bluuebunny/crossref_metadata_2025_split huggingface dataset:
2051-P: Human Autoantibodies to SerpinB13 Are Both Protective and Functional in Type 1 Diabetes
Authors: KRYVALAP YURY , CZYZYK JAN 
Published: June 2020

Our laboratory has identified a novel autoantibody (AA) to serpinB13, a protease inhibitor of cathepsin L, and has found an association of this AA with protection from type 1 diabetes (T1D) in subjects enrolled in the Diabetes Prevention Trial for Type 1 Diabetes (DPT-1). Our additional studies with a monoclonal antibody (mAb) to serpinB13 confirmed these observations in mouse, and implicated this mAb in a protease-mediated cleavage of the Notch 1 receptor as the mechanism of development of pancreatic Ngn3+ endocrine progenitors and resistance to severe diabetes. Whether human AA to serpinB13 plays a similar active role in protection against T1D remains unclear. To examine the potential functionality of human serpinB13 AA, we dialyzed DPT-1 serum samples that were either positive or negative for serpinB13 AA and used them to stimulate mouse embryonic pancreas explants (E12.5) during a 48-hour in vitro culture. Using a quantitative IF microscopy approach, we found that pancreas explants exposed to serpinB13 AA positive serum samples had 50% more Ngn3+ cells compared with pancreas explants incubated with serpinB13 AA negative samples (p=0.0029). This effect was dependent on serpinB13AA rather than other serum factors as positive serum samples immunodepleted of this AA were no longer able to stimulate development of additional Ngn3+ cells (p=0.0016). These data demonstrate that serpinB13 AA is actively involved in regulating the pathways that control development of endocrine cell lineage in the pancreas. Consequently, to our knowledge, serpinB13 AA is the first example of an autoantibody that is not solely a biomarker, but also an active player responsible for improved outcomes in T1D. To examine the role of embryonic serpinB13 AA in human diabetes in future, we aim to measure serpinB13 AA in pregnant women with T1D, or at significant risk for this disease, and assess its impact on the development of diabetes in their offspring after birth.

Disclosure
Y. Kryvalap: None. J. Czyzyk: None.

Funding
American Diabetes Association (I-17-ICTS-083 to J.C.); National Institutes of Health (DK10873901A1)
2068-P: Beta-Cell Compensation to Pubertal Insulin Resistance Is Compromised in High-Fat Fed Rats and Impairs Glucose Homeostasis Later in Life
Authors: CASTELL ANNE-LAURE , FERGUSSON GRACE , ETHIER MÉLANIE , TREMBLAY CAROLINE , GHISLAIN JULIEN , POITOUT VINCENT 
Published: June 2020

Puberty is a time of hormonal changes associated with insulin resistance (IR). Although insulin sensitivity is restored at the end of puberty in healthy youth, it does not resolve in obese adolescents leading to an increased risk of metabolic disease such as T2D. During pregnancy-induced IR, pancreatic β cells increase their functional mass to maintain glucose homeostasis. During puberty, however, the mechanism of β-cell compensation to IR and its role in glucose metabolism in adulthood have not been established. In this study we characterized pancreatic β-cell adaptation to pubertal IR in rats and evaluated the effect of metabolic stress during puberty on glucose homeostasis in adult animals.
Methods: Wistar rats were fed a chow or high fat diet (HFD) during puberty. Fasted plasma insulin and glucose tolerance were determined from weaning to adulthood. β-cell proliferation was assessed by immunostaining of pancreatic cryosections for Ki67 and insulin or Nkx6.1. β-cell mass was measured by morphometric analysis of insulin staining. Isolated islets from Wistar rats were exposed during 72h to the serum of prepubertal, pubertal, or postpubertal animals and β-cell proliferation was evaluated by flow cytometry using insulin and EdU antibodies.
Results: During puberty, glucose intolerance was associated with an increase in insulin levels, suggestive of IR. Accordingly, puberty was characterized by a pulse in β-cell proliferation and a rise in β-cell mass. Pubertal but not pre or post pubertal rat serum induced β-cell proliferation in isolated islets. HFD led to a decrease in β-cell proliferation during puberty with impaired glucose tolerance and defective insulin secretion in adult animals.
Conclusion: During puberty in rats, β cells proliferate to compensate for IR. Metabolic stress during puberty impairs glucose homeostasis later in life. Future studies will identify the circulating factor(s) that trigger β-cell expansion during puberty.

Disclosure
A. Castell: None. G. Fergusson: None. M. Ethier: None. C. Tremblay: None. J. Ghislain: None. V. Poitout: None.
208-OR: Emc10emc10, a Novel Circulating Factor for Systemic Energy and Metabolic Homeostasis
Authors: LI YANLIANG , WANG XUANCHUN , QIANG GUIFEN , MCCANN MAXIMILIAN A. , GIL VICTORIA , BLÜHER MATTHIAS , WEE LIEW CHONG 
Published: June 2020

Proteins secreted from metabolic organs play critical roles in fine-tuning and maintenance of systemic metabolic and energy homeostasis. In this study, we identified a novel obesity-regulated circulating protein, scEmc10 (secreted-isoform of endoplasmic reticulum membrane complex subunit 10), that regulates systemic energy homeostasis. We initially observed that scEmc10 is modulated in adipose tissues in diet-induced obesity (DIO) models and subsequently in obese patients. Using an in-house-developed chemiluminescent immunoassay, we found that circulating Emc10 is significantly upregulated in overweight and obese patients and can be normalized by bariatric surgery-induced weight loss. Subsequently, we observed that ablation of Emc10 protects mice from DIO and associated metabolic dysregulation. Conversely, enhanced circulating Emc10 by expression of secretable AAV-hscEmc10 in the liver promotes obesity and associated metabolic dysfunctions in mice fed with either chow or high fat diet. Subsequent metabolic analysis revealed that Emc10 KO mice on HFD have elevated whole body energy expenditure compared to WT controls via an increase in oxygen consumption in both the brown and inguinal subcutaneous fat. Our mechanistic analysis demonstrated that ablation of scEmc10 promotes adaptive thermogenesis in adipocytes via activation of the PKA signaling pathway and its downstream targets, particularly CREB and p38MAPK. Finally, we also showed that neutralization of scEmc10 in the circulation with a monoclonal antibody reduced body weight and improved diet-induced metabolic dysfunction in obese mice. Our findings implicate Emc10 as a novel therapeutic target for counteracting the development of obesity and obesity-induced metabolic dysfunction.

Disclosure
Y. Li: None. X. Wang: None. G. Qiang: None. M.A. McCann: None. V. Gil: None. M. Blüher: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Novo Nordisk A/S, Sanofi. Speaker’s Bureau; Self; Amgen, Novartis AG. C. Liew: None.

Funding
National Institutes of Health (R01DK090210, R01DK109015); National Natural Science Foundation of China (81070647, 81370936, 81873645, 81873853)
2082-P: A Negative Regulatory Role of Insulinoma Beta Cell–Derived Exosomes in Glucose-Stimulated Insulin Secretion of Beta-Cells by Upregulating Exosomal MicroRNAs-Associated Signals
Authors: LIU SHING-HWA , WU CHENG-TIEN , YU CHIA-CHING 
Published: June 2020

Background: In conditions of diabetes, exosomes have been suggested to play the roles in the pathological processes in both type 1 and type 2 diabetes. Exosomes released from both high-concentration glucose- and cytokine-treated β-cells induced β-cell apoptosis. Pancreatic cancer-derived exosomes have also been found to cause paraneoplastic β-cell dysfunction. Here, we further investigated the effects and mechanisms of insulinoma β-cell-derived exosomes on glucose-stimulated insulin secretion.
Methods: Exosomes from insulinoma β-cell lines RIN-m5f or NIT-1 were prepared by ExoQuick-TC™, and identified its characteristics by Western blotting and nanoparticle tracking analysis. For in vitro study, exosomes at the concentrations of 1-30 μg/mL were used. For in vivo study, ICR mice were treated with PBS or exosomes (30 μg/100 μL) for 4 weeks by tail vein intravenous injection twice a week. The miRNA profile was determined by Next Generation Sequencing (NGS) analysis.
Results: Exosomes from insulinoma β-cells did not affect the cell viability, but significantly inhibited the glucose-stimulated insulin secretion of naïve β-cells. Exogenously administration of exosomes derived from insulinoma β-cells to mice also showed the dysregulation of glucose tolerance and serum insulin levels. Moreover, the results of NGS showed that exosomes may transfer miR-1224, -122-5p, -133a-3p, -10b-5p, -423-5p, -409a-3p to affect insulin secretion-related genes/proteins in β-cells. Exosomes also significantly reduced the protein expressions of phosphorylated Akt, Ca2+/calmodulin-dependent protein kinase-II, and glucose transporter-2 in β-cells, which could be reversed by inhibition of exosome uptake.
Conclusion: Exosomes released from insulinoma β-cells are capable of upregulating exosomal miRNAs-associated signals to dysregulate glucose-stimulated insulin secretion in naïve β-cells.

Disclosure
S. Liu: None. C. Wu: None.

Funding
Ministry of Science and Technology of Taiwan (107-2314-B-002-031-MY3)
2090-P: Noradrenergic Stimulus as a Potential Inducer of Human ß-Cell Dedifferentiation
Authors: CINTI FRANCESCA , FANTUZZI FEDERICA , CARFÍ ANDREA , MEZZA TERESA , ASSUNTA CEFALO CHIARA MARIA , MOFFA SIMONA , SORICE GIAN PIO , GIACCARI ANDREA , CNOP MIRIAM 
Published: June 2020

β dedifferentiation is recognized as one of the main mechanisms responsible for the disappearance of β cells from pancreatic islets of diabetic subjects, but the underlying process is still unclear. Noradrenergic innervation is known to inhibit insulin secretion, and in pancreas of diabetic subjects a 3-fold increase of noradrenergic fibers was observed compared to nondiabetic subjects, directly related to the % of dedifferentiated cells within the islets. The increase in fibers has been associated with worsening β cell function, suggesting a role in the pathogenesis of the disease and perhaps in the process of dedifferentiation. The aim of our study was to use in vitro human β cell models to explore a direct role of innervation in the process of noradrenergic dedifferentiation. The human β cell line EndoC-βH1 was exposed to different concentrations of noradrenaline (NA) (0.4 μM, 4 μM and 40 μM) for 24 and 72 h. The expression of β cell dedifferentiation markers was assessed before and after stimulation of the α2A adrenergic receptor, by morphology (immunofluorescence (IF)), qPCR and ELISA. FGF2 (100 ng/mL), a known inducer of β dedifferentiation in vitro, was used as a positive control. NA treatment did not have any impact on β cell viability, while, as expected, it slightly reduced insulin secretion after 24 h. The expression of β cell dedifferentiation markers (ALDH1a3) was increased by approximately 30% after stimulation with NA, while mature β cell marker were reduced (NKX6.1) up to 60% after 72 h (by IF detection). Moreover, the mRNA expression of FOXO1, the main transcription factor involved in the dedifferentiation process, was reduced up to 35.4% after 72 h stimulation with NA. We observed a dose and time dependent pattern in all the experiments.
In conclusion, the data suggest a direct role of noradrenergic fibers in the dedifferentiation process, potentially identifying a new target for the prevention and therapy of type 2 diabetes.

Disclosure
F. Cinti: None. F. Fantuzzi: None. A. Carfí: None. T. Mezza: None. C. Cefalo: None. S. Moffa: None. G. Sorice: None. A. Giaccari: Speaker’s Bureau; Self; Amgen, AstraZeneca, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mundipharma, Novo Nordisk A/S, Sanofi-Aventis. M. Cnop: None.

Funding
European Foundation for the Study of Diabetes
2093-P: Tetraspanin7-Mediated Inhibition of Beta-Cell Voltage-Dependent Calcium Channels Regulates Glucose-Stimulated Insulin Secretion
Authors: DICKERSON MATTHEW , DADI PRASANNA , BUTTERWORTH REGAN B. , JACOBSON DAVID 
Published: June 2020

Tetraspanin7-mediated inhibition of β-cell voltage-dependent Ca2+ channels regulates glucose-stimulated insulin secretion.
Glucose-stimulated insulin secretion (GSIS) is regulated by Ca2+ influx into pancreatic β-cells through voltage-dependent Ca2+ channels (VDCCs). As some tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling they may also modulate GSIS. TSPAN7 is the most abundant TSPAN in mouse and human islets. TSPAN7 autoantibodies are also found in type 1 diabetic serum, suggesting a link between TSPAN7 and diabetes. The aim herein was to determine if TSPAN7 controls β-cell Ca2+ handling and insulin secretion. Immunofluorescence imaging of mouse and human pancreatic slices indicated that TSPAN7 is highly expressed in β- and α-cells. shRNA-mediated TSPAN7 knockdown (KD) in mouse and human β-cells augmented glucose-stimulated Ca2+ influx (by 32.3±9.5% and 33.8±15.1% respectively) as well as KCl-stimulated Ca2+ influx (45 mM; 34.5±9.6%, human). Mouse β-cell Ca2+ oscillation frequency was also accelerated by TSPAN7 KD (by 29.4±10.1%). As TSPANs interact with and regulate VDCCs, we examined the effect of TSPAN7 on VDCC currents. TSPAN7 KD increased peak L-type VDCC currents in mouse and human β-cells (by 4.1±1.3 pA/pF and 2.0±0.9 pA/pF respectively). Heterologous expression of TSPAN7 with L-type VDCCs in HEK293 cells also decreased VDCC currents (CaV1.2: 4.3±1.8 pA/pF; CaV1.3: 17.5±5.4 pA/pF) and reduced KCl-stimulated (50 mM) Ca2+ influx (CaV1.2: 51.0±7.1%; CaV1.3: 28.4±7.5%). Furthermore, TSPAN7 coimmunoprecipitated with CaV1.2 and CaV1.3 from primary human β-cells and when heterologously expressed in HEK293 cells. Finally, TSPAN7 KD in human β-cells increased basal (5.6 mM glucose; 8.3±5.5 ng*hr-1) and stimulated (45 mM KCl+14 mM glucose; 20.4±17.1 ng*hr-1) insulin secretion. These findings reveal that TSPAN7 modulation of β-cell L-type VDCCs limits glucose-stimulated Ca2+ influx and insulin secretion.

Disclosure
M. Dickerson: None. P. Dadi: None. R.B. Butterworth: None. D. Jacobson: None.

Funding
American Diabetes Association (1-17-IBS-024 to D.J.); National Institutes of Health (T32DK101003, DK097392, DK115620); Vanderbilt University Medical Center Diabetes Research and Training Center (P60DK20593)
2108-P: Complex Posttranslational Processing of Glucagon Prohormone in Human Pancreatic Islets
Authors: ROMANOVA ELENA V. , RUBAKHIN STANISLAV , SWEEDLER JONATHAN V. 
Published: June 2020

An array of signaling peptides and peptide hormones in pancreatic islets results from posttranslational processing of precursor proteins by well-characterized endopeptidases. While the encoded peptide complement can be predicted in silico from the prohormone sequence, actual peptide products often are more diverse due to additional chemical or enzymatic modifications of mature predicted peptides. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for comprehensive molecular characterization, we compiled an experimentally deduced database of endogenous peptides in human pancreatic islets and revealed an unexpected complex processing of several prohormones including proglucagon. Live pancreatic islets from several donors were provided by the Human Pancreas Analysis Program, University of Pennsylvania. Total of 2034 chemically unique peptide sequences were identified and matched to 477 proteins in the human protein database. Among interesting observations, we find differential cleavage of the signal peptide on the glucagon precursor protein, NP_002045.1, and unusual further processing of its encoded mature peptides. Alternate signal peptide cleavage sites are confirmed by detection of both classical GRPP, R[21]-D[50], and its novel variant S[18]-D[50]. An amidated IP-2 D[131]-L[142] and an isoform of MPGF D[131]-D[178], missing the GLP-1 sequence were detected. Previously unreported N-terminus acetylation of glucagon, GLP-1, GLP-1 (7-36), and oxyntomodulin was detected. Novel peptides potentially resulted from variable processing of mature glucagon, GRPP, and oxyntomodulin at internal convention cleavage sites were characterized. New chemically unique peptides originating from proglucagon and documented in this study indicate a more complex posttranslational processing of proglucagon in human islets than previously thought. The finding expands opportunity for mechanistic studies on pathology, progression, and treatments of diabetes.

Disclosure
E.V. Romanova: None. S. Rubakhin: None. J.V. Sweedler: None.

Funding
American Diabetes Association/Pathway to Stop Diabetes (1-18-VSN-19 to J.V.S.)
2114-P: Quantification of GLP-1R Trafficking in Primary Beta Cells in Response to Different Ligands
Authors: GRAY SARAH M. , RAVN PETER , SLOOP KYLE , CAMPBELL JONATHAN , D’ALESSIO JONATHAN 
Published: June 2020

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor that is insulinotropic in β-cells. Multiple endogenous and synthetic ligands exist for the GLP-1R and each ligand interacts with the receptor differently to produce specific activation of intracellular signaling pathways. Ligand binding typically induces receptor internalization, leading to either i) receptor degradation or ii) recycling to the cell membrane. We hypothesized that each specific GLP-1R ligand would produce a unique trafficking profile in primary β-cells. To test this hypothesis, we developed flow cytometry-based assay that can be utilized in primary dispersed mouse islets. First, we validated a GLP-1R antibody (GLP-1R-APC) for flow cytometry application. The GLP-1R-APC stained β-cells, but not α-cells, aligning with the reported expression pattern in islets. Post-sort analysis confirmed GLP-1R expression only in the GLP-1R-APC+ population. Finally, GLP-1R-APC did not stain any cell populations in GLP1R-/- islets. Next, we used this assay to quantify GLP-1R internalization in wild type β-cells. To induce internalization, we treated dispersed islets with either vehicle or a GLP-1R ligand for 30 minutes prior to antibody staining. After vehicle treatment, 90% of the β-cells stained GLP-1R-APC+. Treatment with either GLP-1 or exendin-4 reduced the number of GLP-1R-APC+ β-cells by 97 and 89%, respectively, suggesting near complete internalization of all GLP-1Rs. In contrast, treatment with the GLP-1R antagonist exendin-9 produced staining similar to PBS controls. Finally, we used image-stream technology, a combination of brightfield microscopy and flow cytometry, to visually confirm GLP-1R internalization. Here, vehicle-treated cells produced GLP-1R staining only on the cell membrane, which was nearly ablated by the treatment with a ligand.
In conclusion, we have developed an assay that allows for quantification of GLP-1R trafficking in primary mouse islets.

Disclosure
S.M. Gray: None. P. Ravn: Employee; Self; AstraZeneca. K. Sloop: None. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S.
2120-P: The Protein Phosphatase M1K Connects Glucose Sensing to Protein Translation in Pancreatic Beta Cells
Authors: DELEYE YANN , HERRING JACOB , HESS KAVAN H. , LEININGER BRENNAN , MCGARRAH ROBERT W. , TESSEM JEFFERY S. , WHITE PHILLIP J. 
Published: June 2020

The protein phosphatase m1K (PPm1K) dephosphorylates and activates the rate limiting enzyme in the branched-chain amino acid (BCAA) catabolism pathway, branched chain a-keto acid dehydrogenase (BCKDH). A common single nucleotide polymorphism upstream of the gene for PPm1K is associated with elevated circulating levels of BCAA and increased risk for development of type 2 diabetes; however, the function of PPm1K in the pancreatic beta cell has not been assessed. In line with our previous observations in liver, we found that increasing glucose concentrations in INS1 cells results in elevated expression of the ChREBP β isoform and lower PPM1K mRNA expression. SiRNA-mediated knockdown (KD) of PPm1K to levels achieved by glucose in INS1 cells lead to robust phosphorylation of the ribosomal protein S6 on its regulatory phosphosites ser235/236 and ser240/s244. Notably, this occurred absent any effect on the phosphorylation of the upstream proteins mammalian target of rapamycin (mTOR) or p70 S6 Kinase (S6K) and could not be reversed by inhibiting BCKDH phosphorylation with the small molecule, BT2. Thus Ppm1K appears to regulate S6 independently of mTOR, S6K, and BCKDH. Surface sensing of translation (SUnSET) studies revealed that the increase in S6 phosphorylation in PPm1K KD INS1 cells is associated with a 2-fold increase in protein translation rate. Accordingly, PPm1K KD increased both insulin content and glucose stimulated insulin secretion in INS1 cells. In line with the higher translation rate, PPm1K KD cells displayed signs of endoplasmic reticulum stress including increased expression of spliced xbp1 and Bip mRNA.
In conclusion, our data highlight a new role for PPm1K in pancreatic beta cells, as a mechanism connecting glucose sensing to protein translation via modulation of S6 phosphorylation.

Disclosure
Y. Deleye: None. J. Herring: None. K.H. Hess: None. B. Leininger: None. R.W. McGarrah: None. J.S. Tessem: None. P.J. White: None.

Funding
American Diabetes Association/Pathway to Stop Diabetes (1-16-INI-17 to P.J.W.)
2123-P: Targeted Deletion of Grb10 in Pancreatic ß-Cells Increases ß-Cell Mass via mTORC-Dependent Inhibition of Cell Dedifferentiation
Authors: ZHANG JINGJING , LIU FENG 
Published: June 2020

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that negatively regulates insulin and mTORC1 signaling. Grb10 is highly expressed in mouse islets, but its role in β-cells function remains unclear. Here we show that Grb10 is highly expressed in human islets and the expression levels of Grb10 are markedly induced in the islets of diabetic mice as well as aged humans. β-cell-specific knockout of Grb10 in mice increased β-cell mass, reduced apoptosis, and improved glucose-stimulated insulin secretion. Mechanistically, knockout of Grb10 in β-cells enhanced mTORC1 signaling and inhibited β-cell dedifferentiation, which could be blocked by rapamycin administration in vivo. Our findings revealed a mechanism by which the Grb10/mTORC1 axis regulates β-cell dedifferentiation and function. Identification of key regulators of β-cell dedifferentiation will shed new light on the development of effective therapeutic strategies for treating both type 1 diabetes (T1D) and type 2 diabetes (T2D).

Disclosure
F. Liu: None.

Funding
National Natural Science Foundation of China (91749118, 81770775, 81730022); Planned Science and Technology Project of Hunan Province (2017RS3015)