18th Annual Pioneers in Endocrinology Workshop Program

“Changing the Set Point: Mechanisms of Surgical Versus Pharmacological Weight Loss”

Randy J. Seeley, Ph.D.

Henry King Ransom Endowed Professor of Surgery and Professor of Internal Medicine, Medical School, and Professor of Nutritional Sciences, School of Public Health, University of Michigan
Director, Michigan Nutrition Obesity Research Center

“Gut-Brain GLP1 Signaling in Motivated Behavior”

Linda Rinaman, Ph.D.

Distinguished Research Professor, Department of Psychology, and R. Bruce Masterton
Professor of Neuroscience, Florida State University

Abstracts

 

Cancer Diseases (CD)

CD 1. Steroidogenic Precursors for Evaluation of Adrenocortical Carcinoma: A Case Series
F. Arafa, N. Markovic, D. Cohen, S. Lubtiz, A. Ohri
Rutgers Robert Wood Johnson Medical School

Adrenocortical carcinoma (ACC) is a rare, aggressive endocrine malignancy with poor prognosis. Most cases are functional; adrenal steroid precursors like 11-deoxycortisol aid diagnosis and serve as markers for recurrence, metastasis, and treatment response. We present two metastatic ACC cases with elevated precursors, highlighting their value in diagnosis and management.

A 44-year-old previously healthy man presented with subacute neuropsychiatric changes, weight loss, and new diagnoses of diabetes, hypertension, and an incidentally discovered vertebral compression fracture. Imaging showed a hepatic mass, retroperitoneal adenopathy, and a right adrenal nodule. Biopsy confirmed metastatic ACC (SF-1+). Biochemical evaluation showed markedly elevated cortisol (not suppressed on 1-mg DST), 24-hour urine cortisol and steroid precursors including 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone. He began palliative chemotherapy with mitotane and osilodrostat for hypercortisolism. Despite increasing doses of mitotane, 11-deoxycortisol and 11-deoxycorticosterone levels remained high, causing persistent hypertension and hypokalemia. Rising 17-hydroxyprogesterone level, which was unaffected by osilodrostat, signaled disease progression.

A 50-year-old woman with resistant hypertension and type 2 diabetes presented with worsening blood pressure, hyperglycemia, and weight gain. Testing revealed elevated cortisol, 24-hour urine cortisol, and 11-deoxycortisol with suppressed ACTH consistent with adrenal source. The elevated 11-deoxycortisol raised suspicion for ACC. CT imaging demonstrated a 7.7-cm left retroperitoneal mass suggestive of adrenal origin. She underwent left adrenalectomy and liver mass resection; pathology confirmed metastatic ACC with hepatoid differentiation. Postoperatively, she developed adrenal insufficiency requiring hydrocortisone, but 11-deoxycortisol levels remained elevated. She received palliative chemotherapy and mitotane, which successfully suppressed 11-deoxycortisol. A brief trial of osilodrostat for elevated precursors was discontinued due to insurance barriers. Currently, 11-deoxycortisol is being used as a tumor marker to monitor her biochemical response to mitotane monotherapy.

ACC is an aggressive malignancy with a poor prognosis. Biochemical evaluation for cortisol excess and co-secretion of steroid precursors is critical, as markedly elevated precursors strongly raise suspicion for ACC and guide management. Although not part of the standard adrenal mass work-up, steroid precursors can assist with diagnosis and serve as useful biomarkers of disease activity and treatment response. This case series illustrates the diagnostic and therapeutic relevance of steroidogenic precursors in patients with suspected or confirmed ACC.

CD 2. Previously Unrecognized Thyroid Carcinoma in Patients With Obesity Screened Before GLP-1 Receptor Agonist Therapy
E. Lupiano, S. Ten
Rutgers-Robert Wood Johnson Medical School, Child Health Institute of New Jersey

Background
Routine baseline thyroid imaging is not currently recommended before GLP-1 therapy in patients without specific risk factors. Reports of increased thyroid cancer detection shortly after treatment initiation may reflect surveillance and pre-existing disease rather than a direct drug effect. Obesity itself is associated with increased thyroid nodule prevalence and differentiated thyroid cancer risk, particularly papillary thyroid carcinoma.
Objective
To evaluate the utility of baseline thyroid ultrasound screening before initiation of GLP-1 receptor agonist (GLP-1) therapy in patients with obesity.
Methods
We conducted a retrospective review of patients with obesity who underwent thyroid ultrasound screening before initiation of GLP-1 therapy at our institution. Institutional Review Board approval was obtained. We included 195 patients.
Results
Among 195 patients who underwent baseline thyroid ultrasound, 10 (5.1%) were found to have previously unrecognized thyroid carcinoma. Of these patients, 9 were female and 1 was male, with ages ranging from 14 to 61 years and BMI ranging from 30.5 to 56.9 kg/m². All patients with thyroid carcinoma had obesity with multiple metabolic comorbidities. Histologic evaluation demonstrated papillary thyroid carcinoma in 9 of 10 patients and follicular thyroid carcinoma in 1 patient.
Conclusion
Baseline thyroid ultrasound identified previously unrecognized thyroid carcinoma in 5.1% of patients with obesity undergoing evaluation before GLP-1 therapy. Although routine thyroid ultrasound screening before GLP-1 initiation is not currently recommended, the unexpectedly high prevalence of occult thyroid malignancy in this cohort suggests that the potential role of baseline thyroid ultrasound in selected patients with obesity warrants further investigation.

CD 3. Prenatal alcohol exposure alters the tumor microenvironment in MMTV-Wnt1 mice
S. A. Totilo, K. N. Lambert, A. Messyasz, A. Lemenze, A. E. Jetzt, W. S. Cohick
Rutgers University Department of Animal Sciences, Graduate Program in Endocrinology and Animal Biosciences; Rutgers University Graduate Program in Nutritional Sciences; Rutgers University Molecular and Genomics Informatics Core (MaGIC); Molecular and Genomics Informatics Core (MaGIC), Department of Pathology, Immunology & Laboratory Medicine; Rutgers University Department of Animal Sciences

Breast cancer is the most common cancer among women in the United States. Both modifiable and non-modifiable factors, including alcohol consumption, influence breast cancer risk. Timing and duration of alcohol exposure can impact this risk. The Developmental Origins of Health and Disease (DOHaD) hypothesis states that early-life exposures, such as prenatal alcohol exposure (PAE), can predispose individuals to disease later in life. Using the MMTV-Wnt1 mouse model for basal-like breast cancer, our laboratory has shown PAE altered proportions of the mammary epithelial cell (MEC) subpopulations in preneoplastic mammary glands, as well as tumors formed in adulthood. In our animal studies, we also found that PAE promoted mammary tumorigenesis by decreasing tumor latency. These findings suggest that PAE reprograms the MEC profile potentially contributing to tumorigenesis. The current work investigates PAE-induced changes in tumor gene expression and signaling of individual tumor cell types via single cell transcriptomics. These studies provide further insight into the mechanisms by which PAE affects mammary tumorigenesis, advancing our understanding of the developmental origins of breast cancer risk.

CD 4. Integrative In Silico and In Vitro Analysis of the Association Between Cigarette Smoke and Glioblastoma Progression
N. Jang, W. Huang, H. R. Baik, A. Arumugan, S. L. Chang
Institute of NeuroImmune Pharmacology, Seton Hall University, South Orange, USA

Glioblastoma (GBM), classified as a grade IV glioma, is the most aggressive and common malignant primary brain tumor in adults. Despite its poor prognosis, GBM’s etiology remains incompletely understood, and the environmental factors that may influence its development and progression have not been fully characterized. Cigarette smoke is a major source of exposure to numerous carcinogenic compounds and is a leading cause of preventable disease and death in the United States. Limited studies have investigated a potential association between cigarette smoke exposure and GBM. The molecular pathways through which cigarette smoke exposure may influence GBM-related processes remain largely unexplored. By integrating Ingenuity Pathway Analysis (IPA)-based predictions from the QIAGEN Knowledge Base (QKB), validation through human transcriptomic data from TCGA and GTEx, and experimental testing, this study aims to investigate the molecular and cellular mechanisms through which cigarette smoke exposure may affect GBM progression and to examine the responses of U87 GBM tumor cells to cigarette smoke extract (CSE) and nicotine.

Using IPA, 94 molecules associated with cigarette smoke and GBM were identified within the QKB. A network linking cigarette smoke to GBM through these molecules was constructed, and the Molecular Activity Predictor (MAP) was used to predict the effects of cigarette smoke within the network. Of the 94 shared molecules, 12 were identified as potential mediators linking cigarette smoke exposure to GBM activation. These 12 molecules were significantly associated with GBM (B-H-corrected p = 1.41 x 10^-52) and exhibited a positive activation z-score (z = 1.367). Differential expression analysis in GEPIA3, based on 166 GBM tumor samples and 110 normal brain samples from TCGA and GTEx, followed by IPA’s Canonical Pathway Analysis, identified 795 significantly altered pathways (adjusted p < 0.05). Of the 12 predicted mediators, 9 were significantly differentially expressed, and 8 of these showed expression changes consistent with the IPA predictions.

In U87 cells, a preliminary scratch assay showed that 0.5 mM nicotine was associated with a 51% increase in migration relative to the control (p = 0.08). An effect of 5% CSE on migration was not observed in this preliminary experiment. Additional biological replications are necessary to further evaluate the effects of both nicotine and CSE on U87 cell migration. RT-qPCR validation of the candidate mediators in U87 cells is ongoing to further assess their potential involvement in cigarette smoke-associated GBM processes and to provide a foundation for subsequent mechanistic studies.

 

Endocrinology and Endocrine-Related Disorders (EERD)

EERD 1. Pembrolizumab-Induced Hypoparathyroidism: A Case Report
F. Arafa, S. Lubitz
Rutgers Robert Wood Johnson Medical School

Introduction:
Immune checkpoint inhibitors (ICIs), including programmed cell death protein-1 (PD-1) inhibitors such as pembrolizumab, are associated with immune-related adverse events (irAEs), including endocrinopathies. While thyroid dysfunction, hypophysitis, and insulin-deficient diabetes are well recognized, hypoparathyroidism remains exceedingly rare. We present a case of severe hypocalcemia secondary to pembrolizumab-induced hypoparathyroidism.
Case Presentation:
A 70-year-old male with stage IIIA left upper lobe non-squamous cell lung cancer, status post neoadjuvant therapy and robotic left upper lobectomy with lymph node dissection, was receiving chemotherapy and pembrolizumab. He was referred for severe hypocalcemia. Serum calcium had been normal (9.1 mg/dL) approximately two weeks before pembrolizumab initiation. Two weeks after starting therapy, calcium declined to 7.9 mg/dL and progressively decreased to 6.5 mg/dL. Renal function and magnesium were normal. He was asymptomatic with negative Trousseau and Chvostek signs, although ECG demonstrated a prolonged QTc of 463 ms. He had no history of hypocalcemia, relevant medication exposures, or recent transfusions. Laboratory evaluation revealed an inappropriately normal PTH of 15 pg/mL and elevated phosphorus of 5.3 mg/dL, consistent with hypoparathyroidism. Given the temporal association, pembrolizumab-induced hypoparathyroidism was diagnosed.
Discussion/Conclusion:
ICI-induced hypoparathyroidism is an exceedingly rare endocrine irAE, with limited cases reported. Proposed mechanisms include immune-mediated parathyroid injury involving T-cell activity and autoantibodies against calcium-sensing receptors. This case highlights the importance of recognizing atypical endocrine toxicities following immunotherapy. New hypocalcemia with an inappropriately normal PTH should prompt evaluation for hypoparathyroidism, even in asymptomatic patients. Further research is needed to clarify its incidence, pathophysiology, and optimal management.

EERD 2. The Neonatal Testosterone Surge in Mice and the Programming of Adult Metabolism Across the Sexes
C. Hamson, A. Yasrebi, T.A. Roepke
Rutgers, The State University of New Jersey, New Brunswick, Endocrinology and Animal Biosciences Graduate Program; Department of Animal Sciences, Rutgers University

The neonatal testosterone (T) surge occurs shortly after birth in mice with testes and is responsible for sexual differentiation in the brain. However, the influence of the neonatal T surge in producing the differences across the sexes in the hypothalamic control of adult metabolism is largely unknown. Furthermore, the role of estrogen receptor alpha (ER alpha) signaling in developmental programming during the neonatal T surge is also unclear. Our hypothesis is that ER signaling is involved in, at least, part of the developmental programming that occurs in the hypothalamus when testosterone from the T surge is converted to estrogen in the brain via the enzyme aromatase. To manipulate the T surge, neonatal females (postnatal day 0/1) were injected with testosterone propionate (80 microg/20ml) to simulate a surge of testosterone while neonatal males were injected with formestane (25 microg/ml), an aromatase inhibitor, to prevent the neonatal T surge’s ability to activate ER signaling. Neonates were allowed to mature to early adulthood (PND57) prior to metabolic phenotyping. We found that manipulating the T increased an early developmental marker, anogenital distance, in females and induced a sex reversal in adult locomotor behavior (wheel running) and suppressed metabolic rates in adult females. Similar but inverse effects were observed in males exposed to formestane. Following the same injection paradigm, we also examined gene expression in in the mediobasal hypothalamus (MBH) in PND2 pups. Using bulk RNA sequencing, we found differences in gene expression within the sexes in pathways responsible development. By simulating the T surge in females, and blocking estrogen signaling in males, we demonstrated the crucial role that the neonatal T surge has in early-life programming in the hypothalamus to produce sex-dependent variability in adult metabolism.

EERD 3. Tirzepatide Reverses Extreme Insulin Requirements in BSCL2-Related Congenital Generalized Lipodystrophy
S. Katsoudas, S. Ten
Rutgers-Robert Wood Johnson Medical School Child Health Institute of New Jersey, Rutgers Health/Monmouth Medical Center, NJ

Background
Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by near-total absence of adipose tissue, severe insulin resistance, hypertriglyceridemia, diabetes mellitus, and ectopic lipid accumulation. BSCL2-related CGL (CGL type 2) is often associated with an early and severe metabolic phenotype, including hepatic steatosis and progressive liver disease.
Case Report
19-year-old female with biallelic pathogenic variants in BSCL2 was diagnosed with CGL at age 4 years. Clinical manifestations began with hepatosplenomegaly at 1 year. By age 3 years, she had developed severe insulin-resistant diabetes requiring insulin and hypertension. Generalized loss of subcutaneous fat, hepatosplenomegaly, hepatic steatosis, hypertriglyceridemia, and severe insulin resistance established the diagnosis of CGL.
Her subsequent course was complicated by poorly controlled diabetes, acanthosis nigricans, necrobiosis lipoidica diabeticorum, hyperlipidemia, diabetic nephropathy with proteinuria, neuropathy, ovarian failure, and progressive liver disease with steatosis, cirrhosis, and ascites.
Metreleptin was initiated at age 4 years; however, poor adherence was associated with worsening metabolic control. By age 19, she had extreme insulin resistance requiring 600 units/day of U-500 insulin, with an HbA1c of 10.3% despite intensive insulin therapy.
At age 19, tirzepatide (Mounjaro) was initiated at 2.5 mg once weekly. Glycemic control improved dramatically, allowing complete discontinuation of U-500 insulin, with normalization of glucose levels. Thus, insulin requirements decreased from 600 units/day to zero after initiating Tirzepatide. At age 20, Metreleptin 2 mg daily was added to her treatment regimen.
Conclusion
Low-dose tirzepatide produced a remarkable metabolic response in BSCL2-related CGL with extreme insulin resistance, reducing insulin requirements from 600 units/day to zero and achieving normalization of glucose levels. This response occurred before Metreleptin was reintroduced, suggesting an independent therapeutic effect of Tirzepatide. Dual GIP/GLP-1 receptor agonism may represent a promising therapeutic approach for severe insulin-resistant diabetes in generalized lipodystrophy.

EERD 4. Long-Term Metabolic Benefits of Tirzepatide in AGPAT2-Related Congenital Generalized Lipodystrophy
S. Aulas, S. Ten
Rutgers-Robert Wood Johnson Medical School Child Health Institute of New Jersey, Richmond University Medical Center, Staten Island, NY

Background
Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by a near-total absence of adipose tissue, severe insulin resistance, and early-onset diabetes. We report a sustained metabolic response to tirzepatide in a patient with severe insulin-resistant diabetes due to AGPAT2-related CGL.
Case report
An African American male diagnosed with diabetes at 9 years of age was referred to our center at age 11. Clinical findings included acanthosis nigricans, muscular extremities, and a normal BMI of 23.2 kg/m². Serum leptin was markedly reduced at 0.5 ng/mL (Nl, 1.4–16.5), triglycerides 238 mg/dL, HbA1c 14.7%. ALT 36 U/L and AST 34 U/L were elevated. Islet cell antibodies were negative.
Genetic testing identified a homozygous AGPAT2 c.IVS4-2A>G variant.
MRI revealed generalized muscle hypertrophy with markedly decreased subcutaneous and intra-abdominal fat, periarticular and intramedullary lytic bone lesions, and hepatomegaly.
Treatment with high doses of insulin up to 3 u/kg/day with addition of metformin was unsuccessful. After starting Metreleptin treatment within 1 month, we were able to stop insulin therapy. HbA1c improved from 11 to 5.9% after 6 months of therapy. Triglycerides and liver transaminases normalized.
Because of the burden of daily leptin injections, the patient declined to continue leptin therapy, and his HbA1c subsequently increased to 14%. At 22 years of age, tirzepatide (Mounjaro) therapy was initiated and titrated to 15 mg weekly. Treatment resulted in marked and sustained improvement in glycemic control, normalization of triglyceride levels, and resolution of hepatomegaly. During 3 years of therapy, HbA1c remained stable at 7.0–7.4%, triglycerides normalized, hepatomegaly resolved, and there was no evidence of hepatic steatosis.
Discussion:
Tirzepatide enhances glucose-dependent insulin secretion, suppresses glucagon secretion, and improves glycemic control and insulin sensitivity even in the setting of profound adipose-tissue deficiency.
Conclusion: Tirzepatide therapy may dramatically improve the metabolic complications in patients with CGL

EERD 5. Atypical Presentation of Type 2 Diabetes with Diabetic Ketoacidosis and Response to Tirzepatide
Muhammad Jawad Hassan, MD1, Svetlana B. Ten, MD 2
Richmond University Medical Center. Staten Island, NY 1, Rutgers-Robert Wood Johnson Medical School Child Health Institute of New Jersey, NJ2

Background
Diabetic Ketoacidosis (DKA) is traditionally viewed as the hallmark presentation of autoimmune Type 1 Diabetes; up to 10% to 25% of children and adolescents with newly diagnosed with Type 2 Diabetes present initially with DKA.
Case report
A 13-year-old boy presented with a 1-week history of nausea, three episodes of vomiting, and abdominal pain. He has severe class III obesity, with a weight of 129 kg and BMI of 44.27 kg/m², and has acanthosis nigricans. At presentation, labs showed diabetic ketoacidosis, with a pH of 7.12, bicarbonate of 10.4 mmol/L, β-hydroxybutyrate >6 mmol/L, and glucose of 404 mg/dL.
Following ICU treatment with intravenous insulin, he required insulin replacement more than 1 u/kg: (glargine 80 units/day; carbohydrate ratio 1:5; correction 1 unit:15 mg/dL). C-peptide was 0.81 ng/mL (normal 0.8–3.5). Islet cell, GAD, IA-2, insulin, and ZnT8 antibodies were negative.
One week after DKA resolution, insulin was discontinued, and Tirzepatide (Mounjaro) 2.5 mg weekly was initiated, resulting in normoglycemia without insulin therapy.
He has a strong family history of diabetes. The patient’s family is from Pakistan. His brother, father, mother, MGF, PGF, PGM, and three paternal aunts have diabetes and are treated with Janumet. A 28-gene Invitae monogenic diabetes panel was negative; whole-genome sequencing is planned.
Discussion
Severe glucose toxicity combined with profound insulin resistance can cause a functional failure of the pancreatic beta cells. This mimics the absolute insulin deficiency of Type 1 Diabetes and precipitates severe ketoacidosis. This presentation is increasingly observed in adolescents with morbid obesity and insulin resistance.
Conclusion
This case highlights the potential role of Tirzepatide in improving glycemic control and reducing insulin requirements in adolescents with severe obesity, marked insulin resistance, and ketosis-prone diabetes.

EERD 6. Biphasic MODY7 Due to a Novel KLF11 Variant Responsive to Semaglutide (Ozempic)
A. Makar, J. Matthew, A. Khattab, S. Ten
Rutgers-Robert Wood Johnson Medical School, Child Health Institute of New Jersey

Background
A biphasic glycemic trajectory as early-onset neonatal diabetes with remission in infancy followed by recurrence during adolescence is well described in monogenic KATP-channel disorders (KCNJ11, ABCC8) and transcription factor defects (HNF4A, INS). Our case presents for the first time a biphasic presentation of the novel MODY 7 variant: heterozygous KLF11 c.1259-2_1259-1del splice variant, which may produce a similar remitting–relapsing phenotype.
Case Report
We report a unique case of a 15-year-old girl diagnosed with MODY 7. She was initially seen at 13 yrs. of age. Her BMI was 18.1 kg/m2. She has mild developmental delay, ADHD, tall stature. Physical examination was unremarkable. She has a strong family history of diabetes. Mother has prediabetes, and MGF has type 2 diabetes.
The patient initially presented with neonatal diabetes in Ukraine, treated with insulin until 8 months of age, when she experienced a hypoglycemic coma, after which insulin was discontinued, and normoglycemia persisted for 14 years. At 15.4 years of age, she developed recurrent diabetes (HbA1c 8%), which responded to low-dose Ozempic therapy 0.25 mg weekly, achieving HbA1c of 5.6%. Islet cell autoantibodies were negative. Genetic testing was done as part of the Invitae Monogenic Diabetes Panel and revealed KLF11, lntron 3, c.1259-2_1259-1del (Splice site), heterozygous, Uncertain Significance
The patient additionally has a 47,XXX karyotype associated with developmental delay, ADHD, tall stature, and premature ovarian insufficiency diagnosed at 14 years of age. Gonadotropins were elevated and anti-Müllerian hormone was markedly reduced. She is receiving transdermal estrogen replacement therapy.
Conclusion: The heterozygous KLF11 c.1259-2_1259-1splice del VUS represents a potentially pathogenic splice-altering variant that may contribute to impaired insulin secretion via disruption of the KLF11–PDX1 transcriptional pathway. The marked glycemic response to low-dose Semaglutide further suggests that GLP-1 receptor agonism may preserve and augment residual β-cell function in this remitting–relapsing phenotype.

EERD 7. Semaglutide treatment was associated with ≥5% BMI reduction in 55.7% of adolescents
R. Daly, J. Matthew, E. Lupiano, D. Matthew, I. Mamkin, M. Gangat, I. Marshall, A. Khattab, A. Bhangoo, S.Ten
Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, Unterberg Children’s Hospital, Monmouth Medical Center, RWJBarnabas Health, NJ, Cooperman Barnabas Medical Center, Newark Beth Israel Medical Center, RWJBarnabas Health, NJ, Rutgers-Robert Wood Johnson Medical School and Child Health Institute of New Jersey, CHOC Hospital, Orange, California

Background
Variability in response to Semaglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, remains incompletely understood. Potential contributors include differences in GLP-1R expression and signaling, genetic susceptibility, and metabolic and neuroendocrine pathways regulating appetite, satiety, and energy balance. We evaluated BMI responses in adolescents receiving Semaglutide.
Methods
We conducted an IRB–approved retrospective chart review of 122 adolescents with severe obesity and BMI >30 kg/m² who received Semaglutide for 6 months to 2 years. We stratified patients into four groups by percentage BMI change. We assessed changes in triglycerides, the triglyceride-to-HDL cholesterol (TG/HDL) ratio, and HbA1c.
Results
Mean age was 15 ± 2.3 years, mean baseline BMI was 41.5 ± 8.7 kg/m², and 76 patients (62.3%) were female. Overall, 68 patients (55.7%) achieved ≥5% BMI reduction.
Thirty-five patients (28.7%) achieved ≥10% BMI reduction, with BMI decreasing from 39 ± 7.45 to 31.4 ± 6.6 kg/m². Mean reduction was 19 ± 7.9%; 2 patients had diabetes.
Thirty-three patients (27.1%) achieved 5–9% BMI reduction, with BMI decreasing from 41.6 ± 7.2 to 38.2 ± 6.9 kg/m². Mean reduction was 6.6 ± 1.3%; 7 had diabetes.
Twenty-four patients (19.7%) gained weight, with BMI increasing from 42.6 ± 11.4 to 45.13 ± 12.5 kg/m². Mean increase was 5.7 ± 4.2%; 5 had diabetes.
Thirty patients (24.6%) had <5% BMI reduction, with BMI decreasing from 43.3 ± 8.5 to 42.3 ± 8.4 kg/m². Mean reduction was 2.3 ± 1%; 6 had diabetes.
Greater BMI reductions were associated with significant improvements in triglycerides, TG/HDL ratio, and HbA1c. Some patients who gained weight nevertheless showed improvements in triglycerides and insulin resistance.
Conclusion
Semaglutide produced ≥5% BMI reduction in 55.7% of adolescents. Variable weight responses and metabolic improvements despite weight gain support individualized treatment strategies.

EERD 8. Semaglutide (Wegovy) treatment in Alstrom Syndrome
Joshua Matthew, MS4 1Ahmed Khattab MD2, Svetlana Ten, MD 2
Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ1, Rutgers-Robert Wood Johnson Medical School and Child Health Institute of New Jersey 2

Background
Alstrom syndrome is a rare autosomal recessive ciliopathy caused by biallelic variants in ALMS gene. It is associated with severe early-onset obesity and progressive multisystem involvement. Limited data are available regarding response to GLP-1 receptor agonist therapy in patients with suspected or confirmed Alstrom syndrome.
Case Report
A 15-year-old girl with class III obesity, impaired fasting glucose, and dysmenorrhea demonstrated substantial improvement during Semaglutide (Wegovy) therapy.
Family history was notable for obesity and type 2 diabetes in her father, who previously underwent sleeve gastrectomy and is currently treated with metformin and Semaglutide.
Initial BMI was 41 kg/m2. After 1 yr of treatment, weight decreased from 116.9 kg to 90.9 kg, representing a 26.0-kg loss (22.2%), while BMI decreased from 41.03 to 31.98 kg/m2, a 22.1 % reduction.
Genetic testing through Prevention Genetics identified compound heterozygous ALMS1 variants of uncertain significance.
She has no reported history of visual or hearing impairment. Cardiology evaluation found no evidence of dilated or restrictive cardiomyopathy. Echocardiography identified an incidental aortic valve Lambl’s Excrescence.
Menstrual cycles and impaired fasting glucose normalized during treatment.
Conclusion
This case highlights a strong clinical response to Semaglutide in an adolescent with severe obesity and reported ALMSl variants suggestive of Alstrom syndrome. Although the molecular diagnosis needs further clarification, the response suggests that GLP-1 receptor agonist therapy may be a promising option for obesity management in patients with suspected ciliopathy-related obesity.

EERD 9. Exposure to hog dust extract disrupts cyclic regulation of gut estrogen deconjugation in femal mice.
L. Andrews, J. Cruz-Arriaga, T. Fanslow, M. Crawford
Rutgers University Department of Animal Sciences, Graduate Program in Endocrinology and Animal Biosciences; Rutgers University MBS; Rutgers University School of Arts and Sciences

Concentrated animal feeding operations (CAFOs) have contributed to an increased presence of airborne biological/chemical agents in these facilities and surrounding residential communities, increasing the risk of respiratory, gastrointestinal and metabolic complications. There has been growing public attention on the increasing prevalence of infertility in these areas, along with the suggestion that environmental pollutants are a contributing factor to ovarian dysfunction and reduced sperm quality. The endocrine system relies partly on intestinal homeostasis, with a broad range of gut microbes producing β -glucuronidase (gmGUS), an enzyme essential for estrogen deconjugation and reabsorption. We have previously demonstrated that intranasal exposure to CAFO emissions (hog dust extract; HDE) for 15-days promotes chronic airway inflammation, increases intestinal permeability and alters commensal gut microbial communities in mice, highlighting the importance of the gut-lung axis. Therefore, the aim of our current study is to characterize estrous cycle stage dependent dysregulation of gut luminal β -glucuronidase activity following hog dust extract (HDE) exposure. Briefly, 8-week-old female C57/BL6 mice (n=8) were intranasally exposed to saline (n=4) or HDE (n=4) for 30 days. Bronchoalveolar lavage fluid from HDE-treated mice showed no changes in macrophage cell numbers (unpaired t-test, p=0.1077), but exhibited elevated neutrophil (immature neutrophils, p=0.0033 and mature neutrophils, p<0.0001) and eosinophils (p<0.0001) levels in comparison to saline controls, indicating the presence of an inflammatory response within the lungs. Measurement of intestinal permeability showed a trend in increased FD4 permeability (p=0.0795) and a significant increase in RD70 permeability (p=0.0058) in HDE-treated mice. Moreover, ovary weight was significantly higher in HDE-treated mice (p=0.0121) and HDE exposure disrupted cycle-stage specific fecal estrogen excretion in estrus (p=0.0449) and metestrus (p=0.0110). Additionally, fecal gmGUS activity during the estrous cycle showed an increased trend in HDE-treated mice (Cycle 4; p=0.0948) in comparison to saline controls. Moreover, 16S rRNA sequencing of ileal, cecal and proximal colon luminal contents revealed a shift in several hormone-associated bacterial taxa: Erysipelotrichia relative abundance was significantly reduced by HDE in ileum (p=0.0486) and cecum (p=0.0351); Prevotellaceae relative abundance was nearly abolished by HDE in cecum (p=0.0252) and proximal colon (p=0.0086); Betaproteobacteria relative abundance was significantly reduced by HDE in cecum (p=0.0123); and Lachnospiraceae relative abundance was significantly increased by HDE in ileum (p=0.0373). Together, these findings suggest that airway exposure to CAFOs dusts promotes airway inflammation and can access the gastrointestinal tract to promote dysbiosis and alter levels of fecal estrogen and gmGUS during estrous. Further identification of mechanistic interactions between the gastrointestinal and reproductive system are imperative to understand the complex physiological consequences of chronic exposure to organic dusts derived from CAFOs on downstream reproductive outcomes.

EERD 10. Adipocyte-Specific Overexpression of FGF21 Promotes Lipogenic Gene Expression in Visceral Adipose Tissue
C. Gliniak, T. Kulkarni, M. Vudata, J. Evelyn
Department of Nutritional Sciences, Rutgers, New Brunswick

The mechanisms underlying fat formation and loss can help researchers understand how to treat obesity-related diseases in humans. Chronic obesity leads to unhealthy adipose tissue, which is linked to inflammation and dysregulated metabolism underlying diseases such as obesity, type 2 diabetes, and cardiovascular disease. We study visceral fat depots of white adipose tissue (WAT), which is considered the pathological fat depot, versus subcutaneous fat, which is protective in mice and humans. Adipose tissue not only stores energy but is an endocrine organ. Fibroblast growth factor 21 (FGF21) is secreted by liver and adipose tissue is associated with increased longevity, increased insulin sensitivity, and decreased liver steatosis.

It is unclear whether the benefits come from FGF21 secreted by adipose tissue, resulting in a paracrine effect, or from stress-induced secretion of FGF21 by the liver.

To examine adipocyte-specific FGF21 secretion and its metabolic outcomes, we crossed whole body FGF21KO mice with inducible, adipocyte-specific FGF21 overexpression mice. Adult male and female mice were fed a high-fat diet with doxycycline (to activate FGF21 expression) for 4 weeks.

To study the effect of adipocyte-specific FGF21 secretion, we analyzed gene expression of markers for healthy adipocyte genes in epididymal white adipose tissue (eWAT) using qPCR. Compared to the control group, FGF21 overexpression increased fatty acid synthase (Fasn), adiponectin (Adipoq), sterol-regulating binding protein (Srebp-1c), peroxisome proliferator-activated receptor gamma (Ppary).

Furthermore, there was no change in methionine aminopeptidase 2 (MetAP2) between the groups, indicating that FGF21 specifically increased lipogenic gene expression.

As hypothesized, our findings suggest that, in the absence of liver FGF21, adipose tissue-derived FGF21 significantly increases the expression of these genes in eWAT that promote healthy lipogenesis. We also found that there are no sex differences for these genes. This data supports future study of FGF21’s role in adipose tissue, and the possibility to improve metabolic health by increasing adipose FGF21 by lifestyle or pharmacological means.

EERD 11. Sex-Specific Cellular and Molecular Responses Associated with Binge Ethanol–Induced Splenic Atrophy
W. Huang, M. Bishir, X. Liu, S. Chang
Institute of NeuroImmune Pharmacology, Seton Hall University, South Orange, USA; Huazhong University of Science and Technology, Wuhan, China

Binge ethanol (EtOH) exposure induces splenic atrophy in adolescent F344 rats, with males and females showing differential reductions in spleen size despite identical EtOH exposure. Here, we used single-cell RNA sequencing (scRNA-seq) to define cell-type-specific transcriptional responses associated with the severity of EtOH-induced splenic atrophy. Adolescent F344 rats received binge EtOH (4.8 g/kg/day; 52% w/v; i.g.) for 3 days at pubertal onset and were sacrificed 24 h after the final exposure. Cell-type-specific pseudobulk profiles were analyzed using DESeq with a model incorporating treatment and spleen size as a continuous variable to identify severity-associated genes while accounting for treatment effects. The severity-associated differentially expressed genes were analyzed using Ingenuity Pathway Analysis (IPA). Our analysis revealed distinct sex-specific cellular and molecular responses were associated with splenic atrophy. In males, cytotoxic T cells showed the strongest response, with 182 severity-associated genes, followed by B cells (70 severity-associated genes), CD4 T cells (31 severity-associated genes), and monocytes (26 severity-associated genes). T and B cells exhibited inflammatory and interferon/TLR-related signatures, while cytotoxic T cells showed pro-apoptotic and cytotoxic programs, including necroptosis and TP53-associated responses. These findings suggest that cytotoxic T cells represent a major vulnerable population in male splenic atrophy, whereas B cells showed relatively resistant survival-associated responses. In females, proliferative cells showed the strongest response (138 severity-associated genes), followed by dendritic cells (60 severity-associated genes), plasma cells (31 severity-associated genes), B cells (22 severity-associated genes), and neutrophils (13 severity-associated genes). Interferon/antiviral signaling was prominently activated in proliferative cells, neutrophils, and B cells, accompanied by altered proliferative and cell-death programs. Notably, proliferative cells were observed only in females. These findings reveal sex-specific cellular vulnerabilities associated with EtOH-induced splenic atrophy. In males, greater atrophy was associated primarily with transcriptional changes in cytotoxic T cells and inflammatory pathways, whereas in females, atrophy was more strongly associated with proliferative cell populations and interferon-related responses.

EERD 12. GLP-1 Receptor Agonist Semaglutide Impacts Estrous Cycle in Mice
K. Hines and J.Barker
Drexel University College of Medicine

The glucagon-like peptide-1 (GLP-1) receptor agonist semaglutide is used for the treatment for type 2 diabetes and obesity and is a promising novel option for treating alcohol use disorder. Semaglutide is known to interact with the estrogen system, and the GLP-1 and estrogen hormone systems working in tandem may lead to greater treatment efficacy. Higher plasma GLP-1 and GLP-1 gene expression have been seen during higher estradiol phases in the menstrual cycle in humans and the estrous cycle in rats, suggesting estradiol may modulate GLP-1. However, less is known about potential reciprocal effects where GLP-1 receptor agonists may impact estradiol and the estrous cycle. To begin to understand this, we assessed the impact of semaglutide on the estrous cycle. Female C57BL/6J mice received daily subcutaneous injections of either semaglutide (n = 12; 0.026 mg/kg) or vehicle (n = 12; 1 ml/kg; tris-HCl Buffer and 0.02% Tween 80) for 30 days. Estrous cycles were monitored via daily vaginal smears starting at least three cycles prior to any injections and continued throughout the study. Food and water intake were measured starting one week prior to injections. Semaglutide-treated mice showed a decrease in weight, but no significant differences in water or food intake. Mice that received semaglutide had a greater number long estrous cycles (greater than 6 days) compared to vehicle animals pointing to a possible toward disruption of normal hormonal cyclicity. In order to identify potential brain regions activated by a semaglutide injection, ongoing studies will compare areas including the nucleus accumbens, medial preoptic area, prefrontal cortex, ventral tegmental area, and the hippocampus. These areas have been implicated in the effects of semaglutide and are also sensitive to the estrous cycle. Our current results suggest that semaglutide disrupts the estrous cyclicity in mice which warrant additional studies to explore the interplay between GLP-1 and estrogen systems.

 

Nutrition, Metabolism, and Mental Health (NMMH)

NMMH 1. Investigation of hemp extract for cardiometabolic improvement in postmenopause
H. Palmer, G. Haag, N. Dreher, B. Rosas De Leon, D. Roopchand
Department of Food Science and NJ Institute of Food Nutrition and Health, Rutgers University, New Brunswick, NJ 08901

The reduction of 17β-estradiol (E2) in postmenopause is associated with a range of metabolic disorders. Surveys and online posts reveal that women are using phytocannabinoid preparations to alleviate menopause related symptoms in the absence of clinical support. Cannabidiol (CBD) is the primary phytocannabinoid found in Cannabis sativa L. Our prior work was the first to test the effect of CBD on metabolic health in postmenopause using an ovariectomized (OVX) mouse model. OVX mice supplemented with CBD had improved metabolic and bone phenotypes. The objective of the present work was to investigate how full spectrum hemp extract (containing CBD and other cannabinoids) affects exercise capacity, behavior, and metabolic endpoints in OVX and sham surgery (SS) mice. Following OVX or SS, female C57BL/6J mice were perorally administered either hemp extract (HEMP) containing 25 mg CBD/kg or vehicle (peanut butter and coconut oil) daily for 12 weeks (n = 8-9/group). Exercise power and endurance were tested during treatment weeks 1, 3, 6, and 11. Both OVX and SS HEMP-treated mice showed improved power but not endurance. After 9 weeks of treatment, Y-maze, open field test (OFT), novel object recognition (NOR), and elevated plus maze (EPM) behavioral tests were performed. OVX+HEMP and SS+HEMP mice showed increased locomotor activity compared to their vehicle-treated counterparts. Cognition was unaffected in HEMP-treated OVX mice but reduced in HEMP-treated SS mice. Metabolic phenotyping included weekly body weight measurements and an oral glucose tolerance test conducted during the 12th week of the treatment. Hemp extract reduced OVX-induced weight gain. Oral glucose tolerance decreased in HEMP-treated SS mice but was unaffected in HEMP-treated OVX mice. These results suggest the effects of hemp extract on exercise, behavior, and metabolic health may depend on hormonal status.

NMMH 2. Respiratory Effects of Ethanol and Mitragynine: Changes in Breathing Rate and Minute Ventilation in Male Mice
P. Kesale, S. Fields, N.T. Bello
Rutgers University Department of Animal Sciences, Rutgers University SAS Cell Biology and Neuroscience

Kratom (Mitragyna speciosa) is widely used for pain management and opioid withdrawal, yet its respiratory effects when combined with alcohol remain poorly understood. Because mitragynine and ethanol produce central nervous system depression through distinct pharmacological mechanisms, co-administration may increase the risk of respiratory impairment. This study evaluated the effects of mitragynine and ethanol, administered individually and in combination, on respiratory function in 8 week old male C57BL/6J mice. Mice received an intraperitoneal injection of vehicle or mitragynine (56 mg/kg), followed by oral gavage with distilled water or ethanol (5 g/kg). Respiratory function was assessed using whole-body plethysmography by measuring breathing rate (breaths per minute; BPM) and minute ventilation (MVb) over a 90-minute recording period. Breathing rate and minute ventilation were analyzed using repeated-measures ANOVA or mixed-effects analysis, and area under the curve (AUC) values were analyzed using a two-way general linear model (GLM). Ethanol significantly reduced breathing rate, and combined mitragynine and ethanol treatment produced the greatest reduction in breathing rate, resulting in a significant treatment interaction. Ethanol also significantly reduced overall minute ventilation (MVb AUC; p = 0.0421), whereas mitragynine pretreatment and the interaction between mitragynine and ethanol were not statistically significant. Minute ventilation changed significantly over time, although treatment differences were not statistically significant. These findings suggest that combined exposure to mitragynine and ethanol may produce greater respiratory impairment than either treatment alone and support further investigation into the respiratory effects associated with combined kratom and alcohol use.

NMMH 3. A Genetically Encoded Optical Sensor for Real-Time Detection of GLP-1 Signaling In Vivo
V. Mirabella, A. Saber, F. Luo, Y. Lu, R. Savani, L. Wang, Z.P. Pang
Center for NeuroMetabolism, Child Health Institute of New Jersey, Rutgers University

Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived neuropeptide produced primarily by intestinal L-cells and brainstem nucleus tractus solitarius (NTS) preproglucagon neurons, where it signals satiety following food intake. GLP-1 signals through the GLP-1 receptor (GLP-1R), a G protein-coupled receptor, rendering conventional electrophysiological approaches insufficient for resolving GLP-1 release dynamics with high spatial and temporal resolution. Here, we describe the development and validation of a novel genetically encoded optical sensor, i.e., the Reporter for Transmission mediated by G-protein-coupled Receptor (RTGR), for GLP-1. GLP-1 RTGR was engineered by inserting a circularly permuted GFP (cpGFP) into the stalk domain, a segment linking the extracellular domain to the transmembrane region, of GLP-1R, enabling robust, real-time fluorescence responses to GLP-1. In vitro characterization in a heterologous expression system shows that GLP-1 RTGR localizes to the plasma membrane and exhibits high sensitivity and specificity for GLP-1 relative to other proglucagon-derived peptides. In paraventricular nucleus (PVN) brain slices expressing GLP-1 RTGR, trains of high-frequency electrical stimulation increased fluorescence signal, with no responses observed under control conditions. Using fiber photometry in vivo, GLP-1 RTGR signals were detected in the PVN during prolonged high-frequency optogenetic stimulation of NTS GLP-1 neurons in Gcg-Cre::GLP-1R-Cre mice expressing GLP-1 RTGR in the PVN and a red-shifted opsin in the NTS. No responses were observed under control conditions or following brief stimulation, suggesting sustained neuronal activity is required for GLP-1 release in vivo. In freely moving mice, longitudinal overnight fiber photometry recordings revealed prandial state-dependent changes in GLP-1 signaling in the PVN, with GLP-1 RTGR fluorescence increasing following each eating bout. Notably, preproglucagon knockout mice showed disrupted feeding behavior and lacked the signaling dynamics observed in control animals. These findings support a role for GLP-1 signaling in satiety and refeeding, consistent with previous studies implicating central GLP-1 in feeding regulation. In summary, these results indicate GLP-1 RTGR as a powerful and validated tool for tracking real-time GLP-1 dynamics in vivo, providing new insight into the neuroendocrine mechanisms underlying appetite regulation. Future work will define in vivo spatiotemporal GLP-1 release and mechanisms governing hypothalamic neuropeptide release.

NMMH 4. All-trans-retinoic acid signaling in lung macrophage differentiation and polarization
D. LoBiondo, S. Hajjam, I. Dar, O. Ilnytska, I. O. Shmarakov
Rutgers University Department of Animal Sciences; Rutgers University Graduate Program in Endocrinology and Animal Biosciences

Acute lung injury (ALI) is a pathophysiological condition characterized by excessive inflammation, lung damage, and a high mortality rate. Lung macrophages (resident alveolar and interstitial, as well as recruited populations) are the main drivers of inflammation in ALI that undergo transcriptionally regulated differentiation and polarization within the lung tissue to initiate and resolve inflammation. All-trans-retinoic acid (ATRA), the most active vitamin A metabolite and a ligand for nuclear hormone receptors, retinoic acid receptors (RARs), controls the transcription of more than 500 genes, including ones involved in inflammation. We hypothesize that ATRA-RAR signaling contributes to ALI-associated inflammation by regulating macrophage transcriptional responses. Our goal is to understand the mechanism behind ATRA actions in macrophage differentiation and polarization, ultimately leading to potential therapeutic strategies to lessen the severity of ALI.
To test the hypothesis, we employed 1) pharmacological manipulations of cultured bone marrow-derived macrophages (BMDM), an in vitro model of recruited macrophage differentiation and polarization, using RAR isoform-specific agonists and antagonists, and 2) genetic manipulation of ATRA signaling in alveolar macrophages (AM-Rardn mice), an in vivo model of altered macrophage RAR-mediated signaling (Rardn transgene overexpression using Cd11c-driven Cre) during ALI.
Our data indicate that BMDM cultures significantly upregulate expression of genes necessary for retinoid uptake and ATRA synthesis during differentiation and maintain steady mRNA levels of these genes during polarization into either pro-inflammatory (M1) or pro-resolution (M2) functional phenotypes. Among RAR isoforms, Rarα and Rarγ mRNA expression were significantly higher in fully differentiated naïve BMDMs than in undifferentiated precursors, while Rarβ expression remained unchanged. Furthermore, mature macrophage polarization resulted in significantly upregulated Rarγ expression in proinflammatory macrophages (M1 phenotype), with no changes in Rarα expression and downregulation of Rarβ transcription. Treatment of mature BMDM cultures with Rarγ antagonist during polarization resulted in significantly downregulated expression of M1-associated proinflammatory genes (Tnf-α, Il-1β, Nos2), with no changes in M2 marker gene expression (Arg1, Chil3, Mgl1).
Consistent with studies using pharmacological inhibition of RARs, genetic ablation of ATRA signaling in mature Cd11c-expressing macrophages was associated with significant downregulation of proinflammatory gene expression during polarization of BMDM cultures derived from bone marrow of AM-Rardn mice. Furthermore, during LPS-induced ALI, AM-Rardn mice exhibited improved recovery of lung function, as assessed by whole-body plethysmography, and improved overall survival over the 7-day period following LPS instillation.
Our data provide experimental evidence that retinoid signaling is integral to the transcriptional programs of macrophage differentiation and polarization. Targeting ATRA-RAR-mediated signaling can serve as a strategy to reduce macrophage-mediated acute inflammation during ALI
Supported by the NIH grant HL171112.

NMMH 5. 𝛽-carotene oxygenase 2 deficiency impairs early postnatal cardiac growth through altered retinoic acid signaling and cardiomyocyte cell cycle regulation
E. Madonna, A. Glazer, Y. Kim, C. Holloway, L. Quadro
Nutritional Science Graduate Program, Department of Food Science, Rutgers Center for Lipid Research & Institute of Food Nutrition and Health, Rutgers University, New Brunswick, New Jersey, USA; NIH/National Cancer Institute (NCI), Molecular Targets Program, Center for Cancer Research, NCI, Frederick, MD

Vitamin A is an essential micronutrient that supports many critical biological functions in mammals[1]. Our laboratory demonstrated that three-month-old female mice lacking β-carotene9′,10′-oxygenase (Bco2-/-), the only enzyme of the adult heart that cleaves β-carotene into retinoids (vitamin A and its derivatives), display cardiac retinoic acid (RA) insufficiency [2]. We also found that the hearts of both male and female Bco2-/- mice are smaller than wild-type (WT) controls as early as postnatal day 4 (P4), suggesting impaired postnatal heart growth.
During the first week after birth, cardiomyocytes proliferate before permanently exiting the cell cycle and transitioning to hypertrophic growth [3]. Preliminary analyses revealed that Bco2-/- females’ hearts show a reduced number of nuclei and fewer mitotic events at P4. Furthermore, mRNA analysis of cell cycle checkpoint markers revealed that both cell cycle-promoting cyclins (Ccb1, Ccnd2, Ccne1) and inhibitors (p18, p21, p27) were downregulated in the P4 mutant hearts, suggesting that cell cycle progression may be attenuated, thereby reducing proliferation at this early postnatal age. Moreover, the transcriptional factor Meis1, which promotes cell cycle arrest and regulates p21 [4], was downregulated in Bco2-/- hearts at P4. Interestingly, Meis1 is regulated by RA [5], therefore suggesting that RA levels are reduced in the hearts of the Bco2-/- mice at P4.
Taken together, these and other data suggest that low RA levels in the hearts of Bco2-/- mice may contribute to reduced proliferation P4, providing a potential explanation for the smaller heart phenotype of the mutant mice.

NMMH 6. RETINOL BINDING PROTEIN 4 EXPRESSION IN MACROPHAGE POLARIZATION
S. Hajjam, D. LoBiondo, I. Dar, O. Ilnytska, I.O. Shmarakov
Department of Animal Sciences, Rutgers,The State University of New Jersey, New Brunswick, NJ

Retinol binding protein 4 (RBP4) is a low-molecular-weight (~21 kDa) carrier protein with a characteristic β-barrel structure, primarily responsible for delivering all-trans-retinol (vitamin A) through the bloodstream. While the majority of circulating plasma RBP4 is produced and secreted by hepatocytes, a growing body of evidence indicates that other cell types, including white and brown adipocytes, lung fibroblasts, and intestinal enteroendocrine cells, express RBP4. Moreover, RBP4 has been shown to influence immune and inflammatory mechanisms in adipose tissue and vasculature in a paracrine manner, contributing to the progression of metabolic disease, suggesting that RBP4’s function in the body may extend beyond vitamin A transport. Consistent with these reports, we have identified that during acute lung injury (ALI), RBP4 contributes to inflammatory responses that drive lung inflammation in ALI. Moreover, we have identified two pools of RBP4 in the lung: circulating hepatocyte-derived RBP4 and locally expressed RBP4 by lung cells, including lung macrophages. We hypothesized that RBP4 regulates lung inflammation by affecting the responsiveness of lung macrophages, the predominant cell type that regulates the initiation and resolution of inflammation following lung injury. We tested our hypothesis using bone marrow-derived macrophage (BMDM) cultures and animal models of lung injury.
Using mouse BMDM cultures, we identified Rbp4 expression by macrophages and observed an increase in its expression during differentiation from precursor cells into mature naïve (M0) macrophages. Furthermore, we identified that macrophage polarization is associated with Rbp4 downregulation during the transition from naïve to proinflammatory (M1) state and significant, robust Rbp4 upregulation during the transition from naïve to pro-resolution (M2) macrophages. Moreover, specific M2-associated upregulation of Rbp4 expression and its secretion into the media was further confirmed by ELISA. Further studies using human derived macrophages from the THP-1 cell line confirmed the same rbp4 expression pattern in human polarized macrophages.
Using immunofluorescent staining of fixed mouse lung tissue sections, we identified a colocalization of Rbp4 immunoreactivity associated with cells positive for F4/80, a general macrophage marker.

NMMH 7. ADAD1 forms an undescribed post-meiotic germ cell granule that may regulate transcript-specific translation
E. Snyder, S. Potgieter, A. Badrinath
Rutgers University

Mammalian male germ cells are highly reliant on translation regulation as they undergo genome compaction prior to completing their differentiation. During the later stages of their differentiation, in which they are transcriptionally silent, translation regulation is mediated in large part by RNA binding proteins (RBPs), many of which are poorly understood. ADAD1 is a testis-specific RBP expressed in meiotic and post-meiotic germ cells that is required for male fertility in both mice and men. Previous work has suggested ADAD1 acts as a transcript-specific translation activator, as Adad1 knockout mice (Adad1-/-) exhibit reduced ribosome association of and delayed protein production from select transcripts important for post-meiotic differentiation. This work demonstrates that ADAD1 forms a previously uncharacterized post-meiotic cytoplasmic condensate, which also contains pumilio 1 (PUM1), a known translation regulator. Further, we identify a developmentally dependent association of the ADAD1 granule with another post-meiotic condensate implicated in a wide range of post-transcriptional regulatory events, the chromatoid body. Finally, motif analysis of transcripts impacted by ADAD1 loss revealed a significant enrichment of PUM1 binding sites in their 3’ UTRs. Together, these results suggest that the ADAD1 granule acts to regulate translation of specific transcripts in post-meiotic male germ cells.

NMMH 8. Hepatic Activating Transcription Factor 5 and the Metabolic Response to Dietary Protein Restriction
F. C. Rodriguez-Polanco, E. M. Lopez, R. C. Wek, T. G. Anthony
Department of Nutritional Sciences, Rutgers University, New Brunswick, New Jersey, USA; Indiana University School of Medicine, Indianapolis, Indiana, USA

Dietary protein restriction (PR) is a mild form of amino acid insufficiency (AAI) that is associated with improved metabolic health via fibroblast growth factor 21 (FGF21), a hormone that regulates energy homeostasis and metabolism. AAI activates a cell-protective mechanism known as the integrated stress response (ISR). ISR activation enhances the synthesis of the structurally similar activating transcription factors (ATF) 4 and ATF5. In vivo, the loss of ATF4 delays, but does not prevent, the production of FGF21 associated with PR. The interplay between ATF4 and ATF5 in regulating FGF21 production and resulting effects on metabolism is unknown. We hypothesized that ATF5 supports FGF21 production and associated changes in energy expenditure and body composition during PR. C57BL6/J mice (male and female) with liver-specific deletion of ATF5 (lsAtf5), ATF4 (lsAtf4), and combined (lsAtf4/Atf5), were fed a normal (18% protein by kcal) or PR (4% protein by kcal) diet, for up to 7 days alongside floxed littermates as intact controls. We analyzed biomarkers of the ISR and monitored metabolism by measuring food intake, energy expenditure by indirect calorimetry, and body composition by magnetic resonance imaging. The hepatic Atf5 mRNA abundance was highly increased in lsAtf4 male mice after 4 and 7 days of PR compared to floxed, suggesting a contribution of hepatic Atf5 in the absence of Atf4. In the lsAtf4/Atf5 male mice, the hepatic Fgf21 mRNA was blocked after 12 hours, 4 days and 7 days of PR, but it was not blocked in the lsAtf4 and lsAtf5 mice. In addition, hepatic Atf5 loss partially contributes to phenotypic changes in body weight after 7 days of PR in male mice. Finally, sex differences were observed in the response to PR, with female mice showing less substantial but similar patterns to males. Our results demonstrated that the loss of hepatic Atf4 and Atf5 prevent the production of Fgf21 and its mRNA associated with PR in male mice. These findings demonstrate that hepatic Atf5 plays a complementary role in ISR execution during PR-mediated metabolism and suggest a functional overlap of hepatic Atf4 and Atf5.

NMMH 9. Assessing the Roles of Transcription Factors ATF4 and CHOP in Hepatic Autophagy During Activation of the Integrated Stress Response (ISR) by Endoplasmic Reticulum (ER) Stress
S. A. Farooq, T. J. Franklin, C. T. Bhavsar, E. M. Lopez, R. C. Wek, T. G. Anthony
Department of Endocrinology and Animal Biosciences, Rutgers University; Department of Nutritional Sciences, Rutgers University; Indiana University School of Medicine, Indianapolis, IN

ISR proteins, ATF4 and CHOP, can regulate autophagy during dietary and ER stress. Autophagy activation requires inhibition of the mTORC1 pathway, which can be mediated by ATF4 and CHOP. Using a mouse model of ER stress that mimics features of metabolic dysfunction-associated liver disease (MASLD), this study investigates the roles of ATF4 and CHOP in regulating hepatic autophagy. C57BL/6J mice of both sexes (Ddit3+/+ and Atf4+/+Ddit3+/+) and mice with hepatocyte-specific deletions of Ddit3 (encodes CHOP, Ddit3∆/∆) or both Atf4 and Ddit3 (Atf4∆/∆Ddit3∆/∆) were injected with tunicamycin (TUN, 1 mg/kg BW) to induce ER stress or vehicle (equal volume of 0.3% DMSO). Mice were euthanized 6 hours post-injection, and their livers were snap frozen in liquid nitrogen. ISR activity was determined by immunoblotting for p-PERK and p-eIF2a. Autophagy was assessed via RT-qPCR of Sqstm1/p62 and Map1lc3b, and immunoblotting for p62 and LC3-II. mTORC1 activity was evaluated by immunoblotting for p-S6K1. In all mice, TUN activated the ISR by increasing hepatic p-PERK and p-eIF2a levels compared to DMSO. Sqstm1/p62 expression and p62 protein levels were increased with TUN in both Ddit3+/+ and Atf4+/+Ddit3+/+ mice and this effect was absent in the Ddit3∆/∆ and Atf4∆/∆Ddit3∆/∆ mice. In all mice, TUN did not alter Map1lc3b expression relative to DMSO; however, LC3-II protein was reduced. Together, the p62 and LC3-II data suggest autophagy impairment during TUN. Additionally, TUN increased mTORC1 activity by elevating p-S6K1 levels compared to DMSO in all mice. This study highlights a novel interaction between ER stress and autophagy, where acute TUN exposure perturbs autophagy, presumably, by activating mTORC1. Additionally, TUN-mediated expression of p62, a known ATF4 gene target and involved in the selective autophagy process, also requires CHOP, underscoring the importance of multiple ISR transcription factors in autophagy. Given emerging evidence that autophagy may serve as a therapeutic target for MASLD, future studies will focus on further characterizing the role of these two ISR transcription factors in regulating autophagy.

NMMH 10. Bone, Energy Restriction, and Nuts (BERN) in Older Adults: A Randomized Controlled Trial Study Protocol.
S. Shapses, H. L. Ayvazian, A. Pacheco-Irizarri, Y. Schlussel, A. Spaeth, A. Shah
Department of Nutritional Sciences, Rutgers University
Institute of Food, Nutrition and Health (NJ-IFNH)
Department of Medicine, Rutgers Robert Wood Johnson Medical School; Department of Kinesiology & Health, Rutgers University

Weight loss improves metabolic health in older adults but is often accompanied by reductions in bone mineral density (BMD) and evidence shows it can increase fracture risk. Identifying dietary strategies that preserve bone during energy restriction is critical. In addition, older adults are at increased risk of sleep disturbances. Nuts and legumes are rich in monounsaturated fatty acids and protein and may support metabolic and skeletal health. However, their role during weight loss on bone and sleep outcomes in older adults remains unclear. This study examines skeletal health and other parameters during a modest calorie-restricted 6-month intervention in older adults (50–75 years) with overweight and obesity (BMI 25-42 kg/m²). Participants are randomly assigned to two isocaloric snacks designed to provide approximately 15% of daily energy needs. One snack is higher in fat (72%;peanut-based) and the other is higher in carbohydrate (73%; grain-based). All participants receive counseling on lifestyle changes by a registered dietitian weekly for the first 8 weeks and biweekly thereafter. The primary endpoint is change in total hip BMD assessed by dual-energy x-ray absorptiometry at baseline to 24 weeks. Secondary endpoints include areal BMD at additional sites (femoral neck, lumbar spine and radius), bone quality (using peripheral quantitative computed tomography), bone turnover and metabolic health markers, and sleep/circadian measures. Additional outcomes include physical function and cognition tests. The target sample size is 22 per intervention group, with recruitment of multiple cohorts planned to achieve a total of 44 participants. Recruitment, screening, enrollment and data collection is ongoing, and results will determine whether incorporating a higher in fat and protein snack, during a calorie-restricted diet attenuates skeletal loss and alters sleep and metabolic health in older adults.

NMMH 11. Maternal Exposure to PFAS Mixture Alters Offspring Growth and Adulthood Neurobehavior in Mice
C. Brown, J. LaChow, A. Yasrebi, A. Bernstein, T. Roepke
Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, New Brunswick, NJ; Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with widespread human exposure through consumer products, industrial uses, and drinking water. Although early-life PFAS exposure is increasingly associated with adverse developmental outcomes, its lasting effects on growth and neurobehavior remain incompletely understood, particularly following exposure to environmentally relevant mixtures. This study evaluated the impact of maternal exposure to a mixture of PFAS compounds on offspring growth and adulthood neurobehavioral outcomes in wild-type C57BL/6 mice. Female mice were administered a PFAS mixture containing PFOA, PFOS, PFNA, PFHxS, and GenX (0.32 mg/kg/day) in drinking water beginning one week before breeding and continuing through gestation and lactation to postnatal day (PND) 14. Offspring body weight was measured from birth to PND56, where body composition was evaluated using MRI. At nine weeks, avoidance and anhedonia-like behaviors were assessed using the open field, elevated plus maze, novelty suppressed feeding, and sucrose preference tests. Maternally exposed offspring exhibited significantly reduced body weight at all stages compared with untreated controls. Body weight trajectories as well as body composition showed no change across treatments, suggesting that early size deficits are maintained across development rather than driven by altered postnatal growth rate. Consensus across behavioral assays shows increases in multiple measures of avoidance and anhedonia-like behaviors in maternally exposed male offspring. Exposed males exhibited increased immobile episodes and decreased entries to the center of the open-field test apparatus, increased entries to the closed arms of the elevated plus maze, and increased latency to eat in a novel arena following a fast. These findings suggest a persistent and sex-dependent mechanism underlying altered adult neurobehavioral outcomes following early-life exposure to a PFAS mixture. Ongoing studies are investigating whether these phenotypes are linked to molecular alterations within stress- and reward-related brain regions.

NMMH 12. Adipocyte-Specific FGF21 Overexpression Alters Ceramide Synthase Gene Expression in Mice Fed a High-Fat Diet
C. Gliniak, M. Vudata, T. Kulkarni, J. Evelyn
Rutgers University Department of Nutritional Sciences

Obesity is one of the major drivers of type 2 diabetes and is linked to approximately 30-53% of new diabetic diagnoses yearly, which also significantly increases the chances of cardiovascular disease and stroke. During metabolic syndrome and obesity, dysfunctional adipose tissue loses its ability to store fat safely. This leads to an excess of free fatty acids (FFA) in the bloodstream, which are deposited in vital organs such as the liver, heart, and pancreas, leading to lipotoxicity. In obesity, other bioactive sphingolipids, termed ceramides, accumulate in adipose tissue and are secreted into the circulation, affecting organs such as the heart. These lipids can disrupt glucose uptake, which promotes insulin resistance. Ceramide accumulation reduces energy production efficiency by impairing electron transport chain function and triggering apoptotic pathways, thereby contributing to tissue damage. Fibroblast growth factor 21 (FGF21), a hormone that is produced by the liver and adipocytes, promotes insulin sensitivity in adipose and increases energy expenditure via the brain. Our previous research suggested that FGF21 reduced ceramides in adipose tissue, but it is unknown whether this is mediated by a transcriptional mechanism. Furthermore, it is unknown whether FGF21 secreted by the liver or adipose tissue is driving this effect. Therefore, we hypothesized that FGF21 overexpression specifically induced in adipocytes would lower ceramides in mice fed an HFD for 4 weeks. To explore this specific correlation between FGF21 and ceramide synthesis, we used a transgenic mouse model in which FGF21 overexpression was controlled via doxycycline administration. Using qPCR, we analyzed male versus female visceral epididymal white adipose tissue (eWAT). The female and male mice exhibited FGF21 overexpression and significantly increased expression of the ceramide synthase genes Cers2 and Cers6 compared to the control mice. The increase in Cers 2 and 6 was unexpected in female mice, and we will follow up on tissue ceramide levels. These findings demonstrate that gender plays a key role in FGF21 responses in visceral adipose tissue.

NMMH 13. Dual Mechanisms of Methionine Aminopeptidase 2 Inhibition in Female Murine Adipocytes
C. Gliniak, Y. Chen
Rutgers University Department of Nutritional Science

Methionyl aminopeptidase 2 (METAP2) inhibition is anticarcinogenic and antiobesogenic in mice and humans. It is a multifunctional protein that catalyzes the removal of the N-terminal methionine from newly synthesized proteins and influences cellular stress signaling via the integrated stress response (ISR). However, at the cellular level, it remains unclear which pathway by which METAP2 inhibition alters the phenotype of adipocytes. To determine this mechanism, preadipocytes were isolated from the stromal vascular fraction of subcutaneous adipose tissue, differentiated into mature adipocytes, and treated with the METAP2 inhibitor TNP-470 and the endoplasmic reticulum (ER) stress inducer tunicamycin.
Female mouse preadipocytes and adipocytes were treated with vehicle, 1µM, or 10µM of TNP-470 for 2 hours, followed by qPCR analysis of the expression of the ISR-associated genes. Adipocytes showed significant increases in activating transcription factor 4 (Atf4), C/EBP homologous protein (Chop), and fibroblast growth factor 21 (Fgf21) expression at 10 uM TNP-470. In preadipocytes, Chop and Fgf21 significantly increased TNP-470 vs. vehicle.
To determine whether the transcriptional changes induced by METAP2 inhibition were mediated by the ISR. Adipocytes were treated with TNP-470 (10 µM), tunicamycin (0.5 µg), or a combination of both. Compared to vehicle, METAP2 inhibition increased Atf4, Chop, and Fgf21 and Chop increased with tunicamycin indicating activation of ER stress. In co-treated adipocytes, tunicamycin attenuated TNP-470-induced upregulation of Atf4 and Fgf21, suggesting that ER stress signaling interacts with pathways targeted by METAP2 inhibition. ISR signaling converges on the phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α). Western blot analysis showed tunicamycin reduced phosphorylated and total eIF2α (pEIF/EIFα) in adipocytes, which was unexpected. METAP2 inhibition increased pEIF2a/EIF2a in the tunicamycin-treated adipocytes, indicating that METAP2 inhibition is a dominant activator of ISR. Future experiments will determine whether METAP2 inhibition in adipose tissue is mediated through the ISR or N-terminal protein processing.

NMMH 14. Role of GDF15 in asparaginase-induced food aversion and liver steatosis
D. Bermeo Grajales, E. M. Lopez, M. Gomez Jenkins, M. Ibrahim, C. T. Bhavsar, B. A. Zalma, T. Anthony
Department of Nutirional Sciences, Rutgers University, New Brunswick, New Jersey

Asparaginase is a chemotherapeutic enzyme used to treat pediatric and adult forms of acute lymphoblastic leukemia (ALL). Its enzymatic activity depletes circulating asparagine and glutamine, triggering the Integrated Stress Response (ISR) an adaptive signaling pathway, which induces the transcription of many genes, including Gdf15. GDF15 contributes to anorexia and weight loss by suppressing appetite. Weight loss during asparaginase exposure is linked to hepatic steatosis and toxicity in mice, especially in obesity. Previously, we reported that asparaginase elevates circulating GDF15 in mice and this correlated with reduced body weight. A secondary analysis of an unpublished dataset from lean and obese mice fed either low-fat (10% fat by kcal) or high fat (60% fat by kcal) diets during asparaginase exposure also showed an inverse relationship between hepatic Gdf15 mRNA with body weight change (p= <0.001) and food intake (p=0.0036). Based on these findings, we hypothesized that genetic deletion of Gdf15 would rescue asparaginase-associated food aversion and body weight loss. To test this hypothesis, global Gdf15 knockout (Gdf15KO) mice and their wild type controls (WT) were intraperitoneally injected with pegylated asparaginase (1.5 international units per gram body weight) or equivolume saline vehicle (n=24 mice; 6 males and 6 females per group). Daily body weight and food intake were recorded for four consecutive days after injection, and biospecimens (serum, liver) were collected on the last day. Asparaginase exposure in WT mice significantly reduced food intake (p=0.0363), and body weight ~8% (p= 0.0021) and increased hepatic Gdf15 mRNA levels, as expected. In contrast, Gdf15KO mice maintained normal food intake (p=0.6465) and were resistant to asparaginase-induced weight loss (body weight change ≤4%). We noted that asparaginase-induced activation of the hepatic ISR and liver ISR target genes in Gdf15KO mice were similar to WT mice, indicating that systemic loss of GDF15 did not alter other aspects of the hepatic ISR to asparaginase. Importantly, liver triglyceride accumulation was rescued in asparaginase-exposed Gdf15KO mice. Overall, these data suggest GDF15 is a crucial mediator of asparaginase-associated food aversion and protects against body weight loss and associated liver steatosis by rescuing normal intake.

NMMH 15. Early Ethanol-Induced Neuroepithelial Perturbation and Subsequent Developmental Trajectory of Human Cerebral Organoids
S. L. Chang, M. Bishir, I. K. Sariyer, A. Arumugam, W. Huang
Institute of NeuroImmune Pharmacology, Seton Hall University; Temple University Lewis Katz School of Medicine

Human cerebral organoids (hCOs) provide a physiologically relevant model for investigating how alcohol exposure during early brain development may influence subsequent developmental trajectories. Our preliminary characterization of unguided hCOs demonstrated progressive developmental changes from D10 through D80. D10 hCOs exhibited a predominantly neuroepithelial/progenitor state, whereas D40 hCOs showed enrichment of CNS development, synaptogenesis, and neurotrophic signaling programs. D60 hCOs showed increasing neuroinflammatory and glial-associated signatures, including emergence of an endogenous microglial/myeloid-associated population, while D80 hCOs exhibited more mature signatures involving axonal guidance, myelination, and neurotransmitter signaling.

Spatial Gene Set Enrichment Analysis (SGSEA) at D10 revealed that EtOH (50 mM, 24 h )-exposed D10 hCOs exhibited a spatial developmental shift resembling that was observed in D20 hCOs. This finding indacted an abnormal prematuration of hCOs on Day 10 with EtOH exposure for 24 h. Flow cytometry demonstrated reductions in CD71, SOX2, PAX6, and Ki67, indicating disruption of progenitor-associated cellular programs. Bulk RNA sequencing identified EtOH-associated alterations in Wnt-related signaling pathways consistent with premature progenitor differentiation and altered cortical patterning. These findings establish an early EtOH-induced neuroepithelial perturbation and provide the rationale for examining its later consequences.

Building on these findings, hCOs will be exposed to EtOH at D10 and maintained without EtOH through D40 and D60. Age-matched vehicle-treated hCOs will serve as controls. Flow cytometry will assess neural progenitor maintenance (SOX2, PAX6, Ki67), neuronal differentiation (NeuN), astroglial differentiation (GFAP), and endogenous microglial/myeloid-associated populations (IBA-1, CD11B). Bulk RNA sequencing and Ingenuity Pathway Analysis will identify persistent alterations in developmental, neuronal, glial, and neuroimmune programs.

This study will determine whether an early abnormal EtOH-induced neuroepithelial perturbation is followed by altered cellular and molecular developmental trajectories at D40 and D60.

NMMH 16. Establishing an in vitro human cardiomyocyte model to investigate retinoic acid signaling in maternal cardiac remodeling
A. Glazer, Y. Kim, L. Quadro
Department of Food Science, Rutgers Center for Lipid Research, Rutgers University & Institute of Food Nutrition and Health, Rutgers University, New Brunswick, New Jersey, USA

Retinoic acid is an apocarotenoid that can be derived from oxidative cleavage of provitamin A carotenoids (1). It is a transcriptional regulator that has been shown to control cardiomyocyte differentiation and to play a role in the pathogenesis of maladaptive cardiac hypertrophy (2). Yet its role in physiological heart remodeling, such as that occurring in the maternal heart during pregnancy and postpartum, remains obscure. During pregnancy, hormonal stimuli, along with an increase in blood volume, contribute to an increase in maternal heart size, partially due to enlargement of cardiomyocytes, which reverts to its normal size postpartum (3). Preliminary data from our lab revealed a decrease in retinoic acid concentrations during late pregnancy in the mouse maternal heart which returns to baseline levels postpartum. Based on this and other data, we hypothesized that retinoic acid signaling modulates maternal cardiac hypertrophy and regression. To better understand the molecular mechanisms underlying this modulation, we developed the first in vitro model of maternal cardiomyocyte remodeling using AC16 human immortalized cardiomyocytes (4). Hypertrophy of AC16 cells was induced by various stimuli (Angiotensin II, Phenylephrine, or Progesterone) in the absence of retinoic acid in the culture media. Replenishment of retinoic acid reverted cell growth. Time course and dose response experiments were performed to define the optimal model conditions. Hypertrophy was assessed by cell area and Nppa gene expression. This model provides the framework to investigate the role of retinoic acid signaling in the physiological hypertrophy during pregnancy and regression postpartum of the maternal heart. It also enables comparison and characterization of the molecular pathways that distinguish physiological from pathological heart remodeling.

NMMH 17. Adult Peripheral NPY Deletion Does Not Alter Metabolic or Thermogenic Responses to High-Fat Diet
S. Sefidgari Abrasi, T. Akhayeva, M. A. Butera, C. Buettner
Division of Endocrinology, Metabolism & Nutrition, Department of Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, USA; Graduate Program in Endocrinology and Animal Biosciences, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA

Neuropeptide Y (NPY) is a highly conserved neuropeptide with well-established central roles in feeding, neuroendocrine regulation, and energy balance. NPY is also expressed in peripheral sympathetic nerve fibers, where it is co-stored and co-released with norepinephrine and may influence metabolic responses in peripheral tissues. Previous studies using catecholaminergic Cre models have implicated sympathetic NPY in glucose homeostasis, thermogenesis, blood pressure regulation, and protection from obesity. However, these models may include developmental effects and recombination in central catecholaminergic populations. We therefore investigated the physiological role of adult peripheral NPY using an inducible deletion model.

Npy^fl/fl^ mice were crossed with Rosa26-CreERT2 mice, and recombination was induced with tamoxifen beginning at 5 weeks of age. Following a recovery period, mice were challenged with a 60% high-fat diet and underwent metabolic phenotyping. Peripheral NPY deletion was assessed in multiple tissues, while hypothalamic NPY expression was examined to determine whether central NPY was preserved.

Even though peripheral NPY deletion occurred, knockout mice showed body weight, food intake, body composition, locomotor activity, energy expenditure, and respiratory exchange ratio comparable to control mice. Glucose tolerance, insulin sensitivity, fasting and glucose-stimulated plasma insulin concentrations, and blood pressure were also unchanged. In addition, knockout mice maintained core body temperature during acute cold exposure similarly to controls.

These findings indicate that adult peripheral NPY deletion is insufficient to alter systemic metabolic regulation, blood pressure, or acute cold tolerance under the conditions tested. The difference between these findings and previous studies using catecholaminergic Cre models may reflect differences in the timing and tissue specificity of NPY deletion. Together, our results suggest that adult peripheral sympathetic NPY is not required for maintaining metabolic and thermogenic function during high-fat diet challenge, although it may play a greater role under other physiological or metabolic conditions.