17th Annual Pioneers in Endocrinology Workshop Program

“How Aging Affects Bone and Muscle Crosstalk”

Gustavo Duque, MD, PhD

Dr. Joseph Kaufmann Chair in Geriatric Medicine

Director, The Simone & Edouard Schouela RUISSS McGill Centre of Excellence for Sustainable Health of Seniors

“Weight Loss Induced Bone Loss: Mechanisms and Mitigations”

Clifford J. Rosen, MD

Director of Clinician and Translational Research
Senior Scientist
Maine Medical Center Research Institute

Professor of Medicine
Tufts University School of Medicine

Abstracts

 

Bone (B)

B1. Time-Restricted Eating Differentially Affects Bone and Gut Microbiota in Older Women During Caloric Restriction
S. Shapses, B. McGuire, S. Ghosh, Y. Schlussel, L. Zhao
Rutgers University, Department of Nutritional Sciences, Department of Biochemistry and Microbiology

Purpose: Caloric restriction (CR) is recommended to reduce weight. However, weight loss can negatively affect bone. Bone turnover and the gut microbiota display circadian rhythms, and extended eating windows (>12 hours) can contribute to circadian disruption and negatively affect health. Time-restricted eating (TRE) manipulates meal timing, and circadian clocks are within multiple organs, including bone. Despite this, TRE studies have not examined bone as a primary outcome. We hypothesized that TRE would attenuate diet-induced bone loss. Methods: Postmenopausal women were randomized to six months of CR with or without TRE. Both groups were asked to attend 12 counseling sessions for weight loss. Fasting blood draws and fecal samples were collected at weeks 0, 12, and 24, and oral glucose tolerance tests were administered at weeks 0 and 24. Bone mineral density (BMD) was assessed by dual-energy x-ray absorptiometry and peripheral quantitative computerized tomography at weeks 0 and 24. Results: The women (61 ± 5 years, 72% white, 33 ± 4 kg/m2) had an eating window of 12.3 ± 0.9 hours/day. During the intervention, CR (n=21) women consumed food over 12.6 ± 0.8 hours/day, while TRE+CR (n=26) reduced the eating window to 8.2 ± 0.7 hours (p<0.001). Weight loss did not differ between CR and TRE+CR (-6 ± 5.6 vs -7.6 ± 3.9 kg). Hip BMD decreased over time (p<0.001), and lumbar spine showed a trend to decrease (p<0.08) over time without a difference between groups. A greater decline in 33% radius BMD was seen in the CR compared to the TRE+CR group (p<0.001). HOMA-IR, Matsuda Index, and area under the curve for glucose and insulin improved over time (p<0.05) but showed no between-group differences. Gut microbiota analysis indicated within-group differences in beta diversity for TRE+CR (p < 0.05), which were not observed in the CR group. There were no differences in alpha diversity. Conclusion: This trial is the first time-restricted eating intervention to examine the circadian effect of food intake on bone. Hip and spine BMD decreased, as expected with weight loss, while the addition of TRE to CR attenuated radius (33%) BMD loss and altered gut microbiota diversity in older women. Guild analysis of gut microbiota is ongoing.

B2. Protein Kinase A activity positively regulates osteoclast differentiation
C. Le-Henaff, Y. Qin, L. S. Kirschner, B. Zhao, N. C. Partridge
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, USA, Weill Cornell Medical College, Hospital for Special Surgery, New York, NY 10021, USA, Departments of Cancer Biology and Genetics and the Division of Endocrinology, Diabetes, and Metabolism, Department of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA

Osteoporosis causes a decreased bone mass and altered bone microarchitecture. This is associated with an increase in the risk of fractures. This highly prevalent disease results from an imbalance between bone formation and bone resorption with a decrease in bone formation by osteoblasts and an increase in bone resorption by osteoclasts. We have found that activation of Protein Kinase A (PKA) in the osteoclastic lineage in mice caused osteopenia. PKA action in osteoclasts has not been clearly investigated. Previous studies were mainly done in vitro without clear results yielding controversies in PKA activity in osteoclastogenesis or osteoclast activity. To elucidate the role of PKA activity during osteoclastogenesis, we constitutively modulated this enzyme activity in osteoclastic lineages (from monocytes to mature osteoclasts) using LysM-Cre in mice: first, we deleted the PKA regulatory subunit 1A (Prkar1a) to increase PKA activity (PKAhighOC) and secondly, we hypoactivated PKA using a dominant negative mouse model (PKAlowOC) which results in a mutation in the cAMP binding site of the regulatory subunit and blocks the release of the active catalytic subunit. At 8 weeks of age, hyperactive osteoclastic PKA caused a decrease in trabecular bone volume (-23% in males to -33% in female mice), thickness (-12%) and number (-15% to -24%) and an increase in trabecular separation (14%). These mice showed increased osteoclastogenesis in vivo and in vitro (2-fold increase in osteoclast number). Conversely, we have found that prevention of PKA activity in the osteoclastic lineage increased trabecular bone volume (18%) with increased trabecular thickness (9% to 12%) and separation (23% to 45%). While hyperactivation of PKA induced an increase in expression of several osteoclastic genes (Rank, Acp5, Ctsk) and transcription factors (Pu.1, cFos) involved during osteoclastogenesis, most of them were downregulated when PKA activation was prevented. These results collectively suggest that PKA activity positively regulates steps of osteoclast differentiation and function. These genetic data, for the first time, show that PKA is a key positive regulator in osteoclastogenesis.

B3. Parathyroid Hormone Stimulates Rankl Expression Through Nuclear Translocation of CRTC2 and 3 in Mouse Osteoblasts but Not in UMR 106-01 Cells
N. Partridge, A. Mohapatra, W. Petrosky, C. Le Henaff, J. Joseph
Center for Advanced Biotechnology and Medicine, Rutgers University

Parathyroid hormone (PTH) is a regulator of calcium homeostasis and is used to treat osteoporosis, however, excessive levels or prolonged treatment leads to net bone resorption through receptor activator of nuclear factor kappa-Β ligand (RANKL). Current models indicate nuclear translocation of CREB-regulated transcription coactivators (CRTC1/2/3) are responsible for Rankl transcription in osteoblasts after PTH-treatment. To determine the role of CRTCs in Rankl transcription in these cells, primary mouse calvarial osteoblasts were differentiated in culture or the rat osteosarcoma cell line, UMR 106-01, was plated, the cells treated with PTH and qPCR, quantitative immunofluorescence and Western blots performed. Here, we found that PTH (rat 1-34, 10-8 M) increased nuclear translocation of CRTC2 and 3 in a time-dependent manner in the differentiated mouse osteoblasts, with 72% of nuclei showing immunofluorescence at 30 min for CRTC2, 87% at 45 min, and 90% at 60 min. Control levels varied from 2-7%. In contrast, CRTC3 showed 89-91% at all three times after PTH treatment, while control levels varied from 2-6%. Surprisingly, no translocation into the nucleus of either CRTC2 or 3 occurred in the UMR 106-01 cells after PTH treatment. Measurement of Rankl mRNA abundance showed that PTH stimulated its expression by 15-20-fold at 4 h in the differentiated mouse osteoblasts while there was only a 2-3-fold stimulation in the UMR cells. In contrast, the UMR cells showed a 20-30-fold stimulation by PTH of Mmp13 expression and an 80-90% decrease in Sost expression (an alternative pathway through HDAC4/5). The lack of translocation of CRTCs into the nucleus of the UMR cells explains why there is so little stimulation of Rankl in these cells while transcription of genes regulated by HDAC4/5, Mmp13 and Sost, are highly modified. The differentiated mouse osteoblasts likely serve as a better model for PTH action that mimics the in vivo situation.

B4. Sexual Dimorphism in Osteoblastic Genes Following Early Life Antibiotic Exposure
M. Blaser, M. Hennessy, C. Le Henaff, M. Zhang, N. Partridge
Rutgers Center for Advanced Biotechnology and Medicine, Rutgers SEBS, Dept. of Biochemistry and Microbiology

The gut microbiome plays a central role in host physiology, extending beyond digestion to influence immunity, metabolism, and overall development. Evidence suggests that early life disturbances to the microbiome through antibiotic exposure have consequences for host growth and skeletal health. This project investigates the effects of early life subtherapeutic short term antibiotic treatment (ELST-STAT) on the establishment of the gut microbiome and bone development in mice, with the goal of better understanding the gut-bone axis. Pregnant dams were administered a subtherapeutic dose of penicillin (6.67mg/L) beginning on gestational day 18 through postnatal day 7 (P7), allowing offspring exposure via both gestation and lactation. Pups were euthanized at P7, P14, P21, and P56 to provide a developmental timeline to evaluate both transient and lasting effects of early antibiotic exposure. At each time point, body composition was measured non-invasively using dual-energy X-ray absorptiometry (DEXA-Piximus). Gastrointestinal microbiome samples were collected for sequencing and compositional analysis, while femurs, tibiae, and vertebrae were harvested for assessment of bone mass, microarchitecture, and gene expression. Analyses of DEXA and body length measurements suggest no uniform phenotype for bone mineral composition or size. Gene expression for osteoblast formation (Runx2, Alpl, Col1a1, Bsp, Bglap, Sost, RankL, Opg), mature osteoclasts (Ctsk, Acp5), and chondrocyte differentiation (Mmp13, Col2a1) was measured using quantitative PCR. For Sost, RankL, and Opg, levels generally peaked at P56 with antibiotic-induced sexual dimorphism; in females, early life antibiotic exposure induced greater expression than controls with the opposite effect in males. By integrating microbiome profiling with bone phenotype measurements, this study aims to identify specific developmental windows in which antibiotic-induced microbial disruption may alter skeletal outcomes and affect bone health. Findings will contribute to a deeper understanding of the gut-bone axis and its sensitivity to early life environmental perturbations, and sex-based differences in bone development.

B5. BMI Stratification Reveals Relative Skeletal Muscle Index (RSMI) Differentially Predicts Bone Quality in Older Women
S. Shapses, B. McGuire, Y. Schlussel
Department of Nutritional Sciences, Rutgers University

Purpose: Body mass index (BMI), fat, and lean mass can influence bone mineral density (BMD) and bone quality. However, the differential impact of fat and lean mass on bone quality is unclear. Furthermore, evidence suggests that although BMI positively predicts BMD, a higher BMI may compromise bone quality. We assessed if skeletal muscle mass predicts bone quality in postmenopausal women. Methods: Bone quality and BMD were measured in 91 healthy postmenopausal women (ages 50-75y) with a BMI between 25-41 kg/m2 using peripheral quantitative computed tomography and dual-energy x-ray absorptiometry. Measurements also included relative skeletal muscle index (RSMI) (appendicular lean mass divided by height) and fat mass index (fat divided by height). Women were stratified by the median BMI (31 kg/m2) and multiple regression analyses were adjusted for age, RSMI, and FMI. Results: Women had a BMI of 32 ± 4 kg/m2 and were 59 ± 5 years of age. In the lower and higher median groups, BMI (29 ± 1 vs 35 ± 3 kg/m2) and RSMI (7.3 ± 0.8 vs 8.0 ± 1 kg/m2), differed significantly (p < 0.001). In the higher compared to lower median BMI group, areal BMD (hip and forearm) was higher (p < 0.05) and lumbar spine showed a trend to be higher. RSMI was a predictor of periosteal circumference, polar moment of inertia, and stress-strain index of the tibia in the lower median BMI group (p<0.05) but not for obese women with BMI > 31. Fat mass index did not predict any outcomes. Conclusion: RSMI positively predicts bone quality, but not areal BMD, in postmenopausal women with a BMI of 25-31 kg/m2, but not in those with a BMI > 31. This suggests that skeletal muscle mass may be useful to explain the variance of bone quality in older women who are overweight or slightly obese.

Nutrition, Metabolism, and Health (NMH)

NMH1. Body composition and dietary intake associates with sleep macro-architecture in young adults
A. Spaeth, D. Valencia, C. Chovanes, S. Shapses
Department of Kinesiology and Health, Rutgers University – New Brunswick, Kinesiology and Applied Physiology Graduate Program, Department of Nutritional Sciences, Rutgers University – New Brunswick

Insufficient sleep is associated with excess body fat and daytime sleepiness in adults. Prior work has also shown that body fat percentage associates with increased rapid-eye movement sleep (REM) and that fiber intake associates with increased slow wave sleep (SWS). Here we aimed to examine relationships between body composition and sleep macro-architecture in young adults, who are at increased risk for insufficient sleep. Participants were 72 students enrolled at a large public university (20 ± 2, 61% female) who completed the Epworth Sleepiness Scale (ESS) and dietary screening questionnaire (DSQ), wore an actigraph on their non-dominant wrist 24/7 for 14 days and completed at-home polysomnography on a night where they were instructed to remain in bed for 9h. Body composition and bone metrics were measured by dual X-ray absorptiometry. Pearson’s and Spearman’s correlations analyzed relationships between body composition and sleep variables. Higher BMI associated with poorer actigraphy-measured sleep efficiency (ρ = -0.425, p <0.01) and greater wake after sleep onset (ρ=0.33, p<0.01). Greater fat mass associated with less total sleep time (actigraphy: r=-0.33, p<0.01; PSG: r=-0.28, p<0.05) and greater daytime sleepiness (r=0.33, p<0.01). Greater bone mineral content (BMC) was associated poorer sleep efficiency SE (r=-0.28, p<0.05). Expected fiber (ρ=-0.27, p< 0.05) and added sugar (ρ=-0.25, p<0.05) intake also associated with poorer sleep efficiency and NREM3% respectively, while expected fruit and vegetable intake and fiber intake was associated with poorer sleep quality (ρ=-0.27, p< 0.05; ρ=-0.23, p< 0.05). Findings contribute to growing evidence demonstrating bidirectional relationships between sleep health and body composition, particularly fat mass.

NMH2. Mitragynine Modulates Glucose Metabolism and Inflammation Independent of Insulin Signaling
N. T. Bello, L. Alsarkhi
Rutgers University

Objectives: Kratom (Mitragyna speciosa) is a botanical from Southeast Asia used to manage pain and opioid withdrawal, but with reported effects on glucose metabolism. The bioactive alkaloid in kratom is mitragynine (MTG), which has agonist actions on opioid and adrenergic receptors. This study investigates the dose-dependent effects of MTG and morphine on blood glucose and insulin signaling, the impact of repeated MTG on glucose metabolism and insulin sensitivity in diabetic mice, and the interaction between MTG and insulin. Methods: For the acute experiment, C57BL/6J male mice were divided into two feeding groups: Regular chow (RC) and High-Fat Diet (HFD; 60% fat). After 6 weeks, they received a single acute intraperitoneal (IP) injection of morphine (10 mg/kg), MTG (10 mg/kg), or vehicle. Blood glucose levels were recorded every 15 minutes, and tissues were collected 60 minutes post-injection. For the chronic experiment, after 12 weeks, RC and HFD C57BL/6J male mice received 150 mg/kg of streptozotocin (IP). By day three, both groups showed increased blood glucose levels, and then on day seven, they were treated daily with MTG (1-10 mg/kg) for one week. Plasma, hepatic, and pancreatic tissues were processed for glucose and insulin signaling. To assess the interaction between MTG and insulin during an acute high-dose injection, C57BL/6J male mice received a single acute intraperitoneal (IP) injection of insulin (5 U/kg), MTG (10 mg/kg), insulin (5 U/kg) + MTG (10 mg/kg), or vehicle. Blood glucose levels were recorded every 15 minutes, and tissues were collected 60 minutes post-injection. Results: In acute experiments, mitragynine (10 mg/kg) raised blood glucose, while morphine (10 mg/kg) decreased it; both reduced corticosterone levels. Mitragynine increased blood glucose; morphine did not. In chronic experiments, low-dose MTG (1 mg/kg) improved glucose levels in the RC group; high doses were effective in the HFD group. Liver function improved, as indicated by lower ALT levels, and IL-6 levels decreased, suggesting anti-inflammatory effects. Combining insulin with high-dose mitragynine caused severe hypoglycemia and boosted insulin signaling in the hindbrain nucleus tractus solitarius (NTS) after 60 minutes. Conclusions: The dose-dependent effects of mitragynine are not well understood. MTG effects vary with dose and diet, acutely increasing glucose levels independently of corticosterone or insulin. At the same time, chronic treatment improves glycemia and liver function. A decrease in IL-6 suggests a potential anti-inflammatory effect. Overall, mitragynine may enhance metabolic outcomes in hyperglycemic conditions through insulin-independent and stress-neutral pathways. Funding Sources: USDA-NIFA NJ06220.

NMH 3. The Neonatal Testosterone Surge in Mice and the Programming of Adult Metabolism Across the Sexes
T. Roepke, C. Hamson, A. Yasrebi
Department of Animal Sciences, Endocrinology and Animal Biosciences Graduate Program

The neonatal testosterone (T) surge occurs in males shortly after birth in mice 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 control of adult metabolism is largely unknown. Furthermore, the role of estrogen receptor signaling in developmental programming during the neonatal T surge is also unclear. To manipulate the T surge, neonatal females (postnatal 0/1) were injected with testosterone to simulate the T surge while neonatal males were injected with an aromatase inhibitor, formestane to prevent the neonatal T surge’s activating ERa signaling. Neonates were allowed to mature to early adulthood prior to metabolic phenotyping. We found that manipulating the T increased an early developmental marker like anogenital distance in females and elicited 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. By simulating the T surge in females, and blocking estrogen signaling in males, we are able to show how crucial the neonatal T surge is in programming the hypothalamus to produce sex-dependent variability in adult metabolism.

NMH 4. Investigation of hemp extract’s potential for metabolic health improvement in postmenopause
D. Roopchand, H. Palmer, G. Haag
Department of Food Science and NJ Institute of Food Nutrition and Health, Rutgers University

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. In various preclinical models, CBD was shown to reduce inflammation, improve gut barrier integrity, limit bone loss, and improve cognitive function, suggesting it may be useful for addressing these issues in menopausal and post-menopausal women. Our prior work was the first to demonstrate that ovariectomized (OVX) mice supplemented with CBD had improved metabolic and bone phenotypes, decreased intestinal inflammation, and increased relative abundance of fecal Lactobacillus species. My present work has two objectives: 1) To investigate whether OVX or sham surgery (SS) mice supplemented with a full spectrum hemp extract (containing CBD and other phytocannabinoids) will show improvements in exercise performance and metabolic health endpoints; and 2) To investigate putative interactions between CBD and proteins involved in estrogen signaling using an in silico approach. The data indicate that, compared to vehicle, OXV mice supplemented with hemp extract had improved exercise capacity and reduced body weight. Molecular docking and molecular dynamics simulations suggest that CBD may interact with estrogen receptors alpha (ERα) and beta (ERβ).

NMH5. Peripubertal BPA Exposure Suppresses Motivation via the Orexin System in Female Rats
T. Roepke, M. Atkinson, K. Moran, M. H. James
Department of Animal Sciences, Endocrinology and Animal Biosciences Graduate Program, Robert Wood Johnson Medical School Department of Psychiatry

Bisphenol-A (BPA) is a prevalent endocrine disrupting compound, found in human blood and urine samples. While research has shown that BPA exposure can severely affect neural development during prenatal exposure, there lacks research around peri-pubertal exposure, a critical developmental window. The age of puberty onset in girls has been lowering globally and is linked to negative mental health outcomes. Previous research has shown that peri-natal BPA exposure induces early onset puberty and decreases motivation. Orexin is a key mediator of motivation and has been shown to be decreased in BPA exposed rats. We exposed female rats from PND21 to PND56 with 250ug dose of BPA in their water, or a control group (0.5% Ethanol). We then conducted stereotaxic surgery to inject a viral vector for an excitatory DREADD into the lateral hypothalamus or a control virus on PND57. Rats then underwent between session behavioral economics using sucrose pellets as the reward on five frequency ratios and were tested with the corresponding DREADD agonist (JHU37160), or a vehicle. They were also tested on within session behavioral economics on the same frequency ratios, as well as a saccharin preference test to measure anhedonia and locomotor testing to evaluate the off-target effects of JHU. We saw that BPA exposed rats had an earlier onset of puberty. We also found that BPA lowers motivation and hedonic set point compared to vehicle treated rats. Further, we showed that using DREADDs to excite orexin neurons partially rescues the BPA induced anhedonia.

NMH 6. Postprandial endotoxemia is higher after a saturated than monounsaturated fatty acid enriched meal in older adults: a randomized cross-over study
S. Shapses, J. Chang, A. Ogilvie, R. Serrano, O. Carino, A. Shah
Rutgers University, Department of Nutritional Sciences, Kinesiology and Physiology Graduate Program, Division of Endocrinology, Dept of Medicine, Rutgers-RWJ School of Medicine, Rutgers Environmental Health & Safety

Objective: As the population ages, it is important to identify healthy dietary factors for the elderly. Lipopolysaccharide (LPS), an endotoxin derived from gram-negative bacteria, is associated with inflammation and whose blood concentrations increase following a high-fat diet. One study showed a rise in serum endotoxin after a saturated fat (SFA) meal but not other fatty acids. It remains unclear whether monounsaturated fatty acids (MUFA) differentially influence postprandial endotoxemia. Methods: In a randomized crossover study, older adults with overweight and obesity consumed a high fat (46% /550 kcal) meal enriched with either MUFA (20g MUFA, 5g SFA; peanut foods) and SFA (15g SFA; 11g MUFA; buttered egg sandwich), both matched for polyunsaturated fats. Meals were given in random order on two separate study days, each following a 3-day low-fat diet. Blood samples were collected before and 0.5, 2, 3, 4, 5, and 6 hours after the meal. to assess LPS, triglycerides, and inflammatory markers including lipopolysaccharide-binding protein (LBP), C-reactive protein, tumor necrosis factor-α, interleukin-6, and plasminogen activator inhibitor-1. Results: Healthy older individuals (n=16, 75% female,) were 62.7±4.8 years with a body mass index 33.4±6.3 kg/m2. The postprandial rise for serum endotoxin at 4 hours was twice as high after the SFA than MUFA meal (p=0.028) and LBP was also higher (p=0.024). No other inflammatory markers were higher for SFA compared to MUFA at any time point. Both meals increased serum triglycerides, but there was a greater rise at 2-hours for MUFA than SFA meal (p=0.017). Conclusion: Consumption of the SFA compared to MUFA meal led to a greater rise in postprandial serum endotoxin and LBP concentration. Further studies could examine other unsaturated fatty acids and determine if there is a differential inflammatory response.

NMH 7. FGF21 reduces ceramide levels in visceral adipose tissue independently of adiponectin in aged mice fed a high-fat diet
C. M. Gliniak, P. E. Scherer, R. Gordillo, Q. Lin, B. Field
Dept of Nutritional Sciences, Rutgers, New Brunswick, Touchstone Diabetes Center, University of Texas Southwestern Medical Center, Dallas, TX

Fibroblast growth factor-21 (FGF21) is a hormone that promotes energy balance, insulin sensitivity, and longevity. FGF21 reduces ceramide levels, bioactive sphingolipids that accumulate and contribute to adipose tissue inflammation and dysfunction that underlie cardiometabolic disease and diseases associated with aging. Specific ceramides are linked to disease; however, how FGF21 alters specific ceramide species in adipose tissue has not been investigated. We generated mice with doxycycline-inducible, adipocyte-specific overexpression of FGF21 and fed them a HFD for 1.5 years. LC–MS/MS analysis was performed for adipose tissue sphingolipids. In visceral epididymal (eWAT), ceramide species (C16, C18, C20, C22, and C24:1) were significantly decreased in transgenic vs. control mice. In contrast, in subcutaneous adipose tissue (scWAT), ceramides C24 and C25 increased in transgenic mice. Furthermore, six species of hexosyl- and lactosyl-ceramides species were reduced in eWAT in transgenic vs control mice. Upon FGF21 overexpression, lipids generated from the breakdown of ceramide, sphinganine, and sphingosine were significantly lowered in eWAT, but were not altered in scWAT. Overexpression of FGF21 in adipocytes robustly increases serum adiponectin levels, which is anti-inflammatory in part because adiponectin receptors possess intrinsic ceramidase activity, thereby breaking down ceramides. By crossing mice with adipocyte-specific FGF21 overexpression with adiponectin KO mice, we observed that FGF21 lowers ceramides in eWAT in the absence of adiponectin. In contrast, FGF21 alterations of scWAT ceramide levels depend on the presence of adiponectin. This suggests that FGF21 signaling and crosstalk with adiponectin to lower ceramides in eWAT and scWAT differ. Our findings hold important implications for understanding the role of FGF21 in adipose tissue across lifespan. The reduction in ceramides was most pronounced in eWAT, which may mediate the anti-inflammatory effects of FGF21. Additionally, these results are relevant to the discovery of the mechanisms by which FGF21 increases longevity, since we have recently shown that FGF21 does not increase energy expenditure in aged mice. This indicates that FGF21 ceramide-lowering activity in peripheral tissues may prevent metabolic disease, especially later in life. In essence, our research has unveiled a novel aspect of FGF21 function, shedding light on its differential impact on inflammation and insulin sensitivity in visceral vs subcutaneous adipose tissue.

NMH 8. Amino Acid-Fortified Oral Rehydration Therapy Decreases the Duration of Pediatric Acute Infectious Diarrhea
P. Breslin, P. Harmon, M. Weidner, P. Trivedy, M. Do, E. Lerner, S. Gaur
Rutgers University, Department of Nutritional Sciences, Robert Wood Johnson Medical School, Department of Pediatric Gastroenterology, Hackensack University Medical Center, Robert Wood Johnson Medical School, Pediatric Emergency Department, Robert Wood Johnson Medical School, Department of Pediatrics, Robert Wood Johnson Medical School, PediatricClinical Research Center

Diarrhea is the second-leading cause of death globally in children between 6 months and 5 years of age. The gold standard treatment is Oral Rehydration Therapy (ORT), which provides glucose and electrolytes to replace those lost in diarrhea. While ORT is a lifesaving intervention, it does not reduce the duration or severity of diarrhea, and many children are unable to endure the extended illness. We hypothesized that an amino acid-based formulation of ORT, fortified ORT (fORT) can maintain the rehydration benefits of ORT and decrease the duration and severity of diarrhea by strengthening the gut, inducing a stronger immune response, and providing the enterocyte’s preferred energy source. We are conducting a double-blind, randomized standard-of-care-controlled trial. Patients are recruited at the Pediatric Emergency Department or two Outpatient Pediatric Primary Care Offices when they present with <48 hours of diarrhea that is presumed infectious. Patients are randomly assigned to an intervention: fortified Oral Rehydration Therapy (fORT) or standard Oral Rehydration Therapy (ORT). Diarrhea duration, diarrheal severity, and stool antimicrobial peptide content are the primary outcome measures. Preliminary results indicate that the amino acid-based oral rehydration solution reduces the duration of diarrhea from treatment start to first solid stool by approximately 30% (p < 0.05). There is a trend toward reducing the duration of diarrhea from the start of treatment to the last diarrheal stool, (p = 0.07). To date, no participants have experienced adverse events, indicating that fORT is both safe and effective at managing pediatric acute diarrhea. We are presently enrolling participants and processing stool samples. This study indicates that amino acid-fortified oral rehydration therapy may be superior to standard-of-care glucose-based oral rehydration therapy for children with mild to moderate diarrhea. In the future, we aim to conduct a larger clinical trial in children with moderate to severe diarrhea who may benefit the most from this intervention.

NMH 9. “REACHING FOR THE STRAS”: RETINOID UPTAKE IN ACUTE LUNG INJURY
Ibrahim Dar 1, Olga Ilnytska 1, Lukas Ortega 1, , Nicole Ficken 1, David LoBiondo1, Souhail Hajjam1, Johannes von Lintig 2 and Igor Shmarakov 1
Department of Animal Sciences, Rutgers University School of Environmental and Biological Sciences, New Brunswick, NJ, USA. Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH, USA

We are interested in understanding retinoid (vitamin A and its metabolites) metabolism and signaling in functionally heterogeneous and developmentally diverse adult lung cells. Our research has established that adult lung structural cells containing retinoids, including lipofibroblasts, alveolar epithelial and endothelial cells, predominantly rely on and take up lipoprotein-derived postprandial retinoids. These cells do not utilize a retinoid delivery pathway involving retinol-binding protein 4 (RBP4) and its cell surface signaling receptor and transporter of retinol STRA6 that facilitates retinol (ROH) uptake from the RBP4-ROH complex. Surprisingly, our studies identified that a significant elevation of STRA6 expression in the mouse lung is a characteristic feature of acute lung injury (ALI). We hypothesized that this elevation is associated with immune cells infiltrating lung tissue and that the retinoid uptake via the STRA6-RBP4 axis can drive inflammatory responses during ALI. Our study aimed at testing this hypothesis by characterizing the cell-specific expression of STRA6 in the lung and assessing the contribution that the STRA6-RBP4 axis has in ALI progression. We have discovered that lung macrophages, a key cellular component of the innate immune system, are the cell type that specifically expresses STRA6 in the lung and responds with STRA6 upregulated expression during ALI. Furthermore, using mouse models of whole-body STRA6 (Stra6-/- mice) or RBP4 (Rbp4-/- mice) knockout we showed that the lack of functional STRA6 or RBP4 significantly impacts acute inflammatory response during ALI induced by intranasal lipopolysaccharide (LPS) instillation. Both Stra6-/- and Rbp4-/- mice were characterized by augmented ALI-associated inflammation and were protected from severe ALI. The observed protective phenotypes detected in either STRA6- or RBP4-deficient mouse lungs were due to the differences in the distribution of macrophage populations resulting in lower expression and accumulation of pro-inflammatory cytokines. Our research has identified STRA6 as a novel marker of immune cell activation in the lung. Moreover, our experimental data underscore the critical role of the STRA6-RBP4 axis in lung immune cell responsiveness to inflammatory stimulus and overall recovery during ALI. Our data provide an experimental rationale for targeting the STRA6-RBP4 pathway to mitigate severe ALI. This research was funded by grant R01 HL171112 from the NIH and start-up funds from Rutgers, The State University of New Jersey.

NMH 10. Very early effects of antibiotics on metabolic organs in mice enhancing later adiposity
M. J. Blaser, M. Zhang, H. Li
Center for Advanced Biotechnology and Medicine, Rutgers University

Although obesity is a pandemic, much of the pathogenesis is obscure. For nearly 80 years, antibiotics have been used on farms for the growth promotion of livestock. Prior studies have shown that sub-therapeutic antibiotic treatment (STAT) promoted growth and increased adiposity in mice of both sexes, and early-life short-term (ELST; 4 weeks) STAT was sufficient to induce long-term adiposity. To understand the underlying mechanisms, we focused on the liver, the central metabolic organ. The liver is metabolically active and plastic during early life when the composition and population structure of the microbiome are maturing. We hypothesized that perturbing the gut microbiome would impact hepatic metabolic function in early life, predisposing to obesity development. In this study, we examined the effects of ELST-STAT by administering water or low-dose penicillin (LDP) to pregnant dams 3 days before delivery, with continued treatment for 1, 2, or 4 weeks to perturb the pup’s early-life microbiome, followed by dietary stresses. We found that as early as one week after antibiotic exposure beginning at birth, antibiotic-treated mice grew bigger with increased hepatic and fat mass, with differences persisting after the microbiome largely returned to normal. Hepatic transcription and circulating level of insulin-like growth factor 1 (IGF-1), a hormone regulating growth, were elevated by LDP, whereas LDP decreased its binding protein IGFBP1. IGFBP1 level shows significant inverse correlation with body weight and inguinal white adipose tissue (iWAT) mass. In iWAT, expression of IGF1 receptor (Igfr) and peroxisome proliferator-activated receptor gamma (Pparγ) was upregulated by LDP, indicating an increased adipogenesis in response to higher IGF1. Early antibiotic exposure and a high-fat diet had synergistic effects on promoting the accumulation of iWAT and perigonadal visceral (pgVAT) adipose tissue, the two primary depots for energy storage. Microbiome analysis showed that 1 week of antibiotic treatment since birth altered the microbiome population structure and significantly reduced Lactobacillus abundance. Its abundance at 3 weeks showed a strong inverse correlation with iWAT and pgVAT mass at week 12, suggesting a protective role. These data provide evidence that the ELST-STAT perturbed microbiome has an impact on metabolic organs in early life, which contribute to the increased growth and development of obesity.

NMH 11. Mitragynine’s Acute Effects on Heart Rate, Feeding Behavior, and Temperature in Mice
N. Bello, S. Fields
Animal Sciences, Rutgers University, Nutritional Sciences, Rutgers University

Kratom, a Southeast Asian plant long used for its stimulating and analgesic effects, is gaining attention for its bioactive alkaloids, particularly mitragynine and 7-hydroxymitragynine. These compounds, which interact with opioid and adrenergic systems, are currently under investigation for their ability to mitigate opioid withdrawal. However, their broader physiological effects are unknown, particularly in overweight/obese populations. This study evaluated acute impacts on heart rate, feeding behavior, and body temperature in normal weight and diet-induced C57Bl/6J obese mice. Micro-HRT telemetry loggers (Star-Oddi, Garðabær, Iceland) were surgically implanted for continuous cardiovascular and thermal monitoring, and the BioDAQ system (Research Diets, New Brunswick, NJ) was used to quantify food intake. Mice received intraperitoneal doses of vehicle (20% Tween-80, 80% DI water), 5.6 mg/kg mitragynine, 56 mg/kg mitragynine, and 1 mg/kg 7-hydroxymitragynine, with one-week washouts between doses. Dose*time interactions were observed for heart rate AUC across the 24-hour cycle (p < 0.001), with the most pronounced change following the 5.6 mg/kg mitragynine dose. In contrast, the highest dose (56 mg/kg mitragynine) significantly reduced caloric intake over 24 hours (p = .031), while other doses showed no clear feeding effects. No significant changes in body temperature were measured across doses. These findings reveal a non-linear relationship between alkaloid dose and physiological outcomes. Lower mitragynine doses markedly influenced heart rate, whereas higher doses suppressed food intake. The distinct dose-dependent effects highlight kratom’s complex interaction with autonomic and metabolic systems—and signal the need for more nuanced exploration of its pharmacological profile.

Epigenetics (E)

E1. FETAL ALCOHOL EXPOSURE INDUCED EPIGENETIC TRANSGENERATIONAL INHERITANCE OF X-CHROMOSOME LINKED IQSEC2, WDR13 AND NXF-7 IN FISCHER 344 RAT OFFSPRING FOR THREE GENERATIONS: EVIDENCE FOR MALE GERM LINE TRANSMISSION AND ROLE IN BEHAVIOR ABNORMALITIES
D. Sarkar, O. Gangisetty, L. Gerlikhman, A. Palagani, S. Chaudhary
Rutgers University

Fetal alcohol exposure (FAE) causes various neurodevelopmental deficits in offspring, including an elevated response of stress axis, anxiety and immune dysfunction and have been shown to transmit for multiple generations via male germline. Increased anxiety has an impact on cognitive behavior such as learning and memory. We used Fischer 344 rats fed with 6.7% alcohol liquid diet (AF), pair-fed with an isocaloric liquid diet (PF) or rat chow ad libitum (AD) during gestation day 7-21 and their offspring were used for the study. We generated alcohol fed male germ line (AFM) by breeding AF male rats with control AD female, alcohol fed female germline (AFF) by breeding AF female rats with male control, AD rats for successive F2 and F3 generations. We performed Morris water maze test to study learning and memory behavior. We found that latency time during training period is increased, escaped latency time is reduced significantly in AF rats compared to controls. The same behavior abnormalities were observed in AFM group of F2 and F3 generations. However, the mechanism by which FAE induced male germ line transmission of cognitive behavior abnormalities is not understood. We hypothesize that FAE induced DNA methylation may contribute for behavior abnormalities observed. We employed reduced representation bisulfite sequencing (RRBS) to analyze whole genome wide DNA methylation using sperm DNA samples. Sperm epigenome data revealed that FAE induced a no of differential methylated regions, (DMRs) assigned to different genes. We found 338 hypermethylated DMR assigned genes were unique to AFM but not in AFF group. off these, 24 were linked to X-chromosome, 1 linked to Y chromosome. Gene ontology showed some of these genes are associated with disease, behavior abnormalities. We next measured the expression of Iqsec2, Wdr13, and Nxf7 and found mRNA expression were significantly reduced in hypothalamus of AF rats in F1, AFM group in F2, F3 generations compared to controls. Methylation of DMRs of these genes were hypermethylated in hypothalamus and sperm samples. Our results suggest that FAE induced male germline transmitted hypermethylation of Iqsec2, Wdr13, Nxf7 showed reduced gene expression in hypothalamus may provide a possible role in behavior abnormalities such as learning and memory observed in rat offspring. (Supported by NIH grant R01AA025359).

E2. Crysvita ( Burosumab ) treatment demonstrated efficacy and safety in a Mother and Son with X-linked hypophosphatemic Rickets
S. Ten, J. Matthew
Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, US

Introduction: X-linked hypophosphatemic rickets (XLHR) is caused by PHEX gene mutation, leading to excessive fibroblast growth factor 23 (FGF23) secretion. FGF23 reduces renal phosphate reabsorption and suppresses 1,25-dihydroxyvitamin D synthesis. Crysvita a human monoclonal IgG1 antibody, binds and neutralizes excess FGF23. Patient 1: We present a 12-month-old boy referred for evaluation of short stature and poor growth rate. He was growing at 0.11 % while the target height was at the 50th %. He had delayed motor development, late walking, speech delay. IGF-1, IGFBP3, TFT, and celiac screen were normal. Phosphorus was low at 2.3 mg/dL, ALKP increased at 1200 U/L (117–311), PTH 39 pg/mL, calcium 10.2 mg/dL, Vit D 25 39 ng/mL were normal. FGF23 was elevated to 37 pg/mL (Nl < 22). The diagnosis of XLHR was confirmed by a novel, pathogenic PHEX gene variant c.1309G>T (p.Glu437*), Exon 12. Crysvita was initiated at 20 months, resulting in phosphorus levels of 3.9 mg/dL, ALKP of 552 U/L, and an improved growth rate at 24 months (height 81 cm, 1.3%) and at 7.1 years (height 118 cm, 21.43%). Treatment resulted in normalization of growth rate, muscle tone, and developmental delay. Patient 2: Mother was 35years old, with short stature (60″) and a history of surgery in childhood due to bowed legs. She had fatigue and bone pain, and bowed legs. She had the same pathogenic variant in the PHEX gene. Phosphorus was low 2.2 mg/dl (Nl. 2.5-4.5), ALKP was increased to 158 U/L (NL. 31-125). Mother’s FGF23 was increased to 60 pg/ml (Nl < 22). After Crysvita treatment Phosphorus 2.7 mg/dl and ALKP 31.6 U/L normalized, fatigue and bone pain resolved. Conclusion: Crysvita therapy demonstrated efficacy and safety in both mother and child with XLHR. These cases highlight the importance of genetic testing and phosphate evaluation in patients with unexplained growth failure or skeletal deformities.

Endocrinology Disorders (ED)

ED1. A Combination of Growth Hormone and Voxzogo (Vosoritide) Therapy in a Child with Achondroplasia
S. Ten, J. Matthew
Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, US

Introduction: Voxzogo (vosoritide) is the first precision therapy shown to increase growth velocity in children with achondroplasia by counteracting FGFR3-mediated signaling. We report two cases of children with achondroplasia treated with Voxzogo, one of whom received combination therapy with GH. Case 1: A 9-year-old girl with genetically confirmed achondroplasia (FGFR3 c.1138G>A [p.Gly380Arg], a heterozygous pathogenic variant was treated with Voxzogo. Brain MRI revealed stable mild ventriculomegaly, probably secondary to aqueductal stenosis. At 9.3 years, prior to therapy, her height was 101.5 cm (–5.3 SDS). Voxzogo therapy was initiated, and after one year (at 10.3 years), height increased to 107.5 cm (–5 SDS), corresponding to a growth velocity of 6.0 cm/year. Bone age (BA) was 9 years at a chronological age (CA) of 9.6 years. At 10.3 years, GH therapy (0.3 mg/kg/week) was added to Voxzogo. At this time, BA was 10 years at CA 10.6 years. After one year of combination therapy, at 11.3 years, height was 116 cm (–4.8 SDS), with a growth velocity of 8.5 cm/year. During the second year, growth declined to 6.8 cm/year. At 12 years, her height was 121 cm ( -4.5 SDS). Case 2: A 4.8-year-old girl with achondroplasia (FGFR3 c.1138G>A [p.Gly380Arg]) was born after a normal pregnancy. At 2 years, she underwent foramen magnum decompression. MRI of the pituitary showed postoperative changes consistent with suboccipital craniectomy and C1 laminectomy, moderate supratentorial ventriculomegaly. Bone age was 3.6 years at CA 4.8 years. At the initiation of Voxzogo therapy (at 4.6 years of age), her height was 84 cm (–5.45 SDS); after one year (at 5.7 years), her height increased to 89 cm (–5.38 SDS), corresponding to a growth velocity of 5 cm/year. Conclusion: Combination therapy with Voxzogo and GH demonstrated an additive effect on growth velocity in a child with achondroplasia, particularly during the first year of treatment. However, the response decreased in the second year, underscoring the importance of individualized dosing and the need for further evaluation of efficacy and safety.

ED2. Pseudohypoparathyroidism secondary to the Mutation of PRKAR1A
S. Ten, R. Daly
Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, US

Case Presentation: A 9-year-old girl presented for evaluation of short stature and brachydactyly. Her height was at the 4th percentile (Target Height ~75th%), weight 70th%, and BMI 21.4 kg/m² (94th%). Physical findings included mild midface hypoplasia, brachydactyly, and brachymetacarpia, with radiographs showing shortened 2nd–5th metacarpals. Dental development was delayed. Brain MRI was normal. She had no cognitive impairment. She was found to have PTH resistance, hypothyroidism secondary to TSH resistance and GH deficiency secondary to GHRH resistance. TSH was elevated at 8.34 mIU/L, the free T4 was normal. TSH normalized on Levothyroxine 75 mcg daily. PTH level was elevated at 149 pg/ml (Nl. 9-59), with normal Ca 9.5 mg/dl, Ph 4.9 mg/dl, D25 of 34 ng/ml, D1.25 levels 55 pg/ml. On treatment with Calcitriol 0.75 mcg daily, Calcium 500 mg daily, PTH became normal 60 pg/ml with normal Calcium level. She failed GH stimulation test, secondary to GHRH resistance and was treated with GH. BMI was elevated to 29.4 kg/m2 ( 95.2 %), She developed insulin resistance with elevated Insulin of 30 uIU/ml, elevated Triglyceride 262 mg/dl and low HDL levels. Karyotype, Microarray, SHOX, GNAS/STX16 gene analysis were normal. There was no evidence of GNAS methylation changes. PRKAR1A analysis revealed a heterozygous mutation c.1003C>T (p.Arg335Cys), AD inheritance. Acrodysostosis type 1 was diagnosed secondary to this mutation. No other affected relatives were identified. Mutations in PRKAR1A are also associated with Carney Complex type 1, intracardiac myxoma, and primary pigmented nodular adrenocortical disease (PPNAD-1). Echocardiogram and adrenal function should be monitored every 6 months. Conclusion: This case demonstrates that pseudohypoparathyroidism-like phenotypes are not limited to GNAS defects. While most cases are due to GNAS gene defects, mutations in PRKAR1A, PDE4D, PDE3A can also cause overlapping phenotypes, including brachydactyly, short stature. PRKAR1A mutations should be considered in patients with brachydactyly, multihormonal resistance, and normal GNAS testing, as early diagnosis can guide targeted surveillance for cardiac and adrenal complications.

ED3. Treatment with Letrozole in 2 brothers with Congenital Adrenal Hyperplasia
S. Ten, B. Labib
Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ

Introduction: Patients with congenital adrenal hyperplasia (CAH) frequently develop advanced bone age (BA), which can compromise adult height. Aromatase inhibitors, such as letrozole, reduce estrogen production and slow bone maturation. However, data on long-term safety in CAH remains limited. We present two brothers with CAH treated with Letrozole. Case 1: Male, 18 years old, diagnosed at 4.9 years with CAH, salt-wasting type. He had no functional CYP21A2 gene (Allele 1: 30 kb deletion, Allele 2: 30 kb deletion). He was treated with Cortef 13 mg/m²/day, Florinef 0.2 mg/day, Crenessity 100 mg twice a day. Letrozole 2.5 mg daily was initiated at 6.9 years, when BA was 13 yrs., and discontinued at 15.4 years. On Letrozole bone maturation slowed down, so at 7.9 yrs. BA was 13 yrs. At CA 13.6 yrs. BA was 13.6 yrs. At CA 15.5 yrs. BA was 14.6 yrs. At CA 16.3 yrs. BA was 15 yrs. Final height was 170.5 cm (22%), normal for his target height 175 cm (45%). Bone Mineral Density (BMD): At 11.4 yrs. the Lumbar spine Z-score was normal 0.8 (110%), the subtotal body Z-score was 0 (100%). At 14.5 yrs. the Lumbar spine Z-score was normal -0.5 (93%), the subtotal bone Body Z-score was -1.3 (90%). At 16.6 yrs. the Lumbar spine Z-score was -1.9 (76%), the subtotal body Z-score was -2.4 (83%). Duration of therapy: 8 years. Case 2: Male, 15 years old, diagnosed at birth with CAH, salt-wasting type, He had the same CYP21A2 deletion. He was treated with Cortef 11 mg/m²/day, Florinef 0.1 mg/day and Crenessity 50 mg twice a day. Growth hormone started at 14 years. Letrozole 2.5 mg daily was initiated at 10.6 years when BA was 13 years. On Letrozole bone maturation slowed down, at 14.3 yrs. BA was 13.6 yrs, at 15.3 yrs BA 13.6 yrs. Bone mineral density was obtained 1 yr. after beginning of the therapy at 11.6 yrs. and was normal. The lumbar spine Z-score was -1.5 (83%), the subtotal body Z-score was -1.7 (88%). At 13.6 yrs the lumbar spine Z-score decreased, was -2.2 (73%), the subtotal body Z-score was -3 (79%). Target height 175 cm (45%), at 15.3 years, height 153.5 cm (2%). Conclusion: Letrozole slowed bone maturation in both siblings with CAH. BMD remained within the normal range during the first four years of therapy in the older sibling but declined to –1.9 after 8 years of treatment. In the younger sibling, BMD was -1.7 after one year of therapy (within normal limits) but decreased to -2.2 after three years of treatment. The rate of BMD loss can vary among patients; therefore, annual monitoring of bone mineral density is essential during treatment.

ED4. Branched chain amino acid sufficiency is necessary for proper luteinizing hormone response and testosterone synthesis
E. Snyder, Y. Shamailova, T. Anthony, S. Farooq, M. Gilmore, T. Stanek, E. Lopez
Rutgers University, Graduate Program in Endocrinology and Animal Biosciences, Department of Animal Sciences, Department of Nutritional Sciences, NJ Institute for Food, Nutrition, and Health

Testosterone production by testicular Leydig cells (steroidogenesis) is vital to male fertility and overall male health. Information about how nutrition influences Leydig cell steroidogenesis is lacking. Branched chain amino acids (BCAAs – leucine, isoleucine, and valine) are essential amino acids and important regulators of protein synthesis and energy production. Circulating and tissue BCAA levels are tightly regulated by the enzyme branched chain a-keto acid dehydrogenase kinase (BCKDK), which inhibits their catabolism. This work explored how BCAAs, and especially leucine, modulate male fertility and testosterone production. In a mutant mouse model of Bckdk, breeding analysis showed reduced male fertility and circulating testosterone. Further, morphological evaluation demonstrated testicular and epididymal abnormalities consistent with abnormal testicular androgen signaling. Fertility was partially rescued by feeding a high protein diet while circulating testosterone was not. In wild type testes, Leydig cells were the primary cell type to express BCKDK. Leveraging a primary interstitial cell culture, cell survival and apoptosis analyses demonstrated Leydig cells are highly sensitive to leucine deprivation and this sensitivity is enhanced under steroidogenesis stimulating conditions. Lastly, using the same primary cell culture system, testosterone production was shown to be lost under leucine deprivation. In total, this work demonstrates Leydig cells are uniquely sensitive to BCAA status under steroidogenesis stimulation and that regulated BCAA catabolism may be important for optimal male fertility.

Drug Abuse (DA)

DA1. A Comparative Study of Morphine Tolerance and Withdrawal in F344 and HIV-1Tg Rats
S. Chang, W. Huang, M. Bishir, S. Jawadi, S. Babu Chidambaram, I. Sariyer
Seton Hall University Institute of NeuroImmune Pharmacology, JSS Academy of Higher Education and Research, Temple University Lewis Katz School of Medicine

We previously reported greater incidence of opioid use among the people living with HIV (PLH), but the role of HIV-1 viral proteins in development of opioid tolerance and dependence in PLH remains unclear. Our research was the first to show an increase in mu opioid receptor expression in the presence of HIV viral proteins. We also found overlapping signaling pathways mediated by HIV-1 viral protein and morphine, resulting in increased opioid dependence in PLH. Using HIV-1Tg rats, model to mimic PLH on cART, we compared morphine tolerance and naloxone induced withdrawal between female F344 and HIV-1Tg rats. On Day 1, rats received 2 morphine (75 mg) pellets or placebo, followed by 4 pellets on Day 2 subcutaneously. Tail flick latency was recorded before pelleting, at 2- and 8-hours following pelleting on Days 1 and 2, and once daily on Days 3-7. On Day 7, naloxone (2 mg/kg,i.p.) was injected to induce withdrawal. Both groups given morphine showed increased latency within 2 hours of pelleting. However, by Day 3, HIV-1Tg rats showed a decrease in the latency, whereas F344 rats showed a decrease on Day 4. By Day 7, F344 rats returned to baseline latency, but HIV-1Tg rats failed to recover completely, suggesting greater tolerance in HIV-1Tg rats. Naloxone induced physical withdrawal symptoms including tooth chattering, paw tremor, diarrhea, squeaking and orbital tightening. Episodes of paw tremor were significantly higher in HIV-1Tg compared to F344 rats received morphine pellets. These findings reveal that HIV-1Tg rats develop greater morphine tolerance and withdrawal symptoms.

DA2. Central Opioid and Peripheral Immune Signaling mediates Alcohol’s Transition from Analgesia to Hyperalgesia in Adolescent Mice
S. L. Chang, M. Bishir, W. Huang, S. Jawadi, S. Babu Chidambaram
Institute of NeuroImmune Pharmacology, Seton Hall University, South Orange, NJ, 07079, USA, Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Mysuru 570015, India

Alcohol consumption exerts profound effects in the pain system, being analgesic through acute exposure by modulating the neurons in the pain centers, whereas long term exposure induces chronic pain, chiefly mediated via neuroinflammation. However, no studies have investigated the underlying mechanisms involved in the transition from analgesia to mechanical allodynia and thermal hyperalgesia. In this study, adolescent C57BL/6 (B6) mice, 3-4 weeks old, were exposed to 3-day binge ethanol (EtOH) [3-dBE] 5g/kg; 42% v/v EtOH/ H2O in seven days to mimic weekend binge drinking of the adolescence to investigate EtOH modulation of mechanical and thermal pain responses and the underlying mechanisms. Another set of B6 mice received 7-cycles (7X) of 3-dBE, with daily monitoring of mechanical and thermal pain responses. We found a significant increase in both the mechanical and thermal pain responses in the initial two cycles of 3-dBE and then the animals showed allodynia and hyperalgesia responses throughout the 7X. The 3-dBE on morphine’s anti-nociceptive properties was also examined. We found that 1X 3-dBE significantly increased morphine’s anti-nociception, whereas 7X 3-dBE significantly reduced morphine’s anti-nociception. Opioidergic signaling in the pre-frontal cortex, one of the common areas critically involved in alcohol intoxication, withdrawal and anticipation as well as pain, was found to be altered with 1X and 7X 3-dBE. Oprm1 expression was found to be increased with 1X and decreased with 7X 3-dBE exposure. Similar results were also observed with its transcription factor NfkB1. We then investigated the involvement of peripheral immune signaling in EtOH modulation of pain. With 1X 3-dBE there was no significant difference in the immune cell population in either spleen or blood of B6 mice. However, with 7X 3-dBE, we found a significant increase in the CD8+ circulating cytotoxic T cells, and an increased trend in the circulating levels of T cells, B cells and monocytes. In contrast, the levels of circulating neutrophils were found to be significantly higher in both 1X and 7X 3-dBE treated B6 mice. Taking together, these studies have demonstrated that central opioid signaling and peripheral immune signaling appear to be critically involved in alcohol’s transition from analgesia to mechanical allodynia and thermal hyperalgesia.