Structural determinants of dual incretin receptor agonism by tirzepatide.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.

Intercellular Coupling of the Cell Cycle and Circadian Clock in Adult Stem Cell Culture.

Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted WNT as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.

In Vivo Mechanism of Action of Sodium Caprate for Improving the Intestinal Absorption of a GLP1/GIP Coagonist Peptide.

Sodium caprate (C10) has been widely evaluated as an intestinal permeation enhancer for the oral delivery of macromolecules. However, the effect of C10 on the intestinal absorption of peptides with different physicochemical properties and its permeation-enhancing effect in vivo remains to be understood. Here, we evaluated the effects of C10 on intestinal absorption in rats with a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP-GLP1) dual agonist peptide (LY) and semaglutide with different enzymatic stabilities and self-association behaviors as well as the oral exposure of the LY peptide in minipigs. Furthermore, we investigated the mechanism of action (MoA) of C10 for improving the intestinal absorption of the LY peptide in vivo via live imaging of the rat intestinal epithelium and tissue distribution of the LY peptide in minipigs. The LY peptide showed higher proteolytic stability in pancreatin and was a monomer in solution compared to that in semaglutide. C10 increased in vitro permeability in the minipig intestinal organoid monolayer to a greater extent for the LY peptide than for semaglutide. In the rat jejunal closed-loop model, C10 increased the absorption of LY peptide better than that of semaglutide, which might be attributed to higher in vitro proteolytic stability and permeability of the LY peptide. Using confocal live imaging, we observed that C10 enabled the rapid oral absorption of a model macromolecule (FD4) in the rat intestine. In the duodenum tissues of minipigs, C10 was found to qualitatively reduce the tight junction protein level and allow peptide uptake to the intestinal cells. C10 decreased the transition temperature of the artificial lipid membrane, indicating an increase in membrane fluidity, which is consistent with the above in vivo imaging results. These data indicated that the LY's favorable physicochemical properties combined with the effects of C10 on the intestinal mucosa resulted in an ∼2% relative bioavailability in minipigs.

Wnt/ß-catenin promotes gastric fundus specification in mice and humans.

Despite the global prevalence of gastric disease, there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/ß-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium, and that ß-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology, and also represent a new platform for drug discovery.

Generation of intestinal chemosensory cells from nonhuman primate organoids.

Several gastrointestinal epithelial cells are involved in taste signal transduction. Although rodent tissues are extensively used as a human gut model, recent studies show that the chemical sensing system in rodents differs from that in humans. Nonhuman primates in biomedical research are valuable animal models to advance our understanding of biological responses in humans. The 3D organoid culture produces functional gastrointestinal epithelial cells in vitro and can be generated from animal and human tissues. Here, we report the generation of intestinal chemosensory cells from nonhuman primates, macaques, using an organoid culture system. We were able to maintain macaque intestinal organoids in the proliferation medium for more than six months. Upon switching to differentiation medium, we observed a drastic change in organoid morphology and chemosensory cell marker protein expression. This switch from proliferation to differentiation was confirmed by transcriptome analysis of the duodenum, jejunum, and ileum organoids. We further observed that the supplementation of culture media with interleukin (IL)-4 or the Notch inhibitor dibenzazepine (DBZ) accelerated terminal cell differentiation into chemosensory cells. Overall, we generated monkey intestinal organoids for the first time. These organoids are suitable for studying the function of primate chemosensory cells.

Trefoil Factor Peptides and Gastrointestinal Function.

Trefoil factor (TFF) peptides, with a 40-amino acid motif and including six conserved cysteine residues that form intramolecular disulfide bonds, are a family of mucin-associated secretory molecules mediating many physiological roles that maintain and restore gastrointestinal (GI) mucosal homeostasis. TFF peptides play important roles in response to GI mucosal injury and inflammation. In response to acute GI mucosal injury, TFF peptides accelerate cell migration to seal the damaged area from luminal contents, whereas chronic inflammation leads to increased TFF expression to prevent further progression of disease. Although much evidence supports the physiological significance of TFF peptides in mucosal defenses, the molecular and cellular mechanisms of TFF peptides in the GI epithelium remain largely unknown. In this review, we summarize the functional roles of TFF1, 2, and 3 and illustrate their action mechanisms, focusing on defense mechanisms in the GI tract.

Cell injury triggers actin polymerization to initiate epithelial restitution.

The role of the actin cytoskeleton in the sequence of physiological epithelial repair in the intact epithelium has yet to be elucidated. Here, we explore the role of actin in gastric repair in vivo and in vitro gastric organoids (gastroids). In response to two-photon-induced cellular damage of either an in vivo gastric or in vitro gastroid epithelium, actin redistribution specifically occurred in the lateral membranes of cells neighboring the damaged cell. This was followed by their migration inward to close the gap at the basal pole of the dead cell, in parallel with exfoliation of the dead cell into the lumen. The repair and focal increase of actin was significantly blocked by treatment with EDTA or the inhibition of actin polymerization. Treatment with inhibitors of myosin light chain kinase, myosin II, trefoil factor 2 signaling or phospholipase C slowed both the initial actin redistribution and the repair. While Rac1 inhibition facilitated repair, inhibition of RhoA/Rho-associated protein kinase inhibited it. Inhibitors of focal adhesion kinase and Cdc42 had negligible effects. Hence, initial actin polymerization occurs in the lateral membrane, and is primarily important to initiate dead cell exfoliation and cell migration to close the gap.

Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells.

A self-organizing organoid model provides a new approach to study the mechanism of human liver organogenesis. Previous animal models documented that simultaneous paracrine signaling and cell-to-cell surface contact regulate hepatocyte differentiation. To dissect the relative contributions of the paracrine effects, we first established a liver organoid using human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as previously reported. Time-lapse imaging showed that hepatic-specified endoderm iPSCs (HE-iPSCs) self-assembled into three-dimensional organoids, resulting in hepatic gene induction. Progressive differentiation was demonstrated by hepatic protein production after in vivo organoid transplantation. To assess the paracrine contributions, we employed a Transwell system in which HE-iPSCs were separately co-cultured with MSCs and/or HUVECs. Although the three-dimensional structure did not form, their soluble factors induced a hepatocyte-like phenotype in HE-iPSCs, resulting in the expression of bile salt export pump. In conclusion, the mesoderm-derived paracrine signals promote hepatocyte maturation in liver organoids, but organoid self-organization requires cell-to-cell surface contact. Our in vitro model demonstrates a novel approach to identify developmental paracrine signals regulating the differentiation of human hepatocytes.

Deficient Active Transport Activity in Healing Mucosa After Mild Gastric Epithelial Damage.

BACKGROUND: Peptic ulcers recur, suggesting that ulcer healing may leave tissue predisposed to subsequent damage. In mice, we have identified that the regenerated epithelium found after ulcer healing will remain abnormal for months after healing. AIM: To determine whether healed gastric mucosa has altered epithelial function, as measured by electrophysiologic parameters. METHOD: Ulcers were induced in mouse gastric corpus by serosal local application of acetic acid. Thirty days or 8 months after ulcer induction, tissue was mounted in an Ussing chamber. Transepithelial electrophysiologic parameters (short-circuit current, Isc. resistance, R) were compared between the regenerated healed ulcer region and the non-ulcerated contralateral region, in response to luminal hyperosmolar NaCl challenge (0.5 M). RESULTS: In unperturbed stomach, luminal application of hyperosmolar NaCl transiently dropped Isc followed by gradual recovery over 2 h. Compared to the starting baseline Isc, percent Isc recovery was reduced in 30-day healing mucosa, but not at 8 months. Prior to NaCl challenge, a lower baseline Isc was observed in trefoil factor 2 (TFF2) knockout (KO) versus wild type (WT), with no Isc recovery in either non-ulcerated or healing mucosa of KO. Inhibiting Na/H exchanger (NHE) transport in WT mucosa inhibited Isc recovery in response to luminal challenge. NHE2-KO baseline Isc was reduced versus NHE2-WT. In murine gastric organoids, NHE inhibition slowed recovery of intracellular pH and delayed the repair of photic induced damage. CONCLUSION: Healing gastric mucosa has deficient electrophysiological recovery in response to hypertonic NaCl. TFF2 and NHE2 contribute to Isc regulation, and the recovery and healing of transepithelial function.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

Trefoil factor 2 activation of CXCR4 requires calcium mobilization to drive epithelial repair in gastric organoids.

KEY POINTS: Determining the signalling cascade of epithelial repair, using murine gastric organoids, allows definition of regulatory processes intrinsic to epithelial cells, at the same time as validating and dissecting the signalling cascade with more precision than is possible in vivo Following single cell damage, intracellular calcium selectively increases within cells adjacent to the damage site and is essential for promoting repair. Trefoil factor 2 (TFF2) acts via chemokine C-X-C receptor 4 and epidermal growth factor receptor signalling, including extracellular signal-regulated kinase activation, to drive calcium mobilization and promote gastric repair. Sodium hydrogen exchanger 2, although essential for repair, acts downstream of TFF2 and calcium mobilization. ABSTRACT: The gastric mucosa of the stomach is continually exposed to environmental and physiological stress factors that can cause local epithelial damage. Although much is known about the complex nature of gastric wound repair, the stepwise process that characterizes epithelial restitution remains poorly defined. The present study aimed to determine the effectors that drive gastric epithelial repair using a reductionist culture model. To determine the role of trefoil factor 2 (TFF2) and intracellular calcium (Ca2+ ) mobilization in gastric restitution, gastric organoids were derived from TFF2 knockout (KO) mice and yellow Cameleon-Nano15 (fluorescent calcium reporter) transgenic mice, respectively. Inhibitors and recombinant protein were used to determine the upstream and downstream effectors of gastric restitution following photodamage (PD) to single cells within the gastric organoids. Single cell PD resulted in parallel events of dead cell exfoliation and migration of intact neighbouring cells to restore a continuous epithelium in the damage site. Under normal conditions following PD, Ca2+ levels increased within neighbour migrating cells, peaking at ∼1 min, suggesting localized Ca2+ mobilization at the site of cell protrusion/migration. TFF2 KO organoids exhibit delayed repair; however, this delay can be rescued by the addition of exogenous TFF2. Inhibition of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK)1/2 or a TFF2 receptor, chemokine C-X-C receptor 4 (CXCR4), resulted in significant delay and dampened Ca2+ mobilization. Inhibition of sodium hydrogen exchanger 2 (NHE2) caused significant delay but did not affect Ca2+ mobilization. A similar delay was observed in NHE2 KO organoids. In TFF2 KO gastric organoids, the addition of exogenous TFF2 in the presence of EGFR or CXCR4 inhibition was unable to rescue repair. The present study demonstrates that intracellular Ca2+ mobilization occurs within gastric epithelial cells adjacent to the damage site to promote repair by mechanisms that involve TFF2 signalling via CXCR4, as well as activation of EGFR and ERK1/2. Furthermore NHE2 is shown to be important for efficient repair and to operate via a mechanism either downstream or independent of calcium mobilization.

Helicobacter pylori Uses the TlpB Receptor To Sense Sites of Gastric Injury.

Helicobacter pylori is a pathogen that chronically colonizes the stomachs of approximately half of the world's population and contributes to the development of gastric inflammation. We demonstrated previously in vivo that H. pylori uses motility to preferentially colonize injury sites in the mouse stomach. However, the chemoreceptor responsible for sensing gastric injury has not yet been identified. In this study, we utilized murine gastric organoids (gastroids) and mutant H. pylori strains to investigate the components necessary for H. pylori chemotaxis. High-intensity 730-nm light (two-photon photodamage) was used to cause single-cell damage in gastroids, and repair of the damage was monitored over time; complete repair occurred within ∼10 min in uninfected gastroids. Wild-type H. pylori accumulated at the damage site after gastric damage induction. In contrast, mutants lacking motility (ΔmotB) or chemotaxis (ΔcheY) did not accumulate at the injury site. Using mutants lacking individual chemoreceptors, we found that only TlpB was required for H. pylori accumulation, while TlpA, TlpC, and TlpD were dispensable. All strains that were able to accumulate at the damage site limited repair. When urea (an identified chemoattractant sensed by TlpB) was microinjected into the gastroid lumen, it prevented the accumulation of H. pylori at damage sites. Overall, our findings demonstrate that H. pylori colonizes and limits repair at damage sites via chemotactic motility that requires the TlpB chemoreceptor to sense signals generated by gastric epithelial cells.

Effect of essential amino acids on enteroids: Methionine deprivation suppresses proliferation and affects differentiation in enteroid stem cells.

We investigated the effects of essential amino acids on intestinal stem cell proliferation and differentiation using murine small intestinal organoids (enteroids) from the jejunum. By selectively removing individual essential amino acids from culture medium, we found that 24 h of methionine (Met) deprivation markedly suppressed cell proliferation in enteroids. This effect was rescued when enteroids cultured in Met deprivation media for 12 h were transferred to complete medium, suggesting that Met plays an important role in enteroid cell proliferation. In addition, mRNA levels of the stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) decreased in enteroids grown in Met deprivation conditions. Consistent with this observation, Met deprivation also attenuated Lgr5-EGFP fluorescence intensity in enteroids. In contrast, Met deprivation enhanced mRNA levels of the enteroendocrine cell marker chromogranin A (ChgA) and markers of K cells, enterochromaffin cells, goblet cells, and Paneth cells. Immunofluorescence experiments demonstrated that Met deprivation led to an increase in the number of ChgA-positive cells. These results suggest that Met deprivation suppresses stem cell proliferation, thereby promoting differentiation. In conclusion, Met is an important nutrient in the maintenance of intestinal stem cells and Met deprivation potentially affects cell differentiation.

Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5(+)) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5(+) or Lgr6(+) cells from taste tissue can generate continuously expanding 3D structures ("organoids"). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2'-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5(+) cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6(+) cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5(+) or Lgr6(+) cells, validating the use of this model for the study of taste cell generation.

Motility and chemotaxis mediate the preferential colonization of gastric injury sites by Helicobacter pylori.

Helicobacter pylori (H. pylori) is a pathogen contributing to peptic inflammation, ulceration, and cancer. A crucial step in the pathogenic sequence is when the bacterium first interacts with gastric tissue, an event that is poorly understood in vivo. We have shown that the luminal space adjacent to gastric epithelial damage is a microenvironment, and we hypothesized that this microenvironment might enhance H. pylori colonization. Inoculation with 106 H. pylori (wild-type Sydney Strain 1, SS1) significantly delayed healing of acetic-acid induced ulcers at Day 1, 7 and 30 post-inoculation, and wild-type SS1 preferentially colonized the ulcerated area compared to uninjured gastric tissue in the same animal at all time points. Gastric resident Lactobacillus spp. did not preferentially colonize ulcerated tissue. To determine whether bacterial motility and chemotaxis are important to ulcer healing and colonization, we analyzed isogenic H. pylori mutants defective in motility (ΔmotB) or chemotaxis (ΔcheY). ΔmotB (10(6)) failed to colonize ulcerated or healthy stomach tissue. ΔcheY (10(6)) colonized both tissues, but without preferential colonization of ulcerated tissue. However, ΔcheY did modestly delay ulcer healing, suggesting that chemotaxis is not required for this process. We used two-photon microscopy to induce microscopic epithelial lesions in vivo, and evaluated accumulation of fluorescently labeled H. pylori at gastric damage sites in the time frame of minutes instead of days. By 5 min after inducing damage, H. pylori SS1 preferentially accumulated at the site of damage and inhibited gastric epithelial restitution. H. pylori ΔcheY modestly accumulated at the gastric surface and inhibited restitution, but did not preferentially accumulate at the injury site. H. pylori ΔmotB neither accumulated at the surface nor inhibited restitution. We conclude that bacterial chemosensing and motility rapidly promote H. pylori colonization of injury sites, and thereby biases the injured tissue towards sustained gastric damage.

Helicobacter pylori targets cancer-associated apical-junctional constituents in gastroids and gastric epithelial cells.

OBJECTIVE: Helicobacter pylori strains that express the oncoprotein CagA augment risk for gastric cancer. However, the precise mechanisms through which cag(+) strains heighten cancer risk have not been fully delineated and model systems that recapitulate the gastric niche are critical for understanding pathogenesis. Gastroids are three-dimensional organ-like structures that provide unique opportunities to study host-H. pylori interactions in a preclinical model. We used gastroids to inform and direct in vitro studies to define mechanisms through which H. pylori modulates expression of the cancer-associated tight junction protein claudin-7. DESIGN: Gastroids were infected by luminal microinjection, and MKN28 gastric epithelial cells were cocultured with H. pylori wild-type cag(+) strains or isogenic mutants. ß-catenin, claudin-7 and snail localisation was determined by immunocytochemistry. Proliferation was assessed using 5-ethynyl-2'-deoxyuridine, and levels of claudin-7 and snail were determined by western blot and flow cytometry. RESULTS: Gastroids developed into a self-organising differentiation axis and H. pylori induced mislocalisation of claudin-7 and increased proliferation in a CagA- and ß-catenin-dependent manner. In MKN28 cells, H pylori-induced suppression of claudin-7 was regulated by ß-catenin and snail. Similarly, snail expression was increased and claudin-7 levels were decreased among H. pylori-infected individuals. CONCLUSIONS: H. pylori increase proliferation in a strain-specific manner in a novel gastroid system. H. pylori also alter expression and localisation of claudin-7 in gastroids and human epithelial cells, which is mediated by ß-catenin and snail activation. These data provide new insights into molecular interactions with carcinogenic potential that occur between H. pylori and epithelial cells within the gastric niche.

The use of murine-derived fundic organoids in studies of gastric physiology.

KEY POINTS: An in vitro approach to study gastric development is primary mouse-derived epithelium cultured as three-dimensional spheroids known as organoids. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Organoids maintained in co-culture with immortalized stomach mesenchymal cells express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. We report the use of these models for studies of epithelial cell biology and cell damage and repair. ABSTRACT: Studies of gastric function and disease have been limited by the lack of extended primary cultures of the epithelium. An in vitro approach to study gastric development is primary mouse-derived antral epithelium cultured as three-dimensional spheroids known as organoids. There have been no reports on the use of organoids for gastric function. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Both models were generated from single glands dissociated from whole fundic tissue and grown in basement membrane matrix (Matrigel) and organoid growth medium. Model 1 enriches for a stem cell-like niche via simple passage of the organoids. Maintained in Matrigel and growth medium, proliferating organoids expressed high levels of stem cell markers CD44 and Lgr5. Model 2 is a system of gastric organoids co-cultured with immortalized stomach mesenchymal cells (ISMCs). Organoids maintained in co-culture with ISMCs express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. Thus, we report the use of these models for studies of epithelial cell biology and cell damage and repair.

Glutamine and alanyl-glutamine promote crypt expansion and mTOR signaling in murine enteroids.

L-glutamine (Gln) is a key metabolic fuel for intestinal epithelial cell proliferation and survival and may be conditionally essential for gut homeostasis during catabolic states. We show that L-alanyl-L-glutamine (Ala-Gln), a stable Gln dipeptide, protects mice against jejunal crypt depletion in the setting of dietary protein and fat deficiency. Separately, we show that murine crypt cultures (enteroids) derived from the jejunum require Gln or Ala-Gln for maximal expansion. Once expanded, enteroids deprived of Gln display a gradual atrophy of cryptlike domains, with decreased epithelial proliferation, but stable proportions of Paneth and goblet cell differentiation, at 24 h. Replenishment of enteroid medium with Gln selectively activates mammalian target of rapamycin (mTOR) signaling pathways, rescues proliferation, and promotes crypt regeneration. Gln deprivation beyond 48 h leads to destabilization of enteroids but persistence of EGFP-Lgr5-positive intestinal stem cells with the capacity to regenerate enteroids upon Gln rescue. Collectively, these findings indicate that Gln deprivation induces a reversible quiescence of intestinal stem cells and provides new insights into nutritional regulation of intestinal epithelial homeostasis.

Indian Hedgehog mediates gastrin-induced proliferation in stomach of adult mice.

BACKGROUND & AIMS: Loss of expression of Sonic Hedgehog (Shh) from parietal cells results in hypergastrinemia in mice, accompanied by increased expression of Indian Hedgehog (Ihh) and hyperproliferation of surface mucous cells. We investigated whether hypergastrinemia induces gastric epithelial proliferation by activating Ihh signaling in mice. METHODS: We studied mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and hypergastrinemia, crossed with gastrin-deficient (GKO) mice (PC-Shh(KO)/GKO). When mice were 3-4 months old, gastric tissues were collected and analyzed by histology, for incorporation of bromodeoxyuridine, and for expression of the surface mucous cell marker Ulex europaeus. PC-Shh(KO)/GKO mice were given gastrin infusions for 7 days; gastric surface epithelium was collected and expression of Ihh was quantified by laser capture microdissection followed by quantitative reverse transcriptase polymerase chain reaction. Mouse stomach-derived organoids were incubated with or without inhibitors of WNT (DKK1) or Smoothened (vismodegib) and then cocultured with immortalized stomach mesenchymal cells, to assess proliferative responses to gastrin. RESULTS: Gastric tissues from PC-Shh(KO)/GKO mice with hypergastrinemia had an expanded surface pit epithelium, indicated by a significant increase in numbers of bromodeoxyuridine- and Ulex europaeus-positive cells, but there was no evidence for hyperproliferation. Gastrin infusion of PC PC-Shh(KO)/GKO mice increased expression of Ihh and proliferation within the surface epithelium compared with mice given infusions of saline. In gastric organoids cocultured with immortalized stomach mesenchymal cells, antagonists of WNT and Smoothened inhibited gastrin-induced proliferation and WNT activity. Activity of WNT in media collected from immortalized stomach mesenchymal cells correlated with increased expression of glioma-associated oncogene homolog 1, and was inhibited by DKK1 or vismodegib. CONCLUSIONS: Ihh signaling mediates gastrin-induced proliferation of epithelial cells in stomachs of adult mice.