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High-content screening identifies a role for Na(+) channels in insulin production.

ABSTRACT: Insulin production is the central feature of functionally mature and differentiated pancreatic ?-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote ?-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 ?-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of ?-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect ?-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and ?-cell differentiation status. In particular, our unbiased screen identified a novel role for a ?-cell sodium channel gene in insulin production.

SUBMITTER: Szabat M

PROVIDER: S-EPMC4807443 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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High-content screening identifies a role for Na(+) channels in insulin production.

Szabat Marta M Modi Honey H Ramracheya Reshma R Girbinger Vroni V Chan Forson F Lee Jason T C JT Piske Micah M Kamal Sepehr S Carol Yang Yu Hsuan YH Welling Andrea A Rorsman Patrik P Johnson James D JD

Royal Society open science 20151202 12

Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven ...[more]

PMID: 27019722

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Project description:Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO)--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism-focused investigations within zebrafish and mammalian models.

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High-content screening identifies a role for Na(+) channels in insulin production.

Publications

High-content screening identifies a role for Na(+) channels in insulin production.

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