Signalling pathways involved in the detection of peptones by murine small intestinal enteroendocrine L-cells.

Glucagon like peptide-1 is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. Here, we investigated mechanisms underlying the sensing of peptones by primary small intestinal L-cells. Meat, casein and vegetable-derived peptones (5 mg/ml), the L-amino acids Phe, Trp, Gln and Ala (20 mM each), and the dipeptide glycine-sarcosine (20 mM) stimulated GLP-1 secretion from primary cultures prepared from the small intestine. Further mechanistic studies were performed with meat peptone, and revealed the elevation of intracellular calcium in L-cells. Inhibition of the calcium sensing receptor (CaSR), transient receptor potential (TRP) channels and Q-type voltage gated calcium channels (VGCC) significantly attenuated peptone-stimulated GLP-1 release and reduced intracellular Ca(2+) responses. CaSR inhibition also attenuated the GLP-1 secretory response to Gln. Targeting these pathways in L-cells could be used to increase endogenous production of GLP-1 and offer exploitable avenues for the development of therapeutics to treat diabetes and obesity.This work was funded by grants from the Wellcome Trust (WT088357/Z/09/Z and WT084210/Z/07/Z), the MRC Metabolic Diseases Unit (MRC_MC_UU_12012/3) and Full4Health (FP7/2011-2015, grant agreement n° 266408).This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.peptides.2015.07.01

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

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

Stimulation of incretin secreting cells

The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon like peptide-1 (GLP-1) are secreted from enteroendocrine cells in the gut and regulate physiological and homeostatic functions related to glucose control, metabolism and food intake. This review provides a systematic summary of the molecular mechanisms underlying secretion from incretin cells, and an understanding of how they sense and interact with lumen and vascular factors and the enteric nervous system through transporters and G-protein coupled receptors (GPCRs) present on their surface to ultimately culminate in hormone release. Some of the molecules described below such as sodium coupled glucose transporter 1 (SGLT1), G-protein coupled receptor (GPR) 119 and GPR40 are targets of novel therapeutics designed to enhance endogenous gut hormone release. Synthetic ligands at these receptors aimed at treating obesity and type 2 diabetes are currently under investigation

High fat diet impairs the function of glucagon-like peptide-1 producing L-cells.

UNLABELLED: Glucagon-like peptide-1 (GLP-1) acts as a satiety signal and enhances insulin release. This study examined how GLP-1 production from intestinal L-cells is modified by dietary changes. METHODS: Transgenic mouse models were utilized in which L-cells could be purified by cell specific expression of a yellow fluorescent protein, Venus. Mice were fed on chow or 60% high fat diet (HFD) for 2 or 16 weeks. L-cells were purified by flow cytometry and analysed by microarray and quantitative RT-PCR. Enteroendocrine cell populations were examined by FACS analysis, and GLP-1 secretion was assessed in primary intestinal cultures. RESULTS: Two weeks HFD reduced the numbers of GLP-1 positive cells in the colon, and of GIP positive cells in the small intestine. Purified small intestinal L-cells showed major shifts in their gene expression profiles. In mice on HFD for 16 weeks, significant reductions were observed in the expression of L-cell specific genes, including those encoding gut hormones (Gip, Cck, Sct, Nts), prohormone processing enzymes (Pcsk1, Cpe), granins (Chgb, Scg2), nutrient sensing machinery (Slc5a1, Slc15a1, Abcc8, Gpr120) and enteroendocrine-specific transcription factors (Etv1, Isl1, Mlxipl, Nkx2.2 and Rfx6). A corresponding reduction in the GLP-1 secretory responsiveness to nutrient stimuli was observed in primary small intestinal cultures. CONCLUSION: Mice fed on HFD exhibited reduced expression in L-cells of many L-cell specific genes, suggesting an impairment of enteroendocrine cell function. Our results suggest that a western style diet may detrimentally affect the secretion of gut hormones and normal post-prandial signaling, which could impact on insulin secretion and satiety.This work was funded by grants from the Wellcome Trust (WT088357/Z/09/Z and WT084210/Z/07/Z), the MRC Metabolic Diseases Unit (MRC_MC_UU_12012/3) and Full4Health (FP7/2011-2015, grant agreement n° 266408). GLP-1 immuno-assays were performed by Keith Burling at the MRC-MDU.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.peptides.2015.06.00

Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid. METHODS: GLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter. RESULTS: L-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells. CONCLUSIONS/INTERPRETATION: GPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.This work was funded by grants from the Wellcome Trust (WT088357/Z/09/Z and WT084210/Z/07/Z), the MRC Metabolic Diseases Unit (MRC_MC_UU_12012/3), Full4Health (FP7/2011-2015, grant agreement n° 266408) and a BBSRC/AstraZeneca CASE studentship to CEM.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.peptides.2015.06.01