Regulation of low-density lipoprotein on lipid metabolism in macrophages of large yellow croaker (Larimichthys crocea).

Low-density lipoprotein (LDL) is the main carrier of cholesterol transport in plasma, which participates in regulating lipid homeostasis. Studies in mammals have shown that high levels of LDL in plasma absorbed by macrophages trigger the formation of lipid-rich foam cells, leading to the development of atherosclerotic plaques. Although lipid-rich atherosclerosis-like lesions have been discovered in the aorta of several fish species, the physiological function of LDL in fish macrophages remains poorly understood. In the present study, LDL was isolated from the plasma of large yellow croaker (Larimichthys crocea), and mass spectrometry analysis identified two truncated forms of apolipoprotein B100 in the LDL protein profile. Transcriptomic analysis of LDL-stimulated macrophages revealed that differentially expressed genes (DEGs) were enriched in various pathways related to lipid metabolism, as confirmed by the fact that LDL increased total cholesterol and cholesteryl esters content. Meanwhile, the gene and protein expression levels of perilipin2 (PLIN2), a DEG enriched in the PPAR signaling pathway, were upregulated in response to LDL stimulation. Importantly, knocking down plin2 significantly attenuates LDL-induced cholesterol accumulation and promotes cholesterol efflux. Furthermore, the transcription factor PPARγ, which is upregulated in response to LDL stimulation, can enhance the promoter activity of plin2. In conclusion, this study suggests that LDL may upregulate plin2 expression through PPARγ, resulting in cholesterol accumulation in fish macrophages. This study will facilitate the investigation of the function of LDL in regulating lipid homeostasis in macrophages and shed light on the evolutionary origin of LDL metabolism in vertebrates.

Phosphatidylethanolamine alleviates OX-LDL-induced macrophage inflammation by upregulating autophagy and inhibiting NLRP1 inflammasome activation.

Oxidized low-density lipoprotein (OX-LDL)-induced inflammation and autophagy dysregulation are important events in the progression of atherosclerosis. Phosphatidylethanolamine (PE), a multifunctional phospholipid that is enriched in cells, has been proven to be directly involved in autophagy which is closely associated with inflammation. However, whether PE can influence OX-LDL-induced autophagy dysregulation and inflammation has not been reported. In the present study, we revealed that OX-LDL significantly induced macrophage inflammation through the CD36-NLRP1-caspase-1 signaling pathway in fish. Meanwhile, cellular PE levels were significantly decreased in response to OX-LDL induction. Based on the relationship between PE and autophagy, we then examined the effect of PE supplementation on OX-LDL-mediated autophagy impairment and inflammation induction in macrophages. As expected, exogenous PE restored impaired autophagy and alleviated inflammation in OX-LDL-stimulated cells. Notably, autophagy inhibitors reversed the inhibitory effect of PE on OX-LDL-induced maturation of IL-1ß, indicating that the regulation of PE on OX-LDL-induced inflammation is dependent on autophagy. Furthermore, the positive effect of PE on OX-LDL-induced inflammation was relatively conserved in mouse and fish macrophages. In conclusion, we elucidated the role of the CD36-NLRP1-caspase-1 signaling pathway in OX-LDL-induced inflammation in fish and revealed for the first time that altering PE abundance in OX-LDL-treated cells could alleviate inflammasome-mediated inflammation by inducing autophagy. Given the relationship between OX-LDL-induced inflammation and atherosclerosis, this study prompts that the use of PE-rich foods promises to be a new strategy for atherosclerosis treatment in vertebrates.

Transcription factor EB (TFEB) participates in antiviral immune responses independent of mTORC1 in macrophage of large yellow croaker (Larimichthys crocea).

Transcription factor EB (TFEB) plays an integral role in the production of proinflammatory cytokines and chemokines in response to pathogen stimulation in mammals. However, the role of TFEB in antiviral immune responses and the potential regulatory mechanisms in fish remain poorly understood. Here, we cloned and characterized Larimichthys crocea TFEB (LcTFEB) with 524 amino acids and a typical basic helix-loop-helix-leucine zipper domain. LcTFEB could translocate into the nucleus upon starvation and had a comparatively high expression in immune tissues. Similar to the expression of antiviral immune genes, the transcriptional expression and activity of LcTFEB showed a trend of increasing and then decreasing with the prolongation of stimulation. Inhibition of LcTFEB using siRNA dramatically increased the polyinosinic-polycytidylic acid (poly (I:C))-induced interferon response and pro-inflammatory cytokines mRNA expression levels, whereas pharmacological activation and overexpression of LcTFEB exhibited the reverse effects. Mechanically, LcTFEB might promote the expression of IFNh as negative feedback to limit the virus-induced inflammatory responses. Notably, although inhibition of mTORC1 exacerbated poly (I:C)-triggered inflammatory responses, the effects of LcTFEB were independent of mTORC1. Overall, this study revealed an unidentified critical role of LcTFEB in the regulation of antiviral immune responses and promoted the understanding of TFEB in the antiviral immunity of fish macrophages.

Palmitic acid induces intestinal lipid metabolism disorder, endoplasmic reticulum stress and inflammation by affecting phosphatidylethanolamine content in large yellow croaker Larimichthys crocea.

In the 21st century, intestinal homeostatic imbalance has emerged as a growing health challenge worldwide. Accumulating evidence reveals that excessive intake of saturated fatty acid (SFA) induces intestinal homeostatic imbalance. However, the potential molecular mechanism is still unclear. In the present study, we found that palm oil or palmitic acid (PA) treatment disturbed lipid metabolism homeostasis and triggered endoplasmic reticulum (ER) stress and inflammation in the intestine or intestinal cells of large yellow croaker (Larimichthys crocea). Interestingly, PA treatment significantly decreased phosphatidylethanolamine (PE) content in the intestinal cells. PE supplementation decreased triglyceride content in the intestinal cells induced by PA treatment by inhibiting fatty acid uptake and lipogenesis. PE supplementation suppressed ER stress. Meanwhile, PE supplementation alleviated inflammatory response through p38 MAPK-p65 pathway, reducing the damage of intestinal cells caused by PA treatment to some extent. Our work revealed that intestinal homeostatic imbalance caused by PA treatment was partly due to the decrease of PE content. PE consumption might be a nutritional strategy to regulate intestinal homeostasis in fish and even human beings.

Role of acyl-coenzyme A oxidase 1 (ACOX1) on palmitate-induced inflammation and ROS production of macrophages in large yellow croaker (Larimichthys crocea).

Acyl-coenzyme A oxidase 1 (ACOX1) is the rate-limiting enzyme in peroxisomal ß-oxidation, and it plays an essential role in mediating the inflammatory response and reactive oxygen species (ROS) metabolism in mammals. However, the role of ACOX1 in fish has not been completely elucidated. Herein, this study was conducted to investigate the role of large yellow croaker (Larimichthys crocea) ACOX1 (Lc-ACOX1) on palmitate (PA)-induced inflammation and ROS production. In this study, Lc-ACOX1 was cloned and characterized. The full-length CDS of Lc-acox1 was 1986 bp, encoding 661 amino acids. Tissue distribution results showed that the gene expression of Lc-acox1 was the highest in the intestine and the lowest in the spleen. Moreover, results showed that the mRNA expression of Lc-acox1 was upregulated by PA, with elevated pro-inflammatory gene expression, including il-1ß, il-6, il-8, tnf-α, cox2 and ifn-γ, as well as ROS content in macrophages of large yellow croaker. Furthermore, the role of Lc-ACOX1 in inflammation induced by PA was investigated by using the ACOX1 inhibitor TDYA. Treatment of macrophages with TDYA reduced the mRNA expression of pro-inflammatory genes induced by PA. Moreover, inhibition of ACOX1 reduced the elevated level of ROS caused by PA and increased the mRNA expression of antioxidant genes. In conclusion, this study first identified that fish ACOX1 was involved in the PA-induced inflammatory response and ROS production.

Docosahexaenoic Acid Ameliorates the Toll-Like Receptor 22-Triggered Inflammation in Fish by Disrupting Lipid Raft Formation.

BACKGROUND: Although dietary DHA alleviates Toll-like receptor (TLR)-associated chronic inflammation in fish, the underlying mechanism is not well understood. OBJECTIVES: This study aimed to explore the role of Tlr22 in the innate immunity of large yellow croaker and investigate the anti-inflammatory effects of DHA on Tlr22-triggered inflammation. METHODS: Head kidney-derived macrophages of croaker and HEK293T cells were or were not pretreated with 100 µM DHA for 10 h prior to polyinosinic-polycytidylic acid (poly I:C) stimulation. We executed qRT-PCR, immunoblotting, and lipidomic analysis to examine the impact of DHA on Tlr22-triggered inflammation and membrane lipid composition. In vivo, croakers (12.03 ± 0.05 g) were fed diets containing 0.2% [control (Ctrl)], 0.8%, and 1.6% DHA for 8 wk before injection with poly I:C. Inflammatory genes expression and rafts-related lipids and protein expression were measured in the head kidney. Data were analyzed by ANOVA or Student t test. RESULTS: The activation of Tlr22 by poly I:C induced inflammation, and DHA diminished Tlr22-targeted inflammatory gene expression by 56-73% (P ≤ 0.05). DHA reduced membrane sphingomyelin (SM) and SFA-containing phosphatidylcholine (SFA-PC) contents, as well as lipid raft marker caveolin 1 amounts. Furthermore, lipid raft disruption suppressed Tlr22-induced Nf-κb and interferon h activation and p65 nuclear translocation. In vivo, expression of Tlr22 target inflammatory genes was 32-64% lower in the 1.6% DHA group than in the Ctrl group upon poly I:C injection (P ≤ 0.05). Also, the 1.6% DHA group showed a reduction in membrane SM and SFA-PC contents, accompanied by a decrease in caveolin 1 amounts, compared with the Ctrl group. CONCLUSIONS: The activation of Tlr22 signaling depends on lipid rafts, and DHA ameliorates the Tlr22-triggered inflammation in both head kidney and head kidney-derived macrophages of croaker partially by altering membrane SMs and SFA-PCs that are required for lipid raft organization.

High Fat Activates O-GlcNAcylation and Affects AMPK/ACC Pathway to Regulate Lipid Metabolism.

A high-fat diet often leads to excessive fat deposition and adversely affects the organism. However, the mechanism of liver fat deposition induced by high fat is still unclear. Therefore, this study aimed at acetyl-CoA carboxylase (ACC) to explore the mechanism of excessive liver deposition induced by high fat. In the present study, the ORF of ACC1 and ACC2 were cloned and characterized. Meanwhile, the mRNA and protein of ACC1 and ACC2 were increased in liver fed with a high-fat diet (HFD) or in hepatocytes incubated with oleic acid (OA). The phosphorylation of ACC was also decreased in hepatocytes incubated with OA. Moreover, AICAR dramatically improved the phosphorylation of ACC, and OA significantly inhibited the phosphorylation of the AMPK/ACC pathway. Further experiments showed that OA increased global O-GlcNAcylation and agonist of O-GlcNAcylation significantly inhibited the phosphorylation of AMPK and ACC. Importantly, the disorder of lipid metabolism caused by HFD or OA could be rescued by treating CP-640186, the dual inhibitor of ACC1 and ACC2. These observations suggested that high fat may activate O-GlcNAcylation and affect the AMPK/ACC pathway to regulate lipid synthesis, and also emphasized the importance of the role of ACC in lipid homeostasis.

Early Life Intervention Using Probiotic Clostridium butyricum Improves Intestinal Development, Immune Response, and Gut Microbiota in Large Yellow Croaker (Larimichthys crocea) Larvae.

Marine fish larvae are vulnerable during the early life period. The early intervention using probiotics may be a promising method to improve growth of fish larvae. In this study, a 30-day feeding trial was conducted to evaluate the effects of early life intervention using probiotic Clostridium butyricum (CB) on growth performance, intestinal development, immune response and gut microbiota of large yellow croaker (Larimichthys crocea) larvae. Four isonitrogenous and isolipidic diets were formulated with the supplementation of four different levels of CB (5 × 109 CFU g-1), 0.00% (Control), 0.10% (CB1), 0.20% (CB2), and 0.40% (CB3). Results showed that larvae fed diets with CB had significant higher final length than the control group. Meanwhile, larvae fed the diet with 0.10% CB had significant higher final weight and specific growth rate (SGR) than the control group. However, no significant difference in survival rate was observed among dietary treatments. CB supplementation significantly increased the height of intestinal villus and the length of intestinal enterocyte. Similarly, CB supplementation significantly increased the expression of tight zonula occludens-2 (zo-2) and ornithine decarboxylase (odc) than the control group. Larvae fed the diet with 0.20% CB had significant higher lipase and leucine-aminopeptidase (LAP) activity than the control group. Moreover, CB supplementation significantly improved immune enzyme activities than the control group. Sequencing of bacterial 16S rRNA V4-5 region indicated that dietary CB altered intestinal microbiota profile and decreased intestinal microbial diversities of larvae. CB supplementation could effectively increase the abundance of CB, and decrease the abundance of some potential pathogenic bacteria in larval gut. These results revealed that early life intervention using 0.10-0.20% CB could promote growth of large yellow croaker larvae probably through promoting intestinal development, improving immune enzyme activities and modulating gut microbiota.

Molecular cloning and the involvement of IRE1α-XBP1s signaling pathway in palmitic acid induced - Inflammation in primary hepatocytes from large yellow croaker (Larimichthys crocea).

Apart from mitigating endoplasmic reticulum (ER) stress, vast studies have demonstrated the crucial role of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α) - spliced X-box binding protein 1 (XBP1s) signaling pathway in inflammatory response in mammals. In addition, palmitic acid (PA)-induced inflammation has been verified in large yellow croaker (Larimichthys crocea). However, whether the IRE1α-XBP1s signaling pathway is involved in inflammatory response caused by PA remains poorly studied in fish. The present study was aimed at elucidating the role of the IRE1α-XBP1s signaling pathway in inflammatory response induced by PA in primary hepatocytes from large yellow croaker. In the present study, the full-length cDNA of ire1α and xbp1s were cloned and comprised 3793 bp and 1789 bp with an open reading frame of 3279 bp and 1170 bp, encoding 1093 and 390 amino acids, respectively. IRE1α protein possessed a protein kinase and endoribonuclease domain and XBP1s protein possessed a basic-leucine zipper domain. The IRE1α protein and XBP1s protein located to the ER membrane and nucleus respectively. The ire1α and xbp1s were widely transcribed in various tissues with the higher level in intestine, liver, adipose and head kidney. The ER stress-inducing agent tunicamycin (Tm) and PA treatment significantly activated the IRE1α-XBP1s signaling pathway and increased the pro-inflammatory genes expression including tumor necrosis factor α (tnfα), interleukin 6 (il-6) and interleukin 1ß (il-1ß) (P < 0.05). When KIRA6, the IRE1α kinase inhibitor, was used to block the IRE1α-XBP1s signaling pathway, the Tm and PA-induced pro-inflammatory genes expression was significantly suppressed (P < 0.05). These data indicated that the IRE1α-XBP1s signaling pathway was involved in the PA-induced inflammatory response in large yellow croaker.