Inhibition of GSK3B phosphorylation improves glucose and lipid metabolism disorder.

Hepatic glycolipid metabolism disorder is considered as one of the key pathogenic factors for many chronic diseases. Revealing the molecular mechanism of metabolic disorder and exploring drug targets are crucial for the treatment of glucose and lipid metabolic diseases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to be associated with the pathogenesis of various metabolic diseases. Herein, GAPDH-knockdown ZFL cells and GAPDH-downregulation zebrafish exhibited significant lipid deposition increase and glycogen reduction, thus inducing glucose and lipid metabolism disorders. Using high-sensitivity mass spectrometry-based proteomic and phosphoproteomic analysis, we identified 6838 proteins and 3738 phosphorylated proteins in GAPDH-knockdown ZFL cells. The protein-protein interaction network and DEPPs analyses showed that gsk3baY216 were involved in lipid and glucose metabolism, which was verified by In vitro study. The enzyme activity analysis and cell staining results showed that HepG2 and NCTC-1469 cells transfected with GSK3BY216F plasmid had significantly lower glucose and insulin levels, the decreased lipid deposition, and the increased glycogen synthesis than those transfected with GSK3BY216E plasmid, suggesting that inhibition of GSK3B phosphorylation could significantly improve GSK3B hyperphosphorylation-induced glucose tolerance impairment and insulin sensitivity reduction. To our knowledge, this is the first multi-omic study of GAPDH-knockdown ZFL cells. This study provides insights into the molecular mechanism of glucose and lipid metabolic disorder, and provides potential targets (kinases) for the treatments of human glucose and lipid metabolic diseases.

Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase is essential for the production of DHA in zebrafish.

Compared with other species, freshwater fish are more capable of synthesizing DHA via same biosynthetic pathways. Freshwater fish have a "Sprecher" pathway to biosynthesize DHA in a peroxisome-dependent manner. Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) is involved in the hydration and dehydrogenation reactions of fatty acid ß-oxidation in peroxisomes. However, the role of Ehhadh in the synthesis of DHA in freshwater fish remains largely unclear. In this study, the knockout of Ehhadh significantly inhibited DHA synthesis in zebrafish. Liver transcriptome analysis showed that Ehhadh deletion significantly inhibited SREBF and PPAR signaling pathways and decreased the expression of PUFA synthesis-related genes. Our results from the analysis of transgenic zebrafish (Tg:Ehhadh) showed that Ehhadh overexpression significantly increased the DHA content in the liver and significantly upregulated the expression of genes related to PUFA synthesis. In addition, the DHA content in the liver of Tg:Ehhadh fed with linseed oil was significantly higher than that of wildtype, but the expression of PUFA synthesis-related genes fads2 and elovl2 were significantly lower, indicating that Ehhadh had a direct effect on DHA synthesis. In conclusion, our results showed that Ehhadh was essential for DHA synthesis in the "Sprecher" pathway, and Ehhadh overexpression could promote DHA synthesis. This study provides insight into the role of Ehhadh in freshwater fish.

Cloning and expressions of chop in loach (Misgurnus anguillicaudatus) and its response to hydrogen peroxide (H2O2) stress.

C/EBP [CCAAT/enhancer-binding protein]-homologous protein gene (chop) which plays an important role in endoplasmic reticulum stress-induced apoptosis was investigated here by RACE and qPCR in an aquaculture animal for the first time. The full-length cDNA sequence of loach (Misgurnus anguillicaudatus) chop was 2533 bp, encoding 266 amino acids. The expression level of loach chop changed during different early life stages, with the highest expression at the 8-cell stage. Among different tissues, loach chop predominantly was expressed in gill, spleen, and gonad. We performed a hydrogen peroxide (H2O2, a common-used disinfectant) stress trial to explore the role of loach chop, with three different concentrations (0 µM, 50 µM, and 100 µM) of H2O2. The 100-µM dose was lethal for half the population but the other concentrations did not result in mortality. The activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) in loach gill, liver, and spleen decreased with extended stress time and increased H2O2 concentration. The expression levels of gill chop in loaches from the 100-µM group were significantly higher than those from the other two treatment groups at 12 and 24 h of exposure. atf4 and bax, two proapoptotic genes, were significantly upregulated in gills of loaches from the 100-µM group compared to the other two groups 18 h and 24 h after treatment. bcl2, an antiapoptotic gene, presented an opposite trend. These results indicated a close relationship between H2O2 stress and fish apoptosis with loach chop playing an important role in H2O2 stress response.

C24:0 avoids cold exposure-induced oxidative stress and fatty acid ß-oxidation damage.

Low temperatures can cause severe growth inhibition and mortality in fish. Previous studies about the cold resistance of fish mainly focused on the role of unsaturated fatty acids, rather than saturated fatty acids (SFAs). In this study, the role of very-long-chain SFA synthetized by fatty acyl elongase 1 gene (elovl1) in cold resistance was explored. Both an aggravated liver oxidative stress and a mitochondrial metabolism disorder were observed in elovl1a -/- and elovl1b -/- zebrafish with cold stress. In vitro studies confirmed that high levels of C20:0 and C22:0 obviously increased the hepatocyte oxidative stress and activated the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway to further induce apoptosis and inflammation. We further demonstrated that C24:0 could promote mitochondrial ß-oxidation to improve the cold resistance of zebrafish. Overall, our results define a positive role of C24:0 fatty acids synthetized by elovl1 in the cold resistance of fish.

Evolution and Functional Characteristics of the Novel elovl8 That Play Pivotal Roles in Fatty Acid Biosynthesis.

Elongation of very long-chain fatty acid (Elovl) proteins are key enzymes that catalyze the rate-limiting step in the fatty acid elongation pathway. The most recently discovered member of the Elovl family, Elovl8, has been proposed to be a fish-specific elongase with two gene paralogs described in teleosts. However, the biological functions of Elovl8 are still to be elucidated. In this study, we showed that in contrast to previous findings, elovl8 is not unique to teleosts, but displays a rather unique and ample phylogenetic distribution. For functional determination, we generated elovl8a (elovl8a-/-) and elovl8b (elovl8b-/-) zebrafish using CRISPR/Cas9 technology. Fatty acid composition in vivo and zebrafish liver cell experiments suggest that the substrate preference of Elovl8 overlapped with other existing Elovl enzymes. Zebrafish Elovl8a could elongate the polyunsaturated fatty acids (PUFAs) C18:2n-6 and C18:3n-3 to C20:2n-6 and C20:3n-3, respectively. Along with PUFA, zebrafish Elovl8b also showed the capacity to elongate C18:0 and C20:1. Gene expression quantification suggests that Elovl8a and Elovl8b may play a potentially important role in fatty acid biosynthesis. Overall, our results provide novel insights into the function of Elovl8a and Elovl8b, representing additional fatty acid elongases not previously described in chordates.

A network-based approach to identify protein kinases critical for regulating srebf1 in lipid deposition causing obesity.

Obesity is a rapidly growing health pandemic, underlying a wide variety of disease conditions leading to increases in global mortality. It is known that the phosphorylation of various proteins regulates sterol regulatory element-binding transcription factors 1 (srebf1), a key lipogenic transcription factor, to cause the development of obesity. To detect the key protein kinases for regulating srebf1 in lipid deposition, we established the srebf1 knockout model in zebrafish (KO, srebf1-/-) by CRISPR/Cas9. The KO zebrafish exhibited a significant reduction of total free fatty acid content (fell 60.5%) and lipid deposition decrease compared with wild-type (WT) zebrafish. Meanwhile, srebf1 deletion in zebrafish eliminated lipid deposition induced by high-fat diet feeding. Compared with WT zebrafish, a total of 697 differentially expressed proteins and 316 differentially expressed phosphoproteins with 439 sites were identified in KO by differential proteomic and phosphoproteomic analyses. A significant number of proteins identified were involved in lipid and glucose metabolism. Moreover, some protein kinases critical for regulating srebf1 in lipid deposition, including Cdk2, Pkc, Prkceb, mTORC1, Mapk12, and Wnk1, were determined by network analyses. An in vitro study was performed to verify the network analysis results. Our findings provide potential targets (kinases) for human obesity treatments.

Novel CaCO3/chitin aerogel: Synthesis and adsorption performance toward Congo red in aqueous solutions.

In this study, chitin aerogel doped with nano-calcium carbonate (CaCO3/chitin aerogel) was prepared by dissolving chitin in Na2CO3/Ca (OH)2/urea system followed by epichlorohydrin (ECH) addition as a cross-linker and then freeze-drying of the hydrogel. The as-obtained CaCO3/chitin aerogel showed good adsorption properties toward the adsorption of Congo Red (CR) with maximum adsorption capacity reaching approximately 266.4 mg·g-1. Besides, the adsorption capacity was affected by the initial concentration and pH. The adsorption kinetics revealed a second-order kinetics model of CaCO3/chitin aerogel toward CR, and the adsorption process was controlled by both the liquid film diffusion and intra-particle diffusion. In sum, chitin aerogel looks promising as an effective adsorbent for anion dye adsorption. The cost-effective and eco-friendly developed approaches are also of great interest for future environmental remediation.

Polyunsaturated fatty acids synthesized by freshwater fish: A new insight to the roles of elovl2 and elovl5 in vivo.

At present, fish provide an important supply of long-chain polyunsaturated fatty acids (LC-PUFAs) for human consumption. Previous studies have shown that fatty acyl elongase 2 (elovl2) and elovl5 play important roles in fish LC-PUFA synthesis. Generally, freshwater fish have a stronger ability to synthesize LC-PUFAs than marine fish. However, the roles of elovl2, elovl5 and elovl2 + elovl5 in LC-PUFA synthesis of freshwater fish in vivo are not very clear. In this study, the elovl2 knockout zebrafish (elovl2-/-), elovl5 knockout zebrafish (elovl5-/-) and the double gene knockout zebrafish (DKO) were generated by CRISPR/Cas9 technology for the first time. Compared with wild type zebrafish (WT), elovl5-deletion zebrafish showed a significant increase in C22 PUFA content, which might be due to the up-regulation expressions of elovl4b and elovl2. elovl5 expressed at very low levels in livers of elovl2-/- relative to WT, indicating that elovl5 may be an "assistant attacker" of elovl2 in LC-PUFA synthesis of zebrafish. Moreover, there were no significant differences in levels of C18-C22 PUFAs between DKO and WT, indicating that besides elovl2 + elovl5 path, LC-PUFA synthesis in zebrafish could be performed by other paths. In addition, the hepatic lipidomic analysis results revealed that the contents of C22:6n-3 in phosphatidyl ethanolamine (PE-DHA) and PE-C22 PUFAs were more easily affected by the absence of elovl2 and elovl5. Our results suggest that the elovl2+elovl5 path is not the only path for LC-PUFA synthesis in zebrafish, and provide novel insights into the roles of elovl2 and elovl5 in LC-PUFA synthesis of freshwater fish.

fat-1 transgenic zebrafish are protected from abnormal lipid deposition induced by high-vegetable oil feeding.

High dietary concentration of vegetable oil, particularly those rich in n-6 polyunsaturated fatty acids (PUFAs), can induce negative physiological effects including excessive lipid deposition in teleost fish. Omega-3 desaturase (Fat-1) of Caenorhabditis elegans is able to convert n-6 PUFAs to n-3 PUFAs and thus induces a low n-6/n-3 PUFAs ratio alleviating lipid deposition. In this study, we investigated the effects of dietary n-6 PUFAs on lipid metabolism of fat-1 transgenic zebrafish (Tg:fat-1), to explore the role of fat-1 in fish lipid metabolism. We first generated Tg:fat-1 zebrafish and assayed the effects of a low-fat diet (LFD) and a high-fat diet (HFD) prepared from soybean oil. Wild type zebrafish (WT) fed with HFD (HFD-WT) exhibited increased obesity and lipid deposition, especially in the abdominal cavity and liver. These defects were absent from HFD-Tg:fat-1. For each diet group, Tg:fat-1 exhibited significantly decreased levels of almost all hepatic lipid classes compared with WT. Expression levels of lipid synthesis-related genes and lipid deposition-related genes were markedly lower in the liver of HFD-Tg:fat-1 compared with HFD-WT. In contrast, the steatolysis-related genes significantly upregulated in HFD-Tg:fat-1. Then expression profiles of mitochondrial energy metabolism-related genes and ATP contents in the livers from LFD-WT, LFD-Tg:fat-1, HFD-WT, and HFD-Tg:fat-1 were determined. Our findings suggest that fat-1 protects fish from abnormal lipid deposition induced by high-vegetable oil feeding, through endogenously converting n-6 PUFAs to n-3 PUFAs. KEY POINTS: • fat-1 transgenic zebrafish (Tg:fat-1) can endogenously convert n-6 PUFAs to n-3 PUFAs. • Tg:fat-1 avoid serious abnormal lipid deposition induced by high-vegetable oil feeding. • fat-1 transgenosis effectively improved lipid metabolism and mitochondrial energy metabolism in zebrafish.

Quantitative Phosphoproteomic Analysis Reveals the Regulatory Networks of Elovl6 on Lipid and Glucose Metabolism in Zebrafish.

Elongation of very long-chain fatty acids protein 6 (Elovl6) has been reported to be associated with clinical treatments of a variety of metabolic diseases. However, there is no systematic and comprehensive study to reveal the regulatory role of Elovl6 in mRNA, protein and phosphorylation levels. We established the first knock-out (KO), elovl6-/-, in zebrafish. Compared with wild type (WT) zebrafish, KO presented significant higher whole-body lipid content and lower content of fasting blood glucose. We utilized RNA-Seq, tandem mass tag (TMT) labeling-based quantitative technology and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to perform the transcriptomic, proteomic and phosphoproteomic analyses of livers from WT and elovl6-/- zebrafish. There were 734 differentially expressed genes (DEG) and 559 differentially expressed proteins (DEP) between elovl6-/- and WT zebrafish, identified out of quantifiable 47251 transcripts and 5525 proteins. Meanwhile, 680 differentially expressed phosphoproteins (DEPP) with 1054 sites were found out of quantifiable 1230 proteins with 3604 sites. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis of the transcriptomic and proteomic data further suggested that the abnormal lipid metabolism and glucose metabolism in KO were mainly related to fatty acid degradation and biosynthesis, glycolysis/gluconeogenesis and PPAR signaling pathway. Based on phosphoproteomic analyses, some kinases critical for lipid metabolism and glucose metabolism, including ribosomal protein S6 kinase (Rps6kb), mitogen-activated protein kinase14 (Mapk14) and V-akt murine thymoma viral oncogene homolog 2-like (Akt2l), were identified. These results allowed us to catch on the regulatory networks of elovl6 on lipid and glucose metabolism in zebrafish. To our knowledge, this is the first multi-omic study of zebrafish lacking elovl6, which provides strong datasets to better understand many lipid/glucose metabolic risks posed to human health.

In-situ preparation of biochar-loaded particle electrode and its application in the electrochemical degradation of 4-chlorophenol in wastewater.

In order to effectively degrade 4-chlorophenol in wastewater, a biochar-loaded particle electrode was prepared using an in-situ method. The method employed bagasse as the raw material, KOH as the activator, SnCl4·5H2O, MnCl2·4H2O and SbCl3 as the modifiers. Furthermore, X-ray diffraction, scanning electron microscopy and energy dispersive spectrometer were used to characterize the crystal composition, morphology and elemental compositions of the proposed particle electrode. The electrochemical performance of the proposed particle electrode was analyzed using an electrochemical workstation. A three-dimensional packed-bed electrochemical reactor was constructed using the loaded biomass carbon particle electrodes. The effects of different loaded biomass carbon particle electrodes on the electrochemical degradation of 4-chlorophenol were studied, and the mechanism of electrochemical degradation of 4-chlorophenol was discussed. The results showed that the effect of the supported biomass carbon particle electrode on the degradation of 4-chlorophenol was significantly higher than that of the unsupported biomass carbon particle electrode. Additionally, the electrochemical degradation of 4-chlorophenol was greatly influenced by the biomass carbon particle electrode with different loading and concentration. The removal efficiencies of 4-chlorophenol using the electrochemical treatment under the studied experimental conditions were found in the following descending order: Mn/AC > Sn/AC > Sb/AC. Among them, the biomass carbon particle electrode prepared using 150 g L-1 MnCl2·4H2O showed the best treatment effect for 4-chlorophenol. After electrochemical treatment of 500 mg L-1 of 4-chlorophenol-simulated wastewater for 1 h, the removal efficiency of 4-chlorophenol reached 99.93%.