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

Abstract

Docosahexaenoic acid (DHA) plays a significant role in muscle fiber development in fish, yet the precise mechanisms governing its uptake remain poorly 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. Grass carp myoblasts were isolated and cultured, and immunofluorescence, fatty acid analysis, and gene expression analysis were performed. 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 relative mRNA expression (P < 0.05). Notably, both the DHA + CD36 inhibitor sulfosuccinimidyl oleate sodium (DHA + SSO) group and the DHA + 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 + 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) in 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 suggests the existence of a PPARα-mediated positive feedback loop that promotes DHA uptake, myoblast proliferation, muscle fiber development, and ultimately the growth of grass carp.

1. Introduction

The global shift in dietary patterns has increased the demand for high-quality animal proteins, with aquatic products gaining preference due to their nutritional value (FAO, 2020; Xue et al., 2023). As the aquaculture industry grows rapidly, consumer expectations for aquatic product quality have risen; however, intensive farming has led to a decline in fish flesh quality (Liu et al., 2020; Ji et al., 2024). Therefore, optimizing the growth and quality of fish muscle tissue has become a crucial goal for enhancing aquaculture efficiency.

Docosahexaenoic acid (DHA), a biologically active long-chain polyunsaturated fatty acid (LC-PUFA), plays a vital role in numerous biological processes, including antioxidation (Zou et al., 2023), immune modulation (Williams-Bey et al., 2014), and lipid metabolism regulation (Ding et al., 2021). Recent studies have further elucidated the beneficial effects of DHA on fish muscle growth and quality (Wang et al., 2020). Muscle fibers, the fundamental structural units of muscle, undergo proliferation and differentiation processes that directly determine muscle growth and quality (Oksbjerg and Therkildsen, 2017; Periago et al., 2005; Wang et al., 2024a). Research has shown that DHA significantly promotes the proliferation of primary myoblasts in turbot (Scophthalmus maximus L.) (Gao et al., 2024). Additionally, DHA supplementation in the diet of blunt snout bream (Megalobrama amblycephala) enhances muscle proliferation and the development of muscle fibers (Wang et al., 2020). A previous study further substantiates that DHA stimulates muscle fiber development in grass carp (Ctenopharyngodon idellus) through the activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway (Ji et al., 2024). However, despite the preliminary understanding of the nutritional regulatory mechanisms of DHA in promoting fish muscle growth, the processes by which DHA crosses the membrane to enter myoblasts remain unclear and warrant further investigation.

Cluster of differentiation 36 (CD36), a transmembrane glycoprotein classified within the class B scavenger receptor family, is abundantly expressed in cardiac and skeletal muscle cells, where it primarily functions as a transporter of long-chain fatty acids (LCFAs) (Glatz et al., 2016; Nozaki et al., 1995). Su and Abumrad (2009) identified a direct link between CD36 expression levels and the efficiency of fatty acid uptake in murine muscle, highlighting its central role in lipid metabolism. Beyond its involvement in lipid transport, CD36 also facilitates muscle regeneration by regulating satellite cell activation. Its absence impairs this process and delays myogenic tissue repair (Verpoorten et al., 2020). In both tilapia (Oreochromis niloticus) and zebrafish (Danio rerio), CD36 expression exhibits a sensitive response to changes in dietary fatty acid composition, suggesting that its function is conserved across species (Jia et al., 2020; Wang et al., 2019). The CD36 is localized within the lipid raft microdomains of the cell membrane, where it colocalizes with caveolin-1 (CAV1), a critical structural protein of caveolae, and relies on CAV1-mediated endocytosis to facilitate its fatty acid uptake (Mattern et al., 2009). Downregulation of CAV1 diminishes membrane-associated CD36, thus inhibiting fatty acid uptake, whereas CAV1 overexpression enhances CD36 membrane localization and promotes uptake efficiency (Glatz et al., 2010; Pohl et al., 2004; Ring et al., 2006). Studies in mammals have shown that CD36 has been identified as a downstream target of peroxisome proliferator-activated receptor alpha (PPARα), which regulates its promoter activity (Madonna et al., 2011). Interestingly, similar results were also observed in the large yellow croaker (Larimichthys crocea) (Hao et al., 2020a). DHA, a natural ligand of PPARα, has been shown to facilitate its own accumulation in skeletal muscle through this pathway, enhance exercise endurance (Valentine et al., 2018), and protect cardiac function (Cheng et al., 2024). Thus, it is proposed that DHA could enter the myoblasts of grass carp via the PPARα-CD36-CAV1 pathway, thereby promoting muscle fiber development.

Grass carp (C. idellus) is a vital species in global aquaculture, but intensive production conditions may result in a decline in flesh quality (Ji et al., 2024; Zhao et al., 2018). Although a prior study has demonstrated that DHA enhances the growth performance of grass carp and improves muscle tissue quality (Ji et al., 2025), the molecular mechanisms by which DHA enters myoblasts and promotes muscle fiber development remain unclear. This study presents the first evidence that DHA upregulates CD36 expression through PPARα activation, thereby enhancing DHA absorption via CAV1-mediated endocytosis, which ultimately promotes muscle fiber development in grass carp. This finding provides a theoretical foundation for the precise inclusion of DHA in aquaculture feeds and opens new avenues for advancing nutritional strategies aimed at improving fish muscle quality.

2. Materials and methods

2.1. Animal ethics statement

All experimental procedures were conducted in full compliance with the Guiding Principles for Experimental Animals established by the Animal Care and Use Committee of Northwest A&F University, which provided the necessary approval (approval No. NWAFU-DKXC-20220701).

2.2. In vitro study

2.2.1. Cultivation and treatment of grass carp myoblasts

Grass carp myoblasts were isolated and cultured following the protocol established in a previous study (Ji et al., 2024), with detailed procedures outlined below. Initially, grass carp weighing approximately 40 g were anesthetized and euthanized using an ice-water solution, followed by a 2 min external sterilization with 75% ethanol. Under sterile conditions, white muscle tissue adjacent to the dorsal fin was excised and rinsed three times with phosphate-buffered saline (PBS) containing streptomycin (100 μg/mL) and penicillin (100 IU/mL) (P1400, Beijing Solarbio Technology Co., Ltd., Beijing, China). The tissue was subsequently minced into 1-mm³ fragments and washed three times with M199 medium (SH30253.01B, HyClone Laboratories, Inc., Logan, UT, USA) supplemented with antibiotics. The muscle fragments were carefully placed in a 6-well culture plate with 2 to 3 mm spacing between each fragment. The plate was inverted and pre-incubated at 28 °C in a 5% CO₂ incubator for 4 h. Following this, a myoblast growth medium containing 20% fetal bovine serum (13011–8611, Evergreen Biotechnology Co., Ltd., Guiyang, Guizhou, China), 1% penicillin–streptomycin (P1400), 20 ng/mL basic fibroblast growth factor (bFGF; P00032), and 1 ng/mL epidermal growth factor (EGF; P00033), all obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China), was added to fully submerge the tissue. The cultures were incubated at 28 °C in a 5% CO₂ atmosphere, with the medium replaced every 2 d, until the cells fully covered the culture surface. Subsequently, the muscle fragments were carefully removed using sterile tweezers. The cells were then digested with 0.25% trypsin (SH30042.01B, HyClone Laboratories, Inc., Logan, UT, USA) to isolate myoblasts. Following centrifugation, the resulting cell pellet was resuspended in fresh medium and transferred to new flasks for continued passaging.

To investigate whether DHA promotes myoblast proliferation via the CD36-mediated absorption pathway, this study utilized DHA (HY-B2167) and the CD36 inhibitor sulfosuccinimidyl oleate sodium (SSO; HY-112847A), both from MedChem Express LLC (Monmouth Junction, NJ, USA). The myoblasts were divided into three groups: the control group, the DHA group (50 μmol/L DHA incubated alone for 24 h), and the DHA + SSO group (pre-incubated with 200 μmol/L CD36 inhibitor SSO for 4 h, followed by co-incubation with 50 μmol/L DHA for 24 h). Subsequently, the collected myoblasts were subjected to western blotting, quantitative real-time PCR (qRT-PCR), fatty acid composition analysis, and 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation assay. Furthermore, to examine the role of CAV1 in DHA absorption and its effect on myoblast proliferation, this study employed DHA, the CAV1 inhibitor nystatin (Nys; N8040, Beijing Solarbio Technology Co., Ltd., Beijing, China), and the CD36 inhibitor SSO. The myoblasts were divided into four groups: the control group, the DHA group, the pre-incubated with 75 μmol/L CAV1 inhibitor Nys for 4 h, followed by co-incubation with 50 μmol/L DHA for 24 h (DHA + Nys) group, and the pre-incubated with 200 μmol/L SSO and 75 μmol/L Nys for 4 h, followed by co-incubation with 50 μmol/L DHA for 24 h (DHA + Nys + SSO) group. Subsequently, the collected myoblasts were subjected to western blotting, qRT-PCR, fatty acid composition analysis, and an EdU cell proliferation assay. To further investigate the regulatory role of CAV1 in DHA absorption by grass carp myoblasts, the following experimental procedure was adopted: when the grass carp myoblasts reached 80% confluence, the mCherry-CAV1 eukaryotic expression vector (NC_067243.1, Universal Biotech Co., Ltd., Hefei, Anhui, China) was transfected into the cells following the instructions for the Hieff Trans Liposomal 2000 Transfection Reagent (40802ES03, Yeasen Biotechnology Co., Ltd., Shanghai, China). After 24 h post-transfection, the experimental groups were treated with either DHA alone (DHA + CAV1 overexpression [CAV1 OE]) or DHA combined with SSO for 24 h (DHA + CAV1 OE + SSO). The myoblasts from each group were then collected for subsequent qRT-PCR and fatty acid composition analysis. All experiments were conducted using myoblasts at passages three to five to ensure cellular stability and phenotypic consistency. The DHA concentration was chosen based on a prior study conducted in the same laboratory (Ji et al., 2024), and the concentrations and durations of SSO and Nys treatments were determined according to previous in vitro studies (Gyamfi et al., 2021; Synowiec et al., 2023).

2.2.2. Cultivation and treatment of human embryonic kidney 293T (HEK 293T) cells

HEK 293T cells were cultured following the protocol described in a previous study (Bian et al., 2024). HEK 293T cells were obtained from the China Center for Type Culture Collection (CCTCC; Wuhan, Hubei, China) and cultured in high-glucose DMEM (SH30243.01, HyClone Laboratories, Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; 40131ES76, Yeasen Biotechnology Co., Ltd., Shanghai, China) and 100 U/mL penicillin–streptomycin (P1400, Beijing Solarbio Technology Co., Ltd., Beijing, China) at 37 °C with 5% CO₂. To further elucidate the role of CD36 in DHA uptake, cells were seeded into 24-well plates containing pre-coated culture slides and grown until approximately 70% confluence was reached. At this point, the cells were transiently transfected with pEGFP-CD36 (an enhanced green fluorescent protein-tagged CD36 expression plasmid) and mCherry-CAV1 (a monomeric red fluorescent protein-tagged CAV1 expression plasmid) using Hieff Trans Liposomal 2000 Transfection Reagent (40802ES03) for 24 h. Following transfection, cells were treated with DHA at defined time intervals, and samples were harvested at designated time points. The internalization of CD36 and its co-localization with CAV1 were subsequently visualized and recorded using a confocal laser scanning microscope (Model Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany).

2.3. In vivo study

2.3.1. Experimental design and feeding management

In this study, three iso-nitrogenous and iso-lipidic diets were formulated: a basal diet (control group, without DHA), a basal diet supplemented with 0.5% DHA (DHA), and a basal diet supplemented with 0.5% DHA and 30 mg/kg of the PPARα inhibitor (DHA + GW6471). DHA-enriched oil (90.94%) was sourced from Shaanxi Panier Biotechnology Co., Ltd. (Xian, Shaanxi, China). The treatment concentration was determined based on previous animal studies utilizing DHA and GW6471 (Xue et al., 2025; Wen et al., 2020). The composition and nutritional content of the experimental diets are presented in Table 1, while the fatty acid composition is outlined in Table 2. All experimental feeds were formulated based on prior research from the same laboratory to ensure that the nutritional requirements of the grass carp were met (Xue et al., 2023). A total of 180 grass carp (initial body weight [IBW]: 32.03 ± 0.05 g) were randomly assigned to 9 cages (1.5 m × 1.5 m × 1.8 m), with 20 fish per cage and 3 replicate cages per treatment. During the study, the fish were fed three times daily (08:00, 12:00, and 16:00) for a duration of 8 weeks. The experiment was conducted under natural light conditions, with water temperature, dissolved oxygen, ammonia nitrogen, and nitrite levels monitored twice weekly. The ranges for these parameters were as follows: 25 to 28 °C for temperature, 8 to 11 mg/L for dissolved oxygen, 0.02 to 0.10 mg/L for ammonia nitrogen, and 0.005 to 0.010 mg/L for nitrite.

Table 1.

The formulation of experimental diets and proximate composition of the experimental diets (%, DM basis).

Items	Experimental diets2
	Control	DHA	DHA + GW6471
Ingredients
Casein	34.00	34.00	34.00
Gelatin	8.00	8.00	8.00
Corn starch	15.00	15.00	15.00
α-Starch	15.00	15.00	15.00
Soybean oil	1.05	1.05	1.05
Linseed oil	1.75	1.75	1.75
Lard oil	2.20	1.65	1.65
DHA-enriched oil (90.94%)	0.00	0.55	0.55
Choline chloride	0.50	0.50	0.50
Premix1	1.00	1.00	1.00
Ethoxyquin	0.05	0.05	0.05
Calcium phosphate primary	2.00	2.00	2.00
Carboxy methyl cellulose	2.00	2.00	2.00
Cellulose	17.45	17.45	17.42
Inhibitor	0.00	0.00	0.03
Total	100.00	100.00	100.00
Proximate composition
CP	36.17	36.24	36.48
Crude lipid	5.08	5.04	5.17
Moisture	10.56	10.61	10.40
OM	84.41	84.36	84.50
NDF	14.01	14.80	14.22
ADF	13.22	13.79	13.25
GE, kJ/g	17.31	17.29	17.35

DM = dry matter; CP = prude protein; OM = organic matter; NDF = neutral detergent fiber; ADF = acid detergent fiber; GE = gross energy; DHA = docosahexaenoic acid; GW6471 = peroxisome proliferator-activated receptor α inhibitor.

¹Premix (per kg of diet): vitamin A 67 IU; vitamin D 16.2 IU; vitamin E 7.4 g; vitamin K₃ 340 mg; vitamin B₁ 670 mg; vitamin B₂ 1000 mg; vitamin B₆ 800 mg; vitamin B₁₂ 1.4 mg; vitamin C 10 g; D-pantothenic acid 2.65 g; folic acid 330 mg; nicotinamide 5.35 g; choline chloride 35 g; biotin 34 mg; inositol 8 g, Fe 14 g; Cu 350 mg; Zn 4 g; Mn 1.4 mg; Mg 10 g; Co 30 mg; I 40 mg; Se 35 mg.

²Control, diet without DHA supplementation; DHA, diet supplemented with 0.5% DHA; DHA + GW6471, diet supplemented with 0.5% DHA and GW6471.

Table 2.

Fatty acid composition of experimental diets (% total fatty acids).

Fatty acids	Experimental diets1
	Control	DHA	DHA + GW6471
14:0	1.23	0.40	0.40
16:0	9.07	7.52	7.60
18:0	11.56	8.52	8.60
SFA	21.85	16.43	16.60
16:1n-7	1.40	1.17	1.13
18:1n-9	30.44	25.36	25.56
MUFA	31.84	26.52	26.69
18:2n-6	25.33	23.55	23.65
18:3n-6	0.62	0.57	0.57
20:3n-6	0.74	0.70	0.69
22:4n-6	2.34	2.19	2.16
n-6 PUFA	29.04	27.02	27.08
18:3n-3	13.72	13.48	13.53
20:3n-3	0.65	0.62	0.61
20:5n-3	0.00	2.02	1.95
22:6n-3 (DHA)	0.00	11.40	11.03
n-3 PUFA	14.37	27.52	27.12
PUFA	43.41	54.54	54.20
n-3/n-6 PUFA	0.49	1.02	1.00

SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; DHA = docosahexaenoic acid; GW6471 = peroxisome proliferator-activated receptor α inhibitor.

¹Control, diet without DHA supplementation; DHA, diet supplemented with 0.5% DHA; DHA + GW6471, diet supplemented with 0.5% DHA and GW6471.

2.3.2. Experimental sampling

Prior to sampling, the experimental fish were fasted for 24 h and anesthetized with 100 mg/L tricaine methanesulfonate (MS-222). Two fish from each cage were randomly selected and stored at −20 °C for whole-body proximate composition analysis. Subsequently, 15 fish were randomly chosen, weighed, and their body lengths measured. The internal organs were carefully separated and weighed, followed by the rapid collection of muscle samples from above the lateral line and posterior to the head. A portion of muscle tissue from 3 fish was immediately fixed in 4% formaldehyde for histological analysis. The remaining muscle tissue from 12 fish was rapidly frozen in liquid nitrogen and stored at −80 °C for subsequent analyses. The following equations were applied to determine growth performance, feed efficiency, and biological parameters:

$$\text{Weight}\text{gain}\text{rate}\left(\text{WGR},\%\right)=100\times \left[\text{Final}\text{body}\text{weight}\left(\text{FBW}\right)-\mathrm{IBW}\right]/\text{IBW;}$$

$$\text{Specific}\text{growth}\text{rate}\left(\text{SGR},\%/\mathrm{d}\right)=100\times \left[\ln \left(\text{FBW}\right)-\ln \left(\text{IBW}\right)\right]/\text{Days;}$$

$$\text{Feed}\text{conversion}\text{ratio}\left(\text{FCR}\right)=\text{Total amount of the feed consumed}/\left(\text{FBW}-\text{IBW}\right)\text{;}$$

$$\text{Feed intake (FI},\mathrm{g}/\text{fish)}=\text{Feed consumed}/\left(\text{Number}\text{of}\text{fish}\right)\text{;}$$

$$\text{Condition}\text{factor}\left(\text{CF},\mathrm{g}/{\text{cm}}^3\right)=100\times \text{Body weight}/{\left(\text{Body}\text{length}\right)}^3\text{;}$$

$$\text{Hepatosomatic}\text{index}\left(\text{HSI},\%\right)=100\times \text{Liver}\text{weight}/\text{Body weight;}$$

$$\text{Viscera}\text{index}\left(\text{VSI},\%\right)=100\times \text{Viscera}\text{weight}/\text{Body weight;}$$

$$\text{Protein}\text{retention}\text{efficiency}\left(\text{PRE},\%\right)=100\times \left(\text{Fish}\text{protein}\text{gain}\right)/\left(\text{Total}\text{protein}\text{intake}\right)\text{;}$$

$$\text{Protein}\text{efficiency}\text{ratio}\left(\text{PER}\right)=\left(\text{Wet}\text{weight}\text{gain}\right)/\left(\text{Protein}\text{intake}\right)\text{.}$$

2.4. Sample analysis

2.4.1. Proximate composition

The proximate composition of the diets and muscle tissues was analyzed following the procedures described by AOAC (2005). Crude protein (CP; method 2001.11) content was determined by measuring the nitrogen content using the Kjeldahl method and multiplying the obtained value by 6.25. Crude lipid content (method 920.39) was determined after overnight pretreatment of the samples, followed by continuous extraction with petroleum ether. Moisture content (method 2001.12) was determined by drying the samples at 105 °C to a constant weight. Crude ash content (method 942.05) was determined by combusting the samples at 550 °C in a muffle furnace (Model SX-4-10, Tianjin Taice Instrument Co., Ltd., Tianjin, China). The organic matter (OM) content of the diets was determined by subtracting the crude ash content from the dry matter (DM). The contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in the experimental diets were analyzed according to the method described by Van Soest et al. (1991), using a filter bag and fiber analyzer (Model A2000i, Ankom Technology, Inc., Macedon, NY, USA). The gross energy (GE) content of the experimental diets was analyzed using an automatic oxygen bomb calorimeter (Model A1329DD, Parr Instrument Co., Inc., Moline, IL, USA).

2.4.2. Fatty acid composition

Fatty acids from experimental diets, muscle tissues, and myoblasts were extracted and analyzed according to the procedures described by Shi et al. (2017) and Lei et al. (2018), with adjustments to reagent volumes specific to each sample type. For diet and muscle tissues, approximately 0.2 g of each sample was extracted with 5 mL of methanol–chloroform (1:2, v/v), whereas myoblast pellets, obtained after cell detachment and centrifugation, were mixed with 500 μL of the same solvent mixture. All samples were shaken for 2 h at room temperature and then centrifuged at 940 × g for 5 min after the addition of either 4 mL of deionized water (for diet and muscle samples) or 500 μL of deionized water (for myoblasts). The lower organic layer was collected and evaporated to dryness under vacuum, and the lipid residue was subsequently redissolved in 1 mL (diet and muscle) or 200 μL (myoblast) of hexane. Methylation was performed by adding 1 mL (diet and muscle) or 200 μL (myoblast) of 0.4 mol/L methanolic KOH, followed by incubation for 2 h at room temperature. Following the addition of 2 mL (diet and muscle) or 500 μL (myoblast) of distilled water, the upper organic phase was collected and analyzed using a gas chromatograph equipped with an autosampler (Model Nexis GC-2030, Shimadzu Corp., Kyoto, Japan). Fatty acid composition was quantified based on peak areas normalization and expressed as the percentage of each fatty acid relative to the total identified fatty acids.

2.4.3. Triglyceride (TG) content

The TG content in the muscle was quantified using the tissue TG content assay kit (E1013, Beijing Puli Lai Gene Technology Co., Ltd., Beijing, China). Protein concentration in the homogenate was determined using the bicinchoninic acid (BCA) protein assay kit (PC0020, Beijing Solarbio Technology Co., Ltd., Beijing, China) and was subsequently used for normalization. Absorbance was measured with a multifunctional microplate reader (Model Synergy H1, BioTek Instruments, Inc., Winooski, VT, USA).

2.4.4. Histological analysis

Muscle tissue was stained using the hematoxylin and eosin (H&E) method for histological analysis. H&E staining was performed on muscle tissue sections obtained from two fish per cage. The muscle tissue samples were first rinsed three times with normal saline and subsequently fixed in 4% paraformaldehyde for 24 h. After fixation, the samples underwent a dehydration process in a series of increasing ethanol concentrations, followed by treatment with xylene to remove the ethanol. The tissues were then embedded in paraffin and sectioned into thin slices, measuring 4 to 6 μm in thickness, using a microtome (Model RM2265, Leica Microsystems GmbH, Wetzlar, Germany). The sections were stained with H&E for histological analysis, and the prepared slides were examined under a microscope (Model Eclipse 50i, Nikon Corporation, Tokyo, Japan) for further evaluation.

2.4.5. The EdU cell proliferation assay

Following the manufacturer’s guidelines, the BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 555 (C0075S, Beyotime Biotechnology Co., Ltd., Shanghai, China) was used to evaluate the proliferation of myoblasts isolated from grass carp. The myoblasts were seeded into 24-well plates and cultured until they reached approximately 70% confluence. Following a 24 h treatment with DHA and inhibitors, the myoblasts were exposed to a 10 μmol/L EdU solution for 2 h at 28 °C. Subsequently, the myoblasts were fixed with 4% paraformaldehyde for 10 min to preserve cell morphology, followed by three washes with PBS. The myoblasts were then permeabilized with 0.3% Triton X-100 solution for 15 min to facilitate intracellular staining, after which they were washed 3 times with PBS. Under dark conditions at room temperature, the myoblasts were incubated for 30 min with click reaction solution. After incubation, the myoblasts were washed with PBS and stained with Hoechst 33342 for nuclear visualization. Finally, the myoblasts were observed and imaged using an inverted fluorescence microscope (Model CKX53, Olympus Corporation, Tokyo, Japan), and the total number of nuclei as well as the number of EdU-positive nuclei were quantified using ImageJ software (version 1.54p, National Institutes of Health, Bethesda, MD, USA).

2.4.6. Dual-luciferase reporter assay

The promoter region of the grass carp cd36 gene was cloned from genomic DNA using the method outlined by Bian et al. (2024). The amplified promoter fragment was inserted into the pGL3-Basic vector to construct the reporter plasmid pGL3-CD36. Putative PPARα consensus binding sites within the upstream promoter region of the cd36 gene were identified using the JASPAR database (http://jaspar.genereg.net/). Site-directed mutagenesis of the predicted PPARα binding site was performed using the QuickMutation Site-Directed Mutagenesis Kit (D0206, Beyotime Biotechnology Co., Ltd., Shanghai, China). The recombinant plasmids were transfected into HEK 293T cells seeded in 24-well plates using the Hieff Trans Liposomal 2000 Transfection Reagent (40802ES03). Luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (RG028, Beyotime Biotechnology Co., Ltd., Shanghai, China), and firefly luciferase activity was normalized to that of Renilla luciferase.

2.4.7. Quantitative real-time PCR (qRT-PCR)

The procedures for RNA extraction, reverse transcription, and qRT-PCR followed a previous study (Xue et al., 2023). Briefly, total RNA was extracted from muscles and myoblasts using the TRNzol reagent (DP424, Tiangen Biotech Co., Ltd., Beijing, China). RNA purity and concentration were determined by measuring absorbance at 260 and 280 nm with a spectrophotometer. The PrimeScript RT reagent kit (RR037, Takara Bio Inc., Dalian, Liaoning, China) was subsequently applied to reverse transcribe the RNA into complementary DNA (‌cDNA). qRT-PCR was performed on the CFX Opus 96 real-time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the ChamQ SYBR qPCR Master Mix Kit (Q311, Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China). The amplification efficiencies of all genes were determined from the standard curves generated using the 10-step serial dilution series. Relative mRNA expression levels were then quantified using the 2^−ΔΔCt method, with β-actin as the internal reference gene. The amplification efficiency of each pair of primers ranged from 95% to 105%. The primers employed for this study are provided in Table S1. The full names of the abbreviations of all genes can be found in Table S2.

2.4.8. Western blotting analysis

Western blotting was carried out following the protocol outlined by Jin et al. (2018). In brief, myoblasts were lysed on ice using radio immunoprecipitation assay (RIPA) lysis buffer to extract proteins, and protein concentrations were then determined using a BCA protein assay kit (R0010, Beijing Solarbio Technology Co., Ltd., Beijing, China). The protein samples were subjected to electrophoresis and subsequent transfer to membranes. Following blocking, the membranes were incubated at 4 °C with rabbit polyclonal antibodies against CD36 (1:1000, WL02390, Wanleibio Bio-Tech Co., Ltd., Shenyang, Liaoning, China), CAV1 (1:1000, WL02047, Wanleibio Bio-Tech Co., Ltd., Shenyang, Liaoning, China), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000, GB11002, Beijing Solarbio Technology Co., Ltd., Wuhan, Hubei, China), and then washed. Afterward, the membranes were incubated with secondary anti-rabbit antibodies. Finally, the membranes were developed using enhanced chemiluminescence (ECL; 310212, Zeta Life Inc., Atlanta, GA, USA) reagent and visualized using the ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Relative protein expression levels were calculated using ImageJ software (version 1.54p, National Institutes of Health, Bethesda, MD, USA) by normalizing the optical density of the target protein bands.

2.5. Statistical analyses

All data were presented as means and standard error of the mean (SEM). Statistical analyses were conducted using SPSS software (version 26.0, SPSS Inc., Chicago, IL, USA). The normality and homogeneity of the data were assessed initially, followed by pairwise comparisons between groups using an independent samples t-test. The mathematical model is expressed as follows:

$$Y_{ij}=\mu +T_i+{\epsilon }_{ij,}$$

where Y_ij represents the observed value for the j-th replicate within the i-th treatment group; μ is the overall mean; T_i is the fixed effect of the i-th treatment group; and ε_ij is the random residual error. Statistical significance was determined at P < 0.05. Following the data analysis, all graphical representations were generated using GraphPad Prism software (version 8.0.2, GraphPad Software Inc., San Diego, CA, USA).

3. Results

3.1. Cluster of differentiation 36 mediates the uptake of DHA into the myoblasts of grass carp

Upon DHA treatment of CD36-overexpressing HEK 293T cells, the internalization of CD36 is depicted in Fig. 1A. CD36 was localized to the plasma membrane in the control group. However, after 2 h of DHA exposure, CD36 on the plasma membrane was observed to migrate into the cell, suggesting that DHA is internalized via CD36-mediated endocytosis. Moreover, the DHA + SSO group significantly decreased the protein levels of CD36 in myoblasts compared to the DHA group (Fig. 1B and C, P = 0.038). Table 3 presents the fatty acid composition of grass carp myoblasts. In comparison with the control group, the DHA group demonstrated a marked elevation in the DHA (22:6n-3) level in the myoblasts (P < 0.001). However, the DHA + SSO group showed a marked reduction in the DHA level compared to the DHA group (P = 0.001).

Fig. 1.

Cluster of differentiation 36 (CD36) mediates the uptake of DHA into the myocytes of grass carp. (A) The internalization of CD36 in human embryonic kidney 293T (HEK 293T) cells transfected with CD36 after treated for 2 h. Green fluorescence indicated CD36 and 4′,6-diamidino-2-phenylindole (DAPI) staining marked the nucleus, scale bar = 8 μm. (B) Protein levels of CD36 expression in myoblasts treated with CD36 inhibitor. (C) Quantitative results of CD36 protein levels in myoblasts treated with CD36 inhibitor. Control, untreated; DHA, treated with 50 μmol/L DHA; DHA + SSO, co-treated with 50 μmol/L DHA and 200 μmol/L SSO. DHA = docosahexaenoic acid; SSO = sulfosuccinimidyl oleate sodium (a CD36 inhibitor); GAPDH = glyceraldehyde-3-phosphate dehydrogenase. P-value less than 0.05 indicates a significant difference, n = 3.

Table 3.

Effects of DHA and DHA + SSO on the fatty acid composition of grass carp myoblasts (% total fatty acids).

Fatty acids	Groups1			SEM	P-value
	Control	DHA	DHA + SSO		DHA vs. Control	DHA vs. DHA + SSO
14:0	0.50	0.54	0.85	0.077	0.764	0.181
16:0	9.31	7.01	42.99#	4.981	0.088	<0.001
18:0	0.25	0.07	0.11	0.081	0.461	0.719
SFA	10.06	7.63	43.95#	5.024	0.067	<0.001
16:1n-7	40.11	33.46∗	3.14#	4.898	0.011	<0.001
18:1n-9	27.97	34.31	30.16	1.231	0.072	0.206
MUFA	68.08	67.77	33.31#	5.030	0.920	<0.001
20:2n-6	1.47	0.67	0.48	0.256	0.203	0.754
20:3n-6	15.09	12.67∗	16.66#	0.600	0.046	0.002
20:4n-6	1.30	0.74∗	0.93	0.075	<0.001	0.074
22:4n-6	0.25	0.17	0.25	0.027	0.282	0.253
n-6 PUFA	18.11	14.25∗	18.31#	0.704	0.011	0.007
20:5n-3	2.87	1.64∗	3.28	0.328	0.014	0.088
22:5n-3	0.25	0.37	0.32	0.041	0.289	0.661
22:6n-3 (DHA)	0.62	8.35∗	3.19#	1.001	<0.001	0.001
n-3 PUFA	3.75	10.35∗	6.79#	0.900	<0.001	0.026
PUFA	21.86	24.61	25.10	0.718	0.074	0.791
n-3/n-6 PUFA	0.21	0.72∗	0.37#	0.069	<0.001	0.002

SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; DHA = docosahexaenoic acid; SSO = sulfosuccinimidyl oleate sodium (a cluster of differentiation [CD] 36 inhibitor); SEM = standard error of the mean.

^∗Indicates a significant difference between the DHA group and the control group based on an independent samples t-test (P < 0.05).

^#Indicates a significant difference between the DHA + SSO group and the DHA group based on an independent samples t-test (P < 0.05); n = 3.

¹Control, untreated; DHA, treated with 50 μmol/L DHA; DHA + SSO, co-treated with 50 μmol/L DHA and 200 μmol/L SSO.

3.2. Docosahexaenoic acid enhances myoblast proliferation through the CD36-mediated uptake pathway

The impact of CD36 on myoblast proliferation is illustrated in Fig. 2. In comparison with the DHA group, the mRNA expression levels of proliferation-related genes (cyclin D1 and cyclin E) were markedly reduced in the DHA + SSO group (Fig. 2A and B, P < 0.05). Consistent with these gene expression findings, the percentage of EdU-positive myoblasts relative to the total population of myoblasts was significantly decreased in the DHA + SSO group (Fig. 2C and D, P = 0.003), suggesting that CD36 inhibition attenuates the proliferative effect of DHA on myoblasts.

Fig. 2.

Docosahexaenoic acid (DHA) enhances the proliferation of grass carp myoblasts through the cluster of differentiation 36 (CD36)-mediated uptake mechanism. (A and B) The relative mRNA expression levels of proliferation-related genes (cyclin D1 and cyclin E) in myoblasts treated with DHA for 24 h. (C) Percentage of 5-ethynyl-2′-deoxyuridine (EdU)-positive myoblastsrelative to the total myoblasts. (D) EdU (red fluorescence) and Hoechst (blue fluorescence, nuclei) staining. Scale bar, 200 μm. Control, untreated; DHA, treated with 50 μmol/L DHA; DHA + SSO, co-treated with 50 μmol/L DHA and 200 μmol/L SSO. SSO = sulfosuccinimidyl oleate sodium (a CD36 inhibitor). P-value less than 0.05 indicates a significant difference, n = 3.

3.3. Cluster of differentiation 36 facilitates DHA uptake through CAV1-dependent endocytosis

The modulation of mRNA expression of endocytosis-related genes (cav1, cav2, cav3, and clathrin) by DHA in myoblasts is depicted in Fig. 3A–D. The cav1 and cav2 exhibited significant upregulation in response to DHA, with their mRNA expression levels markedly elevated, in comparison with the control group (Fig. 3A and B, P < 0.05), whereas the mRNA expression levels of cav3 and clathrin in the DHA group showed no significant differences compared to the control group (Fig. 3C and D, P > 0.05). The colocalization of CD36 and CAV1 in HEK 293T cells, as well as the internalization process occurring over time following DHA treatment, is presented in Fig. 3E. CD36 colocalized with CAV1, and both were completely internalized after 2 h of DHA treatment, indicating that CD36 mediates DHA uptake via a CAV1-dependent endocytic mechanism. As shown in Table 4, the DHA group exhibited a significant increase in the DHA level within the myoblasts compared to the control group (P < 0.001). Nevertheless, compared to the DHA group, the DHA + Nys group significantly reduced the DHA level in grass carp myoblasts (P = 0.040).

Fig. 3.

Cluster of differentiation 36 (CD36) facilitates the uptake of docosahexaenoic acid (DHA) in grass carp myoblasts via a CAV1-dependent endocytic pathway. (A-D) The mRNA expression of endocytosis-related genes in myoblasts treated with DHA for 24 h. (E) The colocalization and internalization of CD36 (green fluorescence) and CAV1 (red fluorescence) were observed in human embryonic kidney 293T (HEK 293T) cells transfected with plasmid after being treated with DHA for different time. Scale bar, 8 μm. Control, untreated; DHA, treated with 50 μmol/L DHA. P-value less than 0.05 indicates a significant difference, n = 3.

Table 4.

Effects of DHA, DHA + Nys, and DHA + Nys + SSO on the fatty acid composition of grass carp myoblasts (% total fatty acids).

Fatty acids	Groups1				SEM	P-value
	Control	DHA	DHA + Nys	DHA + Nys + SSO		DHA vs. Control	DHA + Nys vs. DHA	DHA + Nys + SSO vs. DHA + Nys
14:0	13.88	14.62	7.91#	9.25	0.808	0.536	0.001	0.150
16:0	0.54	0.48	6.33#	0.39ˆ	0.656	0.364	<0.001	<0.001
18:0	38.68	34.85	32.79	37.83	0.899	0.082	0.438	0.073
SFA	53.09	49.95	47.04	47.47	0.926	0.059	0.193	0.880
16:1n-7	0.45	1.93∗	3.06#	2.81	0.270	<0.001	0.001	0.331
18:1n-9	7.94	7.62	8.02	9.62ˆ	0.296	0.687	0.639	0.043
MUFA	8.39	9.55	11.08	12.43	0.452	0.203	0.119	0.051
20:2n-6	0.93	0.42∗	1.17#	1.55ˆ	0.111	0.008	0.001	0.008
20:3n-6	0.78	1.19	0.93	1.45	0.103	0.072	0.243	0.138
20:4n-6	21.89	20.46	22.81#	22.7	0.388	0.054	0.029	0.931
22:4n-6	4.36	3.33	3.60	2.13	0.445	0.192	0.846	0.400
n-6 PUFA	27.97	25.41	28.51	27.83	0.653	0.071	0.171	0.776
20:5n-3	8.09	6.17	6.60	5.25	0.461	0.112	0.771	0.350
22:5n-3	1.47	1.32	1.01#	0.99	0.065	0.309	0.047	0.807
22:6n-3 (DHA)	0.99	7.61∗	5.76#	6.03	0.662	<0.001	0.040	0.709
n-3 PUFA	10.55	15.09∗	13.38	12.27	0.593	0.004	0.302	0.501
PUFA	38.52	40.50	41.88	40.10	0.763	0.359	0.579	0.486
n-3/n-6 PUFA	0.38	0.59∗	0.48	0.44	0.027	<0.001	0.152	0.674

SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; DHA = docosahexaenoic acid; SSO = sulfosuccinimidyl oleate sodium (a cluster of differentiation [CD] 36 inhibitor); Nys = nystatin (a CAV1 inhibitor); SEM = standard error of the mean.

^∗Indicates a significant difference between the DHA group and the control group based on an independent samples t-test (P < 0.05).

^#Indicates a significant difference between the DHA + Nys group and the DHA group based on the independent samples t-test (P < 0.05).

^ˆIndicates a significant difference between the DHA + Nys + SSO group and the DHA + Nys group based on an independent samples t-test (P < 0.05); n = 3.

¹Control, untreated; DHA, treated with 50 μmol/L DHA; DHA + Nys, co-treated with 50 μmol/L DHA and 75 μmol/L Nys; DHA + Nys + SSO, co-treated with 50 μmol/L DHA, 75 μmol/L Nys, and 200 μmol/L SSO.

3.4. Caveolin-1 is crucial for the uptake of DHA by myoblasts and their subsequent proliferation

The DHA + Nys group significantly reduced CAV1 protein expression compared to the DHA group (Fig. 4A and B, P < 0.001). The DHA + Nys group significantly decreased the percentage of EdU-positive myoblasts compared to the DHA group (Fig. 4C and D, P < 0.001). Consistent with this, the mRNA expression of the proliferation-associated genes (cyclin D1 and cyclin E) was markedly reduced in the DHA + Nys group compared to the DHA group (Fig. 4E and F, P < 0.05). These findings indicate that CAV1 inhibition diminishes the proliferative effects of DHA on myoblasts. Figure 4G illustrates the transfection efficiency of CAV1 in myoblasts. Both the DHA and CAV1 OE groups significantly increased cav1 mRNA expression in myoblasts compared to the control group (P < 0.05). Additionally, the DHA + CAV1 OE group showed a further significant increase in cav1 mRNA expression compared to the CAV1 OE group (Fig. 4G, P < 0.001). The effects of DHA, DHA + CAV1 OE, and DHA + CAV1 OE + SSO on the fatty acid composition of grass carp myoblasts are presented in Table 5. The DHA + CAV1 OE group did not increase intracellular DHA content compared to the DHA group (P = 0.127). However, the intracellular DHA content in the DHA + CAV1 OE + SSO group was markedly decreased compared with the DHA + CAV1 OE group (P = 0.018).

Fig. 4.

Docosahexaenoic acid (DHA) promotes myoblast proliferation in grass carp via cluster of differentiation 36 (CD36) and CAV1. (A) Protein levels of CAV1 expression in myoblasts treated with CAV1 inhibitor. (B) Quantitative results of CAV1 protein levels in myoblasts treated with CAV1 inhibitor. (C) Percentage of 5-ethynyl-2′-deoxyuridine (EdU)-positive myoblasts relative to the total myoblasts. (D) EdU (red fluorescence) and Hoechst (blue fluorescence, nuclei) staining. Scale bar, 200 μm. (E and F) The mRNA expression levels of proliferation-related genes (cyclin D1 and cyclin E) in myoblasts treated with DHA for 24 h. (G) Overexpression of cav1 in myoblasts. Control, untreated; DHA, treated with 50 μmol/L DHA; DHA + Nys, co-treated with 50 μmol/L DHA and 75 μmol/L Nys; DHA + Nys + SSO, co-treated with 50 μmol/L DHA, 75 μmol/L Nys, and 200 μmol/L SSO; CAV1 OE, myoblasts were transfected with a CAV1-overexpression plasmid; DHA + CAV1 OE, CAV1-overexpressing myoblasts were treated with 50 μmol/L DHA. SSO = sulfosuccinimidyl oleate sodium (a CD36 inhibitor); Nys = nystatin (a CAV1 inhibitor); CAV1 OE = CAV1-overexpressing; GAPDH = glyceraldehyde-3-phosphate dehydrogenase. P-value less than 0.05 indicates a significant difference, n = 3.

Table 5.

Effects of DHA, DHA + CAV1 OE, and DHA + CAV1 OE + SSO on the fatty acid composition of grass carp myoblasts (% total fatty acids).

Fatty acids	Groups1				SEM	P-value
	Control	DHA	DHA + CAV1 OE	DHA + CAV1 OE + SSO		DHA vs. Control	DHA + CAV1 OE vs. DHA	DHA + CAV1 OE + SSO vs. DHA + CAV1 OE
14:0	14.00	13.25	9.85#	7.82ˆ	0.734	0.467	0.010	0.019
16:0	0.55	0.51	0.38	0.43	0.024	0.493	0.119	0.303
18:0	38.30	34.92∗	35.70	33.17	0.600	0.008	0.552	0.073
SFA	52.86	48.67∗	45.93	41.42ˆ	1.191	0.002	0.073	0.009
16:1n-7	0.43	2.21∗	1.53	2.15	0.282	0.004	0.437	0.456
18:1n-9	8.05	6.90	8.55#	8.62	0.236	0.067	0.040	0.878
MUFA	8.47	9.11	10.07	10.78	0.387	0.446	0.447	0.526
20:2n-6	0.91	0.51∗	1.03	0.60	0.108	0.027	0.189	0.246
20:3n-6	0.90	1.24	1.35	2.42ˆ	0.184	0.096	0.721	0.016
20:4n-6	21.76	20.35	22.23	22.43	0.359	0.112	0.078	0.847
22:4n-6	4.45	3.92	2.34#	4.90ˆ	0.308	0.174	0.010	0.001
n-6 PUFA	28.02	26.02	26.96	30.36ˆ	0.577	0.082	0.437	0.032
20:5n-3	8.10	7.08∗	8.69#	9.13	0.235	0.009	0.001	0.297
22:5n-3	1.51	1.39	1.06	2.11ˆ	0.127	0.404	0.269	0.003
22:6n-3 (DHA)	1.04	7.72∗	7.29	6.21ˆ	0.703	<0.001	0.127	0.018
n-3 PUFA	10.65	16.19∗	17.04#	17.45	0.736	<0.001	0.044	0.291
PUFA	38.67	42.22∗	43.99	47.81ˆ	0.986	0.010	0.187	0.034
n-3/n-6 PUFA	0.38	0.62∗	0.63	0.58	0.028	<0.001	0.738	0.065

SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; DHA = docosahexaenoic acid; SSO = sulfosuccinimidyl oleate sodium (a cluster of differentiation [CD] 36 inhibitor); CAV1 OE = CAV1-overexpressing; SEM = standard error of the mean.

^∗Indicates a significant difference between the DHA group and the control group based on an independent samples t-test (P < 0.05).

^#Indicates a significant difference between the DHA + CAV1 OE group and the DHA group based on the independent samples t-test (P < 0.05).

^ˆIndicates a significant difference between the DHA + CAV1 OE + SSO group and the DHA + CAV1 OE group based on an independent samples t-test (P < 0.05); n = 3.

¹Control, untreated; DHA, treated with 50 μmol/L DHA; DHA + CAV1 OE, CAV1-overexpressing myoblasts were treated with 50 μmol/L DHA; DHA + CAV1 OE + SSO, CAV1-overexpressing myoblasts were co-treated with 50 μmol/L DHA and 200 μmol/L SSO.

3.5. Docosahexaenoic acid promotes myofiber proliferation and development in muscle tissue via the PPARα-CD36-mediated uptake pathway

Fluorescence reporter gene assays demonstrated that PPARα regulates the transcription of CD36 (Fig. 5A, P < 0.001). Compared to the DHA group, both the control and DHA + GW6471 groups demonstrated a significant decrease in the mRNA expression of cd36 and cav1 (Fig. 5B, P < 0.05). Histological analysis revealed that, compared to the DHA group, muscle fiber diameter was significantly larger in both the control and DHA + GW6471 groups (Fig. 5C and E, P < 0.05), while muscle fiber density was markedly reduced (Fig. 5D and E, P < 0.05). Additionally, in the DHA group, the mRNA expression of genes related to muscle growth and development (myod, myog, myhc, myf4, and myf5), and fibroblast growth factors (fgf6a and fgf6b) was markedly elevated compared with that in both the control and DHA + GW6471 groups (Fig. 5F, P < 0.05). As shown in Table 6, compared to the control group, the DHA level in the muscle tissue of the DHA group was significantly higher (P = 0.002). Nevertheless, compared to the DHA group, the DHA + GW6471 group significantly reduced the DHA level in grass carp myoblasts (P = 0.004).

Fig. 5.

Docosahexaenoic acid (DHA) promotes myofiber proliferation and development in muscle tissue via the PPARα-cluster of differentiation 36 (CD36)-mediated uptake pathway. (A) Site-directed mutagenesis analysis of PPARα binding sites on the pGL3.0-CD36 vector in human embryonic kidney 293T (HEK 293T) cells. (B) The mRNA expression levels of fatty acid absorption (cd36 and cav1) in muscle tissue. (C) Muscle fiber diameter. (D) Muscle fiber density. (E) Representative hematoxylin and eosin (H&E) staining in muscle tissue. Scale bar, 100 μm. (F) The mRNA expression of muscle growth and development related genes (myog, myod, myhc, mrf4, and myf5) and fiber cell growth factor (fgf6a and fgf6b) in muscle tissue. Control, diet without DHA supplementation; DHA, diet supplemented with 0.5% DHA; DHA + GW6471, diet supplemented with 0.5% DHA and GW6471. GW6471 = peroxisome proliferator-activated receptor α inhibitor; Luc = luciferase. P-value less than 0.05 indicates a significant difference, n = 3.

Table 6.

Effects of the experimental diets on the fatty acid composition of grass carp muscle (% total fatty acids).

Fatty acids	Groups1			SEM	P-value
	Control	DHA	DHA + GW6471		DHA vs. Control	DHA + GW6471 vs. DHA
14:0	1.26	1.17	1.10	0.033	0.122	0.421
16:0	20.41	18.98∗	20.34#	0.241	0.003	0.002
18:0	5.78	5.71	5.62	0.040	0.415	0.398
SFA	27.45	25.86∗	27.06#	0.251	0.001	0.004
16:1n-7	5.10	4.05∗	4.70#	0.162	0.003	0.022
18:1n-9	27.93	23.47∗	25.74#	0.686	0.004	0.047
MUFA	33.03	27.52∗	30.44#	0.846	0.022	0.041
18:2n-6	8.91	8.40	8.45	0.111	0.115	0.853
18:3n-6	0.33	0.27∗	0.26	0.012	<0.001	0.090
20:3n-6	0.99	0.87∗	0.87	0.020	0.001	0.904
20:4n-6	6.33	6.26	5.64#	0.122	0.690	0.026
n-6 PUFA	16.56	15.80∗	15.21#	0.198	0.004	0.003
18:3n-3	3.69	3.71	3.87	0.035	0.760	0.050
20:3n-3	0.14	0.18∗	0.18	0.008	0.013	0.774
20:5n-3	0.72	0.93∗	0.91	0.034	<0.001	0.358
22:6n-3 (DHA)	5.34	12.35∗	8.63#	1.048	0.002	0.004
n-3 PUFA	9.89	17.17∗	13.60#	1.082	0.001	0.004
PUFA	26.44	32.97∗	28.81#	0.979	0.001	0.001
n-3/n-6 PUFA	0.60	1.09∗	0.89#	0.073	0.001	0.012

SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; DHA = docosahexaenoic acid; GW6471 = peroxisome proliferator-activated receptor α inhibitor; SEM = standard error of the mean.

^∗Indicates a significant difference between the DHA group and the control group based on an independent samples t-test (P < 0.05).

^#Indicates a significant difference between the DHA + GW6471 group and the DHA group based on an independent samples t-test (P < 0.05); n = 3.

¹Control, diet without DHA supplementation; DHA, diet supplemented with 0.5% DHA; DHA + GW6471, diet supplemented with 0.5% DHA and GW6471.

3.6. The PPARα plays a critical role in enhancing the growth performance of grass carp by DHA

As indicated in Table 7,no significant differences were observed in the IBW and FI among the groups in grass carp (P > 0.05). Interestingly, although the FCR in the DHA group was slightly reduced relative to the control group, this difference was not statistically significant (P = 0.059). In contrast, the FCR in the DHA + GW6471 group was significantly higher than that in the DHA group (P = 0.011). Compared to the control group, the DHA group exhibited significant increases in FBW, WGR, SGR, PRE, and PER (P < 0.05). However, the DHA + GW6471 group significantly decreased these parameters (P < 0.05). In contrast to the control group, the DHA group demonstrated significant reductions in CF, VSI, and HSI (P < 0.05). Compared to the DHA group, the DHA + GW6471 group showed a significant increase in CF and VSI (P < 0.05), but had no significant effect on HSI (P = 0.881).

Table 7.

Effects of the experimental diets on growth performance and biological parameters of grass carp.

Items	Groups1			SEM	P-value
	Control	DHA	DHA + GW6471		DHA vs. Control	DHA + GW6471 vs. DHA
IBW, g	32.05	32.02	32.03	0.015	0.624	0.681
FBW, g	61.69	66.39∗	60.40#	1.052	0.039	0.012
WGR, %	92.46	107.31∗	88.56#	3.288	0.035	0.012
SGR, %/d	1.17	1.30∗	1.13#	0.030	0.036	0.012
FCR	1.89	1.63	2.00#	0.066	0.059	0.011
FI, g/fish	55.84	55.80	56.50	0.288	0.972	0.424
CF, g/cm3	1.93	1.76∗	1.93#	0.032	0.017	0.014
VSI, %	9.76	8.31∗	8.98#	0.220	0.003	0.005
HSI, %	2.89	2.31∗	2.30	0.100	<0.001	0.881
PRE, %	20.55	26.52∗	18.61#	1.298	0.009	0.008
PER	1.47	1.70∗	1.39#	0.054	0.046	0.008

IBW = initial average body weight; FBW = final body weight; DHA = docosahexaenoic acid; GW6471 = peroxisome proliferator-activated receptor α inhibitor; SEM = standard error of the mean.

^∗Indicates a significant difference between the DHA group and the control group based on an independent samples t-test (P < 0.05).

^#Indicates a significant difference between the DHA + GW6471 group and the DHA group based on an independent samples t-test (P < 0.05); n = 3.

¹Control, diet without DHA supplementation; DHA, diet supplemented with 0.5% DHA; DHA + GW6471, diet supplemented with 0.5% DHA and GW6471.

3.7. The PPARα plays a pivotal role in enhancing lipid utilization in the muscle tissue of grass carp through DHA

As presented in Table 8, compared to the control group, the moisture and crude lipid in the muscles of the DHA group were significantly reduced, whereas these values were markedly elevated following the addition of GW6471 (P < 0.05). Notably, the crude protein in the muscles of the DHA group increased compared to the control group (P = 0.012), but decreased significantly after the addition of GW6471 (P = 0.037). The TG content in the muscles of the DHA group was significantly lower than in the control group (P = 0.003), but was markedly increased after the addition of GW6471 (P = 0.006). Additionally, in the DHA group, the mRNA expression of genes related to lipid lipolysis (atgl and hsl) and fatty acid β-oxidation (pparα and cpt-1a) was markedly higher than that in both the control and DHA + GW6471 groups (Fig. 6, P < 0.05).

Table 8.

Effects of the experimental diets on the proximate composition and TG levels in the muscle of grass carp (%).

Items	Groups1			SEM	P-value
	Control	DHA	DHA + GW6471		DHA vs. Control	DHA + GW6471 vs. DHA
Moisture	80.61	77.66∗	80.66#	0.576	0.026	0.018
CP	15.84	18.68∗	16.71#	0.486	0.012	0.037
Crude lipid	0.77	0.66∗	0.77#	0.022	0.029	0.044
TG, mmol/g	0.11	0.07∗	0.12#	0.007	0.003	0.006

DHA = docosahexaenoic acid; GW6471 = peroxisome proliferator-activated receptor α inhibitor; CP = prude protein; TG = triglyceride; SEM = standard error of the mean.

^∗Indicates a significant difference between the DHA group and the control group based on an independent samples t-test (P < 0.05).

^#Indicates a significant difference between the DHA + GW6471 group and the DHA group based on an independent samples t-test (P < 0.05); n = 3.

¹Control, diet without DHA supplementation; DHA, diet supplemented with 0.5% DHA; DHA + GW6471, diet supplemented with 0.5% DHA and GW6471.

Fig. 6.

Effects of the experimental diets on lipid metabolism-related genes in grass carp muscle. The mRNA expression levels of lipolysis (hsl and atgl) and fatty acid β-oxidation-related genes (pparα and cpt-1a) in muscle tissue. Control, diet without docosahexaenoic acid (DHA) supplementation; DHA, diet supplemented with 0.5% DHA; DHA + GW6471, diet supplemented with 0.5% DHA and GW6471. GW6471 = peroxisome proliferator-activated receptor α inhibitor. P-value less than 0.05 indicates a significant difference, n = 3.

4. Discussion

As the intensification of aquaculture progresses, the decline in the quality of fish meat has grown more pronounced (Ji et al., 2024). Consequently, maintaining the high quality of aquatic products has become a critical challenge for the industry (Yang et al., 2023). Muscle fiber characteristics play a crucial role in determining flesh quality (Periago et al., 2005; Wang et al., 2024a). While it is well-established that DHA promotes myocyte proliferation and muscle fiber development in fish (Gao et al., 2024; Ji et al., 2024; Wang et al., 2020), the exact cellular entry mechanism of DHA remains unclear. This study reveals that DHA enters the myoblasts of grass carp through a CD36-mediated, CAV1-dependent endocytic pathway. Subsequently, DHA activates PPARα, which subsequently upregulates the transcription of CD36, thereby facilitating further DHA uptake by the myoblasts and promoting myoblast proliferation.

Docosahexaenoic acid, a biologically active fatty acid, is essential for supporting fish muscle growth and quality, particularly in the development of muscle fibers (He et al., 2022; Ji et al., 2024). Furthermore, the effectiveness of DHA is contingent upon its intake and accumulation within cells (Akbar et al., 2005). The CD36 protein is a critical fatty acid transporter in myoblasts, playing an essential role in the uptake of LCFAs (Glatz et al., 2016). Moreover, CD36 has been identified as a key mediator of fatty acid uptake and oxidation in cardiac tissue, responsible for approximately 68% of the total fatty acid supply (Habets et al., 2007; Pepino et al., 2014). In both adipose and muscle tissues of mice, CD36 is responsible for about 50% of fatty acid absorption (Coburn et al., 2000). Nickerson et al. (2009) demonstrated that CD36 is the most efficient fatty acid transporter in vivo. Consistent with these findings, our study observed that the CD36-specific inhibitor SSO significantly reduced DHA accumulation in myoblasts, suggesting that CD36 is the primary transporter facilitating DHA entry into myoblasts in grass carp. CD36 facilitates fatty acid absorption in myoblasts, thereby influencing muscle cell proliferation. Previous studies have shown that CD36 promotes skeletal muscle cell proliferation by modulating the cell cycle, and knockdown of CD36 intensifies the inhibitory effect of palmitic acid on cell proliferation and accelerates apoptosis (Sun et al., 2022). After muscle injury, fatty acids are essential for cellular regeneration, but CD36 deficiency impairs fatty acid uptake, resulting in a marked decrease in myoblast proliferation (Wang et al., 2024b). Moreover, this study found that inhibition of CD36 decreased the relative DHA content in myoblasts, significantly downregulated the mRNA expression of cell proliferation-related genes, and markedly reduced the percentage of EdU-positive myoblasts. These results emphasize the pivotal role of CD36 in mediating DHA absorption in myoblasts, thereby enhancing DHA’s capacity to promote myoblast proliferation and growth.

Previous studies have demonstrated that the internalization of CD36 is essential for fatty acid uptake (Xu et al., 2013). In adipocytes, 30 min of oleic acid treatment induced CD36 translocation from the plasma membrane to the intracellular compartment, with CD36 becoming nearly undetectable on the membrane after 2 h (Hao et al., 2020b). Similarly, in this study, after 2 h of DHA treatment, CD36 in HEK 293T cells resulted in pronounced internalization. Endocytosis, a fundamental biological process in eukaryotes, primarily occurs via two classical pathways: clathrin-mediated and caveolin-mediated mechanisms for the uptake of exogenous substances (Kaksonen and Roux, 2018; Kotova et al., 2020; Rothberg et al., 1992). The mRNA expression levels of cav1, cav2, cav3, and clathrin were measured following DHA treatment. After 24 h, only the mRNA levels of cav1 and cav2 were significantly elevated. Previous studies have shown that CAV2 is localized to the surface of lipid droplets and can undergo dynamic redistribution (Fujimoto et al., 2001). Its N-myristoylation is crucial for coordinating its phosphorylation, palmitoylation, and ubiquitination processes, which thereby regulate insulin signaling (Kwon et al., 2020). Moreover, CAV2 is not required for caveolae formation in certain tissues (Chidlow and Sessa, 2010). Caveolae play a critical role in raft-dependent LCFA uptake (Pohl et al., 2004), implying that CAV2 may be involved in lipid metabolism through alternative pathways. In contrast, CAV1 not only constitutes the structural scaffold of caveolae but also directly binds LCFAs, playing a central role in fatty acid uptake and metabolism (Meshulam et al., 2006; Otis et al., 2017; Trigatti et al., 1999). Previous studies have shown that CAV1 overexpression significantly enhances fatty acid absorption, while its deficiency disrupts caveolar integrity and reduces lipid uptake efficiency (Pohl et al., 2004; Razani et al., 2002). In addition, CAV1 is a critical regulatory factor for CD36 membrane localization, likely influencing fatty acid absorption by modulating its distribution on the plasma membrane (Ring et al., 2006). Pohl et al. (2004) demonstrated that in 3T3-L1 mouse preadipocytes cells, the absence of CAV1 led to a 50% reduction in fatty acid uptake and a decrease in CD36 membrane localization. However, overexpression of CAV1 restored CD36 localization to the plasma membrane. These findings suggest that CAV1 in grass carp may also contribute to DHA absorption. Building upon this mechanism, the present study further examined the impact of CAV1 inhibition or overexpression on DHA levels in grass carp myoblasts. The results showed that the DHA + Nys group significantly reduced intracellular DHA levels, whereas the DHA + CAV1 OE group did not enhance DHA uptake, indicating that its function is dependent on CD36. Further experiments revealed that DHA uptake was significantly reduced in the DHA + CAV1 OE + SSO group, highlighting the critical collaborative role of both factors in fatty acid absorption. This finding is consistent with the study by Hao et al. (2020b), which demonstrated that CAV1 knockdown significantly inhibited CD36 endocytosis. In conclusion, DHA uptake in grass carp myoblasts is critically dependent on the coordinated endocytic process mediated by both CD36 and CAV1.

The CD36 gene promoter is a direct target of PPAR (Tontonoz et al., 1998), and this study employed a dual-luciferase reporter gene assay, which demonstrated that PPARα directly activates the transcription of CD36, aligning with the conclusion that PPARα acts as an upstream regulator of CD36 in mammals (Madonna et al., 2011; Sato et al., 2002). PPARα, a natural receptor for DHA, is known to facilitate DHA accumulation in skeletal muscle and enhance muscle function (Casanova et al., 2014; Huang et al., 2016; Hsiao et al., 2019). This in vivo study demonstrated that DHA enhanced the mRNA expression of PPARα. Inhibition of PPARα significantly reduced CD36 expression and DHA content in muscle tissue. These findings align with previous studies in mice (Mata-Sotres et al., 2022), suggesting that DHA upregulates CD36 expression through PPARα activation, thereby promoting DHA uptake. Furthermore, the supplementation of DHA in this study significantly enhanced the FBW, WGR, and SGR of grass carp. This result is consistent with findings from previous studies on common carp (Cyprinus carpio L.) (He et al., 2022) and blunt snout bream (M. amblycephala) (Wang et al., 2020). However, the DHA + GW6471 treatment group reversed these effects, suggesting that PPARα plays a pivotal role in mediating the growth-promoting effects of DHA on grass carp. Body weight gain is strongly correlated with increases in both visceral weight and muscle mass (Du and Turchini, 2021). In this study, the DHA treatment group significantly reduced the CF and VSI in grass carp, while the DHA + GW6471 group reversed this effect, further supporting the idea that DHA promotes weight gain in grass carp by enhancing muscle mass, through the mediation of PPARα. Consequently, DHA may enhance muscle development in grass carp by activating the PPARα pathway, thereby promoting their overall growth.

The growth of the fish is predominantly reflective of muscle tissue development (Weitkunat et al., 2017). Muscle development is regulated by myogenic regulatory factors (MRFs), including myod, myf5, mrf4, and myog (Hu et al., 2023). Myod and myf5 primarily initiate the proliferation phase of myoblasts (Dong et al., 2022; Maguire et al., 2012), whereas myog is crucial in the differentiation of myoblasts (Zhang et al., 2018). DHA significantly upregulated the mRNA expression of these myogenic regulatory factors (myod, myf5, mrf4, and myog), with subsequent downregulation observed following PPARα inhibition. The structure of muscle fibers in fish directly influences meat quality. Typically, denser muscle fibers lead to firmer texture, greater toughness, and enhanced flavor in the flesh (Periago et al., 2005; Wang et al., 2024a). This study observed that DHA significantly increased muscle fiber density in grass carp, whereas PPARα inhibition attenuated this effect. Thus, PPARα plays a crucial role in the DHA-mediated regulation of muscle fiber density and meat quality in grass carp.

5. Conclusion

In conclusion, this study comprehensively investigated the mechanisms underlying DHA absorption in grass carp myoblasts, revealing its cellular uptake via a CD36/CAV1-dependent endocytic pathway. Upon entry into the myoblasts, DHA activates PPARα, thereby enhancing the transcription of the CD36 gene. This process not only facilitates DHA uptake by myoblasts but also regulates lipid metabolism pathways, promoting myocyte proliferation and improving the growth performance and muscle quality of grass carp. The findings provide a novel perspective for nutritional research in aquatic animals, particularly through the enhancement of DHA absorption and metabolism pathways, thereby optimizing the growth performance of grass carp.

Credit Author Statement

Rongrong Xue: Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Lu Zhou: Writing – review & editing, Methodology, Investigation, Formal analysis. Luyao Jia: Writing – review & editing, Formal analysis, Data curation. Jie Zhang: Writing – review & editing, Methodology. Handong Li: Writing – review & editing, Supervision. Tao Zhao: Writing – review & editing, Supervision. Jian Sun: Writing – review & editing, Supervision. Hong Ji: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: