Angiotensin II Type 1 Receptor-Dependent GLP-1 and PYY Secretion in Mice and Humans.

Angiotensin II (Ang II) is the key hormone mediator of the renin angiotensin system, which regulates blood pressure and fluid and electrolyte balance in the body. Here we report that in the colonic epithelium, the Ang II type 1 receptor is highly and exclusively expressed in enteroendocrine L cells, which produce the gut hormones glucagon-like peptide-1 and peptide YY (PYY). Ang II stimulated glucagon-like peptide-1 and PYY release from primary cultures of mouse and human colon, which was antagonized by the specific Ang II type 1 receptor blocker candesartan. Ang II raised intracellular calcium levels in L cells in primary cultures, recorded by live-cell imaging of L cells specifically expressing the fluorescent calcium sensor GCaMP3. In Ussing chamber recordings, Ang II reduced short circuit currents in mouse distal colon preparations, which was antagonized by candesartan or a specific neuropeptide Y1 receptor inhibitor but insensitive to amiloride. We conclude that Ang II stimulates PYY secretion, in turn inhibiting epithelial anion fluxes, thereby reducing net fluid secretion into the colonic lumen. Our findings highlight an important role of colonic L cells in whole-body fluid homeostasis by controlling water loss through the intestine.This work was funded by grants from the Wellcome Trust (106262/Z/14/Z, 106 263/Z/14/Z), the MRC Metabolic Diseases Unit (MRC MC UU 12012/3, MRC MC UU 12012/5) and Full4Health (FP7/ 2011–2015, grant agreement no 266 408).This is the author accepted manuscript. The final version is available from the Endocrine Society via http://dx.doi.org/10.1210/en.2016-138

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

Role of enteroendocrine L-cells in arginine vasopressin-mediated inhibition of colonic anion secretion.

KEY POINTS: Arginine vasopressin (AVP) stimulates the release of enteroendocrine L-cell derived hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) in vitro from mouse and human colons. This is mediated by the AVP receptor 1B, which is highly enriched in colonic L-cells and linked to the elevation of L-cell calcium and cAMP concentrations. By means of Ussing chambers, we show that AVP reduced colonic anion secretion, although this was blocked by a specific neuropeptide Y receptor Y1 receptor antagonist, suggesting that L-cell-released PYY acts locally on the epithelium to modulate fluid balance. In human serum samples, PYY concentrations were higher in samples with raised osmolality and copeptin (surrogate marker for AVP). These findings describe, for the first time, the role of L-cells in AVP regulated intestinal fluid secretion, potentially linking together hormonal control of blood volume and blood glucose levels, and thus adding to our understanding of the complex pathways involved in the gut hormonal response to different stimuli. ABSTRACT: Arginine vasopressin (AVP) regulates fluid balance and blood pressure via AVP receptor (AVPR)2 in the kidney and AVP receptor 1A in vascular smooth muscle. Its role in intestinal function has received less attention. We hypothesized that enteroendocrine L-cells producing glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) may be a target of AVP and contribute to the control of fluid balance. Avpr1b expression was assessed by quantitative RT-PCR on flourescence-activated cell sorting-isolated L- and control cells and was enriched in colonic L-cells. AVP stimulated GLP-1 and PYY release from primary cultured murine and human colonic cells and was associated with elevated calcium and cAMP concentrations in L-cells as measured in cultures from GLU-Cre/ROSA26-GCaMP3 and GLU-Epac2camps mice. An antagonist of AVPR1B reduced AVP-triggered hormone secretion from murine and human cells. In Ussing chambers, basolaterally applied AVP reduced colonic anion secretion and this effect was blocked by a specific neuropeptide Y receptor Y1 (NPY1R) antagonist. In human serum, PYY concentrations were higher in samples with raised osmolality or copeptin (a surrogate marker for AVP). In conclusion, we propose that AVP activates L-cell AVPR1B, causing GLP-1 and PYY secretion. PYY in turn reduces colonic anion secretion via epithelial NPY1R. Our data suggest L-cells are active players in the hypothalamic control of intestinal fluid homeostasis, providing a potential link between the regulation of blood volume/pressure/osmolality and blood glucose.This work was funded by grants from the Wellcome Trust (106262/Z/14/Z, 106263/Z/14/Z), the MRC Metabolic Diseases Unit (MRC_MC_UU_12012/3) and Full4Health (FP7/2011-2015, grant agreement no 266408). Juraj Rievaj was initially supported by an EFSD Albert Renold Travel Fellowship.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1113/JP27205

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

Impact of Driving Techniques on Fuel Consumption

Climatologists constantly point to the change of the climate on Earth. They consider CO2 emissions to be one of the main causes of these changes. Transport is significant producer of the greenhouse gases. If one litre of gasoline is burnt, 2.5 kg of CO2 is released into environment. Incineration of one litre of oil means that the air gets 2.7 kg of CO2 [1]. Moreover, the air gets other pollutants, particularly nitrogen oxides NOx, unburned hydrocarbons, particulates. If we want to stop or delay the climate change, we should minimize production of the greenhouse gases. This objective could be implemented through legislation, designing of more efficient propulsion units, construction of infrastructure or using of alternative fuels. The easiest way is to minimize vehicle fuel consumption. This is dependent on driver and his driving techniques. The technical condition of the vehicle, route selection and the type of the vehicle are also significant [2]. We want to show the influence of the driving technique on the fuel consumption. Article describes four different driving techniques and their impact on the fuel consumption

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