Gamma-Oryzanol Supplementation: Lipid and Metabolic Profiling in Soleus and EDL Muscles of Wistar Rats with Diet-Induced Obesity

Gamma-oryzanol (ORY) is a phytosterol widely recognized for its antioxidant and anti-obesity effects. However, its effects on lipid and metabolic remodeling in skeletal muscle, particularly in oxidative and glycolytic phenotypes, remain poorly understood. Therefore, this study aimed to investigate how ORY modulates metabolic remodeling in soleus (oxidative) and extensor digitorum longus (EDL; glycolytic) muscles in rats with diet-induced obesity. Male Wistar rats were randomly assigned to three groups (n=6/group): Control (CTRL), high sugar-fat (HSF), and HSF supplemented with ORY (0.5% w/w) (HSF+ORY). After 30 weeks, animals were fasted, anesthetized, and euthanized (1458/2024-CEUA). Nutritional parameters were assessed, and untargeted lipidomics and metabolomics were performed. Lipids and metabolites were extracted using a methyl tert-butyl ether protocol and analyzed by liquid chromatography coupled to high-resolution mass spectrometry. Statistical analyses included one-way ANOVA/Tukey’s or Kruskal-Wallis/Dunn’s post-hoc tests (α=5%). Multivariate analyses (PLS-DA) and volcano plots were used for feature selection, and pathway/network integration was performed using Ingenuity Pathway Analysis. Compared to CTRL, the HSF group presented higher caloric intake, body weight, adiposity index, and pronounced lipidomic and metabolomic remodeling in both muscles, confirming the effectiveness of the obesogenic model. ORY supplementation did not alter nutritional status but induced distinct metabolic responses in soleus and EDL. In comparison to HSF, HSF+ORY exhibited significant remodeling of several phospholipid, sphingolipid, diacylglycerol, and triglyceride species, along with modulation of energy metabolism intermediates in both oxidative and glycolytic phenotypes. At the metabolite level, in soleus, ORY affected key stress-responsive metabolites (proline, log2FC = −0.38) and metabolic signaling (leucine,log2FC = −0.34); regarding EDL, the supplementation affected nucleotide-related pathways, including nucleotide catabolism (z-score = −2.00) and salvage (z-score = −2.00), and reduced nucleoside and molecule transport (z-score = −2.24). These findings suggest that ORY exerts targeted effects on lipid metabolism and metabolic pathways without reversing the obese phenotype. The modulation of phospholipid classes and lipid-derived intermediates indicates an impact on membrane remodeling and cellular stress regulation, which may contribute to improved mitochondrial and redox homeostasis. These effects may be partly attributed to the antioxidant properties of ORY, derived from ferulic acid esters, which are known to scavenge reactive oxygen species and preserve phospholipid integrity. Markedly in EDL muscle, the attenuation of oxidative stress and improved metabolic balance markers were more pronounced. Additionally, the glycolytic muscles presented changes in nucleotide metabolism and reduced abundance of purines and nucleosides, possibly reflecting attenuation of metabolic stress and redox imbalance. In conclusion, ORY supplementation modulates key lipid and metabolic pathways in skeletal muscle independently of changes in body weight. These results support a targeted role of ORY in metabolic remodeling and highlight its potential as a therapeutic strategy for metabolic muscle dysfunction associated with obesity.

Acknowledgments: FAPESP (2023/06451-6 / 2024/04007-4).