Biology subjects

Macdonald, P. E.

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

12 recordsLinked to original sources

bioRxiv2026

Functional, transcriptomic, and proteomic profiles of human primary and stem cell-derived beta cells in a state of high insulin production and increased fragility

Insulin production is a cardinal feature of pancreatic {beta} cells. Studies in rodents show that {beta} cells can switch between low and high insulin gene activity states and that elevated insulin production makes {beta} cells more vulnerable to stresses associated with diabetes. In people, genetically elevated insulin production increases the risk of type 1 diabetes. Via effects on obesity, hyperinsulinemia contributes to the pathogenesis of type 2 diabetes. Here, we characterize {beta} cells in low and high INS gene activity states sorted from primary human islets transduced with INS-GFP adenovirus and differentiated INS-EGFP knock-in embryonic stem cells (SC{beta} cells). We profile {beta} cell function, protein synthesis, resilience to diabetes associated stress, single {beta} cell transcriptomes and their co-activity networks, and purified {beta} cell proteomes. We show that human {beta} cells transition between distinct states. High INS cells have elevated maturity marker mRNAs and proteins, increased protein translation, are larger, but also more susceptible to cell death when exposed to diabetes-relevant stresses. We also catalogue thousands of differences in proteins in high INS stem cell-derived {beta} cells compared directly with high INS primary {beta} cells. Our study improves our understanding of the delicate balance between insulin production and {beta} cell resilience and guides the engineering of better {beta} cells. Blurbtranscriptional, proteomic, and functional analyses of insulin gene expression states in human {beta} cells from donor islets and stem cells Key findingsO_LIWe identify high and low INS gene activity states in human insulin-producing cells from donor islets and embryonic stem cell differentiations. C_LIO_LIWe characterize the relationship between insulin production and fragility, demonstrating that increased insulin production comes at a cost of reduced resilience to multiple stresses. C_LIO_LIFunctional, transcriptomic, and proteomic analyses identify similarities and differences between how primary and stem cell-derived {beta} cells manage stress and insulin production. C_LIO_LIWe report a comprehensive side-by-side proteomic analysis of purified primary and stem cell- derived {beta} cells in the high INS state and identify differences in protein production and secretion machinery, providing a roadmap for making better {beta} cells. C_LI

physiology↗

bioRxiv2026

Computational modelling of natural cell-to-cell heterogeneity reveals key parameters that control the diversity of human pancreatic islet β-cell excitability in response to glucose

Insulin-producing {beta}-cells demonstrate remarkable heterogeneity in their individual responsiveness to glucose, and that cellular heterogeneity contributes to coordinating islet activity and glucose homeostasis. Our current understanding of how variation in cell-intrinsic factors control cellular excitability and insulin secretion is informed by foundational experiments conducted on dispersed single {beta}-cells. Such studies are limited in their ability to link multiple electrical or metabolic properties within a single cell and preclude the ability to relate, post hoc, each parameters contribution to glucose responsiveness. Computational modelling represents a unique and underutilized tool to integrate and investigate the role of natural {beta}-cell heterogeneity in physiologic glucose responses. Herein, we utilize a high-volume single-cell electrophysiology "patch-seq" dataset to define the physiologically relevant sources of variability in human {beta}-cell electrophysiology and model their influence on single-cell glucose responses. Three thousand in silico human {beta}-cells were fitted to physiologically relevant variations in glucokinase activity, K+ current, Na+ current, Ca2+ current, and exocytotic function. Four dominant electrical phenotypes arose at low (2 mM) and high (20 mM) glucose: silent, bursting, spiking, and depolarized. Approximately 50% of uncoupled {beta}-cells remained electrically silent at high glucose. Furthermore, Na+ channel half-inactivation voltage was a major predictor of the silent and spiking phenotypes at each glucose concentration, and of cells that transition from silent to spiking when glucose increased. Indeed, experimentally observed variation in Na+ channel voltage dependence was second only to variation in ATP-sensitive potassium channel conductance in determining {beta}-cell excitability. Our data-driven computational modelling highlights the functional importance of electrical heterogeneity in human {beta}-cell glucose responses, and provides a useful tool for generating testable hypotheses.

physiology↗

bioRxiv2025

Supervised machine learning identifies impaired mitochondrial quality control in β cells with development of type 2 diabetes

In type 2 diabetes (T2D), molecular pathways driving {beta} cell failure are difficult to resolve with standard single cell analysis. Here we developed an interpretable, supervised machine learning framework that couples sparse rule-based classification (SnakeClassifier), pathway constrained modelling (BlackSwanClassifier), and {beta} cell mitochondrial fitness stratification (Kolmogorov-Arnold Neural Networks KANN), linking and integrating them into disease mechanisms in single cell RNA sequencing (scRNA-seq) from 52 human donors. SnakeClassifier trained on 50 genes accurately predicted T2D at single cell resolution, outperforming classical ensemble machine learning classifier models, and yielded donor level diabetes scores that correlated with chronic hyperglycemia. The clustering of {beta} cell populations ({beta}1-4) revealed a resilient non-diabetic (ND) {beta}1 subtype characterized by preserved {beta} cell identity genes and lower disease risk, whereas T2D {beta}2-4 subtypes exhibited upregulation of genes involved in cellular and mitochondrial stress and suppression of genes promoting oxidative phosphorylation and insulin secretion. Mitophagy emerged as the dominant program linked to T2D and a mitophagy focused BlackSwanClassifier nominated PINK1, BNIP3, and FUNDC1 as key regulators. PINK1 was enriched in ND {beta}1, decreased with T2D disease score and connected sex stratified mitophagy. We generated a KANN derived mitochondrial fitness index (MFI) integrating mitophagy, mitochondrial proteostasis, biogenesis and oxidative phosphorylation into a single interpretable score (R2 = 0.934 vs module-based mitochondria quality index), which identified mitophagy PINK1, SQSTM1, PRKN and BNIP3 as top contributors to T2D progression. These transparent models unify prediction with T2D disease mechanism and identify the mitophagy receptor PINK1 as a central determinant of {beta} cell metabolic fitness

systems biology↗

bioRxiv2025

Multiplexed Deep Visual Proteomics Unveils Spatial Heterogeneity and Rare Endocrine States in Human Adult Pancreatic Islets

Pancreatic islets are highly specialized tissue compartments that regulate metabolism, and their dysfunction contributes to diseases such as prediabetes and diabetes. Characterizing islet morphology and cell plasticity is essential for understanding these pathophysiological states, yet high-resolution spatial proteomics remains challenging due to the islets small size and cellular complexity. Here, we present multiplexed deep visual proteomics (mxDVP), an approach that integrates high-plex imaging with ultra-sensitive mass spectrometry to achieve deep spatial proteome profiling of defined cell types in tissue sections. Enhanced segmentation, semi- automated annotation, and optimized laser microdissection enable the enrichment of rare endocrine populations that are often overlooked in single-cell analyses. mxDVP achieves deep proteome coverage of >6,000 proteins from as few as 100 cells, including low-abundance transcription factors critical for endocrine cell fate determination. By profiling over 864,000 human pancreatic cells, we identify 12 endocrine subtypes, including polyhormonal hybrids, revealing previously unrecognized islet heterogeneity, metabolic regulation, and cellular adaptability.

molecular biology↗

bioRxiv2024

Single cell multiome profiling of pancreatic islets reveals physiological changes in cell type-specific regulation associated with diabetes risk

Physiological variability in pancreatic cell type gene regulation and the impact on diabetes risk is poorly understood. In this study we mapped gene regulation in pancreatic cell types using single cell multiomic (joint RNA-seq and ATAC-seq) profiling in 28 non-diabetic donors in combination with single cell data from 35 non-diabetic donors in the Human Pancreas Analysis Program. We identified widespread associations with age, sex, BMI, and HbA1c, where gene regulatory responses were highly cell type- and phenotype-specific. In beta cells, donor age associated with hypoxia, apoptosis, unfolded protein response, and external signal-dependent transcriptional regulators, while HbA1c associated with inflammatory responses and gender with chromatin organization. We identified 10.8K loci where genetic variants were QTLs for cis regulatory element (cRE) accessibility, including 20% with lineage- or cell type-specific effects which disrupted distinct transcription factor motifs. Type 2 diabetes and glycemic trait associated variants were enriched in both phenotype- and QTL-associated beta cell cREs, whereas type 1 diabetes showed limited enrichment. Variants at 226 diabetes and glycemic trait loci were QTLs in beta and other cell types, including 40 that were statistically colocalized, and annotating target genes of colocalized QTLs revealed genes with putatively novel roles in disease. Our findings reveal diverse responses of pancreatic cell types to phenotype and genotype in physiology, and identify pathways, networks, and genes through which physiology impacts diabetes risk.

genomics↗

bioRxiv2023

Complete Suspension Differentiation of Human Pluripotent Stem Cells into Pancreatic Islets Using Vertical-Wheel Bioreactors

Advanced protocols to produce human pluripotent stem cell (SC)-derived islets show promise in functional, metabolic, and transcriptional maturation of cell therapy product to treat diabetes. Available protocols are either developed as complete planar (2D) or, in later stages, combined with suspension cultures (3D). Despite marked progress, both approaches have clear limitations for scalability, cell loss and batch to batch heterogeneity during differentiation. Using a Vertical Wheel(R) bioreactor system, we present a highly efficient and scalable complete suspension protocol across all stages for directed differentiation of human pluripotent stem cells into functional pancreatic islets. Here, we generate homogeneous, metabolically functional, and transcriptionally enriched SC-islets and compared against adult donor islets. Generated SC-islets showed enriched endocrine cell composition ([~]63% CPEP+NKX6.1+ISL1+) and displayed functional maturity for glucose stimulated insulin secretion ([~]5-fold) during in vitro and post transplantation. Comprehensive stage-specific single-cell mass flow cytometry characterization with dimensional reduction analysis at stage-4 and -6 confirmed optimal maturation was achieved without heterogeneity. Notably, by 16-weeks transplantation follow-up, normal glycemic homeostasis was restored, and glucose responsive human c-peptide secretion response (2-fold) was achieved. Four months post engraftment, graft-harvested single cells displayed islet hormonal cell composition with flow cytometry, improved functional maturity by in vivo glucose-stimulated insulin secretion (GSIS) and enhanced transcriptional landscape with real-time expression that closely resembled patterns comparable to adult human islets. Our comprehensive evaluation of a complete suspension method applied across all stages using Vertical Wheel(R) bioreactors for SC-islets generation highlight progressive molecular and functional maturation of islets while reducing potential cell loss and cellular heterogeneity. Such a system could potentially be scaled to deliver clinical grade SC-islet products in a closed good manufacturing practice type environment. One Sentence SummaryThis study describes all-stages complete suspension protocol for SC-islets generation.

bioengineering↗

bioRxiv2023

Pharmacological or genetic inhibition of Scn9a protects beta-cells while reducing insulin secretion in type 1 diabetes

Pancreatic {beta} cells are essential for glucose homeostasis and are progressively lost during the development of type 1 diabetes. We previously demonstrated that the use-dependent Na+ channel inhibitor, carbamazepine, protects mouse {beta} cells in vitro and in vivo. Here, we confirmed the protective effects of carbamazepine and other Na+ channel inhibitors in human {beta} cells and investigated the specific role of the Na+ channel {beta} subunit gene Scn9a (Nav1.7) in {beta} cell function and survival. We generated {beta} cell-specific knockout mice on the non-obese diabetic (NOD) background both Ins1Cre knock-in and AAV8-Ins1-Cre approaches resulting in significant reduction of {beta} cell Na+ currents. Ca2+ responses and insulin secretion were significantly reduced, but only under the highest glucose conditions. Notably, carbamazepine treatment did not further alter insulin secretion or {beta} cell survival in Scn9a-knockout islets, indicating that {beta} cell Scn9a primarily mediates this drug's measured effects. Consistent with this, {beta} cell-specific deletion of Scn9a using AAV8-Ins1-Cre significantly reduced diabetes incidence in NOD mice. scRNAseq showed that this protection was associated with reduced Ins2 and increased Cdk8 in {beta} cells. Collectively, our data show that Scn9a plays important roles in {beta} cell excitability and survival during type 1 diabetes, thereby supporting this ion channel as a potential therapeutic target for {beta} cell preservation.

physiology↗

bioRxiv2023

Integration of single-cell multiomic measurements across disease states with genetics identifies mechanisms of beta cell dysfunction in type 2 diabetes

Altered function and gene regulation of pancreatic islet beta cells is a hallmark of type 2 diabetes (T2D), but a comprehensive understanding of mechanisms driving T2D is still missing. Here we integrate information from measurements of chromatin activity, gene expression and function in single beta cells with genetic association data to identify disease-causal gene regulatory changes in T2D. Using machine learning on chromatin accessibility data from 34 non-diabetic, pre-T2D and T2D donors, we robustly identify two transcriptionally and functionally distinct beta cell subtypes that undergo an abundance shift in T2D. Subtype-defining active chromatin is enriched for T2D risk variants, suggesting a causal contribution of subtype identity to T2D. Both subtypes exhibit activation of a stress-response transcriptional program and functional impairment in T2D, which is likely induced by the T2D-associated metabolic environment. Our findings demonstrate the power of multimodal single-cell measurements combined with machine learning for identifying mechanisms of complex diseases.

systems biology↗

bioRxiv2022

FK506-binding protein 2 participates in proinsulin folding

Apart from chaperoning, disulphide bond formation and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94, and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high molecular weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic {beta}-cells increased apoptosis without detectable upregulation of ER-stress response genes. Interestingly, FKBP2 mRNA was overexpressed in {beta}-cells from pancreatic islets of T2D patients. Based on molecular modelling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B chain (P28) is the substrate of FKBP2s isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

molecular biology↗

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↗