The miniature pig is a suitable animal model for investigating human cardiovascular diseases. Nevertheless, the alterations in lipid metabolism within atherosclerotic plaques of miniature pigs, along with the underlying mechanisms, remain to be comprehensively elucidated. In this study, we aim to examine the alterations in lipid composition and associated pathways in the abdominal aorta of atherosclerotic pigs induced by a high-fat, high-cholesterol, and high-fructose (HFCF) diet using lipidomics and RNA-Seq methods. The results showed that the content and composition of aortic lipid species, particularly ceramide, hexosyl ceramide, lysophosphatidylcholine, and triglyceride, were significantly altered in HFCF-fed pigs. Meanwhile, the genes governing sphingolipid metabolism, iron ion homeostasis, apoptosis, and the inflammatory response were significantly regulated by the HFCF diet. Furthermore, C16 ceramide could promote iron deposition in RAW264.7 cells, leading to increased intracellular reactive oxygen species (ROS) production, apoptosis, and activation of the toll-like receptor 4 (TLR4)/nuclear Factor-kappa B (NF-қB) inflammatory pathway, which could be mitigated by deferoxamine. Our study demonstrated that dysregulated ceramide metabolism could increase ROS production, apoptosis, and inflammatory pathway activation in macrophages by inducing iron overload, thus playing a vital role in the pathogenesis of atherosclerosis. This discovery could potentially provide a new target for pharmacological therapy of cardiovascular diseases such as atherosclerosis.

Obesity has emerged as a major risk factor for human health, exacerbated by aging and changes in dietary habits. It represents a significant health challenge, particularly for older people. While numerous studies have examined the effects of obesity and aging on fat metabolism independently, research on their combined effects is limited. In the present study, the protective action against white fat accumulation after a high-fat diet (HFD) exerted by exogenous melatonin, a circadian hormone endowed with antioxidant properties also involved in fat metabolism, was investigated in a mouse model. For this purpose, a battery of tests was applied before and after the dietary and melatonin treatments of the animals, including epididymal white adipose tissue (eWAT) histological evaluations, transcriptomic and lipidomic analyses, real-time PCR tests, immunofluorescence staining, Western blot, the appraisal of serum melatonin levels, and transmission electron microscopy. This study found that aged mice on a high-fat diet (HFD) showed increased lipid deposition, inflammation, and reduced antioxidant glutathione (GSH) levels compared to younger mice. Lipidomic and transcriptomic analyses revealed elevated triglycerides, diglycerides, ceramides, and cholesterol, along with decreased sphingomyelin and fatty acids in eWAT. The genes linked to inflammation, NF-κB signaling, autophagy, and lipid metabolism, particularly the melatonin and glutathione pathways, were significantly altered. The aged HFD mice also exhibited reduced melatonin levels in serum and eWAT. Melatonin supplementation reduced lipid deposition, increased melatonin and GSH levels, and upregulated AANAT and MTNR1A expression in eWAT, suggesting that melatonin alleviates eWAT damage via the MTNR1A pathway. It also suppressed inflammatory markers (e.g., TNF-α, NLRP3, NF-κB, IL-1β, and CEBPB) and preserved mitochondrial function through enhanced mitophagy. This study highlights how aging and HFD affect lipid metabolism and gene expression, offering potential intervention strategies. These findings provide important insights into the mechanisms of fat deposition associated with aging and a high-fat diet, suggesting potential intervention strategies.