NAD (h)和 NADP (h)氧化还原配对与细胞能量代谢


NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism



Significance: The nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) and NADP+/reduced NADP+ (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders.

意义: 烟酰胺腺嘌呤二核苷酸(NAD +)/还原型 NAD + (NADH)和 NADP +/还原型 NADP + (NADPH)氧化还原配对对维持细胞氧化还原稳态和调节包括细胞代谢在内的多种生物事件至关重要。这两个氧化还原配对的不足或不平衡已经与许多病理性疾病相关。

Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD+-consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics.

最新进展: 新发现的生物合成酶和新开发的基因编码生物传感器使我们能够更好地了解细胞如何维持 NAD (h)和 NADP (h)池。由 NAD (h)/NADP (h)变化反映的氧化还原应激(氧化还原应激)的概念越来越引起人们的重视。NAD + 消耗蛋白在调节细胞氧化还原和代谢稳态中的新兴作用是目前研究的热点。

Critical Issues: The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism.

关键问题: 细胞 NAD (h)和 NADP (h)的生物合成和分布是高度区域化的。理解细胞如何维持氧化还原耦合库的稳定水平以确保其正常功能并同时避免诱导氧化还原应激是至关重要的。此外,了解 NAD (h)-和 NADP (h)-利用酶如何与其他信号通路(如缺氧诱导因子调节的信号通路)相互作用,以维持细胞的氧化还原稳态和能量代谢也是非常重要的。

Future Directions: Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD+ precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251–272.

未来发展方向: 需要进一步研究室室化 NAD (h)/NADP (h)池之间的相互关系,以及这两个二核苷酸氧化还原配对在正常和病理条件下如何协同调节细胞氧化还原状态和细胞代谢。此外,最近的研究表明,使用药物干预或营养物质为基础的生物活性 NAD + 前体作为治疗代谢性疾病的干预措施的效用。因此,更好地了解 NAD (h)和 NADP (h)的细胞功能可能有助于有效地解决宿主病理性疾病。抗氧化剂。氧化还原信号。28,251-272.Keywords: 关键词: : cellular metabolism, NAD(H), NADP(H), oxidative stress, redox state, reductive stress 细胞代谢,NAD (h) ,NADP (h) ,氧化应激,氧化还原状态,还原应激Go to: 去:



Nicotinamide adenine dinucleotide (NAD+) is not only a coenzyme for oxidoreductases but also serves as a substrate for three classes of enzymes: sirtuin family deacetylases (SIRT1-7), poly(ADP)-ribosyl polymerases (PARP1-2), and cADP-ribose synthases (CD38 and CD157) (2266). NAD+ can be reduced to NADH via dehydrogenases and can also be phosphorylated to NADP+ via NAD+ kinases (NADKs). The NAD+/NADH redox couple is known as a regulator of cellular energy metabolism, that is, of glycolysis and mitochondrial oxidative phosphorylation. By contrast, NADP+ together with its reduced form, reduced NADPH, is involved in maintaining redox balance and supporting the biosynthesis of fatty acids and nucleic acids (141144). Given the crucial roles of NAD+/NADH and NADP+/NADPH in regulating the cellular redox state, energy metabolism, mitochondrial function, gene expression, and signaling pathways, these redox couples are essential for maintaining a large array of biological processes (2224144). Thus, loss of redox homeostasis of these molecules has been linked to a variety of pathological conditions, such as cardiovascular diseases, neurodegenerative diseases, cancer, and aging (24144).

烟酰胺腺嘌呤二核苷酸(NAD +)不仅是氧化还原酶的辅酶,而且是三类酶的底物: sirtuin 家族去乙酰化酶(SIRT1-7)、 poly (ADP)-ribosyl 聚合酶(PARP1-2)和 cADP-ribose 合酶(CD38和 CD157)(22,66)。NAD + 可以通过脱氢酶还原为 NADH,也可以通过 NAD + 激酶磷酸化为 NADP + 。NAD +/NADH 氧化还原夫妇被认为是细胞能量代谢的调节者,即糖酵解和线粒体氧化磷酸化。相反,NADP + 及其还原形式,还原 NADPH,参与维持氧化还原平衡和支持脂肪酸和核酸的生物合成(141,144)。鉴于 NAD +/NADH 和 NADP +/NADPH 在调节细胞氧化还原状态、能量代谢、线粒体功能、基因表达和信号通路中的关键作用,这些氧化还原配对对维持大量生物过程(22,24,144)至关重要。因此,这些分子失去氧化还原稳态与各种病理状态有关,如心血管疾病、神经退行性疾病、癌症和衰老(24,144)。

In this review, we will examine the biosynthesis of NAD+ and NADP+ with an emphasis on recent discoveries into the extracellular sources of NAD+, the newly identified synthetic enzymes, and the role of nicotinamide nucleotide transhydrogenase-mediated NADPH production. We will also discuss the subcellular distribution of NAD(H) and NADP(H) and their intercompartmental trafficking. In addition, we will extensively discuss the paradoxical functions of these two redox couples in maintaining cellular redox homeostasis as well as their regulation of cellular metabolism under various physiological and pathological conditions.

本文综述了 NAD + 和 NADP + 的生物合成,重点介绍了 NAD + 的胞外来源、新发现的合成酶以及烟酰胺核苷酸转移酶介导的 NADPH 产生的作用。我们还将讨论 NAD (h)和 NADP (h)的亚细胞分布及其相互间隔转运。此外,我们将广泛讨论这两对氧化还原夫妇在维持细胞氧化还原稳态方面的矛盾作用以及他们在各种生理和病理条件下对细胞新陈代谢的调节。Go to: 去:

Biosynthesis of NAD+ in Mammals

哺乳动物 NAD + 的生物合成

In mammalian cells, NAD+ is synthesized from four different precursors (Fig. 1), tryptophan (Trp), nicotinic acid (NA), nicotinamide (NAM), and nicotinamide riboside (NR), through three pathways: the de novo pathway, the Preiss–Handler pathway, and the salvage pathway (246688141) (Fig. 2). The salvage pathway predominates in most cell types.

在哺乳动物细胞中,NAD + 是由色氨酸(Trp)、烟酸(NA)、烟酰胺(NAM)和烟酰胺核苷(NR)四种不同的前体通过三个途径合成的: 从新途径、 Preiss-Handler 途径和补救途径(24,66,88,141)。补救途径在大多数细胞类型中占主导地位。

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FIG. 1. 图1

Schematic structures of NAD+(A), NAD+ precursors, and NAD+ derivatives (B-J).

NAD + (a)、 NAD + 前体和 NAD + 衍生物(B-J)的示意结构。

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Open in a separate window 在单独的窗口中打开FIG. 2. 图2

Biosynthesis of NAD(P)+ in mammalian cells. NAD+ is synthesized by three pathways: the de novo pathway, the Preiss–Handler pathway, and the salvage pathway. De novo NAD+ synthesis from L-Trp is mediated by enzymes in the kynurenine pathway. IDO/TDO catalyzes the first and rate-limiting step by converting L-Trp to N-formylkynurenine. After four enzymatic reactions, the intermediate ACMS undergoes spontaneous cyclization to form QA, which is the second rate-limiting step. QA is then converted to NAMN by QPRT using PRPP as a cosubstrate. In the Preiss–Handler pathway, NA is first metabolized into NAMN by NAPRT. The de novo and Preiss–Handler pathways converge at NAMN, which is further metabolized into NAAD by three NMNATs at the expense of ATP. NAD+ is synthesized from NAAD under the catalysis of NADSYNs. NAM and NR serve as NAD+ precursors for the salvage pathway, in which NAM and NR are initially converted into a common product, NMN, by NAMPT or NRK, respectively. Following that conversion, NMN is metabolized to NAD+ by the same NMNAT enzymes used by the other two pathways. Once formed, NAD+ can be phosphorylated into NADP+ by NADK. ACMS, 2-amino-3-carboxy-muconate-semialdehyde; IDO, indoleamine 2,3-dioxygenase; K3H, kynurenine-3-hydroxylase; KFase, kynurenine formamidase; NA, nicotinic acid; NAAD, NA adenine dinucleotide; NADK, NAD+ kinase; NADSYN, NAD+ synthetase; NAM, nicotinamide; NAMN, nicotinic acid mononucleotide; NAMPT, NAM phosphoribosyltransferase; NAPRT, NA phosphoribosyltransferase; NMNAT, NMN adenylyltransferase; NR, nicotinamide riboside; NRK, NR kinase; PRPP, phosphoribosyl pyrophosphate; QA, quinolinic acid; QPRT, quinolinate phosphoribosyltransferase; TDO, tryptophan 2,3-dioxygenase; Trp, tryptophan.

哺乳动物细胞 NAD (p) + 的生物合成。NAD + 通过三种途径合成: 新途径、 Preiss-Handler 途径和补救途径。L- 色氨酸从头合成 NAD + 是通过犬尿氨酸途径中的酶介导的。IDO/TDO 通过将 l- 色氨酸转化为 n- 甲酰基犬尿氨酸来催化第一步和限速步骤。中间体经过四步酶促反应,自发环合成 QA,为第二限速步骤。然后 QPRT 使用 PRPP 作为辅助底物将 QA 转换为 NAMN。在 Preiss-Handler 通路中,NA 首先被 NAPRT 代谢成 NAMN。新生通路和 Preiss-Handler 通路在 NAMN 汇合,NAMN 通过3个 NMNATs 进一步代谢进入 NAAD,代价是 ATP。以 NAAD 为原料,在 nadsyn 催化下合成 NAD + 。NAM 和 NR 作为 NAD + 的前体,分别通过 NAMPT 和 NRK 将 NAM 和 NR 转化为共同的产物 NMN。在这个转化过程中,NMN 被另外两个途径所使用的同样的 NMNAT 酶代谢成 NAD + 。NAD + 一旦形成,就可以被 NADK 磷酸化为 NADP + 。2- 氨基 -3- 羧基-穆康酸-半醛,IDO,吲哚胺2,3-双加氧酶,K3H,犬尿氨酸 -3- 羟化酶,KFase,犬尿脒甲酸酶,NA,烟酸,NAAD,NA 腺嘌呤二核苷酸,NADK,NAD + 激酶;NADSYN,NAD + 合成酶; NAM,烟酰胺; NAMN,烟酸单核苷酸; NAMPT,NAM 磷酸核糖转移酶; NAPRT,NA 磷酸核糖转移酶; NMNAT,NMN 腺苷酸转移酶; NR,烟酰胺核糖苷; NRK,NR 激酶;磷酸核糖焦磷酸; QA,喹啉酸; QPRT,喹啉酸磷酸核糖转移酶; TDO,色氨酸2,3- 双加氧酶; Trp,色氨酸。

De novo synthesis of NAD+


De novo NAD+ synthesis from L-Trp is mediated by enzymes in the kynurenine pathway via an eight-step process (Fig. 2). Conversion of L-Trp to N-formylkynurenine is the first and rate-limiting step, which is catalyzed by indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) (88). TDO is primarily expressed in the liver (114). IDO expression has been detected in extrahepatic cells, including human vascular endothelial and smooth muscle cells (1530), dermal fibroblasts (47), macrophages (52), neurons, microglia, and astrocytes (53). Human lung, placenta, and small intestine show relatively higher IDO activity than neuronal tissue (137). Since the enzymatic activity of IDO and TDO requires oxygen, their activity is anticipated to be attenuated under low oxygen tension. Surprisingly, current literature shows that hypoxia enhances IDO and TDO activity in vitro and in vivo (4063). This paradoxical phenomenon could be explained by the findings that IDO can utilize superoxide anion (O2•−) as its substrate (88), and O2•− production is increased under hypoxia (98). Future studies are needed to investigate the precise effects of hypoxia on TDO/IDO activity.

L- 色氨酸从头合成 NAD + 是由犬尿氨酸途径中的酶通过一个八步过程介导的(图2)。在吲哚胺2,3-双加氧酶(IDO)和色氨酸2,3-双加氧酶(TDO)(88)的催化下,l- 色氨酸转化为 n- 甲酰基犬尿氨酸是第一步,也是限速步骤。TDO 主要表达在肝脏(114)。IDO 在肝外细胞中有表达,包括人血管内皮细胞和平滑肌细胞(15,30)、真皮成纤维细胞(47)、巨噬细胞(52)、神经元、小胶质细胞和星形胶质细胞(53)。人的肺、胎盘和小肠的 IDO 活性比神经元组织(137)高。由于 IDO 和 TDO 的酶活性需要氧气,预计它们的活性将在低氧张力下衰减。令人惊讶的是,目前的文献表明,缺氧增强 IDO 和 TDO 活性在体外和体内(40,63)。这种矛盾现象可以用以下发现来解释: IDO 可以利用超氧阴离子(O2 · -)作为底物(88) ,O2 ·-的产生在缺氧条件下增加(98)。未来的研究需要调查缺氧对 TDO/IDO 活性的确切影响。

In the next steps of this pathway, kynurenine formamidase (KFase) converts N-formylkynurenine into kynurenine, which is further hydroxylated into 3-hydroxy-kynurenine by kynurenine-3-hydroxylase (K3H). K3H, localized to the mitochondrial outer membrane, is a NADPH-dependent and flavin-adenine dinucleotide (FAD)-containing monooxygenase (88). K3H expression in human tissues is abundant in the liver and placenta, with lower levels in the kidney (5).

在此途径的下一步,犬尿氨酸甲酰胺酶(KFase)将 n- 甲酰基犬尿氨酸转化为犬尿氨酸,犬尿氨酸再被犬尿氨酸 -3- 羟化酶(K3H)羟化为3- 羟基犬尿氨酸。K3H 定位于线粒体外膜,是一种依赖于 nadph 和黄素腺嘌呤二核苷酸的含单加氧酶(88)。K3H 在人体组织中的表达丰富于肝脏和胎盘,在肾脏中的表达较低(5)。

Once formed, hydroxylated kynurenine next undergoes two additional enzymatic reactions forming an unstable intermediate, 2-amino-3-carboxy-muconate-semialdehyde (ACMS). ACMS can be removed from the NAD+ synthetic pathway by its decarboxylation into 2-amino-3-muconate-semialdehyde (AMS) catalyzed by ACMS decarboxylase (ACMSD), a process that ultimately leads to the formation of picolinic acid or CO2 and H2O. ACMS can also undergo spontaneous cyclization to form quinolinic acid (QA), which is then converted to nicotinic acid mononucleotide (NAMN) by quinolinate phosphoribosyltransferase (QPRT) using phosphoribosyl pyrophosphate (PRPP) as a cosubstrate. Of note, the QPRT reaction is not efficient and occurs only when the level of QA exceeds the enzymatic capacity of ACMSD, which renders this reaction as a second rate-limiting step in de novo NAD+ synthesis (88) and helps to explain why Trp-dependent synthesis is less efficient than the other two NAD+ biosynthetic pathways.

一旦形成,羟基化的犬尿氨酸下一步经历两个额外的酶反应,形成一个不稳定的中间体,2- 氨基 -3- 羧基-芥酸-半醛(ACMS)。ACMS 脱羧酶(ACMSD)催化2- 氨基 -3- 葡萄糖酸-半醛(AMS)脱羧反应,最终生成苦味酸或 CO2和 H2O,从而使 ACMS 脱除 NAD + 合成途径。ACMS 也可以自发环化形成喹啉酸(QA) ,然后由喹啉磷酸核糖转移酶(QPRT)以磷酸核糖焦磷酸(PRPP)为辅助底物将其转化为烟酸单核苷酸(NAMN)。值得注意的是,QPRT 反应并不高效,只有当 QA 水平超过 ACMSD 的酶促能力时才会发生,这使得该反应成为新 NAD + 合成(88)中的第二个限速步骤,并有助于解释为什么依赖 trp 的合成比另外两个 NAD + 生物合成路径低效。

NAMN is then adenylated to form NA adenine dinucleotide (NAAD) with the catalysis of ATP by NMN adenylyltransferases (NMNATs). In mammals, three isoforms of NMNATs exist with a distinct tissue- and organelle-specific distribution (3637106149). NMNAT1, an exclusively nuclear enzyme, is ubiquitously expressed in human tissues. It is highly abundant in the heart and skeletal muscle, expressed to a lesser extent in the liver and kidney, and barely detectable in the brain and small intestine (1136). NMNAT2 is located in cytosol and Golgi apparatus, and its expression in human tissues is found principally in the brain and weakly in the heart and skeletal muscle (14106). In contrast, NMNAT3 is found in cytosol and mitochondria (14149). Tissue distribution shows that this enzyme is mostly present in human lung and spleen, where the other two isoforms are barely detectable (14149). The tissue- and organelle-specific expression pattern implies that the function of NMNATs is not redundant and also explains the cellular compartmentalization of NAD+.

在腺苷酸转移酶(NMNATs)的作用下,NAMN 被腺苷酸化形成腺苷酸二核苷酸(NAAD)。在哺乳动物中,存在三种 NMNATs 亚型,具有明显的组织和细胞器特异性分布(36,37,106,149)。NMNAT1是一种特异性的核酶,在人体组织中广泛表达。它在心脏和骨骼肌中含量极高,在肝脏和肾脏中表达较少,在大脑和小肠中几乎无法检测到(11,36)。NMNAT2位于细胞溶胶和 Golgi 器官,在人体组织中主要表达于大脑,在心脏和骨骼肌中弱表达(14,106)。NMNAT3则存在于细胞质和线粒体中(14,149)。组织分布显示,这种酶主要存在于人的肺和脾中,其他两种亚型几乎无法检测到(14,149)。组织和细胞器的特异性表达模式表明,NMNATs 的功能是不多余的,也解释了 NAD + 的细胞防火分区。

The final step in the de novo pathway is the conversion of NAAD into NAD+ by ATP-dependent NAD+synthetases (NADSYNs), which catalyze an amidation reaction using glutamine or ammonia as an amide donor. Two NADSYN isoforms (NADSYN1 and 2) have been identified in humans. NADSYN1 expression is enriched in the liver, kidney, small intestine, and testis, where the expression of NADSYN2 is weak (62). Interestingly, NADSYN1 utilizes both glutamine and ammonia as amide donors, whereas NADSYN2 only catalyzes ammonia-dependent NAD+ synthesis (62).

从新途径的最后一步是由 atp 依赖的 NAD + 合成酶(nadsyn)将 NAAD 转化为 NAD + ,nadsyn 以谷氨酰胺或氨作为酰胺供体催化酰胺化反应。两个 NADSYN 亚型(NADSYN1和2)已经在人体中被确定。NADSYN1在肝脏、肾脏、小肠和睾丸中表达丰富,其中 NADSYN2表达较弱(62)。有趣的是,NADSYN1同时利用谷氨酰胺和氨作为酰胺供体,而 NADSYN2只催化依赖氨的 NAD + 合成(62)。

The Preiss–Handler pathway

Preiss-Handler 路径

This pathway was discovered by Preiss and Handler in human erythrocytes and rat liver, where they found NA could be converted into NAD+ in a three-step process that produces NAMN and NAAD as intermediate metabolites (103104).

Preiss 和 Handler 在人类红细胞和大鼠肝脏中发现了这种途径,他们发现 NA 可以通过三个步骤转化为 NAD + ,产生 NAMN 和 NAAD 作为中间代谢物(103,104)。

In mammals, NA is first metabolized into NAMN by NA phosphoribosyltransferase (NAPRT) at the expense of PRPP (Fig. 2). NAPRT expression is widespread and its messenger RNA (mRNA) has been detected in almost all human tissues tested thus far (35). ATP is a dual allosteric modulator that can stimulate or inhibit NAPRT activity at low (<100 μM) or high (100–640 μM) concentrations, respectively (43). Intriguingly, metabolites of glucose and fatty acids have distinct effects on NAPRT activity, as well. For instance, dihydroxyacetone phosphate (DHAP) and pyruvate stimulate its activity, whereas glyceraldehyde-3-phosphate (G3P), phosphoenolpyruvate, fructose-1,6-biphosphate (F-1,6-BP), acetyl-CoA, succinyl-CoA, and CoA inhibit its activity (43). To date, the mechanisms for these differential regulatory effects are unknown.

在哺乳动物中,NA 首先被 NA 磷酸核糖基转移酶(NAPRT)代谢成 NAMN,代价是 PRPP (图2)。NAPRT 广泛表达,其信使 RNA (mRNA)已在几乎所有人体组织中检测到(35)。ATP 是一种双异位调控因子,分别在低浓度(< 100μM)或高浓度(100-640μM)时刺激或抑制 NAPRT 活性。有趣的是,葡萄糖和脂肪酸的代谢物对 NAPRT 活性也有不同的影响。例如,二羟丙酮磷酸酯(DHAP)和丙酮酸盐刺激其活性,而甘油醛 -3- 磷酸酯(G3P)、磷酸烯醇式丙酮酸、果糖 -1,6-二磷酸酯(F-1,6-BP)、乙酰辅酶 a、琥珀酰辅酶 a 和辅酶 a 抑制其活性(43)。迄今为止,这些差异调节效应的机制尚不清楚。

NAMN serves as the converging point for the Preiss–Handler pathway and the de novo pathway, with NAMN undergoing the same reactions for NAD+ synthesis as described in the aforementioned section. NA seems to be a more efficient precursor for NAD+ synthesis than Trp, as 1 mg NA is equivalent to ∼60 mg dietary Trp (12).

NAMN 作为 Preiss-Handler 路径和从头路径的汇合点,NAMN 在 NAD + 合成方面经历了上述部分所描述的相同反应。NA 对 NAD + 的合成似乎比 Trp 更有效,因为1毫克 NA 相当于∼60毫克膳食 Trp (12)。

The salvage pathway


NAM, a product of niacin and SIRT enzymes, can be used to synthesize NAD+ through the salvage pathway (24141). In mammals, NAM phosphoribosyltransferase (NAMPT), a rate-limiting enzyme, catalyzes the conversion of NAM and PRPP into NMN, which is then converted into NAD+ by NMNATs that catalyze the formation of NAAD from NAMN in the de novo pathway (Fig. 2) (24141). NAMPTmRNA is ubiquitously expressed in all tissues tested, with higher levels present in bone marrow, liver, and muscle than other tissues (41115). Immunocytochemical staining demonstrates that NAMPT protein is found in the nucleus and cytoplasm with a change in its intracellular distribution according to the proliferation state of the cells (72).

NAM 是烟酸和 SIRT 酶的产物,可以通过挽救途径(24,141)合成 NAD + 。在哺乳动物中,NAM 磷酸核糖基转移酶(NAMPT)是一种限速酶,它催化 NAM 和 PRPP 转化为 NMN,然后 NMNATs 在新生途径中催化 NAMN 形成 NAAD + (图2) ,24,141。NAMPT mRNA 在所有被检测的组织中普遍表达,在骨髓、肝脏和肌肉中的表达水平高于其他组织(41,115)。免疫细胞化学染色显示 NAMPT 蛋白主要分布于细胞核和细胞质中,细胞内分布随细胞增殖状态的改变而改变(72)。

Notably, in addition to intracellular NAMPT (iNAMPT) protein, an extracellular form of NAMPT (eNAMPT) has also been identified. The eNAMPT, also denoted pre-B cell colony-enhancing factor (PBEF), was initially recognized as a secreted cytokine, which synergizes cell colony formation induced by stem cell factor and interleukin 7 (115). Later, Rongvaux et al. (112) showed that PBEF is a cytosolic NAMPT involved in NAD+ biosynthesis in mouse liver. Furthermore, eNAMPT was found in human circulation, and leukocytes were also identified as a source for eNAMPT (41). Revollo and colleagues (109) reported that mouse adipocytes also secreted eNAMPT, which exhibits even higher NAD+biosynthetic activity than the intracellular iNAMPT. Consistent with these observations, a high concentration of NMN is present in mouse plasma, and plasma eNAMPT and NMN levels are reduced in NAMPT heterozygous knockout females (109). Recently, Yoon et al. (145) demonstrated that deacetylation of iNAMPT by the SIRT1 deacetylase enhances eNAMPT secretion and activity in murine adipocytes, and that adipocyte-specific knock out or knock in of NAMPT systemically affected plasma eNAMPT levels, hypothalamic NAD+ biosynthesis, SIRT1 function, and exercise capacity.

值得注意的是,除了细胞内的 NAMPT (iNAMPT)蛋白外,一种细胞外形式的 NAMPT (珐琅 pt)也被鉴定出来。前 b 细胞集落增强因子(pre-B cell colony-enhancement factor,PBEF)是一种分泌性细胞因子,协同干细胞因子和白细胞介素7(interleukin 7,115)诱导的细胞集落形成。后来,Rongvaux 等人(112)证明 PBEF 是一种参与小鼠肝脏 NAD + 生物合成的细胞溶质 NAMPT。此外,在人体循环中发现了珐琅质,白细胞也被确定为珐琅质(41)的来源。Revollo 和他的同事(109)报告说,小鼠脂肪细胞也分泌烯胺基转移酶,这种转移酶比细胞内的烯胺基转移酶具有更高的 NAD + 生物合成活性。与这些观察结果一致的是,小鼠血浆中存在着高浓度的 NMN,而 NAMPT 杂合基因敲除雌性小鼠(109)的血浆烯酰胺 pt 和 NMN 水平降低。最近,Yoon 等(145)证实,SIRT1脱乙酰基酶对 iNAMPT 的脱乙酰化作用增强了小鼠脂肪细胞的烯胺铂分泌和活性,脂肪细胞特异性敲除或敲除 NAMPT 可全身影响血浆烯胺铂水平、下丘脑 NAD + 生物合成、 SIRT1功能和运动能力。

NR is a newly discovered NAD+ precursor that feeds into the salvage pathway (16). NR is first phosphorylated into NMN by NR kinases (NRK1-2), after which NMNATs catalyze the production of NAD+ (Fig. 2). NRKs are highly conserved in eukaryotes (16). NRK1 is ubiquitously expressed in mammalian tissues, whereas NRK2 expression is restricted to the heart, brain, and skeletal muscle and not present in the liver, kidney, lung, or pancreas (19). Overexpression of NRK1 in NIH3T3 cells and hepatocytes elevates cellular NAD+ levels in response to NR addition (108). In mice, injection of NR augmented NAD+ levels in the muscle, liver, brain, and brown adipose tissue, an effect that was abrogated in NRK1 knockout mice (108).

NR 是一种新发现的 NAD + 前体,可以进入补救途径(16)。NR 首先被 NRK1-2激酶磷酸化为 NMN,然后 NMNATs 催化 NAD + 的产生(图2)。NRKs 在真核生物中高度保守(16)。NRK1在哺乳动物组织中普遍表达,而 NRK2仅在心脏、脑和骨骼肌中表达,在肝脏、肾脏、肺或胰腺中不表达(19)。NRK1在 NIH3T3细胞和肝细胞中的过表达使细胞 NAD + 水平升高。在小鼠中,注射 NR 增加了肌肉、肝脏、大脑和褐色脂肪组织的 NAD + 水平,这一效应在 NRK1基因敲除小鼠中被消除。

Importantly, NR has been detected in cow’s milk as a natural nutrient (16130). It has been estimated that cow’s milk typically contains about 12 μmol NAD+ vitamin precursor/L, of which 60% is present as NAM and 40% as NR (130). A recent study reported that oral NR supplementation is bioavailable for NAD+biosynthesis in healthy volunteers and that NR exhibits unique and superior pharmacokinetics compared to NA and NAM in mice (128). These lines of evidence highlight the potential use of NR as an NAD+ source and therapeutic agent to treat metabolic diseases.

重要的是,牛奶中已检测出 NR 是一种天然营养素(16,130)。据估计,牛乳中一般含有约12 μmol NAD + 维生素前体/l,其中60% 为 NAM,40% 为 NR (130)。最近的一项研究报道,口服补充硝酸还原酶对健康志愿者 NAD + 生物合成是有效的,并且硝酸还原酶在小鼠体内表现出独特的和优于 NA 和 NAM 的药代动力学。这些证据突出了 NR 作为 NAD + 来源和治疗代谢性疾病的潜在用途。

A systematic regulatory network, denoted “NAD+ World,” has been developed to describe the critical roles of NAMPT-mediated NAD+ biosynthesis and NAD+-dependent SIRT1 in the regulation of metabolism and aging in mammals (70), providing important insights into the systems-level regulatory mechanisms that connect metabolism and aging in mammals. The NAD+ World 2.0 emphasizes the importance of intertissue communication among organs and provides a systems biology approach for modeling and controlling the aging process and longevity in mammals (69). This NAD+-dependent control system consists of two feedback loops involving eNAMPT and iNAMPT, and the NAD+-dependent SIRT1 enzyme.

在哺乳动物体内,nampt 介导的 NAD + 生物合成和 NAD + 依赖的 SIRT1在调节代谢和衰老过程中的关键作用,被称为“ NAD + World” ,为研究哺乳动物代谢和衰老过程中的系统级调控机制提供了重要的信息。NAD + World 2.0强调了器官之间组织间通讯的重要性,并提供了一种用于建模和控制哺乳动物衰老过程和寿命的系统生物学方法(69)。这个依赖 NAD + 的控制系统由两个反馈回路组成,包括 ampt 和 iNAMPT,以及依赖 NAD + 的 SIRT1酶。

Figueiredo and colleagues (31) constructed a metabolic network describing NAD+ biosynthesis and degradation, and used the concept of elementary flux modes to explore the potential routes in the network of NAD+ generation, which include the three known biosynthetic pathways mentioned above. Such a systematic analysis and comparison of metabolic networks specific for yeast and humans highlight the differences across species regarding the use of precursor biosynthetic routes and NAD+-dependent signaling. A network of functional associations among the NAD+-related proteins mentioned above can be derived from String v10 (125), providing a systems-level view of their functional connections (Fig. 3).

Figueiredo 和他的同事构建了一个描述 NAD + 生物合成和降解的代谢网络模型,并使用基本通量模式的概念来探索 NAD + 生成网络中的潜在路径,其中包括上面提到的3个已知的生物合成路径。这种对酵母和人类特有的代谢网络的系统分析和比较突出了不同物种在使用前体生物合成路线和 NAD + 依赖信号方面的差异。上面提到的 NAD + 相关蛋白之间的功能关联网络可以从 String v10(125)中派生出来,提供了它们功能连接的系统级视图(图3)。

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A network of NAD+-related enzymes and proteins derived from String v10. Network nodes represent proteins and edges represent protein–protein functional associations with evidence from different sources, as illustrated in the figure. Those interactions ascertained “from curated databases” and “experimentally determined” are known protein–protein interactions imported from databases of physical interactions and experimental repositories. Those interactions derived from “gene neighborhood,” “gene fusions,” mean that proteins fused in some genomes are very likely to be functionally linked. “Gene co-occurrence,” “gene coexpression,” and “protein homology” denote protein–protein interactions predicted by computational methods using genomic information; “gene neighborhood” assumes that a similar genomic context in different species indicates similar functions of the proteins; “gene fusions” means that proteins fused in some genomes are very likely to be functionally linked; “gene co-occurrence” assumes that proteins with an occurrence in the same pathway tend to have similar functions; “gene coexpression” represents predicted protein–protein associations based on observed patterns of similar expression of genes; and “protein homology” refers to protein–protein association predicted by using homologous relationships. “Text mining” uses a large body of scientific texts to search for statistically relevant co-occurrences of genes. HIF-1α, hypoxia-inducible factor 1α; MNADK, mitochondrial NADK; NRF2, nuclear factor (erythroid-derived 2)-like 2; PARP, poly(ADP)-ribosyl polymerase; SIRT, sirtuin family deacetylase.

由 String v10衍生的 NAD + 相关酶和蛋白组成的网络。网络节点表示蛋白质和边缘表示蛋白质-蛋白质功能联系的证据来自不同的来源,如图所示。这些相互作用从“精选数据库”和“实验确定”是已知的蛋白质-蛋白质相互作用从物理相互作用数据库和实验仓库导入。这些相互作用来源于“基因邻居” ,“基因融合” ,这意味着在一些基因组中融合的蛋白质很可能具有功能性连接。“基因共现”、“基因共表达”和“蛋白质超家族”表示利用基因组信息计算方法预测的蛋白质-蛋白质相互作用; “基因邻域”表示在不同物种中相似的基因组背景表明相似的蛋白质功能; “基因融合”表示在某些基因组中融合的蛋白质很可能具有功能连接; “基因共现”表示在同一通路中出现的蛋白质具有相似的功能; “基因共表达”表示基于观察到的相似基因表达模式预测的蛋白质-蛋白质关联; “蛋白质超家族”表示利用同源关系预测的蛋白质-蛋白质关联。“文本挖掘”使用大量的科学文本来搜索统计上相关的基因共同出现。Hif-1α,缺氧诱导因子1α; MNADK,线粒体 NADK; NRF2,类核因子2; PARP,多聚 ADP-核糖基聚合酶; SIRT,sirtuin 家族去乙酰化酶。Go to: 去:

Biosynthesis of NADP+

生物合成 NADP +

NADP+, a structural analogue of NAD+, is synthesized by transferring a phosphate group from ATP to the 2′-hydroxyl group of the adenosine ribose moiety of NAD+ (Fig. 2) (2144). This reaction is catalyzed by NADKs, the sole enzymes responsible for de novo NADP+ synthesis in both prokaryotic and eukaryotic cells (144), suggesting that NADK is a determinant of cellular NADP+ levels. NADK mRNA was found to be equally expressed in most human tissues with the exception of skeletal muscle and small intestine (79). In Escherichia coli and Salmonella enterica, the NADK enzyme is highly selective for its substrates NAD+and ATP, and its activity requires divalent cations, such as Mg2+, Ca2+, and Mn2+ (5071). These features are conserved in human NADK enzyme (79134). Interestingly, bacterial NADK activity is strongly inhibited by NADPH and NADH, and less strongly by NADP+ (5071), suggesting the existence of a negative feedback mechanism. To date, it is unknown whether these allosteric factors also regulate human NADK activity.

NADP + 是 NAD + 的结构类似物,它是通过将 ATP 的一个磷酸基团转移到 NAD + 核糖结构的2′-羟基上而合成的。这个反应是由 NADKs 催化的,唯一的酶负责从头合成 NADP + 在原核和真核细胞(144) ,表明 NADK 是一个决定性的细胞 NADP + 水平。除骨骼肌和小肠外,大多数人体组织中 NADK mRNA 的表达水平相同。在大肠桿菌和肠道沙门氏菌中,NADK 酶对其底物 NAD + 和 ATP 具有高度选择性,其活性需要二价阳离子,如 Mg2 + ,Ca2 + 和 Mn2 + (50,71)。这些特征在人类 NADK 酶(79,134)中是保守的。有趣的是,NADPH 和 NADH 对细菌 NADK 活性的抑制作用较强,而 NADP + (50,71)的抑制作用较弱,提示存在一种负反馈机制。迄今为止,尚不清楚这些变构因子是否也调节人类 NADK 活动。

For many years, humans were believed to have only one cytosolic NADK protein (102); however, this dogma was challenged by the discovery of a mitochondrial NADK (MNADK) by two different groups (97148). Ohashi et al. (97) discovered that a human protein, C5orf33, catalyzes the formation of NADP+ from NAD+ and ATP, and is localized to mitochondria in HEK293 cells. The MNADK mRNA was detected in all tissues tested, and its levels were found to be much more abundant relative to the cytosolic NADK (97). A separate study confirmed the finding that C5orf33 is a MNADK, and showed that MNADK is evolutionarily conserved in multicellular organisms (148). In mice, its mRNA is expressed most highly in the liver, followed by mitochondrial-rich tissues, for example, heart and skeletal muscle (148).

多年来,人类被认为只有一个细胞内的 NADK 蛋白(102) ,然而,这个教条被两个不同群体(97,148)发现的线粒体 NADK (MNADK)所挑战。Ohashi 等人(97)发现,一种人类蛋白,C5orf33,催化 NAD + 和 ATP 形成 NADP + ,并定位于 HEK293细胞的线粒体。所有检测组织中都检测到 MNADK 的 mRNA,其水平相对于细胞内 NADK (97)更为丰富。一项独立的研究证实了 C5orf33是 MNADK 的发现,并表明 MNADK 在多细胞生物中是进化上保守的。在小鼠中,其 mRNA 在肝脏表达最高,其次是线粒体组织,例如心脏和骨骼肌(148)。

In addition, MNADK mutation was recently discovered in patients with dienoyl-CoA reductase (DECR) deficiency with hyperlysinemia, a rare disorder affecting the metabolism of polyunsaturated fatty acids and lysine (65). Fibroblasts from MNADK mutant patients had decreased DECR activity and reduced mitochondrial NADP(H) levels with no change in cytosolic NADP(H) levels (65). Overexpression of the wild-type MNADK restored DECR activity in patient fibroblasts (65), suggesting that MNADK might be an appealing therapeutic target for this disorder.

此外,最近在双烯酰辅酶 a 还原酶(DECR)缺乏伴高赖氨酸血症的患者中发现了 MNADK 突变,这是一种影响多不饱和脂肪酸和赖氨酸(65)代谢的罕见疾病。来自 MNADK 突变患者的成纤维细胞 DECR 活性降低,线粒体 NADP (h)水平降低,而胞浆 NADP (h)水平没有变化(65)。野生型 MNADK 的过度表达恢复了病人成纤维细胞(65)的 DECR 活性,提示 MNADK 可能是一个有吸引力的治疗目标。Go to: 去:

Metabolic Sources and Cellular Compartmentalization of NAD(H) and NADP(H) Couples

NAD (h)和 NADP (h)夫妇的代谢来源和细胞防火分区

The distribution of the NAD+/NADH and NADP+/NADPH redox couples is highly compartmentalized due to specific localization of NAD+ and NADP+ biosynthetic enzymes and the bioavailability of NAD+precursors (Fig. 4). Importantly, since the mitochondrial inner membrane is impermeable to NAD(H) and NADP(H) (24102141), the mitochondrial and cytosolic NAD(H) and NADP(H) pools are regulated by multiple shuttles, such as the malate–aspartate shuttle for the NAD(H) pools and the isocitrate-α-ketoglutarate (α-KG) shuttle for NADP(H) pools (66). These shuttle mechanisms enable cells to maintain redox and energy homeostasis in normal and stressed states.

NAD +/NADH 和 NADP +/NADPH 氧化还原配对的分布由于 NAD + 和 NADP + 生物合成酶的特异性定位和 NAD + 前体的生物利用度而高度区分(图4)。重要的是,由于线粒体内膜对 NAD (h)和 NADP (h)(24,102,141)是不透性的,因此线粒体和细胞内 NAD (h)和 NADP (h)池是由多种穿梭物调节的,如用于 NAD (h)池的 malate-aspartate 穿梭物和用于 NADP (h)池的异柠檬酸 -α- 酮戊二酸(α-kg)穿梭物(66)。这些穿梭机制使细胞在正常和应激状态下维持氧化还原和能量稳态。

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Compartmentalization of cellular NAD(H) and NADP(H). In the extracellular milieu, NAD+ from exogenous sources or exported from cells by connexin 43 (Cx43) hemichannels undergoes a series of sequential reactions forming NAM, NMN, NR by ectoenzyme CD38, eNAMPT, and CD73. While extracellular NAM, NMN, and NA are membrane permeable and freely enter the cytosol, extracellular NR is imported via a nucleoside transporter (NT). Once in the cytosol, these precursors can generate NAD(H) and NADP(H) as illustrated in Figure 2. Exogenous NADH is imported into the cytosol via P2X7 receptor-mediated endocytosis. NAD+ can be consumed by cytosolic and nuclear NAD+-dependent proteins (SIRT1, 2, 6, and 7 as well as PARPs) to form NAM, which feeds into the salvage pathway to synthesize NAD+. In mitochondria, NAD+ is synthesized from NMN by NMNAT3 and can be consumed by mitochondrial proteins, such as SIRT3-5 and PARP1, forming NAM. It is proposed that NAM can be converted into NMN by iNAMPT in mitochondrial compartment. NADP+ is formed by MNADK-catalyzed phosphorylation of NAD+. eNAMPT, extracellular NAMPT; iNAMPT, intracellular NAMPT; NADH, reduced NAD+.

细胞 NAD (h)和 NADP (h)的防火分区。在细胞外环境中,外源 NAD + 或 connexin 43(Cx43)半纤维通道经过一系列的连续反应,形成 NAM、 NMN、 NR,并与胞外酶 CD38、烯胺铂和 CD73发生反应。细胞外 NAM、 NMN、 NA 具有膜透性,可自由进入胞浆,而 NR 则通过核苷转运蛋白(NT)进入胞浆。一旦进入细胞溶胶,这些前体可以产生 NAD (h)和 NADP (h) ,如图2所示。外源性 NADH 通过 P2X7受体介导的内吞作用进入细胞质。NAD + 可被细胞质和核 NAD + 依赖蛋白质(SIRT1、2、6、7以及 PARPs)消耗,形成 NAM,进入补救途径合成 NAD + 。在线粒体中,NAD + 是由 NMN 通过 NMNAT3合成的,可以被线粒体蛋白如 SIRT3-5和 PARP1消耗,形成 NAM。提示在线粒体腔内,iNAMPT 可将 NAM 转化为 NMN。NADP + 是由 mnadk 催化的 NAD + 磷酸化形成的。细胞外 NAMPT; iNAMPT,细胞内 NAMPT; NADH,减少 NAD + 。

NAD(H) in the extracellular milieu

细胞外环境中的 NAD (h)

Historically, NAD+ and NADH have been viewed as unable to be transported across membranes (73143). This concept has been challenged by recent evidence. First, NAD+ and its precursors have been detected at nanomolar concentrations in plasma and other body fluids (96109120). Moreover, NAD+ can be released into superfusates of mouse urinary bladder and gastrointestinal smooth muscle, as well as canine mesenteric artery, under both basal and stimulated conditions (92120). Finally, addition of exogenous NAD+ or NADH to culture medium augmented intracellular and mitochondrial-specific NAD+ and NADH levels in several experimental systems; however, the mechanisms by which NAD(H) is transported across membranes remain elusive.

历史上,NAD + 和 NADH 被认为是不能跨膜转运的(73,143)。最近的证据对这一概念提出了质疑。首先,在血浆和其他体液(96,109,120)中检测到纳摩尔浓度的 NAD + 及其前体。此外,在基础和刺激条件下(92,120) ,NAD + 均能被释放到小鼠膀胱、胃肠平滑肌和犬肠系膜动脉的超融合区。最后,在培养基中加入外源性 NAD + 或 NADH 可以提高几个实验系统中细胞内和线粒体特异性 NAD + 和 NADH 水平,然而,NAD (h)跨膜转运的机制仍然不清楚。

It has been proposed that extracellular NAD(H) could be released from dying cells; however, recent observations suggest that transmembrane transporters in living cells may mediate active exo/endocytosis of NAD(H) (55). In support of this concept, addition of exogenous NAD+ to culture medium was found to increase intracellular NAD+ and NADH levels in HeLa cells, a process that was abrogated when cells were cultured at 4 °C, suggesting the involvement of a specific and active transporter (101). In addition, connexin 43 hemichannels were reported to mediate transmembrane NAD+ fluxes in a Ca2+-dependent manner in 3T3 fibroblasts (20), and P2X7 receptors were shown to transport NADH across the plasma membranes of astrocytes (86).

已有研究提出细胞外 NAD (h)可以从死亡细胞中释放出来,但最近的研究表明,活细胞中的跨膜转运蛋白可能介导 NAD (h)的活性外分泌/内分泌(55)。为了支持这一观点,在培养基中加入外源性 NAD + 可以提高 HeLa 细胞内 NAD + 和 NADH 水平,这一过程在4 ° c 培养时被消除,提示存在特异性和活性的转运蛋白(101)。此外,连接蛋白43个半通道以钙离子依赖的方式介导3T3成纤维细胞跨膜 NAD + 通量,P2X7受体通过星形胶质细胞质膜转运 NADH (86)。

Alternatively, extracellular NAD+ could be metabolized into its cell-permeable precursors, which then enter cells via specific carrier proteins (Fig. 4). CD38, an NAD+-consuming enzyme, is an ectoenzyme tethered to the outer surface of plasma membrane that converts NAD+ to cyclic ADP ribose (cADPR) and NAM by an ADP-ribosyl cyclase reaction (32). As discussed in the section of Biosynthesis of NAD+ in mammals, NAM can be metabolized by eNAMPT into cell-permeable NMN (41). Moreover, CD73, an ectoenzyme that was previously characterized as a 5′-AMP nucleotidase, can also dephosphorylate NMN to NR (46). The latter, in turn, can be transported into cells through dipyridamole-sensitive nucleoside transporters (94). Furthermore, NAM, NMN, and NR have been found in milk and plasma (109130).

另外,细胞外 NAD + 可代谢成其可渗透细胞的前体,然后通过特定的载体蛋白进入细胞(图4)。CD38是一种耗费 NAD + 的酶,是一种通过 adp 核糖环化酶反应将 NAD + 转化为环ADP核糖和 NAM 的胞外酶。正如在哺乳动物 NAD + 生物合成部分所讨论的那样,NAM 可被烯胺铂代谢成细胞渗透性 NMN (41)。此外,CD73,一种以前被认为是5′-AMP 核苷酸酶的外生酶,也可以将磷酸化 NMN 去除到 NR (46)。后者又可通过双嘧达莫敏感的核苷转运体(94)转运到细胞中。此外,在牛奶和血浆中还发现了 NAM、 NMN 和 NR (109,130)。

Once these precursors are transported into cells, they can be utilized to synthesize NAD+ through the pathways described above. Indeed, exogenous addition of NAD+ precursors (NAM, NR, and NMN) to cells grown in culture increases intracellular NAD+ levels and protects against cell death (51101). Thus, although additional studies are necessary to determine the precise mechanisms involved, evidence suggests that extracellular NAD(H) can directly or indirectly affect intracellular NAD(H) levels to regulate cellular functions.

一旦这些前体被转运到细胞内,它们就可以通过上述途径合成 NAD + 。事实上,外源性添加 NAD + 前体(NAM、 NR 和 NMN)到培养细胞中会增加细胞内 NAD + 水平,并保护细胞免于死亡(51,101)。因此,虽然额外的研究是必要的,以确定涉及的确切机制,证据表明,细胞外 NAD (h)可以直接或间接影响细胞内 NAD (h)水平,以调节细胞功能。

NAD(H) and NADP(H) in tissues and subcellular organelles

组织和亚细胞器中的 NAD (h)和 NADP (h)

In general, NAD(H) and NADP(H) are predominantly bound to intracellular proteins, with free NAD(H) and NADP(H) accounting for only a small proportion of these dinucleotide pools (66). The intracellular content of NAD(H) and NADP(H) differs markedly among tissues and cell types. In rat liver, total NAD(H) (free and bound) and total NADP(H) were reported to be 3166 and 1788 nmol/g dry weight, respectively (126). In rat heart, the amount of total NAD+ was estimated to be 500 nmol/g wet weight (33). A total NAD+ concentration of 368 μM was reported for mouse erythrocytes (136). By contrast, in human erythrocytes from healthy adults, the concentrations of NAD+, NADH, NADP+, and NADPH were found to be 48, 1.4, 26, and 16 μM, respectively (124).

一般来说,NAD (h)和 NADP (h)主要与细胞内蛋白结合,游离 NAD (h)和 NADP (h)只占这些二核苷酸池(66)的一小部分。细胞内 NAD (h)和 NADP (h)含量在不同组织和细胞类型之间存在显著差异。在大鼠肝脏中,总 NAD (游离结合)和总 NADP (h)分别为3166和1788nmol/g 干重(126)。在大鼠心脏中,NAD + 总量估计为500nmol/g 湿重(33)。报道小鼠红细胞 NAD + 总浓度为368μM (136)。正常人红细胞 NAD + 、 NADH、 NADP + 和 NADPH 浓度分别为48、1.4、26和16μM (124)。

Notably, recently developed genetically encoded fluorescent biosensors provide new and accurate measures of cellular NAD(H) and NADP(H) levels and their compartmental pools. Using the Peredox-mCherry fluorescent biosensor, Hung and colleagues (68) found a higher cytosolic NADH/NAD+ ratio in primary astrocytes than primary neurons, suggesting that the redox state in primary astrocytes is more reduced than that in neurons. We recently developed a ratiometric fluorescent biosensor, SoNar, that demonstrates greater variability in cytosolic NADH/NAD+ and greater stability of mitochondrial NADH/NAD+ in live cells in various stress states (150). Another group utilized the RexYFP biosensor and showed that mitochondria contain much higher NADH levels than cytoplasm in HEK293 cells (17). Furthermore, fluorescent lifetime imaging, which can differentiate NADH and NADPH fluorescence, reveals that the enzyme-bound NADPH/NADH ratio is higher (2.2:1) in glia-like outer pillar supporting cells and substantially lower (0.4:1) in outer hair cells of rat cochlea (18).

值得注意的是,最近开发的基因编码荧光生物传感器提供了新的和准确的测量细胞 NAD (h)和 NADP (h)水平及其区室池。利用 Peredox-mCherry 荧光生物传感器,Hung 和他的同事(68)发现原代星形胶质细胞的 NADH/NAD + 比值高于原代神经元,提示原代星形胶质细胞的氧化还原状态比神经元的还要低。我们最近开发了一种比率荧光生物传感器 SoNar,它显示了细胞内 NADH/NAD + 的更大变异性和活细胞内 NADH/NAD + 在不同应力状态下的更大稳定性(150)。另一组使用 RexYFP 生物传感器,发现 HEK293细胞(17)中线粒体的 NADH 水平远高于细胞质。此外,荧光寿命成像技术可以区分 NADH 和 NADPH 荧光,发现胶质细胞样外柱支持细胞的 NADPH/NADH 酶结合比较高(2.2:1) ,而大鼠耳蜗外毛细胞的 NADPH/NADH 酶结合比较低(0.4:1)。

The intracellular distribution of NAD(H) and NADP(H) is highly compartmentalized. For example, in rat liver, ∼40% of total NAD(H) and 59% of total NADP(H) are found in mitochondria (126). In cultured HEK293 cells, the estimated total cellular NAD+ was ∼365 μM and mitochondrial NAD+ is ∼246 μM, suggesting that the majority of intracellular NAD+ is present in the mitochondria (138). While nuclear membranes may be freely permeable to NAD+ and NADP+, the mitochondrial inner membrane is generally impermeable to both dinucleotides [NAD(P)+] (66143). Therefore, while cytosolic and nuclear NAD(P)+ can be maintained through the pathways mentioned in the Biosynthesis of NAD+ in Mammals and Biosynthesis of NADP+ sections, mitochondrial NAD+ and NADP+ levels are maintained through additional organelle-specific mechanisms (Fig. 4). The presence of mitochondrial NMNAT (NMNAT3) suggests that NAD+ can be synthesized from NMN in this organelle (24). The recent identification of MNADK suggests another means by which cells maintain mitochondrial NADP+ levels (97148). In addition, SIRT3-5 and PARP1, mitochondrial NAD+-consuming proteins, degrade NAD+ into NAM, which can replenish the NAD+ pool through the salvage pathway in this organelle (24).

NAD (h)和 NADP (h)在细胞内的分布是高度区域化的。例如,在大鼠肝脏中,线粒体(126)含有ー40% 的 NAD (h)和59% 的 NADP (h)。细胞总 NAD + 约为365μM,线粒体 NAD + 约为246μM,提示细胞内 NAD + 主要存在于线粒体(138)。核膜对 NAD + 和 NADP + 可通透,而线粒体内膜对二核苷酸[ NAD (p) + ](66,143)通常不通透。因此,虽然细胞质和核 NAD (p) + 可以通过哺乳动物 NAD + 生物合成和 NADP + 生物合成中提到的途径维持,但线粒体 NAD + 和 NADP + 水平通过额外的细胞器特异性机制维持(图4)。线粒体 NMNAT (NMNAT3)的存在提示 NMN 在这个细胞器(24)中可以合成 NAD + 。最近对 MNADK 的鉴定提示了细胞维持线粒体 NADP + 水平的另一种途径(97,148)。此外,线粒体 NAD + 消耗蛋白 SIRT3-5和 PARP1降解 NAD + 转化为 NAM,通过挽救途径补充 NAD + 库(24)。

It is interesting to note that cellular NAD(H) and NADP(H) pools possess differential sensitivity in response to diverse stimuli. Cytoplasmic and nuclear total NAD(H) is susceptible to changes in cellular nutrient levels (e.g., glucose, lactate, and pyruvate), whereas total mitochondrial NAD(H) is relatively well maintained and required for cell survival in response to toxic stresses (138151).

有趣的是,注意到细胞 NAD (h)和 NADP (h)池具有不同的灵敏度响应不同的刺激。细胞质和细胞核的总 NAD (h)容易受到细胞营养水平(如葡萄糖、乳酸和丙酮酸)的变化影响,而线粒体的总 NAD (h)则相对维持良好,细胞在应对毒性应激时需要存活(138,151)。

Metabolic sources of NAD(P)H and cytosolic/mitochondrial shuttles

NAD (p) h 的代谢来源及胞质/线粒体梭子

The interconversion between NAD(P)+ and their reduced forms NAD(P)H can occur in cellular energy metabolic pathways such as glycolysis, the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle, and mitochondrial oxidative phosphorylation (Figs. 5 and ​and66).

NAD (p) + 与其还原形式 NAD (p) h 之间的相互转换可以发生在细胞能量代谢途径中,如糖酵解、磷酸戊糖途径蛋白(PPP)、三元羧酸(TCA)循环和线粒体氧化磷酸化(图5和图6)。

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Open in a separate window 在单独的窗口中打开FIG. 5. 图5

Metabolic sources of NAD(H) and cytosolic/mitochondrial NADH shuttles. (A) In the cytosol, interconversion of NAD+ and NADH is mediated by the glycolytic enzymes GAPDH and LDH. In the mitochondrial matrix, PDH, ME2, GLUD, and TCA cycle enzymes (IDH3, KGDH, and MDH2) contribute to NAD(H) production. (B) Cytosolic and mitochondrial NADH are exchanged through two shuttles: the malate–aspartate shuttle and the glycerol-3-phosphate shuttle. In the malate–aspartate shuttle, cytosolic MDH1 and mitochondrial MDH2 catalyze the reversible interconversion of OAA and malate in conjunction with the interconversion of NAD+ and NADH. Cytosolic GOT1 and mitochondrial GOT2 catalyze the reversible conversion between OAA and L-Asp coupled with the interconversion of Glu and α-KG. The α-KG/malate antiporter (encoded by SLC25A11 gene) and aspartate-glutamate antiporter (encoded by SLC25A13) transport intermediate metabolites between cytosol and mitochondria. In this shuttle, NADH is oxidized to NAD+ in cytosol and NAD+ is reduced to NADH in mitochondria. In the glycerol-3-phosphate shuttle, cytosolic GPDH reduces the glycolytic intermediate DHAP into glycerol-3-phosphate and simultaneously oxidizes NADH to NAD+ in the cytoplasm. Mitochondrial GPDH catalyzes the reverse reaction by oxidizing glycerol-3-phosphate into DHAP and transferring electrons to FAD forming FADH2. α-KG, α-ketoglutarate; DHAP, dihydroxyacetone phosphate; FAD, flavin-adenine dinucleotide; G3P, glyceraldehyde-3-phosphate; GAPDH, glyceraldehyde phosphate dehydrogenase; Glu, glutamate; GLUD, glutamate dehydrogenases; Glut, glucose transporters; GOT, glutamate-OAA transaminase; GPDH, glycerol-3-phosphate dehydrogenase; IDH, isocitrate dehydrogenase; KGDH, α-ketoglutarate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; OAA, oxaloacetate; PDH, pyruvate dehydrogenase; SCL25A11 and SCL25A13, solute carrier family 25 member 11 and 13, respectively; TCA, tricarboxylic acid.

NAD (h)的代谢来源与细胞质/线粒体 NADH 的关系。(a)在细胞溶胶中,NAD + 和 NADH 的相互转换是通过糖酵解酶 GAPDH 和 LDH 介导的。在线粒体基质中,PDH、 ME2、 GLUD 和 TCA 循环酶(IDH3、 KGDH 和 MDH2)参与 NAD (h)的生产。(b)细胞质和线粒体 NADH 通过苹果酸-天冬氨酸穿梭和甘油-3- 磷酸穿梭进行交换。在苹果酸-天冬氨酸穿梭中,细胞质 MDH1和线粒体 MDH2催化 OAA 和苹果酸的可逆相互转换,并与 NAD + 和 NADH 的相互转换相结合。胞质 GOT1和线粒体 GOT2催化 OAA 和 L-Asp 之间的可逆转换以及 Glu 和 α-kg 之间的相互转换。α-kg/苹果酸逆向转运蛋白(SLC25A11编码)和天冬氨酸-谷氨酸逆向转运蛋白(SLC25A13编码)在细胞质和线粒体之间运输中间代谢产物。在这种穿梭中,NADH 在细胞质中被氧化成 NAD + ,而在线粒体中 NAD + 被还原成 NADH。在甘油 -3- 磷酸酯穿梭中,胞质内的 GPDH 使糖酵解中间体 DHAP 降解为甘油 -3- 磷酸酯,同时将 NADH 氧化为胞质内的 NAD + 。线粒体 GPDH 通过氧化甘油 -3- 磷酸盐形成 DHAP,将电子转移到 FAD 形成 FADH2来催化反应。α-kg,α- 酮戊二酸; DHAP,二羟丙酮磷酸; FAD,黄素腺嘌呤二核苷酸; G3P,甘油醛 -3- 磷酸; GAPDH,甘油醛磷酸脱氢酶; 谷氨酸; GLUD,谷氨酸脱氢酶; 葡萄糖转运蛋白; GOT,谷氨酸-oaa 转氨酶;GPDH,甘油-3-磷酸脱氢酶[NAD(P)⁺] ; IDH,异柠檬酸脱氢酶; KGDH,α- 酮戊二酸脱氢酶; LDH,乳酸脱氢酶; MDH,苹果酸脱氢酶; ME,苹果酸酶; OAA,草酰乙酸; PDH,丙酮酸脱氢酶; SCL25A11和 SCL25A13,溶质载体家族25个成员11和13,分别; TCA,三元羧酸。

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Metabolic sources of NADP(H) and the cytosolic/mitochondrial NADPH shuttle. In the cytosol, NADPH is primarily produced by G6PD and 6PGD in the pentose phosphate pathway. ME1 also contributes to cytosolic NADPH production. Mitochondrial NADPH is generated by NADP+-dependent IDH2, GLUD, NNT, and ME3. The cytosolic and mitochondrial NADPH is exchanged through the isocitrate-α-KG shuttle, where cytosolic IDH1 and mitochondrial IDH2 catalyze the interconversion of isocitrate and α-KG in conjunction with the interconversion of NADP+ and NADPH. The citrate carrier protein (encoded by SLC25A1 gene) and the α-KG/malate antiporter (encoded by SLC25A11 gene) mediate the transport of isocitrate and α-KG between cytosol and mitochondria, respectively. 6PG, 6-phosphogluconate; 6PGD, 6-phosphogluconate dehydrogenase; G6P, glucose-6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; NNT, nicotinamide nucleotide transhydrogenase; R5P, ribose-5-phosphate; SCL25A1, solute carrier family 25 member 1.

NADP (h)的代谢来源及细胞质/线粒体 NADPH 穿梭。在细胞溶胶中,NADPH 主要由磷酸戊糖途径中的 G6PD 和6PGD 产生。ME1也参与细胞内 NADPH 的产生。线粒体 NADPH 由依赖于 NADP 的 IDH2、 GLUD、 NNT 和 ME3产生。细胞质和线粒体 NADPH 通过异柠檬酸 -α-kg 穿梭进行交换,其中胞质 IDH1和线粒体 IDH2催化异柠檬酸和 α-kg 的相互转换,并与 NADP + 和 NADPH 的相互转换相结合。柠檬酸盐载体蛋白(SLC25A1编码)和 α- 千克/苹果酸逆向转运蛋白(SLC25A11编码)分别介导异柠檬酸和 α- 千克在细胞溶胶和线粒体之间的转运。6PG,6-磷酸葡萄糖酸; 6PGD,6-磷酸葡萄糖酸脱氢酶; G6P,葡萄糖-6-磷酸; G6PD,葡萄糖-6-磷酸脱氢酶; NNT,烟酰胺核苷酸转移酶; R5P,核糖 -5- 磷酸; SCL25A1,溶质载体家族25个成员1。

In the cytoplasm, NADH can be generated as a by-product of glycolysis (Fig. 5A). This occurs at the sixth step of glycolysis, where two molecules of G3P are oxidized to two molecules of 1,3-bisphosphoglycerate coupled with the reduction of NAD+ to NADH by glyceraldehyde phosphate dehydrogenase (GAPDH) (87). Cytosolic NADH can also be produced by lactate dehydrogenase (LDH), which catalyzes a reversible conversion between lactate and pyruvate.

在细胞质中,NADH 可以作为糖酵解的副产物产生(图5A)。这发生在糖酵解的第六步,G3P 的2个分子被氧化成2个1,3-二磷酸甘油酸分子,同时 NAD + 被甘油醛磷酸脱氢酶(GAPDH)还原成 NADH。乳酸脱氢酶还可以产生细胞质 NADH,催化乳酸和丙酮酸之间的可逆转换。

In the mitochondria, the TCA cycle can produce eight molecules of NADH per molecule of glucose under well-oxygenated conditions (141). Once the glycolytic end-product pyruvate is transported into mitochondria, the pyruvate dehydrogenase (PDH) complex decarboxylates it into acetyl-CoA and simultaneously reduces NAD+ to NADH (87). Acetyl-CoA then enters the TCA cycle where NAD+ is reduced to NADH by NAD+-dependent isocitrate dehydrogenase 3 (IDH3), α-ketoglutarate dehydrogenase (KGDH), and malate dehydrogenase (MDH2) (Fig. 5A). Moreover, NAD+-linked malic enzyme (ME2) can produce NADH via conversion of malate to pyruvate (111). Glutamate dehydrogenases (GLUD1-2) can also metabolize glutamate into the TCA cycle intermediate α-KG using NAD+ as a cofactor to produce NADH.

在线粒体中,TCA 循环在充分氧合的条件下每个葡萄糖分子可以产生八个 NADH 分子(141)。一旦糖酵解终产物丙酮酸被转运到线粒体,丙酮酸脱氢酶复合物(PDH)将其脱羧进入乙酰辅酶 a,同时将 NAD + 还原为 NADH (87)。乙酰辅酶 a 进入 TCA 循环,NAD + 通过依赖 NAD + 的异柠檬酸脱氢酶3(IDH3)、 α- 酮戊二酸脱氢酶(KGDH)和苹果酸脱氢酶(MDH2)还原为 NADH。此外,NAD + 连接的苹果酸酶(ME2)可以通过苹果酸转化为丙酮酸(111)来产生 NADH。谷氨酸脱氢酶(GLUD1-2)也可以通过 NAD + 作为辅助因子代谢谷氨酸进入 TCA 循环中间体 α-kg,产生 NADH。

In general, the outer mitochondrial membrane is very porous, enabling NADH to diffuse freely into the intermembrane space; however, the inner mitochondrial membrane is impermeable to NADH (24102141). To circumvent this impediment, two NADH shuttles, the malate–aspartate shuttle and the glycerol-3-phosphate shuttle, can transport NADH into the mitochondrial matrix (Fig. 5B) (6690).

一般来说,线粒体外膜是非常多孔的,使 NADH 能够自由地扩散进入膜间隙; 然而,线粒体内膜是不可渗透的 NADH (24,102,141)。为了绕过这个障碍,NADH 穿梭机,苹果酸-天冬氨酸穿梭机和甘油-3- 磷酸盐穿梭机,可以将 NADH 输送到线粒体基质。

In the first shuttle, the electron carrier malate is imported from the cytosol into the mitochondrial matrix through an α-KG/malate antiporter (encoded by SLC25A11 gene) in conjunction with export of α-KG into the cytosol. Once in the matrix, malate is oxidized into oxaloacetate (OAA) by MDH2, transferring electrons to NAD+ forming NADH. OAA is then transaminated into aspartate by mitochondrial glutamate-OAA transaminase (GOT2). Subsequently, the aspartate/glutamate antiporter (encoded by SLC25A13 gene) exports aspartate into the cytosol where cytosolic GOT (GOT1) converts aspartate back to OAA, which is, in turn, reduced to malate in conjunction with oxidizing NADH to NAD+ by cytosolic MDH (MDH1). Thus, the malate–aspartate shuttle is reversible and requires multiple enzymes. In this shuttle, NADH is oxidized to NAD+ in the cytosol and NAD+ is reduced to NADH in mitochondria. NAD+ is used as an electron acceptor during glycolysis, whereas NADH is used by mitochondrial complex I to drive the mitochondrial electron transport chain (ETC).

在第一艘航天飞机中,电子载体苹果酸通过 α-kg/苹果酸逆向转运蛋白(SLC25A11编码)从细胞溶胶进入线粒体基质,同时将 α-kg 输出到细胞溶胶中。一旦进入基质,MDH2将苹果酸氧化成草酰乙酸(OAA) ,将电子转移到 NAD + 形成 NADH。OAA 通过线粒体谷氨酸转氨酶(GOT2)转氨进入门冬氨酸。随后,天冬氨酸/谷氨酸逆向转运蛋白(SLC25A13编码)将天冬氨酸输送到胞浆中,胞浆内的 GOT (GOT1)将天冬氨酸转化为 OAA,而 OAA 又被胞浆内的 MDH1氧化为 NADH,再被 NADH 氧化为 NAD + 。因此,苹果酸-天冬氨酸穿梭是可逆的,需要多种酶。在这种穿梭中,NADH 在细胞质中被氧化成 NAD + ,而 NAD + 在线粒体中被还原成 NADH。NAD + 在糖酵解过程中被用作电子受体,而 NADH 被线粒体复合体 i 用来驱动线粒体电子传递链(ETC)。

Unlike the malate–aspartate shuttle, electron transfer via the glycerol-3-phosphate shuttle is irreversible and needs only one enzyme, glycerol-3-phosphate dehydrogenase (GPDH) (Fig. 5B) (90). Cytosolic GPDH reduces the glycolytic intermediate DHAP to glycerol-3-phosphate and simultaneously oxidizes NADH to NAD+ in the cytoplasm. Mitochondrial GPDH catalyzes the reverse reaction by oxidizing glycerol-3-phosphate to DHAP and transferring electrons to FAD forming FADH2, which then donates electrons at mitochondrial respiratory complex II (succinate dehydrogenase; SDH) to reduce ubiquinone (Q) to ubiquinol (QH2). The use of FADH2, rather than NADH, as an electron source yields less energy per mole.

与苹果酸-天冬氨酸穿梭机不同,通过甘油-3- 磷酸电子转移穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机穿梭机。胞质内的 GPDH 使糖酵解中间体 DHAP 降解为甘油 -3- 磷酸酯,同时使 NADH 氧化为胞质内的 NAD + 。线粒体 GPDH 通过氧化甘油-3-磷酸酯到 DHAP,将电子转移到 FAD 形成 FADH2,然后在线粒体呼吸复合体 II (琥珀酸脱氢酶; SDH)上供给电子,使泛醌(q)降解为泛醌(QH2) ,从而催化反应。使用 FADH2而不是 NADH 作为电子源,每摩尔产生的能量较少。

Cytosolic NADPH is primarily generated from the PPP by glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) (87141). G6PD catalyzes the conversion of glucose-6-phosphate (G6P) into 6-phosphogluconate (6PG), which can be further metabolized into ribose-5-phosphate (R5P) by 6PGD. Both reactions are coupled to the reduction of NADP+ to NADPH (Fig. 6). In addition, other enzymes also contribute to the cytosolic NADPH pool, such as IDHs and MEs (87139), all of which have both cytosolic and mitochondrial isozymes. Cytosolic IDH (IDH1) catalyzes the same reaction as mitochondrial IDH3 using NADP+ rather than NAD+ as a cofactor, forming NADPH. Cytosolic ME (ME1) catalyzes oxidative decarboxylation of malate to pyruvate with the reduction of NADP+ to NADPH. The relative contribution of these enzymes to NADPH production remains elusive. Fan and colleagues (38) found in proliferating HEK293T cells that the greatest contributor to cytosolic NADPH is the oxidative PPP. In differentiating adipocytes, Liu et al. (82) showed that ME is the primary source of NADPH; however, in hypoxia the main source is the oxidative PPP.

细胞质 NADPH 主要由葡萄糖-6-磷酸脱氢酶(G6PD)和6-磷酸葡萄糖酸脱氢酶(6PGD)(87,141)从 PPP 产生。G6PD 催化葡萄糖-6-磷酸(G6P)转化为6-磷酸葡萄糖酸(6PG) ,6PGD 可进一步代谢为核糖 -5- 磷酸酯(R5P)。这两个反应都耦合到 NADP + 还原为 NADPH (图6)。此外,其他酶也有助于细胞质 NADPH 库,如 IDHs 和 MEs (87,139) ,所有这些都具有胞质和线粒体同工酶。与线粒体 IDH3相同,胞内 IDH1通过 NADP + 而不是 NAD + 作为辅助因子催化反应,形成 NADPH。细胞质微生物 ME (ME1)催化苹果酸的氧化脱羧合成丙酮酸,还原 NADP + 生成 NADPH。这些酶对 NADPH 产生的相对贡献仍然难以捉摸。Fan 和他的同事(38)在增殖的 HEK293T 细胞中发现,对细胞溶质 NADPH 最大的贡献者是氧化的 PPP。在脂肪细胞的分化过程中,Liu 等(82)表明 ME 是 NADPH 的主要来源,而缺氧时主要来源于氧化性 PPP。

Mitochondrial NADPH can be produced by mitochondrial isozymes of IDH (IDH2) and ME (ME3) (Fig. 6) (87139). In addition, NADP+-dependent GLUDs can also generate NADPH through the conversion of glutamate to α-KG (111). Of note, another significant contributor to mitochondrial NADPH is NAM nucleotide transhydrogenase (NNT), which catalyzes the following reversible reaction: NADH + NADP+← → NAD+ + NADPH (66113). NNT resides in the mitochondrial inner membrane and is driven by the electrochemical proton gradient. In the presence of a proton gradient, for example, under physiological conditions, the equilibrium of this reaction moves far to the right, which explains the fact that over 95% of mitochondrial NADP+ is reduced and that the redox potential of NADPH/NADP+ (−400 mV) is more negative than that of NADH/NAD+ (−300 mV) in mitochondria (66113).

线粒体 NADPH 可由 IDH (IDH2)和 ME (ME3)的同工酶产生(图6)(87,139)。此外,依赖 NADP + 的 glud 也可以通过谷氨酸转化为 α- 千克(111)来产生 NADPH。值得注意的是,另一个对线粒体 NADPH 的重要贡献者是 NAM 核苷酸转移酶(NNT) ,它催化以下可逆反应: NADH + NADP + ←→ NAD + + NADPH (66,113)。NNT 主要存在于线粒体内膜中,并受到电化学质子梯度的驱动。在质子梯度的存在下,例如,在生理条件下,这个反应的平衡向右移动,这解释了为什么线粒体 NADP + 95% 以上被降低,而且 nadph/NADP + (- 400mv)的氧化还原电位比线粒体中 NADH/NAD + (- 300mv)的氧化还原电位更为负(66,113)。

As mentioned earlier, the mitochondrial inner membrane is impermeable to NADP(H) (24102141). Communication between cytosolic and mitochondrial NADP(H) pools is conducted by the isocitrate-α-KG shuttle (Fig. 6) (66100). This NADPH shuttle functions through IDH1 and IDH2 isozymes. In the mitochondrial matrix, NADP+-dependent IDH2 converts α-KG into isocitrate by oxidizing NADPH to NADP+. Isocitrate is then pumped into the cytosol in exchange for malate by the citrate carrier protein (encoded by SLC25A1 gene). In the cytosol, IDH1 catalyzes the reverse reaction by transforming isocitrate to α-KG and NADP+ to NADPH. Subsequently, α-KG is transported into mitochondrial matrix by the α-KG/malate antiporter as a carrier in the malate–aspartate shuttle. Thus, the isocitrate-α-KG shuttle plays a pivotal role in maintaining cellular NADPH levels.

如前所述,线粒体内膜是不透 NADP (h)(24,102,141)。细胞质和线粒体 NADP (h)池之间的通讯由 isocitrate-α-KG 穿梭机进行(图6)(66,100)。这种 NADPH 穿梭通过 IDH1和 IDH2同工酶发挥作用。在线粒体基质中,依赖于 NADP + 的 IDH2通过将 NADPH 氧化为 NADP + ,将 α-kg 转化为异柠檬酸盐。然后,异柠檬酸被泵入细胞溶胶中,用柠檬酸载体蛋白(由 SLC25A1基因编码)交换苹果酸。在细胞溶胶中,IDH1通过将异柠檬酸转化为 α-kg 和 NADP + 转化为 NADPH 来催化逆反应。随后,α- 千克被 α- 千克/苹果酸逆向转运线粒体基质作为苹果酸-天门冬氨酸穿梭机的载体运送至大肠杆菌。因此,isocitrate-α-KG 穿梭机在维持细胞 NADPH 水平方面起着关键作用。Go to: 去:

NAD(H) and NADP(H) Regulate Cellular Redox Homeostasis

NAD (h)和 NADP (h)调节细胞氧化还原稳态

NAD(P)H and glutathione (GSH) serve as dual-function participants in maintaining cellular redox homeostasis. GSH is a cosubstrate for hydrogen peroxide (H2O2) removal by glutathione peroxidases (GPxs); and NADP(H) functions as an indispensable cofactor for glutathione reductase (GR) and thioredoxin reductases (TRs) that are essential for GPx- and peroxiredoxin (Prx)-mediated peroxide removal, respectively. Paradoxically, excess accumulation of GSH and/or NAD(P)H leads to reductive stress (60), and may directly contribute to the production of O2•− and H2O2. Excess NAD(P)H, in particular, may be utilized by NADPH oxidases (NOXs) to produce reactive oxygen species (ROS) (59116). Since the critical roles of GSH in redox stress (oxidative and reductive stress) have been extensively discussed elsewhere (6110117), in the context of this review, we primarily focus on the NAD(H) and NADP(H) redox couples.

NAD (p) h 和谷胱甘肽(GSH)具有维持细胞氧化还原稳态的双重功能。GSH 是谷胱甘肽过氧化物酶(GPxs)去除过氧化氢(H2O2)的辅助底物,而 NADP (h)是谷胱甘肽过氧化物酶(GR)和硫氧还蛋白还原酶(TRs)不可缺少的辅助因子,这些辅助因子分别对 GPx-和过氧化物过氧化氢(Prx)介导的过氧化物去除起重要作用。反常的是,谷胱甘肽和/或 NAD (p) h 的过度积累导致还原应激(60) ,并可能直接促进 O2-和 H2O2的产生。尤其是过量的 NAD (p) h,可被 NADPH 氧化酶(NOXs)利用产生活性氧类(ROS)(59,116)。由于 GSH 在氧化还原应激(氧化和还原应激)中的关键作用已在其他文献(6,110,117)中得到广泛讨论,本文主要讨论 NAD (h)和 NADP (h)氧化还原偶联。

NAD(H) and NADP(H) as antioxidant cofactors

NAD (h)和 NADP (h)作为抗氧化辅助因子

As mentioned above, NADPH is an essential cofactor of GR and TRs. GR catalyzes the recycling of GSH from its oxidized form (GSSG) (Fig. 7). In this context, NADPH donates two electrons to reduce GSSG to GSH by GR; the recycled GSH can then be used to reduce H2O2 to water by GPxs (21). In the context of TRs, TRs transfer electrons from NADPH to reduce oxidized thioredoxin (Trx-S2) to its reduced form Trx-(SH)2, which serves as a source of reducing equivalents in the enzymatic removal of H2O2 and other organic hydroperoxides by Prxs (21).

如上所述,NADPH 是 GR 和 TRs 的重要辅助因子。GR 催化 GSH 从氧化态(GSSG)中回收(图7)。在这种情况下,NADPH 捐赠两个电子,以减少 GSSG 到谷胱甘肽 GR,回收的谷胱甘肽可用于还原 H2O2的水由 GPxs (21)。在 TRs 中,TRs 将电子从 NADPH 转移到氧化硫氧还蛋白(Trx-S2)还原为 Trx-(SH)2,这是 Prxs (21)降低酶去除 H2O2和其他有机过氧化氢氧化物当量的来源。

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NADPH and NAD+ function as cofactors in antioxidant defense systems. NADPH is an essential cofactor of GR and TRs. GR catalyzes the recycling of GSH from GSSG, and TRs reduces oxidized Trx-S2 into Trx-(SH)2. Simultaneously, both enzymes require NADPH as an electron donor and oxidize it to NADP+, which can be reduced back to NADPH by ME1, IDH1, G6PD, and G6PD in the cytoplasm, and NNT, ME3, GLUD, and IDH2 in the mitochondria. Once O2•− is formed, for example, from NOXs in the cytosol and from mitochondrial ETC, cytosolic CuZnSOD and mitochondrial MnSOD reduce it to H2O2. GSH can be used by GPx to reduce H2O2 further to water. Trx-(SH)2 provides reducing equivalents for Prx in the removal of H2O2. NAD+ is required for SIRT deacetylase activity. Cytosolic SIRT2 enhances G6PD activity, and mitochondrial SIRT3 enhances IDH2 activity, to enhance the generation of NADPH from NADP+. CuZnSOD, copper zinc superoxide dismutase; ETC, electron transport chain; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, oxidized GSH; H2O2, hydrogen peroxide; MnSOD, manganese superoxide dismutase; NOX, NADPH oxidase; Prx, peroxiredoxin; TR, thioredoxin reductase; Trx-S2, oxidized thioredoxin; Trx-(SH)2, reduced thioredoxin.

NADPH 和 NAD + 在抗氧化防御系统中的作用。NADPH 是糖皮质激素受体(GR)和促肾上腺皮质激素受体(TRs)的重要辅助因子。GR 催化 GSH 从 GSSG 中回收,TRs 还原氧化的 Trx-S2生成 Trx-(SH)2。同时,这两种酶都需要 NADPH 作为电子供体,并将其氧化为 NADP + ,在细胞质中 ME1、 IDH1、 G6PD 和 G6PD 还原为 NADPH,在线粒体中还原为 NNT、 ME3、 GLUD 和 IDH2。一旦 O2•-形成,例如,从胞质中的 NOXs 和线粒体 ETC,胞质 CuZnSOD 和线粒体 MnSOD 将其降低到 H2O2。谷胱甘肽可以被谷胱甘肽过氧化物酶用来将过氧化氢还原成水。Trx-(SH)2为 Prx 提供了去除 H2O2的还原当量。SIRT 脱乙酰酶活性需要 NAD + 。胞质 SIRT2增强 G6PD 活性,线粒体 SIRT3增强 IDH2活性,提高 NADP + 生成 NADPH 的能力。铜锌超氧化物歧化酶; ETC,电子传递链; GPx,谷胱甘肽过氧化物酶; GR,谷胱甘肽还原酶; GSH,还原型谷胱甘肽; GSSG,氧化型谷胱甘肽; H2O2,过氧化氢; MnSOD,锰超氧化物歧化酶; NOX,NADPH 氧化酶; Prx,过氧化还原酶; TR,硫氧还蛋白还原酶; Trx-S2,氧化型硫氧还蛋白; Trx-(SH)2,还原型硫氧还蛋白。

Given that G6PD is the rate-limiting enzyme of the PPP and the principal source of cytosolic NADPH (87141), modulation of G6PD activity is expected to affect cellular NADPH levels and consequently cellular redox state and biological functions. Indeed, G6PD-deficient mice showed decreased levels of NADPH and GSH as well as reduced activity of GPx, which contribute to oxidative damage in the renal cortex (135). Similarly, mouse embryonic stem cells lacking G6PD also had lower cellular NADPH and GSH levels, with enhanced sensitivity to oxidative stress-induced cell death due to the shutdown of the PPP (39). Moreover, our group previously reported that G6PD expression and activity are inhibited by aldosterone in mouse aortic endothelial cells and in mouse aorta (77). This inhibition of G6PD is associated with reduced cellular levels of NADPH, GSH, and nitric oxide (NO), augmented ROS production, and impaired vascular function. Importantly, blockade of aldosterone by a mineralocorticoid receptor antagonist or overexpression of G6PD restored G6PD activity and significantly reversed these adverse effects in vivo(77).

鉴于 G6PD 是 PPP 的限速酶,也是细胞内 NADPH (87,141)的主要来源,因此调节 G6PD 活性可能会影响细胞内 NADPH 水平,进而影响细胞的氧化还原状态和生物学功能。实际上,g6pd 缺陷的小鼠表现出 NADPH 和 GSH 水平的降低以及 GPx 活性的降低,而 g6pd 缺陷导致肾皮质的氧化损伤(135)。同样,缺乏 G6PD 的小鼠胚胎干细胞也有较低的细胞 NADPH 和 GSH 水平,由于 PPP 的关闭,对氧化应激诱导的细胞死亡敏感性增强(39)。此外,我们的研究小组以前报道过醛固酮可以抑制小鼠主动脉内皮细胞和主动脉(77)中 G6PD 的表达和活性。这种 G6PD 的抑制与 NADPH、 GSH 和 NO 的细胞水平降低、 ROS 的产生增加和血管功能的损害有关。重要的是,通过盐皮质激素受体拮抗剂阻断醛固酮或过度表达 G6PD 可以恢复 G6PD 活性,并在体内显著逆转这些不良反应(77)。

Gain-of-function studies further confirmed the protective role of G6PD. For example, the enhancement of G6PD activity by genetic or pharmacological means elevated cellular NAPDH and GSH pools, promoted ROS detoxification, and increased cell viability in primary vascular endothelial and smooth muscle cells in vitro (3478). In transgenic mice, a modest (approximately two-fold) overexpression of G6PD was associated with an increase in NADPH levels in the brain and liver, in protection of these tissues against aging-induced oxidative damage, and an extension of life span (in females) compared to littermates with basal activity of G6PD (95). Thus, these studies clearly support the concept that G6PD is a major source of cytosolic NADPH, and that NADPH is the indispensable reducing agent for ROS elimination and redox homeostasis (Fig. 7).

功能增益研究进一步证实了 G6PD 的保护作用。例如,通过遗传或药物增强 G6PD 活性意味着提高细胞 NAPDH 和 GSH 池,促进活性氧解毒,提高原代血管内皮细胞和平滑肌细胞的细胞活性(34,78)。在转基因小鼠中,与 G6PD (95)基础活性的同胞相比,G6PD 的适度(大约两倍)过表达与脑和肝中 NADPH 水平的升高有关,可以保护这些组织免受老化引起的氧化损伤,延长寿命(女性)。因此,这些研究清楚地支持这样的概念,即 G6PD 是细胞内 NADPH 的主要来源,NADPH 是活性氧清除和氧化还原稳态不可缺少的还原剂(图7)。

In the mitochondrion, IDH2 is a major enzyme for NADPH production (Fig. 7) (113); it catalyzes the oxidative decarboxylation of isocitrate producing α-KG and CO2. Gain- and loss-of function studies confirmed the essential functions of IDH2 in maintaining cellular NADPH levels and cellular redox balance. For example, IDH2 knockout mice exhibit decreased NADPH/NADP+ and GSH/GSSG ratios and increased H2O2 levels and oxidative damage. These changes in redox state are associated with cardiac hypertrophy, accelerated heart failure, and apoptotic cell death in cardiomyocytes (74). In other studies, exposure to 7-ketocholesterol, a major oxidation product of cholesterol, inhibited IDH2 expression and activity via upregulation of microRNA-144 (42). The decrease in IDH activity was accompanied by lower NADPH and GSH levels as well as a decrease in NO production, leading to oxidative stress in human aortic endothelial cells and impaired vascular function in ex vivo murine aortae (42). As expected, IDH2silencing also decreased NADPH levels (147), while IDH2 overexpression increased NADPH levels and protected against oxidative stress-induced cell death in HEK293 cells (121). Taken together, these findings suggest that IDH2-derived NADPH is required for detoxification of peroxides by NADPH-dependent peroxidases in mammalian mitochondria.

在线粒体中,IDH2是产生 NADPH 的主要酶(图7)(113) ,它催化异柠檬酸的氧化脱羧产生 α-kg 和 CO2。功能的获得和丧失研究证实了 IDH2在维持细胞 NADPH 水平和细胞氧化还原平衡方面的基本功能。例如,IDH2基因敲除小鼠表现出 NADPH/NADP + 和 GSH/GSSG 比值降低,H2O2水平和氧化损伤增加。氧化还原状态的这些变化与心肌肥大、心力衰竭加速和心肌细胞的细胞凋亡有关。在其他研究中,暴露于7- 酮胆固醇,胆固醇的主要氧化产物,通过上调 microrna 144(42)抑制 IDH2的表达和活性。IDH 活性的降低伴随着 NADPH 和 GSH 水平的降低以及 NO 生成的减少,导致人主动脉内皮细胞的氧化应激和离体小鼠主动脉血管功能的损害。正如预期的那样,IDH2沉默也降低了 NADPH 水平(147) ,而 IDH2过表达增加了 NADPH 水平,并保护 HEK293细胞免受氧化应激诱导的细胞死亡(121)。综上所述,这些发现提示 idh2衍生的 NADPH 是哺乳动物线粒体中依赖于 NADPH 的过氧化物解毒所必需的。

In addition to IDH2, the mitochondrial enzyme NNT is another major source of mitochondrial NADPH that catalyzes the reversible conversion of NADH and NADP+ to NAD+ and NADPH (Fig. 6) (113). The critical role of NNT in regulating redox status has been well described in humans and animals. For example, the left ventricles of heart failure patients displayed lower NNT activity and NADPH levels, which correlated with decreases in GR activity and GSH levels compared to the left ventricles of non-heart failure patients (118). Under a physiological workload, cardiomyocytes from C57BL/6J mice carrying spontaneous loss-of-function mutations of NNT produced markedly higher levels of H2O2 compared to cardiomyocytes from NNT wild-type C57BL/6N mice (93), suggesting that NNT is required for NADPH regeneration to fuel H2O2 detoxifying enzymes. Indeed, concurrent with their decreased levels of NADPH, isolated liver mitochondria of NNT mutant C57BL/6J mice were less effective at removing exogenous peroxide (111). Finally, inactivation of NNT by short hairpin RNA (shRNA) or a chemical inhibitor decreases cellular NADPH and GSH levels, inhibits cell proliferation, and increases H2O2 accumulation and oxidative damage (4485142). Taken together, these findings indicate that mitochondrial NNT plays an important role in maintaining the necessary pools of cellular reducing equivalents and in preventing mammalian cells from oxidative damage and dysfunction.

除了 IDH2,线粒体 NNT 酶是另一个主要来源的线粒体 NADPH,催化 NADH 和 NADP + 可逆转化为 NAD + 和 NADPH (图6)(113)。NNT 在调节氧化还原状态中的关键作用已经在人类和动物中得到了很好的描述。例如,心力衰竭患者的左心室 NNT 活性和 NADPH 水平较低,这与非心力衰竭患者的 GR 活性和 GSH 水平下降相关(118)。在生理负荷下,携带 NNT 自发性功能缺失突变的 C57BL/6J 小鼠心肌细胞产生的 H2O2水平明显高于 NNT 野生型 C57BL/6N 小鼠(93)的心肌细胞,这表明 NNT 是促进 NADPH 再生为 H2O2解毒酶提供能量所必需的。实际上,随着 NADPH 水平的降低,NNT 突变的 C57BL/6J 小鼠离体肝线粒体去除外源性过氧化物的效率降低(111)。最后,小发夹RNA 或化学抑制剂使 NNT 失活,降低细胞 NADPH 和 GSH 水平,抑制细胞增殖,增加 H2O2积累和氧化损伤(44,85,142)。综上所述,这些发现表明线粒体 NNT 在维持必要的细胞还原当量池和防止哺乳动物细胞氧化损伤和功能障碍方面发挥着重要作用。

It is noteworthy that NADK, the NADP+-producing enzyme, has also been shown to modulate the cellular NADPH pool (Fig. 7). Overexpression of NADK elevated cellular NADPH levels and, thereby, accelerated H2O2 removal in HEK293 cells and rat pancreatic β cells (49102). By contrast, silencing NADK reduced NADPH levels leading to H2O2 accumulation and inhibition of glucose-stimulated insulin secretion in rat pancreatic β cells (49).

值得注意的是,NADK,NADP + 生产酶,也已被证明调节细胞 NADPH 池(图7)。过度表达 NADK 提高细胞 NADPH 水平,从而加速 HEK293细胞和大鼠胰岛 β 细胞(49,102)的 H2O2去除。相比之下,沉默的 NADK 减少 NADPH 水平,导致 H2O2的积累和抑制葡萄糖刺激的胰岛素分泌在大鼠胰岛 β 细胞(49)。

NAD+ is also involved in regulating the cellular redox state through the actions of SIRT enzymes. Recent studies demonstrate that NAD+-dependent SIRT2 and SIRT5 deacetylate and activate G6PD, thus increasing cellular NADPH and antioxidant capacity in vivo and in vitro (133154). The opposite effects were found in HEK293 cells and mouse embryonic fibroblasts lacking SIRT5 activity (154).

NAD + 还通过 SIRT 酶的作用参与调节细胞的氧化还原状态。近年来的研究表明,NAD + 依赖的 SIRT2和 SIRT5脱乙酰基和激活 G6PD,从而提高细胞 NADPH 和体内外抗氧化能力(133,154)。在 HEK293细胞和缺乏 SIRT5活性的小鼠胚胎成纤维细胞中发现相反的效果(154)。

Furthermore, like G6PD, IDH2 is regulated by SIRT proteins, with SIRT5 also increasing IDH2 activity by deacetylation. Consequently, silencing SIRT5 diminished IDH2 activity and cellular NADPH and GSH levels, sensitizing cells to paraquat-induced oxidative cytotoxicity (154). Moreover, mitochondrial SIRT3 has been reported to upregulate IDH2 activity through deacetylation in a mouse model of calorie restriction. Elevated IDH2 activity induced by calorie restriction was associated with increases in NADPH levels and the GSH/GSSG ratio in the inner ear, brain, and liver tissues, enhancing the protection of these tissues against oxidative damage (121).

此外,与 G6PD 一样,IDH2也受到 SIRT 蛋白的调节,SIRT5还通过脱乙酰化增加 IDH2的活性。因此,沉默 SIRT5降低 IDH2活性和细胞 NADPH 和 GSH 水平,增强细胞对百草枯诱导的氧化细胞毒性(154)。此外,据报道,线粒体 SIRT3可以通过去乙酰化上调卡路里限制小鼠模型中 IDH2的活性。卡路里限制诱导的 IDH2活性升高与内耳、脑和肝组织中 NADPH 水平和 GSH/GSSG 比值的升高有关,增强了这些组织对氧化损伤的保护作用(121)。

NAD(H) and NADP(H) are pro-oxidants and induce redox stress

NAD (h)和 NADP (h)是氧化还原应激的促氧化剂

Excess levels of cellular NADH and/or NADPH can lead to reductive stress. NAD(P)H fuels cellular ROS production via its role as a substrate for the NOX family proteins (NOX1-7) that produce H2O2 and O2•−(13). As discussed in the section on NAD(H) and NADP(H) as antioxidant cofactors, G6PD is the major source of the cytosolic NADPH pool. Modulation of G6PD activity can affect cellular NADPH levels and ROS production by NOX proteins. Overexpression of G6PD increased cellular NAD(P)H levels and upregulated NOX gp91phox and p22phox subunit mRNA expression, potentiating ROS production and oxidative damage in mouse pancreatic β cells and thymic lymphoma cells (76127).

过多的细胞 NADH 和/或 NADPH 水平可以导致还原应激。NAD (p) h 通过其作为 NOX 家族蛋白(NOX1-7)的底物的作用促进细胞内 ROS 的产生,这些 NOX 家族蛋白产生 H2O2和 O2 · (13)。正如在 NAD (h)和 NADP (h)作为抗氧化辅助因子一节中所讨论的,G6PD 是细胞内 NADPH 库的主要来源。调节 G6PD 活性可以影响细胞 NADPH 水平和 NOX 蛋白产生活性氧。G6PD 过表达增加了小鼠胰岛 β 细胞和胸腺淋巴瘤细胞 NAD (p) h 水平和 NOX gp91phox 和 p22phox 亚单位 mRNA 的表达,加强了 ROS 的产生和氧化损伤(76,127)。

By contrast, loss of G6PD activity was found to be beneficial in many disease models by opposing high NADPH levels and reductive stress. For example, G6PD deficiency reduced NADPH levels and significantly inhibited angiotensin II (Ang II)-induced O2•− production and decreased medial aortic thickness (89). Similar effects were observed in pacing-induced heart failure, where inhibition of G6PD activity abrogated elevations in NADPH levels and ROS production in failing hearts (54). Furthermore, mice with cardiac-specific overexpression of the mutant human αB-crystallin (R120G mutant) gene recapitulated the pathology of human protein aggregation cardiomyopathy and exhibited reductive stress in the heart as evidenced by increased GSH levels and increased activities of GR, G6PD, catalase, and GPx1 (107). Notably, these pathological changes and reductive stress were significantly reversed by replacing the wild-type G6PD in the R120G mice with a hypomorphic G6PD mutant (107), suggesting that G6PD-mediated reductive stress contributes to the development of this pathophenotype. Therefore, these pieces of evidence support the notion that NADPH pools produced by G6PD can induce redox stress and cellular dysfunction.

相反,在许多疾病模型中,由于反对高 NADPH 水平和还原应激,G6PD 活性的丧失被发现是有益的。例如,G6PD 缺乏可降低 NADPH 水平,显著抑制血管紧张素 II (Ang II)诱导的 O2产生和降低内侧主动脉厚度(89)。在起搏诱导的心力衰竭中也观察到了类似的效果,抑制 G6PD 活性降低了 NADPH 水平的升高和衰竭心脏中活性氧的产生(54)。此外,心脏特异性过表达突变型人 αb-crystallin (R120G 突变型)基因的小鼠重现了人类蛋白质聚集性心肌病的病理过程,并表现出心脏还原性应激,其证据是谷胱甘肽(GSH)水平升高,GR、 G6PD、过氧化氢酶和 GPx1(107)活性增强。这些病理变化和还原应激都被 R120G 小鼠中的野生型 G6PD 替换为低等型 G6PD 突变体(107)而得到明显逆转,提示 G6PD 介导的还原应激促进了 R120G 小鼠的病理变化。因此,这些证据支持的概念,NADPH 池产生的 G6PD 可以诱导氧化还原应激和细胞功能障碍。

Together, these data suggest that cellular redox homeostasis results from a delicate balance between NADPH-dependent protection against oxidant stress and NADPH-dependent reductive stress. This balance can be influenced by the relative activity of NADPH-dependent NOXs and antioxidant enzymes (e.g., Trxs, Prxs, and TRs) that depend directly or indirectly on NADPH, as well as exogenous/endogenous stimuli in a specific cell type or tissue.

总之,这些数据表明,细胞的氧化还原稳态是由于人海万花筒(电影)依赖的氧化还原应激和氧化还原应激之间的相互作用。这种平衡可能受到 NADPH 依赖的 NOXs 和抗氧化酶(如 Trxs、 Prxs 和 TRs)的相对活性的影响,这些酶直接或间接依赖于 NADPH,以及特定细胞类型或组织中的外源/内源刺激。

Mitochondrial NADH is oxidized to NAD+ at mitochondrial respiratory complex I (NADH dehydrogenase). Electrons from NADH, in conjunction with electrons from complex II, are relayed through the mitochondrial ETC to reduce molecular oxygen to water at respiratory complex IV (cytochrome C oxidase; COX) (1091). Under physiological conditions in mammalian cells, approximately 0.1–0.2% of total oxygen consumed is converted to O2•− as a result of electron leakage from the ETC. This electron leakage greatly increases under stress or pathological conditions and is associated with enhanced production of mitochondrial O2•− generation (Fig. 8) (10).

线粒体 NADH 在线粒体呼吸复合体 i 上被氧化为 NAD + (NADH脱氢酶)。来自 NADH 的电子与来自复合体 II 的电子一起,通过线粒体 ETC 中继,在呼吸复合体 IV (细胞色素c氧化酶; COX)(10,91)降低分子氧到水中。在哺乳动物细胞的生理条件下,大约0.1-0.2% 的总耗氧量转化为 O2•-,这是等离子体等离子体电子泄漏的结果。这种电子泄漏在压力或病理条件下大大增加,并与线粒体 O2生成增加有关(图8)(10)。

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NADH as pro-oxidant induces redox stress. (A) ROS production by the mitochondrial ETC under physiological conditions. NADH generated primarily in the TCA cycle is oxidized to NAD+ at mitochondrial respiratory complex I (NADH dehydrogenase). Subsequently, electrons from NADH in conjunction with electrons from the oxidation of succinate at complex II (succinate dehydrogenase) are relayed through the mitochondrial ETC and eventually reduce the oxygen molecule to water at complex IV (cytochrome C oxidase). This process is coupled with pumping protons (H+) from the mitochondrial matrix into the intermembrane space at complex I, III, and IV generating an electrochemical proton gradient, which drives ATP production at complex V (ATP synthase). Under physiological conditions, ∼0.1% to 0.2% of total oxygen consumed gains electrons from mitochondrial complex I and III leakage to form O2•−, which is rapidly converted to the more stable H2O2 by MnSOD or by spontaneous dismutation. A steady level of ROS is beneficial and required for many biological processes. (B) Under stressed states, such as hypoxia, NNT reversal, and RET, the mitochondrial NADH/NAD+ ratio increases leading to complex I dysfunction and ROS production. In addition, the NADH-dependent mitochondrial enzymes KGDH and PDH also contribute to mitochondrial ROS production. Overloaded levels of ROS result in redox stress, which is detrimental to cellular function. Fum, fumarate; PDH, pyruvate dehydrogenase; RET, reverse electron transfer; ROS, reactive oxygen species; Suc, succinate.

NADH 作为氧化促进剂诱导氧化还原应激。(a)生理条件下线粒体 ETC 产生活性氧的研究。在 TCA 循环中主要产生的 NADH 在线粒体呼吸复合物 i 上被氧化成 NAD + (NADH脱氢酶)。随后,来自 NADH 的电子与配合物 II (琥珀酸脱氢酶)中琥珀酸氧化产生的电子通过线粒体 ETC 中继,最终将氧分子还原为配合物 IV (细胞色素c氧化酶)中的水。这个过程与将质子(h +)从线粒体基质泵入复合体 i、 III 和 IV 的膜间隙中耦合,产生电化学质子梯度,从而驱动复合体 v (ATP 合酶)的 ATP 生成。在生理条件下,线粒体复合物 i 和 III 的泄漏会使总耗氧量的ー0.1% ー0.2% 获得电子,形成 O2 ·-,并通过 MnSOD 或自发突变迅速转化为更稳定的 H2O2。稳定的活性氧水平对许多生物过程是有益的,也是必需的。(b)在缺氧、 NNT 逆转、 RET 等应激状态下,线粒体 NADH/NAD + 比值增加,导致复杂性 i 功能障碍和 ROS 产生。此外,nadh 依赖的线粒体酶 KGDH 和 PDH 也有助于线粒体活性氧的产生。过量的活性氧导致氧化还原应激,这是有害的细胞功能。富马酸; PDH,丙酮酸脱氢酶; RET,反向电子转移; 活性氧,活性氧类; Suc,琥珀酸。

Both complex I and complex III have been described as major sites of mitochondrial O2•− under physiological and pathological conditions (Fig. 8) (1091). Since O2•− is highly reactive and has a very short half-life, it is rapidly converted to the more stable H2O2 by manganese superoxide dismutase (MnSOD) (or by spontaneous dismutation).

在生理和病理条件下,复合体 i 和复合体 III 都被描述为线粒体 O2-的主要位点(图8)(10,91)。由于 O2•-具有很高的反应活性并且半衰期非常短,它可以通过锰超氧化物歧化酶(MnSOD)(或者通过自发变异)迅速转化为更稳定的 H2O2。

ROS production by complex I requires a high NADH/NAD+ ratio, whereas its dependence on proton motive force is controversial (175105122132). Under basal conditions, isolated mitochondria generate extremely low levels of ROS when exogenous oxidizable substrates are absent (122132); however, addition of exogenous complex I substrates, such as glutamate, malate, or α-KG, augments the levels of NADH and, simultaneously, stimulates H2O2 production by ∼10-fold in isolated brain mitochondria (122). Interestingly, subsequent addition of ADP results in oxidation of NADH leading to a >50% reduction in H2O2 production (122). In rat L6 myoblasts, treatment with 1 mM antioxidant N-acetyl-L-cysteine increases the NADH/NAD+ ratio, which correlates with increases in mitochondrial H2O2 levels and free radical leak (119). These biochemical and cell culture studies suggest that a high NADH/NAD+ ratio is required for ROS production at complex I.

配合物 i 产生活性氧需要较高的 NADH/NAD + 比值,而它对质子动力的依赖性是有争议的(1,75,105,122,132)。在基础条件下,当缺乏外源可氧化底物(122,132)时,分离的线粒体产生极低水平的活性氧(ROS) ; 然而,添加外源复合物 i 底物,如谷氨酸、苹果酸或 α-kg,增加 NADH 的水平,同时,刺激分离的脑内 H2O2的产生10倍ー122。有趣的是,随后添加 ADP 导致 NADH 的氧化,导致 H2O2产生的超过50% 的还原(122)。在大鼠 L6成肌细胞中,1mm 抗氧化剂 n- 乙酰 -l- 半胱氨酸增加 NADH/NAD + 比值,这与线粒体 H2O2水平增加和自由基泄漏(119)有关。这些生物化学和细胞培养研究表明,在复合体 i 中产生活性氧需要较高的 NADH/NAD + 比例。

This concept is further supported by animal studies (93). Nickel et al. showed that C57BL/6J mice lacking functional NNT activity are protected against cardiac overload-induced heart failure compared to controls (wild-type C57BL/6N mice) (93). Reduced failure of the myocardium in NNT-deficient mice was associated with decreased NADH and H2O2 production as well as reduced oxidative damage (93). These findings suggest that pathological cardiac pressure overload induces reverse flux of NNT to generate NADH at the expense of NADPH resulting in a high NADH/NAD+ ratio, which further promotes ROS generation at complex I leading to oxidative injury and cytotoxicity (Fig. 8B).

这一概念得到动物研究的进一步支持(93)。Nickel 等人的研究表明,与对照组(野生型 C57BL/6N 小鼠)(93)相比,缺乏功能性 NNT 活性的 C57BL/6J 小鼠对心脏超负荷引起的心力衰竭有保护作用。Nnt 缺陷小鼠心肌衰竭的减少与 NADH 和 H2O2的产生减少以及氧化损伤的减少有关(93)。这些结果表明,病理性心脏压力超负荷引起 NNT 反向通量产生 NADH,而 NADPH 的代价是高 NADH/nad + 比值,这进一步促进了复合体 i 活性氧的产生,导致氧化损伤和细胞毒性(图8B)。

Furthermore, complex I can also operate in a reverse electron transfer (RET) mode, which leads to enhanced O2•− production at complex I compared with the forward mode (1). Under physiological conditions, RET is a minor contributor to ROS production; however, RET-induced ROS production is observed in the heart under an ischemia/reperfusion (IR) challenge (26). During ischemia, succinate is selectively increased in various murine tissues, including the heart, and is generated by the NADH-driven reversal of the SDH reaction. After reperfusion, the accumulated succinate is rapidly reoxidized by SDH, leading to ROS generation at complex I through RET to cause IR injury. Blocking ischemic succinate accumulation by inhibiting SDH attenuates mitochondrial ROS production and heart IR injury (Fig. 8B) (26).

此外,复合 i 还可以在反向电子转移模式(RET)下运行,与正向模式(1)相比,这将导致复合 i 的 O2产量增强。在生理条件下,RET 对 ROS 的产生贡献很小,但是,在缺血/再灌注(IR)的刺激下,可以观察到 RET 诱导的 ROS 产生。缺血期间,琥珀酸在包括心脏在内的各种小鼠组织中有选择性地增加,这是通过 nadh 驱动的 SDH 反应逆转而产生的。再灌注后,琥珀酸的积累被 SDH 迅速氧化,导致复合体 i 上活性氧的产生,通过 RET 引起 IR 损伤。通过抑制琥珀酸脱氢酶阻断缺血性琥珀酸蓄积,减少线粒体 ROS 产生和心脏 IR 损伤(图8B)(26)。

Accumulating evidence demonstrates that hypoxia increases cellular NADH levels and ROS production in most mammalian cells. Under hypoxia, mitochondrial NADH and FADH2 are unable to be oxidized by the ETC leading to a buildup of these reducing equivalents and subsequent reductive stress (28). Reductive stress enables one-electron reduction of oxygen to form O2•− and thereby underlies the rise in ROS production under hypoxia (28).

越来越多的证据表明,缺氧增加了大多数哺乳动物细胞的细胞 NADH 水平和活性氧的产生。在低氧条件下,线粒体 NADH 和 FADH2不能被 ETC 氧化,导致这些还原当量的积累和随后的还原应激(28)。还原性压力使单电子还原的氧气能够形成氧气,从而导致缺氧时活性氧的产生增加。

Our recent work demonstrates that primary human lung fibroblasts cultured in hypoxia (0.2% O2) produce significantly higher levels of mitochondrial ROS compared with normoxic cells (21% O2) (98). In particular, the elevation in mitochondrial ROS accumulation is associated with an increase in the NADH/NAD+ ratio and an accumulation of L-2-hydroxyglutarate, a reductive metabolite of α-KG (98), whose increase serves to buffer the reductive stress through inhibiting glycolysis and TCA cycle and, thus, NADH production under hypoxia. In bovine coronary artery smooth muscle cells, hypoxia also increased mitochondrial ROS production and NAD(P)H levels, as well as the cytosolic NADH/NAD+ ratio (indicated by the lactate/pyruvate ratio) (Fig. 8B) (45).

我们最近的工作表明,原代人肺成纤维细胞在缺氧(0.2% O2)条件下产生的线粒体活性氧明显高于正常细胞(21% O2)(98)。尤其是,线粒体内活性氧(ROS)积累的增加与 NADH/NAD + 比值的增加以及 l-2- 羟基戊二酸(l-2- 戊二酸)的积累有关。 l-2- 羟基戊二酸是 α-kg (98)的还原性代谢物,它的增加通过抑制糖酵解和 TCA 循环来缓冲还原应激,从而抑制缺氧条件下 NADH 的产生。在牛冠状动脉平滑肌细胞中,缺氧还增加了线粒体 ROS 的产生和 NAD (p) h 水平,以及细胞内 nadh/NAD + 比值(以乳酸/丙酮酸比值表示)(图8B)(45)。

It is worthwhile to note that other NADH-related mitochondrial enzymes, such as KGDH and PDH, also contribute to mitochondrial ROS production (123131). Two separate studies demonstrate that isolated mitochondrial KGDH and PDH from the brain or heart tissue produce O2•− and H2O2 in the presence of α-KG and redox cofactors (123131). Addition of NAD+ suppresses ROS production by these enzymes by switching KGDH from H2O2 formation mode to catalytic mode (formation of NADH via this reaction: α-KG + NAD+ + CoA → Succinyl-CoA + CO2 + NADH), whereas the addition of NADH stimulates KGDH-mediated ROS production (123131). Interestingly, in the presence of its substrates (α-KG and CoA) and different ratios of NADH to total NAD(H) (NADH and NAD+), a higher NADH/NAD(H) ratio is related to more ROS production by KGDH (131). Together, these data suggest that NADH promotes ROS formation by complex I and III of the ETC as well as via the stimulation of the mitochondrial enzymes KGDH and PHD (Fig. 8B).

值得注意的是,其他 nadh 相关的线粒体酶,如 KGDH 和 PDH,也有助于线粒体 ROS 的产生(123,131)。两项独立的研究表明,在 α-kg 和氧化还原辅助因子(123,131)存在的情况下,从大脑或心脏组织分离的线粒体 KGDH 和 PDH 产生 O2•-和 H2O2。NAD + 的加入通过将 KGDH 从 H2O2生成模式转化为催化模式(此反应形成 NADH: α-kg + NAD + + CoA →琥珀酰辅酶 a + CO2 + NADH) ,抑制这些酶产生 ROS,而 NADH 的加入则刺激 KGDH 介导的 ROS 产生(123,131)。有趣的是,在底物(α-kg 和 CoA)和 NADH 与 NAD (h)(NADH 和 NAD +)比值不同的情况下,较高的 NADH/NAD (h)比值与 KGDH (131)产生较多的 ROS 有关。总之,这些数据表明,NADH 促进活性氧的形成复合物 i 和 III 的 ETC,以及通过刺激线粒体酶 KGDH 和 PHD (图8B)。Go to: 去:

NAD(H) and NADP(H) and Cellular Metabolism

NAD (h)和 NADP (h)与细胞代谢

In addition to their crucial roles in maintaining cellular redox state, the NAD(H) and NADP(H) redox couples are also critical regulators of cellular metabolism (Fig. 9). Typically, NAD+ is necessary for glycolysis and for the biosynthesis of nucleotides and amino acids; NADH provides electrons for mitochondrial oxidative phosphorylation and ATP production (87). NADP+ supports the PPP to generate NADPH that is indispensable for reductive biosynthesis of nucleotides, amino acids, and lipids (87). As described in the Metabolic Sources of NAD(P)H and Cytosolic/Mitochondrial Shuttles section, many metabolic enzymes catalyze the intra/interconversion of NAD(H) and NADP(H). Thus, changes in NAD(H) and NADP(H) levels affect cellular metabolism and vice versa.

除了它们在维持细胞氧化还原状态中的关键作用外,NAD (h)和 NADP (h)氧化还原配对也是细胞代谢的关键调节因子(图9)。通常情况下,NAD + 对于糖酵解和核苷酸和氨基酸的生物合成是必需的; NADH 为线粒体氧化磷酸化和 ATP 的生产提供电子。NADP + 支持 PPP 生成对核苷酸、氨基酸和脂质还原性生物合成不可缺少的 NADPH (87)。正如 NAD (p) h 的代谢来源和胞浆/线粒体梭子部分所描述的那样,许多代谢酶催化 NAD (h)和 NADP (h)的内部/相互转换。因此,NAD (h)和 NADP (h)水平的变化影响细胞代谢,反之亦然。

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NAD+ regulates cellular metabolism. Intracellular NAD+ levels can be increased by supplementing with NAD+ precursors, enhancing the expression and activity of NAD+ biosynthetic enzymes, or by inhibiting NAD+ consumption by PARPs and CD38 enzymes. Increased NAD+ levels further enhance the activity of SIRT proteins. SIRT1, 3, and 6 are deacetylases, whereas SIRT4 is an ADP-ribosylase. SIRT1 deacetylates and activates FOXO1 and PGC-1 resulting in the stimulation of mitochondrial oxidative phosphorylation (OXPHOS) and FA oxidation. SIRT1 can also deacetylate and activate AceCS1 and HIF-2α promoting FA synthesis and glutaminolysis, respectively. By contrast, SIRT1 and SIRT6 deacetylate and inactivate HIF-1α suppressing glycolysis. Similar to SIRT1, SIRT3 deacetylation can also increase mitochondrial OXPHOS and FA synthesis and oxidation. The mitochondria-localized enzyme, SIRT3, can deacetylate mitochondrial complex I and II proteins to enhance their activity; it also targets and activates AceCS2 and LCAD to enhance FA synthesis and oxidation, respectively. Unlike SIRT1, 3, and 6, SIRT4 ADP-ribosylates and inhibits GLUD activity leading to suppression of insulin secretion under basal and stimulated conditions. AceCS, acetyl-CoA synthetase; FA, fatty acids; FOXO1, forkhead box O1; LCAD, long-chain acyl coenzyme A dehydrogenase; OXPHOS, oxidative phosphorylation; PGC-1, peroxisome proliferator-activated receptor γ coactivator 1.

NAD + 调节细胞代谢。通过补充 NAD + 前体,增强 NAD + 生物合成酶的表达和活性,抑制 PARPs 和 CD38酶对 NAD + 的消耗,可以提高细胞内 NAD + 水平。提高 NAD + 水平进一步增强 SIRT 蛋白的活性。SIRT1,3和6是去乙酰化酶,而 SIRT4是 adp 核糖酶。SIRT1去乙酰化并激活 FOXO1和 PGC-1,从而刺激线粒体氧化磷酸化和 FA 氧化。SIRT1还能分别促进 AceCS1和 hif-2α 的合成和谷氨酰胺分解,并能激活 AceCS1和 hif-2α。与此相反,SIRT1和 SIRT6去乙酰化和灭活 hif-1α 抑制糖酵解。与 SIRT1相似,SIRT3脱乙酰基也能增加线粒体 OXPHOS 和 FA 的合成和氧化。线粒体局部化酶 SIRT3可以去乙酰化线粒体复合物 i 和 II 蛋白增强其活性,也可以作用于 AceCS2和 LCAD,分别促进 FA 的合成和氧化。与 SIRT1,3,6不同,SIRT4 adp- 核糖基化,抑制 GLUD 活性,导致抑制基础和刺激条件下的胰岛素分泌。内皮细胞,乙酰辅酶 a 合成酶; 脂肪酸,脂肪酸; FOXO1,叉头盒 O1; LCAD,长链酰基辅酶 a 脱氢酶; OXPHOS,氧化磷酸化; PGC-1,过氧化物酶体增殖物活化受体 γ 辅激活因子1。

Modulating NAD(H) biosynthesis regulates cellular metabolism

调节 NAD (h)生物合成调节细胞代谢

NR, NAM, and NMN are precursors of NAD+ biosynthesis via the salvage pathway (Biosynthesis of NAD+ in Mammals section). Accumulating evidence highlights the importance of these precursors in modulating cellular NAD+ levels and metabolism in various disease models. For example, Canto et al. (23) showed that feeding mice NR elevates cellular NAD+ levels in skeletal muscle and brown adipose tissue and prevents high-fat diet-induced weight gain and obesity, likely due to enhanced insulin sensitivity and energy expenditure. In fact, NR supplementation improves glucose tolerance, mitochondrial biogenesis, and oxygen consumption in mice fed a high-fat diet. Similar protective effects were also observed in prediabetic and diabetic mice that received NR in their diet (129).

NR、 NAM 和 NMN 是通过挽救途径(哺乳动物组织中 NAD + 的生物合成)合成 NAD + 的前体。越来越多的证据强调了这些前体在调节各种疾病模型中细胞 NAD + 水平和代谢的重要性。例如,Canto 等人(23)表明,喂养小鼠 NR 能提高骨骼肌和褐色脂肪组织中的细胞 NAD + 水平,防止高脂肪饮食引起的体重增加和肥胖,这可能是由于增强了胰岛素敏感性和能量消耗。事实上,补充硝酸还原酶可以提高高脂饮食小鼠的葡萄糖耐量、线粒体生物合成和耗氧量。类似的保护作用也观察到在糖尿病前期和糖尿病小鼠接受 NR 的饮食(129)。

In addition, in obese and diabetic mice, NAM supplementation elevates hepatic NAD(H) levels and the NAD+/NADH ratio, resulting in increased mitochondrial content, enhanced mitochondrial glucose metabolism, and improved insulin sensitivity (140). Likewise, administration of NMN ameliorates the decrease in NAD+ levels in the liver and hepatocytes, improves glucose metabolism, and attenuates insulin resistance in high-fat diet-induced type 2 diabetic mice (146). Furthermore, decreases in total and nuclear NAD+ levels are found in skeletal muscles of aged mice, which correlate with compromised mitochondrial function characterized by decreased expression of mitochondrial-encoded respiratory subunits, decreased mitochondrial content, and impairment of ATP production (48). Supplementation of NAM raises cellular NAD+ levels and rescues mitochondrial function in aged mice (48). These lines of evidence suggest that NAD+ precursors can modulate cellular metabolism and reverse metabolic pathophenotypes by promoting NAD+ biosynthesis.

此外,在肥胖和糖尿病小鼠,NAM 补充提高肝脏 NAD (h)水平和 NAD +/NADH 比值,导致线粒体内容物增加,提高线粒体葡萄糖代谢,改善胰岛素敏感性(140)。同样,在高脂饮食诱导的2型糖尿病小鼠(146)中,服用 NMN 可以改善肝脏和肝细胞中 NAD + 水平的降低,改善葡萄糖代谢,减轻胰岛素抵抗。此外,老年小鼠骨骼肌中总 NAD 和核 NAD + 水平下降,这与线粒体功能受损有关,线粒体编码的呼吸亚单位表达减少,线粒体内容减少,ATP 产生受损(48)。补充 NAM 可提高老龄小鼠细胞 NAD + 水平,改善线粒体功能。这些证据表明,NAD + 前体通过促进 NAD + 生物合成调节细胞代谢和逆转代谢途径类型。

Given that NAD+ biosynthesis from its precursors is governed by a cascade of enzymatic reactions, modulating the activity of rate-limiting enzymes can also alter cellular NAD+ levels and consequently cellular metabolism. Pharmacological inhibition of NAMPT with FK866 reduces cellular NAD(H) levels and NAD+/NADH ratio, which are associated with decreases in oxygen consumption and ATP production and upregulation of glycolytic gene expression, indicating a metabolic shift toward glycolysis in rat primary cardiomyocytes (99). Interestingly, supplementation of NMN, the enzymatic product of NAMPT, is able to abrogate the decrease in NAD(H) levels significantly, preventing the metabolic shift to glycolysis (99). In mice, high-fat diet-induced type 2 diabetes suppresses NAMPT expression in the liver and white adipose tissue leading to reduced NAD+ levels, which correlate with insulin resistance and impaired glucose metabolism (146). Dietary administration of NMN in diabetic mice reverses these phenotypes (146). These findings suggest that NAD+ produced by NAMPT is critical for maintaining glucose metabolism. Notably, NAMPT is a target gene of the transcription factor hypoxia-inducible factor 1α (HIF-1α) (7). HIF-1α signaling is known to reprogram cellular metabolism toward glycolysis (60). Therefore, HIF-1α-mediated upregulation of NAMPT is expected to enhance NAD+ biosynthesis and, thus, cellular NAD+ levels, which is required for glycolysis.

鉴于 NAD + 从其前体生物合成是由一系列酶反应控制的,调节限速酶的活性也可以改变细胞 NAD + 水平,从而改变细胞的代谢。FK866对 NAMPT 的药理抑制降低了细胞 NAD (h)水平和 NAD +/NADH 比值,这与降低氧耗、 ATP 产生和糖酵解基因表达上调有关,表明大鼠原代心肌细胞(99)向糖酵解的代谢转移。有趣的是,补充 NMN,NAMPT 的酶产品,能够显著减少 NAD (h)水平的下降,防止代谢转向糖酵解(99)。在小鼠中,高脂肪饮食诱导的2型糖尿病抑制了肝脏和白色脂肪组织中 NAMPT 的表达,导致 NAD + 水平降低,而 NAD + 水平与胰岛素抵抗和糖代谢受损相关(146)。饮食给予糖尿病小鼠 NMN 可逆转这些表型(146)。这些发现表明 NAMPT 产生的 NAD + 对于维持葡萄糖代谢是至关重要的。值得注意的是,NAMPT 是转录因子缺氧诱导因子1α (hif-1α)的靶基因。Hif-1α 信号通过重新编程细胞代谢向糖酵解(60)转变。因此,缺氧诱导因子 -1α 介导的 NAMPT 上调有望促进 NAD + 的生物合成,从而提高糖酵解所需的细胞 NAD + 水平。

The mitochondrial enzyme NNT is an important source of mitochondrial NAD+ and NADPH (Metabolic Sources of NAD(P)H and Cytosolic/Mitochondrial Shuttles section). Knockdown of NNT results in increased cytosolic NAD+/NADH and NADP+/NADPH ratios and promotes a metabolic switch that utilizes glycolysis rather than glutaminolysis as the major anaplerotic reaction to replenish the TCA cycle with anabolic carbons. NNT knockdown correlates with inhibition of cell proliferation and sensitization of melanoma to glucose deprivation-induced cell death (44). By contrast, cells overexpressing NNT switch back to glutaminolysis as their main energy source (44). Genetic or pharmacological blockade of NNTreduces cellular NAD(P)H levels, depolarizes mitochondrial membrane potential, and inhibits mitochondrial oxidative phosphorylation, thereby increasing the susceptibility to oxidative stress-induced cell death (85142). These results suggest that NNT is essential for maintenance of mitochondrial function and redox balance to support cell proliferation and survival.

线粒体 NNT 是线粒体 NAD + 和 NADPH (NAD (p) h 的代谢来源和胞浆/线粒体梭形区)的重要来源。NNT 的降低导致细胞内 NAD +/NADH 和 NADP +/NADPH 比值增加,促进代谢开关,利用糖酵解而不是谷氨酰胺溶解作为主要的代谢反应来补充合成碳。NNT 基因敲除与抑制黑色素瘤细胞增殖和致敏葡萄糖剥夺诱导的细胞死亡相关(44)。相比之下,过度表达 NNT 的细胞转回谷氨酰胺溶解作为它们的主要能量来源(44)。基因或药物阻断 NNT 降低细胞 NAD (p) h 水平,使线粒体膜电位去极化,抑制线粒体氧化磷酸化,从而增加氧化应激诱导细胞死亡的敏感性(85,142)。这些结果提示 NNT 对维持线粒体功能和氧化还原平衡以支持细胞增殖和存活至关重要。

NMNAT also regulates NAD+ biosynthesis. Silencing NMNAT1 decreases nuclear NAD+ levels and SIRT1 activity, resulting in a metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis owing to impaired mitochondrial function in primary mouse myoblasts (48). Overexpression of NMNAT1in these cells restores cellular NAD+ levels and completely abrogates these metabolic changes via a SIRT1-dependent mechanism (48).

NMNAT 还调节 NAD + 的生物合成。沉默 NMNAT1降低了细胞核 NAD + 水平和 SIRT1活性,导致小鼠原始成肌细胞的线粒体功能受损,从线粒体氧化磷酸化转变为有氧糖酵解。NMNAT1在这些细胞中的过度表达可以恢复细胞 NAD + 水平,并通过 sirt1依赖机制完全消除这些代谢变化(48)。

Manipulating NAD(H) consumption regulates cellular metabolism

控制 NAD (h)的摄入调节细胞代谢

In addition to biosynthetic enzymes, cellular NAD(H) levels are also determined by NAD+-consuming enzymes, including SIRT deacetylases (SIRT1-7), PARPs (PARP1-2), and cADP-ribose synthases (CD38 and CD157) (2266).

除了生物合成酶之外,细胞 NAD (h)水平也由 NAD + 消耗酶来测定,包括 SIRT 去乙酰化酶(SIRT1-7)、 PARPs (PARP1-2)和 cADP-ribose 合酶(CD38和 CD157)(22,66)。

SIRTs are mammalian homologues of the yeast silent information regulator 2 (sir2) and are localized in distinct subcellular compartments: SIRT1, SIRT6, and SIRT7 are primarily found in the nucleus; SIRT3, SIRT4, and SIRT5 are located in mitochondria; and SIRT2 is mainly localized to cytosol (245766). SIRTs exhibit both protein deacetylase and mono ADP-ribosyltransferase activity. SIRTs utilize NAD+ as a substrate to catalyze the deacetylation reaction at lysine residue of proteins to produce NAM, deacetylated protein, and 2′-O-acetyl-ADP ribose (245766). These enzymes are involved in regulating numerous biological processes, including cellular metabolism. Changes in NAD+ bioavailability can alter the activity of these enzymes and, thereby, affect energy metabolism.

SIRTs 是酵母沉默信息调节因子2(sir2)的哺乳动物同源基因,定位于不同的亚细胞区域: SIRT1,SIRT6,和 SIRT7主要位于细胞核,SIRT3,SIRT4和 SIRT5位于线粒体,SIRT2主要定位于细胞溶胶(24,57,66)。SIRTs 同时具有蛋白质去乙酰化酶和单 adp- 核糖基转移酶活性。以 NAD + 为底物,在赖氨酸残基上催化脱乙酰化反应生成 NAM、脱乙酰化蛋白和2′-o- 乙酰基 -adp 核糖(24,57,66)。这些酶参与调节许多生物过程,包括细胞代谢。NAD + 生物利用度的变化可以改变这些酶的活性,从而影响能量代谢。

In yeast, calorie restriction lowers cellular NADH levels, an inhibitor of sir2 activity, leading to an increase in the NAD+/NADH ratio and activation of sir2. Concurrently, there is a metabolic shift to mitochondrial oxidative phosphorylation and a resulting extension of life span (8081), suggesting that sir2 regulates cellular metabolism in yeast.

在酵母中,卡路里限制降低细胞 NADH 水平,一种 sir2活性的抑制剂,导致 NAD +/NADH 比率的增加和 sir2的激活。同时,也有一个向线粒体氧化磷酸化的代谢转变,并由此延长寿命(80,81) ,这表明 sir2调节酵母细胞的新陈代谢。

Mammalian homologues of sir2 also regulate metabolism (Fig. 9). In a mitochondrial disease model, COX assembly protein knockout/knockin (SCO2 ko/ki) mice exhibit ubiquitous COX deficiency and exercise intolerance (25). NR administration increases the NAD+/NADH ratio in skeletal muscle and improves motor performance compared to vehicle-fed SCO2 ko/ki mice. This protection is associated with SIRT1-mediated deacetylation and activation of forkhead box O1 (FOXO1) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), thereby upregulating the expression of genes involved in fatty acid oxidation and mitochondrial oxidative phosphorylation to enhance energy production (25). In addition, SIRT1 has been found to activate acetyl-CoA synthetase 1 (AceCS1) by deacetylation, resulting in a pronounced increase in AceCS1-dependent fatty acid synthesis from acetate in Cos-7 cells (58).

Sir2的哺乳动物同源基因也调节新陈代谢(图9)。在线粒体疾病模型中,COX 组装蛋白敲除/敲除(SCO2 ko/ki)小鼠表现出普遍的 COX 缺乏和运动耐受性(25)。硝酸还原酶能提高骨骼肌中 NAD +/NADH 比值,改善运动能力。这种保护作用与 sirt1介导的去乙酰化和叉头盒 O1(FOXO1)和过氧化物酶体增殖物活化受体 γ 辅激活因子1α (pgc-1α)的激活有关,从而提高了与脂肪酸氧化和线粒体氧化磷酸化有关的基因的表达,以提高能量产生(25)。此外,SIRT1已被发现通过脱乙酰化作用激活乙酰辅酶 a 合成酶1(AceCS1) ,导致 Cos-7细胞中来自乙酸酯的 AceCS1依赖性脂肪酸合成显著增加(58)。

Likewise, mitochondrial SIRT3 is also able to activate mitochondrial AceCS2 (58). Furthermore, SIRT3 has been shown to stimulate ATP production by activating proteins in the mitochondrial respiratory chain. In particular, SIRT3-induced deacetylation of complex I proteins (e.g., NDUFA9) and subunit A (SDHA) in complex II augments the activity of both complexes, increasing ATP production (327). Consequently, SIRT3 expression levels positively correlate with ATP production in murine tissues; and tissue ATP production is lower in SIRT3 knockout mice compared with wild-type littermates (3). Moreover, SIRT3 stimulates mitochondrial fatty acid oxidation by deacetylation and activation of long-chain acyl coenzyme A dehydrogenase in murine liver (64).

同样,线粒体 SIRT3也能够激活线粒体 AceCS2(58)。此外,已经证明 SIRT3通过激活线粒体呼吸链中的蛋白质来刺激 ATP 的生产。特别是 sirt3诱导的复合物 i 蛋白(如 NDUFA9)和复合物 II 中的亚单位 a (SDHA)的脱乙酰化,增加了这两个复合物的活性,提高了 ATP 的产量(3,27)。因此,SIRT3的表达水平与小鼠组织中 ATP 的产生呈正相关; 与野生型同胞相比,SIRT3基因敲除小鼠组织中 ATP 的产生较低(3)。此外,SIRT3通过去乙酰化和激活小鼠肝脏长链酰基辅酶 a 脱氢酶(64) ,刺激线粒体脂肪酸氧化。

Finally, unlike SIRT1 and SIRT3, SIRT4 is an ADP-ribosyltransferase, capable of ADP-ribosylating and inactivating mitochondrial GLUD2 in mouse pancreatic β cells to suppress insulin secretion in response to glucose, glutamine, or leucine (56). Consequently, SIRT4 knockout mice exhibit increased insulin secretion under basal and stimulated conditions. In vitro studies further demonstrate that the restoration of insulin secretion requires the normal activity of GLUD2 in SIRT4-deficient pancreatic β cells (56).

最后,不像 SIRT1和 SIRT3,SIRT4是一个 adp- 核糖基转移酶,能够 adp- 核糖基化和失活小鼠胰岛 β 细胞线粒体 GLUD2,以抑制胰岛素分泌的反应葡萄糖,谷氨酰胺,或亮氨酸(56)。因此,SIRT4基因敲除小鼠在基础和刺激条件下表现出胰岛素分泌增加。体外研究进一步表明,sirt4缺陷胰岛 β 细胞(56)恢复胰岛素分泌需要正常的 GLUD2活性。

Emerging evidence suggests that SIRT proteins also interact with HIF signaling pathway in regulating cellular metabolism (Fig. 9). It is noteworthy that different SIRTs exhibit distinct effects on HIF activity. Specifically, SIRT1 and SIRT6 are negative regulators of HIF-1α, while SIRT1 activates HIF-2α activity. For example, in skeletal muscle of aged mice, nuclear NAD+ levels and SIRT1 activity are much lower than in skeletal muscle of young mice (48). The decrease in SIRT1 activity is associated with metabolic reprogramming to aerobic glycolysis due to compromised mitochondrial function evidenced by decreases in mitochondrial content, ATP production, and mitochondria-encoded gene expression of the respiratory complexes (48). The metabolic shift and mitochondrial dysfunction are also correlated with pseudohypoxic activation of HIF-1α but not HIF-2α signaling likely through downregulation of von Hippel Lindau (VHL) protein. Importantly, feeding old mice NMN rescues cellular NAD+ levels and SIRT1 activity, and upregulates VHL expression, leading to an inhibition of age-related activation of HIF-1α signaling under normoxia and a normalization of mitochondrial oxidative phosphorylation (48).

新出现的证据表明 SIRT 蛋白也与 HIF 信号通路相互作用,调节细胞代谢(图9)。值得注意的是,不同的 SIRTs 对 HIF 活性有明显的影响。具体来说,SIRT1和 SIRT6是 hif-1α 的负性调节因子,而 SIRT1激活 hif-2α 活性。例如,在老龄小鼠骨骼肌中,核 NAD + 水平和 SIRT1活性远低于幼龄小鼠(48)。SIRT1活性的下降与有氧糖酵解的代谢重编程有关,这是由于线粒体功能受损,线粒体含量、 ATP 产量和呼吸复合物的线粒体编码基因表达减少所证明的(48)。代谢转移和线粒体功能障碍也与 hif-1α 的假缺氧激活有关,但与 hif-2α 信号通路可能通过下调 von Hippel Lindau (VHL)蛋白而无关。重要的是,给老年小鼠喂 NMN 可以改善细胞 NAD + 水平和 SIRT1活性,并上调 VHL 表达,导致在正常氧下抑制与年龄相关的 hif-1α 信号的激活和线粒体氧化磷酸化的正常化(48)。

Furthermore, cancer cells are known to utilize both glycolysis and glutaminolysis for energy generation. Corbet et al. (29) reported that when human cervical and pharyngeal cancer cells (squamous cell) were cultured in medium at pH 6.5 for 8–10 weeks, they reprogrammed their metabolism from glycolysis to glutaminolysis. This metabolic switch elevated cellular NAD+ levels and enhanced SIRT1 activity in acidic cultures, resulting in deacetylation of HIF-1α and HIF-2α proteins (29). Deacetylation of HIF-1α decreases its activity leading to inhibition of glycolysis, whereas HIF-2α deacetylation boosts its activity to promote glutaminolysis via upregulating glutaminase 1 expression (29). These actions collaboratively ensure the metabolic switch of energy sources to support cell proliferation and tumor growth. Results from these two studies suggest that SIRT1 negatively regulates HIF-1α signaling, but its effects on HIF-2α activity depends on cell context.

此外,已知癌细胞利用糖酵解和谷氨酰胺溶解来产生能量。Corbet 等(29)报道,当人颈部和咽部癌细胞(鳞状细胞)在 pH 6.5的培养基中培养8-10周时,它们的代谢从糖酵解重新编程为谷氨酰胺溶解。这种代谢转换提高了细胞 NAD + 水平,增强了 SIRT1在酸性培养中的活性,导致 hif-1α 和 hif-2α 蛋白的去乙酰化。Hif-1α 的去乙酰化降低了其抑制糖酵解的活性,而 hif-2α 的去乙酰化通过上调谷氨酰胺酶1的表达增强了其促进谷氨酰胺分解的活性(29)。这些行动协同确保能源的代谢开关,以支持细胞增殖和肿瘤生长。这两项研究的结果表明,SIRT1负性调节 hif-1α 信号,但其对 hif-2α 活性的影响取决于细胞环境。

Similar to SIRT1, SIRT6 was reported to be a corepressor of HIF-1α signaling; however, SIRT6 accomplished this effect by deacetylating the chromatin of HIF-1α target gene promoters (153). Embryonic stem cells from SIRT6 knockout mice showed normoxic stabilization of HIF-1α protein that led to a metabolic shift toward aerobic glycolysis as demonstrated by increases in glucose uptake, lactate production, and glycolytic gene expression as well as a decrease in oxygen consumption (153).

与 SIRT1相似,据报道 SIRT6是 hif-1α 信号的辅抑制子,然而,SIRT6通过去乙酰化 hif-1α 靶基因启动子的染色质来实现这一效应(153)。来自 SIRT6基因敲除小鼠的胚胎干细胞显示 hif-1α 蛋白正常的稳定性,导致代谢转向有氧糖酵解,表现为葡萄糖摄取、乳酸产生和糖酵解基因表达增加以及氧消耗减少(153)。

Cellular NAD+ bioavailability can also be affected by the activity of PARPs and CD38 (Fig. 9). Modulation of PARPs or CD38 activity affects cellular metabolism by altering NAD+, thereby affecting SIRT1 activity. Two different groups reported that skeletal muscle of PARP1 or PARP2 knockout mice exhibited higher NAD+ content and SIRT1 activity than wild-type mice (89). In this context, increased SIRT1 activity deacetylates and activates FOXO1 and PGC-1α, which augments energy expenditure and protects against high-fat diet-induced obesity by increasing mitochondrial biogenesis and oxidative phosphorylation (89). These effects were further confirmed in cultured cells with PARP1 or PARP2silencing, but were attenuated by simultaneous knockdown of SIRT1 in PARP1- or PARP2-deficient cells (9), suggesting that enhanced mitochondrial function requires NAD+-dependent SIRT1 activity in PARP-deficient cells.

细胞 NAD + 的生物利用度也可能受到 PARPs 和 CD38活性的影响(图9)。PARPs 或 CD38活性的调节通过改变 NAD + 影响细胞代谢,从而影响 SIRT1活性。两个不同的组报道 PARP1或 PARP2基因敲除小鼠骨骼肌表现出较高的 NAD + 含量和 SIRT1活性比野生型小鼠(8,9)。在这种情况下,增加 SIRT1的活性去乙酰化并激活 FOXO1和 pgc-1α,通过增加线粒体生物合成和氧化磷酸化,增加能量消耗并防止高脂肪饮食诱导的肥胖。这些效应在 PARP1或 PARP2沉默的培养细胞中得到进一步证实,但通过同时击倒 PARP1或 PARP2缺陷细胞(9)中的 SIRT1而减弱,提示 PARP1缺陷细胞的线粒体功能增强需要 NAD + 依赖的 SIRT1活性。

Similarly, tissues from CD38 knockout mice have increased cellular NAD+ levels, which correlate with higher SIRT1 activity in liver nuclear extracts compared to wild-type nuclear extracts (4). By contrast, a decrease in cellular NAD+ levels is found in HEK293 cells overexpressing CD38, resulting in decreased expression of glycolytic genes and attenuated cell proliferation (67). Taken together, these findings highlight the interactions of the three classes of NAD+-consuming proteins in regulating cellular NAD+levels and energy metabolism.

类似地,CD38基因敲除小鼠的组织提高了细胞 NAD + 水平,这与肝核提取物中 SIRT1活性高于野生型核提取物相关(4)。相反,过度表达 CD38的 HEK293细胞 NAD + 水平下降,糖酵解基因表达减少,细胞增殖减弱(67)。综上所述,这些发现强调了三类 NAD + 消耗蛋白在调节细胞 NAD + 水平和能量代谢中的相互作用。Go to: 去:

Concluding Remarks


The NAD(H) and NADP(H) redox couples serve as cofactors or/and substrates for many enzymes to maintain cellular redox homeostasis and energy metabolism. Deficiency or imbalance in cellular NAD(H) and NADP(H) levels perturbs cellular redox state and metabolic homeostasis leading to redox stress, energy stress, and eventually disease states. Thus, maintaining cellular NAD(H) and NADP(H) balance is critical for cellular function. This balance is maintained dynamically and governed by biosynthesis, consumption, and compartmental localization.

NAD (h)和 NADP (h)的氧化还原配对是许多酶维持细胞氧化还原稳态和能量代谢的辅助因子或底物。细胞 NAD (h)和 NADP (h)水平的缺乏或不平衡会扰乱细胞的氧化还原状态和代谢内稳态,导致氧化还原应激、能量应激和最终的疾病状态。因此,维持细胞 NAD (h)和 NADP (h)的平衡是关键的细胞功能。这种平衡是动态维持和调控的生物合成,消费和区室定位。

Newly identified biosynthetic enzymes, for example, eNAMPT and MNADK, and newly developed genetically encoded fluorescent biosensors have improved our understanding of how compartmentalized NAD(H)/NADP(H) pools inter-relate in response to various physiological and pathological stimuli. The use of these newly developed fluorescent tools has been limited to cell cultures until recently, when xenografts expressing these sensors have been used to monitor the NAD+/NADH redox state in murine models (150152). In addition, the metabolism of NAD+ and its precursors in the extracellular compartment and how exogenous NAD+ enters cells have not been well explored and nor understood. Future research on these topics is needed.

新近发现的生物合成酶,例如,烯酰化酶和锰酸钾,以及新近发展的基因编码的荧光生物传感器,提高了我们对 NAD (h)/NADP (h)池如何区域化地响应各种生理和病理刺激的认识。直到最近,这些新开发的荧光工具仅限于细胞培养,当表达这些传感器的异种移植物被用于监测小鼠模型中 NAD +/NADH 的氧化还原状态(150,152)。此外,NAD + 及其前体在细胞外区域的代谢以及外源性 NAD + 如何进入细胞尚未得到很好的研究和理解。今后需要对这些主题进行研究。

The level of cellular NAD(H)/NADP(H) is essential for maintaining redox homeostasis. Deficiency in these redox couples can lead to oxidative or reductive stress, depending on the redox ratio of each. Both oxidative stress and reductive stress are detrimental to normal cell functions. This dual role complicates the use of global antioxidants as rational and effective therapeutic approaches to redox stress disorders.

细胞 NAD (h)/NADP (h)水平对维持氧化还原稳态至关重要。缺乏这些氧化还原配对可导致氧化或还原压力,这取决于每个氧化还原比。氧化应激和还原应激都对正常细胞功能有害。这种双重作用使全球抗氧化剂作为治疗氧化还原应激障碍的理性和有效的方法复杂化。

In addition, it is clear that NAD(H) and NADP(H) are required for maintaining cellular metabolism. Future efforts are still needed to understand how an imbalance of these two redox couples directly affects energy metabolism and how this imbalance alters NAD(H)/NADP(H)-dependent enzymes and, thus, affects their functions in regulating cellular metabolism.

另外,NAD (h)和 NADP (h)显然是维持细胞代谢所必需的。这两对氧化还原配对的不平衡如何直接影响能量代谢,以及这种不平衡如何改变 NAD (h)/NADP (h)依赖的酶,从而影响它们调节细胞代谢的功能,还需要进一步的研究。

Finally, the emergence of bioactive NAD+ precursors, for example, NR, and of specific pharmacological inhibitors, for example, NAMPT inhibitor FK866, provides promising therapeutic approaches for the treatment of NAD(H)- and NADP(H)-related metabolic disorders through modulating cellular NAD(H) levels.

最后,生物活性 NAD + 的前体,如 NR 和特定的药物抑制剂,如 NAMPT 抑制剂 FK866的出现,通过调节细胞 NAD (h)水平,为治疗 NAD (h)和 NADP (h)相关的代谢性疾病提供了有希望的治疗途径。Go to: 去:


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