Anti-miR33 therapy improved hepatopancreatic lipid and immune metabolism disorders in grass carp, Ctenopharyngodon idella.

Lipid metabolism disorders are found ubiquitously in farmed fish and occur as a result of excessive fat accumulation. Previous studies have found that miR-33 is involved in lipid metabolism; however, its role in fish lipid metabolism is unclear. We sought to clarify this relationship in grass carp in vivo and in vitro. Our findings revealed the length of miR-33 to be 65 bp. Phylogenetic tree analysis showed that grass carp miR-33 was most closely related to fish miR-33 (Siganus canaliculatus). Hepatocytes transfected with miR-33 mimic displayed markedly raised TG content (P < 0.05) as well as increased levels of lipid synthesis-related transcription factors (P < 0.05). Compared with blank and saline groups, total serum cholesterol, AST, and LDL levels were suppressed in groups treated with the miR-33 antagomir (P < 0.05). Moreover, the expression levels of PPARγ and SREBP-1c mRNA were significantly decreased in contrast to those found in the control group (P < 0.05). Similar findings were noted in the expression of immune-related proinflammatory molecules (TNFα, IL-1ß, IL-6, and NF-κB), which also demonstrated decreased levels (P < 0.05). Conversely, high expressions of anti-inflammatory factors (TGF-ß1 and IL-10) were noted (P < 0.05). This investigation strongly supports the role of miR-33 in hepatopancreas-based lipid metabolism and immunity. miR-33 may have been highly conserved in early vertebrates in order to facilitate liver-specific metabolic and immunomodulatory functions. Our findings provide a basis for further investigations exploring the mechanisms surrounding fish lipid metabolism and may aid in preventing and treating immunocompromised fish as well as fish with fatty hepatopancreas, and other metabolic diseases.

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.

Grape seed proanthocyanidin extract ameliorates hepatic lipid accumulation and inflammation in grass carp (Ctenopharyngodon idella).

Hepatic lipid metabolism disorder due to excessive fat accumulation in fish is a significant problem in aquaculture. Studies have shown that grape seed procyanidin extract (GSPE) can regulate fish lipid metabolism and improve fish immunity. However, the mechanism is unclear. In this study, we used grass carp that stores excess fat in the liver as a model. In vitro, GSPE treatment of hepatocytes for 3 h significantly decreased TG content, accompanied with decreased expression of SREBP-1c, FAS, and ACC and increased expression of PPARα, ATGL, and LPL. GSPE treatment for 1 h significantly decreased expression of pro-inflammatory cytokines (TNFα, IL-6, IL-1ß, and NF-κB) and increased the expression of anti-inflammatory cytokines (IL-10 and TGF-ß1). In vivo, the administration of GSPE significantly reduced high-fat diet-induced increase of serum CHOL, TG, and HDL, but increased LDL content. GSPE treatment for 3 h increased expression of ATGL and LPL, and significantly decreased the expression of HFD-fed-induced SREBP-1c, ACC, FAS, PPARγ, PPARα, and H-FABP. GSPE treatment for 3 h also significantly decreased the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1ß) and increased the expression of the anti-inflammatory cytokine IL-10. The expression levels of the lipogenic miRNAs, miR-33, and miR-122, were suppressed both in vivo and in vitro by GSPE. In summary, GSPE had hypolipidemic and potential anti-inflammatory effects in the liver, potentially mediated by miR-33 and miR-122.

Regulation of growth performance and lipid metabolism in juvenile grass carp (Ctenopharyngodon idella) with honeysuckle (Lonicera japonica) extract.

This study investigated the effects of honeysuckle extract (Lonicera japonica, HE) on the growth performance and lipid metabolism of juvenile grass carp (Ctenopharyngodon idella). HE at doses of 10 g kg-1 (LHE), 20 g kg-1 (MHE), and 40 g kg-1 (HHE) were individually mixed with the basal diet and fed to grass carp for 10 weeks, and ginseng extract (20 g kg-1, GSE) was used as a positive control. The results showed that HE administration exerted no effect on growth performance, but the hepatosomatic index (HSI) and muscle and liver lipid contents were significantly decreased in the LHE and MHE groups. The serum levels of LDL-c, total triglyceride (TG) and total cholesterol (TC) also declined in the HE-treated groups. Moreover, the disordered vacuolization and nucleus migration in the liver were alleviated in the MHE and HHE groups, and mRNA expressions of lipogenesis-related genes, such as acc1, fas, srebp1, and pparγ decreased. Similarly, the expression of genes related to lipolysis, such as cpt1, atgl, lpl, and pparα, was found to be significantly increased in the MHE and HHE groups compared with the control. Taken together, HE can effectively improve the lipid metabolism and ameliorate the lipid deposition of grass carp and thus may be a promising feed additive in aquaculture.

Intestinal microbiota and lipid metabolism responses in the common carp (Cyprinus carpio L.) following copper exposure.

The present study was conducted to determine the effects of waterborne copper exposure on the lipid metabolism and intestinal microbiota of juvenile common carp (Cyprinus carpio L.). Common carp were exposed to four waterborne copper (Cu) concentrations (0 (control), 0.07 (low), 0.14 (medium), and 0.28 (high) mg Cu/L) for 8 weeks. Exposure to a high concentration of Cu had a negative effect on growth indices (weight gain rate (WGR) and specific growth rate (SGR)). The biochemical indices measured in serum (low-density lipoprotein (LDL) and triglycerides (TGs)) were significantly affected by exposure to medium concentration levels of Cu. The mRNA levels of lipogenic enzymes (acetyl-CoA carboxylase 1 (ACC-1) and fatty acid synthase (FAS)) and sterol-regulator element-binding protein-1 (SREBP-1) in liver tissue and tight binding protein genes (ZO-1 and occludin) in intestinal epithelial tissue were significantly downregulated in the 0.14 and 0.28 mg/L Cu treatment groups, accompanied by upregulated mRNA levels of lipolysis enzymes (lipoprotein lipase (LPL) and carnitine palmitoyl transferase 1 (CPT-1)) in the liver. The data also showed that the composition of intestinal microbiota was changed following Cu exposure and could alter the α-diversity and ß-diversity. The abundances of few putative short-chain fatty acid (SCFA)-producing bacteria, including Allobaculum, Blautia, Coprococcus, Faecalibacterium, Roseburia, and Ruminococcus, decreased significantly. More specifically, Roseburia sequences were positively associated with lipogenic enzymes, total protein (TP), and TGs and negatively associated with lipolysis enzymes. Other sequences related to probiotics (Lactobacillus, Bacillus and Akkermansia) were also found to decrease, accompanied by an increase in sequences related to pathogens (Pseudomonas and Acinetobacter). To the best of our knowledge, the present study provides the first evidence that waterborne, chronic Cu exposure can disturb the composition of intestinal microbiota related to lipid metabolism and immunity in freshwater fish, thereby increasing the risk of pathogen invasion.

A fluorescence method for determination of glucose transport by intestinal BBMV of common carp.

Epithelial brush-border membrane vesicles (BBMVs) were isolated from the intestine of common carp and studied systematically by enzyme activity, transmission electron microscopy and immunoblotting. The uptake time course and the substrate concentration effect were assessed, and then, the ability of phlorizin and cytochalasin B to inhibit uptake was analyzed. The results show that sucrase, alkaline phosphatase and Na+-K+-ATPase activities in these vesicles were enriched 7.94-, 6.74- and 0.42-fold, respectively, indicating a relatively pure preparation of apical membrane with little basolateral contamination. The vesicular structure was in complete closure, as confirmed by electron microscopy. The presence of SGLT1 on the BBMVs was confirmed by Western blot analysis. In the time course experiment, the glucose uptake by BBMVs in Na+ medium displayed an initial accumulation (overshoot) at 5 min followed by a rapid return to equilibrium values at 60 min. Over the 2-NBDG concentration range selected, the external 2-NBDG concentration in NaSCN medium graphed as a curved line. Phlorizin and cytochalasin B had an obvious inhibitory effect on 2-NBDG transport in carp BBMVs, and the detected fluorescence intensity decreased. The inhibition rate in the 1000 µM group was the strongest at 64.18% and 63.61% of phlorizin and cytochalasin B, respectively, indicating the presence of carriers other than SGLT1. This study is the first to demonstrate that 2-NBDG can be used as a convenient and sensitive probe to detect glucose uptake in fish BBMVs. This technology will provide a convenient method to discover new effects and factors in glucose metabolism.

Effects of glucose, insulin and triiodothyroxine on leptin and leptin receptor expression and the effects of leptin on activities of enzymes related to glucose metabolism in grass carp (Ctenopharyngodon idella) hepatocytes.

Leptin is an important regulator of appetite and energy expenditure in mammals, but its role in fish metabolism control is poorly understood. Our previous studies demonstrated that leptin has an effect on the regulation of food intake and energy expenditure as well as lipid metabolism (stimulation of lipolysis and inhibition of adipogenesis) in the grass carp Ctenopharyngodon idella. To further investigate the role of leptin in fish, the effects of glucose, insulin and triiodothyroxine (T3) on the expression levels of leptin and leptin receptor (Lepr) and the effects of leptin on the activities of critical glucose metabolism enzymes in grass carp hepatocytes were evaluated in the present study. Our data indicated that leptin gene expression was induced by glucose in a dose-dependent manner, while Lepr gene expression exhibited a biphasic change. A high dose of insulin (100 ng/mL) significantly up-regulated the expression of leptin and Lepr. Leptin expression was markedly up-regulated by a low concentration of T3 but inhibited by a high concentration of T3. T3 up-regulated Lepr expression in a dose-dependent manner. Together, these data suggest that leptin had a close relationship with three factors (glucose, insulin and T3) and might participate in the regulation of glucose metabolism in grass carp. In addition, we also found that leptin affected the activities of key enzymes that are involved in glucose metabolism, which might be mediated by insulin receptor substrate-phosphoinositol 3-kinase signaling.

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.

Characterization and tissue distribution of leptin, leptin receptor and leptin receptor overlapping transcript genes in yellow catfish Pelteobagrus fulvidraco.

In the present study, full-length cDNA sequences of leptin (LEP), leptin receptor (LEPR) and leptin receptor overlapping transcript (LEPROT) were cloned from yellow catfish Pelteobagrus fulvidraco, and their tissue distribution profiles were determined. The validated cDNA of yellow catfish leptin (ycLEP), leptin receptor (ycLEPR) and LEPROT were 1119, 4195 and 827bp in length, encoding the peptide of 172, 1086 and 130 amino acid residues, respectively. The phylogenetic analysis revealed that fish LEP, LEPR and LEPROT were separated from tetrapod, and also ycLEPS were separated from other fish species. The ycLEP mRNA expression levels were highest in liver, followed by ovary, mesenteric fat and spleen, and lowest in intestine, heart, muscle, pituitary and testis. The ycLEPR mRNA levels were highest in pituitary, intermediate in mesenteric fat, liver, ovary, muscle and spleen, and lowest in heart, intestine and testis. The ycLEPROT mRNA levels were highest in pituitary, followed by spleen, mesenteric fat, heart, ovary, liver, muscle, testis and intestine. Identification and tissue distribution of yellow catfish LEP, LEPR and LEPROT genes provided initial step towards understanding their biological roles in yellow catfish.

The role of leptin in lipid metabolism in fatty degenerated hepatocytes of the grass carp Ctenopharyngodon idellus.

Leptin (Lep) is a key factor in the regulation of energy homeostasis in mammals, but its role in the fatty degenerated hepatocytes of the grass carp Ctenopharyngodon idellus is still unknown. The aim of our study is to determine the underlying mechanism and possible effects of C. idellus Lep function in lipid metabolism in C. idellus fatty degenerated hepatocytes. Fatty degenerated hepatocytes of C. idellus were established through treatment with media containing 0.1 % lipid emulsion (LE). Hepatic triglycerides had markedly accumulated in the treated hepatocytes 48 h later. Furthermore, we demonstrated that Lep dose dependently promoted the release of glycerol, but not FFA, in fatty degenerated hepatocytes. We also found that Lep affected the expression of key genes related to lipid metabolism at the transcriptional and translational levels. A total of ten genes, including HSL, ATGL, PPARα, PPARß, UCP1, UCP2, PGC-1α, and CPTIα-1b, were markedly upregulated, while SCD1a and PPARγ were downregulated with Lep treatment. Moreover, the protein levels of HSL and ATGL and the LPL activity also significantly increased. The Lep-induced lipolysis was disrupted by the JAK-STAT inhibitor AG490, suggesting that JAK-STAT signaling pathways were involved in the process of Lep-induced lipolysis. Using the IRS-PI(3)K-specific inhibitor W1628, we found that only the Lep-induced downregulation of PPARγ was reduced. This result indicated that the IRS-PI(3)K signaling pathway was involved in the regulation of the adipogenic gene PPARγ. Overall, our results provided evidence that Lep directly stimulated JAK-STAT signaling-mediated lipolysis and fatty acid ß-oxidation gene expression in the fatty degenerated hepatocytes of C. idellus and inhibited the adipogenesis mediated by the IRS-PI(3)K signaling pathway.

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.

[Effects of different cryoprotectants on post-thaw viability of porcine preadipocyte].

To explore the optimal cryoprotectant for porcine preadipocyte cryppreservation, the primary pig preadipocytes were cryopreseved with DMSO, ethlyleneglycol(EG), PVP and DMSO+PVP cryoprotective agents (CPA) and without CPA, respectively. After 30d, the cells were recovered, viability of pig preadipocyte cryopreserved with different CPAs was evaluated by the Typlan exclusion, growth characteristic was analyzed with MTT, the differentiation potentials of pig preadipocytes were observed by morphology change from preadipocyte to mature adipocyte and Oil Red O staining. The results were as follows: four cryoprotectants all could protect the pig preadipocyte from frozen at a certain degree, and the efficiency was as follows: PVP > MSO+PVP > DMSO > EG > without cryoprotectant. The growth state and the lipogenic capability of the pig preadipocytes cryopreserved with PVP were similar to those unfreezing cells. Thus, we draw a conclusion: PVP is the optimal cryoprotectant for pig preadipocyte.