bluuebunny/crossref_metadata_2025_split huggingface dataset:
1750-P: Differential Impacts of Two Novel HNF1A Variants Associated with Familial Young-Onset Diabetes on Protein Function and Insulin Secretion In Vitro
Authors: CHERKAOUI INES , DU QIAN , DION CAMILLE , LEITCH HARRY , CHABOSSEAU PAULINE L. , EGLI DIETRICH M. , SACHEDINA DILSHAD , WASTIN JULES , MISRA SHIVANI , RUTTER GUY A. 
Published: June 2023

Introduction: Mutations in the HNF1A are a cause of monogenic diabetes and are usually dominantly inherited. We report in vitro and clinical studies on two novel HNF1A variants to determine pathogenicity: a recessive (homozygous) HNF1A variant (p.A251T) and a dominant HNF1A variant (p.S19L). We also investigated the mechanisms by which the former variant affects insulin secretion using beta cells derived from induced pluripotent stem cells (iPSCs).
Methods: Nuclear localization, DNA binding, and transactivation activity were examined using standard techniques. iPSC lines from proband carrying the p.A251T variant were differentiated into beta-like cells according to Sui et al, CPHG, 2018. Ca2+ dynamics were monitored with Cal-520, and insulin secretion was measured by homogeneous time-resolved fluorescence assay compared to wild-type HNF1A (WT).
Results: HNF1A p.A251T showed a mild (10.4% ± 7.3, p= 0.16) decrease in transactivation activity compared with WT, and decreased (48% of WT, p=0.12) DNA binding. These reductions were significant in p.S19L (DNA binding 57% of WT, p=0.049). Significant impaired nuclear uptake (63% vs 90% cells for WT, p&lt;0.0001) was only apparent for p.S19L. Glucose-stimulated insulin secretion was decreased in A251T iPSCs compared with WT (0.29% vs. 0.46% of total insulin). In contrast, glucose-induced Ca2+ increases were increased in mutant cells (arbitrary units: 7.39 ± 1.23, mean +/− SD, n=725 cells vs. 4.58 ± 0.77, n=724, p= &lt; 0.001)
Conclusion: We demonstrate the value of combining multiple in vitro  functional studies to decipher novel HNF1A variant pathogenicity. The recessive p.A251T variant has relatively mild effects on protein function compared to a dominant p.S19L variant. However, when studying proband iPSC derived beta-cell A251T impairs glucose-stimulated insulin secretion despite preserved Ca2+ entry, suggesting a novel mechanism of action not captured by standard tests of HNF1A function.

Disclosure
I.Cherkaoui: None. G.A.Rutter: Consultant; Sun Pharmaceutical Industries Ltd. Q.Du: None. C.Dion: None. H.Leitch: None. P.L.Chabosseau: None. D.M.Egli: None. D.Sachedina: None. J.Wastin: None. S.Misra: Research Support; Dexcom, Inc.

Funding
Diabetes UK
1761-P: Verapamil Increases ß-Cells Proliferation in MIN6 Cells and ß-Cell Mass in Zebrafish Model
Authors: AREFANIAN HOSSEIN , AL MADHOUN ASHRAF , ALRASHED FATEMA , BAHMAN FATEMAH , ALSAEED HALEMAH , ABUKHALAF NERMEEN A. , SHENOUDA STEVE , WILLIAMS MICHAYLA R. , NIZAM RASHEEBA , JOHN SUMI E. , JACOB SINDHU , ALHUSAYAN MOHAMMED , KURIAN SHIBU J. , KOCHUMON SHIHAB P. , SINDHU SARDAR T.A.K. , ALZAID FAWAZ , ABU-FARHA MOHAMED , ABUBAKER JEHAD , THANGAVEL ALPHONSE T. , AHMAD RASHEED , ALMULLA FAHD 
Published: June 2023

Recent clinical studies highlighted a significant clinical utility for verapamil in both type 1 (T1D) and type 2 (T2D) diabetes. Verapamil treatment in T1D lowered dependency on exogenous insulin. In T2D, addition of verapamil to metformin therapy improved glycemic control. These findings led us to further study the molecular and cellular mechanisms of verapamil in the treatment of T1D and T2D using an in vitro (MIN6 mouse β cell-line) and in vivo zebrafish (Danio rerio) model. In this study, MIN6 cells were cultured in growth media containing 5.6 mM glucose (mimicking normoglycemia) and supplemented with 2% BSA to eliminate any proliferative effect of high glucose in the media, and growth factors in FBS. The cells were treated with different concentrations of verapamil. The levels of cell proliferation (MTT assay), cell growth (cell counts), Ki67 expression (IF), and cell cycle (flow cytometry) were studied in MIN6 cells treated with the optimal dose detected (50 μM) of verapamil. Larvae (24hpf) of the Ins:NfsB-mCherry transgenic zebrafish (expressing mCherry fluorescence reporter under the control of insulin proximal promoter) were treated with 10 uM of verapamil for 3 days. Full larvae images were acquired using a stereo microscope. Our results show that verapamil induced significantly increased proliferation of MIN6 cells. Cell cycle analysis shows initiation of the proliferative effect during an early time point (1-4 hours) after verapamil treatment. Higher Ki67 expression indicated enhanced cellular proliferation at 24 hours and growth curve data confirmed that the proliferative effect of verapamil lasted for 7 days. As evident from increased mCherry fluorescence intensity in zebrafish larvae, verapamil treatment increased the β cell mass during the exposure period of three days. Taken together, our findings support the positive impact of verapamil on mouse β cell proliferation in vitro as well as on increased β cell mass in Zebrafish model.

Disclosure
H.Arefanian: None. S.E.John: None. S.Jacob: None. M.Alhusayan: None. S.J.Kurian: None. S.P.Kochumon: None. S.T.K.Sindhu: None. F.Alzaid: None. M.Abu-farha: None. J.Abubaker: None. A.T.Thangavel: None. A.Al madhoun: None. R.Ahmad: None. F.Almulla: None. F.Alrashed: None. F.Bahman: None. H.Alsaeed: None. N.A.Abukhalaf: None. S.Shenouda: None. M.R.Williams: None. R.Nizam: None.

Funding
Kuwait Foundation for the Advancement of Sciences (RACB-2019-001)
1763-P: Pretreatment with Verapamil Increases MIN6 Mouse ß-Cell Survival after Exposure to Cytotoxic Stressors
Authors: AREFANIAN HOSSEIN , BAHMAN FATEMAH , AL MADHOUN ASHRAF , SINDHU SARDAR T. , ALRASHED FATEMA , KOCHUMON SHIHAB P. , NIZAM RASHEEBA , JOHN SUMI E. , JACOB SINDHU , ALHUSAYAN MOHAMMED , ABUKHALAF NERMEEN A. , SHENOUDA STEVE , ALSAEED HALEMAH , KURIAN SHIBU J. , WILLIAMS MICHAYLA R. , ALZAID FAWAZ , ABU-FARHA MOHAMED , ABUBAKER JEHAD , THANGAVEL ALPHONSE T. , AHMAD RASHEED , ALMULLA FAHD 
Published: June 2023

Verapamil was found to lower the risk of developing new-onset diabetes and to lower fasting glucose levels in persons with diabetes. While previous studies have focused on treatment with verapamil, our objective in this study was to determine the effect of verapamil pre-treatment on MIN6 mouse β cell-line viability in response to different stressors mimicking conditions of in vitro models of type 1 (T1D) and type 2 diabetes (T2D). MIN6 cells were treated with either streptozotocin (STZ), T1D-cytomix containing IL-1β, TNF-α, and INF-γ, or a T2D-cytomix containing IL-1β, TNF-α, and palmitic acid. MTT assay was performed to determine viability of the cells pre-treated with verapamil (50 µM) for 24 hours, followed by each stressor treatment for another 24 hours, under normal (5.6 mM) glucose conditions. For all stressors, pre-treatment with verapamil significantly increased the cell viability, compared to verapamil-untreated controls. To study the synergistic effect of verapamil pre-treatment on the combined treatment of verapamil plus stressors, MIN6 cells were pre-treated with verapamil, then exposed to co-treatment conditions. We found that co-treatment of verapamil with each stressor significantly improved the cell survival, compared to cells treated with the stressor only. In case of STZ and T2D-cytomix, pre-treatment of verapamil significantly increased the cell viability, compared to cells that only received co-treatment alone. No significant synergistic effect of verapamil was observed as pre-treatment on co-treatment of verapamil and T1D-cytomix. In all cases showing protective effect of verapamil pre-treatment, verapamil improved cell viability in a dose-dependent manner. These results suggest that verapamil may have cytoprotective effect in MIN6 β cells under conditions mimicking T1D/T2D insults, and pre-treatment with verapamil, in particular, offers higher protection.

Disclosure
H.Arefanian: None. M.Alhusayan: None. N.A.Abukhalaf: None. S.Shenouda: None. H.Alsaeed: None. S.J.Kurian: None. M.R.Williams: None. F.Alzaid: None. M.Abu-farha: None. J.Abubaker: None. A.T.Thangavel: None. F.Bahman: None. R.Ahmad: None. F.Almulla: None. A.Al madhoun: None. S.T.Sindhu: None. F.Alrashed: None. S.P.Kochumon: None. R.Nizam: None. S.E.John: None. S.Jacob: None.

Funding
Kuwait Foundation for the Advancement of Sciences (RACB-2019-001)
1768-P: Sel1L-Hrd1 Endoplasmic Reticulum-Associated Degradation Regulates PC2 Function and Proglucagon Processing in Islet α-Cells
Authors: REINERT RACHEL B. , ZHU WENZHEN , PAN LINXIU , CHIARA RUSSO ANNA , RAY ROHIT , SHRESTHA NEHA , LINDBERG IRIS , QI LING 
Published: June 2023

Pancreatic islets rely on cellular protein quality control to maintain glucoregulatory hormone production. Proglucagon, precursor of glucagon and glucagon-like peptide 1 (GLP1), is expressed in islet α cells, intestinal L-cells, and some neurons. Islet α cells predominantly convert proglucagon to glucagon through prohormone convertase 2 (PC2). Islet-derived GLP1 is increasingly recognized as an important contributor to islet function, but the factors controlling its production remain unclear. To understand how protein quality control affects proglucagon processing in α cells, we inactivated the highly conserved Sel1L-Hrd1 endoplasmic reticulum-associated degradation (ERAD) system using Cre-loxP recombination in mice. Compared to littermate controls, Sel1LΔGcg mice showed an age-related decline in glucagon secretion in vivo, associated with a 43% decrease in pancreatic glucagon content (76.1 +/- 14.1 vs. 134.4 +/- 14.9 ng/g). Pancreatic GLP1 content was unchanged in Sel1LΔGcg mice (779.4 +/- 60.7 vs. 773.4 +/- 128.6 pg/g), despite a 37% reduction in α cell mass (0.19 +/- 0.04 vs. 0.12 +/- 0.04 mg/pancreas). Expression of the precursor form of PC2 (proPC2) was increased in Sel1LΔGcg α cells, suggesting accumulation of misfolded protein and/or impaired maturation. To investigate the mechanism underlying these changes, we used CRISPR to delete Sel1L in αTC cells. ERAD-deficient αTC-ΔSel1L cells showed 1.75-fold higher expression of proPC2, in addition to appearance of a novel truncated (~56 kD) form of proPC2. Though expression of the mature (64 kD) PC2 form was unchanged in αTC-ΔSel1L cells, PC2 activity was reduced by half in a specific enzyme assay. This was accompanied by decreased expression of 7B2, a chaperone required for proPC2 transit and activation in the secretory pathway. These data show that Sel1L-Hrd1 ERAD is required to ensure proper proPC2 maturation and glucagon production in α cells, thus maintaining classic α cell identity.

Disclosure
R.B.Reinert: None. W.Zhu: None. L.Pan: None. A.Russo: None. R.Ray: None. N.Shrestha: None. I.Lindberg: None. L.Qi: None.

Funding
National Institutes of Health (K08DK129719, T32DK007245)
1769-P: Single-Nucleus Multiomics Reveals Unique Molecular Signatures of ß-Cell Heterogeneity and Compensation in Human Obesity
Authors: KIM HYUNKI , LI YAN , ZHU HOUXIANG , REN ZIYOU , EL JELLAS KHADIJA , SHUKLA NAMRATA , CHEN MENGJIE , KULKARNI ROHIT 
Published: June 2023

The increasing prevalence of obesity and type 2 diabetes (T2D) continues to be a major economic burden worldwide. Healthy humans adapt to metabolic stress by increasing their functional β-cell mass. Failure of this process, called de-compensation, potentially triggers the development of T2D. However, molecular mechanism(s) underlying the ability of heterogenous human β-cells to compensate remain elusive. To interrogate the heterogeneity of gene expression regulation during successful β-cell compensation, we performed single-nucleus multiome (RNA/ATAC) sequencing in human islet cells. In this study, we analyzed a total of 160,954 islet nuclei isolated from nondiabetic lean (LN-ND, n=7), overweight (OW-ND, n=5), obese (OB-ND, n=7), or T2D (n=10) cadaveric donors. Following total cell integration and subclustering, we identified 16 islet/pancreatic cell types including 6 human β-cell subtypes (β1-6). Single-nucleus multiomics revealed unique molecular signatures of the β-cell subtypes that compensate for obesity. We identified unique sets of genes and biological pathways for each β-cell subtype that correlated with the body mass index among the ND individuals. We also identified β-cell subtype-specific transcription factor motifs and intra-islet interactions that are enriched during compensation. Finally, the intersection of publicly available human islet/β-cell-specific chromatin looping data with our datasets revealed compensatory β-cell subtype-specific cis-regulatory elements that overlap with T2D genomic variants. In summary, we report the discovery of unique signatures of chromatin landscape and gene expression regulation in heterogenous β-cells secondary to potential cell-cell interactions during islet compensation in human obesity. These results provide novel insights into the epigenetic regulation in human islet cells that can be harnessed to develop new therapeutic approaches in precision medicine to counter T2D.

Disclosure
H. Kim: None. Y. Li: None. H. Zhu: None. Z. Ren: None. K. El Jellas: None. N. Shukla: None. M. Chen: None. R. Kulkarni: Advisory Panel; Novo Nordisk, Inversago. Research Support; Inversago. Advisory Panel; Biomea Fusion, Inc., REDD Pharma.

Funding
National Institutes of Health (R01067536)
1773-P: Glucocorticoid Receptor Signaling Regulates Pancreatic Beta-Cell Circadian Transcriptome and Function
Authors: SEN SATISH K. , WANG LIU , BROWN MATTHEW , WU JIE , WEI ZONG , MATVEYENKO ALEKSEY 
Published: June 2023

Glucocorticoid (GC) signaling is an important regulator of glucose homeostasis. GC secretion also serves as an entrainment signal that coordinates circadian gene expression of peripheral circadian clocks. Pancreatic beta cells are key biological controllers of glucose homeostasis through the coordinated circadian release of insulin. However, the role of GC signaling in the regulation of beta cell circadian clocks and insulin secretion remains unknown. To address this, we first performed chromatin immunoprecipitation/sequencing (ChIP-seq) in beta cell lines (MIN6 and INS1) and identified &gt;1,000 glucocorticoid receptor (GR)-bound regions (FDR&lt;0.01) in response to the synthetic GC dexamethasone (DEX, 100nM). Annotation of GR-bound regions showed enrichment for pathways regulating insulin secretion and circadian rhythms. To further explore the effects of GC on circadian beta cell gene expression and function in vivo, we used C57BL/6 mice housed under either 12h/12h light-dark cycles (LD) or constant light-mediated circadian disruption (CD). Mice were subsequently administered daily intraperitoneal injections of DEX (2 mg/kg) or vehicle, designed to restore circadian secretion of GC disrupted in CD group. DEX treatment normalized circadian regulation of glucose tolerance (p&lt;0.05 vs. vehicle) and glucose-stimulated insulin secretion (p&lt;0.05 vs vehicle) in CD group. We next utilized RNA sequencing to assess circadian transcriptional profiles in islets isolated from LD, CD, and CD + DEX mice. Consistent with previous studies, CD resulted in abrogation of circadian islet mRNA expression, whereas DEX restored circadian rhythmicity and enhanced expression of core clock genes (e.g., Bmal1, Dbp, Nr1d1) and genes regulating beta cell identity (e.g., Ins1, Nkx6.1) and insulin secretion (e.g., Gck, Irs2). These data highlight the GC/GR axis as a direct regulator of beta cell circadian gene expression, insulin secretion and glucose tolerance.

Disclosure
S.K.Sen: None. L.Wang: None. M.Brown: None. J.Wu: None. Z.Wei: None. A.Matveyenko: None.

Funding
National Institutes of Health (DK098468)
1778-P: Endoplasmic Reticulum Stress Leads to Pancreatic Beta-Cell Failure by Suppressing cAMP Signaling via ATF4
Authors: LEE JAEMIN 
Published: June 2023

Type 2 diabetes (T2D) is characterized by β-cell dysfunction and loss. Endoplasmic reticulum (ER) stress responses, especially the PERK-ATF4 pathway, promote β-cell disorders. The role of the PERK-ATF4 pathway in β-cell dysfunction is still unknown. We report that ATF4 induces PDE4D production which impairs β-cell function by downregulating cAMP signaling. Transgenic expression of ATF4 in β-cells caused early β-cell dysfunction and loss, resembling accelerated T2D. ATF4 expression enhanced PDE4D, decreased cAMP signaling, and inhibited incretin and glucose responses in β-cells. In leptin receptor-deficient diabetic (db/db) mice, β-cells displayed higher nuclear ATF4 localization along with elevated PDE4D expression. Inhibiting PDE4 activity with selective pharmacological agents increased β-cell function in db/db and ATF4 transgenic mice.
In summary, ER stress promotes β-cell failure via ATF4-mediated PDE4D synthesis, proposing PDE4D as a therapeutic target for safeguarding β-cell function in T2D.

Disclosure
J. Lee: None.

Funding
National Research Foundation of Korea (2019R1A4A102972413, 2020M3A9D8038660)
179-LB: Should We Screen with a Fasting Blood Glucose Test? A Novel Two-Step Screening Strategy for Gestational Diabetes—A Large Tertiary Comparative Cohort Study
Authors: DEITCH JESSICA , LEE I-LYNN , YATES CHRISTOPHER J. , KATHPAL ESHA , LAWTON PAUL , SHAHID IBRAHIM , STEELE CHERYL , HAMBLIN PETER S. , KRISHNAMURTHY BALASUBRAMANIAN , SAID JOANNE M. , TEALE GLYN R. , KEVAT DEV 
Published: June 2023

Background: Gestational diabetes mellitus (GDM) diagnosis is resource intensive and controversial. A modified COVID-19 GDM diagnostic method was introduced at Western Health Melbourne, an Australian tertiary hospital caring for a multi-ethnic population with a high GDM prevalence at 20-23%. In place of a universal 2 hour 75g OGTT, a novel two step diagnostic method was instituted. A fasting blood glucose (FBG) was performed with thresholds based on a secondary analysis of HAPO data. A FBG of ≥5.1mmol/L (91.8mg/dL) diagnosed GDM; women with FBG 4.7-5.0mmol/L (84.6-90.0mg/dL) proceeded to an OGTT (IADPSG diagnostic criteria); FBG ≤4.6mmol/L (82.8mg/dL) was considered to exclude GDM.
Methods: A retrospective cohort study of singleton pregnancies birthing after 20 weeks’ gestation compared perinatal outcomes in 6469 women who underwent modified screening (MS) during the COVID-19 pandemic with 8669 women who had conventional screening (CS) prior to COVID-19.
Results: GDM prevalence was the same using either MS or CS (22.9 vs 22.2% p=0.31) with similar rates of insulin use. There was no difference in the prevalence of large for gestational age infants (10.3 vs 9.4% p=0.06), pre-term birth (6.6 vs 6.4% p=0.53) or gestational age at birth (39.2 vs 39.1 weeks). Mean birth weight was increased with MS (3335g vs 3307g) and small for gestational age rate reduced (9.1 vs 10.5% p=0.01). The MS group had fewer inductions (34.2 vs 39.8% p&lt;0.01). Primary caesarean section rate was unchanged (18.4 vs 18.4% p=0.91). MS reduced the need for an OGTT by 76%. Women with a FBG ≤4.6mmol/L by MS had non-inferior outcomes compared to women with a FBG ≤4.6mmol/L with CS irrespective of GDM diagnosis.
Conclusion: In a large multi-ethnic cohort with a high prevalence of GDM, modified screening using FBG greatly reduced the number of resource intensive OGTTs with non-inferior perinatal outcomes. A prospective randomised trial comparing these methods is warranted.

Disclosure
J. Deitch: None. J. M. Said: None. G. R. Teale: None. D. Kevat: Advisory Panel; Novo Nordisk, Speaker's Bureau; Boehringer Ingelheim and Eli Lilly Alliance, Novo Nordisk, Abbott Diabetes, AstraZeneca. I. Lee: None. C. J. Yates: None. E. Kathpal: None. P. Lawton: None. I. Shahid: None. C. Steele: None. P. S. Hamblin: None. B. Krishnamurthy: None.

Funding
Australian Government Research Training Program; Sheppard Millar Lowe Research Trust Scholarship
18-OR: Implementation of a Hybrid Screening and Monitoring Model in Colorado—Autoimmunity Screening for Kids (ASK)
Authors: GENO RASMUSSEN CRISTY , BAUTISTA KIMBERLY A. , KARBAN RACHEL , BEDOY RUTH , FELIPE-MORALES DANIEL , REWERS MARIAN 
Published: June 2023

Objective: Screening for type 1 diabetes (T1D) in the general population is becoming more accessible. We examined facilitators and barriers to implementing a hybrid screening and monitoring model utilizing telehealth in Colorado.
Methods: Qualitative, semi-structured interviews to define key factors for establishing a telehealth T1D screening and monitoring program were conducted with 14 parents who had a child identified as high risk of developing T1D participating in the ASK study and 10 ASK providers. Purposeful sampling was used to identify participants. Interviews explored opinions regarding a hybrid model that includes cloud-based glucose monitoring, education via telehealth, parental placement of continuous glucose monitors, home finger pokes to check for islet autoantibody status and glucose, use of home oral glucose tolerance tests, as well as engagement of primary health care providers for monitoring across stages of T1D.
Results: Between 1/2020 and 9/2022, 133 participants in the ASK study participated in the ASK hybrid screening and monitoring model completing at least two remote telehealth visits. Thematic analysis from parent interviews (n=14; 71% female, 79% had no family history of T1D) identified two key themes:1) appreciation for flexibility offered through hybrid monitoring, and 2) importance of coordinated communication between provider team regardless of location. Provider interviews (n=10; 70% female) identified three key themes:1) interest in hybrid model that was not time intensive to implement, 2) access to monitoring and educational resources to support clinic team and patients with remote care, and 3) for rural areas, potential constraints regarding access to consistent broadband that could interfere with telehealth visits.
Conclusion: The use of a hybrid care model may be warranted to support clinics and families with screening and monitoring for early stages of T1D toward translation into standard practice regardless of location.

Disclosure
C.Geno rasmussen: None. K.A.Bautista: None. R.Karban: None. R.Bedoy: None. D.Felipe-morales: None. M.Rewers: Research Support; Provention Bio, Juvenile Diabetes Research Foundation (JDRF), Hemsley Charitable Trust, Dexcom, Inc., Janssen Research & Development, LLC.

Funding
The Leona M. and Harry B. Helmsley Charitable Trust (2109-04818)