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.
Highlights
Type 2 diabetes is an escalating epidemic involving gene-e nvironment interactions that culminate in β-cell failure and insulin resistance
We further confirmed impaired insulin secretion by reduced bioluminescence in Bmal1-/- compared to WT β-cell lines expressing insulin-N anoLuc in response to stimulatory concentrations of glucose (20 mM), potassium chloride, forskolin, and the phosphodiesterase inhibitor 3-isobutyl-1 -methylxanthine (IBMX) (Figure 1D)
We have identified an unexpected role for the P2Y1 receptor as a BMAL1-c ontrolled insulinotropic factor required for enhanced β-cell glucose-s timulated Ca2+ influx and insulin secretion in response to IVM
Summary
Type 2 diabetes is an escalating epidemic involving gene-e nvironment interactions that culminate in β-cell failure and insulin resistance. Recent chemical screens have identified new factors that modulate the core clock, including casein kinase 1 inhibitors that lengthen the circadian period through stabilizing PER proteins (Hirota et al, 2010; Chen et al, 2012), and a separate series of cryptochrome stabilizer compounds have been discovered that control glucose homeostasis in vivo (Hirota et al, 2012). We reasoned that compounds that enhance insulin secretion in the setting of β-cell clock disruption might in turn uncover therapeutic targets for more common forms of diabetes mellitus (Marcheva et al, 2010; Perelis et al, 2015; Marcheva et al, 2020; Moffat et al, 2017). We further identified the P2Y1 receptor as a direct transcriptional target of the molecular clock factor BMAL1 and a potent regulator of glucose-d ependent calcium signaling. Our findings establish a chemical genetic strategy to identify novel endocrine cell therapeutics
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