Glucose Regulates Cyclin D2 Expression in Quiescent and Replicating Pancreatic β-Cells Through Glycolysis and Calcium Channels

Understanding the molecular triggers of pancreatic β-cell proliferation may facilitate the development of regenerative therapies for diabetes. Genetic studies have demonstrated an important role for cyclin D2 in β-cell proliferation and mass homeostasis, but its specific function in β-cell division and mechanism of regulation remain unclear. Here, we report that cyclin D2 is present at high levels in the nucleus of quiescent β-cells in vivo. The major regulator of cyclin D2 expression is glucose, acting via glycolysis and calcium channels in the β-cell to control cyclin D2 mRNA levels. Furthermore, cyclin D2 mRNA is down-regulated during S-G2-M phases of each β-cell division, via a mechanism that is also affected by glucose metabolism. Thus, glucose metabolism maintains high levels of nuclear cyclin D2 in quiescent β-cells and modulates the down-regulation of cyclin D2 in replicating β-cells. These data challenge the standard model for regulation of cyclin D2 during the cell division cycle and suggest cyclin D2 as a molecular link between glucose levels and β-cell replication.

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NF-κB activity during pancreas development regulates adult β-cell mass by modulating neonatal β-cell proliferation and apoptosis

Cell Death Discovery ◽

10.1038/s41420-020-00386-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Dror Sever ◽

Anat Hershko-Moshe ◽

Rohit Srivastava ◽

Roy Eldor ◽

Daniel Hibsher ◽

...

Keyword(s):

Cell Proliferation ◽

Developmental Stages ◽

Cell Mass ◽

Diabetes Incidence ◽

Β Cells ◽

Β Cell ◽

Cell Dysfunction ◽

Regulatory Circuit ◽

Proliferation And Apoptosis

AbstractNF-κB is a well-characterized transcription factor, widely known for its roles in inflammation and immune responses, as well as in control of cell division and apoptosis. However, its function in β-cells is still being debated, as it appears to depend on the timing and kinetics of its activation. To elucidate the temporal role of NF-κB in vivo, we have generated two transgenic mouse models, the ToIβ and NOD/ToIβ mice, in which NF-κB activation is specifically and conditionally inhibited in β-cells. In this study, we present a novel function of the canonical NF-κB pathway during murine islet β-cell development. Interestingly, inhibiting the NF-κB pathway in β-cells during embryogenesis, but not after birth, in both ToIβ and NOD/ToIβ mice, increased β-cell turnover, ultimately resulting in a reduced β-cell mass. On the NOD background, this was associated with a marked increase in insulitis and diabetes incidence. While a robust nuclear immunoreactivity of the NF-κB p65-subunit was found in neonatal β-cells, significant activation was not detected in β-cells of either adult NOD/ToIβ mice or in the pancreata of recently diagnosed adult T1D patients. Moreover, in NOD/ToIβ mice, inhibiting NF-κB post-weaning had no effect on the development of diabetes or β-cell dysfunction. In conclusion, our data point to NF-κB as an important component of the physiological regulatory circuit that controls the balance of β-cell proliferation and apoptosis in the early developmental stages of insulin-producing cells, thus modulating β-cell mass and the development of diabetes in the mouse model of T1D.

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Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00040.2013 ◽

2013 ◽

Vol 305 (1) ◽

pp. E149-E159 ◽

Cited By ~ 76

Author(s):

Rachel E. Stamateris ◽

Rohit B. Sharma ◽

Douglas A. Hollern ◽

Laura C. Alonso

Keyword(s):

Cell Proliferation ◽

Cell Mass ◽

Extended Period ◽

Time Frame ◽

High Fat ◽

Β Cells ◽

Β Cell ◽

Cyclin D2 ◽

Mass Expansion

Type 2 diabetes (T2D) is caused by relative insulin deficiency, due in part to reduced β-cell mass ( 11 , 62 ). Therapies aimed at expanding β-cell mass may be useful to treat T2D ( 14 ). Although feeding rodents a high-fat diet (HFD) for an extended period (3–6 mo) increases β-cell mass by inducing β-cell proliferation ( 16 , 20 , 53 , 54 ), evidence suggests that adult human β-cells may not meaningfully proliferate in response to obesity. The timing and identity of the earliest initiators of the rodent compensatory growth response, possible therapeutic targets to drive proliferation in refractory human β-cells, are not known. To develop a model to identify early drivers of β-cell proliferation, we studied mice during the first week of HFD exposure, determining the onset of proliferation in the context of diet-related physiological changes. Within the first week of HFD, mice consumed more kilocalories, gained weight and fat mass, and developed hyperglycemia, hyperinsulinemia, and glucose intolerance due to impaired insulin secretion. The β-cell proliferative response also began within the first week of HFD feeding. Intriguingly, β-cell proliferation increased before insulin resistance was detected. Cyclin D2 protein expression was increased in islets by day 7, suggesting it may be an early effector driving compensatory β-cell proliferation in mice. This study defines the time frame and physiology to identify novel upstream regulatory signals driving mouse β-cell mass expansion, in order to explore their efficacy, or reasons for inefficacy, in initiating human β-cell proliferation.

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Impact of Small-Molecule Glucokinase Activator on Glucose Metabolism and β-Cell Mass

Endocrinology ◽

10.1210/en.2008-1183 ◽

2008 ◽

Vol 150 (3) ◽

pp. 1147-1154 ◽

Cited By ~ 50

Author(s):

Akinobu Nakamura ◽

Yasuo Terauchi ◽

Sumika Ohyama ◽

Junko Kubota ◽

Hiroko Shimazaki ◽

...

Keyword(s):

Cell Proliferation ◽

Glucose Metabolism ◽

Cell Mass ◽

Isolated Islets ◽

Brdu Incorporation ◽

Wild Type ◽

Β Cell ◽

Glucokinase Activator ◽

Insulinoma Cells

We investigated the effect of glucokinase activator (GKA) on glucose metabolism and β-cell mass. We analyzed four mouse groups: wild-type mice and β-cell-specific haploinsufficiency of glucokinase gene (Gck+/−) mice on a high-fat (HF) diet. Each genotype was also treated with GKA mixed in the HF diet. Rodent insulinoma cells and isolated islets were used to evaluate β-cell proliferation by GKA. After 20 wk on the above diets, there were no differences in body weight, lipid profiles, and liver triglyceride content among the four groups. Glucose tolerance was improved shortly after the GKA treatment in both genotypes of mice. β-Cell mass increased in wild-type mice compared with Gck+/− mice, but a further increase was not observed after the administration of GKA in both genotypes. Interestingly, GKA was able to up-regulate insulin receptor substrate-2 (Irs-2) expression in insulinoma cells and isolated islets. The administration of GKA increased 5-bromo-2-deoxyuridine (BrdU) incorporation in insulinoma cells, and 3 d administration of GKA markedly increased BrdU incorporation in mice treated with GKA in both genotypes, compared with those without GKA. In conclusion, GKA was able to chronically improve glucose metabolism for mice on the HF diet. Although chronic GKA administration failed to cause a further increase in β-cell mass in vivo, GKA was able to increase beta cell proliferation in vitro and with a 3-d administration in vivo. This apparent discrepancy can be explained by a chronic reduction in ambient blood glucose levels by GKA treatment. Glucokinase activator is able to improve glucose metabolism and has an effect on β cell proliferation.

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Neonatal growth and regeneration of β-cells are regulated by the Wnt/β-catenin signaling in normal and diabetic rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00538.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. E245-E256 ◽

Cited By ~ 34

Author(s):

Florence Figeac ◽

Benjamin Uzan ◽

Monique Faro ◽

Noura Chelali ◽

Bernard Portha ◽

...

Keyword(s):

Cell Proliferation ◽

Wnt Pathway ◽

Diabetic Rats ◽

Β Cells ◽

Β Cell ◽

Ductal Cells ◽

Gsk 3Β ◽

Canonical Wnt

Wnt/β-catenin signaling is critical for a variety of fundamental cellular processes. Here, we investigated the implication of the Wnt/β-catenin signaling in the in vivo regulation of β-cell growth and regeneration in normal and diabetic rats. To this aim, TCF7L2, the distal effector of the canonical Wnt pathway, was knocked down in groups of normal and diabetic rats by the use of specific antisense morpholino-oligonucleotides. In other groups of diabetic rats, the Wnt/β-catenin pathway was activated by the inhibition of its negative regulator GSK-3β. GSK-3β was inactivated by either LiCl or anti-GSK-3β oligonucleotides. The β-cell mass was evaluated by morphometry. β-cell proliferation was assessed in vivo and in vitro by BrdU incorporation method. In vivo β-cell neogenesis was estimated by the evaluation of PDX1-positive ductal cells and GLUT2-positive ductal cells and the number of β cells budding from the ducts. We showed that the in vivo disruption of the canonical Wnt pathway resulted in the alteration of normal and compensatory growth of β-cells mainly through the inhibition of β-cell proliferation. Conversely, activation of the Wnt pathway through the inhibition of GSK-3β had a significant stimulatory effect on β-cell regeneration in diabetic rats. In vitro, GSK-3β inactivation resulted in the stimulation of β-cell proliferation. This was mediated by the stabilization of β-catenin and the induction of cyclin D. Taken together, our results demonstrate the involvement of the canonical Wnt signaling in the neonatal regulation of normal and regenerative growth of pancreatic β-cells. Moreover, we provide evidence that activation of this pathway by pharmacological maneuvers can efficiently improve β-cell regeneration in diabetic rats. These findings might have potential clinical applications in the regenerative therapy of diabetes.

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The small molecule kobusone can stimulate islet β-cell replication in vivo

Journal of International Medical Research ◽

10.1177/03000605211032849 ◽

2021 ◽

Vol 49 (7) ◽

pp. 030006052110328

Author(s):

Jin woo Choi ◽

Jin-deok Joo ◽

Jang hyeok In ◽

Daewoo Kim ◽

Yongshin Kim ◽

...

Keyword(s):

Cell Proliferation ◽

Body Weight ◽

Serum Insulin ◽

Cyclin D3 ◽

Control Group ◽

Cell Replication ◽

Β Cell ◽

Insulin Levels ◽

Glucose Levels

Objective To investigate the ability of kobusone to reduce high glucose levels and promote β-cell proliferation. Methods Four-week-old female db/db mice were assigned to the kobusone (25 mg/kg body weight, intraperitoneally twice a day) or control group (same volume of PBS). Glucose levels and body weight were measured twice a week. After 6 weeks, intraperitoneal glucose tolerance tests and immunohistochemical studies were performed, and insulin levels were determined. The expression of mRNAs involved in cell proliferation, such as PI3K, Akt, cyclin D3 and p57Kip 2 , was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results Kobusone reduced blood glucose levels after 3 weeks and more strongly increased serum insulin levels than the vehicle. Immunohistochemistry illustrated that kobusone increased 5-bromo-2′-deoxyuridine incorporation into islet β-cells, suggesting that it can stimulate islet β-cell replication in vivo. RT-qPCR indicated that kobusone upregulated the mRNA expression of PI3K, Akt, and cyclin D3 and downregulated that of p57Kip2. Conclusion Our findings suggest that kobusone is a potent pancreatic islet β-cell inducer that has the potential to be developed as an anti-diabetic agent.

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Crocins from Crocus sativus L. in the Management of Hyperglycemia. In Vivo Evidence from Zebrafish

Molecules ◽

10.3390/molecules25225223 ◽

2020 ◽

Vol 25 (22) ◽

pp. 5223

Author(s):

Eleni Kakouri ◽

Adamantia Agalou ◽

Charalabos Kanakis ◽

Dimitris Beis ◽

Petros A. Tarantilis

Keyword(s):

Glucose Metabolism ◽

Zebrafish Embryo ◽

Crocus Sativus ◽

Β Cells ◽

High Blood Glucose ◽

Glucose Levels ◽

Blood Glucose Levels ◽

Level Reduction ◽

Crocus Sativus L

Diabetes mellitus is a disease characterized by persistent high blood glucose levels and accompanied by impaired metabolic pathways. In this study, we used zebrafish to investigate the effect of crocins isolated from Crocus sativus L., on the control of glucose levels and pancreatic β-cells. Embryos were exposed to an aqueous solution of crocins and whole embryo glucose levels were measured at 48 h post-treatment. We showed that the application of crocins reduces zebrafish embryo glucose levels and enhances insulin expression. We also examined whether crocins are implicated in the metabolic pathway of gluconeogenesis. We showed that following a single application of crocins and glucose level reduction, the expression of phosphoenolpyruvate carboxykinase1 (pck1), a key gene involved in glucose metabolism, is increased. We propose a putative role for the crocins in glucose metabolism and insulin management.

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Loss of Foxd3 Results in Decreased β-Cell Proliferation and Glucose Intolerance During Pregnancy

Endocrinology ◽

10.1210/en.2010-1462 ◽

2011 ◽

Vol 152 (12) ◽

pp. 4589-4600 ◽

Cited By ~ 22

Author(s):

Jennifer L. Plank ◽

Audrey Y. Frist ◽

Alison W. LeGrone ◽

Mark A. Magnuson ◽

Patricia A. Labosky

Keyword(s):

Cell Proliferation ◽

Cell Mass ◽

Diabetic Patients ◽

Β Cells ◽

Β Cell ◽

Forkhead Box ◽

Physiological Demands ◽

Mass Expansion ◽

Specific Deletion

A complete molecular understanding of β-cell mass expansion will be useful for the improvement of therapies to treat diabetic patients. During normal periods of metabolic challenges, such as pregnancy, β-cells proliferate, or self-renew, to meet the new physiological demands. The transcription factor Forkhead box D3 (Foxd3) is required for maintenance and self-renewal of several diverse progenitor cell lineages, and Foxd3 is expressed in the pancreatic primordium beginning at 10.5 d postcoitum, becoming localized predominantly to β-cells after birth. Here, we show that mice carrying a pancreas-specific deletion of Foxd3 have impaired glucose tolerance, decreased β-cell mass, decreased β-cell proliferation, and decreased β-cell size during pregnancy. In addition, several genes known to regulate proliferation, Foxm1, Skp2, Ezh2, Akt2, and Cdkn1a, are misregulated in islets isolated from these Foxd3 mutant mice. Together, these data place Foxd3 upstream of several pathways critical for β-cell mass expansion in vivo.

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Procedure for Seeding Cells on the Disque Platform v1

10.17504/protocols.io.bvi3n4gn ◽

2021 ◽

Author(s):

Peter Anthony Jones* ◽

Kisuk Yang* ◽

Jeffrey M Karp

Keyword(s):

Cell Proliferation ◽

3D Culture ◽

Zinc Content ◽

Fold Increase ◽

Cell Loss ◽

High Concentration ◽

Β Cells ◽

Β Cell ◽

Culture Systems

Advances in treating β cell loss include islet replacement therapies or increasing cell proliferation rate in type 1 and type 2 diabetes. We previously developed a proliferation-inducing prodrug (ZnPD6) that targets the high concentration of zinc ions in β cells, and which exhibits a 2.4-fold increase in β cell proliferation compared to the DYRK1A inhibitor harmine. These prodrugs were identified through screening on the Disque Platform (DP)—a high-fidelity culture system where stem cell–derived β cells are reaggregated into thin, 3D discs within 2D 96-well plates that mimic in vivo conditions. The Disque Platform allows for the formation of 3D micro-tissues within an automation-friendly design, and is capable of systematically manipulating the cell niche in order to identify chemical and physical cues that enhance β cell proliferation. The Disque Platform better replicates the zinc content of native islets, enabling for the screening of zinc-activated prodrugs whose activity cannot be detected in 2D culture systems, which typically display a markedly lowered zinc content. The Disque Platform is a reliable screening platform that bridges the advantages of 2D and 3D culture systems and responds to interventions when conventional systems cannot produce a clear signal or readout. Here we describe a standard protocol for the formation of 3D micro-tissues in the Disque Platform.

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Role of the G-Protein Coupled Receptor 3-Salt Inducible Kinase 2 Pathway in Human β Cell Proliferation

10.1101/2021.05.31.446443 ◽

2021 ◽

Author(s):

Caterina Iorio ◽

Jillian L Rourke ◽

Lisa Wells ◽

Jun-Ichi Sakamaki ◽

Emily Moon ◽

...

Keyword(s):

Cell Proliferation ◽

G Protein ◽

Cell Mass ◽

Standard Of Care ◽

G Protein Coupled Receptor ◽

Β Cells ◽

Β Cell ◽

Cell Therapies ◽

G Protein Coupled

Loss of pancreatic β cells is the hallmark of type 1 diabetes (T1D), for which provision of insulin is the standard of care. While regenerative and stem cell therapies hold the promise of generating single-source or host-matched tissue to obviate immune-mediated complications, these will still require surgical intervention and immunosuppression. Thus, methods that harness the innate capacity of β cells to proliferate to increase β cell mass in vivo are considered vital for future T1D treatment. However, early in life β cells enter what appears to be a permanent state of quiescence, directed by an evolutionarily selected genetic program that establishes a β cell mass setpoint to guard against development of fatal endocrine tumours. Here we report the development of a high-throughput RNAi screening approach to identify upstream pathways that regulate adult human β cell quiescence and demonstrate in a screen of the GPCRome that silencing G-protein coupled receptor 3 (GPR3) leads to human pancreatic β cell proliferation. Loss of GPR3 leads to activation of Salt Inducible Kinase 2 (SIK2), which is necessary and sufficient to drive cell cycle entry, increase β cell mass, and enhance insulin secretion in mice. Taken together, targeting the GPR3-SIK2 pathway represents a novel avenue to stimulate the regeneration of β cells.

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The two major glucokinase isoforms show conserved functionality in β-cells despite different subcellular distribution

Biological Chemistry ◽

10.1515/hsz-2018-0109 ◽

2018 ◽

Vol 399 (6) ◽

pp. 565-576 ◽

Cited By ~ 3

Author(s):

Brian Lu ◽

Miguel Munoz-Gomez ◽

Yasuhiro Ikeda

Keyword(s):

Cell Proliferation ◽

Subcellular Distribution ◽

Normal Diet ◽

Β Cells ◽

Β Cell ◽

Functional Conservation ◽

Exon 1 ◽

The Impact

Abstract Glucokinase (GCK) is crucial to regulating glucose metabolism in the liver and in pancreatic β-cells. There are two major GCK isoforms, hepatic and pancreatic GCKs, which differ only in exon 1. However, the functional differences between the two GCK isoforms remain poorly understood. Here, we used a β-cell-targeted gene transfer vector to determine the impact of isoform-specific GCK overexpression on β-cells in vitro and in vivo. We showed that pancreatic GCK had a nuclear localization signal unique to the pancreatic isoform, facilitating its nuclear distribution in β-cells. Despite the difference in subcellular distribution, overexpression of GCK isoforms similarly enhanced glucose uptake and β-cell proliferation in vitro. Overexpression of hepatic or pancreatic GCK also similarly enhanced β-cell proliferation in normal diet mice without affecting fasting glucose and intraperitoneal glucose tolerance tests (IPGTT). Our further study on human GCK sequences identified disproportional GCK amino acid variants in exon 1, while mutations linked to maturity onset diabetes of the young type 2 (MODY2) were disproportionally found in exons 2 through 10. Our results therefore indicate functional conservation between the two major GCK isoforms despite their distinct subcellular distribution.

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