A Macrophage NBR1-MEKK3 Complex Triggers JNK-Mediated Adipose Tissue Inflammation in Obesity

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细胞信号转导通路

细胞信号转导通路

Chromatin/Epigenetics Resources
Overview of Chromatin / Epigenetics
Chromatin regulation refers to the events affecting chromatin structure and therefore, transcriptional control of gene expression patterns. Epigenetics, specifically, refers to the heritable modifications which result in altered gene expression and are not known to be encoded in DNA. The nucleosome, made up of four histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have more recently been shown to be dynamic proteins, undergoing multiple types of post-translational modifications (PTMs) and interacting with regulatory proteins to control gene expression. Protein acetylation plays a crucial role in regulating chromatin structure and transcriptional activity. Histone hyperacetylation by histone acetyltransferases (HATs) is associated with transcriptional activation, whereas histone deacetylation by histone deacetylases (HDACs) is associated with transcriptional repression. Hyperacetylation can directly affect chromatin structure by neutralizing the positive charge on histone tails and disrupting nucleosome-nucleosome and nucleosomeDNA interactions. In addition, acetylation creates binding sites for bromodomain-containing chromatin regulatory proteins (histone modification readers). Unlike acetylation, methylation does not alter the charge of arginine and lysine residues and is unlikely to directly modulate nucleosomal interactions required for chromatin folding. Methylated arginine and lysine residues are major determinants for formation of active and inactive regions of the genome. Methylation facilitates binding of chromatin regulatory proteins/histone modification readers that contain various methyl-lysine or methyl-arginine binding domains (PHD, chromo, WD40, Tudor, MBT, Ankyrin repeats, PWWP domains). Recruitment of co-activator and co-repressor proteins is dependent on the specific lysine residue that is modified. The modulation of chromatin structure is an essential component in the regulation of transcriptional activation and repression. One strategy by which chromatin structure can be modulated is through disruption of histone-DNA contacts by ATP-dependent chromatin remodelers, such as the NuRD, Polycomb, and SWI/SNF complexes, which have been shown to regulate gene activation/repression, cell growth, the cell cycle, and differentiation. Chromatin structure is also modulated through other PTMs such as phosphorylation of histone proteins, which affects association with DNA-interacting proteins and has been recently identified to play a role in coordinating other histone modifications. Furthermore, methylation of DNA at cytosine residues in mammalian cells affects chromatin folding and is a heritable, epigenetic modification that is critical for proper regulation of gene silencing, genomic imprinting, and development. Three families of mammalian DNA methyl-transferases have been identified, DNMT1/2/3, that play distinct roles in embryonic stem cells and adult somatic cells. In addition to the core histone proteins, a number of histone variants exist that confer different structural properties to nucleosomes and play a number of specific functions such as DNA repair, proper kinetochore assembly and chromosome segregation during mitosis, and regulation of transcription. Chromatin and epigenetic regulation is crucial for proper programming of the genome during development and under stress conditions, as the misregulation of gene expression can lead to diseased states such as cancer.

聚乙二醇修饰的纳米金三角颗粒对乳腺癌4T1细胞的光热治疗作用

聚乙二醇修饰的纳米金三角颗粒对乳腺癌4T1细胞的光热治疗作用

聚乙二醇修饰的纳米金三角颗粒对乳腺癌4T1细胞的光热治疗作用毕俊;王守巨【摘要】目的光热治疗是乳腺癌治疗研究的前沿热点,纳米金三角用于乳腺癌光热治疗的相关报道较少.文中旨在研究660纳米激光照射下,聚乙二醇修饰的纳米金三角颗粒(PEG-GTN)对乳腺癌4T1细胞的光热治疗效果.方法将制备的GTN表面用巯基聚乙二醇保护后,与乳腺癌4T1细胞共培养,按5、10、20和40μg/mL浓度加入分别含GTN和PEG-GTN的培养基,以未加PEG-GTN者作对照.通过MTT实验检测其对细胞的毒性,并在660纳米激光照射后通过MTT实验检测其对细胞的光热治疗作用.结果 5、10、20和40μg/mL浓度下将GTN与4T1细胞共培养24h 后,细胞毒性检测结果显示细胞存活率分别为(71.2±8.7)%、(72.6±4.6)%、(65.8±2.3)%和(29.9±3.2)%,两两比较差异有统计学意义(P<0.05).PEG-GTN与4T1细胞共培养24h后,5、10、20和40μg/mL细胞毒性检测结果显示细胞的存活率分别为(96.2±4.4)%、(95.9±4.4)%、(95.2±4.8)%和(96.6±4.7)%,两两比较差异有统计学意义(P<0.05);光热治疗结果显示细胞存活率分别为(92.2±6.2)%、(51.6±6.8)%、(25.7±4.5)%和(4.8±2.5)%,两两比较差异有统计学意义(P<0.05),10、20、和40μg/mL时细胞存活率与对照孔[(100±1.8)%]比较差异有统计学意义(P<0.01).结论 PEG-GTN细胞毒性低,在近红外激光照射下,能有效杀灭乳腺癌4T1细胞,为其进一步用于体外和动物光热治疗研究奠定了基础.【期刊名称】《医学研究生学报》【年(卷),期】2019(032)007【总页数】4页(P696-699)【关键词】乳腺癌;聚乙二醇;修饰;纳米金三角颗粒;光热治疗【作者】毕俊;王守巨【作者单位】210002南京,东部战区总医院(原南京军区南京总医院)医学影像科;210002南京,东部战区总医院(原南京军区南京总医院)医学影像科【正文语种】中文【中图分类】R737.90 引言光热治疗具有毒性低、空间选择性高的优点,是近几年肿瘤纳米治疗研究的前沿和热点[1]。

藏红花素介导DKK3调控GSK-3β

藏红花素介导DKK3调控GSK-3β

◇基础研究◇摘要目的:探讨藏红花素(crocin )对阿尔兹海默症(Alzheimer's disease ,AD )小鼠认知能力的改善作用及机制。

方法:SD 大鼠海马区注射A β25-35建立AD 模型,随机分为AD 组、AD+L 、M 、H-crocin 组(10、20、40mg/kg )和AD+donepezil 组(1mg/kg 盐酸多奈哌齐),腹腔注射治疗4周,另设置Sham 组。

采用避暗实验、水迷宫实验评估大鼠学习、记忆能力,ELISA 测定大鼠血清A β含量,HE 染色和Tunel 染色确定大鼠海马区内病理改变及神经元细胞凋亡,免疫组化测定大鼠海马区Brdu 、Dcx 、NeuN 表达,Western blot 测定大鼠脑组织A β、DKK3、β-catenin 、p-GSK-3β/GSK-3β、Caspase-3、Bax 、Bcl-2蛋白表达。

结果:与Sham 组相比,AD 组大鼠的学习、记忆能力下降,血清A β含量升高,且海马区的病理改变严重,神经元细胞凋亡增加,Brdu 、Dcx 、NeuN 含量降低,A β、DKK3、p-GSK-3β/GSK-3β、Caspase-3、Bax 蛋白表达升高,β-catenin 、Bcl-2蛋白表达降低(P <0.01)。

与AD 组相比,给予不同剂量crocin 和donepezil 治疗后,AD 大鼠学习、记忆能力提高,血清A β含量降低,海马区的病理改变减轻,神经元细胞凋亡减少,Brdu 、Dcx 、NeuN 含量升高,A β、DKK3、p-GSK-3β/GSK -3β、Caspase-3、Bax 蛋白表达升高,β-catenin 、Bcl-2蛋白表达降低(P <0.05),crocin 的剂量依赖效应显著。

结论:crocin 通过减少神经元细胞凋亡,介导DKK3调控GSK-3β/β-catenin 通路来改善AD 大鼠认知损伤。

MEKK3 的研究进展

MEKK3 的研究进展

MEKK3 的研究进展摘要:有丝分裂原活化蛋白激酶激酶激酶3即MEKK3(mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase kinase 3)是一种丝氨酸/苏氨酸蛋白激酶,隶属于家族()。

在特定条件下,MEKK3可以通过活化信号通路中下游的信号分子,对细胞的形成、增殖以及凋亡产生一定的调控作用,MEKK3参与包含等信号通路,与众多肿瘤的形成及发展有密切的关系。

MEKK3同时也参与炎症和免疫应答,可促进IL-6等细胞因子的表达,启动及放大炎症反应。

现就MEKK3的分子结构、活化机制、在信号通路及免疫调节中所起的所起的关键作用及相关作用机制行相关综述。

关键词:有丝分裂原活化蛋白激酶激酶激酶3,信号转导,免疫调节将细胞外刺激信号传递到细胞内相应蛋白并作出应答且在细胞凋亡、运动、增殖、分化等众多生理过程中均有参与的信号途径,即为有丝分裂原活化蛋白激酶()信号途径。

是一种丝氨酸/苏氨酸蛋白激酶,属于家族(),全称为有丝分裂原活化蛋白激酶激酶激酶3(),其广泛存在于人体各种组织及器官中。

MEKK3可以通过激活不同的信号通路,有效调控细胞的形成、增殖以及凋亡,在细胞免疫应答以及调节方面发挥重要生理作用。

一、MEKK3的分子结构MEKK3最早于1996年由Johnson等[1]从NIH-3T3细胞中克隆获得。

该激酶由626个氨基酸组成,分子量约为78~80 kD。

目前发现MEEK3蛋白发挥生理功能主要依赖两个结构域,一个是位于氨基端的PB1结构域,另一个是位于羧基端的活化环结构域。

PB1结构域主要是起调节作用,在该结构的影响下,可结合其他分子,构成具有一定特异性特征的二聚体,其作用是提高信号通路中细胞信号传递的准确性[2]。

而位于羧基端的活化环结构域含有特异性的丝氨酸/苏氨酸残基位点,通过这个结构域中不同位点的磷酸化,使MEKK3活化并参与细胞内信号传导,产生不同的生理作用。

T7噬菌体DNA的提取及其反向遗传拯救方法的建立

T7噬菌体DNA的提取及其反向遗传拯救方法的建立

T7噬菌体DNA的提取及其反向遗传拯救方法的建立徐海;王义伟;陈瑾;郑其升;侯继波【摘要】从T7噬菌体培养液中粗提噬菌体颗粒,经热裂解后用苯酚、氯仿抽提进而获得纯净的T7噬菌体DNA.用PCR、酶切法鉴定T7噬菌体DNA的完整性.通过对不同感受态细菌浓度、T7噬菌体DNA用量、电转化电压条件的优化,建立了T7噬菌体反向遗传拯救方法.结果显示,提取的DNA结构完整,能够被特异性酶切割,多克隆位点序列正确.T7噬菌体的反向遗传拯救方法最优化条件为200 ng T7噬菌体DNA、1 ml 5×109感受态细菌、1.5 kV电转化电压,在此条件下获得的拯救效率为3.5×105 PFU/ng (DNA).%The T7 phage DNA was purified with benzene polyphenol and chloroform from T7 phage particles after heat cracking. The integrity of T7 phage DNA was identified by PCR and enzemy digestion, and a reverse genetic rescue system for the purified T7 DNA was established through the optimization of the conditions, such as T7 phage DNA input dosage, density of complete cells, and electrotransformation voltage. The results showed that the purified T7 DNA could be digested with EcoR I or Hind Ⅲ, and the multiclon site was correct. The reverse genetics rescue system was successfully established. The highest rescue efficiency was obtained under the conditions of 200 ng T7 phage DNA,5×l09 complete cell per millili ter and 1.5 kV electrotransformation voltage, with a output of 3.5×l05 PFU/ng (DNA).【期刊名称】《江苏农业学报》【年(卷),期】2012(028)002【总页数】4页(P355-358)【关键词】T7噬菌体;DNA提取;电转化;反向遗传拯救【作者】徐海;王义伟;陈瑾;郑其升;侯继波【作者单位】江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014;江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014;江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014;江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014;江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014【正文语种】中文【中图分类】S432.4+1T7噬菌体是感染大肠杆菌的烈性噬菌体,在大肠杆菌的胞浆内组装,成熟的T7噬菌体通过细胞裂解而释放,现已完成其全序列分析,遗传背景清楚,病毒颗粒结构复杂。

蛋白质尼罗红染色试剂盒 产品说明书(中文版)

蛋白质尼罗红染色试剂盒 产品说明书(中文版)

GENMED SCIENTIFICS INC. U.S.A GMS30007 v.A GENMED蛋白质尼罗红染色试剂盒产品说明书(中文版)主要用途GENMED蛋白质尼罗红染色试剂是一种旨在使用一种荧光染料尼罗红直接在聚丙烯酰胺凝胶上进行染色,数分钟内在清晰背景上产生灵敏度10纳克以下蛋白质,通过紫外光激发显示红色条带的权威而经典的技术方法。

该技术由大师级科学家精心研制、成功实验证明的。

其适用于单向或双向变性或天然蛋白电泳的检测。

产品即到即用,性能稳定,一步操作,敏感度高,显色清晰,重复性好,建立标准化体系。

技术背景尼罗红染色剂(9-diethylamino-5H-benzo[alpha]-phenoxazine-5-one;Nile red)是一种水溶性的荧光染料。

与蛋白质结合后,在紫外光的激发下,显示红色。

无需固着处理。

可以用于考马斯亮蓝染色后的再染色。

可与质谱分析兼容。

SDS将显著增强染色效果。

产品内容GENMED清理液(Reagent A)毫升GENMED染色液(Reagent B)毫升产品说明书保存方式保存在4℃冰箱里, 避免光照,有效保证2月用户自备塑料盘:用于染色的容器无离子水:用于稀释试剂的溶液平式摇荡仪:用于染色操作时的孵育实验步骤实验开始前,完成SDS-PAGE蛋白电泳的运行,取出的胶体,作好定向标记,然后进行下列操作:1.准备一个大小的染色塑料盘2.放入聚丙烯酰胺凝胶3.加入毫升GENMED清理液(Reagent A)在凝胶上,覆盖整个胶体4.小心倒掉染色盘里的GENMED清理液(Reagent A)5.加入毫升用户自备的无离子水6.加入毫升GENMED染色液(Reagent B),强烈混匀,使染色液均匀分布(注意:不要损坏胶体)7.在室温下平式摇荡仪上孵育分钟,速度为RPM,避免光照8.小心倒掉染色盘里的染色液9.加入毫升用户自备的无离子水,用手摇动秒10.小心倒掉无离子水11.重复实验步骤三次12.即刻放在UV透射仪上观察和照相:条带显示红色13.可以继续进行考马斯亮蓝染色注意事项1.本产品为20次操作2.胶体越大,用量相应增加3.建议电泳运行缓冲液中的SDS终浓度为0.05%,以降低背景噪音4.操作时须戴手套5.建议用无离子水稀释或清洗6.孵育温度为室温7.孵育后,即刻进行观察;放置时间过久,影响效果8.本公司提供系列蛋白染色试剂产品质量标准1.本产品经鉴定性能稳定2.本产品经鉴定染色效果出色使用承诺杰美基因秉着“信誉至上、客户满意、质量承诺”的宗旨为我们的用户提供优质产品和服务。

单磷酸鸟苷还原酶在皮肤黑色素瘤诊断及预后中的价值

第35卷第3期长治医学院学报Vol.35No.32021年6月JOURNAL OF CHANGZHI MEDICAI COLLEGE Jun.2021161・基础研究・单磷酸鸟昔还原酶在皮肤黑色素瘤诊断及预后中的价值冀中豪1郑榕静彳摘要目的:分析单磷酸鸟著还原酶(GMPR)在皮肤黑色素瘤中的表达及预后价值,进而通过免疫浸润分析和富集分析揭示其在皮肤黑色素瘤发生发展中的作用机制。

方法:提取癌症基因组图谱(TCGA)中的皮肤黑色素瘤和GTEx中对应的正常组织数据。

利用R语言统计分析GMPR在皮肤黑色素瘤中的表达情况和临床相关性;根据GMPR表达量将470例皮肤黑色素瘤患者划分为GMPR-Low组和GMPR-High组,获取差异表达基因进行GO、KEGG分析和GSEA分析;使用R包中的xGell算法进行免疫浸润分析;进而通过STRING数据构建PPI网络,使用Cytoscape软件分析HUB基因。

结果:GMPR在皮肤黑色素瘤中高表达,其表达与肿瘤T分期有关(P<0.05);ROG曲线结果(AUC=0.826)提示其有较好的诊断效能;K-M法生存分析表明GMPR低表达患者总生存和无病生存优于高表达组(P<0.05),并且其免疫浸润较好;富集分析表明GMPR可能通过白介素信号转导途径发挥作用;构建PPI网络筛选得出HUB基因CDH2和SPP1,二者表达与GMPR均为负相关。

结论:GMPR可能是皮肤黑色素瘤的诊断和预后标志物,其可能通过白介素信号转导途径调节免疫细胞浸润进而影响皮肤黑色素瘤患者的生存和预后。

关键词皮肤黑色素瘤;单磷酸鸟昔还原酶;生物信息学分析中图分类号R739.5文献标识码A文章编号1006(2021)03-161-06Clinical Significance and Prognostic Value of Guanosine Monophosphate Reductase in Cutaneous MelanomaJI Zhonghao,QIE RongjingDepartment of Basic Medicine,Changzhi Medical CollegeAbstract Objective:To analyze the expression and prognostic value of guanosine monophosphate reductase(GMPR)in cuta­neous melanoma,and to reveal its mechanism of action in the occurrence and development of cutaneous melanoma.Methods:The data of skin melanoma(SKCM)in TCGA(The Cancer Genome Atlas)and normal tissue in GTEX(Genotype-Tissue Expression) were extracted.Rlanguage was used to analyze the expression and clinical relevance of GMPR in cutaneous melanoma;470SKCM patients were divided into GMPR-low group and GMPR-high group according to the expression of GMPR,and the differentially ex­pressed genes were obtained for GO,KEGG and GSEA analysis;xcell algorithm in R package was used for immune infiltration anal­ysis;PPI network was constructed by string data and analyzed by Cytoscape software.Results:GMPR was highly expressed in cuta­neous melanoma f and its expression was correlated w让h T stage(P<0.05).The results of ROC curve(AUC=0.826)indicated that GMPR had better diagnostic efficacy;K-M survival analysis showed that the overall survival and disease-free survival of patients w让h low expression of GMPR were better than those of patients with high expression of GMPR(P<0.05),and their immune infil­tration was better;enrichment analysis showed that the expression of GMPR might be through the interleukin signal transduction pathway.The expression of hub genes CDH2and SPP1was negatively correlated with GMPR.Conclusion:GMPR is a diagnostic and prognostic marker of cutaneous melanoma.It may regulate immune cell infiltration through interleukin signal transduction path­way,and then affect the survival and prognosis of patients with cutaneous melanoma.Key words cutaneous melanoma;guanosine monophosphate reductase;bioinformatics analysis皮肤黑色素瘤(skin cutaneous melanoma,件者单位]长治医学院机能综合实验室(W000)2长治市人民医院基金项目长治医学院春晖计划项目(QD201907);山西省高等学校科技创新项目(2020L0378)SKCM)是世界性重大公共卫生问题⑴,SKCM患者死亡率不断上升⑵。

ARTP诱变选育维生素K2高产菌株

ARTP诱变选育维生素K2高产菌株作者:杨林史小利张建国崔凤霞来源:《食品界》2024年第07期维生素K2是一类重要的脂溶性维生素,在凝血、抗骨质疏松等方面具有重要作用。

为了提升纳豆芽孢杆菌发酵过程中维生素K2(MK-7)的产量,以纳豆芽孢杆菌ND09为出发菌株,采用常压室温等离子体(ARTP)诱变技术进行选育,获得一株MK-7高产突变株,产MK-7含量达到26.42mg/L,为出发菌株的2.1倍。

经10代传代培养性能稳定,对其摇瓶发酵装液量、接种量、发酵温度进行优化,MK-7含量最终达到38.7mg/L,为生物发酵生产MK-7提供了有利的微生物资源。

维生素 K2 被称做甲萘醌(Menaquinone,MK),其C3位的侧链是由不同数量的异戊二烯基组成的,存在一系列亚型,可记作 MK-n(n 代表异戊二烯基的个数),其中最为重要的两类亚型是MK-4和MK-7,与其他维生素K2相比,MK-7 在人体中具有半衰期长、亲缘性高的特点,在医药和功能性食品等领域备受关注,并且MK-7是生物活性最高的一种维生素K2。

它在食品中含量极少,可以预防和治疗骨质疏松症,有“铀金维生素”之称,主要通过调节体内钙离子的沉积,从而刺激骨组织内骨钙素的合成。

此外,MK-7是哺乳动物正常血液凝固时不可或缺的营养素。

天然MK-7只能通过微生物发酵法获得,其中,纳豆芽孢杆菌是目前工业化生产MK-7的主要菌种。

因为它不仅容易培养、生长速度快,而且产MK-7含量相对高。

对于野生型的纳豆芽孢杆菌往往需要进行诱变育种提高产量,从而满足工业化生产的需求。

常压室温等离子体诱变(ARTP)是一种通过在常压和室温下产生等离子体来引发基因突变的技术。

ARTP诱变利用高能量等离子体中的化学反应和物理效应,对细胞的DNA进行改变,从而引起基因组的突变。

ARTP作为一种生物育种技术,因其操作便捷、安全、高效的特点,在生物科学领域广泛应用。

本实验以前期分离保存的纳豆芽孢杆菌作为出发菌株,旨在筛选得到MK-7产量高且遗传稳定性好的突变菌株,为该菌株能够进一步应用于生产打下基础。

MEKC血样中药物,外文翻译

MEKC血样中药物.第1页原始文件沃尔夫冈·BuchbergerÆ马蒂亚斯Ferdig鲁道夫·索默Æ翠THANH VO 雷帕霉素在人体血液中的微量分析胶束电动色谱收稿日期:2004年3月16日/日期:11 2004/5月接受日期:2004年5月18号/发表时间:2004年7月20日Ó施普林格出版社2004年摘要毛细管电泳法与UV检测波长为278 nm的分析已经开发在人类的免疫抑制剂雷帕霉素(西罗莫司)血微克每升的水平低。

分离有在酸性载体电解质含有已取得十二烷基硫酸钠和30%(体积/体积)腈。

为样品净化和富集,离线固固相萃取的步骤中使用的基于二氧化硅的反相材料和毛细管聚焦技术采用。

后者允许注射增加扩大样本量,而不会过度带。

虽然这种新方法是不敏感,比现有的质谱联用液相色谱程序法,它是完全适合常规分析的RA-pamycin血液中这种药物治疗的患者。

最后,但并非最不重要的,低的成本使一个有吸引力的建立的方法替代。

关键词雷帕霉素Æ胶束电动血色谱Æ分析介绍雷帕霉素(西罗莫司)是三烯大环内酯类抗生素具有抗真菌,消炎,抗肿瘤和这是目前使用的免疫抑制特性用于预防器官移植排斥反应的反式种植园。

雷帕霉素的结构图给出。

1。

雷怕霉素已被证明是阻止T 细胞活化和扩散以及激活P70 S6文FK-506结合激酶,表现出很强的结合蛋白质。

它也抑制活性的蛋白质哺乳动物雷帕霉素靶蛋白(mTOR)的,该功能蒸发散在促进肿瘤生长的信号转导通路。

雷帕霉素结合到受体蛋白质(FKBP12),和的rapamycin/FKBP12复杂的结合mTOR的并阻止mTOR的互动与目标蛋白质在这一信号通路。

对于栽种的器官的患者是必不可少的监测雷帕霉素在他们的血液中的水平。

决定雷帕霉素在血液中的中断可以确定迄今已完成高效液相色谱紫外检测器[1-4],最近由高效液相色谱法,连字符和质谱(MS)[5-8]。

维生素K3微乳的制备及其抗肿瘤作用的研究

维生素K3微乳的制备及其抗肿瘤作用的研究发表时间:2017-06-15T15:04:45.490Z 来源:《医师在线》2017年4月下第8期作者:任立震陈雪娇李佳凌严娇王俊平[导读] PEG2000-DSPE修饰微乳表面,有可能避免网状内皮系统对微乳的吞噬和破坏作用,有助于提高维生素K3的血药浓度。

(沈阳医学院药理学教研室;沈阳辽宁110034)摘要:目的:维生素K3抗癌作用机制是诱导活性氧的产生同时激活碱性核酸酶,但缺乏足够的肿瘤靶向性,且水中溶解度很低,难以静脉给药。

采用聚乙二醇(PEG)修饰的毫微乳运载维生素K3,有可能解决上述问题。

方法:采用二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(PEG2000-DSPE)、油酸制备维生素K3微乳,建立动物S-180实体肿瘤和艾氏腹水癌模型,评价维生素K3微乳的抗肿瘤作用。

结果:维生素K3微乳静脉给药具有显著的抗肿瘤作用并能显著延长荷瘤动物的生命(P<0.01)。

结论:维生素K3微乳有可能成为一种可以静脉注射的抗癌药物。

关键词:维生素K3;微乳;抗肿瘤作用维生素K3(Vitamin K3)是一种人工合成的维生素K,能够抑制多种肿瘤细胞的生长[1-5]。

维生素K3参与人体细胞的氧化还原循环,通过产生活性氧自由基发挥抗癌作用,其机制包括:谷胱甘肽耗竭,NADPH氧化,大分子损伤和破坏细胞内钙离子平衡,同时还能激活碱性核酸酶。

但是,维生素K3水溶性差,缺乏肿瘤靶向性,不利于其发挥抗肿瘤作用。

采用聚乙二醇(PEG)修饰的微乳具有一定程度的肿瘤靶向性,特别适用于运载水不溶性的抗癌药物[6]。

因此,我们研制了维生素K3微乳,并对其体内抗肿瘤作用进行了评价。

1. 实验材料1.1 药品与仪器维生素K3,美国Sigma公司;油酸,美国Sigma公司,纯度>99.0%;聚乙二醇2000-二硬脂酰基磷脂酰乙醇胺 (PEG2000-DSPE) ,美国Polylipid Co., Ltd.;JSM-T200电子显微镜(日本电子公司)。

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Cell MetabolismArticleA Macrophage NBR1-MEKK3ComplexTriggers JNK-MediatedAdipose Tissue Inflammation in ObesityEloy D.Hernandez,1,4Sang Jun Lee,1,4Ji Young Kim,1Angeles Duran,1Juan F.Linares,1Tomoko Yajima,1Timo D.Mu¨ller,2Matthias H.Tscho¨p,2Steven R.Smith,3Maria T.Diaz-Meco,1and Jorge Moscat1,*1Sanford-Burnham Medical Research Institute,La Jolla,CA92037,USA2Institute for Diabetes and Obesity,Helmholtz Diabetes Center,Helmholtz Zentrum Mu¨nchen&Division of Metabolic Diseases,Department of Medicine,Technische Universita¨t Mu¨nchen,85764Munich,Germany3Translational Research Institute for Metabolism and Diabetes,Florida Hospital,Sanford-Burnham Medical Research Institute,FL32789,USA4Co-first authors*Correspondence:jmoscat@/10.1016/j.cmet.2014.06.008SUMMARYThe c-Jun NH(2)-terminal kinase(JNK)is a critical determinant of obesity-associated inflammation and glucose intolerance.The upstream mechanisms controlling this pathway are still unknown.Here we report that the levels of the PB1domain-containing adaptor NBR1correlated with the expression of proinflammatory molecules in adipose tissue from human patients with metabolic syndrome,suggest-ing that NBR1plays a key role in adipose-tissue inflammation.We also show that NBR1inactivation in the myeloid compartment impairs the function, M1polarization,and chemotactic activity of macro-phages;prevents inflammation of adipose tissue; and improves glucose tolerance in obese mice. Furthermore,we demonstrate that an interaction between the PB1domains of NBR1and the mitogen-activated kinase kinase3(MEKK3)enables the formation of a signaling complex required for the activation of JNK.Together,these discoveries identify an NBR1-MEKK3complex as a key regulator of JNK signaling and adipose tissue inflammation in obesity.INTRODUCTIONObesity is an international healthcare priority due to its increasing prevalence and its association with glucose intoler-ance(Spiegel and Nabel,2006;Yach et al.,2006).The lack of a complete understanding of the precise regulatory networks that control adipogenesis,energy expenditure,and inflammation is a fundamental problem in metabolic research.It is clear also that obesity-induced inflammation underlies critical aspects of glucose metabolism deregulation and insulin resistance(Glass and Olefsky,2012;Gregor and Hotamisligil,2011).We recently identified a signaling molecule that plays important roles in obesity and the inflammation and glucose intolerance that develop in the context of this condition.Specifically,genetic ablation of the signaling adaptor p62(also known as sequesto-some1)in mice resulted in mature-onset obesity,adipose inflammation,and glucose intolerance(Rodriguez et al.,2006). Notably,p62is a member of the PB1domain-containing signaling network,which also includes kinases such as protein kinase C z(PKC z),mitogen-activated protein kinase kinase2 (MEKK2),and MEKK3,as well as adapters such as partition-ing-defective protein6(Par6)and NBR1(Moscat et al.,2006). It is believed that p62can interact alternatively with PKC z or NBR1through their respective PB1domains,but the physiolog-ical role and mechanisms of action of NBR1in vivo have not yet been clarified(Moscat and Diaz-Meco,2011;Moscat et al., 2006,2007,2009).Although PKC z-deficient mice do not show alterations in adiposity as compared to wild-type(WT)mice when both are fed with high-fat diet(HFD),PKC z-deficient mice showed increased adipose inflammation and impaired glucose tolerance(Lee et al.,2010).Our data on p62knockout(KO)mice and cells have demon-strated that p62is a critical negative regulator of white adipose tissue(WAT)adipogenesis but is a positive regulator of brown adipose tissue(BAT)function,through the negative regulation of ERK1and the positive regulation of p38,respectively(Mu¨ller et al.,2013).This model explains why the adipose-specific abla-tion of p62in mice results not only in an increase in adiposity but also impaired nonshivering thermogenesis,which in turn leads to a decrease in the metabolic rate(Mu¨ller et al.,2013).The fact that PKC z is a negative regulator of obesity-induced inflammation is of great functional relevance,since recent studies have high-lighted the importance of inflammation in the induction of glucose intolerance in obese mice(Hotamisligil,2006;Qatanani and Lazar,2007;Schenk et al.,2008;Shoelson et al.,2006;Sol-inas et al.,2007).Also,experiments from a number of research groups have demonstrated that the ablation of macrophages in mice normalizes glucose homeostasis in the context of obesity (Gordon,2003;Gordon and Taylor,2005;Lumeng et al., 2007a,2007b,2007c;Mantovani et al.,2004;Patsouris et al., 2008).Interestingly,selective genetic inactivation of p62in the myeloid compartment using cell-specific Cre mouse lines re-vealed that p62does not have an impact on macrophages in the adipose tissue of obese mice(Mu¨ller et al.,2013).Thisfindingsuggests that the enhanced inflammation in the total body p62-deficient mouse is secondary to increased adiposity and not due to a potential role of p62in the myeloid compartment.The domain organization of NBR1is remarkably similar to that of p62,featuring PB1,zinc-finger,and UBA domains.The outcomes of overexpression and transfection studies have sug-gested that NBR1is involved in growth-factor trafficking (Marda-kheh et al.,2009)and/or p62-mediated processes (Kirkin et al.,2009;Lange et al.,2005;Yang et al.,2010).However,its precise in vivo contribution to the control of metabolic homeostasis and/or the ensuing inflammation in the context of obesity has not been investigated.It is possible that p62,PKC z ,and NBR1play different roles in the control of metabolic homeostasis de-pending on cell type.Here,we have characterized the effect of myeloid-specific NBR1ablation and found that this protein plays a critical role in the regulation of macrophage polarization toward the M1phenotype during obesity-induced inflammation.Ourstudies in human patients with metabolic syndrome revealed a significantly positive correlation between NBR1transcript levels and markers of metabolic alterations and inflammation.These results establish that NBR1plays a critical role in obesity-induced glucose intolerance,and that this is due to its impact on macrophage function.They also highlight the relevance of the PB1network comprised of p62,PKC z and NBR1at the cellular interface between metabolism and inflammation.RESULTSHigh NBR1Levels Correlate with Obesity in Human PatientsThe initial evidence of a possible connection between NBR1and abnormalities in metabolic homeostasis and obesity-induced inflammation came from our analysis of adipose tissues from two cohorts of human patients.The first consisted of 63young and basically healthy men and women with a wide range of body mass index (BMI)and body fat values (see Ukropcova et al.,2007for subject characteristics).We found a positive and significant statistical correlation between NBR1and PPAR g (r =0.36,p =0.003)transcript levels in this cohort (data not shown),which suggested that NBR1and PPAR g could act in the same pathway to control metabolic homeostasis in adipose tissue of obese but generally healthy patients.This conclusion was supported by analysis of a second cohort,which consisted of 44middle-aged men with NCEP-defined ‘‘metabolic syn-drome’’(Grundy et al.,2004)(Table S1available online).This population is characterized by more adipose tissue inflammation than the healthy population in the study described above,and we found a highly significant positive correlation between NBR1transcript levels and not only PPAR g 1and PPAR g 2(Figures 1A and 1B)but also markers of the monocyte and macrophage line-age,such as CD68and CD163(Figures 1C and 1D).Notably,the latter have been validated as markers of macrophage infiltration based on immunohistochemistry (data not shown).Significant correlations were also found with transcripts encoding chemo-kines and proinflammatory proteins like MCP-1and MIP-1(Fig-ures 1E and 1F).These results suggest a potential role of NBR1in obesity-induced inflammation.NBR1in the Myeloid Compartment Plays a Critical Role in Obesity-Induced InflammationIntrigued by the potential of a macrophage-based link between NBR1,adipose tissue inflammation,and metabolic syndrome,we next investigated the expression of NBR1in the adipose tissue of obese WT mice fed a HFD as compared to lean mice fed a regular chow diet (RD).Double immunofluorescence of NBR1and the macrophage marker F4/80showed NBR1staining exclusively in crown-like structures that colocalized with F4/80mostly in the HFD adipose sections,which indicates that NBR1is specifically expressed in macrophages that infiltrate obese adipose tissue (Figure 2A).Consistent with this,NBR1mRNA levels were significantly increased in adipose tissue macrophages (ATMs)isolated from obese as compared to lean mice (Figure 2B).Therefore,to address the role of NBR1in adi-pose tissue inflammation in vivo,we generated mice in which NBR1was genetically and selectively inactivated in the myeloid cell compartment.Specifically NBR1fl/flmice were crossedtoFigure 1.NBR1Transcripts Levels Correlate with Adipose Inflam-mation in Obese Patients(A–F)Positive correlation between transcript levels of NBR1and PPAR g 1(A),PPAR g 2(B),CD68(C),CD163(D),MIP-1(E),and MCP-1(F)in obese patients.See also Table S1.Cell MetabolismNBR1-MEKK3,a Key Regulator of JNKlysozyme M(LysM)cre mice.This mouse line(herein termed NBR1My KO)and their corresponding NBR1fl/flWT controls were fed a HFD for12weeks,and body weight was monitored continuously.The results in Figure2C confirm that NBR1was effectively and selectively deleted from macrophages.The lack of NBR1in the myeloid compartment did not affect the body weight or fat content of these mice,regardless of whether they were fed the HFD(Figures2D and2E)or regular chow diet, even when monitored over a longer period(data not shown). However,in the mutant mice,macrophage recruitment to the adipose tissue was significantly impaired(Figure2F),and the levels of inflammatory markers in the WAT were also reduced (Figure2G).Fluorescence-activated cell sorting(FACS)analysis confirms a reduced presence of ATMs in the WAT of mutant mice (Figures2H and S1A).It has been established that ATMs can differentiate along different lineages,including the so-called M1and M2(Gordon,2003;Kang et al.,2008;Odegaard et al., 2008;Vats et al.,2006),and an increase in the M1/M2ratio has been proposed to be responsible for glucose intolerance responses in obese mice(Kang et al.,2008;Odegaard et al., 2008).When isolated ATMs from WT and NBR1My KO mice were analyzed for several markers of macrophage differentia-tion,it was clear that the loss of NBR1selectively impaired M1polarization with a slight although reproducible increase in M2polarization(Figure2I).Macrophage infiltration was also inhibited in the livers from NBR1My KO mice as compared to iden-tically treated WT controls,as determined by the levels of macro-phage transcript markers CD68and F4/80(Figure S1B).Results of Figure S1C show that livers from KO mice displayed a reduc-tion in the expression of genes associated with M1polarization (TNF a,IL-6,and MCP-1)and an increase in the expression of the M2polarization marker Mgl2.Of note,although not reaching statistical significance,the circulating levels of TNF a and IL-6 were decreased in KO mice(Figure S1D).Furthermore,the levels of circulating MCP1were significantly decreased in the serum of KO mice(Figure S1E),consistent with previously published studies demonstrating the role of this chemokine in the recruit-ment of inflammatory macrophages to obese adipose tissue(Ta-kahashi et al.,2003).NBR1Deficiency in Macrophages Improves Glucose MetabolismIn order to address whether the defects in macrophage polariza-tion of mutant mice affect glucose homeostasis,HFD-fed NBR1My KO mice where compared to identically treated WT con-trols in terms of glucose tolerance and insulin response.As demonstrated in Figure3A,NBR1deficiency in macrophages resulted in improved glucose clearance in glucose tolerance tests.Likewise,NBR1deficiency in macrophages resulted in improved insulin responses in insulin tolerance tests(Figure3B) and activation of Akt in liver,muscle,and WAT(Figure3C).In contrast,no differences were found in glucose tolerance and insulin response assays between both mouse genotypes when fed RD(Figures S2A and S2B).Thesefindings suggest that a lack of NBR1in the myeloid compartment improves glucose metabolism in the context of chronic HFD exposure.Consistent with this,fasting basal glucose(Figure3D)and insulin(Figure3E) levels were significantly reduced in the mutant mice,as were the transcript levels of liver PEPCK and G6Pase(Figure3F).How-ever,and in agreement with the fact that the NBR1My KO mice did not differ from their WT counterparts with respect to body weight and adiposity,the metabolic changes in the context of a HFD were not significantly different from those observed in identically treated WT mice(Figures S2C–S2H).A wide range of metabolic parameters was tested,including the whole-body respiratory exchange ratio(RER),diurnal patterns of food and water intake,locomotor activity,and calorie expenditure.There-fore,although the loss of NBR1in the myeloid compartment had a significant impact on adipose tissue inflammation and glucose homeostasis,it did not affect fat content or energy utilization at the organismal level.NBR1Is Critical for M1Differentiation and Macrophage Activation Ex VivoIn order to gain a more in-depth understanding of the mecha-nisms whereby NBR1regulates macrophage function in obesity, we used an ex vivo system,whereby bone-marrow-derived macrophages(BMDMs)were differentiated along the M1or M2lineage.Adipocytes secrete type2cytokines,such as IL-13and IL-4,that skew ATMs toward the anti-inflammatory M2profile.In contrast to their M1counterparts,which have a proinflammatory profile,M2ATMs counteract inflammation in vivo.ATMs are induced to differentiate along the M1lineage when exposed to IFN g(Gordon,2003;Kang et al.,2008;Ode-gaard et al.,2008;Vats et al.,2006).Therefore,BMDMs of both genotypes were incubated with IFN g or IL-13to promote M1or M2differentiation,respectively.Afterward,the expression of specific markers of each lineage was determined by quantita-tive RT-PCR.Notably,BMDMs from NBR1My KO mice had a dramatically reduced ability to respond to IFN g,as evident from the low levels of induced IL-6,TNF a and NOS transcription, but were sensitive to IL-13,as demonstrated by high levels of Arg1and Mgl2transcription(Figures4A and4B).These results are consistent with NBR1being required for the differentiation of macrophages along the M1,but not the M2,lineage and there-fore being an important transducer of proinflammatory signals. Consistent with this notion,the ability of BMDMs to induce IL-6 production,as measured at either the protein(Figure4C)or mRNA(Figure4D)level,in response to endotoxin exposure(lipo-polysaccharide[LPS])was also severely blunted in NBR1-defi-cient macrophages.In order to confirm these observations and rule out hypothetical developmental defects in KO macro-phages,macrophage-derived Raw cells were treated with either a shRNA that effectively knocked down NBR1levels(shNBR1; Figure4E,inset)or a control shRNA(shNT).Depletion of NBR1 in Raw cells resulted in impaired production of IL-6in response to LPS stimulation(Figure4E),reinforcing the hypothesis that NBR1plays a critical role in the inflammatory activation of mac-rophages.Because the loss of NBR1in the myeloid compart-ment reduced not only WAT inflammation but also the number of macrophages in that tissue(Figures2F–2I),we next tested if the chemotactic response of the mutant macrophages was affected.To this end,we performed migration assays using a Boyden chamber and applying the chemoattractant MCP-1. Both Raw cells depleted of NBR1(Figure4F)and NBR1My KO BMDMs(Figure4G)exhibited severely reduced chemotactic activity.Similar results were obtained with undifferentiated monocytes(Figure S3).Collectively,these results demonstrateCell MetabolismNBR1-MEKK3,a Key Regulator of JNKFigure 2.Myeloid-Specific Deletion of NBR1Reduced ATM and Inflammation in HFD-Fed Mice(A)Immunofluorescence staining for NBR1(green),F4/80(red),and DAPI (blue)in epididymal WAT of RD-fed mice (top)and HFD-fed mice (bottom).Colocal-ization of NBR1and F4/80is shown in yellow in the merged image (n =5mice).(legend continued on next page)Cell MetabolismNBR1-MEKK3,a Key Regulator of JNKthat NBR1is a positive regulator of the cell-autonomous,proin-flammatory functions of macrophages.NBR1Regulates the MEKK2/3-MKK4-JNK Pathway in MacrophagesTo begin to address the signaling pathway whereby NBR1gov-erns macrophage functions,BMDMs were stimulated with LPS for various periods ranging up to 1hr,and a number of signaling parameters were evaluated.Whereas JNK activation was repro-ducibly inhibited in the NBR1My KO BMDMs (Figure 5A),neither the activation of ERK (pERK)nor the phosphorylation and degra-dation of I k B were affected (Figure 5A).NBR1knockdown in the macrophage-derived Raw cell line led to a similar suppression of the JNK response to LPS treatment (Figure 5B).JNK is the main MAPK activated upon inflammatory stress,and it has been asso-ciated with glucose intolerance in HFD-treated mice (Hirosumi et al.,2002).JNK is also required for the polarization of BMDMs to the proinflammatory M1phenotype.Moreover,its deletion in macrophages reduces ATM infiltration as well as inflammation and enhances the efficiency of glucose metabolism in the context of obesity (Han et al.,2013;Sabio et al.,2008;Vallerie et al.,2008).Therefore,our results demonstrating that NBR1deficiency resulted in impaired JNK activation in macrophages and that it improved glucose tolerance of mice with a myeloid-specific KO establish NBR1as a key regulator of JNK function in the context of obesity-induced inflammation.In the next series of experiments we investigated the mech-anisms whereby NBR1regulates JNK activation.Interestingly,whereas activation of the JNK upstream kinase MKK4was severely suppressed in NBR1My KO BMDMs,that of MKK3/6was not affected (Figure 5C).When macrophages were stimu-lated with palmitate,a saturated fatty acid abundant during obesity,we found a robust activation of JNK in the WT cultures but not in NBR1My KO macrophages (Figure 5D).Notably,the synthesis of IL-6and TNF a was likewise induced by palmitate treatment in WT but not in mutant macrophages (Figure 5E).MKK7was not activated in either the KO or in WT macro-phages (data not shown).These results strongly suggest that NBR1positively regulates JNK through a direct or indirect interaction with MKK4and/or any of its upstream activators (MAPKKKs).PB1-Mediated Scaffold Role of NBR1in Macrophages As NBR1contains a PB1domain,we reasoned that its most likely immediate target would be a PB1-containing MAPKKK,MEKK2,or MEKK3.We further hypothesized that such an inter-action might involve direct contact between an acidic residue in the NBR1PB1domain and a basic residue in the N-terminal region of the MEKK2or MEKK3PB1domain (Figure 6A)(Moscat et al.,2006;Nakamura et al.,2010).Interestingly,MEKK3(Fig-ure 6B),similarly to NBR1(Figure 2B),was upregulated in ATMs isolated from HFD-fed mice as compared to those from mice fed a regular chow diet.To test this possibility,we ectopi-cally expressed HA-tagged MEKK2or MEKK3with Flag-tagged NBR1in human HEK293T cells and then assessed interactions between NBR1and these kinases by immunoprecipitation fol-lowed by immunoblotting.The results in Figure 6C show that,whereas Flag-tagged NBR1was efficiently immunoprecipitated with HA-tagged MEKK3,this was not the case for HA-tagged MEKK2,establishing that NBR1interacts specifically with MEKK3.Notably,a D50R mutation in the PB1domain of NBR1completely abolished this interaction (Figure 6D).Figure 6E demonstrates that endogenous NBR1and MEKK3likewise interact physically.In vitro studies using recombinant MKK4protein demonstrate that it was not able to interact with purified NBR1in a pull-down assay but that the addition of recombinant MEKK3allowed the coprecipitation of MKK4and NBR1(Figure S4A).Consistent with the requirement of the PB1for MEKK3interaction with NBR1,the NBR1-D50R mutant completely abolished the in vitro coprecipitation of NBR1with MKK4(Figure S4A).In agreement with these in vitro results,pull-down of ectopically expressed GST-MKK4resulted in coimmunoprecipitation of ectopically expressed NBR1and MEKK3(Figure 6F).This dem-onstrates that MEKK3acts as a bridge to bring MKK4and NBR1together.To test the existence of this complex in an endogenous setting,we next stimulated HEK293cells stably expressing hTLR4,MD2,and CD14with LPS and then immunoprecipitated endogenous NBR1.Western blotting revealed that the interac-tion between NBR1and MEKK3was not inducible,but the inter-action with MKK4was (Figure 6G).Notably,treatment of BMDM with palmitate resulted in enhanced expression of NBR1and MEKK3(Figure 6H),in accordance with the data of Figures 2B and 6B in ATMs from obese mice.More importantly,under these conditions we also found a clear endogenous NBR1-MEKK3interaction (Figure 6H).Thus,NBR1appears to function as an organizer of a MEKK3/MKK4cassette that is required to activate JNK and an obesity-induced inflammatory response.The fact that the simple overexpression of NBR1is sufficient to elevate JNK activation to levels comparable to those observed following stimulation with LPS (Figure 6I)further supports the notion that the ability of NBR1to nucleate a signaling complex is essential(B)Quantitative analysis of relative expression of NBR1,as measured by RT-PCR using total RNA isolated from ATMs of RD-fed mice and HFD-fed mice (n =6–10mice).(C)NBR1expression in BMDMs (Mø),liver,epididymal WAT,and BAT of WT and NBR1My KO mice,as assessed by western blotting.Results are representative of three experiments.(D and E)(D)Body weight and (E)fat mass of WT and NBR1My KO mice fed with HFD (n =6–8mice).(F)Morphology and myeloid infiltration of WAT samples from HFD-fed WT and NBR1My KO mice,as assessed by H&E staining and F4/80immunostaining (n =6–8mice).(G)Quantitative analysis of relative expression of markers of inflammation,as measured by RT-PCR assay using total RNA isolated from WAT of WT and NBR1My KO mice (n =6–8mice).(H)The stromal vascular fraction (SVF)of epididymal adipose tissue was isolated from WT and NBR1My KO mice fed with HFD (4weeks)and examined by flow cytometry to detect the total number of F4/80+ATMs (n =5mice).(I)Quantitative analysis of relative expression of markers of inflammation,as measured by RT-PCR using total RNA isolated from ATMs of WT and NBR1My KO mice (n =5mice).*p <0.05;***p <0.001.Results are presented as mean ±SEM.Scale bars,50m m.See also Figure S1.Cell MetabolismNBR1-MEKK3,a Key Regulator of JNKfor the activation of JNK.In agreement with our hypothesis,the knockdown of MEKK3in BMDM led to reduced JNK activation,as well as of IL-6and TNF a synthesis,in response to palmitate stimulation (Figures 6J and 6K).MLK3,another MAP3K like MEKK3,has been shown be activated by saturated free fatty acids and required for JNK activation in embryo fibroblasts,as well as in the adipose tissue of HFD-fed mice (Holzer et al.,2011;Jaeschke and Davis,2007).However,MLK3deficiency does not affect obesity or glucose homeostasis in HFD-fed mice (Jaeschke and Davis,2007).Our data demonstrate that although MLK3is activated in palmitate-treated macrophages,this is independent of NBR1(Figure S4B).Consistent withaFigure 3.Myeloid-Specific Deletion of NBR1Improved Glucose Clearance in HFD-Fed Mice(A and B)Glucose tolerance test (GTT)(A)and insulin tolerance test (ITT)(B)in 8-week-old WT and NBR1My KO mice fed a HFD for 4weeks (n =6–8mice).(C)WT and NBR1My KO HFD-fed mice were fasted overnight and then treated by intraperitoneal injection with 1U/kg insulin (15min).Representative tissue samples were examined by immunoblot analysis by probing with antibodies to phospho-AKT,AKT,and actin.Quantification of phospho-AKT is shown at bottom (n =5mice).(D and E)Blood concentration of glucose (D)and insulin (E)in overnight fasted mice HFD-fed WT and NBR1My KO mice (n =6–8mice)were measured.(F)Quantitative analysis of relative expression of PEPCK and G6Pase,as measured by RT-PCR using total RNA isolated from livers of HFD-fed WT and NBR1My KO mice (n =6–8mice).*p <0.05;**p <0.01;***p <0.001.Results are presented as mean ±SEM.See also Figure S2.Cell MetabolismNBR1-MEKK3,a Key Regulator of JNKfunctional role for the PB1domains in the interaction between NBR1and MEKK3,the overexpression of NBR1but not of a PB1mutant (D50R)activates JNK in cotransfection with MEKK3(Figure 6L).Interestingly,similar results were obtained in palmitate-activated cells (Figure 6M).Furthermore,the re-expression of NBR1WT but not of NBR1-D50R reconstituted JNK activation in response to palmitate in NBR1-deficient BMDMs (Figure 6N).Figure 4.NBR1Deletion in Macrophages Impaired Macrophage Polarization toward the Proinflammatory M1Phenotype(A and B)Quantitative analysis of relative expression of markers of inflammation.Total RNA was isolated from BMDMs of WT and NBR1My KO mice and incubated with either 100ng/ml of IFN g for 8hr or 10ng/ml of IL-13for 72hr.The relative expression of the indicated markers of M1(A)or M2(B)polarization was measured by quantitative RT-PCR assay (n =6mice).(C)IL-6secretion by BMDMs from WT and NBR1My KO mice fed with HFD for 12weeks after LPS stimulation (100ng/ml)for 3hr (n =4mice).(D)Relative expression of mRNA IL-6by BMDMs from WT and NBR1My KO mice after LPS stimulation for 8hr (n =6mice).(E)IL-6secretion by shNT and shNBR1Raw cells after LPS stimulation for 8hr.(F)Migration of shNT and shNBR1Raw cells after stimulation with the chemoattractant MCP-1for 20hr,as determined by a modified Boyden chamber assay.Left panel:representative filters.Right panel:quantitation of data with total number of cells migrated indicated on y axis.(G)Migration of BMDMs from WT and NBR1My KO mice,treated as in (F).Panels as in (F).Data are representative of three experiments.*p <0.05;**p <0.01;***p <0.001.Results are presented as mean ±SEM.Scale bars,100m m.See also Figure S3.Cell MetabolismNBR1-MEKK3,a Key Regulator of JNKFigure 5.NBR1Deletion in Macrophages Downregulated the MEKK2/3-MKK4-JNK Pathway(A–C)Western blot analysis with the indicated antibodies in BMDMs from WT and NBR1My KO mice ([A]and [C])or in shNT-and shNBR1-treated Raw cells (B)stimulated with LPS (100ng/ml)for the indicated time.Quantification of pJNK (A and B)and pMKK4(C)fold change activation is shown in the right panels.(legend continued on next page)Cell MetabolismNBR1-MEKK3,a Key Regulator of JNKDISCUSSIONMany studies have demonstrated that obesity-induced inflam-mation plays a critical role in the generation of the metabolic syn-drome,including a dysfunctional glucose metabolism and insulin resistance (Glass and Olefsky,2012;Gregor and Hotamisligil,2011).However,the precise signaling cascades accounting for these effects still need to be identified,which is of great impor-tance for the design of more efficacious treatments of type 2diabetes.In this regard,JNK was recently recognized as an important factor in obesity-induced inflammation,a condition that contributes significantly to aberrant glucose metabolism in obesity (Han et al.,2013;Lanuza-Masdeu et al.,2013;Sabio et al.,2008;Vallerie et al.,2008;Zhang et al.,2011).The initial observations were that JNK activity was abnormally elevated in obese mice and that whole-body ablation of JNK1resulted in de-creases in body weight and adiposity in the context of a HFD (Hirosumi et al.,2002;Tuncman et al.,2006).Although of great relevance,those results did not distinguish between a role for JNK activity in controlling the inflammation induced by obesity from those effects specific to the control of adiposity itself and ter studies,in which JNK1was genetically and selectively inactivated in adipocytes,established that this JNK isoform plays a critical role in the inflammatory phenotype of adipocytes and only a minor role in that of macrophages,in the context of obesity-induced glucose intolerance (Sabio et al.,2008).However,the most recent data from the same investiga-tors demonstrated that simultaneous genetic inactivation of both JNK1and JNK2in the myeloid compartment improved glucose tolerance in these mice,although it did not affect the body weight or adiposity (Han et al.,2013).Other potential mech-anisms of action might include the implication of NBR1in the control of inflammation in b cell function.However,the fact that insulin actions in ITT and phospho-Akt induction are enhanced in response to insulin injection suggests that the most likely main mechanism of action of macrophage NBR1is not by controlling insulin secretion.Collectively these data demonstrated that JNK activation in macrophages is a critical step in obesity-induced inflammation and glucose intolerance.However,the upstream activators of this JNK activity in macro-phages remained unknown.Our data in the present paper demonstrate that the loss of NBR1specifically in macrophages largely prevents obesity-induced inflammation in vivo.More-over,our ex vivo and in vitro results link the PB1domain-medi-ated interaction between NBR1and MEKK3to JNK activation and the M1polarization of macrophages.These observations have to be put in the context of the other PB1domain proteins,p62and PKC z ,which have been also implicated in the regulation of obesity and the metabolic alter-ations associated to this physiopathological condition (Moscat et al.,2006,2007,2009).In this regard,it is quite striking that whereas p62deficiency results in obesity and that the inflamma-tion associated to the lack of p62is secondary to increased adiposity,the PKC z loss selectively promotes inflammation inobese mice without effects on adiposity (Mu¨ller et al.,2013;Rodriguez et al.,2006).Interestingly,PKC z antiinflammatory actions are mostly confined to the adipocyte,with no effects on the hematopoietic system (Lee et al.,2010).In contrast,NBR1is a proinflammatory signaling adaptor in macrophages by nucleating a MEKK3-driven JNK cascade,not only in response to inflammatory signals such as LPS,but more impor-tantly in response to saturated fatty acids like palmitate.The mechanisms whereby palmitate promotes the accumulation of NBR1,and MEKK3,are not known but will be very interesting to address in future studies.Overall,these findings reveal a potentially pharmacologically targetable mechanism underlying JNK activation and suggest that blocking NBR1binding to MEKK3may represent an opportunity to prevent type 2diabetes in human obesity.EXPERIMENTAL PROCEDURESAntibodies and ReagentsMurine IFN g ,IL-13,and MCP-1were obtained from Peprotech,Inc.Lipopoly-saccharide from Escherichia coli 0111:B4was from Sigma-Aldrich.Gluta-thione sepharose 4b,protein G sepharose 4fast flow,and protein A sepharose 4fast flow were from GE Healthcare Life Sciences.HisPur Cobalt resin was from Thermo Scientific.EZview red anti-HA affinity gel was from Sigma-Aldrich.Antibodies against HA (sc-805),myc (sc-40),NBR1(sc-130380),MEKK2(sc-1088),MEKK4(sc-166196),MKK4(sc-837),MKK3/6(sc-13069),ERK1(sc-94),p38(sc-728),TAK1(sc-7162),and AKT (sc-5298)were from Santa Cruz Biotechnology.Antibodies against pMKK3/6(#9231S),pMKK4(#9151S),pMKK7(#4171S),pJNK (#4668S),pERK (#4370S,pp38(#9215S),IkBalpha (#4814S),pIkBalpha (#9246S),and pAKT-S473(#4058S)were from Cell Signaling Technology.Flag (F3165)and b -actin (clone AC-74)antibodies were from Sigma-Aldrich.Antibody against MEKK3(m79820)was purchased from BD Biosciences.p62antibody (GP62-C)was from PROGEN Biotechnik GmbH.Antibody against JNK (#551196)was purchased from PharMingen.pCMV5-Flag-NBR1and pWZL-Flag-NBR1plasmids were obtained by sub-cloning mouse NBR1(NM_008676)into the pCMV5-Flag and pWZL-Flag vec-tors,respectively.The point mutation resulting in an aspartic acid to arginine substitution at position 50(D50R)of the NBR1PB1domain was engineered into the pCMV5-Flag-NBR1and pWZL-Flag-NBR1constructs using the QuikChange site-directed mutagenesis kit (Stratagene)and NBR1D50primers (Table S2).pEBG-MKK4(GST-MKK4)plasmid was from Addgene (Plasmid:21563).pCMV-HA-MEKK2and pCMV-HA-MEKK3plasmids were a kind gift from Dr.Frank S.Lee (University of Pennsylvania School of Medicine).MLK3activity reagents were a kind gift from Dr.Ajay Rana (Loyola University Chicago Stritch School of Medicine).MiceNBR1fl/flmice were described previously (Yang et al.,2010).NBR1fl/flmice were bred to LysM-cre mice to generate myeloid-specific NBR1-KO (NBR1My KO).All genotyping was done by PCR.Animals were maintained under controlled temperature (22.5 C)and illumination (12hr dark/light cycle).Mice had free access to water and were fed either standard chow or high-fat diets (45%-HFD;D12451or 60%-HFD,D12492;Research Diets Inc.)ad libi-tum.To assess glucose tolerance and insulin sensitivity,8-week-old mice were fed a standard chow or a 60%high-fat diet for 4weeks.Mice were in-jected intraperitoneally with 2g glucose/kg body weight after overnight fasting (25%D-glucose [Fisher Scientific]in 0.9%saline)for GTT assay.For ITT,mice were injected with 0.5–0.75U insulin/kg body weight (100U/ml Novolin R [Novo Nordisk])after 6hr fasting.Tail-blood glucose levels were measured(D)Western blot analysis with the indicated antibodies in BMDMs from WT and NBR1My KO mice fed a HFD and stimulated with palmitate (0.8mM)for the indicated times.Quantification of pJNK fold change activation is shown in the right panel.(E)Quantitative analysis of relative expression of markers of inflammation as measured by RT-PCR using total RNA isolated from BMDMs of WT and NBR1My KO mice treated as in (D).Data are representative of three experiments.*p <0.05;**p <0.01;***p <0.001.Results are presented as mean ±SEM.Cell MetabolismNBR1-MEKK3,a Key Regulator of JNK。

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