scholarly journals The Tetracycline-Controlled Transactivator (Tet-On/Off) System in Beta Cells Reduces Insulin Expression and Secretion in Mice

2021 ◽  
Author(s):  
Nathalie Jouvet ◽  
Khalil Bouyakdan ◽  
Scott A. Campbell ◽  
Cindy Baldwin ◽  
Shannon E. Townsend ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cell ◽  
Beta Cells ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Ectopic Expression ◽  
Whole Body ◽  
Specific Gene

Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in beta cells, transgene silencing, low inducibility, ectopic expression and off-targets effects are persistent challenges. In this study, we investigated whether an inducible, Tet-Off system with beta-cell specific MIP-itTA driven expression of TetO-Cre<sup>Jaw/J </sup>could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination; beta cell architecture; <i>in vitro</i> and <i>in vivo</i> glucose-stimulated insulin secretion; and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g. itTA, rtTA or tTA) in beta cells significantly reduced <i>Insulin</i> gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with beta cells and highlights the need for consistent and comprehensive controls when using the Tet-On or Tet-Off systems for genome editing.

scholarly journals The Tetracycline-Controlled Transactivator (Tet-On/Off) System in Beta Cells Reduces Insulin Expression and Secretion in Mice

2021 ◽  
Author(s):  
Nathalie Jouvet ◽  
Khalil Bouyakdan ◽  
Scott A. Campbell ◽  
Cindy Baldwin ◽  
Shannon E. Townsend ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cell ◽  
Beta Cells ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Ectopic Expression ◽  
Whole Body ◽  
Specific Gene

Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in beta cells, transgene silencing, low inducibility, ectopic expression and off-targets effects are persistent challenges. In this study, we investigated whether an inducible, Tet-Off system with beta-cell specific MIP-itTA driven expression of TetO-Cre<sup>Jaw/J </sup>could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination; beta cell architecture; <i>in vitro</i> and <i>in vivo</i> glucose-stimulated insulin secretion; and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g. itTA, rtTA or tTA) in beta cells significantly reduced <i>Insulin</i> gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with beta cells and highlights the need for consistent and comprehensive controls when using the Tet-On or Tet-Off systems for genome editing.

scholarly journals Expression of a tetracycline-controlled transactivator (Tet-On/Off system) in beta cells reduces insulin expression and secretion in mice

2021 ◽  
Author(s):  
Nathalie Jouvet ◽  
Khalil Bouyakdan ◽  
Cindy Baldwin ◽  
Jadwiga Marcinkiewicz ◽  
Thierry Alquier ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cell ◽  
Beta Cells ◽  
Glucose Homeostasis ◽  
Cell Biology ◽  
Cell Function ◽  
Genetic Manipulation ◽  
Ectopic Expression ◽  
Whole Body ◽  
Specific Gene

ABSTRACTControllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However, in beta cells, transgene silencing, low inducibility, ectopic expression and off-targets effects on cell function and glucose homeostasis are a persistent challenge. In this study, we investigated whether an inducible, Tet-Off system with beta-cell specific MIP-itTA driven expression of TetO-CreJaw/J could circumvent previous issues of specificity, efficacy and toxicity. Following assessment of tissue-specific gene recombination; beta cell architecture; in vitro and in vivo glucose-stimulated insulin secretion (GSIS); and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g. itTA, rtTA or tTA) in beta cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying a MIP-itTA transgene, independent of Cre-recombinase expression or doxycycline treatment. These results raise significant concern regarding the use of Tet-On or Tet-Off systems for genome editing in beta cells and emphasize the need to control for effects of transactivator expression. Our study echoes ongoing challenges faced by fundamental researchers focused on beta cells and highlights the need for consistent and careful control of experiments using these research tools.

scholarly journals Pancreatic beta cell selective deletion of mitofusins 1 and 2 (Mfn1 and Mfn2) disrupts mitochondrial architecture and abrogates glucose-stimulated insulin secretion in vivo

2020 ◽  
Author(s):  
Eleni Georgiadou ◽  
Charanya Muralidharan ◽  
Michelle Martinez ◽  
Pauline Chabosseau ◽  
Alejandra Tomas ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Membrane Potential ◽  
Beta Cell ◽  
Beta Cells ◽  
Glucose Sensing ◽  
Mitochondrial Fusion ◽  
Intravital Imaging ◽  
Mouse Islets

AbstractBackground and aimsMitochondria are highly dynamic organelles, fundamental to cellular energy homeostasis. Mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cells. Whether mitochondrial ultra-structure, and the proteins controlling fission and fusion, are important for glucose recognition are unclear. Mitochondrial fusion is supported by proteins including mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy (OPA1), and fission by dynamin-related protein 1 (DRP1). Here, we generated mice with beta cell-selective, adult-restricted deletion of Mfn1 and Mfn2 (βMfn1/2-KO), and explored the impact on insulin secretion and glucose homeostasis in vivo and in vitro.Materials and methodsC57BL/6J mice bearing Mfn1 and Mfn2 alleles with loxP sites, were crossed to animals carrying an inducible Cre recombinase at the Pdx1 locus (PdxCreERT). Isolated islets were used for live beta cell fluorescence imaging of cytosolic (Cal-520) or mitochondrial (Pericam) free Ca2+ concentration and membrane potential (TMRE). Mitochondrial network characteristics were quantified using super resolution fluorescence and transmission electron microscopy. Beta cell-beta cell connectivity was assessed using the Pearson (R) analysis and Monte Carlo simulation in intact mouse islets. Intravital imaging was performed in mice injected with an adeno-associated virus to express the cytosolic Ca2+ sensor GCaMP6s selectively in beta cells and TMRM to visualise mitochondria using multiphoton microscopy.ResultsβMfn1/2-KO mice displayed higher fasting glycaemia than control littermates at 14 weeks (8.6 vs 6.4 mmol/L, p>0.05) and a >five-fold decrease in plasma insulin post-intraperitoneal glucose injection (5-15 min, p<0.0001). Mitochondrial length, and glucose-induced Ca2+ accumulation, mitochondrial hyperpolarisation and beta cell connectivity were all significantly reduced in βMfn1/2-KO mouse islets. Examined by intravital imaging of the exteriorised pancreas, antiparallel changes in cytosolic Ca2+ and mitochondrial membrane potential, observed in control animals in vivo, were suppressed after Mfn1/2 deletion.ConclusionMitochondrial fusion and fission cycles are essential in the beta cell to maintain normal mitochondrial bioenergetics and glucose sensing both in vitro and in the living mouse. Such cycles may be disrupted in some forms of diabetes to impair mitochondrial function and, consequently, insulin secretion.

scholarly journals Functional cytochrome P450 1A enzymes are induced in mouse and human islets following pollutant exposure

Diabetologia ◽  
2019 ◽  
Vol 63 (1) ◽  
pp. 162-178 ◽  
Author(s):  
Muna Ibrahim ◽  
Erin M. MacFarlane ◽  
Geronimo Matteo ◽  
Myriam P. Hoyeck ◽  
Kayleigh R. C. Rick ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Insulin Secretion ◽  
Beta Cell ◽  
Human Islets ◽  
High Dose ◽  
Single High Dose ◽  
Direct Exposure ◽  
Mouse Islets

Abstract Aims/hypothesis Exposure to environmental pollution has been consistently linked to diabetes incidence in humans, but the potential causative mechanisms remain unclear. Given the critical role of regulated insulin secretion in maintaining glucose homeostasis, environmental chemicals that reach the endocrine pancreas and cause beta cell injury are of particular concern. We propose that cytochrome P450 (CYP) enzymes, which are involved in metabolising xenobiotics, could serve as a useful biomarker for direct exposure of islets to pollutants. Moreover, functional CYP enzymes in islets could also impact beta cell physiology. The aim of this study was to determine whether CYP1A enzymes are activated in islets following direct or systemic exposure to environmental pollutants. Methods Immortalised liver (HepG2) and rodent pancreatic endocrine cell lines (MIN6, βTC-6, INS1, α-TC1, α-TC3), as well as human islets, were treated in vitro with known CYP1A inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3-MC). In addition, mice were injected with either a single high dose of TCDD or multiple low doses of TCDD in vivo, and islets were isolated 1, 7 or 14 days later. Results CYP1A enzymes were not activated in any of the immortalised beta or alpha cell lines tested. However, both 3-MC and TCDD potently induced CYP1A1 gene expression and modestly increased CYP1A1 enzyme activity in human islets after 48 h. The induction of CYP1A1 in human islets by TCDD was prevented by cotreatment with a cytokine mixture. After a systemic single high-dose TCDD injection, CYP1A1 enzyme activity was induced in mouse islets ~2-fold, ~40-fold and ~80-fold compared with controls after 1, 7 and 14 days, respectively, in vivo. Multiple low-dose TCDD exposure in vivo also caused significant upregulation of Cyp1a1 in mouse islets. Direct TCDD exposure to human and mouse islets in vitro resulted in suppressed glucose-induced insulin secretion. A single high-dose TCDD injection resulted in lower plasma insulin levels, as well as a pronounced increase in beta cell death. Conclusions/interpretation Transient exposure to TCDD results in long-term upregulation of CYP1A1 enzyme activity in islets. This provides evidence for direct exposure of islets to lipophilic pollutants in vivo and may have implications for islet physiology.

scholarly journals miR-11 regulates pupal size of Drosophila melanogaster via directly targeting Ras85D

2017 ◽  
Vol 312 (1) ◽  
pp. C71-C82 ◽  
Author(s):  
Yao Li ◽  
Shengjie Li ◽  
Ping Jin ◽  
Liming Chen ◽  
Fei Ma
Keyword(s):  
Drosophila Melanogaster ◽  
Untranslated Region ◽  
Deletion Mutant ◽  
Ectopic Expression ◽  
Whole Body ◽  
Physiological Processes ◽  
Insulin Producing Cells ◽  
Pupal Size

MicroRNAs play diverse roles in various physiological processes during Drosophila development. In the present study, we reported that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with the following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3′-untranslated region in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, our current work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis.

Comparative toxicity of alloxan, N-alkylalloxans and ninhydrin to isolated pancreatic islets in vitro

1997 ◽  
Vol 155 (2) ◽  
pp. 283-293 ◽  
Author(s):  
A Jorns ◽  
R Munday ◽  
M Tiedge ◽  
S Lenzen
Keyword(s):  
Beta Cell ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Ultrastructural Changes ◽  
Cell Necrosis ◽  
High Concentrations ◽  
Beta Cell Necrosis

The in vitro toxicity of the diabetogenic agent alloxan as documented by the induction of beta cell necrosis was studied in isolated ob/ob mouse pancreatic islets. The effect of alloxan has been compared with that of a number of N-alkyl alloxan derivatives and with that of the structurally related compound, ninhydrin. Alloxan and its derivatives were selectively toxic to pancreatic beta cells, with other endocrine cells and exocrine parenchymal cells being well preserved, even at high concentration. In contrast, ninhydrin was selectively toxic to pancreatic beta cells only at comparatively low concentration, destroying all islet cell types at high concentrations. The ultrastructural changes induced by all the test compounds in pancreatic beta cells in vitro were very similar to those observed during the development of alloxan diabetes in vivo. The relative toxicity of the various compounds to pancreatic beta cells in vitro was not, however, related to their ability to cause diabetes in vivo. Indeed, the non-diabetogenic substances ninhydrin, N-butylalloxan and N-isobutylalloxan were very much more toxic to isolated islets than the diabetogenic compounds alloxan and N-methylalloxan. These results suggest that the differences in diabetogenicity among alloxan derivatives are not due to intrinsic differences in the susceptibility of the pancreatic beta cells to their toxicity, but may reflect differences in distribution or metabolism. High concentrations of glucose protected islets against the harmful effects of alloxan and its derivatives, but not those of ninhydrin. Low levels of glucose, and non-carbohydrate nutrients, afforded little protection, indicating that the effect of glucose is not due to the production of reducing equivalents within the cell, 3-O-Methylglucose, which protects against alloan diabetes in vivo, did not protect against alloxan toxicity in vitro. Since 3-O-methylglucose is known to prevent uptake of alloxan by pancreatic beta cells, it appears that uptake of alloxan by the cell is not a prerequisite for the induction of beta cell necrosis.

scholarly journals Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

2020 ◽  
Author(s):  
Giuliano Giuseppe Stirparo ◽  
Agata Kurowski ◽  
Stanley Eugene Strawbridge ◽  
Hannah Stuart ◽  
Thorsten Edwin Boroviak ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Specific Gene ◽  
List Type ◽  
Early Mouse

AbstractOCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism.Highlights and noveltyMajor pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markersJAK/STAT signalling is defective in OCT4 null embryosOCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identityOCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency

scholarly journals ECM-enriched alginate hydrogels for bioartificial pancreas: an ideal niche to improve insulin secretion and diabetic glucose profile

2019 ◽  
Vol 17 (4) ◽  
pp. 228080001984892 ◽  
Author(s):  
Joana Crisóstomo ◽  
Ana M Pereira ◽  
Sílvia J Bidarra ◽  
Ana C Gonçalves ◽  
Pedro L Granja ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Diabetic Patients ◽  
Bioartificial Pancreas ◽  
Multicellular Spheroids ◽  
Rgd Peptides ◽  
Alginate Hydrogels ◽  
And Function

Introduction: The success of a bioartificial pancreas crucially depends on ameliorating encapsulated beta cells survival and function. By mimicking the cellular in vivo niche, the aim of this study was to develop a novel model for beta cells encapsulation capable of establishing an appropriate microenvironment that supports interactions between cells and extracellular matrix (ECM) components. Methods: ECM components (Arg-Gly-Asp, abbreviated as RGD) were chemically incorporated in alginate hydrogels (alginate-RGD). After encapsulation, INS-1E beta cells outcome was analyzed in vitro and after their implantation in an animal model of diabetes. Results: Our alginate-RGD model demonstrated to be a good in vitro niche for supporting beta cells viability, proliferation, and activity, namely by improving the key feature of insulin secretion. RGD peptides promoted cell–matrix interactions, enhanced endogenous ECM components expression, and favored the assembly of individual cells into multicellular spheroids, an essential configuration for proper beta cell functioning. In vivo, our pivotal model for diabetes treatment exhibited an improved glycemic profile of type 2 diabetic rats, where insulin secreted from encapsulated cells was more efficiently used. Conclusions: We were able to successfully introduce a novel valuable function in an old ally in biomedical applications, the alginate. The proposed alginate-RGD model stands out as a promising approach to improve beta cells survival and function, increasing the success of this therapeutic strategy, which might greatly improve the quality of life of an increasing number of diabetic patients worldwide.

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