PPARα-CD36-CAV1-mediated docosahexaenoic acid (DHA) uptake promotes muscle fiber development in grass carp (Ctenopharyngodon idellus)

Docosahexaenoic acid (DHA) plays a significant role in muscle fiber development in fish, yet the precise mechanisms governing its uptake remain inadequately understood. This study examines the molecular mechanisms underlying exogenous DHA uptake in grass carp ( Ctenopharyngodon idellus ) myoblasts and those promoting myoblast proliferation after DHA entry. By isolating and culturing grass carp myoblasts, employing immunofluorescence, fatty acid analysis, and gene expression analysis, this study found that cluster of differentiation 36 (CD36) facilitates DHA uptake via a caveolin-1 (CAV1)-dependent endocytic pathway, significantly increasing DHA accumulation in myoblasts ( P < 0.05). Elevated intracellular DHA further upregulated cd36 and cav1 mRNA expression ( P < 0.05). Notably, both the DHA and CD36 inhibitor sulfosuccinimidyl oleate sodium (DHA + SSO) group and the DHA and CAV1 inhibitor nystatin (DHA + Nys) group exhibited significant reductions in DHA uptake, 5-ethynyl-2′-deoxyuridine (EDU)-positive myoblast proportion, and proliferation-related gene expression (cyclin D1 and cyclin E) ( P < 0.05). Moreover, DHA uptake in the DHA with CAV1 overexpression group (DHA + CAV1 OE) was not significantly different from the DHA group ( P > 0.05), but was significantly higher than the DHA + CAV1 OE + SSO group ( P < 0.05), highlighting the essential role of CD36 in DHA uptake. Further dual-luciferase reporter assays demonstrated that peroxisome proliferator-activated receptor α (PPARα) directly regulates the transcription of CD36, thereby identifying CD36 as a target gene of PPARα. To further confirm the role of PPARα in CD36-mediated DHA uptake, 180 grass carp (initial body weight: 32.03 ± 0.05 g) were randomly assigned to three groups (three replicates of 20 fish each): without DHA (Control), 0.5% DHA (DHA), and 0.5% DHA with PPARα inhibitor (DHA + GW6471) for an 8-week experiment. The results demonstrated that DHA upregulated pparα and cd36 mRNA expression in muscle tissue, which enhanced DHA accumulation, increased muscle fiber density, and ultimately promoted the final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) of grass carp ( P < 0.05). However, the DHA + GW6471 treatment significantly reversed these effects ( P < 0.05). In summary, DHA is absorbed by grass carp myoblasts via the CD36/CAV1-dependent endocytic pathway. Upon DHA entry, DHA activates PPARα, which increases CD36 transcription, thereby promoting further DHA uptake. This establishes a PPARα-mediated positive feedback loop that promotes DHA uptake, myoblast proliferation, muscle fiber development, and ultimately the growth of grass carp.