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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.

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.

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)

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.

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

AMP-activated protein kinase (AMPK) is an important cellular energy sensor in regulation of metabolic activity. AMPK activation improves glucose homeostasis by increasing muscle glucose uptake and reducing hepatic glucose production. Although the effects on glucose-stimulated insulin secretion in the pancreatic β cells are still controversial. Our aim was to investigate the regulation of insulin secretion in pancreatic β cells using specific pharmacological AMPK activators and in mouse models of constitutive activation. We used a chronic global AMPK (γ1D316A) and a pancreatic β cell specific (β-D316A) activation mouse model that express the AMPK γamma1 isoform with a D316A mutation under the control of β-actin and ins1 promoter respectively. We tested how chronic AMPK activation impacted insulin secretion on C57BL6 mouse isolated islets using direct activators PF-06409577 (β1 isoform-selective) and RA089 (binds both β1 and β2 isoforms). Global activation of AMPK reduced insulin secretion from islets isolated from γ1D316A mice on chow and high-fat diet (2.4-fold, p Disclosure M. Nguyen-Tu: None. J.P. Harris: None. A. Martinez-Sanchez: None. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma.

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

Aim: In-vitro studies reveal a subpopulation of β cells which co-ordinate insulin release from pancreatic islets. This connectivity has not been established in 3 dimensions across the islet or in the living animal. We investigated this in the living zebrafish D. rario or in mouse islets engrafted in the anterior eye chamber (ACE), using the recombinant Ca2+ sensor GCaMP6 under the insulin promoter/C57Bl6, Ins1Cre:GCaM6ff/f. Methods: Whole islet live imaging of D. rario primary islets in situ at an acquisition rate of 0.8Hz, covering 700μm, was achieved using resonant scanner technology with a Leica inverted laser scanning confocal system, using a 25X/N.A. 0.95 water lens. Fully vascularized islets in the murine ACE were imaged under isofluorane anaesthesia on a modified Nikon Ti-E spinning disc confocal microscope (20x 0.75 NA water immersion objective, 3 Hz frame rate). Results: 3-dimensional β cell connectivity (Pearson R statistic) significantly rose to 88% across the zebrafish islet in response to glucose stimulation. Focused ablation of the co-ordinating “hub” (but not follower) β cells significantly attenuated the subsequent response to a glucose challenge. In mouse islets, high circulating glucose levels were also associated with a significant rise in 3D beta cell connectivity from 65 to 86% (n=3; p=0.02). Granger causality analysis on these islets revealed that even on prolonged (>10 minute) glucose stimulation, pan-islet connectivity was sustained and the most highly connected cells were those in the region of the islet from which Ca2+ waves emanated. Binarized signal analysis (n=6) separately confirmed the presence of a highly-connected β cell subpopulation (8.7 ± 3.6% of cells). Conclusions: These studies evidence beta cell “hubs” which are responsible for the initiation and sustained coordination of the Ca2+ response to glucose across fish and mouse islets in vivo. Disclosure V. Salem: None. L.F. Delgadillo Silva: None. K. Suba: None. A. Martin Alonso: None. W. Kim: None. V. Kalogianni: None. N. Akhtar: None. N. Mousavy Gharavy: None. E. Georgiadou: None. I. Leclerc: None. L. Briant: None. D. Hodson: None. W. Distaso: None. N.N. Ninov: None. G.A. Rutter: Consultant; Self; Sun Pharma. Funding Diabetes UK; UK Wellcome Trust

Background: Bariatric surgery is known to improve glucose tolerance and weight loss, but can also cause a postprandial increase in renal glucose excretion following oral glucose tolerance test. Both effects have also been observed following treatment with sodium glucose co-transporter 2 (SGLT2) inhibitors. Aim: To investigate whether there is an effect of bariatric surgery (Vertical Sleeve Gastrectomy; VSG) on renal SGLT2 expression in mice. Methods: Fourteen lean mice underwent VSG (n=8) or sham (n=6) surgery. Glucose Tolerance tests with or without treatment with the SGLT2 inhibitor dapagliflozin were performed four weeks post operatively, in order to assess if exogenous SGLT2 inhibition has the same euglycemic effects as bariatric surgery. Kidneys were harvested from fed mice and analysed using Quantitative real-time PCR (qRT-PCR) and immunofluorescence. Results: VSG did not cause any weight loss when compared to sham operated mice (p=0.37, Mann-Whitney test). However, VSG mice displayed significantly improved glucose tolerance (AUC=103 ±6.8; AUC =66.6 ±2.9 in control and VSG mice, respectively; p0.05). In contrast, treatment of sham mice with dapagliflozin increased glucose tolerance, though to a lesser extent than VSG. Nevertheless, qPCR and immunofluorescence analysis on mouse kidneys demonstrated a significant lowering of SGLT2 expression at both the protein (n=5, p=0.0007) and mRNA (n=7, 0 Conclusions: Both gastric and intestinal surgery in lean animals causes a significant inhibition of SGLT2 in the kidney cortex. These findings are in line with our previous results on the effects of Duodenal Jejunal Bypass in lean rats, and point towards a physiologically-relevant gut-kidney axis. SGLT2 inhibition may thus be an important mechanism through which bariatric surgery improves glucose tolerance in man. Disclosure E. Akalestou: None. L. Lopez Noriega: None. P.L. Chabosseau: None. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma. Funding UK Wellcome Trust

miRNAs are tiny RNAs that repress gene expression post-transcriptionally, necessary for normal endocrine function. AMPK is essential for glucose homeostasis and a suggested target of metformin. β-cell-specific inactivation of AMPK in mice (AMPKdKO) impairs glucose homeostasis and β-cell function. AMPKdKO mice present dysregulation of several miRNAs that may contribute to these defects, including miR-125b (∼2.5 fold upregulated). High levels of miR-125b are associated with hyperglycaemia (HbA1c) in prediabetic, T2D and T1D subjects, suggesting miR-125b as a biomarker or contributor to the development of diabetes. The role of miR-125b in β-cells remains elusive although in other cell types regulates apoptosis, growth and differentiation. We aim to determine miR-125b function in β-cells and the role of glucose and AMPK in the regulation of its expression. miR-125b level in human islets strongly correlates with BMI and is induced by glucose in both mouse and human islets, while feeding mice a ketogenic (low sugar) diet reduced islet miR-125b. These effects disappeared in islets with β-cell specific deletion of AMPK whereas treatment of human islets with highly specific AMPK activators significantly decreased miR-125b, indicating that glucose regulates miR-125b via AMPK in both mice and humans. miR-125b overexpression in MIN6 cells limited cytokine-induced apoptosis whilst strongly reducing insulin content. RNA-Seq on these cells suggests a role for miR-125b in metabolism, respiration and cytokine receptor interactions and RNA-IP of the miRNA-induced silencing complex revealed novel miR-125b targets such as mannose-6-phosphate receptor (MPR), important for enzyme sorting within secretory granules. Preliminary data show that β-cell-specific inhibition of miR-125b using a molecular “sponge” improves glucose tolerance in mice fed a high-fat diet. In summary, we reveal miR-125b as an important regulator of β-cell function that might contribute to the deleterious effects of hyperglycaemia on β-cells. Disclosure R. Cheung: None. G. Pizza: None. D.M. Rolando: None. P.L. Chabosseau: None. M. Nguyen-Tu: None. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma. A. Martinez-Sanchez: None. Funding Medical Research Council UK

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.

Carbohydrate-responsive element-binding protein (ChREBP) is a regulator of pancreatic β-cell gene expression and an important mediator of glucotoxicity. Glucose increases the activity and nuclear localization of ChREBP by still ill-defined mechanisms. Here we reveal, using both MIN6 and primary mouse β-cells, a unique mechanism behind ChREBP nuclear translocation. At low glucose concentrations, ChREBP interacts with sorcin, a penta EF hand Ca2+ binding protein, and is sequestered in the cytosol. Sorcin overexpression inhibits ChREBP nuclear accumulation at high glucose and reduced the activity of L-type pyruvate kinase (L-PK) and TxNIP promoters, two well-characterized ChREBP target genes. Sorcin inactivation by RNA interference increases ChREBP nuclear localization and in vivo binding to the L-PK promoter at low glucose concentrations. Ca2+ influx was essential for this process since Ca2+ chelation with EGTA, or pharmacological inhibition with diazoxide and nifedipine, blocked the effects of glucose. Conversely, mobilization of intracellular Ca2+ with ATP caused the nuclear accumulation of ChREBP. Finally, sorcin silencing inhibited ATP-induced increases in intracellular Ca2+ and glucose-stimulated insulin secretion. We therefore conclude that sorcin retains ChREBP in the cytosol at low glucose concentrations and may act as a Ca2+ sensor for glucose-induced nuclear translocation and the activation of ChREBP-dependent genes.

OBJECTIVE The fuel sensor AMP-activated protein kinase (AMPK) in the hypothalamus regulates energy homeostasis by sensing nutritional and hormonal signals. However, the role of hypothalamic AMPK in glucose production regulation remains to be elucidated. We hypothesize that bidirectional changes in hypothalamic AMPK activity alter glucose production. RESEARCH DESIGN AND METHODS To introduce bidirectional changes in hypothalamic AMPK activity in vivo, we first knocked down hypothalamic AMPK activity in male Sprague-Dawley rats by either injecting an adenovirus expressing the dominant-negative form of AMPK (Ad-DN AMPKα2 [D157A]) or infusing AMPK inhibitor compound C directly into the mediobasal hypothalamus. Next, we independently activated hypothalamic AMPK by delivering either an adenovirus expressing the constitutive active form of AMPK (Ad-CA AMPKα1312 [T172D]) or the AMPK activator AICAR. The pancreatic (basal insulin)-euglycemic clamp technique in combination with the tracer-dilution methodology was used to assess the impact of alternations in hypothalamic AMPK activity on changes in glucose kinetics in vivo. RESULTS Injection of Ad-DN AMPK into the hypothalamus knocked down hypothalamic AMPK activity and led to a significant suppression of glucose production with no changes in peripheral glucose uptake during the clamps. In parallel, hypothalamic infusion of AMPK inhibitor compound C lowered glucose production as well. Conversely, molecular and pharmacological activation of hypothalamic AMPK negated the ability of hypothalamic nutrients to lower glucose production. CONCLUSIONS These data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo.

Accumulation of triglyceride in islets may contribute to the loss of glucose-stimulated insulin secretion (GSIS) in some forms of type 2 diabetes (Diraison et al., Biochem J 373:769–778, 2004). Here, we use adenoviral vectors and oligonucleotide microarrays to determine the effects of the forced expression of SREBP1c on the gene expression profile of rat islets. Sterol regulatory element binding protein-1c (SREBP1c) overexpression led to highly significant ( P < 0.1 with respect to null adenovirus) changes in the expression of 1,238 genes or expressed sequence tags, of which 1,180 (95.3%) were upregulated. By contrast, overexpression of constitutively active AMP-activated protein kinase (AMPK), expected to promote lipolysis, altered the expression of 752 genes, of which 702 (93%) were upregulated. To identify specific targets for SREBP1c or AMPK, we eliminated messages that were 1 ) affected in the same direction by the expression of either protein, 2 ) changed by less than twofold, or 3 ) failed a positive false discovery test; 206 SREBP1c-regulated genes (195; 95% upregulated) and 48 AMPK-regulated genes (33; 69% upregulated) remained. As expected, SREBP1c-induced genes included those involved in cholesterol ([6][1]), fatty acid ([3][2]), and eicosanoid synthesis. Interestingly, somatostatin receptor (sstr1) expression was increased by SREBP1c, whereas AMPK induced the expression of peptide YY, the early endocrine pancreas marker. [1]: #ref-6 [2]: #ref-3