Lipid droplets participate in modulating innate immune genes in Ctenopharyngodon idella kidney cells.

Lipid droplets (LDs) are increasingly being recognized as important immune modulators in mammals, in additional to their function of lipid ester deposition. However, the role of LDs in fish immunity remains poorly understood. In this study, the function of LDs in the innate immune response of Ctenopharyngodon idella kidney (CIK) cells, which are the equivalent of myeloid cells in vertebrates, was investigated. LD number and TG content significantly increased in the CIK cells following exposure to lipopolysaccharide (LPS), peptidoglycan (PGN), and polyriboinosinic-polyribocytidylic acid (Poly [I: C]) for 24 h, accompanied by increases in the relative expression of several innate immune genes. However, fatty acid compositions of the triglycerides were not changed after treatment with these three pathogenic mimics. LPS, PGN, and Poly (I: C) did not alter the relative expressions of lipogenic (FAS, SCD, and DGAT) and lipid catabolic (PPARα, ATGL, and CPT-1) genes. However, these treatments did increase the mRNA levels of lipid transportation genes (FATP/CD36, ACSL1, and ACSL4), and also decreased the non-esterified fatty acid level in the medium. To further explore the role of LDs in the immune response, CIK cells were incubated with different concentrations (0, 100, 200, 300, 400, 500 µM) of exogenous lipid mix (LM; oleic acid [OA]:linoleic acid [LA]:linolenic acid [LNA] = 2:1:1), and were then transferred to a lipid-free medium and incubated for 24 h. LD size and number increased with the increase in lipid levels, and this was accompanied by increased expression of innate immune genes, including MyD88, IRF3, and IL-1ß, which were expressed at their highest levels in 300 µM exogenous lipid mix. Interestingly, after incubating with different fatty acids (LM, OA, LA, LNA, arachidonic acid [ARA], and docosahexaenoic acid [DHA]; 300 µM), ARA and DHA were more potent in inducing LD formation and innate immune gene expression in the CIK cells. Finally, atglistatin, an ATGL inhibitor, effectively attenuated the expression of most genes upregulated by ARA or DHA, suggesting that lipolysis may be involved in the regulation of immune genes at the transcriptional level. Overall, the findings of this study demonstrate that LDs are functional organelles that could act as modulators in the innate immune response of CIK cells. Additionally, long-chain polyunsaturated fatty acid enriched LDs play a unique role in regulating this process.

Lipid accumulation in grass carp (Ctenopharyngodon idellus) fed faba beans (Vicia faba L.).

Feeding faba beans (Vicia faba L.) to grass carp (Ctenopharyngodon idellus) increases muscle compactness but decreases growth and motility. The lipid metabolism of grass carp was examined to assess potential effects of feeding faba beans on physiological properties using a total of 180 fish. The treatment group was fed faba beans for 120 days and a commercial diet for another 30 days. The control group received a commercial diet for 150 days. Fish were sampled every month. Weight gain was significantly lower in the treatment group than in the control. Hardness, springiness, chewiness, cohesiveness, and gumminess of the dorsal muscle increased significantly with the feeding faba beans from 30 to 120 days, which was not reversed by the subsequent feeding of commercial diet. Fat accumulation increased significantly in the treatment group as suggested by the condition factor, viscera index, hepatopancreatic index, and intraperitoneal fat index (IPFI), hepatopancreas, and muscle fat content but was not affected by subsequent feeding with the commercial diet. Serum triglyceride and total cholesterol levels were significantly reduced in the experimental diet group. In the hepatopancreas and intraperitoneal fat IPF, monounsaturated fatty acids showed significantly higher content in faba bean feeding fish, whereas polyunsaturated fatty acid content showed the reversed pattern. In the hepatopancreas, the activities of the lipogenic enzymes malate dehydrogenase and glucose 6-phosphate dehydrogenase were higher in the treatment than in the control group. Moreover, the treatment group showed lower mRNA levels of carnitine palmitoyltransferase-1. Overall, our results clearly demonstrate increasing lipid accumulation in the viscera of faba bean-fed grass carp.

Feeding Faba Beans (Vicia faba L.) Reduces Myocyte Metabolic Activity in Grass Carp (Ctenopharyngodon idellus).

In this study, we aimed to explore the effects of faba bean (Vicia faba L.) on the energy metabolism of grass carp (Ctenopharyngodon idellus). A total of 180 fish (∼2900 g) were randomly assigned to six tanks (2.5 × 2.5 × 1.2 m; 30 individuals per tank) and fed either faba bean (Vicia faba L.) or a commercial diet for 120 days (3% body weight, twice per day). The results showed that faba bean-fed grass carp (FBFG) had significantly lower growth and higher fat accumulation in the mesenteric adipose tissue and hepatopancreas than commercial diet-fed grass carp (CDFG). Compared with CDFG, FBFG exhibited no significant difference in proximate composition of the muscle; however, an obvious decrease in muscle fiber size and significantly higher hardness, chewiness, and gumminess were observed. Transcriptome results showed that a total of 197 genes were differentially regulated in the dorsal muscle. Down-regulated genes included four genes annotated with myocyte development and 12 transcripts annotated with components of myofibrils. In addition, the FBFG group exhibited significantly lower expression of genes associated with oxygen transport, the mitochondrial respiratory chain, and creatine metabolism, suggesting reduced energy availability in the muscle of the FBFG. Moreover, using western-blotting and enzyme assays, we found decreased protein levels in the mitochondrial electron transport respiratory chain and creatine metabolism activities, as well as increased expression of autophagy marker protein levels, in the muscle of FBFG. Overall, our results suggest that an abnormal energy distribution may exist in grass carps after feeding with faba bean, which is reflected by a mass of fat deposition in the adipose tissue and hepatopancreas and subdued metabolic activity in the muscle.

Smad4-dependent regulation of type I collagen expression in the muscle of grass carp fed with faba bean.

Smad4 is the key regulator in the transforming growth factor ß1 (TGF-ß1)/Smads signal pathway, and is also the crux of the regulation of type I collagen expression in mammals. In fish, however, the relationship between Smad4 and type I collagen is still unknown. Given the widely accepted importance of type I collagen in fish muscle hardness, we seek to explore this issue by analyzing the expressions of the TGF-ß1/Smads pathway molecules and type I collagen in the muscle of crisp grass carp fed with faba bean, which shows increased muscle hardness. The study found that (1) in the process of feeding the grass carp with faba bean, the mRNA and protein expressions of TGF-ß1, Smad2 and Smad4 all increased along with the increase of type I collagen expression (Col1α1 and Col1α2); (2) one day after the injection of Smad4 over-expression vector, both mRNA and protein expressions of Col1α1 and Col1α2 significantly increased, reaching the maximum on the 2nd and 5th day, respectively; (3) one day after the injection of Smad4 RNAi interference vector, the mRNA and protein expressions of Col1α1 and Col1α2 decreased, reaching the minimum on the 5th day. These results revealed that Smad4 is the major regulator of type I collagen in the muscle of grass carp fed with faba bean. This study would provide an important mechanistic basis for nutritional regulation of type I collagen in the muscle of fish.

Proteomic signature of muscle fibre hyperplasia in response to faba bean intake in grass carp.

Fish muscle growth is important for the rapidly developing global aquaculture industry, particularly with respect to production and quality. Changes in muscle fibre size are accomplished by altering the balance between protein synthesis and proteolysis. However, our understanding regarding the effects of different protein sources on fish muscle proteins is still limited. Here we report on the proteomic profile of muscle fibre hyperplasia in grass carp fed only with whole faba bean. From the results, a total of 99 significantly changed proteins after muscle hyperplasia increase were identified (p < 0.05, ratio <0.5 or >2). Protein-protein interaction analysis demonstrated the presence of a network containing 56 differentially expressed proteins, and muscle fibre hyperplasia was closely related to a protein-protein network of 12 muscle component proteins. Muscle fibre hyperplasia was also accompanied by decreased abundance in the fatty acid degradation and calcium signalling pathways. In addition, metabolism via the pentose phosphate pathway decreased in grass carp after ingestion of faba bean, leading to haemolysis. These findings could provide a reference for the prevention and treatment of human glucose-6-phosphate dehydrogenase deficiency ("favism").