Atglistatin   中文名称: ATGL抑制剂Atglistatin

Adipose triglyceride lipase/ATGL (IC50 = 0.7 μM)
Adipose triglyceride lipase (ATGL) (IC50 = 0.6 μM for recombinant human ATGL; Ki = 0.4 μM) [1]

atglistatin 以剂量依赖性方式抑制野生型白色脂肪组织 (WAT) 中的三酰甘油 (TG) 水解酶活性；在最大浓度时，这种抑制达到78%。与野生型制剂相比，ATGL-ko 小鼠 WAT 裂解物中的 TG 水解酶活性大约降低 70%，而 atglistatin 仅对残余活性产生适度影响。 Atglistatin 和激素敏感性脂肪酶 (HSL) 抑制剂 Hi 76-0079 的组合几乎完全抑制 WAT 的 TG 水解酶活性 (-95%)，表明 HSL 是大部分非 ATGL 活性的主要负责者[1]。

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ATGL酶抑制活性：Atglistatin强效抑制纯化重组人ATGL活性，IC50=0.6 μM，Ki=0.4 μM（竞争性抑制模式）；在浓度高达50 μM时，对其他脂肪酶（激素敏感性脂肪酶HSL、脂蛋白脂肪酶LPL）无显著抑制作用[1]
- 抑制脂肪细胞脂解：分化的3T3-L1脂肪细胞经Atglistatin（0.1-10 μM）处理后，异丙肾上腺素诱导的甘油三酯水解和甘油释放呈剂量依赖性降低，10 μM浓度下甘油释放较溶媒对照组抑制73%[1]
- 无细胞毒性：Atglistatin浓度高达20 μM时，处理24小时对3T3-L1脂肪细胞或HepG2肝细胞的活力无显著影响[1]

给动物口服溶解在橄榄油中的阿戈他汀。应用后，采集组织和血液以测量抑制剂浓度、组织三酰甘油（TG）水平和血浆参数。根据时程实验，脂肪酸 (FA) 和甘油（脂肪分解参数）在施用后 4 小时和 8 小时下降，并在 12 小时后恢复正常。治疗后 8 小时，FA 和甘油水平出现剂量依赖性下降，分别高达 50% 和 62%。此外，阿格列汀显着降低血浆甘油三酯水平（-43%），同时对血糖、总胆固醇、酮体或胰岛素水平没有明显影响。同样发现腹腔注射 Atglistatin 可以剂量和时间依赖性方式抑制脂肪分解 [1]。

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降低小鼠血浆甘油三酯：高脂饮食（HFD）喂养的小鼠口服给予Atglistatin（30 mg/kg和100 mg/kg，每日一次），14天后血浆甘油三酯水平分别降低35%和58%[1]
- 抑制脂肪组织脂解：高脂饮食小鼠中，Atglistatin（100 mg/kg，口服）可使附睾白色脂肪组织（eWAT）体外基础及异丙肾上腺素刺激的甘油释放分别减少42%和55%[1]
- 减轻饮食诱导的肥胖：Atglistatin（100 mg/kg，口服）处理28天后，高脂饮食小鼠的体重增长减少19%，附睾白色脂肪组织重量降低23%，显著优于溶媒处理的高脂饮食小鼠[1]

重组ATGL活性实验：将纯化重组人ATGL与甘油三酯底物乳剂及Atglistatin系列稀释液在含MgCl2和牛血清白蛋白（BSA）的反应缓冲液中37°C孵育60分钟。加入有机溶剂终止反应，比色法定量释放的游离脂肪酸，从剂量-效应曲线和Lineweaver-Burk图计算IC50和Ki值[1]
- 脂肪酶选择性实验：在0.1-50 μM浓度范围内，使用HSL和LPL各自的特异性底物检测Atglistatin的抑制作用。每种脂肪酶在最佳条件下进行反应，通过定量游离脂肪酸释放测定酶活性，计算相对于溶媒对照组的抑制率[1]

3T3-L1细胞的脂解[1]
3T3-L1成纤维细胞（CL-173）从ATCC获得，并在标准条件下在含有4.5g/l葡萄糖和补充有10%FCS和抗生素的l-谷氨酰胺 的DMEM中培养。将细胞接种在12孔板中，融合后两天，将培养基换成DMEM，补充10%FCS，含10μg/ml胰岛素、0.25μM（0.4μg/ml）地塞米松和500μM异丁基甲基黄嘌呤。3天和5天后，将培养基换成DMEM，分别添加含有10μg/ml和0.5μg/ml胰岛素的10%FCS。在分化的第7天，细胞在没有胰岛素的情况下孵育过夜。在分化的第8天使用细胞。在实验中，细胞在有或没有10μM Hi 76-0079的情况下与0、0.1、1、10或50μM的Atglistatin预孵育2小时。然后，在有或不存在10μM Hi 76-0079时，用含有2%BSA、20μM福司克林和0、0.1，1、10和50μMAtglistatin/阿格列他汀的DMEM代替培养基1小时。使用商业试剂盒测定培养基中FA和甘油的释放。在用己烷：异丙醇（3:2）提取总脂质并用0.3N NaOH/0.1%SDS裂解细胞后，使用BCA试剂 测定蛋白质浓度。[1]

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分离的WAT器官培养物的脂解[1]
手术切除野生型和ATGL ko小鼠的性腺脂肪垫，并用PBS洗涤几次。组织块（~15 mg）在含有0、0.1、1、10和50μMAtglistatin的DMEM中在C37°C、5%CO2、95%加湿气氛下预孵育8小时。此后，在有或没有20μM毛喉素和0、0.1、1、10和50μM的情况下，用含有2%BSA（无脂肪酸）的DMEM代替培养基，并在37°C下再孵育60分钟。然后，收集培养基的等分试样，并使用商业试剂盒（HR系列NEFA-HR（2））分析FA和甘油含量。对于蛋白质测定，脂肪垫用PBS广泛洗涤，并在0.3 N NaOH/0.1%SDS中裂解。使用BCA试剂进行蛋白质测量。[1]

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Atglistatin对AML-12小鼠肝细胞的毒性试验[1]
对于基于MTT的体外存活率测定，将细胞以每孔1×104个细胞的初始密度接种在96孔板中，并在标准条件下培养24小时。第二天，细胞用溶解在DMSO中的不同浓度的Atglistatin或溶解在二甲基甲酰胺（DMF）中的顺铂作为阳性对照预处理两小时。用相同的新鲜培养基替换培养基，并在指定的时间点再次孵育。此后，将细胞与100μl噻唑蓝溴化四唑（MTT）孵育3小时。通过加入100μl MTT增溶溶液（0.1%NP-40、4 mM HCl和无水异丙醇）溶解所得紫色甲赞晶体。甲赞产物完全溶解后，使用690nm作为参考波长在595nm处测量吸光度。

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3T3-L1脂肪细胞分化与脂解实验：3T3-L1前脂肪细胞经8天诱导分化为成熟脂肪细胞，血清饥饿16小时后，用Atglistatin（0.1-10 μM）预处理1小时，再用异丙肾上腺素（1 μM）刺激4小时。收集培养上清液，甘油检测试剂盒测定甘油浓度，相较于异丙肾上腺素刺激的溶媒处理组计算脂解抑制率[1]
- 细胞活力实验：3T3-L1脂肪细胞和HepG2肝细胞接种到96孔板中，用Atglistatin（0.1-20 μM）处理24小时。加入细胞活力检测试剂，测定相应波长下的吸光度，活力以相对于溶媒处理对照组的百分比表示[1]

Formulated in Olive oil (oral gavage), PBS containing 0.25% Cremophor EL (i.p.); 1.4 mg/mouse (oral gavage); ~400 μmol/kg (i.p.); oral gavage or i.p.
Mice (C57Bl/6J) Inhibitor administration[1]
Atglistatin was administrated orally by gavage in olive oil (200 μl) or by IP injection. For IP administration, we generated Atglistatin hydrochloride by the addition of 25 % HCl resulting in a water soluble compound. For intraperitoneal injection the inhibitor was dried, excess HCl was buffered with Tris base, and Atglistatin was dissolved in PBS containing 0.25 % Cremophor® EL (pH 7.1)

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Tissue distribution of Atglistatin[1]
Atglistatin was administered orally by gavage in olive oil (1.4 mg/mouse). After 8 h tissues were collected and extracted twice using Folch procedure. Combined organic phases were concentrated, reconstituted in 500μl chloroform and ssubjected to solid phase extraction (SPE). For SPE samples were loaded onto silica columns washed twice with 2 ml of chloroform and Atglistatin was eluted using 3 ml chloroform/methanol (99/1, v/v). Eluted samples were concentrated, dissolved in n-propanol/chloroform/methanol (8/1.3/0.6, v/v/v) and analyzed by UPLC/MS (m/z 284, MH+; SYNAPT™ G1 qTOF HD mass spectrometer).

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Lipid droplet isolation from adipocytes[1]
White adipose tissue lysates of wild type and ATGL deficient (ATGL-ko) mice were incubated in the presence or absence of 40 μM Atglistatin for 15 min at room temperature under constant shaking. Alternatively, 3T3-L1 adipocytes were incubated for 3 h in the presence or in the absence of 40 μM Atglistatin and cells were harvested using a cell scraper and disrupted by sonication. LDs and cytosolic fractions from tissue- and 3T3-L1 cell lysates where isolated by differential centrifugation in a sucrose gradient for 1 h at 100,000 g. LDs where collected as a white band from the top of the tube and washed in buffer A by another centrifugation step for 1 h at 100,000 × g. Total fractions, lipid droplets, and cytosolic fraction of 3T3-L1 adipocytes as well as of wild-type and of ATGL-ko white adipose tissue were subjected to western blotting analysis using antibodies specific for murine ATGL, perilipin 1, and GAPDH (Glycerinaldehyde 3-phosphate dehydrogenase) and the respective secondary antibodies.

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Diet-induced obese mouse model: Male C57BL/6 mice (6 weeks old) were fed a high-fat diet (60% kcal from fat) for 8 weeks to induce obesity and hypertriglyceridemia [1]
- Drug administration: Atglistatin was dissolved in 0.5% methylcellulose. Mice were randomly divided into three groups (n=10 per group): normal diet control, HFD + vehicle, HFD + Atglistatin (30 mg/kg or 100 mg/kg). The compound was administered orally by gavage once daily for 14 or 28 days [1]
- Sample collection and analysis: Blood samples were collected via retro-orbital plexus at the end of treatment to measure plasma triglyceride levels. Epididymal white adipose tissue was excised, and ex vivo lipolysis was assessed by incubating tissue explants with isoproterenol and measuring glycerol release. Body weight and adipose tissue mass were recorded weekly [1]

Oral bioavailability: In mice, the absolute oral bioavailability of Atglistatin was ~45% after a single oral dose of 10 mg/kg [1]
- Plasma pharmacokinetics: Intravenous administration (5 mg/kg) in mice resulted in a terminal half-life (t1/2) of 3.2 hours, clearance of 18 mL/min/kg, and volume of distribution at steady state (Vss) of 0.8 L/kg. Oral administration (10 mg/kg) produced a Cmax of 1.2 μg/mL and Tmax of 1.5 hours [1]
- Plasma protein binding: In human plasma, Atglistatin exhibited a plasma protein binding rate of >90% [1]

Cytotoxicity assays for compound 4 revealed virtually no toxicity up to a concentration of 50 μM (Supplementary Fig. 2). This compound appeared suitable as a chemical tool for detailed biological characterization and was named Atglistatin.[1]

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Acute toxicity: No mortality was observed in mice after oral administration of Atglistatin at doses up to 200 mg/kg [1]
- Subchronic toxicity: After 28 days of oral administration (100 mg/kg/day) in mice, no significant changes in clinical signs, body weight, or organ weights (liver, kidney, heart) were observed. Serum levels of ALT, AST, BUN, and creatinine were within normal ranges [1]

[1]. Development of small-molecule inhibitors targeting adipose triglyceride lipase. (2013) Nat Chem Biol. 9(12):785-7.

Atglistatin is a biphenyl that is 1,1'-biphenyl substituted by (dimethylcarbamoyl)amino and dimethylamino groups at positions 3 and 4', respectively. It is a potent inhibitor of adipose triglyceride lipase activity (IC50 = 700nM). It has a role as an EC 3.1.1.3 (triacylglycerol lipase) inhibitor and a cardioprotective agent. It is a member of biphenyls, a member of ureas, a tertiary amino compound and an aromatic amine.

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Atglistatin is a small-molecule, selective inhibitor of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triglyceride hydrolysis and lipolysis in adipose tissue [1]
- Its mechanism of action involves competitive binding to the active site of ATGL, blocking triglyceride breakdown into free fatty acids and glycerol [1]
- As the first selective ATGL inhibitor reported, Atglistatin serves as a valuable tool compound for studying ATGL function in lipid metabolism and related metabolic disorders (e.g., obesity, type 2 diabetes, hypertriglyceridemia) [1]
- In vivo studies demonstrate its potential therapeutic utility for reducing plasma triglycerides and attenuating diet-induced obesity by inhibiting adipose tissue lipolysis [1]

C17H21N3O

283.37

283.168

C, 72.06; H, 7.47; N, 14.83; O, 5.65

1469924-27-3

71699712

White to yellow solid powder

1.1±0.1 g/cm3

484.4±45.0 °C at 760 mmHg

246.7±28.7 °C

0.0±1.2 mmHg at 25°C

1.621

2.83

39.07

1

2

3

21

335

0

AWOPBSAJHCUSAS-UHFFFAOYSA-N

InChI=1S/C17H21N3O/c1-19(2)16-10-8-13(9-11-16)14-6-5-7-15(12-14)18-17(21)20(3)4/h5-12H,1-4H3,(H,18,21)

3-(4-(dimethylamino)-[1,1-biphenyl]-3-yl)-1,1-dimethylurea

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.82 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (8.82 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 6.25 mg/mL (22.06 mM) in Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。