TGR5 (GPCR19); Microbial Metabolite; Human Endogenous Metabolite

Hyodeoxycholic acid is a secondary hydrophilic bile acid formed in the small intestine by disrupted bacterial flora. As a TGR5 agonist, its EC50 in CHO cells is 31.6 μM [1]. Hyodeoxycholic acid (50, 100 μM) increases the expression of genes (Abca1, Abcg1 and Apoe) involved in RAW 264.7 cell resection [2].

---

HDCA increased the expression of genes involved in cholesterol efflux in RAW 264.7 cells [2]
We examined whether HDCA influences the expression of genes involved in cholesterol efflux in a macrophage cell line, RAW 264.7. The concentrations of HDCA used in treating the RAW cells in our study, 50 and 100 μM, were close to the circulating HDCA levels of LDLRKO mice that received the 1.25% HDCA supplementation in diet (mean 42.4 μM, range 31–66 μM). HDCA treatment significantly increased the expression of ATP-binding cassette subfamily A member 1 (Abca1), ATP-binding cassette subfamily G member 1 (Abcg1), and apolipoprotein E (Apoe) in RAW cells in a dose-response way. Thus, at a dose of 50 μM HDCA, the expression of Abca1, Abcg1, and Apoe was significantly increased by 57, 54, and 106%, respectively, as compared with the vehicle-treated group (Fig. 5E). Furthermore, at a dose of 100 μM HDCA, the expression of Abca1, Abcg1, and Apoe was significantly increased by 201, 112, and 189%, respectively, as compared with the vehicle-treated group (Fig. 5E). Expression of Srb1 was similar between the vehicle-treated and HDCA-treated groups (Fig. 5E). Expression of the nuclear receptor Lxrα, but not Lxrβ or peroxisome proliferator-activated receptor γ1 (Pparγ1), was modestly increased by HDCA treatment by 36%, at both the 50 and 100 μM doses, as compared with the control group (Fig. 5E).

Hyodeoxycholic acid (HDCA; 1.25% (wt/wt)) significantly reduced fat mass and increased lean body mass in LDLRKO but did not increase serum levels of any organ toxicity markers. Hyodeoxycholic acid blocks atherosclerotic lymphoma lesions at multiple sites in LDLRKO, improves constipation lipoprotein profiles, reduces cholesterol levels and blocks cholesterol absorption efficiency, and increases daily cholesterol excretion through fecal respiration. Hyodeoxycholic acid also improves HDL function, which can be accomplished simply by cholesterol interruption assay [2].

---

We examined the effects of a natural secondary bile acid, hyodeoxycholic acid (HDCA), on lipid metabolism and atherosclerosis in LDL receptor-null (LDLRKO) mice. Female LDLRKO mice were maintained on a Western diet for 8 wk and then divided into 2 groups that received chow, or chow + 1.25% HDCA, diets for 15 wk. We observed that mice fed the HDCA diet were leaner and exhibited a 37% (P<0.05) decrease in fasting plasma glucose level. HDCA supplementation significantly decreased atherosclerotic lesion size at the aortic root region, the entire aorta, and the innominate artery by 44% (P<0.0001), 48% (P<0.01), and 94% (P<0.01), respectively, as compared with the chow group. Plasma VLDL/IDL/LDL cholesterol levels were significantly decreased, by 61% (P<0.05), in the HDCA group as compared with the chow diet group. HDCA supplementation decreased intestinal cholesterol absorption by 76% (P<0.0001) as compared with the chow group. Furthermore, HDL isolated from the HDCA group exhibited significantly increased ability to mediate cholesterol efflux ex vivo as compared with HDL of the chow diet group. In addition, HDCA significantly increased the expression of genes involved in cholesterol efflux, such as Abca1, Abcg1, and Apoe, in a macrophage cell line. Thus, HDCA is a candidate for antiatherosclerotic drug therapy [2].

---

HDCA supplementation significantly increased circulating HDCA levels and did not affect general health of the mice. HDCA inhibited atherosclerotic lesion formation in LDLRKO at multiple sites. HDCA improved plasma lipoprotein profiles and decreased plasma glucose level. HDCA supplementation decreased intestinal cholesterol absorption efficiency and increased daily cholesterol excretion through fecal output. Effects of HDCA on liver lipid content, hepatic gene expression, and bile composition[2].

Cell culture and treatment conditions [2]
RAW 264.7 cells, a mouse macrophage cell line, were cultured in growth medium containing DMEM supplemented with 10% FBS (Hyclone, South Logan, UT, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin. For treatment, RAW 264.7 cells were plated in 6-well plates (7.5×10−5 cells/well) in growth medium for 2 d. After being washed with PBS, the cells were incubated for 24 h in treatment medium (DMEM supplemented with 1% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin) containing various chemicals or dimethyl sulfoxide (DMSO) as the vehicle control. The cells were then washed with PBS before total RNA was isolated as described below.

---

Cholesterol efflux assay [2]
RAW 264.7 cells were plated in 24-well plates (300,000 cells/well) and grown for 1 d. Cells in each well were then incubated with 1 ml of growth medium containing 25 μg/ml human acetylated LDL and 1 μCi 3H-cholesterol/ml for 48 h in a CO2 incubator. After being washed with PBS, the cells were then incubated with DMEM + 0.2% fatty acid-free bovine serum albumin overnight. After being washed with PBS, the cells were incubated with 0.5 ml of test samples (mouse HDL in DMEM+0.2% BSA) for 4 h at 37°C. Afterward, the supernatant was collected, and the cells were lysed with 0.1 N NaOH. Radioactivity that was associated with the supernatant and the cells, respectively, was then measured by liquid scintillation.

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].

---

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]

---

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.

---

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]

---

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

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

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.37 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.37 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.37 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)