Pomegranate peel extract counteracts palmitate-induced insulin resistance by inducing mitophagy, reducing inflammation, and regulating MAPK signaling in C2C12 myotubes

Pomegranate peel, a by-product rich in ellagitannin-type polyphenols, was dried at 60 °C and extracted (ethanol:water, 38:62 v/v) to yield an extract characterized for total polyphenols, punicalagin, ellagic acid (Folin–Ciocalteu assay and HPLC), and antioxidant capacity (DPPH, FRAP, ORAC). To assess its protective effects against insulin resistance induced by palmitic acid, differentiated C2C12 skeletal muscle myotubes were treated for 6 h with vehicle (0.25 % bovine serum albumin), palmitic acid (0.2 mM), or palmitic acid plus pomegranate peel extract (equivalent to 100 μM punicalagin). Insulin signaling (p-AKT^Ser473), mechanistic target of rapamycin pathway activity, mitogen-activated protein kinase signaling, and NF-κB activation (IκBα^Ser32) were quantified by Western blotting; autophagic flux was assessed via the LC3 II/I ratio; mitochondrial morphology by transmission electron microscopy; and mitochondrial reactive oxygen species by MitoSOX™ flow cytometry. Data were analyzed by one-way ANOVA and Tukey's test or Friedman test with Dunn's post hoc (p < 0.05), according to data distribution. Compared to palmitic acid alone, pomegranate peel extract fully restored insulin-stimulated AKT phosphorylation, attenuated mitochondrial reactive oxygen species accumulation, increased the LC3 II/I ratio, induced mitophagosome formation and mitochondrial elongation, and modulated MAPK activation. In conclusion, pomegranate peel extract prevented palmitate-induced insulin resistance in muscle cells by activating mitophagy and rebalancing key signaling pathways, highlighting its potential as a functional ingredient to enhance muscle insulin sensitivity and mitochondrial health.