Nitrosation of CD36 Regulates Endothelial Function and Serum Lipids
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Abstract
During obesity, endothelial cells (ECs) become lipid laden, leading to endothelial dysfunction. We tested posttranslational modification on CD36 that may regulate EC lipid accumulation.
METHODS:
We used an EC-specific Cav1 (caveolin-1) knockout mouse, nitrosation and palmitoylation assays, and whole animal Nγ-nitro-l-arginine methyl ester administration to examine blood lipids.
RESULTS:
EC-specific Cav1 knockout male mice are hyperlipidemic regardless of diet but retain endothelial cell function. We found these mice have significantly increased NO in response to the lack of Cav1, and the presence or absence of NO toggled inversely EC lipid content and plasma lipid in mice. The NO nitrosated the fatty acid translocase CD36 at the same cysteines that are palmitoylated on CD36. The nitrosation of CD36 prevented its trafficking to the plasma membrane and decreased lipid accumulation. The physiological effect of this mechanism was a reliance on NO for endothelial function and not dilation.
CONCLUSIONS:
This work suggests that CD36 nitrosation occurs as a protective mechanism to prevent EC lipotoxicity.
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Review coordinated by Life Science Editors Foundation Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors Potential Conflicts of Interest: None
PUNCHLINE This preprint reveals a novel mechanism by which nitric oxide (NO) regulates lipid uptake in endothelial cells through nitrosation of the fatty acid transporter CD36. In conditions where endothelial NO is elevated, CD36 is modified at key cysteine residues, which prevents its localization to the plasma membrane and thus inhibits lipid uptake. This nitrosation-based regulation protects endothelial cells from lipid overload while increasing circulating serum lipids. The findings establish a dynamic and reversible regulatory axis—Cav1 → NO → CD36—that links vascular signaling to metabolic control.
BACKGROUND Endothelial cells (ECs), which line the …
Review coordinated by Life Science Editors Foundation Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors Potential Conflicts of Interest: None
PUNCHLINE This preprint reveals a novel mechanism by which nitric oxide (NO) regulates lipid uptake in endothelial cells through nitrosation of the fatty acid transporter CD36. In conditions where endothelial NO is elevated, CD36 is modified at key cysteine residues, which prevents its localization to the plasma membrane and thus inhibits lipid uptake. This nitrosation-based regulation protects endothelial cells from lipid overload while increasing circulating serum lipids. The findings establish a dynamic and reversible regulatory axis—Cav1 → NO → CD36—that links vascular signaling to metabolic control.
BACKGROUND Endothelial cells (ECs), which line the blood vessels, are uniquely positioned as gatekeepers of metabolic exchange—controlling the delivery of nutrients, including fatty acids, from the bloodstream into peripheral tissues. In the setting of metabolic syndrome, a state of chronic nutrient excess, this finely tuned lipid transport system becomes dysregulated. Excessive lipid uptake by ECs leads to lipid accumulation, mitochondrial dysfunction, and progressive endothelial impairment, which in turn contributes to the pathogenesis of cardiovascular and metabolic diseases.
While ECs appear to possess intrinsic protective mechanisms to buffer against lipid overload, the molecular basis of these protective responses is poorly understood. The authors sought to uncover how ECs might actively limit lipid uptake under stress, and what upstream signals trigger this protective state.
QUESTIONS ADDRESSED How do endothelial cells protect themselves from lipid-induced dysfunction during nutrient excess? What regulatory mechanisms allow them to limit lipid uptake under stress?
SUMMARY Reduced endothelial Cav1 leads to increased NO production and, in turn, nitrosation of CD36. This modification prevents CD36 from reaching the plasma membrane, blocking lipid uptake into ECs. As a result, circulating serum lipids increase, but endothelial function is preserved. These findings define NO-mediated nitrosation as a new mechanism of post-translational regulation of CD36, with implications for endothelial health in metabolic disease.
KEY RESULTS Cav1 is downregulated in endothelial cells in obesity Figures 1A–F, Supplementary Fig. 1A–F Goal: Identify genes affected by obesity that regulate endothelial lipid uptake. Outcome: Single-cell RNA-seq of mouse and human adipose tissues reveals consistent downregulation of Cav1 in all endothelial subtypes during obesity (Fig. 1A–F). Supplementary Fig. 1 shows quality control, cell-type identification, and confirmation of downregulated Cav1 expression in both species (Supp. Fig. 1A–F).
Loss of Cav1 increases circulating lipids and decreases EC lipid uptake Figures 2A–K, Supplementary Fig. 2A–E Goal: Determine the physiological effect of Cav1 loss on lipid homeostasis. Outcome: EC-specific Cav1 knockout mice have elevated serum triglycerides, cholesterol, and LDL (Fig. 2E–G), but reduced lipid droplet accumulation in ECs (Fig. 2J). They maintain normal weight and show improved glucose tolerance (Fig. 2H–L). Supplementary Fig. 2 confirms successful endothelial deletion of Cav1 (Supp. Fig. 2A–C) and shows that hyperlipidemia is not due to differences in dietary intake or lipid absorption (Supp. Fig. 2D–E).
Loss of Cav1 elevates NO and suppresses lipid uptake Figures 3A–J, 4A–F, Supplementary Fig. 3A–E Goal: Test whether elevated NO mediates lipid uptake defects. Outcome: Cav1 knockout increases serum nitrate/nitrite (Fig. 3J), reflecting elevated NO. Pharmacologic NO inhibition with L-NAME restores lipid uptake in HAMECs (Fig. 4A) and mouse aorta (Fig. 4B), and reduces serum lipids (Fig. 4C–D). Deletion of eNOS in ECs—but not in RBCs—rescues the phenotype (Fig. 4E–F). Supplementary Fig. 3 shows that Cav1 knockout does not impair vasodilatory responses to acetylcholine and confirms NO elevation by multiple readouts (Supp. Fig. 3A–E).
CD36 mediates endothelial lipid uptake and is regulated by NO Figures 5A–F, Supplementary Fig. 4A–D Goal: Identify whether CD36 is necessary and sufficient for NO-regulated lipid uptake. Outcome: CD36 localizes to Cav1-enriched domains (Fig. 5A) and is required for lipid uptake (Fig. 5C). NO donors suppress CD36-mediated lipid uptake in HEK293T cells (Fig. 5D), and NO induces CD36 nitrosation (Fig. 5E). Pharmacologic CD36 inhibition abolishes the effect of eNOS deletion (Fig. 5F). Supplementary Fig. 4 confirms that CD36 expression is unaltered by NO or Cav1 loss, suggesting the effect is post-translational (Supp. Fig. 4A–D).
CD36 is nitrosated at cysteines 3 and 466, disrupting palmitoylation and lipid uptake Figures 6A–D, 7A–F, Supplementary Fig. 5A–C Goal: Identify the functional nitrosation sites and their impact on trafficking. Outcome: CD36 is nitrosated at C3 and C466. Mutating these residues abolishes nitrosation and restores lipid uptake despite NO exposure (Fig. 6C–D). Nitrosation prevents palmitoylation of CD36 (Fig. 7D), explaining the loss of plasma membrane localization (Fig. 7B–F). Supplementary Fig. 5 shows quantification of CD36 localization shifts (Supp. Fig. 5A–C).
Nitrosation restricts CD36 to the ER and blocks its trafficking Figures 7A–F, Supplementary Fig. 6A–D Goal: Understand the subcellular localization of nitrosated CD36. Outcome: In Cav1-deficient or NO-treated ECs, CD36 remains in the endoplasmic reticulum (ER) (Fig. 7B, 7E). L-NAME restores membrane localization (Fig. 7C, 7F). Supplementary Fig. 6 provides further co-localization data with ER and Golgi markers and quantifies trafficking defects (Supp. Fig. 6A–D).
NO protects ECs from lipid-induced mitochondrial dysfunction and impaired vasodilation Figures 8A–C, Supplementary Fig. 7A–E Goal: Determine the physiological consequence of NO-CD36 signaling. Outcome: Lipid exposure combined with L-NAME leads to mitochondrial dysfunction (Fig. 8A) and impaired vasodilation (Fig. 8C), both rescued by NO. Supplementary Fig. 7 shows full mitochondrial stress test profiles and validation of mitochondrial protein levels (Supp. Fig. 7A–E).
STRENGTHS Defines a novel mechanism linking Cav1, NO, and CD36 to lipid homeostasis
Broadens our understanding of endothelial metabolic self-regulation
Identifies post-translational nitrosation as a reversible toggle on lipid uptake
Uses elegant genetic models, in vivo functional assays, and biochemical rigor
Links vascular NO signaling to metabolic adaptation
Highly relevant to metabolic syndrome, lipedema, and vascular disease
FUTURE WORK & EXPERIMENTAL DIRECTIONS Investigate whether modulating CD36 nitrosation could be therapeutic in hyperlipidemia
Study whether this mechanism contributes to sex differences in metabolic disease
Explore its role in other vascular beds and tissue-specific lipid handling
Test implications for lipedema, a fat-distribution disorder involving endothelial dysfunction
Define how palmitoylation and nitrosation are balanced or dynamically regulated
AUTHORSHIP NOTE This review was drafted with the assistance of ChatGPT (OpenAI) to help organize and articulate key ideas clearly and concisely. I provided detailed prompts, interpretations, and edits to ensure the review reflects an expert understanding of the biology and the paper’s contributions. The final version has been reviewed and approved by me.
FINAL TAKEAWAY This preprint reframes endothelial cells as active regulators of systemic metabolism. By showing that nitrosation of CD36 suppresses lipid uptake and preserves endothelial function under metabolic stress, the authors reveal a previously unrecognized mechanism of cellular protection. This discovery expands our understanding of how ECs maintain homeostasis in nutrient-rich environments and opens new directions for treating lipid-associated diseases like obesity, lipedema, and atherosclerosis.
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