Biology subjects

Akerman, I.

Publications and source records attributed to Akerman, I..

11 recordsLinked to original sources

bioRxiv2026

High-purity stem cell-derived β-cells recapitulate key transcriptional and functional features of human islets

Human pluripotent stem cell-derived islets (SC-islets) offer an excellent medium for human pancreatic disease modelling and mechanistic studies into diabetes. While substantial progress has been made in differentiation protocols, their implementation in different laboratories result in variable {beta}-cell proportions with contaminant non-endocrine and proliferative cell types. To date, no facility-level implementation exists for producing SC-islets that can be shipped and benchmarked across multiple sites. Here, we describe the scalable optimisation, standardization, and facility-level implementation of an established human stem cell differentiation strategy that consistently results in a high proportion of {beta}-cells, with up to 75% of cells co-expressing C-peptide and the pancreatic endocrine marker, ISL1. Functionally, SC-islets exhibit glucose-responsive calcium influx and insulin secretion, recapitulating key physiological {beta}-cell functions. Single-cell transcriptomic profiling reveals a simplified endocrine landscape dominated by {beta}-cells, with a striking transcriptional similarity to human primary {beta}-cells (Pearsons r2[~]0.9). We observe smaller fractions of - and enterochromaffin-like cells with very low levels of poly-hormonal or proliferating cell types (<3%). Taken together, we provide a well-defined, reproducible and accessible in vitro SC-islet platform benchmarked for functionality at multiple recipient sites.

cell biology↗

bioRxiv2024

Chromatin folding motifs define the basic grammar of TAD architecture in single alleles

Chromatin folding exhibits extensive cell-to-cell variability, yet whether this variability is dominated by continuously varying conformations or by recurrent organizational patterns remains unclear. Here, we combined high-resolution chromatin tracing with a novel machine learning algorithm to show that chromatin organization can be compactly represented by a limited repertoire of recurrent folding motifs. Using polymer simulations and cohesin depletion experiments, we further show that these motifs arise primarily from loop extrusion. Analysis of distinct tissues reveals that a shared motif repertoire is sufficient to describe diverse structural ensembles, with biological differences arising primarily through changes in motif occupancy and combinatorial usage. More generally, rather than generating entirely new conformations, transcriptional and disease-associated changes, including those occurring during disease onset, act primarily by redistributing motif occupancy within this common repertoire. In particular, gene activation is associated with a shift toward decompacted states and reduced contact frequencies, revealing a quantitative link between transcription and chromatin organization at the single-allele level. Together, these findings reveal that single-allele chromatin variability is structured by a constrained repertoire of recurrent folding motifs whose usage is modulated by regulatory context, providing a new framework to interpret how chromatin organization relates to gene activity.

genomics↗

bioRxiv2023

The macronuclear genomic landscape within Tetrahymena thermophila

The extent of intraspecific genomic variation is key to understanding species evolutionary history, including recent adaptive shifts. Intraspecific genomic variation remains poorly explored in eukaryotic microorganisms, especially in the nuclear dimorphic ciliates, despite their fundamental role as laboratory model systems and their ecological importance in many ecosystems. We sequenced the macronuclear genome of 22 laboratory strains of the oligohymenophoran Tetrahymena thermophila, a model species in both cellular biology and evolutionary ecology. We explored polymorphisms at the junctions of programmed eliminated sequences, and reveal their utility to barcode very closely related cells. As for other species of the genus Tetrahymena, we confirm micronuclear centromeres as gene diversification centres in T. thermophila, but also reveal a two-speed evolution in these regions. In the rest of the genome, we highlight recent diversification of genes encoding for extracellular proteins and cell adhesion. We discuss all these findings in relation with ciliates ecology and cellular characteristics. Impact StatementThis is the first study of population genomics in the ciliate Tetrahymena thermophila. This bacterivore species plays an important role in aquatic trophic chains and is widely used as a model in cell and molecular biology, ecology, evolution or toxicology. As all ciliates, it contains a germline micronucleus and a somatic macronucleus. Sequencing of the macronucleus reveals that the centromeric region of the micronucleus are simultaneously a region of new gene diversification, as observed in other Tetrahymena species, and a region containing highly conserved genes. The results also confirm that the formation of the macronucleus from the micronucleus is highly imprecise. Interestingly, this process generates a genomic barcode that can discriminate cells derived from a given sexual reproduction event, allowing to study more finely population dynamics/history in nature. Data summaryAll data are fully provided in Supplementary Materials. The raw data of the 22 Tetrahymena genomes have been deposited in the Sequence Read Archive (https://www-ncbi-nlm-nih-gov.inee.bib.cnrs.fr/bioproject/PRJNA1012331). Accession numbers are listed in Table S1 (available in the online version of this article).

genomics↗

bioRxiv2023

A heterozygous CEBPA mutation disrupting the bZIP domain causes MDS disease progression

Myelodysplastic syndrome disease (MDS) has a variable risk for progression to AML. Mutations in CEBPA are associated with a high risk of disease progression, but whether this mutation is causative for AML development is unclear. To answer this question, we generated patient-derived, MDS-specific iPSCs recapitulating the patient disease phenotype upon differentiation to blood, with hematopoietic progenitor cells showing erythroid and myeloid-dysplasia. Introduction of a frameshift mutation affecting the C/EBP bZIP domain led to disease progression, with a reduction in clonogenic potential, block in granulocyte development and increased self-renewal capacity of erythroid progenitors. ATAC-seq revealed that the acquisition of this mutation reshaped the chromatin landscape at distal cis-regulatory regions, promoting changes in clonal composition as observed by single cell RNAseq. Our work identifies mutant CEBPA as causative for MDS disease progression, providing a new isogenic MDS experimental model for drug screening to improve diagnostic and therapeutic strategies. HighlightsO_LIDevelopment of isogenic iPSC model of clonal evolution of MDS C_LIO_LIMonoallelic disruption of CEBPA bZIP domain is causative for MDS disease progression C_LIO_LIMonoallelic disruption of CEBPA bZIP reshapes chromatin landscape C_LIO_LIPatient derived iPSCs recapitulate drug responsiveness C_LI

cancer biology↗

bioRxiv2022

High-resolution glucose fate-mapping reveals LDHB-dependent lactate production by human pancreatic β cells

Using 13C6 glucose labeling coupled to GC-MS and 2D 1H-13C HSQC NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning {beta} cell function. In both mouse and human islets, the contribution of glucose to the TCA cycle is similar. Pyruvate-fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is six-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and {beta} cell-specific LDHB expression. LDHB inhibition increases glucose-dependent lactate generation in mouse and human {beta} cells, and decreases Ca2+-spiking frequency without affecting ATP/ADP levels. Thus, we show that LDHB limits glucose-stimulated lactate generation in {beta} cells. Further studies are warranted to understand how lactate impacts {beta} cell metabolism and/or function. HIGHLIGHTSO_LIHuman and rodent islets generate lactate following glucose stimulation. C_LIO_LI{beta} cells specifically express LDHB, which acts to limit lactate generation. C_LIO_LILDHB inhibition influences Ca2+ spiking frequency without affecting ATP/ADP ratio. C_LI eTOCCuozzo et al show that glucose-stimulated rodent and human islets generate lactate. Transcriptomic and imaging analyses reveal that LDHB is specifically expressed in {beta} cells and unexpectedly restrains lactate production. LDHB expression and thus regulated lactate generation might reflect a key mechanism underlying {beta} cell metabolism, function and survival.

physiology↗

bioRxiv2020

Predominant activation of 11-oxygenated androgens by peripheral blood mononuclear cells

ContextAndrogens are important modulators of immune cell function impacting proliferation, differentiation and cytokine production. The local generation of active androgens from circulating androgen precursors is an important mediator of androgen action in peripheral androgen target cells or tissue. ObjectiveTo characterize the activation of classic and 11-oxygenated androgens in human peripheral blood mononuclear cells (PBMCs). MethodsPBMCs were isolated from healthy male donors and incubated ex vivo with precursors and active androgens of the classic and 11-oxygenated androgen pathways. Steroids were quantified by liquid chromatography-tandem mass spectrometry. The expression of genes encoding steroid-metabolizing enzymes was assessed by quantitative PCR. ResultsPBMCs generated 8-fold higher amounts of the active 11-oxygenated androgen 11-ketotestosterone than the classic androgen testosterone from their respective precursors. We identified the enzyme AKR1C3 as the major reductive 17{beta}-hydroxysteroid dehydrogenase in PBMCs responsible for both conversions and found that within the PBMC compartment natural killer cells are the major site of AKRC13 expression and activity. Steroid 5-reductase type 1 catalyzed the 5-reduction of classic but not 11-oxygenated androgens in PBMCs. Lag time prior to the separation of cellular components from whole blood increased 11KT serum concentrations in a time-dependent fashion, with significant increases detected from two hours after blood collection. Conclusions11-oxygenated androgens are the preferred substrates for androgen activation by AKR1C3 in PBMCs, primarily conveyed by natural killer cell AKR1C3 activity, yielding 11KT the major active androgen in PBMCs. Androgen metabolism by PBMCs can affect the measurement results of serum 11-ketotestosterone concentrations, if samples are not separated in a timely fashion.

biochemistry↗

bioRxiv2020

Neonatal diabetes mutations disrupt a chromatin pioneering function that activates the human insulin gene

Despite the central role of chromosomal context in gene transcription, human noncoding DNA variants are generally studied outside of their endogenous genomic location. This poses major limitations to understand the true consequences of causal regulatory variants. We focused on a cis-regulatory mutation (c.-331C>G) in the INS gene promoter that is recurrently mutated in unrelated patients with recessive neonatal diabetes. We created mice in which a ~3.1 kb human INS upstream region carrying -331C or -331G alleles replaced the orthologous mouse Ins2 region. This human sequence drove cell-specific transcription in mice. It also recapitulated poised chromatin during pancreas development and active chromatin in differentiated {beta}-cells. The c.-331C>G mutation, however, blocked active chromatin formation in differentiated b-cells. We further show that another neonatal diabetes gene product, GLIS3, had a singular pioneer-like ability to activate INS chromatin in non-pancreatic cells, which was hampered by the c.-331C>G mutation. This in vivo analysis of human regulatory defects, therefore, uncovered cis and trans components of a mechanism that is essential to activate the endogenous INS gene.

genomics↗

bioRxiv2020

The prolyl hydroxylase PHD3 maintains β-cell glucose metabolism during fatty acid excess

The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a hypoxia-inducible factor target that uses molecular oxygen to hydroxylate proline. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting {beta}-cells. Here, we show that deletion of PHD3 specifically in {beta}-cells ({beta}PHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, {beta}PHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift away from glycolysis. However, {beta}PHD3KO islets are able to maintain oxidative phosphorylation and insulin secretion by increasing utilization of fatty acids to supply the tricarboxylic acid cycle. This nutrient-sensing switch cannot be sustained and {beta}PHD3KO islets begin to show signs of failure in response to prolonged metabolic stress, including impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes and insulin secretion. Thus, PHD3 might be a pivotal component of the {beta}-cell glucose metabolism machinery by suppressing the use of fatty acids as a primary fuel source, under obesogenic and insulin resistant states. SIGNIFICANCE STATEMENTProlyl-4-hydroxylase 3 (PHD3) is involved in the oxygen-dependent regulation of cell phenotype. A number of recent studies have shown that PHD3 might operate at the interface between oxygen availability and metabolism. To understand how PHD3 influences insulin secretion, which depends on intact glucose metabolism, we generated mice lacking PHD3 specifically in pancreatic {beta}-cells. These mice, termed {beta}PHD3KO, are apparently normal until fed high fat diet at which point their {beta}-cells switch to fatty acids as a fuel source. This switch cannot be tolerated and {beta}-cells in {beta}PHD3KO mice eventually fail. Thus, PHD3 maintains glucose-stimulated insulin secretion in {beta}-cells during states of fatty acid excess, such as diabetes and obesity.

physiology↗

bioRxiv2019

Vitamin D-binding protein is required for the maintenance of α-cell function and glucagon secretion

Vitamin D-binding protein (DBP) or GC-globulin carries vitamin D metabolites from the circulation to target tissues. DBP expression is highly-localized to the liver and pancreatic -cells. While DBP serum levels, gene polymorphisms and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates -cell morphology, -cell function and glucagon secretion. Deletion of DBP led to smaller and hyperplastic -cells, altered Na+ channel conductance, impaired -cell activation by low glucose, and reduced rates of glucagon secretion. Mechanistically, this involved reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects were also seen in {beta}-cell and {delta}-cell function. Immunostaining of human pancreata revealed generalized loss of DBP expression as a feature of late-onset and longstanding, but not early-onset type 1 diabetes. Thus, DBP is a critical regulator of -cell phenotype, with implications for diabetes pathogenesis. HIGHLIGHTSO_LIDBP expression is highly-localized to mouse and human -cells C_LIO_LILoss of DBP increases -cell number, but decreases -cell size C_LIO_LI-cells in DBP knockout islets are dysfunctional and secrete less glucagon C_LIO_LIDBP expression is decreased in -cells of donors with late-onset or longstanding type 1 diabetes C_LI

physiology↗

bioRxiv2019

Nicotinamide riboside augments the human skeletal muscle NAD+ metabolome and induces transcriptomic and anti-inflammatory signatures in aged subjects: a placebo-controlled, randomized trial

NAD+ is modulated by conditions of metabolic stress and has been reported to decline with aging, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome, and questioned if tissue NAD+ levels are depressed with aging. We supplemented 12 aged men with NR 1g per day for 21-days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways. NR also depressed levels of circulating inflammatory cytokines. In an additional study, 31P magnetic resonance spectroscopy-based NAD+ measurement in muscle and brain showed no difference between young and aged individuals. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR, while suggesting that NAD+ decline is not associated with chronological aging per se in human muscle or brain.

clinical trials↗

bioRxiv2016

Human pancreatic β cell lncRNAs control cell-specific regulatory networks

Recent studies have uncovered thousands of long non-coding RNAs (IncRNAs) in human pancreatic {beta} cells. {beta} cell lncRNAs are often cell type-specific, and exhibit dynamic regulation during differentiation or upon changing glucose concentrations. Although these features hint at a role of lncRNAs in {beta} cell gene regulation and diabetes, the function of {beta} cell lncRNAs remains largely unknown. In this study, we investigated the function of {beta} cell-specific lncRNAs and transcription factors using transcript knockdowns and co-expression network analysis. This revealed lncRNAs that function in concert with transcription factors to regulate {beta} cell-specific transcriptional networks. We further demonstrate that lncRNA PLUTO affects local three-dimensional chromatin structure and transcription of PDX1, encoding a key {beta} cell transcription factor, and that both PLUTO and PDX1 are downregulated in islets from donors with type 2 diabetes or impaired glucose tolerance. These results implicate lncRNAs in the regulation of {beta} cell-specific transcription factor networks.

genomics↗