NCI-H716 Cells Research Articles

Chronic Exposure to TNFα Impairs Secretion of Glucagon-Like Peptide-1.

Obesity is associated with systemic inflammation and elevated levels of TNFα, leading to impaired glucose tolerance. In humans, obesity is also associated with reduced nutrient-stimulated secretion of the intestinal incretin hormone, glucagon-like peptide-1 (GLP-1). We hypothesized that TNFα plays a direct role in the impairment of GLP-1 secretion from the enteroendocrine L-cell and that blocking TNFα can restore both GLP-1 secretion and glucose homeostasis. Expression of the TNFα receptor subytpe-1 was detected in the human NCI-H716 and murine GLUTag L-cell models and in mouse ileal sections. Although TNFα acutely increased GLP-1 release from NCI-H716 cells (P < .05-.001), preincubation with TNFα for 24 hours reduced proglucagon mRNA (P < .05) and GLP-1 cellular (P < .05) levels without affecting cell viability. Furthermore, both NCI-H716 and GLUTag cells pretreated with TNFα for 24 hours no longer responded to known GLP-1 secretagogues, an effect that was reversed by coincubation with the Nuclear Factor Kappa B inhibitor, 5-aminosalicylic acid, in the NCI-H716 cells. Mice given a high-fat diet (HFD) for 12 weeks developed impaired glucose tolerance, hyperinsulinemia, and increased TNFα mRNA expression in fat and ileal tissue. Hyperglycemia and hyperinsulinemia were reduced in HFD mice treated with the anti-TNFα biological, etanercept, for 2 weeks. In primary intestinal cultures from these animals, HFD control mice had impaired GLP-1 secretion, and this was not observed in the HFD etanercept-derived cultures (P < .05). In conclusion, chronic exposure to TNFα directly impairs GLP-1 secretion at the level of the intestinal L-cell, an effect that is reversed by anti-TNFα therapy in association with improved glucose tolerance.

Berberine induces GLP-1 secretion through activation of bitter taste receptor pathways

Our previous studies revealed that berberine-mediated GLP-1 secretion was a possible mechanism for berberine exerting good effects on hyperglycemia. This study was designed to ascertain whether berberine-induced secretion of GLP-1 was related with activation of bitter taste receptors expressed in gastrointestinal tract. Western blotting results showed that TAS2R38, a subtype of bitter taste receptor, was expressed on human enteroendocrine NCI-H716 cells. GLP-1 secretion induced by berberine from NCI-H716 cells was inhibited by incubation with anti-TAS2R38 antibody. We further performed gene silencing using siRNA to knockdown TAS2R38 from NCI-H716 cells, which showed that siRNA knockdown of the TAS2R38 reduced berberine-mediated GLP-1 secretion. We adopted inhibitors of PLC and TRPM5 known to be involved in bitter taste transduction to investigate the underlying pathways mediated in berberine-induced GLP-1 secretion. It was found that PLC inhibitor U73122 inhibited berberine-induced GLP-1 release in NCI-H716 cells, while TRPM5 blocker quinine failed to attenuate berberine-induced secretion of GLP-1. The present results demonstrated that berberine stimulated GLP-1 secretion via activation of gut-expressed bitter taste receptors in a PLC-dependent manner. Because berberine was found to be a ligand of bitter taste receptor, the results of present study may provide an explanation for some bitter taste substance obtain hypoglycemic effect.

A bitter herbal medicine Gentiana scabra root extract stimulates glucagon-like peptide-1 secretion and regulates blood glucose in db/db mouse

Ethnopharmacological relevanceGentiana scabra root extract (GS) is frequently prescribed as an internal remedy in traditional Korean medicine for treatment of diabetes mellitus. GS contains bitter iridoid glycosides including loganic acid, gentiopicrin, trifloroside, and rindoside. We previously reported that the intestinal bitter taste sensation stimulates GLP-1 secretion, and thereupon hypothesized that the blood glucose regulatory effect of GS is due to its GLP-1 secreting effect in enteroendocrine L cells. Materials and methodWe studied GLP-1 secreting effect of GS treatment and its cellular downstream mechanism in human enteroendocrine NCI-H716 cells using the G protein-coupled receptor (GPCR) pathway inhibitors. Intracellular calcium assay also demonstrated the signal transduction pathway stimulated by the GS treatment. Using db/db mice, we performed oral glucose tolerance test (OGTT) to examine the blood glucose lowering effect of GS administration. We also collected the mouse plasma during the OGTT to measure the GLP-1 and insulin levels. ResultWe demonstrated dose-dependent GLP-1 secreting effect of GS on the NCI-H716 cells. The GLP-1 secreting effect of GS is mediated by the G protein βγ-subunit and inositol triphosphate. Using db/db mice, we found that the effect of GS on lowering blood glucose is due to its GLP-1 secretion, and consequential insulinotropic effect. The chemical fingerprint of GS was obtained through a direct analysis in realtime mass spectrometry (DART-MS) and high-performance liquid chromatography (HPLC)/MS. Through the GLP-1 secretion study, we found that loganic acid, an iridoid glycoside, contributes to the GLP-1 secreting effect of GS. ConclusionThe findings of this study highlight the potential of exploiting the antidiabetic effect of GS on type 2 diabetes mellitus patients.

AZD-4547 exerts potent cytostatic and cytotoxic activities against fibroblast growth factor receptor (FGFR)-expressing colorectal cancer cells.

Colorectal cancer (CRC) causes significant mortalities worldwide. Fibroblast growth factor (FGF) receptor (FGFR) signaling is frequently dysregulated and/or constitutively activated in CRCs, contributing to cancer carcinogenesis and progression. Here, we studied the activity of AZD-4547, a novel and potent FGFR kinase inhibitor, on CRC cells. AZD-4547 inhibited CRC cell growth in vitro, and its activity correlated with the FGFR-1/2 expression level. AZD-4547 was cytotoxic and pro-apoptotic in FGFR-1/2-expressed CRC cell lines (NCI-H716 and HCT-116), but not in FGFR-1/2 null HT-29 cells. Further, AZD-4547 inhibited cell cycle progression and attenuated the activation of FGFR1-FGFR substrate 2 (FRS-2), ERK/mitogen-activated protein kinase (MAPK), and AKT/mammalian target of rapamycin (AKT/mTOR) signalings in NCI-H716 and HCT-116 cells. In vivo, AZD-4547 oral administration at effective doses inhibited NCI-H716 (high FGFR-1/2 expression) xenograft growth in nude mice. Phosphorylation of FGFR-1, AKT, and ERK1/2 in xenograft specimens was also inhibited by AZD-4547 administration. Thus, our preclinical studies strongly support possible clinical investigations of AZD-4547 for the treatment of CRCs harboring deregulated FGFR signalings.

Ezetimibe Stimulates Intestinal Glucagon-Like Peptide 1 Secretion Via the MEK/ERK Pathway Rather Than Dipeptidyl Peptidase 4 Inhibition

ObjectiveEzetimibe is known as a Niemann-Pick C1-Like 1 (NPC1L1) inhibitor and has been used as an agent for hypercholesterolemia. In our previous study, ezetimibe administration improved glycemic control and increased glucagon like peptide-1 (GLP-1), an incretin hormone with anti-diabetic properties. However, the mechanisms by which ezetimibe stimulates GLP-1 secretion are not fully understood. Thus, the specific aim of this study was to investigate the mechanism(s) by which ezetimibe stimulates GLP-1 secretion. Materials/methodsMale KK/H1J mice were divided into following groups: AIN-93G (NC), NC with ezetimibe (10mg/kg/day), 45% high fat (HF) diet, and HF diet with ezetimibe. To investigate the role of ezetimibe in glucose homeostasis and GLP-1 secretion, an insulin tolerance test was performed and serum and intestinal GLP-1 levels and intestinal mRNA expression involved in GLP-1 synthesis were measured after 6weeks of ezetimibe treatment. In vivo and in vitro dipeptidyl peptidase-4 (DPP-4) inhibition assays were employed to demonstrate the association between ezetimibe-induced GLP-1 change and DPP-4. The molecular mechanism by which ezetimibe affects GLP-1 secretion was evaluated by using human enteroendocrine NCI-H716 cells. ResultsEzetimibe supplementation significantly ameliorated HF-increased glucose and insulin resistance in the type 2 diabetic KK/H1J mouse model. Serum and intestinal active GLP-1 levels were significantly increased by ezetimibe in HF-fed animals. However, mRNA expression of genes involved in intestinal GLP-1 synthesis was not altered. Furthermore, ezetimibe did not inhibit the activity of either in vivo or in vitro dipeptidyl peptidase-4 (DPP-4). The direct effects of ezetimibe on GLP-1 secretion and L cell secretory mechanisms were examined in human NCI-H716 intestinal cells. Ezetimibe significantly stimulated active GLP-1 secretion, which was accompanied by the activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK). Ezetimibe-increased GLP-1 secretion was abrogated by inhibiting the MEK/ERK pathway with PD98059. ConclusionThese findings suggest a possible novel biological role of ezetimibe in glycemic control to stimulate intestinal GLP-1 secretion via the MEK/ERK signaling pathway.

Ingestion of coffee polyphenols increases postprandial release of the active glucagon-like peptide-1 (GLP-1(7-36)) amide in C57BL/6J mice.

The widespread prevalence of diabetes, caused by impaired insulin secretion and insulin resistance, is now a worldwide health problem. Glucagon-like peptide 1 (GLP-1) is a major intestinal hormone that stimulates glucose-induced insulin secretion from β cells. Prolonged activation of the GLP-1 signal has been shown to attenuate diabetes in animals and human subjects. Therefore, GLP-1 secretagogues are attractive targets for the treatment of diabetes. Recent epidemiological studies have reported that an increase in daily coffee consumption lowers diabetes risk. The present study examined the hypothesis that the reduction in diabetes risk associated with coffee consumption may be mediated by the stimulation of GLP-1 release by coffee polyphenol extract (CPE). GLP-1 secretion by human enteroendocrine NCI-H716 cells was augmented in a dose-dependent manner by the addition of CPE, and was compatible with the increase in observed active GLP-1(7-36) amide levels in the portal blood after administration with CPE alone in mice. CPE increased intracellular cyclic AMP (cAMP) levels in a dose-dependent manner, but this was not mediated by G protein-coupled receptor 119 (GPR119). The oral administration of CPE increased diet (starch and glyceryl trioleate)-induced active GLP-1 secretion and decreased glucose-dependent insulinotropic polypeptide release. Although CPE administration did not affect diet-induced insulin secretion, it decreased postprandial hyperglycaemia, which indicates that higher GLP-1 levels after the ingestion of CPE may improve insulin sensitivity. We conclude that dietary coffee polyphenols augment gut-derived active GLP-1 secretion via the cAMP-dependent pathway, which may contribute to the reduced risk of type 2 diabetes associated with daily coffee consumption.

Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography–tandem mass spectrometry (LC–MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-11–37, GLP-17–37, and GLP-17–36amide from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1pM, respectively. Meanwhile, the ratios of GLP-11–37, GLP-17–37, and GLP-17–36amide to total GLP-1 peptides were similar (6±3, 26±3, and 78±5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells.

Spergularia marina induces glucagon-like peptide-1 secretion in NCI-H716 cells through bile acid receptor activation.

Spergularia marina Griseb. (SM) is a halophyte that grows in mud flats. The aerial portions of SM have been eaten as vegetables and traditionally used to prevent chronic diseases in Korea. However, there has been no scientific report that demonstrates the pharmacological effects of SM. Glucagon-like peptide-1 (GLP-1) is important for the maintenance of glucose and energy homeostasis through acting as a signal in peripheral and neural systems. To discover a functional food for regulating glucose and energy homeostasis, we evaluated the effect of an aqueous ethanolic extract (AEE) of SM on GLP-1 release from enteroendocrine NCI-H716 cells. In addition, we explored the Takeda G-protein-coupled receptor 5 (TGR5) agonist activity of AEE-SM in Chinese hamster ovary (CHO)-K1 cells transiently transfected with human TGR5. As a result, treatment of NCI-H716 cells with AEE-SM increased GLP-1 secretion and intracellular Ca(2+) and cyclic AMP (cAMP) levels in a dose-dependent manner. Transfection of NCI-H716 cells with TGR5-specific small interference RNA inhibited AEE-SM-induced GLP-1 secretion and the increase in Ca(2+) and cAMP levels. Moreover, AEE-SM showed that the TGR5 agonist activity in CHO-K1 cells transiently transfected with TGR5. The results suggest that AEE-SM might be a candidate for a functional food to regulate glucose and energy homeostasis.

Denatonium induces secretion of glucagon-like peptide-1 through activation of bitter taste receptor pathways.

This study was designed to ascertain whether human enteroendocrine cells express bitter taste receptors, and whether activation of these receptors with bitter-tasting ligands induces secretion of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). We used human enteroendocrine NCI-H716 cells, isolated duodenal segments from mice, and whole mice as our experimental systems for investigating stimuli and mechanisms underlying GLP-1- and PYY-stimulated release. We measured hormone levels by ELISA and determined bitter taste receptor expression by real-time quantitative PCR. We adopted a pharmacological approach using inhibitors and enhancers of downstream signalling pathways known to be involved in bitter taste transduction in taste bud cells to investigate these pathways in NCI-H716 cells. Using a pharmacological approach, we identified signalling pathways triggered by the denatonium benzoate (DB)-activated bitter receptors. This involved activation of α-gustducin (Gαgust)-the specific G-protein subunit that is also present in taste bud cells-reduction of intracellular cAMP levels and enhancement of phospholipase C (PLC) activity, which ultimately led to increased intracellular calcium concentrations and hormone release. Gavage of DB, followed by gavage of glucose, to db/db mice stimulated GLP-1 and subsequent insulin secretion, leading to lower blood glucose levels. Our study demonstrates that activation of gut-expressed bitter taste receptors stimulates GLP-1 secretion in a PLC-dependent manner. In diabetic mice, DB (a ligand of bitter taste receptor cells), when given via gavage, lowers blood glucose levels in diabetic mice after oral glucose administration, through increased secretion of GLP-1.

FGFR2 is amplified in the NCI-H716 colorectal cancer cell line and is required for growth and survival.

Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. FGFR2 is amplified in breast and gastric cancer, and we report here the first characterization of FGFR2 gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. FGFR2 is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability in vitro, indicating “addiction” of NCI-H716 cells to FGFR2. NCI-H716 growth in a xenograft model was also inhibited by an FGFR small molecule inhibitor. FGFR2 was required for activation of multiple downstream signaling proteins including AKT, ERK, S6RP and NFKB. Inhibition of downstream kinases such as AKT or ERK alone had modest effects on proliferation, whereas combined inhibition of AKT and ERK signaling resulted in a loss of viability similar to FGFR2 inhibition. We identified elevated FGFR2 expression in a small subset of primary colorectal cancer, however FGFR2 amplification was not observed. Although FGFR2 amplification is not common in primary colon cancer or lymph node and liver metastases, other subsets of colorectal cancer such as ascites, from which the NCI-H716 cell line was derived, have yet to be tested. These results suggest that emerging FGFR inhibitor therapeutics may have efficacy in a subset of colon cancer driven by FGFR2 amplification.

Association of GLP-1 secretion with anti-hyperlipidemic effect of ginsenosides in high-fat diet fed rats

ObjectiveGinsenosides, major bioactive constituents in Panax ginseng, have been shown to exert anti-hyperlipidemia effects. However, the underlying mechanism was not well-elucidated due to the low bioavailability of ginsenosides. Glucagon-like peptide-1 (GLP-1) was considered to be a critical regulator of energy homeostasis. Our previous studies have showed that ginseng total saponins (GTS) exhibited antidiabetic effects partly via modulating GLP-1 release. The aim of this study was to investigate the potential role of GLP-1 in anti-hyperlipidemia effect of GTS in rats fed with high-fat diet. Material and methodsMale Sprague–Dawley rats were fed with normal diet (CON) or high-fat diet (HFD) for 4weeks. Then, the HFD rats orally received vehicle (HFD), 150mg/kg/day (HFD-GL) and 300mg/kg/day of GTS (HFD-GH) for another 4weeks, respectively. ResultsFour-week GTS treatment significantly ameliorated hyperlipidemia, decreased body fat, liver weight and improved insulin resistance. It was found that high-dose GTS treatment increased portal GLP-1 level induced by glucose loading, accompanied by increased intestinal GLP-1 content, L-cell number and prohormone convertase 3 mRNA expression. Data from NCI-H716 cells showed that both GTS and ginsenoside Rb1 significantly increased GLP-1 secretion as well as proglucagon mRNA level in NCI-H716 cells supplemented with 10% HFD-rat serum. ConclusionsHyperlipidemia and insulin resistance were attenuated effectively in response to GTS treatment. These improvements may be associated with the increased secretion of GLP-1.

Tastant quantitative analysis from complex mixtures using taste cell-based sensor and double-layered cascaded series stochastic resonance

In this paper, tastant quantitative analysis from complex mixtures using taste cell-based sensor and double-layered cascaded series stochastic resonance (DCSSR) method has been investigated. Taste cells, NCI-H716 cells and STC-1 cells, are cultured on carbon screen printed electrode (CSPE) to fabricate integrated sensing devices. Cell culture status on CSPE is observed by scanning electron microscope (SEM) method. Molecular components referring to taste receptor protein and signal transduction (α-gustducin) in taste cells are identified by immunocytochemistry. The chemical mixtures containing sweet/bitter tastants in 7 concentrations are measured by corresponding cell-based sensor. Real-time EIS measurement data of taste cell-based sensor is recorded and processed by DCSSR. Tastant mixtures containing the same chemical components share the same eigen peak located noise intensities (EPLNIs). Correlations and statistical tests on DCSSR signal-to-noise ratio (SNR) maximums (Max-SNR) have been conducted to give a clearly comparison with stochastic resonance (SR) method. Results demonstrate that DCSSR method presents better quantitative perception abilities for sucrose/quinine tastants than SR. Sucrose/quinine concentrations can be discriminated by Max-SNR values. The proposed method provides a promising way for the construction of a novel biological tongue.

Ginsenoside metabolite compound K stimulates glucagon-like peptide-1 secretion in NCI-H716 cells via bile acid receptor activation

Compound K (CK) is a major metabolite of ginsenosides that is absorbed. CK has antidiabetic effects, although the mechanisms underlying the effects of CK have not fully been known. To elucidate the mechanisms underlying the antidiabetic effects of CK, we studied the effects of CK on GLP-1 secretion from NCI-H716 cells, and explored the mechanisms underlying CK-induced GLP-1 secretion. Treatment of NCI-H716 cells with 10, 50, and 100 μM CK significantly increased GLP-1 secretion, and intracellular Ca²⁺ and cAMP levels in a dose-dependent manner. Transfection of NCI-H716 cells with siRNA specific to α-gustducin and siRNA specific to TAS1R3 had no effect on CK-induced GLP-1 secretion and Ca²⁺ increase. However, transfection of NCI-H716 cells with TGR5-specific siRNA significantly inhibited CK-induced GLP-1 secretion and the increase in Ca²⁺ and cAMP levels. Moreover, CK showed human TGR5 agonist activity in CHO-K1 cells transiently transfected with human TGR5. Our data provide a novel mechanism of CK for antidiabetic effects. Moreover, the findings might suggest that CK is a potential agent that has multiple biological functions in the body via GLP-1 secretion and TGR5 activation.

GLP-1 amidation efficiency along the length of the intestine in mice, rats and pigs and in GLP-1 secreting cell lines

xxxMeasurements of plasma concentrations of the incretin hormone GLP-1 are complex because of extensive molecular heterogeneity. This is partly due to a varying and incompletely known degree of C-terminal amidation. Given that virtually all GLP-1 assays rely on a C-terminal antibody, it is essential to know whether or not the molecule one wants to measure is amidated. We performed a detailed analysis of extractable GLP-1 from duodenum, proximal jejunum, distal ileum, caecum, proximal colon and distal colon of mice (n=9), rats (n=9) and pigs (n=8) and determined the degree of amidation and whether this varied with the six different locations. We also analyzed the amidation in 3 GLP-1 secreting cell lines (GLUTag, NCI-H716 and STC-1). To our surprise there were marked differences between the 3 species with respect to the concentration of GLP-1 in gut. In the mouse, concentrations increased continuously along the intestine all the way to the rectum, but were highest in the distal ileum and proximal colon of the rat. In the pig, very little or no GLP-1 was present before the distal ileum with similar levels from ileum to distal colon. In the mouse, GLP-1 was extensively amidated at all sampling sites, whereas rats and pigs on average had around 2.5 and 4 times higher levels of amidated compared to non-amidated GLP-1, although the ratio varied depending upon the location. GLUTag, NCI-H716 and STC-1 cells all exhibited partial amidation with 2–4 times higher levels of amidated compared to non-amidated GLP-1.

Hexane Fractions of Bupleurum falcatum L. Stimulates Glucagon‐Like Peptide‐1 Secretion through Gβγ‐Mediated Pathway

Bupleurum falcatum L. has been used traditionally as a medicinal herb in Korean medicine. The hexane fraction of BF (HFBF), which was profiled with Direct Analysis in Real Time-Mass Spectrometry (DART-MS), activates the secretion of glucagon-like peptide-1 (GLP-1) in NCI-H716 cells significantly. We performed a microarray analysis and GLP-1 ELISA assay, as well as calcium imaging experiments with inhibitors, to investigate the mechanism of action of the HFBF. Through the microarray analysis, it was found that the ITPR2 gene that encodes the inositol 1,4,5-trisphosphate (IP3) receptor is up-regulated and the HFBF induces cell depolarization by inhibiting the voltage-gated channel expression in NCI-H716 cells. In addition, we found that the intracellular calcium in NCI-H716 cells, with Gallein, U73122, and 2APB as inhibitors, was decreased. These results suggest that the HFBF activates the GLP-1 secretion through the Gβγ pathways in the enteroendocrine L cells after treatment with the HFBF.

Foods for appetite control: Can carbohydrates stimulate the ileal brake and enhance satiety?

New therapeutic approaches targeting appetite suppression for the management of body weight and prevention of obesity are currently of widespread interest. The research within this thesis has focused on the ileal brake, which is an endogenous appetite suppression mechanism that is activated by the presence of macronutrients in the ileum. The aim of the thesis was to investigate whether this mechanism can be activated firstly by direct delivery of glucose, and if so, secondly through oral delivery of protected available carbohydrate (CHO) from foods into the ileum. If resulting in suppression of appetite and food intake, it was anticipated that the findings in this thesis may have important implications for the development of food-derived appetite suppressants and/or therapeutics. The first clinical trial (Chapter 4) in this thesis used a model in which 14 healthy male volunteers were intubated with a 3.8 m long naso-ileal (NI) tube, the aim of which was to deliver glucose directly to the ileum, the distal small intestine (SI). The trial was a 5-day, randomised, residential, cross-over design investigating delivery of glucose (15 g) and saline to both the ileum and the duodenum. In this carefully controlled NI tube feeding study, there was evidence that the direct delivery of glucose into the ileum enhanced aspects of subjectively-rated satiety,glucagon-like-peptide-1 (GLP-1) and peptide YY (PYY) release, and decreased short-term energy intake (EI) compared with the direct delivery of saline into the ileum (-10%, -481 kJ, trend) and glucose into the duodenum (-22%, -988 kJ, Tukey???s post-hoc, P < 0.05) at an ad libitum lunch meal. An in vitro study (Chapter 5) was conducted to investigate the mechanisms by which intestinal enteroendocrine L-cells in the ileum couple glucose sensing to GLP-1 secretion using NCI-H716 cells, a human L-cell line model. The findings that GLP-1 secretion was triggered by glucose and inhibited by a well-established sGLT-1inhibitor and the presence of sGLT-1 transcript in the NCI-H716 cells suggest that the action of sGLT-1 is involved in glucose sensing. The findings that GLP-1 secretion was triggered by fructose and the presence of TAS1R2 receptor and ??-gustducin transcripts also suggest that the activation of sweet taste receptors may participate in the regulation of the secretion of GLP-1. In light of these results, it is possible to infer that glucose-induced GLP-1 secretion from L-cells in the ileum is a consequence of the combined effect of sGLT-1 and activation of the sweet taste receptors. The aim of the second clinical trial (Chapter 6) was to compare the effects of low-dose (500 mg) and high-dose (1500 mg) commercially available grape seed extract (GSE), hypothesised to induce post-meal CHO malabsorption in the proximal SI with a placebo control, consumed at the same time as a standardised high-starch breakfast meal on appetite over the subsequent 3-4 hours and EI at a subsequent ad libitum lunch meal in 20 healthy males. The trial was a randomised, double blind, placebo…

Transcriptomic analysis of the bitter taste receptor-mediated glucagon-like peptide-1 stimulation effect of quinine

Quinine is a bitter taste receptor agonist that has been studied its anti-pyretic, anti-malarial, anti-pain, and anti-inflammatory activity. In this study, glucagon-like peptide-1 (GLP-1) stimulation effect of quinine was investigated. Bitter taste receptors are G protein-coupled receptor (GPCR), which transfer the molecular signal through its downstream cascade. The activation of bitter taste receptor, which expressed in the enteroendocrine L cells, stimulates the GLP-1 secretion and therefore can be a therapeutic target of the type-2 diabetes mellitus (T2DM). Here, we studied GLP-1 stimulation effect of quinine on the endocrine differentiated NCI-H716 cells. To investigate the molecular mode-of-action of the GLP-1 stimulation effect of quinine in the enteroendocrine L cells, transcriptomic analysis was performed. Our data suggest that quinine stimulates the GLP-1 secretion through the bitter taste receptor-signaling pathway, and thus has the possibility of therapeutic agent of T2DM.

Sweet and bitter tastant discrimination from complex chemical mixtures using taste cell-based sensor

In this paper, sweet and bitter tastants discrimination from complex chemical mixtures using taste cell-based sensor is investigated. The taste receptor cells (NCI-H716 and STC-1 cells) are cultured on carbon screen-printed electrodes to fabricate integrated taste sensors. NCI-H716 cell-based sensor is utilized to discriminate thirteen selected sweetener mixtures and seven tastant mixtures. STC-1 cell-based sensor is used in seven tastant mixtures discrimination. Real-time electrochemical impedance spectrum data is measured when the sensor is stimulated by tastant mixtures. Measurement data is analyzed by non-linear bistable stochastic resonance. Signal-to-noise ratio maximums and eigen peak located noise intensities are used to characterize tastant mixture information. Experiment results indicate that the studied taste receptor cell-based sensors are capable to discriminate some tastant concentrations from complex chemical mixtures. Correlation between sensor responses and tastant concentration has been conducted. Statistical tests are conducted to confirm statistical significance of the signal-to-noise ratio parameters of various tastant mixtures. Results indicate that responses of STC-1 cell-based sensor have high positive correlation with quinine concentrations. Tastant perception abilities of STC-1 cell-based sensor are much better than that of NCI-H716 cell-based sensor. This study validates that the living taste receptor cell-based sensor presents its potential in specific tastant information measurement in the presence of other tastants in solution environment, which provides a promising way to construct a novel biological electric tongue.

Abstract A278: ARQ 087, a multi-tyrosine kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models.

Abstract Fibroblast growth factors (FGF) and their receptors (FGFR) play important roles in cell proliferation, cell differentiation, cell migration, cell survival, protein synthesis, and angiogenesis. The FGFR family consists of four genes encoding tyrosine kinase receptors (FGFR1, FGFR2, FGFR3, and FGFR4). Dysregulation of FGFR signaling has been implicated in a number of developmental syndromes as well as cancers, e.g., squamous non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, liver, breast, ovarian, endometrial, and bladder carcinomas, fueling significant interest in FGFRs as targets for therapeutic intervention. ArQule, Inc. has discovered a novel, ATP competitive class of FGFR inhibitors from which ARQ 087 emerged as a candidate for advancement into clinical development. ARQ 087 is a potent multi-kinase inhibitor with pan-FGFR activity against FGFR1, FGFR2, mutant FGFR2 (N549H), FGFR3, and FGFR4 kinases, all exhibiting IC50 values in the low nanomolar range in biochemical assays. Ki versus FGFR1 and FGFR2 were 2.8nM and 0.68nM, respectively. ARQ 087 inhibited ectopically expressed FGFR1, 2, and 3 in COS-1 cells as well as, to a varying extent, the proliferation of BaF3 cells expressing the FGFR family of receptors (FGFR2≫FGFR1/FGFR3≫FGFR4). Cell proliferation studies suggested a correlation of FGFR2 mRNA amplification in gastric and other cancers with associated sensitivity to treatment with ARQ 087. Along these lines, ARQ 087 demonstrated potent inhibition of FGFR2 phosphorylation in NCI-H716, Kato III, SNU-16 and MFM223 cells; all demonstrated to be driven by FGFR2. The inhibition of cell growth was associated with an ARQ 087-induced G1 cell cycle arrest and subsequent induction in apoptosis that appears to be related to the levels of FGFR2 protein. Cell lines driven by FGFR2 activating mutations did not undergo apoptosis but did accumulate in G1 following ARQ 087 treatment. In vivo, ARQ 087 induced regressions in FGFR2-driven xenograft models (SNU-16, NCI-H716 and BaF3/FGFR2) and inhibited tumor progression in a model harboring an FGFR2-activating mutation (AN3CA). In addition, concentration-dependent inhibition of phosphorylation of FGFR2 and the downstream FGFR pathway signals (FRS2α, MEK, ERK, and AKT) was evident in response to ARQ 087 treatment in both in vitro and in vivo pharmacodynamic assays. In summary, ARQ 087 is an orally bioavailable kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models, possessing good drug-like properties. A clinical development plan including a patient selection strategy is defined and the drug is currently in Phase I clinical studies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A278. Citation Format: Daniel Dransfield, Jennifer Lee, Carol Waghorne, Cathy Bull, Ronald E. Savage, Xiaolan Zhao, Shipeng Yuan, Edward Chang, Enkeleda Nakuci, Sudharshan Eathiraj, Susan Cornell-Kennon, Xiubin Gu, Syed Ali, Chang-Rung Chen. ARQ 087, a multi-tyrosine kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A278.

Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from malignant tumor cells

Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM-carried genes remains to be clarified. The human colorectal cancer cell line NCI-H716 and human malignant primitive neuroectodermal tumor cell line SK-PN-DW are known to contain many DMs. To examine the amplification of DM-carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI-H716 and SK-PN-DW tumor cells. We identified the amplification regions and the DM-carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM-carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM-carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM-carried genes led to a decrease in the number of DMs and reduced the amplification of the DM-carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM-carried genes resulted in a decrease in cell proliferation and invasion ability.