Your browser doesn't support javascript.
loading
: 20 | 50 | 100
1 - 12 de 12
1.
bioRxiv ; 2024 Apr 18.
Article En | MEDLINE | ID: mdl-38659945

ß cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting ß cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that ß cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched EVs that could impact surrounding ß cells. Consistent with this, proinflammatory cytokine treatment of INS-1 ß cells and human islets concurrently increased ß cell nSMase2 and ceramide expression, as well as EV ceramide staining. Direct chemical activation or genetic knockdown of nSMase2, or treatment with a GLP-1 receptor agonist also modulated cellular and EV ceramide. Small RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to ß cell function and identity. Coculture experiments using CD9-GFP tagged INS-1 cell EVs demonstrated that either cytokine treatment or chemical nSMase2 activation increased EV transfer to recipient cells. Children with recent-onset T1D showed no abnormalities in circulating ceramide-enriched EVs, suggesting a localized, rather than systemic phenomenon. These findings highlight nSMase2 as a regulator of ß cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of ß cell inflammatory stress. Article Highlights: a. Why did we undertake this study?: Mechanisms connecting ß cell stress to changes in extracellular vesicle (EV) cargo and potential impacts on diabetes are poorly defined.b. What is the specific question we wanted to answer?: Does ß cell inflammatory stress engage neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways to generate ceramide-enriched EVs.c. What did we find?: Proinflammatory cytokine treatment of ß cells increased ß cell ceramide expression, along with EV ceramide in part via increases in nSMase2. Ceramide-enriched EVs housed a distinct set of miRNAs linked to insulin signaling. Both cytokine treatment and nSMase2 activation increase EV transfer to other ß cells.d. What are the implications of our findings?: Our findings highlight nSMase2 as a regulator of ß cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of ß cell inflammatory stress.

2.
Development ; 151(9)2024 May 01.
Article En | MEDLINE | ID: mdl-38587174

The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.


Gene Expression Regulation, Developmental , Homeodomain Proteins , Organoids , Regulatory Factor X Transcription Factors , Trans-Activators , Humans , Regulatory Factor X Transcription Factors/genetics , Regulatory Factor X Transcription Factors/metabolism , Animals , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Trans-Activators/metabolism , Trans-Activators/genetics , Organoids/metabolism , Organoids/embryology , Duodenum/metabolism , Duodenum/embryology , Intestines/embryology , Intestinal Atresia/genetics , Induced Pluripotent Stem Cells/metabolism , Body Patterning/genetics , Signal Transduction/genetics , Mutation/genetics
3.
Accid Anal Prev ; 195: 107100, 2024 Feb.
Article En | MEDLINE | ID: mdl-38154856

OBJECTIVE: Several studies have documented the relative risk or odds of injury and fatality for females versus males in motor vehicle crashes (Parenteau et al. 2013, Forman et al. 2019, Brumbelow and Jermakian, 2022; Noh et al. 2022). Though, none combined National Automotive Sampling System-Crashworthiness Data System (NASS-CDS) and Crash Investigation Sampling System (CISS). The aim of this study was to document the relative odds of various injury outcomes for females versus males while considering a broad range of crash types, pre-crash and crash variables, and occupant characteristics. METHODS: Multivariable logistic regression was carried out to study the odds of injury for females versus males. A select imputation method (Hot Deck, Approximate Bayesian Bootstrap) was applied as part of efforts to create multivariable logistic regression models for 25 different injury outcomes associated with occupants (age 13 years and older) involved in passenger vehicle crashes published in NASS-CDS (2000 to 2015) and CISS (2017-2021). Both pre-crash (n=7) and crashworthiness (n=22) predictor variables were considered, but only significant variables at p≤0.10 level were retained in final models. Six crash-type models were produced for each injury outcome; one that included all crashes, one for each of four different planar crash types (frontal, near-side, far-side, rear), and one for crashes involving rollover. These six sets of crash-type models were expanded further to include a model version that included both pre-crash/environment and crashworthiness predictor variables and one model limited to crashworthiness predictors only. Different than other recent studies, all crash types, occupant restraint conditions, and seating positions were considered. Occupant sex was retained in all models to facilitate female versus male injury outcome odds ratio (OR) assessments. RESULTS: Female versus male injury OR estimates for 300 unique models are presented. Females had significantly higher odds of injury than males in 36 models (OR>1.0, p-value ≤0.05). This contrasts with 43 models where females had significantly lower odds (OR<1.0, p≤0.05). For the remaining 221 models, there was a near even split in how often the odds of injury were non-significantly higher (n=103) and non-significantly lower (n=114) for females as compared to males (p>0.05). In four cases, the OR estimate was 1.00. Amongst the results, there was a trend for females to have higher odds of AIS 2+ injuries (MAIS 2+ OR=1.75 and 1.69 for Full and Crashworthiness models, respectively for the All Crashes dataset). These increases included higher estimates for lower extremity injuries in frontal crashes, consistent with earlier studies (e.g., Forman et al. 2019). However, for certain AIS 2+ (neck, thorax) and AIS 3+ injuries (head, neck, thorax), females had significantly lower odds of injury (p≤0.05). The trends for reduced odds of injury for females were most prevalent in non-frontal crash models.


Accidents, Traffic , Wounds and Injuries , Humans , Male , Female , Adolescent , Bayes Theorem , Logistic Models , Motor Vehicles , Wounds and Injuries/epidemiology , Wounds and Injuries/etiology , Abbreviated Injury Scale
4.
Cell Rep Med ; 4(11): 101261, 2023 11 21.
Article En | MEDLINE | ID: mdl-37918404

In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing ß cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with ß cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during ß cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000 mg/m2) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve ß cell function in T1D through islet cell-autonomous effects.


Diabetes Mellitus, Type 1 , Humans , Mice , Animals , Diabetes Mellitus, Type 1/drug therapy , Ornithine Decarboxylase/genetics , Ornithine Decarboxylase/metabolism , Ornithine Decarboxylase Inhibitors/pharmacology , Eflornithine/pharmacology , Eflornithine/therapeutic use , Putrescine/metabolism
5.
Cell Stem Cell ; 30(11): 1434-1451.e9, 2023 11 02.
Article En | MEDLINE | ID: mdl-37922878

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.


Pluripotent Stem Cells , Humans , Mice , Animals , Hematopoietic Stem Cells , Colon , Organoids , Macrophages
6.
Cell Mol Gastroenterol Hepatol ; 15(6): 1293-1310, 2023.
Article En | MEDLINE | ID: mdl-36608902

BACKGROUND & AIMS: The intestinal stem cell niche is exquisitely sensitive to changes in diet, with high-fat diet, caloric restriction, and fasting resulting in altered crypt metabolism and intestinal stem cell function. Unlike cells on the villus, cells in the crypt are not immediately exposed to the dynamically changing contents of the lumen. We hypothesized that enteroendocrine cells (EECs), which sense environmental cues and in response release hormones and metabolites, are essential for relaying the luminal and nutritional status of the animal to cells deep in the crypt. METHODS: We used the tamoxifen-inducible VillinCreERT2 mouse model to deplete EECs (Neurog3fl/fl) from adult intestinal epithelium and we generated human intestinal organoids from wild-type and NEUROGENIN 3 (NEUROG3)-null human pluripotent stem cells. We used indirect calorimetry, 1H-Nuclear Magnetic Resonance (NMR) metabolomics, mitochondrial live imaging, and the Seahorse bioanalyzer (Agilent Technologies) to assess metabolism. Intestinal stem cell activity was measured by proliferation and enteroid-forming capacity. Transcriptional changes were assessed using 10x Genomics single-cell sequencing. RESULTS: Loss of EECs resulted in increased energy expenditure in mice, an abundance of active mitochondria, and a shift of crypt metabolism to fatty acid oxidation. Crypts from mouse and human intestinal organoids lacking EECs displayed increased intestinal stem cell activity and failed to activate phosphorylation of downstream target S6 kinase ribosomal protein, a marker for activity of the master metabolic regulator mammalian target of rapamycin (mTOR). These phenotypes were similar to those observed when control mice were deprived of nutrients. CONCLUSIONS: EECs are essential regulators of crypt metabolism. Depletion of EECs recapitulated a fasting metabolic phenotype despite normal levels of ingested nutrients. These data suggest that EECs are required to relay nutritional information to the stem cell niche and are essential regulators of intestinal metabolism.


Pluripotent Stem Cells , Stem Cell Niche , Mice , Humans , Animals , Enteroendocrine Cells/metabolism , Intestines , Nutrients , Mammals
7.
Cell Rep ; 41(7): 111641, 2022 11 15.
Article En | MEDLINE | ID: mdl-36384107

Long-term impacts of diet have been well studied; however, the immediate response of the intestinal epithelium to a change in nutrients remains poorly understood. We use physiological metrics and single-cell transcriptomics to interrogate the intestinal epithelial cell response to a high-fat diet (HFD). Within 1 day of HFD exposure, mice exhibit altered whole-body physiology and increased intestinal epithelial proliferation. Single-cell transcriptional analysis on day 1 reveals a cell-stress response in intestinal crypts and a shift toward fatty acid metabolism. By 3 days of HFD, computational trajectory analysis suggests an emergence of progenitors, with a transcriptional profile shifting from secretory populations toward enterocytes. Furthermore, enterocytes upregulate lipid absorption genes and show increased lipid absorption in vivo over 7 days of HFD. These findings demonstrate the rapid intestinal epithelial response to a dietary change and help illustrate the essential ability of animals to adapt to shifting nutritional environments.


Diet, High-Fat , Intestinal Mucosa , Mice , Animals , Diet, High-Fat/adverse effects , Intestinal Mucosa/metabolism , Lipid Metabolism , Adaptation, Physiological , Lipids
8.
Gastroenterology ; 163(4): 1053-1063.e7, 2022 10.
Article En | MEDLINE | ID: mdl-35803312

BACKGROUND & AIMS: Two patients with homozygous mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea, and poor weight gain, the causes of which were not identified through routine clinical testing. We aimed to perform a deep analysis of the stomach and intestine using organoids derived from induced pluripotent stem cells from PDX1188delC/188delC patients. METHODS: Gastric fundic, antral, and duodenal organoids were generated using induced pluripotent stem cell lines from a PDX1188delC/188delC patient and an isogenic induced pluripotent stem cell line where the PDX1 point mutation was corrected. RESULTS: Patient-derived PDX1188delC/188delC antral organoids exhibited an intestinal phenotype, whereas intestinal organoids underwent gastric metaplasia with significant reduction in enteroendocrine cells. This prompted a re-examination of gastric and intestinal biopsy specimens from both PDX1188delC/188delC patients, which recapitulated the organoid phenotypes. Moreover, antral biopsy specimens also showed increased parietal cells and lacked G cells, suggesting loss of antral identity. All organoid pathologies were reversed upon CRISPR-mediated correction of the mutation. CONCLUSIONS: These patients will now be monitored for the progression of metaplasia and gastrointestinal complications that might be related to the reduced gastric and intestinal endocrine cells. This study demonstrates the utility of organoids in diagnosing uncovered pathologies.


Induced Pluripotent Stem Cells , Organoids , Cell Differentiation , Humans , Induced Pluripotent Stem Cells/metabolism , Metaplasia/metabolism , Mutation , Organoids/metabolism , Stomach
9.
Curr Opin Endocrinol Diabetes Obes ; 29(2): 169-176, 2022 04 01.
Article En | MEDLINE | ID: mdl-35066539

PURPOSE OF REVIEW: The intestinal enteroendocrine cells (EECs) are specialized hormone-secreting cells that respond to both circulating and luminal cues. Collectively, EECs constitute the largest endocrine organ of the body and signal to a multitude of targets including locally to neighboring intestinal cells, enteric neurons, as well as systemically to other organs, such as the pancreas and brain. To accomplish their wide range of downstream signaling effects, EECs secrete multiple hormones; however, the mechanisms that influence EEC development in the embryo and differentiation in adults are not well defined. RECENT FINDINGS: This review highlights the recent discoveries in EEC differentiation and function while also discussing newly revealed roles of transcription factors and signaling networks involved in the allocation of EEC subtypes that were discovered using a combination of novel intestinal model systems and genetic sequencing. We also discuss the potential of these new experimental models that study the mechanisms regulating EEC function and development both to uncover novel therapeutic targets. SUMMARY: Several EEC hormones are being used to treat various metabolic disorders, such as type 2 diabetes and obesity. Therefore, understanding the signaling pathways and gene regulatory networks that facilitate EEC formation is paramount to the development of novel therapies.


Diabetes Mellitus, Type 2 , Cell Differentiation , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Enteroendocrine Cells/physiology , Hormones , Humans , Intestines
10.
Nat Commun ; 11(1): 4791, 2020 09 22.
Article En | MEDLINE | ID: mdl-32963229

The ability to absorb ingested nutrients is an essential function of all metazoans and utilizes a wide array of nutrient transporters found on the absorptive enterocytes of the small intestine. A unique population of patients has previously been identified with severe congenital malabsorptive diarrhea upon ingestion of any enteral nutrition. The intestines of these patients are macroscopically normal, but lack enteroendocrine cells (EECs), suggesting an essential role for this rare population of nutrient-sensing cells in regulating macronutrient absorption. Here, we use human and mouse models of EEC deficiency to identify an unappreciated role for the EEC hormone peptide YY in regulating ion-coupled absorption of glucose and dipeptides. We find that peptide YY is required in the small intestine to maintain normal electrophysiology in the presence of vasoactive intestinal polypeptide, a potent stimulator of ion secretion classically produced by enteric neurons. Administration of peptide YY to EEC-deficient mice restores normal electrophysiology, improves glucose and peptide absorption, diminishes diarrhea and rescues postnatal survival. These data suggest that peptide YY is a key regulator of macronutrient absorption in the small intestine and may be a viable therapeutic option to treat patients with electrolyte imbalance and nutrient malabsorption.


Enteroendocrine Cells/metabolism , Intestinal Absorption/physiology , Ion Transport/physiology , Nutrients/metabolism , Animals , Enterocytes , Glucose/metabolism , Human Embryonic Stem Cells , Humans , Intestine, Small , Intestines , Mice , Mice, Inbred C57BL , Peptide YY , Receptors, Gastrointestinal Hormone/metabolism , Receptors, Vasoactive Intestinal Peptide/metabolism , Sodium-Hydrogen Exchanger 3 , Water/metabolism
11.
Development ; 145(19)2018 10 01.
Article En | MEDLINE | ID: mdl-30143540

Enteroendocrine cells (EECs) are a minor cell population in the intestine yet they play a major role in digestion, satiety and nutrient homeostasis. Recently developed human intestinal organoid models include EECs, but their rarity makes it difficult to study their formation and function. Here, we used the EEC-inducing property of the transcription factor NEUROG3 in human pluripotent stem cell-derived human intestinal organoids and colonic organoids to promote EEC development in vitro An 8-h pulse of NEUROG3 expression induced expression of known target transcription factors and after 7 days organoids contained up to 25% EECs in the epithelium. EECs expressed a broad array of human hormones at the mRNA and/or protein level, including motilin, somatostatin, neurotensin, secretin, substance P, serotonin, vasoactive intestinal peptide, oxyntomodulin, GLP-1 and INSL5. EECs secreted several hormones including gastric inhibitory polypeptide (GIP), ghrelin, GLP-1 and oxyntomodulin. Injection of glucose into the lumen of organoids caused an increase in both GIP secretion and K-cell number. Lastly, we observed formation of all known small intestinal EEC subtypes following transplantation and growth of human intestinal organoids in mice.


Enteroendocrine Cells/cytology , Enteroendocrine Cells/metabolism , Pluripotent Stem Cells/cytology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Count , Cell Differentiation , Hormones/metabolism , Humans , Intestines/cytology , Nerve Tissue Proteins/metabolism , Organoids/cytology , Pluripotent Stem Cells/metabolism , Time Factors , Transcription Factors/metabolism
12.
Dev Biol ; 424(2): 147-161, 2017 04 15.
Article En | MEDLINE | ID: mdl-28279709

Vasculogenesis involves the differentiation of vascular endothelial progenitors de novo from undifferentiated mesoderm, their migration and coalescence to form the major embryonic vessels and the acquisition of arterial or venous identity. Vascular Endothelial Growth Factor (Vegf) signaling plays multiple roles during vascular development. However, its function during embryonic vasculogenesis has been controversial. Previous studies have implicated Vegf signaling in either regulating arteriovenous specification or overall vascular endothelial differentiation. To clarify the role of Vegf in embryonic vasculogenesis and identify its downstream targets, we used chemical inhibitors of Vegf receptor (Vegfr) signaling in zebrafish embryos as well as zebrafish genetic mutants. A high level of chemical inhibition of Vegfr signaling resulted in the reduction of overall vascular endothelial marker gene expression, including downregulation of both arterial and venous markers, ultimately leading to the apoptosis of vascular endothelial cells. In contrast, a low level of Vegfr inhibition specifically blocked arterial specification while the expression of venous markers appeared largely unaffected or increased. Inhibition of Vegfr signaling prior to the initiation of vasculogenesis reduced overall vascular endothelial differentiation, while inhibition of Vegfr signaling starting at mid-somitogenesis stages largely inhibited arterial specification. Conversely, Vegf overexpression resulted in the expansion of both arterial and pan-endothelial markers, while the expression of several venous-specific markers was downregulated. We further show that Vegf signaling affects overall endothelial differentiation by modulating the expression of the ETS transcription factor etv2/ etsrp. etv2 expression was downregulated in Vegfr- inhibited embryos, and expanded in Vegfaa-overexpressing embryos. Furthermore, vascular-specific overexpression of etv2 in Vegfr-inhibited embryos rescued defects in vascular endothelial differentiation. Similarly, vegfaa genetic mutants displayed a combination of the two phenotypes observed with chemical Vegfr inhibition: the expression of arterial and pan-endothelial markers including etv2 was downregulated while the expression of most venous markers was either expanded or unchanged. Based on these results we propose a revised model which explains the different phenotypes observed upon inhibition of Vegf signaling: low levels of Vegf signaling promote overall vascular endothelial differentiation and cell survival by upregulating etv2 expression, while high levels of Vegf signaling promote arterial and inhibit venous specification.


Cell Differentiation , Endothelial Cells/cytology , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Zebrafish Proteins/genetics , Animals , Arteries/drug effects , Arteries/metabolism , Biomarkers/metabolism , Cell Count , Cell Differentiation/drug effects , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Gene Expression Regulation, Developmental/drug effects , Gene Knockdown Techniques , Indoles/pharmacology , Models, Biological , Morpholinos/pharmacology , Mutation/genetics , Pyrroles/pharmacology , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor/metabolism , Signal Transduction/drug effects , Somites/drug effects , Somites/metabolism , Veins/drug effects , Veins/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/metabolism
...