Dynamic nitrosation - palmitoylation of CD36 regulates serum lipids
Regulation of endothelial cell (EC) lipid content is crucial for cell and organ function. During obesity, ECs become lipid laden leading to lipotoxicity and endothelial dysfunction which further contribute to metabolic syndrome progression. Here, we demonstrate a novel pathway by which the endothelium, via eNOS-dependent nitrosation, inhibits excess lipid accumulation during hyperlipidemic conditions in obesity. In the vasculature, nitric oxide has been reported as a potent vasodilator. However, we highlight a new role for nitric oxide as a modulator of serum lipids. We show this occurs as a result of the downregulation of Cav1, a potent negative regulator of endothelial nitric oxide synthase, increasing EC endogenous nitric oxide synthesis. Using EC-specific Cav1 knockout mice, we are able to increase nitric oxide in vivo. This increased nitric oxide leads to nitrosation of cysteines 3 and 466 on the cytoplasmic tails of CD36, a fatty acid translocase, disrupting palmitoylation of these residues and subsequently inhibiting trafficking of CD36 to the plasma membrane. Together, this work suggests that CD36 nitrosation occurs as a protective mechanism to prevent lipotoxicity and EC dysfunction during the progression of metabolic syndrome. Overall design: Sixteen hours prior to assessment HAMECs were treated with 1mM L-NAME and subsequently four hours prior to assessment HAMECs were treated with 50 µM lipids as well as 50 µM DETA NONOate. RNA was isolated using Quick-RNA MiniPrep Kit (Genesee Scientific: 11-328). When necessary, samples were further concentrated and purified with the RNeasy MinElute Cleanup Kit (Qiagen: 74804). PE150 reads were generated with the Illumina NovoSeq platform. The paired-end reads were mapped to the hg38 reference genome using STAR (v 2.7.9a). Gene counts were generated with FeatureCounts in the Rsubread (v. 2.8.8) package. DESeq2 (v. 1.34.0) was used to calculate log fold change and adjusted p-values. The adjusted p-values were then adjusted for multiple comparisons between groups by the Benjamini-Hochberg procedure using p.adjust(). Ensemble IDs were converted to gene symbols using the EnsDb.Hsapiens.v75 and org.Hs.eg.db packages.