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Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic β-cells. Whether mitofusin gene expression, and hence, mitochondrial network integrity, is important for glucose or incretin signaling has not previously been explored. Here, we generated mice with β-cell-selective, adult-restricted deletion knock-out (dKO) of the mitofusin genes Mfn1 and Mfn2 (βMfn1/2 dKO). βMfn1/2-dKO mice displayed elevated fed and fasted glycemia and a more than fivefold decrease in plasma insulin. Mitochondrial length, glucose-induced polarization, ATP synthesis, and cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in βMfn1/2-dKO mice, and glucagon-like peptide 1 or glucose-dependent insulinotropic peptide receptor agonists largely corrected defective glucose-stimulated insulin secretion through enhanced EPAC-dependent signaling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps-based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the β-cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in β-cells, the potential contributions of altered mitochondrial dynamics to diabetes development, and the impact of incretins on this process. G.A.R. was supported by a Wellcome Trust Senior Investigator Award (098424AIA) and Wellcome Trust Investigator Award (212625/Z/18/Z), Medical Research Council Programme grants (MR/R022259/1, MR/J0003042/ 1, MR/L020149/1), an Experimental Challenge Grant (DIVA, MR/L02036X/1), a Medical Research Council grant (MR/N00275X/1), and Diabetes UK grants (BDA/11/0004210, BDA/15/0005275, BDA16/0005485). I.L. was supported by a Diabetes UKD project grant (16/0005485). This project has received funding from the European Commission Innovative Medicines Initiative 2 Joint Undertaking, under grant agreement no. 115881 (RHAPSODY). This Joint Undertaking receives support from the European Union’s Horizon 2020 Research and Innovation Programme. This work is supported by the Swiss State Secretariat for Education, Research and Innovation (SERI), under contract no. 16.0097. A.T. was supported by Medical Research Council project grant MR/R010676/ 1. Intravital imaging was performed using resources and/or funding provided by National Institutes of Health grants R03 DK115990 (to A.K.L.), Human Islet Research Network UC4 DK104162 (to A.K.L., RRID:SCR_014393). BJ acknowledges support from the Academy of Medical Sciences, Society for Endocrinology, The British Society for Neuroendocrinology, the European Federation for the Study of Diabetes, an Engineering and Physical Sciences Research Council capital award, and the Medical Research Council (MR/R010676/1). S.A.S. was supported by the JDRF (CDA-2016-189, SRA-2018-539, COE-2019-861), the National Institutes of Health (R01 DK108921, U01 DK127747), and the U.S. Department of Veterans Affairs (I01 BX004444).

Background and Aims: Mitochondrial metabolism of glucose is vital for the initiation of insulin secretion from pancreatic β-cells. Whether mitochondrial ultra-structure, and the proteins controlling mitochondrial fission and fusion, are essential for glucose recognition is less clear. Here, β-cell-restricted deletion of Mfn1 and Mfn2 in adult mice was used to investigate the role of mitochondrial dynamics in insulin secretion and glucose homeostasis in vivo and in vitro. Methods: C57BL6 mice bearing Mfn1 and Mfn2 alleles with FloxP sites, were crossed to animals carrying an inducible Cre recombinase expressed under Pdx1 promoter control (PdxCreERT). Following tamoxifen induction, Mfn1 and Mfn2 knockdown was confirmed in KO islets. Live β-cell fluorescence imaging of Ca2+ accumulation and membrane potential were performed on isolated islets and in the living mouse. Mitochondrial ultra-structure was measured using super resolution fluorescence imaging and EM. Results: Mitochondrial length was strongly and selectively diminished in β-cells from βMfn1/2 KO mice. KO animals displayed higher fasting and fed glycaemia than control animals at 14 weeks and a considerable (>5-fold) decrease in plasma insulin post-IP glucose injection. Additionally, mitochondrial length, glucose-induced hyperpolarization, ATP synthesis, Ca2+ accumulation, O2 consumption and β-cell connectivity were all significantly reduced in βMfn1/2-KO mouse islets. Ca2+ dynamics and mitochondrial membrane potential changes were also suppressed in vivo. Remarkably, defective GSIS in islets isolated from βMfn1/2-KO mice was normalised by the addition of GLP-1 receptor agonists. Conclusions: Mitochondrial fusion and fission cycles are indispensable in the β-cell for normal glucose sensing in vitro and in vivo. GLP-1 receptor agonism may mitigate the effects of mitochondrial abnormalities in some forms of diabetes. Disclosure E. Georgiadou: None. M. Ibberson: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. A. K. Linnemann: None. Y. Ali: None. T. Rodriguez: None. G. A. Rutter: Advisory Panel; Self; Sun Pharmaceutical Industries Ltd. C. Muralidharan: None. P. L. Chabosseau: None. A. Tomas: Other Relationship; Self; Sun Pharmaceutical Industries Ltd. T. Stylianides: None. C. Legido-quigley: None. N. Alsabeeh: None. C. Cruciani-guglielmacci: Consultant; Self; The Healthy Aging Company. C. Magnan: None. Funding UK Wellcome Trust (WT098424AIA, 212625/Z/18/Z); Medical Research Council (MR/R022259/1, MR/J0003042/1, MR/L020149/1, MR/N00275X/1); Experimental Challenge Grant (DIVA, MR/L02036X/1); Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA16/0005485)

Background Bariatric surgery is an effective treatment for type 2 diabetes. Early post-surgical enhancement of insulin secretion is key for diabetes remission. The full complement of mechanisms responsible for improved pancreatic beta cell functionality after bariatric surgery is still unclear. Our aim was to identify pathways, evident in the islet transcriptome, that characterize the adaptive response to bariatric surgery independently of body weight changes. Methods We performed entero-gastro-anastomosis (EGA) with pyloric ligature in leptin-deficient ob/ob mice as a surrogate of Roux-en-Y gastric bypass (RYGB) in humans. Multiple approaches such as determination of glucose tolerance, GLP-1 and insulin secretion, whole body insulin sensitivity, ex vivo glucose-stimulated insulin secretion (GSIS) and functional multicellular Ca2+-imaging, profiling of mRNA and of miRNA expression were utilized to identify significant biological processes involved in pancreatic islet recovery. Findings EGA resolved diabetes, increased pancreatic insulin content and GSIS despite a persistent increase in fat mass, systemic and intra-islet inflammation, and lipotoxicity. Surgery differentially regulated 193 genes in the islet, most of which were involved in the regulation of glucose metabolism, insulin secretion, calcium signaling or beta cell viability, and these were normalized alongside changes in glucose metabolism, intracellular Ca2+ dynamics and the threshold for GSIS. Furthermore, 27 islet miRNAs were differentially regulated, four of them hubs in a miRNA-gene interaction network and four others part of a blood signature of diabetes resolution in ob/ob mice and in humans. Interpretation Taken together, our data highlight novel miRNA-gene interactions in the pancreatic islet during the resolution of diabetes after bariatric surgery that form part of a blood signature of diabetes reversal. Funding European Union's Horizon 2020 research and innovation programme via the Innovative Medicines Initiative 2 Joint Undertaking (RHAPSODY), INSERM, Societe Francophone du Diabete, Institut Benjamin Delessert, Wellcome Trust Investigator Award (212625/Z/18/Z), MRC Programme grants (MR/R022259/1, MR/J0003042/1, MR/L020149/1), Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA 16/0005485) project grants, National Science Foundation (310030–188447), Fondation de l'Avenir.

Objective Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-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 β-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation. Methods We used isolated islets from mice deleted selectively in the β-cell for Stard10 (βStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein. Results βStard10KO 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 versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3’ position. Lipidomic analysis of βStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in βStard10KO islets. Conclusion Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing., Highlights • βStard10KO β-cells show altered granule morphology. • Deletion of Stard10 increased basal secretion of newly synthesised proinsulin. • βStard10KO islets had an altered lipidomics profile, including phosphatidylinositols. • STARD10 bound to phosphoinositides in a lipid overlay assay. • STARD10 structure reveals that its cavity readily accommodates phosphatidylinositols.

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 (βMfn1/2 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.ResultsβMfn1/2 dKO mice displayed elevated fed and fasted glycaemia (pfive-fold decrease (p2+ increases were all reduced (p2+ dynamics were suppressed in vivo (pMfn1/2 dKO mice (pConclusions/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 abstractImpact 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 Δψm), 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 (ψm), 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 (βMfn1/2 dKO), highly fragmented mitochondria were associated with reduced mitochondrial Ca2+ ([Ca2+]m) accumulation, leading to a less polarised Δψm, weaker OCR, lower mtDNA copy number and decreased ATP synthesis. This is expected to result in weaker ψm 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 βMfn1/2 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/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, Δψm, ATP production and beta cell connectivity are impaired in βMfn1/2 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.

Aim: Risk alleles for type 2 diabetes (T2D) in the STARD10 locus, impair insulin secretion and are associated with decreased proinsulin:insulin ratios. We have shown that the T2D risk associated with variation at this locus is likely to be mediated through lowered STARD10 expression in the β cell. Here, we investigate the mechanisms by which STARD10 may regulate insulin secretion. Materials and Methods: A 3-dimensional model of STARD10 was constructed from the structure of STARD2, using the modelling tools Chimera and Modeller. Islets were isolated from StarD10fl/fl-Ins1Cre male mice (βStarD10KO). Electron Microscopy (EM) images were obtained from isolated islets after chemical fixation. Total zinc content was measured by inductively coupled plasma mass spectrometry. Pulse-chase analysis of proinsulin processing was performed using 35S-labelled amino acids. RNA-Seq was done on polyadenylated transcripts selected during the preparation of paired-end, directional RNAseq libraries, sequenced on an Illumina HiSeq 4000 machine. Results: Molecular modelling indicated that STARD10 binds either phosphatidylcholine or phosphatidylethanolamine. EM analysis of islets from βStarD10KO mice revealed a markedly altered dense core granule appearance, with a dramatic increase (Fold change = 4.3; p Conclusion: We identify STARD10 as a critical regulator of insulin granule biogenesis and β cell zinc homeostasis. Our data also suggest that increased β cell Zn2+ secretion in risk allele carriers may decrease the clearance of mature insulin to lower plasma proinsulin:insulin ratio. Disclosure G. Carrat: None. E. Haythorne: None. L. Haataja: None. P. Arvan: None. A. Tomas: None. A. Piunti: None. T.J. Pullen: None. E. Georgiadou: None. T. Stylianides: None. V. Salem: None. W. Distaso: None. A. Cakebread: None. D. Hodson: None. A.C. Fung: None. R.B. Sessions: None. F. Alpy: None. A.P. Kong: Advisory Panel; Self; Lilly Diabetes. Research Support; Self; AstraZeneca, Lilly Diabetes. Speaker's Bureau; Self; Abbott. Other Relationship; Self; AstraZeneca, Novartis Pharmaceuticals Corporation, Sanofi. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma. Funding Medical Research Council UK; UK Wellcome Trust; Royal Society; Societe Francophone du Diabete

Pancreatic β-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca2+ sensor GCaMP6 to assess glucose-regulated connectivity in living zebrafish Danio rerio, and in murine or human islets transplanted into the anterior eye chamber. In each setting, Ca2+ waves emanated from temporally defined leader β-cells, and three-dimensional connectivity across the islet increased with glucose stimulation. Photoablation of zebrafish leader cells disrupted pan-islet signalling, identifying these as likely pacemakers. Correspondingly, in engrafted mouse islets, connectivity was sustained during prolonged glucose exposure, and super-connected ‘hub’ cells were identified. Granger causality analysis revealed a controlling role for temporally defined leaders, and transcriptomic analyses revealed a discrete hub cell fingerprint. We thus define a population of regulatory β-cells within coordinated islet networks in vivo. This population may drive Ca2+ dynamics and pulsatile insulin secretion., info:eu-repo/semantics/published

The deterioration of highway filter drains (HFDs) is driven by the intrusion of foreign particles within the drainage trench. Being a porous material that offers high water removal capacity at the beginning of its service life, the drainage performance of the backfill can be significantly reduced in time by the introduced fouling material. This poses a serious safety hazard for road users (standing water on the carriageway), and can potentially have an effect in the structural capacity of the pavement. With currently limited approaches to methodically evaluate the physical condition of such assets, the Ground Penetrating Radar (GPR) offers an effective, non-destructive and continuous way to achieve this at both the project and network level. The laboratory calibration study carried out to support its adoption in a condition assessment system, builds upon the evaluation of a HFD-specific condition index aligned to permeability trials and the extraction of dielectric properties of the granular backfill material at different fouled states. The paper thus discusses what kind of HFD distress information are to be collected (condition data) and how this can be achieved (data collection methods), and defines four distinctive HFD media condition bands (Excellent to Very Poor) based on the proposed free voids ratio ( R FV ) ranges and extracted relative permittivity values.

Pancreatic β-cells are responsible for production and secretion of insulin in response to increasing blood glucose levels. Defects in β-cell function lead to hyperglycemia and diabetes mellitus. Here, we show that CNOT3, a CCR4–NOT deadenylase complex subunit, is dysregulated in islets in diabetic db/db mice, and that it is essential for murine β cell maturation and identity. Mice with β cell-specific Cnot3 deletion (Cnot3βKO) exhibit impaired glucose tolerance, decreased β cell mass, and they gradually develop diabetes. Cnot3βKO islets display decreased expression of key regulators of β cell maturation and function. Moreover, they show an increase of progenitor cell markers, β cell-disallowed genes, and genes relevant to altered β cell function. Cnot3βKO islets exhibit altered deadenylation and increased mRNA stability, partly accounting for the increased expression of those genes. Together, these data reveal that CNOT3-mediated mRNA deadenylation and decay constitute previously unsuspected post-transcriptional mechanisms essential for β cell identity., Dina Mostafa et al. report that β cell function and identity depends on the deadenylase complex subunit CNOT3. The authors found that Cnot3 was dysregulated in diabetic mice and, when knocked out in wild type, mice have impaired glucose tolerance and gradually develop diabetes.

Imaging of Ca2+ dynamics in the intact islet in vitro has recently been subjected to connectivity analysis informed by similar approaches in neuronal systems. This has revealed the existence of intercellular networks between individual β cells which are increased by high glucose. Here, we sought to investigate if network connectivity also exists in islets in vivo under conditions of near-normal vascularisation and innervation. Donor mouse (C57BL/6J) islets were infected with adenovirus expressing the Ca2+ sensor GCaMP6m and transplanted into the anterior chamber of the eye in syngeneic recipients. Under isofluorane anaesthesia, fully implanted islets were imaged using a spinning disk confocal microscope (Nikon Ti-E, 25 x water dipping 1.0 NA objective, 300ms exposure time, 1 frame per second, 488nm excitation). Mean baseline blood glucose was 19mM and rose to >30mM following an IP bolus of 1.5g/L glucose. Human islets were infected with AV-GCaMP6m, transplanted into a nu/nu mouse recipient and imaged under conditions of normoglycaemia (blood glucose 3.9-4.9 mM followed by hyperglycaemia (blood glucose >30 mM). As circulating blood glucose increased, the proportion of connected cells in mouse islets rose from 65.5% to 88.3% (n=3 islets in 3 different animals, p=0.02) whilst the coefficient of correlation (a measure of the strength of the connectivity) showed a tendency to increase from 0.34 to 0.44 (p=NS). Data from human islets also revealed a tendency towards a rise in connectivity from 58.3% to 63.9% in response to rising circulating glucose. These data demonstrate that functional connectivity of Ca2+ dynamics between β cells is conserved in vivo in both mouse and human islets, with enhanced connectivity patterns observed in response to rising blood glucose levels. This technique, which allows β cell connectivity to be examined prospectively over time, will provide a powerful new approach for assessing its significance in health and disease. Disclosure V. Salem: None. K. Suba: None. A. Martin Alonso: None. P.L. Chabosseau: None. E. Georgiadou: None. T. Stylianides: None. L. Briant: None. D. Hodson: None. G. Carrat: None. I. Leclerc: None. D.C. Gaboriau: None. S.M. Rothery: None. G.A. Rutter: Research Support; Self; Servier.

Pancreatic β-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca