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Bariatric surgery improves glucose homeostasis but the underlying mechanisms are not fully elucidated. Here, we show that the expression of sodium glucose cotransporter-2 (SGLT2/Slc5a2) is reduced in the kidney of lean and obese mice following vertical sleeve gastrectomy (VSG). Indicating an important contribution of altered cotransporter expression to the impact of surgery, inactivation of the SGLT2/Slc5a2 gene by CRISPR/Cas9 attenuated the effects of VSG, with glucose excursions following intraperitoneal injection lowered by ~30% in wild-type mice but by ~20% in SGLT2 null animals. The effects of the SGLT2 inhibitor dapaglifozin were similarly blunted by surgery. Unexpectedly, effects of dapaglifozin were still observed in SGLT2 null mice, consistent with the existence of metabolically beneficial off-target effects of SGLT2 inhibitors. Thus, we describe a new mechanism involved in mediating the glucose lowering effects of bariatric surgery.

Aims/hypothesis Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site. Methods AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-βH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for β1- vs β2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell. Results Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (ppp2+ dynamics in response to 991 (AUC, ppppp Conclusions/interpretation AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators. Graphical abstract

Background Soluble Resistance Related Calcium Binding Protein (sorcin) is a calcium (Ca2+) binding protein which has been shown to play a role in maintaining intracellular endoplasmic reticulum (ER) Ca2+ stores and lowering ER stress. Recently, our lab has demonstrated that sorcin expression was downregulated in the islets of Langerhans of mice fed a high-fat diet or in human islets incubated with the saturated fatty acid palmitate. We also showed that overexpression of sorcin under control of the rat insulin promoter (RIP7) in C57BL/6J mice, or whole body sorcin deletion in 129S1/SvImJ mice, improves or impairs insulin secretion and pancreatic β-cell function respectively. The mechanisms behind this beneficial role of sorcin in the pancreatic β-cell might depend on protection against lipotoxic endoplasmic reticulum (ER) stress through improved ER Ca2+ dynamics and activation of the Activating Transcription Factor 6 (ATF6) branch of the unfolded protein response (UPR). Whether sorcin is also implicated in hypothalamic ER stress during the progression of obesity is unknown. Aim To investigate a potential role of sorcin in hypothalamic ER stress, leptin resistance, hyperphagia and obesity. Methods Whole-body sorcin null mice, backcrossed onto the C57BL/6J genetic background, were used. Body weight, food intake and EchoMRI body composition were measured in vivo whereas qRT-PCR analysis of sorcin and ER stress markers expression were performed on the arcuate nucleus of the hypothalamus. Leptin signalling through STAT3 phosphorylation was measured by Western blots on sorcin-null HEK293 cells, engineered by CRISPR/Cas9, and transfected with leptin receptor (LepRb). Results Sorcin expression was not influenced in the arcuate nucleus (ARC) of the hypothalamus by diet-induced obesity. Whole-body sorcin ablation did not cause ARC ER stress nor changes in body weight, body composition or food intake in C57BL/6 male mice exposed to a high-fat, high-sugar diet. STAT3 phosphorylation (Y705) in response to leptin was not impaired in sorcin-null HEK293 cells. Conclusion In our model, whole body sorcin ablation did not increase hypothalamic ER stress nor influenced food intake or body weight.

BackgroundSoluble Resistance Related Calcium Binding Protein (sorcin) is a calcium (Ca2+) binding protein which has been shown to play a role in maintaining intracellular endoplasmic reticulum (ER) Ca2+ stores and lowering ER stress. Recently, our lab has demonstrated that sorcin expression was downregulated in the islets of Langerhans of mice fed a high-fat diet or in human islets incubated with the saturated fatty acid palmitate. We also showed that overexpression of sorcin under control of the rat insulin promoter (RIP7) in C57BL/6J mice, or whole body sorcin deletion in 129S1/SvImJ mice, improves or impairs insulin secretion and pancreatic β-cell function respectively. The mechanisms behind this beneficial role of sorcin in the pancreatic β-cell might depend on protection against lipotoxic endoplasmic reticulum (ER) stress through improved ER Ca2+ dynamics and activation of the Activating Transcription Factor 6 (ATF6) branch of the unfolded protein response (UPR). Whether sorcin is also implicated in hypothalamic ER stress during the progression of obesity is unknown. This could potentially contribute to the diminished satiety typically observed in overweight individuals.AimTo investigate a potential role of sorcin in hypothalamic ER stress, leptin resistance, hyperphagia and obesity.MethodsWhole-body sorcin null mice, backcrossed onto the C57BL/6J genetic background, were used. Body weight, food intake and EchoMRI body composition were measured in vivo whereas qRT-PCR analysis of sorcin and ER stress markers expression were performed on the arcuate nucleus of the hypothalamus. Leptin signalling through STAT3 phosphorylation was measured by Western blots on sorcin-null HEK293 cells, engineered by CRISPR/Cas9, and transfected with leptin receptor (LepRb).ResultsSorcin expression was not influenced in the arcuate nucleus (ARC) of the hypothalamus by diet-induced obesity. Whole-body sorcin ablation did not cause ARC ER stress nor changes in body weight, body composition or food intake in C57BL/6 male mice exposed to a high-fat, high-sugar diet. STAT3 phosphorylation (Y705) in response to leptin was not impaired in sorcin-null HEK293 cells.ConclusionIn our model, whole body sorcin ablation did not increase hypothalamic ER stress nor influenced food intake or body weight.

AimsSpatially-organised increases in cytosolic Ca2+within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including “leaders” which initiate Ca2+waves. Whether leader cells possess unique molecular features, or localisation, is unknown.Main methodsHigh speed confocal Ca2+imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected “hub” cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing (“Flash-Seq”).Key findingsDistinct Ca2+wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29% of islets, though “hub” cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padjvsfollower cells. Leader cells were also located significantly closer to delta cells in Euclidian space than were follower cells.SignificanceThe existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. Specifically, these data raise the possibility of altered signalling from delta cells towards somatostatin receptors present on leader cell cilia.

Pulsatile increases in Ca2+ within beta cells underlie the stimulation of insulin secretion at elevated glucose concentrations. Recent data obtained by imaging these transients in intact islets have revealed the existence of subpopulations of beta cells (“leaders”) which initiate Ca2+ waves across the syncytium and are critical for these dynamics. Whether leader cells possess unique transcriptional features is unknown. By combining Ca2+ imaging with cell labelling and isolation, we sought to address this question. Method: Islets were isolated from C57BL6/J mice expressing the Ca2+ probe GCaMP6f selectively in beta cells. After infection with an adenovirus expressing photo-activable- (PA) mCherry, Ca2+ imaging was performed at 11mM glucose at 5 Hz. Leader (or follower) cells were identified and photopainted by PA-mCherry activation at 405nm. After islet dispersal, cell sorting was used to isolate single cells. cDNA libraries were obtained using NEBNext Single cell kit and used for sequencing (Illumina HiSeq 4000) . Reads were aligned to the mmtranscriptome with Salmon v1.3 and differential expression analysis performed with DESeq2. Differentially expressed genes were subjected to Gene Ontology analysis. Results: cDNA was obtained from 13 leader cells, identified as those exhibiting the first Ca2+ signal increase during Ca2+ waves, and 9 follower cells. Of 96 differentially expressed genes, the most strongly affected included cell adhesion proteins (Cdh22; Scarf2) and the calcium sensor Mctp1. Other genes identified were involved in mitochondrial Ca2+ uptake (Mcub) , cytochrome assembly (Tmem177) and type 2 diabetes risk (Tcf7l2) . GO analysis identified 9 enriched functional clusters, including protein ubiquitination and metal ion binding. Conclusion: Leader cells possess a stably altered transcriptome, and the affected genes include several which may regulate cell adhesion. The extent to which the identified genes reflect the contribution of these cells to Ca2+ wave initiation requires further study. Disclosure P.L.Chabosseau: n/a. S.Yong: None. L.Lopez: None. I.Leclerc: None. Y.Ali: n/a. A.Martinez-sanchez: None. G.A.Rutter: Advisory Panel; Sun Pharmaceutical Industries Ltd., Research Support; Sun Pharmaceutical Industries Ltd.

Aims: To describe the in vitro characteristics and antidiabetic in vivo efficacy of the novel glucagon-like peptide-1 receptor agonist (GLP-1RA) GL0034. Materials and Methods: Glucagon-like peptide-1 receptor (GLP-1R) kinetic binding parameters, cyclic adenosine monophosphate (cAMP) signalling, endocytosis and recycling were measured using HEK293 and INS-1832/3 cells expressing human GLP-1R. Insulin secretion was measured in vitro using INS-1832/3 cells, mouse islets and human islets. Chronic administration studies to evaluate weight loss and glycaemic effects were performed in db/db and diet-induced obese mice. Results: Compared to the leading GLP-1RA semaglutide, GL0034 showed increased binding affinity and potency-driven bias in favour of cAMP over GLP-1R endocytosis and β-arrestin-2 recruitment. Insulin secretory responses were similar for both ligands. GL0034 (6 nmol/kg) led to at least as much weight loss and lowering of blood glucose as did semaglutide at a higher dose (14 nmol/kg). Conclusions: GL0034 is a G protein-biased agonist that shows powerful antidiabetic effects in mice, and may serve as a promising new GLP-1RA for obese patients with type 2 diabetes.

AIMS: Spatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including "leaders" which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. MAIN METHODS: High speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected "hub" cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing ("Flash-Seq"). KEY FINDINGS: Distinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though "hub" cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj

Aims/hypothesis Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. Methods CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. Results Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. Conclusions/interpretation Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that 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 humans. Data availability The datasets generated and/or analysed during the current study are available in the Biorxiv repository (www.biorxiv.org/content/10.1101/2020.05.18.099200v1). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576) and ProteomeXchange (www.ebi.ac.uk/pride/archive/projects/PXD021597) repositories, respectively. Graphical abstract

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).

Bariatric surgery improves both insulin sensitivity and secretion and can induce diabetes remission. However, the mechanisms and time courses of these changes, particularly the impact on β cell function, are difficult to monitor directly. In this study, we investigated the effect of Vertical Sleeve Gastrectomy (VSG) on β cell function in vivo by imaging Ca2+ dynamics in islets engrafted into the anterior eye chamber. Mirroring its clinical utility, VSG in mice results in significantly improved glucose tolerance, and enhanced insulin secretion. We reveal that these benefits are underpinned by augmented β cell function and coordinated activity across the islet. These effects involve changes in circulating GLP-1 levels which may act both directly and indirectly on the β cell, in the latter case through changes in body weight. Thus, bariatric surgery leads to time-dependent increases in β cell function and intra-islet connectivity which are likely to contribute to diabetes remission., Bariatric surgery procedures reduce weight and improve metabolic diseases. Here the authors investigate the effects of Vertical Sleeve Gastrectomy (VSG) on beta-cell function in mice using in vivo Ca2 + imaging and show that VSG increases beta-cell function and intra-islet connectivity.

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)

β-arrestin-2 (Barr2) is typically associated with G protein-coupled receptor desensitization and endocytosis, and reduced Barr2 recruitment is thought to enable prolongation of pharmacodynamic effects of biased glucagon-like peptide-1 receptor (GLP-1R) agonists. However, the precise role of Barr2 in GLP-1R function in β-cells is poorly understood. To study this role, a tamoxifen-inducible β-cell specific Barr2 knockout mouse model was generated (Pdx1CreERT/Barr2 fl/fl, referred to as β-Barr2 KO). β-Barr2 KO animals on high-fat high-sucrose diet were less glucose tolerant and tended to respond worse to acute exendin-4 (Ex4) treatment vs. controls (Barr2 fl/fl). However, Ex4 effects on blood glucose were more pronounced in KO mice 6h post-agonist injection [IPGTT AUC (6h) for Ex4/ vehicle: 0.84 ± 0.04 vs. 1.08 ± 0.05, p Disclosure S. Bitsi: None. K. Suba: None. N. Mohamed: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. G. A. Rutter: Advisory Panel; Self; Sun Pharmaceutical Industries Ltd. V. Salem: None. B. Jones: Research Support; Self; Sun Pharmaceutical Industries Ltd. A. Tomas: Other Relationship; Self; Sun Pharmaceutical Industries Ltd. Funding Medical Research Council (MR/R010676/1)

Background and Aims: To characterize a novel incretin analogue which engages glucagon-like peptide-1 (GLP1R) and glucose-dependent insulinotropic peptide (GIPR) receptors. Methods: Potencies for cAMP production were determined at GLP-1R and GIPR in Flp-In T-REx-293 cells. Assessment of SNAP-tagged GLP-1R internalisation by confocal microscopy, incorporation of GLP-1R into lipid rafts, Gs recruitment to GLP-1R and GIPR with NanoBiT™ used INS-1(832/3) beta cells. Ca2+ responses were recorded in mouse islets by time-lapse confocal microscopy using Cal520 AM. Results: HISHS-2001 displayed higher potency versus tirzepatide for cAMP production at both receptors (Table). GLP-1R beta arrestin-2 recruitment was lower with HISHS-2001, indicating increased bias. Efficacy and potency of recruitment of Gs protein to the GLP-1R were lower for HISHS-2001 vs. tirzepatide (Table). HISHS-2001 showed increased potency for Gs recruitment to the GIPR. GLP-1R endocytosis was lower for HISHS-2001 vs. tirzepatide but both agonists showed increased recruitment of GLP-1R to lipid rafts vs. semaglutide. Ca2+ responses, and potentiation of insulin secretion from mouse islets tended to be higher for HISHS-2001 vs. tirzepatide. Both displayed enhanced insulin secretion from human islets vs. semaglutide (Table). Conclusions: HISHS-2001 may provide a useful new GLP1R-GIP dual agonist. Disclosure Y. Manchanda: None. G. A. Rutter: Advisory Panel; Self; Sun Pharmaceutical Industries Ltd. B. Jones: Research Support; Self; Sun Pharmaceutical Industries Ltd. G. Carrat: None. Z. Ramchunder: None. P. Marchetti: Consultant; Self; Menarini Group. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. R. Thennati: None. V. S. Burade: None. A. Tomas: Other Relationship; Self; Sun Pharmaceutical Industries Ltd. Funding Sun Pharmaceutical Industries Ltd. (P79787)

Pancreatic beta cells form a highly connected network within the islet, with transcriptomic and functional heterogeneity existing between individual cells. A sub-population of leader cells has been proposed to act as functional hubs, controlling islet responses to glucose and other secretagogues. Using high speed confocal Ca2+ imaging, we aimed here to examine (a) the role of these cells in wave propagation and (b) their stability over time. Methods: Islets were isolated from C57BL6 male mice expressing the recombinant Ca2+ probe GCaMP6f selectively in the beta cell (Ins1Cre:GCaMP6f/f). Islets were then infected with an adenovirus expressing photo-activatable- (PA) mCherry. Ca2+ imaging was performed across a single islet plane in modified KREBS-Ringer medium at 11mM glucose using a Zeiss LSM 810 laser-scanning microscope and data acquisition at 5 Hz. Cells were photopainted by excitation of PA-mCherry at 405nm. Results: Leader cells were identified as those exhibiting the first detectable increase in Ca2+ signal during subsequent Ca2+ waves. The great majority of islets (85%, n=26, three independent experiments) displayed repetitive Ca2+ waves (1.10±0.26 min-1), and 82.9±5.0% of waves propagated directly from an identifiable leader cell. There was an average of 2.5±0.4 leader cells per islet with 62.9±0.70% of waves emanating from a “principal” leader cell. Ca2+ imaging experiments performed 16 h after the original session revealed that 37±0.08% (n=4) of waves emanated from the same, now photopainted, leader cell. Conclusions: These data suggest that (a) a complex, non-binary, heterogeneity exists between leader and follower beta cells, (b) leader behaviour is an intrinsic phenotype of a sub-population of cells with a half-life in this role of ∼12 h. Disclosure P. L. Chabosseau: None. A. Martinez-sanchez: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. V. Salem: None. G. A. Rutter: Advisory Panel; Self; Sun Pharmaceutical Industries Ltd.

Impaired pancreatic {beta}-cell function and insulin secretion are hallmarks of type 2 diabetes. MicroRNAs are short non-coding RNAs that silence gene expression, vital for the development and function of endocrine cells. MiR-125b is a highly conserved miRNA abundant in {beta}-cells, though its role in these cells remains unclear. Here, we show that miR-125b expression in human islets correlates with body mass index (BMI) of the donors and is regulated by glucose in an AMP-activated protein kinase-dependent manner in both mice and humans. Using and unbiased high-throughput approach, we identify dozens of direct gene targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Whereas inactivation of miR-125b in human beta cells led to shorter mitochondria and improved glucose stimulated insulin secretion, mice over-expressing mir-125b selectively in {beta}-cells displayed defective insulin secretion and marked glucose intolerance. Moreover, the {beta}-cells of these transgenic animals showed strongly reduced insulin content and secretion and contained enlarged lysosomal structures. Thus, miR125b provides a glucose-controlled regulator of organelle dynamics that negatively regulates insulin secretion in {beta}-cells. HighlightsO_LIIslet miR125b correlates with BMI and is regulated by glucose via AMP-activated protein kinase in {beta}-cells C_LIO_LImiR125b targets dozens of genes including those involved in the regulation of mitochondrial (i.e. Mtfp1) and lysosomal (i.e. M6pr) morphology/function C_LIO_LIDeletion of miR125b results in shorten mitochondria and improves glucose stimulated insulin secretion (GSIS) C_LIO_LISelective overexpression of miR125b in {beta}-cells causes the appearance of enlarged lysosomes, reduced GSIS and insulin content, loss of {beta}-cell identity and impaired insulin secretion and glucose tolerance in vivo C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/444559v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1772ccdorg.highwire.dtl.DTLVardef@11e6e76org.highwire.dtl.DTLVardef@e62b81org.highwire.dtl.DTLVardef@132405c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Since 2008, cancer became the first cause of death in France, exceeding cardiovascular diseases. The part of occupational cancers is close to 5% of all cancers but may still be widely underestimated. The detection of occupational cancer risk factors remains complicated. Indeed, most occupational risk factors are also present in the general environment. Thus far, 36 substances and 13 activities have been classified as carcinogenic (Group 1) and 37 substances and 6 activities have been classified as probably carcinogenic (Group 2A) by the International Agency for Research on Cancer (IARC). The most common cancer site is the lung, followed by bladder and skin (except melanoma). The most frequently observed occupational exposure was chemicals, building and construction, mining, metal production and coal transformation. Thus far, preventive actions are mandatory in occupational health in France, and are about occupational exposure monitoring, protection of vulnerable populations and reinforced medical supervision. Research must be continued to improve the knowledge on occupational carcinogens, in order to better protect the health of workers.

Sorcin is a calcium-binding protein involved in maintaining endoplasmic reticulum (ER) Ca

Aims/hypothesis Transcription factor 7-like 2 (TCF7L2) is a downstream effector of the Wnt/β-catenin signalling pathway implicated in type 2 diabetes risk through genome-wide association studies. Although its expression is critical for adipocyte development, the potential roles of changes in adipose tissue TCF7L2 levels in diabetes risk are poorly defined. Here, we investigated whether forced changes in Tcf7l2 expression in adipocytes affect whole body glucose or lipid metabolism and crosstalk between disease-relevant tissues. Methods Tcf7l2 was selectively ablated in mature adipocytes in C57BL/6J mice using Cre recombinase under Adipoq promoter control to recombine Tcf7l2 alleles floxed at exon 1 (referred to as aTCF7L2 mice). aTCF7L2 mice were fed normal chow or a high-fat diet for 12 weeks. Glucose and insulin sensitivity, as well as beta cell function, were assessed in vivo and in vitro. Levels of circulating NEFA, selected hormones and adipokines were measured using standard assays. Results Reduced TCF7L2 expression in adipocytes altered glucose tolerance and insulin secretion in male but not in female mice. Thus, on a normal chow diet, male heterozygote knockout mice (aTCF7L2het) exhibited impaired glucose tolerance at 16 weeks (p = 0.03) and increased fat mass (1.4 ± 0.1-fold, p = 0.007) but no changes in insulin secretion. In contrast, male homozygote knockout (aTCF7L2hom) mice displayed normal body weight but impaired oral glucose tolerance at 16 weeks (p = 0.0001). These changes were mechanistically associated with impaired in vitro glucose-stimulated insulin secretion (decreased 0.5 ± 0.1-fold vs control mice, p = 0.02) and decreased levels of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (0.6 ± 0.1-fold and 0.4 ± 0.1-fold vs control mice, p = 0.04 and p p = 0.03 and p = 0.05, respectively). Following exposure to a high-fat diet for 12 weeks, male aTCF7L2hom mice exhibited reduced in vivo glucose-stimulated insulin secretion (0.5 ± 0.1-fold vs control mice, p = 0.02). Conclusions/interpretation Loss of Tcf7l2 gene expression selectively in adipocytes leads to a sexually dimorphic phenotype, with impairments not only in adipocytes, but also in pancreatic islet and enteroendocrine cells in male mice only. Our findings suggest novel roles for adipokines and incretins in the effects of diabetes-associated variants in TCF7L2, and further illuminate the roles of TCF7L2 in glucose homeostasis and diabetes risk.

BackgroundGlucocorticoids produced by the adrenal cortex are essential for the maintenance of metabolic homeostasis. Glucocorticoid activation is catalyzed by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) and signalling is achieved through the glucocorticoid receptor (GR), a ligand-dependent transcription factor. Excess glucocorticoids are associated with insulin resistance and hyperglycaemia. A small number of studies have investigated the effects of bariatric surgery, a gastrointestinal procedure known to improve insulin sensitivity, on glucocorticoid metabolism, but the hypothesised mechanism is assumed to be via weight loss.AimTo investigate the effect of bariatric surgery on glucocorticoid metabolism in lean and obese mice.MethodsLean mice and HFD mice underwent Vertical Sleeve Gastrectomy (VSG) or sham surgery. Glucose and insulin tolerance tests were performed at four and ten weeks post operatively and circulating corticosterone was measured. Liver and adipose tissues were harvested from fed mice and 11β-HSD1 and GR levels were measured by quantitative RT-PCR or Western (immuno-) blotting, respectively.ResultsVSG did not cause excess weight loss in lean mice when compared to sham operated mice. However, both lean and HFD VSG mice displayed significantly improved glucose clearance and insulin sensitivity. Remarkably, VSG restores physiological corticosterone production in HFD mice and reduces11β-HSD1 levels at four and ten weeks post-surgery. Additionally, lean mice displayed significantly lowered mRNA levels of 11β-HSD1 in subcutaneous adipose tissue and GR in liver.ConclusionsBariatric surgery improves insulin sensitivity and reduces glucocorticoid activation at tissular level, under physiological and pathophysiological (obesity) conditions, irrespective of weight loss. The reduction of glucocorticoid exposure may represent an additional contribution to the health benefits of bariatric surgery. These findings point towards a physiologically relevant gut-adrenal axis.

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of {beta}-cell development and function. Consequently, the mis-expression of members of this group may contribute to the risk of type 2 diabetes (T2D). Here, we investigate roles for an antisense lncRNA expressed from the Pax6 locus (annotated as Pax6os1 in mice and PAX6-AS1 in humans) in {beta}-cell function. The transcription factor Pax6 is required for the development of pancreatic islets and maintenance of a fully differentiated {beta}-cell phenotype. Pax6os1/PAX6-AS1 expression was increased in pancreatic islets and {beta}-cell lines at high glucose concentrations, in islets from mice fed a high fat diet, and in those from patients with type 2 diabetes. Silencing or deletion of Pax6os1/PAX6-AS1 in MIN6 cells and EndoC-{beta}H1cells, respectively, upregulated {beta}-cell signature genes, including insulin. Moreover, shRNA-mediated silencing of PAX6-AS1 in human islets not only increased insulin mRNA, but also enhanced glucose-stimulated insulin secretion and calcium dynamics. In contrast, inactivation of Pax6os1 in mice was largely without effect on glucose homeostasis, though female Pax6os1 null mice on high fat diet (HFD) showed a tendency towards enhanced glucose clearance. Together, our results suggest that increased expression of PAX6-AS1 at high glucose levels may contribute to {beta}-cell dedifferentiation and failure in some forms of type 2 diabetes. Thus, targeting PAX6-AS1 may provide a promising strategy to enhance insulin secretion and improve glucose homeostasis in type 2 diabetes.

Background: Glucocorticoids (GC) produced by the adrenal cortex are essential for the maintenance of metabolic homeostasis. GC activation is catalyzed by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) and signalling is achieved through the glucocorticoid receptor (GR), a ligand-dependent transcription factor. Excess glucocorticoids are associated with insulin resistance and hyperglycaemia. A small number of studies have investigated the effects of bariatric surgery, a gastrointestinal procedure known to improve insulin sensitivity, on glucocorticoid metabolism, but the hypothesised mechanism is assumed to be via weight loss. Aim: To investigate the effect of bariatric surgery on glucocorticoid metabolism in lean and obese mice. Methods: Fourteen lean mice and twelve HFD mice underwent Vertical Sleeve Gastrectomy (VSG) (n=7-8) or sham (n=5-6) surgery. Glucose and insulin tolerance tests were performed four weeks post operatively. Liver, gut and adipose tissue were harvested from fed mice and analysed using Quantitative real-time PCR (qRT-PCR) and Western Blot (WB). Results: VSG did not cause excess weight loss in lean mice when compared to sham operated mice (p=0.37). However, both lean and HFD VSG mice displayed significantly improved glucose and insulin tolerance (p Conclusions: Bariatric surgery improves insulin sensitivity and reduces glucocorticoid activation physiologically and in the obese state, irrespective of weight loss. The reduction in glucocorticoid signalling may represent an additional contribution to the health benefits of bariatric surgery. These findings point towards a physiologically-relevant gut-adrenal axis. Disclosure E. Akalestou: None. L. Lopez Noriega: None. I. Christakis: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. Funding Rosetrees Foundation (M825); British Society for Neuroendocrinology; Society for Endocrinology (19ES001); UK Wellcome Trust (212625/Z/18/Z); Medical Research Council UK (MR/R022259/1); European Union (115881)

Aims: Long-term outcomes for islet transplantation remain suboptimal. An important determinant of islet transplantation success is vascularisation of engrafted islets. The epidermal growth factor receptor (EGFR) is a key angiogenic and pro-survival factor. In an in vivo mouse model, we investigated if EGFR overexpression in pancreatic islets improves engraftment. Methods: Donor islets from C57BL/6 mice were infected with adenoviruses generated to encoding human EGFR alongside a GFP reporter. To assess the effects of EGFR over-expression on beta-cell identity and function, qPCR was run to determine expression of angiogenesis, beta-cell enriched and beta-cell disallowed genes compared with islets infected with control (empty vector) virus. To assess the effects of EGFR-overexpression on implantation in vivo, EGFR-overexpressing or control (empty vector) islets were transplanted into the anterior chambers of the eyes of syngeneic recipients. Using confocal microscopy, islet volume (GFP signal) and blood vessel density (following IV administration of dextran conjugated Texas Red) were assessed regularly and longitudinally over 30 days. Results: qPCR results showed that EGFR-overexpressing islets had greater gene expression of angiogenic factors. Consistent with preserved beta-cell identity, beta-cell enriched genes were upregulated in EGFR-overexpressing islets compared to control islets, whilst beta-cell disallowed genes were repressed. EGFR-overexpressing islets demonstrating greater density compared to control islets at all time points (n=5 animals per group, 1-5 islets per eye, p Conclusion: We are the first to demonstrate that EGFR overexpression can promote the vascularisation of transplanted islets in vivo. The transcriptional profiling of these islets suggests an improved potential for beta-cell regenerative capacity and function. Disclosure V. Salem: None. U. Ali: None. K. Suba: None. S. Bitsi: None. T. Lopes: None. A. Martin Alonso: None. Y.S. Patel: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. B. Owen: None. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. S.M. Rothery: None. A. Tomas: None. Funding Diabetes UK (15/0005317)

Aims: GLP-1 (GLP-1R) and glucagon (GCGR) receptor co-agonists are in development to treat type 2 diabetes. The effect of chronic GCGR agonism on weight loss is well-documented, but its direct influence on islet function remains poorly understood. We investigated the effects of a long-acting glucagon analogue G778 on pancreatic islet calcium dynamics. Methods: C57Bl/6 (n=9 per group) mice with diet-induced obesity (DIO) received daily subcutaneous injections of G778 100nmol/kg or saline (and calorie restricted to achieve weight matching) for 40 days. All mice had syngeneic islets with beta-cell specific expression of the calcium fluorophore GCaMP6f implanted in the anterior chamber of the eye, providing a direct, longitudinal readout of beta-cell function in vivo using confocal microscopy. Such islets were tested in vitro to measure the effects of G778 on whole islet calcium dynamics. Another experiment measured the effects of G778 administration on oxygen consumption in a CLAMS system. Results: G778 produced a small but significant rise in energy expenditure (EE) in mice. At 40 days, there was non-significant difference in body weight loss between the saline and G778-treated groups (0.9g ±1.1 vs. 3.4g ±0.7 p=0.07), but a marked improvement in glucose tolerance in mice treated long term with the glucagon analogue (15 min glucose on a 2mg/kg IPGTT 15.9 mmol/L vs. 9.2 mmol/L p Summary: Our data suggest that the glucagon element of a glucagon/GLP-1 analogue contributes to weight-loss partly through an elevation in EE but also has independent effects on insulin secretory function, promoting more robust and sustained beta-cell calcium responses. Disclosure K. Suba: None. Y.S. Patel: None. A. Martin Alonso: None. R. Scott: None. J.S. Minnion: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. B. Owen: None. W. Distaso: None. T.M. Tan: None. K. Murphy: None. S. Bloom: None. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. V. Salem: None. Funding Diabetes UK (15/0005317)

Background and Aims: We have sought to develop novel GLP-1R agonists (RA) with improved insulinotropic effects on the pancreatic beta cell, and extended duration of action. Here, we have explored the affinity for the receptor, and action on signalling and insulin secretion, of three novel compounds. Methods: Agonist dissociation kinetics were assessed by time-resolved FRET using SNAP-tagged GLP-1R-expressing HEK293 cells and fluorescent exendin-(9-39)-FITC. Signal bias was quantified by comparing the generation of cAMP and beta-arrestin recruitment to the GLP-1R. Recycling was measured in INS-1(832/3) cells expressing SNAP-GLP-1R, and time-lapse confocal imaging on a Zeiss LSM-780 inverted microscope. Insulin secretion was measured in vitro in islets isolated from lean C57BL6 J mice. Time-resolved FRET was used to quantify released and total hormone. Semaglutide (sema.) was supplied by Sun Pharma. Results: GL0001, GL0028 and GL0034 showed similar binding but slower dissociation kinetics at the GLP-1R, leading to a higher binding affinity (pKd ca. 7.6) vs. semaglutide (pKd ca. 7.4). A slightly higher bias (1.3-, 1.5- and 1.5-fold, respectively, n=5) towards beta-arrestin vs. cAMP signalling was also apparent for each RA vs. sema). Coupling to cAMP generation (pEC50-pKd) was lower (1.7-, 2.0- and 1.8-fold, respectively) than sema. for each RA. Whereas endocytosis of SNAP-GLP-1R was essentially complete in 5 min. after RA (100 nM) addition for sema. and GL0034, uptake of labelled receptor was barely detectable even at 10 min. for GL0028. Efficacy in potentiating insulin secretion from isolated mouse islets at 11 mM glucose, assessed after 30 min., was similar for GL0028 and sema. Conclusion: The novel agents GL0001 and GL0034 show similar binding properties, and impact on GLP-1R uptake, compared to sema. The more limited receptor uptake for GL0028 vs. other RA may influence the balance of central vs. pancreatic actions on insulin secretion and glucose homeostasis. Disclosure G. Carrat: None. M. Nguyen-Tu: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. R. Thennati: None. B. Jones: None. A. Tomas: None. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. Funding Medical Research Council (R022259/1); UK Wellcome Trust (212625/Z/18/Z); European Union (115881)

Background: Bariatric surgery improves both insulin sensitivity and secretion in T2D. However, the impact on insulin secretion is difficult to monitor directly, requiring correction for insulin sensitivity. The degree to which surgery impacts β-cell function versus mass, and the time course over which these changes occur, also remain unclear. Aim: To investigate the effect of bariatric surgery on β-cell function in vivo, we imaged Ca2+ dynamics prospectively at the cellular level in islets engrafted into the anterior eye chamber. Methods: Ins1Cre mice were crossed to animals that express the genetic calcium indicator GCaMP6f behind a Lox PSTOPLoxP cassette. Isolated islets were engrafted into male C57BL6 mice maintained for 12 weeks on high fat/high sucrose diet (HFD). At pre- (-1) and post-operative weeks 4, 8 and 12, Vertical Sleeve Gastrectomy (VSG) (n=7) and sham (n=6) groups were anaesthetized, and islets imaged in the eye using a Nikon microscope equipped with a spinning disc, 488 nm laser and 20x/1.0 water immersion objective. Islets were scored as positive for wave activity when these were recurrent, had a defined origin and covered >75 % of the image plane. Glucose (3g/kg) tolerance and insulin secretion were assessed in parallel. Results: The VSG group demonstrated substantial weight loss during the first 4 weeks post-operatively but regained pre-operative weight by week 12. Nonetheless, VSG mice displayed significantly improved glucose tolerance and insulin secretion (p Conclusions: By imaging islet function in the eye in vivo we demonstrate that bariatric surgery leads to a time-dependent increase in glucose-induced Ca2+ dynamics of individual islets, likely to underlie increased insulin secretion. Disclosure E. Akalestou: None. K. Suba: None. L. Lopez Noriega: None. P.L. Chabosseau: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. V. Salem: None. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. Funding UK Wellcome Trust (212625/Z/18/Z); Rosetrees Trust (M825); UK Medical Research Council (MR/R022259/1); Harry Keen Diabetes UK Fellowship (BDA 15/0005317); European Union

Bariatric surgery improves both insulin sensitivity and secretion and can induce diabetes remission. However, the mechanisms and time courses of these changes, particularly the impact on β cell function, are difficult to monitor directly. In this study, we investigated the effect of Vertical Sleeve Gastrectomy (VSG) on β cell function in vivo by imaging Ca

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 β cell function, versus mass, have been difficult to establish. In this study, we investigated the effect of bariatric surgery on β 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 β 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 β cell performance. We thus demonstrate that bariatric surgery leads to time-dependent increases in individual β 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 β cells both implicated. Whilst the speed of remission – usually within a few days – argues for improvements in β cell function rather than increases in mass, a direct demonstration of changes at the level of individual β 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 β cell - β cell connectivity, both of which likely underlie increased insulin release.

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.

Levels of the transcription factor ATF6α, a key mediator of the unfolded protein response, that provides cellular protection during the progression endoplasmic reticulum (ER) stress, are markedly reduced in the pancreatic islet of patients with type 2 diabetes and in rodent models of the disease, including ob/ob and high fat-fed mice. Sorcin (gene nameSRI) is a calcium (Ca2+) binding protein involved in maintaining ER Ca2+homeostasis.We have previously shown that overexpressing sorcin under the rat insulin promoter in transgenic mice was protective against high fat diet-induced pancreatic beta cell dysfunction, namely preserving intracellular Ca2+homeostasis and glucose-stimulated insulin secretion during lipotoxic stress. Additionally, sorcin overexpression was apparently activating ATF6 signalling in MIN6 cells despite lowering ER stress.Here, in order to investigate further the relationship between sorcin and ATF6, we describe changes in sorcin expression during ER and lipotoxic stress and changes in ATF6 signalling after sorcin overexpression or inactivation, both in excitable and non-excitable cells.Sorcin mRNA levels were significantly increased in response to the ER stress-inducing agents thapsigargin and tunicamycin, but not by palmitate. On the contrary, palmitate caused a significant decrease in sorcin expression as assessed by both qRT-PCR and Western blotting despite inducing ER stress. Moreover, palmitate prevented the increase in sorcin expression induced by thapsigargin. In addition, sorcin overexpression significantly increased ATF6 transcriptional activity, whereas sorcin inactivation decreased ATF6 signalling. Finally, sorcin overexpression increased levels of ATF6 immunoreactivity and FRET imaging experiments following ER stress induction by thapsigargin showed a direct sorcin-ATF6 interaction.Altogether, our data suggest that sorcin down-regulation during lipotoxicity may prevent full ATF6 activation and a normal UPR during the progression of obesity and insulin resistance, contributing to beta cell failure and type 2 diabetes.

Aims/hypothesis 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 Ca2+ 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. Methods Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. Results Glucose-stimulated mitochondrial Ca2+ accumulation (pppMcu-null (βMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (pMcu-KO animals. βMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (ppMcu-KO ( Conclusions/interpretation MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in βMcu-KO mice remain to be established.

Global rates of diabetes mellitus are increasing, and treatment of the disease consumes a growing proportion of healthcare spending across the world. Pancreatic β-cells, responsible for insulin production, decline in mass in Type 1 and, to a more limited degree, in Type 2 diabetes. However, the extent and rate of loss in both diseases differs between patients resulting in the need for the development of novel diagnostic tools, which could quantitatively assess changes in mass of β-cells over time and potentially lead to earlier diagnosis and improved treatments. Exendin-4, a potent analogue of glucagon-like-peptide 1 (GLP-1), binds to the receptor GLP-1R, whose expression is enriched in β-cells. GLP-1R has thus been used in the past as a means of targeting probes for a wide variety of imaging modalities to the endocrine pancreas. However, exendin-4 conjugates designed specifically for MRI contrast agents are an under-explored area. In the present work, the synthesis and characterization of an exendin-4-dota(ga)-Gd(III) complex, GdEx, is reported, along with its in vivo behaviour in healthy and in β-cell-depleted C57BL/6J mice. Compared to the ubiquitous probe, [Gd(dota)](-), GdEx shows selective uptake by the pancreas with a marked decrease in accumulation observed after the loss of β-cells elicited by deleting the microRNA processing enzyme, DICER. These results open up pathways towards the development of other targeted MRI contrast agents based on similar chemistry methodology.

Variants in the transcription factor-7-like 2 (TCF7L2/TCF4) gene, involved in Wnt signaling, are associated with type 2 diabetes. Loss of Tcf7l2 selectively from the β cell in mice has previously been shown to cause glucose intolerance and to lower β cell mass. Deletion of the tumor suppressor liver kinase B1 (LKB1/STK11) leads to β cell hyperplasia and enhanced glucose-stimulated insulin secretion, providing a convenient genetic model for increased β cell growth and function. The aim of this study was to explore the possibility that Tcf7l2 may be required for the effects of Lkb1 deletion on insulin secretion in the mouse β cell. Mice bearing floxed Lkb1 and/or Tcf7l2 alleles were bred with knockin mice bearing Cre recombinase inserted at the Ins1 locus (Ins1Cre), allowing highly β cell-selective deletion of either or both genes. Oral glucose tolerance was unchanged by the further deletion of a single Tcf7l2 allele in these cells. By contrast, mice lacking both Tcf7l2 alleles on this background showed improved oral glucose tolerance and insulin secretion in vivo and in vitro compared with mice lacking a single Tcf7l2 allele. Biallelic Tcf7l2 deletion also enhanced β cell proliferation, increased β cell mass, and caused changes in polarity as revealed by the "rosette-like" arrangement of β cells. Tcf7l2 deletion also increased signaling by mammalian target of rapamycin (mTOR), augmenting phospho-ribosomal S6 levels. We identified a novel signaling mechanism through which a modifier gene, Tcf7l2, lies on a pathway through which LKB1 acts in the β cell to restrict insulin secretion.

Resume Les medicaments sont des substances chimiques tres largement utilisees en milieu de soin. Ils echappent a la reglementation europeenne CLP (Classification Labelling Packaging) sur le risque chimique mais doivent pourtant etre pris en compte dans l’evaluation du risque chimique des professionnels. Parmi ces medicaments, les anticancereux font l’objet d’une attention particuliere depuis plusieurs annees, compte tenu de leurs effets potentiels sur la sante. Nos connaissances sur le danger de ces molecules viennent a la fois des etudes menees chez l’animal, chez les patients traites, mais pour lesquels les doses absorbees sont d’un niveau tres largement superieur a celles des professionnels, et des etudes menees chez ces derniers dont certaines sont maintenant anciennes. Outre les effets aigus lies a des niveaux d’exposition importants dans des contextes surtout accidentels actuellement, l’attention des preventeurs est essentiellement tournee vers les effets cancerogenes et reprotoxiques de certaines molecules, les assimilant ainsi a un risque chimique CMR (cancerogene, mutagene ou toxique pour la reproduction). Face a ce danger, il convient de developper l’evaluation des expositions pour mieux caracteriser le risque pour le personnel. La surveillance biologique de l’exposition professionnelle aux medicaments anticancereux developpee depuis quelques annees et les frottis de surface ont permis d’identifier les sources et voies d’exposition a tous les stades de leur manipulation, mais egalement lors de contacts avec les patients traites ou de facon indirecte a partir d’un environnement contamine. Compte tenu de ces elements, des mesures de prevention sont a mettre en œuvre. Outre la mise en place depuis une vingtaine d’annees d’unites de reconstitution centralisee des chimiotherapies, les donnees actuelles de contamination montrent la necessite de reflechir a des mesures de prevention lors des autres phases d’activites exposantes. Des recommandations existent mais restent pour certaines encore imprecises. Cet article fait le point sur les donnees de la litterature actuellement disponibles.

Introduction Le cancer broncho-pulmonaire est le deuxieme cancer le plus frequent chez l’homme, et la premiere cause de mortalite en France. Le principal facteur de risque identifie est le tabac, associe a 8 cas sur 10. Des facteurs professionnels ont egalement ete identifies, et seraient susceptibles d’expliquer 15 % a 30 % des cas. Un depistage par scanner thoracique annuel a ete propose aux Etats-Unis (National Lung Screnning Trial, NLST) selon trois criteres d’inclusion : l’âge (55-74 ans), et le tabagisme (30 paquets-annee ou PA) non sevre (depuis moins de 15 ans). En France, le depistage n’est pas recommande chez les fumeurs par la Haute Autorite de sante (HAS), mais les recommandations emises en 2015 par les societes savantes et labelisees par la HAS et l’INCA preconisent un depistage chez les agents exposes professionnellement. La faisabilite de ces recommandations doit etre evaluee par l’etude LUCSO (LUng Cancer Screnning with low-dose computed tomography in a smokers population exposed to Occupational lung carcinogens). Objectif Decrire une population repondant aux criteres de LUCSO et du NLST dans une cohorte de patients pris en charge au CHU de Bordeaux pour un cancer broncho-pulmonaire. Materiel et methode Tous les patients suivis pour un cancer broncho-pulmonaire dans les services de pneumologie et chirurgie thoracique beneficient d’une enquete medico-socio-professionnelle au lit du malade, secondairement tele-expertisee, par un specialiste des risques professionnels (preventeur et/ou medecin) suivant une methode formalisee. Ont ete decrits les pourcentages de patients repondant aux criteres du NLST et de LUCSO : exposition au moins 10 ans a un cancerogene et tabac ≥ 30 PA, ou co-exposition a deux cancerogenes et tabac ≥ 20 PA, ou co-exposition a trois cancerogenes et tabac ≥ 10 PA, chez les patients âges de 55 a 74 ans. Resultats Parmi les 318 cas expertises, 38 % auraient ete eligibles aux criteres du NLST, et 23 % selon les criteres de LUCSO. Parmi les patients non eligibles, 26 % sont exclus sur criteres d’âge, 48 % sur criteres d’exposition (non expose ou exposition inferieure a 10 ans), et 46 % sur critere tabagique. Plus d’un tiers des patients exclus a ete co-expose a des agents cancerogenes pulmonaires au moins 10 ans. Conclusion Sur l’ensemble des patients suivis pour cancers broncho-pulmonaires au CHU de Bordeaux, 38 % selon le NLST et 23 % selon LUCSO seraient eligibles au depistage. Ces resultats sont a corroborer a des analyses sur une population plus representative de la population generale.