Intestinal Knockout Mice Research Articles

Intestinal Knockout of Peroxisome Proliferator-Activated Receptor-Alpha Affects Structural Adaptation but not Liver Injury Following Massive Enterectomy

BackgroundResection-associated liver steatosis, injury, and fibrosis is a devastating complication associated with massive small bowel resection (SBR). Peroxisome proliferator-activated receptor-alpha (PPARα) is a key regulator of intestinal lipid transport and metabolism whose expression is selectively increased after SBR. Here we asked if attenuating intestinal PPARα signaling would prevent steatosis and liver injury after SBR. MethodsPparα was deleted selectively in adult mouse intestine using a tamoxifen-inducible Cre-LoxP breeding schema. Mice underwent 50% SBR. At 10 weeks post-operatively, metabolic phenotyping, body composition analysis, in vivo assessment of lipid absorption and intestinal permeability, and assessment of adaptation and liver injury was completed. ResultsPparα intestinal knockout and littermate control mice were phenotypically similar in terms of weight trends and body composition after SBR. All mice demonstrated intestinal adaptation with increased villus height and crypt depth; however, Pparα intestinal knockout mice exhibited decreased villus growth at 10 weeks compared to littermate controls. Liver injury and fibrosis were similar between groups as assessed by serum AST and ALT levels, Sirius Red staining, and hepatic expression of Col1a1 and Acta2. ConclusionsInducible intestinal deletion of Pparα influences structural adaptation but does not mitigate liver injury after SBR. These findings suggest that enterocyte PPARα signaling in adult mice is dispensable for resection-induced liver injury. The results are critical for understanding the contribution of intestinal lipid metabolic signaling pathways to the pathogenesis of hepatic injury associated with short bowel syndrome.

1895-P: Knocking Out Intestinal GPR17 Improves Glucose Homeostasis and Increases Satiety during Fasting-Refeeding Challenge

Aims: G protein-coupled receptors (GPCRs) in the intestinal enteroendocrine cells (EECs) regulate gut hormone secretion by responding to nutritional, microbial, and inflammatory factors, which contributes to the maintenance of glucose and energy homeostasis. We investigated the expression of GPR17, an orphan GPCR, in human and murine gastrointestinal tract and determined its metabolic roles using intestinal specific Gpr17 knockout mice. Methods: Gpr17 expression was profiled by quantitative RT-PCR, immunohistochemistry and flow cytometry. To elucidate the metabolic function of intestinal Gpr17, we generated constitutive (Vil1Cre,Gpr17-/-) and inducible (Vil1Cre-ERT2,Gpr17-/-) intestine-specific Gpr17 knockout mice and analyzed their metabolic features, including glucose tolerance, indirection calorimetry, feeding and microbiome composition. We measured nutrient-stimulated incretin secretion in Gpr17 knockout mice and cultured intestinal organoids. Results: We found that Gpr17 was enriched in Glucagon-like peptide 1 (GLP-1) expressing EECs in human and rodent intestine. Both constitutive and inducible Gpr17 intestinal knockout mice displayed improved glucose tolerance without alterations in pancreatic function and microbiota. In addition, Gpr17 intestinal knockout mice exhibited reduced foraging activity and less rebound hyperphagia in a fasting-refeeding paradigm. Notably, Gpr17 deficient mice and intestinal organoids responded to nutritional cues with enhanced GLP-1, but not GIP, secretion. Forskolin induced cAMP production was significantly inhibited by a synthetic Gpr17 agonist in GLUTag cells overexpressing Gpr17. Conclusions: Our work suggests GPR17 signaling in the enteroendocrine cells fine-tunes GLP-1 secretion. Targeting intestinal GPR17 for appetite and glucose metabolism represents a potential therapeutic strategy for type 2 diabetes and obesity. Disclosure S. Yan: None. A. Reilly: None. J.M. Conley: None. H. Ren: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK120772, R00DK098294); Indiana Diabetes Center (P30DK097512); Indiana Clinical and Translational Sciences Institute (UL1TR002529)

Plasma membrane-localized TMEM16 proteins are indispensable for expression of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is the secretory chloride channel in epithelial tissues that has a central role in cystic fibrosis (CF) lung and gastrointestinal disease. A recent publication demonstrates a close association between CFTR and TMEM16A, the calcium-activated chloride channel. Thus, no CFTR chloride currents could be detected in airways and large intestine from mice lacking epithelial expression of TMEM16A. Here, we demonstrate that another plasma membrane-localized TMEM16 paralogue, TMEM16F, can compensate for the lack of TMEM16A. Using TMEM16 knockout mice, human lymphocytes, and a number of human cell lines with endogenous protein expression or heterologous expression, we demonstrate that CFTR can only function in the presence of either TMEM16A or TMEM16F. Double knockout of intestinal epithelial TMEM16A/F expression did not produce offsprings, suggesting a lethal phenotype in utero. Plasma membrane-localized TMEM16A or TMEM16F is required for exocytosis and expression of CFTR in the plasma membrane. TMEM16A/F proteins may therefore have an impact on disease severity in CF. KEY MESSAGES: • Cystic fibrosis is caused by the defective Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR). • A close relationship exists between CFTR and the calcium-activated chloride channels TMEM16A/TMEM16F. • In conditional airway and intestinal knockout mice, lymphocytes from Scott disease patients and in overexpressing cells, CFTR is not functional in the absence of TMEM16A and TMEM16F. • TMEM16A and TMEM16F support membrane exocytosis and are essential for plasma membrane insertion of CFTR.

Hypoxia Could Induce Endoplasmic-Reticulum Stress by Regulating the Expression of β-Catenin Via Low-Density Lipoprotein Receptor-Related Protein 6 Pathway

Background: Wnt/β-catenin was reported as an important pathway regulated by hypoxia, however, the exact mechnism has not been elucidated. Methods: Fecal 16S RNA sequencing and microarray were performed to selected the potential biomarks of LPR6 during the regulation of hypoxia on Wnt/β-catenin. Phosphorylated elF2α was detected for endoplasmic-reticulum stress. The ubiquitylation of β-catenin was determined by Immunoprecipitation. LRP6 was added to investigate the biological role on β-catenin. LRP6 intestinal knockout mice and LRP6 overexpression mice was established to verify the effects of LPR6 on tight junction proteins, endoplasmic-reticulum stress, and β-catenin expression. Two drugs curcumin and auranofin were used to explore the effects of LRP6 on the intestinal tight junction proteins. Findings: Inhibition of β-catenin and activation of ER stress induced by hypoxia. ER stress could decrease the expression of β-catenin through ubiquitylation via LRP6. Increased expression level of β-catenin, DKK1, and tight junction proteins, and decreased phosphorylated elF2α were found in LRP6 transgenic mice, while opposite results was found in the group of LRP6 intestinal knockout mice. Administration of curcumin and auranofin further verify the effects of LRP6 on the intestinal tight junction proteins. Interpretation: Hypoxia could induce endoplasmic-reticulum stress by regulating the expression of β-catenin via LRP6 pathway, leading to the intestinal barrier dysfunction. Funding Statement: This work was financially supported by National Natural Science Foundation of China (No. 81670480, 81570189), Training Program of the Major Research Plan of the Natural Science Foundation of Guangdong Province (2014A030308004), and High level talents in Guangdong Province special support plan - Youth Science and technology innovation talents (2015TQ01R334), Major Scientific Research Projects at Provincial Level in Guangdong General University (2017KZDXM068). Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: The study protocol and experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee of Guangzhou Medical University and were approved by the Institutional Ethics Committee of Guangzhou Medical University.

Lack of iron-related phenotype in Sp6 intestinal knockout mice

Based on a microarray study, which demonstrated the upregulation of Sp6 transcriptional factor in iron deficient rat duodenum, we reasoned that SP6 could regulate iron absorption by controlling the expression of iron absorption genes (Collins,2006). For that, we generated Sp6 specific intestinal knockout mice. Our data suggest a lack of transcriptional upregulation of Sp6 in mice in conditions where iron absorption is promoted (phlebotomy, iron deficiency, hpx mouse), and an absence of iron-related phenotype in Sp6 intestinal knockout model. We propose that other Sp6 orthologues may be involved in the genetic response to increase iron absorption, possibly in co-operation with hypoxia inducible factor 2 alpha (HIF-2α)—a newly discovered regulator of iron absorption.

Integrin-linked kinase regulates cell proliferation and tumour growth in murine colitis-associated carcinogenesis

Background:Integrins are transmembrane cell surface receptors that mediate cell–cell and cell–matrix contacts. Integrin-linked kinase (ILK) is the binding partner of β1 and β3 integrins, and has been ascribed essential roles...