Biology subjects

Georgiadou, E.

Publications and source records attributed to Georgiadou, E..

8 recordsLinked to original sources

bioRxiv2025

Serine palmitoyltransferase-mediated de novo sphingolipid biosynthesis is required for normal insulin production and glucose tolerance

Aims/HypothesisThe importance for normal insulin secretion of ceramide synthesis is unclear. De novo ceramide synthesis requires serine palmitoyl transferase, SPT2, encoded by Sptl2. MethodsWe generated {beta}-cell-selective Sptl2 null mice by crossing animals with floxed alleles to mice expressing Cre recombinase from the Ins1 locus. Metabolic phenotyping, transcriptomic, functional analyses and histology were performed using standard approaches. ResultsIslets from Sptlc2{Delta}Ins1 mice displayed marked alterations in ceramide and sphingomyelin levels: ceramide content: p=0.016 and p=0.109; sphingomyelin content: p=0.016 and p=0.004 in Sptlc2{Delta}Ins1 vs Sptlc2CTL mice under regular and high fat diet, respectively, despite compensatory increases in the expression of enzymes in the salvage and sphingomyelinase pathways. Correspondingly, profound abnormalities were observed in glucose-regulated insulin secretion and glucose tolerance in vivo, both on a regular chow and high fat diet. These changes were associated with a drastic ([~]80%) lowering in {beta}-cell numbers, and a more minor increase in delta cell numbers. They were also preserved in animals maintained on a ketogenic diet, consistent with a cell autonomous effect on the {beta}-cell. Despite normal glucose-regulated intracellular calcium dynamics and insulin secretion, marked transcriptomic changes were observed in Sptlc2{Delta}Ins1 mouse islets, with affected GO terms including lysosome organisation and regulation of autophagy. Consistent with roles for compromised SPT2 function in diseased {beta}-cells, Sptl2 expression in Balbc and DBA2J mouse islets was lowered by a high fat-diet. Moreover, SPTLC2 mRNA tended to be lower, and SPTLC1 mRNA was significantly decreased, in islets from human subjects with type 2 diabetes versus normoglycemic individuals. ConclusionsPreserved de novo ceramide synthesis is required to maintain normal {beta}-cell mass and thus insulin secretion in mice. Therapeutic approaches which seek to target this process systemically using pharmacological SPT2 inhibitors should thus be treated with caution. Research in contextO_ST_ABS- What is already known about this subject?C_ST_ABSCeramides are key components of sphingolipid metabolism. Excess ceramide levels contribute to lipotoxicity and {beta}-cell apoptosis. -cell-restricted deletion of Cers2, which is responsible for the synthesis of very long ceramide chains, alters the insulin content of pancreatic islets and modifies glucose tolerance. Deletion of Cers 5 or 6, responsible for the synthesis of the long chains, has no effect. - What is the key question?What is the importance of de novo ceramide synthesis in {beta}-cells for the normal regulation of insulin production and glucose homeostasis? - What are the new findings?Inhibition of the de novo ceramide synthesis pathway in {beta}-cells, achieved by selective deletion of Sptlc2, encoding subunit 2 of the serine palmitoyltransferase (SPT) enzyme, induces a major alteration of glucose tolerance and insulin secretion. This is accompanied by a drastic reduction in {beta}-cell mass and islet insulin content. The remaining islets of Sptlc2{Delta}Ins1 display normal glucose-regulated intracellular calcium dynamics and insulin secretion despite imbalances in ceramide and sphingomyelin levels and substantial transcriptomic changes. Expression of SPTLC1, which encodes the other subunit of the SPT heterodimer, is reduced in islets from humans with type 2 diabetes, and a trend is observed towards lowered SPTLC2 expression. Taken together, these findings highlight the importance of de novo ceramide synthesis for normal {beta}-cell survival and function - How might this impact on clinical practice in the foreseeable future?By suppressing insulin production, global blockade or inhibition of SPT2, achieved with pharmacological approaches which seek to rescue insulin sensitivity in T2D, may be deleterious for glucose tolerance.

physiology↗

bioRxiv2023

Establishment of beta cell heterogeneity via differential CpG methylation at Nnat

Aims/hypothesisBeta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly-connected hub cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility which we explore here by focussing on the imprinted gene neuronatin (Nnat), which is required for normal insulin synthesis and secretion. MethodsSingle cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing eGFP under the control of the Nnat enhancer/promoter regions were generated for fluorescence-activated cell (FAC) sorting of beta cells and downstream analysis of CpG methylation by bisulphite and RNA sequencing, respectively. Animals deleted for the de novo methyltransferase, DNMT3A from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 and Cal-590. Insulin secretion was measured using Homogeneous Time Resolved Fluorescence Imaging. ResultsNnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic data sets demonstrated the early establishment of Nnat-positive and negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a sub-population specialised for insulin production, reminiscent of recently-described "{beta}HI" cells and were diminished in db/db mice. Hub cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialization. Conclusions/interpretationThese findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may thus contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. Research in contextWhat is already known about this subject? - Neuronatin (Nnat/NNAT) is an imprinted gene in humans and mice and is required for glucose-stimulated insulin secretion in vivo - Pancreatic beta cells are functionally heterogeneous with specific highly-connected subpopulations known to coordinate islet wide Ca2+ dynamics - Functional subpopulations have been described in human beta cells and their distribution is altered in type 2 diabetes What is the key question? - Does NNAT mark a discrete subpopulation of functional beta cells and which epigenetic pathways coordinate its formation and maintenance? What are the new findings? - A subpopulation of NNAT+ beta cells is established prior to the first week of postnatal life in mice via de novo DNA methylation at the Nnat promoter - NNAT+ beta cells are transcriptionally highly differentiated and appear to be functionally specialised for insulin production, possibly corresponding to recently-described "{beta}HI" and "CD63hi" beta cells. NNAT is expressed in a subset of beta cells across the human islet, and its deficiency in human beta cells diminishes glucose-stimulated insulin secretion - NNAT+ cells are likelier to belong to the population of follower, rather than hub cells, consistent with a role in insulin production rather than glucose detection How might this impact on clinical practice in the foreseeable future? - Epigenome-modifying compounds may provide a way of enhancing beta cell function and the ensemble behaviour of the islet to stimulate insulin secretion

cell biology↗

bioRxiv2020

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis

Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. Whilst previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. Here, we show that systemic inactivation of C2cd4b in mice leads to marked, but highly sexually dimorphic, changes in body weight and glucose homeostasis. Female C2cd4b mice display unchanged body weight but abnormal glucose tolerance and defective in vivo, but not in vitro, insulin secretion, associated with a marked decrease in follicle stimulating hormone levels. In sharp contrast, male C2cd4b null mice displayed normal glucose tolerance but an increase in body weight and fasting glycemia after maintenance on high fat diet. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic {beta} cell function at larval stages in C2cd4ab null zebrafish. These studies suggest that C2cd4b may act centrally to influence sex-dependent circuits which control pancreatic {beta} cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in man.

genetics↗

bioRxiv2020

Intravital imaging of islet Ca2+ dynamics reveals enhanced β cell connectivity after bariatric surgery in mice

Bariatric surgery improves both insulin sensitivity and secretion in type 2 diabetes. However, these changes are difficult to monitor directly and independently. In particular, the degree and the time course over which surgery impacts {beta} cell function, versus mass, have been difficult to establish. In this study, we investigated the effect of bariatric surgery on {beta} cell function in vivo by imaging Ca2+ dynamics prospectively and at the single cell level in islets engrafted into the anterior eye chamber. Islets expressing GCaMP6f selectively in the {beta} cell were transplanted into obese male hyperglycaemic mice that were then subjected to either vertical sleeve gastrectomy (VSG) or sham surgery. Imaged in vivo in the eye, VSG improved coordinated Ca2+ activity, with 90% of islets observed exhibiting enhanced Ca2+ wave activity ten weeks post-surgery, while islet wave activity in sham animals fell to zero discernible coordinated islet Ca2+ activity at the same time point. Correspondingly, VSG mice displayed significantly improved glucose tolerance and insulin secretion. Circulating fasting levels of GLP-1 were also increased after surgery, potentially contributing to improved {beta} cell performance. We thus demonstrate that bariatric surgery leads to time-dependent increases in individual {beta} cell function and intra-islet connectivity, together driving increased insulin secretion and diabetes remission, in a weight-loss independent fashion. Significance StatementUsed widely to treat obesity, bariatric surgery also relieves the symptoms of type 2 diabetes. The mechanisms involved in diabetes remission are still contested, with increased insulin sensitivity and elevated insulin secretion from pancreatic {beta} cells both implicated. Whilst the speed of remission - usually within a few days - argues for improvements in {beta} cell function rather than increases in mass, a direct demonstration of changes at the level of individual {beta} cells or islets has been elusive. Here, we combine vertical sleeve gastrectomy with intravital imaging of islets engrafted into the mouse anterior eye chamber to reveal that surgery causes a time-dependent improvement in glucose-induced Ca2+ dynamics and {beta} cell - {beta} cell connectivity, both of which likely underlie increased insulin release.

cell biology↗

bioRxiv2020

Pancreatic beta cell selective deletion of mitofusins 1 and 2 (Mfn1 and Mfn2) disrupts mitochondrial architecture and abrogates glucose-stimulated insulin secretion in vivo

Aims/hypothesisMitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic beta cells. Whether mitochondrial networks may be important for glucose or incretin sensing has yet to be determined. MethodsHere, we generated mice with beta cell-selective, adult-restricted deletion of the mitofusin genes Mfn1 and Mfn2 ({beta}Mfn1/2 dKO). Whole or dissociated pancreatic islets were used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production or GSIS in response to increasing glucose concentrations or GLP-1 receptor agonists. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. Results{beta}Mfn1/2 dKO mice displayed elevated fed and fasted glycaemia (p<0.01, p<0.001) and a >five-fold decrease (p<0.0001) in plasma insulin. Mitochondrial length, glucose-induced polarisation, ATP synthesis and cytosolic Ca2+ increases were all reduced (p<0.05,p<0.01,p<0.0001) in dKO islets, and beta cell Ca2+ dynamics were suppressed in vivo (p<0.001). In contrast, oral glucose tolerance was near normal in {beta}Mfn1/2 dKO mice (p<0.05, p<0.01) and GLP-1 or GIP receptor agonists largely corrected defective GSIS from isolated islets through an EPAC-dependent signalling activation. Conclusions/interpretationMitochondrial fusion and fission cycles are thus essential in the beta cell to maintain normal glucose, but not incretin, sensing. Defects in these cycles in some forms of diabetes might therefore provide opportunities for novel incretin-based or other therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/055384v3_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@258053org.highwire.dtl.DTLVardef@c07cborg.highwire.dtl.DTLVardef@1d3bb1forg.highwire.dtl.DTLVardef@1a1f128_HPS_FORMAT_FIGEXP M_FIG Impact of Mfn1/2 deletion on glucose and incretin stimulated-insulin secretion in beta cells. (A) In control animals, glucose is taken up by beta cells through GLUT2 and metabolised by mitochondria (elongated structure) through the citrate (TCA) cycle, leading to an increased mitochondrial proton motive force (hyperpolarised {Delta}{psi}m), accelerated ATP synthesis and O2 consumption rate (OCR). Consequently, the cytoplasmic ATP:ADP ratio rises, which causes closure of KATP channels, depolarisation of plasma membrane potential ({psi}m), opening of VDCCs and influx of cytosolic Ca2+. Elevated [Ca2+]cyt triggers a number of ATP-dependent processes including insulin secretion and improved beta-beta cell communication through connexin 36 (Cx36). (B) Following Mfn1/2 deletion (Mfn1/2 dKO), highly fragmented mitochondria were associated with reduced mitochondrial Ca2+ ([Ca2+]m) accumulation, leading to a less polarised {Delta}{psi}m, weaker OCR, lower mtDNA copy number and decreased ATP synthesis. This is expected to result in weaker {psi}m depolarisation, cytosolic Ca2+ influx and beta-beta cell connectivity due to lower expression of Cx36. Despite observing a higher number of docked insulin granules on the plasma membrane, insulin secretion was highly suppressed in these animals. This was also associated with increased beta cell death and reduced beta cell mass. (C) In response to incretins, insulin secretion is potentiated through the activation of GLP1-R and cAMP signalling involving PKA- and EPAC2-dependent pathways. Elevated [Ca2+]cyt triggers a number of ATP-dependent processes including insulin secretion and Ca2+ removal into the endoplasmic reticulum (ER).(D) In Mfn1/2 dKO cells, activation of the GLP1-R was shown to be linked with a potentiation of the EPAC2 pathway that is PKA independent, along with an increased ER Ca2+ uptake and improved beta-beta cell communication. How these amplifying signals of glucose metabolism for insulin secretion are linked with fragmented mitochondria remains unknown. Red and bold arrows represent enhanced pathways; dashed arrows represent impaired pathways. This figure was produced using illustrations from Servier Medical Art, http://smart.servier.com/ C_FIG Research in contextWhat is already known about this subject? Mitochondrial ultrastructural variations and number are altered in beta cells of human T2D patients [1]. Mice lacking Opa1, which controls mitochondrial fusion and inner membrane cristae structure, in beta cells, develop hyperglycaemia and defects in GSIS [2]. What is the key question? Is an interconnected mitochondrial network essential in primary mouse beta cells for normal insulin secretion and glucose homeostasis? What are the new findings? We generated mice with beta cell-selective, adult-restricted deletion of the mitofusin genes Mfn1 and Mfn2 and show that insulin secretion and glucose homeostasis are strongly reduced in vivo. Cytosolic and mitochondrial Ca2+ increases, {Delta}{psi}m, ATP production and beta cell connectivity are impaired in {beta}Mfn1/2 dKO animals. Incretins bypass the above defects through an exchange protein directly activated by cAMP (EPAC)-dependent signalling mechanism. How might this impact on clinical practice in the foreseeable future? The ability of incretins to bypass defects in mitochondrial function might be exploited by the design of new agonists which target this pathway.

molecular biology↗

bioRxiv2020

The type 2 diabetes gene product STARD10 is a phosphoinositide binding protein that controls insulin secretory granule biogenesis

ObjectiveRisk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the {beta}-cell, impaired glucose-induced insulin secretion and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids, and thus the pathways through which STARD10 regulates {beta}-cell function, are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the {beta}-cell and its effect on proinsulin processing and insulin granule biogenesis and maturation. MethodsWe used isolated islets from mice deleted selectively in the {beta}-cell for Stard10 ({beta}StarD10KO) and performed electron microscopy, pulse-chase, RNA sequencing and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay were performed on purified STARD10 protein. Results{beta}StarD10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased. Amongst the differentially expressed genes in {beta}StarD10KO islets, expression of the phosphoinositide binding proteins Pirt and Synaptotagmin 1 were decreased while lipidomic analysis demonstrated changes in phosphatidyl inositol levels. The inositol lipid kinase PIP4K2C was also identified as a STARD10 binding partner. STARD10 bound to inositides phosphorylated at the 3 position and solution of the crystal structure of STARD10 to 2.3 [A] resolution revealed a binding pocket capable of accommodating polyphosphoinositides. ConclusionOur data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis and insulin processing.

cell biology↗

bioRxiv2020

Survival and Proliferation of T-cell Acute Lymphoblastic Leukaemia Depends on mTOR-regulated Glutamine Uptake and EAAT1 Activity

T-cell acute lymphoblastic leukaemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients have refractory disease or relapse and die from the disease. To improve patient outcome new therapeutics are needed. With the aim to identify new therapeutic targets, we analysed the metabolic adaptations that T-ALL cells exhibit and found that glutamine uptake is essential for their proliferation. Isotope tracing experiments showed that glutamine fuels aspartate synthesis through the TCA cycle and that glutamine and glutamine-derived aspartate together supply three nitrogen atoms in purines and all but one atom in pyrimidine rings. We show that the glutamate-aspartate transporter EAAT1, which is normally only expressed in the CNS, is crucial for glutamine conversion to nucleotides and that T-ALL cell proliferation depends on EAAT1 function. Through this work, we identify EAAT1 as a novel therapeutic target for T-ALL treatment.

cancer biology↗

bioRxiv2019

The mitochondrial Ca2+ uniporter MCU is required for normal glucose-stimulated insulin secretion in vitro and in vivo

Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic {beta}-cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in {beta}-cells has not previously been examined in vivo. Here, we inactivated the pore-forming subunit MCUa (MCU) selectively in the {beta}-cell in mice using Ins1Cre-mediated recombination. Glucose-stimulated mitochondrial Ca2+ accumulation, ATP production and insulin secretion were strongly (p<0.05 and p<0.01) inhibited in MCU null animals ({beta}MCU-KO) in vitro. Interestingly, cytosolic Ca2+ concentrations increased (p<0.001) whereas mitochondrial membrane depolarisation improved in {beta}MCU-KO animals. Male {beta}MCU-KO mice displayed impaired in vivo insulin secretion at 5 (p<0.001) but not 15 min. post intraperitoneal (IP) injection of glucose while the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p<0.05) in young {beta}MCU-KO (<12 weeks), but not older animals. We conclude that MCU is crucial for mitochondrial Ca2+ uptake in pancreatic {beta}-cells and is required for normal GSIS. The apparent compensatory mechanisms which maintain glucose tolerance in {beta}MCU-KO mice remain to be established.

physiology↗