Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors.

Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca(2+). In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca(2+) response to TDCA. Using small-volume Ussing chambers optimized for measuring GLP-1 secretion, we found that both a GPBAR1 agonist and TDCA stimulated GLP-1 release better when applied from the basolateral than from the luminal direction and that luminal TDCA was ineffective when intestinal tissue was pretreated with an ASBT inhibitor. ASBT inhibition had no significant effect in nonpolarized primary cultures. Studies in the perfused rat gut confirmed that vascularly administered TDCA was more effective than luminal TDCA. Intestinal primary cultures and Ussing chamber-mounted tissues from GPBAR1-knockout mice did not secrete GLP-1 in response to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms.Mesoscale GLP-1 immuno assays were performed by Keith Burling and colleagues at the Medical Research Council Metabolic Diseases Unit, Cambridge. Thisworkwas supported by the Wellcome Trust (grants 084 210/Z/07/Z, 088 357/Z/09/Z and 099 825/Z/12/Z) and the MRC (grant MRC_MC_UU_12012/ 3), the Novo Nordisk Center for Basic Metabolic Research (Novo Nordisk Foundation, Denmark) and the European Union’s Seventh Framework Programme for Research, Technological Development, and Demonstration Activities (Grant No. 266 408) Juraj Rievaj was supported by an EFSD Albert Renold Travel Fellowship. Ussing chamber equipment was initially kindly lent by Dr. Todd Alexander, Departments of Pediatrics& Physiology, University of Alberta, Canada.This is the final version of the article. It first appeared from Endocrine Society via http://dx.doi.org/10.1210/en.2015-132

Adenosine triphosphate is co-secreted with glucagon-like peptide-1 to modulate intestinal enterocytes and afferent neurons.

Enteroendocrine cells are specialised sensory cells located in the intestinal epithelium and generate signals in response to food ingestion. Whilst traditionally considered hormone-producing cells, there is evidence that they also initiate activity in the afferent vagus nerve and thereby signal directly to the brainstem. We investigate whether enteroendocrine L-cells, well known for their production of the incretin hormone glucagon-like peptide-1 (GLP-1), also release other neuro-transmitters/modulators. We demonstrate regulated ATP release by ATP measurements in cell supernatants and by using sniffer patches that generate electrical currents upon ATP exposure. Employing purinergic receptor antagonists, we demonstrate that evoked ATP release from L-cells triggers electrical responses in neighbouring enterocytes through P2Y2 and nodose ganglion neurones in co-cultures through P2X2/3-receptors. We conclude that L-cells co-secrete ATP together with GLP-1 and PYY, and that ATP acts as an additional signal triggering vagal activation and potentially synergising with the actions of locally elevated peptide hormone concentrations.Wellcome Trust joint investigator award (106262/Z/14/Z and 106263/Z/14/Z); MRC programme within the Metabolic Diseases Unit (MRC_MC_UU_12012/3); MRC Metabolic Diseases Unit [MRC_MC_UU_12012/5] ; Wellcome Trust Strategic Award [100574/Z/12/Z

Characterisation of proguanylin expressing cells in the intestine – evidence for constitutive luminal secretion

Abstract: Guanylin, a peptide implicated in regulation of intestinal fluid secretion, is expressed in the mucosa, but the exact cellular origin remains controversial. In a new transgenic mouse model fluorescent reporter protein expression driven by the proguanylin promoter was observed throughout the small intestine and colon in goblet and Paneth(-like) cells and, except in duodenum, in mature enterocytes. In Ussing chamber experiments employing both human and mouse intestinal tissue, proguanylin was released predominantly in the luminal direction. Measurements of proguanylin expression and secretion in cell lines and organoids indicated that secretion is largely constitutive and requires ER to Golgi transport but was not acutely regulated by salt or other stimuli. Using a newly-developed proguanylin assay, we found plasma levels to be raised in humans after total gastrectomy or intestinal transplantation, but largely unresponsive to nutrient ingestion. By LC-MS/MS we identified processed forms in tissue and luminal extracts, but in plasma we only detected full-length proguanylin. Our transgenic approach provides information about the cellular origins of proguanylin, complementing previous immunohistochemical and in-situ hybridisation results. The identification of processed forms of proguanylin in the intestinal lumen but not in plasma supports the notion that the primary site of action is the gut itself

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Vehicle's technical condition and emission

Artykuł prezentuje wyniki badań mających na celu znalezienie różnic w emisji spalin dwóch silników o zapłonie iskrowym pojazdów osobowych o różnym roku produkcji. Porównania dokonano podczas poziomego ruchu pojazdów ze stałą prędkością. Podczas prowadzonych badań silnik samochodu pokonywał tylko następujące opory: układu napędowego, toczenia oraz powietrza. Do badań wybrano samochody o porównywalnych masach i powierzchni czołowej. Były to Škoda 105 L wyprodukowana w 1983 roku oraz Toyota Yaris 1,0 VVTi z roku 2003.The article presents the research results aimed to get true differences of pollution production between two cars with spark ignition engine depending on the car's age. A car movement by constant speed on horizontal plane was realised for comparison. The vehicle engine must overcome only mechanical transmission losses, air resistance and rolling resistance, too. The air resistance size depends on the speed, the vehicle frontal area and the air resistance coefficient. It was chosen vehicles with approximated equal weight and approximated equal vehicle frontal area for comparison. These conditions fulfil vehicles Škoda 105 L, made in 1983, and Toyota Yaris 10,0 VVTi, made in 200

Determination of trace Mn(II) in pharmaceutical diet supplements by cathodic stripping voltammetry on bare carbon paste electrode

A simple electroanalytical method for quantification of Mn in pharmaceutical diet supplements has been developed. The method is based on the formation of insoluble MnO2 on the electrode during deposition step at the potential of +0.85V vs SCE, and its consecutive reductive dissolution back to Mn(II) during differential pulse voltammetry scan. Pure carbon paste was found to be a suitable material for this kind of determination because of its easy preparation, good accessibility, low cost, as well as its appropriate analytical properties. In 0.1 mol L-1 phosphate buffer (pH 7.4) supporting electrolyte and at optimised deposition time (120 s) the linear calibration plot was obtained in the concentration range 1 × 10 -6-12 × 10-6 mol L-1; the corresponding detection limit was 1 × 10-7 mol L-1. The method was applied to the analysis of pharmaceutical dosage forms containing manganese as an essential trace element

Characterisation of proguanylin expressing cells in the intestine – evidence for constitutive luminal secretion

Abstract: Guanylin, a peptide implicated in regulation of intestinal fluid secretion, is expressed in the mucosa, but the exact cellular origin remains controversial. In a new transgenic mouse model fluorescent reporter protein expression driven by the proguanylin promoter was observed throughout the small intestine and colon in goblet and Paneth(-like) cells and, except in duodenum, in mature enterocytes. In Ussing chamber experiments employing both human and mouse intestinal tissue, proguanylin was released predominantly in the luminal direction. Measurements of proguanylin expression and secretion in cell lines and organoids indicated that secretion is largely constitutive and requires ER to Golgi transport but was not acutely regulated by salt or other stimuli. Using a newly-developed proguanylin assay, we found plasma levels to be raised in humans after total gastrectomy or intestinal transplantation, but largely unresponsive to nutrient ingestion. By LC-MS/MS we identified processed forms in tissue and luminal extracts, but in plasma we only detected full-length proguanylin. Our transgenic approach provides information about the cellular origins of proguanylin, complementing previous immunohistochemical and in-situ hybridisation results. The identification of processed forms of proguanylin in the intestinal lumen but not in plasma supports the notion that the primary site of action is the gut itself

Ileo-colonic delivery of conjugated bile acids improves glucose homeostasis via colonic GLP-1-producing enteroendocrine cells in human obesity and diabetes.

BACKGROUND: The bile acid (BA) pathway plays a role in regulation of food intake and glucose metabolism, based mainly on findings in animal models. Our aim was to determine whether the BA pathway is altered and correctable in human obesity and diabetes. METHODS: We conducted 3 investigations: 1) BA receptor pathways were studied in NCI-H716 enteroendocrine cell (EEC) line, whole human colonic mucosal tissue and in human colonic EEC isolated by Fluorescence-activated Cell Sorting (ex vivo) from endoscopically-obtained biopsies colon mucosa; 2) We characterized the BA pathway in 307 participants by measuring during fasting and postprandial levels of FGF19, 7αC4 and serum BA; 3) In a placebo-controlled, double-blind, randomised, 28-day trial, we studied the effect of ileo-colonic delivery of conjugated BAs (IC-CBAS) on glucose metabolism, incretins, and lipids, in participants with obesity and diabetes. FINDINGS: Human colonic GLP-1-producing EECs express TGR5, and upon treatment with bile acids in vitro, human EEC differentially expressed GLP-1 at the protein and mRNA level. In Ussing Chamber, GLP-1 release was stimulated by Taurocholic acid in either the apical or basolateral compartment. FGF19 was decreased in obesity and diabetes compared to controls. When compared to placebo, IC-CBAS significantly decreased postprandial glucose, fructosamine, fasting insulin, fasting LDL, and postprandial FGF19 and increased postprandial GLP-1 and C-peptide. Increase in faecal BA was associated with weight loss and with decreased fructosamine. INTERPRETATIONS: In humans, BA signalling machinery is expressed in colonic EECs, deficient in obesity and diabetes, and when stimulated with IC-CBAS, improved glucose homeostasis. ClinicalTrials.gov number, NCT02871882, NCT02033876. FUNDING: Research support and drug was provided by Satiogen Pharmaceuticals (San Diego, CA). AA, MC, and NFL report grants (AA- C-Sig P30DK84567, K23 DK114460; MC- NIH R01 DK67071; NFL- R01 DK057993) from the NIH. JR was supported by an Early Career Grant from Society for Endocrinology

Electroolfactogram responses from organotypic cultures of the olfactory epithelium from postnatal mice

Organotypic cultures of the mouse olfactory epithelium connected to the olfactory bulb were obtained with the roller tube technique from postnatal mice aged between 13 and 66 days. To test the functionality of the cultures, we measured electroolfactograms (EOGs) at different days in vitro (DIV), up to 7 DIV, and we compared them with EOGs from identical acute preparations (0 DIV). Average amplitudes of EOG responses to 2 mixtures of various odorants at concentrations of 1 mM or 100 \u3bcM decreased in cultures between 2 and 5 DIV compared with 0 DIV. The percentage of responsive cultures was 57%. We also used the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to trigger the olfactory transduction cascade bypassing odorant receptor activation. Average amplitudes of EOG responses to 500 \u3bcM IBMX were not significantly different in cultures up to 6 DIV or 0 DIV, and the average percentage of responsive cultures between 2 and 5 DIV was 72%. The dose-response curve to IBMX measured in cultures up to 7 DIV was similar to that at 0 DIV. Moreover, the percentage of EOG response to IBMX blocked by niflumic acid, a blocker of Ca-activated Cl channels, was not significantly different in cultured or acute preparations. \ua9 The Author 2008. Published by Oxford University Press. All rights reserved