Abundant and constant expression of uncoupling protein 2 in the liver of red sea bream Pagrus major

Four overlapping cDNA fragments encoding a partial sequence for uncoupling protein 2 (UCP2) were amplified by PCR using degenerate primers from the liver of a marine teleost fish, red sea bream (Pagrus major). The partial sequence was 674 bp long, encoding 224 amino acids. The deduced amino acid sequence from the cDNA partial sequence contained the signature motifs for mitochondrial transporter protein and revealed positional identity higher than 72.8% with UCP2 from mammals. The fish UCP2 gene was highly expressed in the liver but almost undetectable in the visceral mesenteric adipose tissue. Using beta-actin as control, the UCP2 mRNA level was determined to be at least 20-fold higher in the liver than in the visceral mesenteric adipose tissues. Neither 48 h starvation nor high lipid diet had any significant effect on liver UCP2 gene expression, indicating that the abundant UCP2 gene expression was stable and might have some basic function in a fish liver that always contains high lipid content. The striking contrast of UCP2 gene expression in the two fish fat-depot organs is consistent with their large differences in oxidative capacity. We suggest that the fish liver may adapt to a constantly high fat deposit by maintaining high UCP2 expression to constrain reactive oxygen species (ROS) production and protect hepatocytes from apoptosis.     

High Cholesterol Diet Induces IL-1β Expression in Adult but Not Larval Zebrafish

Recently, it has been demonstrated that high cholesterol diet induced hypercholesterolemia and vascular lipid oxidation and accumulation in zebrafish larvae, suggesting that zebrafish is a new promising atherosclerosis model in addition to mouse models. However, up to date, there was no report regarding inflammatory cytokine expression during the lipid accumulation in zebrafish larva and adult fish. In this study, we first demonstrated the expression levels of IL-1β and TNF-α in high cholesterol diet (HCD)-fed zebrafish larvae, and found that although HCD induced vascular lipid accumulation, the cytokine expressions in the larvae were not changed by HCD. Furthermore, there was no significant difference in leukocyte accumulation in vessels between control and HCD fed group. But prolonged HCD induced IL-1β expression in spleen and liver compared to those of control zebrafish, and produced very early stage of fatty streak lesion in dorsal aorta of 19 week HCD-fed zebrafish. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis.     

斑马鱼fem-1c cDNA克隆与表达分析

为进一步探究鱼类性别决定的相关机理, 增加对鱼类性控基因表达和功能的认识, 克隆斑马鱼fem-1c 基因并对其进行表达分析。研究采用RACE-PCR方法从斑马鱼卵巢组织cDNA中克隆了fem-1c的cDNA全长序列, 其大小为2701 bp, 编码618个氨基酸。生物信息学分析显示, 斑马鱼FEM-1C蛋白包含9个ANK结构域、2个TPR结构域和2个低复杂性区域, 与其他脊椎动物的FEM-1C蛋白序列保守性较高。脊椎动物的fem-1c与tmed7、trim36等邻近的45个基因具有保守的同线性关系。半定量RT-PCR实验结果显示斑马鱼fem-1c在受精后17d开始表达, 并特异地表达于成体卵巢组织中。RNA原位杂交结果显示, fem-1c基因mRNA定位于卵巢组织的Ⅰ期和Ⅱ期卵母细胞胞质中。fem-1c的时空表达特征暗示其在斑马鱼卵巢分化中具有重要作用。       

The effects of starvation and force-feeding on the metabolism of the Northern pike, Esox lucius L.

The effects of starvation and force-feeding on certain tissue and blood constituents were studied in the Northern pike, Esox lucius L. Starvation resulted in a reduction of liver and muscle glycogen and liver lipid. Blood glucose concentration and haematocrit were reduced, total plasma cholesterol levels were increased, while the levels of plasma free fatty acids (FFA), amio acid nitrogen and protein remained unaltered. No significant changes were observed in either muscle protein, muscle water or the response to amino acid loading during the starvation period.
The force-feeding of pike starved for 3 months resulted in liver lipid and muscle glycogen being increased to levels higher than those observed in freshly-captured fish. Liver glycogen, however, increased to values only slightly higher than those of starved animals. Furthermore, while force-feeding had little effect on plasma FFA or protein concentrations, blood glucose, plasma cholesterol and haematocrit returned to the levels found in freshlycaptured fish and those of amino acid nitrogen were higher.
The results indicate that pike are well adapted for periods of prolonged starvation and that hepatic and extra-hepatic lipid and glycogen stores serve for metabolic needs during food shortage, while body protein is conserved. The endocrine basis for these changes in the tissue and blood constituents is discussed.     

Structure, evolution, and liver-specific expression of sterol 12alpha-hydroxylase P450 (CYP8B).

The rat CYP8B cDNA encoding sterol 12alpha-hydroxylase was cloned and sequenced. The amino acid sequence of the heme-binding region of CYP8B was close to those of CYP7A (cholesterol 7alpha-hydroxylase) and CYP7B (oxysterol 7alpha-hydroxylase). Molecular phylogenetic analysis suggests that CYP8B and the CYP7 family derive from a common ancestor. The P450s of the CYP7 and CYP8 families, except for CYP8A (prostacyclin synthase), catalyze the oxygenation of sterols from an alpha surface in the middle of the steroid skeleton. These facts suggest that CYP8B is a P450 closely linked to those of the CYP7 family. CYP8B was expressed specifically in liver. Hepatic CYP8B mRNA level and the 12alpha-hydroxylase activity were altered by cholestyramine feeding, starvation, streptozotocin-induced diabetes mellitus, and administration of clofibrate, dexamethasone or thyroxin, indicating the pretranslational regulation of CYP8B expression. The enhanced CYP8B mRNA expression in streptozotocin-induced diabetic rats was significantly decreased by insulin within 3 h of its administration. These facts demonstrate a regulatory role of insulin in CYP8B expression as a suppressor.     

Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary

In the present study, two gonad cDNA libraries from zebrafish testes and ovaries were constructed and a total of 1025 expressed sequence tag (EST) clones were generated from the two libraries: 501 from the testis library and 524 from the ovary library. A total of 641 of the EST clones were identified to share significant sequence identity with known sequences in GenBank, representing at least 478 different zebrafish genes. In order to understand the molecular compositions of the two gonad organs, the expression profiles of the identified clones in these two gonad cDNA libraries were analyzed. Both gonad libraries have a higher portion of clones for nuclear proteins and a lower portion for proteins in translational machinery, cytoskeleton and mitochondria than our previously characterized whole-adult cDNA library. Most abundant cDNA clones in the two gonad libraries were identified and over 10% of ovary clones were found to encode egg membrane proteins (zona pellucida or ZP proteins). Furthermore, the testis library showed a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST projects; thus, the testis library can supply more unique and novel cDNA sequences in a zebrafish EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the gonad development in zebrafish. Eleven potential clones were selected to analyze their expression patterns by Northern blot hybridization. Most of them showed a specific or predominant expression in the expected testis or ovary tissue. At last, four of the clones were found, by section in situ hybridization, to be expressed specifically in the germ cells of the testis or ovary and thus they are suitable molecular markers for analyses of spermatogenesis and oogenesis.     

Lipolysis and lipophagy play individual and interactive roles in regulating triacylglycerol and cholesterol homeostasis and mitochondrial form in zebrafish

Neutral lipases-mediated lipolysis and acid lipases-moderated lipophagy are two main processes for degradation of lipid droplets (LDs). However, the individual and interactive roles of these metabolic pathways are not well known across vertebrates. This study explored the roles of lipolysis and lipophagy from the aspect of neutral and acid lipases in zebrafish. We established zebrafish strains deficient in either adipose triglyceride lipase (atgl^?/?; AKO fish) or lysosomal acid lipase (lal^?/?; LKO fish) respectively, and then inhibited lipolysis in the LKO fish and lipophagy in the AKO fish by feeding diets supplemented with the corresponding inhibitors Atglistatin and 3-Methyladenine, respectively. Both the AKO and LKO fish showed reduced growth, swimming activity, and oxygen consumption. The AKO fish did not show phenotypes in adipose tissue, but mainly accumulated triacylglycerol (TAG) in liver, also, they had large LDs in the hepatocytes, and did not stimulate lipophagy as a compensation response but maintained basal lipophagy. The LKO fish reduced total lipid accumulation in the body but had high cholesterol content in liver; also, they accumulated small LDs in the hepatocytes, and showed increased lipolysis, especially Atgl expression, as a compensatory mechanism. Simultaneous inhibition of lipolysis and lipophagy in zebrafish resulted in severe liver damage, with the potential to trigger mitophagy. Overall, our study illustrates that lipolysis and lipophagy perform individual and interactive roles in maintaining homeostasis of TAG and cholesterol metabolism. Furthermore, the interactive roles of lipolysis and lipophagy may be essential in regulating the functions and form of mitochondria.     

Cellular retinol-binding protein type II (CRBPII) in adult zebrafish (Danio rerio). cDNA sequence, tissue-specific expression and gene linkage analysis.

We have determined the nucleotide sequence of a zebrafish cDNA clone that codes for a cellular retinol-binding protein type II (CRBPII). Radiation hybrid mapping revealed that the zebrafish and human CRBPII genes are located in syntenic groups. In situ hybridization and emulsion autoradiography localized the CRBPII mRNA to the intestine and the liver of adult zebrafish. CRBPII and intestinal fatty acid binding protein (I-FABP) mRNA was colocalized to the same regions along the anterior-posterior gradient of the zebrafish intestine. Similarly, CRBPII and I-FABP mRNA are colocalized in mammalian and chicken intestine. CRBPII mRNA, but not I-FABP mRNA, was detected in adult zebrafish liver which is in contrast to mammals where liver CRBPII mRNA levels are high during development but rapidly decrease to very low or undetectable levels following birth. CRBPII and I-FABP gene expression appears therefore to be co-ordinately regulated in the zebrafish intestine as has been suggested for mammals and chicken, but CRBPII gene expression is markedly different in the liver of adult zebrafish compared to the livers of mammals. As such, retinol metabolism in zebrafish may differ from that of mammals and require continued production of CRBPII in adult liver. The primary sequence of the coding regions of fish and mammalian CRBPII genes, their relative chromosomal location in syntenic groups and possibly portions of the control regions involved in regulation of CRBPII gene expression in the intestine appear therefore to have been conserved for more than 400 million years.     

斑马鱼nr1d4a和nr1d4b基因的表达及其对不同环境刺激的响应

研究获得了斑马鱼nr1d4a和nr1d4b基因的cDNA,进行了序列比对和系统进化分析,并采用实时定量RT-PCR（qPCR）方法研究了其表达模式及对不同环境刺激的转录反应。研究发现,斑马鱼nr1d4a和nr1d4b是由基因复制产生的旁系同源基因,具有高度保守的DNA结合结构域和配体结合结构域。斑马鱼nr1d4a和nr1d4b的表达模式具有明显的差别。nr1d4a在胚胎发育早期的表达量很低,72 hpf时开始显著升高;而nr1d4b具有较高水平的母源性表达,6 hpf时的表达量明显降低,但也在72 hpf显著回升。nr1d4a在脑和肾脏中表达量最高,其次是鳃、卵巢、精巢和眼,在肝脏中的表达量最低;nr1d4b在卵巢中表达量最高,其次是精巢和脑,在肠道和心脏中表达量最低。斑马鱼nr1d4a和nr1d4b都能被多种环境刺激瞬时诱导表达。16℃低温处理0.5h就能显著诱导斑马鱼nr1d4a和nr1d4b基因的表达,但处理6h后其诱导效应开始下降并逐渐消失。除低温外,重金属（2 mol/L镉）、缺氧（5%氧气）和盐度（5）处理均能瞬时诱导nr1d4a和nr1d4b的表达,说明nr1d4a和nr1d4b基因可能参与斑马鱼对多种环境刺激的适应性反应。研究为深入揭示鱼类nr1d4a和nr1d4b基因的生物学功能及其表达调控机制奠定了基础。       

Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages

We have cloned the full-length cDNA for the human ATP binding cassette transporter 1 (hABC1). The 6603-bp open reading frame encodes a polypeptide of 2201 amino acids resulting in a deduced molecular weight of 220 kDa. The hABC1 cDNA is highly homologous (62%) to the human rim ABC transporter (ABCR). hABC1 is expressed in a variety of human tissues with highest expression levels found in placenta, liver, lung, adrenal glands, and fetal tissues. We demonstrate that the hABC1 expression is induced during differentiation of human monocytes into macrophages in vitro. In macrophages, both the hABC1 mRNA and protein expression are upregulated in the presence of acetylated low-density lipoprotein (AcLDL). The AcLDL-induced increase in hABC1 expression is reversed by cholesterol depletion mediated by the addition of high-density lipoprotein (HDL3). Our data, demonstrating sterol-dependent regulation of hABC1 in human monocytes/macrophages, suggest a novel role for this transporter molecule in membrane lipid transport.