Visfatin: a protein secreted by visceral fat that mimics the effects of insulin

Fat tissue produces a variety of secreted proteins (adipocytokines) with important roles in metabolism. We isolated a newly identified adipocytokine, visfatin, that is highly enriched in the visceral fat of both humans and mice and whose expression level in plasma increases during the development of obesity. Visfatin corresponds to a protein identified previously as pre-B cell colony-enhancing factor (PBEF), a 52-kilodalton cytokine expressed in lymphocytes. Visfatin exerted insulin-mimetic effects in cultured cells and lowered plasma glucose levels in mice. Mice heterozygous for a targeted mutation in the visfatin gene had modestly higher levels of plasma glucose relative to wild-type littermates. Surprisingly, visfatin binds to and activates the insulin receptor. Further study of visfatin's physiological role may lead to new insights into glucose homeostasis and/or new therapies for metabolic disorders such as diabetes.     

短期氟哌啶醇、利培酮、阿立哌唑作用对小鼠糖脂代谢及行为学影响

探究不同抗精神病药物(APDs)短期作用对MK801诱导的精神分裂症模型小鼠体质量、摄食、糖脂代谢以及动物行为学的影响.将60只雄性昆明小鼠随机分成5组,分别给予连续7 d的生理盐水, MK801, MK801加氟哌啶醇(2.0 mg/kg)(HAL), MK801加利培酮(1.0 mg/kg)(RIS), MK801加阿立哌唑(4.0 mg/kg)(ARI)处理.结果显示, MK801诱导的精神分裂症模型组自发活动(旷场实验)较正常组显著增加.模型组小鼠体质量、摄食及糖脂代谢参数均无显著性变化.加入APDs处理后,与模型组比较, 3种药物均能显著降低小鼠的自发活动,该参数与血脂中的甘油三酯(TG)和总胆固醇(TC)的浓度呈显著的负相关;此外, TG浓度还与肝脏组织中重要的转录因子SREBP1c的表达增加呈显著的正相关,与组蛋白受体H_1R的表达增加也呈显著的正相关.其中,利培酮表现出较强的引发代谢紊乱的趋势,该药物促进了肝脏组织中SREBP1c和SREBP-2以及H_1R的表达显著升高.因此得出,在APDs短期作用下,阿立哌唑无明显糖脂代谢紊乱,具有安全性高、不良反应小的特点.氟哌啶醇的过度镇静效应,可能是这类药物诱发一定程度的代谢性副反应的主要原因;而利培酮作为临床治疗重型精神疾病的一线用药,能较好地发挥疗效,但因其与其他靶点的亲和效应,可能引起更为严重的代谢紊乱副反应,并独立于体质量的增加.     

Diet-induced hypercholesterolemia in mice: prevention by overexpression of LDL receptors

The current studies were designed to determine whether chronic overexpression of low density lipoprotein (LDL) receptors in the liver would protect mice from the increase in plasma LDL-cholesterol that is induced by high-fat diets. A line of transgenic mice was studied that express the human LDL receptor gene in the liver under control of the transferrin promoter. When fed a diet containing cholesterol, saturated fat, and bile acids for 3 weeks, the transgenic mice, in contrast to normal mice, did not develop a detectable increase in plasma LDL. The current data indicate that unregulated overexpression of LDL receptors can protect against diet-induced hypercholesterolemia in mice.     

Role of brain insulin receptor in control of body weight and reproduction

Insulin receptors (IRs) and insulin signaling proteins are widely distributed throughout the central nervous system (CNS). To study the physiological role of insulin signaling in the brain, we created mice with a neuron-specific disruption of the IR gene (NIRKO mice). Inactivation of the IR had no impact on brain development or neuronal survival. However, female NIRKO mice showed increased food intake, and both male and female mice developed diet-sensitive obesity with increases in body fat and plasma leptin levels, mild insulin resistance, elevated plasma insulin levels, and hypertriglyceridemia. NIRKO mice also exhibited impaired spermatogenesis and ovarian follicle maturation because of hypothalamic dysregulation of luteinizing hormone. Thus, IR signaling in the CNS plays an important role in regulation of energy disposal, fuel metabolism, and reproduction.     

Modification of ocular defects in mouse developmental glaucoma models by tyrosinase

Mutations in the cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1) gene are a common cause of human primary congenital glaucoma (PCG). Here we show that Cyp1b1-/- mice have ocular drainage structure abnormalities resembling those reported in human PCG patients. Using Cyp1b1-/- mice, we identified the tyrosinase gene (Tyr) as a modifier of the drainage structure phenotype, with Tyr deficiency increasing the magnitude of dysgenesis. The severe dysgenesis in eyes lacking both CYP1B1 and TYR was alleviated by administration of the tyrosinase product dihydroxyphenylalanine (l-dopa). Tyr also modified the drainage structure dysgenesis in mice with a mutant Foxc1 gene, which is also involved in PCG. These experiments raise the possibility that a tyrosinase/l-dopa pathway modifies human PCG, which could open new therapeutic avenues.     

苜蓿总黄酮对小鼠脂类代谢及氧自由基的影响

试验结果显示：灌胃给予苜蓿总黄酮（TFA）的小鼠全血中甘油三酯（TG）和低密度脂蛋白胆固醇（LDL—C）的含量明显降低（P〈0．05）,而高密度脂蛋白胆固醇（HDL—C）含量升高,而且其肝脏和肾脏组织匀浆中过氧化物歧化酶（SOD）的活性显著升高（P〈0．05）,丙二醛（MDA）的含量降低。因此得出结论：TFA具有降血脂、降低低密度脂蛋白胆固醇含量、预防和减轻动脉硬化的作用;同时也具有抑制氧自由基损伤、防止脂质过氧化的作用。     

玉竹多糖对高脂膳食小鼠糖脂代谢调节作用的研究

采用高脂饲料联合玉竹多糖喂养小鼠,连续观察1个月,研究灌胃300 mg/kg的玉竹多糖对高脂膳食小鼠的体重、Lee's指数、肝指数、脂肪指数、血糖水平和血脂代谢等的影响。结果表明:玉竹多糖能明显改善高脂膳食小鼠紊乱的血糖、血脂代谢,显著降低高脂膳食小鼠的体重、Lee's指数、脂肪指数、血糖水平、甘油三酯(TG)、血清总胆固醇(TC)和低密度脂蛋白(LDL-C)(P0.05)。可见,玉竹多糖具有较好的调节糖脂作用,并可抑制小鼠肥胖的形成。     

PNPLA5-knockout rats induced by CRISPR/Cas9 exhibit abnormal bleeding and lipid level

Patatin-like phospholipase domain containing 5(PNPLA5) is a neotype neutral lipase with dual activity of anabolism and catabolism in vitro and in vivo,which has a low mRNA expression level in humans and mice.PNPLA5,which is localized to lipid droplets and required for efficient autophagy by optimal initiation,has been speculated to possess triglyceride hydrolase activity,and has been associated with low density lipoprotein cholesterol(LDL-C).Above all,PNPLA5 is a relatively new gene,which is reported less about its biological function research,especially the function research in the rats is still blank.In this study,we examined the spatiotemporal expression profile of PNPLA5 and found that it was expressed at low levels in most organs of Sprague Dawley(SD) rats,but was present at very high levels in the skin and testes.To further determine the biological function of PNPLA5 in mammals,we generated PA/PL45-knockout SD rats using the clustered regularly-interspaced short palindromic repeats(CRISPR)/Cas9 system.PA/PL45-null rats were viable,but showed a variety of phenotypic abnormalities,such as abnormal bleeding,and varied hematobiochemical parameters including increased serum total cholesterol(TC),triglycerides and high density lipoprotein cholesterol(HDL-C) level,and reduced LDL-C level,compared with wild-type control rats.These data are consistent with an important role for PNPLA5 in lipid metabolism,providing a new target gene and animal model for treatment of cardiovascular diseases in the future.     

猪OLR1基因对肌内前脂肪细胞分化的影响

研究氧化型低密度脂蛋白受体1(oxidized low-density lipoprotein receptor1, OLR1)基因在猪前脂肪细胞分化过程中的作用,为深入研究OLR1基因在脂肪沉积中的功能奠定基础。根据GenBank中猪OLR1基因序列(登录号:NM_213805),构建OLR1基因过表达载体OLR1-pcDNA3.1,同时合成干扰OLR1表达的siRNA。从恩施黑猪背最长肌中分离前脂肪细胞,分别将OLR1-pcDNA3.1和OLR1-siRNA转染前脂肪细胞,并对细胞进行诱导分化,至第6天,通过油红O染色、甘油三酯含量测定和荧光定量PCR检测OLR1基因对前脂肪细胞分化的影响。结果显示,OLR1过表达组脂滴明显多于对照组,甘油三酯含量显著(P<0.05)增加,成脂分化标志基因PPARγ的表达量极显著(P<0.01)升高、C/EBPα和FAS的表达量显著(P<0.05)升高;干扰OLR1后,细胞内脂滴减少,甘油三酯含量显著(P<0.05)下降,成脂分化标志基因PPARγ的表达量极显著(P<0.01)下降,C/EBPα和FAS的表达量显著(P<0.05)降低。综上表明,OLR1基因具有促进前脂肪细胞成脂分化的作用。     

鸸鹋油纳米乳剂对急性高脂血症小鼠血脂的影响

采用微射流技术制备鸸鹋油纳米乳,研究其对高脂饲料诱导的急性高脂血症模型小鼠血脂的影响,并评价鸸鹋油纳米乳的辅助降血脂功能。以昆明种小鼠为实验动物,采用腹腔注射75%蛋黄乳剂建立高脂血症模型,口服灌胃不同剂量的鸸鹋油纳米乳（按鸸鹋油剂量计,分别为1 mL/kg、2 mL/kg、3 mL/kg）,给药结束后分离小鼠血清,测定血清中甘油三酯（TG）、总胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）含量。结果显示采用微射流方法成功制备得到粒径较小且粒度分布均一的鸸鹋油纳米乳;服药后,高脂血模型组与正常对照组相比较, TG、TC明显升高（P＜0.001)、HDL-C变化不明显（P＞0.05);与模型组比较,鸸鹋油纳米乳剂组TG、TC含量降低,且呈剂量依赖性,高剂量组最为明显（P＜0.01),而且与鸸鹋油对照组比较,作用更为明显,表明制备的鸸鹋油纳米乳具有辅助降血脂功能。     

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Butyrylcholinesterase (BCHE) is a natural bioscavenger that protects humans against organophosphate toxicity. Due to the limited yield of human BCHE (hBCHE) when purifying from human plasma, it is necessary to find an alternative method to produce this protein. One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE. In this study, we cloned the hBCHEgene into a human lactoferrin (hLF) bacterial artificial chromosome (BAC) construct to make a hLF-hBCHE BAC construct. Subsequently, we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice. Expression analysis showed that recombinant hBCHE (rhBCHE) was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 76±12 to 159±28 mg·L^-1. Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE. Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.     

Krüppel-Like Factor 3（KLF3）基因对牛脂肪沉积的作用

【目的】通过研究牛KLF3基因对牛脂肪分化和脂肪酸代谢关键基因表达量的作用,进而探讨KLF3 基因对脂质沉积的影响。【方法】合成干扰siRNA,筛选得到KLF3基因干扰效率最高SiRNA,分离培养秦川牛肉用新品系新生牛前脂肪细胞生长至70%-90%汇合度时转染筛选得到的KLF3基因SiRNA和阴性对照SiRNA（NC）,并进行诱导分化培养至第0天和第4 天时,利用荧光定量PCR方法研究分化标志基因过氧化物酶体增殖物激活受体γ（peroxisome proliferator activated receptor γ, PPARγ）、CCAAT增强子结合蛋白（CCAAT/enhancer-binding proteins α, C/EBPα）,脂肪酸代谢关键基因脂肪酸合成酶（fatty acid synthase,FAS）、乙酰辅酶A羧化酶（acetyl-CoA carboxylase α, ACCα）和脂肪酸结合蛋白4（fatty acid binding protein 4, FABP4）基因在牛脂肪细胞分化不同时期干扰KLF3基因处理组和对照组之间表达量的变化,同时在干扰KLF3基因处理并进行诱导脂肪细胞分化的第 4天采用油红O染色法观察干扰KLF3基因处理组和对照组之间脂滴差异及采用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)方法测定甘油三酯含量进一步确定KLF3基因对牛脂肪细胞分化和脂肪酸代谢的作用。【结果】KLF3-Si2 具有最高的干扰效率达到73%。转染KLF3-Si2后PPARγ的表达量在牛脂肪细胞诱导分化的第0天 和第4 天与对照组相比分别下调了58% 和37%,达到了差异极显著（P<0.01）,C/EBPα 的表达量也分别下调了64% 和41%,达到了差异极显著（P<0.01）。脂质代谢的关键基因FABP4的表达量与对照组相比在牛脂肪细胞诱导分化的第0天和第4 天分别下调了89% 和60%,而ACCα的表达量干扰KLF3处理组与对照组相比在脂肪细胞诱导分化第0天和第4天分别下调了50% 和37%, 而FAS的表达量则分别下调了73% 和19%,都分别达到了差异极显著（P<0.01）。在牛脂肪细胞诱导分化第4天油红O染色和甘油三酯含量测定也发现干扰KLF3基因处理组与对照组相比脂滴减少,甘油三酯含量显著下降（P<0.05）。【结论】干扰牛KLF3基因可以抑制牛脂肪细胞分化和脂肪酸代谢关键基因表达量下调,进而影响脂肪的沉积。     

Neonatal hepatitis induced by alpha 1-antitrypsin: a transgenic mouse model

Transgenic mouse lineages were established that carry the normal (M) or mutant (Z) alleles of the human alpha 1-antitrypsin (alpha 1-Pi) gene. All of the alpha 1-Pi transgenic mice expressed the human protein in the liver, cartilage, gut, kidneys, lymphoid macrophages, and thymus. The human M-allele protein was secreted normally into the serum. However, the human Z-allele protein accumulated in several cell types, but particularly in hepatocytes, and was found in serum in tenfold lower concentrations than the M-allele protein. Mice in one lineage carrying the mutant Z allele expressed high levels of human alpha 1-Pi RNA and displayed significant runting (50% of normal weight) in the neonatal period. This lineage was found to have alpha 1-Pi-induced liver pathology in the neonatal period, concomitant with the accumulation of human Z protein in diastase-resistant cytoplasmic globules that could be revealed in the Periodic acid-Schiff reaction (PAS). The phenotype of mice in the strain expressing high levels of the Z allele is remarkably similar to human neonatal hepatitis, and this strain may prove to be a useful animal model for studying this disease.     

白藜芦醇和EGCG联用对小鼠骨骼肌抗氧化能力的影响

【目的】研究白藜芦醇（Resveratrol,RES）和表没食子儿茶素没食子酸酯（Epigallocatechin-3-Gallate, EGCG）联合使用对小鼠骨骼肌抗氧化能力影响的机制,为开发能通过提高畜禽骨骼肌抗氧化能力进而改善肉品质的新型、绿色、安全的饲料添加剂提供理论依据。【方法】将体重18～22 g的清洁级昆明种小鼠40只,随机分为4组,即二甲基亚砜（DMSO）对照组（CK）、RES组、EGCG组和RES+EGCG联用组,每组10只,采取灌胃的方式对应给药,剂量为每天400 mg/kg,连续给药28 d后测定小鼠骨骼肌中抗氧化酶活力指标,并通过荧光定量PCR方法测定小鼠骨骼肌中抗氧化相关基因的表达量。【结果】RES和EGCG联用小鼠的采食量、体重和脏器指数无显著影响（P>0.05）。与CK相比,RES组、EGCG组及RES+EGCG组血清和肌肉中丙二醛（MDA）含量显著降低（P<0.05,下同）,谷胱甘肽过氧化物酶（GSH-Px）、总超氧化物歧化酶（T-SOD）活力显著升高,小鼠腓肠肌中GSH-Px、超氧化物歧化酶1(SOD1)、超氧化物歧化酶2(SOD2)和核...     

高能营养液对游泳小鼠抗疲劳效果的研究

以负重游泳的方法制备疲劳模型,观察小鼠力竭性游泳时间和游泳后的血糖、尿素氮及肝糖原水平.结果表明:高能营养液组、葡萄糖氨基酸组、葡萄糖组动物力竭性游泳时间比游泳对照组分别延长142.42%、83.23%和37.37%,高能营养液组和葡葡糖氨基酸组动物力竭性游泳时间与游泳对照组比较,差异极显著(P<0.01);游泳对照组与正常对照组比较,血糖、肝糖原含量均降低,尿素氮水平升高.差异均极显著(P<0.01);与游泳对照组相比,高能营养液组、葡萄糖氨基酸组、葡萄糖组的血糖和肝糖原水平均有所升高,尿素氮水平卜升幅度减小.其中,高能营养液中高剂量组的血糖、血尿素氮、肝糖原与游泳对照组比较,差异极显著(P<0.01).表明该宠物用高能营养液能有效维持游泳动物血糖、肝糖原水平,抑制蛋白质分解,增强体能,具有一定的抗疲劳作用.     

油脂来源及使用量对小鼠生长、健康、血脂和肝脏胆固醇代谢指标的影响

【目的】观察饲料中添加不同种类和不同剂量的油脂对小鼠生长性能、健康状态、血脂指标和肝脏胆固醇代谢关键基因相对表达量的影响,探讨日粮中不同油脂来源及使用量对肝脏胆固醇代谢的作用机制,为哺乳动物筛选较为适宜的油脂类型及其添加量提供理论参考。【方法】试验选取3周龄体重16-19 g的健康昆明种小鼠48只,随机分为4组,每组4个重复,每个重复3只。4组分别饲喂正常小鼠饲料（对照组）和添加4%豆油（B组）、4%乳化椰子粉（L组）以及8%乳化椰子粉（H组）的饲料14 d。试验期间,记录每天的饲喂次数、每次饲喂量、饲料剩余量,所有试验动物均自由采食和饮水。分析试验期间小鼠的体重变化和平均日采食量（ADFI）、平均日增重（ADG）以及料重比（F/G）等生长性能指标,分析小鼠的血液分布和健康指数等健康状态指标,测定小鼠的肝脏重以及甘油三酯（TG）、总胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）等血清脂质指标,运用实时荧光定量PCR技术检测小鼠肝脏组织中胆固醇7-α羟化酶（CYP7A1）、低密度脂蛋白受体（LDLR）和3-羟-3-甲基戊二酸单酰辅酶A还原酶（HMGCR）这3个基因的mRNA表达水平。【结果】①生长性能方面,添加4%豆油组的小鼠体重变化、平均日采食量和肝脏脏器指数与对照组差异显著（P＜0.05）,而平均日增重、料重比和肝脏重与对照组差异不显著（P＞0.05）;添加4%乳化椰子粉组的小鼠各生长性能指标与对照组差异不显著（P＞0.05）;8%乳化椰子粉显著提高小鼠的平均日采食量（P＜0.05）,其他指标差异不显著（P＞0.05）。②健康状态方面,各试验组小鼠的血液分布和健康指数与对照组相比均无显著性差异（P＞0.05）。③血脂指标方面,与对照组相比,添加4%豆油显著降低了小鼠血清中TC和HDL-C含量（P＜0.05）,对TG和LDL-C的影响差异不显著（P＞0.05）,添加4%乳化椰子粉对小鼠的各项血脂指标均无显著性影响（P＞0.05）,添加8%乳化椰子粉能显著降低血清TC含量（P＜0.05）,但对TG、HDL-C和LDL-C无显著性影响（P＞0.05）;④肝脏胆固醇相关基因方面,添加8%乳化椰子粉组肝脏中CYP7A1基因mRNA表达量显著高于对照组（P＜0.05）,而4%豆油组和4%乳化椰子粉组与对照组相比无显著差异（P＞0.05）。肝脏HMGCR基因和LDLR基因mRNA表达水平与对照组差异不显著（P＞0.05）。【结论】日粮中添加豆油增加了小鼠日采食量和体重等指标,降低了血清总胆固醇和高密度脂蛋白胆固醇;高剂量乳化椰子粉的添加提高了小鼠日采食量,降低了血清总胆固醇,并增加了肝脏CYP7A1基因mRNA的表达。相比于添加高剂量乳化椰子粉,低剂量乳化椰子粉在改善小鼠脂代谢方面应用效果并不好。添加油脂可能是通过调控肝脏中胆固醇代谢相关酶的基因表达量来影响小鼠肝脏胆固醇的代谢,从而维持体内的胆固醇代谢的稳态。     

Synthesis of functional human hemoglobin in transgenic mice

Human alpha- and beta-globin genes were separately fused downstream of two erythroid-specific deoxyribonuclease (DNase) I super-hypersensitive sites that are normally located 50 kilobases upstream of the human beta-globin gene. These two constructs were coinjected into fertilized mouse eggs, and expression was analyzed in transgenic animals that developed. Mice that had intact copies of the transgenes expressed high levels of correctly initiated human alpha- and beta-globin messenger RNA specifically in erythroid tissue. An authentic human hemoglobin was formed in adult erythrocytes that when purified had an oxygen equilibrium curve identical to the curve of native human hemoglobin A (Hb A). Thus, functional human hemoglobin can be synthesized in transgenic mice. This provides a foundation for production of mouse models of human hemoglobinopathies such as sickle cell disease.     

树鼩脂蛋白脂肪酶的cDNA克隆、组织分布及活性测定

目的了解不易感动脉粥样硬化动物树鼠句的脂蛋白脂肪酶的结构和活性特点,并与其它种属比较,探讨脂蛋白脂肪酶在树鼠句独特脂代谢中的作用。方法以树鼠句脂肪细胞mRNA反转录获得的cDNA一链为模板,应用SMART-RACE技术获得树鼠句脂蛋白脂肪酶的cDNA序列并推导出其氨基酸序列;实时定量PCR方法检测树鼠句脂蛋白脂肪酶mRNA在各组织中的分布;真核表达树鼠句和人脂蛋白脂肪酶蛋白,放射性同位素法测定二者的活性并比较。结果树鼠句脂蛋白脂肪酶cDNA序列全长为3293bp,其中编码区1437bp,编码20个氨基酸的信号肽和458个氨基酸的成熟蛋白,该成熟蛋白N-端比人脂蛋白脂肪酶多10个氨基酸。树鼠句脂蛋白脂肪酶与其它哺乳类动物的氨基酸序列同源性大于90%,功能域高度保守。树鼠句脂蛋白脂肪酶的组织表达谱比较广泛,在心肌和脂肪组织中大量表达,但在骨骼肌中未检测到,却在肝脏中有中低水平表达,与人脂蛋白脂肪酶在这两种组织中的表达相反。活性测定结果显示,树鼠句脂蛋白脂肪酶活性显著高于人(7.67倍),这与测定的树鼠句血浆低甘油三酯水平相符。结论树鼠句的高活性脂蛋白脂肪酶可能是其血浆低水平甘油三酯的主要原因。     

Overexpression of low density lipoprotein (LDL) receptor eliminates LDL from plasma in transgenic mice

A complementary DNA encoding the human low density lipoprotein (LDL) receptor under control of the mouse metallothionein-I promoter was injected into fertilized mouse eggs, and a strain of mice expressing high levels of LDL receptors was established. After administration of cadmium, these mice cleared intravenously injected 125I-labeled LDL from blood eight to ten times more rapidly than did normal mice. The plasma concentrations of apoproteins B-100 and E, the two ligands for the LDL receptor, declined by more than 90 percent after cadmium treatment, but the concentration of another apoprotein, A-I, was unaffected. Therefore, overexpression of an endocytotic receptor can dramatically lower the ambient concentration of its ligand in vivo.