Adipogenesis in murine preadipocyte 3T3L-1 has been used as a model system to study anti-obese bioactive molecules. During adipogenesis in 3T3-L1 preadipocytes, we found that capsanthin inhibited adipogenesis (IC50; 2.5 μM) and also showed lipolytic activity in differentiated adipocytes from the preadipocytes (ED50; 872 nM). We identified that the pharmacological activity of capsanthin on adipogenesis in 3T3-L1 was mainly due to its adrenoceptor-β2-agonistic activity. In high-fat diet animal model study, capsanthin significantly enhanced spontaneous locomotive activities together with progressive weight-loss. The capsanthin-induced activation of kinetic behavior in mice was associated with the excessive production of ATP initiated by both the enhanced lipolytic activity together with accelerated oxidation of fatty acids due to the adrenoceptor β2-agonistic activity of capsanthin. Capsanthin also dose-dependently increased adiponectin and p-AMPK activity in high fat diet animals, suggesting that capsanthin has both anti-obesity and insulin sensitizing activities.
The use of red pepper (
This study was directed to elucidate whether capsanthin affects adipocyte-related biological functions like capsaicin. The effect of capsanthin on adipocyte functions was determined in the adipogenesis model of murine preadipocyte cell line 3T3-L1. In addition, the pharmacological activity of capsanthin was validated with high-fat diet-induced obesity mouse models. In this study, we first demonstrated that capsanthin may have both anti-obese and anti-diabetic potentials.
Red pepper powder (10 kg) was extracted 3 times with ethyl acetate (EtOAc, 50 L) by refluxing under nitrogen replacement for 3 hours for each extraction processes and vacuum evaporated to give 1.53 kg of EtOAc extract
Capsanthin di-fatty-acyl-ester 10 mg dissolved in absolute ethanol was treated by heated trans-esterification with catalytic amount of NaOCH3 under N2-replacement. After appropriate dilution of the reaction mixture with hexane, the hexane layer was analyzed by gas liquid chromatography (GLC) to find the composition of fatty-acyl-ethyl esters; lauric acid 8.7%, myristic acid 50.6%, palmitic acid 40.7%. GLC conditions; HP-5890-II Series, detector; FID, column; DB-23 capillary (Agilent technologies [Santa Clara, CA, USA], 60 m, 0.25 mm ID, 0.25 μm), Oven Temp. initial 130°C, 2.7°C/min gradient to ∼230°C, 3 min, inlet Temp.; 270°C, detector Temp.; 300°C, carrier gas; N2, 30psi, split ratio; 1/50, flow rate; 30 ml/min.
The cell line 3T3-L1 pre-adipocytes was purchased from the Korean Cell Line Bank. Cells were cultured (Zebisch
The fully differenciated adipocytes under the presence of capsanthin was centrifuged at 3000 g for 10 min to harvest the cultured cells. Total RNA was isolated from cultured cells using GeneAll Ribosspin (Cat. No. 304-150; GeneAll Biotechnology, Co., ltd., Seoul, Korea). The extracted RNA was quantitated by absorbance using a nanodrop spectrophotometer (Maestrogen nanodrop) and processed for RT-PCR (Kong and Park, 2008). All PCR primers were obtained from BioNEER (Daejeon, Korea) which included (Farmer, 2006; Lin and Lane, 1994; Fig. 2A)
PPARγ (F:GAGATGCCATTCTGGCCCACCAACTTCGG, R:TATCATAAATAAGCTTCAATCGGATGGT TC)
C/EBPα (F:TCATCCACTTCACCAGTGACAA, R:AAACCATCCTCTGGGTCTCC).
Cellular proteins were extracted from control and capsanthin treated 3T3-L1 cells. Cells were collected by centrifugation and washed once with phosphate buffered saline (PBS). The washed cell pellets were resuspened in extraction-RIPA-lysis buffer and incubated for 60 min at 4°C, with gentle shaking. Cell debris was removed by centrifugation (15000 g), followed by quick freezing of the supernatants for 10 min at 4°C. The cellular protein from treated and untreated cell extracts were electroblotted onto a nitrocellulose membrane following separation on 10% SDS-polyacrylamide gel electrophoresis (Mahmood and Yang, 2012). The immunoblot was incubated overnight with blocking by 5% skim milk at 4°C, followed by incubation with diluted polyclonal antibodies against p-AMPK (Lizcano
All of the isolated four carotenoids from the extract of red pepper together with other four purchased carotenoids were subjected to in vitro evaluation of anti-adipogenic activity by using 3T3-L1 cell culture system to obtain IC50-values for the anti-adipogenic activities and ED50-values for the lipolytic potentials. The cells were cultured in the same way as described above and after 4 times replacement of induction media (8 days incubation), the culture system was treated with 10 μM capsanthin in 0.5% DMSO and incubated for two days. Following that lipolytic activity of capsanthin were assessed by staining triglycerides in the attached cells on plate with ORO. The attached cells (capsanthin treated) were fixed with 10% paraformaldehyde for 1 h. After being washed well with PBS (pH 6.8), cells were incubated with ORO for 2 h at 37°C under 5% CO2. Then, the plate was rinsed thoroughly with PBS at least 5 times to remove unbound ORO. ORO was washed with PBS buffer 3 times. The triglyceride bound ORO was extracted with isopropanol and assayed the ORO-content. The extracted ORO was transferred to 96 well plates and absorbances were measured using ELISA reader (at 510 nm). Potent lipolytic activity was observed, hence ED50-value was determined as 872 nM for free capsanthin and 9.80 μM for ester-form capsanthin by serial dilution assay techniques (Table 1).
The lipolytic activities of 20 μM capsanthin under the presence of each 5 μM of various adrenoceptor-antagonist i.e.; α1-antagonist (doxazosin-mesyate), α2-antagonist (yohimbrin. HCl), β1-antagonist (metoprolol-tartrate), β2-antagonist (C118,551.HCl) and β3-antagonist (SR59230A) (Sigma Aldrich Co.), were assayed by incubation overnight at 37°C, 5% CO2 atmosphere. Triglycerides in adipocyte were stained by incubation with ORO. Unbound ORO was washed with PBS buffer 3 times. The triglyceride bound ORO was extracted with isopropanol and assayed the ORO content by HPLC (Fig. 2B). HPLC (waters 486) condition: wavelength 450 nm, eluent acetonitrile : water (9:1), column Mightysil RP-18 GP (150×4.6 mm, 5 μM; Kanto Co., Inc., Tokyo, Japan; Fig. 2B).
Female C57BL/6C mice (four weeks) were obtained from Orientbio Co (Seongnam, Korea). The mice were fed a laboratory chow and water
Single mouse was accommodated in a cage which was equipped with a wheel on which one way running exercise is possible together with automatic digital counting system. Each mouse in the cage is freely accessible to water and high fat diet. Animal cages equipped with same facilities were order-made to test the spontaneous locomotive activities of each mouse. Thirty five mice were allocated to five specific groups consisting of seven mice per group; Group-1 (control group); high fat diet (HFD)+cooking oil 50 μL, Group-2 (positive control group); HFD+capsaicin 1 μmol, Group-3; HFD+capsanthin 1 μmol, Group-4; HFD+capsanthin 5 μmol and Group-5; HFD+10 μmol capsanthin per day, respectively. All capsanthin samples were orally administered as the cooking oil solution. The test was conducted during 14 days. Every each other day afternoon at 1:00 PM the cumulative data of spontaneous locomotive activities of each mouse (running score) during past 24 h together with the body weight of each mouse were recorded and the digital counters were reset. The average values for the seven mice/group for running score (Fig. 3A) and average body weights were recorded on (Fig. 3B).
After the completion of running experiments the mice were anesthetized with diethyl ether by the bell-jar technique and sacrificed by decapitation to collect trunk blood. To prepare serum, the blood was clotted at 4°C overnight and the clotted blood was centrifuged at 3000 g for 20 min. Serums were stored at −80°C until analysis. At the end of feeding experiment the weight of abdominal fat pads were measured after laparotomy operation (Fig. 3C).
LDL-cholesterol, HDL-cholesterol, total cholesterol, ketone body, adiponectin, alanine-aminotransferase (ALT) TNF-α and p-AMPK content in the mouse serum participated to the spontaneous locomotive activity tests were assayed according to the instruction manuals of respective assay kits; LDL-cholesterol assay kit (#5607-02, Bio Scientific, MD, USA), HDL-cholesterol assay kit (#5607-01, Bio Scientific, MD, USA), total cholesterol assay kit (STA-390, Cell Biolabs, San Diego, CA, USA), ketone body assay kit (EKBD-100, Bioassay system, Hayward, CA, USA), adiponectin assay kit (R&D system, Minneapolis, MN, USA) and TNF-α ELISA Kit (KMC3012, Invitrogen, Carlsbad, CA, USA).
All data are recorded with mean value ± standard deviation calculated by Origin Program (Origin ver. 8.0, Origin Lab, Northampton, MA, USA).
EtOAc extract of red pepper is composed of large amount of triglycerides, fairly good amount of capsaicin and a little amount of unstable carotenoids. After evaporation of EtOAc, intense red oily liquid was obtained. We could eliminate capsaicin which has already been reported as a potent anti-adipogenic substance (Diepvens
C/EBPα and PPARγ are well known as the most reliable gene-expression factors for the adipocyte differentiation. It is well known fact that down regulation of both expression factor C/EBPα and PPARγ (Kong and Park, 2008) are concerned with the inhibition of differentiation from preadipocyte to adipocyte (Fig. 2A, Table 1). As shown in Table 1, capsanthin (free form) shows the most potent anti-adipogenic and lipolytic activity, however, esterified form capsanthin showed very weak activity. The other xanthophyll components showed very weak activities for both the inhibition of adipocyte differentiation and for the lipolytic potencies. During the incubation of 3T3-L1 cells with free capsanthin, we found a thick layer of free fatty acids floating on the surface of cell culture system due to the powerful lipolytic activity of capsanthin.
In order to see the mode of capsanthin-action on the lipolytic activity, 3T3-L1-cell cultures were treated with capsanthin under the presence of various adrenoceptor-antagonists as doxazocin (α1), yohimbrin·HCl (α2), metoprolol.tartrate (β1), ICI118,551·HCl (β2) and SR59230A (β3) (Otton
Capsanthin fed mice showed highly enhanced spontaneous locomotive activities with dose dependency (Fig. 3A), and continuously progressing weight loss during 14 days of experimental period (Fig. 3B), however, capsaicin fed animals (positive control group) showed unexpectedly no spontaneous locomotive activity rather obvious sleeping behavior all through the experimental period (Fig. 3A) and body weight change showed first 4days slight loss and thereafter until the end of experimental period the positive control group showed highest increase of body weight (Fig. 3B). Hence, the highly activated spontaneous locomotive activity of capsanthin group must be due to the increased production of ATP as the result of increased oxidation of fatty acids in muscle cell (Otton
At the end of capsanthin feeding experiments, all mice were sacrificed to obtain mice serum and to see the laparotomic view for visceral fat contents and found that capsaicin fed mice (positive control group) showed large size of white adipose tissue pad (WAT), however, capsanthin fed mice showed only highly shrinked brown adipose tissue (BAT) instead of WAT as shown in Fig. 3C. These results suggest that the anti-adipogenic activity of capsanthin may be directly concerned with anti-obesity activity of red pepper, however, capsaicin’s thermogenic propertiy is not concerned with anti-obesity activity in our present experiments.
Enhanced lipolytic activity of capsanthin will induce temporary overflow of fatty-acids in the blood stream which might influence negatively due to cyto-toxicities (Shimabukuro
Capsanthin is contained in red pepper or paprika in the form of long chain fatty-acyl-esters (diacyl-ester; 80.8%, monoacylester; 17.2%, free-form; 2.0%) (Schweiggert
By the way to the capsanthin studies, we found an unexpected example in which the well known thermogenic (Diepvens
Capsanthin the red xanthophyll pigment shows potent anti-adipogenic, lipolytic and fatty-acid burning activities due to its potent adrenoceptor-β2-agonistic activity. In the animal feeding experiment, mice showed highly enhanced spontaneous locomotive activity due to the excessive production of ATP from activated burning fatty acid. Together with sustained weight loss. Capsaicin which was adopted as the positive control in our experiment is highly anti-adipogenic and thermogenic substance, hence, showed no spontaneous locomotive activity and showed rather sleeping behaviors due to the deficit of ATP and showed no weight-loss, but rather weight increase. Capsanthin is potent anti-adipogenic but not thermogenic substance, hence it may be a good candidate for the development of new bioactive agent effective as a new anti-obese or insulin sensitivity enhancing substance.
The authors are deeply appreciating to our government, The Ministry of Science and Technology, Republic of Korea, for the financial support (Grant No. A004400069).