Chronic noncancer pain management: Integration of a nurse-led program in primary care.

PROBLEM ADDRESSED: Chronic noncancer pain is often excessively managed with medications (most notably opioids) instead of nonpharmacologic options or multidisciplinary care-the gold standards. OBJECTIVE OF PROGRAM: To offer an effective alternative to pharmacologic management of chronic noncancer pain in primary care. PROGRAM DESCRIPTION: Patients 18 years of age or older with chronic noncancer pain were referred by family physicians or nurse practitioners in a family health team (outpatient, multidisciplinary clinic) in Ottawa, Ont. A registered nurse used the Pain Explanation and Treatment Diagram with patients, taught self-management skills (related to habits [smoking, consumption of alcohol, diet], exercise, sleep, ergonomics, and psychosocial factors), and referred patients to relevant resources. CONCLUSION: A nurse-led chronic pain program, initiated without extra funding, was successfully integrated into a primary care setting. Among the participating patients in the pilot project, outcomes related to pain intensity, pain interference with daily living, and opioid use were encouraging. This program could serve as a model for improving chronic noncancer pain management in primary care.

Opposing effects on regulated insulin secretion of acute vs chronic stimulation of AMP-activated protein kinase.

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-ßH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for ß1- vs ß2-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 (p<0.01, p<0.05 and p<0.001, respectively), despite a lowering of glucose-induced intracellular free Ca2+ dynamics in response to 991 (AUC, p<0.05) and to RA089 at the highest dose (25 µmol/l) at 5.59 min (p<0.05). Although abolished in the absence of AMPK, the effects of 991 were observed in the absence of the upstream kinase, liver kinase B1, further implicating 'amplifying' pathways. In marked contrast, chronic activation of AMPK, either globally or selectively in the beta cell, achieved using a gain-of-function mutant, impaired insulin release in vivo (p<0.05 at 15 min following i.p. injection of 3 mmol/l glucose) and in vitro (p<0.01 following incubation of islets with 17 mmol/l glucose), and lowered glucose tolerance (p<0.001). 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.

In vivo and in vitro characterization of GL0034, a novel long-acting glucagon-like peptide-1 receptor agonist.

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 ß-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.

Adipocyte-specific deletion of Tcf7l2 induces dysregulated lipid metabolism and impairs glucose tolerance in mice.

AIMS/HYPOTHESIS: Transcription factor 7-like 2 (TCF7L2) is a downstream effector of the Wnt/ß-catenin signalling pathway implicated in type 2 diabetes risk through genome-wide association studies. Although its expression is critical for adipocyte development, the potential roles of changes in adipose tissue TCF7L2 levels in diabetes risk are poorly defined. Here, we investigated whether forced changes in Tcf7l2 expression in adipocytes affect whole body glucose or lipid metabolism and crosstalk between disease-relevant tissues. METHODS: Tcf7l2 was selectively ablated in mature adipocytes in C57BL/6J mice using Cre recombinase under Adipoq promoter control to recombine Tcf7l2 alleles floxed at exon 1 (referred to as aTCF7L2 mice). aTCF7L2 mice were fed normal chow or a high-fat diet for 12 weeks. Glucose and insulin sensitivity, as well as beta cell function, were assessed in vivo and in vitro. Levels of circulating NEFA, selected hormones and adipokines were measured using standard assays. RESULTS: Reduced TCF7L2 expression in adipocytes altered glucose tolerance and insulin secretion in male but not in female mice. Thus, on a normal chow diet, male heterozygote knockout mice (aTCF7L2het) exhibited impaired glucose tolerance at 16 weeks (p = 0.03) and increased fat mass (1.4 ± 0.1-fold, p = 0.007) but no changes in insulin secretion. In contrast, male homozygote knockout (aTCF7L2hom) mice displayed normal body weight but impaired oral glucose tolerance at 16 weeks (p = 0.0001). These changes were mechanistically associated with impaired in vitro glucose-stimulated insulin secretion (decreased 0.5 ± 0.1-fold vs control mice, p = 0.02) and decreased levels of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (0.6 ± 0.1-fold and 0.4 ± 0.1-fold vs control mice, p = 0.04 and p < 0.0001, respectively). Circulating levels of plasma NEFA and fatty acid binding protein 4 were increased by 1.3 ± 0.1-fold and 1.8 ± 0.3-fold vs control mice (p = 0.03 and p = 0.05, respectively). Following exposure to a high-fat diet for 12 weeks, male aTCF7L2hom mice exhibited reduced in vivo glucose-stimulated insulin secretion (0.5 ± 0.1-fold vs control mice, p = 0.02). CONCLUSIONS/INTERPRETATION: Loss of Tcf7l2 gene expression selectively in adipocytes leads to a sexually dimorphic phenotype, with impairments not only in adipocytes, but also in pancreatic islet and enteroendocrine cells in male mice only. Our findings suggest novel roles for adipokines and incretins in the effects of diabetes-associated variants in TCF7L2, and further illuminate the roles of TCF7L2 in glucose homeostasis and diabetes risk. Graphical abstract.

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis.

AIMS/HYPOTHESIS: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. METHODS: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. RESULTS: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. CONCLUSIONS/INTERPRETATION: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. DATA AVAILABILITY: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.

The pore-forming subunit MCU of the mitochondrial Ca2+ uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice.

AIMS/HYPOTHESIS: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. METHODS: Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. RESULTS: Glucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (ßMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in ßMcu-KO animals. ßMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young ßMcu-KO (<12 weeks), but not in older animals vs WT mice. CONCLUSIONS/INTERPRETATION: MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in ßMcu-KO mice remain to be established.

Decreased STARD10 Expression Is Associated with Defective Insulin Secretion in Humans and Mice.

Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in ß cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, ß-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca2+ dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult ß cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in ß cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the ß cell.

Transcription factor-7-like 2 (TCF7L2) gene acts downstream of the Lkb1/Stk11 kinase to control mTOR signaling, ß cell growth, and insulin secretion.

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

MiR-184 expression is regulated by AMPK in pancreatic islets.

AMPK is a critical energy sensor and target for widely used antidiabetic drugs. In ß cells, elevated glucose concentrations lower AMPK activity, and the ablation of both catalytic subunits [ß-cell-specific AMPK double-knockout (ßAMPKdKO) mice] impairs insulin secretion in vivo and ß-cell identity. MicroRNAs (miRNAs) are small RNAs that silence gene expression that are essential for pancreatic ß-cell function and identity and altered in diabetes. Here, we have explored the miRNAs acting downstream of AMPK in mouse and human ß cells. We identified 14 down-regulated and 9 up-regulated miRNAs in ßAMPKdKO vs. control islets. Gene ontology analysis of targeted transcripts revealed enrichment in pathways important for ß-cell function and identity. The most down-regulated miRNA was miR-184 (miR-184-3p), an important regulator of ß-cell function and compensatory expansion that is controlled by glucose and reduced in diabetes. We demonstrate that AMPK is a potent regulator and an important mediator of the negative effects of glucose on miR-184 expression. Additionally, we reveal sexual dimorphism in miR-184 expression in mouse and human islets. Collectively, these data demonstrate that glucose-mediated changes in AMPK activity are central for the regulation of miR-184 and other miRNAs in islets and provide a link between energy status and gene expression in ß cells.-Martinez-Sanchez, A., Nguyen-Tu, M.-S., Cebola, I., Yavari, A., Marchetti, P., Piemonti, L., de Koning, E., Shapiro, A. M. J., Johnson, P., Sakamoto, K., Smith, D. M., Leclerc, I., Ashrafian, H., Ferrer, J., Rutter, G. A. MiR-184 expression is regulated by AMPK in pancreatic islets.

The transcription factor Pax6 is required for pancreatic ß cell identity, glucose-regulated ATP synthesis, and Ca2+ dynamics in adult mice.

Heterozygous mutations in the human paired box gene PAX6 lead to impaired glucose tolerance. Although embryonic deletion of the Pax6 gene in mice leads to loss of most pancreatic islet cell types, the functional consequences of Pax6 loss in adults are poorly defined. Here we developed a mouse line in which Pax6 was selectively inactivated in ß cells by crossing animals with floxed Pax6 alleles to mice expressing the inducible Pdx1CreERT transgene. Pax6 deficiency, achieved by tamoxifen injection, caused progressive hyperglycemia. Although ß cell mass was preserved 8 days post-injection, total insulin content and insulin:chromogranin A immunoreactivity were reduced by ∼60%, and glucose-stimulated insulin secretion was eliminated. RNA sequencing and quantitative real-time PCR analyses revealed that, although the expression of key ß cell genes, including Ins2, Slc30a8, MafA, Slc2a2, G6pc2, and Glp1r, was reduced after Pax6 deletion, that of several genes that are usually selectively repressed ("disallowed") in ß cells, including Slc16a1, was increased. Assessed in intact islets, glucose-induced ATP:ADP increases were significantly reduced (p < 0.05) in ßPax6KO versus control ß cells, and the former displayed attenuated increases in cytosolic Ca2+ Unexpectedly, glucose-induced increases in intercellular connectivity were enhanced after Pax6 deletion, consistent with increases in the expression of the glucose sensor glucokinase, but decreases in that of two transcription factors usually expressed in fully differentiated ß-cells, Pdx1 and Nkx6.1, were observed in islet "hub" cells. These results indicate that Pax6 is required for the functional identity of adult ß cells. Furthermore, deficiencies in ß cell glucose sensing are likely to contribute to defective insulin secretion in human carriers of PAX6 mutations.

Local and regional control of calcium dynamics in the pancreatic islet.

Ca2+ is the key intracellular regulator of insulin secretion, acting in the ß-cell as the ultimate trigger for exocytosis. In response to high glucose, ATP-sensitive K+ channel closure and plasma membrane depolarization engage a sophisticated machinery to drive pulsatile cytosolic Ca2+ changes. Voltage-gated Ca2+ channels, Ca2+ -activated K+ channels and Na+ /Ca2+ exchange all play important roles. The use of targeted Ca2+ probes has revealed that during each cytosolic Ca2+ pulse, uptake of Ca2+ by mitochondria, endoplasmic reticulum (ER), secretory granules and lysosomes fine-tune cytosolic Ca2+ dynamics and control organellar function. For example, changes in the expression of the Ca2+ -binding protein Sorcin appear to provide a link between ER Ca2+ levels and ER stress, affecting ß-cell function and survival. Across the islet, intercellular communication between highly interconnected "hubs," which act as pacemaker ß-cells, and subservient "followers," ensures efficient insulin secretion. Loss of connectivity is seen after the deletion of genes associated with type 2 diabetes (T2D) and follows metabolic and inflammatory insults that characterize this disease. Hubs, which typically comprise ~1%-10% of total ß-cells, are repurposed for their specialized role by expression of high glucokinase (Gck) but lower Pdx1 and Nkx6.1 levels. Single cell-omics are poised to provide a deeper understanding of the nature of these cells and of the networks through which they communicate. New insights into the control of both the intra- and intercellular Ca2+ dynamics may thus shed light on T2D pathology and provide novel opportunities for therapy.

Molecular phenotyping of single pancreatic islet leader beta cells by "Flash-Seq".

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 < 0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Providing some support for these findings, ADCY6 immunoreactivity tended to be higher in leader than follower cells, whereas cilia number and length tended to be lower in the former. Finally, leader cells were located significantly closer to delta, but not alpha, cells in Euclidian space than were follower cells. SIGNIFICANCE: The existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. These data also raise the possibility that localised signalling between delta and leader cells contributes to the initiation and propagation of islet Ca2+ waves.

Impact of an SLC30A8 loss-of-function variant on the pancreatic distribution of zinc and manganese: laser ablation-ICP-MS and positron emission tomography studies in mice.

Introduction: Common 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]. Methods: Following 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. Results: Our 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. Discussion: These 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 ß-cell zinc and manganese content and impact the levels of these metals in the exocrine pancreas to improve insulin secretion.

Identification of biomarkers for glycaemic deterioration in type 2 diabetes.

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.

Vertical Sleeve Gastrectomy Lowers SGLT2/Slc5a2 Expression in the Mouse Kidney.

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 clustered regularly interspaced short palindromic repeats (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.

Vertical sleeve gastrectomy normalizes circulating glucocorticoid levels and lowers glucocorticoid action tissue-selectively in mice.

Objectives: Glucocorticoids produced by the adrenal cortex are essential for the maintenance of metabolic homeostasis. Glucocorticoid activation is catalysed by 11ß-hydroxysteroid dehydrogenase 1 (11ß-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. Methods: Glucose 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 11ß-HSD1 levels were measured by quantitative RT-PCR or Western (immuno-) blotting, respectively. 11ß-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. Results: Under 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 11ß-HSD1 expression in liver and adipose tissue post-surgery. Elimination of the 11ß-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. Conclusions: Bariatric 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.

Mitofusins Mfn1 and Mfn2 Are Required to Preserve Glucose- but Not Incretin-Stimulated ß-Cell Connectivity and Insulin Secretion.

Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic ß-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 ß-cell-selective, adult-restricted deletion knock-out (dKO) of the mitofusin genes Mfn1 and Mfn2 (ßMfn1/2 dKO). ßMfn1/2-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 ßMfn1/2-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 ß-cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in ß-cells, the potential contributions of altered mitochondrial dynamics to diabetes development, and the impact of incretins on this process.

Glucose-Dependent miR-125b Is a Negative Regulator of ß-Cell Function.

Impaired pancreatic ß-cell function and insulin secretion are hallmarks of type 2 diabetes. miRNAs are short, noncoding RNAs that silence gene expression vital for the development and function of ß cells. We have previously shown that ß cell-specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in ß cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycemia in ß cells. Here, we show that islet miR-125b-5p expression is upregulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose-stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased, high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human ß-cell line EndoCß-H1 shortened mitochondria and enhanced GSIS, whereas mice overexpressing miR-125b-5p selectively in ß cells (MIR125B-Tg) were hyperglycemic and glucose intolerant. MIR125B-Tg ß cells contained enlarged lysosomal structures and had reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.