Eicosapentaenoic acid (EPA) improves grass carp (Ctenopharyngodon idellus) muscle development and nutritive value by activating the mTOR signaling pathway.

Skeletal muscle is the mainly edible part of fish. Eicosapentaenoic acid (EPA) is a crucial nutrient for fish. This study investigated the effect of EPA on the muscle development of grass carp along with the potential molecular mechanisms in vivo and in vitro. Muscle cells treated with 50 µM EPA in vitro showed the elevated proliferation, and the expression of mammalian target of rapamycin (mTOR) signaling pathway-related genes was upregulated (P < 0.05). In vivo experiments, 270 grass carp (27.92 g) were fed with one of the three experimental diets for 56 days: control diet (CN), 0.3% EPA-supplement diet (EPA), and the diet supplemented with 0.3% EPA and 30 mg/kg rapamycin (EPA + Rap). Fish weight gain rate (WGR) was improved in EPA group (P < 0.05). There was no difference in the viscerosomatic index (VSI) and body height (BH) among all groups (P > 0.05), whereas the carcass ratio (CR) and body length in the EPA group were obviously higher than those of other groups (P < 0.05), indicating that the increase of WGR was due to muscle growth. In addition, both muscle fiber density and muscle crude protein also increased in EPA group (P < 0.05). The principal component analysis showed that total weight of muscle amino acid in EPA group ranked first. Dietary EPA also increased protein levels of the total mTOR, S6k1, Myhc, Myog, and Myod in muscle (P < 0.05). In conclusion, EPA promoted the muscle development and nutritive value via activating the mTOR signaling pathway.

Molecular cloning and characterization of BNIP3 and NIX1/2 and their role in DHA-induced mitophagy and apoptosis in grass carp (Ctenopharyngodon idellus) adipocytes.

B-cell lymphoma-2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and BNIP3 like (BNIP3L or NIX) play a vital role in regulating mitophagy and the intrinsic apoptosis in mammals, but their gene characterizations remain unclear in fish. Herein, bnip3, nix1 and nix2 were isolated and characterized from grass carp (Ctenopharyngodon idellus), which encode peptides of 194, 233 and 222 amino acids, respectively. As typical BH3-only proteins, grass carp BNIP3, NIX1 and NIX2 proteins contain BH3 and C-terminal transmembrane domains for inducing apoptosis. Moreover, the LC3-interacting region motif of BNIP3, NIX1 and NIX2 is also conserved in grass carp. Phylogenetic analyses also demonstrated that nix1 and nix2 may have originated from the genome duplication event. Expression pattern analysis indicated that bnip3, nix1 and nix2 were highest expressed in brain, followed by eye (bnip3) and liver (nix1 and nix2). BNIP3, NIX1 and NIX2 localized to the nucleus and the cytoplasm, with a predominant localization to mitochondria within the cytoplasm. In the present study, we found that 200 µM DHA impaired the mitochondrial function, manifested as the decreased antioxidant ability, cellular ATP content and mitochondrial membrane potential in grass carp adipocytes. In addition, the gene expression and enzyme activities of caspase family were significantly increased in 200 µM DHA group, indicating that adipocyte apoptosis was induced. Meanwhile, DHA increased the gene expression of bnip3, nix1 and nix2 in a dose-dependent manner in grass carp adipocytes. The colocalization of mitochondria and lysosomes was promoted by 200 µM DHA treatment, implying that BNIP3/NIX-related mitophagy was activated in adipocytes. Based on these findings, it can be inferred that BNIP3/NIX-related mitophagy may be involved in the adipocyte apoptosis induced by DHA in grass carp.

DHA induces adipocyte lipolysis through endoplasmic reticulum stress and the cAMP/PKA signaling pathway in grass carp (Ctenopharyngodon idella).

Docosahexaenoic acid (DHA) is a biologically active fatty acid that reduces the accumulation of lipids. However, the molecular mechanism underlying this process, particularly in fish, is not well understood. Recent studies show that endoplasmic reticulum (ER) stress triggers the activation of the unfolded protein response, which has been revealed to play an essential role in lipid metabolism. In this study, we explored the effect of DHA on ER stress and investigated the potential molecular mechanisms underlying DHA-induced adipocyte lipolysis in grass carp (Ctenopharyngodon idella) both in vivo and in vitro. We found that DHA remarkably reduced the triglyceride content, increased the secretion of glycerol, promoted lipolysis in adipocytes and evoked ER stress, whereas inhibiting ER stress using 4-phenyl butyric acid (4-PBA) inhibited the effects of DHA (P < 0.05). These results implied that ER stress potentially participates in DHA-induced adipocyte lipolysis. Additionally, STF-083010, a specific inositol-requiring enzyme 1α (IRE1α)-inhibitor, attenuated the effects of DHA on lipolysis, demonstrating that IRE1α and X-box binding protein 1 potentially participate in DHA-induced lipolysis. DHA also activated the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway by increasing the level of cAMP and activating the PKA enzyme (P < 0.05). Nevertheless, H89, a PKA inhibitor, weakened DHA-induced lipolysis by inhibiting the cAMP/PKA signaling pathway. Furthermore, inhibiting ER stress using 4-PBA also inhibited lipolysis and alleviated DHA-induced activation of the cAMP/PKA signaling pathway, suggesting that ER stress may participate in DHA-induced lipolysis through the activation of the cAMP/PKA signaling pathway. Our data illustrate that DHA supplementation can be a promising nutritional strategy for ameliorating lipid accumulation in grass carp. The present study elucidated the molecular mechanism for DHA-induced lipolysis in grass carp adipocytes and emphasized the importance of ER stress and the cAMP/PKA pathway in DHA-induced lipolysis. These results deepen our understanding of ameliorating lipids deposition in freshwater fish by targeting DHA.

The endoplasmic reticulum stress and B cell lymphoma-2 related ovarian killer participate in docosahexaenoic acid-induced adipocyte apoptosis in grass carp (Ctenopharyngodon idellus).

Docosahexaenoic acid (DHA) lessens adipose tissue lipid deposition partly by inducing adipocyte apoptosis in grass carp, but the underlying mechanism remains unclear. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) is the novel pathway for inducing apoptosis. This study aimed to explore the potential role of ER stress in DHA-induced apoptosis in grass carp (Ctenopharyngodon idellus) adipocytes. DHA induced apoptosis by deforming the nuclear envelope, condensing the chromatin, and increasing the expression of apoptosis-related proteins and genes in vivo and in vitro (P < 0.05). However, the ER stress inhibitor, 4-phenylbutyric acid (4-PBA), effectively suppressed DHA-induced apoptosis (P < 0.05), indicating that ER stress mediates DHA-induced adipocyte apoptosis. Furthermore, we observed that 200 µM DHA significantly up-regulates the transcripts of B cell lymphoma-2 (BCL-2) related ovarian killer (BOK) in vitro (P < 0.05). BOK is a pro-apoptotic protein in the BCL-2 family, which governs the mitochondria apoptosis pathway. Hence, we hypothesized that BOK might be an important linker between ER stress and apoptosis. We cloned and identified two grass carp BOK genes, BOKa and BOKb, which encode peptides of 213 and 216 amino acids, respectively. BOKa primarily localizes in ER and mitochondria in the cytoplasm, while BOKb localizes in the nucleus and cytoplasm of grass carp adipocytes. Moreover, 200 µM DHA treatment up-regulated the mRNA expression of BOKa and BOKb, whereas 4-PBA suppressed the DHA-induced expressions. These results raised the possibility that BOK participates in DHA-induced adipocyte apoptosis through ER stress signaling, in line with its localization in ER and mitochondria. Two UPR branches, the inositol-requiring enzyme 1 (IRE1α) and activating transcription factor 6 (ATF6) signaling pathways, are possibly important in DHA-induced adipocyte apoptosis, unlike protein kinase RNA-activated-like ER kinase. The study also emphasized the roles of BOKa and BOKb in IRE1α- and ATF6-mediated apoptosis. This work is the first to elucidate the importance of the ER stress-BOK pathway during adipocyte apoptosis in teleost.

Cultivated fish accumulates excessive inedible fats in the abdominal cavity, thus wasting energy and causing metabolic disease. This study showed that docosahexaenoic acid (DHA) induces apoptosis in grass carp adipocytes through endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Therefore, DHA possibly alleviates lipid accumulation partly by reducing the number of adipocytes. Furthermore, transcriptome analysis demonstrated that B cell lymphoma-2-related ovarian killer (BOK) is probably an important linker between ER stress and apoptosis. We first cloned and identified the grass carp BOK genes, BOKa and BOKb. Next, we preliminarily confirmed that BOKa and BOKb participate in DHA-induced apoptosis mainly through activating transcription factor 6 and inositol-requiring enzyme 1 α signaling pathways, the branches of UPR. This study provides a theoretical foundation for the lipid-reduction role of DHA in teleost.

PKA/ATGL signaling pathway is involved in ER stress-mediated lipolysis in adipocytes of grass carp (Ctenopharyngodon idella).

The relationship between endoplasmic reticulum stress (ER stress) and lipolysis in mammals has been widely studied, but it is relatively scarce in fish. The present study used grass carp Ctenopharyngodon idella as a model to investigate the effect of ER stress on lipolysis in adipocytes of fish. We found that ER stress evoked by tunicamycin (TM) treatment significantly induced lipolysis in adipocytes. Subsequently, in order to further investigate whether protein kinase A (PKA) is involved in ER stress-induced lipolysis, we treated adipocytes with PKA activator forskolin and inhibitor H89. The results showed that the mechanism was related to the activation of PKA, especially the catalytic subunit PRKACBa. Notably, we also found that PKA regulates lipolysis by targeting mRNA level and protein and enzyme activities of adipotriglyceride lipase (ATGL). Taken together, our findings suggest that PKA/ATGL signaling pathway is involved in ER stress-mediated lipolysis of grass carp adipocytes. It provides a theoretical basis for further study on the mechanism of lipolysis in fish and other vertebrates.

Docosahexaenoic acid lessens hepatic lipid accumulation and inflammation via the AMP-activated protein kinase and endoplasmic reticulum stress signaling pathways in grass carp (Ctenopharyngodon idella).

The liver is the primary organ for frontline immune defense and lipid metabolism. Excessive lipid accumulation in the liver severely affects its metabolic homeostasis and causes metabolic diseases. Docosahexaenoic acid (DHA) is known for its beneficial effects on lipid metabolism and anti-inflammation, but its molecular mechanism remains unknown, especially in fish. In this study, we evaluated the protective effects of DHA on hepatic steatosis of grass carp (Ctenopharyngodon idella) in vivo and in vitro and mainly focused on the AMP-activated protein kinase (AMPK) and endoplasmic reticulum stress (ER stress) signaling pathway analysis. Grass carp were fed with purified diets supplemented with 0%, 0.5% and 1% DHA for 8 weeks in vivo. 1% DHA supplementation significantly decreased the liver triglyceride (TG), malondialdehyde (MDA), serum tumor necrosis factor α (TNFα) and nuclear factor kappa B (NFκB) contents. DHA administration suppressed ER stress and decreased the mRNA expressions related to hepatic inflammation and lipogenesis, accompanied by the activation of AMPK. Correspondingly, DHA activated the AMPK signaling pathway, and inhibited palmitic acid (PA)-evoked ER stress and lipid accumulation and inflammation of grass carp hepatocytes in vitro. In contrast, the inhibitor of AMPK (compound C, CC) abrogated the effects of DHA to improve PA-induced liver injury and ER stress. In conclusion, DHA inhibits ER stress in hepatocytes by the activation of AMPK and exerts protective effects on hepatic steatosis in terms of improving antioxidant ability, relieving hepatic inflammation and inhibiting hepatic lipogenesis. Our findings give a theoretical foundation for further elucidation of the beneficial role of DHA in vertebrates.

Endoplasmic reticulum stress is involved in lipid accumulation induced by oleic acid in adipocytes of grass carp (Ctenopharyngodon idella): focusing on the transcriptional level.

It has been extensively claimed that endoplasmic reticulum stress (ER stress) is related to lipid accumulation in mammals, but little is known in fish. This study aims at elucidating the role of ER stress in mediating lipid accumulation induced by monounsaturated oleic acid (OA) with a focus on the transcriptional level. We treated the adipocytes of grass carp with 200 µM and 400 µM OA, respectively, while the control group was treated with 2% bovine serum albumin (BSA). The results showed that cell viability was significantly improved, while 400 µM OA treatment promoted neutral lipid accumulation along with stimulating ER stress more obviously. Although lipolysis and fatty acid ß-oxidation were activated simultaneously, the primary effect of OA seems to be promotion of lipid accumulation. To further explore whether ER stress affects lipid accumulation, 4-phenyl butyric acid (4-PBA), an effective inhibitor of ER stress, was used to pretreat the cells for 4 h. Unsurprisingly, it was found that the mRNA expressions of genes linked with ER stress were decreased. Intracellular triglyceride (TG) content was also decreased, which was in accordance with the mRNA expressions of adipogenic and lipogenic transcription factors as well as their target genes. Collectively, our data shows that ER stress may take part in OA-induced lipid accumulation in adipocytes via activating adipogenesis and lipogenesis. Based on this, strategies for protecting ER could be used to alleviate excessive accumulation of lipid in grass carp adipose tissue.

Perilipin 1-3 in grass carp Ctenopharyngodon idella: molecular characterization, gene structure, tissue distribution, and mRNA expression in DHA-induced lipid droplet formation in adipocytes.

Perilipin family is the main structural proteins of lipid droplet (LD) that is intracellular neutral lipid store ponds, and regulates LD assembly and formation, and is crucial for lipid metabolism. Here three paralogs of perilipin family were characterized from grass carp and their complete coding sequences (CDS) were obtained, including perilipin1, perilipin2, and perilipin3, coding peptides of 492, 454, and 419 amino acids, respectively. The alignment of the homology of grass carp perilipin deduced amino acid sequences with other teleost species showed that the homology with mammalian was less than 55%. PAT (perilipin) domain in mammalian was also predicted in grass carp perilipin 1-3 proteins. Genomic organization analysis revealed that grass carp perilipin1 contained 6 coding exons, while both perilipin2 and perilipin3 consisted of 7 coding exons. The mRNA encoding three paralogs were expressed in a wide range of tissues; perilipin1-3 were primarily expressed in adipose tissue and liver; besides, perilipin3 was also highly expressed in the heart. In vitro, 200 µM DHA increased the proportion of smaller lipid droplets effectively in fully differentiated adipocytes of grass carp. The mRNA expression of perilipin1, perilipin2, and perilipin3 was significantly increased in the adipocytes treated with DHA (P < 0.05, P < 0.01). The same responses of different paralogs in the adipocytes during DHA treatment suggest that they might play synergistic roles in the formation of LDs.

cAMP-dependent protein kinase A in grass carp Ctenopharyngodon idella: Molecular characterization, gene structure, tissue distribution and mRNA expression in endoplasmic reticulum stress-induced adipocyte lipolysis.

Protein kinase A (PKA), one of the most widely studied protein kinases, has many functions in cells, including regulating the metabolism of sugar and lipid. Here we identified nine isoforms of the PKA family in grass carp Ctenopharyngodon idella and obtained their complete coding sequences (CDS), including PRKACAa, PRKACAb, PRKACBa, PRKACBb, PRKAR1A, PRKAR1B, PRKAR2Aa, PRKAR2Ab and PRKAR2B, and PRKACA, PRKACB and PRKAR2A, which may experience fish-specific genome duplication. Sequence analysis showed that the predicted protein structures of PKA gene family members in grass carp were different. Grass carp PRKACAa, PRKACAb, PRKACBa, and PRKACBb contained serine/threonine protein kinases, while PRKAR1A, PRKAR1B, PRKAR2Aa, PRKAR2Ab and PRKAR2B contained two cyclic nucleotide-monophosphate binding domains. PRKACAa, PRKACBa, PRKACBb, PRKAR1A, PRKAR1B and PRKAR2Aa contained 10 coding exons, while PRKACAb and PRKAR2Ab consisted of 12 coding exons and 5 coding exons, respectively. The messenger RNA (mRNA) of the nine PKA isoforms was detected in a wide range of tissues, but their abundance showed tissue-dependent expression patterns. In tunicamycin-induced adipocyte lipolysis, only the mRNA levels of PRKACAb and PRKACBa showed a significant increase in adipocyte (p < .05), indicating that nine PKA isoforms may serve somewhat different roles in endoplasmic reticulum (ER) stress-mediated lipolysis in fish. These results suggested that nine grass carp PKA isoforms may play different roles in tissues, and their expression levels were differently modulated by ER stress in adipocyte.

Nuclear factor-κB subunit p65 is involved in lipopolysaccharide-induced lipid accumulation via regulating DGAT1b in Ctenopharyngodon idellus kidney cells.

Lipopolysaccharide (LPS) can promote the accumulation of triglycerides (TGs) in CIK (Ctenopharyngodon idellus kidney) cells, but the underlying mechanism is unclear. In this study, two genes involved TG synthesis, DGAT1a and DGAT1b, were isolated and characterized from grass carp Ctenopharyngodon idella, which encode peptides of 498 and 501 amino acids, respectively. Phylogenetic and synteny analyses indicated that DGAT1a and DGAT1b could have originated from the teleost-specific genome duplication event. Analysis of the exon-intron structures clarified that genomic structures of all DGAT1 proteins are conserved in vertebrates. DGAT1a mRNA was highly expressed in gut, adipose tissue and heart, while DGAT1b mRNA was highly expressed in liver and kidney. After LPS treatment, only expression of DGAT1b was up-regulated and knockdown of DGAT1b reduced the content of TG, suggesting that DGAT1b is involved in LPS-induced lipid accumulation. To explore the mechanism underlying the transcriptional regulation of DGAT1b in response to LPS, we cloned DGAT1b promoter sequence. Its promoter sequence consists of IRF7, RelA (p65) and RelB binding elements. Dual luciferase assay and q-PCR suggested that the promoter of DGAT1b can be activated by the overexpression of p65, but cannot be triggered by IRF7 and RelB. Mutational analysis shows that the potential p65 binding sites may locate in the region -111/-100 bp of the DGAT1b promoter. These results indicated that DGAT1b is the target gene of NF-κB p65. Finally, inhibiting p65 effectively decreased LPS-induced lipid accumulation. Taken together, we demonstrate that NF-κB p65 takes part in the lipid accumulation by regulating DGAT1b-induced TG synthesis in LPS signalling in CIK cells. The finding that NF-κB p65 links LPS signalling and TG synthesis adds to our growing appreciation of the interplay between immunity and lipid metabolism in fish.

Glycogen synthase kinase-3ß (GSK-3ß) of grass carp (Ctenopharyngodon idella): Synteny, structure, tissue distribution and expression in oleic acid (OA)-induced adipocytes and hepatocytes.

Glycogen synthase kinase-3ß (GSK-3ß) plays a crucial role in regulating lipid metabolism, endoplasmic reticulum (ER) stress and apoptosis in mammals, however, its gene structure and function is little known about in fish. Here, its two paralogs, grass carp (Ctenopharyngodon idella) GSK-3ß1 and GSK-3ß2 were isolated and characterized, encoding peptides of 421 and 457 amino acids, respectively. These two paralogs may have originated from the teleost-specific genome duplication (TSGD) event. Alignment of grass carp GSK-3ß deduced amino acid sequences with select teleost species showed that the protein is conserved. However, two paralogs of the GSK-3ß had great variation in gene structure: the GSK-3ß1 contained eleven exons while the GSK-3ß2 contained nine exons. The two paralogs were expressed in a wide range of tissues, GSK-3ß1 was most expressed in adipose tissue, GSK-3ß2 was most expressed in liver. In OA-induced adipocytes and hepatocytes, we found that GSK-3ß1 mRNA expression was significantly increased only in adipocytes, while the mRNA expression of GSK-3ß2 was dramatically increased both in adipocytes and hepatocytes. These results provide evidence that the GSK-3ß may participate in the process of lipid accumulation in OA-induced adipocytes and hepatocytes of grass carp.

Greater potency of adipocytes compared with preadipocytes under lipopolysaccharide exposure in grass carp Ctenopharyngodon idella.

Excessive body fat is a chronic inflammatory disorder. In this process, white adipose tissue (WAT) performs immune activities because of the dysregulated expression of adipokines. Excessive fat is accumulated in farmed fish, thereby threatening fish health. Studies have shown that adipose tissue is also an active immune organ in fish, capable of participating in and influencing immune responses. Adipocytes are the main cellular component of adipose tissue; however, little is known about the relationship between adipocyte and inflammation in fish. In this study, we analyzed transcriptome changes during adipogenesis in the primary culture of grass carp adipocytes using bioinformatics. The results showed that inflammatory signaling pathway may be activated during grass carp adipocyte differentiation, such as NFκB signaling pathway, Toll-like receptor signaling pathway and Adipocytokine signaling pathway, indicating that grass carp adipocytes have immune activities. Exposure to LPS induced expression of adipokines genes in adipocytes and preadipocytes, including NF-kB, IL-6, MCP-1 and TNFα, suggesting that preadipocytes and adipocytes both have immune response and the immune activity is conserved in vertebrates white adipocytes. Further study found that these immune marker genes were higher expressed in adipocytes compared with preadipocytes in LPS-induced inflammation. In summary, adipocyte should be considered as an active immune site in fish. Adipocytes have greater potency compared with preadipocytes in LPS-induced inflammation. This study indicated that adipocytes and preadipocytes may have different contribution in inflammation.

Two faces of PPARα/NFκB signaling pathway in inflammatory responses to adipocytes lipolysis in grass carp Ctenopharyngodon idella.

Adipose tissue plays an important role in energy reservation, also be considered as vital immunological organ in animals. Adipocytes are the basic unit of adipose tissue, while little is known about the relationship between lipid metabolism and inflammatory response in fish adipocytes so far. In this study, forskolin was used to induce adipocyte lipolysis, and 5 µM forskolin and 30 µM forskolin both triggered lipolysis by increasing ATGL expression. Consequently, 30 µM Forskolin instead of 5 µM Forskolin induced the expression of NF-κB and its target pro-inflammatory cytokine genes including MCP-1, IL-6 and TNF-α. Further study found that low grade rate of lipolysis activated PPARα gene, and its inhibitory effect on the mRNA expression of NF-κB and its target genes inhibited the adipocyte inflammation. On the contrary, high grade rate of lipolysis increased the expression levels of NF-κB and its target genes, while their expression were attenuated by inhibition of reactive oxygen species (ROS) using α-tocopherol, suggesting that ROS generated due to the PPARα-mediated oxidation of released fatty acids from lipolysis may contribute to adipocyte inflammation. These results indicated that PPARα has dose effect in inflammatory responses to adipocyte lipolysis in grass carp. Taken together, grass carp adipocytes have immune activity. The inflammatory response is linked to the grade rate of adipocyte lipolysis in grass carp adipocytes, and excessive adipocyte lipolysis may promote a dynamic immune response in adipose tissue. This is the first study showing the regulatory effects of lipolysis on immune functions in fish adipocytes.