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Summary: Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning β cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and β cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human β cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in β cells to maintain appropriate insulin release.

IntroductionProinflammatory cytokines are implicated in pancreatic ß cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of nonsense-mediated RNA decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß cells.MethodsA luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. The gain- or loss-of-function of two key NMD components, UPF3B and UPF2, is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques.ResultsCyt attenuate NMD activity in insulin-producing cell lines and primary human ß cells. These effects are found to involve ER stress and are associated with the downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing raise or lower Cyt-induced cell death, respectively, in EndoC-ßH3 cells and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increases alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in the extracellular matrix (ECM), including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitizes ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signaling, potentially serving as a protective response against Cyt-induced NMD component expression.ConclusionOur findings highlight the central importance of RNA turnover in ß cell responses to inflammatory stress.

IntroductionType 2 diabetes (T2D) onset, progression and outcomes differ substantially between individuals. Multi-omics analyses may allow a deeper understanding of these differences and ultimately facilitate personalised treatments. Here, in an unsupervised “bottom-up” approach, we attempt to group T2D patients based solely on -omics data generated from plasma.MethodsCirculating plasma lipidomic and proteomic data from two independent clinical cohorts, Hoorn Diabetes Care System (DCS) and Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS), were analysed using Similarity Network Fusion. The resulting patient network was analysed with Logistic and Cox regression modelling to explore relationships between plasma -omic profiles and clinical characteristics.ResultsFrom a total of 1,134 subjects in the two cohorts, levels of 180 circulating plasma lipids and 1195 proteins were used to separate patients into two subgroups. These differed in terms of glycaemic deterioration (Hazard Ratio=0.56;0.73), insulin sensitivity and secretion (C-peptide, p=3.7e-11;2.5e-06, DCS and GoDARTS, respectively; Homeostatic model assessment 2 (HOMA2)-B; -IR; -S, p=0.0008;4.2e-11;1.1e-09, only in DCS). The main molecular signatures separating the two groups included triacylglycerols, sphingomyelin, testican-1 and interleukin 18 receptor.ConclusionsUsing an unsupervised network-based fusion method on plasma lipidomics and proteomics data from two independent cohorts, we were able to identify two subgroups of T2D patients differing in terms of disease severity. The molecular signatures identified within these subgroups provide insights into disease mechanisms and possibly new prognostic markers for T2D.

Abstract We identify biomarkers for disease progression in three type 2 diabetes cohorts encompassing 2,973 individuals across three molecular classes, metabolites, lipids and proteins. Homocitrulline, isoleucine and 2-aminoadipic acid, eight triacylglycerol species, and lowered sphingomyelin 42:2;2 levels are predictive of faster progression towards insulin requirement. Of ~1,300 proteins examined in two cohorts, levels of GDF15/MIC-1, IL-18Ra, CRELD1, NogoR, FAS, and ENPP7 are associated with faster progression, whilst SMAC/DIABLO, SPOCK1 and HEMK2 predict lower progression rates. In an external replication, proteins and lipids are associated with diabetes incidence and prevalence. NogoR/RTN4R injection improved glucose tolerance in high fat-fed male mice but impaired it in male db/db mice. High NogoR levels led to islet cell apoptosis, and IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. This comprehensive, multi-disciplinary approach thus identifies biomarkers with potential prognostic utility, provides evidence for possible disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

IntroductionCommon variants in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8 (expressed largely in pancreatic islet alpha and beta cells), are associated with altered risk of type 2 diabetes. Unexpectedly, rare loss-of-function (LoF) variants in the gene, described in heterozygous individuals only, are protective against the disease, even though knockout of the homologous SLC30A8 gene in mice leads to unchanged or impaired glucose tolerance. Here, we aimed to determine how one or two copies of the mutant R138X allele in the mouse SLC30A8 gene impacts the homeostasis of zinc at a whole-body (using non-invasive 62Zn PET imaging to assess the acute dynamics of zinc handling) and tissue/cell level [using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to map the long-term distribution of zinc and manganese in the pancreas].MethodsFollowing intravenous administration of [62Zn]Zn-citrate (~7 MBq, 150 μl) in wild-type (WT), heterozygous (R138X+/−), and homozygous (R138X+/+) mutant mice (14–15 weeks old, n = 4 per genotype), zinc dynamics were measured over 60 min using PET. Histological, islet hormone immunohistochemistry, and elemental analysis with LA-ICP-MS (Zn, Mn, P) were performed on sequential pancreas sections. Bulk Zn and Mn concentration in the pancreas was determined by solution ICP-MS.ResultsOur findings reveal that whereas uptake into organs, assessed using PET imaging of 62Zn, is largely unaffected by the R138X variant, mice homozygous of the mutant allele show a substantial lowering (to 40% of WT) of total islet zinc, as anticipated. In contrast, mice heterozygous for this allele, thus mimicking human carriers of LoF alleles, show markedly increased endocrine and exocrine zinc content (1.6-fold increase for both compared to WT), as measured by LA-ICP-MS. Both endocrine and exocrine manganese contents were also sharply increased in R138X+/− mice, with smaller increases observed in R138X+/+ mice.DiscussionThese data challenge the view that zinc depletion from the beta cell is the likely underlying driver for protection from type 2 diabetes development in carriers of LoF alleles. Instead, they suggest that heterozygous LoF may paradoxically increase pancreatic β-cell zinc and manganese content and impact the levels of these metals in the exocrine pancreas to improve insulin secretion.

BackgroundSARS-CoV-2 infection during pregnancy may cause adverse maternal, neonatal and placental outcomes. While tissue hypoxia is often reported in COVID-19 patients, pregnant women with anemia are suspected to be more prone to placental hypoxia-related injuries.MethodsThis hospital-based cross-sectional study was conducted between August-November 2021, during COVID-19 second wave in India. Term pregnant women (N=212) admitted to hospital for delivery were enrolled consecutively. Since hospital admission mandated negative RT-PCR test for SARS-CoV-2 virus, none had active infection. Data on socio-demography, COVID-19 history, maternal, obstetric, and neonatal outcomes were recorded. Pre-delivery maternal and post-delivery cord blood samples were tested for hematological parameters and SARS-CoV-2 IgG. Placentae were studied for histology.ResultsOf 212 women, 122 (58%) were seropositive for SARS-CoV-2 IgG, but none reported COVID-19 history; 134 (63.2%) were anemic. In seropositive women, hemoglobin (p=0.04), total WBC (p=0.009), lymphocytes (p=0.005) and neutrophils (p=0.02) were significantly higher, while ferritin was high, but not significant and neutrophils to lymphocytes (p=0.12) and platelets to lymphocytes ratios (p=0.03) were lower. Neonatal outcomes were similar. All RBC parameters and serum ferritin were significantly lower in anemic mothers but not in cord blood, except RDW that was significantly higher in both, maternal (p=0.007) and cord (p=0.008) blood from seropositive anemic group compared to other groups. Placental histology showed significant increase in villous hypervascularity (p=0.000), dilated villous capillaries (p=0.000), and syncytiotrophoblasts (p=0.02) in seropositive group, typically suggesting placental hypoxia. Maternal anemia was not associated with any histological parameters. Univariate and multivariate logistic regression analyses of placental histopathological adverse outcomes showed strong association with SARS-CoV-2 seropositivity but not with maternal anemia. When adjusted for several covariates, including anemia, SARS-CoV-2 seropositivity emerged as independent risk factor for severe chorangiosis (AOR 8.74, 95% CI 3.51-21.76, p

ObjectivesGlucocorticoids produced by the adrenal cortex are essential for the maintenance of metabolic homeostasis. Glucocorticoid activation is catalysed by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). Excess glucocorticoids are associated with insulin resistance and hyperglycaemia. A small number of studies have demonstrated effects on glucocorticoid metabolism of bariatric surgery, a group of gastrointestinal procedures known to improve insulin sensitivity and secretion, which were assumed to result from weight loss. In this study, we hypothesize that a reduction in glucocorticoid action following bariatric surgery contributes to the widely observed euglycemic effects of the treatment.MethodsGlucose and insulin tolerance tests were performed at ten weeks post operatively and circulating corticosterone was measured. Liver and adipose tissues were harvested from fed mice and 11β-HSD1 levels were measured by quantitative RT-PCR or Western (immuno-) blotting, respectively. 11β-HSD1 null mice (Hsd11b1-/-) were generated using CRISPR/Cas9 genome editing. Wild type and littermate Hsd11b1-/- mice underwent Vertical Sleeve Gastrectomy (VSG) or sham surgery. ResultsUnder the conditions used, no differences in weight loss were observed between VSG treated and sham operated mice. However, both lean and obese WT VSG mice displayed significantly improved glucose clearance and insulin sensitivity. Remarkably, VSG restored physiological corticosterone production in HFD mice and reduced 11β-HSD1 expression in liver and adipose tissue post-surgery. Elimination of the 11β-HSD1/Hsd11b1 gene by CRISPR/Cas9 mimicked the effects of VSG on body weight and tolerance to 1g/kg glucose challenge. However, at higher glucose loads, the euglycemic effect of VSG was superior to Hsd11b1 elimination.ConclusionsBariatric surgery improves insulin sensitivity and reduces glucocorticoid activation at the tissular level, under physiological and pathophysiological (obesity) conditions, irrespective of weight loss. These findings point towards a physiologically relevant gut-glucocorticoid axis, and suggest that lowered glucocorticoid exposure may represent an additional contribution to the health benefits of bariatric surgery.

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.

Objective: β cell dedifferentiation may underlie the reversible reduction in pancreatic β cell mass and function in type 2 diabetes (T2D). We previously reported that β cell-specific Sirt3 knockout (Sirt3f/f;Cre/+) mice developed impaired glucose tolerance and glucose-stimulated insulin secretion after feeding with high fat diet (HFD). RNA sequencing showed that Sirt3-deficient islets had enhanced expression of Enpp2 (Autotaxin, or ATX), a secreted lysophospholipase which produces lysophosphatidic acid (LPA). Here, we hypothesized that activation of the ATX/LPA pathway contributed to pancreatic β cell dedifferentiation in Sirt3-deficient β cells. Methods: We applied LPA, or lysophosphatidylcoline (LPC), the substrate of ATX for producing LPA, to MIN6 cell line and mouse islets with altered Sirt3 expression to investigate the effect of LPA on β cell dedifferentiation and its underlying mechanisms. To examine the pathological effects of ATX/LPA pathway, we injected the β cell selective adeno-associated virus (AAV-Atx-shRNA) or negative control AAV-scramble in Sirt3f/f and Sirt3f/f;Cre/+ mice followed by 6-week of HFD feeding. Results: In Sirt3f/f;Cre/+ mouse islets and Sirt3 knockdown MIN6 cells, ATX upregulation led to increased LPC with increased production of LPA. The latter not only induced reversible dedifferentiation in MIN6 cells and mouse islets, but also reduced glucose-stimulated insulin secretion from islets. In MIN6 cells, LPA induced phosphorylation of JNK/p38 MAPK which was accompanied by β cell dedifferentiation. The latter was suppressed by inhibitors of LPA receptor, JNK, and p38 MAPK. Importantly, inhibiting ATX in vivo improved insulin secretion and reduced β cell dedifferentiation in HFD-fed Sirt3f/f;Cre/+ mice. Conclusions: Sirt3 prevents β cell dedifferentiation by inhibiting ATX expression and upregulation of LPA. These findings support a long-range signaling effect of Sirt3 which modulates the ATX-LPA pathway to reverse β cell dysfunction associated with glucolipotoxicity.

The rare SLC30A8 mutation encoding a truncating p.Arg138* variant (R138X) in zinc transporter 8 (ZnT8) is associated with a 65% reduced risk for type 2 diabetes. To determine whether ZnT8 is required for beta cell development and function, we derived human pluripotent stem cells carrying the R138X mutation and differentiated them into insulin-producing cells. We found that human pluripotent stem cells with homozygous or heterozygous R138X mutation and the null (KO) mutation have normal efficiency of differentiation towards insulin-producing cells, but these cells show diffuse granules that lack crystalline zinc-containing insulin granules. Insulin secretion is not compromised in vitro by KO or R138X mutations in human embryonic stem cell-derived beta cells (sc-beta cells). Likewise, the ability of sc-beta cells to secrete insulin and maintain glucose homeostasis after transplantation into mice was comparable across different genotypes. Interestingly, sc-beta cells with the SLC30A8 KO mutation showed increased cytoplasmic zinc, and cells with either KO or R138X mutation were resistant to apoptosis when extracellular zinc was limiting. These findings are consistent with a protective role of zinc in cell death and with the protective role of zinc in T2D.

Beta cell subpopulations with low expression in PDX1, MAFA, and insulin might contribute to islet function and insulin release. Here the authors show that altering the proportion of PDX1LOW MAFALOW to PDX1HIGH MAFAHIGH cells impairs islet function.

Objective: β-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. We previously showed that increased miR-7 levels trigger β-cell dedifferentiation and diabetes. We used β-cell-specific miR-7 overexpressing mice (Tg7) to test the hypothesis that loss of β-cell identity triggered by miR-7 overexpression alters islet gene expression and islet microenvironment in diabetes. Methods: We performed bulk and single-cell RNA sequencing (RNA-seq) in islets obtained from β-cell-specific miR-7 overexpressing mice (Tg7). We carried out loss- and gain-of-function experiments in MIN6 and EndoC-bH1 cell lines. We analysed previously published mouse and human T2D data sets. Results: Bulk RNA-seq revealed that β-cell dedifferentiation is associated with the induction of genes associated with epithelial-to-mesenchymal transition (EMT) in prediabetic (2-week-old) and diabetic (12-week-old) Tg7 mice. Single-cell RNA-seq (scRNA-seq) indicated that this EMT signature is enriched specifically in β-cells. These molecular changes are associated with a weakening of β-cell: β-cell contacts, increased extracellular matrix (ECM) deposition, and TGFβ-dependent islet fibrosis. We found that the mesenchymal reprogramming of β-cells is explained in part by the downregulation of Pdx1 and its inability to regulate a myriad of epithelial-specific genes expressed in β-cells. Notable among genes transactivated by Pdx1 is Ovol2, which encodes a transcriptional repressor of the EMT transcription factor Zeb2. Following compromised β-cell identity, the reduction in Pdx1 gene expression causes a decrease in Ovol2 protein, triggering mesenchymal reprogramming of β-cells through the induction of Zeb2. We provided evidence that EMT signalling associated with the upregulation of Zeb2 expression is a molecular feature of islets in T2D subjects. Conclusions: Our study indicates that miR-7-mediated β-cell dedifferentiation induces EMT signalling and a chronic response to tissue injury, which alters the islet microenvironment and predisposes to fibrosis. This research suggests that regulators of EMT signalling may represent novel therapeutic targets for treating β-cell dysfunction and fibrosis in T2D.

Mounting evidence suggests a role for alternative splicing (AS) of transcripts in the normal physiology and pathophysiology of the pancreatic β-cell. In the apparent absence of RNA repair systems, RNA decay pathways are likely to play an important role in controlling the stability, distribution and diversity of transcript isoforms in these cells. Around 35% of alternatively spliced transcripts in human cells contain premature termination codons (PTCs) and are targeted for degradation via nonsense-mediated decay (NMD), a vital quality control process. Inflammatory cytokines, whose levels are increased in both type 1 (T1D) and type 2 (T2D) diabetes, stimulate alternative splicing events and the expression of NMD components, and may or may not be associated with the activation of the NMD pathway. It is, however, now possible to infer that NMD plays a crucial role in regulating transcript processing in normal and stress conditions in pancreatic β-cells. In this review, we describe the possible role of Regulated Unproductive Splicing and Translation (RUST), a molecular mechanism embracing NMD activity in relationship to AS and translation of damaged transcript isoforms in these cells. This process substantially reduces the abundance of non-functional transcript isoforms, and its dysregulation may be involved in pancreatic β-cell failure in diabetes.

Numerous studies have sought to decipher the genetic and other mechanisms contributing to β-cell loss and dysfunction in diabetes mellitus. However, we have yet to fully understand the etiology of the disease or to develop satisfactory treatments. Since the majority of diabetes susceptibility loci are mapped to non-coding regions within the genome, understanding the functions of non-coding RNAs in β-cell biology might provide crucial insights into the pathogenesis of type 1 (T1D) and type 2 (T2D) diabetes. During the past decade, numerous studies have indicated that long non-coding RNAs play important roles in the maintenance of β-cell mass and function. Indeed, lncRNAs have been shown to be involved in controlling β-cell proliferation during development and/or β-cell compensation in response to hyperglycaemia. LncRNAs such as TUG-1 and MEG3 play a role in both β-cell apoptosis and function, while others sensitize β-cells to apoptosis in response to stress signals. In addition, several long non-coding RNAs have been shown to regulate the expression of β-cell-enriched transcription factors in cis or in trans. In this review, we provide an overview of the roles of lncRNAs in maintaining β-function and mass, and discuss their relevance in the development of diabetes.

Summary: Pancreatic β cell failure is key to type 2 diabetes (T2D) onset and progression. Here, we assess whether human β cell dysfunction induced by metabolic stress is reversible, evaluate the molecular pathways underlying persistent or transient damage, and explore the relationships with T2D islet traits. Twenty-six islet preparations are exposed to several lipotoxic/glucotoxic conditions, some of which impair insulin release, depending on stressor type, concentration, and combination. The reversal of dysfunction occurs after washout for some, although not all, of the lipoglucotoxic insults. Islet transcriptomes assessed by RNA sequencing and expression quantitative trait loci (eQTL) analysis identify specific pathways underlying β cell failure and recovery. Comparison of a large number of human T2D islet transcriptomes with those of persistent or reversible β cell lipoglucotoxicity show shared gene expression signatures. The identification of mechanisms associated with human β cell dysfunction and recovery and their overlap with T2D islet traits provide insights into T2D pathogenesis, fostering the development of improved β cell-targeted therapeutic strategies.

Cystathionine beta synthase (CBS) catalyzes the first step of the transsulfuration pathway from homocysteine to cystathionine, and its deficiency leads to hyperhomocysteinemia (HHcy) in humans and rodents. To date, scarce information is available about the HHcy effect on insulin secretion, and the link between CBS activity and the setting of type 2 diabetes is still unknown. We aimed to decipher the consequences of an inborn defect in CBS on glucose homeostasis in mice. We used a mouse model heterozygous for CBS (CBS+/−) that presented a mild HHcy. Other groups were supplemented with methionine in drinking water to increase the mild to intermediate HHcy, and were submitted to a high-fat diet (HFD). We measured the food intake, body weight gain, body composition, glucose homeostasis, plasma homocysteine level, and CBS activity. We evidenced a defect in the stimulated insulin secretion in CBS+/− mice with mild and intermediate HHcy, while mice with intermediate HHcy under HFD presented an improvement in insulin sensitivity that compensated for the decreased insulin secretion and permitted them to maintain a glucose tolerance similar to the CBS+/+ mice. Islets isolated from CBS+/− mice maintained their ability to respond to the elevated glucose levels, and we showed that a lower parasympathetic tone could, at least in part, be responsible for the insulin secretion defect. Our results emphasize the important role of Hcy metabolic enzymes in insulin secretion and overall glucose homeostasis.

Covid-19 is a recently-emerged infectious disease caused by the novel severe acute respiratory syndrome coronavirus SARS-CoV2. SARS-CoV2 differs from previous coronavirus infections (SARS and MERS) due to its high infectivity (reproduction value, R0, typically 2–4) and pre- or asymptomatic transmission, properties that have contributed to the current global Covid-19 pandemic. Identified risk factors for disease severity and death from SARS-Cov2 infection include older age, male sex, diabetes, obesity and hypertension. The reasons for these associations are still largely obscure. Evidence is also emerging that SARS-CoV2 infection exacerbates the underlying pathophysiology of hyperglycemia in people with diabetes. Here, we discuss potential mechanisms through which diabetes may affect the risk of more severe outcomes in Covid-19 and, additionally, how diabetic emergencies and longer term pathology may be aggravated by infection with the virus. We consider roles for the immune system, the observed phenomenon of microangiopathy in severe Covid-19 infection and the potential for direct viral toxicity on metabolically-relevant tissues including pancreatic beta cells and targets of insulin action.

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.

The inheritance of variants that lead to coding changes in, or the mis-expression of, genes critical to pancreatic beta cell function can lead to alterations in insulin secretion and increase the risk of both type 1 and type 2 diabetes. Recently developed clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene editing tools provide a powerful means of understanding the impact of identified variants on cell function, growth, and survival and might ultimately provide a means, most likely after the transplantation of genetically “corrected” cells, of treating the disease. Here, we review some of the disease-associated genes and variants whose roles have been probed up to now. Next, we survey recent exciting developments in CRISPR/Cas9 technology and their possible exploitation for β cell functional genomics. Finally, we will provide a perspective as to how CRISPR/Cas9 technology may find clinical application in patients with diabetes.

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

Objective: The objective of this study was to determine how pharmacokinetically advantageous acylation impacts on glucagon-like peptide-1 receptor (GLP-1R) signal bias, trafficking, anti-hyperglycaemic efficacy, and appetite suppression. Methods: In vitro signalling responses were measured using biochemical and biosensor assays. GLP-1R trafficking was determined by confocal microscopy and diffusion-enhanced resonance energy transfer. Pharmacokinetics, glucoregulatory effects, and appetite suppression were measured in acute, sub-chronic, and chronic settings in mice. Results: A C-terminally acylated ligand, [F1,G40,K41.C16 diacid]exendin-4, was identified that showed undetectable β-arrestin recruitment and GLP-1R internalisation. Depending on the cellular system used, this molecule was up to 1000-fold less potent than the comparator [D3,G40,K41.C16 diacid]exendin-4 for cyclic AMP signalling, yet was considerably more effective in vivo, particularly for glucose regulation. Conclusions: C-terminal acylation of biased GLP-1R agonists increases their degree of signal bias in favour of cAMP production and improves their therapeutic potential.

Glucocorticoids are steroid hormones produced by the adrenal cortex and are essential for the maintenance of various metabolic and homeostatic functions. Their function is regulated at the tissue level by 11β-hydroxysteroid dehydrogenases and they signal through the glucocorticoid receptor, a ligand-dependent transcription factor. Clinical observations have linked excess glucocorticoid levels with profound metabolic disturbances of intermediate metabolism resulting in abdominal obesity, insulin resistance and dyslipidaemia. In this review, we discuss the physiological mechanisms of glucocorticoid secretion, regulation and function, and survey the metabolic consequences of excess glucocorticoid action resulting from elevated release and activation or up-regulated signaling. Finally, we summarize the reported impact of weight loss by diet, exercise, or bariatric surgery on circulating and tissue-specific glucocorticoid levels and examine the therapeutic possibility of reversing glucocorticoid-associated metabolic disorders.

Objective: The metabolic role of d-serine, a non-proteinogenic NMDA receptor co-agonist, is poorly understood. Conversely, inhibition of pancreatic NMDA receptors as well as loss of the d-serine producing enzyme serine racemase have been shown to modulate insulin secretion. Thus, we aim to study the impact of chronic and acute d-serine supplementation on insulin secretion and other parameters of glucose homeostasis. Methods: We apply MALDI FT-ICR mass spectrometry imaging, NMR based metabolomics, 16s rRNA gene sequencing of gut microbiota in combination with a detailed physiological characterization to unravel the metabolic action of d-serine in mice acutely and chronically treated with 1% d-serine in drinking water in combination with either chow or high fat diet feeding. Moreover, we identify SNPs in SRR, the enzyme converting L-to d-serine and two subunits of the NMDA receptor to associate with insulin secretion in humans, based on the analysis of 2760 non-diabetic Caucasian individuals. Results: We show that chronic elevation of d-serine results in reduced high fat diet intake. In addition, d-serine leads to diet-independent hyperglycemia due to blunted insulin secretion from pancreatic beta cells. Inhibition of alpha 2-adrenergic receptors rapidly restores glycemia and glucose tolerance in d-serine supplemented mice. Moreover, we show that single nucleotide polymorphisms (SNPs) in SRR as well as in individual NMDAR subunits are associated with insulin secretion in humans. Conclusion: Thus, we identify a novel role of d-serine in regulating systemic glucose metabolism through modulating insulin secretion. Keywords: d-serine, Diabetes, Obesity, Insulin secretion

Glucagon-like peptide-1 receptor (GLP-1R) promotes insulin secretion from pancreatic beta cells and undergoes agonist-mediated endocytosis. Here, authors study GLP-1R endocytosis caused by different agonists and show that a longer plasma membrane retention time of GLP-1R results in greater long-term insulin release.

Objective: In type 2 diabetes (T2D), pancreatic β cells become progressively dysfunctional, leading to a decline in insulin secretion over time. In this study, we aimed to identify key genes involved in pancreatic beta cell dysfunction by analyzing multiple mouse strains in parallel under metabolic stress. Methods: Male mice from six commonly used non-diabetic mouse strains were fed a high fat or regular chow diet for three months. Pancreatic islets were extracted and phenotypic measurements were recorded at 2 days, 10 days, 30 days, and 90 days to assess diabetes progression. RNA-Seq was performed on islet tissue at each time-point and integrated with the phenotypic data in a network-based analysis. Results: A module of co-expressed genes was selected for further investigation as it showed the strongest correlation to insulin secretion and oral glucose tolerance phenotypes. One of the predicted network hub genes was Elovl2, encoding Elongase of very long chain fatty acids 2. Elovl2 silencing decreased glucose-stimulated insulin secretion in mouse and human β cell lines. Conclusion: Our results suggest a role for Elovl2 in ensuring normal insulin secretory responses to glucose. Moreover, the large comprehensive dataset and integrative network-based approach provides a new resource to dissect the molecular etiology of β cell failure under metabolic stress. Keywords: Diabetes, Pancreas, Beta cell dysfunction, Network analysis, Molecular phenotyping, Metabolic stress

Abstract Photopharmacology describes the use of light to precisely deliver drug activity in space and time. Such approaches promise to improve drug specificity by reducing off-target effects. As a proof-of-concept, we have subjected the fourth generation photoswitchable sulfonylurea JB253 to comprehensive toxicology assessment, including mutagenicity and maximum/repeated tolerated dose studies, as well as in vivo testing in rodents. Here, we show that JB253 is well-tolerated with minimal mutagenicity and can be used to optically-control glucose homeostasis in anesthetized mice following delivery of blue light to the pancreas. These studies provide the first demonstration that photopharmacology may one day be applicable to the light-guided treatment of type 2 diabetes and other metabolic disease states in vivo in humans.

Diabetes mellitus now affects more than 400 million individuals worldwide, with significant impacts on the lives of those affected and associated socio-economic costs. Although defects in insulin secretion underlie all forms of the disease, the molecular mechanisms which drive them are still poorly understood. Subsets of specialised beta cells have, in recent years, been suggested to play critical roles in “pacing” overall islet activity. The molecular nature of these cells, the means through which their identity is established and the changes which may contribute to their functional demise and “loss of influence” in both type 1 and type 2 diabetes are largely unknown. Genomic imprinting involves the selective silencing of one of the two parental alleles through DNA methylation and modified imprinted gene expression is involved in a number of diseases. Loss of expression, or loss of imprinting, can be shown in mouse models to lead to defects in beta cell function and abnormal insulin secretion. In the present review we survey the evidence that altered expression of imprinted genes contribute to loss of beta cell function, the importance of beta cell heterogeneity in normal and disease states, and hypothesise whether there is a direct link between the two.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.

Aims/hypothesis: The adipose tissue-derived hormone leptin plays an important role in the maintenance of body weight and glucose homeostasis. Leptin mediates its effects by interaction with leptin receptors (LepRb), which are highly expressed in the hypothalamus and other brain centres, and at lower levels in the periphery. Previous studies have used relatively promiscuous or inefficient Cre deleter strains, respectively, to explore the roles of LepR in pancreatic β and α cells. Here, we use two newly-developed Cre lines to explore the role of leptin signalling in insulin and proglucagon-expressing cells. Methods: Leptin receptor expression was measured in isolated mouse islets and highly-purified islet cells by RNASeq and quantitative RT-PCR. Mice lacking leptin signalling in pancreatic β, or in α and other proglucagon-expressing cells, were generated using Ins1Cre- or iGluCre-mediated recombination respectively of flox'd leptin receptor alleles. In vivo glucose homeostasis, changes in body weight, pancreatic histology and hormone secretion from isolated islets were assessed using standard techniques. Results: Leptin receptor mRNA levels were at or below the level of detection in wild-type adult mouse isolated islets and purified cells, and leptin signalling to Stat3 phosphorylation was undetectable. Whereas male mice further deleted for leptin receptors in β cells exhibited no abnormalities in glucose tolerance up to 16 weeks of age, females transiently displayed improved glucose tolerance at 8 weeks (11.2 ± 3.2% decrease in area under curve; p

Aims/Hypothesis: Glucagon release from pancreatic alpha cells is required for normal glucose homoeostasis and is dysregulated in both Type 1 and Type 2 diabetes. The tumour suppressor LKB1 (STK11) and the downstream kinase AMP-activated protein kinase (AMPK), modulate cellular metabolism and growth, and AMPK is an important target of the anti-hyperglycaemic agent metformin. While LKB1 and AMPK have emerged recently as regulators of beta cell mass and insulin secretion, the role of these enzymes in the control of glucagon production in vivo is unclear. Methods: Here, we ablated LKB1 (αLKB1KO), or the catalytic alpha subunits of AMPK (αAMPKdKO, -α1KO, -α2KO), selectively in ∼45% of alpha cells in mice by deleting the corresponding flox'd alleles with a preproglucagon promoter (PPG) Cre. Results: Blood glucose levels in male αLKB1KO mice were lower during intraperitoneal glucose, aminoimidazole carboxamide ribonucleotide (AICAR) or arginine tolerance tests, and glucose infusion rates were increased in hypoglycemic clamps (p

Increasing evidence suggests that loss of β cell characteristics may cause insulin secretory deficiency in diabetes, but the underlying mechanisms remain unclear. Here, we show that Rfx6, whose mutation leads to neonatal diabetes in humans, is essential to maintain key features of functionally mature β cells in mice. Rfx6 loss in adult β cells leads to glucose intolerance, impaired β cell glucose sensing, and defective insulin secretion. This is associated with reduced expression of core components of the insulin secretion pathway, including glucokinase, the Abcc8/SUR1 subunit of KATP channels and voltage-gated Ca2+ channels, which are direct targets of Rfx6. Moreover, Rfx6 contributes to the silencing of the vast majority of “disallowed” genes, a group usually specifically repressed in adult β cells, and thus to the maintenance of β cell maturity. These findings raise the possibility that changes in Rfx6 expression or activity may contribute to β cell failure in humans.

Liver kinase B1 (LKB1; also known as STK11) is a serine/threonine kinase and tumour suppressor that is mutated in Peutz-Jeghers syndrome (PJS), a premalignant syndrome associated with the development of gastrointestinal polyps. Proglucagon-expressing enteroendocrine cells are involved in the control of glucose homeostasis and the regulation of appetite through the secretion of gut hormones such as glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY). To determine the role of LKB1 in these cells, we bred mice bearing floxed alleles of Lkb1 against animals carrying Cre recombinase under proglucagon promoter control. These mice (GluLKB1KO) were viable and displayed near-normal growth rates and glucose homeostasis. However, they developed large polyps at the gastro-duodenal junction, and displayed premature mortality (death from 120 days of age). Histological analysis of the polyps demonstrated that they had a PJS-like appearance with an arborising smooth-muscle core. Circulating GLP-1 levels were normal in GluLKB1KO mice and the polyps expressed low levels of the peptide, similar to levels in the neighbouring duodenum. Lineage tracing using a Rosa26tdRFP transgene revealed, unexpectedly, that enterocytes within the polyps were derived from non-proglucagon-expressing precursors, whereas connective tissue was largely derived from proglucagon-expressing precursors. Developmental studies in wild-type mice suggested that a subpopulation of proglucagon-expressing cells undergo epithelial-mesenchymal transition (EMT) to become smooth-muscle-like cells. Thus, it is likely that polyps in the GluLKB1KO mice developed from a unique population of smooth-muscle-like cells derived from a proglucagon-expressing precursor. The loss of LKB1 within this subpopulation seems to be sufficient to drive tumorigenesis.

SUMMARY Axon degeneration is observed in neurodegenerative diseases and neuroinflammatory disorders, such as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. The molecular basis of this process remains largely unknown. Here, we show that mice deleted for the tumour suppressor LKB1 (also called STK11) in the spinal cord, some parts of the brain and in the endocrine pancreas (βLKB1KO mice) develop hind-limb dysfunction and axon degeneration at about 7 weeks. Demyelination and macrophage infiltration are observed in the white matter of these mice, predominantly in the bilateral and anterior funiculi of the thoracic segment of the spinal cord, suggesting damage to the ascending sensory signalling pathway owing to LKB1 deletion in the brain. Microtubule structures were also affected in the degenerated foci, with diminished neurofilament and tubulin expression. Deletion of both PRKAA1 genes, whose products AMPKα1 and AMPKα2 are also downstream targets of LKB1, with the same strategy was without effect. We thus define LKB1 as an intrinsic suppressor of axon degeneration and a possible target for strategies that can reverse this process.

Previous studies have reported both positive and negative effects of culture of islets at high glucose concentrations on regulated insulin secretion. Here, we have reexamined this question in mouse islets and determined the role of changes in lipid synthesis in the effects of glucose. Glucose-stimulated insulin secretion (GSIS) and gene expression were examined in islets from C57BL/6 mice or littermates deleted for sterol-regulatory element binding protein-1 (SREBP1) after 4 days of culture at high glucose concentrations. Culture of control islets at 30 versus 8 mmol/l glucose led to enhanced secretion at both basal (3 mmol/l) and stimulatory (17 mmol/l) glucose concentrations and to enhanced triacylglycerol accumulation. These changes were associated with increases in the expression of genes involved in glucose sensing (glucose transporter 2, glucokinase, sulfonylurea receptor 1, inwardly rectifying K+ channel 6.2), differentiation (pancreatic duodenal homeobox 1), and lipogenesis (Srebp1, fatty acid synthase, acetyl-coenzyme A carboxylase 1, stearoyl-coenzyme A desaturase 1). When cultured at either 8 or 30 mmol/l glucose, SREBP1-deficient (SREBP1−/−) islets displayed reduced GSIS and triacylglycerol content compared with normal islets. Correspondingly, glucose induction of the above genes in control islets was no longer observed in SREBP1−/− mouse islets. We conclude that enhanced lipid synthesis mediated by SREBP1c-dependent genes is required for the adaptive changes in islet gene expression and insulin secretion at high glucose concentrations.

Pancreatic β-cell dysfunction is central to the pathogenesis of type 2 diabetes and may involve secretory failure through glucolipotoxity. The relative importance of the transcription factors carbohydrate-responsive element binding protein (ChREBP), sterol-responsive element binding protein-1c (SREBP-1c), and upstream stimulatory factor (USF) in the induction of lipogenic genes by glucose remains unclear. By confocal imaging, we show that ChREBP translocates to the nucleus in MIN6 β cells in response to glucose. Both ChREBP and SREBP-1c were required for the induction of the fatty acid synthase (FAS) promoter by glucose, and chromatin immunoprecipitation (ChIP) assay revealed that glucose induced the binding of both ChREBP and SREBP-1c to the FAS promoter without affecting USF2 binding. By contrast, ChIP assay revealed that high glucose prompted direct binding of ChREBP, but not SREBP-1c or USF2, to the liver-type pyruvate kinase (L-PK) promoter. This event was indispensable for the induction of the L-PK gene by glucose, as demonstrated by RNA silencing, single-cell promoter analysis, and quantitative real-time PCR. We conclude that ChREBP is a critical regulator of lipogenic genes in the β cell and may play a role in the development of glucolipotoxicity and β cell failure through alteration of gene expression in type 2 diabetes.

More than 65 loci, encoding up to 500 different genes, have been implicated by genome-wide association studies (GWAS) as conferring an increased risk of developing type 2 diabetes (T2D). Whilst mouse models have in the past been central to understanding the mechanisms through which more penetrant risk genes for T2D, for example, those responsible for neonatal or maturity-onset diabetes of the young, only a few of those identified by GWAS, notably TCF7L2 and ZnT8/SLC30A8, have to date been examined in mouse models. We discuss here the animal models available for the latter genes and provide perspectives for future, higher throughput approaches towards efficiently mining the information provided by human genetics.