Structure, anti-inflammatory and anti-bacterial activities of novel pentacyclic triterpenoids and other constituents from the leaves of Pittosporum elevaticostatum.

The investigation of the leaves of Pittosporum elevaticostatum Chang et Yan led to the isolation of fifteen pentacyclic triterpenoids (1-15), including five previously undescribed ones (1-5), and nine others (16-24). The structures of compounds 1-5 were elucidated based on comprehensive spectroscopic techniques, including one dimension (1D) and 2D nuclear magnetic resonance (NMR), high resolution electrospray ionization mass spectroscopy (HR-ESI-MS), and other methods. Compounds 2 and 13 demonstrated significant inhibitory activity against Listeria monocytogenes (L. monocytogenes) with minimum inhibitory concentration (MIC) values of 32 µM. Scanning electron microscopy (SEM) observations revealed insights into the antibacterial mechanism, indicating that compounds 2 and 13 either prevent biofilm formation of dispersed the preformed cell membranes. Additionally, compounds 1, 5, 7, and 12 exhibited anti-inflammatory activity on lipopolysaccharide (LPS)-stimulated BV-2 microglial cells with IC50 values ranging from 11.27 to 17.80 µM.

Ultrathin and High-Modulus LiBO2 Layer Highly Elevates the Interfacial Dynamics and Stability of Lithium Anode under Wide Temperature Range.

Lithium (Li) metal batteries (LMBs) face huge challenges to achieve long cycling life at wide temperature range owing to the severe dendrite growth at subambient temperature and the intense side reactions with electrolyte at high temperature. Herein, an ultrathin LiBO2 layer with an extremely high Young's modulus of 8.0 GPa is constructed on Li anode via an in situ reaction between Li metal and 4,4,5,5-tetramethyl-1,3,2-dioxa-borolane (TDB) to form LiBO2 @Li anode, which presents two times higher exchange current density than pristine Li anode. The LiBO2 layer presents a strong absorption to Li ions and greatly improves the interfacial dynamics of Li-ion migration, which induces homogenous lithium nucleation and deposition to form a dense lithium layer. Consequently, the Li dendrite growth during cycling at subambient temperature and the side reactions with electrolyte at high temperature are simultaneously suppressed. The LiBO2 @Li/LiNi0.8 Co0.1 Mn0.1 O2 (NCM811) full batteries with limited Li capacity and high cathode mass loading of 9.9 mg cm-2 can steadily cycle for 300 cycles with a capacity retention of 86.6%. The LiBO2 @Li/NCM811 full batteries and LiBO2 @Li/LiBO2 @Li symmetric batteries also present excellent cycling performance at both -20 and 60 °C. This work develops a strategy to achieve outstanding performance of LMBs at wide working temperature-range.

GATA3 functions downstream of BRCA1 to suppress EMT in breast cancer.

Purpose: Functional loss of BRCA1 is associated with poorly differentiated and metastatic breast cancers that are enriched with cancer stem cells (CSCs). CSCs can be generated from carcinoma cells through an epithelial-mesenchymal transition (EMT) program. We and others have previously demonstrated that BRCA1 suppresses EMT and regulates the expression of multiple EMT-related transcription factors. However, the downstream mediators of BRCA1 function in EMT suppression remain elusive. Methods: Depletion of BRCA1 or GATA3 activates p18INK4C , a cell cycle inhibitor which inhibits mammary epithelial cell proliferation. We have therefore created genetically engineered mice with Brca1 or Gata3 loss in addition to deletion of p18INK4C , to rescue proliferative defects caused by deficiency of Brca1 or Gata3. By using these mutant mice along with human BRCA1 deficient as well as proficient breast cancer tissues and cells, we investigated and compared the role of Brca1 and Gata3 loss in the activation of EMT in breast cancers. Results: We discovered that BRCA1 and GATA3 expressions were positively correlated in human breast cancer. Depletion of BRCA1 stimulated methylation of GATA3 promoter thereby repressing GATA3 transcription. We developed Brca1 and Gata3 deficient mouse system. We found that Gata3 deficiency in mice induced poorly-differentiated mammary tumors with the activation of EMT and promoted tumor initiating and metastatic potential. Gata3 deficient mammary tumors phenocopied Brca1 deficient tumors in the induction of EMT under the same genetic background. Reconstitution of Gata3 in Brca1-deficient tumor cells activated mesenchymal-epithelial transition, suppressing tumor initiation and metastasis. Conclusions: Our finding, for the first time, demonstrates that GATA3 functions downstream of BRCA1 to suppress EMT in controlling mammary tumorigenesis and metastasis.

Novel insights for SREBP-1 as a key transcription factor in regulating lipogenesis in a freshwater teleost, grass carp Ctenopharyngodon idella.

Disturbances in lipid metabolism are at the core of several health issues facing modern society, including fatty liver and obesity. The sterol regulatory element-binding protein 1 (SREBP-1) is one important transcription factor regulating lipid metabolism, but the relevant mechanism still remains unknown. The present study determined the transcriptional regulation of SREBP-1 and its target genes (including acetyl-CoA carboxylase α (accα), fatty acid synthase (fas) and stearoyl-CoA desaturase 1 (scd1)) in a freshwater teleost, grass carp Ctenopharyngodon idella. We cloned and characterised the 1988 bp, 2043 bp, 1632 bp and 1889 bp sequences of srebp-1, accα, scd1 and fas promoters, respectively. A cluster of putative binding sites of transcription factors, such as specific protein, yin yang 1, nuclear factor Y, sterol response elements (SRE) and enhancer box (E-box) element, were predicted on their promoter regions. Overexpression of nSREBP-1 reduced srebp-1 promoter activity, increased scd1 and fas promoter activity but did not influence accα promoter activity. The site-mutation and electrophoretic mobility shift assay analysis indicated that srebp-1, fas and scd1 promoters, but not accα promoter, possessed SRE. In Ctenopharyngodon idella kidney (CIK) cells of grass carp, nSREBP-1 overexpression significantly reduced srebp-1 mRNA expression and up-regulated miR-29 mRNA expression. The 3'UTR of srebp-1 possessed the potential miR-29 binding site and miR-29 up-regulated the luciferase activity of srebp-1 3'UTR and srebp-1 mRNA expression, implying a self-activating loop of SREBP-1 and miR-29 in grass carp. Based on the above-mentioned results, we found two novel transcriptional mechanisms for SREBP-1 in grass carp: (1) the auto-regulation sited on the SREBP-1 promoter regions was suppressive and (2) there was a self-activating loop of SREBP-1 and miR-29.

Estrogen promotes estrogen receptor negative BRCA1-deficient tumor initiation and progression.

BACKGROUND: Estrogen promotes breast cancer development and progression mainly through estrogen receptor (ER). However, blockage of estrogen production or action prevents development of and suppresses progression of ER-negative breast cancers. How estrogen promotes ER-negative breast cancer development and progression is poorly understood. We previously discovered that deletion of cell cycle inhibitors p16Ink4a (p16) or p18Ink4c (p18) is required for development of Brca1-deficient basal-like mammary tumors, and that mice lacking p18 develop luminal-type mammary tumors. METHODS: A genetic model system with three mouse strains, one that develops ER-positive mammary tumors (p18 single deletion) and the others that develop ER-negative tumors (p16;Brca1 and p18;Brca1 compound deletion), human BRCA1 mutant breast cancer patient-derived xenografts, and human BRCA1-deficient and BRCA1-proficient breast cancer cells were used to determine the role of estrogen in activating epithelial-mesenchymal transition (EMT), stimulating cell proliferation, and promoting ER-negative mammary tumor initiation and metastasis. RESULTS: Estrogen stimulated the proliferation and tumor-initiating potential of both ER-positive Brca1-proficient and ER-negative Brca1-deficient tumor cells. Estrogen activated EMT in a subset of Brca1-deficient mammary tumor cells that maintained epithelial features, and enhanced the number of cancer stem cells, promoting tumor progression and metastasis. Estrogen activated EMT independent of ER in Brca1-deficient, but not Brca1-proficient, tumor cells. Estrogen activated the AKT pathway in BRCA1-deficient tumor cells independent of ER, and pharmaceutical inhibition of AKT activity suppressed EMT and cell proliferation preventing BRCA1 deficient tumor progression. CONCLUSIONS: This study reveals for the first time that estrogen promotes BRCA1-deficient tumor initiation and progression by stimulation of cell proliferation and activation of EMT, which are dependent on AKT activation and independent of ER.

Fatty Acid ß-Oxidation Is Essential in Leptin-Mediated Oocytes Maturation of Yellow Catfish Pelteobagrus fulvidraco.

Although several studies have been conducted to study leptin function, information is very scarce on the molecular mechanism of leptin in fatty acid ß-oxidation and oocytes maturation in fish. In this study, we investigated the potential role of fatty acid ß-oxidation in leptin-mediated oocytes maturation in Pelteobagrus fulvidraco. Exp. 1 investigated the transcriptomic profiles of ovary and the differential expression of genes involved in ß-oxidation and oocytes maturation following rt-hLEP injection; rt-hLEP injection was associated with significant changes in the expression of genes, including twenty-five up-regulated genes (CPT1, Acsl, Acadl, Acadm, Hadhb, Echsl, Hsd17b4, Acca, PPARα, CYP8B1, ACOX1, ACBP, MAPK, RINGO, Cdc2, MEK1, IGF-1R, APC/C, Cdk2, GnRHR, STAG3, SMC1, FSHß and C-Myc) and ten down-regulated gene (PPARγ, FATCD36, UBC, PDK1, Acads, Raf, Fizzy, C3H-4, Raf and PKC), involved in fatty acid ß-oxidation and oocytes maturation. In Exp. 2, rt-hLEP and specific inhibitors AG490 (JAK-STAT inhibitor) were used to explore whether leptin induced oocytes maturation, and found that leptin incubation increased the diameters of oocytes and percentage of germinal vesicle breakdown (GVBD)-MII oocytes, up-regulated mRNA levels of genes involved in oocytes maturation and that leptin-induced oocyte maturation was related to activation of JAK-STAT pathway. In Exp. 3, primary oocytes of P. fulvidraco were treated with (R)-(+)-etomoxir (an inhibitor of ß-oxidation) or l-carnitine (an enhancer of ß-oxidation) for 48 h under rt-hLEP incubation. Exp. 3 indicated that the inhibition of fatty acid ß-oxidation resulted in the down-regulation of gene expression involved in oocytes maturation, and repressed the leptin-induced up-regulation of these gene expression. Activation of fatty acid ß-oxidation improved the maturation rate and mean diameter of oocytes, and up-regulated gene expression involved in oocytes maturation. Leptin is one of the main factors that links fatty acid ß-oxidation with oocyte maturation; ß-oxidation is essential for leptin-mediated oocyte maturation in fish.

Cancer cell-derived von Willebrand factor enhanced metastasis of gastric adenocarcinoma.

Cancer prognosis is poor for patients with blood-borne metastasis. Platelets are known to assist cancer cells in transmigrating through the endothelium, but ligands for the platelet-mediated cancer metastasis remain poorly defined. von Willebrand factor (vWF) is a major platelet ligand that has been widely used as a biomarker in cancer and associated inflammation. However, its functional role in cancer growth and metastasis is largely unknown. Here we report that gastric cancer cells from patients and cells from two well-established gastric cancer lines express vWF and secrete it into the circulation, upon which it rapidly becomes cell-bound to mediate cancer-cell aggregation and interaction with platelets and endothelial cells. The vWF-mediated homotypic and heterotypic cell-cell interactions promote the pulmonary graft of vWF-overexpressing gastric cancer BGC823 cells in a mouse model. The metastasis-promoting activity of vWF was blocked by antibodies against vWF and its platelet receptor GP Ibα. It was also reduced by an inhibitory siRNA that suppresses vWF expression. These findings demonstrate a causal role of cancer-cell-derived vWF in mediating gastric cancer metastasis and identify vWF as a new therapeutic target.

Structure and Functional Analysis of Promoters from Two Liver Isoforms of CPT I in Grass Carp Ctenopharyngodon idella.

Carnitine palmitoyltransferase I (CPT I) is a key enzyme involved in the regulation of lipid metabolism and fatty acid ß-oxidation. To understand the transcriptional mechanism of CPT Iα1b and CPT Iα2a genes, we cloned the 2695-bp and 2631-bp regions of CPT Iα1b and CPT Iα2a promoters of grass carp (Ctenopharyngodon idella), respectively, and explored the structure and functional characteristics of these promoters. CPT Iα1b had two transcription start sites (TSSs), while CPT Iα2a had only one TSS. DNase I foot printing showed that the CPT Iα1b promoter was AT-rich and TATA-less, and mediated basal transcription through an initiator (INR)-independent mechanism. Bioinformatics analysis indicated that specificity protein 1 (Sp1) and nuclear factor Y (NF-Y) played potential important roles in driving basal expression of CPT Iα2a gene. In HepG2 and HEK293 cells, progressive deletion analysis indicated that several regions contained cis-elements controlling the transcription of the CPT Iα1b and CPT Iα2a genes. Moreover, some transcription factors, such as thyroid hormone receptor (TR), hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor (PPAR) family, were all identified on the CPT Iα1b and CPT Iα2a promoters. The TRα binding sites were only identified on CPT Iα1b promoter, while TRß binding sites were only identified on CPT Iα2a promoter, suggesting that the transcription of CPT Iα1b and CPT Iα2a was regulated by a different mechanism. Site-mutation and electrophoretic mobility-shift assay (EMSA) revealed that fenofibrate-induced PPARα activation did not bind with predicted PPARα binding sites of CPT I promoters. Additionally, PPARα was not the only member of PPAR family regulating CPT I expression, and PPARγ also regulated the CPT I expression. All of these results provided new insights into the mechanisms for transcriptional regulation of CPT I genes in fish.

Magnesium Reduces Hepatic Lipid Accumulation in Yellow Catfish (Pelteobagrus fulvidraco) and Modulates Lipogenesis and Lipolysis via PPARA, JAK-STAT, and AMPK Pathways in Hepatocytes.

Background: Magnesium influences hepatic lipid deposition in vertebrates, but the underlying mechanism is unknown.Objective: We used yellow catfish and their isolated hepatocytes to test the hypothesis that magnesium influences lipid deposition by modulating lipogenesis and lipolysis.Methods: Juvenile yellow catfish (mean ± SEM weight: 3.43 ± 0.02 g, 3 mo old, mixed sex) were fed a 0.14- (low), 0.87- (intermediate) or 2.11- (high) g Mg/kg diet for 56 d. Primary hepatocytes were incubated for 48 h in control or MgSO4-containing medium with or without 2-h pretreatment with an inhibitor (AG490, GW6471, or Compound C). Growth performance, cell viability, triglyceride (TG) concentrations, and expression of enzymes and genes involved in lipid metabolism were measured. Results: Compared with fish fed low magnesium, those fed intermediate or high magnesium had lower hepatic lipids (18%, 22%) and 6-phosphogluconate dehydrogenase (6PGD; 3.7%, 3.8%) and malic enzyme (ME; 35%, 48%) activities and greater mRNA levels of the lipolytic genes adipose triacylglyceride lipase (atgl; 82% and 1.7-fold) and peroxisome proliferator-activated receptor (ppara; 18% and 1.0-fold), respectively (P < 0.05). Relative mRNA levels of AMP-activated protein kinase (ampk) a1, ampka2, ampkb1, ampkb2, ampkg1a, ampkg1b, Janus kinase (jak) 2a, jak2b, and signal transducers and activators of transcription (stat) 3 in fish fed high magnesium were higher (24% to 3.1-fold, P < 0.05) than in those fed low or intermediate magnesium. Compared with cells incubated with MgSO4 alone, those incubated with MgSO4 and pretreated with AG490, GW6471, or Compound C had greater TG concentrations (42%, 31%, or 56%), g6pd (98%, 59%, or 51%), 6pgd (68%, 73%, or 32%) mRNA expression, and activities of G6PD (35%, 45%, or 16%) and ME (1.5-fold, 1.3-fold, or 13%), and reduced upregulation (61%, 25%, or 45%) of the lipolytic gene, atgl (P < 0.05).Conclusions: Magnesium reduced hepatic lipid accumulation in yellow catfish and the variation might be attributed to inhibited lipogenesis and increased lipolysis. PPARA, JAK-STAT, and AMPK pathways mediated the magnesium-induced changes in lipid deposition and metabolism. These results offer new insight into magnesium nutrition in vertebrates.

AKTs/PKBs: molecular characterization, tissue expression and transcriptional responses to insulin and/or wortmannin in yellow catfish Pelteobagrus fulvidraco.

In the present study, four AKT isoforms termed AKT1, AKT2, AKT3a and AKT3b were isolated and characterized from yellow catfish. Their molecular characterizations, tissue expressions and transcriptional responses to insulin and/or wortmannin were determined. The validated complementary DNA (cDNA) of yellow catfish AKT1, AKT2, AKT3a and AKT3b were 1422, 1431, 1389 and 1440 bp in length, encoding the peptide of 472, 475, 462 and 479 amino acid residues, respectively. The amino acid sequences of yellow catfish AKTs possessed all the characteristics of AKTs in other species. AKT1, AKT2 and AKT3b contained a conserved domain structure including a specific PH domain, a central catalytic domain and a C-terminal regulatory domain, while AKT3a lacked the C-terminal regulatory domain. All mRNAs of AKTs were expressed at the highest levels in the ovary. Among other tissues, the messenger RNA (mRNA) of AKT1 was widely distributed in all tested tissues, and AKT2 mRNA was more abundant in the muscle, liver and fat and lowest in other tested tissues, while AKT3a mRNA was predominant in the brain and showed no significant difference among other tested tissues, and AKT3b mRNA was highly expressed in the ovary, followed by the brain, muscle and fat and was relatively low in other tissues. Intraperitoneal insulin injection and incubation increased the mRNA expression of AKT1 and AKT2, but not that of AKT3a and AKT3b in the liver and hepatocytes of yellow catfish. Wortmannin reduced the mRNA level of all AKT isoforms and also alleviated the insulin-induced changes of AKT2 expression. The present study cloned full-length cDNA sequences of four AKTs in fish and determined their tissue expression profiles and studied their transcriptional responses to insulin and/or wortmannin, which serves to increase our understanding of their physiological function in lipid metabolism in fish.

IRS1 and IRS2: molecular characterization, tissue expression and transcriptional regulation by insulin in yellow catfish Pelteobagrus fulvidraco.

The insulin receptor substrate (IRS) proteins, in particular, IRS1 and IRS2, are the key downstream players of insulin signaling pathway and the regulation of lipid metabolism. In the present study, two genes of IRS (IRS1 and IRS2) were isolated and characterized from yellow catfish Pelteobagrus fulvidraco. Their molecular characterizations, tissue expressions, and transcriptional levels by insulin both in vivo and in vitro were determined. The validated complementary DNAs encoding for IRS1 and IRS2 were 3693 and 3177 bp in length, encoding proteins of 1230 and 1058 amino acid residues, respectively. Similarly to mammals, amino acid sequence alignment revealed that IRSs contained an N-terminal pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and several C-terminal multiple sites of tyrosine phosphorylation. Both IRS1 and IRS2 were widely expressed across the ten tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, anterior intestine, heart, mid-kidney, and ovary), but at the variable levels. Insulin injection at 1 µg/g in vivo significantly stimulated the messenger RNA (mRNA) expression of IRS2, but not IRS1 mRNA expression levels in the liver of yellow catfish after 48 h. In hepatocytes of yellow catfish, insulin incubation significantly stimulated the IRS1 (at a 1000 nM insulin group) and IRS2 (at both 100 and 1000 nM insulin groups) mRNA expressions, which indicated that IRS2 was more sensitive than IRS1 to insulin stimulation in the liver of yellow catfish, and IRS2 played a more important role in mediating insulin's effects on the liver metabolism. The present study serves to increase our understanding into the function of IRS in fish.

Effects and mechanisms of waterborne copper exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to determine the effects and mechanism of waterborne copper (Cu) exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco. To this end, two experiments were conducted. In Exp. 1, the partial cDNA sequences of three steroidogenesis-related genes (androgen receptor (ar), steroidogenic factor 1 (sf-1) and steroidogenic acute regulatory protein (star)) were firstly characterized from P. fulvidraco. The predicted amino acid sequences for the P. fulvidraco ar, sf-1 and star contained the main structural features characteristic in other species. In Exp. 2, P. fulvidraco were exposed to three waterborne Cu concentrations (control, 30µg/l and 60µg/l, respectively) for 56days. Sampling occurred on day 28 and day 56, respectively. On day 28, the levels of serum sex-steroid hormones (FSH and LH) and the mRNA levels of steroidogenesis-related genes (3ß-hsd, cyp11a1, cyp17, cyp19a, sf-1 and star) were significantly increased in ovary of P. fulvidraco exposed to 30µg Cu/l. The immunohistochemical analysis showed the positive reaction of ER, VTG and aromatase in low dose exposure group. These indicated that in low dose and relative short-term exposure, Cu was beneficial. In contrast, 60µg Cu/l exposure significantly reduced the levels of serum FSH, LH, E2 and P, and the mRNA levels of ovarian 20ß-hsd, cyp19a and erα in P. fulvidraco. On day 56, waterborne Cu concentration exposure reduced the levels of serum gonadotropins and sex hormones, and down-regulated the mRNA levels of steroidogenesis-related genes, indicating long-term Cu exposure had toxic effect on the secretion of sex-steroid hormone in P. fulvidraco. For the first time, our study cloned cDNA sequences of ar, sf-1 and star in P. fulvidraco, and demonstrated the effects and mechanism of waterborne Cu exposure influencing hormones secretion and synthesis in dose- and time-dependent manner in P. fulvidraco, which will help to understand the Cu-induced reproductive toxicity at both protein and transcriptional levels in fish.

Five metal elements homeostasis-related genes in Synechogobius hasta: Molecular characterization, tissue expression and transcriptional response to Cu and Fe exposure.

Two isoforms of Cu transporter (CTR1 and CTR2) and metallothionein (MT1 and MT2), and divalent metal ion transporter 1 (DMT1) were cloned and characterized in Synechogobius hasta, respectively. The protein sequences of S. hasta CTRs possessed two methionine-rich regions (MxM and MxxxM) and three transmembrane regions. At the C-terminus, CTR1 contained a sequence of conserved cysteine and histidine residues (HCH), while CTR2 did not contain the conserved sequence. The protein sequence of S. hasta DMT1 possessed all the characteristic features of DMT1, including twelve conserved hydrophobic cores of transmembrane domains. The protein sequences of S. hasta MTs were highly conserved in the total number of cysteine residues and their locations. mRNA of the five genes were expressed in a wide range of tissues but the levels were relatively higher in the liver. Cu exposure tended to up-regulate the mRNA expressions of CTR2, DMT1, MT1 and MT2. However, Fe down-regulated the Cu-induced increase of CTR2 and DMT1 mRNA levels. For the first time, our study cloned and characterized CTR1, CTR2, DMT1, MT1 and MT2 genes in S. hasta and determined their tissue-specific expression, and also the transcriptional change by Cu and Fe exposure, which shed new light on the CuFe relationship and help to understand the basic mechanisms of Cu and Fe homeostasis in fish.

Enhanced autophagy reveals vulnerability of P-gp mediated epirubicin resistance in triple negative breast cancer cells.

Epirubicin (EPI) is widely used for triple negative breast cancer (TNBC), but a substantial number of patients develop EPI resistance that is associated with poor outcome. The underlying mechanism for EPI resistance remains poorly understood. We have developed and characterized an EPI-resistant (EPI-R) cell line from parental MDA-MB-231 cells. These EPI-R cells reached stable growth in the medium containing 8 µg/ml of EPI. They overexpressed P-glycoprotein (P-gp) and contained numerous autophagic vacuoles. The suppression of P-gp overexpression and/or autophagy restored the sensitivity of these EPI-R cells to EPI. We further show that autophagy conferred resistance to EPI on MDA cells by blocking the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated pro-apoptotic signals. Together, these results reveal a synergistic role of P-gp, autophagy, and NF-κB pathways in the development of EPI resistance in TNBC cells. They also suggest that blocking the P-gp overexpression and autophagy may be an effective means of reducing EPI resistance.

Cloning, mRNA expression and transcriptional regulation of five retinoid X receptor subtypes in yellow catfish Pelteobagrus fulvidraco by insulin.

Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and mediate development, reproduction, homeostasis and cell differentiation processes in vertebrates. In this study, full-length cDNA sequences of five rxr subtypes from yellow catfish Pelteobagrus fulvidraco were cloned. Their mRNA expression patterns in different tissues and transcriptional regulation by insulin were determined. Five P. fulvidraco rxr (Pf-rxr) subtypes differed in the length of cDNA sequence and the open reading frame, but shared the similar domain structures as in typical nuclear receptors. Phylogenetic analysis revealed that the five Pf-rxr subtypes were paralogous genes, and that Pf-rxrßa and Pf-rxrßb had arisen during a teleost-specific genome duplication event. Five subtypes of Pf-rxr were detected in all the tested tissues. Overlapping and distinct expression patterns were found for different Pf-rxr subtypes, suggesting functional redundancy and divergence of these duplicates. Intraperitoneal insulin injection and incubation reduced the mRNA expression of Pf-rxrgb, but not other subtypes, in the liver and hepatocytes of P. fulvidraco, respectively, suggesting that Pf-rxrgb is the dominant rxr subtype involved in the insulin signaling pathway in P. fulvidraco.

Endoplasmic reticulum stress and disturbed calcium homeostasis are involved in copper-induced alteration in hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to investigate the effect of Cu exposure on ER stress and Ca(2+) homeostasis, and explore the underlying mechanism of the ER stress and disturbed Ca(2+) homeostasis in the regulation of hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco. To this end, three experiments were conducted. In experiment 1, P. fulvidraco were exposed to three waterborne Cu concentrations for 56 days. Waterborne Cu exposure evoked ER stress and SREBP-1c activation and resulted in dysregulation of hepatic lipid metabolism in liver of P. fulvidraco in a time-dependent manner. In experiment 2, specific inhibitors 2-APB (IP3 receptor inhibitor) and dantrolene (RyR receptor inhibitor) were used to explore whether Ca(2+) release from ER was involved in the Cu-induced ER stress change. Dantrolene and 2-APB prevented Cu-induced intracellular Ca(2+) elevation, demonstrating that the release of Ca(2+) from the ER, mediated by both RyR and IP3R, contributed to dysregulation of lipid metabolism. In experiment 3, a chemical chaperone (PBA) was used to demonstrate whether Cu-induced alteration in lipid metabolism was suppressed through the attenuation of ER stress. PBA attenuated the Cu-induced elevation of mRNA expression of SREBP-1c, SCAP, ACC, FAS, GRP78/BiP, GRP94, CRT, eIF2α and XBP-1, and alleviated the Cu-induced downregulation of Insig-1. Based on these observations, these results reveal a link between ER stress and the change of lipid metabolism induced by Cu, which will help to understand the Cu-induced toxicity on cellular and molecular level, and provide some novel insights into the regulation of lipid metabolism in fish.

Effects of insulin and its related signaling pathways on lipid metabolism in the yellow catfish Pelteobagrus fulvidraco.

The influence of insulin on hepatic metabolism in fish is not well understood. The present study was therefore conducted to investigate the effects of insulin on lipid metabolism, and the related signaling pathways, in the yellow catfish Pelteobagrus fulvidraco. Hepatic lipid and intracellular triglyceride (TG) content, the activity and expression levels of several enzymes and the mRNA expression of transcription factors (PPARα and PPARγ) involved in lipid metabolism were determined. Troglitazone, GW6471, fenofibrate and wortmannin were used to explore the signaling pathways by which insulin influences lipid metabolism. Insulin tended to increase hepatic lipid accumulation, the activity of lipogenic enzymes (6PGD, G6PD, ME, ICDH and FAS) and mRNA levels of FAS, G6PD, 6PGD, CPT IA and PPARγ, but down-regulated PPARα mRNA level. The insulin-induced effect could be stimulated by the specific PPARγ activator troglitazone or reversed by the PI3 kinase/Akt inhibitor wortmannin, demonstrating that signaling pathways of PPARγ and PI3 kinase/Akt were involved in the insulin-induced alteration of lipid metabolism. The specific PPARα pathway activator fenofibrate reduced insulin-induced TG accumulation, down-regulated the mRNA levels of FAS, G6PD and 6PGD, and up-regulated mRNA levels of CPT IA, PPARα and PPARγ. The specific PPARα pathway inhibitor GW6471 reduced insulin-induced changes in the expression of all the tested genes, indicating that PPARα mediated the insulin-induced changes of lipid metabolism. The present results contribute new knowledge on the regulatory role of insulin in hepatic metabolism in fish.

Regulation and mechanism of leptin on lipid metabolism in ovarian follicle cells from yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to determine the effect of leptin on lipid metabolism in ovarian follicle cells of yellow catfish Pelteobagrus fulvidraco. For that purpose, primary ovarian follicle cells were isolated from yellow catfish, cultured and subjected to different treatments (control, 0.1% DMSO, 500ng/ml leptin, 500ng/ml leptin plus 100µM wortmannin, 500ng/ml leptin plus 50nM AG490, respectively) for 48h. Intracellular triglyceride (TG) content, the activities (CPT I, FAS, G6PD, and 6PGD) and/or expression level of several enzymes (CPT I, FAS, G6PD, 6PGD, ACCa and ACCb), as well as the mRNA expression of transcription factors (PPARα, PPARγ and SREBP-1) involved in lipid metabolism were determined. Recombinant human leptin (rt-hLEP) incubation significantly reduced intracellular TG content, activities and mRNA levels of FAS, G6PD and 6PGD, SREBP-1 and PPARγ, but enhanced activity and mRNA level of CPT I, PPARα and ACCa. Specific inhibitors AG490 and wortmannin of JAK-STAT and IRS-PI3K signaling pathways prevented leptin-induced changes, indicating that JAK-STAT and IRS-PI3K signaling pathways were involved in the process of leptin-induced changes of lipid metabolism. Based on these observations above, for the first time, our study indicated that leptin reduced lipid deposition by activating lipolysis and suppressing lipogenesis in ovarian follicles of yellow catfish, and both JAK-STAT and IRS-PI3K signaling pathways were involved in the changes of leptin-induced lipid metabolism.

Effects of recombinant human leptin administration on hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco: in vivo and in vitro studies.

The present study was conducted to investigate the effects and mechanism of leptin influencing lipid metabolism in yellow catfish Pelteobagrus fulvidraco. To this end, hepatic lipid (in vivo experiment) and intracellular triglyceride (TG) (in vitro experiment) content, the activities and/or expression level of several enzymes (CPT-1, 6PGD, G6PD, FAS, ME and ICDH) as well as the mRNA expression of transcription factors (PPARα, PPARγ and SREBP-1) involved in lipid metabolism were determined. Using the primary hepatocytes of yellow catfish, specific inhibitors AG490 (JAK-STAT inhibitor) and wortmannin (IRS-PI3K inhibitor) were used to explore the signaling pathways of leptin effects on lipid metabolism. Intraperitoneal injection of recombinant human leptin (rt-hLEP) significantly reduced hepatic lipid content, activities of lipogenic enzymes (6PGD, G6PD, ME, ICDH and FAS) as well as mRNA levels of 6PGD, G6PD, FAS, PPARγ and SREBP-1 genes, but up-regulated activity and mRNA level of CPT-1 and PPARα. Using primary hepatocytes, rt-hLEP incubation also reduced intracellular TG content, mRNA levels of G6PD and PPARγ genes, but enhanced mRNA levels of PPARα, CPT-1 and SREBP-1. Leptin-induced effects could partially be reversed by specific inhibitors AG490, suggesting that JAK-STAT signaling pathways played important roles in the process of leptin-induced changes in lipid metabolism. Wortmannin significantly suppressed the decrease of TG content induced by leptin, reflecting that IRS-PI3K was involved in the leptin-mediate changes as well. To our knowledge, the present study provides, for the first time, evidence that rt-hLEP can increase lipolysis and reduce lipogenesis at the both enzymatic and molecular levels in fish with the combination of in vivo with in vitro studies, which serves to increase our understanding into the roles and mechanisms of leptin regulating lipid metabolism in fish.

Three unsaturated fatty acid biosynthesis-related genes in yellow catfish Pelteobagrus fulvidraco: Molecular characterization, tissue expression and transcriptional regulation by leptin.

Three key genes, stearoyl-CoA desaturase-1 (SCD1), Δ6 fatty acyl desaturases (Fads2 (Δ6)) and elongase of very long-chain fatty acid 5 (ELOVL5) were firstly isolated and characterized from yellow catfish, Pelteobagrus fulvidraco. The protein sequence of yellow catfish SCD1 and Fads2 (Δ6) possessed all the characteristics of microsomal fatty acid Fads2 (Δ6), including three histidine boxes, two transmembrane regions and one N-terminal cytochrome b5 domain containing heme-binding motif. The protein sequence of yellow catfish ELOVL5 possessed characteristic features of ELOVL5, including multiple transmembrane regions, endoplasmic reticulum retention signal and a single histidine box. mRNAs of the three genes were expressed in various tissues, but their mRNA levels varied among tissues. Intraperitoneal injection of recombinant human leptin (rb-hLEP) inhibited mRNA expression of SCD1, but not Fads2 (Δ6) and ELOVL5. For the first time, our study cloned SCD1, Fads2 (Δ6) and ELOVL5 genes in Siluriformes and demonstrated their differential expression among tissues, and also differential regulation by leptin, which serves to increase our understanding on their physiological function in fish.