Studying Nutrient Absorption, Malabsorption and Drug Bioavailability in Intestinal Organoids T. Zietek 1*, E. Rath 2, H. Kessler 3, F. Reichart 4, M. Metzger 5, M. Schweinlin 6, D. Haller2, H. Daniel1 1 Dept.
of Nutritional Physiology, TUM, Freising; 2 ZIEL Institute for Food & Health, TUM, Freising; 3 Institute for Advanced Study, TUM, Garching 4 Organic Chemistry Dept., TUM, Garching; 5 Fraunhofer IGB, Translational Center, Würzburg; 6 University Hospital Würzburg, TERM, Würzburg *
[email protected]
Intestinal Organoids for Biomedical Research Research on intestinal transport processes and on incretin hormone secretion is of high interest for the therapy of malabsorption syndromes or metabolic disorders such as obesity and diabetes. We generated organoids derived from wild type mice and from mice lacking different intestinal transporters and conducted functional studies on nutrient transport, hormone secretion and intracellular signaling pathways. We also use organoids for screenings on bioavailability of drugs and prodrugs such as PEPT1-mediated absorption of the antibiotic cefadroxil or transport of novel peptidomimetics binding to specific integrin subtypes such as αVβ6. These novel compounds are promising targets for cancer therapy and tumor characterization. We are currently investigating if our functional studies are transferable to human organoids.
Drug Screening
Nutrient & Drug Transport
Chemical properties of peptidomimetics PM3 (left)4 and PM4 (right)5, tested in transport studies using intestinal organoids.
Transport studies in organoids from WT and KO mice using radiolabeled substrates1. PEPT1: peptide/drug transporter, SGLT1: glucose transporter, GLUT5: fructose transporter2.
Transport studies of peptidomimetics (PM) as targets for cancer therapy6, competitive inhibition of Gly-Sar transport.
Transporter Localization
Polarization
Small-intestinal organoid cultures (mouse) after
1st
SGLT1
passage1.
GIP
Glucose → GLP-1 release
GLUT2
wt
TGR5
wt
pept1 -/-
Intracellular Signaling
GIP mRNA
BCECF
Staining of enteroendocrine cells in small-intestinal organoids1. GLP-1 and GIP gene expression patterns in organoids derived from different parts of the small intestine. Peptides → GLP-1 release
wt
Organoids derived from wildtype (wt) or transporter knockout mice1. SGLT1: Sodium-dependent glucose transporter 1, PEPT1: Proton-coupled peptide transporter 1, GLUT2: Facilitated glucose transporter 2, TGR5: Bile acid receptor2.
Incretin Hormone Secretion GLP-1 mRNA
PEPT1
sglt1 -/-
Polarized organoid epithelium, immunofluorescent stainings1. Blue: nuclei, red: villin, green: e-cadherin.
GLP-1
wt
Fura-2
Glucose
Gly-Sar
Glucose → GIP release
Live cell imaging, organoids loaded with the fluorescent calcium indicator Fura-2 or pH indicator BCECF (upper panel). Glucose-induced calcium signal (left) and intracellular acidification upon glycyl-sarcosine treatment (right)1.
GLP-1 and GIP secretion induced by glucose or peptides in organoids derived from WT or transporter KO mice1,3. F/I: Forskolin/IBMX (maximum hormone output).
Human Intestinal Organoids
References 1 Zietek et al. Intestinal organoids for assessing nutrient transport, sensing and incretin secretion. Sci Rep 2015 2 Daniel & Zietek. Taste and move: glucose and peptide transporters in the gastrointestinal tract. Exp Physiol 2015
Healthy donors
Malabsorption patients
We are transferring our transport studies to human organoids (healthy donors). Patientderived organoids will allow studying malabsorption syndromes such as Fackoni-Bickl syndrome (GLUT2 deficiency) or glucose-galactose malabsorption (SGLT1 deficiency).
3 Zietek & Daniel. Intestinal nutrient sensing and blood glucose control. Curr Opin Clin Nutr Metab Care 2015 4 Ovadia et al. The effect of multiple N-methylation on intestinal permeability of cyclic hexapeptides. Mol Pharm 2011 5 Biron et al. Improving oral bioavailability of peptides by multiple N-methylation: somatostatin analogues. Angew Chem Int Ed Engl 2008 6 Maltsev et al. Stable peptides instead of stapled peptides: highly potent αVβ6-selective integrin ligands. Angew Chem Int Ed Engl 2016
