早期补充 PQQ 对肥胖小鼠肝脏脂毒性和炎症的发育规律具有长期的保护作用

0 Comments

Early PQQ supplementation has persistent long-term protective effects on developmental programming of hepatic lipotoxicity and inflammation in obese mice

Karen R. Jonscher 作者: Karen r. Jonscher,*,1Michael S. Stewart 迈克尔 · s · 斯图尔特,Alba Alfonso-Garcia 阿尔巴 · 阿方索-加西亚,Brian C. DeFelice 布莱恩 · c · 德费利斯,§Xiaoxin X. Wang 王,Yuhuan Luo 罗,Moshe Levi 莫舍 · 利瓦伊,Margaret J. R. Heerwagen 玛格丽特 · j · r · 赫尔瓦根,Rachel C. Janssen 雷切尔 · c · 詹森,Becky A. de la Houssaye 贝基 · a · 德拉 · 胡塞,Ellen Wiitala 艾伦 · 维塔拉,Garrett Florey 加勒特 · 弗洛里,Raleigh L. Jonscher 罗利 · l · 琼舍,Eric O. Potma 埃里克 · o · 波特马,#Oliver Fiehn 奥利弗 · 费恩,§** and 及Jacob E. Friedman 雅各布 · e · 弗里德曼Author information 作者信息Article notes 文章注释Copyright and License information 版权和许可证信息Disclaimer 免责声明This article has been 这篇文章已经被cited by 引用 other articles in PMC. 在 PMC 的其他文章

Abstract

摘要

Nonalcoholic fatty liver disease (NAFLD) is widespread in adults and children. Early exposure to maternal obesity or Western-style diet (WD) increases steatosis and oxidative stress in fetal liver and is associated with lifetime disease risk in the offspring. Pyrroloquinoline quinone (PQQ) is a natural antioxidant found in soil, enriched in human breast milk, and essential for development in mammals. We investigated whether a supplemental dose of PQQ, provided prenatally in a mouse model of diet-induced obesity during pregnancy, could protect obese offspring from progression of NAFLD. PQQ treatment given pre- and postnatally in WD-fed offspring had no effect on weight gain but increased metabolic flexibility while reducing body fat and liver lipids, compared with untreated obese offspring. Indices of NAFLD, including hepatic ceramide levels, oxidative stress, and expression of proinflammatory genes (Nos2Nlrp3Il6, and Ptgs2), were decreased in WD PQQ-fed mice, concomitant with increased expression of fatty acid oxidation genes and decreased Pparg expression. Notably, these changes persisted even after PQQ withdrawal at weaning. Our results suggest that supplementation with PQQ, particularly during pregnancy and lactation, protects offspring from WD-induced developmental programming of hepatic lipotoxicity and may help slow the advancing epidemic of NAFLD in the next generation.—Jonscher, K. R., Stewart, M. S., Alfonso-Garcia, A., DeFelice, B. C., Wang, X. X., Luo, Y., Levi, M., Heerwagen, M. J. R., Janssen, R. C., de la Houssaye, B. A., Wiitala, E., Florey, G., Jonscher, R. L., Potma, E. O., Fiehn, O. Friedman, J. E. Early PQQ supplementation has persistent long-term protective effects on developmental programming of hepatic lipotoxicity and inflammation in obese mice.

非酒精性脂肪性肝病(NAFLD)广泛存在于成人和儿童。早期暴露于母体肥胖或西式饮食(WD)会增加胎儿肝脏的脂肪变性和氧化应激,并且与后代终生患病的风险有关。吡咯并喹啉醌是一种存在于土壤中的天然抗氧化剂,富含人类母乳,对哺乳动物的发育至关重要。我们研究了在妊娠期饮食诱导肥胖小鼠模型胎儿期补充 PQQ 剂量是否可以保护肥胖后代免于 NAFLD 的进展。与未经治疗的肥胖子代相比,在出生前和出生后给予母乳喂养的子代 PQQ 治疗对体重增加没有影响,但增加了代谢灵活性,同时减少了体脂和肝脂。NAFLD 的各项指标,包括肝神经酰胺水平、氧化应激和促炎症基因(Nos2,Nlrp3,Il6和 Ptgs2)的表达,在 WD PQQ-fed 小鼠中下降,伴随脂肪酸氧化基因表达增加和 Pparg 表达减少。值得注意的是,这些变化甚至在断奶后仍然存在。我们的结果表明,补充 PQQ,特别是在妊娠和哺乳期,可以保护后代免受 wd 引起的肝脂毒性的发育程序,并可能有助于减缓 NAFLD 在下一代的流行。ー jonscher,k. r. ,Stewart,M.s. ,Alfonso-Garcia,a. ,DeFelice,B.c. ,Wang,x. ,Luo,y. ,Levi,m. ,Heerwagen,M.j,houssaye,b. a. ,Wiitala,e. ,Florey,g. ,Jonscher,r. l. ,Potma,早期补充 PQQ 对肥胖小鼠肝脏脂毒性和炎症的发育过程具有持久的长期保护作用。Keywords: 关键词:antioxidant, PGC-1α, ceramide, lipidomics, CARS 抗氧化剂,pgc-1,神经酰胺,脂质组学,CARS

Nonalcoholic fatty liver disease (NAFLD) describes a broad spectrum of chronic liver abnormalities, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH, the more severe form of the disease), characterized by different degrees of inflammation and fibrosis. NAFLD is the most common liver disease in the world and affects 20–30% of all adults in the United States and well over 60% of adults with obesity, increasing the risk of cardiovascular events, type 2 diabetes and hepatocellular carcinoma (1). There is compelling evidence from nonhuman primate and rodent models linking a maternal obesogenic environment and development of NAFLD in offspring (23). Indeed, exposure to a high-fat diet (HFD) or Western-style diet (WD) in utero, compared with only postnatal high-energy diet feeding, rapidly worsens the liver phenotype to fibrosis in rodents (245).

非酒精性脂肪性肝病(NAFLD)描述了一系列广泛的慢性肝脏异常,从单纯性脂肪变性到非酒精性脂肪性肝炎(NASH,一种更严重的疾病) ,拥有属性和纤维化程度不同。非酒精性脂肪肝是世界上最常见的肝脏疾病,影响美国20-30% 的成年人和超过60% 的肥胖成年人,增加心血管事件、2型糖尿病和肝细胞性肝癌的风险。非人类的灵长类动物和啮齿动物模型有令人信服的证据表明,产妇的肥胖环境与后代发生 NAFLD 有关(2,3)。事实上,在子宫内暴露于高脂肪饮食(HFD)或西式饮食(WD) ,与只在产后喂养高能量饮食相比,会迅速恶化啮齿动物的肝脏表型,导致肝纤维化(2,4,5)。

Periconception obesity in both parents affects reproductive health (reviewed in ref. 6), potentially by affecting embryonic mitochondrial metabolism, which may underlie the progression of liver damage observed in offspring born to obese mothers (79). How oxidative metabolism, including that of offspring exposed to a maternal HFD, may be restored and whether restoration will effectively blunt development of NAFLD, remain open questions. Therapeutic approaches targeting mitochondrial oxidative function, delivered both during gestation and lactation, may prevent metabolic complications in offspring in later life. Sen and Simmons (10) demonstrated that high doses of vitamins A, E, and C and selenium provided during pregnancy and lactation to WD-fed rats, effectively decreased adiposity and improved glucose tolerance in offspring switched to a lower fat control diet after birth; however, effects on liver were not tested. Likewise, we recently established that supplementing the diet of obese nonhuman primates fed an HFD with the antioxidant resveratrol prevented development of fatty liver in the fetus (11); however, it altered fetal pancreatic morphology, suggesting the need for further studies.

父母双方的全套叠肥胖会影响性健康。6) ,可能通过影响胚胎线粒体代谢,这可能是在肥胖母亲所生后代中观察到的肝损伤进展的基础(7-9)。如何恢复唿吸作用,包括暴露在母体 HFD 中的后代,以及恢复是否会有效地阻碍 NAFLD 的发展,仍然是一个悬而未决的问题。针对线粒体氧化功能的治疗方法,在妊娠期和哺乳期同时进行,可以预防后代代谢并发症。Sen 和 Simmons (10)证明,高剂量的维生素 a、 e、 c 和硒在孕期和哺乳期提供给 wd- 喂养的大鼠,能有效地降低肥胖症和提高后代的葡萄糖耐量,在出生后转换到低脂肪控制饮食,但是,对肝脏的影响没有测试。同样,我们最近证实,给肥胖的非人类灵长类动物喂食抗氧化剂白藜芦醇,可以防止胎儿脂肪肝的发展(11) ; 然而,它改变了胎儿的胰腺形态,表明需要进一步的研究。

Pyrroloquinoline quinone (PQQ) is a ubiquitous natural bacterial cofactor found in soil, plants, and interstellar dust and is essential for reproductive health and normal development in mammals (1213). PQQ is a powerful antioxidant. In the reduced form, its aroxyl radical-scavenging activity is 7.4-fold higher than that of vitamin C, the most active water-soluble antioxidant (14). PQQ stimulates mitochondrial biogenesis in vitro by activating peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC)-1α (15), an important regulator of metabolism and mitochondrial oxidative defense. PQQ supplementation increased the concentration of lysozyme in plasma (a component of the innate immune system) in broiler chicks (16), mitigated streptozotocin-induced oxidative damage and diabetes in mice (17), and protected mice from thioacetamide-induced liver fibrosis (18), whereas diets devoid of PQQ resulted in elevated plasma lipids in rats (19), suggesting PQQ targets the lesions of NAFLD. The major source of this important antioxidant in mammals is dietary (20), and PQQ is highly enriched in human breast milk (21), making it an intriguing dietary therapeutic for preventing excess maternal obesity-induced oxidative stress or ameliorating mitochondrial dysfunction and inflammation caused by exposure to an overabundance of toxic lipids during development.

吡咯并喹啉醌是一种普遍存在于土壤、植物和星际尘埃中的天然细菌辅助因子,对哺乳动物的性健康和正常发育至关重要。PQQ 是一种强效的抗氧化剂。在还原型中,其清除芳氧基自由基的活性比最活跃的水溶性抗氧化剂维生素 c 高7.4倍。PQQ 通过激活 PPAR-辅激活因子(PGC)-1(15)刺激线粒体生物合成,PQQ 是一种重要的新陈代谢调节因子和线粒体氧化防御过氧化物酶体增殖物活化受体。补充 PQQ 可以增加肉鸡(16只)血浆中溶菌酶(先天免疫系统的一种成分)的浓度,减轻链脲佐菌素诱导的氧化损伤和小鼠(17只)的糖尿病,保护小鼠免受硫代乙酰胺诱导的肝纤维化(18只) ,而缺乏 PQQ 的饮食导致大鼠(19只)血脂升高,提示 PQQ 的目标是 NAFLD 的损害。哺乳动物体内这种重要的抗氧化剂的主要来源是饮食(20) ,而且 PQQ 富含人类母乳(21) ,这使得它成为一种有趣的饮食疗法,可以预防母体过度肥胖引起的氧化应激或减轻线粒体功能障碍和发育过程中暴露于过多的有毒脂质引起的炎症。

The goal of this study was to test the hypothesis that PQQ supplementation, provided prenatally at conception and through lactation in obese pregnancy, improves offspring metabolic outcomes, enhances oxidative defense, and prevents NAFLD in the livers of obese offspring exposed to HFD both prenatally and after weaning. Furthermore, using coherent anti-Stokes Raman spectroscopy (CARS) imaging and lipidomics profiling, we sought to determine whether levels of bioactive lipids, frequently linked to hepatic apoptosis and metabolic dysfunction, are reduced in livers of obese mice given supplemental PQQ.

本研究的目的是检验 PQQ 补充剂的假设,即 PQQ 补充剂在受孕前和肥胖妊娠期通过哺乳提供,改善后代代谢结果,增强氧化防御,并防止肥胖后代在断奶前和断奶后接触 HFD 的肝脏 NAFLD。此外,通过相干的反斯托克斯拉曼光谱学显像和脂质组学分析,我们试图确定服用 PQQ 补充剂的肥胖小鼠肝脏中的生物活性脂质水平是否降低,这些脂质经常与肝细胞凋亡和代谢紊乱有关。

MATERIALS AND METHODS

物料及方法

Animals and diets

动物和饮食

All experiments were reviewed, approved, and monitored by the University of Colorado Institutional Animal Care and Use Committee in accordance with the Guide for Care and Use of Laboratory Animals(National Research Council, Washington DC, USA). C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and used for 2 different studies: a maternal–fetal study (study 1) and a developmental programming study in offspring (study 2), as outlined in Supplemental Fig. S1. For study 1, 8-wk-old females were randomly assigned to either a low-fat diet (LFD; 3.85 kcal/g, 10% kcal from fat) (D12450B; Research Diets, New Brunswick, NJ, USA) or a high-fat diet (HFD; 4.73 kcal/g, 45% kcal from fat) (D12451; Research Diets) matched for micronutrients. The diets were fed to the mice ad libitumfor 8 wk before mating and throughout gestation. At ∼16 wk of age, females were mated with standard chow-fed males, and PQQ supplementation was initiated. PQQ (obtained as a gift from Dr. Robert Rucker, University of California, Davis) was dissolved in water at a concentration of 1.25 mg/l (3.8 μM) providing ∼7.5 μg/d based on standard C57BL/6J mouse water consumption (22). This daily dose was derived from human studies (23) and adjusted for increased caloric expenditure in mice vs. humans. Successful pregnancy was confirmed by the presence of a vaginal plug. Mothers were individually housed during gestation, and mice and food were weighed weekly.

所有的实验都是由科罗拉多大学博尔德分校动物护理和使用委员会根据实验动物护理和使用指南(美国华盛顿特区国家研究委员会)进行审查、批准和监测的。C57BL/6J 小鼠购自美国杰克逊纪念实验室,用于2个不同的研究: 母胎研究(研究1)和后代发育规划研究(研究2) ,概述在补充图。中一。在研究1中,8周大的女性被随机分配到低脂肪饮食(LFD; 3.85 kcal/g,10% kcal from fat)(D12450B; 美国新泽西州新不伦瑞克的研究饮食)或高脂肪饮食(HFD; 4.73 kcal/g,45% kcal from fat)(D12451; 研究饮食)配合微量营养素。这些饲料在交配前和整个妊娠期自由喂养小鼠8周。在∼16周龄时,雌性与标准喂食的雄性交配,并开始补充 PQQ。PQQ (由加州大学戴维斯分校 Robert Rucker 博士赠送)溶于浓度为1.25 mg/l (3.8 m)的水中,以标准 C57BL/6J 小鼠耗水量(22)提供∼7.5 g/d。这个每日剂量来源于人类研究(23) ,并根据小鼠与人类相比增加的热量消耗进行调整。成功的怀孕是由阴道塞的存在证实的。母鼠在妊娠期间被单独安置,每周称量老鼠和食物的重量。

Blood and tissues of mothers and pups were harvested in very late gestation [embryonic d (E)18.5] (24). The animals were unfed for 4 h before glucose analysis and then were euthanized. Maternal blood and tissues were quickly collected with simultaneous dissection and isolation of fetal–placental units. Each fetus and placenta was weighed separately, and each fetus was subsequently euthanized by decapitation and exsanguination. Fetal blood was pooled between littermates for subsequent analysis. Fetal livers were dissected, snap frozen, and stored at −80°C for later analysis; stabilized in RNALater (Qiagen, Valencia, CA, USA); or embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) and used for cryosections. Livers from 1 fetus per litter were selected for analysis; therefore nrepresents the number of litters in study 1. Maternal weight was determined as gross weight before harvest minus aggregate weight of fetal–placental units. Placentas were measured with a micrometer, lengthwise and across the placental diameter after dissection (24). The placental surface area was calculated as length × width.

母体和幼体的血液和组织在非常晚的妊娠期[胚胎 d (e)18.5](24)获得。动物在葡萄糖分析前停食4h,然后实施安乐死。同时解剖分离胎盘单位,快速采集母血和组织。每个胎儿和胎盘分别称重,然后对每个胎儿进行斩首和放血处死。胎儿血液在同一胎儿之间汇集以便随后分析。胎儿肝脏被解剖、冷冻,并保存在 -80 ° c 以备后续分析; 在 RNALater (美国加利福尼亚州巴伦西亚市)稳定存放; 或在最佳切割温度(OCT)复合物(美国加利福尼亚州托兰斯市樱花 Finetek)中埋藏,用于低温切片。选取每窝1个胎儿的肝脏进行分析,因此 n 代表研究1中胎儿的数量。母体体重以收获前的总体重减胎盘单位的总体重为准。解剖后用千分尺、纵向和横向测量胎盘直径(24)。胎盘表面积以长宽计算。

For study 2, sibling offspring of breeders (fed a standard chow diet) were randomly stratified into breeding groups and fed standard chow [CH; 22% kcal from fat, 23% protein, 55% carbohydrate, 3.3 kcal/g; (CH) 2019, Teklad; Envigo, Indianapolis, IN, USA] or WD [TD.88137, Envigo; 42% kcal from fat, 15% protein, 43% carbohydrate (34% sucrose by weight), 0.2% cholesterol, and 4.5 kcal/g], with or without PQQ supplementation. In the maternal–fetal studies, dams were fed an HFD to focus on the effects of a high lipid load on fetal hepatic metabolism. We changed from HFD to WD for the postnatal studies, because WD accelerates steatosis and mild fibrosis (25) and at 20 wk of age, the WD replicates a phenotype similar to human NASH (26). In previous studies, we found that long-term feeding of mice with an LFD adversely affected their health and led to an increased rate of early death from undetermined causes in the vivarium. The standard chow diet represented a more physiologically relevant diet that reflected a healthier level of fat consumption; therefore, we decided to use that diet, even though it was not matched for micronutrients with WD. Second-generation offspring were either continued on the maternal diet or were switched after weaning to WD without PQQ (WD PQQ/WD). Last, we tested whether a subset of WD-fed offspring supplemented with PQQ after weaning at a higher concentration of 12.1 μM, providing ∼24 μg/d per mouse, provided increased protection from weight gain (WD PQQ 3X). Tissues from offspring were either harvested at the time of weaning [postnatal d (PND) 21] or at 20 wk of age. Offspring were unfed for 4 h and then were euthanized via isoflurane anesthetic followed by cervical dislocation and cardiac exsanguination. Livers were dissected, weighed, snap frozen in liquid nitrogen, and stored at −80°C until they were used or were embedded in OCT compound and used for cryosections. The n in study 2 represents the number of animals studied. The study mice were derived from repeated multiple litters, representing 2 or more breeding pairs. The data presented herein include males (except for E18.5 mice), because they have been shown to respond more robustly to diet-induced obesity than females (2427).

对于研究2,饲养者的兄弟后代(饲喂标准饲料)被随机分成饲养组,饲喂标准饲料(CH; 22% 脂肪,23% 蛋白质,55% 碳水化合物,3.3千卡/克; (CH)2019,Teklad; Envigo,IN,美国)或 WD (TD. 88137,go; 42% 脂肪,15% 蛋白质,43% 蔗糖(34% 体重) ,0.2% 蔗糖,4.5千卡/克) ,同时添加或不添加 PQQ。在母胎研究中,母鼠被喂以高脂肪负荷对胎儿肝脏代谢的影响。因为 WD 加速脂肪变性和轻度纤维化(25) ,在20周龄时,WD 复制出类似于人类 NASH (26)的表型。在以前的研究中,我们发现长期喂养 LFD 小鼠会对它们的健康产生不利影响,并导致由于未确定的病因导致的早期死亡率增加。标准的食谱代表了一种生理上更相关的饮食,反映了脂肪摄入量的健康水平; 因此,我们决定使用这种饮食,即使它与患有 WD 的微量营养素不匹配。第二代子代要么继续采用母体饲料,要么在断奶后转换为无 PQQ (WD PQQ/WD)的 WD。最后,我们测试了在断奶后添加 PQQ (12.1 m,每只小鼠提供ー24 g/d)的 wdfed 后代子集是否增加了对体重增加的保护(WD PQQ 3X)。后代组织要么在断奶时[出生后 d (PND)21]收获,要么在20周龄收获。子代停食4小时后行异氟醚麻醉,随后行颈椎脱位、心脏放血。肝脏解剖、称重、液氮速冻,-80 °c 保存,直至使用或置于 OCT 复合物中用于低温切片。研究2中的 n 代表所研究的动物数量。这项研究的小鼠来源于重复多胎,代表2个或更多的繁殖对。这里提供的数据包括雄性(E18.5小鼠除外) ,因为已经证明它们对饮食诱导的肥胖反应比雌性更强烈(24,27)。

Indirect calorimetry

间接量热法

Energy balance (energy intake, resting and nonresting energy expenditure, and fuel utilization) was determined via indirect calorimetry at PND 21 or 20 wk. The mice were individually placed in an 8-chamber system (Oxymax Comprehensive Lab Animal Monitoring System; Columbus Instruments, Columbus, OH, USA), equipped with control of temperature, humidity, and light/dark cycle. The mice were acclimated to the system for 2–3 d before data acquisition and then monitored for 3–5 d, during which time food and water intake were measured, as well as oxygen consumption (Vo2) and carbon dioxide production (Vco2). The respiratory quotient (RQ) was calculated as the ratio of CO2 production to O2consumption (Vco2/Vo2) (2830). Activity was measured and thermic energy from food (TEF; kilocalories above resting energy expenditure that are necessary to metabolize or store foods consumed) was inferred from total energy expenditure and activity (31). After monitoring, the mice were returned to group housing for up to 48 h and then euthanized.

采用间接量热法测定能量平衡(能量摄入、静息和非静息能量消耗、燃料利用)。这些小鼠被分别放置在一个8室的系统中(Oxymax 综合实验室动物监测系统; Columbus Instruments,OH,USA) ,该系统配备有温度、湿度和光/暗循环控制装置。小鼠在数据采集前适应该系统2-3天,然后监测3-5天,在此期间测量食物和水的摄入量,以及氧耗量(Vo2)和二氧化碳产量(Vco2)。呼吸商(RQ)以 CO2产生量与 O2消耗量(Vco2/Vo2)的比值(28ー30)计算。测量活动量,并从总能量消耗和活动量(31)推断食物的热量(TEF; 千卡路里,高于代谢或储存食物所需的静息能量消耗)。在监测后,将小鼠送回组房长达48小时,然后实施安乐死。

Body composition

身体成分

Body composition was determined in adult male mice by quantitative magnetic resonance imaging (qMRI; Echo MRI Whole Body Composition Analyzer; Echo Medical Systems, Houston, TX, USA). Body composition was measured either before acclimation to the calorimeter or 24–48 h before death.

采用定量磁共振成像技术(qMRI; Echo MRI 全身成分分析仪; Echo Medical Systems,休斯顿,TX)测定成年雄性小鼠的体成分。在适应热量计前或死亡前24-48小时测量体成分。

Analysis of metabolic markers

代谢标志物分析

Maternal and fetal glucose concentrations were determined at death with a OneTouch Ultra Meter (Lifescan, Milpitas, CA, USA); unfed offspring glucose levels were quantified using blood from the tail vein 1–2 wk before euthanasia. Blood was collected via cardiac puncture for the mother/offspring or by decapitation of the fetus after euthanasia. Fetal blood was pooled from each litter and treated as a single sample for analysis. After incubation at either room temperature for 15 min (fetuses) or on ice for 30 min (offspring), blood samples were centrifuged for 10 min at 4000 g. Insulin concentration was determined in serum using an Ultrasensitive Insulin ELISA (Crystal Chem, Downers Grove, IL, USA), according to the manufacturer’s instructions. Maternal homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as fasting blood glucose (in mg/dl) × fasting insulin (in microU/ml) divided by 405. Serum triglycerides (TGs) were determined with the Infinity Triglycerides Liquid Stable Reagent (Thermo Fisher Scientific, Waltham, MA, USA) with an internal glycerol standard and quantified with a spectrophotometer reading at 540 nm. Liver TGs were extracted from ∼50 mg of frozen tissue (3) and quantified as for serum. Final concentrations were normalized to starting tissue weight at homogenization.

死亡时用 OneTouch Ultra Ultra Meter (米尔皮塔斯 Lifescan)测定母亲和胎儿血糖浓度,安乐死前用尾静脉1-2周的血液测定未喂食子女的血糖水平。血液采集通过心脏穿刺为母亲/子女或安乐死后的胎儿断头。胎儿的血液从每窝中汇集起来,作为单一样本进行分析。在室温孵育15分钟(胎儿)或冰孵育30分钟(子代)后,血液样本在4000g 胰岛素浓度下离心10分钟,用超灵敏的胰岛素酶联免疫吸附试验(Crystal Chem,Downers Grove,IL,USA)按照制造商的说明书进行检测。以空腹血糖(mg/dl)为空腹胰岛素(microU/ml)除以405。血清甘油三酯(TGs)用无限大甘油三酯液体稳定试剂(沃尔瑟姆 Thermo Fisher Scientific,MA,USA)测定,甘油内标,并在540nm 处用光谱仪读数进行定量。肝组织总黄酮从冰冻组织中提取大约50毫克,作为血清测定。最终浓度均匀化为起始组织重量。

CARS imaging

车辆成像

CARS images of 12-μm-thick liver cryosections from selected adult mice were obtained by combining 2 laser beams: a Stokes beam fixed at 1064 nm and a pump beam tuned at 817 nm. The CARS microscope has been described (32). We also analyzed E18.5 and PND 21 liver sections using an Olympus FV1000 FCS/RICS microscope (Olympus, Tokyo, Japan) in the Advanced Light Microscopy Core at the University of Colorado Anschutz Medical Campus.

结合固定在1064nm 处的斯托克斯光束和调谐在817nm 处的泵浦光束,从选定的成年小鼠肝脏冷冻切片中获得了12m 厚的 CARS 图像。描述了 CARS 显微镜(32)。我们还使用奥林巴斯 FV1000 FCS/RICS 显微镜(日本东京奥林巴斯)在科罗拉多大学博尔德分校安舒茨医学院的高级光学显微镜核心中分析了 E18.5和 PND 21肝脏切片。

Lipidomics

Sample

样本

An additional WD-fed cohort of male mice was studied, with or without constant exposure to PQQ (n = 6–7 mice/group, mean age 5.7 ± 0.2 wk). These mice were slightly older than PND 21 because they were subjected to glucose tolerance testing, qMRI, and metabolic chamber analysis for 5–7 d after weaning. Allowing for 2–3 d between tests for stress reduction and accounting for availability in the metabolic chamber, which accommodates only 8 cages, led to a delay in tissue harvesting. Liver tissue samples were harvested and kept at −80°C before analysis. Lipids were extracted from 5 mg of tissue (33), albeit slightly modified. In brief, tissues were homogenized in ice-cold methanol containing deuterated and odd-chain lipid standards. Subsequently, ice-cold methyl tert-butyl ether was added to each sample, followed by vortexing and addition of water. After centrifugation, the upper organic phase was removed, dried, and resuspended in 9:1 methanol:toluene. A portion of each resuspended sample was removed and diluted 20-fold for triacylglycerol analysis.

本文研究了有无持续暴露于 PQQ (n = 6ー7只/组,平均年龄5.7 ± 0.2周)的 WD-fed 雄性小鼠队列。这些小鼠比 PND 21稍老一些,因为它们在断奶后5-7天接受了葡萄糖耐量试验、 qMRI 和代谢室分析。允许2-3天之间的测试压力减少和解释的可用性,在新陈代谢室,其中只容纳8个笼子,导致延迟组织收获。取肝组织标本,保存在 -80 ° c,然后进行分析。脂质提取自5毫克的组织(33) ,虽然略有改变。简单地说,组织匀浆在含有氘化和奇链脂质标准的冰冷甲醇中。随后,在每个样本中加入冰冷的甲基叔丁基醚,然后是涡流和加水。离心后,上层有机相被去除,干燥,并复苏在9:1甲醇: 甲苯。每个复苏样本的一部分被取出并稀释20倍用于三酰甘油分析。

Liquid chromatography and mass spectrometry

液相色谱和质谱法

Chromatographic separation was performed with a 1290 Infinity Binary LC (Agilent Technologies, Santa Clara, CA, USA) equipped with a charged surface hybrid (CSH) C18 column (Acquity CSH C18 column; 1.7 µm 2.1 × 100 mm; Waters, Milford, MA, USA). Mobile phase A consisted of 60:40 v/v acetonitrile:water and mobile phase B consisted of 90:10 v/v isopropanol:acetonitrile. Ammonium formate (10 mM) and formic acid (0.1% v/v) were added equally to both mobile phases as positive-ion mode modifiers, and ammonium acetate (10 mM) was used for negative-ion mode analysis. Gradient program for both positive- and negative-ionization mode was as follows: 15% B (initial), gradient to 30% B at 2.0 min, gradient to 48% B at 2.5 min, gradient to 82% B at 11.0 min, gradient to 99% B at 11.5 min, hold at 99% B until 12.0 min, gradient to initial conditions at 12.1 min, and hold until 15 min. Flow rate was 0.6 ml/min. Sample (1.67 μl) was injected in positive mode 2 times: once at standard resuspension concentration and a second time 20-fold diluted for triacylglycerol analysis. Five microliters of sample were injected in negative mode. All mass spectrometry data were collected on a 6530 Accurate Mass quadrupole time-of-flight (QTOF; Agilent Technologies) system, using both positive and negative mode electrospray ionization. Pools of each sample group (WD PQQ and WD) were created and injected to collect tandem MS data for annotation purposes.

色谱分离采用1290 Infinity Binary LC (美国加利福尼亚州圣克拉拉的安捷伦科技有限公司) ,配备了带电表面混合(CSH) C18柱(Acquity CSH C18柱; 1.7 m 2.1100 mm; 米尔福德 Waters)。流动相 a 为60:40 v/v 乙腈: 水,流动相 b 为90:10 v/v 异丙醇: 乙腈。甲酸铵(10mm)和甲酸(0.1% v/v)均加入到两种流动相中作为正离子模式改进剂,乙酸铵(10mm)用于负离子模式分析。正负电离模式的梯度方案为: 15% b (初始) ,梯度为30% b (2.0 min) ,梯度为48% b (2.5 min) ,梯度为82% b (11.0 min) ,梯度为99% b (11.5 min) ,梯度为99% b (12.0 min) ,梯度为12.1 min (12.1 min)。流速为0.6 ml/min。样品(1.67 l)以阳性方式注射2次: 一次在标准悬浮浓度,第二次20倍稀释用于甘油三酯分析。以阴性方式注射5微升样品。所有质谱法的数据都是在一个6530准确的质量四极飞行时间系统上收集的,这个系统使用了正模和负模电喷雾离子法。创建并注入每个样本组(WD PQQ 和 WD)的池,以收集串联的 MS 数据用于注释目的。

Data processing

数据处理

Samples were annotated via in-house accurate-mass/retention-time libraries previously generated from tandem mass spectrometry (MS/MS) data collected by using the specific liquid chromatography (LC)/MS method implemented and via MS/MS data collected from treatment pools and analyzed using LipidBlast and NIST MS Search 2.2 (34). MassHunter Workstation Software Quantitative Analysis B.07.00 (Agilent Technologies) was used for targeted peak picking and integration.

样品通过先前使用特定液相色谱/质谱方法收集的串联质谱(MS/MS)数据生成的内部精确质量/保留时间库进行注释,并通过从处理池收集的 MS/MS 数据进行分析,使用 LipidBlast 和 NIST MS Search 2.2(34)进行分析。MassHunter Workstation Software Quantitative Analysis b. 07.00(安捷伦科技有限公司)被用于有针对性的峰值采集和集成。

Total RNA extraction and real-time quantitative PCR

总 RNA 提取及实时定量 PCR

RNA isolation from snap-frozen liver tissue from fetuses and offspring was performed with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Real-time quantitative PCR (qPCR) was performed on an iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) or an ABI Prism Step One (Thermo Fisher Scientific) (2435). Gene expression of each target sequence was normalized to expression of the reference genes Ppia and Sdha in fetal livers and 18S rRNA in 20 wk livers. Forward and reverse sequences of primers are summarized in Table 1. Equal efficiency of the reverse transcription of RNA was confirmed through quantification of expression of the reference genes, which did not differ between groups (for diet, PQQ, and interaction, all P > 0.1).

根据制造商的说明书,用 RNeasy 迷你试剂盒(Qiagen)从胎儿及子代冻存的肝组织中提取 RNA。采用 iQ5实时 PCR 检测系统(Bio-Rad,Hercules,CA,USA)和 ABI Prism 第一步(Thermo Fisher Scientific)(24,35)进行实时定量 PCR (qPCR)。每个靶序列的基因表达被规范化为参考基因 Ppia 和 Sdha 在胎儿肝脏和18S rRNA 在20周的肝脏。引物的正向和反向序列总结在表1中。通过对参考基因表达量的定量分析,证实了 RNA 逆转录的效率相等,各组间差异不显著(饮食、 PQQ、相互作用均 p > 0.1)。

TABLE 1.

表一

Forward and reverse sequences of primers used to probe expression of selected genes

用于探测所选基因表达的引物的正向和反向序列

Primer, 5′–3′ 底漆,5′-3′
Gene 基因Forward 前进Reverse 倒车
18STCCGATAACGAACGAGAC (美国)《行动计划》季刊CTAAGGGCATCACAGACC
AcadmGGCGATGAAGGTTGAACTC 中文名称: ggcgatgaaggtgaactcTACTCTGTGTTGAATCCATAGC 加拿大电力公司
Cpt1aaAGAAGAAGTTCATCCGATTCAAG 阿加加加特卡特卡特卡特卡特卡特卡亚格CCACCACCACGATAAGCC 加拿大农业协会
Cpt1abGTCCCTCCAGCTGGCTTATC 中国科学技术协会CATGCGGCCAGTGGTGTCTA 计算机辅助语言学
Il1bAGTTGACGGACCCCAAAAGA 加拿大农业研究所GGACAGCCCAGGTCAAAGG (美国)《公民权利和政治权利法案》
Il6TCCATCCAGTTGCCTTCTTG 中国科学技术进步协会TTTCTCATTTCCACGATTTCCC 公司名称:
Il10GGTTGCCAAGCCTTATCG (美国)《中国日报》周刊TCTTCACCTGCTCCACTG 中国科学技术协会
Nfkb1TGTAGATAGGCAAGGTCAGAATG TgtaggcaaggtcagatgAACACTGGAAGCACGGATG (美国)《公民权利和政治权利国际公约》
Nlrp3GCCTTGAAGAAGAGTGGATGG GccttgaagagagtggatggGCGTGTAGCGACTGTTGAG Gctgtagctgttgag
Nos2 第二名AGAGAGATCCGATTTAGAGTCTTGGT (美国)《公民权利和政治权利国际公约》TGACCCGTGAAGCCATGAC 加拿大空气污染防治委员会
Ppara 女名女子名aTCGCTATCCAGGCAGAAGG (美国)《公民权利和政治权利公约》AACAACAATAACCACAGA 阿卡塔阿卡卡加加
Ppara 女名女子名bTGCCCTGAACATCGAGTGTCGAAT 全球变暖TCGTACACCAGCTTCAGCCGAATA (美国)《中国日报》周刊
Pparg1TGTTGACCCAGAGCATGGTG (美国)《高速列车》季刊TGCGAGTGGTCTTCCATCAC 中文翻译
Pparg2CTGACGGGGTCTCGGTTG 中文名称:AACCATGGTAATTTCAGTAAAGGGT 阿卡塔塔塔塔塔加加加加加加
Ppargc1aaGTCAGAGTGGATTGGAGTTG GtcagagtgattgagttgAAGTCATTCACATCAAGTTCAG (美国)《全球变化趋势》季刊
Ppargc1abACCCACAGGATCAGAACAAACCCT 卡加加卡卡卡卡卡卡卡卡卡卡卡卡卡TTGGTGTGAGGAGGGTCATCGTTT (英国)《时代周刊》
Ppia 女名女子名GTCTCCTTCGAGCTGTTTGC 国家地理空间信息中心TAAAGTCACCACCCTGGCAC 加拿大国际贸易有限公司
Ptgs2GGTCTGGTGCCTGGTCTG 中国科学技术大学CTCTCCTATGAGTATGAGTCTGC (美国)《联合国气候变化框架公约》
SdhaTGCAGGCCTGGAGATAAAGTTCCT 美国国家航空航天局AACACTGCAGCATGGTTCTGCATC 美国农业科学学会
Sod1CTTCTCGTCTTGCTCTCTC 中国科学技术委员会TCCTGACAACACAACTGG 美国航空航天局
Sod2ATGTAGGTGTCTTCAGCCACA AtgtagtcttcagccacaAATTCCCAGCAAACACAGAGT (美国)《公民权利和政治权利国际公约》
Tlr4TTCAAGACCAAGCCTTTCAG (英国)《新闻报道》月刊GTCACATCACATAGTCCTTCC 全球气候变化专门委员会
Tnf 肿瘤坏死因子GGTTCTGTCCCTTTCACTCAC 中国电信集团有限公司TGCCTCTTCTGCCAGTTCC 高速公路交通管理委员会

aPrimer used for analysis of mRNA from 20-wk-old mouse livers. 20周龄小鼠肝脏 mRNA 分析的引物bPrimer used for analysis of mRNA from fetal livers. 用于分析胎儿肝脏 mRNA 的引物

SOD activity assay

超氧化物歧化酶活性测定

Mitochondrial-enriched supernatants were isolated from 30 mg frozen liver samples in buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, and 1 mM EGTA (pH 7.4) at 5% w/v. After homogenization, the samples were centrifuged 5 min at 600 g. Superoxide dismutase (SOD) activity was measured in a 10 μg sample (protein content measured by bicinchoninic acid assay) with a kit from Cayman Chemical (Ann Arbor, MI, USA), according to the manufacturer’s directions.

以210mm 甘露醇、70mm 蔗糖、5mm HEPES 和1mm EGTA (ph7.4)为缓冲溶液,在5% w/v 条件下,从30mg 冰冻肝脏标本中分离线粒体富集培养上清液。匀浆后,样品以600g 超氧化物歧化酶离心5分钟后,根据制造商的说明,在一个10g 的样品中用 Cayman Chemical 公司(安阿伯,MI,USA)的试剂盒测定蛋白质含量。

Western blot analysis

免疫印迹分析

Frozen livers (∼50 mg) were homogenized with tissue protein extraction reagent buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors, and equivalent amounts of total extracted proteins (determined by Bradford assay) were separated on 4–20% SDS-polyacrylamide gel and then transferred onto a PVDF membrane. Anti-MnSOD antibody (Cell Signaling Technology, Danvers, MA, USA) was used to probe the blot, and a UVP BioSpectrum Imaging System (Upland, CA, USA) was used to acquire the chemiluminescent signal. β-Actin (Cell Signaling Technology) was used as the control. Pixel densities in bands were quantified with ImageJ (National Institutes of Health, Bethesda, MD, USA).

用含有蛋白酶和磷酸酶抑制剂的组织蛋白提取试剂缓冲液(Thermo Fisher Scientific)匀浆冷冻肝脏(∼50mg) ,并用4ー20% sds- 聚丙烯酰胺凝胶分离等量的总提取蛋白,然后转移到 PVDF 膜上。用细胞信号转导技术(Cell signal Technology,丹弗斯)和 UVP 生物光谱成像系统(UVP BioSpectrum Imaging System,UVP,CA,USA)获得化学发光信号。以肌动蛋白(细胞信号转导技术)为对照。像素密度在波段用 ImageJ 量化(国立卫生研究院,贝塞斯达,MD,美国)。

Histology

组织学

Samples from snap-frozen livers were fixed for 24 h in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin by the Pathology Core at the University of Colorado Anschutz Medical Campus.

将冻存的肝脏标本用4% 的多聚甲醛固定24小时,石蜡包埋,切片,用苏木精和伊红染色,并在科罗拉多大学博尔德分校安舒茨医学院的病理核心进行染色。

Statistics

统计数字

Data are expressed as means ± sem. The significance of differences between groups was determined by 2-way ANOVA or Mann-Whitney U test with Prism 6.0 Software (GraphPad, La Jolla, CA, USA). A Student’s t test was performed on LC/MS data, to determine statistical significance of peak heights. Differences were deemed significant at P < 0.05. Principle component analysis was performed on LC/MS data by using MetaboAnalyst 3.0 (36).

数据表示为 ± sem。两组间差异的显著性用 Prism 6.0软件(GraphPad,La Jolla,CA,USA)进行2因素方差分析或 Mann-Whitney u 检验。对 LC/MS 数据进行学生 t 检验,以确定峰高的统计学意义。差异有统计学意义(p < 0.05)。主成分分析采用代谢分析3.0(36)对 LC/MS 数据进行分析。

RESULTS

结果

Treating with PQQ during pregnancy does not affect maternal weight gain but increases placental growth

在怀孕期间使用 PQQ 治疗不会影响母亲的体重增加,但会增加胎盘的生长

We tested the hypothesis that PQQ supplementation, provided both prenatally (at the time of mating) and after weaning, would improve metabolic outcomes of offspring of obese dams. Female mice were fed an LFD or HFD before mating with LFD-fed males and continued to be fed the same diet throughout gestation and lactation, adding PQQ supplementation at mating. Body weight of HFD-fed mice was higher before mating, and this difference was maintained during pregnancy, with no effect of PQQ supplementation in LFD- or HFD-fed mothers (Fig. 1A). The increased energy density of the HFD (4.73 kcal/g) as compared with the LFD (3.85 kcal/g) led to increased energy intake for the obese group, although food consumption (in grams per day) did not differ between groups (Supplemental Fig. S2). Neither diet nor PQQ treatment affected the number of pups per litter (Fig. 1B), and the average fetal weight was not different between groups (Fig. 1C). However, PQQ treatment led to a significant increase in placental weight (Fig. 1D) and placental surface area (Fig. 1E), regardless of maternal diet.

我们检验了这样的假设,即 PQQ 补充,在产前(交配时)和断奶后都提供,可以改善肥胖母鼠后代的代谢结果。雌性小鼠在与 LFD 喂养的雄性小鼠交配前喂食 LFD 或 HFD,并在整个妊娠期和哺乳期继续喂食相同的饲料,在交配时加入 PQQ 补充剂。高密度脂蛋白饲料喂养的小鼠在交配前体重较高,这种差异在妊娠期保持不变,在低密度脂蛋白饲料或高密度脂蛋白饲料喂养的母鼠中没有 PQQ 补充的影响(图1A)。与 LFD (3.85 kcal/g)相比,HFD (4.73 kcal/g)的能量密度增加,导致肥胖组的能量摄入增加,尽管食物消耗量(以每天克计)在各组之间没有差异(补充图1)。中二)。饮食和 PQQ 处理均不影响每窝幼崽的数量(图1B) ,各组胎儿的平均体重无差异(图1C)。然而,PQQ 治疗导致显着增加胎盘重量(图1D)和胎盘表面积(图1E) ,无论母亲饮食。

An external file that holds a picture, illustration, etc.
Object name is fasebj201600906Rf1.jpg

Figure 1. 图1

PQQ supplementation does not protect against maternal weight gain but increases placental size in pregnant obese mice. A) Maternal weights for matched groups were recorded from the initiation of experimental diet to E18.5. PQQ supplementation began at mating. Weights at 8 and 12 wk after diet initiation were compared. B) Number of pups per litter. CD) Fetal (C) and matched placental (D) weights were measured. E) Placenta length and width were measured with a micrometer, and placental surface area was calculated. Data are means ± sem (n = 5–7/group). *P < 0.05, with 2-way ANOVA comparing diets in A and PQQ treatment in D and E.

PQQ 补充剂不能防止母鼠体重增加,但可以增加肥胖孕鼠的胎盘大小。从实验开始到 E18.5,记录配对组孕妇体重。PQQ 补充剂开始于交配时。比较日粮启动后8周和12周的重量。B)每窝幼崽数目。测量胎儿(c)和配对胎盘(d)重量。用微米测量胎盘长度和宽度,计算胎盘表面积。数据均值为 ± sem (n = 5-7/组)。* p < 0.05,方差分析比较 a 组和 PQQ 组在 d 组和 e 组的日粮。

Metabolic phenotypes of male offspring of obese dams are modulated by PQQ throughout development

肥胖母鼠雄性后代的代谢表型在整个发育过程中受 PQQ 的调控

The effect of PQQ supplementation on diet-induced obesity had not been studied, to our knowledge, prompting us to measure levels of glucose, insulin, and TGs in both lean and obese dams and offspring in our model. Results are summarized in Table 2. Short-term supplementation with PQQ during pregnancy increased maternal glucose levels significantly, in both LFD- (control) and HFD-fed mothers, without changing fasting insulin. We investigated cohorts of offspring at E18.5 (fetuses), PND 21 (weanlings), and 20 wk (adults). In contrast to mothers, glucose levels of E18.5 fetuses of HFD-fed dams exposed to PQQ were not changed; nor was glucose affected by PQQ treatment at PND 21. At 20 wk, glucose was markedly increased by the WD in the group without PQQ. Although glucose and insulin levels were lower in the WD PQQ-fed mice, as compared to levels in the WD-fed cohort, the effect on HOMA-IR reduction did not reach significance (assessed by t test). Fetuses from HFD-fed dams and offspring fed the WD had significantly increased hepatic TGs vs. control-fed offspring, whereas PQQ supplementation significantly reduced TG accumulation in both control and HFD- or WD-fed mice, with the exception of PND 21 WD-fed mice. Twenty-wk-old offspring of WD-fed dams given PQQ only during gestation and lactation showed significant reductions in glucose and hepatic TGs compared to WD-fed offspring without maternal PQQ.

据我们所知,PQQ 补充剂对饮食诱导的肥胖的影响还没有研究,这促使我们在我们的模型中测量瘦母鼠和肥胖母鼠及其后代的葡萄糖、胰岛素和 TGs 的水平。结果摘要载于表2。在没有改变空腹胰岛素的情况下,LFD-(对照组)和 hfd 喂养的母亲在怀孕期间短期补充 PQQ 可显著提高孕妇的血糖水平。我们调查了 E18.5(胎儿)、 PND 21(断奶仔鱼)和20周(成年)的后代群落。与母亲相比,在 PND-21期间,HFD-fed 大鼠胎儿 E18.5的血糖水平没有改变,PQQ 治疗对胎儿血糖水平也没有影响。在20周时,无 PQQ 组的葡萄糖含量明显升高。虽然 WD PQQ-fed 小鼠的血糖和胰岛素水平低于 WD-fed 小鼠,但对 HOMA-IR 降低的影响并未达到显著水平(t 检验)。与对照喂养的子代相比,高密度脂蛋白饲料喂养的母鼠和喂食高密度脂蛋白饲料的子代的胎儿肝脏 TG 含量显著增加,而 PQQ 的补充显著降低了对照组和高密度脂蛋白饲料喂养的小鼠的 TG 含量,只有 PND 21 WD- 喂养的小鼠例外。仅在妊娠和哺乳期给予 PQQ 的20周龄 WD-fed 母鼠的后代,与未给予 PQQ 的 WD-fed 母鼠相比,其血糖和肝脏 tg 显著降低。

TABLE 2.

表二

Metabolic phenotype of mothers and offspring throughout development

母亲和后代在整个发育过程中的代谢表型

Phenotype 表型CTL/CTL 煤化油/煤化油a n = 5–11 = 5-11HFD/HFD 手足口病/手足口病n = 7–9 = 7-9CTL PQQ/CTL PQQCtlpqq/ctlpqqn= 5–7 = 5-7HFD PQQ/HFD PQQ HFD PQQ/HFDn = 5–7 = 5-7
Maternal 母性
 Glucose (mg/dl) 葡萄糖(毫克/分升)77.5 ± 5.590.3 ± 6.3119.8 ± 10.4106.2 ± 13.0#
 Insulin (ng/ml) 胰岛素(ng/ml)0.83 ± 0.050.77 ± 0.030.86 ± 0.130.68 ± 0.01
 HOMA-IR4.57 ± 0.434.94 ± 0.397.17 ± 1.255.13 ± 0.63
 Serum TGs (mg/dl) 血清总黄酮(mg/dl)23.3 ± 5.823.7 ± 6.020.0 ± 10.321.8 ± 2.3
E18.5 (averaged per litter) E18.5(平均每窝)
 Glucose (mg/dl) 葡萄糖(毫克/分升)72.4 ± 13.8116.9 ± 21.785.2 ± 21.692.7 ± 9.2
 Insulin (ng/ml) 胰岛素(ng/ml)1.27 ± 0.130.81 ± 0.05* 0.81 ± 0.05 *1.02 ± 0.080.78 ± 0.07
 HOMA-IR6.54 ± 1.416.73 ± 1.326.05 ± 1.615.14 ± 0.69
 Hepatic TGs (mg/dl) 肝脏总胆固醇(mg/dl)10.9 ± 0.822.4 ± 2.9* 22.4 ± 2.9 *7.3 ± 1.58.5 ± 0.8#
PND 21b
 Glucose (mg/dl) 葡萄糖(毫克/分升)193.3 ± 10.7200.6 ± 9.3179.2 ± 18.5197.0 ± 18.5
 Hepatic TGs (mg/dl) 肝脏总胆固醇(mg/dl)14.9 ± 1.517.3 ± 1.5* 17.3 ± 1.5 *11.6 ± 0.924.3 ± 2.6
20 wk 20周
 Glucose (mg/dl) 葡萄糖(毫克/分升)154.8 ± 8.7172.9 ± 10.6* 172.9 ± 10.6 *124.3 ± 10.9167.8 ± 14.4
 Insulin (ng/ml) 胰岛素(ng/ml)b0.87 ± 0.111.34 ± 0.310.84 ± 0.19
 HOMA-IRb9.58 ± 1.3216.48 ± 3.9410.02 ± 2.42
 Hepatic TGs (mg/dl) 肝脏总胆固醇(mg/dl)14.4 ± 4.563.8 ± 5.0* 63.8 ± 5.0 *9.3 ± 0.737.0 ± 4.0#
20 wk: WD PQQ/WD supplemented during gestation and lactation only 20周: 仅在妊娠和哺乳期补充 WD pqq/WD
 Glucose (mg/dl) 葡萄糖(毫克/分升)172.9 ± 10.6125.2 ± 8.8#
 Hepatic TGs (mg/dl) 肝脏总胆固醇(mg/dl)63.8 ± 5.044.8 ± 5.0#

Data are means ± sem.

数据是手段 ± sem。aControl diet: LFD for materna/fetal studies, CH for offspring studies; HFD: high-fat-diet for maternal/fetal studies, WD for offspring studies. 对照饮食: 母胎研究用 LFD,后代研究用 CH,母胎研究用高脂肪饮食,后代研究用 WDbInsulin concentrations were not measured for PND 21 or 20 wk CH-PQQ/CH-PQQ-fed mice. * Pnd21和20wk CH-PQQ/CH-PQQ-fed 小鼠未测定胰岛素浓度P < 0.05 vs. CTL for diet effect; . 细胞毒性 t 淋巴细胞对饮食的影响;#P < 0.05 vs. CTL PQQ for PQQ effect. . ctlpqq 的 PQQ 效果

Prolonged treatment with PQQ improves body composition but does not protect male offspring of obese dams from WD-induced weight gain

长期使用 PQQ 治疗可以改善身体组成,但不能保护肥胖大坝的雄性后代免受 wd 引起的体重增加

We charted growth through adulthood for offspring continued with WD feeding and for a subset of WD-PQQ–fed offspring with PQQ withdrawn after weaning (WD PQQ/WD). WD-fed mice supplemented with PQQ throughout pregnancy and postnatally were not protected from weight gain (PQQ supplementation similarly did not affect weight gain of CH-fed mice; Supplemental Fig. S3 and Fig. 2A). Mice with PQQ withdrawn postnatally gained weight at the same rate as their WD-PQQ– and WD-fed counterparts; however, they were smaller at weaning and maintained the same weight difference throughout life (Fig. 2B). WD-fed litters were not significantly smaller than WD PQQ-fed litters (4.4 ± 0.3 vs. 5.0 ± 0.4 pups/litter); thus, litter size was unlikely to contribute to this disparity. Increasing the concentration of PQQ after weaning did not alter growth trajectories (Supplemental Fig. S4). PQQ did not affect energy or drink intake (Supplemental Fig. S5). Body composition was measured by ECHO-MRI. The ratio of fat mass to body mass of CH-fed mice (0.176 ± 0.02) was similar to that obtained with PQQ supplementation (CH PQQ/CH PQQ 0.128 ± 0.03; P = 0.3); however, PQQ treatment reduced WD-induced increases in fat mass (Fig. 2C) and liver hypertrophy (Fig. 2D) at 20 wk, even when PQQ was provided only during gestation and lactation.

我们绘制了成年后继续进行 WD 喂养的后代和断奶后停用 PQQ 的 WD-PQQ 喂养的后代的生长图(WD PQQ/WD)。WD-fed 小鼠在整个妊娠期和出生后补充 PQQ 不能防止体重增加(PQQ 补充剂同样不影响 ch 喂养小鼠的体重增加;。中三及图2A)。生后撤除 PQQ 的小鼠与喂食 wd-PQQ-和 WD-fed 的小鼠体重增长率相同,但断奶时体重较小,且终生体重差异保持不变(图2B)。WD-fed 的窝仔数并不显著小于 WD PQQ-fed 的窝仔数(4.4 ± 0.3与5.0 ± 0.4) ,因此,窝仔数不可能导致这种差异。断奶后增加 PQQ 浓度不改变生长轨迹(补充图。S4).PQQ 没有影响能量或饮料的摄入(补充图。中五)。采用回波磁共振成像技术测量体成分。补充 PQQ 组小鼠脂肪质量与体重的比值(0.176 ± 0.02)与补充 PQQ 组相似(0.128 ± 0.03; p = 0.3) ,但 PQQ 组即使只在妊娠和哺乳期补充 PQQ 组,20wk 时仍能减少 wd- 所致的脂肪质量增加(图2C)和肝脏肥大(图2D)。

An external file that holds a picture, illustration, etc.
Object name is fasebj201600906Rf2.jpg

Figure 2. 图2

Growth and energy metabolism characteristics of male offspring continued on the maternal diet or with PQQ removed after weaning. Mice were weighed weekly after weaning. A) Growth curves for mice continued to 20 wk of age on the maternal diet. B) Effect of removing PQQ after weaning. PQQ was removed from offspring of WD PQQ-fed dams after weaning, and they were fed the WD for an additional 17 wk. CD) At death, the percentage of fat mass (C) was measured, and the ratio of liver mass to body mass (D) was determined. EF) Before death, mice were placed in a metabolic chamber and the RQ (E) and TEF (F) consumption and storage on total energy expenditure (TEF_TEE) were calculated. Data are means ± sem (n = 3–10/group). *P < 0.05 vs. CH mice in A vs. drink-switched mice (WD PQQ/WD) in B and vs. WD-fed mice in CF.

断奶后母体日粮或去除 PQQ 后雄性后代生长和能量代谢特征继续。小鼠断奶后每周称体重。在母体饮食中,小鼠的生长曲线持续到20周龄。B)断奶后去除 PQQ 的效果。在断奶后,PQQ 从 WD PQQ-fed 母鼠的后代身上移除,再给它们喂食 WD 17周。死亡时测定肝脏脂肪质量(c)百分比,测定肝脏脂肪质量与体重(d)的比值。E,f)死亡前将小鼠置于代谢室内,计算总能量消耗量(RQ (e)和总能量消耗量(TEF)和储存量(TEF _ tee)。数据均值为 ± sem (n = 3-10/组)。* p < 0.05: a 组小鼠、 b 组小鼠、 c 组小鼠、饮水开关组小鼠(WD pqq/WD)。

“Metabolic flexibility” defines the ability to switch between lipid and glucose oxidation, depending on substrate availability, and is measured by a change in the RQ. Impaired metabolic flexibility may result in poor glycemic control, reduced energy expenditure, and weight gain. To elucidate whether PQQ reduces liver TGs and fat mass through a change in energy metabolism or metabolic flexibility, we measured CO2production and O2 consumption in a metabolic chamber at 20 wk and calculated the RQ. RQs were affected by both diet and PQQ, with the WD reducing RQ and PQQ increasing RQ in WD-fed animals (Fig. 2E). The increase in RQ suggests that PQQ treatment improves the ability of WD-fed mice to switch from lipid to glucose oxidation during a meal, thus increasing their metabolic flexibility (37), even when they received PQQ supplementation only during gestation and lactation. Energy expenditure for mice in the metabolic chamber was calculated and, notably, the TEF was significantly increased for WD-fed mice treated with PQQ (Fig. 2F). Taken together, these results suggest that although PQQ does not protect from overall weight gain, it may increase metabolic flexibility in a high fat/high carbohydrate environment.

“代谢灵活性”定义了在脂质和葡萄糖氧化之间切换的能力,这取决于底物的可用性,并通过 RQ 的变化来衡量。受损的代谢灵活性可能会导致血糖控制不良,减少能量消耗,体重增加。为了阐明 PQQ 是否通过改变能量代谢或代谢灵活性来降低肝脏 TGs 和脂肪质量,我们在一个代谢室中测量了20周的 CO2产生量和 O2消耗量,并计算了 RQ。饲料和 PQQ 均影响 RQ,WD 降低的 RQ 和 PQQ 增加 WD-fed 动物的 RQ (图2E)。RQ 的增加提示 PQQ 治疗提高了 wd- 喂养小鼠在进食过程中从脂质转化为葡萄糖氧化的能力,从而提高了它们的代谢灵活性(37) ,即使它们只在妊娠和哺乳期间补充 PQQ。能量消耗的小鼠代谢室,尤其是显着增加毒性当量因子的 WD-fed 小鼠处理 PQQ (图2F)。综上所述,这些结果表明,虽然 PQQ 不能保护整体体重增加,但是它可以在高脂肪/高碳水化合物的环境中增加代谢灵活性。

PQQ treatment increases expression of regulators of lipid catabolism in WD-fed offspring

PQQ 治疗增加 WD-fed 后代脂质分解代谢调节因子的表达

Given the striking reduction of hepatic TGs and potential changes in fuel metabolism associated with PQQ supplementation, we next investigated the effect of PQQ on expression of genes that regulate lipid metabolism. PGC-1α mediates regulation of both PPARα and -γ (reviewed in refs. 3839). We therefore determined the effects of WD and PQQ on those gene sets in fetal and male adult offspring. At E18.5, maternal feeding of the HFD did not significantly alter fetal gene expression; however, maternal supplementation with PQQ modestly decreased expression of Ppargc1aPparg2Ppara, and Cpt1a, regardless of diet (Fig. 3A). In adults maintained on the maternal diet, PQQ treatment significantly increased Ppargc1a expression (Fig. 3B). Pparg1 mediates lipogenesis, whereas Pparg2 in the liver is protective and prevents lipotoxicity (40). Expression levels of both isoforms were significantly increased by both maternal and postnatal exposure to the WD, although Pparg1 expression was more highly up-regulated than Pparg2. PQQ supplementation significantly decreased expression levels of both isoforms in the WD PQQ/WD PQQ-fed group compared with levels in the WD/WD-fed group (Fig. 3B). PQQ treatment did not affect Pparg1 expression in CH-fed mice. PQQ significantly altered Pparg2, increasing expression in the CH-fed group and decreasing it in obese adult offspring (Fig. 3B). Unlike E18.5, Pparaand Cpt1a mRNA expression in adult offspring were significantly increased in WD PQQ/WD PQQ-fed animals (Fig. 3C). We also investigated expression of Acadm, another gene involved in fatty acid oxidation and found similar significant up-regulation in WD PQQ/WD PQQ-fed offspring (Fig. 3C). We again investigated persistent effects in our cohort with PQQ withdrawn at weaning (Fig. 3D) and found that mRNA expression levels of the WD PQQ/WD-fed group followed the same trends as WD-fed offspring maintained with continuous PQQ treatment. Taken together, these data suggest that PQQ up-regulates catabolic and oxidative genes in the livers of animals exposed to excess lipids during early life, and this altered response persists into adulthood.

考虑到 PQQ 补充剂引起的肝组织 TGs 的显著减少和燃料代谢的潜在变化,我们接下来研究 PQQ 对调节脂质代谢基因表达的影响。Pgc-1介导 ppar 和-(的调节。38,39).因此,我们确定了 WD 和 PQQ 对胎儿和雄性成年后代这些基因组的影响。在 E18.5,母乳喂养的 HFD 没有显著改变胎儿的基因表达,然而,母乳喂养中添加 PQQ 适度降低 Ppargc1a,Pparg2,Ppara 和 Cpt1a 的表达,无论饮食(图3A)。在维持母体饮食的成年人中,PQQ 处理显著增加了 Ppargc1a 的表达(图3B)。Pparg1介导脂肪生成,而 Pparg2在肝脏中具有保护作用并防止脂毒性(40)。两种亚型的表达水平在母亲和出生后暴露于 WD 时都显著增加,尽管 Pparg1表达比 Pparg2高度上调。PQQ 补充显著降低了 WD PQQ/WD PQQ-fed 组与 WD/WD fed 组两种异构体的表达水平(图3B)。PQQ 治疗对慢性乙型肝炎小鼠 Pparg1基因表达无影响。PQQ 显著改变 Pparg2,增加了 ch- 联合喂养组的表达,降低了肥胖成年后代的表达(图3B)。与 E18.5不同,WD pqq/WD PQQ-fed 动物后代 Ppara 和 Cpt1a mRNA 表达显著增加(图3C)。我们还研究了另一个参与脂肪酸氧化的基因 Acadm 的表达,发现在 WD pqq/WD PQQ-fed 后代中也有类似的显著上调(图3C)。我们在我们的队列中再次调查了断奶时撤除 PQQ 后的持续效应(图3D) ,发现 WD PQQ/WD-fed 组的 mRNA 表达水平与 WD-fed 后代持续 PQQ 治疗后的表达水平相同。综上所述,这些数据表明 PQQ 在早期生命期间暴露于过量脂质的动物肝脏中上调了分解代谢和氧化基因,这种改变的反应持续到成年期。

An external file that holds a picture, illustration, etc.
Object name is fasebj201600906Rf3.jpg

Figure 3. 图3

Regulation of hepatic metabolism is shifted toward catabolic mechanisms by PQQ treatment. A–C) Real-time quantitative PCR was used to measure mRNA expression of key metabolic transcription factors in fetal livers (A) and livers (B) from male adult (20 wk) offspring, and expression of fatty acid oxidation genes was measured in adult males (C). Gene expression was normalized to LFD-fed results and Ppia and Sdhaexpression levels (fetal) and CH-fed results and 18S rRNA expression levels (adult). Data are means ± sem (n= 3–9/group). *P < 0.05, effect of diet, for WD-fed compared with CH-fed animals; #P < 0.05, main effect of supplementation with PQQ. D) Persistent effects were assessed in offspring of WD-fed mothers with either no PQQ (WD/WD), the WD-fed cohort with PQQ withdrawn at weaning (WD PQQ/WD), or those maintained on the maternal diet (WD PQQ/WD PQQ) (n = 5–6/group). Gene expression was normalized to WD-fed results and 18S rRNA expression levels. *P < 0.05 compared with WD PQQ/WD-fed mice by Mann-Whitney U test.

PQQ 治疗使肝脏代谢调节向分解代谢机制转变。采用 a-c)实时定量 PCR 技术,检测雄性成年子代胎儿肝脏(a)和肝脏(b)中关键代谢转录因子的 mRNA 表达,并测定脂肪酸氧化基因在成年雄性子代(c)中的表达。基因表达与 LFD-fed 结果、 Ppia 和 Sdha 表达水平(胎儿)和 CH-fed 结果及18S rRNA 表达水平(成人)相一致。数据均值为 ± sem (n = 3-9/组)。* p < 0.05,饲料效应,喂喂大剂量维生素 c 组与喂喂大剂量维生素 c 组比较,# p < 0.05,补喂 PQQ 的主要效应。D)无 PQQ (WD/WD)、有 PQQ (WD PQQ/WD)的母乳喂养组(WD PQQ/WD)和母乳喂养组(WD PQQ/WD PQQ)组(n = 5-6)的后代持续效应评估。基因表达恢复到 WD-fed 结果和18S rRNA 表达水平。* Mann-Whitney u 检验与 WD pqq/WD 喂养小鼠比较 p < 0.05。

Hepatic lipid composition and lipid droplet size is affected by PQQ treatment

PQQ 治疗对肝脏脂质组成和脂滴大小有影响

The reduction of total hepatic TGs after PQQ treatment in E18.5 and 20-wk-old obese offspring was consistent with gene expression patterns; however, the slight increase in TGs in PND 21 obese offspring on PQQ was unexpected; therefore, we used CARS microscopy to further investigate lipid droplet accumulation in liver cryosections from all offspring groups. Few lipid droplets were observed in livers from chow-fed mice (data not shown). Displayed in Fig. 4A are representative CARS images of livers from HFD-exposed fetuses and WD-fed offspring (2 mice/group). At E18.5, lipid droplet accumulation was lower in mice exposed to HFD PQQ compared with HFD-exposed fetuses, in agreement with the total TG measurements in Table 2 for these groups. By PND 21, offspring had accumulated much more fat, likely through suckling from the dams. Although variability was observed between mice, liver sections from offspring treated with PQQ appeared to have larger lipid droplets, on average, than offspring without PQQ. Fat accretion continued into adulthood; however, by 20 wk, droplets were much larger in the WD-fed mice with no PQQ treatment as compared to those with long-term consumption of WD with PQQ, and fewer and smaller droplets were observed in the WD PQQ/WD images as compared with those of the WD-fed mouse livers, which again is consistent with the total TG measurements above. Similar patterns were observed in hematoxylin and eosin -stained images (Supplemental Fig. S6).

PQQ 治疗后 E18.5和20周龄肥胖子代肝总 tg 的降低与基因表达模式一致,但 PND 21肥胖子代肝总 tg 的轻微增加是意外的,因此,我们利用 CARS 显微镜进一步研究了所有子代组肝冷冻切片中脂滴的积累情况。在喂食食物小鼠的肝脏中观察到少量脂滴(数据未显示)。图4A 中显示的是典型的高手足口病暴露胎儿和 wd- 喂养后代(2只小鼠/组)的肝脏 CARS 图像。在 E18.5,与 HFD PQQ 暴露的胎儿相比,HFD PQQ 暴露的小鼠脂滴积累较低,这与表2中这些组的总 TG 测量结果一致。到了 PND 21,后代的脂肪积累量要多得多,很可能是通过母乳喂养的。虽然在小鼠之间观察到了变异性,但 PQQ 治疗后代的肝脏切片似乎平均比没有 PQQ 的后代有更大的脂滴。脂肪增长持续到成年期,然而,到20周时,未经 PQQ 治疗的 WD-fed 小鼠的液滴比长期服用 WD-qq 的小鼠的液滴大得多,而且与 WD-fed 小鼠肝脏的液滴相比,WD PQQ/WD 图像中的液滴越来越少,越来越小,这也与上述总 TG 测量结果一致。在苏木精和伊红染色图像中也观察到了类似的模式。中六)。

An external file that holds a picture, illustration, etc.
Object name is fasebj201600906Rf4.jpg

Figure 4. 图4

PQQ treatment alters hepatic lipid profiles around the time of weaning, increasing protective TGs and decreasing SM and ceramide concentrations. A) CARS microscopy was used to visualize lipid droplets in 12-μm-thick cryosections from mouse livers at E18.5, PND 21 (males), and 20 wk (males). Representative images from 2 mice are shown for offspring of dams treated with or without PQQ. Mat PQQ, 20-wk-old WD PQQ/WD-fed mice that received PQQ via maternal supplementation only. Magnification, ×60; field of view, 350 μm. HFD*, E18.5 fetuses were from HFD-fed mothers, PND 21 and 20-wk-old offspring were WD fed. B) Untargeted lipidomics analysis was performed on 6–7 mice/group (WD- and WD PQQ-fed) from 6-wk-old males. When multiple instances of a compound were present, unique ions were selected based on highest mean abundance and were used for subsequent analysis. Lipids were organized into general classes, with percentage of total identified shown for each class. Student’s t test was performed to determine significance. The percentage of compounds with significant changes are shown in parentheses for each class. P < 0.05. CD) A subset of the most abundant significantly changed TGs (C) and the total PQQ-induced change in TGs (D). EF) A subset of the most abundant significantly changed SMs and ceramides (E) and the total PQQ-induced change in SMs and ceramide concentrations from the lipidomics analysis (F) are plotted. Data are means ± sem. *P < 0.05, by Mann-Whitney U test.

PQQ 治疗在断奶前后改变肝脏脂质分布,增加保护性 TGs,降低 SM 和神经酰胺浓度。A) CARS 显微镜观察了 E18.5、 PND 21(雄性)和20周(雄性)小鼠肝脏12m 厚的冷冻切片中的脂滴。这是两只老鼠的代表性图片,显示的是用 PQQ 处理或不用 PQQ 处理的水坝的后代。Matpqq,20周龄 WD PQQ/WD-fed 小鼠,仅通过母体补充 PQQ。放大率: 60; 视野: 350米。HFD * 、 E18.5胎儿来源于 HFD 喂养的母亲,PND 21和20周龄后代采用 WD 喂养。B)对6-7周龄雄性小鼠进行非靶向脂质组学分析(WD-和 WD PQQ-fed)。当一个化合物存在多个实例时,根据最高的平均丰度选择独特的离子,并用于后续分析。油脂被组织成一般的类,与百分比的总识别显示为每个类。进行学生 t 检验以确定显著性。每类化合物中变化显著的百分比见括号内。P < 0.05.最丰富的子集显著改变 TGs (c)和总 pqq 诱导的 TGs (d)变化。用脂质组学方法分析了脂质体和神经酰胺含量的变化,得到了脂质体和神经酰胺含量变化最显著的子集。数据是手段 ± sem。* p < 0.05,采用 Mann-Whitney u 测试。

Given that TGs and lipid droplet sizes were increased in WD PQQ-fed PND 21 mice, we were curious as to whether PQQ treatment results in altered lipid composition in the liver. We performed untargeted MS-based lipidomics in liver tissue from the additional WD-fed cohort of ∼6-wk-old mice, with or without constant exposure to PQQ. Differences in lipidomics data were examined by principle component analysis (Supplemental Fig. S7), highlighting the multivariate separation between treatment classes (WD vs. WD PQQ). Summarized in Fig. 4B is the relative distribution of classes of unique compounds identified in both positive and negative ion mode. The percentage of compounds within each class that were significantly different between groups is indicated within each section of the chart in parentheses. Most of the identified ions were phosphoglycerides (38%); however, abundance of only 1% of the >150 phosphoglycerides identified changed significantly between groups. Lipid classes showing the most significant changes were the TGs and the sphingomyelins (SMs)/ceramides, representing 18 and 22% of the compounds detected, respectively. Of the lipid classes, TGs were the most affected by maternal PQQ treatment, with 29% of the unique TG moieties changing significantly. The next most altered class of lipids was the combined SMs/ceramides, with 14% of unique compounds changing significantly. A subset of the significantly changed TGs is plotted in Fig. 4C, and, consistent with the total TG data for WD-fed PND 21 offspring, peak abundance was increased for age-matched offspring of WD PQQ-treated dams as compared with untreated counterparts (summed TG intensity; Fig. 4D). Notably, TG (54:3), identified as triolein, a bioactive lipid associated with protection from oxidative stress (41), was increased in WD PQQ-treated mice. In contrast, concentrations of SMs and ceramides, associated with lesions of NAFLD such as insulin resistance, oxidative stress, and inflammation (4245), were decreased with PQQ treatment in mice fed the WD (Fig. 4E; summed intensity in Fig. 4F). Thus, PQQ supplementation of WD-fed mothers during pregnancy and lactation appeared to shift the liver lipid composition of offspring around the time of weaning to increase lipid mediators of oxidative defense and decrease proinflammatory mediators. Whether this protective lipid profile persists in adults remains to be determined.

考虑到 WD PQQ-fed PND 21小鼠 TGs 和脂滴大小增加,我们对 PQQ 治疗是否导致肝脏脂质成分改变感到好奇。我们在肝组织中进行非靶向的基于 MS-based 的脂质组学,这些脂质组来自于额外的 WD-fed 队列的∼6周龄小鼠,不论是否持续暴露于 PQQ。脂质组学数据的差异通过主成分分析进行检验(补充图。S7) ,强调治疗类之间的多元分离(WD 与 WD PQQ)。图4B 总结了在正离子和负离子模式下鉴定的独特化合物类别的相对分布。在括号中的图表的每一部分中都标明了每一类中各组之间有显著差异的化合物的百分比。大多数确定的离子是磷酸甘油酯(38%) ,然而,丰度只有1% 的大于150磷酸甘油酯确定明显改变之间的群体。脂类变化最明显的是 TGs 和鞘磷脂/神经酰胺,分别占所检测化合物的18% 和22% 。在脂类中,母体 PQQ 处理对 TG 的影响最大,其中29% 的 TG 变化明显。脂类的变化次之的是 sms/神经酰胺化合物,其中14% 的独特化合物发生了显著变化。在图4C 中绘制了一个显著变化的 TGs 子集,并且与 WD-fed PND 21子代的总 TG 数据一致,WD pqq 处理的母鼠与未处理的母鼠相比,年龄匹配的子代峰值丰度增加(TG 总强度; 图4D)。值得注意的是,经鉴定为 triolein 的 TG (54:3) ,一种与氧化应激(41)的保护作用有关的生物活性脂质,在 WD pqq 治疗小鼠中增加。相反,在喂食 WD 的小鼠中,与 NAFLD 损害相关的 SMs 和神经酰胺浓度,如胰岛素抵抗、氧化应激和炎症(42-45) ,在 PQQ 治疗下降(图4E; 总强度见图4F)。因此,在妊娠期和哺乳期补充 PQQ 可使断奶前后子代肝脏脂质组成发生变化,增加氧化防御的脂质介质,降低促炎症介质。这种保护性脂质在成年人中是否持续存在还有待确定。

Oxidative defense response of obese offspring is differentially affected by PQQ throughout development

PQQ 在肥胖子代发育过程中对氧化防御反应的影响不同

The significant increase of triolein, a marker for protective response to oxidative stress (41), in mice supplemented with PQQ prompted us to investigate the activity and expression of the oxidative defense enzyme SOD. The activity of mitochondrial manganese-dependent (Mn)SOD in fetal livers was significantly increased in HFD-exposed E18.5 fetuses, whereas maternal supplementation with PQQ significantly decreased SOD activity for HFD- but not LFD-exposed fetuses (Fig. 5A). We examined mRNA expression levels of Sod1 (cytoplasmic) and Sod2 (mitochondrial) in adult mouse livers and found that, after birth, expression of Sod1 was markedly increased in WD/WD-fed mice, as compared with CH/CH-fed animals and was reduced in PQQ-supplemented mice. Notably, Sod1 expression was significantly decreased in WD-fed animals with PQQ withdrawn at weaning (WD PQQ/WD), demonstrated a persistent effect of PQQ on Sod1 (Fig. 5B). Expression of Sod2 was unchanged by diet or PQQ treatment (Fig. 5C). We further investigated mitochondrial MnSOD protein expression by Western blot analysis. A representative blot shown in Fig. 5D and quantified in Fig. 5E, shows that expression of the mitochondrial gene product, MnSOD, was significantly decreased in adult male offspring fed a WD diet and increased by PQQ treatment in the WD-fed offspring, suggesting changes in MnSOD protein abundance, which may not be reflected by mRNA expression levels, are affected by both diet and PQQ.

在补充 PQQ 的小鼠体内,三油酸甘油酯的显著增加,促使我们研究氧化防御酶 SOD 的活性和表达。HFD 暴露胎儿肝脏线粒体锰依赖性 SOD 活性显著升高,而 PQQ 显著降低 HFD 暴露胎儿 SOD 活性(图5A)。我们检测了成年小鼠肝脏中 Sod1(细胞质)和 Sod2(线粒体)的 mRNA 表达水平,发现 WD/WD-fed 小鼠肝脏中 Sod1的表达明显高于 ch/ch 喂养小鼠,而且在 pqq 喂养小鼠肝脏中表达下降。结果显示,在断奶时撤除 PQQ 的 WD-fed 动物中,Sod1的表达显著降低,表明 PQQ 对 Sod1的持续作用(图5B)。日粮和 PQQ 处理均未改变 Sod2的表达(图5C)。我们进一步研究了线粒体 MnSOD 蛋白的表达。图5D 中有代表性的蛋白质印迹,图5E 中有量化表达,表明线粒体基因产物 MnSOD 在摄食 WD 饲料的成年雄性后代中显著降低,而在摄食 WD 饲料的雄性后代中通过 PQQ 处理增加,提示 MnSOD 蛋白质丰度的变化可能不会被 mRNA 表达水平所反映,这是受饲料和 PQQ 共同作用的结果。

An external file that holds a picture, illustration, etc.
Object name is fasebj201600906Rf5.jpg

Figure 5. 图5

Response of obese offspring to oxidative stress in early life and in later life is altered by PQQ treatment. Offspring of dams fed a LFD or HFD were assessed for response to oxidative stress by measuring activity or expression of SOD and Nos2A) MnSOD activity was measured in E18.5 fetuses (n = 5/group). BC) Expression levels of cytoplasmic Sod1 (B) and mitochondrial Sod2 (C) were measured by qPCR for male adult offspring continued on the maternal diet or drink switched after weaning (n = 3–9/group). *P < 0.05, compared with WD/WD determined by unpaired Student’s t test with Welch’s correction. D) Protein expression was determined by Western blot, and a representative blot from n = 2/group is shown. E) Image analysis was performed using ImageJ, and the densitometry was measured. After background subtraction, integrated pixel densities were normalized to control values (CH/CH) (n = 4 mice/group). F) mRNA expression of Nos2 was measured for the same groups as in B and C. *P < 0.05; #P < 0.1 compared with WD/WD feeding, determined by unpaired Student’s t test with Welch’s correction. Data are means ± sem.

肥胖后代对早期和晚期氧化应激的反应是通过 PQQ 治疗改变的。通过测量 SOD 和 Nos2的活性或表达,评估喂食 LFD 或 HFD 的母鼠的后代对氧化应激的反应。A)测定 E18.5胎儿(n = 5/组)的 MnSOD 活性。用 qPCR 方法检测断奶后母体饮食或饮水转换的雄性后代细胞质 Sod1(b)和线粒体 Sod2(c)的表达水平(n = 3-9/组)。* p < 0.05,与未配对学生 t 测验和韦尔奇修正后的 WD/WD 比较。D)蛋白表达采用 Western blot 检测,发现 n = 2/组有代表性的蛋白表达。E)使用 ImageJ 进行图像分析,并测量密度。减背景后,将积分像素密度归一化为对照值(CH/CH)(n = 4只小鼠/组)。F)用不配对学生 t 检验和 Welch 校正法测定2型糖尿病患者 b 组和 c 组 Nos2 mRNA 表达水平,p < 0.05,p < 0.1。数据是手段 ± sem。

Inducible NOS-2 is up-regulated in the liver in response to injury, oxidative stress, and inflammation (4647), which are lesions of NAFLD in addition to TG accretion. In 20-wk-old offspring, we found that the WD resulted in significant up-regulation of Nos2 expression as compared to the CH diet, whereas PQQ treatment selectively diminished hepatic Nos2 expression in WD-fed offspring (Fig. 5F). Furthermore, Nos2 expression in WD-fed mice with PQQ withdrawn at weaning (WD PQQ/WD) was even lower than that of the WD PQQ/WD PQQ-fed group. Taken together, these data demonstrate that subpharmacological supplementation with PQQ can robustly reverse lipid-induced increases in expression of enzymes associated with oxidative stress and inflammation, which manifest even before birth. Supplementation with this potent antioxidant is effective in adulthood, even when provided only during gestation and lactation.

可诱导的 NOS-2在肝脏对损伤、氧化应激和炎症的反应上调,这些是 NAFLD 的损伤和甘油三酯增加。在20周龄后代中,我们发现 WD 导致 Nos2表达显著上调,而 PQQ 处理选择性地降低 WD-fed 后代肝脏 Nos2的表达(图5F)。此外,断奶后撤除 PQQ 的 WD-fed 小鼠(WD PQQ/WD)中 Nos2的表达甚至低于 WD PQQ/WD PQQ-fed 组。综上所述,这些数据表明,亚药物补充 PQQ 可以有力地逆转脂质诱导的与氧化应激和炎症相关的酶的表达增加,这种增加甚至在出生前就表现出来。补充这种有效的抗氧化剂是有效的在成年,即使只提供在怀孕和哺乳期。

PQQ supplementation reduces expression of proinflammatory genes in adult WD-fed offspring

PQQ 补充降低 wd- 喂养成年子代促炎症基因的表达

The marked reversal of Nos2 mRNA expression in PQQ-treated WD-fed offspring suggested that the action of PQQ is targeted to inflammatory pathways. Therefore, we used qPCR to probe mRNA expression levels of proinflammatory genes in livers of adult male offspring maintained with the maternal diet. Results are summarized in Table 3, where we observed a general pattern of increased inflammatory gene expression associated with consumption of WD. Supplementing with PQQ inhibited WD-induced up-regulation of these proinflammatory mediators. Expression of the p50 subunit of NFκB (Nfkb1) and IL10 did not change significantly with diet or PQQ. TLR4, TNF, and IL1β gene expression did not show significant effects of PQQ, although significance was reached in some cases when compared pair-wise by ttest. A significant effect of both diet (increase) and PQQ (decrease) was observed for the inflammasome pathway genes NLRP3 and IL6, and cyclooxygenase (COX)-2 (Ptgs2) gene expression was markedly and specifically modulated by PQQ. Figure 6 shows, again, that PQQ supplementation given only during gestation and lactation persistently protected WD-fed offspring from up-regulation of inflammatory programs into adulthood.

PQQ 处理的 WD-fed 后代 Nos2 mRNA 表达明显逆转,提示 PQQ 的作用是针对炎症通路的。因此,本研究利用定量 pcr 技术,探讨母体饮食对成年男性子代肝脏促炎症基因表达水平的影响。结果总结在表3,其中我们观察到一个普遍的模式增加炎症基因表达与消费 WD。补充 PQQ 可抑制 wd- 诱导的这些前炎症介质的上调。Nfb1和 IL10的 p50亚基表达量随日粮和 PQQ 浓度的增加无明显变化。TLR4、 TNF 和 il1基因的表达对 PQQ 没有显著影响,虽然在某些情况下通过 t 检验比较达到了显著性。观察到饮食(增加)和 PQQ (减少)对炎症途径基因 NLRP3和 IL6均有显著影响,且 PQQ 对环氧化酶 -2(Ptgs2)基因表达有明显的特异性调节作用。图6再次显示,只在妊娠和哺乳期补充 PQQ 持续保护 wd- 喂养的后代免受炎症程序上调进入成年期。

TABLE 3.

表三

mRNA expression of proinflammatory genes in 20-wk-old male mice

20周龄雄性小鼠促炎症基因的 mRNA 表达

Gene 基因CH 甲烷n = 4–6 = 4-6WD 四轮驱动n = 4–6 = 4-6CH PQQ n = 4–5 = 4-5WD PQQ n = 5–7 = 5-7
Nlrp31.00 ± 0.114.67 ± 0.73* 4.67 ± 0.73 *1.52 ± 0.142.12 ± 0.25#
Tlr41.00 ± 0.062.51 ± 0.33* 2.51 ± 0.33 *1.38 ± 0.271.66 ± 0.26
Nfkb11.00 ± 0.131.38 ± 0.141.19 ± 0.141.29 ± 0.13
Tnf 肿瘤坏死因子1.00 ± 0.1710.51 ± 3.8* 10.51 ± 3.8 *1.55 ± 0.653.15 ± 0.67
Il101.00 ± 0.311.98 ± 0.691.58 ± 0.651.55 ± 0.10
Il61.00 ± 0.224.10 ± 0.90* 4.10 ± 0.90 *0.54 ± 0.151.21 ± 0.18#
Il1b1.00 ± 0.223.65 ± 1.05* 3.65 ± 1.05 *0.81 ± 0.310.93 ± 0.07
Ptgs21.00 ± 0.091.09 ± 0.040.71 ± 0.090.76 ± 0.06#

Data are means ± sem normalized to CH. *P < 0.05 vs. CH for diet effect; #P < 0.05 vs. CH PQQ for PQQ effect.

数据均值 ± sem 归一化为 CH。* p < 0.05与 CH 比较,p < 0.05与 CH PQQ 比较,p < 0.05。

An external file that holds a picture, illustration, etc.
Object name is fasebj201600906Rf6.jpg

Figure 6. 图6

PQQ supplementation during gestation and lactation only leads to mRNA expression profiles of proinflammatory genes in adults similar to those of WD-fed offspring receiving PQQ throughout their lifespan. mRNA expression of proinflammatory genes was measured in livers of adult male offspring. Mothers were fed a WD, with or without PQQ, and and either offspring continued on the maternal diet to 20 wk (WD/WD- or WD PQQ/WD PQQ-fed), or PQQ was withdrawn at weaning (WD PQQ/WD). Data are means ± sem (n = 5–6/group). Mann-Whitney U test was used in pair-wise comparison of groups. *P < 0.05 compared with WD/WD-fed mice; #P < 0.05 compared with WD PQQ/WD-fed mice.

在妊娠期和哺乳期补充 PQQ 只能导致致炎基因的 mRNA 表达谱在成年人中的表达与那些在整个寿命期间接受 PQQ 的 wd- 喂养的后代相似。测定成年雄性子代肝脏中促炎症基因的 mRNA 表达。母亲喂食 WD,无论有无 PQQ,要么继续母亲饮食至20周(WD/WD-或 WD PQQ/WD PQQ-fed) ,要么在断奶时停用 PQQ (WD PQQ/WD)。数据均值为 ± sem (n = 5-6/组)。对个组进行成对比较,使用 Mann-Whitney u 检验。* WD/WD-fed 小鼠与 WD pqq/WD-fed 小鼠比较 p < 0.05;。

DISCUSSION

讨论

Collectively, multiple lines of evidence from human and animal studies have converged to suggest that pregnancy and the postnatal period are uniquely crucial windows of opportunity for prevention of metabolic disease in this and subsequent generations (24850). However, nontoxic, nonsynthetic compounds that can be safely used in pregnancy to target specific pathways for disease risk are few and far between. In the current study, supplementation with PQQ, a naturally occurring antioxidant found in foods and enriched in human breast milk, given at subpharmacological levels throughout pregnancy and lactation, protected obese mouse offspring from hepatic lipotoxicity, increased metabolic flexibility, and suppressed liver inflammation without affecting growth or weight gain.

来自人类和动物研究的多条证据汇集在一起,表明怀孕和产后期是这一代和后代预防代谢疾病的唯一重要机会窗口(2,48-50)。然而,能够在怀孕期间安全地用于针对疾病风险的特定途径的无毒、非合成化合物很少。在目前的研究中,补充 PQQ—- 一种存在于食物中的天然抗氧化剂,富含在人类母乳中,在整个怀孕和哺乳期以亚药理水平给予,保护肥胖小鼠后代免受肝脏脂肪中毒,增加代谢灵活性,抑制肝脏炎症,而不影响生长或体重增加。

PQQ, given at the time of conception, had no effect on maternal weight gain but increased maternal glucose. PQQ at the molecular level enhances the production of pyruvate from lactate (51), possibly changing the cellular redox state and favoring increased gluconeogenesis (52) in the mothers. However, there was no effect of PQQ on fetal glucose, litter size, or fetal weights, suggesting that although statistically significant, maternal glucose had no effect on fetal growth, despite increasing placental weight. The lower fetal insulin levels in the HFD-fed group may have been caused by impaired insulin secretion or by the immaturity of the islets at E18.5 (53). With no significant changes observed in insulin levels in adulthood, we suspect that this is a transient effect; however, follow-up studies are necessary to pursue this notion further. Notably, PQQ given to mothers fed both LFD and HFD led to significantly reduced levels of fetal hepatic TGs compared to unexposed mice at E18.5. More studies are needed to identify whether the mechanisms underlying this change reflect effects on the mother or the placenta or represent a direct effect of PQQ on fetal development in utero.

PQQ 在受孕时服用,对母体体重增加无影响,但增加了母体血糖。PQQ 在分子水平上提高了从乳酸(51)生成丙酮酸,可能改变了细胞的氧化还原状态,有利于增加母体的糖异生(52)。然而,PQQ 对胎儿血糖、胎仔数或胎儿体重没有影响,这表明尽管有统计学意义,母亲血糖对胎儿生长没有影响,尽管胎盘体重增加。Hfd 喂养组较低的胎儿胰岛素水平可能是由于胰岛素分泌受损或在 E18.5(53)胰岛的不成熟所致。成年人胰岛素水平没有明显变化,我们怀疑这是一个短暂的影响,然而,后续研究是必要的,以进一步追求这一概念。值得注意的是,给同时喂食 LFD 和 HFD 的母鼠 PQQ,在 E18.5时,与未暴露的小鼠相比,显著降低了胎儿肝脏 TGs 的水平。需要进行更多的研究,以确定这种变化背后的机制是否反映了对母亲或胎盘的影响,还是代表 PQQ 对子宫内胎儿发育的直接影响。

PQQ reduced expression of both Ppara and Pparg2 in the livers of E18.5 mice compared to HFD-fed controls, an effect that persisted for Pparg2 but not Ppara in adult mice fed WD and exposed to PQQ. PQQ activates PGC-1α, a transcriptional coactivator implicated in regulation of reactive oxygen species–detoxifying genes, such as SOD, as well as many other metabolic mediators, including PPARα and -γ (as reviewed in ref. 36). In adult but not fetal livers, Ppara expression was significantly increased by PQQ supplementation, but only for obese offspring; diet did not have a significant effect. This result is consistent with findings reported by Bauerly et al. (19), where PQQ supplementation did not elevate PPARα expression in chow-fed rat livers or in Hepa 1–6 cells. We also observed similar up-regulation in expression of the fatty acid oxidation genes Cpt1a and Acadm at 20 wk, suggesting that PQQ increases fatty acid oxidation under conditions of overnutrition to reduce hepatic TGs. Notably, we observed a significant increase in expression of Pparg isoforms with WD feeding that was normalized by PQQ treatment in adult obese offspring, consistent with Sen and Simmons (10), who observed a similar trend for total Pparg expression in adipose tissue from offspring of obese dams treated with antioxidants. PPAR-γ2 is ectopically induced in liver in response to overnutrition and may prevent lipotoxicity by facilitating TG accumulation and increasing the lipid-buffering capacity of the liver (40); PQQ may activate similar pathways.

与 HFD-fed 对照组相比,PQQ 降低了 E18.5小鼠肝脏 Ppara 和 Pparg2的表达,这种效应对于 Pparg2持续存在,而对于摄食 WD 和暴露于 PQQ 的成年小鼠则没有。PQQ 激活 pgc-1,一个转录辅激活因子,涉及调节活性氧类解毒基因,如 SOD,以及许多其他代谢调节因子,包括 ppar 和-(参见参考文献。36).Ppara 在成年胎儿肝脏中的表达明显增加,但仅在肥胖后代中表达增加,饮食对 Ppara 表达没有显著影响。这一结果与 Bauerly 等人(19)的研究结果一致,即 PQQ 补充剂不能提高食物喂养大鼠肝脏或 Hepa 1-6细胞中 ppar 的表达。我们还观察到脂肪酸氧化基因 Cpt1a 和 Acadm 在20周时表达类似的上调,提示 PQQ 在过度营养条件下增加脂肪酸氧化以减少肝组织总黄酮。值得注意的是,我们观察到 Pparg 异构体的表达在 WD 喂养的成年肥胖后代中显著增加,这与 Sen 和 Simmons (10)观察到的肥胖母鼠后代脂肪组织中总 Pparg 表达的类似趋势一致。Ppar-2是应对营养过剩而在肝脏外源性诱导的,可能通过促进甘油三酯的积累和提高肝脏的脂质缓冲能力来防止脂毒性(40) ; PQQ 可能激活类似的通路。

In adult mice, we measured significant decreases in WD-induced hepatic TGs and expression of proinflammatory cytokines associated with PQQ supplementation, particularly for NLRP-3, a major component of the inflammasome; NOS-2; and IL-6. We also observed PQQ-specific up-regulation of COX-2 gene expression (Ptgs2). The significant effect of PQQ on IL-6, as compared with that of TNF and IL-1β, is similar to that observed in a thioacetamide model of induced fibrosis, where PQQ normalized expression of IL6 more effectively than other genes (18). These results also complement observations by Tchaparian et al. (54) and suggest that PQQ activates IL-6 to affect Jak-Stat signaling. IL-6 production was also significantly diminished by PQQ treatment in synoviocytes, via attenuating phosphorylation of p38 and JNK (55). In addition, an in vivo model of osteoarthritis has demonstrated an effect of PQQ in decelerating inflammatory responses via inhibition of NO production by suppressing IκBα degradation (56), suggesting PQQ acts systemically to modulate IL-6 and NO levels through different pathways. Notably, the ceramide content in livers of WD-fed weanling mice was lowered by PQQ, suggesting that the disease-causing effects of excess lipids on inflammation and oxidative stress had already been mitigated in early life. Taken together, these data show that PQQ targets multiple proinflammatory pathways in the liver to protect against WD-induced liver lipotoxicity, although there was no change in WD-induced weight gain in the offspring of WD-fed mothers.

在成年小鼠中,我们测量了 wd 诱导的肝组织 TGs 和 PQQ 补充相关的促炎细胞因子的表达显著下降,特别是 NLRP-3,炎症组的主要成分,NOS-2和 IL-6。我们还观察到 pqq 特异性上调 COX-2基因表达(Ptgs2)。PQQ 对 IL-6的显著影响,与 TNF 和 il-1相比,与硫代乙酰胺诱导的肝纤维化模型相似,PQQ 对 IL-6的规范化表达比其他基因更有效(18)。这些结果也补充了 Tchaparian 等人的观察(54) ,并表明 PQQ 激活 IL-6影响 Jak-Stat 信号。PQQ 通过减弱 p38和 JNK 的磷酸化作用,显著降低滑膜细胞 IL-6的产生。此外,骨关节炎体内模型证实 PQQ 通过抑制 i b 降解(56)抑制 NO 的产生而减慢炎症反应,提示 PQQ 通过不同途径系统调节 IL-6和 NO 水平。值得注意的是,PQQ 降低了 WD-fed 断奶小鼠肝脏中的神经酰胺含量,这表明过量的脂类对炎症和氧化应激的致病作用在小鼠早期就已经得到了缓解。综上所述,这些数据显示 PQQ 的目标是肝脏中的多种促炎症途径,以防止 wd- 诱导的肝脏脂肪毒性,尽管 wd- 喂养的母亲的后代在 wd- 诱导的体重增加方面没有变化。

As reviewed by Akagawa et al. (14), in numerous in vitro and in vivo models, PQQ acts as an antioxidant by scavenging O2 and protects mitochondria from oxidative stress-induced damage. Whether PQQ acts preconception to reduce oxidative stress in oocytes or sperm cells or improves placental function by reducing oxidative stress, indirectly protecting the fetus, is beyond the scope of the present study but is important to determine. The progressive development of NAFLD involves excessive TG accumulation in hepatocytes and results in cellular dysfunction caused by oxidative stress, lipotoxicity, or bacterial endotoxin activation of liver resident macrophages (Kupffer cells), inducing secretion of proinflammatory cytokines that activate neighboring stellate cells (57). Inflammatory cells, particularly the Kupffer cells, are recruited to the liver in response to liver injury or after exposure to danger signals. PQQ reduced macrophage infiltration in thioacetamide-induced injury (18) and inhibited osteoclast formation (58), suggesting that PQQ targets macrophage activity. Oxidative stress, associated with maternal obesity (5960), may connect the liver to intrauterine programming sequelae in the macrophage, particularly viaepigenetic mechanisms (6164). Whether PQQ simply limits macrophage infiltration or actually blocks the impact of maternal nutrition on developmental programming in the macrophage to reduce inflammation in the adult offspring is a critical area for future study.

正如 Akagawa 等人回顾(14) ,在众多的体外和体内模型,PQQ 作为一个抗氧化剂清除氧-和保护线粒体免受氧化应激诱导的损伤。是否 PQQ 作用于预先受孕以减少卵母细胞或精子细胞的氧化应激,或者通过减少氧化应激来提高胎盘功能—- 间接保护胎儿—- 超出了目前研究的范围,但是确定这一点很重要。NAFLD 的进行性发展包括肝细胞内过多的 TG 蓄积,导致细胞功能障碍,这些障碍是由氧化应激、脂毒性或肝驻留巨噬细胞(Kupffer 细胞)的细菌内毒素激活引起的,诱导分泌促炎细胞因子激活邻近的星状细胞(57)。炎症细胞,特别是 Kupffer 细胞,在肝脏损伤或暴露于危险信号后,被招募到肝脏。PQQ 减少了硫代乙酰胺诱导的损伤(18)中巨噬细胞的浸润,抑制了破骨细胞的形成(58) ,提示 PQQ 作用于巨噬细胞的活性。氧化应激与母体肥胖有关(59,60) ,可能通过表观遗传机制(61-64)将肝脏与巨噬细胞的宫内编程后遗症联系起来。PQQ 是否仅仅限制了巨噬细胞的浸润,或者实际上阻止了母体营养对巨噬细胞发育规划的影响,以减少成年后代的炎症,是未来研究的一个关键领域。

We tested the effect of early life PQQ supplementation on metabolic flexibility in adult mice. We measured an increase in RQ and TEF for PQQ-supplemented mice that may be interpreted as improvement in metabolic flexibility (37). Insulin resistance and altered glucose disposal rate, as well as dysfunction in fatty acid oxidation in mitochondria, impair metabolic flexibility in patients with type 2 diabetes (65). Decreased RQ has also been associated with impaired insulin-mediated suppression of lipolysis in adipose tissue (66); however, the action of PQQ in adipose tissue has not yet been established. The RQ for animals in which PQQ was removed after weaning was increased similarly to that for the continually supplemented mice, suggesting that PQQ supplementation during gestation and lactation permanently alters fuel utilization in the offspring. The further increase in TEF for the WD-fed animals with PQQ withdrawn at weaning may account for their decreased weight, although the reason underlying the increase remains unclear. Nevertheless, PQQ supplementation during this early-life period conferred persistent effects into adulthood, reducing the accumulation of TGs, suppressing induction of proinflammatory pathways, and increasing fatty acid oxidation. PQQ in early life protected against diet-induced lipotoxicity and diminished NAFLD, suggesting that it might be useful in pregnancy and lactation for conferring future health benefits for offspring. Whether these effects are related to persistent epigenetic changes in genes that regulate liver health or adipose tissue remains to be determined.

我们测试了早期补充 PQQ 对成年小鼠代谢灵活性的影响。我们测量了 pqq 补充小鼠的 RQ 和 TEF 的增加,这可以解释为代谢灵活性的改善(37)。胰岛素抵抗和葡萄糖处理率的改变,以及线粒体脂肪酸氧化功能障碍,损害了2型糖尿病患者的代谢灵活性(65)。RQ 下降也与胰岛素介导的脂肪组织脂解抑制受损有关(66) ,然而,PQQ 在脂肪组织中的作用尚未确定。断奶后去除 PQQ 的小鼠的呼吸商与不断补充 PQQ 的小鼠相似,表明 PQQ 在妊娠期和哺乳期的补充永久性地改变了后代的燃料利用。在断奶时停用 PQQ 的 wd- 喂养的动物血清总毒性当量的进一步增加可能是它们体重下降的原因,尽管增加的原因仍不清楚。尽管如此,在这个早期生命阶段补充 PQQ 可以持续影响成年期,减少 TGs 的积累,抑制促炎症途径的诱导,并增加脂肪酸氧化。PQQ 在早期生活中可以防止饮食引起的脂肪毒性和减少 NAFLD,这表明 PQQ 在妊娠和哺乳期可能有益于为后代提供未来的健康益处。这些影响是否与调节肝脏健康或脂肪组织的基因中的持续表观遗传变化有关还有待确定。

CONCLUSIONS

结论

We have demonstrated for the first time, to our knowledge, that subpharmacological supplementation of PQQ, when provided prenatally in obese pregnant mice, protects against disease pathways involved in NAFLD and NASH, decreasing hepatic PPARγ expression in early life. Continued treatment after weaning showed similar trends and results in reduced hepatic TG accumulation, inhibition of proinflammatory programs, and increased oxidative defense. Protection against weight gain was not achieved; however, PQQ altered fuel utilization and increased metabolic flexibility in obese animals and significantly reduced adiposity, resulting in an overall healthier metabolic state in offspring exposed to the WD. Our novel observation of a reduction in ceramides in the postnatal livers of mice and normalization of PPARγ by PQQ in adults suggests that prolonged treatment with PQQ protects against hepatic lipotoxicity, as suggested in other studies (18). In vivo tests in preclinical models of diabetes and NASH and in vitro tests have reported no genotoxic potential of PQQ (67) and recent short-term human studies have shown PQQ-related improvements in cognitive function (68), dry skin (69), and lipid metabolism (70). Preclinical and human studies have also demonstrated that supplemental PQQ improves oxidative stress (2371), suggesting that dietary supplementation with PQQ is an emerging therapeutic target worthy of further study in the battle to reduce the risks of NAFLD in infants exposed to maternal overnutrition.

我们首次证明,在肥胖孕鼠出生前补充 PQQ,可以防止 NAFLD 和 NASH 相关的疾病通路,减少早期肝脏 ppar 的表达。断奶后继续治疗显示出相似的趋势,结果是减少肝脏 TG 的积累,抑制促炎症程序,增加氧化防御。然而,PQQ 改变了肥胖动物的燃料利用率,提高了它们的代谢灵活性,显著降低了肥胖症的发生率,从而使得后代暴露在 WD 中的后代总体上处于更健康的代谢状态。我们对小鼠出生后肝脏神经酰胺含量减少和 PQQ 使成年小鼠 ppar 正常化的新观察表明,PQQ 延长治疗对抗肝脏脂毒性,正如其他研究(18)所建议的那样。糖尿病和 NASH 临床前模型的体内试验和体外试验报告 PQQ (67)没有基因毒性潜能,最近的短期人体研究显示 PQQ 相关的认知功能改善(68)、皮肤干燥(69)和脂质代谢(70)。临床前和人体研究也表明补充 PQQ 可以改善氧化应激,这表明在降低暴露于孕产妇过度营养的婴儿患非酒精性脂肪肝风险的斗争中,饮食补充 PQQ 是一个值得进一步研究的新兴治疗目标。

发表评论

您的电子邮箱地址不会被公开。 必填项已用*标注