Diet-induced obesity in mice impairs host defense against Klebsiella pneumonia in vivo and glucose transport and bactericidal functions in neutrophils in vitro

2021 ◽  
Author(s):  
Peter Mancuso ◽  
Jeffrey L Curtis ◽  
Anne Marie Weitzel ◽  
Cameron A Griffin ◽  
Benjamin Bouchard ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Host Defense ◽  
Lung Homogenate ◽  
Klebsiella Pneumonia ◽  
Oxygen Intermediates ◽  
Bacterial Killing ◽  
Bacterial Colony ◽  
Diet Induced Obesity ◽  
The Impact

Obesity impairs host defense against Klebsiella pneumoniae but responsible mechanisms are incompletely understood. To determine the impact of diet-induced obesity on pulmonary host defense against K. pneumoniae, we fed 6-week-old male C57BL/6j mice a normal (ND) or high fat diet (HFD) (13% versus 60% fat, respectively) for 16 weeks. Mice were intratracheally infected with Klebsiella, assayed at 24 or 48 h for bacterial colony-forming units, lung cytokines, and leukocytes from alveolar spaces, lung parenchyma, and gonadal adipose tissue were assessed using flow cytometry. Neutrophils from uninfected mice were cultured with and without 2-deoxy-D-glucose (2-DG) and assessed for phagocytosis, killing, reactive oxygen intermediates (ROI), transport of 2-DG, and glucose transporter (GLUT1-4) transcripts, and protein expression of GLUT1 and GLUT3. HFD mice had higher lung and splenic bacterial burdens. In HFD mice, baseline lung homogenate concentrations of IL-1β, IL-6, IL-17, IFN-ɣ, CXCL2, and TNF-ɑ were reduced relative to ND mice, but following infection were greater for IL-6, CCL2, and CXCL2 and IL-1β (24 h only). Despite equivalent lung homogenate leukocytes, HFD mice had fewer intra-alveolar neutrophils. HFD neutrophils exhibited decreased Klebsiella phagocytosis and killing, and reduced ROI to heat-killed Klebsiella in vitro. 2-DG transport was lower in HFD neutrophils, with reduced GLUT1 and GLUT3 transcripts and protein (GLUT3 only). Blocking glycolysis with 2-DG impaired bacterial killing and ROI production in neutrophils from mice fed ND but not HFD. Diet-induced obesity impairs pulmonary Klebsiella clearance and augments blood dissemination by reducing neutrophil killing and ROI due to impaired glucose transport.

scholarly journals Once-daily gentamicin therapy for experimental Escherichia coli meningitis.

1997 ◽  
Vol 41 (1) ◽  
pp. 49-53 ◽  
Author(s):  
A Ahmed ◽  
M M París ◽  
M Trujillo ◽  
S M Hickey ◽  
L Wubbel ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Bacterial Killing ◽  
Once Daily ◽  
E Coli ◽  
Aminoglycoside Therapy ◽  
Gentamicin Concentration ◽  
Gentamicin Therapy ◽  
The Impact

In vitro and in vivo studies have demonstrated that the bacteriologic efficacy of once-daily aminoglycoside therapy is equivalent to that achieved with conventional multiple daily dosing. The impact of once-daily dosing for meningitis has not been studied. Using the well-characterized rabbit meningitis model, we compared two regimens of the same daily dosage of gentamicin given either once or in three divided doses for 24 or 72 h. The initial 1 h mean cerebrospinal fluid (CSF) gentamicin concentration for animals receiving a single dose (2.9 +/- 1.7 micrograms/ml) was threefold higher than that for the animals receiving multiple doses. The rate of bacterial killing in the first 8 h of treatment was significantly greater for the animals with higher concentrations in their CSF (-0.21 +/- 0.19 versus -0.03 +/- 0.22 log10 CFU/ml/h), suggesting concentration-dependent killing. By 24h, the mean reduction in bacterial titers was similar for the two regimens. In animals treated for 72 h, no differences in bactericidal activity was noted for 24, 48, or 72 h. Gentamicin at two different dosages was administered intracisternally to a separate set of animals to achieve considerably higher CSF gentamicin concentrations. In these animals, the rate of bacterial clearance in the first 8 h (0.52 +/- 0.15 and 0.58 +/- 0.15 log10 CFU/ml/h for the lower and higher dosages, respectively) was significantly greater than that in animals treated intravenously. In conclusion, there is evidence of concentration-dependent killing with gentamicin early in treatment for experimental E. coli meningitis, and once-daily dosing therapy appears to be at least as effective as multiple-dose therapy in reducing bacterial counts in CSF.

scholarly journals Cooperation between Reactive Oxygen and Nitrogen Intermediates in Killing of Rhodococcus equi by Activated Macrophages

2000 ◽  
Vol 68 (6) ◽  
pp. 3587-3593 ◽  
Author(s):  
Patricia A. Darrah ◽  
Mary K. Hondalus ◽  
Quiping Chen ◽  
Harry Ischiropoulos ◽  
David M. Mosser
Keyword(s):  
Nitric Oxide ◽  
Prolonged Exposure ◽  
Rhodococcus Equi ◽  
Activated Macrophages ◽  
Intracellular Killing ◽  
Oxygen Intermediates ◽  
Bacterial Killing ◽  
Ifn Γ

ABSTRACT Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-γ), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO−), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS−/−) or the oxidative burst (gp91 phox−/− ) are more susceptible to lethalR. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O2 −) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO− efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO− in situ. Inhibition of nitric oxide production by activated macrophages by usingN G-monomethyl-l-arginine blocks their production of ONOO− and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equiby IFN-γ-activated macrophages.

scholarly journals Impact of Hypothermia on Differentiation and Maturation of Neutrophils

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2393-2393
Author(s):  
Yusuke Torikoshi ◽  
Asumi Yokota ◽  
Naoka Kamio ◽  
Atsushi Sato ◽  
Tsukimi Shouji ◽  
...  
Keyword(s):  
Body Temperature ◽  
Host Defense ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Environmental Temperature ◽  
Cecal Ligation ◽  
The Body ◽  
Neutrophil Differentiation ◽  
The Impact

Abstract Accumulating evidence has suggested that low body temperature is associated with the risk of infection. Unintentional drops in the body temperature known as "accidental hypothermia" are occasionally accompanied with infections. Patients under therapeutic hypothermia for post-cardiac arrest care are also susceptible to infections. In addition, secondary hypothermia caused by severe sepsis is significantly associated with higher mortality. These observations suggest the negative impact of hypothermia on host defense. Neutrophils are continuously produced in the bone marrow (BM) and supplied to the peripheral blood (PB) or tissues, where they fight against microorganisms. In addition to the neutrophil functions, sufficient supply of neutrophils is a critical determinant of host defense. However, little is known about the impact of hypothermia on granulopoiesis, the process of neutrophil production in the BM. In this study, we investigated the changes in granulopoiesis under hypothermic conditions. We first analyzed the neutrophils in the PB of mice exposed to low environmental temperature (4 °C). Under this condition, rectal temperature of the mice significantly declined from 36.7±0.4 °C to 35.5±0.4 °C. After 72-hour exposure to the low environmental temperature, PB neutrophil counts were significantly decreased. In order to understand the reason for the decrease, we analyzed their BMs by flow cytometry. Previously we developed a unique strategy to divide cells undergoing granulopoiesis into 5 subpopulations based on the expression of c-kit and Ly6G, which reflect successive differentiation/maturation from #1 (c-kithi Ly6G-) to #5 (c-kit- Ly6Ghi) (Satake S and Hirai H et al. J Immunol, 2012). In BM cells of the mice exposed to the low environmental temperature, a significant decrease in mature neutrophils (#5) and a significant increase in cellular intermediates (#3 and #4) were observed, while total BM cell numbers were unchanged. In order to clarify whether these changes were cell-intrinsic or -extrinsic, total BM cells were cultured in vitro at either 35 °C or 37 °C in the presence of G-CSF. Flow cytometric analysis of these cultured BM cells at 72 hours revealed the increase in the intermediates (#2 to #4) and a decrease in the mature subpopulation (#5), suggesting that these alterations were cell-intrinsic phenomena. When neutrophil precursors (#1 or #2) were purified by cell sorter and subjected to in vitro culture at 35 °C for 48 hours, the number of resultant mature neutrophils (#5) were significantly less than those induced at 37 °C. These results clearly indicate that hypothermia delayed neutrophil differentiation/maturation. Interestingly, mice with sepsis induced by cecal ligation and puncture (CLP) accompanied with lower body temperature revealed significantly fewer PB granulocytes and shorter survival when compared to those mice which maintained normal body temperature after CLP. In order to understand the molecular mechanisms underlying the differentiation/maturation delay induced by hypothermia, we performed RNA sequencing of purified neutrophil precursors (#2) after 24-hour culture either at 35 °C or 37 °C. Interestingly, we found alterations in amino acid metabolic pathways and target genes of C/EBP, which is the transcription factor family required for granulopoiesis and cellular metabolism. Collectively, these results indicate hypothermia causes neutropenia through delayed neutrophil differentiation/maturation. We are currently analyzing metabolic changes to understand more precise molecular mechanisms by which hypothermia regulates granulopoiesis. This study will facilitate the understanding of host defense at low body temperature, and shed novel insight into the management of hypothermia in patients. Disclosures Kashiwagi: Takara Bio Inc.: Employment. Hirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding.

scholarly journals Impact of thrombocytes, on bacterial growth and antimicrobial activity of selected antibiotics

2019 ◽  
Vol 39 (3) ◽  
pp. 593-597 ◽  
Author(s):  
Alina Karoline Nussbaumer-Pröll ◽  
Sabine Eberl ◽  
Birgit Reiter ◽  
Thomas Stimpfl ◽  
Walter Jäger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Limiting Factor ◽  
Growth Media ◽  
Healthy Human ◽  
Bacterial Killing ◽  
Bacteria Growth ◽  
The Impact

AbstractIn vitro pharmacodynamic models are used to optimize in vivo dosing regimens in antimicrobial drug development. One limiting factor of such models is the lack of host factors such as corpuscular blood components as erythrocytes which have already been shown to impact activity of antibiotics and/or growth of the pathogen. However, the impact of thrombocytes has not previously been investigated. We set out to investigate if the addition of thrombocytes (set to physiological concentrations in blood of healthy human, i.e., 5 × 105 thrombocytes/μL standard growth media Mueller Hinton Broth, MHB) has an influence on bacterial growth and on the efficacy of antibiotics against Gram+ and Gram− bacteria. Growth assays and time-killing-curves (TKC) were performed with ATCC-strains of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in triplicate over 24 h. The same approach was followed for 5 clinical isolates of Escherichia coli. Meropenem, ciprofloxacin, and tigecycline were tested as representatives of broad-spectrum antibiotics, and concentrations several-fold above and below the minimal inhibitory concentration (MIC) were simulated. No significant impact of thrombocytes was found on bacterial growth or antimicrobial stability for the investigated agents. Bacteria reduced thrombocyte content to different degree, indicating direct interaction of pathogens and thrombocytes. Impact on bacterial killing was observed but was not fully reproducible when thrombocytes from different donors where used. While interaction of bacteria and thrombocytes was evident in the present study, interaction between antibiotic activity and thrombocytes seems unlikely. Whether variability was caused by different thrombocyte concentrates needs further investigation.

Metallo-β-lactamase resistance in Enterobacteriaceae is an artefact of currently utilized antimicrobial susceptibility testing methods

2020 ◽  
Vol 75 (4) ◽  
pp. 997-1005 ◽  
Author(s):  
Tomefa E Asempa ◽  
Kamilia Abdelraouf ◽  
David P Nicolau
Keyword(s):  
Susceptibility Testing ◽  
Bacterial Killing ◽  
Phenotypic Profile ◽  
Fold Reduction ◽  
Pharmacodynamic Profile ◽  
Zinc Depletion ◽  
Zinc Concentrations ◽  
The Impact

Abstract Background MBLs are a major contributor to β-lactam resistance when tested using CAMHB. Despite in vitro resistance, positive outcomes have been reported in MBL-infected patients following carbapenem treatment. The impact of physiological zinc concentrations on this in vitro–in vivo MBL discordance warrants investigation. Objectives To evaluate meropenem in vitro activity against MBL-producing Enterobacteriaceae in zinc-depleted broth (Chelex-CAMHB, EDTA-CAMHB) and assess meropenem efficacy in murine infection models. Methods Neutropenic mice received a meropenem human-simulated regimen of 2 g q8h or levofloxacin 750 mg q24h (for model validation). Zinc concentrations were determined in conventional CAMHB, zinc-depleted CAMHB and epithelial lining fluid (ELF) of lung-infected mice. Results All MBL-producing isolates (NDM, n = 25; VIM, n = 3; IMP, n = 2) examined were meropenem resistant in CAMHB and susceptible in zinc-depleted CAMHB (5- to 11-fold reduction), with zinc depletion having no impact on levofloxacin MICs. Zinc concentrations (mean ± SD) in CAMHB were 0.959 ± 0.038 mg/L and in both zinc-depleted CAMHB and ELF were <0.002 mg/L. In vivo, levofloxacin displayed predictable efficacy consistent with its phenotypic profile, while meropenem produced >1 log unit bacterial killing despite in vitro resistance in conventional CAMHB. Conclusions Results indicate that meropenem in vivo efficacy is best represented by the pharmacodynamic profile generated using MICs determined in zinc-depleted media for MBL-producing Enterobacteriaceae. These translational data suggest that the use of conventional CAMHB for MBL susceptibility testing is inappropriate in distinguishing meaningful in vivo resistance given that zinc concentrations are supraphysiological in conventional CAMHB and negligible at infection sites.

scholarly journals Plasma gelsolin improves lung host defense against pneumonia by enhancing macrophage NOS3 function

2015 ◽  
Vol 309 (1) ◽  
pp. L11-L16 ◽  
Author(s):  
Zhiping Yang ◽  
Terry Ting-Yu Chiou ◽  
Thomas P. Stossel ◽  
Lester Kobzik
Keyword(s):  
Host Defense ◽  
Bacterial Pneumonia ◽  
Secondary Infection ◽  
Bacterial Clearance ◽  
Bacterial Killing ◽  
Plasma Gelsolin ◽  
Patients At Risk ◽  
Secondary Bacterial Pneumonia

Plasma gelsolin (pGSN) functions as part of the “extracellular actin-scavenging system,” but its potential to improve host defense against infection has not been studied. In a mouse model of primary pneumococcal pneumonia, recombinant human pGSN (rhu-pGSN) caused enhanced bacterial clearance, reduced acute inflammation, and improved survival. In vitro, rhu-pGSN rapidly improved lung macrophage uptake and killing of bacteria ( Streptococcus pneumoniae, Escherichia coli, and Francisella tularensis). pGSN triggers activating phosphorylation (Ser1177) of macrophage nitric oxide synthase type III (NOS3), an enzyme with important bactericidal functions in lung macrophages. rhu-pGSN failed to enhance bacterial killing by NOS3−/− macrophages in vitro or bacterial clearance in NOS3−/− mice in vivo. Prophylaxis with immunomodulators may be especially relevant for patients at risk for secondary bacterial pneumonia, e.g., after influenza. Treatment of mice with pGSN challenged with pneumococci on postinfluenza day 7 (the peak of enhanced susceptibility to secondary infection) caused a ∼15-fold improvement in bacterial clearance, reduced acute neutrophilic inflammation, and markedly improved survival, even without antibiotic therapy. pGSN is a potential immunomodulator for improving lung host defense against primary and secondary bacterial pneumonia.

scholarly journals Pharmacodynamic Evaluation of the Activities of Six Parenteral Vancomycin Products Available in the United States

2014 ◽  
Vol 59 (1) ◽  
pp. 622-632 ◽  
Author(s):  
Arnold Louie ◽  
Michael T. Boyne ◽  
Vikram Patel ◽  
Clayton Huntley ◽  
Weiguo Liu ◽  
...  
Keyword(s):  
United States ◽  
Drug Exposure ◽  
The United States ◽  
Mouse Serum ◽  
Infection Model ◽  
Population Pharmacokinetic ◽  
Bacterial Killing ◽  
The Impact

ABSTRACTA recent report found that generic parenteral vancomycin products may not havein vivoefficacies equivalent to those of the innovator in a neutropenic murine thigh infection model despite having similarin vitromicrobiological activities and murine serum pharmacokinetics. We compared thein vitroandin vivoactivities of six of the parenteral vancomycin products available in the United States. Thein vitroassessments for the potencies of the vancomycin products included MIC/minimal bactericidal concentration (MBC) determinations, quantifying the impact of human and murine serum on the MIC values, and time-kill studies. Also, the potencies of the vancomycin products were quantified with a biological assay, and the human and mouse serum protein binding rates for the vancomycin products were measured. Thein vivostudies included dose-ranging experiments with the 6 vancomycin products for three isolates ofStaphylococcus aureusin a neutropenic mouse thigh infection model. The pharmacokinetics of the vancomycin products were assessed in infected mice by population pharmacokinetic modeling. No differences were seen across the vancomycin products with regard to anyin vitroevaluation. Inhibitory sigmoid maximal bacterial kill (Emax) modeling of the relationship between vancomycin dosage and the killing of the bacteria in micein vivoyielded similarEmaxand EC50(drug exposure driving one-halfEmax) values for bacterial killing. Further, there were no differences in the pharmacokinetic clearances of the 6 vancomycin products from infected mice. There were no important pharmacodynamic differences in thein vitroorin vivoactivities among the six vancomycin products evaluated.

scholarly journals Geroscience approaches to obesity

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. 830-830
Author(s):  
Alessandro Bitto ◽  
Matt Kaeberlein
Keyword(s):  
Adefovir Dipivoxil ◽  
Fatty Acid Biosynthesis ◽  
Transcriptase Inhibitor ◽  
Nutrient Metabolism ◽  
Mtor Inhibition ◽  
Diet Induced Obesity ◽  
Expression Of Genes ◽  
Reverse Transcriptase Inhibitor ◽  
The Impact

Abstract Besides aging, obesity is the greatest risk factor for numerous chronic pathologies, including metabolic syndrome, type 2 diabetes, cardiovascular disease, hypertension, and cancer. Preventing and treating obesity would greatly reduce healthcare costs and the impact of the aging process, with estimated savings up to $145,000,000,000. Pharmacological interventions identified by geroscience may prove effective against diet-induced obesity. To test this hypothesis, we fed a 66% kcal/fat diet to nine-month-old C57Bl6/N mice for 6 weeks and treated them with either rapamycin, acarbose, or a combination thereof. Rapamycin, and to a lesser extent acarbose, prevented weight gain and fat accumulation in these mice. We detected increased expression of the Liver Activating Protein (LAP) isoform of the transcription factor CCAT/Enhancer Binding Protein β (C/EBPβ) in the liver of mice treated with rapamycin. C/EBPβ-LAP mediates some of the effects of caloric restriction on nutrient metabolism and increases lifespan in a mouse transgenic model. We tested whether independent activation of C/EBPβ-LAP would recapitulate the effects of rapamycin by treating mice on a high-fat diet with adefovir dipivoxil, a reverse transcriptase inhibitor that can activate LAP in vitro independently of mTOR inhibition. Adefovir dipivoxil reduced weight and fat mass accumulation in mice over the course of 6 weeks. Mice treated with adefovir dipivoxil showed increased expression of genes involved in β-oxidation and lipids mobilization, and reduced activation of fatty acid biosynthesis and lipid storage pathways. Our results identify C/EBPβ-LAP as a potential new target to improve lipid homeostasis in both aging and obesity.

scholarly journals New Dosing Strategies for an Old Antibiotic: Pharmacodynamics of Front-Loaded Regimens of Colistin at Simulated Pharmacokinetics in Patients with Kidney or Liver Disease

2013 ◽  
Vol 58 (3) ◽  
pp. 1381-1388 ◽  
Author(s):  
Gauri G. Rao ◽  
Neang S. Ly ◽  
Curtis E. Haas ◽  
Samira Garonzik ◽  
Alan Forrest ◽  
...  
Keyword(s):  
Renal Failure ◽  
Viable Count ◽  
Infection Model ◽  
Combination Regimen ◽  
Time Curve ◽  
Bacterial Killing ◽  
Content Type ◽  
Dosing Regimens ◽  
The Impact

ABSTRACTIncreasing evidence suggests that colistin monotherapy is suboptimal at currently recommended doses. We hypothesized that front-loading provides an improved dosing strategy for polymyxin antibiotics to maximize killing and minimize total exposure. Here, we utilized anin vitropharmacodynamic model to examine the impact of front-loaded colistin regimens against a high bacterial density (108CFU/ml) ofPseudomonas aeruginosa. The pharmacokinetics were simulated for patients with hepatic (half-life [t1/2] of 3.2 h) or renal (t1/2of 14.8 h) disease. Front-loaded regimens (n= 5) demonstrated improvement in bacterial killing, with reduced overall free drug areas under the concentration-time curve (fAUC) compared to those with traditional dosing regimens (n= 14) with various dosing frequencies (every 12 h [q12h] and q24h). In the renal failure simulations, front-loaded regimens at lower exposures (fAUC of 143 mg · h/liter) obtained killing activity similar to that of traditional regimens (fAUC of 268 mg · h/liter), with an ∼97% reduction in the area under the viable count curve over 48 h. In hepatic failure simulations, front-loaded regimens yielded rapid initial killing by up to 7 log10within 2 h, but considerable regrowth occurred for both front-loaded and traditional regimens. No regimen eradicated the high bacterial inoculum ofP. aeruginosa. The current study, which utilizes anin vitropharmacodynamic infection model, demonstrates the potential benefits of front-loading strategies for polymyxins simulating differential pharmacokinetics in patients with hepatic and renal failure at a range of doses. Our findings may have important clinical implications, as front-loading polymyxins as a part of a combination regimen may be a viable strategy for aggressive treatment of high-bacterial-burden infections.

scholarly journals Impact of Diet-Induced Obesity on Intestinal Stem Cells: Hyperproliferation but Impaired Intrinsic Function That Requires Insulin/IGF1

Endocrinology ◽  
2014 ◽  
Vol 155 (9) ◽  
pp. 3302-3314 ◽  
Author(s):  
Amanda T. Mah ◽  
Laurianne Van Landeghem ◽  
Hannah E. Gavin ◽  
Scott T. Magness ◽  
P. Kay Lund
Keyword(s):  
Stem Cells ◽  
Nutrient Intake ◽  
Intestinal Stem Cells ◽  
Exogenous Insulin ◽  
Reporter Mouse ◽  
Diet Induced Obesity ◽  
Egfp Reporter ◽  
The Impact

Abstract Nutrient intake regulates intestinal epithelial mass and crypt proliferation. Recent findings in model organisms and rodents indicate nutrient restriction impacts intestinal stem cells (ISC). Little is known about the impact of diet-induced obesity (DIO), a model of excess nutrient intake on ISC. We used a Sox9-EGFP reporter mouse to test the hypothesis that an adaptive response to DIO or associated hyperinsulinemia involves expansion and hyperproliferation of ISC. The Sox9-EGFP reporter mouse allows study and isolation of ISC, progenitors, and differentiated lineages based on different Sox9-EGFP expression levels. Sox9-EGFP mice were fed a high-fat diet for 20 weeks to induce DIO and compared with littermates fed low-fat rodent chow. Histology, fluorescence activated cell sorting, and mRNA analyses measured impact of DIO on jejunal crypt-villus morphometry, numbers, and proliferation of different Sox9-EGFP cell populations and gene expression. An in vitro culture assay directly assessed functional capacity of isolated ISC. DIO mice exhibited significant increases in body weight, plasma glucose, insulin, and insulin-like growth factor 1 (IGF1) levels and intestinal Igf1 mRNA. DIO mice had increased villus height and crypt density but decreased intestinal length and decreased numbers of Paneth and goblet cells. In vivo, DIO resulted in a selective expansion of Sox9-EGFPLow ISC and percentage of ISC in S-phase. ISC expansion significantly correlated with plasma insulin levels. In vitro, isolated ISC from DIO mice formed fewer enteroids in standard 3D Matrigel culture compared to controls, indicating impaired ISC function. This decreased enteroid formation in isolated ISC from DIO mice was rescued by exogenous insulin, IGF1, or both. We conclude that DIO induces specific increases in ISC and ISC hyperproliferation in vivo. However, isolated ISC from DIO mice have impaired intrinsic survival and growth in vitro that can be rescued by exogenous insulin or IGF1.

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