Biology subjects

Hanyaloglu, A.

Publications and source records attributed to Hanyaloglu, A..

8 recordsLinked to original sources

bioRxiv2026

A fluorescent reporter system for scalable and live detection of PYY production from enteroendocrine cells with single-event resolution

Peptide YY (PYY) comprises the secretory repertoire of enteroendocrine L-cells alongside glucagon-like peptide-1 (GLP-1), and positively modulates postprandial satiety, digestion mechanics, and regeneration of the intestinal epithelium. Whereas immortalised GLP-1-secreting human L-cell models support pre-clinical drug discovery, comparable human lines that robustly secrete PYY are lacking, hindering mechanistic studies of its release. We present a biosensor for scalable detection of PYY production and secretion from human enteroendocrine cells in vitro. Guided by in silico structural prediction, we engineer a superecliptic phluorin (SEP)-tagged PYY, SEP-PYY, that engages native hormone processing machinery and is responsive to canonical nutrient stimuli when expressed in a human enteroendocrine cell line, NCI-H716. SEP-PYY production and secretion can be measured by optimised flow cytometry and spectrofluorometric plate readouts respectively, methods with superior time- and cost-efficacy to current hormone detection methods. Leveraging the pH sensitivity of SEP, we use this reporter system in detection of single-event hormone exocytosis by total internal reflection microscopy. Finally, we demonstrate the application of our system in screening ligands of metabolite-sensing G-protein coupled receptors that drive SEP-PYY secretion, and supporting discovery of druggable pathways in metabolic disease.

cell biology↗

bioRxiv2025

D- and L-Lactate enhance intestinal barrier function via activation of an apical HCAR1/Giα pathway in a human colonic epithelial cell model

The stereoisomers of lactate, L- and D- are not only metabolic substrates but also signalling molecules, capable of activating and signalling through its G protein-coupled receptor, Hydroxycarboxylic acid receptor 1 (HCAR1). These stereoisomers are both produced by the gut microbiota at millimolar concentrations creating a physiological environment for lactate-sensing unique to the gut yet, poorly understood. Here we identify a role for D-/L-lactate on intestinal barrier function. A human colonic epithelial cell model, Caco2, activated Gi signalling in response to both L- and D-lactate, although L-lactate exhibited a more potent and rapid Gi signal profile. When differentiated, apically but not basally treated D-/L-lactate enhanced tight junctions and reduced cell permeability, consistent with the apical localization of HCAR1. This improved barrier function occurred in a Gi-dependent manner. In addition, apical lactate rescued the reduced intestinal barrier function induced by lipopolysaccharides. This work highlights the potential for D-/L-lactate supplementation in improving gut health. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/685091v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@28fa82org.highwire.dtl.DTLVardef@56a75borg.highwire.dtl.DTLVardef@10578c6org.highwire.dtl.DTLVardef@3c3cd2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

A selective and augmentable FFAR2 signal circuitry programs a butyrate-induced cellular identity of enteroendocrine L-cells

Activation of free fatty acid receptor 2 (FFAR2) on enteroendocrine L-cells mediates secretion of glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), key regulators of central appetite control with therapeutic relevance to obesity. Here, we show that butyrate, a metabolite derived from fermentation of dietary fibre and an FFAR2 agonist, stimulates a PYY-biased profile in a human L-cell model at the transcriptional, morphological and secretory level via an FFAR2-Gi axis that does not require dynamin-dependent receptor internalization. We observe that butyrate modulates active Notch cascades within a Hes1-GFP mouse organoid model, which are antagonistic to secretory differentiation, and identify butyrate-dependent regulation of late-stage human enteroendocrine maturation markers, NeuroD1 and Pax6. Butyrate-mediated upregulation of Pyy and Pax6 is enhanced by the FFAR2-selective Gi biased allosteric agonist AZ-1729. Our study reveals functions of spatiotemporally-regulated butyrate-activated FFAR2 signalling mechanisms that could be pharmacologically amplified to fine-tune L-cell populations in the human colon. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/659710v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@14b9620org.highwire.dtl.DTLVardef@16d4c10org.highwire.dtl.DTLVardef@9cb031org.highwire.dtl.DTLVardef@f3a0e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Enteropancreatic neurons drive the glucoregulatory response to ingested lipid

Enteropancreatic neurons project from the small intestinal wall to the pancreas. Though well positioned to mediate the effects of ingested nutrients on pancreatic function, the metabolic role of these neurons is unclear. Diets rich in olive oil promote weight loss and improve remission rates in patients with T2DM. Here, we show that olive oil improves acute glucose tolerance by stimulating insulin release via neurotensin receptor type 1 (NTSR1)-expressing enteropancreatic neurons. These neurons are necessary for the effects of olive and neurotensin on glucose tolerance, and their activation is sufficient to improve glucose tolerance. These findings suggest a mechanism by which dietary olive oil regulates blood glucose levels and present a novel functional role for enteropancreatic neurons in regulating glucose homeostasis.

physiology↗

bioRxiv2025

A single domain intrabody as a novel tool to bias the subcellular trafficking of the follicle-stimulating hormone receptor

Intracellular variable fragments from heavy-chain only antibodies of camelids (intra-VHH) have been successfully used for their stabilizing properties to solve the 3D structure of active G protein-coupled receptors (GPCRs) bound to their cognate transducers. They also provide tools to link a given conformation of a GPCR to the signalling network engaged, thus allowing extensive structure/activity studies. Recently, they have been instrumental in tracking active GPCRs in various subcellular compartments. Here, we report the isolation and characterization of iPRC2, an intra-VHH recognizing the 1st and 3rd intracellular loops of the FSHR, but not of the luteinizing hormone/choriogonadotropin receptor close relative. Its expression in the cell decreases the cAMP production in response to hormone binding, and requires Gs for optimal interaction with the receptor. Importantly, iPRC2 increases the FSHR accumulation in the early endosomes, and consequently, diminishes its recycling to the cell surface. Hence, in contrast to previously described intra-VHH that disclose active GPCR intracellular location, iPRC2 provokes per se a location bias, through its ability to reroute the FSHR. Thus, it is an innovative tool to examine the functional consequences of GPCR accumulation in various sub-cellular compartments.

pharmacology and toxicology↗

bioRxiv2024

Stabilised D2R G-protein coupled receptor oligomers identify multi-state β-arrestin complexes

The G-protein coupled receptor (GPCR) superfamily directs central roles in many physiological and pathophysiological processes via diverse and complex mechanisms. GPCRs can exhibit signal pleiotropy via formation of di/oligomers both with themselves and other GPCRs. A deeper understanding of the molecular basis and functional role of oligomerization would facilitate rational design of activity-selective ligands. A structural model of the D2 dopamine receptor (D2R) homomer identified distinct combinations of substitutions likely to stabilize protomer interactions. Molecular modelling of {beta}-arrestin-2 ({beta}arr2) bound to predicted dimer models suggests a 2:2 receptor:{beta}arr2 stoichiometry, with the dimer favouring {beta}arr2 over Gi coupling. A combination of biochemical, biophysical and super-resolution, single molecule imaging approaches demonstrated that the D2R mutant homomers exhibited greater stability. The mutant D2R homomers also exhibited bias towards recruitment of the GPCR adaptor protein {beta}arr2 with either faster or ligand-independent {beta}arr2 recruitment, increased internalization and reprogrammed regulation of ERK signaling. Through GPCR dimer-stabilization, we propose that D2R di/oligomerization has a role in {beta}arr2-biased signaling.

cell biology↗

bioRxiv2020

Endosomal free fatty acid receptor 2 signaling is essential for propionate-induced anorectic gut hormone release

The ability of propionate, a short chain fatty acid produced from the fermentation of non-digestible carbohydrates in the colon, to stimulate the release of anorectic gut hormones, such as glucagon like peptide-1 (GLP-1), is an attractive approach to enhance appetite regulation, weight management and glycaemic control. Propionate induces GLP-1 release via its G protein-coupled receptor (GPCR), free fatty acid receptor 2 (FFA2); a GPCR that activates Gi and Gq/11 pathways. However, how pleiotropic GPCR signaling mechanisms in the gut regulates appetite is poorly understood. Here, we identify propionate-mediated G protein signaling is spatially directed within the cell via the targeting of FFA2 to very early endosomes. Furthermore, propionate activates an endosomal Gi/p38 signaling pathway, which is essential for propionate-induced GLP-1 release in enteroendocrine cells and colonic crypts. Our study reveals that intestinal metabolites can engage membrane trafficking pathways and endosomal signaling platforms to orchestrate complex GPCR pathways within the gut.

cell biology↗

bioRxiv2019

Genetically encoded intrabody sensors illuminate structural and functional diversity in GPCR-β-arrestin complexes

Interaction of {beta}-arrestins ({beta}arrs) upon agonist-stimulation is a hallmark of G protein-coupled receptors (GPCRs) resulting in receptor desensitization, endocytosis and signaling. Although overall functional roles of {beta}arrs are typically believed to be conserved across different receptors, emerging data now clearly unveils receptor-specific functional contribution of {beta}arrs. The underlying mechanism however remains mostly speculative and represents a key missing link in our current understanding of GPCR signaling and regulatory paradigms. Here, we develop synthetic intrabody-based conformational sensors that help us visualize the assembly and trafficking of GPCR-{beta}arr1 complexes in cellular context for a broad set of receptors with spatio-temporal resolution. Surprisingly, these conformational sensors reveal a previously unappreciated level of diversity in GPCR-{beta}arr complexes that extends beyond the current framework of affinity-based classification and phosphorylation-code-based interaction patterns. More importantly, this conformational diversity arising from spatial signature of phosphorylation sites manifests directly in the form of distinct functional outcomes, including even opposite contribution of {beta}arrs in signal-transduction for different receptors. Taken together, these findings uncover that despite an overall similar interaction and trafficking patterns; critical structural and functional differences exist in {beta}arr complexes for different GPCRs that define and fine-tune receptor-specific downstream responses.

cell biology↗