类黄酮芹菜素对 NAD + 酶 CD38的抑制作用

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Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38

Implications for Cellular NAD + Metabolism, Protein Acetylation, and Treatment of Metabolic Syndrome 细胞 NAD 的意义,代谢,蛋白质乙酰化和代谢症候群的治疗

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Abstract

摘要

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD+metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD+ levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD+ase in mammals. Moreover, CD38 knockout mice have higher NAD+ levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD+ levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD+ levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD+ levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD+-dependent pathways.

代谢症候群是一个日益严重的全球性健康问题。因此,发展新的策略来治疗这种疾病势在必行。在过去的几年里,对 NAD + 代谢的调控已经成为改善代谢症候群的一个可信的策略。特别是,细胞 NAD + 水平的增加具有有益的作用,可能是因为去乙酰化酶的激活。以前,我们报道过 CD38是哺乳动物中主要的 NAD + 酶。此外,CD38基因敲除小鼠具有更高的 NAD + 水平,并且能够抵抗肥胖和代谢症候群。这里,我们表明 CD38通过改变 NAD + 水平和 sirtuin 活性来调节整体蛋白质乙酰化。另外,我们还对两种 CD38抑制剂槲皮素和芹菜素进行了表征。我们发现,药物抑制 CD38可导致细胞内 NAD + 水平升高,芹菜素处理细胞可降低 p53和 RelA-p65的整体乙酰化水平。最后,给予芹菜素可以提高 NAD + 水平,降低整体蛋白乙酰化,改善糖脂代谢平衡。我们的结果表明 CD38是通过 NAD + 依赖途径治疗代谢性疾病的一个新的药理学靶点。

Obesity is a disease that has reached epidemic proportions in developed and developing countries (13). In the U.S., >60% of the population is overweight (1,3,4). Obesity is a feature of metabolic syndrome, which includes glucose intolerance, insulin resistance, dyslipidemia, and hypertension. These pathologies are well-documented risk factors for cardiovascular disease, type 2 diabetes, and stroke (4). It is therefore imperative to envision new strategies to treat metabolic syndrome and obesity.

肥胖症是一种在发达国家和发展中国家达到流行程度的疾病(1-3)。在美国,超过60% 的人口超重(1,3,4)。肥胖是代谢症候群的一个特征,包括葡萄糖耐受不良、胰岛素抵抗、血脂异常和高血压。这些病理是心血管疾病、2型糖尿病和中风的危险因素。因此,设想治疗代谢症候群和肥胖症的新策略势在必行。

Recently, the role of NAD+ as a signaling molecule in metabolism has become a focus of intense research. It was shown that an increase in intracellular NAD+ levels in tissues protects against obesity (5,6), metabolic syndrome, and type 2 diabetes (57). Our group was the first to demonstrate that an increase in NAD+ levels protects against high-fat diet–induced obesity, liver steatosis, and metabolic syndrome (5). This concept was later expanded by others using different approaches, including inhibition of poly-ADP-ribose polymerase (PARP)1 (6) and stimulation of NAD+ synthesis (7).

The ability of NAD+ to affect metabolic diseases seems to be mediated by sirtuins (8). This family of seven NAD+-dependent protein deacetylases, particularly SIRT1, SIRT3, and SIRT6, has gained significant attention as candidates to treat metabolic syndrome and obesity (9). Sirtuins use and degrade NAD+ as part of their enzymatic reaction (8), which makes NAD+ a limiting factor for sirtuin activity (9). In particular, silent mating information regulation 2 homolog 1 (SIRT1) has been shown to deacetylate several proteins, including p53 (10), RelA/p65 (11), PGC1-α (12), and histones (13), among others. In addition, increased expression of SIRT1 (14), increased SIRT1 activity (15), and pharmacological activation of SIRT1 (16) protect mice against liver steatosis and other features of metabolic syndrome when mice are fed a high-fat diet. Given the beneficial consequences of increased SIRT1 activity, great efforts are being directed toward the development of pharmacological interventions aimed at activating SIRT1.

We previously reported that the protein CD38 is the primary NAD+ase in mammalian tissues (17). In fact, tissues of mice that lack CD38 contain higher NAD+ levels (17,18) and increased SIRT1 activity compared with wild-type mice (5,17). CD38 knockout mice are resistant to high-fat diet–induced obesity and other aspects of metabolic disease, including liver steatosis and glucose intolerance, by a mechanism that is SIRT1 dependent (5). These multiple lines of evidence suggest that pharmacological CD38 inhibition would lead to SIRT1 activation through an increase in NAD+ levels, resulting in beneficial effects on metabolic syndrome.

我们以前报道过 CD38蛋白是哺乳动物组织中主要的 NAD + 酶(17)。事实上,与野生型小鼠(5,17)相比,缺乏 CD38的小鼠组织中有更高的 NAD + 水平(17,18)和更高的 SIRT1活性。CD38基因敲除小鼠通过一种 SIRT1依赖的机制,对高脂肪饮食诱导的肥胖和代谢疾病的其他方面,包括肝脏脂肪变性和葡萄糖耐受不良有抵抗力。这些证据表明,药理性的 CD38抑制作用会通过增加 NAD + 水平导致 SIRT1的激活,从而对代谢症候群有益。

Recently, it was shown that in vitro, CD38 is inhibited by flavonoids, including quercetin (19). Flavonoids are naturally occurring compounds present in a variety of plants and fruits (20). Among them, quercetin [2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one] and apigenin [5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] have been shown to have beneficial effects against cancer (2124). In fact, apigenin and quercetin ameliorate atherosclerosis (25) and reduce inflammation (2628). However, the mechanisms of action of flavonoids remain largely unknown. We hypothesized that the effect of some flavonoids in vivo may occur through inhibition of CD38 and an increase in NAD+ levels in tissues, which lead to protection against metabolic syndrome.

近年来研究表明,类黄酮(包括槲皮素(19))对 CD38有抑制作用。类黄酮是存在于各种植物和水果中的天然化合物(20)。其中槲皮素[2-(3,4-二羟基苯基)-3,5,7-三羟基 -4h-chromen-4-one ]和芹菜素[5,7-二羟基 -2-(4-羟基苯基)-4 h-1-苯并吡喃 -4- 酮]具有良好的抗癌作用(21-24)。实际上,芹菜素和槲皮素可以改善动脉粥样硬化(25)和减少炎症(26-28)。然而,黄酮类化合物的作用机制仍然很大程度上是未知的。我们假设一些黄酮类化合物在体内的作用可能是通过抑制 CD38和增加组织中 NAD + 的水平而发生的,这些都导致对代谢症候群的保护作用。

Here, we show that CD38 expression and activity regulate cellular NAD+ levels and global acetylation of proteins, including SIRT1 substrates. We confirmed that quercetin is a CD38 inhibitor in vitro and in cells. Importantly, we demonstrate that apigenin is a novel inhibitor of CD38 in vitro and in vivo. Treatment of cells with apigenin or quercetin inhibits CD38 and promotes an increase in intracellular NAD+ levels. An increased NAD+ level decreases protein acetylation through sirtuin activation. Finally, treatment of obese mice with apigenin results in CD38 inhibition, higher NAD+ levels in the liver, and a decrease in protein acetylation. Apigenin treatment improves glucose homeostasis, glucose tolerance, and lipid metabolism in obese mice. Our results clearly demonstrate that CD38 is a novel therapeutic target for the treatment of metabolic diseases and that apigenin and quercetin as well as other CD38 inhibitors may be used to treat metabolic syndrome.

这里,我们表明 CD38的表达和活性调节细胞 NAD + 水平和整体乙酰化的蛋白质,包括 SIRT1底物。我们证实槲皮素在体外和细胞中都是 CD38抑制剂。重要的是,我们证明芹菜素在体内外都是一种新的 CD38抑制剂。芹菜素或槲皮素治疗细胞抑制 CD38和促进细胞内 NAD + 水平升高。NAD + 水平升高可通过 sirtuin 活化降低蛋白质乙酰化水平。最后,用芹菜素治疗肥胖小鼠会导致 CD38抑制、肝脏 NAD + 水平升高和蛋白质乙酰化水平降低。芹菜素治疗改善肥胖小鼠的葡萄糖稳态、葡萄糖耐量和脂质代谢。我们的结果清楚地表明 CD38是治疗代谢性疾病的一个新的治疗靶点,芹菜素和槲皮素以及其他 CD38抑制剂可用于治疗代谢症候群。

RESEARCH DESIGN AND METHODS

研究设计与方法

Reagents and antibodies.

试剂和抗体

All reagents and chemicals were from Sigma-Aldrich. Antibodies for human SIRT1, mouse SIRT1, p65, acetylated p53 (K382), phosphorylated AMP-activated protein kinase (AMPK) (Thr172), AMPK, and acetyl-lysine were from Cell Signaling Technology. Antibody against Nampt was from Bethyl Laboratories. Anti-human CD38 antibody was from R&D Biosystems, and anti-mouse CD38 was from Epitomics.

所有的试剂和化学品都来自 Sigma-Aldrich 公司。针对人类 SIRT1,小鼠 SIRT1,p65,乙酰化 p53(K382) ,磷酸化 AMP活化蛋白激酶(AMPK)(th172) ,AMPK,和乙酰赖氨酸的抗体来自细胞信号技术。抗 Nampt 抗体来自 Bethyl 实验室。抗人 CD38抗体来自 r & d Biosystems,抗鼠 CD38抗体来自 Epitomics。

Cell culture.

细胞培养

A549 cells were kept in RPMI 1640 media supplemented with 10% FBS and penicillin/streptomycin (Invitrogen). Primary CD38 wild-type and knockout mouse embryonic fibroblasts (MEFs) were kept in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, penicillin/streptomycin, and glutamine. Primary MEFs were isolated form embryos (E18) from wild-type and CD38 knockout mice. Primary MEFs were used between passages 2 and 5. 293T and hepatocellular carcinoma (Hep)G2 cells were kept in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and penicillin/streptomycin.

将 A549细胞置于10% FBS 和青霉素/链霉素(Invitrogen)的 RPMI 1640培养基中培养。将初级 CD38野生型和基因敲除小鼠胚胎成纤维细胞(MEFs)置于 Dulbecco 的改良 Eagle’s 培养基中,添加10% FBS、青霉素/链霉素和谷氨酰胺。从野生型和 CD38基因敲除小鼠胚胎(E18)中分离出初级 MEFs。在第2代和第5代之间使用了主要的 MEFs。293T 细胞和肝细胞性肝癌 G2细胞分别置于含10% FBS 和青霉素/链霉素的 Dulbecco 改良 Eagle 培养基中。

Overexpression and small interfering RNA.

过度表达和小干扰RNA

Full-length human CD38 was subcloned into a modified pIRES2–enhanced green fluorescent protein vector. For overexpression, 293T cells were transfected for 48 h with Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.

将人 CD38全长基因亚克隆到改良的 pIRES2增强型绿色荧光蛋白载体中。对于过度表达,按照制造商的说明,用 Lipofectamine 2000(Invitrogen)转染293T 细胞48h。

For CD38 knockdown experiments, probe no. 2 of a TriFECTa kit against human CD38 was used (cat. no. HSC.RNAI.N001775.12.2; IDT). A549 cells were transfected with 40 nmol small interfering RNA (siRNA) duplex using Lipofectamine 2000 according to the manufacturer’s instructions.

在 CD38基因敲除实验中,采用 TriFECTa 试剂盒探针2号对人 CD38基因进行敲除实验。没有。001775.12.2; IDT).利用 Lipofectamine 2000,根据生产商的说明,用40nmol 的 siRNA 转染 A549细胞。

Determination of CD38 activity.

CD38活性的测定

Determination of CD38 activity in cells and tissues was performed as previously described (17). In vitro CD38 activity was measured using 0.1 unit of recombinant human CD38 (R&D Systems) in 0.25 mol/L sucrose and 40 mmol/L Tris-HCl (pH 7.4). The reaction was started by addition of 0.2 mmol/L substrate. Nicotinamide 1,N6-ethenoadenine dinucleotide was used to determine NAD+ase activity and nicotinamide guanine dinucleotide to determine cyclase activity. CD38 activity was expressed as arbitrary fluorescent units per minute (AFU/min).

细胞和组织中 CD38活性的测定如前所述(17)。在0.25 mol/L 蔗糖和40 mmol/L Tris-HCl (ph7.4)条件下,用重组人 CD38(r & d 系统)的0.1单位测定 CD38的体外活性。反应以0.2 mmol/L 底物的加入为开始。用烟酰胺1,n6- 乙基腺嘌呤二核苷酸测定 NAD + 酶活性和烟酰胺鸟嘌呤二核苷酸测定环化酶活性。CD38活性以每分钟任意荧光单位(AFU/min)表示。

NAD+ quantification.

量化

NAD+ extraction and quantification was performed as previously described (17). In brief, cells were lysed by sonication in ice-cold 10% trichloroacetic acid, and then the trichloroacetic acid was extracted with two volumes of an organic phase consisting of 1,1,2-trichloro-1,2,2-trifluroethane and trioctylamine. NAD+ concentration was measured by means of an enzymatic cycling assay (18).

NAD + 萃取和定量进行了如前所述(17)。简而言之,细胞在冰冷的10% 三氯乙酸中被超声裂解,然后用含有1,1,2-三氯 -1,2,2-三氟乙烷和三辛胺的有机相萃取三氯乙酸。用酶循环法测定 NAD + 浓度(18)。

Determination of SIRT1 activity.

SIRT1活性的测定

SIRT1 activity was measured with a fluorimetric assay (Enzo) as previously described (15). One unit of human recombinant SIRT1 was incubated with different concentrations of apigenin plus 100 μmol/L Fluor-de-Lys p53 tetra peptide and 100 μmol/L NAD+. Fluor-de-Lys developer was prepared according to the manufacturer’s recommendations and added to the reactions for 1 h. Fluorescence was read with an excitation of 360 nm and emission at 460 nm.

SIRT1活性用荧光分析法(Enzo)测定,如前所述(15)。用不同浓度的芹菜素 + 100mol/l Fluor-de-Lys p53四肽 + 100mol/l NAD + 培养一个单位的重组人 SIRT1。荧光显影剂是根据制造商的建议制备的,并添加到反应1h 荧光读取与激发波长360nm 和发射波长460nm。

Mouse studies.

老鼠研究

All mice used in this study were maintained in the Mayo Clinic Animal facility. All experimental protocols were approved by the institutional animal care and use committee at Mayo Clinic (protocol no. A33209), and all studies were performed according to the methods approved in the protocol. For generation of obese mice, twelve 20-week-old C57BL/6 mice were placed on a high-fat diet (AIN-93G, modified to provide 60% of calories from fat; Dyets) ad libitum for 4 weeks. Body weight was recorded weekly. After 4 weeks of high-fat diet, mice were randomly divided in two groups and injected daily with 100 mg/kg i.p. apigenin or vehicle for 7 consecutive days while remaining on the high-fat diet. During the treatments, food intake and body weight were monitored daily. There was no difference in these parameters between groups. For the glucose tolerance experiments, mice were housed for 24 h without food, but with water ad libitum, and challenged with one dose of 1.5 g/kg i.p. dextrose. Area under the curve was calculated by the net incremental method (with baseline) and presented as incremental area under the curve.

这项研究中使用的所有小鼠都在梅奥诊所动物实验室进行维护。所有的实验方案都得到了梅奥诊所动物护理和使用委员会的批准(第号方案)。A33209) ,所有研究都按照方案中批准的方法进行。为了生成肥胖小鼠,十二只20周大的 C57BL/6小鼠被放置在高脂肪饮食(AIN-93G,经过改良从脂肪中提供60% 的卡路里; Dyets)自由饮食4周。每周记录体重。高脂饮食4周后,将小鼠随机分为两组,连续7天每天注射100mg/kg 芹菜素或载体,同时保持高脂饮食。在治疗期间,每天监测食物摄入量和体重。这些参数在不同组之间没有差异。在葡萄糖耐量实验中,小鼠不进食,但随意饮水24小时,每公斤静脉注射葡萄糖1.5 g。曲线下面积采用净增量法(带基线)计算,并以曲线下面积增量表示。

Gene expression analysis.

基因表达分析

RNA from flash-frozen liver tissue was extracted with an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized with the iSCRIP cDNA synthesis kit (BioRad) using 600 ng RNA. Quantitative RT-PCR reactions were performed using 1 µmol/L primers and LightCycler 480 SYBR Green Master (Roche) on a LightCycler 480 detection system (Roche). Calculations were performed by a comparative method (2-ΔCT) using 18S rRNA as an internal control. Primers were designed using the IDT software, and the primer sequences were as follows: long-chain acyl-CoA dehydrogenase (LCAD), forward (Fw) GGTGGAAAACGGAATGAAAGG, reverse (Rv) GGCAATCGGACATCTTCAAAG; medium-chain acyl-CoA dehydrogenase (MCAD), Fw TGTTAATCGGTGAAGGAGCAG, Rv CTATCCAGGGCATACTTCGTG; CPT1a, Fw AGACAAGAACCCCAACATCC, Rv CAAAGGTGTCAAATGGGAAGG; and 18S, Fw CGGCTACCACATCCAAGGAA, Rv GCTGGAATTACCGCGGCT.

根据制造商的说明,用 RNeasy Mini Kit (Qiagen)从速冻肝组织中提取 RNA。用600ng 的 RNA 合成 iscripcdna 合成试剂盒(BioRad)合成 cDNA。用1 mol/l 引物和 LightCycler 480 SYBR Green Master (Roche)在 LightCycler 480检测系统上进行定量 RT-PCR 反应。以18S rRNA 作为内部控制,由比照法进行计算。用 IDT 软件设计引物,得到的引物序列为: 长链酰基辅酶 a 脱氢酶(LCAD) ,前向(Fw) GGTGGAAAACGGAATGAAAGG,反向(Rv) ggcaatcggcttcaaag;中链酰基辅酶 a 脱氢酶(MCAD) ,Fw TGTTAATCGGTGAAGGAGCAG,Rv ctatccagctctcgcgtg,CPT1a,Fw AGACAAGAACCCCAACATCC,Rv caaagtgtgtcaatgggaagg,Fw CGGCTACCACATCCAAGGAA,Rv gctggaatccgggggggct。

Lipid treatment.

脂质治疗

Cells were incubated with a mixture of oleic acid and palmitic acid in a 2:1 ratio in culture media supplemented with 1% fatty acid–free BSA (Sigma-Aldrich). Lipids were used at concentrations shown to induce steatosis but not apoptosis (15). Incubations with lipids were performed for 16–24 h.

细胞与油酸和棕榈酸按2:1的比例在添加1% 无脂肪酸 BSA (Sigma-Aldrich)的培养基中孵育。脂类被用于诱导脂肪变性而不是细胞凋亡的浓度(15)。用脂质进行培养16-24小时。

Statistics.

统计数字

Values are presented as means ± SEM of three to five experiments unless otherwise indicated. The significance of differences between means was assessed by ANOVA or two-tailed Student ttest. A P value <0.05 was considered significant.

除非另有说明,否则数值以三到五次实验的平均值表示。采用方差分析和双尾学生 t 检验两种方法比较两种方法的差异。P 值 < 0.05被认为是显著的。

RESULTS

结果

CD38 overexpression decreases NAD+ and promotes protein acetylation.

CD38过表达降低 NAD + ,促进蛋白质乙酰化

We have previously shown that CD38 is the primary NAD+ase in mammalian tissues (17). CD38-deficient mice have increased NAD+ levels in multiple tissues (5,17). To further characterize the role of CD38 in the regulation of NAD+-dependent cellular events, we studied the effect of CD38 manipulation in cells. We found that cells that overexpress CD38 show a significant increase in NAD+ase and ADP ribosyl cyclase activities (Fig. 1A and B) and a consistent decrease in intracellular NAD+ levels (Fig. 1C). Interestingly, we found that overexpression of CD38 also led to an increase in global protein acetylation (Fig. 1E). The pattern of acetylated proteins was analyzed by plotting an intensity profile of the lanes in the Western blots (Fig. 1F). It is worth noting that CD38 overexpression promotes changes in the level of acetylation of several proteins (red arrows in Fig. 1E and F), while other bands remain unchanged. This is consistent with the fact that only sirtuin deacetylases depend on NAD+ for their activity (8); histone deacetylases of classes I and II have a different enzymatic mechanism that does not require NAD+ (29).

我们以前已经证明 CD38是哺乳动物组织中主要的 NAD + 酶(17)。Cd38缺陷小鼠多种组织中 NAD + 水平升高(5,17)。为了进一步研究 CD38在 NAD + 依赖性细胞事件调控中的作用,我们研究了 CD38对细胞的调控作用。我们发现过度表达 CD38的细胞 NAD + 酶和 ADP 核糖环化酶活性显著增加(图1A 和 b) ,细胞内 NAD + 水平持续下降(图1C)。有趣的是,我们发现过度表达 CD38也导致整体蛋白乙酰化的增加(图1E)。乙酰化蛋白的模式分析通过绘制一个强度剖面的车道在西部 blots (图1F)。值得注意的是,CD38过度表达促进了几种蛋白质(图1E 和 f 中的红色箭头)乙酰化水平的变化,而其他条带保持不变。这与只有 sirtuin 去乙酰化酶的活性依赖 NAD + 的事实是一致的(8) ; i 类和 II 类组蛋白去乙酰化酶有不同的酶机制,不需要 NAD + (29)。

FIG. 1.

FIG. 1. 图1

CD38 overexpression decreases NAD+ and promotes protein acetylation in cells. 293T cells were transfected with empty vector or human CD38-coding vector. After 48 h, cells were harvested, and NAD+ase activity (A), ADP-ribosyl-cyclase activity (B), and total intracellular NAD+ levels (C) were measured in cell lysates. *P < 0.05, n = 3. D: Western blot for CD38 in 293T cells transfected with empty vector or with human CD38. E: Western blot showing total protein acetylation in cells transfected with empty vector or with human CD38. Anti–acetylated (Ac) lysine antibody was used. Red arrows highlight the main bands that showed variations in intensity. F: Intensity profile of the Western blot shown in E. Western blots were scanned and intensity profile was obtained using ImageJ. Red arrows correspond with intensity of the same bands shown in E.

CD38过表达降低细胞内 NAD + ,促进蛋白质乙酰化。用空载体或人 cd38编码载体转染293T 细胞。48h 后取细胞,测定细胞裂解液中 NAD + 酶活性(a)、 adp 核糖周期酶活性(b)和总的细胞内 NAD + 水平(c)。* p < 0.05,n = 3.D: 空载体转染293T 细胞或人 CD38转染293T 细胞后进行 CD38蛋白印迹检测。E: 蛋白质印迹显示空载体转染细胞或人 CD38转染细胞总蛋白乙酰化。抗乙酰化赖氨酸抗体。红色箭头突出显示了在强度变化的主要乐队。F: 在 e. Western blots 显示的强度轮廓扫描和强度轮廓获得使用 ImageJ。红色箭头表示 e 中同一条带的强度。

CD38 downregulation increases NAD+ and decreases protein acetylation.

CD38下调提高 NAD + ,降低蛋白质乙酰化水平

Next, we examined whether CD38 downregulation promotes the opposite effect on cellular NAD+ levels and global protein acetylation. This is of key relevance, since we (5) and other investigators (6,7) have shown that an increase in intracellular NAD+ levels protects against metabolic diseases and aging. We transfected cells with control or CD38 siRNA. Cells treated with CD38 siRNA had decreased NAD+ase and ADP ribosyl-cyclase activities (Fig. 2A and B) and a significant increase in intracellular NAD+ levels (Fig. 2C), consistent with the diminished CD38 NAD+ase activity. Moreover, the increase in NAD+ levels was accompanied by a decrease in global protein acetylation (Fig. 2D and E). Finally, we isolated primary MEFs from wild-type and CD38 knockout mice and measured NAD+ levels and protein acetylation. We found that CD38 knockout MEFs have increased NAD+ levels (Fig. 2F) and decreased global protein acetylation (Fig. 2G). We also analyzed p65/RelA acetylation at K310, a site that is an accepted target for cellular SIRT1 activity (7,11). We found that CD38 knockout MEFs show no detectable p65/RelA (K310) acetylation compared with wild-type cells, despite having similar total p65/RelA protein (Fig. 2H) and similar SIRT1 levels. This indicates that SIRT1 activity is increased in the CD38 knockout MEFs.

接下来,我们研究了 CD38下调是否促进了细胞 NAD + 水平和整体蛋白质乙酰化的相反效应。这是关键的相关性,因为我们(5)和其他研究人员(6,7)已经表明,增加细胞内 NAD + 水平可以防止代谢性疾病和衰老。我们用对照或 CD38 siRNA 转染细胞。CD38 siRNA 处理细胞后,NAD + 酶和 ADP 核糖环化酶活性下降(图2A 和 b) ,胞内 NAD + 水平显著升高(图2C) ,与 CD38 NAD + 酶活性下降相一致。此外,NAD + 水平的增加伴随着整体蛋白乙酰化水平的下降(图2D 和 e)。最后,我们从野生型和 CD38基因敲除小鼠中分离出初级 MEFs,并测定 NAD + 水平和蛋白质乙酰化水平。我们发现 CD38基因敲除 MEFs 可增加 NAD + 水平(图2F) ,降低整体蛋白乙酰化水平(图2G)。我们还分析了 K310位点的 p65/RelA 乙酰化,该位点是细胞 SIRT1活性的公认靶点(7,11)。我们发现,与野生型细胞相比,CD38基因敲除 MEFs 没有检测到 p65/RelA (K310)乙酰化,尽管总 p65/RelA 蛋白(图2H)和 SIRT1水平相似。这表明在 CD38基因敲除 MEFs 中 SIRT1活性增加。

FIG. 2.

FIG. 2. 图2

CD38 downregulation increases NAD+ and decreases protein acetylation in cells. A549 cells were transfected with a scrambled siRNA (control siRNA) or human CD38 siRNA. After 72 h, cells were harvested and NAD+ase activity (A), ADP-ribosyl-cyclase activity (B), and total intracellular NAD+ levels (C) were measured from cell lysates. *P < 0.05, n = 3. D: Western blot showing total protein acetylation in cells transfected with control siRNA or with human CD38 siRNA. Anti–acetylated (Ac) lysine antibody was used. Red arrows highlight the main bands that showed variations in intensity. E: Intensity profile of the Western blot shown in D. Western blots were scanned and intensity profile was obtained using Image J. Red arrows correspond with intensity of the same bands showed in DFH: Primary MEFs were purified and cultured from wild-type (WT) and CD38 knockout (KO) mice. F: Intracellular NAD+ levels (*P < 0.05, n = 3). G: Western blot from wild-type and CD38 knockout MEFs showing total protein acetylation in these cells. H: Representative Western blot in wild-type and CD38 knockout MEFs. Acetylated RelA/p65 (K310), total RelA/p65, SIRT1, CD38, and actin antibodies were used.

CD38的下调使细胞中 NAD + 增加,蛋白质乙酰化降低。用干扰 siRNA (control siRNA)或 CD38 siRNA 转染 A549细胞。72h 后,取细胞,测定细胞内 NAD + 酶活性(a)、 adp 核糖环化酶活性(b)和总的细胞内 NAD + 水平(c)。* p < 0.05,n = 3.D: 蛋白质印迹显示总蛋白乙酰化的细胞转染对照 siRNA 或人 CD38小干扰 rna。抗乙酰化赖氨酸抗体。红色箭头突出显示了在强度变化的主要乐队。E: 扫描 d. Western blot 的强度图谱,用图像 j. Red 箭头对应 d. f-h 的同一条带的强度,得到强度图谱: 从野生型(WT)和 CD38基因敲除(KO)小鼠中纯化和培养初级 MEFs。F: 细胞内 NAD + 水平(* p < 0.05,n = 3)。野生型蛋白和 CD38基因敲除 MEFs 的蛋白质印迹显示这些细胞的总蛋白乙酰化。H: 野生型和 CD38基因敲除 MEFs 的典型 Western blot。应用乙酰化 RelA/p65(K310)、 RelA/p65总量、 SIRT1、 CD38和肌动蛋白抗体。

Apigenin and quercetin inhibit CD38 activity in vitro.

芹菜素和槲皮素体外抑制 CD38活性的研究

By use of high-throughput analysis to search for inhibitors of CD38, we found that several flavonoids, including quercetin and apigenin, inhibit CD38 in vitro. The complete screen will be published elsewhere. Recently, Kellenberger et al. (19) also published a list of flavonoids that act as CD38 inhibitors in vitro, many of which were also confirmed by our analysis. Quercetin was one of the compounds found by Kellenberger et al. (19) to inhibit CD38 in vitro. Apigenin, however, was demonstrated to be a novel CD38 inhibitor. We proceeded to further characterize these compounds in vitro and in cells.

通过高通量分析寻找 CD38抑制剂,我们发现几种黄酮类化合物,包括槲皮素和芹菜素,在体外对 CD38有抑制作用。完整的屏幕将在其他地方发布。最近,Kellenberger 等人(19)也发表了一份类黄酮的清单,在体外作为 CD38抑制剂,其中许多也被我们的分析证实。槲皮素是 Kellenberger 等人(19)发现的抑制 CD38的化合物之一。芹菜素是一种新型的 CD38抑制剂。我们进一步在体外和细胞中对这些化合物进行了表征。

The effect of apigenin and quercetin on CD38 activity in vitro was studied using the soluble ectodomain of human CD38 (17). We found that apigenin (Fig. 3A) inhibits in vitro CD38 activity with a half-maximal inhibitory concentration (IC50) of 10.3 ± 2.4 μmol/L for the NAD+ase activity and an IC50 of 12.8 ± 1.6 μmol/L for the ADP-ribosyl-cyclase activity (Fig. 3B and C). In vitro, quercetin (Fig. 3D) inhibits CD38 NAD+ase activity with an IC50 of 13.8 ± 2.1 μmol/L (Fig. 3E) and ADP-ribosyl-cyclase activity with an IC50 of 15.6 ± 3.5 μmol/L (Fig. 3F).

利用人 CD38(17)可溶性胞外结构域研究了芹菜素和槲皮素对 CD38活性的影响。我们发现芹菜素(图3A)抑制体外 CD38活性,半最大抑制浓度(IC50)为10.3 ± 2.4 mol/l,抑制 NAD + 酶活性的 IC50为12.8 ± 1.6 mol/l (图3B 和 c)。在体外,槲皮素抑制 cd38nad + 酶活性,IC50为13.8 ± 2.1 mol/l (图3E) ,adp-ribosyl-cyase 活性为15.6 ± 3.5 mol/l (图3F)。

FIG. 3.

FIG. 3. 图3

The flavonoids apigenin and quercetin inhibit CD38 activity in vitro. A: Chemical structure of apigenin. B and C: In vitro NAD+ase (B) and ADP-ribosyl-cyclase (C) activity using human recombinant-purified CD38 and different concentrations of apigenin. D: Chemical structure of quercetin. E and F: In vitro CD38 NAD+ase activity (E) and ADP-ribosyl-cyclase activity (F) using human recombinant-purified CD38 and different concentrations of apigenin. In all the measurements, compounds were used in the 0.5–100 μmol/L concentration range. Each measurement was done by triplicate. Data points were fitted to a standard competitive inhibition curve using a nonlinear regression program (GraphPad Prism) to yield the IC50 value.

黄酮类芹菜素和槲皮素体外抑制 CD38的活性。答: 芹菜素的化学结构。B 和 c: 利用人重组纯化 CD38和不同浓度的芹菜素对 NAD + 酶(b)和 adp 核糖环化酶(c)的体外活性进行研究。槲皮素的化学结构。E 和 f: 利用重组人 CD38和不同浓度的芹菜素进行 CD38 NAD + 酶活性(e)和 adp 核糖基环化酶活性(f)的体外测定。在所有的测量中,化合物均在0.5-100 mol/l 的浓度范围内使用。每次测量用一式三份。数据点被用一个竞争性抑制剂/非线性回归程序(GraphPad Prism)拟合到一个标准曲线上,从而得到 IC50值。

CD38 inhibition by quercetin and apigenin increases NAD+ levels in cells.

槲皮素和芹菜素抑制 CD38细胞增加 NAD + 水平

Although several flavonoids can inhibit purified recombinant CD38 in vitro (19), it is not known what effects these compounds have in cells. First, we measured the effect of quercetin on endogenous cellular CD38 activity. Inhibition of CD38 activity by quercetin in cells (IC50 = 16.4 ± 1.8 μmol/L) resembles the effect on the recombinant protein (Fig. 4A). Furthermore, we found that quercetin promotes an increase in intracellular NAD+ in a dose-dependent manner (Fig. 4B). To further confirm this effect, we incubated cells in PBS and measured intracellular NAD+levels over time. We found that in untreated cells, NAD+ levels decrease with time (Fig. 4C), probably as a result of the removal of NAD+ precursors from the culture media. However, when the cells were treated with quercetin, NAD+ levels were stable over time, suggesting that inhibition of CD38 is enough to maintain intracellular NAD+ levels in the absence of NAD+precursors. Finally, in order to confirm that the effect of quercetin on cellular NAD+ levels was dependent on CD38, we measured NAD+ after incubation with quercetin in wild-type and CD38 knockout MEFs. We found that quercetin promotes an increase in NAD+ in the wild-type MEFs but does not further increase NAD+ levels in CD38 knockout MEFs (Fig. 4D), indicating that the effect of quercetin on NAD+ levels is CD38 dependent.

虽然几种黄酮类化合物在体外能抑制纯化的重组 CD38(19) ,但尚不清楚这些化合物在细胞中具有什么作用。首先,我们测定了槲皮素对内源性细胞 CD38活性的影响。槲皮素对细胞内 CD38活性的抑制作用(IC50 = 16.4 ± 1.8 mol/l)类似于对重组蛋白的抑制作用(图4A)。此外,我们发现槲皮素以剂量依赖的方式促进细胞内 NAD + 的增加(图4B)。为了进一步证实这一效应,我们在 PBS 中培养细胞,并随时间测量细胞内 NAD + 水平。我们发现,在未经处理的细胞中,NAD + 水平随时间而降低(图4C) ,这可能是由于培养基中去除了 NAD + 前体的结果。然而,当用槲皮素处理细胞时,NAD + 水平随着时间的推移是稳定的,这表明 CD38的抑制足以在没有 NAD + 前体的情况下维持细胞内 NAD + 水平。最后,为了证实槲皮素对细胞 NAD + 水平的影响是依赖于 CD38,我们测定了槲皮素在野生型和 CD38敲除 MEFs 孵育后的 NAD + 。我们发现槲皮素促进了野生型 MEFs 中 NAD + 的增加,但不进一步增加 CD38基因敲除 MEFs 中 NAD + 的水平(图4D) ,这表明槲皮素对 NAD + 水平的影响是依赖于 CD38的。

FIG. 4.

FIG. 4. 图4

CD38 inhibition by quercetin increases NAD+ levels in cells. A: Endogenous CD38 NAD+ase activity was measured in protein lysates from A549 cells. Quercetin was used in the 0.5–100 μmol/L concentration range. Each measurement was done in triplicate. Data points were fitted to a standard competitive inhibition curve using a nonlinear regression program (GraphPad Prism) to yield the IC50 value. B: NAD+ dose-response curve in A549 cells treated with quercetin. Cells were incubated with quercetin for 6 h before NAD+ extraction. *P < 0.05, n = 3. C: NAD+ time course in A549 cells incubated in PBS (●) or in PBS plus quercetin (50 μmol/L) (■). *P < 0.05, n = 3. D: Intracellular NAD+ levels in wild-type (WT) and CD38 knockout (KO) MEFs treated with vehicle (control) (■) or with quercetin (50 μmol/L) (□) for 6 h. NAD+ levels were expressed as percent of change with respect to the control for both cells. Total NAD+ levels were significantly higher in CD38 knockout MEFs. (See Fig. 2F.) *P < 0.05, n = 3.

槲皮素抑制 CD38增加细胞内 NAD + 水平。答: 测定 A549细胞蛋白裂解物中内源性 CD38 NAD + 酶活性。槲皮素在0.5ー100 mol/l 浓度范围内使用。每次测量一式三份。数据点被用一个竞争性抑制剂/非线性回归程序(GraphPad Prism)拟合到一个标准曲线上,从而得到 IC50值。槲皮素对 A549细胞 NAD + 剂量-反应曲线的影响。细胞与槲皮素孵育6h 后,提取 NAD + 。* p < 0.05,n = 3.C: NAD + 时间进程: A549细胞在 PBS 中孵育(●)或在 PBS 中加槲皮素(50 mol/l)(■)。* p < 0.05,n = 3.D: 用载体(对照)(■)或槲皮素(50 mol/l)(□)处理野生型(WT)和 CD38基因敲除(KO) MEFs 6 h 后,NAD + 水平均表达为对照细胞的变化百分率。CD38基因敲除的 MEFs 中 NAD + 总水平显著升高。(见图2F。)* p < 0.05,n = 3.

Apigenin also inhibits CD38 activity in cells (Fig. 5A). In fact, inhibition of cellular CD38 was very similar to that observed with the purified recombinant protein (IC50 = 14.8 ± 2.2 μmol/L in cells and 10.3 ± 2.4 μmol/L in vitro). Apigenin treatment increased NAD+ levels in cells in a dose-dependent manner (Fig. 5B) and protected against NAD+ depletion when cells were incubated in PBS (Fig. 5C). Furthermore, treatment of CD38 knockout MEFs with apigenin had no effect on NAD+ levels (Fig. 5D), indicating that the effect of apigenin on NAD+ levels is mediated by CD38. Interestingly, we found that treatment of wild-type MEFs with apigenin decreased acetylation of RelA/p65 (Fig. 5E). However, in the CD38 knockout MEFs, RelA/p65 acetylation levels were undetectable in the control, and therefore we could not determine the effect of apigenin (Fig. 5E). These results are consistent with the effect of apigenin in intracellular NAD+ levels in these cells (Fig. 5D). Quercetin has been shown to activate SIRT1 in vitro (30), suggesting that it may activate SIRT1 activity by two different mechanisms. To rule out a possible direct effect of apigenin on SIRT1 activity, we measured in vitro recombinant SIRT1 activity in the presence of different concentrations of apigenin. We observed that apigenin does not activate SIRT1 directly (Fig. 5F). Combined, these results clearly demonstrate that apigenin inhibits CD38 in cells and by doing so promotes an increase in NAD+ levels that stimulates NAD+-dependent deacetylases.

芹菜素还抑制细胞中 CD38的活性(图5A)。实际上,CD38对细胞的抑制作用与纯化的重组蛋白相似(IC50 = 14.8 ± 2.2 mol/l,体外为10.3 ± 2.4 mol/l)。芹菜素处理以剂量依赖的方式增加细胞中 NAD + 水平(图5B) ,并在 PBS 中保护细胞免受 NAD + 损耗(图5C)。此外,用芹菜素处理 CD38基因敲除 MEFs 对 NAD + 水平没有影响(图5D) ,表明芹菜素对 NAD + 水平的影响是通过 CD38介导的。有趣的是,我们发现用芹菜素处理野生型 MEFs 可降低 RelA/p65的乙酰化水平(图5E)。然而,在 CD38基因敲除的 MEFs 中,RelA/p65乙酰化水平在对照组中检测不到,因此我们不能确定芹菜素的影响(图5E)。这些结果与芹菜素对这些细胞内 NAD + 水平的影响是一致的(图5D)。槲皮素已被证明在体外激活 SIRT1(30) ,这表明它可能通过两种不同的机制激活 SIRT1活性。为了排除芹菜素对 SIRT1活性的直接影响,我们在不同浓度的芹菜素存在下测定了 SIRT1的体外重组活性。我们观察到芹菜素不直接激活 SIRT1(图5F)。综上所述,这些结果清楚地表明芹菜素抑制细胞中的 CD38并通过这样做促进 NAD + 水平的增加,从而刺激 NAD + 依赖的去乙酰化酶。

FIG. 5.

FIG. 5. 图5

CD38 inhibition by apigenin increases NAD+ and decreases protein acetylation in cells. A: Endogenous CD38 NAD+ase activity was measured in protein lysates from A549 cells. Apigenin was used in the 2.5–100 μmol/L concentration range. Each measurement was done in triplicate. Data points were fitted to a standard competitive inhibition curve using a nonlinear regression program (GraphPad Prism) to yield the IC50 value. B: NAD+ dose-response curve in A549 cells treated with apigenin. Cells were incubated with apigenin for 6 h before NAD+ extraction. C: NAD+ time course in A549 cells incubated in PBS (●) or in PBS plus 25 μmol/L apigenin (■) (*P< 0.05, n = 3). D: Intracellular NAD+ levels in wild-type (WT) and CD38 knockout (KO) MEFs treated with vehicle (control) (■) or with apigenin (25 μmol/L) (□) for 6 h. NAD+ levels were expressed as percent change with respect to the control for both cells. Total NAD+ levels were significantly higher in CD38 knockout MEFs. (See Fig. 2F.) *P < 0.05, n = 3. E: Western blot of wild-type and CD38 knockout MEFs that were treated with vehicle or apigenin as described in D. Samples were immunoblotted for acetylated (Ac)-p65 (K310), total p65, CD38, SIRT1, and actin. F: In vitro SIRT1 activity using recombinant-purified human SIRT1. SIRT1 activity was measured in the presence of different concentrations of apigenin (0–100 μmol/L). Activity was determined in the linear portion of the reaction.

芹菜素抑制 CD38增加细胞 NAD + ,减少蛋白质乙酰化。答: 测定 A549细胞蛋白裂解物中内源性 CD38 NAD + 酶活性。芹菜素在2.5ー100 mol/l 的浓度范围内使用。每次测量一式三份。数据点被用一个竞争性抑制剂/非线性回归程序(GraphPad Prism)拟合到一个标准曲线上,从而得到 IC50值。芹菜素对 A549细胞 NAD + 剂量-反应曲线的影响。在 NAD + 提取前,用芹菜素孵育细胞6h。C: NAD + 在 PBS (●)或 PBS + 25 mol/l 芹菜素(■)培养的 A549细胞中的时程(* p < 0.05,n = 3)。D: 用载体(对照)(■)或芹菜素(25mol/l)(□)处理野生型(WT)和 CD38基因敲除(KO) MEFs 6小时,细胞内 NAD + 水平均表达为对照细胞的百分比变化。CD38基因敲除的 MEFs 中 NAD + 总水平显著升高。(见图2F。)* p < 0.05,n = 3.E: 用载体或芹菜素处理过的野生型和 CD38基因敲除 MEFs 的 Western blot 检测样品中乙酰化(Ac)-p65(K310)、总 p65、 CD38、 SIRT1和肌动蛋白。F: 用重组纯化的人 SIRT1进行体外 SIRT1活性测定。在不同浓度的芹菜素(0-100 mol/l)存在下测定 SIRT1的活性。活性测定在反应的线性部分。

CD38 inhibition by apigenin increases NAD+ and decreases protein acetylation in mice.

芹菜素抑制 CD38增加小鼠 NAD + ,降低蛋白质乙酰化

Apigenin and quercetin have been shown to ameliorate atherosclerosis in mice (25) and to protect against lipid accumulation in cells (25). However, the mechanism of action has not been elucidated. In fact, while most flavonoids activate AMPK, which could explain some of the metabolic effects observed, apigenin is a very poor AMPK activator (25).

芹菜素和槲皮素已被证明可以改善小鼠动脉粥样硬化(25)和防止脂质在细胞中积累(25)。然而,其作用机制尚未阐明。事实上,虽然大多数黄酮类化合物都能激活 AMPK,这可以解释所观察到的一些代谢效应,但芹菜素是一种非常差的 AMPK 激活剂(25)。

Based on the results obtained in cells, we tested whether apigenin inhibits CD38 in vivo using a model of high-fat diet–induced obesity (5,15). We fed adult mice a high-fat diet for 4 weeks. After, we divided the mice randomly in two groups. Each group was injected daily with apigenin (100 mg/kg) or vehicle (DMSO) for a week. We found that mice that had been injected with apigenin had decreased CD38 activity in the liver (Fig. 6A), which correlated with an increase in hepatic NAD+ levels compared with control mice (Fig. 6B). We then examined whether the apigenin treatment had an effect on the level of expression of several proteins involved in NAD+ metabolism. As shown in Fig. 6C, we found no significant differences in the expression of CD38, SIRT1, or Nampt, the primary regulator of the NAD+ salvage pathway. Furthermore, we did not see any changes in phosphorylation or total levels of AMPK (Fig. 6C). However, when we analyzed the liver samples using an acetyl-lysine antibody, we found that the apigenin treatment resulted in a statistically significant decrease in global acetylation of proteins (Fig. 6Dand E).

基于细胞实验的结果,我们用高脂饮食诱导的肥胖模型(5,15)检测芹菜素是否在体内抑制 CD38。我们用高脂肪饲料喂养成年小鼠4周。之后,我们将老鼠随机分成两组。每组每日注射芹菜素(100毫克/千克)或车用二甲基亚砜(DMSO)一周。我们发现注射芹菜素的小鼠肝脏 CD38活性降低(图6A) ,这与肝脏 NAD + 水平的升高相关(图6B)。然后,我们研究了芹菜素处理是否对几种参与 NAD + 代谢的蛋白质的表达水平有影响。如图6 c 所示,我们发现 CD38、 SIRT1或 Nampt 的表达没有显著差异,而 Nampt 是 NAD + 挽救途径的主要调节因子。此外,我们没有看到任何改变磷酸化或总水平的 AMPK (图6C)。然而,当我们使用乙酰赖氨酸抗体分析肝脏样本时,我们发现芹菜素处理导致了蛋白质整体乙酰化的显著降低(图6D 和 e)。

FIG. 6.

FIG. 6. 图6

CD38 inhibition by apigenin increases NAD+ and decreases protein acetylation in vivo. AE: Mice were fed a high-fat diet (HFD) for 4 weeks and then split in two groups. One group was injected with apigenin (100 mg/kg i.p.) and the other with vehicle (DMSO) with a single dose daily for 1 week. A: CD38 activity in the liver at the end of the treatment with apigenin (*P < 0.05, n = 6 animals per group). B: NAD+ levels in the liver after the treatment (*P < 0.05, n = 6 animals per group). C: At the end of the treatment, liver samples were obtained and immunoblotted for CD38, phosphorylated (p)-AMPK (Thr172), AMPK, SIRT1, Nampt, and actin. D: Liver samples were immunoblotted for global acetylation of proteins using an anti–acetylated (Ac) lysine (Lys) antibody. Western blots were scanned and an intensity profile was obtained using Image J. The area under the curve is shown. (*P < 0.05, n = 3 per group.) F: Human HepG2 cells were incubated with vehicle (DMSO), apigenin (25 μmol/L), or apigenin plus EX527 (10 μmol/L) for 6 h. Cell lysates were immunoblotted for acetylated lysine to determine total protein acetylation levels (left panel). The intensity profile of the Western blot was obtained using Image J (right panel).

芹菜素抑制 CD38增加 NAD + ,减少蛋白质乙酰化。A-e: 小鼠喂食高脂肪饲料(HFD)4周,然后分成两组。一组每天注射芹菜素100mg/kg,另一组每天注射载体(DMSO)1次,为期1周。A: 芹菜素治疗结束时肝脏 CD38活性(* p < 0.05,n = 6)。B: 治疗后肝脏中 NAD + 水平(* p < 0.05,n = 6)。C: 在治疗结束时,获得肝脏样本,并对 CD38、磷酸化(p)-AMPK (Thr172)、 AMPK、 SIRT1、 Nampt 和 actin 进行免疫印迹。D: 用抗乙酰化赖氨酸(赖氨酸)抗体对肝样品进行免疫印迹,以进行蛋白质的整体乙酰化。扫描蛋白斑点,并获得一个强度剖面使用图像 j。曲线下面的面积如图所示。(* p < 0.05,n = 3)F: 将人 HepG2细胞与载体(vehicle,DMSO)、芹菜素(25 mol/l)或芹菜素加 EX527(10 mol/l)共孵育6h。应用图像 j (右面板)获得蛋白印迹的强度分布图。

To determine the relevance of SIRT1 in the deacetylation of proteins triggered by apigenin treatment, we used human HepG2 cells: a well-accepted cellular model for studying hepatic cellular signaling (15,25,31). We found that treatment with apigenin decreases total protein acetylation—an effect that is lost in the presence of the SIRT1 inhibitor EX527 (Fig. 6F). Furthermore, treatment of HepG2 cells with apigenin decreased acetylation of p53 at K382 and also of RelA/p65 at K310: sites that are deacetylated by SIRT1 (Supplementary Fig. 1). This effect was reverted when cells were also incubated with the sirtuin inhibitor nicotinamide (Supplementary Fig. 1). Taken together, these results show that apigenin inhibits CD38 in vivo and is associated with increased NAD+ and decreased protein acetylation, likely through the activation of SIRT1.

为了确定 SIRT1在芹菜素治疗引发的蛋白质脱乙酰化中的相关性,我们使用人肝细胞 HepG2细胞(15,25,31) ,这是一个公认的研究肝细胞信号传导的细胞模型。我们发现,芹菜素处理降低了总蛋白乙酰化作用——这种作用在 SIRT1抑制剂 EX527的存在下丧失(图6F)。此外,用芹菜素处理 HepG2细胞,可降低 p53在 K382位点的乙酰化程度,也可降低 RelA/p65在 K310位点的乙酰化程度。当细胞也与去乙酰化酶抑制剂烟酰胺一起孵育时,这种效应被逆转(补充图1)。综上所述,这些结果表明芹菜素在体内抑制 CD38,并且可能通过 SIRT1的激活而与 NAD + 的增加和蛋白质乙酰化的降低有关。

CD38 inhibition by apigenin improves glucose homeostasis in vivo and promotes fatty acid oxidation in the liver.

芹菜素抑制 CD38可改善体内葡萄糖稳态,促进肝脏脂肪酸氧化

Finally, we tested whether apigenin protects against high-fat diet–induced hyperglycemia. We found that after 4 days of treatment, the mice treated with apigenin had significantly lower blood glucose levels compared with the control mice (Fig. 7A). Fasting blood glucose levels were also significantly lower after 1 week of treatment with apigenin (Fig. 7B). Moreover, we found that 1 week of treatment with apigenin was enough to improve glucose homeostasis in the mice (Fig. 7C and D). SIRT1 activation promotes fatty acid oxidation in the liver by inducing the expression of several enzymes involved in fatty acid and cholesterol metabolism (32). In fact, SIRT1 activation in the liver prevents liver steatosis (5,14,15). Mice treated with apigenin had increased expression of the enzymes MCAD and LCAD in the liver (Fig. 8A), suggesting that apigenin treatment enhanced fatty acid oxidation. We confirmed these data by measuring total triglyceride content in the liver. Indeed, we found that the mice treated with apigenin had lower triglyceride levels in the liver compared with control mice (Fig. 8B), showing that apigenin promotes hepatic lipid oxidation. To further confirm this finding, we measured lipid accumulation in cells, using an in vitro model of hepatic steatosis (15). We found that apigenin decreases lipid accumulation in cells and that this effect was completely blocked by the SIRT1 inhibitor EX527 (Fig. 8C). Together, these results show that inhibition of CD38 by apigenin, and perhaps by other flavonoids, constitutes a pharmacological approach to activate sirtuins and treat high-fat diet–induced metabolic disorders. Furthermore, our results point to CD38 as a novel pharmacological target to treat metabolic diseases.

最后,我们测试了芹菜素是否对高脂饮食诱导的高血糖有保护作用。我们发现经过4天的治疗后,用芹菜素治疗的小鼠的血糖水平明显低于对照组小鼠(图7A)。用芹菜素治疗1周后,空腹血糖水平也明显降低(图7B)。此外,我们发现芹菜素处理1周足以改善小鼠的葡萄糖稳态(图7C 和 d)。SIRT1激活通过诱导与脂肪酸和胆固醇代谢有关的几种酶的表达促进肝脏中的脂肪酸氧化(32)。事实上,SIRT1在肝脏的激活可以防止肝脏脂肪变性(5,14,15)。用芹菜素处理的小鼠肝脏中 MCAD 和 LCAD 的表达增加,提示芹菜素处理增强了脂肪酸的氧化。我们通过测量肝脏中总甘油三酯的含量来确认这些数据。实际上,我们发现用芹菜素治疗的小鼠肝脏甘油三酯水平低于对照组小鼠(图8B) ,表明芹菜素促进肝脏脂质氧化。为了进一步证实这一发现,我们使用肝脏脂肪变性的体外模型(15)测量细胞中的脂质积累。我们发现芹菜素可以减少细胞内脂质的积累,SIRT1抑制剂 EX527可以完全阻断这种作用(图8C)。总之,这些结果表明,芹菜素抑制 CD38,或许还有其他类黄酮,构成了激活去乙酰化酶和治疗高脂饮食引起的代谢紊乱的药理学途径。此外,我们的研究结果指出 CD38作为一个新的药理作用靶点治疗代谢性疾病。

FIG. 7.

FIG. 7. 图7

CD38 inhibition by apigenin improves glucose homeostasis in vivo and improves lipid metabolism in the liver. Mice were fed a high-fat diet (HFD) for 4 weeks and then split in two groups. One group was injected with apigenin (100 mg/kg i.p.) and the other with vehicle (DMSO) with a single dose daily for 1 week. A: Blood glucose levels were measured during the week of apigenin treatment in ad libitum feeding conditions (*P < 0.05, n = 6 per group). ●, HFD; ■, HFD plus apigenin. B: Blood glucose levels were measured after 24 h of fasting on day 7 of treatment with apigenin (*P < 0.05, n = 6 per group). C: Glucose tolerance test in mice after 7 days of treatment with apigenin (■) or vehicle (●) (*P < 0.05, n = 6 per group). D: Area under the curve (AUC) calculated for the glucose tolerance test shown in C. ■, HFD; □, HFD plus apigenin.

芹菜素抑制 CD38可改善体内葡萄糖稳态,改善肝脏的脂质代谢。小鼠被喂以高脂肪饮食(HFD)4周,然后分成两组。一组注射芹菜素(100mg/kg i.p.) ,另一组注射载体(DMSO) ,每日一次,为期1周。答: 在自由采食条件下,在芹菜素治疗的一周内测定血糖水平(* p < 0.05,n = 6)。● ,HFD; ■ ,HFD + 芹菜素。B: 用芹菜素治疗第7天禁食24h 后测定血糖(* p < 0.05,n = 6)。C: 芹菜素(■)或载体(●)治疗7天后小鼠的糖耐力测试(* p < 0.05,n = 6)。D: 曲线下面积(AUC) ,用 c ■ ,HFD 表示,□ ,HFD 加芹菜素表示。

FIG. 8.

FIG. 8. 图8

CD38 inhibition by apigenin promotes fatty acid oxidation in the liver. A: mRNA expression of lipid oxidation markers LCAD, MCAD, and CPT1a in the liver measured by RT-PCR in mice treated with apigenin (□) or vehicle (■) (*P < 0.05, n = 6 per group). B: Total triglyceride (TG) content in the liver of mice treated with apigenin or vehicle (*P < 0.05, n = 6 per group). HFD, high-fat diet. C: Total triglycerides levels in HepG2 cells incubated with 0.5 mmol/L oleate/palmitate (O/P) (2:1 ratio), oleate/palmitate plus 25 μmol/L apigenin (API), or oleate/palmitate plus apigenin plus 10 μmol/L EX527 (*P < 0.05, n = 3). D: Working model for apigenin and quercetin effect on CD38. In cells, CD38 maintains low intracellular NAD+ levels with a consequent low sirtuin activity. The inhibition of CD38 in different subcellular compartments leads to an increase in NAD+ levels, which becomes available for sirtuin activation. We propose that the effect of apigenin will activate nuclear and cytoplasmic and also mitochondrial sirtuins, where CD38 has been shown to be expressed.

芹菜素抑制 CD38促进肝脏脂肪酸氧化。A: 逆转录-聚合酶链反应(RT-PCR)检测芹菜素(□)或载体(■)处理小鼠肝脏脂质氧化标志物 LCAD、 MCAD 和 CPT1a 的 mRNA 表达(* p < 0.05,n = 6)。B: 芹菜素组和变应原组小鼠肝脏总甘油三酯(TG)含量(* p < 0.05,n = 6)。高脂肪饮食。C: 0.5 mmol/L 油酸酯/棕榈酸酯(O/P)(2:1) ,油酸酯/棕榈酸酯 + 25 mol/l 芹菜素(API) ,或油酸酯/棕榈酸酯 + 芹菜素 + 10 mol/l EX527(* p < 0.05,n = 3)孵育 HepG2细胞总甘油三酯水平。D: 芹菜素和槲皮素对 CD38的作用模型。在细胞中,CD38维持低的细胞内 NAD + 水平,从而产生低的去乙酰化酶活性。CD38在不同亚细胞区室中的抑制导致 NAD + 水平的升高,这种升高可用于激活 sirtuin。我们认为芹菜素的作用将激活细胞核和细胞质以及线粒体去乙酰化酶,其中 CD38已被证明表达。

DISCUSSION

讨论

The alarming expansion of metabolic diseases has triggered a considerable effort in the development of pharmacological strategies to prevent and treat them. In this regard, the study of sirtuins and specifically SIRT1 has become of great relevance due to the many beneficial effects of their action (8,9). In fact, how to achieve SIRT1 activation in vivo is a subject of intense investigation. One of the strategies to achieve such activation has been the use of drugs that directly target SIRT1. Resveratrol (16) and SRT1720 (33) are two of the early SIRT1-activating compounds that improve metabolism and protect against metabolic disorders, although there is a debate about their mechanism of action (3436). Another mechanism to achieve SIRT1 activation in vivo is to raise intracellular levels of NAD+ either by increased synthesis or diminished degradation (57,18). Previously, we have shown that the enzyme CD38 is the principal regulator of intracellular NAD+ levels in mammalian tissues (17). In fact, we were the first to show that increasing NAD+ levels by deletion of CD38 protects against diet-induced obesity through SIRT1 activation (5). Other research groups later confirmed the importance of NAD+ in the prevention of metabolic diseases. Yoshino et al. (7) showed that administration of nicotinamide mononucleotide (a NAD+ precursor) to mice protects against high-fat diet–induced metabolic disorders. Bai et al. (6) obtained similar results using PARP1 knockout mice. Taken together, the evidence shows that pharmacological interventions that increase NAD+ are a promising avenue for treating metabolic disorders. However, the mechanism by which cellular NAD+ is increased may have different long-term outcomes. We followed survival of wild-type, CD38 knockout, and PARP1 knockout mice fed a high-fat diet. Preliminary studies with small numbers of mice suggest that CD38 knockout mice have increased average and maximum life span compared with wild-type mice when they are fed a high-fat diet. However, in the PARP1 knockout mice, which also have increased cellular NAD+ levels (6), the outcome was opposite this (Supplementary Fig. 2), with the PARP1 knockout mice having a decreased life span compared with the wild-type mice. This difference in survival could be explained by the fact that PARP1 is involved in genomic stability (37) and DNA repair both in the nucleus (37) and in mitochondria (38,39). This suggests that although CD38 and PARP1 knockout mice have similar protection against metabolic disorders, they may have distinct effects on longevity.

代谢性疾病的惊人扩散促使人们在制定预防和治疗这些疾病的药理战略方面作出了相当大的努力。在这方面,去乙酰化酶,特别是 SIRT1的研究已经变得非常重要,因为它们的作用有许多有益的影响(8,9)。实际上,如何在体内实现 SIRT1的激活是一个热门的研究课题。实现这种激活的策略之一是使用直接针对 SIRT1的药物。白藜芦醇(16)和 SRT1720(33)是两个早期 sirt1激活化合物,改善代谢和保护代谢紊乱,虽然有一个关于其作用机制的争论(34-36)。SIRT1在体内激活的另一个机制是通过增加合成或减少降解来提高细胞内 NAD + 水平(5-7,18)。以前,我们已经证明 CD38是哺乳动物组织中细胞内 NAD + 水平的主要调节因子(17)。事实上,我们是第一个证明通过删除 CD38来增加 NAD + 水平可以通过 SIRT1激活来防止饮食诱导的肥胖(5)。其他研究小组随后证实了 NAD + 在预防代谢性疾病方面的重要性。吉野等人(7)表明,给予尼克酰胺单核苷酸(NAD + 前体)的小鼠保护对抗高脂肪饮食引起的代谢紊乱。Bai 等人(6)用 PARP1基因敲除小鼠得到了类似的结果。综上所述,有证据表明,增加 NAD + 的药理干预是治疗代谢紊乱的一个有希望的途径。然而,细胞 NAD + 增加的机制可能有不同的长期结果。我们跟踪了野生型、 CD38基因敲除和 PARP1基因敲除小鼠的存活情况。对少数小鼠的初步研究表明,当喂食高脂肪食物时,CD38基因敲除小鼠与野生型小鼠相比,平均最长寿命增加。然而,在 PARP1基因敲除小鼠中,也增加了细胞 NAD + 水平(6) ,结果与此相反(补充图2) ,与野生型小鼠相比,PARP1基因敲除小鼠的寿命缩短了。这种存活率的差异可以用 PARP1参与细胞核(37)和线粒体(38,39)的基因组稳定性(37)和 DNA 修复来解释。这表明,虽然 CD38和 PARP1基因敲除小鼠对代谢紊乱有类似的保护作用,但它们可能对长寿有不同的影响。

Here, we describe for the first time that the flavonoid apigenin is a CD38 inhibitor, and both apigenin and quercetin promote changes in intracellular NAD+ levels. This increase in NAD+levels leads to changes in protein acetylation likely due to an increase in sirtuin activity. Furthermore, we show that apigenin improves glucose homeostasis and reduces lipid content in the liver in a model of high-fat diet–induced obesity. Our results suggest that lipid oxidation is increased by a SIRT1-dependent mechanism. However, it could also happen that fatty acid synthesis or export is altered, since SIRT1 has been shown to regulate both processes (40,41). Our results demonstrate that CD38 is a promising pharmacological target to promote sirtuin actions and to treat metabolic diseases.

在这里,我们首次描述了黄酮类芹菜素是 CD38抑制剂,芹菜素和槲皮素都促进细胞内 NAD + 水平的变化。NAD + 水平的增加导致了蛋白质乙酰化的改变,这可能与 sirtuin 活性的增加有关。此外,在高脂饮食诱导的肥胖模型中,我们发现芹菜素改善了葡萄糖稳态和降低了肝脏中的脂质含量。我们的结果表明,脂质氧化是通过一个 sirt1依赖的机制增加。然而,它也可能发生,脂肪酸合成或出口被改变,因为 SIRT1已被证明调节这两个过程(40,41)。我们的结果表明,CD38是一个有希望的药理靶点,促进去乙酰化酶的作用和治疗代谢性疾病。

Flavonoids, including apigenin and quercetin, have broad beneficial effects (20). These two flavonoids ameliorate atherosclerosis in mouse genetic models (25). Although some of the beneficial effects of flavonoids on metabolism are believed to be AMPK mediated (25), this has not been clearly elucidated. Indeed, apigenin is a very weak AMPK activator in vivo (25), which suggests an additional mechanism of action. Our findings provide mechanistic evidence that flavonoids can promote an increase in NAD+ levels through inhibition of CD38, resulting in changes in protein acetylation, most likely through stimulation of SIRT1 (Fig. 8D). Although we show here that CD38 inhibition affects SIRT1 activity, it is likely that other sirtuins will also be stimulated by CD38 inhibition. Interestingly, CD38 is also present and active in the mitochondria (42,43), where it may regulate mitochondrial NAD+ levels and mitochondrial sirtuin activity.

类黄酮,包括芹菜素和槲皮素,具有广泛的有益作用(20)。这两种黄酮类化合物改善小鼠遗传模型中的动脉粥样硬化(25)。虽然黄酮类化合物对新陈代谢的一些有益作用被认为是 AMPK 介导的(25) ,但这还没有得到明确的阐明。实际上,芹菜素在体内是一个非常弱的 AMPK 激活剂(25) ,这提示了一个额外的作用机制。我们的研究结果提供了黄酮类化合物通过抑制 CD38而促进 NAD + 水平增加的机制证据,导致蛋白质乙酰化的改变,最有可能是通过刺激 SIRT1(图8D)。虽然我们在这里表明,CD38抑制影响 SIRT1的活性,它可能是其他 sirtuins 也将刺激的 CD38抑制。有趣的是,CD38在线粒体(42,43)中也存在和活跃,它可能调节线粒体 NAD + 水平和线粒体去乙酰化酶活性。

It is likely that, similar to what happens with many other natural compounds, apigenin and quercetin have other cellular targets besides CD38. However, we clearly show that the increase in cellular NAD+ levels promoted by these compounds depends on CD38. More importantly, our findings support the idea that pharmacological inhibition of CD38 can be achieved as a strategy to treat obesity and obesity-related diseases. Further research will help to develop highly selective CD38 inhibitors that may be used as an approach to treat metabolic syndrome in humans.

类似于许多其他天然化合物,芹菜素和槲皮素除了 CD38还有其他的细胞靶点。然而,我们清楚地表明,这些化合物促进的细胞 NAD + 水平的增加依赖于 CD38。更重要的是,我们的发现支持药物抑制 CD38可以作为一种治疗肥胖和肥胖相关疾病的策略。进一步的研究将有助于开发高选择性的 CD38抑制剂,可用于治疗人类的代谢症候群。

ACKNOWLEDGMENTS

鸣谢

This work was supported in part by grants from the American Federation for Aging Research and from the Mayo Foundation; by the Strickland Career Development Award; by the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), grant DK-084055; by Mayo-UOFM Decade of Discovery Grant 63-01; and by Minnesota Obesity Council Grant DK-50456-15. D.A.S. is supported by grants from the NIH/National Institute on Aging, the Juvenile Diabetes Research Foundation, the United Mitochondrial Disease Foundation, and the Glenn Foundation for Medical Research. C.E. is supported by American Heart Association postdoctoral fellowship award 11POST7320060 and A.P.G. by a fellowship from the Portuguese Foundation for Science and Technology (SFRH/BD/44674/2008).

这项研究得到了美国老龄化研究联合会和梅奥基金会的部分资助; 斯特里克兰职业发展奖; 美国国家卫生研究院糖尿病、消化和肾脏疾病研究所(NIH) ,DK-084055的资助; 梅奥-uofm 发现资助十年63-01的资助; 以及明尼苏达州肥胖委员会资助 DK-50456-15的资助。是由美国国立卫生研究院/国家衰老研究所、1型糖尿病研究基金会、联合线粒体疾病基金会和格伦医学研究基金会资助的。美国心脏协会博士后奖学金11POST7320060和葡萄牙科学技术基金会的奖学金(SFRH/BD/44674/2008)。

E.N.C. and M.T.B. are inventors in a patent for CD38 and obesity (U.S. patent no. 8143014). E.N.C. and D.A.S. are inventors on a provisional patent for apigenin as a CD38 inhibitor to treat metabolic syndrome. D.A.S. is a consultant for Sirtris, a GlaxoSmithKline company aiming to develop medicines that target sirtuins. No other potential conflicts of interest relevant to this article were reported.

E.n.c. 和 m.t.b. 是 CD38和肥胖症专利的发明者(美国专利号8143014)。和 d.a.s 是芹菜素作为 CD38抑制剂治疗代谢症候群的临时专利的发明者。是葛兰素史克公司 Sirtris 的顾问,该公司旨在开发针对去乙酰化酶的药物。没有其他与本文有关的潜在利益冲突的报道。

C.E. measured CD38 activity, measured effect of compounds in NAD+ levels, evaluated the effect of CD38 and CD38 inhibitors on protein acetylation, performed in vivo experiments, analyzed tissue samples, performed the lipid measurements, wrote the manuscript, designed experiments, discussed and analyzed data, and corrected the manuscript. V.N. measured CD38 activity, evaluated the effect of CD38 and CD38 inhibitors on protein acetylation, performed in vivo experiments, analyzed tissue samples, performed the longevity studies, designed experiments, discussed and analyzed data, and corrected the manuscript. N.L.P. performed the library screening, performed qPCR, designed experiments, discussed and analyzed data, and corrected the manuscript. V.C. measured CD38 activity and evaluated the effect of CD38 and CD38 inhibitors on protein acetylation. A.P.G. performed qPCR. M.T.B. performed the longevity studies. L.O. analyzed tissue samples. T.A.W. measured CD38 activity. D.A.S. and E.N.C. developed the original idea, designed experiments, discussed and analyzed data, and corrected the manuscript. E.N.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

C.e. 测定了 CD38的活性,测定了化合物在 NAD + 水平上的作用,评价了 CD38和 CD38抑制剂对蛋白质乙酰化的影响,进行了体内实验,分析了组织样品,进行了脂质测定,写了原稿,设计了实验,讨论和分析了数据,并对原稿进行了校正。测定 CD38活性,评价 CD38和 CD38抑制剂对蛋白质乙酰化的影响,进行体内实验,分析组织标本,进行寿命研究,设计实验,讨论和分析数据,并修正原稿。进行了文库筛选,进行了 qPCR,设计了实验,对数据进行了讨论和分析,并对原稿进行了校正。测定了 CD38的活性,并评价了 CD38和 CD38抑制剂对蛋白质乙酰化的影响。进行了 qPCR。进行了长寿研究。L.o. 分析了组织样本。测量了 CD38的活性。和 e.n.c. 发展了最初的想法,设计了实验,讨论和分析了数据,并修改了手稿。是这项工作的保证人,因此,完全可以访问研究中的所有数据,并负责数据的完整性和数据分析的准确性。

The authors thank Caroline Shamu and the staff at Harvard’s Institute of Chemistry and Cell Biology facility, where the small-molecule screen was conducted.

作者感谢 Caroline Shamu 和哈佛大学化学与细胞生物学研究所的工作人员进行了小分子筛选。

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