Additive effect of leptin and palm-LEAP2(1-14) ameliorates obesity-induced metabolic stress in ob/ob mice.

Food texture influences eating rate (ER), and slower ERsare associated with reduced energy intake within a meal. However, it remains unclear whether this acute effect of ER on intake is sustained over time. We investigated whether texture-based differences in meal ER can have a sustained effect on food and energy intake across 11 consecutive days. In a randomised cross-over feeding trial, Dutch adults (n 20) were randomised to an 11-d 'fast' and an 11-d 'slow' ER diet, followed by a 17-d washout period before completing the alternate diet-arm. Participants consumed ad libitum breakfast and dinners of which ER was manipulated using food texture and received the same lunch meals on both diets served in regular-sized fixed portions. Diets were matched for served total weight (gram), energy (kcal) and energy density (kcal/gram) and were equivalent for visual volume, meal liking and meal variety. Meal ER on the 'slow diet' was on average 32 % slower compared with the 'fast diet' (P < 0·01). On days when texture led to significant differences in ER, food intake was reduced by 121 (se 24) g/d (P < 0·001), and this effect did not attenuate over time (P = 0·25). Cumulative food intake was 6 % lower for the slow compared with the fast diet (P < 0·001) with no significant difference in energy intake. On 8 of the 11 test days, meal texture reduced ER and supported a consistent reduction in food intake. Further research should test whether a whole diet approach to lowering ER by modifying meal textures could help to moderate food and energy intakes.

Perinatal leptin effects on hypothalamic brain-derived neurotrophic factor and energy balance-related gene regulation.

Acute/chronic exposure to nicotine modulates feeding behavior in experimental animals and humans. However, how nicotine modulates food intake remains unclear. This study examined the acute effects of the peripheral administration of nicotine on food intake in adult male rats, focusing on the possible involvement of the anorectic peptide nesfatin-1/nucleobindin-2 (NucB2) in the central nervous system (CNS). Initially, cumulative food intake, but not water intake, was significantly decreased 0.5, 1, and 1.5 h after the intraperitoneal administration of nicotine (0.5 mg/kg) in24h-fasted rats. Subsequently, the double-labeled immunohistochemical study revealed that nesfatin-1/NucB2-immunoreactive (ir) neurons expressed Fos-ir in various nuclei of the hypothalamic and brainstem areas, including the supraoptic nucleus and ventral tegmental areas, 90 min after the intraperitoneal administration of nicotine. Finally, pretreatment with intracerebroventricular administration of antisense RNA against nesfatin-1/NucB2 significantly attenuated the suppression of food intake induced by intraperitoneal nicotine administration. The results indicated that the acute effects of peripherally administered nicotine on the suppression of food intake may be partially involved in nesfatin-1/NucB2-containing neurons in the CNS in male adult rats.

Introduction and Objective: Diabetic nephropathy (DN) develops in roughly 30% of diabetic patients &amp; is the leading cause of chronic kidney disease. Characterized by irreversible deterioration of renal function, DN lacks effective therapeutic approaches for treatment. This study explores the renoprotective effects of the dual glucagon-like peptide-1 (GLP-1)/gastric inhibitory polypeptide (GIP) receptor agonist, tirzepatide, in a db/db mouse model of DN to explore its underlying mechanisms. We assessed glucose levels &amp; kidney function markers following treatment with semaglutide &amp; tirzepatide. Methods: Experimental groups consisted of 6-to-8 week-old C57BL/KsJ Cay db/db &amp; db/+ mice randomly assigned based on baseline blood glucose, HbA1c, &amp; body weight: the db/db model group &amp; treatment groups of semaglutide (30 nmol/kg), tirzepatide (7.5 nmol/kg), &amp; tirzepatide (20 nmol/kg). The normal control group consisted of eight age-matched lean db/+ control mice. Drug administration was performed via subcutaneous injection once daily (QD). Body weight, food &amp; water intake, blood glucose, HbA1c, kidney function markers, &amp; histological changes were evaluated. Results: Compared to the GLP-1 receptor agonist semaglutide, the dual GIPR &amp; GLP-1R agonist tirzepatide exhibited superior efficacy in improving glycemic control parameters, including blood glucose, HbA1c, &amp; insulin secretion function. Both tirzepatide (7.5 nmol/kg &amp; 20 nmol/kg) &amp; semaglutide (30 nmol/kg) significantly reduced body weight &amp; body weight change rates in db/db model mice. All three treatment groups showed notable reduction in both daily &amp; cumulative food intake. Tirzepatide showed a more pronounced effect on diabetic nephropathy-related markers, including BUN, UACR, &amp; UAER, compared to semaglutide. Conclusion: Our findings confirm that db/db mice develop hyperglycemia &amp; kidney function impairment, resembling DN. Further, dual GIPR &amp; GLP-1R agonism with tirzepatide demonstrates superior therapeutic potential for managing both glycemic control &amp; renal complications in diabetes. Disclosure A. Wu: None.

Introduction and Objective: The prevalence of obesity and associated disorders necessitates innovative approaches for safe and efficacious therapies. This study aims to characterize PTT-A, a novel unimolecular tetra-agonist in DIO rats and assess its efficacy against Tirzepatide. Methods: DIO rats were dosed for 3 weeks with PTT-A and Tirzepatide and multiple endpoints were examined, including body weight (BW), food intake (FI), body composition by carcass analysis and MRI, metabolic parameters and liver health. Results: Treatment with PTT-A at 10 and 30 nmol/kg over 21 days resulted in significant reductions in cumulative FI and BW (14.3±1.52% and 19.2±0.89% vs vehicle, respectively) driven by reductions in fat mass with no loss of muscle mass. Tirzepatide at 30 nmol/kg reduced BW only by 12.1±0.81% vs vehicle with a decrease in both fat and muscle mass. Gastrocnemius and soleus muscle weights were reduced by approximately 26% with Tirzepatide, with no reduction in muscle weight with PTT-A at same dose. Additionally, PTT-A exhibited robust efficacy in triglycerides lowering, insulin sensitization and liver health, as evidenced by reductions in plasma aminotransferase, plasma lipids, hepatic triglycerides, and liver histology, that surpassed Tirzepatide’s effects at equivalent doses. Conclusion: PTT-A represents a promising novel tetra-agonist that demonstrated enhanced efficacy in weight loss, insulin sensitization, lowering lipids and liver health relative to Tirzepatide. The profound BW reduction after 3 weeks treatment was through fat loss while preserving the muscle. These findings show the potential of tetra-agonists as multifaceted therapeutic options for addressing the challenges of obesity-related metabolic disorders. Disclosure S. Ghosh: Consultant; Pep2Tango Therapeutics, Abvance Therapeutics. P. Valdecantos: Research Support; Pep2Tango Therapeutics. P. Rada: Research Support; Pep2Tango Therapeutics. A.M. Valverde: Research Support; Pep2Tango Therapeutics. C.M. Rondinone: Employee; Pep2Tango Therapeutics.

The interplay of oxytocin and dopaminergic system: Effects on food consumption in broiler chickens.

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is strongly associated with insulin resistance (IR), which leads to a range of PCOS-associated cardiometabolic complications, such as obesity and type 2 diabetes. Glucose transporter type 4 (GLUT4) is a crucial glucose transporter that facilitates insulin-stimulated glucose uptake in white adipose tissue and muscle. Impaired GLUT4 translocation or expression is a hallmark of insulin resistance. In this study, we investigated the effects of the adiponectin receptor agonist AdipoRon, an insulin-sensitizing agent, on cardiometabolic dysfunction and insulin signaling in a PCOS model. Hypothesis: We tested the hypothesis that AdipoRon attenuates metabolic complications via modifications in GLUT4 expression in insulin target tissues in a PCOS model. Methods: To induce PCOS, four-week-old female Sprague Dawley rats were implanted with dihydrotestosterone (DHT) silastic tubes (7.5 mg) or placebo (PBO) for 90 days (n=7-12/group). During the final six weeks of DHT treatment, rats received AdipoRon (50 mg/kg/day) orally in food. Fat and lean mass were determined by Echo-MRI. Food intake was measured weekly. Fasting serum insulin, leptin, and adiponectin were measured by ELISA. Retroperitoneal fat (RPF), subcutaneous fat (SCF), and soleus muscle (SM) ADIPOQ (adiponectin) and SLC2A4 (GLUT4) mRNAs were quantified by digital PCR. Results: DHT significantly (p&lt;0.05) increased BW (315.38 ± 4.02 vs 241.67 ± 3.05 g), fat mass (14.80 ± 0.99 vs 10.94 ± 0.80 g), lean mass (289.91 ± 4.04 vs 222.07 ± 2.80 g), and cumulative food intake (24.26 %) compared to PBO group. DHT significantly increased fasting serum insulin and leptin by 21.41% and 23.90%, respectively, while decreasing fasting serum adiponectin by 27.92% compared to the PBO group. DHT had no impact on ADIPOQ and GLUT4 mRNAs expression in RPF and SM while upregulating SCF ADIPOQ and GLUT4 mRNAs expression by 3.07- and 2.13- fold respectively, compared to the PBO group. AdipoRon significantly decreased BW, fat and lean mass, and cumulative food intake. AdipoRon significantly decreased fasting serum insulin and leptin levels by 26.63% and 47.52% respectively, with no impact on adiponectin levels in PCOS rats. Interestingly, AdipoRon significantly (p&lt;0.05) downregulated SCF GLUT4 mRNA expression by 44.40% with no impact on ADIPOQ compared to the vehicle-treated group. AdipoRon had no effect on the mRNA expression of both genes in RPF and SM in PCOS rats. Conclusion: Obesity and decreased circulating adiponectin levels are present in the PCOS model. The upregulation of the GLUT4 mRNA expression in SCF in PCOS rats may indicate different insulin sensitivity in the different insulin target tissues in the PCOS model. On the other hand, activating adiponectin receptors with AdipoRon attenuated PCOS-associated obesity and hyperinsulinemia. Significance: Targeting adiponectin signaling could be a novel and effective therapeutic approach to mitigate metabolic dysfunction in women with PCOS. Supported by NIH grants R01HL171494 (LLYC), P50MD017338 (LLYC), R01HL144847 (DGR), P20GM121334 (DGR), P20GM104357, and P01HL51971, AHA fellowship 24PRE1198302 (AA). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: Obesity leads to sleep-disordered breathing (SDB), including obesity hypoventilation syndrome (OHS), which is characterized by daytime hypercapnia and hypoventilation during sleep. Current management of SDB is largely non-pharmacological, focusing on lifestyle modifications and continuous positive airway pressure (CPAP) therapy. While beneficial, these approaches have low adherence limiting their long-term effectiveness. Leptin, a hormone with known respiratory stimulatory effects, has shown potential as a pharmacological treatment for OHS, yet leptin resistance poses a significant barrier to its therapeutic use. A novel LEPRb agonist, E1/Aca, overcomes this limitation by crossing the blood-brain barrier (BBB) and bypassing leptin resistance mechanisms. Objective: This study investigates leptin and its analog E1/Aca as potential therapeutic agents to treat OHS. Methods: Male leptin-deficient (ob/ob) mice (n = 12) received vehicle, 1 mg/kg of leptin (positive control), or the same dose of E1/Aca intraperitoneally (IP) twice a day for 3 days in a crossover design, with a one-week washout period. Food intake and body weight were measured daily. Respiratory recordings were assessed using a modified plethysmography setup, focusing on the baseline breathing and the hypercapnic ventilatory response (HCVR). Results: As expected, leptin treatment induced weight loss and reduced food intake. Mice treated with E1/Aca also showed initial weight loss in the first days but then regained weight. Both E1/Aca and leptin reduced cumulative food intake. Chronic treatment with leptin or E1/Aca did not affect baseline breathing. However, E1/Aca and leptin significantly enhanced the HCVR, increasing ventilation and tidal volume in 8% CO2 compared to the vehicle. Conclusions: E1/Aca increased the HCVR in leptin-deficient ob/ob mice and represents a promising therapeutic candidate for OHS. Grant support: R01 HL128970, R41 DA056239, R41 HL167326, 24CDA1270910 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Cancer cachexia (CC) is a multifactorial syndrome affecting up to 80% of advanced cancer patients. Characterized by unintentional weight loss, skeletal muscle wasting, and fat depletion, CC significantly impairs quality of life, treatment tolerance, and survival. Despite its prevalence, effective therapeutic options remain limited, underscoring the urgent need for preclinical models that closely mimic clinical symptoms to support drug development. Towards this end, we developed and validated a CC preclinical model with HT-1080 xenograft in NCG mice, which demonstrated hallmark CC features, including body weight loss, fat and muscle loss, as well as impaired physical performance. The tumor-bearing mice also displayed elevated hGDF15 levels (undetectable in non-tumor-bearing (NTB) mice), and significantly reduced cumulative food intake. In this study, we used Ponsegromab (anti-GDF15 monoclonal antibody) as a positive treatment for CC starting when mice had lost ∼7% body weight. At the end of the study, mice in hIgG1-treated control group suffered over 15% body weight loss, while the Ponsegromab treatment restored body weight to baseline levels. Although Ponsegromab treated mice remained lighter than NTB PBS controls, our data suggested that Ponsegromab treatment could significantly improve food intake and increase survival rates. Fat and muscle tissue weights, including gonadal, peri-renal, and inguinal subscapular white fat, as well as gastrocnemius and soleus muscles, were significantly reduced in the hIgG1-treated control group compared to baseline controls. Ponsegromab treatment preserved fat and muscle weights to levels similar to the baseline group. Motor function assessed via rotarod performance, and grip strength showed coordination and strength impairments in tumor-bearing mice, while Ponsegromab significantly improved grip strength and physical performance in comparison to the hIgG1-treated control group. In conclusion, our validated preclinical cachexia models closely align with clinical CC manifestations, providing a robust platform for the evaluation of novel therapeutic strategies. These models enable detailed exploration of CC mechanisms and support the development of effective interventions to mitigate cachexia and improve patient outcomes. Citation Format: Hongyan Sun, Dan Zhou, Yunlong Jiang, Jianming Xu, Jing Zhao, Xiang Gao, Jun Wang. Preclinical models of cancer cachexia: Bridging the gap to clinical applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3930.

Mitochondria play key roles in neuronal activity, particularly in modulating agouti-related protein (AgRP) and proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC), which regulates food intake. FAM163A, a newly identified protein, is suggested to be part of the mitochondrial proteome, though its functions remain largely unknown. This study aimed to investigate the effects of Fam163a knockdown and mitochondrial dysfunction on food intake, AgRP neuron activity, and mitochondrial function in the hypothalamus. Male C57BL/6 and AgRP-Cre mice received intracranial injections of either Fam163a shRNA, rotenone, or appropriate controls. Behavioral assessments included food intake, locomotor activity, and anxiety-like behaviors. qRT-PCR was used to quantify the expression of the genes related to food intake, mitochondrial biogenesis, dynamics, and oxidative stress. Blood glucose, serum insulin, and leptin levels were measured. Electrophysiological patch-clamp recordings were used to assess the AgRP neuronal activity. Fam163a knockdown in the ARC increased the cumulative food intake in short term (first 7 days) without altering the 25-day food intake and significantly increased the Pomc mRNA expression. Fam163a silencing significantly reduced leptin levels. Both Fam163a knockdown and rotenone significantly reduced the firing frequency of AgRP neurons. Neither Fam163a silencing nor rotenone altered locomotor or anxiety-like behaviors. Fam163a knockdown and rotenone differentially altered the expression of mitochondrial biogenesis-, mitophagy-, fusion-, and oxidative stress-related genes. Hypothalamic FAM163A may play a role in modulating AgRP neuronal activity through regulating mitochondrial biogenesis, dynamics, and redox state. These findings provide insights into the role of FAM163A and mitochondrial stress in the central regulation of metabolism.

BackgroundNumerous physiological properties have been documented in the literature pertaining to spexin, such as its role in appetite regulation. Therefore, the present research aimed to assess the impacts of centrally administering spexin and its interaction with opioid and melanocortin receptors on feeding behavior in neonatal broiler chicks. In experiment 1, the chickens were administered intracerebroventricular (ICV) injection of saline and spexin at varying doses of 5, 7.5 and 10 nmol. During the experiment 2, the broilers were subjected to an ICV injection containing saline, β-FNA (µ-opioid receptor antagonist, 5 µg), spexin (10 nmol), and a mixture of β-FNA plus spexin. Experiments 3 to 6 bore resemblance to experiment 2, with the exception that NTI (δ-opioid receptor antagonist, 5 µg), nor-BNI (κ-opioid receptor antagonist, 5 µg), SHU9119 (MC3/MC4 receptor antagonist, 0.5 nmol) and MCL0020 (MC4 receptor antagonist, 0.5 nmol) were administered instead of β-FNA. Then, the birds were promptly placed back into their individual cages and cumulative food intake was assessed at intervals of 30, 60, and 120 min postinjection.ResultsSpexin demonstrated a significant and dose-dependent decrease in food intake when compared to the control group (P < 0.05). Co-injection of β-FNA + spexin diminished spexin-induced hypophagia (P < 0.05) whereas concurrent administration of NTI and nor-BNI with spexin led to an amplification of the decrease in food consumption induced by spexin (P < 0.05). Additionally, anorexigenic impact of spexin was reversed by combined injection of SHU9119 and MCL0020 with spexin (P < 0.05).ConclusionThese observations suggest that both opioid and melanocortin receptors play crucial role in spexin-induced hypophagia.

Exploring a novel toxicological mechanism: Ammonia-N induces neurotoxicity, oxidative damage in brain tissue, and modulates the expression of central appetite factors to suppress feeding in juvenile Siberian sturgeon (Acipenser baerii Brandt)

Abstract Disclosure: A. Yasrebi: None. K. Otersen: None. O. Groh: None. E. Guthman: None. I. Shmarakov: None. S. Campbell: None. T.A. Roepke: None. The long-term metabolic effects of gender-affirming hormone therapy (GAHT) is largely unknown, in part, due to the lack of properly designed preclinical animal models. Here we propose a rodent study design, beyond the classical endocrine model of gonadectomy and steroid treatment, to accurately reflect the reality of the trans experience. Specifically, trans women do not all seek or undergo orchidectomy (ORX) instead take estrogen supplementation along with an androgen blocker such as finasteride. In this study, 40 WT C57 male mice were divided into 4 groups of 10 mice - 1) intact with oil vehicle, 2) intact with estradiol benzoate (EB, 150 μg/kg/d) and finasteride (F, 0.25 mg/kg/d), 3) ORX with oil; and ORX with EB (150 μg/kg/d). Each mouse was dosed daily for 8 weeks while on a standard chow diet. Mice were pair housed and food intake and body weight were collected weekly prior to behavioral (open field, elevated plus, Y-maze) and metabolic (body composition, metabolic rates, activity, tolerance testing) phenotyping. Intact mice treated with EB+F exhibited less avoidance behaviors, potentially due to differences in movement, with no differences between treatments in the Y-maze. Cumulatively, Intact:EB+F gained less weight than Intact:oil (3.4 ± 0.4 g vs 5.3 ± 0.4 g), while ORX:oil mice gained less than ORX:EB (2.2 ± 0.5 g vs 5.5 ± 0.7 g). These interactive effects were due to differences in the gain of fat or lean mass between ORX and intact and lower cumulative food intake in ORX:oil mice (136 ± 3.8 g vs Intact:oil: 159 ± 4.9 g; Intact:EB+F: 167 ± 6.1 g; ORX:EB: 171 ± 3.4 g). Metabolic assessments found that Intact:EB+F exhibited elevated V.O2, V.CO2, and heat production in the light and dark cycle compared to Intact:oil, while ORX:EB only exhibited elevated heat production compared to ORX:oil. Similarly, Intact:EB+F and ORX:EB moved more than their counterparts during the dark cycle; however, only Intact:EB+F exhibited elevated wheel running. Tolerance tests (i.p.) found that glucose clearance was augmented in ORX:EB mice while insulin-induced glucose clearance was lower in Intact:EB+F compared to Intact:oil but modestly elevated by EB in ORX mice. These data, along with suppressed fasting blood glucose in ORX:EB mice, suggest that glucose homeostasis is differentially modulated by GAHT paradigms in mice. Analysis of hypothalamic, hepatic, adipose, and intestinal gene expression is currently underway. Collectively, these data, generated by using a translationally-relevant preclinical animal model, are the first to establish that metabolism is differentially affected by both steroid treatments and ORX and suggesting that the long-term metabolic effects of GAHT are dependent on the type of treatment. The underlying mechanisms and sites of action remain to be investigated. Presentation: 6/3/2024

γ-aminobutyric acid improves the growth performance, food intake and glucose homeostasis of Micropterus salmoides fed high-carbohydrate diets

Time-restricted feeding modulates gene expression related with rhythm and inflammation in Mongolian gerbils

The objective of this study was to ascertain the interaction between muscarinic, NMDA glutamate, and glycine receptors on sericin-induced hypophagia in neonatal chickens. This study comprised six experiments, with four groups in each and 11 repeats per group. In Experiment 1, the chicken received an ICV injection of saline, sericin (0.5 nmol), or Telenzepine (an M1 muscarinic receptor antagonist, 125 nmol). Group 4 received a co-injection of sericin and Telenzepine. In experiments 2-6, the chicken received ICV injections of AF-DX116 (an M2 muscarinic receptor antagonist, 125 nmol), 4-DAMP (an M3 muscarinic receptor antagonist, 125 nmol), PD102807 (an M4 muscarinic receptor antagonist, 125 nmol), MK-801 (an NMDA glutamate receptor antagonist, 15 nmol), and strychnine (a glycine receptor antagonist, 100 nmol) instead of Telenzepine. Subsequently, the cumulative food intake was quantified at 30, 60, and 120 minutes post-injection. The results demonstrated that the ICV injection of sericin (0.5 nmol) led to a notable reduction in cumulative food intake when compared to the control group (P<0.05). The co-injection of telenzepine and sericin resulted in a notable reduction in the hypophagia induced by sericin, as evidenced by a statistically significant difference (P<0.05). The co-injection of MK-801 and sericin resulted in a reduction of the hypophagic effects of sericin when compared to the control group (P<0.05). The co-injection of strychnine and sericin resulted in a notable reduction in the hypophagic effects of sericin, as evidenced by a statistically significant difference when compared to the control group (P<0.05). These findings suggest that sericin-induced hypophagia is mediated via M1 muscarinic, NMDA glutamate, and glycine receptors in neonatal chickens.

Liver-expressed antimicrobial peptide-2 (LEAP-2) has mutual antagonism with ghrelin, which evokes food intake under a freely fed state. Nevertheless, the impact of LEAP-2 on ghrelin under time-restricted feeding (TRF), which has benefits in the context of metabolic disease, is still unknown. This study aims to explore the impact of central administration of LEAP-2 on the ingestion behavior of rats, which was evaluated using their cumulative food intake in the TRF state. Before intracerebroventricular (ICV) administration of O-n-octanoylated ghrelin (0.1 nmol/rat), as a food-stimulatory model, the rats received various doses of LEAP-2 (0.3, 1, 3 nmol/rat, ICV). Cumulative food intake was recorded at 1, 2, 4, 8, 12, and 24 h after ICV injection under 12 h freely fed and TRF states in a light phase. In 12 h freely fed and TRF states, central administration of ghrelin alone induced feeding behavior. Pre-treatment with LEAP-2 (1 and 3 nmol/rat, ICV) suppressed ghrelin-induced food intake in a dose-dependent manner in a 12 h freely fed state instead of a TRF state, which may have disturbed the balance of ghrelin and LEAP-2. This study provides neuroendocrine-based evidence that may explain why TRF sometimes fails in fighting obesity/metabolic dysfunction-associated steatotic liver disease in clinics.

Objective: The burgeoning prevalence of obesity and associated co-morbidities necessitates innovative approaches for safe and efficacious therapies. We describe the characterization of PTT-A, a novel long-acting peptide agonist for GLP-1, GIP, Amylin and Calcitonin Receptors, in rodent models and assessed its efficacy against the dual GIPR/GLP-1R agonist Tirzepatide. Methods: In vitro assays used recombinant cell lines expressing individual receptors to assess GLP-1, GIP, Amylin and Calcitonin receptor agonism. Multiple metabolic endpoints were examined in rodents, including acute food intake and calcium regulation effects of PTT-A in lean rats, acute glucose-lowering effects in lean mice, and its chronic effects in diet-induced obesity (DIO) rats compared to Tirzepatide. Results: PTT-A exhibited potent GIP, GIP, Amylin, and Calcitonin receptor agonism in cAMP assays, and induced decreases in blood glucose and calcium levels in acute studies in lean rodents. In lean rats, PTT-A dose-dependently reduced cumulative food intake. Chronic studies in DIO rats revealed significant reduction in cumulative food intake and body weight, driven by decreases in fat mass without loss of muscle mass, unlike that seen with Tirzepatide. PTT-A demonstrated robust efficacy for glucose and plasma lipid lowering, insulin sensitization and liver benefits, as indicated by lowered plasma insulin, alanine aminotransferase and liver triglycerides, while outperforming Tirzepatide at equivalent doses. Conclusion: PTT-A is a novel tetra-receptor agonist, offering superior outcomes for weight loss, glycemic control, insulin sensitization, and liver health compared to Tirzepatide. These findings underscore the promise of such poly-pharmacological agents to tackle the complex challenges associated with obesity-related metabolic disorders. Disclosure S. Ghosh: Consultant; Abvance Therapeutics, Akero Therapeutics, Inc., Fractyl Health, Inc., Takeda Pharmaceutical Company Limited. P. Valdecantos: Research Support; Pep2Tango Therapeutics INC. P. Rada: Research Support; Pep2Tango Therapeutics INC. A.M. Valverde: Research Support; Pep2Tango Therapeutics INC. C.M. Rondinone: Board Member; Pep2tango Therapeutics Inc. Stock/Shareholder; Pep2tango Therapeutics Inc.

Objective: Advancements in GLP-1RA research, shifting from daily to weekly injectables, have improved therapeutic outcomes and expanded market potential, paving the way for monthly injectable drug development. This study evaluates the clinical potential of PLGA-encapsulated once-monthly injectable semaglutide through extensive non-clinical assessments, including safety profiling and pharmacokinetic/pharmacodynamic evaluations. Methods: Semaglutide was solubilized in acetic acid with a diverse biodegradable PLGA polymer mixture and processed using ultrasonic spray-drying to create microspheres encapsulating semaglutide. The optimized formulation, PT403, with a proper drug loading and no initial burst was developed. Subsequent assessments, including PK, PD, and toxicology, were conducted in mouse, rat, and/or minipig. Results: PT403 exhibits favorable physicochemical properties, including uniform microsphere size (20μm), one-year stability, and extended release up to 70 days in minipigs. In a Single Ascending Dose (SAD) study in mice, PT403 induced a 20% body weight reduction at doses ranging from 24 to 192 mg/kg. No other notable critical signs were observed, ensuring a safe profile even up to 192 mg/kg. Moreover, simulated release profiles suggest PT403's potential as a once-a-month injectable, with a Peak-to-Trough ratio of 1.4. Both semaglutide and PT403 demonstrated consistent reductions in body weight (30%) and cumulative food intake across 28 repeated injections of semaglutide and double injections of PT403. Conclusion: PT403, featuring uniform microsphere size and favorable drug loading, exhibited safety in mice at the highest dose. Simulations support this formulation, and consistent pharmacodynamic effects across semaglutide and PT403 groups in repeated injections suggest its promise for clinical development as a monthly injectable for diabetes and obesity. Disclosure J. Lee: None. J. Cho: None. E. Jang: None. M. Kim: None. J. Bae: None. M. Choi: None. S. Chang: None. J. Jung: None.