bluuebunny/crossref_metadata_2025_split huggingface dataset:
236-OR: Molecular and Biological Properties of Insulin Icodec, a New Insulin Analog Designed to Give a Long Half-Life Suitable for Once-Weekly Dosing
Authors: NISHIMURA ERICA , KJELDSEN THOMAS , HUBALEK FRANTISEK , GLENDORF TINE , STIDSEN CARSTEN E. , HANSEN BO F. , PEDERSEN THOMAS A. , LUETZEN ANNE , PRIDAL LONE , MADSEN PETER 
Published: June 2020

Insulin icodec is a novel insulin analog with a terminal elimination half-life of ∼196 hours currently in clinical development, designed to be able to cover a full week’s basal insulin requirements with a single subcutaneous injection. To attain such a long half-life, the insulin molecule was modified to achieve an albumin-bound circulating depot of icodec which acts just as human insulin (HI) but is more slowly cleared.Addition of a C20 fatty diacid containing side chain at B29K via a hydrophilic linker imparts strong but reversible albumin binding (10x that of insulin detemir). Three amino acid substitutions (A14E, B16H and B25H) ensure reduced enzymatic degradation of icodec and contribute to attenuating insulin receptor (IR) binding and clearance, further prolonging the half-life. In vitro studies demonstrated that icodec is a specific and full agonist of the human IR, and that it displays the same dose-dependent mode of action as HI, exemplified by its ability to phosphorylate the IR and stimulate intracellular signaling pathways (phospho-AKT and -ERK). Functional assays have demonstrated that icodec elicits the same pattern of metabolic effects as HI, including glucose uptake and lipogenesis in fat cells, and stimulation of glycogen synthesis in liver cells. The affinity of icodec for the IGF-1 receptor was found to be proportionately lower than its binding to the IR. The in vitro mitogenic effect of icodec in primary human mammary cells (HMEC), as well as in mammary and colon carcinoma cells (MCF-7 and COLO 205) was found to be low relative to that of HI.
In conclusion, icodec is a new insulin analog designed to achieve a slowly cleared, albumin-bound circulating depot which results in a long half-life suited for once weekly injections, covering the basal insulin requirements for a full week. *Insulin icodec (proposed INN).

Disclosure
E. Nishimura: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. T. Kjeldsen: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. F. Hubalek: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. T. Glendorf: Employee; Spouse/Partner; Novo Nordisk A/S. Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. C.E. Stidsen: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. B.F. Hansen: Stock/Shareholder; Self; Novo Nordisk A/S. T.A. Pedersen: Employee; Self; Novo Nordisk A/S. A. Luetzen: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. L. Pridal: None. P. Madsen: Employee; Self; Novo Nordisk A/S.
237-LB: Cellular Heterogeneity in Brown, Dermal, and Bone Marrow Adipose Depots
Authors: POWELL NOAH D. , SHARMA RITA , HUANG JUN , LEE KEVIN Y. 
Published: June 2020

Recent studies have shown that heterogeneity among adipocytes exists even within a single white adipose tissue depot. Previous studies from our laboratory have identified three functionally distinct subpopulations of adipocytes with different gene expression and metabolic properties. These adipocyte subpopulations differentially contribute to the individual white adipose tissue depots and can be distinguished by their specific expression of three marker genes: Wilms Tumor 1 (Wt1), Transgelin (Tagln), and Myxovirus 1 (Mx1). In this study, we extend these observations by determining the contributions of these adipocyte subpopulations to other adipose tissue depots including the interscapular brown, dermal, and bone marrow adipose tissue depots. Lineage tracing analysis of Wt1-cre and Tagln-cre crossed to a dual-fluorescent reporter mice which mark type 1 and type 2 preadipocytes and adipocytes, respectively, was performed in two to four month old mice and preadipocytes and adipocytes were quantified by FACS analysis and confocal microscopy. In all depots analyzed in this study, little to no type 1 preadipocytes or adipocytes were present in any of these depots. On the other hand, a significant number of type 2 preadipocytes and adipocytes were found in all depots. In dermal tissue, type 2 adipocytes comprised 9±2% of adipocytes and 45±7% of preadipocytes. Type 2 adipocytes significantly contributed to the bone marrow adipose tissue as 51±4% of adipocytes present and 83±3% of preadipocytes were type 2 cells. Lastly, in brown adipose tissue, type 2 cells comprised 15±1% of adipocytes and 10±3% of preadipocytes. Further studies investigating the metabolism and thermogenic properties of these type 2 brown adipocytes is currently being performed, as a more thorough understanding these adipocyte subpopulations may lead to novel, targeted treatments for obesity and diabetes.

Disclosure
N.D. Powell: None. R. Sharma: None. J. Huang: None. K.Y. Lee: None.
241-OR: Causal Gene Candidates for Type 2 Diabetes Based on Protein-Coding Variants in 127,676 Individuals
Authors: DORNBOS PETER , RAFFIELD LAURA , YIN XIANYONG , FLANNICK JASON 
Published: June 2020

Genome-wide association studies (GWAS) have identified &gt;250 genetic loci associated with type 2 diabetes (T2D), but, currently, few associations have been translated to causal genes. To directly implicate genes in T2D pathogenesis, we analyzed coding variants from the largest whole exome sequencing (WES) study to date, consisting of 36,652 cases and 91,024 controls from the Accelerating Medicines Partnership-T2D-GENES, Cohorts for Heart and Aging Research in Genomic Epidemiology, Myocardial Infarction Genetics, and NHLBI Trans-Omics for Precision Medicine consortia. Through single-variant and gene-level meta-analysis, we identify three evidence tiers of T2D-susceptibility genes. First, 11 genes harbor exome-wide significant missense associations (p≤4.3x10-7) and four genes have significant gene-level associations (p≤2.5x10-6). Two of these associations are novel: a missense mutation in MET (p=2.33x10-7), a gene with no previous genetic link to T2D, and, while presence of misdiagnosed MODY cases cannot be excluded, a series of rare alleles in GCK providing the first exome-wide significant association with common T2D. Second, we apply a gene-level Bayesian statistical approach to identify 29 genes with posterior probabilities (PPA) of ≥50% of explaining signals at GWAS loci. Twenty-one of these genes lie within loci where no causal gene is known, including SGP7 (PPA=0.85), a zinc metalloprotease that contains a previously reported independent common missense variant associated with T2D. Finally, we extend this Bayesian approach to another 1,069 genes previously studied in mice. This analysis identifies 17 genes with gene-level association PPA ≥50%, including 11 genes with no prior human genetic link to T2D such as ONECUT1 (PPA=0.54) which is exclusively expressed in liver and pancreas. Our results further illustrate the value of WES in identifying genes that may be future T2D drug targets. All results presented will be available through the AMP-T2D Knowledge Portal.

Disclosure
P. Dornbos: None. L. Raffield: None. X. Yin: None. J. Flannick: None.
245-OR: CIS-Regulatory Architecture of Human ESC-Derived Hypothalamic Neuron Differentiation Aids in Variant-to-Gene Mapping of Type 2 Diabetes Related Traits
Authors: PAHL MATTHEW C. , LITTLETON SHERIDAN H. , DOEGE CLAUDIA , HODGE KENYAITA M. , LEONARD MICHELLE , LU SUMEI , HAMMOND REZA , BOEHM KEITH , LASCONI CHIARA , SU CHUN , CHESI ALESSANDRA , PIPPIN JAMES A. , JOHNSON MATTHEW , WELLS ANDREW D. , VOIGHT BENJAMIN F. , LEIBEL RUDOLPH , GRANT STRUAN F. , COUSMINER DIANA L. 
Published: June 2020

Dysregulation of hypothalamus-mediated metabolic homeostasis influences risk factors for T2D, such as BMI, pubertal timing and sleep. However, the gene regulatory landscape of the hypothalamus remains unclear. We characterized the physical relationships among gene promoters and their corresponding cis-regulatory elements in human embryonic stem cell (ESC)-derived hypothalamic cells by integrating RNA-seq, ATAC-seq and promoter-focused Capture C profiles. We conducted this effort in an in vitro model of hypothalamic neurons using human ESCs at 3 stages of differentiation: ESCs, hypothalamic progenitors (HPs) and hypothalamic-like neurons (HNs). We identified 87,170 open chromatin regions (OCRs) contacting promoters in at least 1 stage of HN differentiation. Interestingly, we observed that regardless of gene expression pattern during differentiation, overall accessibility of interacting OCRs first increased during the transition to HP, then decreased following differentiation to HN. In order to relate these findings to GWAS data, we tested for enrichment of SNPs within promoter-interacting OCRs. We found significant associations with BMI, sleep traits, age at menarche (AAM) and major depressive disorder (MDD). We mapped these trait-associated variants to their physically interacting genes. We identified 2 putative effector genes for AAM (RPS26 and SUOX at chr12q13.2) and 1 for BMI (DHRS11), which were also supported by colocalized hypothalamus eQTLs in GTEx. Additionally, HyPrColoc analyses revealed several putative effector genes associated with &gt;1 trait, including ZRANB2 for AAM, BMI and MDD, and FEZF1 for AAM and BMI, the latter being known to harbor Mendelian mutations that impair puberty. Taken together, our analyses provide insight into cis-regulatory architecture during hypothalamic development and how it may contribute to phenotypes associated with T2D risk.

Disclosure
M.C. Pahl: None. S.H. Littleton: None. C. Doege: None. K.M. Hodge: None. M. Leonard: None. S. Lu: None. R. Hammond: None. K. Boehm: None. C. Lasconi: None. C. Su: None. A. Chesi: None. J.A. Pippin: None. M. Johnson: None. A.D. Wells: None. B.F. Voight: None. R. Leibel: None. S.F. Grant: None. D.L. Cousminer: None.

Funding
American Diabetes Association (1-17-PDF-077 to D.L.C.); National Institutes of Health (K99HD099330-01); National Center for Research Resources (UL1RR024134); National Center for Advancing Translational Sciences (UL1TR000003); Institute for Translational Medicine and Therapeutics; Children’s Hospital of Philadelphia
249-LB: Effects of Oxidative Phosphorylation Complex Defects on Beta-Cell Biology
Authors: LANG ANNA L. , CAICEDO ALEJANDRO 
Published: June 2020

Background: The insulin-secreting pancreatic beta cell is a highly metabolic cell type relying on mitochondrial metabolism for stimulus-secretion coupling. Specifically, the ATP-producing oxidative phosphorylation (OXPHOS) pathway is central to insulin secretion. Although disruptions in ATP production occur in beta cell dysfunction, the exact mechanisms remain unknown. We have generated beta cell specific OXPHOS complex inducible knock out mouse models of Complex I, III, and IV to determine the direct effects OXPHOS complexes on beta cell function. We hypothesize that individual complexes contribute distinctly to beta cell function.
Methods: Eight-week-old, male OXPHOSf/f - MIPcreERT mice were administered tamoxifen (150mg/kg) or vehicle by I.P. injection for 5 consecutive days. Non-fasting glycemia and body weight were measured one week prior and once per week up to 5 weeks after induction. Glucose tolerance was assessed by glucose tolerance tests (IP-GTT) 1 week before and 3 weeks after induction for all groups. Mice were anesthetized with ketamine/xylazine and perfused with sterile PBS followed by 4% PFA. Pancreata were dissected, embedded in OCT, and cut at 40µM sections for immunofluorescent staining of insulin, glucagon, and somatostatin.
Results: No changes in body weight were observed throughout the experimental period for any group. Complex III knockout animals developed severe hyperglycemia and significant glucose intolerance at 3 weeks after induction. Although the other models display a minor trend towards hyperglycemia starting at 5 weeks after induction, they retain their ability to regulate glucose at 3 weeks as seen by IP-GTT. Interestingly, no changes in islet cytoarchitecture are observed in any of the groups at the 3-week timepoint.
Conclusions: Mice with Complex III deficient beta cells show a robust diabetic phenotype compared to Complex I and IV models. These results support the hypothesis that specific OXPHOS complexes contribute differentially to beta cell function.

Disclosure
A.L. Lang: None. A. Caicedo: None.

Funding
National Institute of Environmental Health Sciences (R21ES025673-01)
251-OR: Cardiac Autoantibodies (AB) Predict Cardiovascular Disease (CVD) in T1D and Are Associated with Cytokine Signatures Linked to CVD Risk
Authors: SOUSA GIOVANE R. , NIEWCZAS MONIKA , LIPES MYRA A. 
Published: June 2020

CVD is the leading cause of mortality in T1D; however, only ∼50% of this risk is explained by traditional risk factors. Recent studies show that subjects with T1D in the DCCT developed cardiac autoimmunity, defined by the presence of ≥2 Ab. We tested the hypothesis that some of the unexplained CVD risk in T1D is mediated by Ab-linked cytokine pathways. We measured Ab in serum from 892 post-DCCT EDIC subjects (mean age 49 and T1D duration 28 years), with no CVD events at baseline (Figure). Cumulative incidence of a first CVD event in subjects with 0, 1, and &gt;2 Ab was 2.0%, 8.9%, and 43%, respectively, over 4-year median follow-up. In Cox regression models, ≥2 Ab predicted CVD events with minimal attenuation by known risk factors. Using a 42-plex array to measure baseline serum cytokines in subjects with ≥2 Ab and matched controls without Ab, the ≥2 Ab group showed 3.4-fold odds (95% CI, 1.6-6.1; P &lt; 0.0001)of having a 1 tertile increase in a proinflammatory component comprising IFN-γ, TNF-α, G-CSF, MDC, IL1-RA, IL-8, and EGF. Risk of CVD events was also independently associated with IFN-γ, TNF-α, and G-CSF levels (HR, 5.4; 95% CI, 1.5-18.9; P = 0.009).
In conclusion, in this cohort with longstanding T1D, cardiac Ab identified subjects at high risk for CVD events, potentially through Ab-linked cytokine pathways. These findings raise the possibility to immunologically predict and prevent CVD in T1D.

Disclosure
G.R. Sousa: None. M. Niewczas: None. M.A. Lipes: None.

Funding
National Institutes of Health (DK1036909)
290-OR: Diabetes Progression in IFG-Receptor Deficient NOD Mice Is Associated with Increased CD8+ T Lymphocyte Sensitivity to Common Gamma Chain Cytokines
Authors: KAY THOMAS , JHALA GAURANG , KRISHNAMURTHY BALASUBRAMANIAN , THOMAS HELEN E. 
Published: June 2020

IFN-γ is a potential drug target in type 1 diabetes and this is part of the rationale for trialing JAK inhibitors (JAKi) for treatment of new onset T1D. It regulates migration and adhesion of leucocytes, as well as expression of MHC class I on islet beta cells. However, neither genetic deficiency of IFN-γ nor its receptor in NOD mice modifies disease progression. To understand this paradox we characterised islet antigen-specific T cells in NOD mice lacking the IFN-γ receptor (Ifngr) generated directly in NOD mouse embryos using CRISPR/Cas9 gene editing. Islet antigen-specific CD8+ T cells (proinsulin B15-23 and IGRP206-214) and CD4+ T cells (proinsulin B10-23and BDC2.5 mimotope) were quantified using tetramer enrichment and flow cytometry. At 125 days of age, insulin-specific (177±41 cells in wild type and 1799±1123 cells in Ifngr mutant, p=0.036) and IGRP-specific CD8+ T cells (2524±735 cells in wild type and 30598±10426 cells in Ifngr mutant, p&lt;0.0001) were present in significantly greater numbers in Ifngr mutant NOD mice. There were no differences in antigen-specific CD4+ T cells. Islet infiltrating T cells from Ifngr mutant mice had increased responses to common gamma chain (γc) cytokines, prevented by a JAKi, and reduced expression of suppressor of cytokine signalling-1 (Socs1) mRNA. Our data suggest that as well as its pro-inflammatory effects, IFN-γ homeostatically controls the expansion of antigen-specific CD8+ T cells by regulating their response to γc cytokines, by mechanisms that include regulation of SOCS-1 expression. Expansion of antigen-specific CD8+ T cells is likely to explain the lack of protection from diabetes in Ifngr mutant NOD mice. JAKi therapy experimentally controlled this increased responsiveness to γc cytokines. Our data show mechanisms underlying protection from diabetes in NOD mice by JAKi and the lack of protection by blocking IFN-γ alone.

Disclosure
T. Kay: None. G. Jhala: None. B. Krishnamurthy: None. H.E. Thomas: None.
291-OR: Deletion within the Helicase 1 Domain of MDA5 Dampens Immune Cell Activation and Delays Type 1 Diabetes
Authors: BLUM SAMUEL I. , CHEN YI-GUANG , TSE HUBERT M. 
Published: June 2020

Antiviral responses are associated with triggering type 1 diabetes (T1D) including the synthesis of reactive oxygen species, proinflammatory cytokines, and autoreactive T cell activation. We previously demonstrated that Coxsackievirus B3 (CVB3) infection of Non-Obese Diabetic (NOD) mice accelerates T1D, partly due to the expression of melanoma differentiation-associated protein 5 (MDA5) antiviral sensor. Genetic mutations within human IFIH1, the gene encoding MDA5, are correlated with T1D susceptibility potentially due to increased IFN-α/β synthesis. However, the role of MDA5 in immune cell activation during T1D is unknown. By utilizing mice that lack (NOD.Ifih1KO) or contain a deletion within the helicase 1 domain of MDA5 (NOD.Ifih1ΔHel1), we can test the hypothesis that reduced MDA5 signaling delays T1D by decreasing IFN-α/β synthesis and debilitating islet-infiltrating macrophage and T cell effector responses. We observed that NOD and NOD.Ifih1KO mice develop T1D similarly, but NOD.Ifih1ΔHel1 were significantly protected from spontaneous and CVB3-accelerated T1D. During spontaneous T1D at 12 weeks of age, pancreatic macrophages from NOD.Ifih1ΔHel1 mice have a significant reduction in MHC-II (∼41%) and CD86 (∼43%) expression compared to NOD and NOD.Ifih1KO mice. CVB3-infected NOD.Ifih1ΔHel1 mice exhibited a significant decrease in pancreatic IFN-α (∼54%) and IFN-β (∼67%) synthesis at day 3 post-infection compared to NOD mice. Pancreatic NOD.Ifih1ΔHel1 CD4 and CD8 T cells had reduced T cell activation via CD25 (∼76 and ∼57%) and PD-1 (∼65 and ∼100%) synthesis, respectively, compared to NOD and NOD.Ifih1KO T cells following CVB3 infection. The Ifih1ΔHel1 mutation, but not the Ifih1KO mutation dampened IFN-α/β synthesis, reduced macrophage and T cell activation, and delayed both spontaneous and CVB3-accelerated T1D. Future studies will examine immune differences between NOD.Ifih1ΔHel1 and NOD.Ifih1KO mice that delay or promote autoimmunity, respectively.

Disclosure
S.I. Blum: None. Y. Chen: None. H.M. Tse: None.

Funding
American Diabetes Association (7-12-CD-11 to H.M.T.); National Institute of Diabetes and Digestive and Kidney Diseases (DK099550)
299-OR: Loss of Insulin and IGF1 Receptors in Muscle Impairs Complex-I Dependent Mitochondrial Bioenergetics and Supercomplex Formation via Foxo Transcription Factors
Authors: BHARDWAJ GOURAV , PENNIMAN CHRISTIE M. , BELTRAN PABLO A. SUAREZ , JENA JAYASHREE , FOSTER COLLIN M. , PORO KENNEDY , JUNCK TAYLOR , HINTON ANTENTOR , SOUVENIR RHONDA , FUQUA JORDAN , MORALES PABLO E. , BRAVO-SAGUA ROBERTO , LIRA VITOR A. , ABEL EVAN D. , ONEILL BRIAN T. 
Published: June 2020

Diabetes is associated with decreased muscle strength and mitochondrial metabolism which can lead to disability. Recently, we reported that insulin receptor (IR) and IGF-1 receptor (IGF1R) prevent muscle atrophy via suppression of FoxOs, but how FoxOs interact with mitochondrial function remains unclear. To test whether IR/IGF1R suppression of FoxOs coordinates muscle strength with mitochondrial function, we measured mitochondrial bioenergetics and RNA-Seq in mice lacking IR, IGF1R, and/or FoxO1/3/4 in muscle. Perinatal muscle-specific deletion of IR (MIRKO) showed a mild 20% decrease in pyruvate-driven respiratory capacity in soleus permeabilized fibers, whereas succinate/Rotenone respiration was not different. Deletion of both IR/IGF1R (MIGIRKO) showed a marked 60% decrease in pyruvate respiration and ATP production in soleus fibers. These mitochondrial abnormalities were reversed in muscle-specific IR/IGF1R and FoxO1/3/4 quintuple knockout (QKO), suggesting FoxO activation mediates the mitochondrial decline. FoxOs are known inducers of autophagy, but mitophagy quantification using MitoTIMER or mitoKeima biosensors was not altered in Day 21 Tamoxifen-inducible IR/IGF1R KO in adult muscle (IND-IGIRKO) or by acute in-vitro deletion of IR/IGF1R primary myotubes. Complex-I activity was decreased 50-70% in MIGIRKO and Day 21 IND-IGIRKO muscle, but other complex activities were unchanged. Furthermore, 14 complex-I subunits were decreased in MIGIRKO by RNA-Seq, and 13 of these were reversed in QKO. IND-IGIRKO also showed a decrease in pyruvate respiration, ATP production, complex-I activity, and complex-I/supercomplex content by blue-native gels 21 days after tamoxifen. Thus, loss of IR or both IR/IGF1R in muscle decreases complex-I driven mitochondrial respiration and supercomplex assemblies, at least in part by FoxO-mediated downregulation of mitochondrial genes.

Disclosure
G. Bhardwaj: None. C.M. Penniman: None. P.A. Suarez Beltran: None. J. Jena: None. C.M. Foster: None. K. Poro: None. T. Junck: None. A. Hinton: None. R. Souvenir: None. J. Fuqua: None. P.E. Morales: None. R. Bravo-Sagua: None. V.A. Lira: None. E.D. Abel: None. B.T. ONeill: None.