bluuebunny/crossref_metadata_2025 huggingface dataset:
1655-P: Multiomics Analysis of Sarcopenic Obesity
Authors: OKAMURA TAKURO , HASEGAWA YUKA , SENMARU TAKAFUMI , HAMAGUCHI MASAHIDE , FUKUI MICHIAKI
Published: June 2024

Background: Sarcopenic obesity is a critical characteristic of type 2 diabetes. This study aimed to investigate its pathological basis using integrated omics and db/db/mice, a model representing this condition.

Methods: The study involved housing 8-week-old db/m and db/db/mice for 8 weeks. Various analyses were conducted, including gene expression in skeletal muscle and small intestine using next-generation sequencing, cytokine arrays of serum, assessment of metabolites in skeletal muscle, stool, and serum, and analysis of the gut microbiota. Histone modifications in small intestinal epithelial cells were profiled using CUT&Tag.

Results: db/db/mice exhibited significantly lower grip strength and higher visceral fat weight (p < 0.0001). Serum cytokine arrays demonstrated higher relative concentrations of VEGF-A (p < 0.0001) and lower concentrations of several other cytokines in db/db/mice. Moreover, mRNA sequencing revealed downregulation of Myh expression in skeletal muscle and upregulation of lipid and glucose transporters and downregulation of amino acid transporter in the small intestine of db/db/mice. The concentrations of saturated fatty acids in skeletal muscle were significantly higher, and the levels of essential amino acids were lower in db/db/mice. Gut microbiota analysis showed lower levels of Bacteroidetes and higher levels of Firmicutes in db/db/mice (p = 0.003). The integrated signal of histone modifications of lipid and glucose transporters was higher, while the integrated signal of histone modifications of amino acid transporter was lower in the db/db/mice.

Conclusion: The multi-omics approach provided insights into the epigenomic alterations in the small intestine, suggesting their involvement in the pathogenesis of sarcopenic obesity induced by overeating and inactivity.

Disclosure

T. Okamura: Research Support; The Japan Diabetes Society, Suzuken Memorial Foundation, Nakatomi Foundation, Public Promoting Association Asano Foundation for Studies on Medicine, Uehara Memorial Foundation, Yakult Bio-Science Foundation, Japan Diabetes Foundation, Grants-in-Aid for Scientific Research. Y. Hasegawa: None. T. Senmaru: None. M. Hamaguchi: None. M. Fukui: None.
1656-P: In Vivo Downregulation of Transglutaminase 2 in High Fat Diet (HFD)-Induced Obese Mice Reduces Metabolic Syndrome–Associated Factors
Authors: ELIZONDO DIANA , HOLLIS MELANIE , PATEL TUSHAR P. , ROBERSON ROBIN , CHEN JOCELYN , YANOVSKI JACK A.
Published: June 2024

Introduction and Objective: Metabolic syndrome (MetS) in obesity increases the risk for type 2 diabetes, stroke, and heart disease. Fundamental gaps in the field remain to establish specific molecular targets to counteract MetS. Transglutaminase 2 (TGM2), a multifunctional enzyme known to crosslink proteins important for GTPase, cell adhesion, and scaffold activities has been implicated in adipose tissue growth and functioning. The objective of this study was to investigate TGM2 effects in MetS in a diet-induced obese mouse model.

Methods: C57BL/6 mice were placed on HFD for 10 weeks and silenced for TGM2 via in vivo RNAi silencing or received a scramble sequence control. Readout indices to evaluate MetS included glucose and insulin tolerance tests, body weight and food consumption monitoring, body DEXA scan, excised tissue phenotyping via histology, qPCR, RNAseq and western blot. Serum was assessed for adiponectin, leptin, free fatty acids and inflammatory parameters.

Results: Successful TGM2 silencing was found predominantly in epididymal white adipose tissue (eWAT) and liver. Diet-induced obese mice silenced for TGM2 had lower total body weight that was accompanied with improved glucose sensitivity, lower serum leptin and free fatty acid concentrations, reduced mean adipocyte size and increased adipocyte number in eWAT, along with reduced expression of molecular pathways associated with adipose tissue growth. Furthermore, there was an observed reduction in liver weight and hepatic steatosis and increased total lean mass.

Conclusion: Results suggest that reducing TGM2 expression reduced obesity and improved glucose homeostasis, making TGM2 modulation in obesity a promising therapeutic approach targeting MetS.

Disclosure

D. Elizondo: None. M. Hollis: None. T.P. Patel: None. R. Roberson: None. J. Chen: None. J.A. Yanovski: Research Support; Rhythm Pharmaceuticals, Inc., Soleno Pharmaceuticals, Inc, Versanis Bil, Hikma Pharmaceuticals, Inc., National Institutes of Health.

Funding

National Institute of Child Health and Human Development (ZIAHD00641); National Institute of Diabetes and Digestive and Kidney Diseases (1K99DK136921)
1659-P: Tirzepatide Compared with Semaglutide for the Functions and Mechanisms of Ameliorating Lipid-Metabolism Disorders in Type 2 Diabetic Mice
Authors: CHEN LIMING
Published: June 2024

Objective: To reveal the role and different underlying mechanisms of tirzepatide (Tzp) and semaglutide (Sema) in ameliorating lipid-metabolism disorders.

Methods: High-fat diet, streptozotocin-induced mice and db/db mice were received a daily injection of vehicle or Sema (40.79nmol/kg) or Tzp (16.87nmol/kg) for 5 weeks. Then biochemical indexes and fat content were detected, and histological staining was performed. Lastly, the transcriptome sequencing was performed in all groups of eWAT and liver tissues.

Results: Tzp or Sema reduced fasting blood-glucose, body weight, food intake, triglyceride, cholesterol levels and liver, eWAT, iWAT, BAT weights and alleviated liver pathology in both type 2 diabetic mouse models. Notably, in comparison with Sema, Tzp treatment showed lower body weight, triglyceride levels, iWAT and eWAT weights and adipocyte diameters. But the effects of BAT and liver did not differ between Tzp and Sema. Tzp and Sema regulated many common pathways and specific pathways. Additionally, in the same effect of regulating mitochondrial function, Tzp preferentially regulated endoplasmic reticulum stress, while Sema preferentially regulated inflammation. In the mechanisms of alleviating liver steatosis between Tzp and Sema, NF-kappaB and calcium-mediated signaling were enriched in the Tzp-treated group and the pathways related to cell cycle process and apoptosis were heavily enriched in Sema-treated mice.

Conclusion: The mechanism of attenuating abnormal lipid accumulation of Tzp was attribute to affecting endoplasmic reticulum stress, NF-kappaB and calcium-mediated signaling in eWAT and liver tissues while Sema preferentially regulated inflammation, cell cycle process and apoptosis. Remarkably, a smaller dosage of Tzp delivered more pronounced weight loss compared with high-dose Sema. Our study provided new insights into comparing the effects in reducing lipid-metabolism disorders between Tzp and Sema.

Disclosure

L. Chen: None.
1668-P: Novel Once-Weekly Amylin Analog Petrelintide (ZP8396) Is Well Tolerated with Improved GI Tolerability after Multiple Dosing
Authors: GRIFFIN JON , HÖVELMANN ULRIKE , MELGAARD ANITA E. , MACURA STANISLAVA , HAMMER METTE , JOHANSEN THUE , OLSEN MINNA B.
Published: June 2024

Petrelintide (ZP8396) has demonstrated potential to reduce body weight after single and multiple dosing (SD and MD) in humans. The tolerability of doses, with similar exposure levels, was compared.

SAD and MAD trials assessed safety, PK, and PD of petrelintide in healthy, lean and overweight males. 56 participants (mean BMI 25.6 kg/m2) were randomized to a SD of petrelintide or placebo (6:2) across seven dose cohorts (0.04 to 2.4 mg). 20 participants (mean BMI 25.4 kg/m2) were randomized to six once-weekly doses (MD) of petrelintide or placebo (7:3) across two dose cohorts (0.6 and 1.2 mg), without dose titration.   Body weight reduction and adverse events (AEs) from Gastrointestinal (GI) and Metabolism and Nutrition SOC are compared descriptively.

SD of 1.4 and 2.4 mg resulted in similar exposures in terms of Cmax and AUCtau as MD doses of 0.6 and 1.2 mg at steady state, respectively. All AEs were mild to moderate, with no serious or severe AEs reported, and no treatment discontinuations. No moderate GI AEs were seen after MD. The most frequently related AEs were decreased appetite, nausea and vomiting. A lower percentage of participants reported nausea or vomiting after MD compared to a SD. For 0.6 mg MD, 14% reported nausea vs 67% for 1.4 mg SD. For 1.2 mg MD, 29% reported nausea vs 83% for 2.4 mg SD. Reporting of vomiting was similarly low for 0.6 mg MD and 1.4 mg SD cohorts, 1 participant in each cohort. For 1.2 mg MD, none reported vomiting whilst 67% reported after 2.4 mg SD. The percentage of participants reporting either decreased appetite and/or early satiety were similar after SD and MD. The mean body weight reduction was 3.6% and 4.2% in SAD cohorts and 5.3% and 5.1% in MAD cohorts, after one or six weeks of dosing, respectively.

Results indicate improved GI tolerability of petrelintide when exposure is gradually increased. AEs of decreased appetite and early satiety were similar after SD and MD. Higher doses are being explored to assess the potential for the management of obesity.

Disclosure

J. Griffin: Employee; Zealand Pharma A/S. U. Hövelmann: None. A.E. Melgaard: Employee; Zealand Pharma A/S. Stock/Shareholder; Zealand Pharma A/S. S. Macura: Employee; Novo Nordisk A/S, Zealand Pharma A/S. M. Hammer: Employee; Zealand Pharma A/S. T. Johansen: Employee; Zealand Pharma A/S. Stock/Shareholder; Zealand Pharma A/S. M.B. Olsen: None.
1672-P: Weight Changes after Tirzepatide Cessation in Chinese Adults with Overweight/Obesity—A Real-World Follow-up Study of SURMOUNT-CN Trial
Authors: CHEN YING , CHEN CONGLING , ZHAO LIN , LU YIBING , FAN XIAOFANG , XU LAN , WANG YIHUA , DONG YU , LI YUANYUAN , XU JIAWEI , SI SI , ZHANG XUE , LI XIAOYING
Published: June 2024

Introduction & Objective: The GIP/GLP-1 receptor agonist tirzepatide (TZP) achieved body weight loss near 20% in Chinese adults with overweight (24 kg/m2≤BMI<28 kg/m2) with at least one weight-related comorbidity or obesity (BMI ≥28 kg/m2) in SURMOUNT-CN. Weight regain post-TZP cessation in Chinese population is unknown. Hence, we examined the changes in body weight (BW) and waist circumference (WC) after TZP cessation in the trial extension.

Methods: SURMOUNT-CN randomized (1:1:1) Chinese adults (n=210) with a BMI of ≥28 kg/m2, or ≥24 kg/m2 and weight-related comorbidities, excluding diabetes, to receive TZP 10 mg / 15 mg / placebo (PBO) for 52 weeks. Our study enrolled those who completed 52 weeks of TZP / PBO and followed them for 6 months. Participants with neither anti-obesity medication (AOM) nor bariatric procedures during follow-up (FU) were eligible analysis population (EAP). Changes of BW and WC at 3- and 6-month FU (±6 weeks) were analyzed using descriptive statistics.

Results: Among 160 enrolled, 152 (TZP 10 mg: 57; TZP 15 mg: 51; PBO: 44) constituted EAP, excluding 8 for AOM use. For EAP, from trial baseline to week 52, TZP showed marked BW and WC reduction: -15.33% (SD: 8.04%) and -12.82 cm (SD: 6.86) for 10 mg, -19.88% (SD: 8.78%) and -15.59 cm (SD: 7.26) for 15 mg, against PBO’s -3.80% (SD: 6.08%) and -3.70 cm (SD: 4.51). From week 52 to 6-month FU, BW and WC increased for TZP 10 mg (9.06% [SD: 7.41%], 2.89 cm [SD: 7.48]), and 15 mg (12.33% [SD: 9.88%], 5.53 cm [SD: 6.52]), while PBO showed slight change (1.83% [SD: 5.17%] and -0.52 cm [SD: 5.58]). Overall, from trial baseline to 6-month FU, changes in BW and WC with TZP 10 mg, 15 mg and PBO were -8.74% (SD: 6.93%) and -10.50 cm (SD: 8.07), -10.57% (SD: 10.23%) and -10.61 cm (SD: 9.29), -2.47% (SD: 7.03%) and -4.02 cm (SD: 7.21).

Conclusion: While over 50% of net weight loss was maintained after 6 months of TZP cessation, the increase in BW and WC from drug cessation supports continued weight management in clinical practice.

Disclosure

Y. Chen: None. C. Chen: None. L. Zhao: None. Y. Lu: None. X. Fan: None. L. Xu: None. Y. Wang: Employee; Eli Lilly and Company. Y. Dong: Employee; Eli Lilly and Company. Y. Li: None. J. Xu: None. S. Si: None. X. Zhang: Employee; Eli Lilly and Company. X. Li: None.
1686-P: Liver Fat as a Potential Marker for Subclinical Myocardial Remodeling and Dysfunction in Obese Adults
Authors: WAN XU , YUE JIANG , ZHAO HANG , HE SHENGYUN , QI YICHENG , YANG MINGLAN , CHE JIE , LU QING , JIANG MENG , BU JUN , MA JING
Published: June 2024

Background: Obesity has been recognized as a risk factor for cardiovascular disease. The association between fat distribution and subclinical myocardial remodeling and dysfunction among the obese population remains unclear.

Methods: A total of 114 participants were included in this study. Liver and pancreas were assessed using MRI-proton density fat fraction (PDFF) technology, and subclinical myocardial fibrosis were evaluated using cardiac magnetic resonance derived extracellular volume fraction (ECV) and native T2 values. Systolic and diastolic dysfunction was defined using values of global longitudinal peak systolic (GLS) and early diastolic longitudinal strain rates.

Results: Patients with NAFLD, defined as liver fat content ≥6%, had a higher prevalence of metabolic disorders, myocardial fibrosis and worse absolute GLS compared to those without NAFLD. Among multiple indicators of visceral fat, liver fat content (LFC) was significantly correlated with epicardial adipose tissue, and subclinical myocardial remodeling markers. This association remained significant after further adjustment for age, sex, waist, drugs and other potential HF risk factors. Multivariable linear regression and moderator analyses revealed that BMI might positively moderate the effect between liver fat and fibrosis indicators, for increased

LFC with higher ECV in highest tertile BMI group (β= 0.130 per SD, p=0.008), while lower ECV in lowest tertile group (β=-0.110 per SD), after adjusting for potential confounders.

Conclusion: Ectopic fat accumulation in liver may be significant risk factor to predict the occurrence and future development of myocardial fibrosis in obese adults.

Disclosure

X. Wan: None. J. Yue: None. H. Zhao: None. S. He: None. Y. Qi: None. M. Yang: None. J. Che: None. Q. Lu: None. M. Jiang: None. J. Bu: None. J. Ma: None.
1695-P: The Evaluation of Clinical Outcomes Assessments and Digital Health Technologies in Obesity Clinical Trials
Authors: GOETZ IRIS A. , SUTTER CAROLYN , ABRAHAM TRACI H. , KANU CHISOM , BOYE KRISTINA , SYMONDS TARA
Published: June 2024

Introduction & Objective: Trials in obesity have traditionally focused on weight loss and resolution of comorbidities as primary outcomes. However, secondary outcomes, such as the impact of weight reduction on the patient experience like health-related quality of life (HRQoL), have increasingly been recognized as important. Therefore, a review was conducted to determine the clinical outcome assessments (COAs) and digital health technologies (DHTs) used in clinical trials to assess patient experience.

Methods: Two clinical trial databases (US & EU) were reviewed to identify phase 2-4 obesity clinical trials (2018-2023). A literature review was also conducted using the OVID database to identify obesity clinical trial publications which included COAs/DHTs (2010-2023).

Results: Trials from the databases (n=53) and publications (n=42) were included in data extraction. This resulted in identification of 111 COAs, the majority being patient-reported outcomes (PROs; n=86), and 2 DHTs. The most frequent COAs were the Short Form 36 (SF-36v2) and Impact of Weight on Quality of Life-Lite Clinical Trials (IWQoL-Lite-CT), although 24 performance outcomes (PerfOs) were also identified, with the 6-minute walk test (6MWT) being most common. These measures were used as either confirmatory or supportive secondary endpoints, with physical function (PF) being the most frequently specified domain. Measures of eating-related thoughts and behaviours and disordered eating were also frequently included, although individual measures varied widely across trials.

Conclusion: Review of COAs/DHTs in registered clinical trials and publications found that multidimensional PRO measures assessing HRQoL were most often used in obesitytrials with limited use currently of DHTs. PROs were typically used as secondary (confirmatory and supportive) endpoints to assess PF. Further work is warranted to assess how these PRO data were viewed by regulators and payers.

Disclosure

I.A. Goetz: Employee; Eli Lilly and Company. C. Sutter: Consultant; Eli Lilly and Company. T.H. Abraham: Consultant; Eli Lilly and Company. C. Kanu: None. K. Boye: Employee; Eli Lilly and Company. T. Symonds: None.
1696-P: Early Predictors of Maternal Metabolic Health—Utility of Regional and Overall Body Composition Measures
Authors: INGRAM KATHERINE , EHO AMI , MCDONALD BREANNA , AMASON JANEEN
Published: June 2024

Pregnancy is characterized by reduced insulin sensitivity, which can advance to gestational diabetes in at-risk individuals. Identifying early predictors of GDM would help pregnancy outcomes by improving the time to intervention in this group.

Objective: To test body composition measures as potential early predictors of gestational insulin resistance.

Methods: At 19-20 weeks gestation, 32 nulliparous women (aged 27.2± 4.5 yrs) received body composition measures including percent body fat (PBF), fat and lean mass by bioimpedance; abdominal adipose via ultrasound (intra-abdominal, IAAT; subcutaneous, SAT); and skinfolds. Homeostasis assessment model of insulin resistance (HOMA-IR) was calculated from fasting glucose and insulin at 24-26 weeks and log-transformed for normality. Missing data was imputed by linear trend. One individual was excluded due to extreme BMI (>50). RESULTS: In analyses controlled for age and race, pre-pregnancy BMI and most measures of overall and regional body composition were associated with HOMA-IR (BMI: r=.523; PBF: r=.481; IAAT: r=.421; SAT: r=.611; fat mass: r=.569; lean mass: r=.434; p<.05 for all). In a regression model with age, race, and lean mass, fat mass was the only significant predictor of HOMA-IR (Model: R2= .345, p=.022; Fat Mass: Std. β= .501, p=.025; Lean Mass: β= .131, p=.535). Replacing lean mass with IAAT and SAT improved the association with HOMA-IR (Model R2=.559, p=.004), with SAT as the only significant predictor (β =.479, p<.01). Skinfold measures did not improve the model or achieve significance.

Conclusion: Body composition measures, especially SAT, may serve as potential early predictors of gestational insulin resistance. Adopting such early predictors may have a significant impact on maternal health by facilitating the implementation of earlier interventions for metabolic health.

Disclosure

K. Ingram: Stock/Shareholder; Eli Lilly and Company, Novo Nordisk, AstraZeneca, Dexcom, Inc. A. Eho: None. B. McDonald: None. J. Amason: None.

Funding

National Institutes of Health (3R15HD102957-01S1)
1698-P: Partial Least Squares Discrimination Analysis of Cytokine Profiles in T Cells with Inflammation in Subjects with Obesity
Authors: SARASWAT SHUBH , SANTACRUZ CALVO SARA , KERN PHILIP A. , NIKOLAJCZYK BARBARA , ZHANG XIAOHUA D.
Published: June 2024

Introduction & Objective: We aims to use Partial Least Squares-Discrimination Analysis (PLS-DA) to analyze cytokine profiles in T cells in the context of obesity.

Methods: PLS-DA is a robust statistical method that facilitates the identification of intricate patterns within complex cytokine data sets. We applied PLS-DA to investigate combinatorial cytokine profiles that differentiate T cells of old and young subjects with obesity. Additionally, we evaluated the effect of metformin on these profiles, focusing on its potential impact across age groups in individuals with obesity.

Results: Our PLS-DA analysis distinguished cytokine profiles produced by T cells from old and young subjects with obesity with a 74% accuracy. PLS-DA indicated a borderline effect of metformin on cytokine profiles generated by T cells from older subjects with obesity, with a model accuracy of 67%. Lastly, PLS-DA indicated no impact of metformin on cytokine profiles produced by T cells from younger subjects with obesity, with a model accuracy of 52%. The outcomes contrast with the strong impact of metformin on cytokine profiles produced by T cells from lean older and younger adults (Bharath et al, Cell Metab 2020).

Conclusion: PLS-DA analysis revealed differences in cytokine profiles among old and young subjects with obesity. This analytical method highlighted the differential impact of metformin on cells from these groups, showing a moderate effect on cells from older subjects with obesity and no significant impact on cells from younger subjects with obesity. These findings emphasize the importance of age and obesity status in cytokine profile alterations and impact of these variables on efficacy of treatments like metformin.

Disclosure

S. Saraswat: None. S. Calvo: None. P.A. Kern: None. B. Nikolajczyk: None. X.D. Zhang: None.

Funding

National Institutes of Health (UL1TR001998, 1U01DK135111 and OT2HL161847) and by the DRC at Washington University (P30 DK020579).
170-OR: Case-Control Study on Whole-Methylome Analysis of Monocytes in Acute Diabetic Charcot Foot—Identifying Differentially Methylated Genes
Authors: KUMAR NAWEEN , BHARTI BISHWA B. , KARN JYOTI P.L. , JHA SUDHIR C.
Published: June 2024

Introduction: Diabetic Charcot Foot (DCF) is a serious diabetes complication that damages bones and joints. Monocyte epigenetic modifications, notably DNA methylation, during DCF are unresolved. This study aims to analyze the whole-methylome of monocytes in acute DCF to identify differentially methylated genes involved in cell migration, differentiation, and osteoclast formation.

Methods: In this case-control study, 30 patients with acute DCF and 30 matched healthy controls were recruited. Monocytes were isolated from peripheral blood samples, and DNA was extracted. Whole-methylome analysis was performed using bisulfite sequencing. Differentially methylated regions (DMRs) were identified and associated with genes involved in monocyte migration, differentiation, and osteoclast formation. Gene expression was further confirmed through qPCR.

Results: Significant differences in methylation patterns were observed between DCF patients and controls. A total of 150 DMRs were identified, with 90 being hypomethylated and 60 hypermethylated in DCF patients. These regions were associated with genes crucial for monocyte function, including those involved in migration (e.g., CXCR4), differentiation (e.g., MAFB), and osteoclast formation (e.g., NFATC1). Gene expression analysis confirmed the upregulation of these genes in DCF patients compared to controls.

Conclusion: This study determined that DCF monocytes had differently methylated genes involved in cell migration, differentiation, and osteoclast formation. These epigenetic alterations may contribute to DCF pathophysiology, providing treatment targets. Further study is needed to understand how these epigenetic modifications affect DCF progression and their potential as biomarkers or therapeutic targets.

Disclosure

N. Kumar: None. B.B. Bharti: None. J.P.L. Karn: None. S.C. Jha: None.
171-OR: Tissue Nanotransfection–Based Endothelial-Targeted Epigenetic Gene Editing In Vivo to Rescue Diabetic Ischemic Wounds
Authors: SINGH KANHAIYA , VERMA SUMIT S. , GNYAWALI SURYA C. , SEN CHANDAN K.
Published: June 2024

Our previous work (PMID: 35192691) has identified that genetically silenced phospholipase Cγ2 (PLCγ2) hinders VEGF therapy of the diabetic ischemic limb. Hyperglycemia caused hypermethylation of endothelial specific gene promoter. Given that epigenetic changes are reversible, this work tests the significance of gene-targeted therapeutic DNA demethylation in improving blood flow to diabetic ischemic wounds. Bipedicle ischemic wounds were placed in streptozotocin (STZ) induced acute diabetic C57BL6 mice. In wound tissue, PLCγ2 promoter CpG methylation levels were analyzed using bisulfite sequencing. Next, to specifically demethylate PLCγ2 promoter in endothelial cells, a demethylation cocktail was designed: (i) (scFv)-TET1 catalytic domain (TET1CD) system capable of inducing targeted DNA methylation, and (ii) endothelial specific guide RNAs. This demethylation cocktail was delivered at the diabetic ischemic wound-edge employing topical tissue nano-transfection (TNT) technology. PLCγ2 protein expression outcomes were assessed using flow cytometry. Functional outcome of such demethylation was assessed using Laser Speckle Perfusion imaging and ultrasonography (Vevo 2100) at days 3, 7&10 post-surgery. Overall DNA hyper methylation was prominent in murine ischemic flaps as demonstrated by the increased ratio of 5-methylcytosine (5-mc, methylation mark) to 5-hydroxymethylcytosine (5-hmC, demethylation mark) (n = 5). Specifically, the PLCγ2 promoter was hypermethylated (n=5). TNT mediated endothelial-targeted demethylation of the PLCγ2 promoter increased the expression of this gene in endothelial cells (n=4). Such demethylation-based upregulation of PLCγ2 improved wound tissue blood flow with increased abundance of VWF+/PLCG2+ vascular elements (n=4). Taken together, topical TNT-based endothelial demethylation of the PLCγ2 gene promoter improved perfusion of cutaneous diabetic wounds resulting in improved closure.

Disclosure

K. Singh: None. S.S. Verma: None. S.C. Gnyawali: None. C.K. Sen: Consultant; SouthWest Technologies. Board Member; VisopalExo. Consultant; Vomaris Inc.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (DK136814, DK128845, DK135447, DK125835); Department of Defense (W81XWH-21-1-0033, W81XWH-22-1-0146)
1723-P: ATF6α Is Required for Pancreatic Beta-Cell Proliferation during Acute Severe Insulin Resistance
Authors: DARKO CHRISTINE O. , WANG YING , CASTRO THALIA A. , ALONSO LAURA C.
Published: June 2024

Introduction: Developing tools to expand beta cell mass is a critical goal towards a true solution to insulin-dependent diabetes. Sub-threshold activation of the UPR increases beta cell proliferation in hyperglycemic conditions via the Activating Transcription Factor 6 (ATF6) arm of UPR. ATF6α overexpression is sufficient to increase beta cell proliferation in high glucose in ex vivo islet cell cultures, and knockdown of ATF6α reduces glucose-induced proliferation. However, chronic beta cell deletion of ATF6α has been reported to cause minimal disruption to healthy biology. It is unknown if ATF6α participates in stress-induced beta cell proliferation in vivo.

Methods: Healthy C57BL/6N, or conditional beta cell ATF6α deletion (with Ins1cre: +/+Cre+, F/+Cre+ or F/FCre+ genotypes studied) male and female mice aged 10-15 weeks were implanted with Alzet minipumps containing 10nmol of S961 (insulin receptor antagonist) or vehicle for 7 days. Metabolic testing was performed, and pancreatic islets were isolated for qPCR analysis.

Results: Under unstressed conditions, beta cell ATF6α deletion did not alter systemic glucose metabolism. Healthy C57BL/6N mice treated with S961 developed marked hyperinsulinemia, hyperglycemia, and glucose intolerance. RNA from pancreatic islets isolated after 7 days of S961 showed molecular evidence of ER stress and increased proliferation markers; male and female mice were similar. Beta cell ATF6α deletion did not impact circulating metabolic parameters during severe insulin resistance, or surprisingly, the islet response to insulin resistance stress. However, the induction of proliferation markers in islets by S961 was significantly impaired in ATF6α deleted mice.

Conclusion: Chronic deletion of ATF6α in beta cells from development through adulthood does not impair beta cell homeostasis under unstressed conditions, but beta cell ATF6α is required for the proliferation response to acute severe insulin resistance in live mice.

Disclosure

C.O. Darko: None. Y. Wang: None. T.A. Castro: None. L.C. Alonso: None.

Funding

National Institutes of Health (R01DK135304, R01DK124906, and R01DK114686)
1728-P: The Nkx6.1 Binding Partner Mef2D Modulates Beta-Cell Insulin Secretion and Cell Survival
Authors: TESSEM JEFFERY S. , CRABTREE JACKIE
Published: June 2024

We have explored Nkx6.1 binding partners and have shown that the transcription factor Mef2D is expressed in the beta cell and interacts with Nkx6.1. Interestingly, Mef2D mRNA and protein levels decrease as a function of aging in primary rat islets (77±27.3% reduction in mRNA and 62±13.4% reduction in protein). We sought to understand how changes in Mef2D levels impinge on beta cell insulin secretion, and survival. We observed that Mef2D overexpression impaired mitochondrial respiration at complex I (41±9.6% decrease), complex II (53±6.4% decrease), complex V (43±5.2% decrease), complex II linked oxidative phosphorylation (47±3.1% decrease) and maximal respiration (41±6.1% decrease), while Mef2D knockdown increased complex II linked oxidative phosphorylation (42±11.4%, increase) and maximal respiration (34±13.0%, increase). Mef2D overexpression decreased glucose stimulated insulin secretion (43±4% decreases) and cell survival with etoposide, camptothecin, and thapsigargin treatment (32±4.1%, 38±6.2%, and 16±4.8% decreases, respectfully). Conversely, Mef2D knockdown increased glucose stimulated insulin secretion (75±7% increase) and cell survival with etoposide, and thapsigargin treatment (25±3.7% and 168±11.4% increases, respectfully). The observed changes in respiration, insulin secretion, and cell survival corresponded with modulated electron transport chain component expression, including expression of complex I component mtND6, with Mef2D overexpression decreasing mtND6 mRNA (49±9.7% decrease) and protein (a 37±6.6% decrease). Our findings define Mef2D as a critical factor for modulating insulin secretion and beta cell survival through modulation of beta cell mitochondrial function.

Disclosure

J.S. Tessem: None. J. Crabtree: None.

Funding

National Institutes of Health (R15DK124835)
173-OR: A Clinical Study of Novel Antimicrobial Peptide PL-5 Topical Spray for Mild to Moderate Infectious Wounds of Diabetic Foot
Authors: QIAN LI , WANG AIPING , HU WEN , WANG GUOFENG , SUN XINJUAN , LIU YU
Published: June 2024

Background: Antimicrobial peptide PL-5 spray is a topical antimicrobial peptide drug with novel mechanism of action and may improve the cure efficiency of diabetic foot infections. We studied the clinical efficacy and safety of investigational PL-5 spray in the treatment of mild to moderate diabetic foot ulcers in combined with standard debridement procedure.

Methods: A multicenter, randomized, double-blind, placebo-controlled clinical trial was conducted in four hospitals in China. Qualified subjects with mild to moderate diabetic foot ulcers were randomly assigned (2:1 ratio) into PL-5 and placebo groups plus a standard debridement. The primary endpoint of the study was the clinical efficacy at day 1 after the end of treatment (EOT1); the secondary endpoints included the clinical efficacy of treatment at day 7 after EOT(EOT7), microbiological efficacy, drug-resistant bacteria clearance rate, wound healing rate, and safety outcomes evaluated at EOT1 and EOT7, respectively.

Results: Subjects in PL-5 and placebo groups (32 vs 15 cases) showed no statistical differences in demographic and clinical characteristics. The clinical efficacy (cure/improvement ratio) in PL-5 group at EOT1 was higher than that in control group (1.33 vs 0.55, p=0.0764). Among the secondary endpoints, the clinical efficacy at EOT7 in PL-5 and control groups were 1.6 vs 0.86, the microbial eradication rates were 57.89% vs 33.33% (EOT1) and 64.71% vs 40.00% (EOT7); the drug-resistant bacteria clearance rates were 71.43% vs 50% (EOT1), respectively. The safety parameters showed no difference between two groups (24.24% vs 33.33%).

Conclusion: With limited samples, antimicrobial peptide PL-5 spray still shows strong potentials on efficacy and safety against the wound infections caused by drug-resistant bacteria. It can be anticipated that topical PL-5 treatment significantly improved the recovery speed of the diabetic foot wounds in further amplified study.

Disclosure

L. Qian: None. A. Wang: None. W. Hu: None. G. Wang: None. X. Sun: None. Y. Liu: None.
1735-P: AKT Modulates Glucose Sensing and Glucagon Secretion in Alpha Cells
Authors: NEVES AMANDA F. , LUBACZEUSKI CAMILA , BOZADJIEVA KRAMER NADEJDA , ANDRADE LOUZADA NETO RUY , BERNAL-MIZRACHI ERNESTO
Published: June 2024

In both T1DM and T2DM, glucagon secretion is impaired and no longer inhibited by hyperglycemia nor insulin, suggesting a defect in the insulin signaling via IRS/PI3k/Akt on pancreatic α-cells. Using a mouse model with constitutive activation of Akt in α-cells (αpCall), we investigated the impact of insulin signaling activation on glucagon secretion at different glucose concentrations. The αpCall mice exhibited lower body weight and hypoglycemia in early life. Given the mosaic expression of the transgene in α-cells, αpCall mice displayed normal glucose homeostasis, glucagon, and insulin secretion. In isolated islets, α-cells from αpCall mice showed increased expression of glucagon and the activation of mTORC1 signaling. In addition, α-cells from αpCall mice exhibited increased exocytosis and glucagon secretion stimulated by low glucose. This result was accompanied by elevated expression levels of Na+ and Ca2+ channels, along with an increase in Na+ and Ca2+ currents. Conversely, exposure of Akt-expressing α-cells to escalating glucose concentrations demonstrated a better suppression of glucagon secretion at high glucose levels. Throughout these experiments, insulin expression and secretion remained consistent under all glucose-stimulated conditions. In summary, our findings highlight the crucial role of AKT signaling activation in α-cells: it suppresses glucagon secretion in hyperglycemic conditions while enhancing glucagon secretion in low-glucose environments. This suggests that insulin signaling through the Akt pathway regulates α-cell sensitivity and glucagon secretion in response to glucose.

Disclosure

A.F. Neves: None. C. Lubaczeuski: Employee; Fractyl Health, Inc. N. Bozadjieva Kramer: None. R. Andrade Louzada Neto: None. E. Bernal-Mizrachi: None.

Funding

National Institutes of Health (5I01BX002728-08)
174-OR: Genetic Variation in GLP-1R Modify the Risk of Hypoglycemia in Response to Intensive Glycemic Control in Adults with Type 2 Diabetes
Authors: XUE QIAOCHU , LI PIAOPIAO , JAGANNATHAN RAM , PASQUEL FRANCISCO J. , UMPIERREZ GUILLERMO , PHILLIPS LAWRENCE S. , ALI MOHAMMED K. , SHAO HUI
Published: June 2024

Introduction: Hypoglycemia is a potential side effect of Intensive Glycemic Control (IGC) in diabetes management. We aimed to investigate whether individuals with certain GLP1R gene variations were at greater hypoglycemia risk with IGC.

Methods: A dynamic cohort design with time-varying exposures was employed using electronic health records linked to survey data from 8,848 individuals in the All-of-Us database. Subjects with type 2 diabetes and antidiabetic medication use could transition between the IGC exposure group (A1c < 7.0%) and the moderate glycemic control group (A1c 7%-8.5%) during follow-up or were censored at their last visit, death, end of the study, or if A1c exceeded 8.5%. Per-protocol analysis was conducted using mixed-effects Cox proportional regression models, adjusting for demographics and a comprehensive set of time-varying variables, including disease histories, lab values, medications, and patient-reported conditions.

Results: During a median follow-up period of 6.2 years, we documented 841 incident cases of hypoglycemia (incidence rate:1.34 per 100 person-years). Compared to the moderate GC group, the IGC group had a higher hypoglycemia risk (hazard ratio (HR): 1.06, 95% CI:1.00-1.12). Thirteen out of 15 GLP1R genetic variants significantly modified such risk, demonstrating a notable variation in risk across different genotypes, with HR ranging from 0.46 to 1.19. Combining these variants in a polygenic risk score showed that higher scores were associated with higher IGC-associated hypoglycemia risk (high score: HR 1.27, 95% CI [1.13-1.43] vs Low score: HR 0.57, 95% CI [0.43, 0.77). Sensitivity analyses excluding all GLP-1RA users yielded consistent results.

Conclusion: Our findings indicate that GLP1R variants play a modulating role in IGC-associated hypoglycemia risk, independent of GLP-1RA use. This effect suggests the prominent role of GLP1R genes in safe glucose-lowering among patients with type 2 diabetes.

Disclosure

Q. Xue: None. P. Li: None. R. Jagannathan: None. F.J. Pasquel: Research Support; Tandem Diabetes Care, Inc., Insulet Corporation, Dexcom, Inc., Ideal Medical Technologies, Novo Nordisk. Consultant; Dexcom, Inc., Medscape. G. Umpierrez: Research Support; Abbott, Bayer Inc., Dexcom, Inc., AstraZeneca. Advisory Panel; Dexcom, Inc. L.S. Phillips: Other Relationship; Diasyst, Inc. Research Support; Kowa Pharmaceuticals America, Inc., Janssen Pharmaceuticals, Inc., AbbVie Inc., Novo Nordisk, GlaxoSmithKline plc, Abbott, Sanofi-Aventis U.S., Pfizer Inc. M.K. Ali: Advisory Panel; Eli Lilly and Company. H. Shao: Consultant; Eli Lilly and Company.
1747-P: Glucolipotoxicity Inhibits Proinsulin Processing through Disrupting V-ATPase Function Leading to Impaired Acidification within Secretory Granules
Authors: IIDA HITOSHI , TAKIGUCHI MAMI , NISHIDA YUYA , WATADA HIROTAKA
Published: June 2024

Increase in serum proinsulin level reflects dysfunction of pancreatic beta cells. Insulin is secreted by pancreatic beta cells after the precursor proinsulin is processed by prohormone convertase (PC) 1/3 in secretory granules (SGs). In the progression of diabetes where pancreatic beta cells are under excess fatty acids and high glucose, insulin biosynthesis is impaired and proinsulin is secreted as an unprocessed form of insulin. However, the exact mechanism of the impairment of proinsulin processing is unclear. As a first step to elucidate its mechanism, we used islets isolated from db/db mice and islets treated with high glucose and palmitate (HGPal), as a model of glucolipotoxicity. As PC1/3 enzyme function is known to be dependent on the luminal acidity in the SGs, we investigated the pH in both islets and found decrease in the acidity of the entire cells by labeling cells with fluorogenic pH indicator with flow cytometry. We also detected decrease in the protein level of insulin and change in the protein expression pattern of PC1/3 by immunoblotting. Morphological analysis with electron microscope revealed that accumulation of immature SGs in these islets probably due to the accumulation of proinsulin inside. Given that the V-ATPase plays an essential role to maintain the acidity of SGs as a proton pump, both fractionation assay and proximity ligation assay revealed that HGPal stimuli inhibit the assembly formation of two major domain V1 and V0 in of V-ATPase Our results demonstrate that glucolipotoxicity altered the luminal acidity in SGs and impaired the proinsulin processing through disrupting V-ATPase function. We anticipate these results may explain underlying molecular mechanisms of the increase in the serum proinsulin level reflecting dysfunction of pancreatic beta cells.

Disclosure

H. Iida: Research Support; Sumitomo Dainippon Pharma Co., Ltd. M. Takiguchi: Research Support; Sumitomo Dainippon Pharma Co., Ltd. Y. Nishida: Research Support; Sumitomo Dainippon Pharma Co., Ltd. H. Watada: Speaker's Bureau; Bayer Inc., Sanofi, Novo Nordisk, Sumitomo Dainippon Pharma Co., Ltd., Abbott Japan Co., Ltd., Eli Lilly and Company, Boehringer-Ingelheim, Daiichi Sankyo, Taiho Pharmaceutical Co. Ltd., Kowa Company, Ltd., Teijin Pharma Limited, Mitsubishi Tanabe Pharma Corporation. Research Support; Sumitomo Dainippon Pharma Co., Ltd., Boehringer-Ingelheim, Mitsubishi Tanabe Pharma Corporation, LifeScan Diabetes Institute, Abbott Japan Co., Ltd., Teijin Pharma Limited, Takeda Pharmaceutical Company Limited, Taiho Pharmaceutical Co. Ltd., Sanwa Kagaku, Souiken.

Funding

JSPS KAKENHI (JP 20K17501)
1754-P: Investigating the Interplay between T-Cell Infiltration, ROS Detoxification, and Pancreatic Beta-cell Dysfunction in Type 1 Diabetes Pathogenesis
Authors: LAZIMI CHARLES , MATHEWS CLAYTON E. , PHELPS EDWARD
Published: June 2024

Introduction & Objective: Reactive oxygen species (ROS), byproducts of cellular metabolism, are implicated in T1D progression, causing lipid, protein, and DNA damage, along with impaired β-cell function and apoptosis. Notably, at the onset of T1D, β-cells exhibit dysregulated glucose metabolism, resulting from decreased glucose uptake and impaired utilization. This metabolic perturbation diminishes NADPH production, a critical antioxidant cofactor from glycolysis and the pentose phosphate pathway (PPP). Consequently, islets in T1D individuals may exhibit compromised ROS-neutralizing abilities, heightening susceptibility to oxidative damage. The objective of this study is to determine glucose metabolism's role in β-cell antioxidant defense and T1D-related dysfunction. We hypothesize that insulitic islets in T1D are more vulnerable to damage from ROS compared to islets in individuals without diabetes and that diminished ROS detoxification contributes significantly to β-cell loss and dysfunction in T1D.

Methods: Islets in live pancreatic tissue slices from both nondiabetic and recent-onset diabetic donors were acutely challenged with hydrogen peroxide (H2O2), and palmitate, followed by glucose stimulations. Real-time monitoring of ROS levels, utilizing ROS biosensor GRX1-roGFP2 and chemical ROS indicators, were conducted.

Results & Conclusion: While it is generally the case that chronic hyperglycemia induces oxidative β-cell damage, our observations suggest that acute glucose stimulation reduces baseline ROS levels by promoting increased GSH in healthy β-cells. Following various ROS challenges including, high glucose stimulations quickly reverted their oxidative effects in healthy β-cells. These results support the concept that poor beta cell glucose responsiveness in T1D increases β-cell susceptibility to ROS.

Disclosure

C. Lazimi: None. C.E. Mathews: None. E. Phelps: Research Support; Immunocore, Ltd, MESO SCALE DIAGNOSTICS, LLC.
1755-P: The Development of Antagonists Targeting Olfr109 to Treat Diabetes
Authors: GE XIAOYAN , CHENG JIE , YANG ZHAO , YU XIAO , SUN JINPENG
Published: June 2024

The olfactory receptor Olfr109 is activated in diabetic models and then impairs islet β cell functions. Antagonizing Olfr109 is a new potential therapeutic strategy to treat diabetes. Previous studies showed that pepducin o109-i2-2 targeting Olfr109 improved islet function and glucose homeostasis. To further obtain the higher potency antagonists, we rationally designed the peptide antagonists targeting Olfr109. We successfully convert the peptide agonist insB 9:23 to the antagonist Ssc-S(R) by combining mutations and non-natural amino acid substitution at specific positions. Importantly, the combined antagonist of Ssc-S(R) had a more significant effect on the improvement of glucose metabolism in diabetic mice. In response to Ssc-S(R), the decreased insulin secretion and increased endoplasmic reticulum stress were significantly alleviated. Therapeutic administration of Ssc-S(R) could significantly ameliorate intra-islet inflammatory responses and improved glucose homeostasis in both Akita and HFD-fed mice. Notably, no significant differences were observed in ins2 WT/Akita Olfr109-/- mice and HFD-fed Olfr109-/- mice. Taken together, we have developed the Olfr109-specific antagonist Ssc-S(R), which may provide new clues for the treatment of islet inflammation and diabetes.

Disclosure

X. Ge: None. J. Cheng: None. Z. Yang: None. X. Yu: None. J. Sun: None.
1767-P: A Modifying Effect of Genetic Background on S64F MAFA-Induced Dysglycemia
Authors: LOYD ZACHARY , LEE DONGSOO , GUO MIN , STEIN ROLAND , CHA JEEYEON
Published: June 2024

Introduction & Objective: The MAFA transcription factor is an essential regulator of mature pancreatic islet β cell function. A rare missense variant of human MAFA, Ser64Phe (S64F), was identified as a MODY gene in patients with familial insulinomatosis or diabetes mellitus (1). S64F MAFA carriers either became hypoglycemic due to multifocal pancreatic insulinomas (insulinomatosis) or develop diabetes, with men more likely to become diabetic and women develop insulinomatosis. A mouse model harboring this mutation recapitulated these sex-dependent glycemic phenotypes (2). Here we sought to investigate the impact of mouse genetic background on S64F MAFA-induced dysglycemia.

Methods: S64F MAFA mutant mice were bred either on a mixed C57/Bl6J (C57) and SJL background, or C57 alone. Glucose tolerance testing assessed serum glucose at t=0 (fasting), 15, 30, 60, and 90 minutes post intraperitoneal injection (0.2mg/kg dextrose). Statistical analysis was performed by Two-way ANOVA. Glucose-stimulated insulin secretion was also assessed at 0, 15 and 30 minute time points. Cut&Run determined DNA-protein interactions to help identify binding targets of MAFA in MIN6 β cells.

Results: Mixed background S64F MAFA male mice were diabetic while females showed improved tolerance compared to WT siblings. In the C57 background, males were euglycemic while females showed improved glycemia. Insulin secretion was impaired only in mixed background mutant males. Cut&Run analysis performed for endogenous MAFA in MIN6 cells identified enrichment at known MAFA targets such as Ins1, Ins2, and Slc2a2, among others, thus verifying our test system.

Conclusion: Genetic background influenced S64F MAFA-dependent glycemia in a sex-dependent manner. Ongoing studies investigate differential targets of this mutant MAFA compared to those of the wild-type protein by Cut&Run analysis within each sex and validate differential targets by RNAScope.

Disclosure

Z. Loyd: None. D. Lee: None. M. Guo: None. R. Stein: None. J. Cha: None.

Funding

National Institutes of Health (5K08DK132507), Burroughs Wellcome Fund Career Award for Medical Scientists
1768-P: The Impact of IL-1 in Islet Antiviral Responses
Authors: BARTOSIAK JACOB , CORBETT JOHN A.
Published: June 2024

Introduction & Objective: Viruses, particularly members of the picornavirus family, are correlated with the onset of T1D. For this reason, viral infection has been identified as an environmental trigger for T1D onset, specifically due to its ability to stimulate the production and release of pro-inflammatory cytokines into the islet. The purpose of this study is to define physiological functions of cytokine signaling in β-cells that may contribute to reduced virus-mediated damage and destruction of β-cells.

Methods: C57BL/6J and DBA/2J mice were infected with encephalomyocarditis virus (EMCV), a highly inflammatory picornavirus that causes dysglycemia in genetically susceptible mouse strains. Pancreas and serum were collected from the animals throughout the course of infection. Insulitis and β-cell morphology were evaluated by H&E and immunofluorescence and serum cytokine concentrations were analyzed by LEGENDplex anti-virus response assay. Furthermore, isolated mouse islets were treated with cytokines and gene responses and EMCV viral replication were measured by qRT-PCR.

Results: We show that C57BL/6J mice are resistant to EMCV infection and maintain islet architecture and glycemic control, despite immune cell infiltration. In contrast, EMCV infection of genetically susceptible DBA/2J mice results in destruction of β-cells, deterioration of islet morphology, and loss of glycemic control. Transiently elevated serum cytokines in C57BL/6J mice suggest successful restriction of EMCV replication whereas sustained elevation of these cytokines in DBA/2J mice suggests a failure of these mice to mount an effective antiviral response. Additionally, islets treated with IL-1β ex-vivo upregulate several antiviral genes and treatment of isolated islets with IL-1β prior to infection attenuates EMCV mRNA accumulation.

Conclusion: These studies suggest IL-1β signaling in β-cells during viral infection functions to activate protective antiviral defense mechanisms designed to limit β-cell damage.

Disclosure

J. Bartosiak: None. J.A. Corbett: None.

Funding

AI2212769