P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian β-cell failure
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Evaluation Summary:
Circadian disruption is widespread in our modern 24/7 society, leading to an increased prevalence of common diseases including type 2 diabetes. The authors conducted an unbiased screen for small-molecule compounds that can restore the attenuated insulin secretion from pancreatic beta cells caused by a disrupted circadian clock. They identified ivermectin and its clock-controlled target, the P2Y1 receptor, which regulate glucose-stimulated Ca2+ influx and insulin secretion in beta cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. Here, we developed a genetically sensitized small-molecule screen to identify druggable proteins and mechanistic pathways involved in circadian β-cell failure. Our approach was to generate β-cells expressing a nanoluciferase reporter within the proinsulin polypeptide to screen 2640 pharmacologically active compounds and identify insulinotropic molecules that bypass the secretory defect in CRISPR-Cas9-targeted clock mutant β-cells. We validated hit compounds in primary mouse islets and identified known modulators of ligand-gated ion channels and G-protein-coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets revealed ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacological analyses established the P2Y1 receptor as a clock-controlled mediator of the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a diabetes target based upon a genetically sensitized phenotypic screen.
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Evaluation Summary:
Circadian disruption is widespread in our modern 24/7 society, leading to an increased prevalence of common diseases including type 2 diabetes. The authors conducted an unbiased screen for small-molecule compounds that can restore the attenuated insulin secretion from pancreatic beta cells caused by a disrupted circadian clock. They identified ivermectin and its clock-controlled target, the P2Y1 receptor, which regulate glucose-stimulated Ca2+ influx and insulin secretion in beta cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The study by Dr. Marcheva and colleagues aims at identification of the pathways and specific proteins involved in regulation of circadian metabolic disruption. With the idea of discovering novel therapeutic targets for beta cell failure in diabetic context, the authors launch high-throughput screening of 2640 pharmacologically active compounds employing beta cell line that is lacking function Bmal1 protein and is thus arrhythmic (clonal line of Bmal1 deficient Beta-TC-6). Insulin NanoLuciferase has been introduced to the cells via lentiviral transduction and served a readout for insulin secretion capacity. The authors validate functionality of this elegant system by correlating bioluminescence signals emanating from insulin driven NanoLuc and actual levels of insulin secretion, at basal conditions and in …
Reviewer #1 (Public Review):
The study by Dr. Marcheva and colleagues aims at identification of the pathways and specific proteins involved in regulation of circadian metabolic disruption. With the idea of discovering novel therapeutic targets for beta cell failure in diabetic context, the authors launch high-throughput screening of 2640 pharmacologically active compounds employing beta cell line that is lacking function Bmal1 protein and is thus arrhythmic (clonal line of Bmal1 deficient Beta-TC-6). Insulin NanoLuciferase has been introduced to the cells via lentiviral transduction and served a readout for insulin secretion capacity. The authors validate functionality of this elegant system by correlating bioluminescence signals emanating from insulin driven NanoLuc and actual levels of insulin secretion, at basal conditions and in response to high glucose concentration. Insulin-stimulating capacity of each of 2640 screened small molecules has been evaluated in these clock-deficient Beta-TC cells. The authors identified 19 initial hits that strongly increased secretion of insulin in this system. Majority of these initial hits targeted ion channels or GPCRs. Most of thus identified small molecules were not further considered due to either high dosage of the compound that was required to obtain the effect, hepatotoxicity, or effects on insulin secretion in the presence of basal levels of glucose when tested on WT mouse islet cells. Among these primary hits, the authors thus focused on ivermectin (IVM), by validating its insulin secretion stimulatory effects on mouse islets isolated from arrhythmic Bmal1KO mice. Furthermore, the authors reported that IVM conferred improved glucose tolerance in vivo in Akita mice, and enhanced insulin secretion in response to glucose by the islets isolated from these mice. Importantly, the authors provide mechanistic insights into the effect of IVM on insulin exocytosis by demonstrating that it modulates glucose stimulated flux of Ca and increases capacitance in Bmal1KO primary beta cells, as well as in human beta cells. Moreover, the authors conducted extensive search for the factors that promote peptide exocytosis, hypothesizing that those might be efficient in rescuing insulin secretion perturbed upon circadian disruption. Employing RNAseq, ChIP analyses and additional approaches, the authors identified purinergic receptor P2Y1 as a target of IVM, that was also rhythmically expressed. Indeed, by blocking P2Y1 signaling pharmacologically, the effects of IVM on insulin secretion were abrogated. In line with this finding, beta cells lacking P2Y1 failed to respond to IVM application. To summarize, based on the large-scale drug screening, the authors identify novel function of the P2Y1 receptor that is driven by Bmal1, and that regulates glucose stimulated Ca influx and insulin exocytosis in mouse and human beta cells in response to IVM. This discovery represents an important advance in our understanding of regulatory machinery of insulin secretion by cell-autonomous clocks operative in beta cell in mice and in humans, and it is of fundamental clinical relevance in context of novel therapeutic targets for diabetes management.
Overall, the study is very well designed and carefully controlled, the authors used state-of-the art approaches, and the work has an important translational potential. The manuscript is very structured, and it is written in a clear and concise manner.
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Reviewer #2 (Public Review):
In this study Marcheva, Weidemann, Taguchi et al. aimed to identify pharmacologically-active compounds that augment insulin secretion in a model of ß cell failure related to circadian disruption (i.e. in pancreatic ß cells and islets that lack the circadian core clock gene Bmal1). To this end they performed an unbiased high-throughput screen, including 2,640 small molecule compounds, in Bmal1 knockout (KO) ß cells expressing an insulin-Nanoluciferase reporter gene. Bioluminescence intensity following simultaneous treatment with glucose and pharmacological compounds was used to quantify changes in insulin secretion and identify those compounds that rescue insulin secretion from ß cells upon Bmal1 KO. They were able to narrow down a single compound hit by additional rounds of validation in ß cells, mouse and …
Reviewer #2 (Public Review):
In this study Marcheva, Weidemann, Taguchi et al. aimed to identify pharmacologically-active compounds that augment insulin secretion in a model of ß cell failure related to circadian disruption (i.e. in pancreatic ß cells and islets that lack the circadian core clock gene Bmal1). To this end they performed an unbiased high-throughput screen, including 2,640 small molecule compounds, in Bmal1 knockout (KO) ß cells expressing an insulin-Nanoluciferase reporter gene. Bioluminescence intensity following simultaneous treatment with glucose and pharmacological compounds was used to quantify changes in insulin secretion and identify those compounds that rescue insulin secretion from ß cells upon Bmal1 KO. They were able to narrow down a single compound hit by additional rounds of validation in ß cells, mouse and human islets and finally uncovered a potential mode of action by which ivermectin positively regulates insulin secretion. They suggest that the purinergic P2Y1 receptor is regulated by the circadian clock (via BMAL1 dependent transcription) and mediates ivermectin's insulinotropic activity. These data add an interesting way to the ongoing connection between circadian disruption and the development of type 2 diabetes based on ß cell failure resulting from perturbed circadian oscillation in these cells.
Overall, the authors present a scientifically sound study. In some cases the conclusions are supported by the data, in other cases hypothesis and experimental approaches need to be clarified and extended and some additional controls need to be provided.
Strengths:
The authors developed an insulin-nanoluciferase reporter system that allows to monitor changes in insulin secretion in a sensitive, cost-effective, and high-throughput manner. This system can be useful to study ß cell function in different context than circadian disruption or pharmacological compound screens. They use a variety of different methods (e.g. bioluminescence and calcium imaging, membrane capacitance measurement, RNA-Seq, in vivo ivermectin treatment and glucose tolerance test) and tools (e.g. Bmal1 KO ß cells, P2ry1 KO ß cells, mouse and human wild type islets, and pancreatic Bmal1 KO mouse islets) to characterize the mode of action of the pharmacological compound they identified. In many parts of the manuscript, the conceptualization and methodology is logical and comprehensible.Weaknesses:
While the authors initially set out to identify pharmacological compounds that are able to enhance insulin secretion in a context of circadian disruption (Bmal1 KO ß cells recapitulate secretory defects observed in primary clock-deficient islets), they later also focus on the activity of ivermectin in wild type ß cells and islets to show that this compound enhances insulin secretion. Thus, the question whether the compound hit and mode of action they identified are specific to "circadian ß cell failure" remains. Moreover, in 2015, Burns et al. (Cell Metabolism) published a similar study using an insulin-gaussia luciferase reporter system to measure changes in insulin secretion in a model of ß cell failure by a 1,600-pilot compound screen. Why their nanoluciferase system is be advantageous over the previously published gaussia luciferase reporter system needs to be clarified by the authors. Lastly, even though Bmal1 KO is a commonly used model for circadian clock disruption, BMAL1 transcription factor may also exhibit biological functions independent of its role as circadian clock component. It would interesting to see if other models of circadian ß cell failure (e.g. Clock∆19 or Cry1,2 dKO) recapitulate the effects of ivermectin treatment. -
