Molecular characterization of ER Stress sensor IRE1α and IRE1β and their expression analysis in response to different fatty acid in grass carp (Ctenopharyngodon idellus)

2021 ◽  
Author(s):  
Zhiguang Chang ◽  
Minghui Yang ◽  
Hong Ji
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Er Stress ◽  
Grass Carp ◽  
Transmembrane Helix ◽  
Specific Inhibitor ◽  
Ctenopharyngodon Idellus ◽  
Cik Cells ◽  
Physiological Processes ◽  
Evolutionarily Conserved

Abstract Inositol-requiring enzyme 1 (IRE1) is the most evolutionarily conserved endoplasmic reticulum (ER) membrane protein. Here, we identified and analyzed two IRE isoforms (IRE1α and IRE1β) in grass carp ( Ctenopharyngodon idellus ). The coding sequences of IRE1α and IRE1β were 3096 and 2835 nucleotides, and they encoded proteins of 1031 and 944 amino acids, respectively. Each of the two IRE1s proteins had four PPQ domains, a transmembrane helix region, an S_TKc and a PUG domain, which were relatively conserved in comparison to mammals. qRT-PCR revealed that IRE1α was the highest in liver and brain, whereas the highest expression of IRE1β expressed was found in the hindgut, which may be closely related with their role. Moreover, we have compared the effect of saturated (palmitic acid, PA) , monounsaturated fatty acid (oleic acid, OA) and polyunsaturated fatty acids (Docosahexaenoic acid, DHA) on ER stress in CIK cells, and found that PA and DHA induced UPR-related gene expression in dose-dependent , and OA increased expression of IRE1α and decreased expression of IRE1β, ATF4 and ATF6 at lower doses. Additionally, our studies revealed that blocking IRE1α pathway using specific inhibitor 4μ8c, subsequently reversed PA-induced autophagy and inflammation, indicating the role of IRE1α in mediating PA-induced CIK cells. Taken together, the study demonstrated the IRE1α and IRE1β that likely have an important role in physiological processes induced by fatty acids in grass carp.

scholarly journals Grass Carp (Ctenopharyngodon idellus) NIMA-Related Kinase 6 Blocks dsRNA-Induced IFN I Response by Targeting IRF3

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaowen Xu ◽  
Meifeng Li ◽  
Zeyuan Deng ◽  
Jihuan Hu ◽  
Zeyin Jiang ◽  
...  
Keyword(s):  
Grass Carp ◽  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Poly I:C ◽  
Ctenopharyngodon Idellus ◽  
Cell Viability Assay ◽  
Antiviral Responses ◽  
Cik Cells ◽  
Viability Assay ◽  
High Level

Accumulating evidence indicates that mammalian NIMA (never in mitosis, gene A)-related kinase 6 (NEK6) plays potential roles during the course of tumorigenesis, but little is known about NEK6 in lower vertebrates. Herein, we reported a mammalian ortholog of NEK6 in grass carp (Ctenopharyngodon idellus) (CiNEK6). Multiple alignment of amino acid sequences and phylogenetic analysis showed that CiNEK6 shares a high level of sequence similarity with its counterparts in birds. CiNEK6 was ubiquitously expressed in all tested tissues, and its expression level was increased under treatment with GCRV (dsRNA virus) or poly I:C (dsRNA analog). Q-PCR and dual-luciferase assays suggested that CiNEK6 overexpression suppressed IFN I activity in CIK cells treated with poly I:C. Knockdown of CiNEK6 resulted in a higher level of IFN I expression in CIK cells treated with poly I:C compared to those which received PBS. Interestingly, analysis of subcellular localization demonstrated that CiNEK6 protein scattered throughout the cytoplasm is gradually congregated together at the edges of karyotheca upon stimulation with poly I:C. Co-IP and co-localization assays suggested that CiNEK6 interacts with CiIRF3 after poly I:C challenge. In poly I:C-treated cells, the phosphorylation of CiIRF3 was increased by CiNEK6 knockdown, but was suppressed by CiNEK6 overexpression, suggesting that CiNEK6 decreases IFN I expression through inhibiting CiIRF3 activity. Cell viability assay, crystal violet staining, and detection of Vp5 also showed that CiNEK6 plays an inhibitory role in IRF3-mediated antiviral responses.

scholarly journals Palmitate and oleate exert differential effects on insulin signalling and glucose uptake in human skeletal muscle cells

2017 ◽  
Vol 6 (5) ◽  
pp. 331-339 ◽  
Author(s):  
Selina Mäkinen ◽  
Yen H Nguyen ◽  
Paulina Skrobuk ◽  
Heikki A Koistinen
Keyword(s):  
Insulin Resistance ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Er Stress ◽  
Glucose Uptake ◽  
Insulin Signalling ◽  
Ros Production ◽  
Human Myotubes ◽  
Human Skeletal Muscle Cells ◽  
Concomitant Exposure

Saturated fatty acids are implicated in the development of insulin resistance, whereas unsaturated fatty acids may have a protective effect on metabolism. We tested in primary human myotubes if insulin resistance induced by saturated fatty acid palmitate can be ameliorated by concomitant exposure to unsaturated fatty acid oleate. Primary human myotubes were pretreated with palmitate, oleate or their combination for 12 h. Glucose uptake was determined by intracellular accumulation of [3H]-2-deoxy-d-glucose, insulin signalling and activation of endoplasmic reticulum (ER) stress by Western blotting, and mitochondrial reactive oxygen species (ROS) production by fluorescent dye MitoSOX. Exposure of primary human myotubes to palmitate impaired insulin-stimulated Akt-Ser473, AS160 and GSK-3β phosphorylation, induced ER stress signalling target PERK and stress kinase JNK 54 kDa isoform. These effects were virtually abolished by concomitant exposure of palmitate-treated myotubes to oleate. However, an exposure to palmitate, oleate or their combination reduced insulin-stimulated glucose uptake. This was associated with increased mitochondrial ROS production in palmitate-treated myotubes co-incubated with oleate, and was alleviated by antioxidants MitoTempo and Tempol. Thus, metabolic and intracellular signalling events diverge in myotubes treated with palmitate and oleate. Exposure of human myotubes to excess fatty acids increases ROS production and induces insulin resistance.

scholarly journals Estimation of peroxisomal and mitochondrial fatty acid oxidation in rat hepatocytes using tritiated substrates

1991 ◽  
Vol 279 (1) ◽  
pp. 147-150 ◽  
Author(s):  
R Rognstad
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Specific Inhibitor ◽  
Rat Hepatocytes ◽  
Acid Oxidation ◽  
Relative Distribution ◽  
Mitochondrial Fatty Acid Oxidation ◽  
Mitochondrial Fatty Acid

The pathways of peroxisomal and mitochondrial fatty acid oxidation were monitored with the use of substrates which produce NAD3H. I used as marker substrates: D-[3-3H]3-hydroxybutyrate for mitochondrial NAD3H production, [2-3H]glycerol for cytosolic NAD3H production, and [2-3H]acetate to measure carbon-bound 3H which was also generated by the metabolism of the commercial 9,10-3H-labelled fatty acids. The assumption that peroxisomal NAD3H can be considered to be equivalent to cytosolic NAD3H was supported using a specific inhibitor of mitochondrial fatty acid oxidation. The approach involves determination of the specific yields, and the relative distribution on carbons 4 and 6, of 3H in glucose from the marker substrates and the labelled fatty acids. In hepatocytes from clofibrate-treated rats, the amount of palmitate or oleate oxidation which starts in the peroxisomes is comparable with that which starts in the mitochondria.

scholarly journals Modification of the content of n-3 highly unsaturated fatty acid, chemical composition, and lipid nutritional indices in the meat of grass carp (Ctenopharyngodon idella) fed alfalfa (Medicago sativa) pellets

2020 ◽  
Vol 98 (4) ◽  
Author(s):  
Luciano F Montenegro ◽  
Adriana M Descalzo ◽  
Sebastián A Cunzolo ◽  
Carolina D Pérez
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Chemical Composition ◽  
Medicago Sativa ◽  
Grass Carp ◽  
Fatty Acid Content ◽  
Ctenopharyngodon Idella ◽  
Nutritional Indices ◽  
Δ9 Desaturase ◽  
Grass Carp Ctenopharyngodon Idella

Abstract A 120-d feeding trial was conducted to determine the effect of alfalfa (Medicago sativa) feeding on growth and chemical composition, fatty acid content, and nutritional and lipid indices of the meat of grass carp (Ctenopharyngodon idella). Two experimental diets were used: alfalfa pellet (AP) diet and artificial grain diet (GD). Final weight, feed conversion rate, and protein efficiency ratio were significantly greater in the GD group (P < 0.05). However, no differences in the length and condition factor were observed. The composition of the meat differed between treatments. The protein content was significantly greater in the AP group (P < 0.05), while the lipid and cholesterol contents were significantly greater in the GD group (P < 0.05). A greater proportion of saturated, n-6 polyunsaturated, and n-6 highly unsaturated fatty acids was obtained in the GD group. The AP group accumulated a greater concentration of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic (DHA) acids (P < 0.05). The fatty acid composition of the meat determined a significant decrease in the thrombogenicity index and saturation index (S/P) in the AP group (P < 0.05). The Elongase index was greater in the GD group (P < 0.05). In contrast, the AP group had a greater index of Δ9 Desaturase and Δ5 + Δ6 Desaturase for n-3 and n-6 fatty acids (P < 0.05). These results suggest that alfalfa feeding decreases the growth of C. idella but improves the quality of meat by increasing the protein, EPA, and DHA contents. It also reduces cholesterol content and improves nutritional indices.

Impacts of diet on hindgut microbiota and short-chain fatty acids in grass carp (Ctenopharyngodon idellus)

2017 ◽  
Vol 48 (11) ◽  
pp. 5595-5605 ◽  
Author(s):  
Yao Tong Hao ◽  
Shan Gong Wu ◽  
Ivan Jakovlić ◽  
Hong Zou ◽  
Wen Xiang Li ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Grass Carp ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Ctenopharyngodon Idellus ◽  
Chain Fatty Acids

scholarly journals Differential Lipotoxic Effects of Palmitate and Oleate in Activated Human Hepatic Stellate Cells and Epithelial Hepatoma Cells

2016 ◽  
Vol 39 (4) ◽  
pp. 1648-1662 ◽  
Author(s):  
Alexandra M. Hetherington ◽  
Cynthia G. Sawyez ◽  
Emma Zilberman ◽  
Alexandra M. Stoianov ◽  
Debra L. Robson ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Er Stress ◽  
Hepatic Stellate Cells ◽  
Hepg2 Cells ◽  
Stellate Cells ◽  
Hepatoma Cells ◽  
Surrounding Tissue ◽  
High Oleate ◽  
Activated Hepatic Stellate Cells

Background/Aims: Nonalcoholic fatty liver disease (NAFLD) progression to fibrosis, cirrhosis and hepatocellular carcinoma, alters the cellular composition of this organ. During late-stage NAFLD, fibrotic and possibly cancerous cells can proliferate and, like normal hepatocytes, are exposed to high concentrations of fatty acids from both surrounding tissue and circulating lipid sources. We hypothesized that primary human activated hepatic stellate cells and epithelial hepatoma (HepG2) cells respond differently to lipotoxic conditions, and investigated the mechanisms involved. Methods: Primary activated hepatic stellate cells and HepG2 cells were exposed to pathophysiological concentrations of fatty acids and comparative studies of lipid metabolic and stress response pathways were performed. Results: Both cell types remained proliferative during exposure to a combination of palmitate plus oleate reflective of the general saturated versus unsaturated fatty acid composition of western diets. However, exposure to either high palmitate or high oleate alone induced cytotoxicity in activated stellate cells, while only palmitate caused cytotoxicity in HepG2 cells. mRNA microarray and biochemical comparisons revealed that stellate cells stored markedly less fatty acids as neutral lipids, and had reduced capacity for beta-oxidation. Similar to previous observations in HepG2 cells, palmitate, but not oleate, induced ER stress and actin stress fiber formation in activated stellate cells. In contrast, oleate, but not palmitate, induced the inflammatory signal TXNIP, decreased cytoskeleton proteins, and decreased cell polarity preceding cell death in activated stellate cells. Conclusions: Palmitate-induced lipotoxicity was associated with ER stress pathways in both primary activated hepatic stellate cells and epithelial hepatoma cells, whereas high oleate caused lipotoxicity only in activated stellate cells, possibly through a distinct mechanism involving disruption of cytoskeleton components. This may have implications for optimal dietary fatty acid compositions during various stages of NAFLD.

Carnitine palmitoyltransferase 1A prevents fatty acid-induced adipocyte dysfunction through suppression of c-Jun N-terminal kinase

2011 ◽  
Vol 435 (3) ◽  
pp. 723-732 ◽  
Author(s):  
Xuefei Gao ◽  
Kuai Li ◽  
Xiaoyan Hui ◽  
Xiangping Kong ◽  
Gary Sweeney ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Cell Lines ◽  
Specific Inhibitor ◽  
Physiological Role ◽  
Carnitine Palmitoyltransferase ◽  
Acid Uptake ◽  
Pharmacological Inhibition

The adipocyte is the principal cell type for fat storage. CPT1 (carnitine palmitoyltransferase-1) is the rate-limiting enzyme for fatty acid β-oxidation, but the physiological role of CPT1 in adipocytes remains unclear. In the present study, we focused on the specific role of CPT1A in the normal functioning of adipocytes. Three 3T3-L1 adipocyte cell lines stably expressing hCPT1A (human CPT1A) cDNA, mouse CPT1A shRNA (short-hairpin RNA) or GFP (green fluorescent protein) were generated and the biological functions of these cell lines were characterized. Alteration in CPT1 activity, either by ectopic overexpression or pharmacological inhibition using etomoxir, did not affect adipocyte differentiation. However, overexpression of hCPT1A significantly reduced the content of intracellular NEFAs (non-esterified fatty acids) compared with the control cells when adipocytes were challenged with fatty acids. The changes were accompanied by an increase in fatty acid uptake and a decrease in fatty acid release. Interestingly, CPT1A protected against fatty acid-induced insulin resistance and expression of pro-inflammatory adipokines such as TNF-α (tumour necrosis factor-α) and IL-6 (interleukin-6) in adipocytes. Further studies demonstrated that JNK (c-Jun N terminal kinase) activity was substantially suppressed upon CPT1A overexpression, whereas knockdown or pharmacological inhibition of CPT1 caused a significant enhancement of JNK activity. The specific inhibitor of JNK SP600125 largely abolished the changes caused by the shRNA- and etomoxir-mediated decrease in CPT1 activity. Moreover, C2C12 myocytes co-cultured with adipocytes pre-treated with fatty acids displayed altered insulin sensitivity. Taken together, our findings have identified a favourable role for CPT1A in adipocytes to attenuate fatty acid-evoked insulin resistance and inflammation via suppression of JNK.

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