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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.

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).

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