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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)

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

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.