Insulin Resistance, Anti-inflammatory, and Antioxidant In vitro Activity of Heterologously Expressed Arenin from Dryophytes arenicolor

Amphibian-derived peptides are promising therapeutics for metabolic dysfunction, oxidative stress, and immune imbalance. We investigated arenin, a Kunitz-type peptide from <i>Dryophytes arenicolor</i>, produced heterologously in <i>E. coli</i>, for anti-metabolic, antioxidant, and immunomodulatory activities. In insulin-resistant hepatocytes, arenin increased glucose uptake from 23.97 ± 2.79% (IR control) to 51.38 ± 2.95% at 250 µg/mL. Arenin also reduced intracellular reactive oxygen species (ROS) in a time-dependent manner. The most significant early effect (20 min) occurred at higher doses (500-1000 µg/mL), whereas at 24 h the effect shifted to lower-intermediate concentrations (15.62-250 µg/mL), with 15.62 µg/mL yielding the lowest ROS levels (15.78 ± 1.66%). In macrophages, nitric oxide (NO) production showed a concentration-dependent, multi-layered response, indicating immunomodulation. These data position arenin as a multifunctional bioactive capable of improving metabolic homeostasis, strengthening antioxidant defenses, and tuning innate immune responses, laying groundwork for peptide-based interventions against metabolic disturbances.