A Combination of Capsaicin and Capsiate Induces Browning in 3T3

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Bioactive Constituents, Metabolites, and Functions

A Combination of Capsaicin and Capsiate Induces Browning in 3T3-L1 White Adipocytes via Activation of the PPAR#/ #3-AR Signaling Pathways Li Fan, Haiyan Xu, Rengui Yang, Yufan Zang, Jingfang Chen, and Hong Qin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02191 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 12, 2019

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Journal of Agricultural and Food Chemistry

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A Combination of Capsaicin and Capsiate Induces Browning

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in 3T3-L1 White Adipocytes via Activation of the

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PPARγ/ β3-AR Signaling Pathways

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Li Fan1, Haiyan Xu1, Rengui Yang2, Yufan Zang1, Jingfang Chen2, Hong Qin1,*

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1 Department of Nutrition Science and Food Hygiene, Xiangya School of Public

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Health, Central South University, 110 Xiangya Road, Changsha, Hunan

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Province, China, 410078

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2 Changsha Center for Disease Control and Prevention, Changsha, Hunan

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Province, China, 410004

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*Corresponding

author:

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[email protected]

Hong

Qin,

Tel:

+8615974222668,

1

ACS Paragon Plus Environment

E-mail:


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Abstract: This study investigated the effects and molecular mechanism of a

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combination of capsaicin and capsiate on promoting lipid metabolism and

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inducing browning in 3T3-L1 white adipocytes. The combination significantly

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suppressed lipid accumulation in adipocytes (P = 0.0190) and robustly

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improved lipid metabolic profiles, including the decreased TG (0.6703 ± 0.0385

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vs 0.2849 ± 0.0188 mmol/g of protein; P ＜ 0.0010), TC (0.1282 ± 0.0241 vs

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0.0651 ± 0.0178 mmol/g of protein; P = 0.0030) and LDL-C (0.0021 ± 0.0017

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vs 0.0005 ± 0.0002 mmol/g of protein; P = 0.0240) and increased HDL-C

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(0.0162 ± 0.0141 vs 0.1002 ± 0.0167 mmol/g of protein; P = 0.0120).

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Furthermore, this combination markedly upgraded the protein levels of CD36

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(P = 0.0070), ATGL (P = 0.0130) and phosphorylation of HSL at Ser660, 565

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and 563 (P ＜ 0.0010, P = 0.0270 and 0.0020, respectively), indicating

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increases of fatty acid transport and lipolysis. The levels of lipid metabolism

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regulators, phosphorylation of AMPKα and β (P = 0.0110 and P ＜ 0.0010,

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respectively), SIRT1 (P = 0.0040) and TRPV1 (P = 0.0140) were also increased

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by the combination. Moreover, the combination greatly activated the browning

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program in adipocytes, as demonstrated by increases in beige specific gene

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and protein. Further research found that the protein levels of PPARγ (P = 0.0010)

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and β3-AR (P = 0.0260) were elevated by the combination, and most of the

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beige specific markers were abolished by pretreatment of antagonists of PPARγ

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or β3-AR. In conclusion, these results indicated that a combination of capsaicin

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and capsiate could induce browning in white adipocytes via activation of the 2


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PPARγ/ β3-AR signaling pathway, and this combination might be worth

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investigating as a potential cure for obesity.

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Keywords: capsaicin, capsiate, combination, lipid metabolism, browning

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INTRODUCTION

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The globally increasing prevalence of obesity and its associated metabolic

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syndrome draw great attention to the need for developing effective strategies

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for obesity treatment. In recent years, an increasing number of reports have

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shown that promoting brown adipose tissue (BAT) like function in white adipose

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tissue (WAT) has therapeutic potential to combat obesity1-3. The “brown-like”

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cell within WAT has been called beige adipocyte. The beige adipocyte is

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characterized as high expression levels of uncoupling protein 1 (UCP1) gene

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and protein, which let it show the similar function of brown adipocyte in

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increasing thermogenesis and energy consumption4-5. Beige adipocytes

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emerge within the WAT in response to certain environmental cues, such as

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exercises, chronic cold exposure, peroxisome proliferator-activated receptor γ

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(PPARγ) agonists, and β adrenergic receptor (β-AR) agonists

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the “beige” phenotype using various dietary compounds is emerging as an

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alternative strategy to increase energy expenditure and obesity treatment10-11.

6-9.

Induction of

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Evidences have shown that some food agents naturally occurs in peppers

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seem to be promising candidates to target different medicinal strategies in the

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treatment of obesity12-15, even more, some of them have the potential ability to

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promote browning program in WAT16-18. Capsaicin and capsiate are derived

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from hot red peppers and sweet pepper fruit (CH-19 Sweet) respectively, and

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they have similar chemical structures19. The latest research showed that

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capsaicin inhibits adipogenesis via PPARγ activation and induces “beige” 4


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phenotype in 3T3-L1 cells20. However, its pungency and propensity for eliciting

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gastrointestinal side effects limited its use in clinical trials. Compared with

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capsaicin, capsiate has less pungent irritancy, while it showed similar or even

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more effective biological activity, such as improving insulin sensitivity, lipid and

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glucose metabolism, etc21-22. There were also studies showing that capsiate

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reducing body fat in humans was associated with activating brown fat

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thermogenesis18. However, the use and popularity of capsiate is not feasible

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due to its high cost and complicated extraction process.

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The current studies found that quite a few factors combining with capsiate

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or capsaicin could be more effective than them alone in promoting energy

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consumption and weight loss, such as cold exposure, exercises or some natural

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compounds23-25. Based on the above, we surmised that whether capsaicin

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combining with capsiate could exert synergistic effects in promoting lipid

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metabolism and browning of WAT. We therefore compared the effects of the

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combination of capsaicin and capsiate to each of them alone on lipid

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metabolism regulating and browning promotion in 3T3-L1 white adipocytes, and

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investigated the underlying mechanism.

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MATERIALS AND METHODS

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Chemicals.

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dexamethasone, 3-isobutyl-1-methylxanthine (IBMX) and SR 59230A (a kind of

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β3-AR antagonist) were purchased from Sigma Aldrich (St. Louis, MO).

Capsaicin

(97%

pure),

capsiate

5


(97.3%

pure),

insulin,


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T0070907 (a kind of PPARγ antagonist) was purchased from MedChem

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Express (Monmouth Junction, NJ). 3T3-L1 cells were obtained from the Peking

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Union Cell Center (Beijing, China). Triacylglycerol (TG), total cholesterol (TC),

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low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein

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cholesterol (HDL-C) kits were obtained from Jiancheng Bioengineering Institute

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(Nanjing, China). The goat anti-rabbit IgG antibody combined with horseradish

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peroxidase and the MTT assay kit were purchased from Ding Guo Changsheng

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Biotechnology Co., Ltd. (Beijing, China). Oil red O was purchased from Solarbio

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Science & Technology Co., Ltd. (Beijing, China). Antibodies against hormone

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sensitive lipase (HSL, A15686), phosphorylated HSL at Ser563 (pHSL-Ser563,

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AP0851), pHSL at Ser565 (pHSL-Ser565, AP0852), pHSL at Ser660 (pHSL-

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Ser660, AP0853), Sirtuin 1 (SIRT1, A0230), Adipose triglyceride lipase (ATGL,

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A6245), cluster of differentiation 36 (CD36, A1470), peroxisome proliferator-

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activated receptor γ coactivator 1-α (PGC-1α, A12348), PR domain containing

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16 (PRDM16, A11581), the vanilloid transient receptor subtype I (TRPV1,

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A8564), β3-AR (A8607) and β-actin (AC026) were purchased from ABclonal

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(Boston, MA). Antibodies against UCP1 (14670S), PPARγ (2443S), AMP-

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activated protein kinase α (AMPKα, 5831), AMP-activated protein kinase β1/2

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(AMPKβ1/2,

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phosphorylated AMPK β1 (pAMPKβ1, 4181) were purchased from Cell

4150),

phosphorylated AMPK

α

(pAMPKα,

2535)

and

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Signaling Technology, Inc. (Boston, MA).

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Cell Culture and Differentiation. 3T3-L1 cells were cultured in DMEM 6


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containing 10% (v/v) newborn calf serum and 1% penicillin-streptomycin

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solution at 37 °C under 5% CO2 atmosphere. Differentiation was induced in

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confluent cells by replacing newborn calf serum with fetal bovine serum.

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Confluent cells were maintained in differentiation induction medium consisting

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of 8 μg/mL of insulin, 1 μM dexamethasone, and 0.5 mM IBMX in DMEM. After

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48 h, cells were switched to the second differentiation medium consisting of 8

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μg/mL of insulin only for another 48 h. Then, cells were switched to

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maintenance medium for another 72 h with the medium replaced every 36 h

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until mature adipocytes formed. The mature adipocytes were treated with

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capsaicin (50 μM), capsiate (50 μM) or a combination of capsaicin (25 μM) and

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capsiate (25 μM) for 48 h. Then the cells were collected for further detection. In

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the experiments with the PPARγ and β3-AR antagonists, 3T3-L1 white

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adipocytes were pretreated with 10 μM T0070907 or 10 μM SR 59230A for 2 h

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prior to the combination of capsaicin and capsiate treatment.

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Cell Viability Assay. Pre-adipocytes were seeded in a 96-well plate at a density

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of 5.0 103 cells/well and incubated until mature adipocytes formed as

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described above. Then cells were treated with different concentrations of

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capsaicin (25, 50 and 100 μM), capsiate (25, 50 and 100 μM) or the combination

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of capsaicin and capsiate (12.5: 12.5, 25: 25 and 50: 50 μM) for 48 h. At the

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end of the incubation, 20 μL MTT solution was added to each well, and the cells

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were further incubated for 4 h. Then those media were removed and 150 μL

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DMSO was added for 10 min with shaking to resuspend the cells. Absorbance 7



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was measured at 570 nm by a microplate reader (Power Wave XS2, BioTek

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Instruments, Inc., Winooski, VT). Six replicate wells were used for each data

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point in the experiment.

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Lipid Content Assays. Intracellular lipid accumulation was quantified by

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staining with oil red O. The mature cells treated with capsaicin and/ or capsiate

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for 48 h were washed twice with PBS, fixed with 4% paraformaldehyde for 10

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min, and stained with oil red O working solution for 30 min. After the staining

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solution was removed, the cells were washed with 60% isopropanol and

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distilled water. The stained lipid droplets were visualized by an inverted

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microscope. The stained lipid droplets were dissolved in isopropanol and

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quantified for absorbance (540 nm) measurement. The levels of TG, TC, LDL-

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C and HDL-C were measured by commercially available kits.

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Western Blot Analysis. The mature cells treated with capsaicin and/ or

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capsiate for 48 h were harvested with RIPA buffer containing protease inhibitors.

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Protein contents were determined by an ultramicro spectrophotometer. Then,

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the proteins (40 μg) were separated by 8% or 10% SDS–PAGE and transferred

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to PVDF membranes. After being blocked with 5% skim milk or 3% BSA in TBST

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at room temperature for 1 h, the membranes were incubated with primary

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antibody overnight at 4 °C specific to TRPV1 (1:1000), β3-AR (1:1000), CD36

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(1:1000), ATGL (1:1000), HSL (1:1000), pHSL-Ser660 (1:1000), pHSL-Ser565

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(1:1000), pHSL-Ser563 (1:1000), UCP1 (1:1000), PPARγ (1:1000), SIRT1

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(1:10000), PGC-1α (1:10000), AMPKα (1:1000), pAMPKα (1:1000), AMPKβ1/2 8


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(1:1000), pAMPKβ1 (1:1000) and PRDM16 (1:1000). Membranes were washed

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in TBST and then incubated with the goat anti-rabbit IgG antibody (1:6000) for

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1 h at room temperature. The signal was detected by using the

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chemiluminescence imager (Tanon-5500, Tanon Science & Technology Co.,

150

Ltd., Shanghai, China). The relative expression of proteins was quantified by

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Tanon Gis software and calculated according to the reference bands of β-actin

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(1:300000). The data represented for the western blot analyses represent

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repetitions of the experiments using biologically different samples (n = 3).

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Quantitative real-time RT-PCR. Total RNA was isolated from mature cells

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treated with capsaicin and/ or capsiate for 48 h using a PureLink® RNA Mini Kit

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purchased from the Thermo Fisher Scientific (Waltham, MA). RNA (1μg) was

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converted to cDNA using HiScript® Ⅱ Q RT SuperMix for qPCR (Vazyme,

158

Nanjing, China). ChamQTM Universal SYBR® qPCR Master Mix (Vazyme,

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Nanjing, China) was employed to quantitatively determine transcription levels

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of genes with RT-PCR (LightCycler 480 Ⅱ, Roche ). The cycling conditions

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were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 60 °C

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for 30 s. The melting curve was also analyzed to ensure that only a single

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product was amplified and the conditions were as follows: 95 °C for 15 s, 60 °C

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for 1 min, and 95 °C for 15 s. PCR reactions were run in triplicate for each

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sample, and relative mRNA expression levels were calculated after

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normalization of values to that of β-actin. Sequences of primer sets used in this

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study are listed in Table 1. 9



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Statistical Analysis. All analyses were performed with SPSS 18.0 software

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(SPSS Inc., Chicago, IL). The data were expressed as means ± the standard

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deviation (SD). Differences among groups were calculated by one-way analysis

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of variance (ANOVA) or independent sample t test (two groups). Values of p

A Combination of Capsaicin and Capsiate Induces Browning in 3T3

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