Biology subjects

Muralidharan, C.

Publications and source records attributed to Muralidharan, C..

13 recordsLinked to original sources

bioRxiv2026

Rapid direct neuronal reprogramming of human dental pulp stem cells

Direct neuronal reprogramming offers an alternative to induced pluripotent stem cell-based differentiation by converting somatic cells directly into neurons without passage through pluripotency. However, commonly used fibroblast-based protocols are often slow and inefficient. Here, we evaluated human dental pulp stem cells (DPSCs), which originate from the cranial neural crest and possess intrinsic neurogenic potential, as a developmentally relevant source of induced neurons (iNs). Using an all-in-one lentiviral vector, we converted DPSCs into iNs within 17 days, compared with 28 days for fibroblasts reprogrammed with the same vector, and achieved significantly higher neuronal purity under the respective established protocols. Multi-omic profiling revealed coordinated suppression of mesenchymal and cell-cycle programs and induction of neuronal, synaptic, and metabolic pathways. Single-nucleus RNA sequencing resolved fibroblast-like, transitional, maturing neuronal, GABAergic-like, and alternative fates, while trajectory inference suggested divergent neuronal and non-neuronal conversion paths. Whole-cell recordings showed that a subset of DPSC-iNs developed early neuronal excitability and voltage-gated inward and outward currents. Together, our findings establish DPSCs as an accessible and developmentally relevant source for rapid direct neuronal conversion. This integrated molecular, single-nucleus, and electrophysiological characterization defines the cellular heterogeneity of DPSC-to-neuron reprogramming and provides a framework for protocol refinement and future patient-specific disease modeling.

neuroscience↗

bioRxiv2025

Retroviral insertions contributed to the divergence of human and chimpanzee brains

Over the past 5-7 million years, humans and chimpanzees have diverged in brain size, structural complexity, and cognitive abilities despite high conservation of protein-coding genes. Notably, the endogenization and proliferation of retroviral infections within host genomes has introduced numerous species-specific regulatory elements that have the potential to influence gene regulation. However, the role of these endogenous retroviruses in hominoid brain evolution remains unclear. A burst of lineage-specific PTERV1 retroviruses recently invaded the chimpanzee genome but are absent in humans. We conducted an epigenomic analysis of PTERV1 insertions in chimpanzee neural organoids and found that they are heavily covered by DNA methylation, representing more than 150 species-specific heterochromatin domains with the capacity to influence gene regulatory networks. We identified one such chimpanzee-specific PTERV1 insertion on chromosome 19 that blocks the expression of the long noncoding RNA LINC00662, via DNA methylation spread to the adjacent genomic region. The expression of LINC00662 was restored in chimpanzee induced pluripotent stem cells when we deleted the PTERV1 insertion using CRISPR editing. We found that LINC00662, a human-specific RNA, is highly expressed in the developing brain and plays an important role in the posttranscriptional control of neuronal maturation, axon outgrowth, and neural organoid development. In summary, our findings describe how endogenous retroviral insertions contributed to the functional divergence of the human and chimpanzee brains. This provides a new mechanism by which retroviral pandemics influenced primate brain speciation.

evolutionary biology↗

bioRxiv2025

Phosphoproteomic profiling reveals post-translational dysregulation in Huntington's disease patient-derived neurons

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin (HTT) gene. Although transcriptomic and proteomic changes have been characterized in patient-derived neurons, the contribution of post-translational modifications (PTMs), such as phosphorylation, remains poorly understood. Here, we present the first phosphoproteomic analysis by mass spectrometry (P-MS) of human induced neurons (iNs) directly reprogrammed from HD patient fibroblasts. We identified 177 phosphopeptides with significantly altered abundance in HD-iNs, mapping to phosphoproteins associated with key signaling pathways known to be affected in HD, such as splicing and autophagy. By integrating P-MS data with previously published proteomic and transcriptomic data from the same donors, we identified distinct subsets of ON-OFF phosphopeptides that exhibited a complete loss of phosphorylation in either HD- or Ctrl-iNs, without corresponding changes at the RNA or protein level. An exception was MXRA8, previously described in glial cells as a mediator of blood-brain barrier integrity and astrocyte-mediated neuroinflammation. This protein showed increased protein abundance despite the absence of phosphorylation in HD- iNs, suggesting a compensatory mechanism - a pattern also observed in human post mortem cortical HD tissue. Additionally, MXRA8 showed altered protein-protein interactions with lysosomal and metabolic regulators in HD-iNs, highlighting its potential role in autophagy impairment as well as in neurovascular dysfunction. These findings uncover a distinct layer of post-translational dysregulation in HD, suggesting that phospho-switch proteins such as MXRA8 may be candidate effectors of pathology and thus site-specific phosphorylation loss may contribute to impaired signaling and proteostasis in human HD neurons.

neuroscience↗

bioRxiv2025

The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

In type 1 diabetes (T1D), the innate and adaptive immune systems attack and eventually destroy the insulin-secreting pancreatic {beta} cells. During this process, {beta} cells activate inflammatory signaling pathways that augment the dysfunction and destruction imposed by cellular autoimmunity. The 12-lipoxygenase (12-LOX) pathway produces the pro-inflammatory eicosanoid 12-HETE, which induces oxidative and endoplasmic reticulum stress and results in diminished insulin secretion and apoptosis. The G protein-coupled receptor GPR31 has been identified as a putative receptor for 12-HETE. In this study, we generated conventional GPR31 knockout (KO) mice on the C57BL/6J background. To interrogate the role of GPR31 in {beta} cells, we treated islets from wildtype and Gpr31b KO mice with pro-inflammatory cytokines and subjected the islets to RNA sequencing. Differentially expressed pathways in Gpr31b KO islets included those pertaining to inflammation and oxidative stress, consistent with functional studies that demonstrated reduced cytokine-induced oxidative stress in Gpr31b KO islets compared to wildtype controls. Bone marrow-derived macrophages from Gpr31b KO mice showed reduced macrophage migration and decreased inflammatory IFN- and IFN-{gamma} signaling by RNA sequencing. To mimic islet and macrophage inflammation as seen in T1D, wildtype and Gpr31b KO mice were treated with the diabetogenic toxin streptozotocin. Compared to wildtype, Gpr31b KO mice had improved glucose tolerance and preserved {beta}-cell mass. siRNA knockdown of Gpr31b in non-obese diabetic (NOD) mice reduced insulitis, macrophage infiltration, and oxidative stress. Collectively, these findings are consistent with previously published data using 12/15-LOX KO mice and suggest that GPR31 mediates the pro-inflammatory responses of 12-HETE in both {beta} cells and macrophages.

molecular biology↗

bioRxiv2025

Human brain cell-type-specific aging clocks based on single-nuclei transcriptomics

Aging is the primary risk factor for most neurodegenerative diseases, yet the cell-type-specific progression of brain aging remains poorly understood. Here, we developed human cell-type-specific transcriptomic aging clocks using high-quality single-nucleus RNA sequencing data from post mortem human prefrontal cortex tissue of 31 donors aged 18 - 94 years, encompassing 73,941 high-quality nuclei. We observed distinct transcriptomic changes across major cell types, including upregulation of inflammatory response genes in microglia from older samples. Aging clocks trained on each major cell type accurately predicted chronological age and remained robust in independent single-nucleus RNA-sequencing datasets, underscoring their broad applicability. These findings demonstrate the feasibility of cell-type-specific transcriptomic clocks to measure biological aging in the human brain and highlight potential mechanisms of selective vulnerability in neurodegenerative diseases. We anticipate these clocks will serve as a basis for further studies in other brain regions and more diverse populations, ultimately advancing our understanding of age-related neurodegenerative processes at the single-cell level.

neuroscience↗

bioRxiv2025

IFN-α Induces Heterogenous ROS Production in Human β-Cells

Type 1 diabetes (T1D) is a multifactorial disease involving genetic and environmental factors, including viral infection. We investigated the impact of interferon alpha (IFN-), a cytokine produced during the immune response to viral infection or the presence of un-edited endogenous double-stranded RNAs, on human {beta}-cell physiology. Intravital microscopy on transplanted human islets using a {beta}-cell-selective reactive oxygen species (ROS) biosensor (RIP1-GRX1-roGFP2), revealed a subset of human {beta}-cells that acutely produce ROS in response to IFN-. Comparison to Integrated Islet Distribution Program (IIDP) phenotypic data revealed that healthier donors had more ROS accumulating cells. In vitro IFN- treatment of human islets similarly elicited a heterogenous increase in superoxide production that originated in the mitochondria. To determine the unique molecular signature predisposing cells to IFN- stimulated ROS production, we flow sorted human islets treated with IFN-. RNA sequencing identified genes involved in inflammatory and immune response in the ROS-producing cells. Comparison with single cell RNA-Seq datasets available through the Human Pancreas Analysis Program (HPAP) showed that genes upregulated in ROS-producing cells are enriched in control {beta}-cells rather than T1D donors. Combined, these data suggest that IFN- stimulates mitochondrial ROS production in healthy human {beta}-cells, potentially predicting a more efficient antiviral response.

cell biology↗

bioRxiv2024

The integrated stress response promotes macrophage inflammation and migration in autoimmune diabetes

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin producing pancreatic {beta}-cells. Macrophages infiltrate islets early in T1D pathogenesis, preceding the influx of T- and B-lymphocytes. The integrated stress response (ISR), a cellular pathway activated during stress, coordinates adaptive changes in gene expression to maintain cell function and survival. To study the ISR in macrophages, bone-marrow derived macrophages were treated with a pharmacological inhibitor of the ISR (ISRIB) and polarized to a proinflammatory M1-like state. We found a reduction in the number of proinflammatory macrophages as well as a reduction in iNOS mRNA and protein levels with ISRIB treatment. RNA-sequencing revealed a reduction in pathways related to stress responses, including ER stress, reactive oxygen species (ROS) regulation, and autophagy as well as migration pathway genes. ISRIB treatment led to decreased macrophage migration after stimulation in vitro and decreased macrophages to the site of injury after tailfin cut in zebrafish in vivo. Pre-diabetic female non-obese diabetic (NOD) mice administered ISRIB demonstrated an overall reduction in the macrophage numbers in the pancreatic islets. Additionally, the insulitic area of pancreata from ISRIB treated NOD mice had increased PD-L1 levels. Our study provides new insights into ISR signaling in macrophages and demonstrates that the ISR might serve as a potential target for intervention in macrophages during early T1D pathogenesis.

immunology↗

bioRxiv2024

12-Lipoxygenase Inhibition Suppresses Islet Immune and Inflammatory Responses and Delays Autoimmune Diabetes in Human Gene Replacement Mice

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing {beta} cells and involves an interplay between {beta} cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in {beta} cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our finding demonstrated that VLX-1005, a potent 12-LOX inhibitor, effectively delayed the onset of autoimmune diabetes in human gene replacement non-obese diabetic mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecule PD-L1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets and polarized proinflammatory macrophages revealed significant alteration of cytokine-responsive pathways and a reduction in interferon response after VLX-1005 treatment. Our studies demonstrated that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.

molecular biology↗

bioRxiv2023

Suppression of the Integrated Stress Response in Islet β Cells Decreases Risk of Autoimmune Diabetes

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing {beta} cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2 kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves {beta} cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in {beta} cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in {beta} cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-{beta}H1 human {beta} cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances {beta} cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

cell biology↗

bioRxiv2023

Real Time In vivo Analysis of Pancreatic Beta-cell Autophagic Flux Reveals Impairment Before Onset of Autoimmune Diabetes

The catabolic pathway of autophagy is critical for pancreatic beta-cell function and is defective in established type 1 diabetes (T1D). However, it is unclear when and how this critical cell process becomes defective during diabetes pathogenesis. To study the nature of autophagy dysfunction in the context of autoimmune diabetes, we utilized intravital microscopy to study autophagic flux in vivo in real time. We generated a custom AAV8- packaged mCherry-eGFP-LC3B biosensor driven by the insulin promoter for beta-cell-selective expression. For real time autophagic flux evaluation, fluorescent signal from eGFP and mCherry fluorophores was correlated in space and time to follow the process of autophagosome-lysosome fusion. We observed autophagic flux defects in the beta-cells of non-obese diabetic (NOD) mouse model of T1D prior to hyperglycemia onset that were less apparent in mice without a functional immune system. We also evaluated autophagic flux in human donor islets that were transplanted under the kidney capsule of immune incompetent mice. Collectively, we provide the first evaluation of autophagic flux in vivo in 4D and demonstrate that autophagy defects precede hyperglycemia in NOD mice suggesting a potential causative role for these defects in beta-cell demise during T1D pathogenesis.

cell biology↗

bioRxiv2021

Loss of Secretory Pathway Ca2+ ATPase (SPCA1) Impairs Insulin Secretion and Reduces Autophagy in the Pancreatic Islet

The {beta} cell Golgi apparatus serves as a significant store of intracellular Ca2+ and an important site of proinsulin maturation. However, the contribution of Golgi Ca2+ to diabetes pathophysiology is unknown. The Golgi primarily utilizes the Secretory Pathway Ca2+ ATPase (SPCA1) to maintain intraluminal Ca2+ stores, and loss of SPCA1 has been linked to impaired Golgi function in other cell types. Here, we demonstrated that SPCA1 expression is decreased in islets from diabetic mice and human organ donors with type 2 diabetes, suggesting SPCA1 may impact diabetes development. INS-1 {beta} cells lacking SPCA1 (SPCA1KO) showed reduced intraluminal Golgi Ca2+ levels, reduced glucose-stimulated insulin secretion (GSIS), and increased insulin content. Islets from SPCA1 haploinsufficient mice (SPCA1+/-) exhibited reduced GSIS, altered glucose-induced Ca2+ oscillations, and altered insulin granule maturation. Autophagy can regulate granule homeostasis, therefore we induced autophagy with Torin1 and found that SPCA1KO cells and SPCA1+/- islets had reduced levels of the autophagosome marker LC3-II. Furthermore, SPCA1KO LC3-II were unchanged after blocking autophagy initiation or autophagolysosome fusion and acidification. Thus, we concluded that {beta} cell SPCA1 plays an important role in the maintenance of Golgi Ca2+ homeostasis and reduced Golgi Ca2+ impairs autophagy initiation and may impact insulin granule homeostasis.

cell biology↗

bioRxiv2020

Pancreatic Beta Cell Autophagy is Impaired in Type 1 Diabetes

Aims/hypothesisPancreatic beta cells are highly metabolic secretory cells that are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species (ROS) during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce ROS to protect against apoptosis both in vitro and in vivo. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesized that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development. MethodsMouse pancreata were collected from chloroquine injected and non-injected NOR, nondiabetic NOD, and diabetic NOD mice. Age and BMI-matched pancreas tissue sections from human organ donors (n=34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). To assess autophagic flux, we injected the mice with chloroquine to inhibit the final stages of autophagy. We analyzed tissues for markers of autophagy via immunofluorescence analysis. Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), LC3A/B (autophagosome marker), Lamp1 (lysosome marker), and p62 (autophagy adaptor protein and marker for autophagic flux). Images were collected on a scanning laser confocal microscope then analyzed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes (formerly called lipofuscin bodies, residual bodies or tertiary lysosomes) were analyzed in electron micrographs of pancreatic tissue sections from human organ donors (nPOD; n=12) deposited in www.nanotomy.org/OA/nPOD. Energy Dispersive X-ray (EDX) analysis was also performed on these tissues to analyze distribution of elements such as nitrogen, phosphorus, and osmium in secondary lysosomes and telolysosomes of nondiabetic and autoantibody positive donor tissues (n=5). ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p=0.0077) and increased p62 in islets of both nondiabetic and diabetic NOD mice (p<0.0001 in both cases) when compared to NOR mice. There was also a significant reduction in autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to both nondiabetic NOD mice (p=0.0004) and NOR mice (p=0.0003). Chloroquine infusions elicited accumulation of autophagosomes in the islets of NOR (p=0.0029) and nondiabetic NOD mice (p<0.0001), but not in the islets of diabetic NOD mice. Chloroquine also stimulated an accumulation of the autophagy adaptor protein p62 in the islets of NOR mice (p<0.001), however this was not observed in NOD mice (regardless of diabetes status). In the human pancreata, we observed significantly reduced autophagosome:lysosome colocalization (p=0.0002) in the residual beta cells of donors with type 1 diabetes compared to nondiabetic controls. We also observed reduced colocalization of proinsulin with lysosomes in the residual beta cells of donors with type 1 diabetes compared to both nondiabetic (p<0.0001) and autoantibody positive donors (p<0.0001). Electron microscopy based analysis of human pancreas sections also revealed accumulation of telolysosomes in beta cells of autoantibody positive donors (p=0.0084), the majority of which had an nitrogen dense ring outside a phospholipid core. Conclusions/interpretationCollectively, we provide evidence of impairment in the final degradation stages of islet macroautophagy and crinophagy in human type 1 diabetes. We also document an accumulation of telolysosomes with nitrogen accumulation at their periphery in the beta cells of autoantibody positive donors. This demonstrates clear differences in the lysosome contents of autoantibody positive donors that may be associated with lysosome dysfunction prior to clinical hyperglycemia. We observe similar impairments in macroautophagy in the diabetic NOD mouse, a model of type 1 diabetes, suggesting that this mouse model can be appropriately used to study the pathogenesis of autophagy/crinophagy loss and how it relates to disease initiation and progression. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development. Research in contextWhat is already known about this subject? O_LIAutophagy confers a cytoprotective role in the beta cell. C_LIO_LIAutophagy is reduced in type 2 diabetes. C_LIO_LIAutophagy in the context of type 1 diabetes is unexplored. C_LI What is the key question? O_LIIs autophagy reduced during the pathogenesis of human type 1 diabetes? C_LI What are the new findings? O_LIWe provide evidence of reduced autophagy and crinophagy in human type 1 diabetes. C_LIO_LIThese data are supported by observations of reduced autophagy in a mouse model of autoimmune diabetes. C_LI How might this impact on clinical practice in the foreseeable future? O_LIThis study provides evidence that autophagy is impaired in human type 1 diabetes. Prior studies have shown that loss of autophagy in the islet is associated with increased beta cell apoptosis, therefore we propose that therapeutic targeting of autophagy pathways may reduce beta cell death in type 1 diabetes development. C_LI

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↗