CD36 facilitates fatty acid uptake by dynamic palmitoylation-regulated endocytosis

Fatty acids are critical for energy production, membrane synthesis, storage, and cellular signaling, yet the mechanism mediating their entry into cells is not fully defined. While passive diffusion has been proposed, increasing evidence supports protein-facilitated uptake in metabolic tissues. CD36 is a key FA uptake protein enriched in caveolae and essential for FA uptake in adipose tissue and muscle, but whether and how it transports FAs across the plasma membrane remains unclear. Protein palmitoylation by DHHC acyltransferases regulates protein localization and trafficking; prior work showed DHHC4/5 palmitoylate CD36 to target it to the plasma membrane, but dynamic regulation of CD36 palmitoylation had not been defined. Endocytosis transports macromolecules across the plasma membrane, suggesting a potential mechanism for hydrophobic FA entry. Here, the authors investigate whether CD36 mediates FA uptake via caveolar endocytosis and how dynamic palmitoylation governs this process, with implications for lipid storage and obesity.

• FAs have low aqueous solubility and are bound to albumin and FABPs; passive diffusion versus protein-mediated uptake remains debated.
• CD36 is implicated in FA uptake in adipose tissue, muscle, and heart; CD36 deficiency lowers FA uptake in humans and mice. CD36 possesses an FA-binding pocket and localizes to caveolae.
• Prior work demonstrated CD36 palmitoylation by DHHC4 (Golgi sorting) and DHHC5 (cell surface maintenance) is required for membrane localization and FA uptake; dynamic regulation was unknown.
• Caveolae and caveolin-1 (CAV1) are required for CD36 localization and function; endocytosis is a major route for entry of hydrophobic cargos (e.g., LDL, cholesterol), suggesting a possible analogous route for FAs.
• Signaling via Src family kinases (SFKs), SYK, VAVs, and JNK has been linked to CD36 functions (e.g., oxLDL phagocytosis), hinting at conserved machinery for endocytic processes.

• Cell models: 3T3-L1 preadipocytes differentiated into adipocytes; primary stromal vascular fraction (SVF)-derived adipocytes from WT, Cd36-/-, Cav1-/-, and Cav1-/-;Cd36-/- mice.
• FA treatments: BSA-conjugated long-chain FAs (oleate, myristate, palmitate, stearate, linoleate, arachidonate). Photoactivable/clickable FA analog PacFA used to visualize and capture FA-protein complexes.
• Imaging and localization: 3D confocal immunofluorescence for CD36 and CAV1; LipidTOX/BODIPY for lipid droplets; surface biotinylation assays for plasma membrane proteins; electron microscopy to visualize caveolae internalization; recycling assays after FA withdrawal.
• PacFA assays: UV crosslinking and click chemistry (Alexa Fluor 488 or biotin azide) to detect colocalization with CD36 and pull-down PacFA-bound proteins (streptavidin capture, immunoblot for CD36/FABP4).
• FA uptake assays: BODIPY 493/503 fluorescence quantification in preadipocytes; 3H-oleate scintillation counting in adipocytes normalized to protein content.
• Palmitoylation analysis: Acyl-RAC to detect palmitoylated CD36 and DHHC5; hydroxylamine controls. Inhibitors used: palmostatin B (general depalmitoylation inhibitor), ML348 (APT1 inhibitor).
• Genetic perturbations: shRNA knockdowns of CD36, CAV1, APT1, LYN, SYK, and other SFKs; overexpression of APT family members; CD36 K164R mutant; DHHC5 mutants Y91E (phospho-mimetic) and Y91F (non-phosphorylatable); DHHC5-/- HEK293T for reconstitution assays.
• Kinase signaling: SFK inhibitor PP2; SYK inhibitor piceatannol; dynamin inhibitor Dyngo4a; VAV inhibitor azathioprine; JNK inhibitor SP600125. Western blots for p-LYN (Y396), p-SYK (Y525/526), p-VAV (Y174), p-JNK (T183/Y185).
• Protein interactions: Immunoprecipitation of DHHC5 for phospho-tyrosine detection; mass spectrometry to identify DHHC5 phosphorylation (Y91) and depalmitoylated CD36 interactors (including SYK).
• In vivo studies: WT and Cd36-/- male mice fed HFD for 8 weeks, daily oral inhibitors bafetinib (dual Bcr-Abl/LYN) or entospletinib (SYK). Body weight monitoring, glucose tolerance tests, food intake, adipose histology (H&E), adipocyte size quantification, plasma free FA/TG and liver TG measurements.
• Statistics: Mean ± SEM; two-sided Student’s t-tests; details in figure legends.

• Long-chain FAs trigger CD36 internalization via caveolae in adipocytes. CD36 and CAV1 co-internalize; knockdown of either CD36 or CAV1 prevents internalization and reduces surface CD36.
• All tested long-chain FAs (C14:0, C16:0, C18:0, C18:2, C20:4) induce CD36 endocytosis; CD36 recycles back to the plasma membrane upon FA withdrawal (within 1–4 h).
• CD36 K164R (defective FA binding) fails to internalize upon oleate, indicating FA binding to CD36 is required to trigger endocytosis.
• PacFA experiments show intracellular colocalization of FA with CD36; ~56% of intracellular PacFA colocalizes with CD36, and PacFA pull-down retrieves CD36, indicating CD36 carries FAs into cells.
• Cav1-/- adipocytes exhibit ~40% decreased 3H-oleate uptake, similar to Cd36-/-; double knockout does not further reduce uptake, supporting caveolar endocytosis as necessary for CD36-dependent FA uptake.
• Oleate rapidly decreases palmitoylation of CD36 (within 5–30 min) and DHHC5; palmitoylation recovers after FA removal, indicating dynamic palmitoylation during uptake.
• APT1 is identified as the depalmitoylase for CD36: APT1 knockdown or ML348 treatment blocks oleate-induced CD36 depalmitoylation and internalization and reduces CD36-dependent FA uptake (ML348 lowers uptake by ~50% in WT preadipocytes; no additional effect in Cd36-/-; ML348 abolishes ~35% CD36-dependent 3H-oleate uptake in adipocytes).
• DHHC5 is phosphorylated at Tyr91 upon oleate (5 min), downstream of CD36. Y91E (phospho-mimetic) inactivates DHHC5, Y91F remains active. Neither Y91E nor Y91F rescues FA uptake in DHHC5-deficient cells, indicating a requirement for dynamic (on/off) regulation of DHHC5 activity.
• LYN (an SFK) phosphorylates DHHC5 Y91: PP2 suppresses pY91; LYN knockdown reduces pY91 and blocks CD36 depalmitoylation/internalization. LYN activation (pY396) peaks at 5 min after oleate and requires CD36. PP2 abolishes CD36-dependent FA uptake in preadipocytes and adipocytes.
• Depalmitoylated CD36 recruits SYK; SYK activation (pY525/526) upon oleate requires CD36 depalmitoylation (blocked by ML348). Piceatannol (SYK inhibitor) blocks VAV and JNK phosphorylation, CD36 internalization, and CD36-dependent FA uptake.
• Additional endocytic machinery requirements: inhibitors of dynamin (Dyngo4a), VAV (azathioprine), and JNK (SP600125) all block oleate-induced CD36 endocytosis.
• Functional outcomes: In live-cell imaging, oleate doubles lipid droplet volume per cell by ~105 min in WT; this growth is suppressed by PP2, ML348, or piceatannol and is minimal in Cd36-/- cells.
• In vivo, bafetinib (LYN inhibitor) or entospletinib (SYK inhibitor) reduce HFD-induced weight gain and adipose mass in WT but not Cd36-/- mice; glucose tolerance improves in treated WT mice without changes in food intake; slight increases in liver triglycerides suggest redistribution of lipids when adipose uptake is reduced.

This study resolves how CD36 facilitates cellular FA uptake: FAs binding to CD36 initiates a caveolar endocytic program rather than simple transmembrane translocation. The mechanism hinges on dynamic palmitoylation of CD36 controlled by DHHC5 and APT1. FA-bound CD36 activates LYN, which phosphorylates and inactivates DHHC5 at Tyr91, enabling APT1-mediated depalmitoylation of CD36. Depalmitoylated CD36 recruits SYK, leading to activation of VAV and JNK, dynamin engagement, and caveolae scission, delivering FA cargo to ER/lipid droplets. Blocking any node (LYN, APT1, SYK, dynamin, VAV, JNK) disrupts endocytosis and abrogates CD36-dependent FA uptake. The findings reconcile CD36’s high surface localization in caveolae with efficient FA delivery and link extracellular FA sensing to intracellular endocytic machinery via kinase signaling. Physiologically, inhibiting LYN or SYK blunts lipid storage in adipocytes and reduces HFD-induced obesity, underscoring the relevance of this pathway to whole-body energy balance and offering therapeutic entry points.

The work identifies CD36-mediated caveolar endocytosis as a major pathway for cellular FA uptake and establishes dynamic palmitoylation of CD36—regulated by LYN-dependent phosphorylation/inactivation of DHHC5 and APT1-mediated depalmitoylation—as essential for this process. Depalmitoylated CD36 recruits SYK to activate downstream effectors (VAV, JNK) that drive endocytosis. Pharmacologic inhibition of LYN or SYK suppresses adipocyte lipid storage and HFD-induced weight gain in mice. Future research could define structural determinants of CD36-SYK interactions, assess tissue-specific roles across metabolic organs, explore DHHC regulation by SFKs more broadly, and evaluate targeted modulation of this pathway for treating obesity and metabolic disease.

• Most mechanistic experiments were performed in 3T3-L1 and primary adipocytes; generalizability to other FA-consuming tissues (e.g., muscle, liver, heart) requires further validation.
• Pharmacological inhibitors (PP2, piceatannol, ML348, bafetinib, entospletinib) may have off-target effects; although genetic knockdowns support conclusions, comprehensive off-target profiling in vivo is limited.
• The precise structural basis and dynamics of CD36’s interaction with SYK and other endocytic components were not delineated.
• Quantitative contribution of this endocytic pathway versus other FA uptake mechanisms under varying physiological conditions remains to be fully established.