bluuebunny/crossref_metadata_2025 huggingface dataset:
354-OR: Physiologic Pathways in Pregnancy Glycemic Regulation Implicated through Genetic Clustering Analysis
Authors: POWE CAMILLE E. , UDLER MIRIAM , ALLARD CATHERINE , KIM JAEGIL , PERRON PATRICE , BOUCHARD LUIGI , FLOREZ JOSE C. , HIVERT MARIE-FRANCE
Published: June 2019

Background: Genetic determinants of glycemic regulation in pregnancy are poorly understood.

Methods: We studied 35 glycemia-related traits measured during pregnancy and 191 genetic variants identified in prior genome-wide association studies of type 2 diabetes (T2D) or gestational glycemia. Using linear regression, we quantified associations between glucose-raising alleles and traits measured in 582 women at 24-28 weeks gestation. We clustered traits and variants using Bayesian nonnegative matrix factorization (bNMF) to identify physiologic pathways involved in pregnancy glucose metabolism.

Results: In a plurality of bNMF iterations (22/50), 5 clusters emerged, with highly weighted traits and loci suggesting distinct physiologic pathways. Cluster 1: reduced insulin secretory response (lower insulin/c-peptide, greater insulin sensitivity); loci included several known or suspected to affect beta-cell function (ABO, CDKN2B, SLC30A8, CDKN1B). Cluster 2: obesity-related hyperglycemia (higher percent body fat, BMI, fasting/post-load glucose); loci included known obesity/T2D loci (MCR4, FTO) and loci previously tied to beta-cell function that associated with higher glucose and greater adiposity pregnant women (MTNR1B, GLP2R). Cluster 3: insulin resistance (reduced insulin sensitivity, higher fasting insulin/c-peptide, greater insulin secretory response); loci included LYPLAL1 and ANKRD55 (known to harbor insulin resistance variants). Cluster 4 traits suggested reduced adiposity with post-load glucose intolerance. Cluster 5 traits suggested a favorable metabolic profile (lower cholesterol and glucose, higher disposition index and insulin sensitivity).

Conclusion: Genetic variants can be grouped to identify physiologic mechanisms at play in gestational glycemic regulation. Associations between physiologically-informed variant clusters, gestational diabetes, and related perinatal outcomes await testing in well-powered cohorts.

Disclosure

C.E. Powe: None. M. Udler: None. C. Allard: None. J. Kim: None. P. Perron: None. L. Bouchard: None. J.C. Florez: None. M. Hivert: None.

Funding

American Diabetes Association/Pathway to Stop Diabetes (1-15-ACE-26 to M-F.H.); National Institute of Diabetes and Digestive and Kidney Diseases (K23DK113218, K24DK110550); Robert Wood Johnson Foundation; Fonds de la recherche du Québec en santé
357-OR: Early Pregnancy Trace Element Mixtures and Gestational Glucose Levels Late in the 2nd Trimester
Authors: ZHENG YINNAN , ZHANG CUILIN , WILLIAMS PAIGE L. , WEISSKOPF MARC , PARSONS PATRICK J. , PALMER CHRISTOPHER D. , BUCK LOUIS GERMAINE M. , JAMES-TODD TAMARRA
Published: June 2019

Background: A number of essential trace elements are involved in glucose metabolism, but the associations between these elements and glucose levels among pregnant women are unclear. Furthermore, little is known about the joint association of these trace elements.

Methods: We used data from 1857 women enrolled in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Fetal Growth Study, for whom trace element status (Zn, Se, Cu, Mo) was determined using plasma collected during the 1st trimester. Non-fasting glucose levels were measured during the GDM screening test. Three approaches were used to assess the joint effects of these trace elements on glucose levels. First, all four trace elements were included in a multivariable linear model and adaptive lasso was performed for model selection. Second, generalized additive models (GAMs) with model selection were fitted to allow non-linear associations. Lastly, a novel method, Bayesian Kernel Machine Regression (BKMR) was used to model the dose-response relationships of the trace element mixture in a non-parametric fashion.

Results: In the multivariable linear model, we observed significant associations of glucose levels with plasma Cu (2.4 mg/dL increase per 1 SD increase on the log scale; 95% CI: 1.1, 3.7) and plasma Mo (-1.4 mg/dL decrease per 1 SD increase on the log scale; 95% CI: -2.6, -0.1). The GAM showed suggestive evidence of non-linear positive interaction between Zn and Cu in plasma (p-value = 0.052). Similar results were observed using the BKMR approach.

Conclusion: Employing different statistical methods to study the effect of early pregnancy trace element mixtures on glucose levels, we found consistent evidences of hyperglycemic effect of Cu and hypoglycemic effect of Mo, as well as potential synergism between Zn and Cu. Future work is needed to better understand the joint effects of these elements in the context of the increasingly insulin-resistant state of pregnancy.

Disclosure

Y. Zheng: None. C. Zhang: None. P.L. Williams: None. M. Weisskopf: None. P.J. Parsons: None. C.D. Palmer: None. G.M. Buck Louis: None. T. James-Todd: None.

Funding

Eunice Kennedy Shriver National Institute of Child Health and Human Development
360-OR: A Novel Genetic Syndrome of Early-Onset Diabetes, Microcephaly, and Epilepsy Due to Homozygous YIPF5 Mutations
Authors: LYTRIVI MARIA , FRANCO ELISA DE , PATEL KASHYAP A. , ESTEVE MARIANA IGOILLO , COSENTINO CRISTINA , WAKELING MATTHEW N. , HALILOGLU BELMA , YILDIZ MELEK , GODBOLE TUSHAR , HATTERSLEY ANDREW T. , CNOP MIRIAM
Published: June 2019

Neonatal diabetes is caused by single gene mutations affecting fundamental β-cell function pathways. 20% of cases of neonatal diabetes remain genetically unexplained. Our aim was to explore the genetic basis of a syndrome characterized by neonatal diabetes, microcephaly and epilepsy, present in 2 unrelated patients. The Turkish and Indian patients were born to consanguineous parents. Using whole genome sequencing, we found that they had homozygous likely deleterious variants (missense, p.(Ala181Val) and in-frame deletion p.(Lys106del)) in YIPF5. We then performed replication studies in 112 patients with neonatal diabetes by targeted next generation sequencing. These identified 2 homozygous YIPF5 mutations (p.(Trp218Arg) and p.(Ile98Ser)) in 3 patients (2 siblings) with early-onset diabetes, epilepsy and microcephaly. In functional studies, YIPF5 was expressed in human islets and fetal brain cortex, as evaluated by qPCR and in situ hybridization. Because YIPF5 is involved in endoplasmic reticulum (ER)-to-Golgi trafficking, we examined the impact of YIPF5 loss-of-function on β-cell survival during ER stress. YIPF5 was silenced in the human β-cell line EndoC-βH1 and in human islets. YIPF5 silencing sensitized β cells to apoptosis induced by the ER stressors thapsigargin and brefeldin A. The ER stressors enhanced CHOP, BiP and spliced XBP1 expression in YIPF5-depleted cells, indicating increased ER stress signaling. Expression of the proapoptotic proteins PUMA and DP5 was also enhanced by YIPF5 silencing. CHOP and DP5 knockdown partially protected YIPF5-deficient cells from apoptosis, suggesting that they mediate apoptosis.

In conclusion, homozygous loss-of-function mutations in YIPF5 are a novel cause of early-onset diabetes, microcephaly and epilepsy. This syndrome unveils a critical role of YIPF5 and ER-to-Golgi trafficking in the function and survival of human β-cells and neurons.

Disclosure

M. Lytrivi: None. E. De Franco: Other Relationship; Self; Novo Nordisk Foundation. K.A. Patel: None. M. Igoillo Esteve: None. C. Cosentino: None. M.N. Wakeling: None. B. Haliloglu: None. M. Yildiz: None. T. Godbole: None. A.T. Hattersley: None. M. Cnop: None.

Funding

Fonds Erasme for Medical Research; European Union (667191); Fonds National de la Recherche Scientifique; Brussels-Capital Region Innoviris
363-OR: ChREBP-Beta Controls Both Proliferation and Cell Death of Pancreatic Beta Cells
Authors: KATZ LIORA S. , BRILL GABRIEL , HERMAN MARK A. , SCOTT DONALD
Published: June 2019

Glucose is a key physiological driver of adaptive beta cell mass expansion, promoting beta cell proliferation both in vitro and in vivo. Carbohydrate response element binding protein β (ChREBPβ) is upregulated in response to glucose and is required for glucose-stimulated beta cell proliferation. To study the role of ChREBPβ in beta cells, we knocked out (KO) or overexpressed (OE) ChREBPβ in mouse pancreatic beta cells using Cre LoxP approaches with either MIP-CreERT (induced by tamoxifen at 3 months of age), or the RIP-Cre model which leads to recombination specifically in β cells, early during embryogenesis (E9). We performed intraperitoneal glucose tolerance tests (IPGTTs) and assessed pancreata for proliferation (Ki67), cell death (TUNEL) and beta cell mass in the 4 mouse models. ChREBPβ MIP-CreERT KO mice displayed impaired glucose tolerance, with an increase of 25.3 ± 7.9% (N=3-4, P<0.05) in the area under the curve (AUC) of IPGTT. Heterozygous or complete Chrebpβ KOs in the RIP-Cre model resulted in an increase in the AUC of IPGTT compared with control littermates (20.1 ± 13.7% and 56 ± 7.1%, respectively; n=2-4). Beta cells from isolated ChREBPβ KO islets had 60.4 ± 17.4% less proliferation compared to control islets in 6 mM glucose, and 22.9 ± 2.4% (P<0.05, N=5) less in 20 mM glucose. Inducible OE of ChREBPβ resulted in 97.7 ± 6% increase in AUC of IPGTT (N=3, p<0.01) and fasting blood glucose levels were increased by 89 ± 13.2% (P<0.01). Similarly, when ChREBPβ was OE early in the embryogenesis of β cells, we observed AUC for GTT was increased by 2.4 ± 0.5 fold compared to non-Cre expressing litter mates (N=5, p<0.05). In both mouse models, the OE of ChREBPβ resulted in β cell death as assessed by TUNEL along with increased in Ki67 staining. Remarkably, the RIP-Cre mice had almost no remaining β cells at 4 months of age and were overtly diabetic. Taken these data together, we propose a molecular regulatory mechanism where ChREBPβ controls both beta cell proliferation and cell death depending on its abundance.

Disclosure

L.S. Katz: None. G. Brill: None. M.A. Herman: None. D. Scott: None.

Funding

National Institutes of Health
372-P: Activation of the Nrf2 Pathway Provides Protection against Hypoglycaemia-Induced Cognitive Impairment in a Rodent Model of Type 1 Diabetes
Authors: MCNEILLY ALISON D. , GALLAGHER JENNIFER , DINKOVA-KOSTOVA ALBENA T. , MCCRIMMON RORY J.
Published: June 2019

Whether recurrent mild-moderate hypoglycaemia (RH) has pathological effects on the brain in the long-term remains unknown, but is of major concern to people with type 1 diabetes (T1D). We have previously demonstrated in a rodent model of T1D that RH is associated with impaired cognitive function resulting from a reduced ability to cope with enhanced oxidative stress and that this was associated with activation of the Nrf2 pathway. This study sought to investigate whether pre-treatment with sulforaphane (SFN), a potent Nrf2 inducer would ameliorate the impact of RH on cognitive function in the T1D rodent model. A chronic stable model of chronic insulin-treated T1D was achieved using streptozotocin (125mg/kg i.p) and insulin implants (Linbit®). Diabetic (male C57bl6 mice n=8-10/group) mice were randomly allocated to one of 3 groups: (i) T1D, (ii) T1D+RH, (iii) T1D+RH+SFN and subjected to repeated episodes of insulin-induced moderate hypoglycemia (3 episodes/week for 4 weeks; mean glucose 2.9±0.6 mmol/l). SFN (50mg/kg i.p.) or Vehicle (1% DMSO/PBS) was administered 24 hour prior to each hypoglycemic episode. Cognition was assessed by novel object recognition (NOR) and spontaneous alternation tasks. Data are mean±SEM. Pre-treatment with SFN had no impact upon body weight (p=ns) or fasting blood glucose (p=ns). SFN significantly improved cognitive performance in the 24 hour NOR task in T1D+RH animals when compared to vehicle (80% improvement; p<0.01). Furthermore, performance in the spontaneous alternation task was enhanced to Control levels in T1D+RH following treatment with SFN (25% improvement; p<0.01). The beneficial effects of SFN on cognition were not observed in Nrf2 null mice. Treatment with the SFN significantly improves RH-induced cognitive impairments in a rodent model of T1D. Therefore, activation of the Nrf2 antioxidant pathway offers a novel therapeutic target for the treatment of cognitive impairments associated with RH in T1D.

Disclosure

A.D. McNeilly: None. J. Gallagher: None. A.T. Dinkova-Kostova: None. R.J. McCrimmon: Advisory Panel; Self; Eli Lilly and Company, Novo Nordisk A/S, Sanofi-Aventis. Research Support; Self; The Leona M. and Harry B. Helmsley Charitable Trust.

Funding

Diabetes UK
373-P: Recurrent Insulin-Induced Hypoglycaemia Leads to Weight Gain in Association with Increased Adiposity and Reduced Basal Metabolic Rate
Authors: MCNEILLY ALISON D. , GALLAGHER JENNIFER , MCCRIMMON RORY J.
Published: June 2019

Intensification of insulin therapy in type 1 diabetes is associated with significant weight gain. Several mechanisms have been proposed to account for this weight gain including reduced glucosuria and suppression of hepatic gluconeogenesis and glucose production however the precise mechanisms responsible for this remain unclear. This study examined the hypothesis that recurrent hypoglycaemia (RH) might contribute, at least in part, to this phenomenon.

Male C57BL6J mice (n=12/group) received either RH (1mU/g insulin i.p. 3 per week for 12 weeks to achieve glucose of approximately 2.8mmol/l) or Vehicle (saline i.p in equivalent volume). Body composition and energy homeostasis were assessed using EchoMRI and CLAMS systems respectively. Hypothalamic transcript abundance was assessed using Taqman real time PCR. Data are expressed as mean+/-SEM.

RH animals were 43% heavier and had increased fat mass compared to Control animals (8.49+/-0.64 v 16.6+/-1.38% body weight) despite comparable food intake (p=ns). Energy expenditure was significantly lower (53% decrease) in RH animal (0.414+/-0.006 v 0.218+/-0.004 kcal/min; p<0.01) and was associated with a decrease in the respiratory exchange ratio (RER: 0.98+/-0.01 v 0.96+/-0.01;p<0.05). There was no difference in locomotor activity. These changes were associated with suppression of genes involved in thermogenesis, mitochondrial energetics and lipolysis within the brown adipose tissue.

Consistent with our hypothesis, RH leads to increased accumulation of fat stored which is associated with reduced energy expenditure and RER. These results suggest that RH induces a state of hypometabolism that may contribute to weight gain observed with intensification of insulin therapy in type 1 diabetes.

Disclosure

A.D. McNeilly: None. J. Gallagher: None. R.J. McCrimmon: Advisory Panel; Self; Eli Lilly and Company, Novo Nordisk A/S, Sanofi-Aventis. Research Support; Self; The Leona M. and Harry B. Helmsley Charitable Trust.

Funding

JDRF; Diabetes UK
378-P: Human Voice Is Modulated by Hypoglycemia and Hyperglycemia in Type 1 Diabetes
Authors: CZUPRYNIAK LESZEK , SIELSKA-BADUREK EWELINA , NIEBISZ AGNIESZKA , SOBOL MARIA , KMIECIK MAGDALENA , JEDRA KATARZYNA , SZYMANSKA-GARBACZ ELEKTRA , NIEMCZYK KAZIMIERZ
Published: June 2019

Anecdotal evidence suggests that high pitched voice may be a clinical symptom of hypoglycemia. We conducted a pioneer study aiming at assessing voice variables during hypoglycemia and hyperglycemia. We recruited 10 non-smoking individuals with type 1 diabetes (4 women, 6 men; mean [±SD] age 40.2±10.4 years, diabetes duration 21.4±9.9 years, HbA1c 8.0±1.8%) who were using real time continuous glucose monitoring system (rt-CGM, Guardian Connect, Medtronic) for mean 5.2±0.6 days in controlled hospital setting. At regular intervals during normoglycemia and additionally at blood glucose <70 mg/dl or >200 mg/dl each patient was instructed to record with a professional voice recorder (LS-14, Olympus) a standardized sentence and the sound of vowel ‘a’ for 3 seconds. The voice samples (n=177) were analyzed against CGMS data with the DiagnoScope software (DiagNova, Wroclaw, Poland). Number of fundamental PERiods (PER), time of fundamental PERiods (PERTime), fundamental frequency (F0), energy (E), amplitude of fundamental frequency (AF0), indicator of voiced/phonation probability (Voiced), simple voice quality (SimpleQ), relative average perturbation (RAP), shimmer (Shimm), amplitude perturbation quotient (APQ), four formant frequencies (F1-F4), harmonic perturbation quotients (HPQ), residual to harmonic ratio (R2H), unharmonic to harmonic ratios (U2H), subharmonic to harmonic ratio (S2H), noise to harmonics ratio (NHR), 1st to 4th harmonic to all energy ratio (Fx1-Fx4) were analyzed. In women during hypoglycemia E, AF0, Voiced, F1, F4, R2H, Fx3, Fx4, while in hyperglycemia - RAP and F2 were significantly altered when compared to normoglycemia. In men in hypoglycemia the differences were found in PER, PERTime, Voiced, SimpleQ, Shimm, APQ, F2, U2H, S2H, Fx2, NHR, while in hyperglycemia - PERTime, F1, HPQ, U2H, Fx2 (all p<0.05). We report for the first time that hypoglycemia and hyperglycemia modulate human voice. This phenomenon may offer potential for early detection and prevention of hypoglycemia.

Disclosure

L. Czupryniak: None. E. Sielska-Badurek: None. A. Niebisz: None. M. Sobol: None. M. Kmiecik: None. K. Jedra: None. E. Szymanska-Garbacz: None. K. Niemczyk: None.

Funding

Polish Ministry of Science and Higher Education
38-LB: Discovery Analysis of MicroRNAs (miRs) Associated with Microvascular Complications in Adults with Type 1 Diabetes
Authors: JANUSZEWSKI ANDRZEJ S. , SCOTT EMMA S. , JOGLEKAR MUGDHA , CARROLL LUKE , FARR RYAN , WONG WILSON , O’NEAL DAVID N. , KARSCHIMKUS CONNIE , FULCHER GREG , HARDIKAR ANAND , JENKINS ALICIA J.
Published: June 2019

Objective: Despite therapeutic advances, type 1 diabetes (T1D) is associated with a risk of development of micro- and macrovascular complications. Early treatment of complications is associated with a more favorable prognosis, hence biomarkers to enable early detection and to monitor treatments may be useful.

Methods: Plasma (EDTA) samples were obtained from 30 adult (M/F = 20/10) individuals with T1D, age 42±16 years, T1D duration 24±11 years, HbA1c 7.5±1.2% and BMI 26.5±4.0 kg/m2 (15 with microvascular complications (retinopathy or nephropathy) - CX+, and 15 without complications - CX-) and compared with 15 nondiabetic controls (CON, matched age, gender, smoking and BMI). T1D CX+ and CX- individuals were matched for age, gender, diabetes duration, smoking, BMI, HbA1c and insulin therapy modality (CSII/MDI). In these discovery cohort samples, profiling of 754 miRs was done using a high throughput probe based quantitative PCR on nanofluidics Open Array platform . Results were analyzed using independent T test, Mann-Whitney test and classification trees. Statistical analysis was undertaken using R (ver. 3.5.1) and XLStat (ver. 2018.6).

Results: Of 754 available miRs on the array, 186 miRs were detectable in plasma. Using an unbiased approach expression of 32 miRs differed significantly in CON vs. T1D and 16 miRs were differentially expressed between CX+ and CX- T1D subjects. Using a Random Forest wrapper algorithm 5 miRs were identified with importance for segregating CX+ vs. CX- status: miR-483-5p, miR-328, miR-1274B, miR-7 and miR-30a-5p. In logistic regression those 5 miRs provided a ROC curve with AUC=0.90 for the CX+ vs. CX- discrimination.

Conclusion: In a discovery analysis of well- matched CX+ and CX- selected miRs provided good discrimination between T1D CX status. Validation studies in a larger cohort of cross-sectional and longitudinal samples are in progress.

Disclosure

A.S. Januszewski: None. E.S. Scott: None. M. Joglekar: None. L. Carroll: None. R. Farr: None. W. Wong: None. D.N. O’Neal: None. C. Karschimkus: None. G. Fulcher: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Research & Development, Merck Sharp & Dohme Corp., Novo Nordisk Inc. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Research & Development, Merck Sharp & Dohme Corp., Novo Nordisk Inc. Research Support; Self; Novo Nordisk Inc. A. Hardikar: None. A.J. Jenkins: Advisory Panel; Self; Abbott, Australian Diabetes Society, Medtronic. Research Support; Self; Abbott, GlySens Incorporated, Medtronic, Mylan. Speaker’s Bureau; Self; Eli Lilly and Company, Novo Nordisk Inc.

Funding

Sydney Medical School Foundation; National Health and Medical Research Council of Australia
386-P: Chronic and Acute Consumption of a Ketogenic Diet Leads to Deficits in Hypoglycemic Counterregulation in Mice
Authors: DUVALL MARINA A. , COULTER CAROLYN , HILL CRISTAL M. , MORRISON CHRISTOPHER , SIKAFFY HUNTER X. , MCDOUGAL DAVID
Published: June 2019

Background: Due to their metabolic benefits, ketogenic diets (KDs) are growing increasingly popular as a dietary strategy for augmenting treatment of type 1 and type 2 diabetes. Although much is known regarding the metabolic benefits of KDs, little is known about their effect on hypoglycemic counter-regulation. We tested whether mice fed a KD have normal hypoglycemic counter-regulation.

Methods: B6 mice were fed a KD (Bio-Serv; F3666) ad lib for either 28 days (chronic) or 7 days (acute). On the final day of the diet, hypoglycemic counter-regulation was assessed via glucoprivic challenge (ICV 2DG, 1 mg/mouse, or saline) in the chronic KD group or via a hypoglycemic insulin tolerance test (ITT) in the acute KD group. Deficits in the counter-regulatory response were determined by comparing glucose and glucagon levels to control mice simultaneously fed a standard chow diet (LabDiet; 5001). ITTs consisted of a variable dose of IP insulin which produced equivalent hypoglycemia in all groups (∼45 mg/dL).

Results: As expected, mice fed a KD either acutely or chronically had lower fasting blood glucose levels relative to controls (chronic KD: 132.4 and 167.5mg/dL respectively, p = 0.0011, n= 12-17/group; Acute KD: 167.3 and 200.7 mg/dL respectively, p = 0.026; n = 7-10/group). In response to glucoprivic challenge, chronic KD mice had a blunted rise in both glucose and glucagon relative to chow fed mice (Delta glucose: 84.2 and 135.8 mg/dL respectively, p = 0.017; Glucagon: 32.7 and 73.8 pg/mL respectively, p = 0.027; n = 5-8/group). Acute KD mice also had reduced serum glucagon levels in response to the ITT (28.8 and 50.1 pg/mL respectively, p = 0.044).

Conclusions: We show that consuming a KD reduces glucose counter-regulation in mice. If a similar effect exists in persons with diabetes it could blunt a homeostatic response which is critical for counteracting treatment-induced hypoglycemic crises. Further characterization of the effects of KDs on glucose counter-regulation is warranted.

Disclosure

M.A. DuVall: None. C. Coulter: None. C.M. Hill: None. C. Morrison: None. H.X. Sikaffy: None. D. McDougal: None.

Funding

American Diabetes Association (1-15-JF-37 to D.M.)
39-OR: VPS41 Contributes to Insulin Granule Biogenesis
Authors: BURNS CHRISTIAN H. , YAU BELINDA , KEBEDE MELKAM A. , ASENSIO CEDRIC S.
Published: June 2019

Insulin secretory granules (ISGs) mediate the regulated secretion of insulin. The basic machinery responsible for this regulated exocytosis consists of specific membrane proteins present both at the plasma membrane and on ISGs. The protein composition of ISGs thus dictates their release properties, yet the mechanisms controlling ISG budding from the trans-Golgi network (TGN), which partly determines this molecular composition, remain poorly understood. The cytosolic protein VSP41 was recently identified as a potential coat protein at the TGN involved in the formation of neuroendocrine/neuronal granules. To test whether this protein might contribute to ISG biogenesis, we have generated VPS41 KO Ins1 cells using CRISPR/Cas9. Loss of VPS41 leads to reduced cellular insulin content with a defect in regulated insulin secretion. We also find, using live-imaging and a pHluorin based reporter, that VPS41 KO cells exhibit less stimulated exocytotic events. At the cellular level, insulin appears to be retained in a perinuclear compartment, and cell fractionation and electron microscopy analysis further reveal a change in ISG protein composition and number. Finally, mice with a beta-cell specific deletion of VPS41 are glucose intolerant and display a defect in insulin secretion in response to glucose. Altogether our data suggest that VPS41 is an important player in the formation or maintenance of ISG.

Disclosure

C.H. Burns: None. B. Yau: None. M.A. Kebede: None. C.S. Asensio: None.

Funding

American Diabetes Association (1-17-JDF-064 to C.S.A.); National Institutes of Health (R15GM116096, R01GM124035)
394-P: Nonsevere Hypoglycemia Treatment in Adults with Type 1 Diabetes: A Validation Study of Carbohydrate Intake at Different Glucose Thresholds
Authors: TALEB NADINE , MESSIER VIRGINIE , PARENT VALERIE , BOVAN DANIJELA , BRAZEAU ANNE-SOPHIE , RABASA-LHORET RÉMI
Published: June 2019

Background: For nonsevere hypoglycemia (NS-H), treatment guidelines recommend 15-20g of rapidly absorbed carbohydrates (CHO) and repeat intake at 15 min until recovery (glucose above 70 mg/dL). The adequacy of this one size fits all approach is questioned. Our group reported an average intake of 32 ± 24g CHO in a type 1 diabetes (T1D) outpatient study. We aim to validate N-SH treatment guidelines in the context of modern therapies.

Methods: Randomized four way cross-over trial to assess treatment with 16g vs. 32 g CHO (glucose tablets) for fasting subcutaneous insulin induced NS-H at two glucose thresholds: a) below 54 mg/dL and b) between 54 to 63 mg/dL. Plasma glucose is measured every 5 min till recovery then at 15-30 min intervals.

Results: Preliminary data was collected for 12 of 33 subjects (53.2 ± 16.8 years old, 58.3% females, HbA1c 56 ± 7.7 mmol/mol, diabetes duration 31.9 ± 15.8 years). Comparison of 16 vs. 32g CHO for each threshold revealed the following change in glucose 15 min post treatment (primary outcome): a) below 54 mg/dL: 12.0 ± 21.8 vs. 17.6± 28.4 mg/dL, p=0.4; b) between 54 to 63 mg/dL: -1.6 ± 19.7 vs. 12 ± 22.7 mg/dL, p=0.06; time needed to recovery (above70 mg/dL): a) below 54 mg/dL: 30 ± 13.6 vs. 29.6 ±14.4 min, p= 0.9; b) between 54 to 63 mg/dL: 37.5 ±14.4 vs. 28.7 ± 17.2 min, p= 0.1 and time needed for disappearance of symptoms: a) below 54 mg/dL: 27.8± 22.3 vs. 22.8 ± 9.5 min, p=0.8; b) between 54 to 63 mg/dL: 24.2 ± 10.7 vs. 16.7± 11.5 min, p=0.5. Regression analysis will be conducted to study response predictors (plasma insulin and catecholamines levels, body composition, etc.)

Conclusion: The response to nonsevere hypoglycemia treatment is extremely variable. A one size fits all recommendation is unlikely to be appropriate. Data of this trial will add to the knowledge of hypoglycemia treatment in T1D.

Disclosure

N. Taleb: None. V. Messier: Other Relationship; Self; Eli Lilly and Company. V. Parent: None. D. Bovan: None. A. Brazeau: None. R. Rabasa-Lhoret: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Janssen Pharmaceuticals, Inc., Lilly Diabetes, Merck & Co., Inc., Novo Nordisk A/S, Sanofi. Research Support; Self; AstraZeneca, Janssen Pharmaceuticals, Inc., Novo Nordisk A/S.

Funding

Canadian Institutes of Health Research (354024); JDRF (4-SRA-2018-651-Q-R)
396-P: SGLT2 Inhibition Suppresses Glucagon Response to Hypoglycemia in Male but Not Female Rats with Streptozotocin-Induced Type 1 Diabetes
Authors: GREENBERG CAYLEE A. , MANDEL ERIN R. , PASIEKA AOIBHE M. , JAHANGIRIESMAILI MAHSA , TREMBLAY JULIE , RIDDELL MICHAEL
Published: June 2019

Sodium glucose co-transporter 2 inhibitors (SGLT2i) show promise as an add-on to insulin therapy in patients with type 1 diabetes (T1D), however their effect on glucagon secretion in T1D is not yet known. Increases in glucagon have the potential to protect against hypoglycemia that may develop from insulin overtreatment or exercise. We investigated whether 8 days of SGLT2i treatment (10mg/kg empagliflozin, 2x/day) altered plasma glucagon concentrations in response to voluntary running wheel activity and insulin-induced hypoglycemia in male and female Wistar rats with streptozotocin-induced T1D. SGLT2 inhibition did not alter basal or post-exercise glucagon concentrations and did not affect exercise-associated changes in blood glucose (BG) levels. Additionally, SGLT2 inhibition did not alter voluntary activity levels. However, in males, SGLT2 inhibition lowered plasma glucagon response during an insulin tolerance test (+22.7±30.8 pg/mL vs. +40.4±48.6 pg/mL, p=0.02) and decreased the time taken to reach hypoglycemia (30±13.4 min vs. 63.8±17.2 min, p<0.0001), as compared to placebo. We also observed novel sex differences in the response of BG and glucagon concentrations to voluntary exercise. In female rats alone, voluntary exercise decreased BG concentrations (-2.9±2.6 mM vs. +1.7±1.8 mM, p=0.0003) and led to lower post-exercise glucagon concentrations (37.3±47.9 pg/mL vs. 117.9±86.0 pg/mL, p=0.01), as compared to inactive animals. On average, voluntary activity levels were higher in females than in males (539.2±199.4 m vs. 308.4±215.6 m, p=0.02), however running distances were not correlated with changes in BG levels during exercise (r2=0.02). Our finding of increased hypoglycemia risk with SGLT2i has concerning implications for its use as an adjunct to insulin therapy in T1D. The novel sex differences that we observed should be investigated in individuals with T1D to determine their human applicability and potential mechanisms.

Disclosure

C.A. Greenberg: None. E.R. Mandel: None. A.M. Pasieka: None. M. Jahangiriesmaili: None. J. Tremblay: None. M. Riddell: Advisory Panel; Self; Xeris Pharmaceuticals, Inc. Research Support; Self; Dexcom, Inc. Speaker's Bureau; Self; Insulet Corporation, Medtronic MiniMed, Inc. Stock/Shareholder; Self; Zucara Therapeutics Inc.
40-LB: Association between Complement and Severe Retinal Complications in Type 1 Diabetes—An 18-Year Follow-Up Study
Authors: HOLT CHARLOTTE B. , HOFFMANN-PETERSEN INGEBORG TORP , øSTERGAARD JAKOB A. , PARVING HANS- HENRIK D. , THIEL STEFFEN , HOVIND PETER , TARNOW LISE , ROSSING PETER , HANSEN TROELS K.
Published: June 2019

Introduction: Diabetic retinopathy (DR) remains the leading cause of preventable vision loss in developed countries. Adverse activation of the complement system plays an essential role in development of complications in diabetes and has been demonstrated in eyes from patients with DR. We hypothesized that complement proteins of lectin pathway can predict severe DR, defined as proliferative DR and macular edema, in newly diagnosed type 1 diabetes.

Methods: In this explorative study, incidence of severe DR was monitored in a cohort of newly diagnosed type 1 diabetes patients (n=270) with median follow-up time of 18 years (range 1-22 years). Baseline complement proteins MBL, M-ficolin, H-ficolin, MASP-1, MASP-2, MASP-3, MAp44, and MAp19 were quantified using immunoassays. A cox proportional hazards regression model was applied to test for difference between quantile groups and adjusted for age and HbA1c.

Results: During follow-up, 28 patients developed severe DR. A tendency arose when testing association between MAp44 levels and severe DR in an unadjusted model (HR, 4th quartile vs. the remaining, 1.3; 95% CI 0.99, 1.6), p = 0.06), which became significant in the adjusted model (HR, 1.33; 95% CI 1.0, 1.7), p = 0.04), Fig. 1.

Conclusion: MAp44 was significantly associated with development of severe DR, whereas other complement factors were not. Further analysis in a larger cohort is warranted.

Disclosure

C.B. Holt: None. I.T. Hoffmann-Petersen: None. J.A. østergaard: None. H.D. Parving: None. S. Thiel: None. P. Hovind: None. L. Tarnow: Stock/Shareholder; Self; Novo Nordisk A/S. Other Relationship; Self; Novo Nordisk Foundation. P. Rossing: Advisory Panel; Self; AstraZeneca, Bayer AG, Boehringer Ingelheim Pharmaceuticals, Inc. Speaker’s Bureau; Self; Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novartis AG, Novo Nordisk A/S. T.K. Hansen: None.

Funding

Aarhus University; Foundation of Skibsredder Per Henriksen
40-OR: The Role of TALK-1 K+ Channels in Pancreatic ß-Cell Insulin Secretion, Mitochondrial Function, and the ER Stress Response
Authors: GRAFF SARAH M. , DADI PRASANNA , IBSEN CHLOE E. , DICKERSON MATTHEW , JORDAN KELLI L. , JACOBSON DAVID
Published: June 2019

During Glucose-Stimulated Insulin Secretion (GSIS), β-cells exhibit oscillations in cytosolic Ca2+ ([Ca2+]c) and endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER), which are regulated by the two-pore domain K+ channel, TALK-1. TALK-1 is the most abundant β-cell K+ channel and it controls Ca2+ homeostasis by modulating K+ permeability at both the plasma and ER membrane. However, how TALK-1 modulates ER stress and β-cell failure during the pathogenesis of diabetes has not been determined. To understand how TALK-1 controls β-cell function under diabetic conditions, we assessed how a mutation in TALK-1 (ND) which causes neonatal diabetes, impacts TALK-1 channel activity and [Ca2+]ER handling. While plasmallemal TALK-1 activity was inhibited with the ND mutation, [Ca2+]ER storage was significantly reduced by the ND mutation compared to control TALK-1. This suggests that TALK-1 ND enhances TALK-1 activity on the ER membrane leading to reduced [Ca2+]ER storage. Reducing [Ca2+]ER enhances ER stress, which would be expected to cause β-cell dysfunction. Indeed, TALK-1 ND significantly reduces β-cell insulin secretion compared to control. To further investigate the role of TALK-1 we developed a β-cell-specific TALK-1 KO mouse model (β-TALK1-KO). On a high fat diet, β-TALK1-KO mice show improved glucose tolerance compared to controls. Mitochondrial calcium ([Ca2+]mito) was also examined because it is modulated by [Ca2+]ER; we found that β-TALK1-KO mice have increased [Ca2+]mito. As reactive oxygen species (ROS) production is a byproduct of mitochondrial respiration, we investigated if TALK-1 affects ROS production. TALK1 KO islets were found to have reduced ROS production compared to wild type islets. Together, these data indicate that increased TALK-1 channel activity reduces [Ca2+]ER, [Ca2+]mito, and increases ROS; suggesting that TALK-1 plays an important role in modulating the β-cell responses to stress associated with diabetic conditions.

Disclosure

S.M. Graff: None. P. Dadi: None. C.E. Ibsen: None. M. Dickerson: None. K.L. Jordan: None. D. Jacobson: None.
411-P: PRMT1 Upregulation and ADMA Accumulation Contribute to the High Sensitivity of Zucker Diabetic Fatty Rats to Myocardial Ischemia/Reperfusion Injury
Authors: XIONG YAN , LIN YUAN , LEI YANPING , HE ZHI-MIN , ZHOU XIN-KE
Published: June 2019

Diabetic patients show a high sensitivity to myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism has remained obscure. Increasing evidence indicates that protein arginine methyltransferase1 (PRMT1) is implicated in modulation of cellular processes including gene transcription, energy metabolism, apoptosis and generation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS). ADMA as an intermediate of protein arginine methylation catalyzed by PRMT1 is associated with diabetic cardiovascular complications. This study was to examine whether PRMT1-ADMA disorder is involved in the high sensitivity to myocardial I/R injury in Zucker diabetic fatty (ZDF) rats. Myocardial I/R injury was induced by 30min of ligation in left anterior descending coronary artery followed by 2∼24h reperfusion. Cardiac function was measured by M-echocardiography, and ADMA signaling pathway was evaluated by expressions, activities or contents of signal molecules. ZDF rats showed severer cardiac dysfunction and larger area of infarction after myocardial I/R injury compared with Zucker-control rats. Expression and activity of PRMT1 and ADMA content were higher while expression and activity of NOS and nitric oxide (NO) content were lower in ZDF rats. Treatment with PRMT1 inhibitor TC-E or knockdown of PRMT1 with shRNA-PRMT1-LV reduced myocardial ADMA accumulation and protected cardiac function of ZDF rats from I/R injury. Exogenous ADMA perfusion of isolated heart from control rats could induce similar cardiac dysfunction to that of I/R injury. Treatment with TC-E or NO donor protected the isolated heart from I/R injury or ADMA-induced impairment. These results indicate that PRMT1 upregulation and ADMA accumulation contribute to the high sensitivity of ZDF rats to myocardial I/R injury and reveal the novel target for prevention and treatment of myocardial I/R injury in diabetes mellitus.

Disclosure

Y. Xiong: None. Y. Lin: None. Y. Lei: None. Z. He: None. X. Zhou: None.

Funding

National Natural Science Foundation of China (81570751); Guangdong Province (2016A030311050)
413-P: SIRT1 Overexpression Restores the Protective Effects of Ischemic Post-conditioning in Diabetic Mice via Modulating Akt Signaling Pathway
Authors: DING MINGGE , ZENG KE , YU MINGZHE , LIU CHAOYANG , FU FENG
Published: June 2019

Ischemic post-conditioning (IPO) activates Akt signaling to confer cardioprotection. The responsiveness of diabetic hearts to IPO is impaired. We hypothesized that decreased cardiac SIRT1, a positive regulator of Akt, may be responsible for the impaired responsiveness of diabetic hearts to IPO-mediated cardioprotection. High-fat diet and streptozotocin-induced diabetic mice were subjected to myocardial ischemia/reperfusion (MI/R, 30 minutes ischemia and 180 minutes reperfusion) or IPO (three cycles of 10 s of reperfusion and ischemia at the onset of reperfusion). Adenoviral vectors encoding GFP or SIRT1 (Ad-SIRT1) were administered by direct injection into the left ventricular. IPO activated the Akt signaling pathway and reduced MI/R injury in nondiabetic hearts but not in diabetic hearts, in which reduced expression of SIRT1 and increased Akt acetylation were observed. Delivery of Ad-SIRT1 into the diabetic hearts reduced Akt acetylation and restored the cardioprotective effects of IPO. In in vitro study, transfection with wild type SIRT1 but not inactive mutant SIRT1 reduced the expression of Akt acetylation and restored the protective effects of hypoxic post-conditioning in high glucose-incubated primary cardiomyocytes. Moreover, the cardiomyocytes transfected with Akt acetylation-defective mutant (K20R) showed increased Akt phosphorylation and enhanced protective effects against hypoxia/reoxygenation injury. These findings demonstrate that SIRT1 up-regulation restores IPO-mediated cardioprotection in diabetic mice via deacetylation-dependent activation of Akt signaling pathway. It is suggested that targeting up-regulation of SIRT1 may be a potential therapeutic strategy to restore myocardial responsiveness to post-conditioning in diabetic patients.

Disclosure

M. Ding: None. K. Zeng: None. M. Yu: None. C. Liu: None. F. Fu: None.

Funding

National Natural Science Foundation of China (81600235, 81670354); Natural Science Foundation of Shaanxi (2018JM7061)
427-P: The Prevalence of Asymptomatic Cardiac Dysfunction Diagnosed by Tissue Doppler Echocardiography in Subjects with Type 2 Diabetes
Authors: TAKATA YASUNORI , TAKAKADO MISAKI , HADATE TOSHIMI , KAWAMURA RYOICHI , OSAWA HARUHIKO
Published: June 2019

Background: The sodium-glucose transporter 2 inhibitors have been found to reduce the risk of hospitalization for heart failure (HF). However, in the majority of clinical studies of diabetes, HF is usually defined as administration to hospital with typical symptoms without measurements of cardiac-function using tissue Doppler echocardiography (TDE). Therefore, the incidence of cardiac-dysfunction in diabetic subjects would be underestimated, leading to a delay in the initiation of treatment. Thus, we analyzed the prevalence of asymptomatic cardiac-dysfunction (ACD) diagnosed by TDE in subjects with diabetes.

Methods: We recruited inpatients or outpatients with type 2 diabetes at university hospital. Subjects with symptomatic HF, atrial fibrillation, myocardial infarction, valvular disease, and hypertrophic cardiomyopathy were excluded from this study. Finally, 343 diabetic subjects were enrolled. Cardiac-dysfunction was defined as left ventricular ejection fraction <50% (systolic dysfunction) and/or the ratio of early transmitral flow velocity to TDE diastolic mitral annular velocity>15 (diastolic dysfunction). To determine optimum cut-off values for BNP and eGFR associated with ACD, we performed ROC curve analyses.

Results: Twenty-four % of subjects had ACD. Seventy-eight % of these subjects had diastolic dysfunction. Age, the proportion of females, pulse wave velocity and BNP were higher, and eGFR was lower in subjects with cardiac-dysfunction than those without cardiac-dysfunction. ROC curve analyses revealed that AUCROC for BNP was 0.66 with a sensitivity of 57% and a specificity of 70% at a cut-off value of 32 mg/gCr, and eGFR was 0.53 with a sensitivity of 74% and a specificity of 70% at a cut-off value of 62 mL/min/1.73m2 (both p<0.001).

Conclusions: About a quarter of diabetic subjects had ACD, and the majority of them were diastolic dysfunction. Therefore, TDE would be needed to accurately assess ACD in subjects with diabetes.

Disclosure

Y. Takata: None. M. Takakado: None. T. Hadate: None. R. Kawamura: None. H. Osawa: None.
43-LB: Characteristics of Statin Initiation and Its Association with Mortality in Diabetic and Nondiabetic U.S. Elderly Veterans
Authors: SOOHOO MELISSA , STREJA ELANI
Published: June 2019

Background: Trials examining statin benefit often exclude elderly patients over age 75 years; thus specific guidelines on statin initiation for this group are lacking. Among U.S. veterans, there is a growing elderly population with a high prevalence of diabetes and cardiovascular (CV) disease. We sought to characterize which elderly diabetic and nondiabetic U.S. veterans initiated a statin and the related all-cause mortality risk.

Methods: We identified 204,217 veterans who were not on statin at baseline (2004-2006) and were more than 75 years old at the time of initial lipid measurement between 2006-2008. Cox models were used to examine associations of statin initiation within 6 months of lipid measurement with all-cause mortality after adjustment for demographics, Charlson Index, CV comorbidites, liver disease, cancer, smoking, and kidney disease.

Results: Cohort mean age was 81±4 years, and comprised 2% female, 8% blacks, 20% diabetics, and 43% with CV disease. Statin initiation was more common in diabetics vs. nondiabetics (11% vs. 7%, respectively) and decreased with higher age. Among diabetics, there was little difference in CV comorbidities for statin initiation vs. not; whereas in nondiabetics, statin initiators were more likely to have ischemic heart disease. For both groups, statin initiators had higher triglyceride and LDL levels. Simvastatin was the most frequently prescribed statin in both groups (over 80%), followed by atorvastatin (3%) in diabetics. Over median follow-up of 9 (7,10) years, statin initiation was associated with a lower adjusted all-cause mortality risk in diabetics (HR: 0.91, 95% CI: 0.88, 0.95), with a similar association in nondiabetics (HR: 0.95, 95% CI: 0.92, 0.97).

Conclusions: Elderly U.S. veterans with diabetes who initiated statin therapy had a moderately lower risk of all-cause mortality. Further analyses will examine considerations for additional predictors and differences in cause of death.

Disclosure

M. Soohoo: None. E. Streja: None.

Funding

U.S. Department of Veteran Affairs
440-P: The Effects of DPP-4 Inhibitors on Plasma Catecholamines and Their Metabolites in Patients with Type 2 Diabetes
Authors: PARK IE BYUNG , LEE DAEHO , LEE KIYOUNG
Published: June 2019

Previous preclinical and clinical studies have raised a concern that dipeptidyl peptidase 4 inhibitors (DPP-4i) increased sympathetic activity, which may be related with increased risk of heart failure in DPP-4i-treated type 2 diabetics. In this study, we aimed to evaluate the relationship between DPP-4 activity and catecholamine secretion in patients with type 2 diabetes. Patients with type 2 diabetes (n=119) and normal healthy control subjects (n=25) participated in this cross-sectional study which included the measurement of serum DPP-4 activity and plasma levels of epinephrine (Epi), norepinephrine (NE), metanephrine (MET) and normetanephrine (NMET), other blood and urine biochemical tests, and the collection of demographic and clinical data. The median ages of patients with type 2 diabetes and healthy control subjects were 61 and 56 years, respectively. Compared with the control group, patients with type 2 diabetes were associated with lower plasma levels of catecholamines and their metabolites. In DPP-4i-treated diabetic patients (n=86) had lower plasma NMET level compared with other antidiabetic treatment group (n=36) [0.31 (IQR: 0.21, 0.43)] nmol/L vs. 0.41 (0.29, 0.46) nmol/L, p<0.05], while the plasma concentrations of the two catecholamines and MET were comparable. The difference in plasma NMET level between the two treatment groups was not affected by beta-blocker and angiotensin II receptor blocker therapy, respectively. Additionally, in DPP-4i-treated type 2 diabetics, DPP-4 activity showed inverse correlations with plasma levels of NE (r=-0.312, p<0.01) and NMET (r=-0.275, p<0.05). However, those inverse correlations were not observed in the healthy control group and type 2 diabetics treated with non-DPP-4i antidiabetic agents. Our results suggest that DPP-4i treatment in type 2 diabetics affects NE release from sympathetic nerve in a DPP-4 inhibitory action-dependent manner.

Disclosure

I. Park: None. D. Lee: None. K. Lee: None.
450-P: Impact of Prior Coronary Artery Disease (CAD)/Cerebrovascular Disease (CVD) and Diabetes Mellitus (DM) on Incident CAD/CVD in Japanese
Authors: FUJIHARA KAZUYA , MATSUBAYASHI YASUHIRO , HARADA MAYUKO , OSAWA TAEKO , YAMAMOTO MASAHIKO , KITAZAWA MASARU , KANEKO MASANORI , SEIDA HIROYASU , YAMANAKA NAUTA , KODAMA SATORU , SONE HIROHITO
Published: June 2019

Although DM and prior CAD/CVD are established risk factors for subsequent CAD and CVD, which has the higher impact on future events is unclear. Moreover, few studies have evaluated combined risks of a history of CAD/CVD on subsequent events in the same cohort. Thus, we analyzed a nationwide claims-based database of 292477 people in 2008-17. Participants had either non-DM or DM based on blood tests and prescriptions (non-DM 274860, DM 17617). They were also classified as with or without prior CAD/CVD based on ICD-10 codes and questionnaire. Cox regression model showed that compared with DM(-)CAD(-), DM(+)CAD(-), DM(+)CAD(-), and DM(+)CAD(+) had 5.2(4.1-6.6), 2.6(2.3-3.0), and 8.9(6.7-11.8) times increased risk of CAD, respectively. Compared with DM(-)CVD(-), DM(-)CVD(+), DM(+)CVD(-), and DM(+)CVD(+) had 5.8(4.6-7.4), 1.4(1.2-1.6), and 5.8(4.0-8.6) times increased risk of CVD, respectively. A stratified analysis of 8 groups of combinations of DM status and prior CAD/CVD showed that those with prior CVD had a significantly increased risk of future CVD and those with prior CAD and DM(+) had a high risk of subsequent CAD (Table). This shows that prior CAD/CVD confers a far higher risk than DM status alone for future CAD/CVD. Prior CAD and DM additively increased the risk of future CAD, while prior CVD alone increased the risk of future CVD without additional effects by diabetes.

Disclosure

K. Fujihara: None. Y. Matsubayashi: None. M. Harada: None. T. Osawa: None. M. Yamamoto: None. M. Kitazawa: None. M. Kaneko: None. H. Seida: None. N. Yamanaka: None. S. Kodama: None. H. Sone: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited.