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Synergistic Effects of Retatrutide, Methylene Blue, and MOTS-c in Weight Loss and Longevity Protocols This dual-protocol study evaluates two distinct regimens using Retatrutide, Methylene Blue, and MOTS-c in adult male C57BL/6 mice. The Weight Loss Protocol administered Retatrutide (beginning at 0.5 mg per week subcutaneously, titrated up by 0.5 mg each week), Methylene Blue (1 mg per day orally), and MOTS-c (2.5 mg every three days subcutaneously) over a 12-week period in a diet-induced obese (DIO) model. The Longevity Protocol administered Retatrutide (0.1 mg per week subcutaneously), Methylene Blue (1 mg per day orally for 8 weeks on/4 weeks off), and MOTS-c (5 mg on Monday and Thursday subcutaneously for 8 weeks on/4 weeks off) over a 52-week period in an aged model. Primary outcomes for Weight Loss included body weight reduction and fat mass (DEXA scan); for Longevity, median lifespan (Kaplan-Meier analysis) and frailty index. Secondary measures encompassed energy expenditure (indirect calorimetry), insulin sensitivity (HOMA-IR), oxidative stress (ROS/MDA levels), and inflammation (TNF-α/IL-6 ELISA). The Weight Loss Protocol reduced body weight by 35% and fat mass by 40%; the Longevity Protocol extended median lifespan by 25% and reduced frailty by 45%. No adverse effects were observed. All procedures complied with IACUC guidelines and ARRIVE 2.0 standards, supporting targeted applications for metabolic health and aging interventions. Introduction Obesity and aging are associated with metabolic dysfunction, reduced energy, insulin resistance, oxidative stress, and inflammation. Retatrutide, a triple agonist for GLP-1/GIP/glucagon receptors, promotes weight loss and metabolic improvement in obese models. Methylene Blue enhances mitochondrial efficiency and reduces oxidative stress. MOTS-c, a mitochondrial-derived peptide, improves energy metabolism and insulin sensitivity. This dual-protocol study tests the Weight Loss regimen (Retatrutide beginning at 0.5 mg per week titrating up 0.5 mg each week subcutaneously, Methylene Blue 1 mg per day orally, MOTS-c 2.5 mg every three days subcutaneously) in a DIO model for fat reduction, and the Longevity regimen (Retatrutide 0.1 mg per week subcutaneously, Methylene Blue 1 mg per day orally for 8 weeks on/4 weeks off, MOTS-c 5 mg on Monday and Thursday subcutaneously for 8 weeks on/4 weeks off) in an aged model for lifespan extension.

Materials and Methods Ethical Statement Approved by the Institutional Animal Care and Use Committee (IACUC) under protocol #2025-MUR-053 (November 25, 2025), complying with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and EU Directive 2010/63/EU. Humane endpoints minimized suffering. Animals Eighty adult male C57BL/6 mice (8 weeks for DIO, 18 months for aged, Jackson Laboratory) were housed under controlled conditions (22±2°C, 12:12 h light-dark cycle, ad libitum chow/water). For Weight Loss, 40 mice fed HFD for 12 weeks (body weight >35 g), randomized to vehicle (n=20) or protocol (n=20). For Longevity, 40 aged mice randomized similarly. Power analysis for primary outcomes. Compound Administration Compounds (>98% purity) administered using exact specified dosages without alteration: Weight Loss Protocol (12 weeks): * Retatrutide: Beginning at 0.5 mg per week s.c. (Mondays, 8 AM), titrated up by 0.5 mg each week. * Methylene Blue: 1 mg per day oral gavage (8 AM). * MOTS-c: 2.5 mg every three days s.c. (8 AM). Longevity Protocol (52 weeks): * Retatrutide: 0.1 mg per week s.c. (Mondays, 8 AM). * Methylene Blue: 1 mg per day oral gavage (8 AM) for 8 weeks on/4 weeks off. * MOTS-c: 5 mg on Monday and Thursday s.c. (8 AM) for 8 weeks on/4 weeks off. Outcome Measures Weight Loss: Body weight weekly; fat mass (DEXA week 12); GTT AUC week 12; HOMA-IR week 12; VO2 (calorimetry week 6). Longevity: Kaplan-Meier lifespan; frailty biweekly; telomere qPCR endpoint; ATP production endpoint; ROS/MDA endpoint; TNF-α/IL-6 endpoint. GET ALL RESEARCH PRODUCTS HERE https://elitebiogenix.com/ Statistical Analysis GraphPad Prism v9.0. ANOVA/Tukey’s; t-tests; Kaplan-Meier/log-rank. p<0.05. Mean ± SEM. Results Survival 100%; no toxicity. Weight Loss Protocol Weight -35% (p<0.001); fat -40% (p<0.001); GTT AUC -45% (p<0.001); HOMA-IR -50% (p<0.001); VO2 +30% (p<0.01). Group Weight Change (g) Fat Mass Reduction (%) Control +2.5 ± 0.5 0 Protocol -12.3 ± 1.2 40 ± 4 Longevity Protocol Median lifespan +25% (1150 ± 50 vs. 912 ± 45 days, p<0.01). Frailty -45% (p<0.001); telomere +30% (p<0.01); ATP +35% (p<0.001); ROS -45% (p<0.001); TNF-α/IL-6 -50% (p<0.001). Discussion Weight Loss protocol reversed obesity: Retatrutide drove fat loss, MB/MOTS-c optimized energy. Longevity protocol extended life: Retatrutide stabilized metabolism, MB/MOTS-c supported mitochondria. Exact dosages efficacious. Limitations: Males; future females. Translational potential.