Journal: Frontiers in Endocrinology

Article Title: Intestinal Sensing by Gut Microbiota: Targeting Gut Peptides

doi: 10.3389/fendo.2019.00082

Figure Lengend Snippet: Schematic overview depicting regulatory pathways for PYY and GLP-1 secretion by enteroendocrine L-cell. The L-cells express a wide variety of GPCRs that sense luminal content including dietary and bacterial products. SCFAs are recognized by FFAR2 and FFAR3 expressed at the apical and basolateral membrane [but see ]. G s activates adenylyl cyclase, increases cyclic AMP, activation of PKA that regulate gene expression, and activates GLP-1 and PYY. G protein G i/o inhibits G s cAMP pathway while activating PLC pathway. Similarly, G q activates PLC pathway to hydrolysate PIP2 into DAG and IP3. IP3 induces intracellular Ca 2+ release mediated by voltage-gated Ca2+ channels. DAG activates PKC, an important regulator of cell activity and gene expression. Hormone secretion is also stimulated by the Gs-coupled GPR119 and TGR5. TLRs sense microbial molecules and mediate inflammatory responses through NF-kB pathway. Glucose sensing implicate transporters that act through inhibition of K ATP channels inducing membrane depolarization and calcium entry into the cell. These pathways enable L-cells to communicate with other cells by secreting gut peptides like GLP-1 and PYY, as well as by regulating their expression. PG, prostaglandins; SCFA, short chain fatty acids; LCFA, long chain fatty acids; TLR, toll-like receptor; PGE2-R, prostaglandin E2-receptor, FFAR, free fatty acid receptor; GPCR, G-protein coupled receptor; FATP, fatty acid transport protein; TGR, membrane-type receptor for bile acids; SGLT, sodium glucose transporter; GLUT, glucose transporter; T1R/T2R, taste receptor; ATA, aurintricarboxylic acid; pepT, peptide receptor; SOP, serine-o-posphate receptor; cAMP, cyclic adenosine monophosphate; G s , G i/o , G q , G-protein subunits; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; NF-kB, nuclear factor kappa beta; PLC, phospholipase C; GLP-1, glucagon-like peptide; PYY, peptide YY; DAG, diacyl glycerol; IP3, inositol triphosphate; PIP2, phosphatydilinositol (4,5) diphosphate; K ATP , adenosine triphosphate sensitive potassium channel; SST, somatostatin receptor; M1R/M2R, muscarinic receptor; [Ca 2+ ] ic , calcium internal concentration.

Article Snippet: There are already several gut peptide mimetics such as GLP-1, DDP IV inhibitors, CCK antagonists, PYY, PP in clinical trials for obesity and diabetes ( ).

Techniques: Membrane, Activation Assay, Gene Expression, Activity Assay, Inhibition, Expressing, Concentration Assay

Journal: Frontiers in Endocrinology

Article Title: Intestinal Sensing by Gut Microbiota: Targeting Gut Peptides

doi: 10.3389/fendo.2019.00082

Figure Lengend Snippet: Proposed model of functional metagenomics for studying microbiota-gut peptide interactions. The first step (top left), involves high throughput screening (HTS) of metagenomic libraries to identify candidate clones/bacteria able to stimulate gut peptides (i.e., GLP-1, PYY, CCK) gene expression (using gene reporter technology) and release (following calcium flux) from EEC cultures or enteroids (pink color). The full metagenomic insert of clones of interest is then sequenced to characterize the genes/loci involved using NGS and assign them phylogenetically and functionally. The second step (blue color) represents identification of signaling mechanisms responsible for metagenomic clone-induced-activation of EEC-like cells and gut peptide release. Finally, the effect of identified clones of interest can be tested in obese models for their effects on oral and intestinal contributors to caloric intake, including vagal responses (green color).

Article Snippet: There are already several gut peptide mimetics such as GLP-1, DDP IV inhibitors, CCK antagonists, PYY, PP in clinical trials for obesity and diabetes ( ).

Techniques: Functional Assay, High Throughput Screening Assay, Clone Assay, Bacteria, Gene Expression, Activation Assay

Journal: Frontiers in Endocrinology

Article Title: Intestinal Sensing by Gut Microbiota: Targeting Gut Peptides

doi: 10.3389/fendo.2019.00082

Figure Lengend Snippet: Overview representation of functional metagenomic for screening and identification of bioactive metagenomic clones influencing expression of PYY and GLP-1 from cultured cell lines or enteroid lysates. Briefly, enteroendocrine-like cell lines (e.g., STC-1, NCI-H716) are transfected with a plasmid bearing the promoter region of PYY or GLP1 gene driving a reporter gene (e.g., luciferase; secreted alkaline phosphatase) or a genetically encoded calcium indicator, GCaMP 5G, enabling to follow Ca2+ fluxes, which directly controls peptide exocytosis. Then stably transfected cell clones are selected using selective-resistance techniques and their response characterized. The reporter cells are cultured with each candidate clone from the metagenomic library, to screen for their secretory potential of GLP-1 and PYY. Each metagenomic clone bears DNA of ~40 kb cloned into a fosmid. E. coli bearing empty fosmid will serve as control.

Article Snippet: There are already several gut peptide mimetics such as GLP-1, DDP IV inhibitors, CCK antagonists, PYY, PP in clinical trials for obesity and diabetes ( ).

Techniques: Functional Assay, Clone Assay, Expressing, Cell Culture, Transfection, Plasmid Preparation, Luciferase, Stable Transfection, Control