Biology subjects

Macdonald, P. E.

Publications and source records attributed to Macdonald, P. E..

3 recordsLinked to original sources

bioRxiv2022

HNF1α transcriptional activation and repression maintain human islet α and β cell function

HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A-MODY) and hypomorphic HNF1A variants confer type 2 diabetes risk, but a lack of experimental systems has limited our understanding of how the transcription factor HNF1 regulates adult human islet function. Here, we combined human islet genetics, RNA sequencing, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) chromatin mapping, patch-clamp electrophysiology and transplantation-based assays to elucidate HNF1-regulated mechanisms in mature pancreatic and {beta} cells. shRNA-mediated suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion both in vitro and after transplantation into immunocompromised mice, recapitulating phenotypes observed in HNF1A-MODY patients. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATP-transporters and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional de-repression through HNF1. CUT&RUN mapping of HNF1 DNA-binding sites in primary human islets verified that a subset of HNF1-regulated genes were direct targets. These data provide unprecedented mechanistic links between HNF1A loss and diabetic phenotypes in mature human and {beta} cells.

genetics↗

bioRxiv2021

Ca2+ oscillations, waves, and networks in islets from human donors with and without type 2 diabetes

Pancreatic islets are highly interconnected structures that produce pulses of insulin and other hormones, maintaining normal homeostasis of glucose and other nutrients. Normal stimulus-secretion and intercellular coupling are essential to regulated secretory responses and these hallmarks are known to be altered in diabetes. In the present study, we used calcium imaging of isolated human islets to assess their collective cell behavior. The activity occurred in the form of calcium oscillations, was synchronized across different regions of islets through calcium waves, and was glucose-dependent: higher glucose enhanced the activity, elicited a greater proportion of global calcium waves, and led to denser and less fragmented functional networks. Hub regions were identified in stimulatory conditions, and they represented the most active islet regions. Moreover, calcium waves were found to be initiated in different subregions and the roles of initiators and hubs did not overlap. In type 2 diabetes, glucose-dependence was retained, but a reduced activity, locally restricted waves, and more segregated networks were detected compared with control islets. Interestingly, hub regions seemed to suffer the most by losing a disproportionately large fraction of connections. These changes affected islets from donors with diabetes in a heterogeneous manner.

physiology↗

bioRxiv2021

Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

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. However, identification of druggable proteins and pathways to ameliorate the burden of circadian metabolic disease remains a challenge. Here, we generated {beta} cells expressing a nano-luciferase reporter within the proinsulin polypeptide to screen 2,640 pharmacologically-active compounds and identify insulinotropic molecules that bypass the secretory defect in clock mutant {beta} cells. We validated lead compounds in primary mouse islets and identified known modulators of ligandgated ion channels and G-protein coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets validated ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacologic analyses established that the molecular clock controls the expression of the purinergic P2Y1 receptor to mediate the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a target to rescue circadian {beta}-cell failure and establish a chemical genetic screen for endocrine therapeutics.

cell biology↗