TGR5 (GPCR19); Microbial Metabolite; Human Endogenous Metabolite

猪去氧胆酸是由破坏菌群在小肠中形成的次级亲水性胆汁酸，作为TGR5的激动剂，在CHO细胞中的EC50为31.6 μM[1]。猪去氧胆酸(50，100 μM)增加参与RAW 264.7 细胞切除的基因 (Abca1、Abcg1 和 Apoe) 的表达[2]。

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HDCA增加了RAW 264.7细胞中与胆固醇外流相关的基因的表达[2]
我们研究了HDCA是否影响巨噬细胞系RAW 264.7中胆固醇流出相关基因的表达。在我们的研究中，用于治疗RAW细胞的HDCA浓度为50和100μM，接近在饮食中补充1.25%HDCA的LDLRKO小鼠的循环HDCA水平（平均42.4μM，范围31-66μM）。HDCA处理以剂量反应的方式显著增加了RAW细胞中ATP结合盒亚家族A成员1（Abca1）、ATP结合盒子家族G成员1（Abcg1）和载脂蛋白E（Apoe）的表达。因此，与赋形剂治疗组相比，在50μM HDCA剂量下，Abca1、Abcg1和Apoe的表达分别显著增加了57%、54%和106%（图5E）。此外，与赋形剂治疗组相比，在100μM HDCA剂量下，Abca1、Abcg1和Apoe的表达分别显著增加了201%、112%和189%（图5E）。载体处理组和HDCA处理组之间Srb1的表达相似（图5E）。与对照组相比，在50和100μM剂量下，HDCA治疗使核受体Lxrα的表达适度增加了36%，但Lxrβ或过氧化物酶体增殖物激活受体γ1（Pparγ1）的表达没有增加（图5E）。

猪去氧胆酸 (HDCA；1.25% (wt/wt)) 在 LDLRKO 中明显减少脂肪量并增加瘦体重，但不会提高任何器官毒性标志物的血清水平。猪去氧胆酸在 LDLRKO 的多个部位阻断动脉粥样淋巴瘤病变的，改善便秘脂蛋白谱系，降低胆固醇水平并阻止胆固醇吸收效率，并通过粪便呼吸增加每日胆固醇排泄。猪去氧胆酸还可以改善HDL功能，这可通过胆固醇中断测定法来简单[2]。

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研究了天然次生胆汁酸猪去氧胆酸（HDCA）对LDL受体缺失（LDLRKO）小鼠脂质代谢和动脉粥样硬化的影响。雌性LDLRKO小鼠在西方饮食中维持8周，然后分为2组，接受食物或食物+1.25%HDCA饮食15周。我们观察到，喂食HDCA饮食的小鼠更瘦，空腹血糖水平降低了37%（P<0.05）。与周粮组相比，补充HDCA显著降低了主动脉根部、整个主动脉和无名动脉的动脉粥样硬化病变大小，分别降低了44%（P<0.0001）、48%（P<0.01）和94%（P<0.01）。与周粮组相比，HDCA组血浆VLDL/IDL/LDL胆固醇水平显著降低了61%（P<0.05）。与食物组相比，补充HDCA使肠道胆固醇吸收降低了76%（P<0.0001）。此外，与饮食组的HDL相比，从HDCA组分离的HDL在体外介导胆固醇流出的能力显著增强。此外，HDCA显著增加了巨噬细胞系中与胆固醇流出相关的基因的表达，如Abca1、Abcg1和Apoe。因此，HDCA是抗动脉粥样硬化药物治疗的候选者[2]。

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补充HDCA显著提高了循环中的HDCA水平，并没有影响小鼠的整体健康。 HDCA抑制LDLRKO多部位动脉粥样硬化病变的形成。 HDCA改善了血浆脂蛋白谱，降低了血糖水平。 补充HDCA会降低肠道胆固醇吸收效率，并通过粪便排出增加每日胆固醇排泄量。 HDCA对肝脏脂质含量、肝脏基因表达和胆汁成分的影响[2]。

细胞培养和处理条件[2]
RAW 264.7细胞是一种小鼠巨噬细胞系，在含有DMEM的生长培养基中培养，DMEM补充了10%FBS（Hyclone，South Logan，UT，USA）、100 U/ml青霉素和100μg/ml链霉素。治疗时，将RAW 264.7细胞铺在生长培养基中的6孔板（7.5×10−5个细胞/孔）中2天。用PBS洗涤后，将细胞在含有各种化学物质或二甲亚砜（DMSO）作为载体对照的治疗培养基（DMEM补充了1%FBS、100 U/ml青霉素和100μg/ml链霉素）中孵育24小时。然后用PBS洗涤细胞，然后如下所述分离总RNA。

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胆固醇外排测定[2]
将RAW 264.7细胞铺在24孔板中（300000个细胞/孔）并生长1天。然后将每个孔中的细胞与1ml含有25μg/ml人乙酰化LDL和1μCi 3H-胆固醇/ml的生长培养基在CO2培养箱中孵育48小时。用PBS洗涤后，将细胞与DMEM+0.2%无脂肪酸的牛血清白蛋白一起孵育过夜。用PBS洗涤后，将细胞与0.5ml测试样品（DMEM中的小鼠HDL+0.2%BSA）在37°C下孵育4小时。然后，收集上清液，用0.1N NaOH裂解细胞。然后通过液体闪烁测量分别与上清液和细胞相关的放射性。

Mice: Eight weeks of a Western diet (21% fat, 0.15% cholesterol; TD.88137) is given to eight-week-old female LDLRKO mice in order to conduct atherosclerosis studies. In order to measure the lesions in the innominate artery and the aortic root region, one group of mice (the baseline group) is put to death at this time. In the baseline group, an atherosclerotic lesion involving the entire aorta is not investigated. Prior to their demise, the surviving mice are split into two groups and given the subsequent diets for an additional 15 weeks: group 1, which is a chow diet with 5% fat (AIN-76A Rodent Diet); and group 2, which is a chow diet plus 1.25% (wt/wt) hydroxycholic acid. For other studies, 8-wk-old female LDLRKO mice are fed a chow diet or chow diet + 1.25% Hyodeoxycholic acid for 3 wk before phenotype measurements. Weekly records are kept on food intake and body weight. By using Bruker Minispec software with Eco Medical Systems software, magnetic resonance imaging (MRI) is used to measure the lean mass and total body fat mass of animals[2].

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Female LDLRKO mice were used. For atherosclerosis studies, 8-wk-old female LDLRKO mice were fed a Western diet (21% fat, 0.15% cholesterol) for 8 wk. One group of mice (baseline group) was euthanized at this time point for lesion measurement in the aortic root region and in the innominate artery. Atherosclerotic lesion in the whole aorta was not examined in the baseline group. The remaining mice were then divided into 2 groups and fed the following diets for another 15 wk before euthanasia: group 1, chow diet (5% fat); and group 2, chow diet + 1.25% (wt/wt) HDCA. For other studies, 8-wk-old female LDLRKO mice were fed a chow diet or chow diet + 1.25% HDCA for 3 wk before phenotype measurements. The HDCA used in the study was purchased from xxx. Food consumption and body weight were recorded weekly. [2]

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Lipid, total bile acids, HDCA assays, serum chemistry tests, gel filtration chromatography, dichlorofluorescein (DCF) assay, and immunoblotting [2]
For plasma lipid and lipoprotein level determinations, mice were denied access to food for 16 h before bleeding. Total cholesterol, HDL cholesterol, free cholesterol, triglycerides, and free fatty acid levels were determined by enzymatic colorimetric assays. Phosphatidylcholine levels were assayed using an enzymatic colorimetric assay. Plasma samples were fractionated by fast-performance liquid chromatography (FPLC) as described previously. To determine the extent of lipid oxidation of HDL samples, 2 μg of HDL cholesterol in 175 μl phosphate-buffered saline (PBS) was added to each well of a 96-well plate, followed by 1 h incubation at 37°C. DCFH (5 μg) in 25 μl PBS was then added to each well, followed by an additional 1 h of incubation at 37°C. The DCF fluorescence intensity was then determined with a plate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm, as described previously. For immunoblotting, FPLC fractions or HDL samples were fractionated by SDS-PAGE; transferred onto a nylon membrane; incubated with a rabbit antibody against mouse apolipoprotein A1 (apoA1), apo B-48/100, or apoE; washed; incubated with a secondary antibody; and detected using electrochemiluminescence. Total bile acid levels were assayed using a kit from Diazyme Laboratories according to the manufacturer's protocol. For determination of total bile acid levels in HDL, FPLC-isolated HDL was concentrated by using Amicon centrifugal filter units. HDL samples carrying plasma-equivalent amount of HDL cholesterol were assayed together with plasma samples (the sources of the HDL preparation) for comparison of total bile acid level. Plasma HDCA levels were determined using a LC/MS/MS method as described below. Standards were prepared in methanol:water (2:1) at HDCA concentrations of 1.00–1000 ng/ml. Samples and standards were extracted by protein precipitation using 100 μl sample or standard and addition of 400 μl methanol containing the internal standard d4-ursodeoxycholic acid (UDCA; 100 ng/ml). The samples were vortexed and centrifuged, and the supernatant (400 μl) was combined with water (400 μl). HDCA was separated on a Supelco Ascentis Express C-18 column (50×2.1 mm, 2.7 μm) at a flow rate of 0.200 ml/min using 2 mobile phases: 10 mM ammonium acetate in water with 0.1% ammonium hydroxide (pH 9); and 10 mM ammonium acetate in methanol with 0.1% ammonium hydroxide. Elution was started with 50% B initially and held for 0.5 min, followed by a linear gradient to 80% B at 4 min and a second gradient to 95% B at 5 min. This composition was held for 2 min, followed by reequilibration to 50% B and a total run time of 8 min. Eluent was directly introduced into a SciexAPI5000, and HDCA was detected by MS/MS monitoring m/z 391.1 in both Q1 and Q3 with a 40 eV collision voltage to lower background interference. The internal standard was detected at m/z 395.1. HDCA eluted at 4.26 min, and the internal standard was detected at 4.08 min. With the use of this system, HDCA was baseline separated from the isobaric species UDCA, deoxycholic acid (5.3 min), and chenodeoxycholic acid (5.1 min).

Metabolism / Metabolites
6alpha-Hydroxylithocholic acid is a known human metabolite of Lithocholic Acid.

[1]. Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure-activity relationships, and molecular modeling studies. J Med Chem. 2008 Mar 27;51(6):1831-41.

[2]. Hyodeoxycholic acid improves HDL function and inhibits atherosclerotic lesion formation in LDLR-knockout mice. FASEB J. 2013 Sep;27(9):3805-17.

Hyodeoxycholic acid is a member of the class of 5beta-cholanic acids that is (5beta)-cholan-24-oic acid substituted by alpha-hydroxy groups at positions 3 and 6. It has a role as a human metabolite and a mouse metabolite. It is a bile acid, a member of 5beta-cholanic acids, a 6alpha,20xi-murideoxycholic acid and a C24-steroid. It is functionally related to a cholic acid. It is a conjugate acid of a hyodeoxycholate.
Hyodeoxycholic Acid has been used in trials studying the treatment of Hypercholesterolemia.

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TGR5, a metabotropic receptor that is G-protein-coupled to the induction of adenylate cyclase, has been recognized as the molecular link connecting bile acids to the control of energy and glucose homeostasis. With the aim of disclosing novel selective modulators of this receptor and at the same time clarifying the molecular basis of TGR5 activation, we report herein the biological screening of a collection of natural occurring bile acids, bile acid derivatives, and some steroid hormones, which has resulted in the discovery of new potent and selective TGR5 ligands. Biological results of the tested collection of compounds were used to extend the structure-activity relationships of TGR5 agonists and to develop a binary classification model of TGR5 activity. This model in particular could unveil some hidden properties shared by the molecular shape of bile acids and steroid hormones that are relevant to TGR5 activation and may hence be used to address the design of novel selective and potent TGR5 agonists.[1]

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Bile acids have been shown to activate the G-protein-coupled receptor TGR5, leading to increased energy expenditure, decreased obesity, and improved insulin sensitivity . HDCA has been shown to activate TGR5 but not FXR in vitro. We found that after 15 wk of HDCA supplementation, the mice were less obese (Table 2) and exhibited significantly lower fasting glucose levels as compared with the chow diet group (Table 4). Expressions of gluconeogenesis genes, Pepck and G6pase, whose expression are known to be decreased by both TGR5 and SHP through the activation of FXR, were significantly lower in the HDCA group as compared with the chow group (Fig. 4C). Since Shp mRNA levels were significantly lower in the livers of the HDCA group than in those of the control group, it is unlikely that the decreased expression of Pepck and G6pase in the HDCA-treated mice was due to the action of FXR and SHP. More likely, this is due to the activation of TGR5 by HDCA. In agreement, hepatic expression of Cyp7a1 and Bsep, 2 genes known to be down- and up-regulated by FXR, respectively, was not altered by HDCA supplementation (Fig. 4C), suggesting HDCA did not activate FXR in the mouse liver. In the small intestine, HDCA did not significantly increase or decrease the expression of FXR target genes Ostα, Ostβ, and Mrp2, either (Fig. 3C). Therefore, our data suggest that HDCA is an agonist for TGR5 but not FXR in vivo. Our data do not support HDCA as an antagonist for FXR.
In summary, our study demonstrates that HDCA influences cholesterol and glucose homeostasis in LDLRKO mice. HDCA supplementation inhibited intestinal cholesterol absorption, lowered plasma VLDL/IDL/LDL cholesterol levels, improved HDL function, and decreased obesity and plasma glucose levels in mice. The glucose-lowering and obesity-preventing effects of HDCA are most likely due to the activation of TGR5. Furthermore, HDCA not only significantly decreased the atherosclerotic lesion size but also decreased the contribution of inflammatory components, macrophages, and incidence of calcification within lesions. Our findings suggest that HDCA is an attractive candidate for the treatment of obesity, diabetes, and atherosclerosis.[1]

C24H40O4

392.5720

392.292

83-49-8

Murideoxycholic acid; 668-49-5; Hyodeoxycholic acid sodium; 10421-49-5; Hyodeoxycholic acid-d5

5283820

White to off-white solid powder

1.1±0.1 g/cm3

547.1±25.0 °C at 760 mmHg

200-201 °C(lit.)

298.8±19.7 °C

0.0±3.3 mmHg at 25°C

1.543

5

77.76

3

4

4

28

605

10

C[C@H](CCC(=O)O)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2C[C@@H]([C@H]4[C@@]3(CC[C@H](C4)O)C)O)C

DGABKXLVXPYZII-SIBKNCMHSA-N

InChI=1S/C24H40O4/c1-14(4-7-22(27)28)17-5-6-18-16-13-21(26)20-12-15(25)8-10-24(20,3)19(16)9-11-23(17,18)2/h14-21,25-26H,4-13H2,1-3H3,(H,27,28)/t14-,15-,16+,17-,18+,19+,20+,21+,23-,24-/m1/s1

(4R)-4-[(3R,5R,6S,8S,9S,10R,13R,14S,17R)-3,6-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid

HDCA; HYODEOXYCHOLIC ACID; 83-49-8; Hyodesoxycholic acid; Iodeoxycholic acid; 7-Deoxyhyocholic acid; Hyodesoxycholsaeure; HYODEOXYCHOLIC_ACID; 3alpha,6alpha-Dihydroxy-5beta-cholan-24-oic acid; Hyodeoxycholic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

DMSO: 78~100 mg/mL (198.7~254.7 mM)
Ethanol: ~78 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.37 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 (6.37 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.37 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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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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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