Effects of Phosphatidic Acid on Mammalian Target of Rapamycin (mTOR) and Metabolic Pathways in Bivalve Mollusk <i>Sinonovacula constricta</i>

Liao, Kai; Zhang, Qian; Qiu, Yangyang; Zhu, Yuxiang; Liu, Yang; Huang, Hailong; Chen, Deshui; Ma, Bin; Zhang, Lin; Ran, Zhaoshou; Yan, Xiaojun; Xu, Jilin

doi:https://doi.org/10.1155/2023/6666946

Aquaculture Research

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Abstract Introduction Materials and Methods Results Discussion Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 6666946 | https://doi.org/10.1155/2023/6666946

Effects of Phosphatidic Acid on Mammalian Target of Rapamycin (mTOR) and Metabolic Pathways in Bivalve Mollusk Sinonovacula constricta

Kai Liao,¹Qian Zhang,¹Yangyang Qiu,¹Yuxiang Zhu,¹Yang Liu,¹Hailong Huang,¹Deshui Chen,²Bin Ma,³Lin Zhang,¹Zhaoshou Ran,¹Xiaojun Yan,¹and Jilin Xu^1,2

Academic Editor: Houguo Xu

Received26 Apr 2023

Revised23 Jun 2023

Accepted03 Jul 2023

Published22 Jul 2023

Abstract

It is of great significance for bivalve aquaculture to promote the growth through nutritional strategy. Phosphatidic acid (PA) is a potential growth-promoting nutraceutical that targets for mammalian target of rapamycin (mTOR) in vertebrates, but its role in invertebrates remains largely unknown. Here, the effects of PA on mTOR and metabolic pathways in bivalve mollusk Sinonovacula constricta were determined by intramuscular injection with di-18 : 2 PA and di-16 : 0 PA. PA (both di-18 : 2 and di-16 : 0 PA) was found to increase the glycogen concentration in the muscle of S. constricta. Di-16 : 0 PA decreased the triglyceride concentration from 0.143 ± 0.04 mmol/g protein to 0.040 ± 0.018 mmol/g protein. The concentration of Asp, Glu, Ala, Cys, Val, Met, Ile, Leu, Phe, Arg, and Pro was reduced by di-18 : 2 PA or/and di-16 : 0 PA. PA increased the mRNA level of mTOR and the phosphorylation levels of eIF4E binding protein 1 and p70S6 kinase 1. Furthermore, PA decreased the protein level of microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ and the mRNA level of AMP-activated protein kinase. The mRNA expressions of two key enzymes of glycolysis (pyruvate kinase and glucokinase) were also upregulated by both PA, while the mRNA level of glucose transporter 1 was increased by di-18 : 2 PA. Di-16 : 0 PA decreased the mRNA level of phosphoenolpyruvate carboxykinase. The mRNA levels of sterol responsive element binding protein, fatty acid synthase, and acetyl-CoA carboxylase were increased by both PA. The mRNA level of stearoyl-CoA desaturase was increased by di-18 : 2 PA. Both PA species increased the mRNA levels of key enzymes involved in tricarboxylic acid cycle (citrate synthase and NADP-isocltrate debydrogenase). Our results indicated that PA activated mTOR signaling pathway, subsequently leading to the increase of anabolism and the inhibition of catabolism in S. constricta.

1. Introduction

Increasing or promoting growth is an important target for bivalve nutrition research. Growth in terms of biomass depends on the accumulation of proteins and lipids, and the redirection of nutrients from catabolism to anabolism [1]. A key regulator of growth from yeast to mammals is the mammalian target of rapamycin (mTOR), which promotes anabolic processes, including protein and lipid synthesis, and suppresses catabolic processes, such as autophagy [2]. First, mTOR stimulates protein synthesis mainly through eIF4E binding protein 1 (4EBP1) and p70S6 kinase 1 (S6K1). The activation of 4EBP1 triggers 5′cap-dependent mRNA translation, while S6K1 enhances the translation efficiency of spliced mRNAs. Second, mTOR drives de novo lipid synthesis through the sterol responsive element binding protein (SREBP), which activates the transcription of genes involved in lipid biosynthesis. Third, mTOR also facilitates growth by promoting a shift in glucose metabolism from oxidative phosphorylation to glycolysis, which likely enhances the incorporation of nutrients into new biomass. Apart from these anabolic processes, mTOR also promotes cell growth by suppressing protein catabolism, most notably autophagy. An important early step of autophagy is the activation of Unc-51-like kinases 1 (ULK1), a kinase that drives autophagosome formation [3]. mTOR phosphorylates ULK1, thereby disrupting the interaction between ULK1 and AMP activated protein kinase (AMPK, a known activator of autophagy) [4].

In bivalve, genes involved in protein synthesis are key regulators of growth [5, 6]. For example, the expression of ribosomal proteins shows linear relationships with growth rates in bivalve larvae (Crassostrea gigas) [5]. Previous studies have demonstrated that mTOR regulates autophagy in response to the environmental contaminants in mussels (Mytilus galloprovincialis Lam.) [7, 8]. Recently, we found that mTOR plays a conserved role in controlling protein synthesis, glucose metabolism, lipid synthesis, and autophagy in razor clam (Sinonovacula constricta) [9]. Thus, mTOR may be an attractive target for promoting bivalve growth.

Phosphatidic acid (PA), a diacyl-glycerophospholipid, is a precursor of membrane phospholipid and triglyceride synthesis and can serve as a signaling lipid. PA is implicated in the control of cell cycle progression and cell growth [10]. Most significantly, PA can directly activate mTOR signaling via binding to the FKBP12-rapamycin binding (FRB) domain of mTOR [11]. It has been shown that exogenous PA from egg or soy lecithin can activate mTOR signaling and stimulate muscle protein synthesis in mammals [12, 13]. Feeding PA improves feed conversion ratio and increases live bird weight and breast fillet weight in broilers [14]. Exogenous PA improves the growth performance of turbot [15] and mechanically PA activates mTOR signaling, increases protein synthesis, lipogenesis, and glycolysis, and stimulates primary muscle cell proliferation in turbot [16]. Given that PA is a potential growth promoting supplement that can be incorporated in artificial diet of bivalves, this study is particularly interested in examining the effects of PA on mTOR and its metabolic pathways in bivalve species such as razor clam, which is an important mariculture bivalve that widely distributed in China, Japan, and Korea.

2. Materials and Methods

2.1. Animals and Experimental Treatments

The procedures for animal care and handling described herein were approved by Ningbo University’s Institutional Animal Care and Use Committee (protocol# 2022–055). Adult S. constricta (shell length, 40–45 mm) collected from the intertidal zone at Sanmen Bay, Ningbo, China. The clams were maintained in an aerated aquarium (100 L) for 7-days starvation prior to the experiments. The seawater was filtered and maintained at 22–25°C, and the salinity was 20%–21%. A pilot experiment was conducted, and the dosage and timing of PA administration were determined per the phosphorylation levels of 4EBP1 (Thr^36/47). The clams that received intramuscularly with 200 μM di-18 : 2 PA (Cat No. L130340, Aladdin) after 6 hr showed a higher level of 4EBP1 phosphorylation (Figure S1). At the beginning of the experiment, one group of clams (30 clams) was uninjected while three groups of clams were injected intramuscularly with, i.e., solvent control (0.02% dimethyl sulfoxide (DMSO); 30 clams, 50 μL/clam); 200 μM di-18 : 2 PA (200 μM di-18 : 2 PA resolved in DMSO; 30 clams, 50 μL/clam); 200 μM di-16 : 0 PA (Cat No. D274831, Aladdin, 200 μM di-16 : 0 PA resolved in DMSO; 30 clams, 50 μL/clam). After 6 hr of treatment, muscles were rapidly removed, pooled into tubes, chilled in liquid nitrogen, and stored at 80°C.

2.2. Biochemical Assays

Muscle glycogen concentration was tested according to the instructions of the Muscle Glycogen Assay Kit (Nanjing Jiancheng, China). In brief, lye (saline) was added to the muscle samples, boiled for 20 min, then cooled to glycogen hydrolysis solution. Glycogen test solution was prepared by adding appropriate amount of distilled water according to muscle weight. Distilled water, glycogen detection solution, and color developing solution were added into the tube to be tested, and then heated in boiling water for 5 min. After cooling, the absorbance at 620 nm was measured by an enzyme marker. Glycogen content was calculated based on the glycogen standard curve.

Triglyceride levels were measured using the TG kit (Nanjing Jiancheng, China) according to the manufacturer’s instructions. In brief, samples were homogenized and followed by centrifugation at 2,500 rpm for 10 min, and the supernatant was taken to be measured. Then, 250 μL working solution was added to the supernatant and incubated at 37°C for 10 min. Triglyceride levels were determined using a microplate reader at 500 nm.

Amino acid content was determined using the L-8900 amino acid analyzer (Hitachi, Tokyo, Japan). In brief, samples were freeze-dried and hydrolyzed with HCL (6 mol/L) at 110°C for 24 hr. After cooling, the volume was fixed, dry, and then dissolved by 0.02 mol/L HCl. Finally, the amino acid content of the samples was determined by aqueous filtration membrane.

2.3. Real-Time RT–PCR

Muscle RNA was obtained using TRIzol (Invitrogen, USA) per the manufacturer’s recommendations. Total RNA concentration and purity were analyzed using a NanoDrop® ND-1000 (NanoDrop, USA), and 1 μg of cDNA was synthesized with a commercial PrimeScript^TM RT Reagent Kit (Takara, Japan) following the manufacturer’s instructions. Real-time RT–PCR was conducted with gene specific primers (Table 1). Real-time RT–PCR was carried out in a 20 μL reaction system containing 1 μL of first-strand cDNA, 10 μL of SYBR Premix Ex Taq (TliRNase Plus) (TaKaRa, Japan), 5 pmol each of forward and reverse primers, and 5 μL of nuclease-free water. Cycling conditions were as follows: an initial denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 94°C for 10 s, annealing at 60°C for 20 s, and extensions at 72°C for 20 s, followed by melting curve analysis of the PCR product. The relative gene expression levels were analyzed by the 2^−ΔΔCT method [17], and data were normalized to the expression of β-actin.

2.4. Western Blotting

The muscle samples were lysed in RIPA buffer (Cowin Biotech, China) with protease (Cowin Biotech, China) and phosphatase inhibitors (Thermo Fisher Scientific, USA). Protein concentrations were determined using a BCA Protein Assay Kit (Sangon Biotech, China). Lysates (10 μg of total protein per lane) were separated by denaturing SDS-PAGE, transferred onto a PVDF membrane and blocked with 5% nonfat milk. Membranes were incubated with antibodies (4°C overnight) against β-actin (Cat No. EM21002, HuaBio, 1 : 10,000), phosphor-4EBP1 (Thr^37/46) (Cat No. 236B4, Cell Signal Technology, 1 : 1,000), phosphor-p70S6K (Thr³⁸⁹) (Cat No. 28735-1-AP, Proteintech, 1 : 2,000), LC3A/B (Cat No. 4108, Cell Signal Technology, 1 : 3,000) and visualized with the corresponding secondary antibody: antimouse or antirabbit (Beyotime Biotechnology, China). The target protein was measured by a chemiluminescence imager (ChemiScope Capture Image System).

2.5. Statistical Analysis

All data were expressed as mean ± SEMs. The density of protein bands was quantified by Image J software (NIH, USA). Difference between control and PA treatment group was analyzed by t-tests using SPSS 16.0 for Windows (SPSS Inc, USA), and P values of less than 0.05 were considered statistically significant.

3. Results

3.1. PA Influenced Glycogen, Triglyceride, and Amino Acid Concentrations in Muscle

The glycogen concentration in the muscle of razor clam was significantly increased by PA (Figure 1). The glycogen concentration was increased from 9 ± 1.12 to 15.6 ± 1.59 mg/g or 14.2 ± 2.30 mg/g after clam injected intramuscularly with 200 μM di-18 : 2 PA or di-16 : 0 PA for 6 hr. Di-16 : 0 PA significantly decreased the triglyceride concentration from 0.143 ± 0.04 mmol/g protein to 0.040 ± 0.018 mmol/g protein, while di-18 : 2 PA did not affect the triglyceride concentration (Figure 2). The concentration of Asp, Glu, Val, and Arg in the muscle of clam was significantly reduced by di-18 : 2 PA and di-16 : 0 PA (Table 2). The concentration of Ala, Cys, Met, Ile, and Pro was reduced by di-18 : 2 PA, while the concentration of Leu and Phe was reduced by di-16 : 0 PA (Table 2).

3.2. PA Activated the mTOR Signaling Pathway

The mRNA level of mTOR was significantly upregulated by di-18 : 2 PA and di-16 : 0 PA (Figure 3(a)). The phosphorylation levels of 4EBP1 and p70S6K were significantly increased by di-18 : 2 PA and di-16 : 0 PA, but to a lesser extent than di-18 : 2 PA (Figures 3(b) and 3(c).

(a)

(b)

(c)

3.3. PA Suppressed Autophagy

The protein level of microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ (LC3-Ⅱ/Ⅰ) was significantly reduced by di-18 : 2 PA, and by di-16 : 0 PA, but to a lesser extent than di-18 : 2 PA (Figure 4(a)). The mRNA level of AMPK was significantly decreased by di-18 : 2 PA, but not by di-16 : 0 PA (Figure 4b).

(a)

(b)

3.4. PA-Modulated Glucose Metabolism, Lipid Synthesis, and Tricarboxylic Acid Cycle

The mRNA levels of pyruvate kinase (PK) and glucokinase (GK) were significantly upregulated by di-18 : 2 PA and di-16 : 0 PA (Figures 5(a) and 5(b)). The mRNA level of glucose transporter 1 (GLUT1) was significantly increased by di-18 : 2 PA, while di-16 : 0 PA did not influence GLUT1 mRNA abundance (Figure 5(c)). Di-16 : 0 PA significantly decreased the mRNA level of phosphoenolpyruvate carboxykinase (PEPCK) compared with control, while di-18 : 2 PA did not affect the mRNA level of PEPCK (Figure 5(d)). There was no effect of PA on the mRNA level of glucose-6-phosphatase (G6Pase) mRNA (Figure 5(e)).

(a)

(b)

(c)

(d)

(e)

The mRNA levels of SREBP, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were significantly increased by di-18 : 2 PA and by di-16 : 0 PA (Figure 6(a)–6(c)). The mRNA level of stearoyl-CoA desaturase (SCD) was significantly increased by di-18 : 2 PA, however di-16 : 0 PA did not influence the mRNA level of SCD (Figure 6(d)).

(a)

(b)

(c)

(d)

PA treatment significantly increased the mRNA levels of citrate synthase (CS) and NADP-isocltrate debydrogenase (NADP) compared with control (Figures 7(a) and 7(b)).

(a)

(b)

4. Discussion

As a master regulator of growth and metabolism, mTOR integrates nutrient signal, especially amino acids, to mediate the metabolic pathways that control growth [18]. In this study, the downstream targets of mTOR (the phosphorylation levels of 4EBP1 and p70S6K) were significantly increased by an intermediate of lipid metabolism, PA. These results indicate that PA induces the activation of mTOR signaling pathway and protein synthesis in the muscle of razor clam. This is similar to previous results observed in mammalian [12, 13] and fish [16]. Mechanistically, the stability and activity of mTOR complex require the interaction of PA with the FRB domain of mTOR [11]. In addition, the subcellular localization of mTOR is also modulated by PA [18]. Endogenous suppressing PA production prevents the activation of mTOR signaling pathway in mammal cell lines [11]. However, several studies have shown that exogenous PA activates mTOR signaling through multiple indirect mechanism, rather than through direct binding to mTOR. For example, results from Winter et al. [19] suggested that exogenous PA is metabolized to lysophosphatidic acid (LPA), which subsequently activates the LPA receptor, then leading to activation of mTOR signaling via ERK pathway. Pioneering works also indicated that PA with some degree of unsaturation is required for a functional interaction with mTOR [20, 21]. In this study, both PA species containing 16 : 0–16 : 0 and 18 : 2–18 : 2 stimulated mTOR activity, but di-16 : 0 PA did show less potential for mTOR activation than di-18 : 2 PA. Whether there is a mechanism for mTOR to distinguish between PA species with some degree of unsaturation remains to be determined in razor clam.

Though the role of mTOR signaling in suppressing catabolism, such as autophagy has been well elucidated, few studies have focused on the effects of exogenous PA on autophagy. The results from Dall’Armi et al. [22] suggested that PA is required for autophagy, and pharmacological and genetic inhibition of PA production decrease the starvation-induced autophagy in mouse cells. In this study, the protein level of LC3-Ⅱ/Ⅰ, a marker for autophagy, was significantly downregulated by PA, suggesting exogenous PA treatment decreased autophagy and catabolism in the muscle of razor clam, potentially due to induction of mTOR signaling. In support of this hypothesis, the mRNA level of another autophagy activator, AMPK, was significantly decreased by di-18 : 2 PA in the muscle of razor clam. Meanwhile the reduced mRNA level of AMPK after di-18 : 2 PA treatment probably indicates a higher energy status in the muscle of razor clam.

In this study, the glycogen concentration in the muscle of razor clam was significantly increased by PA. Glycogen is the primary metabolic reserve in bivalves, and its concentration is positively related to the growth, gonad development, and gametogenesis [23, 24]. In fact, glycogen concentration is a physiological marker of health in bivalves [25]. Moreover, the glycogen concentration is closely associated with the flavor and the quality of the bivalve [26]. PA supplement may therefore represent a nutritional strategy to promote the growth while increasing its glycogen content in bivalves, such as razor clam. In addition to replenishing muscle glycogen, PA supplement can promote the uptake of circulating glucose and stimulate glycolysis. Previous study in mammals showed that PA supplementation increases the mRNA level of glucose transporter, potentially increasing glucose sensitivity in an mTOR-dependent manner [13]. In primary muscle cells of turbot, the mRNA expressions of two key enzymes of glycolysis (GK and PK) are also upregulated by PA, potentially providing intermediates for anabolism and ATP production [16]. In this study, we did observe an increased in the mRNA level of GLUT1 (di-18 : 2 PA), GK and PK in the muscle of razor clam after PA treatment. Furthermore, the mRNA level of the rate enzymes (PEPCK) in gluconeogenesis was also reduced by di-16 : 0 PA. Thus, the results in the current study suggest that PA can increase the uptake of excess circulating glucose, stimulate glycolysis, while inhibit gluconeogenesis in the muscle of razor clam. This synchronized glucose metabolism may be crucial for providing intermediates and ATP for anabolism after PA treatment.

Our findings that PA increased the mRNA levels of SREBP, FAS, ACC, and SCD support that PA promotes lipogenesis in an mTOR-dependent manner. Previous study in primary muscle cells of turbot also showed that the mRNA levels of genes involved in lipogenesis (SREBP1 and FAS) are upregulated by PA [16]. For more than a decade, it has been well-documented that mTOR can regulate the transcription of lipogenic genes such as FAS, ACC, and SCD through SREBP [27]. On the one hand, mTOR regulates the expression of SREBP at the level of transcription and translation, subsequently controlling lipogenesis [28]. On the other hand, mTOR mediates SREBP activity by modulation nuclear localization of lipin 1, which is a PA phosphatase that converts PA to diacylglycerol and serves as a repressor of SREBP activity [29]. Interestingly, di-16 : 0 PA significantly decreased the triglyceride content, though the lipogenesis was upregulated by di-16 : 0 PA in the muscle of razor clam. Researches conducted by Joy et al. [12] and Escalante et al. [30] have demonstrated that there is a trend for PA to decrease body fat in human. Indirectly, it is widely reported that mTOR inhibition induces triglyceride accumulation in yeast and fruit flies [31, 32] and in nutrient starved mammalian cells [33, 34]. The recent research indicates that cells might channel de novo synthesized lipids toward new membrane biogenesis when mTOR is active, while mTOR inhibition leads to a shift of lipids from membrane phospholipid to triglyceride [35]. Thus, this lipid-lowering effect of PA might also be mTOR-dependent in razor clam.

In this study, the mRNA levels of key enzymes involved in tricarboxylic acid (TCA) cycle (CS and NADP) were upregulated by PA, which supports the findings in primary muscle cells of turbot that PA promotes TCA cycle or mitochondrial metabolism [16]. In fact, TCA cycle intermediates and ATP are required for macromolecular biosynthesis and cell growth [36], and mTOR inhibition impairs mitochondrial activity and results in a decrease in the amounts of TCA cycle intermediates [37]. Besides pyruvate from the above-mentioned glycolysis, amino acids are also important source of TCA cycle intermediates. In this study, the concentration of several amino acids, including Asp, Glu, Ala, Cys, Val, Met, Ile, Leu, Phe, Arg, and Pro was reduced by di-18 : 2 PA or/and di-16 : 0 PA. In Jurkat cells, Val, Met, Leu, Phe, Arg, and Pro levels are upregulated by rapamycin, a well-known mTOR inhibitor [38]. In proliferating mammalian cells, mTOR promotes glutaminolysis which converts Glu into TCA cycle intermediate, α-ketoglutarate [39, 40]. Taken together, our results raise the possibility that as with glycolysis, PA enhances the utilization of amino acids as TCA cycle intermediates for macromolecular biosynthesis and energy production through mTOR.

In conclusion, PA activated mTOR signaling pathway, and resulted in the increase of anabolism and the inhibition of catabolism in S. constricta. Our results indicate that PA is a potential growth promoting supplement that might be incorporated in artificial diet of S. constricta.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Kai Liao contributed in validation, data curation, and writing-original draft and review. Qian Zhang, Yangyang Qiu, Yuxiang Zhu, Yang Liu, and Hailong Huang contributed in investigation. Deshui Chen, Bin Ma, Lin Zhang, and Zhaoshou Ran contributed in writing—review and editing. Kai Liao, Xiaojun Yan, and Jilin Xu contributed in project administration and funding acquisition. All authors contributed to the article and approved the submitted version.

Acknowledgments

This work received funding from the Ningbo Science and Technology Research Projects, China (2019B10006), the National Key Research and Development Program of China (2019YFD0900400), the Ningbo Public Welfare Science and Technology Plan Project (2022S157), and the earmarked fund for CARS-49. We thank Peng Shi of Ningbo University for his help with RNA extractions.

Supplementary Materials

Figure S1: the dosage and timing effects of phosphatidic acid on the expression of p-4EBP1 in the muscle of S. constricta. (Supplementary Materials)

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Copyright © 2023 Kai Liao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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