An Assay for Small Scale Screening of Candidate β Cell Proliferative Factors Using Intact Islets

Mosser, Rockann E.; Gannon, Maureen

doi:10.2144/000114115

Cited by 8 publications

(12 citation statements)

References 12 publications

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“…To investigate this, β-cell proliferation was measured in young (8–10 weeks old) WT mouse islets treated ex vivo with the EP3 agonist sulprostone, the EP3 antagonist DG-041, the EP4 agonist CAY10598, or the EP4 antagonist L-161,982 using a proliferation assay developed by our laboratory [38]. After four days of treatment, PL increased β-cell proliferation 3–4-fold over vehicle-treated islets (Figure 2 A - B ).…”

Section: Resultsmentioning

confidence: 99%

“…Ex vivo mouse β-cell proliferation was performed as previously described [38] , [39] . Briefly, 40 mouse islets per replicate were cultured for four days in mouse proliferation media in the presence of 0.1 mM EGTA, which mildly loosens cell–cell contacts to enhance nutrient and compound accessibility to the core of the islet [38] and one or more of the following compounds: vehicle (phosphate buffered saline (PBS)), 0.5 μg/mL recombinant human placental lactogen (PL; Harbor-UCLA Research and Education Institute), 30 nM sulprostone (Cayman Chemical), 30 nM DG-041 (Vanderbilt Institute of Chemical Biology Synthesis Core [40] ), 10 nM CAY10598 (Cayman Chemical), 100 nM L-161,982 (Cayman Chemical), 1 μM U-73122 (Sigma), or 30 nM rapamycin (Calbiochem). All compounds were diluted in PBS to obtain desired concentrations.…”

Section: Methodsmentioning

confidence: 99%

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Opposing effects of prostaglandin E 2 receptors EP3 and EP4 on mouse and human β-cell survival and proliferation

Carboneau

Allan

Townsend

et al. 2017

Molecular Metabolism

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ObjectiveHyperglycemia and systemic inflammation, hallmarks of Type 2 Diabetes (T2D), can induce the production of the inflammatory signaling molecule Prostaglandin E2 (PGE2) in islets. The effects of PGE2 are mediated by its four receptors, E-Prostanoid Receptors 1-4 (EP1-4). EP3 and EP4 play opposing roles in many cell types due to signaling through different G proteins, Gi and GS, respectively. We previously found that EP3 and EP4 expression are reciprocally regulated by activation of the FoxM1 transcription factor, which promotes β-cell proliferation and survival. Our goal was to determine if EP3 and EP4 regulate β-cell proliferation and survival and, if so, to elucidate the downstream signaling mechanisms.Methodsβ-cell proliferation was assessed in mouse and human islets ex vivo treated with selective agonists and antagonists for EP3 (sulprostone and DG-041, respectively) and EP4 (CAY10598 and L-161,982, respectively). β-cell survival was measured in mouse and human islets treated with the EP3- and EP4-selective ligands in conjunction with a cytokine cocktail to induce cell death. Changes in gene expression and protein phosphorylation were analyzed in response to modulation of EP3 and EP4 activity in mouse islets.ResultsBlockade of EP3 enhanced β-cell proliferation in young, but not old, mouse islets in part through phospholipase C (PLC)-γ1 activity. Blocking EP3 also increased human β-cell proliferation. EP4 modulation had no effect on ex vivo proliferation alone. However, blockade of EP3 in combination with activation of EP4 enhanced human, but not mouse, β-cell proliferation. In both mouse and human islets, EP3 blockade or EP4 activation enhanced β-cell survival in the presence of cytokines. EP4 acts in a protein kinase A (PKA)-dependent manner to increase mouse β-cell survival. In addition, the positive effects of FoxM1 activation on β-cell survival are inhibited by EP3 and dependent on EP4 signaling.ConclusionsOur results identify EP3 and EP4 as novel regulators of β-cell proliferation and survival in mouse and human islets ex vivo.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Opposing effects of prostaglandin E 2 receptors EP3 and EP4 on mouse and human β-cell survival and proliferation

Carboneau

Allan

Townsend

et al. 2017

Molecular Metabolism

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“…First, a blinded, small-scale ex vivo assay was performed on intact islets isolated from WT mice as previously described47. Compounds were initially screened at multiple concentrations (0.1–5 mM) to assess dose-response.…”

Section: Methodsmentioning

confidence: 99%

Loss of Free Fatty Acid Receptor 2 leads to impaired islet mass and beta cell survival

Villa

Priyadarshini

Fuller

et al. 2016

Sci Rep

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The regulation of pancreatic β cell mass is a critical factor to help maintain normoglycemia during insulin resistance. Nutrient-sensing G protein-coupled receptors (GPCR) contribute to aspects of β cell function, including regulation of β cell mass. Nutrients such as free fatty acids (FFAs) contribute to precise regulation of β cell mass by signaling through cognate GPCRs, and considerable evidence suggests that circulating FFAs promote β cell expansion by direct and indirect mechanisms. Free Fatty Acid Receptor 2 (FFA2) is a β cell-expressed GPCR that is activated by short chain fatty acids, particularly acetate. Recent studies of FFA2 suggest that it may act as a regulator of β cell function. Here, we set out to explore what role FFA2 may play in regulation of β cell mass. Interestingly, Ffar2−/− mice exhibit diminished β cell mass at birth and throughout adulthood, and increased β cell death at adolescent time points, suggesting a role for FFA2 in establishment and maintenance of β cell mass. Additionally, activation of FFA2 with Gαq/11-biased agonists substantially increased β cell proliferation in in vitro and ex vivo proliferation assays. Collectively, these data suggest that FFA2 may be a novel therapeutic target to stimulate β cell growth and proliferation.

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“…Islets were treated with vehicle or recombinant human CTGF ( 22 ). β-Cell proliferation was assayed as in Mosser and Gannon ( 23 ), with a minimum of 2,500 cells quantified per animal.…”

Section: Methodsmentioning

confidence: 99%

Connective Tissue Growth Factor Modulates Adult β-Cell Maturity and Proliferation to Promote β-Cell Regeneration in Mice

et al. 2014

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Stimulation of endogenous β-cell expansion could facilitate regeneration in patients with diabetes. In mice, connective tissue growth factor (CTGF) is expressed in embryonic β-cells and in adult β-cells during periods of expansion. We discovered that in embryos CTGF is necessary for β-cell proliferation, and increased CTGF in β-cells promotes proliferation of immature (MafA−) insulin-positive cells. CTGF overexpression, under nonstimulatory conditions, does not increase adult β-cell proliferation. In this study, we tested the ability of CTGF to promote β-cell proliferation and regeneration after partial β-cell destruction. β-Cell mass reaches 50% recovery after 4 weeks of CTGF treatment, primarily via increased β-cell proliferation, which is enhanced as early as 2 days of treatment. CTGF treatment increases the number of immature β-cells but promotes proliferation of both mature and immature β-cells. A shortened β-cell replication refractory period is also observed. CTGF treatment upregulates positive cell-cycle regulators and factors involved in β-cell proliferation, including hepatocyte growth factor, serotonin synthesis, and integrin β1. Ex vivo treatment of whole islets with recombinant human CTGF induces β-cell replication and gene expression changes consistent with those observed in vivo, demonstrating that CTGF acts directly on islets to promote β-cell replication. Thus, CTGF can induce replication of adult mouse β-cells given a permissive microenvironment.

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An Assay for Small Scale Screening of Candidate β Cell Proliferative Factors Using Intact Islets

Cited by 8 publications

References 12 publications

Opposing effects of prostaglandin E 2 receptors EP3 and EP4 on mouse and human β-cell survival and proliferation

Opposing effects of prostaglandin E 2 receptors EP3 and EP4 on mouse and human β-cell survival and proliferation

Loss of Free Fatty Acid Receptor 2 leads to impaired islet mass and beta cell survival

Connective Tissue Growth Factor Modulates Adult β-Cell Maturity and Proliferation to Promote β-Cell Regeneration in Mice

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