Ublituximab versus Teriflunomide in Relapsing Multiple Sclerosis.

BACKGROUND: The monoclonal antibody ublituximab enhances antibody-dependent cellular cytolysis and produces B-cell depletion. Ublituximab is being evaluated for the treatment of relapsing multiple sclerosis. METHODS: In two identical, phase 3, double-blind, double-dummy trials (ULTIMATE I and II), participants with relapsing multiple sclerosis were randomly assigned in a 1:1 ratio to receive intravenous ublituximab (150 mg on day 1, followed by 450 mg on day 15 and at weeks 24, 48, and 72) and oral placebo or oral teriflunomide (14 mg once daily) and intravenous placebo. The primary end point was the annualized relapse rate. Secondary end points included the number of gadolinium-enhancing lesions on magnetic resonance imaging (MRI) by 96 weeks and worsening of disability. RESULTS: A total of 549 participants were enrolled in the ULTIMATE I trial, and 545 were enrolled in the ULTIMATE II trial; the median follow-up was 95 weeks. In the ULTIMATE I trial, the annualized relapse rate was 0.08 with ublituximab and 0.19 with teriflunomide (rate ratio, 0.41; 95% confidence interval [CI], 0.27 to 0.62; P<0.001); in the ULTIMATE II trial, the annualized relapse rate was 0.09 and 0.18, respectively (rate ratio, 0.51; 95% CI, 0.33 to 0.78; P = 0.002). The mean number of gadolinium-enhancing lesions was 0.02 in the ublituximab group and 0.49 in the teriflunomide group (rate ratio, 0.03; 95% CI, 0.02 to 0.06; P<0.001) in the ULTIMATE I trial and 0.01 and 0.25, respectively (rate ratio, 0.04; 95% CI, 0.02 to 0.06; P<0.001), in the ULTIMATE II trial. In the pooled analysis of the two trials, 5.2% of the participants in the ublituximab group and 5.9% in the teriflunomide group had worsening of disability at 12 weeks (hazard ratio, 0.84; 95% CI, 0.50 to 1.41; P = 0.51). Infusion-related reactions occurred in 47.7% of the participants in the ublituximab group. Serious infections occurred in 5.0% in the ublituximab group and in 2.9% in the teriflunomide group. CONCLUSIONS: Among participants with relapsing multiple sclerosis, ublituximab resulted in lower annualized relapse rates and fewer brain lesions on MRI than teriflunomide over a period of 96 weeks but did not result in a significantly lower risk of worsening of disability. Ublituximab was associated with infusion-related reactions. (Funded by TG Therapeutics; ULTIMATE I and II ClinicalTrials.gov numbers, NCT03277261 and NCT03277248.).

Dietary selenium sources differentially regulate selenium concentration, mRNA and protein expression of representative selenoproteins in various tissues of yellow catfish Pelteobagrus fulvidraco.

The study was conducted to determine the effects of three dietary Se sources, such as sodium-selenite (S-S), seleno-yeast (S-Y) and seleno-methionine (S-M), on Se concentration, glutathione peroxidase (GPX) and TXNRD activities, and mRNA expression of fifteen representative selenoproteins, and protein expression of four endoplasmic reticulum-resided selenoproteins in a wide range of tissues of yellow catfish. Compared with S-S and S-M groups, dietary S-Y significantly decreased growth performance and feed utilisation of yellow catfish. Dietary Se sources significantly influenced Se contents in the spleen, dorsal muscle and the kidney, GPX activities in spleen, kidney, intestine, muscle and mesenteric fat, and TXNRD activities in the heart, intestine and mesenteric fat. Among ten tested tissues, dietary Se sources influenced mRNA expression of GPX4 and SELENOK in three tissues; GPX3, SELENOS and TXNRD2 in four tissues; SELENOF, SELENON and DIO2 in five tissues; SELENOM, GPX1/2 and TXNRD3 in six tissues; SELENOW in seven tissue and SELENOP and SELENOT in eight tissues. Based on these observations above, S-S and S-M seem to be suitable Se sources for improving growth performance and feed utilisation of yellow catfish. Dietary Se sources differentially influence the expression of selenoproteins in various tissues of yellow catfish. For the first time, we determined the expression of selenoproteins in fish in responses to dietary Se sources, which contributes to a better understanding of the functions and regulatory mechanisms of selenoporteins.

Sirt3-Sod2-mROS-Mediated Manganese Triggered Hepatic Mitochondrial Dysfunction and Lipotoxicity in a Freshwater Teleost.

Exposure to excessive manganese (Mn) is toxic to humans and animals. However, the toxic effects and mechanisms of excessive Mn influencing the vertebrates have been highly overlooked. In the present study, dietary Mn overload significantly increased hepatic lipid and Mn contents, decreased superoxide dismutase 2 (Sod2) activity, increased the Sod2 acetylation level, and induced mitochondrial dysfunction; Mn induced mitochondrial dysfunction through Mtf1/sirtuin 3 (Sirt3)-mediated acetylation of Sod2 at the sites K55 and K70. Meanwhile, mitochondrial oxidative stress was involved in Mn-induced lipotoxicity. Mechanistically, Mn-induced lipotoxicity was via oxidative stress-induced Hsf1 nucleus translocation and its DNA binding capacity to the regions of a peroxisome proliferator-activated receptor g (pparg) promoter, which in turn induced the transcription of lipogenic-related target genes. For the first time, our study demonstrated that Mn-induced hepatic lipotoxicity via a mitochondrial oxidative stress-dependent Hsf1/Pparg pathway and Mtf1/sirt3-mediated Sod2 acetylation participated in mitochondrial dysfunction. Considering that lipid metabolism and lipotoxicity are widely used as the biomarkers for environmental assessments of pollutants, our study provided innovative and important insights into Mn toxicological and environmental evaluation in aquatic environments.

Phospholipase C signal mediated the glucose-induced changes of glucose absorption and lipid accumulation in the intestinal epithelial cells of yellow catfish Pelteobagrus fulvidraco.

In present study, we explored the effects and the underlying mechanisms of phospholipase C (PLC) mediating glucose-induced changes in intestinal glucose transport and lipid metabolism by using U-73122 (a PLC inhibitor). We found that glucose incubation activated the PLC signal and U-73122 pre-incubation alleviated the glucose-induced increase in plcb2, plce1 and plcg1 mRNA expression. Meanwhile, U-73122 pre-treatment blunted the glucose-induced increase in sodium/glucose co-transporters 1/2 mRNA and protein expressions. U-73122 pre-treatment alleviated the glucose-induced increase in TAG content, BODIPY 493/503 fluorescence intensity, lipogenic enzymes (glucose 6-phospate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), malic enzyme and fatty acid synthase (FAS)) activity and the mRNA expressions of lipogenic genes and related transcription factors (6pgd, g6pd, fas, acca, srebp1 and carbohydrate response element-binding protein (chrebp)) in intestinal epithelial cells of yellow catfish. Further research found that U-73122 pre-incubation mitigated the glucose-induced increase in the ChREBP protein expression and the acetylation level of ChREBP in HEK293T cells. Taken together, these data demonstrated that the PLC played a major role in the glucose-induced changes of glucose transport and lipid metabolism and provide a new perspective for revealing the molecular mechanism of glucose-induced changes of intestinal glucose absorption, lipid deposition and metabolism.

Lipophagy mediated carbohydrate-induced changes of lipid metabolism via oxidative stress, endoplasmic reticulum (ER) stress and ChREBP/PPARγ pathways.

High-carbohydrate diets (HCD) can induce the occurrence of nonalcoholic fatty liver disease (NAFLD), characterized by dramatic accumulation of hepatic lipid droplets (LDs). However, the potential molecular mechanisms are still largely unknown. In this study, we investigated the role of autophagy in the process of HCD-induced changes of hepatic lipid metabolism, and to examine the process of underlying mechanisms during these molecular contexts. We found that HCD significantly increased hepatic lipid accumulation and activated autophagy. Using primary hepatocytes, we found that HG increased lipid accumulation and stimulated the release of NEFA by autophagy-mediated lipophagy, and that lipophagy significantly alleviated high glucose (HG)-induced lipid accumulation. Oxidative and endoplasmic reticulum (ER) stress pathways played crucial regulatory roles in HG-induced lipophagy activation and HG-induced changes of lipid metabolism. Further investigation found that HG-activated lipophagy and HG-induced changes of lipid metabolism were via enhancing carbohydrate response element-binding protein (ChREBP) DNA binding capacity at PPARγ promoter region, which in turn induced transcriptional activation of the key genes related to lipogenesis and autophagy. The present study, for the first time, revealed the novel mechanism for lipophagy mediating HCD-induced changes of lipid metabolism by oxidative stress and ER stress, and ChREBP/PPARγ pathways. Our study provided innovative evidence for the direct relationship between carbohydrate and lipid metabolism via ChREBP/PPARγ pathway.

Dietary Glucose Increases Glucose Absorption and Lipid Deposition via SGLT1/2 Signaling and Acetylated ChREBP in the Intestine and Isolated Intestinal Epithelial Cells of Yellow Catfish.

BACKGROUND: Dietary carbohydrate affects intestinal glucose absorption and lipid deposition, but the underlying mechanisms are unknown. OBJECTIVES: We used yellow catfish and their isolated intestinal epithelial cells (IECs) to test the hypothesis that sodium/glucose cotransporters (SGLTs) 1/2 and acetylated carbohydrate response element binding protein (ChREBP) mediated glucose-induced changes in glucose absorption and lipid metabolism. METHODS: Yellow catfish (mean ± SEM weight: 4.68 ± 0.02 g, 3 mo old, mixed sex) were fed diets containing 250 g carbohydrates/kg from glucose (G, control), corn starch (CS), sucrose (S), potato starch (PS), or dextrin (D) for 10 wk. IECs were isolated from different yellow catfish and incubated for 24 h in a control or glucose (15 mM) solution with or without a 2-h pretreatment with an inhibitor [sotagliflozin (LX-4211) or tubastatin A (TBSA)]. Human embryonic kidney cells (HEK293T cells) were transfected with a Flag-ChREBP plasmid to explore ChREBP acetylation. Triglyceride (TG) and glucose concentrations and enzymatic activities were measured in the intestine and IECs of yellow catfish. They also were subjected to immunofluorescence, immunoprecipitation, qPCR, and immunoblotting. Immunoblotting and immunoprecipitation were performed with HEK293T cells. RESULTS: The G group had greater intestine TGs (0.99- to 2.30-fold); activities of glucose 6-phospate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase (0.12- to 2.10-fold); and expression of lipogenic genes (0.32- to 2.34-fold) than the CS, PS, and D groups. The G group had greater intestine sglt1/2 mRNA and protein expression than the CS, S and D groups (0.35- to 1.12-fold and 0.40- to 4.67-fold, respectively), but lower mRNA amounts of lipolytic genes (48.6%-65.8%) than the CS and PS groups. LX-4211 alleviated the glucose-induced increase in sglt1/2 mRNA (38.2%-47.4%) and SGLT1 protein (48.0%) expression, TGs (29.3%), and lipogenic enzyme activities (27.7%-42.1%) and gene expression (38.0%-55.5%) in the IECs. TBSA promoted the glucose-induced increase in TGs (11.3%), fatty acid synthase activity (32.6%), and lipogenic gene expression (21.6%-34.4%) in the IECs and acetylated ChREBP (10.5%) in HEK293T cells. CONCLUSIONS: SGLT1/2 signaling and acetylated ChREBP mediated glucose-induced changes in glucose absorption and lipid metabolism in the intestine and IECs of yellow catfish.

Waterborne copper exposure up-regulated lipid deposition through the methylation of GRP78 and PGC1α of grass carp Ctenopharyngodon idella.

Early molecular events after the exposure of heavy metals, such as aberrant DNA methylation, suggest that DNA methylation was important in regulating physiological processes for animals and accordingly could be used as environmental biomarkers. In the present study, we found that copper (Cu) exposure increased lipid content and induced the DNA hypermethylation at the whole genome level. Especially, Cu induced hypermethylation of glucose-regulated protein 78 (grp78) and peroxisome proliferator-activated receptor gamma coactivator-1α (pgc1α). CCAAT/enhancer binding protein α (C/EBPα) could bind to the methylated sequence of grp78, whereas C/EBPß could not bind to the methylated sequence of grp78. These synergistically influenced grp78 expression and increased lipogenesis. In contrast, DNA methylation of PGC1α blocked the specific protein 1 (SP1) binding and interfered mitochondrial function. Moreover, Cu increased reactive oxygen species (ROS) production, activated endoplasmic reticulum (ER) stress and damaged mitochondrial function, and accordingly increased lipid deposition. Notably, we found a new toxicological mechanism for Cu-induced lipid deposition at DNA methylation level. The measurement of DNA methylation facilitated the use of these epigenetic biomarkers for the evaluation of environmental risk.

Isolation and Characterization of Three Sodium-Phosphate Cotransporter Genes and Their Transcriptional Regulation in the Grass Carp Ctenopharyngodon idella.

It is important to explore the regulatory mechanism of phosphorus homeostasis in fish, which help avoid the risk of P toxicity and prevent P pollution in aquatic environment. The present study obtained the full-length cDNA sequences and the promoters of three SLC20 members (slc20a1a, slc20a1b and slc20a2) from grass carp Ctenopharyngodon idella, and explored their responses to inorganic phosphorus (Pi). Grass carp SLC20s proteins possessed conservative domains and amino acid sites relevant with phosphorus transport. The mRNAs of three slc20s appeared in the nine tissues, but their expression levels were tissue-dependent. The binding sites of three transcription factors (SREBP1, NRF2 and VDR) were predicted on the slc20s promoters. The mutation and EMSA analysis indicated that: (1) SREBP1 binding site (-783/-771 bp) negatively but VDR (-260/-253 bp) binding site positively regulated the activities of slc20a1a promoter; (2) SREBP1 (-1187/-1178 bp), NRF2 (-572/-561 bp) and VDR(615/-609 bp) binding sites positively regulated the activities of slc20a1b promoter; (3) SREBP1 (-987/-977 bp), NRF2 (-1469/-1459 bp) and VDR (-1124/-1117 bp) binding sites positively regulated the activities of the slc20a2 promoter. Moreover, Pi incubation significantly reduced the activities of three slc20s promoters, and Pi-induced transcriptional inactivation of slc20s promoters abolished after the mutation of the VDR element but not SREBP1 and NRF2 elements. Pi incubation down-regulated the mRNA levels of three slc20s. For the first time, our study elucidated the transcriptional regulatory mechanisms of SLC20s and their responses to Pi, which offered new insights into the Pi homeostatic regulation and provided the basis for reducing phosphorus discharge into the waters.

Functional Analysis of Steroidogenic Factor 1 (sf-1) and 17α-Hydroxylase/Lyase (cyp17α) Promoters in Yellow Catfish Pelteobagrus fulvidraco.

The present study was performed to clone and characterize the structures and functions of steroidogenic factor 1 (sf-1) and 17α-hydroxylase/lyase (cyp17α) promoters in yellow catfish Pelteobagrus fulvidraco, a widely distributed freshwater teleost. We successfully obtained 1981 and 2034 bp sequences of sf-1 and cyp17α promoters, and predicted the putative binding sites of several transcription factors, such as Peroxisome proliferator-activated receptor alpha (PPARα), Peroxisome proliferator-activated receptor gamma (PPARγ) and Signal transducer and activator of transcription 3 (STAT3), on sf-1 and cyp17α promoter regions, respectively. Overexpression of PPARγ significantly increased the activities of sf-1 and cyp17α promoters, but overexpression of PPARα significantly decreased the promoter activities of sf-1 and cyp17α. Overexpression of STAT3 reduced the activity of the sf-1 promoter but increased the activity of the cyp17α promoter. The analysis of site-mutation and electrophoretic mobility shift assay suggested that the sf-1 promoter possessed the STAT3 binding site, but did not the PPARα or PPARγ binding sites. In contrast, only the PPARγ site, not PPARα or STAT3 sites, was functional with the cyp17α promoter. Leptin significantly increased sf-1 promoter activity, but the mutation of STAT3 and PPARγ sites decreased leptin-induced activation of sf-1 promoter. Our findings offered the novel insights into the transcriptional regulation of sf-1 and cyp17α and suggested leptin regulated sf-1 promoter activity through STAT3 site in yellow catfish.

Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

Zinc (Zn) deficiency is the most consistently discovered nutritional manifestations of fatty liver disease. Although Zn is known to stimulate hepatic lipid oxidation, little is known about its underlying mechanism of action in lipolysis. Given the potential role of lipophagy in lipid metabolism, the purpose of this study was to test the hypothesis that Zn attenuates hepatic lipid accumulation by modulating lipophagy. The present study indicated that Zn is a potent promoter of lipophagy. Zn administration significantly alleviated hepatocellular lipid accumulation and increased the release of free fatty acids in association with enhanced fatty acid oxidation and inhibited lipogenesis, which was accompanied by activation of autophagy. Moreover, Zn reduced lipid accumulation and stimulated lipolysis by autophagy-mediated lipophagy. Zn-induced up-regulation of autophagy and lipid depletion is free Zn2+-dependent in the cytosols. Zn-induced autophagy and lipid turnover involved up-regulation of the calcium/calmodulin-dependent protein kinase kinase-ß (Ca2+/CaMKKß)/AMPK pathway. Meanwhile, Zn2+-activated autophagy and lipid depletion were via enhancing metal response element-binding transcription factor (MTF)-1 DNA binding at PPARα promoter region, which in turn induced transcriptional activation of the key genes related to autophagy and lipolysis. Zn activated the pathways of Zn2+/MTF-1/ Peroxisome proliferator-activated receptor (PPAR)α and Ca2+/CaMKKß/AMPK, resulting in the up-regulation of lipophagy and accordingly reduced hepatic lipid accumulation. Our study, for the first time, provided innovative evidence of the direct relationship between metal elements (Zn) and lipid metabolism. The present study also indicated the novel mechanism for Zn-induced lipolysis by the activation of Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways, which induced the occurrence of lipophagy. These results provide new insight into Zn nutrition and its potential beneficial effects on the prevention of fatty liver disease in vertebrates.-Wei, C.-C., Luo, Z., Hogstrand, C., Xu, Y.-H., Wu, L.-X., Chen, G.-H., Pan, Y.-X., Song, Y.-F. Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

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.

Functional Analysis of Promoters from Three Subtypes of the PI3K Family and Their Roles in the Regulation of Lipid Metabolism by Insulin in Yellow Catfish Pelteobagrus fulvidraco.

In the present study, the length of 360, 1848 and 367 bp sequences of promoters from three subtypes of PI3K family (PI3KCa, PI3KC2b and PI3KC3) of yellow catfish Pelteobagrus fulvidraco were cloned and characterized. Bioinformatics analysis revealed that PI3KCa, PI3KC2b and PI3KC3 had different structures in their core promoter regions. The promoter regions of PI3KCa and PI3KC2b had CpG islands but no CAAT and TATA box. In contrast, the promoter of PI3KC3 had the canonical TATA and CAAT box but no CpG island. The binding sites of several transcription factors, such as HNF1, STAT and NF-κB, were predicted on PI3KCa promoter. The binding sites of transcription factors, such as FOXO1, PPAR-RXR, STAT, IK1, HNF6 and HNF3, were predicted on PI3KC2b promoter and the binding sites of FOXO1 and STAT transcription factors were predicted on PI3KC3 promoter. Deletion analysis indicated that these transcriptional factors were the potential regulators to mediate the activities of their promoters. Subsequent mutation analysis and electrophoretic mobility-shift assay (EMSA) demonstrated that HNF1 and IK1 directly bound with PI3KCa and PI3KC2b promoters and negatively regulated the activities of PI3KCa and PI3KC2b promoters, respectively. Conversely, FOXO1 directly bound with the PI3KC2b and PI3KC3 promoters and positively regulated their promoter activities. In addition, AS1842856 (AS, a potential FOXO1 inhibitor) incubation significantly reduced the relative luciferase activities of several plasmids of PI3KC2b and PI3KC3 but did not significantly influence the relative luciferase activities of the PI3KCa plasmids. Moreover, by using primary hepatocytes from yellow catfish, AS incubation significantly down-regulated the mRNA levels of PI3KCa, PI3KC2b and PI3KC3 and reduced triacylglyceride (TG) accumulation and insulin-induced TG accumulation, as well as the activities and the mRNA levels of several genes involved in lipid metabolism. Thus, the present study offers new insights into the mechanisms for transcriptional regulation of PI3Ks and for PI3Ks-mediated regulation of lipid metabolism by insulin in fish.

Characterization and mechanism of phosphoinositide 3-kinases (PI3Ks) members in insulin-induced changes of protein metabolism in yellow catfish Pelteobagrus fulvidraco.

In the present study, seven phosphoinositide 3-kinase (PI3K) members (PI3KCa, PI3KCb, PI3KCd, PI3KCg, PI3KC2a, PI3KC2b and PI3KC3, respectively) were isolated and characterized from yellow catfish Pelteobagrus fulvidraco, and their roles in insulin-induced changes of protein metabolism were determined. These seven PI3Ks can be divided into three classes, class I (including PI3KCa, PI3KCb, PI3KCd and PI3KCg), class II (including PI3KC2a and PI3KC2b) and class III (only including PI3KC3). Compared with mammals, all of these members share similar domain structure. Their mRNAs were widely expressed across ten tested tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, intestine, heart, kidney and ovary), but at variable levels. In the in vivo study, insulin treatment significantly increased hepatic protein content at 3h, accompanied with reduced plasma total amino acid contents and liver ALT activity, and with increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in liver. At 6h and 12h, insulin injection showed no significant effect on liver protein content and plasma total amino acid, but reduced liver ALT activity and increased liver total RNA and the mRNA levels of AKT2, mTORC1 and S6K1 in liver at 6h. In the in vitro study, insulin incubation also tended to increase protein content of hepatocytes, accompanied with reduced cell medium total amino acid contents and hepatocytes ALT activity, and increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in hepatocytes. However, insulin treatment showed no significant effect on GDH activity and mRNA expression of PI3KCa, PI3KCd, PI3KCg, PI3KC2b, PI3KC3 and eEF2 both in the in vivo and in vitro studies. Effects of insulin on the mRNA levels of eIF-4E and 4E-BP1 were different between the in vivo and in vitro studies, and also time-dependent. Compared to single insulin group, insulin+wortmannin group increased ALT activity at 6h but reduced T-RNA content at 6 and 12h. AKT2 and S6K1 mRNA levels at 6 and 12h, mRNA levels of mTORC1, 4E-BP1 and eEF2 at 3 and 6h, and EIF-4E mRNA levels at 3 and 12h, PI3KCb and PI3KC2a mRNA levels were significantly lower in insulin+wortmannin group than those in single insulin group. Thus, our study demonstrated that among seven PI3K members, PI3KCb and PI3KC2a were more sensitive to the insulin signaling pathway, and insulin stimulated hepatic protein synthesis in yellow catfish through PI3K signaling pathway.

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.

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.

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.

Safety and pharmacokinetics of ramucirumab in combination with docetaxel in Japanese patients with locally advanced or metastatic breast cancer: a Phase Ib study.

OBJECTIVE: The primary objective of this study was to investigate the safety and tolerability and to confirm the recommended dose of the anti-vascular endothelial growth factor receptor 2 monoclonal antibody ramucirumab in combination with docetaxel in Japanese patients with metastatic/locally advanced breast cancer. METHODS: In this multicenter, single-arm, Phase Ib trial, eligibility criteria included: 20 years or older, Eastern Cooperative Oncology Group performance status of 0/1 and confirmed diagnosis of human epidermal growth factor receptor 2-negative metastatic/locally recurrent inoperable breast adenocarcinoma. Patients received docetaxel (75 mg/m2) followed by ramucirumab (10 mg/kg) on Day 1 of 21-day cycles. Recommended dose was defined as <33% dose-limiting toxicities in dose-limiting toxicity-evaluable patients in Cycle 1. The safety, pharmacokinetics, immunogenicity and antitumor activity were examined. RESULTS: Seven patients were treated. Most adverse events were mild to moderate. Two patients during Cycle 1 experienced a dose-limiting toxicity; one patient each experienced Grade 3 febrile neutropenia and Grade 3 gingivitis. Both dose-limiting toxicities subsequently resolved. No patients discontinued study therapies during Cycle 1. Four serious adverse events were possibly related to ramucirumab in combination with docetaxel. Anti-ramucirumab antibodies were not detected. Pharmacokinetic analysis revealed low total body clearance and long apparent terminal elimination half-life (~7-12 days). Partial response was reported in four patients. CONCLUSIONS: The combination of ramucirumab and docetaxel was tolerable in female Japanese patients with breast cancer. Ramucirumab 10 mg/kg in combination with docetaxel (75 mg/m2) was confirmed as the recommended dose among Japanese patients, supporting its use in future studies.

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.

JAK and STAT members of yellow catfish Pelteobagrus fulvidraco and their roles in leptin affecting lipid metabolism.

The present study clones and characterizes the full-length cDNA sequences of members in JAK-STAT pathway, explores their mRNA tissue expression and the biological role in leptin influencing lipid metabolism in yellow catfish Pelteobagrus fulvidraco. Full-length cDNA sequences of five JAKs and seven STAT members, including some splicing variants, were obtained from yellow catfish. Compared to mammals, more members of the JAKs and STATs family were found in yellow catfish, which provided evidence that the JAK and STAT family members had arisen by the whole genome duplications during vertebrate evolution. All of these members were widely expressed across the eleven tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, anterior intestine, heart, mid-kidney, testis and ovary) but at the variable levels. Intraperitoneal injection in vivo and incubation in vitro of recombinant human leptin changed triglyceride content and mRNA expression of several JAKs and STATs members, and genes involved in lipid metabolism. AG490, a specific inhibitor of JAK2-STAT pathway, partially reversed leptin-induced effects, indicating that the JAK2a/b-STAT3 pathway exerts main regulating actions of leptin on lipid metabolism at transcriptional level. Meanwhile, the different splicing variants were differentially regulated by leptin incubation. Thus, our data suggest that leptin activated the JAK/STAT pathway and increases the expression of target genes, which partially accounts for the leptin-induced changes in lipid metabolism in yellow catfish.

A phase 2, single-arm study of ramucirumab in patients with metastatic renal cell carcinoma with disease progression on or intolerance to tyrosine kinase inhibitor therapy.

BACKGROUND: Multitargeted tyrosine kinase inhibitors (TKIs) have antitumor activity in metastatic renal cell carcinoma (mRCC). Resistance to these agents develops frequently, and their use is often limited by intolerance. Ramucirumab is a recombinant human monoclonal antibody directed against human vascular endothelial growth factor receptor-2. For this study, the authors investigated the clinical efficacy and safety of ramucirumab in patients with TKI-resistant/intolerant mRCC. METHODS: In this single-arm phase 2 trial, patients received ramucirumab 8 mg/kg every 2 weeks until they developed disease progression or intolerable toxicity. The primary endpoint was the best objective response rate (ORR); additional endpoints included the disease control rate (DCR), progression-free survival (PFS), the median duration of overall response, and safety. RESULTS: Thirty-nine patients with RCC received ramucirumab monotherapy. Prior TKI therapy included sunitinib (59% of patients), sunitinib and sorafenib (30.8% of patients), and sorafenib (10.3% of patients). The ORR was 5.1% (95% confidence interval [CI], 0.6%-17.3%). The 12-week DCR was 64.1% (95% CI, 47.2%-78.8%). The median PFS was 7.1 months (95% CI, 4.1-9.7 months), and the median overall survival was 24.8 months (95% CI, 18.9-32.6 months). Grade 3 or higher adverse events that occurred in ≥5% of patients included grade 3 hypertension (7.7%) and proteinuria (5.1%). There was 1 on-study death from multiorgan failure. CONCLUSIONS: Although the study did not meet its primary endpoint of ≥15% ORR, ramucirumab was associated with evidence of antitumor activity in patients with TKI-resistant/intolerant mRCC. Ramucirumab was safe and well tolerated.