Fatty hepatocytes-derived exosomal miR-122 reduces immune function and antioxidant defence in Ctenopharyngodon idella kidney (CIK) cells.

Exosomes are important for intercellular "cross talk", but the role of exosomes in communication between hepatocytes and C. idella kidney (CIK) cells remains unknown. In this study, we detected the changes in factors related to immune and oxidative stress to investigate the molecular mechanism by which fatty hepatocyte-derived exosomes (OA-Exos) reduced immunity and induced oxidative stress in CIK cells. After incubation of CIK cells by OA-Exos for 24 h, tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB) and interleukin-1ß (IL-1ß) were significantly upregulated in the OA-Exos group (P < 0.05), and Mn superoxide dismutase (Mn-SOD) and heme oxygenase-1 (HO-1) were significantly downregulated (P < 0.05). Surprisingly, miR-122 expression was also significantly elevated after OA-Exos incubation. We further identified the expression of miR-122 and found that it was notably increased in OA-Exos compared to hepatocyte-derived exosomes (Exos). Then we transfected CIK cells with miR-122 mimic, consistently, the expression of inflammatory cytokines was also significantly elevated (P < 0.05), and the expression of glutathione peroxidase (GPx), HO-1, and Mn-SOD were dramatically decreased (P < 0.05). Furthermore, HO-1 was improved to be a direct target of miR-122, and transfection with HO-1 siRNA indicated that changes in inflammatory cytokines and genes related to oxidative stress were consistent with the above results of CIK cells incubated with OA-Exos and miR-122 mimic. We concluded that OA-Exos may, through the miR-122/HO-1 pathway, reduce immune function and antioxidant defence in CIK cells.

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.

Comparative analysis of the hepatopancreas transcriptome of grass carp (Ctenopharyngodon idellus) fed with lard oil and fish oil diets.

n-3 highly unsaturated fatty acids (n-3 HUFAs) have been shown to suppress lipid accumulation and improve protein utilization in grass carp; however, little is known about the underlying molecular mechanism. Hence, we analyzed the hepatopancreas transcriptome of grass carp (Ctenopharyngodon idellus) fed either lard oil (LO) or fish oil (FO) diets. RNA-seq data showed that 125 genes were significantly up-regulated and 107 were significantly down-regulated in the FO group. Among them, 17 lipid metabolism related genes, 12 carbohydrate metabolism related genes, and 34 protein metabolism related genes were selected. Lipid metabolism related genes, such as very long-chain acyl-CoA synthetase (ACSVL),carnitine O-palmitoyltransferase 1 (CPT1) and carnitine-acylcarnitine translocase (CACT), were up-regulated in the FO group. But the genes of diacylglycerol O-acyltransferase 2 (DGAT2) and stearoyl-CoA desaturase (SCD) were down-regulated. Down-regulation of glycolysis related genes, such as 6-phosphofructokinase (PFK), phosphoglycerate kinase (PGK) and pyruvate dehydrogenase kinase (PDK), added with up-regulation of gluconeogenesis related genes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), suggests lower utilization of carbohydrate of the FO group. Besides, dietary FO also influenced the protein metabolism related genes, such as up-regulation of genes involved in digestion of dietary protein, mRNA transcription, protein translation and amino acid utilization, down-regulation of genes involved in mRNA degradation and ubiquitination of protein. Interestingly, the up-regulation of mitochondrial uncoupling protein 2 (UCP2) and down-regulation of oxidative phosphorylation related genes (cytochrome c oxidase subunit 4 isoform 2 [COX4I2], HIG1 domain family member 1A [HIGD1A] and cytochrome-b5 reductase [CYB5R]) suggest that energy metabolism may be also influenced by dietary fatty acid composition. These findings presented here provide a comprehensive understanding of the molecular mechanisms governing the effects of fish oil in grass carp.

Hepatitis C virus-related heat-insoluble cryoglobulinemia and thrombotic microangiopathy.

Heat-insoluble cryoglobulinemia is rare, and its pathogenesis and comorbidities remain poorly understood. Here, the authors report a case of hepatitis C virus (HCV)-related heat-insoluble cryoglobulinemia associated with thrombotic microangiopathy and cryoglobulin-occlusive membranoproliferative glomerulonephritis. The patient, a 57-year-old woman, presented with acute kidney injury, thrombocytopenia, anemia with schistocytes, high levels of serum HCV RNA of HCV genotype 2a, rheumatoid factor positivity and high levels of serum immunoglobulin (Ig) M and Igκ. The patient's serum was positive for cryoglobulin at 4°C, and the precipitate required heating to 47°C for dissolution. Cryoglobulin immunofixation was positive for monoclonal IgM and Igκ and polyclonal IgG. However, immunofixation of the cryoglobulin supernatant was negative. Histological examination of renal biopsy revealed a membranoproliferative type I glomerulonephritis. The patient was treated with plasmapheresis, corticosteroids and antiviral therapy of peginterferon plus ribavirin, but symptoms only partially resolved.

Mitochondrial development and the influence of its dysfunction during rat adipocyte differentiation.

Mitochondrial biogenesis is inherent to adipocyte differentiation. Mitochondrial dysfunction leads to abnormal lipid accumulation or the deterioration of the differentiation process. The aim of this study is to investigate the mitochondrial development during the differentiation of rat primary adipocytes and the effect of mitochondrial dysfunction on this process. We found, for the first time, that the number of mitochondria markedly increased during adipocyte differentiation by transmission electron microscopy. By immunofluorescence staining that the protein content of Cyt c increased in differentiated adipocyte in comparison with preadipocyte. The mRNA expression levels of mitochondrial gene including cytochromes c (Cyt c), malate dehydrogenases (MDH), and peroxisome proliferator activated receptor (PPAR) gamma coactivator-1beta (PGC-1beta) significantly increased along with the proceeding of adipocyte differentiation. The damage to mitochondrial respiratory chain function by rotenone caused significant decrease in gene expressions including mitochondrial MDH and PGC-1beta, and PPARgamma, CAAT/enhancer binding protein alpha (C/EBPalpha) and sterol regulatory element binding protein-1c (SREBP-1c), which are known as transcription factors of differentiation, and differentiation marker gene named fatty acid synthetase. Moreover, an apparent decrease was found in the synthesis of triglyceride and ATP due to the damage to mitochondria by rotenone. Based on the above results, our present study revealed that the density and oxidative capacity of mitochondrial markedly increased during primary adipocyte differentiation, and on the other hand, we suggested that mitochondria dysfunction might inhibit the differentiation process.

[Effects of baicalein on the proliferation and differentiation of pig preadipocyte].

To investigate the effects of Baicalein (BAI) on the proliferation and differentiation of pig preadipocytes, and elucidate its potential mechanism. Primary preadipocytes of pig were cultured in vitro. The morphologic changes of preadipocytes differentiation were observed by Oil Red O staining. Status of cell proliferation was detected by MTT assay. The degree of adipogenesis and differentiation were measured by Oil Red O staining extraction assay. The activity of fatty acid synthase (FAS) was detected by spectrophotometry. The mRNA expression of special peroxisome proliferation activated receptor-gamma2 gene (PPARgamma2) was detected by reverse transcriptase polymerase chain reaction (RT-PCR). When preadipocytes differentiated into adipocytes, the preadipocytes were changed from shuttle shape to oval or round, in which big and small lipid droplets were filled. The proliferation of preadipocytes was inhibited by the treatment of 160-640 micromol/L BAI (P < 0.05). The mRNA expression of PPARgamma2 and FAS activity and the differentiation of preadipocytes was repressed by 40-320 micromol/L BAI treatment (P < 0.05). It is concluded that the proliferation and differentiation of preadipocytes is inhibited by BAI in some degree. The effect of BAI on differentiation of preadipocytes may be resulted from inhibiting the mRNA expression of PPARgamma2 and reducing FAS activity.

Nrf1 is critical for redox balance and survival of liver cells during development.

The Nrf1 transcription factor belongs to the CNC subfamily of basic leucine zipper proteins. Knockout of Nrf1 is lethal in mouse embryos, but nothing is known about the cell types that absolutely require its function during development. We show by chimera analysis that Nrf1 is essential for the hepatocyte lineage. Mouse embryonic stem cells lacking Nrf1 developed normally and contributed to most tissues in adult chimeras where Nrf1 is normally expressed. Nrf1-deficient cells contributed to fetal, but not adult, liver cells. Loss of Nrf1 function resulted in liver cell apoptosis in late-gestation chimeric fetuses. Fetal livers from mutant embryos exhibited increased oxidative stress and impaired expression of antioxidant genes, and primary cultures of nrf1(-/-) fetal hepatocytes were sensitive to tert-butyl hydroperoxide-induced cell death, suggesting that impaired antioxidant defense may be responsible for the apoptosis observed in the livers of chimeric mice. In addition, cells deficient in Nrf1 were sensitized to the cytotoxic effects of tumor necrosis factor (TNF). Our results provide in vivo evidence demonstrating an essential role of Nrf1 in the survival of hepatocytes during development. Our results also suggest that Nrf1 may promote cell survival by maintaining redox balance and protecting embryonic hepatocytes from TNF-mediated apoptosis during development.