G protein-coupled bile acid receptor 1 (TGR5) serves a key function in regulating glycometabolism. TGR5 is highly expressed in the mitochondria of brown adipose tissue (BAT) and downregulates adenosine triphosphate synthesis via the bile acid-TGR5-cyclic adenosine monophosphate-2-iodothyronine deiodinase (D2)-triiodothyronine-uncoupling protein pathway, thus regulating energy homeostasis and reducing body weight. Chenodeoxycholic acid (CDCA), the primary bile acid, is a natural ligand of TGR5. The present study aimed to characterize the ability of CDCA to reduce high-fat diet-induced obesity and improve glucose tolerance. A mouse model of diet-induced obesity was constructed. The results demonstrated that a high-fat diet significantly increased the weight of mice after 10 weeks (P<0.05), but following the addition of CDCA and continued feeding for another 10 weeks, a decrease in weight was detected and no significant difference in final weight was observed between the high fat diet group treated with CDCA and the group fed a normal diet. Furthermore, CDCA treatment significantly increased glucose tolerance (P<0.001, P<0.01 and P<0.01 at 15, 40 and 60 min after glucose injection, respectively) and significantly decreased serum insulin levels compared with mice fed a high-fat diet alone. Staining of the liver with hematoxylin and eosin and oil red O revealed that the CDCA-treated group exhibited significantly lower fat accumulation in BAT and WAT compared with mice fed a high-fat diet alone (P<0.001). Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that the expression of D2 activation system-related factors was significantly increased in BAT from mice treated with CDCA (P<0.001), confirming the role of TGR5 in modulating high-fat diet-induced obesity. In addition, CDCA inhibited adipocyte differentiation in 3T3-L1 cells and inhibited ligand-stimulated peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity. These results suggest that CDCA may prevent high-fat diet-induced obesity and hyperglycemia, and that these beneficial effects are mediated via the activation of TGR5 and inhibition of PPARγ transcriptional activity.
The obesity epidemic has become a major public health crisis, contributing to an upsurge in cases of hypertension, coronary heart disease and diabetes (
Mammalian adipose tissue is comprised predominantly of WAT, with a proportionally smaller amount of brown adipose tissue (BAT) (
G protein-coupled bile acid receptor 1 (TGR5), a G protein-coupled receptor highly expressed in the mitochondria of BAT, serves a key function in regulating glycometabolism (
Another cellular receptor that serves an important role in regulating glycometabolism is the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ). It has been demonstrated that the transcription factor PPARγ is essential for adipogenesis, coordinating the expression of hundreds of genes responsible for the development of mature adipocytes (
In the present study, a diet-induced obesity mouse model was used to assess the ability of bile acid ligands to reduce obesity induced by a high-fat diet and improve glucose tolerance. Immunohistochemical staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and ELISA assays were performed to analyze the effects of these ligands on body weight, glucose tolerance, serum insulin levels, hepatic fat tissue and the expression of cAMP, UCP2 and D2 in mouse fat tissue. The results of the current study suggest that TGR5 serves a key function in modulating high-fat diet-induced obesity.
CDCA was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Hematoxylin and eosin (H&E) and the serum insulin detection ELISA kit (cat. no. CSB-E05071m) were purchased from Roche Diagnostics (Indianapolis, IN, USA). All other reagents were purchased from Sigma-Aldrich; Merck KGaA.
A total of 15C57BL/6 wild-type male mice (age, 6 weeks; weight, 15–20 g) were purchased from the Model Animal Research Center of Nanjing University (MARC, Nanjing, China) were maintained in the Animal Resource Facility of the Animal Experiment Center at Fujian Medical University (Fuzhou, China). The mice were housed in a temperature-, humidity and light-controlled environment (25°C; 5.6%; 12-h light/dark cycle). For alimentary-induced obesity rodent models, mice in the high-fat diet (HF) group (n=10) were gavaged with high-lipid food (carbohydrate, 40%; protein, 13%; fat, 40%; other, 7%) and mice in the normal food diet (NF) group (n=5), which acted as the control, were fed with standard rodent chow (carbohydrate, 60%; protein, 22%; fat, 10%; other, 8%). Food and water were supplied
3T3-L1 cells were obtained from Procell Life Science Co., Ltd. (Wuhan, China). 3T3-L1 preadipocytes were cultured in medium A (Dulbecco's modified Eagle's medium; Sigma-Aldrich; Merck KGaA; supplemented with 15% fetal bovine serum; Sigma-Aldrich; Merck KGaA) at 37°C in an atmosphere containing 5% CO2. A total of 2 days after confluence was reached, cells were differentiated into adipocytes following the addition of differentiation medium (medium A containing 0.5 mM 3-isobuthyl-1-methylxantine, 1 mM dexamethasone, 10 mg/ml insulin and 5 mM pioglitazone hydrochloride) in the presence or absence of 5 µg/ml CDCA (day 0). After 2 days, the 3T3-L1 cells were transferred to adipocyte-growing medium (medium A containing 5 mg/ml insulin and 5 mM pioglitazone hydrochloride) in the presence or absence of 5 µg/ml CDCA, which was replenished every 2 days. Dimethyl sulfoxide was used as the vehicle control for the test compounds. On day 8, the differentiated adipocytes were stained with oil red O at room temperature for 10 min and photographed at ×40 magnification using an SP350 digital camera (Olympus Corporation, Tokyo, Japan) and optical microscope (Olympus BX51; Olympus Corporation).
To test the effect of different ligands on blood glucose metabolism, mice from each group that had been gavage for 20 weeks were administered with an intraperitoneal injection of glucose (2 g/kg). A micro blood glucose instrument (Ningbo Kingkerry Medical Instrument Co., Ltd., Ningbo, China) was used to monitor the changes in blood sugar between 0 and 120 min. Serum insulin levels were measured using an ELISA kit. A total of 20 µl serum was obtained at 0 and 120 min after intraperitoneal injection of glucose and processed via centrifuging 200 µl whole mouse blood obtained from the tail vein at 2,000 × g at 4°C for 10 min as described previously (
All mice were humanely euthanized with CO2 gas following 20 weeks treatment with the drug and gavage. Livers were harvested and a tissue sample from the right lobe of each liver was fixed in 4% PBS-buffered paraformalin at room temperature for 24 h. Livers were prepared as either paraffin sections or frozen sections and stained with H&E or oil red O, as described previously (
Total RNA was prepared from the tissue samples using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). First-strand cDNA was synthesized from the total RNA using Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Gene expression of cAMP, UCP2 and D2 was quantified by qPCR using an Applied Biosystems 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 20 ml reaction mixture contained 7.0 ml nuclease-free water, 1.0 ml cDNA (1 mg/ml), 1.0 ml (10 mM) each primer and 10.0 ml Maxima SYBR-Green/ROX qPCR Master Mix (2X) (Thermo Fisher Scientific, Inc.) and underwent the following thermocycling conditions: 95°C for 10 sec followed by 40 cycles of 94°C for 15 sec, annealing at 55°C for 30 sec, and a final extension at 70°C for 30 sec. The data was determined using default threshold settings and the mean Cq was calculated from the quintuplicate PCRs. The ratio of mRNA was calculated by using the equation 2−ΔCq, in which ΔCq=Cqtreatment-Cqcontrol (
Samples from 3T3-L1 cells lysates were isolated using radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Protein concentration was determined using the bicinchoninic acid protein assay. Subsequently, 60 µg/ml protein were subjected to 10% SDS-PAGE and transferred to a polyvinylidenedifluoride membrane. Membranes were blocked with TBST containing 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20, and incubated at 4°C overnight. Subsequently membranes were incubated with goat monoclonal anti-mouse PPARγ (1:1,000; sc-22020 P; Santa Cruz Biotechnology, Inc.), β-actin (1:5,000; sc-58673; Santa Cruz Biotechnology, Inc.). β-actin was used as an internal control for protein loading. The membrane was further incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000; sc-34665; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Membranes visualized using the enhanced chemiluminescence system. Densitometric analysis was performed using Scion Image 3.0 software (Scion Corporation, Frederick, MD, USA).
All data are presented as the mean ± standard error of the mean. The two-tailed Student's t-test was used to evaluate differences between data groups using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
To analyze the effect of different ligands on fat accumulation in mice fed a high-fat diet, a 20-week feeding program was followed. Mice were fed an NF or HF diet for 10 weeks, then some of the HF mice were treated with CDCA (5 g/kg) and feeding was continued for all mice for another 10 weeks. The body weights of the mice were measured each week for 20 weeks (
After 20 weeks feeding, the mice received an intraperitoneal injection with glucose (2 g/kg). Subsequently, blood glucose levels were monitored over a 120-min period to determine glucose tolerance (
Serum insulin levels were determined using ELISA (
To analyze fat deposition in the livers of the mice, liver sections taken from mice euthanized after 20 weeks feeding were stained with H&E and representative images are presented in
To determine the effect of TGR5 ligand treatmenton fat accumulation, WAT and BAT from liver sections taken from mice after 20 weeks of feeding were separated and lipid droplets were detected by oil red O staining. Representative images of liver sections from the different treatment groups (n=10) are presented in
To determine the effect of the TGR5 ligand CDCA at the molecular level, the mRNA levels of the oxidation-related factors UCP2, D2 and cAMP in the WAT and BAT of mice were measured by RT-qPCR (
The effect of CDCA on the transcriptional activity of PPARγ, a master regulator of adipogenesis, was evaluated in differentiating 3T3-L1 cells. As presented in
TGR5 is a membrane receptor that mediates bile acid signaling. In a previous study by the current authors, it was reported that TGR5 regulates glucose and energy metabolism and is therefore a candidate to combat obesity (
In a previous study by the current authors, it was hypothesized that different TGR5 ligands may trigger different downstream pathways and distinct gene expression profiles, leading to specific functional outcomes (
Another effect of TGR5 activation is the stimulation of glucagon-like peptide 1 (GLP1) secretion (
It is imperative that careful consideration is given to selecting the most suitable TGR5 ligand for drug development to ensure safety and efficacy. In the present study, the effects of CDCA was considered for weight reduction. It was determined that CDCA is effective at reducing weight, however optimizing the safety of CDCA is essential before it can be considered an anti obesity drug candidate.
A previous study has investigated the physiological role of FXR, another bile acid-activated receptor, and FXR agonists have been investigated for their potential in treating metabolic disorders (
The present study was supported by the National Natural Science Foundation of China (grant no. 81372092), Fujian Municipal Natural Science Foundation (grant no. 2011Y0029), Fujian Health Department Foundation (grant no. 2013-ZQN-ZD-15), Fujian Finance Department Foundation (grant no. 010110002), Research Foundation of Fujian Provincial Department of Science & Technology (grant no. 2016Y4003), Research Foundation of Fujian Development and Reform Commission (grant no. 201603), Key Program of National Clinical Specialty Discipline Construction of China and Key Clinical Specialty Discipline Construction Program of Fujian, China.
Effect of CDCA on mouse body weight, glucose tolerance and serum insulin levels in the drug-treated mice. Mice were fed with an NF or HF diet for 10 weeks before the addition of CDCA (5 g/kg) to the HF diet food. All mice were then fed for a further 10 weeks (n=5). (A) Body weight was measured each week from 0 to 20 weeks. (B) Body weights of each group at the end of the 20-week feeding period. *P<0.05. (C) After 20 weeks feeding, the mice were fasted for 16 h prior to intraperitoneal injection with glucose (2 g/kg), then blood glucose levels were measured over a 120-min time course by ELISA (n=5). *P<0.05, **P<0.01 and ***P<0.001 vs. NF. #P<0.05, ##P<0.01 and ###P<0.001 vs. HF. (D) Following the measurement of glucose tolerance, endpoint serum insulin levels were detected by ELISA. Data are presented as the mean ± standard error of the mean (n=5). *P<0.05. NF, normal food diet; HF, high-fat diet; CDCA, chenodeoxyclic acid.
Lipid deposition in the liver and adipose tissue. (A) Representative hematoxylin and eosin staining of liver sections from the mice following 20 weeks of feeding and drug treatment. Magnification, ×100 (top row); ×400 (bottom row). Black arrows indicate accumulated fat particles. (B) Liver weight analysis of the mice from different treatment groups. The results are presented as the mean ± standard error of the mean (n=5). (C) WAT and BAT in the livers of mice were separated and lipid droplets were detected by oil red O staining (magnification, ×400). (D and E) Relative lipid content in the liver for each of the treatment groups. Results are presented as mean ± standard error of the mean (n=10). *P<0.05, **P<0.01, ***P<0.001. NF, normal food diet; HF, high-fat diet; CDCA, chenodeoxyclic acid; WAT, white adipose tissue; BAT, brown adipose tissue.
RT-qPCR analysis of oxidation-related factors (D2 activation system) from WAT and BAT. The mRNA levels of fatty acid synthesis and oxidation-related factors cAMP, D2 and UCP2 from (A-C) WAT and (D-F) BAT were measured by RT-qPCR. Expression was measured relative to β-actin. Results are presented as mean ± standard error of the mean (n=5). ***P<0.001 vs. NF. NF, normal food diet; HF, high-fat diet; CDCA, chenodeoxyclic acid; WAT, white adipose tissue; BAT, brown adipose tissue; cAMP, cyclic adenosine monophosphate; D2, 2-iodothyronine deiodinase; UCP2, uncoupled protein 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
CDCA inhibits ligand-activated PPARγ transcription and lipid accumulation in differentiating adipocytes. (A) Results of western blotting indicating the expression of PPARγ in BAT. Results are presented as the mean ± standard error of the mean (n=5). (B) Western blot analysis indicating the effect of CDCA on PPARγ expression during adipogenesis in 3T3-L1 cells. The results are presented as the mean ± standard error of the mean (n=5). (C) Representative images of the differentiated adipocytes on day 8. Cells were stained with oil red O. Magnification, ×40. ***P<0.001. NF, normal food diet; HF, high-fat diet; CDCA, chenodeoxyclic acid; Control, without CDCA treatment; PPARγ, peroxisome proliferator-activated receptor γ.
Primers used in the present study.
Gene name | Forward primer | Reverse primer |
---|---|---|
cAMP | 5′-TATCACTGCTGCTGCTACTG-3′ | 5′-GCGGAGAAGTCCAGCCAGCC-3′ |
Ucp2 | 5′-GTGGTGGTCGGAGATACCAGA-3′ | 5′-GGGCAACATTGGGAGAAGTCC-3′ |
D2 | 5′-AGGACTGGAAGGGGTGATCC-3′ | 5′-CCGACCTGGACCTCAAAGC-3′ |
β-actin | 5′-GGCTGTATTCCCCTCCATCG-3′ | 5′-CCAGTTGGTAACAATGCCATGT-3′ |