scholarly journals Ins1-Cre and Ins1-CreER Gene Replacement Alleles Are Susceptible To Silencing By DNA Hypermethylation

Endocrinology ◽  
2020 ◽  
Vol 161 (8) ◽  
Author(s):  
Elham Mosleh ◽  
Kristy Ou ◽  
Matthew W Haemmerle ◽  
Teguru Tembo ◽  
Andrew Yuhas ◽  
...  
Keyword(s):  
Beta Cell ◽  
Endocrine Cells ◽  
Target Genes ◽  
Cell Function ◽  
Gene Replacement ◽  
Wild Type ◽  
Dna Hypermethylation ◽  
Gene Ablation ◽  
Low Levels ◽  
The Brain

Abstract Targeted gene ablation studies of the endocrine pancreas have long suffered from suboptimal Cre deleter strains. In many cases, Cre lines purportedly specific for beta cells also displayed expression in other islet endocrine cells or in a subset of neurons in the brain. Several pancreas and endocrine Cre lines have experienced silencing or mosaicism over time. In addition, many Cre transgenic constructs were designed to include the hGH mini-gene, which by itself increases beta-cell replication and decreases beta-cell function. More recently, driver lines with Cre or CreER inserted into the Ins1 locus were generated, with the intent of producing β cell-specific Cre lines with faithful recapitulation of insulin expression. These lines were bred in multiple labs to several different mouse lines harboring various lox alleles. In our hands, the ability of the Ins1-Cre and Ins1-CreER lines to delete target genes varied from that originally reported, with both alleles displaying low levels of expression, increased levels of methylation compared to the wild-type allele, and ultimately inefficient or absent target deletion. Thus, caution is warranted in the interpretation of results obtained with these genetic tools, and Cre expression and activity should be monitored regularly when using these lines.

Persistent expression of HNF6 in islet endocrine cells causes disrupted islet architecture and loss of beta cell function

Development ◽  
2000 ◽  
Vol 127 (13) ◽  
pp. 2883-2895 ◽  
Author(s):  
M. Gannon ◽  
M.K. Ray ◽  
K. Van Zee ◽  
F. Rausa ◽  
R.H. Costa ◽  
...  
Keyword(s):  
Beta Cell ◽  
Beta Cells ◽  
Endocrine Cells ◽  
Spatial Organization ◽  
Glucose Transporter ◽  
Cell Function ◽  
Regulatory Element ◽  
Cell Physiology ◽  
Pancreatic Ducts ◽  
And Function

We used transgenesis to explore the requirement for downregulation of hepatocyte nuclear factor 6 (HNF6) expression in the assembly, differentiation, and function of pancreatic islets. In vivo, HNF6 expression becomes downregulated in pancreatic endocrine cells at 18. 5 days post coitum (d.p.c.), when definitive islets first begin to organize. We used an islet-specific regulatory element (pdx1(PB)) from pancreatic/duodenal homeobox (pdx1) gene to maintain HNF6 expression in endocrine cells beyond 18.5 d.p.c. Transgenic animals were diabetic. HNF6-overexpressing islets were hyperplastic and remained very close to the pancreatic ducts. Strikingly, alpha, delta, and PP cells were increased in number and abnormally intermingled with islet beta cells. Although several mature beta cell markers were expressed in beta cells of transgenic islets, the glucose transporter GLUT2 was absent or severely reduced. As glucose uptake/metabolism is essential for insulin secretion, decreased GLUT2 may contribute to the etiology of diabetes in pdx1(PB)-HNF6 transgenics. Concordantly, blood insulin was not raised by glucose challenge, suggesting profound beta cell dysfunction. Thus, we have shown that HNF6 downregulation during islet ontogeny is critical to normal pancreas formation and function: continued expression impairs the clustering of endocrine cells and their separation from the ductal epithelium, disrupts the spatial organization of endocrine cell types within the islet, and severely compromises beta cell physiology, leading to overt diabetes.

scholarly journals The Cell-Specific Pattern of Cholecystokinin Peptides in Endocrine Cells Versus Neurons Is Governed by the Expression of Prohormone Convertases 1/3, 2, and 5/6

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1600-1608 ◽  
Author(s):  
Jens F. Rehfeld ◽  
Jens R. Bundgaard ◽  
Jens Hannibal ◽  
Xiaorong Zhu ◽  
Christina Norrbom ◽  
...  
Keyword(s):  
Endocrine Cells ◽  
Peptide Hormone ◽  
Specific Pattern ◽  
Molecular Forms ◽  
Wild Type ◽  
Prohormone Convertases ◽  
Cerebral Neurons ◽  
Neuronal Processing ◽  
The Brain ◽  
Translational Product

Most peptide hormone genes are, in addition to endocrine cells, also expressed in neurons. The peptide hormone cholecystokinin (CCK) is expressed in different molecular forms in cerebral neurons and intestinal endocrine cells. To understand this difference, we examined the roles of the neuroendocrine prohormone convertases (PC) 1/3, PC2, and PC5/6 by measurement of proCCK, processing intermediates and bioactive, α-amidated, and O-sulfated CCK peptides in cerebral and jejunal extracts of null mice, controls, and in the PC5/6-expressing SK-N-MC cell-line. In PC1/3 null mice, the synthesis of bioactive CCK peptide in the gut was reduced to 3% of the translational product, all of which was in the form of α-amidated and tyrosine O-sulfated CCK-22, whereas the neuronal synthesis in the brain was largely unaffected. This is opposite to the PC2 null mice in which only the cerebral synthesis was affected. SK-N-MC cells, which express neither PC1/3 nor PC2, synthesized alone the processing intermediate, glycine-extended CCK-22. Immunocytochemistry confirmed that intestinal endocrine CCK cells in wild-type mice express PC1/3 but not PC2. In contrast, cerebral CCK neurons contain PC2 and only little, if any, PC1/3. Taken together, the data indicate that PC1/3 governs the endocrine and PC2 the neuronal processing of proCCK, whereas PC5/6 contributes only to a modest endocrine synthesis of CCK-22. The results suggest that the different peptide patterns in the brain and the gut are due to different expression of PCs.

scholarly journals Aquaporin 1 Is Important for Maintaining Secretory Granule Biogenesis in Endocrine Cells

2008 ◽  
Vol 22 (8) ◽  
pp. 1924-1934 ◽  
Author(s):  
Irina Arnaoutova ◽  
Niamh X. Cawley ◽  
Nimesh Patel ◽  
Taeyoon Kim ◽  
Trushar Rathod ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Endocrine Cells ◽  
Cell Function ◽  
Hormone Secretion ◽  
Prohormone Convertase ◽  
Protein Levels ◽  
Prohormone Convertase 1 ◽  
Aqp1 Expression ◽  
Low Levels ◽  
Made In

Abstract Aquaporins (AQPs), a family of water channels expressed in epithelial cells, function to transport water in a bidirectional manner to facilitate transepithelial fluid absorption and secretion. Additionally, AQP1 and AQP5 are found in pancreatic zymogen granules and synaptic vesicles and are involved in vesicle swelling and exocytosis in exocrine cells and neurons. Here, we show AQP1 is in dense-core secretory granule (DCSG) membranes of endocrine tissue: pituitary and adrenal medulla. The need for AQP1 in endocrine cell function was examined by stable transfection of AQP1 antisense RNA into AtT20 cells, a pituitary cell line, to down-regulate AQP1 expression. These AQP1-deficient cells showed more than 60% depletion of DCSGs and significantly decreased DCSG protein levels, including proopiomelanocotin/pro-ATCH and prohormone convertase 1/3, but not non-DCSG proteins. Pulse-chase studies revealed that whereas DCSG protein synthesis was unaffected, approximately 50% of the newly synthesized proopiomelanocortin was degraded within 1 h. Low levels of ACTH were released upon stimulation, indicating that the small number of DCSGs that were made in the presence of the residual AQP1 were functionally competent for exocytosis. Analysis of anterior pituitaries from AQP1 knockout mice showed reduced prohormone convertase 1/3, carboxypeptidase E, and ACTH levels compared to wild-type mice demonstrating that our results observed in AtT20 cells can be extended to the animal model. Thus, AQP1 is important for maintaining DCSG biogenesis and normal levels of hormone secretion in pituitary endocrine cells.

scholarly journals Characterization of 5-(2-18F-fluoroethoxy)-L-tryptophan for PET imaging of the pancreas

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 1851 ◽  
Author(s):  
Ahmed Abbas ◽  
Christine Beamish ◽  
Rebecca McGirr ◽  
John Demarco ◽  
Neil Cockburn ◽  
...  
Keyword(s):  
Beta Cell ◽  
Er Stress ◽  
Beta Cell Function ◽  
Cell Function ◽  
Cell Mass ◽  
Beta Cell Mass ◽  
Wild Type ◽  
Time Activity ◽  
System L ◽  
Akita Mice

Purpose: In diabetes, pancreatic beta cell mass declines significantly prior to onset of fasting hyperglycemia. This decline may be due to endoplasmic reticulum (ER) stress, and the system L amino acid transporter LAT1 may be a biomarker of this process. In this study, we used 5-(2-18F-fluoroethoxy)-L-tryptophan (18F-L-FEHTP) to target LAT1 as a potential biomarker of beta cell function in diabetes. Procedures: Uptake of 18F-L-FEHTP was determined in wild-type C57BL/6 mice by ex vivo biodistribution. Both dynamic and static positron emission tomography (PET) images were acquired in wild-type and Akita mice, a model of ER stress-induced diabetes, as well as in mice treated with streptozotocin (STZ). LAT1 expression in both groups of mice was evaluated by immunofluorescence microscopy. Results: Uptake of 18F-L-FEHTP was highest in the pancreas, and static PET images showed highly specific pancreatic signal. Time-activity curves showed significantly reduced 18F-L-FEHTP uptake in Akita mice, and LAT1 expression was also reduced. However, mice treated with STZ, in which beta cell mass was reduced by 62%, showed no differences in 18F-L-FEHTP uptake in the pancreas, and there was no significant correlation of 18F-L-FEHTP uptake with beta cell mass. Conclusions: 18F-L-FEHTP is highly specific for the pancreas with little background uptake in kidney or liver. We were able to detect changes in LAT1 in a mouse model of diabetes, but these changes did not correlate with beta cell function or mass. Therefore, 18F-L-FEHTP PET is not a suitable method for the noninvasive imaging of changes in beta cell function during the progression of diabetes.

Coumestrol Antagonizes Neuroendocrine Actions of Estrogen via the Estrogen Receptor α

2001 ◽  
Vol 226 (4) ◽  
pp. 301-306 ◽  
Author(s):  
Dena A. Jacob ◽  
Jennifer L. Temple ◽  
Heather B. Patisaul ◽  
Larry J. Young ◽  
Emilie F. Rissman
Keyword(s):  
Estrogen Receptor ◽  
Progesterone Receptors ◽  
Estrogen Receptor Α ◽  
Ventromedial Nucleus ◽  
Wild Type ◽  
Treatment Groups ◽  
Estrogenic Effects ◽  
Low Levels ◽  
The Brain ◽  
All Treatment

The phytoestrogen coumestrol has estrogenic actions on peripheral reproductive tissues. Yet in the brain this compound has both estrogenic and anti-estrogenic effects. We used estrogen receptor α knockout mice (ERαKO) to determine whether coumestrol has estrogenic actions in mice and also if these effects are mediated by the classic ERα. Female wild-type (WT) and ERαKO mice were ovariectomized and treated with estradiol (E2), dietary coumestrol, both, or neither compound. Ten days later the animals were sacrificed, blood was collected, and brain tissues were perfused. Fixed brains were sectioned and immunocytochemistry was employed to quantify progesterone receptors (PR) in the medial preoptic (POA) and ventromedial nucleus of the hypothalamus (VMN). Plasma was assayed for luteinizing hormone (LH). Estrogen treatment induced PR immunoreactivity in both regions in brains of WT females. In ERαKO mice, lower levels of PR were induced. The stimulatory effects of E2 on PR were attenuated in the POA by cotreatment with coumestrol, and the same trend was noted in the VMN. WT ovariectomized females treated with E2 had low levels of LH, while LH was high in untreated females and even higher in ovariectomized females treated with coumestrol. ERαKO females in all treatment groups had high levels of LH. Taken together, the results show that coumestrol has anti-estrogenic actions in the brain and pituitary and that ERα mediates these effects.

Cell autonomous and non-cell autonomous functions of Otx2 in patterning the rostral brain

Development ◽  
1999 ◽  
Vol 126 (19) ◽  
pp. 4295-4304 ◽  
Author(s):  
M. Rhinn ◽  
A. Dierich ◽  
M. Le Meur ◽  
S. Ang
Keyword(s):  
Cell Adhesion ◽  
Target Genes ◽  
Homeobox Gene ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Candidate Target ◽  
Important Regulator ◽  
Mutant Cells ◽  
The Brain ◽  
Brain Patterning

Previous studies have shown that the homeobox gene Otx2 is required first in the visceral endoderm for induction of forebrain and midbrain, and subsequently in the neurectoderm for its regional specification. Here, we demonstrate that Otx2 functions both cell autonomously and non-cell autonomously in neurectoderm cells of the forebrain and midbrain to regulate expression of region-specific homeobox and cell adhesion genes. Using chimeras containing both Otx2 mutant and wild-type cells in the brain, we observe a reduction or loss of expression of Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2 in mutant cells, whereas expression of En2 and Six3 is rescued by surrounding wild-type cells. Forebrain Otx2 mutant cells subsequently undergo apoptosis. Altogether, this study demonstrates that Otx2 is an important regulator of brain patterning and morphogenesis, through its regulation of candidate target genes such as Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2.

scholarly journals Congenital Hyperinsulinism and Hypopituitarism Attributable to a Mutation in FOXA2

2018 ◽  
Vol 103 (3) ◽  
pp. 1042-1047 ◽  
Author(s):  
Mary Ellen Vajravelu ◽  
Jinghua Chai ◽  
Bryan Krock ◽  
Samuel Baker ◽  
David Langdon ◽  
...  
Keyword(s):  
Target Genes ◽  
Cell Function ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Congenital Hyperinsulinism ◽  
Wild Type ◽  
Β Cells ◽  
Pituitary Development ◽  
Forkhead Box ◽  
Β Cell Function

Abstract Context Persistent hypoglycemia in the newborn period most commonly occurs as a result of hyperinsulinism. The phenotype of hypoketotic hypoglycemia can also result from pituitary hormone deficiencies, including growth hormone and adrenocorticotropic hormone deficiency. Forkhead box A2 (Foxa2) is a transcription factor shown in mouse models to influence insulin secretion by pancreatic β cells. In addition, Foxa2 is involved in regulation of pituitary development, and deletions of FOXA2 have been linked to panhypopituitarism. Objective To describe an infant with congenital hyperinsulinism and hypopituitarism as a result of a mutation in FOXA2 and to determine the functional impact of the identified mutation. Main Outcome Measure Difference in wild-type (WT) vs mutant Foxa2 transactivation of target genes that are critical for β cell function (ABCC8, KNCJ11, HADH) and pituitary development (GLI2, NKX2-2, SHH). Results Transactivation by mutant Foxa2 of all genes studied was substantially decreased compared with WT. Conclusions We report a mutation in FOXA2 leading to congenital hyperinsulinism and hypopituitarism and provide functional evidence of the molecular mechanism responsible for this phenotype.

scholarly journals CREB Promotes Beta Cell Gene Expression by Targeting Its Coactivators to Tissue-Specific Enhancers

2019 ◽  
Vol 39 (17) ◽  
Author(s):  
Sam Van de Velde ◽  
Ezra Wiater ◽  
Melissa Tran ◽  
Yousang Hwang ◽  
Philip A. Cole ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Rna Polymerase Ii ◽  
Target Genes ◽  
Cell Function ◽  
Pancreatic Beta Cell ◽  
Specific Gene ◽  
Distal Enhancer ◽  
Determining Factors ◽  
Cell Gene Expression

ABSTRACT CREB mediates effects of cyclic AMP on cellular gene expression. Ubiquitous CREB target genes are induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell-specific genes by targeting CBP/p300 to promoter-distal enhancer regions. Subsequent increases in histone acetylation facilitate recruitment of the coactivators CRTC2 and BRD4, leading to release of RNA polymerase II over the target gene body. Indeed, CREB-induced hyperacetylation of chromatin over superenhancers promoted beta cell-restricted gene expression, which is sensitive to inhibitors of CBP/p300 and BRD4 activity. Neurod1 appears critical in establishing nucleosome-free regions for recruitment of CREB to beta cell-specific enhancers. Deletion of a CREB-Neurod1-bound enhancer within the Lrrc10b-Syt7 superenhancer disrupted the expression of both genes and decreased beta cell function. Our results demonstrate how cross talk between signal-dependent and lineage-determining factors promotes the expression of cell-type-specific gene programs in response to extracellular cues.

scholarly journals Agonist-independent Gαz activity negatively regulates β-cell compensation in a diet-induced obesity model of type 2 diabetes

2020 ◽  
pp. jbc.RA120.015585
Author(s):  
Michael D. Schaid ◽  
Cara L Green ◽  
Darby C. Peter ◽  
Shannon J Gallagher ◽  
Erin Guthery ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cell ◽  
Pancreatic Beta Cells ◽  
Cell Function ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Beta Cell Proliferation ◽  
Wild Type ◽  
Β Cell ◽  
Full Body

The inhibitory G protein alpha subunit, Gαz, is an important modulator of beta-cell function. Full-body Gαz-null mice are protected from hyperglycemia and glucose intolerance after long-term high-fat diet (HFD) feeding. In this study, at a time point in the feeding regimen where wild-type mice are only mildly glucose intolerant, transcriptomics analyses reveal islets from HFD-fed Gαz KO mice have a dramatically altered gene expression pattern as compared to WT HFD-fed mice, with entire gene pathways not only being more strongly up- or down-regulated vs. control-diet fed groups, but actually reversed in direction. Genes involved in the “Pancreatic Secretion” pathway are the most strongly differentially regulated: a finding that correlates with enhanced islet insulin secretion and decreased glucagon secretion at study end. The protection of Gαz-null mice from HFD-induced diabetes is β-cell autonomous, as β-cell-specific Gαz-null (βKO) mice phenocopy the full-body knockouts. The glucose-stimulated and incretin-potentiated insulin secretion response of islets from HFD-fed βKO mice is significantly improved as compared to islets from HFD-fed wild-type controls, which, along with no impact of Gαz loss or HFD feeding on beta-cell proliferation or surrogates of beta-cell mass supports a secretion-specific mechanism. Gαz is coupled to the Prostaglandin EP3 receptor in pancreatic beta-cells. We confirm the EP3γ splice variant has both constitutive and agonist-sensitive activity to inhibit cyclic AMP production and downstream β-cell function, with both activities being dependent on the presence of beta-cell Gαz.

Sign in / Sign up

Export Citation Format

Share Document