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G0-G1 Transition and the Restriction Point in Pancreatic B-Cells in Vivo

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G0-G1 Transition and the Restriction Point in Pancreatic B-Cells in Vivo 578 Diabetes Volume 63, February 2014 Ayat Hija,1 Seth Salpeter,1 Agnes Klochendler,1 Joseph Grimsby,2 Michael Brandeis,3 Benjamin Glaser,4 and Yuval Dor1 G0-G1 Transition and the Restriction Point in Pancreatic b-Cells In Vivo Most of our knowledge on cell kinetics stems from in levels. Insufficient mass of b-cells is a central factor in vitro studies of continuously dividing cells. In this human diabetes, and the identification of methods to study, we determine in vivo cell-cycle parameters of expand b-cellmassisaprimechallengeforregenerative pancreatic b-cells, a largely quiescent population, biology. Similarly to most differentiated cell types, using drugs that mimic or prevent glucose-induced b-cells in the adult organism are largely quiescent. replication of b-cells in mice. Quiescent b-cells However, b-cells do divide rarely, and their duplication exposed to a mitogenic glucose stimulation require is key for the maintenance of b-cell mass homeostasis 8 h to enter the G1 phase of the cell cycle, and this during healthy adult life (1–5) as well as after a di- time is prolonged in older age. The duration of G1,S, abetogenic injury (6). We have recently shown that the and G2/M is ~5, 8, and 6 h, respectively. We further b ISLET STUDIES key physiological trigger for -cell proliferation is glu- provide the first in vivo demonstration of the cose (7). Mitogenic signaling is transduced by glucoki- restriction point at the G -G transition, discovered 0 1 nase, catalyzing the first step of glycolysis, followed by by Arthur Pardee 40 years ago. The findings may have pharmacodynamic implications in the design of closure of ATP-dependent potassium channels, leading regenerative therapies aimed at increasing b-cell to membrane depolarization. Indeed, small-molecule replication and mass in patients with diabetes. glucokinase activators (GKAs), being developed to aug- Diabetes 2014;63:578–584 | DOI: 10.2337/db12-1035 ment insulin secretion in diabetes (8), double the frac- tion of replicating b-cells when administered to mice (7). Coadministration of diazoxide, a drug preventing Most of our knowledge about the mammalian cell-division membrane depolarization, cancels the mitogenic effect cycle is based on studies of cultured cells. Whether basic of GKA (7). cell-cycle concepts and quantitative parameters of the cell In this study, we use these drugs to probe b-cell ki- cycle are conserved between cells growing on plastic in netics in vivo by taking advantage of the ability to time artificial medium and cells in their natural niche is hard the administration of a direct mitogen. This enabled the to determine. One reason is the difficulty in applying and timing of the transition from quiescence to G1 phase of withdrawing specific, direct mitogens in vivo. the cell cycle, the duration of G1, S, and G2/M, as well the Insulin-producing b-cells reside in the islets of Lang- duration of continued mitogen activity that is required erhans and are essential for maintaining normal glucose for b-cells to commit to the cell cycle. 1Department of Developmental Biology and Cancer Research, Institute for Corresponding author: Yuval Dor, [email protected]. Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, Israel Received 2 August 2012 and accepted 7 October 2013. 2 Department of Metabolic Diseases, Hoffmann-La Roche, Nutley, NJ This article contains Supplementary Data online at http://diabetes 3 Department of Genetics, The Alexander Silberman Institute of Life Sciences, .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1035/-/DC1. The Hebrew University of Jerusalem, Jerusalem, Israel 4Endocrinology and Metabolism Service, Department of Internal Medicine, © 2014 by the American Diabetes Association. See http://creativecommons Hadassah-Hebrew University Medical Center, Jerusalem, Israel .org/licenses/by-nc-nd/3.0/ for details. diabetes.diabetesjournals.org Hija and Associates 579 RESEARCH DESIGN AND METHODS Statistical Analyses Mice and Drugs Statistical analyses were performed using a two-tailed t P , P , GKA was dissolved in 79% saline, 20% DMSO (Sigma- Student test. In all graphs: * 0.05, ** 0.01, P , P . fi Aldrich), 1% polysorbate–Tween 80, and injected in- *** 0.005, and 0.05 is not signi cant. Results are 6 traperitoneally at 20 or 50 mg/kg. Control mice received reported as means SE. the same volume of DMSO (20% of total volume). The injection of DMSO did not affect replication rates of RESULTS b-cells when compared with mice that were injected with Time From Mitogenic Stimulus to Cell‐Cycle Entry of saline. Diazoxide was dissolved similarly to GKA and b-Cells In Vivo injected intraperitoneally at 40 mg/kg. BrdU, 5-chloro-29- Most b-cells reside in a quiescent state in vivo. A small deoxyuridine (CldU; MD-Biomedical), and iododeoxyuridine fraction of these cells is engaged in the cell cycle under (IdU; Sigma-Aldrich) were dissolved in PBS (10 mg/mL) and baseline conditions, and an additional fraction can re- injected intraperitoneally at 10 mg/kg. spond to mitogenic stimuli and enter the cell-division We used ICR male mice aged 5 weeks or 6 months. cycle. Cycling b-cells can be identified by staining for cell- Injections of drug or vehicle were typically performed at 4 cycle markers such as Ki67 (marking all stages in the cell P.M., and the animals were killed the next morning at 9 A.M. cycle, but not G0 quiescent cells), PH3 (marking cells in For the G0-G1 experiments, mice were injected with GKA late G2 and mitosis), Cdc47 (marking cycling cells start- fi at 8 A.M. and killed at different time points. At sacri ce, ing in late G1), and the incorporation of thymidine the pancreas was fixed in formalin and embedded in analogs such as BrdU, CldU, and IdU (occurring during paraffin, and 4-mm thick sections were immunostained. S phase and maintained in the DNA permanently). By b Immunostaining and Analysis timing the administration of a -cell mitogen to adult mice, one can assess the time it takes a quiescent b-cell, Images were captured on a Nikon C1 confocal microscope residing in G , to reach the G phase of the cell cycle. (Nikon). For each mouse, .2,000 b-cells (defined as 0 1 This can be defined at the first point after injection of Insulin+Pdx1+ cells) were counted from multiple islets in amitogenwhenasignificant increase in the pro- nonadjacent sections. For each data point, we used three portion of replicating b-cells is observed. In other to five mice. Primary antibodies were: guinea pig anti- words, this will reflect the time it took competent insulin (1:200; DakoCytomation), rabbit anti-Ki67 quiescent b-cells to enter the cell cycle (assuming that (1:200; NeoMarkers), mouse anti-BrdU (Cell Pro- the G state is homogenous with regard to the dis- liferation Kit; 1:300; Amersham Biosciences), rabbit anti– 0 tance from G ). phosphorylated histone H3 (PH3) Ser10 (1:100; Cell 1 As previously shown, injection of GKA causes rapid Signaling Technology), mouse anti-Cdc47 (1:100; Thermo onset of hypoglycemia due to an increase in b-cell gly- Fisher Scientific), goat anti-Pdx1 (1:250; a generous gift colysis and insulin secretion (Fig. 1A). A significant in- from Dr. Christopher Wright, Vanderbilt University), rat crease in the fraction of b-cells engaged in the cell cycle anti-CldU (1:200; AbD Serotec), and mouse anti-IdU (expressing Ki67) occurs 8 h after injection of GKA, but (1:100; BD Biosciences). Secondary antibodies were from not earlier (Fig. 1B and C). This suggests that the tran- all from Jackson ImmunoResearch Laboratories. b ; sition of -cells from G0 to G1 in vivo takes 8h. D E RT-PCR Staining for Cdc47 (Fig. 1 and ) and quantitative RT-PCR for cyclin A2, Top2A, and Ki67 (Supplemen- Total RNA was prepared using Qiagen RNeasy microkit ’ tary Fig. 1) provided results consistent with this (Qiagen) according to the manufacturer s protocol. Total conclusion. RNA (50 ng) was used for first-strand cDNA synthesis using random primers (Roche) and reverse transcriptase Duration of G1 (ImProm-II; Promega). Quantitative real-time PCR was Following a similar reasoning, the duration of G1 can be performed with SYBR Green PCR master mix (Applied calculated from the time after injection of a mitogen Biosystems) in 96-well plates using the 7900HT in- when a significant increase in the proportion of b-cells strument (Applied Biosystems). All reactions were per- that incorporate the thymidine analog BrdU is observed. formed in triplicates. The relative amount of mRNA was This reflects the time required by competent b-cells to calculated using the comparative threshold cycle move from G0 to the DNA synthesis phase. The time of method after normalization to b-actin. The following G1 can therefore be defined as (time from G0 to S) 2 primers were used: b-actin, 59-CACAGCTTCTTTGCAG (time from G0 to G1). Mice were administered with GKA CTCCT-39 and 59-GTCATCCATGGCGAACTGG-39;Ki67, and killed at different time points after pulsing with 59-TTGACCGCTCCTTTAGGTATGAA-39 and 59-TTCCAAG BrdU for 2 h. Thirteen hours (but not 10 h) after in- GGACTTTCCTGGA-39;Top2A,59-AGCAGATTAGCTTC jection of GKA, the proportion of b-cells incorporating GTCAACAGC-39 and 59-ACATGTCTGCCGCCCTTAGA-39; the thymidine analog BrdU is increased. This suggests b ; and CcnA2, 59-CAAGACTCGACGGGTTGCTC-39 and that the duration of G1 in -cells in vivo is 5 h (13 h 59-GAAGGACCAGCAGTGACATGC-39. from G0 to S 2 8 h from G0 to G1) (Fig. 2A). 580 Restriction Point in b-Cells In Vivo Diabetes Volume 63, February 2014 Figure 1—Using GKA to define the time from quiescence to G1 in pancreatic b-cells in vivo.
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