streptozotocin (STZ)-induced diabetic mice model after subcutaneous administration of - MS. --zinc complexation significantly reduced initial burst release from 37.2 to 7.5%. The controlled release bioactive - in vitro was achieved for 4-week period by zinc complexation and addition of ZnCO₃. The optimal and complete cumulative release of - from MS was increased from 23 to 63% in 28 d by using low MW PLGA (MW 14000). The in vivo testing in normal mice and diabetic mice suggest that this zinc-stabilized technique combined with S/O/O method in the presence of water insoluble antacid additive ZnCO₃ preserve the biological activity of -. - MS formulation achieved controlled released in vivo for 28 d and exhibit sustained long term pharmacological efficacy to decrease blood glucose level in diabetic mice. This - MS formulation provides a practical formulation for long-term sustained delivery of - to treat Type II diabetes.

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Glucagon-like peptide-1 (-) induces pancreatic insulin secretion via the cAMP-dependent protein kinase (PKA) pathway. However, the - concentration used in the previous in vitro experiments was far from the in vivo concentrations. Alteration of plasma - concentration at pM order lowers blood glucose concentration. In this study, we examined the - action mechanism at a physiological concentration on insulin secretion. A high concentration of - (10 nM) stimulated intracellular cAMP accumulation and insulin secretion was significantly inhibited by KT5720, a selective inhibitor of PKA. Low - concentrations (1 pM) also increased insulin secretion without significant accumulation of intracellular cAMP, and KT5720 did not affect insulin secretion. Insulin secretion stimulated by 1 pM - was reduced by inhibitors of calcium action, including verapamil, dantrolene, and BAPTA. Thus, we concluded that relatively low - concentrations–comparable to in vivo blood concentrations–promoted insulin secretion independent of the cAMP-PKA pathway. This effect was dependent on intracellular Ca²⁺ concentration. The results of the present study may further the understanding of the dose-dependent response of - signal transducing pathways and the complicated mechanism of insulin secretion. Studies of - at physiologic concentrations may lead to new developments in studies of pancreatic β-cell function.

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Many types of endocrine cells have been identified in the gastroenteropancreatic system of vertebrates, which have subsequently been named with alphabet (s). L cells which secrete the glucagon-like peptide (

)- are scattered in the intestinal epithelium. This review discusses the morphological features of chicken L cells and - secretion from intestinal L cells. L cells, identified using - immunohistochemistry, are open-type endocrine cells that are distributed in the jejunum and ileum of chickens. - co-localizes with GLP-2 and neurotensin in the same cells of the chicken ileum. Intestinal L cells secrete - in response to food ingestion. Proteins and amino acids, such as lysine and methionine, in the diet trigger - secretion from the chicken intestinal L cells. The receptor that specifically binds chicken - is expressed in pancreatic D cells, implying that the physiological functions of chicken - differ from its functions as an incretin in mammals.

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It is well known that glucagon-like peptide 1 (

-) can bind to the - receptor of pancreatic islet to enhance insulin secretion through a cAMP-dependent pathway. However, little is known about the effects of - on the pancreatic exocrine gland. In the gland, a signal transduction of amylase release is evoked mainly by an increase in intracellular Ca²⁺ levels and activation of PKC. Myristoylated alanine-rich C kinase substrate (MARCKS) is known as a major substrate for PKC. We previously demonstrated that MARCKS is involved in pancreatic amylase release through the Ca²⁺-dependent pathway. Here, we studied the effects of - on MARCKS phosphorylation and amylase release through the cAMP-dependent pathway in rat pancreatic acini. By the organ bath technique, - did not induce amylase release in the intact pancreas. In contrast, it induced amylase release and MARCKS phosphorylation in isolated pancreatic acini. An inhibitor of PKA suppressed those effects. Furthermore, a MARCKS-related peptide inhibited the --induced amylase release. These findings suggest that - induces amylase release through MARCKS phosphorylation via activation of PKA in the isolated acini, but not in the intact pancreas.

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In vitro studies in isolated pancreas and islets have shown that glucagon-like peptide-1 (-) promotes insulin release in a typical concentration-dependent manner. In contrast, the relationship between plasma - and insulin concentrations in vivo is complicated, because --promoted insulin release lowers blood glucose, which influences glucose-dependent insulinotropic ability of -. - also stimulates insulin release via hepatoportal neuronal mechanism. Hence, the dynamic relationship between plasma active - vs. insulin and glucose concentrations is obscure. In this study, we aimed to determine in vivo relationships between these parameters in rats. To mimic postprandial state, intraduodenal glucose challenge in anesthetized rats was performed, which can minimize the release of endogenous -. The glucose challenge induced the 1st phase and 2nd phase insulin release. - infusion from jugular vein significantly and concentration-dependently enhanced area under the curve (AUC) of the 1st phase insulin, in which the minimum effective active - concentration was 6.6 pmol/l. In contrast, bell-shaped dose responses were observed for both the 2nd phase and total insulin AUCs, in which a significant increase was obtained only with 11 pmol/l of active - for total insulin AUC. A statistically significant reduction in the plasma glucose AUC was observed when active - concentration was 11 pmol/l and 21 pmol/l. These results indicate that - markedly enhances the 1st phase insulin release while less potently the 2nd phase insulin release, possibly due to a negative feedback regulation of β-cells via reduced plasma glucose levels by the enhanced 1st phase insulin release.

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Exendin-4 is an agonist of the glucagon-like peptide 1 receptor (-R) and is used in the treatment of type 2 diabetes. Since human -R has been identified in various cells besides pancreatic cells, exendin-4 is expected to exert extrapancreatic actions. It has also been suggested to affect gene expression through epigenetic regulation, such as DNA methylation and/or histone modifications. Furthermore, the expression of extracellular-superoxide dismutase (EC-SOD), a major SOD isozyme that is crucially involved in redox homeostasis, is regulated by epigenetic factors. In the present study, we demonstrated that exendin-4 induced the demethylation of DNA in A549 cells, which, in turn, affected the expression of EC-SOD. Our results showed that the treatment with exendin-4 up-regulated the expression of EC-SOD through -R and demethylated some methyl-CpG sites (methylated cytosine at 5'-CG-3') in the EC-SOD gene. Moreover, the treatment with exendin-4 inactivated DNA methyltransferases (DNMTs), but did not change their expression levels. In conclusion, the results of the present study demonstrated for the first time that exendin-4 regulated the expression of EC-SOD by reducing the activity of DNMTs and demethylation of DNA within the EC-SOD promoter region in A549 cells.

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Both nutritive and non-nutritive sweeteners may influence energy and glucose metabolism differently. The hypothesis that sucrose, fructose, aspartame, and sucralose intake differently modulate energy and glucose metabolism was tested in an estrogen-deficient animal model. At 30 min after giving aspartame and sucralose (10 mg/kg body weight), an oral glucose tolerance test (OGTT) was conducted with glucose, sucrose, and fructose in ovariectomized (OVX) rats. After OGTT, they were continuously fed high fat diets including either 10% corn starch (Control), 10% sucrose (Sucrose), 10% fructose (Fructose), 0.05% aspartame + 9.95% starch (Aspartame) or 0.05% sucralose + 9.95% starch (Sucralose) for 8 week. During 30 min after acute administration of aspartame and sucralose, serum glucose concentrations increased despite slightly increased serum insulin levels before glucose infusion. However, glucose tolerance was not significantly different among the groups. In chronic study, serum glucose concentrations were lowest and insulin highest at the overnight-fasted state in Aspartame and Sucralose. Postprandial serum glucagon-like peptide-1 (

-) and insulin levels were higher in Aspartame and Sucralose than Control. Hepatic insulin signaling (pAkt → pGSK-3β) and phosphoenolpyruvate carboxykinase (PEPCK) expression were lower in Sucralose and Aspartame than the Fructose. Serum acetate levels produced by gut microbiota were higher were lower in the fructose group than Aspartame and Sucralose groups. In conclusion, aspartame and sucralose with a meal might be preferable sweeteners to fructose and sucrose in estrogen deficient rats, and possibly post-menopausal women; however, this needs to be confirmed in human studies.

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