bluuebunny/crossref_metadata_2025_split huggingface dataset:
1449-P: Reducing Ghrelin Levels as a Novel Method to Increase Pancreatic Beta-Cell Mass
Authors: GUPTA DEEPALI , BURSTEIN AVI W. , SHANKAR KRIPA , VARSHNEY SALIL , SINGH OMPRAKASH , PAUL SUBHOJIT , OGDEN SEAN B. , OSBORNE-LAWRENCE SHERRI , METZGER NATHAN , RICHARD CORINE P. , ZIGMAN JEFFREY M. 
Published: May 2022

Objective: Exogenous insulin remains the leading therapy for type 1 diabetes mellitus, T1D. However, current insulin formulations and delivery methods do not match the precision of functioning beta-cells, and even intensive monitoring of blood glucose and use of insulin pumps are not enough to halt frequent hypoglycemia, hyperglycemia and morbidity. Thus, regenerating beta-cells has become a major target of diabetes research to treat T1D. While the hormone ghrelin is well known to increase blood glucose, at least in part by inhibiting insulin secretion, ghrelin’s action on beta-cell mass and proliferation has heretofore not been explored in detail. Here, we sought to test the hypothesis that reducing ghrelin levels increases beta-cell mass.
Methods: Using histologic methods, we compared islet anatomy in ghrelin-knockout (ghrelin-KO) mice vs. wildtype littermates at three different ages: newborns (P0-P2) , juveniles (4-week-old) , and adults (10-12-week-old) . We also compared islet anatomy in adult wildtype mice, 4 weeks following conditional ghrelin-cell ablation vs. wildtype mice with intact ghrelin-cells. n= 4-8 mice per group and 33-433 islets per mouse.
Results: Ghrelin gene deletion or ghrelin-cell ablation increased mean islet area and mean islet insulin-immunoreactive (insulin-IR) area by 52-82 % and by 45-51 %, respectively, in juveniles and adults. Increased islet size was driven by an increased % of large islets. Increased insulin-IR area was associated with increased beta-cell number per islet. An increased % of large islets was also observed in newborn ghrelin-KO mice.
Conclusions: In mice, ghrelin plays an important role in controlling beta-cell mass. Reducing ghrelin levels for even short periods can increase islet size by increasing beta-cell numbers.

Disclosure
D.Gupta: None. C.P.Richard: None. J.M.Zigman: Research Support; Novo Nordisk Research Center Indianapolis, Novo Nordisk Research Center Indianapolis, Stock/Shareholder; Medtronic, Medtronic. A.W.Burstein: None. K.Shankar: None. S.Varshney: None. O.Singh: None. S.Paul: None. S.B.Ogden: None. S.Osborne-lawrence: None. N.Metzger: None.
1452-P: PDX1 and CKas Markers of Cell Maturation in the Human Pancreas
Authors: PROSHCHINA ALEXANDRA , KRIVOVA YULIYA , OTLYGA DMITRY , SAVELIEV SERGEY 
Published: May 2022

Regeneration of beta-cell mass is one of the fundamental problems in diabetology. Embryonic source of all pancreatic cell types is the epithelial cells of the primitive ducts. During pancreas development, epithelial progenitors differentiate in three directions, which is accompanied by changes in cell phenotype, including the expression of various types of cytokeratins, transcription factors, as well as hormones and some other antigens. The early markers of pancreatic epithelial progenitors are the homeodomain transcription factor Pdx 1 (pancreatic and duodenal homeobox 1) and an intermediate filament protein cytokeratin (CK19) . The aim of our work was to analyze the distribution of Pdx1 and CKduring endocrine pancreas development in humans. The study was performed on the pancreatic samples from 27 fetuses (gestational age 14-40 weeks) using double and triple immunohistochemistry with antibodies to Pdx1, CK19, and insulin or glucagon. During human prenatal development, the co-expression of CKand Pdx1 was detected in several cell types. Firstly, it was observed in the epithelial cells of the pancreatic ducts. During the branching of pancreatic ducts, CKexpression decreased in outgrows, while the Pdx1 expression was lowered in large ducts. Secondly, co-expression of CKand Pdx1 was detected in endocrine cells (mainly in alpha) . However, the intensity of the reaction to both markers in these cells was low. Phenotype of beta cells was Pdx1+/ CK19−. At the same time, during development, some cells with an intense reaction to CKand Pdx1 were detected in the forming islets. Apparently, an intense reaction to CK19/Pdx1 may characterized low-differentiated (progenitor) cells that are present in the epithelium of ducts and in the forming islets during the development of the human pancreas.

Disclosure
A.Proshchina: n/a. Y.Krivova: None. D.Otlyga: None. S.Saveliev: None.
1454-P: A Transcriptomic Signature of Pancreatic Islet Leader Beta Cells
Authors: CHABOSSEAU PAULINE L. , YONG SU WERN FIONA , LOPEZ LIVIA , LECLERC ISABELLE , ALI YUSUF , MARTINEZ-SANCHEZ AIDA , RUTTER GUY A. 
Published: May 2022

Pulsatile increases in Ca2+ within beta cells underlie the stimulation of insulin secretion at elevated glucose concentrations. Recent data obtained by imaging these transients in intact islets have revealed the existence of subpopulations of beta cells (“leaders”) which initiate Ca2+ waves across the syncytium and are critical for these dynamics. Whether leader cells possess unique transcriptional features is unknown. By combining Ca2+ imaging with cell labelling and isolation, we sought to address this question.
Method: Islets were isolated from C57BL6/J mice expressing the Ca2+ probe GCaMP6f selectively in beta cells. After infection with an adenovirus expressing photo-activable- (PA) mCherry, Ca2+ imaging was performed at 11mM glucose at 5 Hz. Leader (or follower) cells were identified and photopainted by PA-mCherry activation at 405nm. After islet dispersal, cell sorting was used to isolate single cells. cDNA libraries were obtained using NEBNext Single cell kit and used for sequencing (Illumina HiSeq 4000) . Reads were aligned to the mmtranscriptome with Salmon v1.3 and differential expression analysis performed with DESeq2. Differentially expressed genes were subjected to Gene Ontology analysis.
Results: cDNA was obtained from 13 leader cells, identified as those exhibiting the first Ca2+ signal increase during Ca2+ waves, and 9 follower cells. Of 96 differentially expressed genes, the most strongly affected included cell adhesion proteins (Cdh22; Scarf2) and the calcium sensor Mctp1. Other genes identified were involved in mitochondrial Ca2+ uptake (Mcub) , cytochrome assembly (Tmem177) and type 2 diabetes risk (Tcf7l2) . GO analysis identified 9 enriched functional clusters, including protein ubiquitination and metal ion binding.
Conclusion: Leader cells possess a stably altered transcriptome, and the affected genes include several which may regulate cell adhesion. The extent to which the identified genes reflect the contribution of these cells to Ca2+ wave initiation requires further study.

Disclosure
P.L.Chabosseau: n/a. S.Yong: None. L.Lopez: None. I.Leclerc: None. Y.Ali: n/a. A.Martinez-sanchez: None. G.A.Rutter: Advisory Panel; Sun Pharmaceutical Industries Ltd., Research Support; Sun Pharmaceutical Industries Ltd.
1456-P: Molecular Architecture of AR Action in Insulin Secretion in Male Pancreatic ß-Cells
Authors: MOTA DE SA PAULA , QADIR MIRZA MUHAMMAD FAHD , BLANDINO-ROSANO MANUEL , FAN JIA , XU WEIWEI , BERNAL-MIZRACHI ERNESTO , MAUVAIS-JARVIS FRANCK 
Published: May 2022

Male mice lacking the androgen receptor (AR) in islet β cells exhibit blunted glucose-stimulated insulin secretion (GSIS) , leading to hypoinsulinemia and hyperglycemia. We showed that dihydrotestosterone (DHT) activation of an extranuclear AR in β cells potentiates GSIS by amplifying the insulinotropic action of GLP-1 in mouse and human islets. To gain information on DHT-stimulated signaling networks promoting GSIS, we combined functional proteomics with highly selective pharmacological inhibitors in male human islets, and islets from genetic mouse models. We found that DHT-stimulated GSIS in human islets involved a signaling cascade including focal adhesion kinases, the nonreceptor tyrosine kinase SRC, phosphoinositide 3-kinase, and the activation of mTOR complex 2. To define the early network of AR interacting proteins, we performed DHT-stimulated AR immunoprecipitation in INS1 832/3 cells followed by mass spectrometry-based proteomics. We will discuss the AR interactome including: 1) adenylate cyclase-modulating G proteins Gαs and Gαi, cAMP-dependent PKA, and the cAMP-dependent protein LASP1 regulating actin-based cytoskeletal activities; 2) Actin cytoskeleton remodeling proteins promoting actin depolymerization, and 3) Vesicular maturation including the lysosomal vacuolar-type ATPase acidifying insulin secretory granules and the prohormone convertase 2 involved in proinsulin processing. Notably, GLP-1 addition to DHT decreased AR interaction with all these proteins and promoted AR interaction with microtubule motor proteins promoting vesicular transport, proteins involved in exocytic vesicle docking to the plasma membrane, as well as small GTPase activating proteins and guanine nucleotide exchange factors involved in actin remodeling, as well as vesicle trafficking. Together these experiments provide novel insights into the architecture of AR action in enhancing GSIS.

Disclosure
P.Mota de sa: None. M.Qadir: None. M.Blandino-rosano: None. J.Fan: None. W.Xu: None. E.Bernal-mizrachi: None. F.Mauvais-jarvis: Consultant; Mithra Pharmaceutical.

Funding
National Institutes of Health (DK107444 and DK074970) U.S. Department of Veterans Affairs Merit Award (BX003725)
146-OR: Loss of Hydroxycarboxylic Acid Receptor 2 (HCA2) Affects Adipose Tissue Homeostasis in a Sex-Specific Manner during Prolonged Fasting
Authors: MCCAFFERTY KAYLEEN J. , BRINKER EMILY , GRAFF EMILY , STEURY TODD D. , GREENE MICHAEL W. , JUDD ROBERT L. 
Published: May 2022

Hydroxycarboxylic acid receptor 2 (HCA2) , formally known as GPR109A, is a metabolite-sensing receptor expressed predominately on adipocytes, immune cells and intestinal epithelial cells. The primary endogenous ligand of HCA2 is β-hydroxybutyrate (βOHB) , which is elevated during periods of nutrient deprivation including fasting. Previous in vitro studies have suggested that βOHB activation of HCA2 on adipocytes inhibits lipolysis and serves as a negative feedback mechanism to prevent excess fat loss during fasting. However, the metabolic implications of this feedback mechanism have not been tested in vivo during a prolonged fast. To address this question, male and female C57BL/6J (WT) and HCA2 knockout (KO) mice were fasted in metabolic cages for 36 hours. No significant differences were observed for genotype or sex in RER, EE, or VO2. Following the fast, KO males exhibited a significant decrease in body weight compared to their WT counterparts. However, body weight was not significantly different between female WT and KO mice after fasting. Gonadal white adipose tissue (gWAT) depot weights were significantly lower in KO male mice compared to WT male mice, with no significant difference in gWAT weight between female WT and KO mice. Inguinal white adipose tissue (iWAT) depot weights were significantly lower for both sexes in KO mice compared to WT mice. KO males displayed significantly lower levels of circulating NEFAs compared to WT males. However, no differences in NEFAs were observed between female WT and KO mice. No significant differences were shown between groups for glucose, insulin, or βOHB. Therefore, this study demonstrates that mice lacking HCA2 possess a diminished capacity to regulate body and adipose tissue weight during a fast, which is observed to a greater extent in male mice.

Disclosure
K.J.Mccafferty: None. E.Brinker: None. E.Graff: None. T.D.Steury: None. M.W.Greene: None. R.L.Judd: None.
148-OR: Partial Knockout of Skeletal Muscle Dynamin-Related Protein 1 Improves Insulin Sensitivity in Diet-Induced Insulin Resistant Mice, but Not in Normal Control Mice
Authors: KUGLER BENJAMIN A. , LIN NANA , NGUYEN PAUL D. , ALI ABIR , SESAY AMIRA , KALEMBA BABY LENGA , SESAKI HIROMI , ZOU KAI 
Published: May 2022

Dynamin-related protein 1 (Drp1) is the key regulator of mitochondrial fission. We and others have reported excessive Drp1 activity in skeletal muscle from both animals and humans with insulin resistance, which is strongly correlated to impaired skeletal muscle insulin sensitivity. To examine whether Drp1 directly regulates skeletal muscle insulin sensitivity and whole-body glucose homeostasis, we generated tamoxifen-inducible skeletal muscle-specific heterozygous Drp1 partial knockout mice (mDrp1+/-) . Male mDrp1+/- and wildtype (WT) mice were fed with either a high-fat diet (HFD) or low-fat diet (LFD) for four weeks, received tamoxifen injections for five consecutive days, and remained on their respective diet for another four weeks. Improvements in whole-body glucose tolerance and insulin sensitivity were observed in HFD-fed mDrp1+/- mice (P&lt;0.05) , but not in LFD-fed mDrp1+/- mice, when compared to their respective WT controls. Further, HFD-induced impairment in skeletal muscle insulin-stimulated Akt Ser473 phosphorylation (P&lt;0.05) was mitigated in HFD-fed mDrp1+/- mice. In contrast, insulin-stimulated Akt Ser473 phosphorylation was reduced in LFD-fed mDrp1+/- mice compared to LFD-fed WT mice (P&lt;0.05) . mDrp1+/- mice exhibited more fused and interconnected mitochondrial networks in skeletal muscle than WT mice. The examination of mitochondrial function revealed no change in mitochondrial respiration but reductions in mitochondrial Complex I and II and pyruvate dehydrogenase derived H2O2 production in HFD-fed mDrp1+/- mice compared to HFD-fed WT mice (P&lt;0.05) .
In conclusion, our data suggest that partial knockout of skeletal muscle Drp1 improves skeletal muscle insulin sensitivity and whole-body glucose homeostasis in diet-induced insulin-resistant mice but not in normal control mice. These improvements are, at least in part, due to reduced mitochondrial-derived H2O2 production.

Disclosure
B.A.Kugler: None. N.Lin: None. P.D.Nguyen: None. A.Ali: None. A.Sesay: None. B.Kalemba: None. H.Sesaki: None. K.Zou: None.

Funding
National Institutes of Health (R15DK131512) Diabetes Action Research and Education Foundation Grant (#505)
150-LB: Diabetes and School Health (DASH) Program—An Innovative School-Based Health Model to Optimize Care for Youth with Type 1 Diabetes
Authors: WOOD AURELIA C.H. , MOFFETT AMY M. , SIMMS KATHRYN , MOORE MATT , SULI ELVIA , IRWIN MARY KAY K. , EDWARDS ELIZABETH , GANDHI KAJAL 
Published: May 2022

Objectives: Pediatric type 1 diabetes (T1D) care requires collaboration of families, diabetes team and caregivers. Youth spend most of the time in school so care optimization in school is crucial. There are no evidence-based guidelines for school-based care, and management varies based on local legislation, school district, and staffing. An innovative school-based program provides an opportunity to standardize care, improve outcomes, reduce inequities, and inform guidelines for school T1D care.
Methods: The Nationwide Children's Hospital (NCH) Diabetes and School Health (DASH) program is a novel school-based intervention. The intradisciplinary DASH team provides on-site education to students/staff, addresses medical/psychosocial concerns, coordinates delivery of medications/supplies, and facilitates communication. The pilot aimed to enroll 50 students identified as high risk based on their Diabetes Composite Score, a risk assessment tool developed by NCH. Qualitative and quantitative data was obtained for students, caregivers, and staff, including A1C, acute care utilization, educational mastery, quality of life, psychosocial, and health equity measures.
Results: 50 students over 35 schools enrolled in the pilot. The majority used injections, 50% never used a continuous glucose monitor, and the average baseline A1C was 12%. More than 60% identified as an ethnic minority. Survey data showed over 50% of students felt others did not appreciate the burden of T1D care and endorsed T1D "burn out." Over 50% of caregivers endorsed feeling defeated/discouraged by T1D care, and worry about T1D related complications.
Conclusions: DASH is a novel school-based program that provides support, education, and oversight to students at high risk of T1D complications. The program aims to standardize school-based care, empower school staff, improve self-efficacy, address inequities, decrease T1D complications and provide a framework for school based care.

Disclosure
A. C. H. Wood: None. A. M. Moffett: None. K. Simms: None. M. Moore: None. E. Suli: None. M. K. Irwin: None. E. Edwards: None. K. Gandhi: None.
155-OR: Brown Adipose Tissue Leptin Feedback Regulates Feeding Behavior
Authors: MUNOZ MARCOS D. , IRIZARRY SR., VALERIA C. TORRES , XU PINGWEN , LIEW CHONG WEE 
Published: May 2022

Obesity is a consequence of chronic disruption in energy homeostasis. This phenomenon is controlled by systemic and local signals such as leptin, a nutrient-sensitive signal secreted primarily by the white adipose tissue (WAT) . In contrast, interscapular brown adipose tissue (iBAT) has been shown to express undetectable or very low levels of leptin. In this study, unexpectedly, we observed overnight fasting decreased leptin expression in iBAT, and 2-hour refeeding was able to restore the iBAT leptin levels. This nutritional status-dependent fluctuation suggests a vital role of iBAT leptin in the acute regulation of energy homeostasis. Consistently, we found that mice with leptin selectively overexpressed in the UCP1-positive cells of iBAT (LEP-OEiBAT/UCP1) showed reduced fast-induced food intake. This hypophagia phenotype was associated with increased satiation as reflected by decreased meal numbers and increase postmeal interval. Notably, LEP-OEiBAT/UCP1 selectively increased the mRNA expression of leptin in the iBAT without affecting circulating leptin levels, indicating a possible paracrine role of iBAT leptin. In supporting this point of view, we showed that chemical ablation of sensory nerves (SS) in iBAT significantly increased fast-induced food intake, associated with altered meal frequency. Additionally, we also found that iBAT-specific infusion of leptin increased Stat3 phosphorylation (pStat3) in the upstream iBAT-innervating sensory neurons, suggesting leptin-mediated sensory detection. Notably, the increased pStat3 was associated with neural activation in the arcuate nucleus of the hypothalamus in the brain, and chemical ablation of iBAT SS blunted this stimulation. Our results support a model that feeding-induced iBAT leptin modulates iBAT-specific sensory inputs to the brain to regulate food intake and feeding patterns.

Disclosure
M.D.Munoz: None. V.C.Torres irizarry: None. P.Xu: None. C.Liew: None.

Funding
RDK109015A1
157-LB: Scanning Frequency and Glucose Variability in the First Trimester of T1DM Pregnancies Predict Time Spent in Very Low Glucose Range in the Last One—Analysis of Intermittently Scanned Continuous Glucose Monitoring
Authors: BARETIC MAJA , LEKSIC GLORIA , IVANISEVIC MARINA 
Published: May 2022

Scanning frequency and glucose variability in the first trimester of T1DM pregnancies predict time spent in very low glucose range in the last one: analysis of intermittently scanned continuous glucose monitoring
Hypoglycemia is inevitable side effect of tight glycemic control in type 1 diabetes mellitus (T1DM) pregnancies. Using technology novel risk factors are revealed. Total of 34 patients (mean age 29.8 ± 6.1 yrs, BMI 23.6± 3.6 kg/m2, disease duration 15.2± 8.9 yrs) having very low glycemia in third trimester (&lt; 3 mmol/L or 55 mg/dL) were included in the study. Patients used intermittently scanned continuous glucose monitoring (isCGM) FreeStyle Libre, Abott for a minimum of 3 months before conception and had glucose data availability &gt; 80% of the time. Data from first trimester showed mean scanning frequency 12.17 ± 6.5 scans/day, estimated average glucose 7.2 ± 1.4 mmol/L, CV% 41.9 ± 6.5, GMI 6.4 ± 0.5, time below range &lt;3.5 mmol/L or 64 mg/dL 10.7 ± 8.0 % and time spent in very low glucose range &lt;3mmol/L or 55 mg/dL 5.4 ± 4.9 %. In last trimester mean scanning frequency was 16.3 ± 7.6 scans/day, estimated average glucose 6.1 ± 0.9 mmol/L, CV% 34.6 ±6.0 , GMI 5.9 ± 0.4 , time below range 11.0 ±9.3 %, time spent in very low glucose range 5.2 ± 4.8 %. Parameters from the first trimester of pregnancy significantly correlating with time spent in very low glucose range in the third trimester were scanning frequency (r −0.365, p 0.37) and CV % (r 0.465, p 0.006) . Linear regression analysis performed for CV% and scanning frequency showed R square 0.434 and p 0.002 in ANOVA. Two isCGM parameters from first trimester of pregnancy that influence on very low glucose rate in the third one are low scanning frequency and higher %CV. Combined they add to 43% of very low glucose occurrence. Patients who scan frequently in the first trimester of pregnancy and have low glucose variability spend less time in very low glucose range in the third trimester. For the others continuous glucose monitoring with hypo alerts should be offered.

Disclosure
M. Baretic: None. G. Leksic: None. M. Ivanisevic: None.
158-OR: Perinatal Hypercaloric Diet Leads to Cardiometabolic Imbalance via Hypothalamic Methylation Events in Male Mice
Authors: ELGAZZAZ MONA , BERDASCO CLARA , LAZARTIGUES ERIC D. 
Published: May 2022

Cardiometabolic diseases are among the highest prevalent diseases worldwide. We have reported that perinatal maternal programming through hypercaloric diet (HCD) leads to an elevation in systolic BP and blood glucose levels exclusively in male mice offspring. These findings led us to investigate epigenetic modulations in the brain of the offspring that support the cardiometabolic phenotype. To elucidate the underlying mechanisms, we challenged the programmed offspring with postnatal HCD, speculating that it would exaggerate the cardiometabolic phenotype. C57BL6/J dams were fed on HCD or regular diet (RD) for 1 month then mated with RD-fed males. From weaning, all pups were maintained on HCD for 3 months resulting in RD-HCD and HCD-HCD groups of male mice. Results showed that HCD-HCD mice have higher systolic BP when compared to RD-HCD (125 ±1.9 vs. 115 ±2.8 mmHg) . Surprisingly, a glucose tolerance test showed that HCD-HCD mice were more glucose tolerant when compared with RD-HCD mice (AUC 29632 ±3516 vs. 604±4724, p=0.003, n=6-10/group) . Whole-genome methylation sequencing of the hypothalamus in offspring revealed differentially methylated regions (DMR) in HCD-HCD mice. Pathway analysis showed that DMR are annotated to genes involved in insulin signaling, neurotransmitter binding, and inflammatory pathways. Insulin receptor substrate (Irs1) gene exon was hypomethylated in HCD-HCD mice when compared to RD-HCD (DMR= -0.12, p=0.03) . Protein expression showed an increase in Irs1 protein fold change (FC) levels in both HCD-HCD and RD-HCD (FC=1.45 and 1.33 p=0.005, 0.03 respectively) compared to RD-fed mice. However, serine phosphorylated Irs1 (pSer307-Irs1) , an indicator for disturbed insulin signaling, showed decreased levels only in HCD-HCD mice compared to RD-HCD males (p=0.003) . We conclude that perinatal HCD programming leads to methylation events in the hypothalamus that modulate the expression of key genes regulating cardiometabolic functions.

Disclosure
M.Elgazzaz: None. C.Berdasco: None. E.D.Lazartigues: None.

Funding
American Diabetes Association (1-19-IBS-291) ;