Three-dimensional analysis of β-cell proliferation by a novel mouse model

Pancreatic β-cell proliferation has been gaining much attention as a therapeutic target for prevention and treatment of diabetes. In order to evaluate potential β-cell mitogens, accurate and reliable methods for detection and quantification of the β-cell proliferation rate are indispensable. In this study, we developed a novel tool that specifically labels replicating β cells as mVenus+ cells by using RIP-Cre;R26Fucci2aR mice expressing the fluorescent ubiquitination-based cell cycle indicator Fucci2a in β cells. In response to β-cell proliferation stimuli such as insulin receptor antagonist S961 and diet-induced obesity (DIO), the number of EdU+ insulin+ cells per insulin+ cells and the number of mVenus+ cells per <a>mCherry+ mVenus- cells + mCherry- mVenus+ cells</a> were similarly increased in these mice. Three-dimensional imaging of optically cleared pancreas tissue from these mice enabled quantification of replicating β cells in the islets and morphometric analysis of the islets following known mitogenic interventions such as S961, DIO, pregnancy and partial pancreatectomy. Thus, this novel mouse line is a powerful tool for spatiotemporal analysis and quantification of β-cell proliferation in response to mitogenic stimulation.

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Generation and characterization of a novel mouse model that allows spatiotemporal quantification of pancreatic β-cell proliferation

10.2337/figshare.12753533 ◽

2020 ◽

Author(s):

Ada Admin ◽

Shinsuke Tokumoto ◽

Daisuke Yabe ◽

Hisato Tatsuoka ◽

Ryota Usui ◽

...

Keyword(s):

Cell Proliferation ◽

Three Dimensional ◽

Spatiotemporal Analysis ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Partial Pancreatectomy ◽

Diet Induced Obesity ◽

Treatment Of Diabetes

Pancreatic β-cell proliferation has been gaining much attention as a therapeutic target for prevention and treatment of diabetes. In order to evaluate potential β-cell mitogens, accurate and reliable methods for detection and quantification of the β-cell proliferation rate are indispensable. In this study, we developed a novel tool that specifically labels replicating β cells as mVenus+ cells by using RIP-Cre;R26Fucci2aR mice expressing the fluorescent ubiquitination-based cell cycle indicator Fucci2a in β cells. In response to β-cell proliferation stimuli such as insulin receptor antagonist S961 and diet-induced obesity (DIO), the number of EdU+ insulin+ cells per insulin+ cells and the number of mVenus+ cells per <a>mCherry+ mVenus- cells + mCherry- mVenus+ cells</a> were similarly increased in these mice. Three-dimensional imaging of optically cleared pancreas tissue from these mice enabled quantification of replicating β cells in the islets and morphometric analysis of the islets following known mitogenic interventions such as S961, DIO, pregnancy and partial pancreatectomy. Thus, this novel mouse line is a powerful tool for spatiotemporal analysis and quantification of β-cell proliferation in response to mitogenic stimulation.

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A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

Molecular and Cellular Biology ◽

10.1128/mcb.00103-16 ◽

2016 ◽

Vol 36 (23) ◽

pp. 2918-2930 ◽

Cited By ~ 10

Author(s):

Heather L. Hayes ◽

Lu Zhang ◽

Thomas C. Becker ◽

Jonathan M. Haldeman ◽

Samuel B. Stephens ◽

...

Keyword(s):

Cell Proliferation ◽

Islet Cell ◽

Transforming Growth Factor ◽

Cell Function ◽

Transforming Growth Factor Β ◽

Human Islet ◽

Soluble Factor ◽

Β Cells ◽

Β Cell ◽

Insulin Promoter

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation.

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The protein tyrosine phosphatase-BL, modulates pancreatic β-cell proliferation by interaction with the Wnt signalling pathway

Journal of Endocrinology ◽

10.1677/joe-07-0262 ◽

2008 ◽

Vol 197 (3) ◽

pp. 543-552 ◽

Cited By ~ 16

Author(s):

Hannah J Welters ◽

Alina Oknianska ◽

Kai S Erdmann ◽

Gerhart U Ryffel ◽

Noel G Morgan

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Wnt Signalling ◽

Signalling Pathway ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Wnt Signalling Pathway

In pancreatic β-cells, increased expression of the MODY5 gene product, HNF1β, leads to enhanced rates of apoptosis and altered regulation of the cell cycle, suggesting that control of HNF1β expression may be important for the control of β-cell proliferation and viability. It is unclear how these effects of HNF1β are mediated, but previously we have identified a protein tyrosine phosphatase, (PTP)-BL, as an HNF1β-regulated protein in β-cells and have now studied the role of this protein in INS-1 β-cells. Stably transfected cells were generated, which express either wild-type (WT) or a phosphatase-deficient mutant (PTP-BL-CS) of PTP-BL conditionally under the control of a tetracycline-regulated promoter. Enhanced expression of WT PTP-BL inhibited INS-1 cell growth dose dependently, but this effect was not observed when PTP-BL-CS was expressed. Neither construct altered the rate of apoptosis. PTP-BL has been reported to interact with components of the Wnt signalling pathway, and we observed that addition of exogenous Wnt3a resulted in an increase in cell proliferation and a rise in β-catenin levels, consistent with the operation of this pathway in INS-1 cells. Up-regulation of WT PTP-BL antagonised these responses but PTP-BL-CS failed to inhibit Wnt3a-induced proliferation. The rise in β-catenin caused by Wnt3a was also suppressed by over-expression of HNF1β, suggesting that HNF1β may interact with the Wnt signalling pathway via an increase in PTP-BL levels. We conclude that PTP-BL plays an important role in the regulation of cell cycle progression in pancreatic β-cells, and that it interacts functionally with components of the Wnt signalling pathway.

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cMyc Is a Principal Upstream Driver of β-Cell Proliferation in Rat Insulinoma Cell Lines and Is an Effective Mediator of Human β-Cell Replication

Molecular Endocrinology ◽

10.1210/me.2011-1074 ◽

2011 ◽

Vol 25 (10) ◽

pp. 1760-1772 ◽

Cited By ~ 32

Author(s):

Esra Karslioglu ◽

Jeffrey W. Kleinberger ◽

Fatimah G. Salim ◽

Amy E. Cox ◽

Karen K. Takane ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Death ◽

Cell Lines ◽

Cell Cycle Control ◽

Diabetes Research ◽

Cell Replication ◽

Β Cells ◽

Β Cell ◽

Normal Rat

Adult human β-cells replicate slowly. Also, despite the abundance of rodent β-cell lines, there are no human β-cell lines for diabetes research or therapy. Prior studies in four commonly studied rodent β-cell lines revealed that all four lines displayed an unusual, but strongly reproducible, cell cycle signature: an increase in seven G1/S molecules, i.e. cyclins A, D3, and E, and cdk1, -2, -4, and -6. Here, we explore the upstream mechanism(s) that drive these cell cycle changes. Using biochemical, pharmacological and molecular approaches, we surveyed potential upstream mitogenic signaling pathways in Ins 1 and RIN cells. We used both underexpression and overexpression to assess effects on rat and human β-cell proliferation, survival and cell cycle control. Our results indicate that cMyc is: 1) uniquely up-regulated among other candidates; 2) principally responsible for the increase in the seven G1/S molecules; and, 3) largely responsible for proliferation in rat β-cell lines. Importantly, cMyc expression in β-cell lines, although some 5- to 7-fold higher than normal rat β-cells, is far below the levels (75- to 150-fold) previously associated with β-cell death and dedifferentiation. Notably, modest overexpression of cMyc is able to drive proliferation without cell death in normal rat and human β-cells. We conclude that cMyc is an important driver of replication in the two most commonly employed rat β-cell lines. These studies reverse the current paradigm in which cMyc overexpression is inevitably associated with β-cell death and dedifferentiation. The cMyc pathway provides potential approaches, targets, and tools for driving and sustaining human β-cell replication.

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Involvement of the STAT5-cyclin D/CDK4-pRb pathway in β-cell proliferation stimulated by prolactin during pregnancy

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00242.2018 ◽

2019 ◽

Vol 316 (1) ◽

pp. E135-E144 ◽

Cited By ~ 3

Author(s):

Xin Zhao ◽

Yili Xu ◽

Ya Wu ◽

Hui Zhang ◽

Houxia Shi ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Cycle Progression ◽

Cell Mass ◽

Cyclin D ◽

Β Cell ◽

Cycle Progression ◽

Rb Phosphorylation ◽

Insulinoma Cells

During pregnancy, maternal pancreatic β-cells undergo a compensatory expansion in response to the state of insulin resistance, where prolactin (PRL) plays a major role. Retinoblastoma protein (Rb) has been shown to critically regulate islet proliferation and function. The aim of the study was to explore the role of Rb in β-cell mass expansion during pregnancy. Expression of pocket protein family and E2Fs were examined in mouse islets during pregnancy and in insulinoma cells (INS-1) stimulated by PRL. PRL-stimulated INS-1 cells were used to explore the signaling pathway that regulates Rb downstream of the PRL receptor. Pancreas-specific Rb-knockout (Rb-KO) mice were assessed to evaluate the in vivo function of Rb in β-cell proliferation during pregnancy. During pregnancy, expression of Rb, phospho-Rb (p-Rb), p107, and E2F1 increased, while p130 decreased in maternal islets. With PRL stimulation, induction of Rb expression occurred mainly in the nucleus, while p-Rb was predominantly in the cytoplasm. Inhibition of STAT5 significantly restrained the expression of CDK4, Rb, p-Rb, and E2F1 in PRL-stimulated INS-1 cells with attenuation in cell cycle progression. Reduction of Rb phosphorylation by CDK4 inhibition blocked PRL-mediated proliferation of INS-1 cells. On the other hand, knockdown of Rb using siRNA led to an induction in E2F1 leading to cell cycle progression from G1 to S and G2/M phase, similar to the effects of PRL-mediated induction of p-Rb that led to cell proliferation. With Rb knockdown, PRL did not lead to further increase in cell cycle progression. Similarly, while Rb-KO pregnant mice displayed better glucose tolerance and higher insulin secretion, they had similar β-cell mass and proliferation to wild-type pregnant controls, supporting the essential role of Rb suppression in augmenting β-cell proliferation during pregnancy. Rb-E2F1 regulation plays a pivotal role in PRL-stimulated β-cell proliferation. PRL promotes Rb phosphorylation and E2F1 upregulation via STAT5-cyclin D/CDK4 pathway during pregnancy.

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Development of a reliable automated screening system to identify small molecules and biologics that promote human β-cell regeneration

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00515.2015 ◽

2016 ◽

Vol 311 (5) ◽

pp. E859-E868 ◽

Cited By ~ 16

Author(s):

Kristie I. Aamodt ◽

Radhika Aramandla ◽

Judy J. Brown ◽

Nathalie Fiaschi-Taesch ◽

Peng Wang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cyclin D3 ◽

Cell Counting ◽

Cell Cycle Regulators ◽

Β Cells ◽

Β Cell ◽

Ki 67 ◽

Proliferation Markers

Numerous compounds stimulate rodent β-cell proliferation; however, translating these findings to human β-cells remains a challenge. To examine human β-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human β-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled β-cells with high specificity using both nuclear and cytoplasmic markers. β-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1β signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ β-cells, whereas treatment with other compounds had limited to no effect on human β-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human β-cell proliferation, thus allowing for increased testing of candidate human β-cell mitogens.

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SNAPIN Regulates Cell Cycle Progression to Promote Pancreatic β Cell Growth

Frontiers in Endocrinology ◽

10.3389/fendo.2021.624309 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mengxue Jiang ◽

Zhijian Kuang ◽

Yaohui He ◽

Yin Cao ◽

Tingyan Yu ◽

...

Keyword(s):

Diabetes Mellitus ◽

Cell Cycle ◽

Cell Proliferation ◽

Insulin Secretion ◽

Cell Cycle Progression ◽

Cell Number ◽

S Phase ◽

Mrna Levels ◽

Β Cell ◽

Cycle Regulation

In diabetes mellitus, death of β cell in the pancreas occurs throughout the development of the disease, with loss of insulin production. The maintenance of β cell number is essential to maintaining normoglycemia. SNAPIN has been found to regulate insulin secretion, but whether it induces β cell proliferation remains to be elucidated. This study aimed to explore the physiological roles of SNAPIN in β cell proliferation. SNAPIN expression increases with the age of mice and SNAPIN is down-regulated in diabetes. KEGG pathway and GO analysis showed that SNAPIN- interacting proteins were enriched in cell cycle regulation. B cell cycle was arrested in the S phase, and cell proliferation was inhibited after SNAPIN knockdown. The expression of CDK2, CDK4 and CCND1 proteins in the S phase of the cell cycle were reduced after SNAPIN knockdown, whereas they were increased after overexpression of SNAPIN. In addition, insulin protein and mRNA levels also increased or decreased after SNAPIN knockdown or overexpression, respectively. Conclusions: Our data indicate that SNAPIN mediates β cells proliferation and insulin secretion, and provide evidences that SNAPIN might be a pharmacotherapeutic target for diabetes mellitus.

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Decreased IRS Signaling Impairs β-Cell Cycle Progression and Survival in Transgenic Mice Overexpressing S6K in β-Cells

Diabetes ◽

10.2337/db09-0851 ◽

2010 ◽

Vol 59 (10) ◽

pp. 2390-2399 ◽

Cited By ~ 43

Author(s):

Lynda Elghazi ◽

Norman Balcazar ◽

Manuel Blandino-Rosano ◽

Corentin Cras-Méneur ◽

Szabolcs Fatrai ◽

...

Keyword(s):

Cell Cycle ◽

Transgenic Mice ◽

Cell Cycle Progression ◽

Β Cells ◽

Β Cell ◽

Cycle Progression

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STAU2 protein level is controlled by caspases and the CHK1 pathway and regulates cell cycle progression in the non-transformed hTERT-RPE1 cells

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00352-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Lionel Condé ◽

Yulemi Gonzalez Quesada ◽

Florence Bonnet-Magnaval ◽

Rémy Beaujois ◽

Luc DesGroseillers

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

Dna Damage ◽

Steady State ◽

Posttranscriptional Regulation ◽

Cell Cycle Progression ◽

Rna Binding ◽

Regulation Of Gene Expression ◽

Cycle Progression

AbstractBackgroundStaufen2 (STAU2) is an RNA binding protein involved in the posttranscriptional regulation of gene expression. In neurons, STAU2 is required to maintain the balance between differentiation and proliferation of neural stem cells through asymmetric cell division. However, the importance of controlling STAU2 expression for cell cycle progression is not clear in non-neuronal dividing cells. We recently showed that STAU2 transcription is inhibited in response to DNA-damage due to E2F1 displacement from theSTAU2gene promoter. We now study the regulation of STAU2 steady-state levels in unstressed cells and its consequence for cell proliferation.ResultsCRISPR/Cas9-mediated and RNAi-dependent STAU2 depletion in the non-transformed hTERT-RPE1 cells both facilitate cell proliferation suggesting that STAU2 expression influences pathway(s) linked to cell cycle controls. Such effects are not observed in the CRISPR STAU2-KO cancer HCT116 cells nor in the STAU2-RNAi-depleted HeLa cells. Interestingly, a physiological decrease in the steady-state level of STAU2 is controlled by caspases. This effect of peptidases is counterbalanced by the activity of the CHK1 pathway suggesting that STAU2 partial degradation/stabilization fines tune cell cycle progression in unstressed cells. A large-scale proteomic analysis using STAU2/biotinylase fusion protein identifies known STAU2 interactors involved in RNA translation, localization, splicing, or decay confirming the role of STAU2 in the posttranscriptional regulation of gene expression. In addition, several proteins found in the nucleolus, including proteins of the ribosome biogenesis pathway and of the DNA damage response, are found in close proximity to STAU2. Strikingly, many of these proteins are linked to the kinase CHK1 pathway, reinforcing the link between STAU2 functions and the CHK1 pathway. Indeed, inhibition of the CHK1 pathway for 4 h dissociates STAU2 from proteins involved in translation and RNA metabolism.ConclusionsThese results indicate that STAU2 is involved in pathway(s) that control(s) cell proliferation, likely via mechanisms of posttranscriptional regulation, ribonucleoprotein complex assembly, genome integrity and/or checkpoint controls. The mechanism by which STAU2 regulates cell growth likely involves caspases and the kinase CHK1 pathway.

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