bluuebunny/crossref_metadata_2025 huggingface dataset:
206-OR: A Randomized Controlled Trial to Improve Self-Monitoring of Blood Glucose among Youth with Type 1
Authors: WAGNER JULIE , WEYMAN KATE , TICHY EILEEN M. , CENGIZ EDA , ZAJAC KRISTYN , TAMBORLANE WILLIAM V.
Published: June 2019

Objective: This randomized, controlled trial evaluated a monetary-based reinforcement intervention for increasing self-monitoring blood glucose (SMBG) among youth with poorly controlled type 1 diabetes. SMBG frequency was the primary outcome, A1c was the secondary outcome.

Methods: After a 2-week baseline, 60 youth age 12-19 years with <4 BG checks per day and A1c >7.5% but ≤13% were randomized to enhanced usual care (EUC) or Reinforcers. The Reinforcers group earned monetary rewards for SMBG and associated behaviors such as uploading glucose meters, completing pattern recognition forms, and reviewing results with clinicians. Reinforcers were withdrawn at 24 weeks. A follow-up evaluation occurred at 36 weeks.

Results: Randomization produced groups that did not differ on any demographic or clinical characteristics. Participants in the Reinforcers group increased the proportion of days they completed ≥4 SMBG from 14.6% at baseline to 64.4%, 47.5%, and 37.8% at 6, 12, and 24 weeks, respectively. In contrast EUC participants declined from 22.7% at baseline to 17.5%, 10.5%, and 11.1% (ps<0.01 versus EUC at all time points). Group differences were attenuated but remained significant after withdrawal of reinforcers. Effect sizes for SMBG were very large during reinforcement and large after withdrawal of reinforcers. In the Reinforcers group, mean A1c dropped from 9.5±1.2% at baseline to 9.0±1.3% at week 6 and 9.0±1.4% at week 12. For EUC, A1c was 9.2%±0.2 at baseline and ranged from 9.2±1.5% to 9.6±1.6% throughout the study (p<0.05 versus EUC). Group differences in A1c were no longer significant at weeks 24 and 36. Effect sizes for A1c were small during reinforcement and also after withdrawal of reinforcement.

Conclusions: Monetary-based reinforcement of adolescents with type 1 diabetes caused durable increases in SMBG. Modification of the reinforcement structure may be needed to sustain improved metabolic control in this challenging age group.

Disclosure

J. Wagner: None. K. Weyman: None. E.M. Tichy: None. E. Cengiz: Advisory Panel; Self; Abvance, ADOCIA, MannKind Corporation, Novo Nordisk Inc. Speaker's Bureau; Self; Novo Nordisk Inc. K. Zajac: None. W.V. Tamborlane: Consultant; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Medtronic MiniMed, Inc., Novo Nordisk Inc., Sanofi, Takeda Pharmaceutical Company Limited.
2068-P: Alpha Klotho Modulation with Remission of Prediabetes in Human Subjects
Authors: STENTZ FRANKIE B. , AMMONS ANN
Published: June 2019

Alpha klotho (αK) a protein named for its severe aging effects when under expressed has one of its primary sites of synthesis in the kidneys. Therefore, anything resulting in kidney damage may result in lower αK levels. Diabetes is one of the most common causes of kidney damage; thus, assessing αK levels in relation to glycemic control was the objective of this study.

Twelve obese subjects, age 43.1±1.3 years, with prediabetes were studied at baseline and 6 months (mo) after 10% weight loss on a diet consisting of 30% protein, 30% fat, 40% carbohydrates which resulted in 100% remission of their prediabetes to normal glucose. Glucose, insulin and αK levels were determined during a 75 gm glucose tolerance test (OGTT) at baseline and 6 mo. Soluble αK levels increased significantly from baseline to six mo. Although the microalbumin/creatinine ratio was within normal range at baseline indicating normal kidney function, the ratio improved significantly at 6 mo.

Levels of soluble αK were determined to positively covary with the glucose (r2 = 0.83) and insulin levels (r2 = 0.74) during the OGTT at both baseline and at 6 mo.

These results suggest that αK levels may be markers of very early stages of renal damage in prediabetic subjects and that glucose and insulin interact with soluble αK in a significant manner. These results imply that poor glycemic control can have earlier and more systemic effects that previously thought via altering circulating αK levels.

Disclosure

F.B. Stentz: None. A. Ammons: None.

Funding

AD Baskin
2072-P: Alterations in Fat Mass and Bone Mineral Density Are Associated with Decreased Lipocalin-2 after Laparoscopic Sleeve Gastrectomy in Obese Chinese Women
Authors: WEN XIN , MEI FANGYUN , ZHOU DONGLEI , BU LE , QU SHEN
Published: June 2019

Objective: Lipocalin-2 (LCN2) plays an important role in the regulation of the obesity and obesity-related dysmetabolic state. This study aimed to analyze serum LCN2 level in obese Chinese women before and after laparoscopic sleeve gastrectomy (LSG) and determine the association between alteration in fat mass and bone mineral density (BMD) and LCN2 level.

Methods: Fifty-two women (38 obese and 14 with normal body mass index (BMI)) were enrolled in this study. All obese patients underwent LSG. BMDs of the arm, leg, thoracic and lumbar spine, and pelvis were measured by dual-energy X-ray absorptiometry. Body fat mass and distribution were measured by dual-energy X-ray absorptiometry, and routine anthropometric/laboratory biochemical parameters at baseline and 3 and 12 months after LSG were recorded. Serum LCN2 levels were measured using an enzyme-linked immunosorbent assay.

Results: Serum LCN2 level was significantly higher in obese women than in the controls with normal BMI (102.70±27.19 vs. 80.66±19.55, P = 0.009). LCN2 level was significantly decreased at 3 and 12 months after LSG (86.73±26.79, P<0.001, and 64.79±28.39, P<0.001, respectively). LSG led to marked body fat mass and slight BMD decrease. Decreased LCN2 level was significantly correlated with alterations in left and right leg BMDs and trunk fat mass at 12 months after LSG.

Conclusions: Obesity was associated with up-regulated serum LCN2 level. Decreased LCN2 level was positively correlated with changes in BMD and fat mass at 12 months after LSG in Chinese women.

Disclosure

X. Wen: None. F. Mei: None. D. Zhou: None. L. Bu: None. S. Qu: None.

Funding

Shanghai Sailing Science Fund (14YF1412400)
2075-P: Insulin Resistance Improved at Six Months following Bariatric Surgery Despite Accelerated Lipolysis and Accumulation of Lipid-Derived Acylcarnitines
Authors: TAN HONG CHANG , HSU JEAN W. , KOVALIK JEAN-PAUL , KHOO CHIN MENG , CHACKO SHAJI , GANGULY SONALI , TAI E. SHYONG , LAI OI FAH , LIM EUGENE , JAHOOR FAROOK
Published: June 2019

Background: Accelerated lipolysis and the accumulation of incomplete products of fatty acid metabolism may contribute to insulin resistance (IR). It remains unclear whether the improvement in IR during the early period following bariatric surgery could be related to changes in lipid metabolism.

Aims and Hypothesis: In this study, we compared the rates of lipolysis, lipid-related acylcarnitines (ACs) and IR in morbidly obese individuals at baseline, 6-months after bariatric surgery and with lean controls.

Methods: 10 morbidly obese individuals scheduled for sleeve gastrectomy and 14 lean controls were recruited. IR was quantified using OGTT while body composition was measured using the 2H2O technique or DXA scans. Glycerol flux represents whole-body lipolysis and was measured using 2H5-glycerol infusion. Plasma lipid-derived ACs were measured using mass spectrometry as an indicator of complete fatty acid oxidation. Obese subjects were reassessed 6-months after bariatric surgery.

Results: Compared to lean controls, obese subjects had significantly higher fat mass (50.7 ± 1.9 vs. 16.8 ± 1.0 kg) and were significantly more IR. They had higher HOMA-IR (6.6 ± 1.0 vs. 1.43 ± 0.31), glucose and insulin AUC after OGTT. Glycerol flux (16098.1 ± 1136.2 vs. 7266.20± 497.66 umol/h), plasma glycerol (117.13 ± 10.96 vs, 46.79± 3.15 umol/L), C14 (0.225 ± 0.079 vs. 0.042± 0.012) and C16 (0.136 ± 0.016 vs. 096 ± 0.003 umol/L) ACs were significantly higher in the obese individuals. 8 subjects returned at 6-months and lost 19% of their total weight, 29% of fat mass and had a 68% decrease in HOMA-IR. Glycerol flux, plasma glycerol, C14 and C18 ACs however did not show any difference compared to baseline and remained significantly higher compared to the lean controls.

Conclusion: IR improved 6-months following sleeve gastrecotmy despite accelerated lipolysis and accumulation of byproducts of incomplete fatty acid metabolism.

Disclosure

H. Tan: None. J.W. Hsu: None. J. Kovalik: None. C. Khoo: None. S. Chacko: None. S. Ganguly: None. E. Tai: Advisory Panel; Self; Amgen Inc., BASF. O. Lai: None. E. Lim: None. F. Jahoor: None.
2077-P: Effect of Laparoscopic Sleeve Gastrectomy on Sex Hormone Level and Ovarian Volume in Female Obesity with PCOS
Authors: GAO JINGYANG , ZHANG MANNA , ZHU CUILING , WANG XINGCHUN , MEI FANGYUN , QU SHEN
Published: June 2019

Objective: This study was designed to examine changes in body fat distribution after laparoscopic sleeve gastrectomy (LSG) and to confirm whether these changes are correlated with increased testosterone.

Methods: A retrospective study was performed in 51 obese female patients with BMI 30-45 kg/m2 who underwent LSG from Shanghai Tenth People’s Hospital, 38 of whom were PCOS patients. The anthropometric indexes, sex hormone and ovarian volume of ultrasound examinations include both sides ovarian volume, mean ovarian volume (MOV) and maximal ovarian volume (MaxOV) were observed preoperative and follow-up at 3 and 6 months after LSG. The metabolic indexes and ovarian volume were compared before and after surgery.

Results: Compared with preoperative, PCOS patients' weight, BMI, waist circumference and hip circumference were significantly lower at 3 and 6 months after operation (P<0.05). Percentage of excess body weight loss(%EWL) for 3 months and 6 months after LSG was 56.32% and 75.98%. TT and cFT showed a downward trend at 3 and 6 months after surgery while SHBG showed an upward trend (P<0.05). Both sides ovary volume and MOV, MaxOV all decreased at 6 months after LSG, but there was no statistically significant difference compared to baseline. Compared with patients without PCOS, the preoperative MOV, MaxOV, and left ovary volume were significantly larger (P<0.05). At 6 months after surgery, the MOV, MaxOV, left ovary volume and right ovarian volume of PCOS and non-PCOS patients were all reduced. The differences in MOV, MaxOV, left ovary volume and right ovarian volume were no longer statistically significant between the two groups.

Conclusions: TT and cFT decreased and SHBG increased significantly after LSG. MOV shrinked at 6 months after LSG and was no longer different from non-PCOS patients. LSG is not only an important treatment for weight loss, but also improve PCOS and its clinical symptoms effectively in severe obese women.

Disclosure

J. Gao: None. M. Zhang: None. C. Zhu: None. X. Wang: None. F. Mei: None. S. Qu: None.

Funding

National Natural Science Foundation of China (81601269)
208-OR: Which Diabetes Strengths Matter Most? Associations with Key Outcomes
Authors: CAO VIENA T. , ESHTEHARDI SAHAR S. , THOMPSON DEBORAH , KARAVITI LEFKOTHEA , HILLIARD MARISA E.
Published: June 2019

Objectives: Many adolescents use adaptive type 1 diabetes (T1D)-related behaviors, or “strengths,” to overcome challenges and achieve resilient diabetes outcomes (i.e., good self-management, general and T1D-specific quality of life (QoL), and glycemic control). Overall, strengths are linked to these key outcomes, but identifying which individual positive behaviors are most associated may assist care teams in promoting the most important strengths in their patients.

Methods: Participants were n=82 adolescents (age 12-17, M age=15.3±1.5 years, 59% female, M HbA1C=8.9±2.0%, 55% pump) with T1D enrolled in a behavioral intervention. Baseline data included youth self-report on the Diabetes Strengths and Resilience (DSTAR) measure (12 items, 2 scales) and measures of self-management and QoL, and chart-confirmed HbA1c.

Results: Significant correlations between DSTAR items/scales and diabetes outcomes are summarized in the Table.

Conclusions: T1D strengths are related to all key outcomes. The items about overall T1D-related self-efficacy and prioritizing T1D management are related to multiple resilient outcomes and may be important intervention targets. Both DSTAR scales and 9 of their items correlated with self-management, suggesting strengths-based interventions may benefit treatment adherence. Youth with better outcomes may also feel more capable. Unexpected inverse associations for 3 items should be further studied.

Disclosure

V.T. Cao: None. S.S. Eshtehardi: None. D. Thompson: None. L. Karaviti: None. M.E. Hilliard: None.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (1R21DK107951)
2082-P: Pharmacological Induction of Brown and Beige Adipose Tissue
Authors: FINLIN BRIAN , MEMETIMIN HASIYET , CONFIDES AMY L. , ZHU BEIBEI , JOHNSON ZACHARY R. , DUPONT-VERSTEEGDEN ESTHER E. , KERN PHILIP A.
Published: June 2019

Brown adipose tissue (BAT) is associated with improved metabolic homeostasis in humans, and studies in rodents indicate that subcutaneous white adipose tissue (SC WAT) beiging, which involves increased uncoupling protein 1 (UCP1) expression, also is associated with improved metabolism. The goal of this study was to determine the ability of mirabegron (a β3AR agonist), pioglitazone (a thiazolidinedione), or a combination of the drugs to induce brown and beige adipose tissue and to determine the effects on glucose and lipid homeostasis. We randomized obese, insulin-resistant (IR) research participants to mirabegron (50 mg/day), pioglitazone (30 mg/day), or combination therapy treatment groups. Euglycemic clamping, oral glucose tolerance tests, adipose tissue biopsies, and PET-CT scans were performed at baseline and after 10 weeks of treatment. Mirabegron improved glucose homeostasis (reduced HbA1c and improved oral glucose tolerance) to a similar extent as pioglitazone without side effects or weight gain. Although mirabegron treatment increased insulin sensitivity, the effect size was much smaller than pioglitazone, yet mirabegron treatment significantly increased the insulinogenic and disposition indexes, suggesting that a major part of the mechanism of mirabegron action involved improving β-cell function. Mirabegron treatment consistently induced SC WAT beiging as evidenced by increased UCP1 expression (2.4 fold increase; P<0.0001), but did not induce BAT, suggesting that induction of beige adipose may be part of the mechanism responsible for improved glucose homeostasis. Pioglitazone also induced beiging (1.6 fold increase in UCP1; P<0.01), but not BAT. The combination of mirabegron plus pioglitazone was not additive, with similar effects on glucose tolerance as single drug. These results suggest that mirabegron treatment has significant beneficial metabolic effects in obese, IR subjects through a distinct mechanism involving SC WAT beiging and improved pancreatic beta cell function.

Disclosure

B. Finlin: None. H. Memetimin: None. A.L. Confides: None. B. Zhu: None. Z.R. Johnson: None. E.E. Dupont-Versteegden: None. P.A. Kern: None.

Funding

National Institutes of Health (R01DK112282)
2086-P: The Interaction of Fitness, Body Mass Index, and Mortality Risk in Type 2 Diabetes
Authors: NYLEN ERIC , KOKKINOS PETER , FASELIS CHARLES , NARAYAN PUNEET , KARASIK PAMELA , MYERS JONATHAN N.
Published: June 2019

Introduction: Obesity and poor cardiorespiratory fitness (CRF) commonly coexists in type 2 diabetes (T2DM). Moreover, CRF has been associated with lower mortality risk in T2DM patients. In this study, we assessed the modulatory impact of fitness on mortality risk in T2DM patients across the obesity spectrum.

Methods: We identified 6,888 T2DM patients (age 58.3+10.1 years) with no evidence of ischemia as indicated by a maximal standardized exercise treadmill test. We established two CRF categories based on age-adjusted median peak metabolic equivalents (METs) achieved: Unfit (5.4±1.3 METs; n=3,474) and Fit (9.0±2.3 METs; n=3414). We also defined the cohort based on body mass index (BMI) normal-weight (BMI 18.5-24.9 kg/m2); over-weight (BMI 25.0-29.9 kg/m2); and obese (BMI ≥30 kg/m2). We then combined the BMI and CRF categories to form a total of six groups CRF-BMI categories. Cox proportional hazards models, adjusted for known risk factors were constructed to assess the association between CRF-BMI interaction and mortality risk.

Results: During a follow-up of 14.2+6.9 years, 2,495 patients died, accounting for 25.5 events/1,000 person-years of observation. When CRF-BMI groups were considered, the normal-weight-Fit category was used as the referent. Mortality risk in the normal-weight but Unfit patients was 64% higher compared to their Fit counterparts (HR 1.64; 95% CI: 1.35-2.00). In the overweight category, mortality risk was 20% lower in the Fit (HR 0.80; 95% CI: 0.67-0.94) and 28% higher (HR 1.28; 95% CI: 1.09-1.51) in Unfit patients. In obese category the risk was 29% lower for the Fit (HR 0.71; 95% CI: 0.60-0.86) and 23% higher for the Unfit patients (HR 1.23; 95% CI: 1.05-1.45).

Conclusion: Increased CRF favorably modulates mortality risk across the obesity spectrum in T2DM patients. Poor fitness had a substantially greater and negative impact on Normal than over-weight and obese individuals.

Disclosure

E. Nylen: None. P. Kokkinos: None. C. Faselis: None. P. Narayan: Research Support; Self; AstraZeneca, Eli Lilly and Company. P. Karasik: None. J.N. Myers: None.
210-OR: A Combined Method Improves Risk Prediction for Childhood Type 1 Diabetes in the TEDDY Study
Authors: FERRAT LAURIC A. , VEHIK KENDRA , SHARP SETH A. , LERNMARK ÅKE , ZIEGLER ANETTE , REWERS MARIAN , SHE JIN-XIONG , TOPPARI JORMA , AKOLKAR BEENA , KRISCHER JEFFREY , WEEDON MICHAEL N. , RICH STEPHEN S. , ORAM RICHARD A. , HAGOPIAN WILLIAM ,   TEDDY STUDY GROUP
Published: June 2019

Background: There is an unmet need for accurate cost-effective estimation of future T1D risk.

Methods: We derived a combined T1D prediction model using 7883 children followed closely from birth for a median of 9 yr, considering T1D genetic risk score (GRS), family T1D history (FH), standard islet autoantibodies (IA), race, birth circumstances, early growth and nutrient status. T1D developed in 326.

Results: Machine learning and traditional methods (Cox models) performed equivalently as measured by time-dependent AUC ROC. Future T1D was accurately predicted by combining only 3 variables: GRS, FH and IA status. Accuracy of combined scores increased with age at scoring. By age 2, they were highly predictive for T1D in the next 5 years (AUC >0.91, 95% CI 0.88-0.95) (see X on Figure). A 2-yr-old with 2 IA, positive FH and high GRS (>12) would have T1D risk over the next 1, 3 and 5 years of 14% (8-19%), 36% (25-45%) and 51% (39%-61%) respectively. A 2-yr-old with 1 IA, no FH and moderate GRS (10-11) has T1D risk of 0.8% (0.6-1.2%), 2.6% (1.9-3.2%) and 4.3% (3.4%-5.2%) respectively. After newborn genetic screening, only simple venous sampling in routine healthcare settings is required.

Conclusion: This approach allows updated individual risk estimates by age, and in the future may enable release of low risk individuals from surveillance long after initial newborn screening for more cost-efficient population based pediatric T1D prediction.

Disclosure

L.A. Ferrat: None. K. Vehik: None. S.A. Sharp: None. Å. Lernmark: None. A. Ziegler: None. M. Rewers: None. J. She: None. J. Toppari: None. B. Akolkar: None. J. Krischer: None. M.N. Weedon: None. S.S. Rich: None. R.A. Oram: Other Relationship; Self; Randox Laboratories Ltd. W. Hagopian: Research Support; Self; Novo Nordisk A/S.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Allergy and Infectious Diseases; Eunice Kennedy Shriver National Institute of Child Health and Human Development; National Institute of Environmental Health Sciences; JDRF; Centers for Disease Control and Prevention
2106-P: Subcutaneous Abdominal Fat Has a Stronger Association with Prediabetes than Visceral Fat in Young Indians
Authors: WAGH RUCHA H. , BAWDEKAR RUPALI U. , ALENAINI WAREED , THOMAS ELIZABETH L. , BELL JIMMY DAVID , YAJNIK CHITTARANJAN S.
Published: June 2019

Background: India is the world’s capital of ‘thin-fat’ phenotype and young onset diabetes. Abdominal adiposity is a possible explanation. We studied the association of abdominal fat distribution by MRI with glucose-insulin metabolism.

Methods: Young adults [18 years, n=595 (308 boys), PMNS cohort] were classified by ADA 75 g OGTT as NGT and prediabetic. We measured anthropometry, total body fat (DXA), and glucose-insulin parameters (HOMA, Insulinogenic and Matsuda index), and visceral (VAT) and subcutaneous (SAT) abdominal adiposity by MRI.

Results: Prediabetic boys were shorter, had a higher BMI, WHR, VAT and SAT compared to NGT boys. Conversely, prediabetic girls had similar BMI, VAT and SAT. SAT was twice as strongly associated than VAT with glucose, insulin resistance, triglycerides and cholesterol concentrations. On multiple regression SAT remained strongly and independently associated.

Conclusion: Unlike in Europeans, SAT has a stronger ‘detrimental’ association with cardio-metabolic risk factors compared to VAT. This may indicate a different adipose biology in Indians and needs further investigation.

Disclosure

R.H. Wagh: None. R.U. Bawdekar: None. W. Alenaini: None. E.L. Thomas: None. J.D. Bell: None. C.S. Yajnik: None.
2110-P: Increased Proinflammatory Eosinophils in Adipose Tissue of Metabolic Syndrome
Authors: MOUSSA KARINE , GURUNG PURNIMA , DEVARAJ SRIDEVI , JIALAL ISHWARLAL
Published: June 2019

Metabolic Syndrome (MetS) is a common disorder that predisposes to type 2 diabetes mellitus (T2DM) and cardiovascular disease (ASCVD). Adipose tissue (AT) contributes significantly to increased inflammation and insulin resistance (IR) in MetS. Many studies illustrate the role of macrophages and lymphocytes in human SAT, but there is sparse data on other immune cells, especially eosinophils. Hence, in this report we investigated the abundance of eosinophils (EOS) in subcutaneous AT (SAT) of 20 patients with MetS without diabetes, ASCVD, smoking or any inflammatory condition, and matched controls. SAT EOS were quantified by immunochemistry using the Vital New Red stain (ABCAM). Both circulating and SAT EOS were significantly increased 2-fold in MetS (p<0.007) and correlated with each other (r=0.40, p=0.03). Circulating EOS correlated significantly (p<0.05) with triglycerides (TG) (r=0.56), HDL-cholesterol (r=-0.45), IL-6 (r=0.57) and chemerin (r=0.61). SAT EOS correlated significantly (p<0.05) with cardio-metabolic factors including glucose (r=0.42), TG (r=0.39), and adipose IR(r=0.64), biomarkers of inflammation, including leptin (r=0.41), IL-6 (r=0.44), adiponectin (r=-0.39), endotoxin (r=0.56), and chemerin, (r=0.57), and SAT markers of fibrosis (collagen (r=0.42) and Sirius red (r=0.44)).

In conclusion, we make the novel observation that eosinophils are increased in MetS and that SAT EOS are pro-inflammatory and promote fibrosis in SAT. Hence, they contribute to the dysregulation of SAT biology in MetS.

Disclosure

K. Moussa: None. P. Gurung: None. S. Devaraj: None. I. Jialal: None.
2120-P: Knockdown of ETV5 Enhances Nitric Oxide (NO) Production and Caspase-Independent Cell Death in Pancreatic Beta Cells
Authors: LIU FUPENG , PÄTH GÜNTER , LAUBNER KATHARINA , SEUFERT JOCHEN
Published: June 2019

ETV5 (E-twenty six variant gene 5) belongs to the E-twenty six transcription factor family that regulates various biological processes including proliferation, differentiation and apoptosis. Genome-wide association studies linked ETV5 with human obesity. ETV5 is expressed in insulin producing pancreatic beta cells and its deletion in Etv5-/- mice results in less weight gain during high fat diet and impaired glucose tolerance due to reduced insulin exocytosis and less β-cell mass. Against this background we investigated whether ETV5 is regulated by nutritional states in pancreatic beta cells and whether ETV5 plays a role in proliferation and survival during diabetogenic stresses. Insulin producing rat insulinoma-derived INS-1E cells and generated shEtv5-INS1E cells with Dox-inducible Etv5 knockdown served as models. The obtained results demonstrate that ETV5 production is not regulated by low and high glucose or serum deprivation. ETV5 is time and dose-dependently induced by streptozotocin (STZ) and palmitate (PA) to significant levels after 24 h while alloxan has no effect. STZ and PA induce Etv5 gene expression also in primary mouse islets. Knockdown of Etv5 did not change proliferation in expanding INS-1E β-cells but significantly further enhanced STZ- and PA-mediated loss of viability despite same activation of apoptotic effector caspases-3/7 as in control cells. Instead, Etv5 knockdown increased NO production under basal conditions and enhanced necrotic cell death in response to STZ and PA.

In conclusion, this study provided initial evidence that ETV5 plays a currently not completely characterized role in the survival of pancreatic insulin producing β-cell during diabetogenic stress.

Disclosure

F. Liu: None. G. Päth: None. K. Laubner: Research Support; Self; Boehringer Ingelheim International GmbH, GI Dynamics, Inc. Speaker's Bureau; Self; AstraZeneca, Berlin-Chemie AG, Boehringer Ingelheim International GmbH, Merck Sharp & Dohme Corp., Novartis AG. J. Seufert: None.

Funding

Brigitte Bull Foundation; China Scholarship Council (201509370027 to F.L.)
2123-P: Peroxiredoxin6 Is a Novel Promoter of Pancreatic Beta-Cell Survival
Authors: PACIFICI FRANCESCA , DELLA-MORTE DAVID , CAPUANI BARBARA , PASTORE DONATELLA , ARRIGA ROBERTO , COPPOLA ANDREA , DONADEL GIULIA , BELLIA ALFONSO , LAURO DAVIDE
Published: June 2019

Background: Diabetes is a multifactorial disease with low levels of inflammation and loss of functional pancreatic β-cell mass. Pancreatic β-cells have a reduced antioxidant capacity with an increased vulnerability to oxidative stress. Pro-inflammatory cytokines increasing reactive oxygen species production lead to cell death. Peroxiredoxin6 (Prdx6), an antioxidant enzyme, is highly expressed in beta cells compared to other antioxidants. We previously reported as Prdx6 knockout (Prdx6-/-) mice have a mild diabetes mellitus phenotype due to a reduction of pancreatic β-cell mass. Moreover, the higher levels of TNFα in Prdx6-/- mice may induce β-cell loss, contributing to the reduction in β-cell mass. However, the intracellular apoptotic pathway leading to cell death have not been investigated, yet.

Aims and Results: The objective of this study was to analyze the role of Prdx6 in the modulation of apoptosis in response to TNFα. To achieve this goal, Prdx6 was stably silenced (Prdx6KD) in murine pancreatic beta cell line (βTC6), and, the activation of intrinsic and/or extrinsic apoptotic pathway in response TNFα was analyzed in Prdx6KD and control (Scramble) cell lines. We demonstrated that TNFα administration (50 ng/ml for 24h), induced a two folds increase in apoptosis evaluated by FACS analysis, in Prdx6KD cells compared to scramble cells. Furthermore, by using ELISA assay, we measured the activity of caspase 2, 8, and 10 for the extrinsic pathway and caspase 9 for the intrinsic one. We found that after TNFα treatment, Prdx6KD cells exhibited significant higher levels (p<0.05) of caspase 8 and 9 compared to control cells. Moreover, also the activation of caspase 3 and PARP1 cleavage were significantly augmented in Prdx6KD cells compared to scramble cells, both treated with TNF-α as reported above.

Conclusion: These results suggest that Prdx6 can regulate pancreatic β-cells apoptosis.

In conclusion, Prdx6 can be considered a novel potential therapeutic target for diabetes, preserving pancreatic β-cells survival.

Disclosure

F. Pacifici: None. D. Della-Morte: None. B. Capuani: None. D. Pastore: None. R. Arriga: None. A. Coppola: None. G. Donadel: None. A. Bellia: None. D. Lauro: None.
2135-P: Genetic Ablation of CB1R Protects against Streptozotocin-Induced ß-Cell Destruction in Mice
Authors: ASEER KANIKKAI RAJA , EGAN JOSEPHINE M.
Published: June 2019

Approximately 1.25 million Americans have type 1 diabetes mellitus (T1DM), resulting from β-cell destruction. There is no known way to prevent it or to stop destruction of any remaining β-cells. Although the cannabinoid system, specifically cannabinoid 1 receptor (CB1R), is implicated in obesity and T2DM, its role, if any, in T1DM has not been studied. To address this, we induced β-cell destruction in wild type mice (WT: n=8) and mice in which CB1R was genetically nullified in β-cells only (β-CB1R-/-: n=8) in adulthood by five injections of streptozotocin (50 mg/kg intraperitoneally [IP]), a toxin selective for β-cells. We found that β-CB1R-/- mice had random blood glucose (BG) levels in the range of 137-289 mg/dL throughout the study period (28 days) as well as preserved insulin secretion with superior glucose tolerance after IP glucose (2g/kg) at day 8, 15 or 27, relative to their (random BG 500-600 mg/dL) WT littermates. CB1R knockout drove significant increases in pancreatic islet autophagic activity based on increased accumulation of autophagosome hallmarks (LC3B, Beclin-1, ATG5 and ATG7) and reduced p62 expression. Insulin and LC3B were colocalized in CB1R-deficient β-cells. Remarkably, the islets did not show the upregulation of ER unfolded protein response, which was present in β-cells of WT mice, while exerting the induction of protective chaperones. These anti-stress functions were accompanied by increased phosphorylation of AKT (ser) 473 and ERK in β-cells, which reveals a previously unrecognized route for PI3K-AKT pathway to activate the anti-apoptotic NFkB/p65 signaling that coordinately is permissive to β-cell survival. We conclude that CB1R nullification restores autophagy machinery as a novel signaling pathway to counteract ER stress-mediated cell death and confers resistance to β-cell destruction. We propose that CB1R is a potential therapeutic target during the honeymoon phase of T1DM.

Disclosure

K. Aseer: None. J.M. Egan: None.
2137-P: Cholecystokinin Protects Mouse Pancreatic Beta Cells against Cytokine Insult through the Cholecystokinin A Receptor
Authors: KIM HUNG TAE , SACOTTE STEVE , WILLIAMS RASHAUN A. , DE SOUZA ARNALDO H. , HAN JEE YOUNG , BARTOSIAK JACOB T. , YANG GRACE H. , DAVIS DAWN B.
Published: June 2019

Cholecystokinin (CCK) is an incretin-like hormone that is also produced by pancreatic β-cells under conditions of stress and obesity. In the past, we have established a role for CCK in protection from β-cell apoptosis. However, the specific CCK receptor responsible for this protective effect was unknown. Both Cckar and Cckbr mRNA are expressed in mouse islet, although Cckbr expression is very low. Incubation of WT mouse pancreatic islets with CCK-8 (100nM) increases the transcript levels of Cckar but does not impact Cckbr expression. Conversely, 24-hour treatment with proinflammatory cytokine cocktail decreases CCKAR transcript levels but again does not modulate Cckbr expression. Therefore, we hypothesized that the CCKA receptor was the most likely mediator of CCK signaling in the β-cell. We used isolated islets from mice with germline knockout of CCK receptors to determine which receptor was required for the pro-survival effects of CCK on the β-cell. Using imaging flow cytometry of dissociated islet cells stained for annexin V and propidium iodide (PI) we find that islet cells from CCKBR knockout mice have a 19.3% (p<0.005) reduction in cytokine-mediated cell death when treated with CCK-8 (100 nM), similar to the protection seen in wild type islets. This protective effect is lost in the CCKAR and double knockout mouse islets. We also confirm this finding using insulin and TUNEL immunohistochemistry to specifically identify β-cell apoptosis. In CCKBR knockout mouse islets, CCK-8 (100 nM) reduces the % TUNEL positive β-cells from 12.98% to 4.39% (p<0.05) after cytokine exposure, similar to protection seen in WT mouse islets (10.47% to 4.48%). Again, CCK-8 does not protect β-cells from cytokine-induced apoptosis in CCKAR knockout or double CCKR null mouse islets. Taken together, CCKAR, but not CCKBR, is required for CCK-mediated protection of pancreatic β-cells from cytokine-induced stress.

Disclosure

H. Kim: None. S. Sacotte: None. R.A. Williams: None. A.H. de Souza: None. J. Han: None. J.T. Bartosiak: None. G.H. Yang: None. D.B. Davis: None.

Funding

National Institutes of Health
2143-P: GLP-1 Conjugation Enhances the Activity of Antisense Oligonucleotides Targeting Islet Amyloid Polypeptide (IAPP) or Kelch-Like ECH Associated Protein 1 (Keap1) in Isolated Pancreatic Islets
Authors: FU WUXIA , LEE RICHARD , PIRIE ELAINE C. , PRAKASH THAZHA , MURRAY SUE , POWERS ANDREW F. , CROOKE ROSANNE M.
Published: June 2019

The incidence of type 2 diabetes (T2D) is increasing rapidly and is currently one of the 21st century’s major challenges to human health. While T2D has a complex etiology, beta cell dysfunction is widely regarded as a key contributor to T2D disease progression. Antisense oligonucleotides (ASOs) have been shown to provide substantial therapeutic benefit for numerous unmet medical needs. However, unconjugated ASOs have shown minimal activity in pancreatic beta cells, limiting their use as therapeutics in these cells. It has recently been shown that conjugation of glucagon like peptide 1 receptor (GLP-1R) ligands (GLP-1R-L) to ASOs led to significant, substantial increases of ASO activity in the pancreatic islet beta cell, making them a viable therapeutic option for treatment of beta cell dysfunction.1 Two factors that contribute to beta cell dysfunction are IAPP and oxidative stress. A key inhibitor of the intracellular anti-oxidative response is Keap1. We treated isolated islets with parent or GLP-1R-L conjugated ASOs targeting either Keap1 or IAPP. At the highest dose, the parent ASOs reduced their respective islet target mRNAs by up to 20%, while the GLP-1 conjugated ASOs reduced target mRNA by 85%-95%. Furthermore, the GLP-1R-L conjugated Keap1 ASO treatment increased mRNA expression of the anti-oxidative response gene NAD(P)H quinone dehydrogenase 1 (Nqo1) by 390% when compared to PBS controls. Systemic treatment of C57BL/6 mice with the GLP-1R-L conjugated IAPP ASO reduced islet IAPP mRNA by 79% ± 3.7% when compared to saline treated animals. These data suggest that GLP-1R-L conjugated ASOs show exciting potential as a therapeutic platform for the treatment of beta cell dysfunction.

Disclosure

W. Fu: None. R. Lee: Employee; Self; Ionis Pharmaceuticals, Inc. E.C. Pirie: Employee; Self; Ionis Pharmaceuticals, Inc. T. Prakash: Employee; Self; Ionis Pharmaceuticals, Inc. S. Murray: Employee; Self; Ionis Pharmaceuticals, Inc. A.F. Powers: Employee; Self; Ionis Pharmaceuticals, Inc. Stock/Shareholder; Self; Ionis Pharmaceuticals, Inc. R.M. Crooke: Employee; Self; Ionis Pharmaceuticals, Inc. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc.
215-OR: Safety and Performance of the Omnipod Hybrid Closed-Loop System in Young Children Aged 2-6 Years with Type 1 Diabetes
Authors: BUCKINGHAM BRUCE A. , FORLENZA GREGORY P. , SHERR JENNIFER , GALDERISI ALFONSO , EKHLASPOUR LAYA , LEE JOON BOK , OCONNOR JASON B. , DUMAIS BONNIE , HUYETT LAUREN M. , LAYNE JENNIFER E. , LY TRANG T.
Published: June 2019

T1D therapies must be proven safe for young children, who face unique diabetes management challenges. The safety and performance of the Omnipod® hybrid closed-loop (HCL) personalized model predictive control algorithm were assessed in those with T1D aged 2-5.9 y with A1C <10% using an investigational device in a supervised hotel setting. A 7-day at-home standard therapy (ST) phase with CGM use preceded the 48-72 h HCL phase. Meals during HCL were unrestricted, with boluses administered per usual routine. Exercise was performed for ≥30 min/d. The 14 participants were age 4.2 ± 0.9 y, T1D duration 2.4 ± 1.0 y, A1C 7.4 ± 1.0%, and TDD 0.77 ± 0.13 U/kg (mean ± SD). Mean glucose was lower during HCL vs. ST, 148 ± 16 mg/dL vs. 172 ± 27 mg/dL overall and 135 ± 17 mg/dL vs. 167 ± 28 mg/dL overnight. The percent time 70-180 mg/dL was 32% higher during HCL vs. ST overall, 72.6 ± 8.4% vs. 55.2 ± 13.1%, respectively, and 47% higher overnight, 85.3 ± 9.4% vs. 58.2 ± 16.4%. Percent time <70 mg/dL was reduced nearly 2-fold overall and overnight. Glycemic outcomes are reported in the Table. There was 1 severe hypoglycemic event after exercise which was treated with oral carbohydrates. The Omnipod HCL system was safe and performed well in these very young children in a supervised setting, similar to the performance previously observed in older pediatric, adolescent and adult participants.

Disclosure

B.A. Buckingham: Advisory Panel; Self; ConvaTec Inc., Novo Nordisk Inc., Profusa, Inc. Consultant; Self; Medtronic MiniMed, Inc. Research Support; Self; Beta Bionics, ConvaTec Inc., Dexcom, Inc., Insulet Corporation, Medtronic MiniMed, Inc., Tandem Diabetes Care. Other Relationship; Self; Insulet Corporation, Tandem Diabetes Care. G.P. Forlenza: Advisory Panel; Self; Dexcom, Inc. Consultant; Self; Medtronic MiniMed, Inc., Tandem Diabetes Care. Research Support; Self; Dexcom, Inc., Insulet Corporation, Medtronic MiniMed, Inc., Tandem Diabetes Care. J. Sherr: Advisory Panel; Self; Bigfoot Biomedical, Eli Lilly and Company, Insulet Corporation. Consultant; Self; Lexicon Pharmaceuticals, Inc., Medtronic, Sanofi. Research Support; Self; Dexcom, Inc., Insulet Corporation. A. Galderisi: None. L. Ekhlaspour: None. J. Lee: Employee; Self; Insulet Corporation. J.B. OConnor: Employee; Self; Insulet Corporation. B. Dumais: None. L.M. Huyett: Employee; Self; Insulet Corporation. Stock/Shareholder; Self; Insulet Corporation. J.E. Layne: Employee; Self; Insulet Corporation. T.T. Ly: Employee; Self; Insulet Corporation.

Funding

Insulet Corporation
2152-P: Impaired Iron-Sulfur-Cluster Redox Regulation in Monogenic Ncb5or Diabetes
Authors: ZHU HAO , MUTO AZUSA , HOME TRISHA , WANG WENFANG , URANO FUMIHIKO
Published: June 2019

NADH cytochrome b5 oxidoreductase (Ncb5or) is a ferric reductase widely expressed in animal cells. Ncb5or deficiency results in altered iron metabolism, mitochondrial dysfunction and proliferation, and increased oxidative and endoplasmic reticulum (ER) stress. Ncb5or null (KO) mice present early-onset lean diabetes, ataxia and hearing loss. Similar clinical manifestations are observed in patients with type 2 Wolfram Syndrome that is caused by mutations in CISD2 (CDGSH iron-sulfur domains 2). Its [2Fe-2S] iron-sulfur-cluster (ISC) is redox sensitive and becomes labile when oxidized. Because both proteins are associated with ER, Ncb5or may function as a CISD2 reductase for ISC redox regulation. This hypothesis appears to be supported by our recent data. When fed a chow admixture with 400 ppm pioglitazone, a FDA-approved drug capable of stabilizing FeII-ISC in vitro, Ncb5or KO mice exhibit a 4-week delay in developing overt diabetes, specifically, at age 11 weeks vs. 7 weeks under chow. Pioglitazone results in a significant increase in the content of FeII-ISC CISD2 among the total in the liver of prediabetic KO mice when compared to wild type (WT) counterparts, namely, a KO/WT ratio of 0.6 post pioglitazone vs. 0.3 under chow (by native gel electrophoresis and immuno-blot analyses with antibodies that preferentially recognize CISD2 containing FeII-ISC). Pioglitazone also reduces oxidative and ER stress response in the liver of KO mice (qRT-PCR of MT’s, ATF3, and XBP1-spliced transcripts) and attenuates mitochondrial dysfunction in KO mouse embryonic fibroblasts (extracellular flux analysis). However, iron dyshomeostasis, as indicated by transcript profiles of iron genes in KO cells and splenomegaly in KO mice, is not rescued by pioglitazone. Our findings suggest that Ncb5or maintains the redox status of ISC in CISD2 and that, through stabilizing ferrous ISC, pioglitazone can partially rescue defects in Ncb5or-null cells. Studies are underway to characterize the Ncb5or-CISD2 axis in beta-cells.

Disclosure

H. Zhu: None. A. Muto: None. T. Home: None. W. Wang: None. F. Urano: Board Member; Self; Healthbeat. Research Support; Self; Aetas, Amylyx, JDRF, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Stock/Shareholder; Self; CytRx. Other Relationship; Self; Novus Biologicals.

Funding

University of Kansas Medical Center Research Institute
2159-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 , ETHIER MÉLANIE , FERGUSSON GRACE , GHISLAIN JULIEN , POITOUT VINCENT
Published: June 2019

Background: Puberty is a time of hormonal changes that are 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 type 2 diabetes. In pregnancy and obesity-induced IR, β-cells increase their functional mass to maintain glucose homeostasis. During puberty, however, the mechanism of pancreatic β-cell compensation to IR and its role in glucose metabolism later in life have not been established.

Objective: To characterize pancreatic β-cell adaptation to pubertal IR in rats and study the effect of metabolic stress during puberty on glucose homeostasis in adult animals.

Methods: Male and female Wistar rats were fed a chow diet or a high fat diet (HFD) during puberty. Body weight, fasted plasma insulin, glucose tolerance and hormone levels (estradiol, testosterone, insulin growth factor-1 (IGF1), growth hormone (GH)) were determined every 5 days from weaning to adulthood. β-cell proliferation was assessed by immunostaining of pancreatic cryosections for Ki67 and insulin to mark β-cells and β-cell mass by morphometric analysis of insulin staining.

Results: During puberty, glucose intolerance was associated with an increase in insulin levels in both sexes, suggestive of IR. Correspondingly, β-cell proliferation increased, as did islet size and β-cell mass. β-cell expansion correlated with a rise in IGF1/GH levels. HFD during puberty impaired glucose tolerance in adults.

Conclusion: During puberty in rats, β-cells compensate for increased IR. Metabolic stress during puberty impairs glucose homeostasis later in life. Future studies will address whether β-cell expansion during puberty is under control of the IGF1/GH axis.

Disclosure

A. Castell: None. M. Ethier: None. G. Fergusson: None. J. Ghislain: None. V. Poitout: None.

Funding

National Institutes of Health; Societé Francaise d'Endocrinologie Diabetologie Pediatrique
2165-P: In Vivo Islet Imaging Reveals that GLP-1 Paradoxically Increases Insulin Content
Authors: CRAS-MÉNEUR CORENTIN M.H. , FRIKKE-SCHMIDT HENRIETTE , ARVAN PETER , SEELEY RANDY J.
Published: June 2019

Glucagon-like peptide-1 (GLP-1) is an incretin, secreted in response to a meal that promotes glucose-stimulated insulin secretion in animal models and diabetic and nondiabetic humans. GLP-1 analogues have been successfully used as an effective treatment for both obesity and type 2 diabetes. While the general mechanisms of actions of GLP-1 on β-cell growth and function have been extensively studied, a number of questions regarding the dynamic and heterogeneity of the response of individual islets to GLP-1 in vivo remain. Using a fluorescent reporter mouse model originally created by the Arvan Lab, we have optimized a platform allowing us to monitor the content of individual islets in live animals. In this model, a super-folded GFP is bound to the C-Peptide, allowing the fluorescent visualization and quantification of insulin content within the islets without altering their normal physiology. After an overnight fast, the pancreas is exposed by laparotomy in anesthetized mice and the reporter in individual islets can be imaged repeatedly to quantify their insulin content. Upon glucose challenge, the GFP intensity within the islets progressively decreases in control mice, correlating with the insulin secretion within the bloodstream. In order to study the effects of GLP-1 on individual islet secretion, mice were either treated either with Liraglutide, a stable GLP-1 analog, or subjected to Vertical Sleeve Gastrectomy (VSG), a surgical procedure known to increase GLP-1 levels in obese mice. Surprisingly, with both approaches, in addition to increasing insulin secretion upon a glucose challenge, we could also observe insulin content actually increasing within the islets over the course of the excursion. This finding was later confirmed by measuring total insulin content in liraglutide-treated mice. This data indicates that despite causing enhanced insulin secretion, GLP-1 can also very rapidly increase insulin content within islets.

Disclosure

C.M.H. Cras-Méneur: None. H. Frikke-Schmidt: None. P. Arvan: None. R.J. Seeley: Consultant; Self; Ironwood Pharmaceuticals, Inc., Novo Nordisk A/S, Scohia Pharma Inc. Research Support; Self; MedImmune, Novo Nordisk A/S, Zafgen, Inc. Stock/Shareholder; Self; Zafgen, Inc.
2172-P: Effects of Glucocorticoid on Gene Expression and Secretion of Insulin in Pancreatic ß Cells
Authors: MURASHIMA TOSHITAKA , KONDO TAKUMA , KONDO KEN , MORITA NARU , KITAHARA ATSUKO , SUMITANI YOSHIKAZU , TAKAHASHI KAZUTO , TANAKA TOSHIAKI , HOSAKA TOSHIO , ISHIDA HITOSHI
Published: June 2019

Glucocorticoids are widely used in clinical practice, while the chronic administration at a high dose often leads to the progression of steroid-induced diabetes. This hyperglycemic state results mainly from the suppression of insulin secretion due to pancreatic β-cell dysfunction and the increment of systemic insulin resistance. However, the mechanisms which underlie glucocorticoid action on expression and secretion of insulin in pancreatic β-cells have not been clarified. The aim of this study is to elucidate the effects of glucocorticoid on insulin synthesis and secretion in pancreatic β-cells. Insulin gene expression was significantly increased in 30nM of dexamethasone and decreased in 300 and 3000 nM of dexamethasone. These changes in the expression were canceled by RU486, a GR antagonist, at any concentrations of dexamethasone. Meanwhile, insulin and proinsulin secretion were significantly reduced at any concentrations of dexamethasone. Interestingly, the proinsulin/insulin ratio was significantly decreased at 30 nM, and increased at 300 and 3000 nM. The changes were completely restored by RU486 at 30 nM of dexamethasone, while the reduction of insulin and proinsulin secretion by 300 or 3000 nM of dexamethasone was not released by RU486. In addition, similar results were obtained in experiments using knockdown of GR expression. Interestingly, the short-term induction of insulin secretion by membrane depolarization treated with 30 mM of KCl was rather enhanced by 3000 nM of dexamethasone, and this enhancement was not canceled by RU486.

In conclusion, glucocorticoid regulates insulin gene expressions via the genomic pathway containing GR in pancreatic β-cells, while particularly high-dose glucocorticoid regulates insulin secretion principally via non-genomic pathway. Since the proinsulin/insulin ratio increased under chronic administration of high-dose glucocorticoid, it was assumed that the non-genomic pathway was involved in insulin processing.

Disclosure

T. Murashima: None. T. Kondo: None. K. Kondo: None. N. Morita: None. A. Kitahara: None. Y. Sumitani: None. K. Takahashi: None. T. Tanaka: None. T. Hosaka: Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Ono Pharmaceutical Co., Ltd. H. Ishida: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim International GmbH, Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Kowa pharmaceutical Co. Ltd., MSD, Ono Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho.

Funding

Japan Ministry of Education, Culture, Sports, Science and Technology