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Repurposing cAMP-modulating medications to promote β-cell replication.

ABSTRACT: Loss of β-cell mass is a cardinal feature of diabetes. Consequently, developing medications to promote β-cell regeneration is a priority. cAMP is an intracellular second messenger that modulates β-cell replication. We investigated whether medications that increase cAMP stability or synthesis selectively stimulate β-cell growth. To identify cAMP-stabilizing medications that promote β-cell replication, we performed high-content screening of a phosphodiesterase (PDE) inhibitor library. PDE3, -4, and -10 inhibitors, including dipyridamole, were found to promote β-cell replication in an adenosine receptor-dependent manner. Dipyridamole's action is specific for β-cells and not α-cells. Next we demonstrated that norepinephrine (NE), a physiologic suppressor of cAMP synthesis in β-cells, impairs β-cell replication via activation of α(2)-adrenergic receptors. Accordingly, mirtazapine, an α(2)-adrenergic receptor antagonist and antidepressant, prevents NE-dependent suppression of β-cell replication. Interestingly, NE's growth-suppressive effect is modulated by endogenously expressed catecholamine-inactivating enzymes (catechol-O-methyltransferase and l-monoamine oxidase) and is dominant over the growth-promoting effects of PDE inhibitors. Treatment with dipyridamole and/or mirtazapine promote β-cell replication in mice, and treatment with dipyridamole is associated with reduced glucose levels in humans. This work provides new mechanistic insights into cAMP-dependent growth regulation of β-cells and highlights the potential of commonly prescribed medications to influence β-cell growth.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC4179632 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Repurposing cAMP-modulating medications to promote β-cell replication.

Zhao Zhenshan Z Low Yen S YS Armstrong Neali A NA Ryu Jennifer Hyoje JH Sun Sara A SA Arvanites Anthony C AC Hollister-Lock Jennifer J Shah Nigam H NH Weir Gordon C GC Annes Justin P JP

Molecular endocrinology (Baltimore, Md.) 20140801 10

Loss of β-cell mass is a cardinal feature of diabetes. Consequently, developing medications to promote β-cell regeneration is a priority. cAMP is an intracellular second messenger that modulates β-cell replication. We investigated whether medications that increase cAMP stability or synthesis selectively stimulate β-cell growth. To identify cAMP-stabilizing medications that promote β-cell replication, we performed high-content screening of a phosphodiesterase (PDE) inhibitor library. PDE3, -4, an ...[more]

PMID: 25083741

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Project description:ObjectivePancreatic islets are nutrient sensors that regulate organismal blood glucose homeostasis. Glucagon release from the pancreatic α-cell is important under fasted, fed, and hypoglycemic conditions, yet metabolic regulation of α-cells remains poorly understood. Here, we identified a previously unexplored role for physiological levels of leucine, which is classically regarded as a β-cell fuel, in the intrinsic regulation of α-cell glucagon release.MethodsGcgCreERT:CAMPER and GcgCreERT:GCaMP6s mice were generated to perform dynamic, high-throughput functional measurements of α-cell cAMP and Ca2+ within the intact islet. Islet perifusion assays were used for simultaneous, time-resolved measurements of glucagon and insulin release from mouse and human islets. The effects of leucine were compared with glucose and the mitochondrial fuels 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH, non-metabolized leucine analog that activates glutamate dehydrogenase), α-ketoisocaproate (KIC, leucine metabolite), and methyl-succinate (complex II fuel). CYN154806 (Sstr2 antagonist), diazoxide (KATP activator, which prevents Ca2+-dependent exocytosis from α, β, and δ-cells), and dispersed α-cells were used to inhibit islet paracrine signaling and identify α-cell intrinsic effects.ResultsMimicking the effect of glucose, leucine strongly suppressed amino acid-stimulated glucagon secretion. Mechanistically, leucine dose-dependently reduced α-cell cAMP at physiological concentrations, with an IC50 of 57, 440, and 1162 μM at 2, 6, and 10 mM glucose, without affecting α-cell Ca2+. Leucine also reduced α-cell cAMP in islets treated with Sstr2 antagonist or diazoxide, as well as dispersed α-cells, indicating an α-cell intrinsic effect. The effect of leucine was matched by KIC and the glutamate dehydrogenase activator BCH, but not methyl-succinate, indicating a dependence on mitochondrial anaplerosis. Glucose, which stimulates anaplerosis via pyruvate carboxylase, had the same suppressive effect on α-cell cAMP but with lower potency. Similarly to mouse islets, leucine suppressed glucagon secretion from human islets under hypoglycemic conditions.ConclusionsThese findings highlight an important role for physiological levels of leucine in the metabolic regulation of α-cell cAMP and glucagon secretion. Leucine functions primarily through an α-cell intrinsic effect that is dependent on glutamate dehydrogenase, in addition to the well-established α-cell regulation by β/δ-cell paracrine signaling. Our results suggest that mitochondrial anaplerosis-cataplerosis facilitates the glucagonostatic effect of both leucine and glucose, which cooperatively suppress α-cell tone by reducing cAMP.

Project description:UnlabelledRetinal pigment epithelium (RPE) cell integrity is critical to the maintenance of retinal function. Many retinopathies such as age-related macular degeneration (AMD) are caused by the degeneration or malfunction of the RPE cell layer. Replacement of diseased RPE with healthy, stem cell-derived RPE is a potential therapeutic strategy for treating AMD. Human embryonic stem cells (hESCs) differentiated into RPE progeny have the potential to provide an unlimited supply of cells for transplantation, but challenges around scalability and efficiency of the differentiation process still remain. Using hESC-derived RPE as a cellular model, we sought to understand mechanisms that could be modulated to increase RPE yield after differentiation. We show that RPE epithelialization is a density-dependent process, and cells seeded at low density fail to epithelialize. We demonstrate that activation of the cAMP pathway increases proliferation of dissociated RPE in culture, in part through inhibition of transforming growth factor-β (TGF-β) signaling. This results in enhanced uptake of epithelial identity, even in cultures seeded at low density. In line with these findings, targeted manipulation of the TGF-β pathway with small molecules produces an increase in efficiency of RPE re-epithelialization. Taken together, these data highlight mechanisms that promote epithelial fate acquisition in stem cell-derived RPE. Modulation of these pathways has the potential to favorably impact scalability and clinical translation of hESC-derived RPE as a cell therapy.SignificanceStem cell-derived retinal pigment epithelium (RPE) is currently being evaluated as a cell-replacement therapy for macular degeneration. This work shows that the process of generating RPE in vitro is regulated by the cAMP and transforming growth factor-β signaling pathway. Modulation of these pathways by small molecules, as identified by phenotypic screening, leads to an increased efficiency of generating RPE cells with a higher yield. This can have a potential impact on manufacturing transplantation-ready cells at large scale and is advantageous for clinical studies using this approach in the future.

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Repurposing cAMP-modulating medications to promote β-cell replication.

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Repurposing cAMP-modulating medications to promote β-cell replication.

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