京尼平对小鼠棕色和白色脂肪组织UCP1表达的影响

目的:棕色脂肪组织活化和白色脂肪组织棕化是改善减肥的良好策略。本研究利用冷刺激作为阳性对照,观察京尼平对小鼠脂肪组织活化与棕化的作用。方法:8周龄雄性C57BL/6J小鼠30只,随机分为正常对照组、京尼平组、冷刺激组, 每组10只。京尼平组小鼠腹腔注射给予京尼平处理(15 mg/(kg·d),连续9 d),对照组用生理盐水处理,冷刺激组小鼠在室温(22℃±2℃)下处理4 d后,置于4℃环境中进行冷刺激处理5 d(24 h/d)。检测各组小鼠每天摄食量、体重和体温变化,取肩胛下区、腹股沟区及附睾周围部分脂肪组织观察形态学的变化,测定棕色脂肪组织、皮下白色脂肪组织以及内脏白色脂肪组织解偶联蛋白1(UCP1)的表达。结果:与正常对照组相比,京尼平组小鼠白色脂肪湿重下降16%,冷刺激组下降28%,均有明显差异(P＜0.05);京尼平组和冷刺激组白色脂肪组织颜色变深,HE染色显示脂肪细胞内的脂滴变小,数量增加;京尼平组小鼠的皮下、内脏白色脂肪组织和棕色3种脂肪组织中的UCP1表达量均明显增加(P＜0.05)。结论:京尼平通过上调UCP1的表达促进棕色脂肪组织活化和白色脂肪组织棕化,此效应是京尼平降脂减轻体重的作用机制之一。     

Plant uncoupling protein in mitochondria from aged-dehydrated slices of Jerusalem artichoke tubers becomes sensitive to superoxide and to hydrogen peroxide without increase in protein level

We investigated the occurrence of the plant Uncoupling Protein (UCP) in mitochondria isolated from both fresh (f-JAM) and aged-dehydrated (a-d-JAM) slices of Jerusalem artichoke tubers (Helianthus tuberosus L.). The presence of UCP was shown by immunological analysis and its function was investigated by measuring the decrease of the mitochondrial membrane potential due to linoleic acid (LA) and its inhibition by purine nucleotides under conditions in which the adenine nucleotide translocator (ANT) was inhibited by atractyloside (Atr). f-JAM and a-d-JAM had the same protein content, but differed from one another with respect to purine nucleotide inhibition, substrate specificity, and sensitivity to ROS. Hydrogen peroxide and superoxide anion, generated in situ by xanthine plus xanthine oxidase, caused a significant increase in the UCP function in a-d-JAM, but not in f-JAM. This occurred in a manner sensitive to ATP, but not to Atr, thus showing that ANT has no role in the process. The dependence of the rate of membrane potential decrease on increasing LA concentrations, either in the absence or the presence of ROS, showed a sigmoidal saturation both in f-JAM and a-d-JAM. However, addition of ROS in a-d-JAM resulted in about 40% increase of the Vmax value, with no change in the K0.5 (about 20 microM), whereas in f-JAM no effect on either the Vmax or K0.5 (about 28 microM) was found. Furthermore, a decreased ROS production as a result of LA addition was found in both f-JAM and a-d-JAM, the effect being more marked in a-d-JAM.     

Overexpression of wheat mitochondrial uncoupling protein in rice plants confers tolerances to oxidative stresses promoted by exogenous hydrogen peroxide and low temperature

To obtain a better understanding of the function of mitochondrial uncoupling protein (UCP) in higher plants, the wheat gene for mitochondrial uncoupling protein (WhUCP) in rice was overexpressed by Agrobacterium-mediated transformation with a construct containing the WhUCP ORF under control of the 35S promoter. The transgenic rice plants showed a significant increase in tolerance against oxidative stress promoted by exogenous hydrogen peroxide at the seedling stage. The transgenic rice plants overexpressing WhUCP also exhibited greater tolerance against cold stress than did the wild-type plants. These results demonstrated that the mitochondrial UCP in higher plants is positively involved in the pathway for abiotic stress tolerance, probably through a decrease in cellular oxidative damage, and that controlled uncoupling by UCP could be used for improvement of stress tolerance in higher plants.Kenjirou Ozawa and Seiji Murayama are contributed equally to the work     

The role of uncoupling protein 2 in macrophages and its impact on obesity-induced adipose tissue inflammation and insulin resistance

The development of a chronic, low-grade inflammation originating from adipose tissue in obese subjects is widely recognized to induce insulin resistance, leading to the development of type 2 diabetes. The adipose tissue microenvironment drives specific metabolic reprogramming of adipose tissue macrophages, contributing to the induction of tissue inflammation. Uncoupling protein 2 (UCP2), a mitochondrial anion carrier, is thought to separately modulate inflammatory and metabolic processes in macrophages and is up-regulated in macrophages in the context of obesity and diabetes. Here, we investigate the role of UCP2 in macrophage activation in the context of obesity-induced adipose tissue inflammation and insulin resistance. Using a myeloid-specific knockout of UCP2 (Ucp2^ΔLysM), we found that UCP2 deficiency significantly increases glycolysis and oxidative respiration, both unstimulated and after inflammatory conditions. Strikingly, fatty acid loading abolished the metabolic differences between Ucp2^ΔLysM macrophages and their floxed controls. Furthermore, Ucp2^ΔLysM macrophages show attenuated pro-inflammatory responses toward Toll-like receptor-2 and -4 stimulation. To test the relevance of macrophage-specific Ucp2 deletion in vivo, Ucp2^ΔLysM and Ucp2^fl/fl mice were rendered obese and insulin resistant through high-fat feeding. Although no differences in adipose tissue inflammation or insulin resistance was found between the two genotypes, adipose tissue macrophages isolated from diet-induced obese Ucp2^ΔLysM mice showed decreased TNFα secretion after ex vivo lipopolysaccharide stimulation compared with their Ucp2^fl/fl littermates. Together, these results demonstrate that although UCP2 regulates both metabolism and the inflammatory response of macrophages, its activity is not crucial in shaping macrophage activation in the adipose tissue during obesity-induced insulin resistance.     

菠萝磷转运蛋白1家族基因鉴定及特征分析

磷转运蛋白1(phosphate transporter protein 1, PHT1)家族在植物对磷的吸收及再利用过程中发挥重要作用。该研究对菠萝PHT1基因(AcoPHT1)进行全基因组鉴定,并对基因结构、编码蛋白保守功能域和基因表达进行了分析。结果表明:(1)共鉴定到9个AcoPHT1基因,位于基因组7个连锁群上,所有基因均含有1～3个内含子,内含子相位类型多样。(2)除AcoPHT1.8外,AcoPHT1蛋白均为碱性蛋白,所有蛋白属于亲水性蛋白,且含有10～13个跨膜功能域,均具有保守的PHT1蛋白标签序列GGDYPLSATIxSE,主要定位于叶绿体和细胞质中。(3)AcoPHT1蛋白聚类在单子叶植物组和单双子叶植物混合组中,相对于拟南芥,水稻PHT1与菠萝PHT1相似度更高。(4)AcoPHT1基因启动子区含有P1BS、W-box等与磷吸收和响应胁迫有关的多个顺式作用元件。(5)靶基因预测分析显示,基因AcoPHT1.2、AcoPHT1.8和AcoPHT1.9受多个miRNA调控。(6)AcoPHT1基因表达存在组织特异性和功能冗余性,不同PHT1基因可能在菠萝不同组织或发育阶段发挥作用。该研究结果为菠萝PHT1家族基因的功能鉴定和育种应用奠定理论基础。     

Selective expression of an aP2/Fatty Acid Binding Protein4-Cre transgene in non-adipogenic tissues during embryonic development

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, β-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of β-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.Lucy Liaw and Deena Small contributed equally to this work     

Expression pattern of Wnt inhibitor factor 1(Wif1) during the development in mouse CNS

Wnt inhibitor factor-1 (WIF-1) is an extracellular antagonist of Wnts secreted proteins. Here we describe the expression pattern of Wif1 throughout the development of the mouse central nervous system (CNS). Wif1 mRNA can be detected as early as the developmental stage E11, and expression persists to adulthood. In embryonic stages, the level of Wif1 expression was very prominent in several areas including the cerebral cortex, the diencephalon and the midbrain, with the strongest level in the hippocampal plate and the diencephalon. However, after birth, the expression level of Wif1 decreased in the cortex and diencephalon. By adulthood, Wif1 is mainly expressed in the medial habenular nucleus (MHb) in the epithalamus, the mitral layer cells in the olfactory bulb and a few nuclei in the hypothalamus. Our data shows that the expression of Wif1 was very strong during embryonic development of the CNS and suggests that Wif1 may play an essential role in the spatial and temporal regulation of Wnt signals.     

Expression of Crip2, a LIM-domain-only protein, in the mouse cardiovascular system under physiological and pathological conditions

LIM domain-containing proteins mediate protein–protein interactions and play regulatory roles in various physiopathological processes. The mRNA of Crip2, a LIM-only gene, has been detected abundantly in developing and adult hearts but its cell-type specific expression profile has not been well characterized. In this study, we showed that Crip2 is highly expressed in the myocardium, moderately expressed in the endocardium and absent from the epicardium of the developing mouse heart. Interestingly, Crip2 expression is present in the endocardial cells that line both endocardial cushions, whereas it is markedly reduced in the cushion mesenchymes during valve leaflet formation. In the developing vascular system, Crip2 is detected in the endothelial cells of both blood and lymphatic vessels. Consistent with the expression pattern observed in embryos, Crip2 is also highly expressed in the myocardium, endocardium and coronary vascular endothelial cells of the adult heart. In the cardiomyocytes, Crip2 is colocalized with cardiac troponin T in the thin-filaments of sarcomeres. Nonetheless, experimental studies revealed that the expression level of Crip2 is not altered in the isoproterenol (ISO) induced hypertrophic heart. Moreover, Crip2 is detected in endothelial cells of the neovasculature during wound healing and tumor growth. The persistence of Crip2 expression in cardiovascular tissues implies that Crip2 might exert an important impact on the cardiovascular development, maintenance and homeostasis.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.     

Expression of myostatin in the spotted rose snapper Lutjanus guttatus during larval and juvenile development under cultured conditions

In this study, the developmental expression pattern of myostatin (mstn) in the spotted rose snapper Lutjanus guttatus under culture conditions is presented. The full coding sequence of mstn from L. guttatus was isolated from muscle tissue, obtaining 1134 nucleotides which encode a peptide of 377 amino acids. The phylogenetic analysis indicated that this sequence corresponds to mstn‐1. mstn expression was detected in embryonic stages, and maintained at low levels until 28 days post‐hatch, when it showed a significant increase, coinciding with the onset of metamorphosis. After that, expression was fluctuating, coinciding probably with periods of rapid and slow muscle growth or individual growth rates. mstn expression was also analysed by body mass with higher levels detected in smaller animals, irrespective of age. mstn was also expressed in other tissues from L. guttatus, presenting higher levels in brain, eye and gill. In brain for instance, two variants of mstn were isolated, both coding sequences were identical to muscle, except that one of them contained a 75 nucleotide deletion in exon 1, maintaining the reading frame but deleting two conserved cysteine residues. Phylogenetic analysis indicated that this brain variant was also mstn‐1. The function of this variant is not clear and needs further investigation. These results indicate that mstn‐1 participates in different physiological processes other than muscle growth in fishes.     

Identification and analysis of stage-specific expression of lysosome-associated protein transmembrane 4alpha gene during development of preimplantation rabbit nuclear transfer embryo

The stage-specific expression of Lysosome-associated protein transmembrane 4alpha (LAPTM4alpha) in preimplantation rabbit nuclear transfer (NT) embryo was identified with the DDRT-PCR and reverse Northern Blot. The full length (1,364 bp) cDNA of LAPTM4alpha was screened out from cDNA library constructed with rabbit ovary and in situ hybridization (ISH) was used to trace the distribution of the LAPTM4alpha mRNA in intra-ovary, especially the follicle which proved that the LAPTM4alpha gene expression is involved in the follicles development, maturation, ovulation, luteinization, and preimplantation development in the rabbit (Oryctolagus cuniculus domestica). To our knowledge, this is the first characterization of LAPTM4alpha gene expression and mRNA distribution in the rabbit ovary and first evidence for this gene involving in follicle development and rabbit preimplantation development.     

Demonstration of the existence of a single morphological type of gonadotrophic cell in Ellobius lutescens (Microtinae) by an ultrastructural analysis of their development under various physiological and experimental conditions

Summary Ultrastructural studies of pituitaries from Ellobius lutescens (immature males and females, adult hypogonadic males, and virgin and pregnant females) show that the gonadotrophic cells are characterized by a lamellar or vacuolar rough endoplasmic reticulum (RER), a spirally-arranged Golgi apparatus, elongated mitochondria and secretory granules of variable density and size (150–500 m). Ultrastructural differences between gonadotrophic cells previously determined by light microscopy correspond to changes in the development of the protein synthetic apparatus and in the intensity of hormonal discharge. Type 2 gonadotrophs always appear to be more active than type 1 gonadotrophs. After castration, all gonadotrophic cells develop into the same form of castration cell, although type 1 gonadotrophs change more slowly than type 2 cells. Treatment with testosterone induces an inverse development of the gonadotrophic cells which take on the appearance of resting cells similar to those found in immature animals, where the two cell types are also identical. Thus, only one morphological type of gonadotrophic cell can be identified in Ellobius lutescens. Moreover, the gonadotrophic cells of the hypogonadic adult male have the same appearance as those of the female two months after castration, which proves that the negative feedback mechanism which regulates gonadotrophic function is defective in adult male Ellobius lutescens.     

Uncoupling protein mRNA, mitochondrial GTP-binding, and T4 5′-deiodinase activity of brown adipose tissue in Daurian ground squirrel during hibernation and arousal

The mRNA level of uncoupling protein (UCP) specific for brown adipose tissue (BAT) in Daurian ground squirrel, was detected by using a [32P]-labeled oligonucleotide probe. The UCP concentration in mitochondria was indirectly determined by titration with its specific ligand [H3]-labeled GTP. Type II T4 5'-deiodinase of BAT was assayed concomitantly. We found two species of mRNA for UCP with lengths of about 1.9 and 1.5 kb, respectively, both occurring in almost the same concentration. UCP mRNA content was elevated significantly during hibernation, but the UCP concentration did not change compared with that of nonhibernating controls kept at room temperature. When hibernating squirrels were aroused, the UCP mRNA remained at the elevated level as during hibernation, but the UCP concentration increased in comparison with that of nonhibernating controls or during hibernating. Changes in T4 5'-deiodinase activity in BAT were similar to the variations of the UCP mRNA level. These results suggest that the activation of T4 5'-deiodinase in BAT may be an important factor for the up-regulation and maintenance of UCP mRNA content needed for the synthesis of sufficient UCP to acquire the thermogenic capacity for arousal from hibernation.     

Quantitative analysis of protein changes during meristem intiation and bud development in protoplast-derived Petunia hybrida callus

Small calluses from Petunia hybrida leaf protoplasts differentiated into meristems and then vegetative buds in the presence of high cytokinin concentrations. Total proteins from calluses grown at two cytokinin concentrations, of which only the highest induced bud formation, were analysed for molecular markers of cytokinin action by two-dimensional electrophoresis. The protein patterns were also compared to those of differentiated shoots and roots and to those of calluses grown under various hormonal conditions. Five percent of the total proteins detected by this method varied significantly in their level of expression according to the treatment. Quantification by image analysis demonstrated the occurrence of five groups of polypeptides with different expression patterns. Polypeptides from group 1 marked the development of buds and were abundant in young photosynthetic organs from whole plants. Polypeptides from group 5 increased only in calluses grown on control medium, i.e. in the presence of the lowest cytokinin concentration. No protein increased specifically during meristem formation. The possible action of cytokinin on regeneration is discussed in relation to these results.     

Uncoupling protein mRNA, mitochondrial GTP-binding, and T4 5′-deiodinase activity of brown adipose tissue in Daurian ground squirrel during hibernation and arousal

The mRNA level of uncoupling protein (UCP) specific for brown adipose tissue (BAT) in Daurian ground squirrel, was detected by using a [³²P]-labeled oligonucleotide probe. The UCP concentration in mitochondria was indirectly determined by titration with its specific ligand [³H]-labeled GTP. Type II T₄ 5′-deiodinase of BAT was assayed concomitantly. We found two species of mRNA for UCP with lengths of about 1.9 and 1.5 kb, respectively, both occurring in almost the same concentration. UCP mRNA content was elevated significantly during hibernation, but the UCP concentration did not change compared with that of nonhibernating controls kept at room temperature. When hibernating squirrels were aroused, the UCP mRNA remained at the elevated level as during hibernation, but the UCP concentration increased in comparison with that of nonhibernating controls or during hibernating. Changes in T₄ 5′-deiodinase activity in BAT were similar to the variations of the UCP mRNA level. These results suggest that the activation of T₄ 5′-deiodinase in BAT may be an important factor for the up-regulation and maintenance of UCP mRNA content needed for the synthesis of sufficient UCP to acquire the thermogenic capacity for arousal from hibernation.     

Uncoupling protein1 mRNA, mitochondrial GTP-binding, and T4 5′-deiodinase of brown adipose tissue in euthermic Daurian ground squirrel during cold exposure

Regulation of thermogenic activity and uncoupling protein1 (UCP1) expression in brown adipose tissue (BAT) were studied in euthermic Daurian ground squirrel after acute and chronic cold exposure at 4°C. The UCP1 concentration was indirectly determined by titration with its specific ligand [³H]-labeled GTP, and Ucp1 mRNA was detected by using a [³²P]-labeled antisense oligonucleotide probe. Both acute and chronic cold exposure stimulated up-regulation of Ucp1 mRNA. Although UCP1 concentration is not significantly increased after 24 h of cold exposure, it is markedly elevated by 75% in squirrels after 4-week cold adaptation compared with controls raised at 22°C. Changes in T₄ 5′-deiodinase activity were closely associated with variations of Ucp1 mRNA level. Ucp1 gene expression is significantly affected by cold exposure in BAT from euthermic Daurian ground squirrels. In addition, the activation of T₄ 5′-deiodinase may be an important regulatory factor in cold-induced Ucp1 expression.