Activation of transient receptor potential vanilloid 3 channel suppresses adipogenesis.

ENERGY

BALANCE-OBESITY

Activation of transient receptor potential vanilloid 3 channel (TRPV3) suppresses adipogenesis Sin Ying Cheung1; Yu, Huang2; Hiu Yee,Kwan3; Hau Yin, Chung1*; Xiaoqiang Yao2* 1 Food and Nutritional Sciences, School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China.; 2 Li Ka Shing Institute of Health Sciences and School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, China.; 3Centre for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China. Key terms: TRP channels, adipogenesis, insulin

The present study shows activation of the transient receptor potential vanilloid 3 channel (TRPV3) suppresses adipocyte differentiation. We also identified that a major functional catechin compound in green tea and cocoa, (-)-epicatechin, exerts anti-adipogenic effects in the adipocytes through direct activation of TRPV3. TRPV3 was detected in the 3T3-L1 adipocytes using immunohistochemistry and semi-quantitative PCR. TRPV3 activation by activators (-)-epicatechin and diphenylborinic anhydride (DPBA) was determined using live cell fluorescent Ca2⫹ imaging and patch clamp electrophysiology. Using RNA interference, immunoblotting and Oil-red-O staining, we found that the TRPV3 agonists prevented adipogenesis by inhibiting phosphorylation of insulin receptor substrate 1 (IRS-1), downstream phosphoinositide 3-kinase (PI3K)/Akt/forkhead box protein O1 (FOXO1) axis, and the expression of adipogenic genes peroxisome proliferator-activated receptor gamma (PPAR␥) and CCAAT/enhancer-binding protein alpha (C/EBP␣). TRPV3 overexpression hindered adipogenesis in the 3T3-L1 cells. In vivo studies showed that chronic treatment of the TRPV3 activators prevented adipogenesis and weight gain in the mice on high fat diets. Moreover, TRPV3 expression was reduced in the visceral adipose tissue from mice on high fat diets, obese (ob/ob) and diabetic (db/m⫹) mice. In conclusion, our study illustrates the anti-adipogenic role of TRPV3 in the adipocytes.

O

besity is a public health concern and is defined as increased fat mass due to hypertrophy and hyperplasia of adipocytes (1). Adipogenesis plays a key role in the growth of fat mass. It is defined as a process by which undifferentiated preadipocytes in the white adipose tissue convert to differentiated adipocytes. The white adipose tissue serves as energy storage for triglycerides. It secretes adipokines and regulates energy homeostasis (2). Therefore, it is pivotal to understand signal pathways that regulate adipogenesis. It assists the development of clinical therapies that target obesity and its associated pathologies (3). Emerging evidences suggest the importance of Ca2⫹ signaling in the adipocytes (4, 5). In particular, members

of the transient receptor potential channel (TRP) family, a class of Ca2⫹-permeable channels; are known to play regulatory roles in adipocyte function and cardiovascular health. For instance, TRPV1 was suggested to prevent adipogenesis (6). A role in the adipose sensory nerve was also reported (7). The heteromeric TRPC1/C5 channel negatively regulated adiponectin secretion in the adipocytes (8). TRPM7 facilitated adipocyte proliferation and differentiation in the 3T3-L1 preadipocytes (9). A previous report also demonstrated that the 3T3-L1 preadipocytes expressed higher levels of TRPV1, TRPV3, TRPM8, TRPC4 and TRPC6 than the differentiated adipocytes (10). The authors hypothesized that these channels may function to regulate adipogenesis in the adipocytes.

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2015 by the Endocrine Society Received October 11, 2014. Accepted March 11, 2015.

Abbreviations:

doi: 10.1210/en.2014-1831

Endocrinology

endo.endojournals.org

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 March 2015. at 19:39 For personal use only. No other uses without permission. . All rights reserved.

1

2

TRPV3 and adipogenesis

(-)-Epicatechin is a green tea polyphenol. Although its health benefits have not been extensively investigated, various sources of evidences from in vitro and in vivo models suggested that administration of tea catechins comprising (-)-catechin 3-gallate, (-)-epicatechin, (-)-epigallocatechin and (-)-epigallocatechin-gallate promoted antiadipogenic and antiobesity effects (11, 12). Despite downregulation of PPAR␥ and C/EBP␣ was preliminarily suggested, the exact mechanism of action was not thoroughly investigated (13). Our study proposed that the Ca2⫹-permeable nonselective cation channel TRPV3 (14) plays a role in the adipocyte by suppressing adipogenesis. We showed that (-)-epicatechin and an established TRPV3 agonist diphenylborinic anhydride (DPBA), a synthetic phenyl borate derivative (15); promoted antiadipogeneic effects in vitro and in vivo through TRPV3 activation. We also extensively validated the downstream antiadipogenic pathway of TRPV3.

Materials and Methods Cell culture, adipocyte differentiation and RNA interference (RNAi) HEK293 cells (passage 8 –20) (R70507, Life Technologies) and 3T3-L1 preadipocytes (passage 5–14) (93061524, Sigma) were cultured in full Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) containing DMEM, 10% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies). All cells were maintained at 37°C in a humidified incubator supplying 95% O2 and 5% CO2, and subcultured at ⬍ 80% confluence. At 100% confluence, the 3T3-L1 cells were induced to differentiate in full DMEM supplemented with 10 ␮g/mL insulin, 0.5 mmol/L 3-isobutyl-1-methylxanthine and 0.25 mmol/L dexamethasone for 48 hours. Medium was replaced with 10 ␮g/mL insulin in full DMEM for another 48 hours. Medium was renewed with full DMEM every 2 days until day 8 postdifferentiation. Lipid accumulation was detected using Oil-red-O staining (O0625; Sigma). For spectrophotometric analysis of Oil-red-O staining, Oil-red-O was eluted with 100% isopropanol and quantified at an emission wavelength of 580 nm. TRPV3 siRNA (RiboBio) was transfected into the 3T3-L1 cells using Lipofectamine®2000 (Life Technologies) and OptiMEM® (Life Technologies). The cells were at 70% confluence. Experiments were conducted 24 –72 hrs after transfection. To construct 3T3-L1 cells stably expressing TRPV3, the cells were transfected at 70% confluence with TRPV3-PCDNA6/V5His utilizing Lipofectamine®2000 and Opti-MEM®. The cells were selected in full DMEM containing 5 ␮g/mL blasticidin (Life Technologies) for 6 days. LY294002 (#9901) was from Cell Signaling Technology. (-)Epicatechin (E4018), diphenylborinic anhydride (358835) and 2,5-dimethyltetrahydrofuran (D187208) were from Sigma.

Lentiviral vector production and transduction A lentiviral expression vector containing TRPV3 (pGPU6/ GFP/Neo-TRPV3) (GenePharma), the human immunodefi-

Endocrinology

ciency virus (HIV) packaging plasmid p8.2 and the vesicular stomatitis virus glycoprotein envelope plasmid pMD. G were cotransfected into HEK293 cells. Viruses were harvested and titers were calculated as described (16). For viral transduction, the lentiviral vectors were added into the 3T3-L1 cells with 8 ␮g/mL Polybrene (Sigma). Cell cultures were subsequently centrifuged for 60 minutes at 300 x g, 30°C; incubated for 18 hrs at 37°C in a humidified incubator and washed twice with PBS. After 5 days of transduction, 450 ␮g/mL G418 geneticin (Invitrogen) was added into the culture medium for selection of infected cells. Adipocyte differentiation was performed 7 days after. In a typical experiment, approximately 100% of the cells were transduced.

Quantitative real-time PCR Total RNA was extracted from the 3T3-L1 adipocytes using Trizol reagent (Invitrogen) and synthesized into cDNA using Superscript II reverse transcriptase (Invitrogen). SYBR Green (Applied Biosystems) was used for qPCR array. TRPV1, TRPV2, TRPV3, TRPV4, TRPV5 and TRPV6 expressions were normalized with ␤-actin. Primer sequences were as follows (10, 13): TRPV1: Forward 5⬘-GCATCTTCTACTTCAACTTCTTCGTC-3⬘; Reverse 5⬘-CCAATACTCCTTGCGATGGC-3⬘. TRPV2: Forward 5⬘-AGCAGTGGGATGTGGTAAGCTA-3⬘; Reverse 5⬘TTTGTTCAGGGGCTCCAAAACG-3⬘. TRPV3: Forward 5⬘CAAGGACTGCCACCACCATC-3⬘; Reverse 5⬘CATCACAGTTGCCAGAGAGG-3⬘; TRPV4: Forward 5⬘GAGTCCTCAGTAGTGCCTGG-3⬘; Reverse 5⬘CAACAAGAAGAAGAGAGCAGTC-3⬘. TRPV5: Forward 5⬘CGTTGGTTCTTACGGGTTGAA-3⬘; Reverse 5⬘GTTTGGAGAACCACAGAGCCTCTA-3⬘. TRPV6: Forward 5⬘-TTGAGCATGGAGCTGACATC-3⬘; Reverse 5⬘-TCTG␤-Actin: Forward 5⬘CATCAGGTGCTGAAAC-3⬘. GACAACGGCTCCGGCATGTGCAAAG-3⬘; Reverse 5⬘-TTCACGGTTGGCCTTAGGGTTCAG-3⬘.

Histology Visceral adipose tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The tissue sections were stained with hematoxylin-eosin. Images were analyzed using ImageJ software (National Institute of Health).

Immunoprecipitation and immunoblotting Immunoprecipitation experiments were performed using protein-G agarose beads (Roche) in accordance with the manufacturer’s instructions. In immunoblotting analysis, whole-cell lysates were extracted using standard radio-immunoprecipitation assay buffer with proteinase cocktail inhibitors (Roche). Samples were loaded in SDS-PAGE gels, transferred to polyvinyl difluoride membrane (GE Healthcare) and blocked in 2% BSA. Membranes were incubated with primary antibodies at 4°C overnight, washed and incubated with horseradish peroxidaseconjugated secondary antibodies (Santa Cruz Biotechnology). ECLTM Plus Western blotting detection system (GE Healthcare) and the X-ray film processor (Fujifilm) was used for image detection. Protein band intensities were analyzed using ImageJ software (National Institute of Health). Antibodies were purchased from Cell Signaling Technology, Abcam or Santa Cruz Biotechnology.


doi: 10.1210/en.2014-1831

Electrophysiology Patch clamp recordings from HEK293 and 3T3-L1 cells were made in whole-cell configuration (17, 18). The extracellular solution consisted of (mM): 140 NaCl, 10 glucose, 1 CaCl2, 0.5 MgCl2 and 10 HEPES, pH 7.4 (adjusted with NaOH). The intracellular solution consisted of (mM): 120 cesium aspartate, 10 CsCl, 1 MgCl2, 5 EGTA and 10 HEPES, pH 7.4 (adjusted with CsOH). Currents were recorded using a ramp protocol lasting 200 milliseconds from –100 to ⫹100 mV. The holding potential was – 60 mV. Currents were sampled at 5 kHz and filtered at 1 kHz. Data acquisition was performed using ‘PulseFit’ software linked to an EPC-10 amplifier (HEKA Elektronik) and analyzed with PulseFit 8.7 software.

Ca2ⴙ imaging HEK293 and 3T3-L1 cells were seeded on glass cover slips and cultured in full DMEM. On the following day, cells on cover slips were loaded with 5 ␮M Fluo-4 AM (Life Technologies) and 0.02% Pluronic acid F-127 (Life Technologies) for 30 minutes in normal physiological saline solution (NPSS) containing (mM): 1 CaCl2, 140 NaCl, 5 KCl, 1 MgCl2, 10 glucose, and 5 HEPES, pH 7.4 (adjusted with NaOH). Cover slips were mounted individually onto imaging chambers. Cells were illuminated under an excitation wavelength of 488 nm using a confocal fluorescence system (Fluoview F1000, Olympus). Changes in intracellular Ca2⫹ ([Ca2⫹]i) fluorescence intensity were monitored real time at room temperature (20 - 22°C). Ratios of [Ca2⫹]i fluorescence over baseline (F1/F0) were calculated. F1 denotes [Ca2⫹]i fluorescence during recording. F0 denotes [Ca2⫹]i fluorescence at baseline. 0Ca2⫹-PSS was similar to NPSS, excluding Ca2⫹ and added with 0.2 mM EGTA.

Mice This study was approved by the Animal Experimentation Ethics Committee, Chinese University of Hong Kong (CUHK). All mice were supplied by the CUHK Laboratory Animal Service Centre. Three weeks old male wild-type adolescent C57BL/6J mice were separated into 4 treatment groups: (1) control crude diet (D12490B; Research diets); vehicle solution containing tap water and 0.05% tween 80 (Sigma), (2) High fat diet (HF) (D12492; Research diets); vehicle solution containing tap water and 0.05% tween 80, (3) HF, 1 mg/kg/d (-)-epicatechin in tap water and 0.05% tween 80 and, (4) HF, 0.1 mg/kg/d DPBA in tap water and 0.05% tween 80. Drugs were administered daily using an oral gavage. Body weight and food intake were measured every 3 days. Nine weeks old male ob/ob and db/m⫹ mice were supplied by the CUHK Laboratory Animal Service Centre and euthanized together with the treated mice after six weeks of drug treatment. Euthanasia was performed using carbon dioxide asphyxiation. Tissues specified were collected and processed immediately. In accordance with the manufacturers’ instructions, free fatty acid levels in the plasma were measured by Free Fatty Acid Quantification Kit (ab65341; Abcam). Triglyceride levels in the plasma and the liver were measured by Triglyceride Quantification Kit (ab65336; Abcam).

Statistical analysis We utilized unpaired t tests for two group comparison and one-way ANOVA with Newman-keuls for multiple group comparison. Statistical analysis was performed using GraphPad


3

Prism 5.01. Values were calculated as mean ⫾ SD unless specified. Values of P ⬍ .05 were considered significant.

Results TRPV3 is expressed in the 3T3-L1 preadipocytes We detected TRPV3 expression in the 3T3-L1 preadipocytes and differentiated adipocytes using immunohistochemistry (Figure 1A) and qPCR (Figure 1B). In agreement with a previous report (10), our qPCR results indicated greater expression of TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6, and comparable expression of TRPV5 in the 3T3-L1 preadipocytes than the differentiated adipocytes (Figure 1B). (-)-Epicatechin (EC) activates TRPV3 We speculate that some tea catechin compounds may influence the activity of particular TRP channels and facilitate antiadipogenic effects in the adipocytes. By using Ca2⫹ imaging, we screened individual tea catechins and observed whether they are modulators of TRP channel activity. In particular, we identified that (-)-epicatechin enhanced intracellular Ca2⫹ fluorescence ([Ca2⫹]i) in the HEK293 cells expressing TRPV3 (NPSS) but not in the cells expressing TRPV2 (PCDNA6-TRPV2), TRPV4 (PCDNA6-TRPV4) or the empty vector construct (PCDNA6). The TRPV3 inhibitor 2,5-dimethyltetrahydrofuran (DPTHF, 100 ␮M) (17) suppressed the effects of (-)-epicatechin in the TRPV3-transfected HEK293 cells. Perfusion of the cells in bathing solution devoid of extracellular Ca2⫹ (0Ca2⫹-PSS) also prevented the (-)-epicatechin-mediated responses in the adipocytes (Figure 1C, D). Consistent with Ca2⫹ imaging, whole-cell patch clamp recordings suggested that (-)-epicatechin evoked cationic whole-cell currents in the TRPV3-transfected HEK293 cells (TRPV3 ⫹ EC (30 ␮M)) but not in the vector-transfected cells (PCDNA6 ⫹ EC (30 ␮M)). In agreement with previous reports (17, 18), the currents were TRPV3-like and outwardly rectifying. The (-)-epicatechin-mediated whole-cell currents were inhibited by DPTHF (100 ␮M) (Figure 1E, F). Thus, our results demonstrated that (-)epicatechin activates TRPV3. TRPV3 activators enhanced [Ca2ⴙ]i and whole-cell currents in the 3T3-L1 preadipocytes Current knowledge regarding the regulation of adipogenesis is primarily investigated through the use of cultured cell models, such as the 3T3-L1 fibroblasts (19). Hence, by utilizing the 3T3-L1 cell model, we sought to determine whether TRPV3 participates in the regulation


4


of adipogenesis in vitro. We used Ca2⫹ imaging and whole-cell patch clamping to validate the functional prop-

Endocrinology

erties of TRPV3 in the preadipocytes. DPBA is an established TRPV3-specific agonist (17, 18). Both (-)-epicat-

Figure 1. (-)-Epicatechin is a TRPV3 agonist. A, Immunostaining of TRPV3 (green) (n ⱖ 4) in the 3T3–L1 cells, preadipocytes (PA) and differentiated adipocytes (A). The scale bar is 10 ␮M. B, A summary of the relative expression of TRPV channels (PA/A) in the 3T3–L1 cells (n ⫽ 6). C. Representative [Ca2⫹]i response showing (-)-epicatechin (EC, 30 ␮M) evoked [Ca2⫹]i rise in the HEK293 cells expressing TRPV3 ([Ca2⫹]i F1/F0) (n ⫽ 5 cells). D. A summary of data showing fold increase in [Ca2⫹]i (F1/F0) in the HEK293 cells transfected with TRPV3 or vector PCDNA3. Mean ⫾ SD (n ⱖ 5 experiments, ⱖ 20 cells per experiment). *** P ⬍ .001 vs. NPSS ⫹ 30 ␮M EC. E. A representative trace showing EC-activated TRPV3-like whole-cell current in a TRPV3-overexpressing HEK293 cell. F. A summary of data showing the changes in current density amplitudes at ⫹80 mV. EC did not have an effect on the PCDNA3-transfected cells. EC significantly elicited whole-cell currents in the HEK293 cells expressing TRPV3. Mean ⫾ SD (n ⫽ 5 - 6). ***P ⬍ .001.


doi: 10.1210/en.2014-1831

echin (30 ␮M) and DPBA (50 ␮M) enhanced [Ca2⫹]i in the 3T3-L1 preadipocytes. The effects were abrogated by the TRPV3 blocker DPTHF (100 ␮M), TRPV3 siRNAs, or in


5

the absence of extracellular Ca2⫹ (0Ca2⫹-PSS) (Figure 2A-C). EC and DPBA elicited whole-cell currents in the

Figure 2. TRPV3 activators in the 3T3–L1 cells. A and B, Representative [Ca2⫹]i response traces recorded from the 3T3–L1 preadipocytes (n ⫽ 5 cells). C. A summary of data showing fold increase in [Ca2⫹]i (F1/F0) in the 3T3–L1 cells. Mean ⫾ SD (n ⱖ 5 experiments, ⱖ 20 cells per experiment). ***P ⬍ .001. D and E. A representative trace showing EC or DPBA-mediated whole-cell current in a 3T3–L1 preadipocyte. F. A summary of data showing mean current density amplitudes at ⫹80 mV. Mean ⫾ SD (n ⫽ 4 - 5). ***P ⬍ .001.


6


3T3-L1 preadipocytes. The current-voltage relationships (I-V) were less outwardly rectifying than that in the HEK293 cells. It was possibly due to TRPV3 activation by saturating concentrations of the agonists, which resulted in relatively linear current-voltage relationships (I-V) (also known as I2 mode) in the 3T3-L1 cells (17) (Figure 2D, E). The (-)-epicatechin and DPBA-mediated TRPV3 currents were abolished by DPTHF (100 ␮M) and TRPV3-siRNAs (Figure 2F). TRPV3 activators prevented lipid accumulation and adipogenesis in the 3T3-L1 preadipocytes To determine the function of TRPV3 in adipogenesis, the 3T3-L1 preadipocytes were induced to differentiate in the presence of the TRPV3 activators. The cells were treated with 0, 1, 3, 10 and 30 ␮M (-)-epicatechin or 0, 1, 5, 10 and 50 ␮M DPBA every 2 days up to day 8 postdif-

Endocrinology

ferentiation. The TRPV3 activators (-)-epicatechin and DPBA inhibited adipogenesis in a dose-dependent manner. The responses were reversed by DPTHF (100 ␮M) and TRPV3 shRNA (Figure 3 A, B). The TRPV3 shRNA was highly effective in suppressing TRPV3 expression in the 3T3-L1 cells (Figure 5A, B) TRPV3 activators downregulated the phosphoinositide 3-Kinase (PI3K)/Akt pathway and the expression of adipogenic genes The TRPV3 agonists did not alter TRPV3 expression in the adipocytes (Figure 4A). However, they dose-dependently downregulated the PI3K/Akt pathway (Figure 4B, C). The effects were reversed by TRPV3 shRNA (Figure 5A, C, D). Moreover, pharmacological inhibition of PI3K with LY294002 (20 ␮M) attenuated antiadipogeneic effects of the TRPV3 activators (Figure 4D).

Figure 3. The TRPV3 activators prevented differentiation in the 3T3–L1 cells. A. Representative Oil-red-O staining images showing TRPV3 activators-mediated dose-dependent inhibition in adipogenesis in the 3T3–L1 cells (n ⫽ 6). The scale bar was 100 ␮M. B. Spectophotometric analysis of Oil-red-O staining. Mean ⫾ SD (n ⫽ 8 culture wells). ***P ⬍ .001.


doi: 10.1210/en.2014-1831

FOXO1 is a mediator of adipogenesis in the adipocytes.


7

FOXO1 phosphorylation is downstream of the PI3K/Akt

Figure 4. The TRPV3 activators inhibited the PI3K/Akt pathway. A. The TRPV3 agonists did not alter TRPV3 expression in the adipocytes (representative immunoblot from 4 experiments). B and C. The TRPV3 agonists caused dose-dependent decrease in PI3K and Akt phosphorylation in the 3T3–L1 cells. Representative immunoblots (upper panels) and summary of protein band intensities relative to housekeeping gene GAPDH (lower panels). D. The PI3K inhibitor LY294002 (20 ␮M) reversed antiadipogenic effects of the TRPV3 activators. The scale bar is 300 ␮M. Mean ⫾ SD (n ⫽ 4 in B and C; n ⫽ 6 in D). **P ⬍ .05, ***P ⬍ .001.


8


pathway during insulin signaling (19, 20). The TRPV3 agonists prevented FOXO1 phosphorylation, which in turn downregulated the adipogenic genes C/EBP␣ and PPAR␥. Both TRPV3 shRNA and TRPV3 siRNAs reversed the effects of the TRPV3 agonists (Figure 5C-G, Supplemental Figure 1). Therefore, we concluded that inhibition of the PI3K/Akt/FOXO1 pathway is downstream of TRPV3 activation, leading to antiadipogenic effects in the 3T3-L1 cells. TRPV3 and insulin receptor substrate 1 (IRS-1) are associated in the 3T3-L1 preadipocytes The regulatory subunits of phosphorylated PI3K are associated with upstream phosphorylated residues of the insulin receptor substrate (IRS) (19). Thus, we questioned whether the antiadipogenic function of TRPV3 is linked to IRS-1 or IRS-2 in the adipocytes. The TRPV3 agonists

Endocrinology

(-)-epicatechin and DPBA dose-dependently inhibited IRS-1 phosphorylation at Ser612, a pivotal phosphorylation site mediating the effects of insulin in the insulin pathway (21) (Figure 6A). Immunoprecipitation studies showed that TRPV3 and IRS-1, but not IRS-2, physically coupled in the 3T3-L1 preadipocytes (Figure 6B). TRPV3 upregulation suppresses adipogenesis Compared with vector-transfected 3T3-L1 cells (Control), TRPV3 overexpression (TRPV3) in the 3T3-L1 cells suppressed differentiation (Figure 6C). Immunoblotting showed that TRPV3 overexpression downregulated the expression of the adipogenic genes C/EBP␣ and PPAR␥ (Figure 6D-G).

Figure 5. TRPV3 RNAi reversed the TRPV3 activators-mediated inhibition of the PI3K/Akt/FOXO1 pathway and the expression of adipogenic genes PPAR␥ and C/EBP␣. A. Representative immunoblots. B - G. Summary of protein band intensities relative to GAPDH. Mean ⫾ SD (n ⫽ 5). ***P ⬍ .001.


doi: 10.1210/en.2014-1831

TRPV3 activation prevented fat accumulation in vivo We administered daily treatments of the TRPV3 agonists for 6 weeks in the mice. After the treatment, the mice


9

on high fat diets had significantly greater body weight, waist circumference, and amount of visceral, subcutaneous and brown adipose tissue compared with control mice and the mice on high fat diets supplemented with the

Figure 6. TRPV3 and IRS-1 are associated in the 3T3–L1 cells. A. The TRPV3 activators abrogated IRS-1 phosphorylation. Representative immunoblots (upper panels) and summary of relative protein expression (lower panels) (n ⫽ 4). B. TRPV3 coimmunoprecipitated with IRS-1, but not IRS-2, in the cell lysate of the 3T3–L1 preadipocytes. The immunoprecipitate (IP) was concentrated 5-folds with respect to the original cell extract (Lysate). All samples were run on the same gel (n ⫽ 4). C. Decreased adipogenesis was seen in the 3T3–L1 cells stably overexpressing TRPV3 (TRPV3) relative to the vector-transfected cells (Control). D-G. Overexpression of TRPV3 in the 3T3–L1 cells (TRPV3) decreased C/EBP␣ and PPAR␥ expression compared with the vector-transfected cells (Control) (n ⫽ 5). Mean ⫾ SD (n ⫽ 5). **P ⬍ .05, ***P ⬍ .001 vs. Control.


10


TRPV3 activators (Figure 7A-C, F-I). Liver weights, liver and plasma triglyceride levels, and plasma free fatty acid (FFA) levels were similar among all treatment groups (Figure 7J-M). During the first 10 days of treatment, we observed a slight decrease in food intake in the mice on high

Endocrinology

fat diets supplemented with the TRPV3 agonists. However, food consumption was comparable among all treatment groups after 10 days. This indicated that long term feeding of the TRPV3 activators did not alter food intake (Figure 7D-E).

Figure 7. The TRPV3 activators suppressed adipogenesis and weight gain in vivo. A. Representative images of mice showing back, abdominal situs and dissected visceral adipose tissues. B-E. Average body weight and food intake in mice under different treatments. F. Waist circumference. G-J. Weight of visceral, brown, subcutaneous fat and liver. K-M. Plasma and liver triglyceride, plasma FFA levels in the mice. Mean ⫾ SD (n ⫽ 7/ group). ***P ⬍ .001.


doi: 10.1210/en.2014-1831

Mice on high fat diets showed greater increase in adipocyte size in the visceral adipose tissue compared with mice on control diets or high fat diets supplemented with the TRPV3 activators (Figure 8A). Immunoblotting results demonstrated that TRPV3 expression was lower in the visceral and subcutaneous adipose tissue from mice on high fat diets compared with mice on control diets. We also showed that obese (ob/ob) and diabetic (db/m⫹) mice expressed lower levels of TRPV3 than wild-type (WT) mice (Figure 8B).

Discussion Several lines of evidences highlighted the importance of [Ca2⫹]i in the regulation of adipogenesis in the adipocytes. [Ca2⫹]i dysregulation leads to obesity and insulin resistance. Elevation in [Ca2⫹]i markedly suppressed adipogenesis by inhibiting the insulin pathway, triglyceride accumulation and PPAR␥ expression (22, 23). Intracellular Ca2⫹ chelator 1,2-bis(o-aminophenoxy) ethaneN,N,N,N-tetraacetic acid, sodium (BAPTA-AM) enhanced insulin-activated protein-protein interaction, PI3K activity and adipogenesis (5). It is also believed that some members of the TRP channel family play important roles in adipogenesis (6 –10). The present study unravels a novel function of TRPV3 in the adipocytes. TRPV3 activation prevents adipogenesis in the adipocytes. The evidences supporting our claim are as follows: (1) we identified a novel TRPV3 activator (-)-epicatechin (2). We showed that TRPV3 is expressed in the 3T3-L1 preadipocytes and differentiated adipocytes (3). The TRPV3 activators (-)-epicatechin and DPBA enhanced TRPV3-mediated [Ca2⫹]i elevation and whole-cell currents in the 3T3-L1 preadipocytes (4). The TRPV3 activators inhibited the PI3K/Akt/FOXO1 pathway, which in turn downregulated expression of the adipogenic genes PPAR␥ and C/EBP␣ (5). The TRPV3 activators prevented phosphorylation of IRS-1, an upstream modulator of the PI3K/Akt pathway, and prevented lipid accumulation and differentiation in the 3T3-L1 cells (Figure 8C) (6). TRPV3 and IRS-1 are associated in the 3T3-L1 preadipocytes (7). TRPV3 overexpression in the 3T3-L1 cells suppressed adipogenesis (8). The TRPV3 activators prevented high fat diet-induced enlargement of fat depots and weight gain (9). Visceral and subcutaneous adipose tissue from mice on chronic high fat diets, ob/ob and db/m⫹ mice expressed lower levels of TRPV3 than wild-type mice. Adipogenesis is mediated by several pathways in the adipocytes (24 –26). In particular, insulin triggers adipogenesis through phosphorylation of the insulin receptor tyrosine protein kinase, leading to downstream phosphor-


11

ylation of IRS-1 and IRS-2. As a result, IRS-1 and/or IRS-2 phosphorylate PI3K, causing Akt phosphorylation at Thr308 and Ser473. Phosphorylated Akt influences a number of genes mediating insulin signaling and adipogenesis (24, 27, 28). For instance, phosphorylated Akt prevents FOXO1 from entering the cell nucleus by phosphorylating it (29, 30). Alternatively, when FOXO1 is not phosphorylated, it enters the cell nucleus, binds to the promoter of PPAR␥, and inhibits PPAR␥ transcription. Consequently, adipogenesis is suppressed in the cell (31). Furthermore, it has been known that the adipogenic genes PPAR␥ and C/EBP␣ positively regulate the expression of each other. Thus during adipogenesis, C/EBP␣ expression is also upregulated (32, 33). Our study showed that TRPV3 overexpression hindered adipogenesis and adipocyte differentiation in the 3T3-L1 cells. In vivo studies on adolescent mice showed that chronic treatment of the TRPV3 activators suppressed high fat diet-induced adipogenesis and obesity. Hence, given the results from our experiments, TRPV3 may become a potential target for the treatment of obesity. However, it is crucial to note that clinical manipulation of TRPV3 activation must be managed with great care as excessive suppression of adipogenesis may reduce the body’s ability to safely store fat. We hypothesize that this may lead to lipotoxicity, insulin resistance and type 2 diabetes resulting from overaccumulation of ectopic fat in the liver and skeletal muscles (34) In conclusion, the present study elucidates the antiadipogenic function of TRPV3. Activation of TRPV3 prevents adipogenesis in the adipocytes by inhibiting the insulin pathway. We identified a well-known tea catechin (-)-epicatechin as a novel activator of TRPV3. In the future, it is encouraged to examine TRPV3 in the adipocytes as a novel therapeutic target against obesity, obesity-associated insulin resistance and diabetes.

Acknowledgments This work is financially supported by T13–706/11, AoE/M-05/ 12, CUHK478011, and CUHK478413 of the Hong Kong Research Grant Council. Address all correspondence and requests for reprints to: Xiaoqiang YAO, PhD., School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, China., Phone: 852– 39 436 877, Fax: 852–26 035 123, Email: [email protected]. Hau Yin, CHUNG, PhD., Food and Nutritional Sciences, School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China., Phone: 852–39 436 149., Fax: 852–26 036 149, Email: [email protected]


12


Endocrinology

Disclosure Summary: The authors have nothing to disclose.

Figure 8. TRPV3 plays an antiadipogenic role in the adipocytes. A. Representative hematoxylin-eosin staining images (left panel) and summary of data (right panel) showing adipocytes size from visceral fat. Mean ⫾ SD (n ⫽ 7). ***P ⬍ .001. B. Representative images (left panel) and summary of data (right panel) showing TRPV3 expression in visceral and subcutaneous adipose tissue from mice on control and HF diets, ob/ob and db/m⫹ mice. Mean ⫾ SD (n ⫽ 7). ***P ⬍ .001. C. An illustration on the TRPV3-mediated signaling pathway in the adipocytes. Activation of the Ca2⫹permeable TRPV3 channel increases [Ca2⫹]i, which in turn suppresses adipogenesis in the cell by inhibiting the activity of IRS-1, the PI3K/Akt/FOXO1 axis, and the expression of adipogenic genes PPAR␥ and C/EBP␣.


doi: 10.1210/en.2014-1831

Contribution statement: Cheung SY: data acquisition and analysis, article drafting and revision. Yao X and Chung HY: grant support and article revision. Huang Y and Kwan HY: provided necessary reagents, animals and cell line. All authors contributed to the conception and design of the work, as well as final approval of the version to be published. This work was supported by .

References


16.

17.

18. 19.

1. Kopelman PG. Obesity as a medical problem. Nature. 2000;404: 635– 643. 2. Galic S, Oakhill JS, Steinberg GR. Adipose tissue as an endocrine organ. Mol Cell Endocrinol. 2010;316:129 –139. 3. Wang T, Wang Y, Kontani Y, Kobayashi Y, Sato Y, Mori N, Yamashita H. Evodiamine improves diet-induced obesity in a uncoupling protein-1-independent manner: involvement of antiadipogenic mechanism and extracellularly regulated kinase/mitogen-activated protein kinase signaling. Endocrinology. 2008;149:358 –366. 4. Cammisotto PG, Bukowiecki LJ. Role of calcium in the secretion of leptin from white adipocytes. Am K Physiol Regul Integr Comp Physiol. 2004;287:R1380 –R1386. 5. Worral DS, Olefsky JM. The effects of intracellular calcium depletion on insulin signaling in 3T3–L1 adipocytes. Mol Endocrinol. 2002;16:378 –389. 6. Zhang LL, Liu DY, Ma LQ, Luo ZD, Cao TB, Zhong J, Yan ZC, Wang LJ, Zhao ZG, Zhu SJ, Schrader M, Thilo F, Zhu ZM, Tepel M. Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity. Circ Res. 2007;100:1063– 1070. 7. Motter AL, Ahern GP. TRPV1-null mice are protected from dietinduced obesity. FEBS Lett. 2008;582:2257–2262. 8. Sukumar P, Sedo A, Li J, Wilson LA, O’Regan D, Lippiat JD, Porter KE, Kearney MT, Ainscough JFX, Beech DJ. Constitutively active TRPC channels of adipocytes confer a mechanism for sensing dietary fatty acids and regulating adiponectin. Circ Res. 2012;111:191– 200. 9. Chen K, Xu X, Liu Y, Hu Y, Jin M, Li G. TRPM7 channels regulate proliferation and adipogenesis in 3T3–L1 Preadipocytes. J Cell Physiol. 2013;229:60 – 67. 10. Bishnoi M, Kondepudi KK, Gupta A, Karmase A, Boparai RK. Expression of multiple transient receptor potential channel genes in murine 3T3–L1 cell lines and adipose tissue. Pharmacol Rep. 2013; 65(3):751–755. 11. Lee M, Kim C, Kim I, Kim Y. Inhibitory effects of green tea catechin on the lipid accumulation in 3T3–L1 adipocytes. Phytother Res. 2009;23:1088 –1091. 12. Kao YH, Hiipakka RA, Liao S. Modulation of obesity by a green tea catechin. Am J Clin Nutr. 2000;72:1232–1233. 13. Furuyashiki T, Nagayasu H, Aoki Y, Bessho H, Hashimoto T, Kanazawa K, Ashida H. Tea Catechin suppresses adipocyte differentiation accompanied by down-regulation of PPARgamma2 and C/EBPalpha in 3T3–L1 cells. Biosci Biotechnol Biochem. 2004; 68(11):2353–2359. 14. Yang P, Zhu MX. TRPV3. Handb Exp Pharmacol. 2014;222:273– 291. 15. Juvin V, Penna A, Chemin J, Lin YL, Rassendren FA. Pharmaco-

20.

21.

22.

23.

24.

25. 26. 27. 28.

29.

30.

31.

32. 33.

34.

13

logical characterization and molecular determinant of the activation of transient receptor potential V2 channel orthologs by 2-aminoethoxydiphenyl borate. Mol Pharmacol. 2007;72(5):1258 –1268. Naldini L, Blomer U, Gage FH, Trono D, and Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 1996;93:11382–11388. Chung MK, Guler AD, Caterina MJ. Biphasic currents evoked by chemical or thermal activation of the heat-gated ion channel, TRPV3. J Biol Chem. 2005;280(16):15928 –15941. Liu B, Yao J, Zhu M, Qin F. Hysteresis of gating underlines sensitization of TRPV3 channels. J Gen Physiol. 2011;138(5):509 –520. Moreno-Navarrete JM, Fernandez-Real JM. 2012 Adipocyte differentiation. In: Symonds ME (ed) Adipose Tissue Biology. Springer New York, NY, pp 17–38. Penkov DN, Egorov AD, Mozgovaya MN, Tkachuk VA. Insulin Resistance and adipogenesis: role of transcription and secreted factors. Biochemistry (Mosow). 2013;78(1):8 –18. Fafalios A, Ma J, Tan X, Stoops J, Luo J, DeFrances MC, Zarnegar RA. Hepatocyte growth factor receptor (Met)-insulin receptor hybrid governs hepatic glucose metabolism. Nat Med. 2011;17(12): 1577–1585. MB Zemel. Nutritional and endocrine modulation of intracellular calcium: Implications in obesity, insulin resistance and hypertension. Mol Cell Biochem. 1998;188:129 –136. Shi H, Halvorsen YD, Ellis PN, Wilkison WO, Zemel MB. The role of intracellular calcium in human adipocyte differentiation. Physiol Genomics. 2000;3:75– 82. Penkov DN, Egorov AD, Mozgovaya MN, Tkachuk VA. Insulin Resistance and adipogenesis: role of transcription and secreted factors. Biochemistry (Mosow). 2013;78(1):8 –18. Gregoire FM, Smas CM, Sul HS. Understanding adipocyte differentiation. Physio Rev. 1998;78(3):783– 809. Tang QQ, Lane MD. Adipogenesis: from stem cell to adipocyte. Annu Rev Biochem. 2012;81:715–736. Bertrand L, Horman S, Beauloye C, Vanoverschelde JL. Insulin signaling in the heart. Cardiovasc Res. 2008;79:238 –248. Kim Y, Kim H, Chung KY, Choi I, Kim SH. 2014 Transcriptional activation of PIK3R1 by PPAR␥ in adipocytes. Mol Biol Rep Epub ahead of print Yu W, Chen Z, Zhang J. Critical role of phosphoinositide 3-kinase cascade in adipogenesis of human mesenchymal stem cell. Mol Cell Biochem. 2008;310:11–18. Guo S. Insulin signaling, resistance, and metabolic syndrome: insights from mouse models into disease mechanisms. J Endocrinol. 2014;220(2):T1–T23. Armoni M, Harel C, Karni S, Chen H, Bar-Yoseph F, Ver MR. FOXO1 represses peroxisome proliferator-activated receptor- ␥1 and –␥2 gene promoters in primary adipocytes: a novel paradigm to increase insulin sensitivity. J Biol Chem. 2006;281:19881–19891. Lehrke M, Lazar MA. The many faces of PPARgamma. Cell. 2005; 123(6):993–999. Kawai M, Rosen CJ. PPAR␥: a circadian transcription factor in adipogenesis and osteogenesis. Nat Rev Endocrinol. 2010;6:629 – 636. Brandao de Oliverira CI, Chaim EA, Borges da Silva B. Impact of rapid weight reduction on risk of cholelithiasis after bariatric surgery. Obes Surg. 2003;13(4):625– 628.


Activation of mitochondrial transient receptor potential vanilloid 1 channel contributes to microglial migration.

Conjugated polymers mediate effective activation of the Mammalian Ion Channel Transient Receptor Potential Vanilloid 1.

Phenylarsine oxide as a redox modulator of transient receptor potential vanilloid type 1 channel function.

Transient receptor potential vanilloid type-1 channel regulates diet-induced obesity, insulin resistance, and leptin resistance.

Transient receptor potential vanilloid type 1 channel may modulate opioid reward.

A functional transient receptor potential vanilloid 4 (TRPV4) channel is expressed in human endothelial progenitor cells.

The transient receptor potential vanilloid-4 channel: detecting body temperatures that drive defences against mild warmth.

Role of transient receptor potential vanilloid 4 activation in indomethacin-induced intestinal damage.

Vascular Expression of Transient Receptor Potential Vanilloid 1 (TRPV1).

reoxygenation injury in cardiomyocytes.

Olmsted syndrome with erythromelalgia caused by recessive transient receptor potential vanilloid 3 mutations.

Transient Receptor Potential Vanilloid 2 Regulates Myocardial Response to Exercise.

Modulation of transient receptor vanilloid 1 activity by transient receptor potential ankyrin 1.

Furanocoumarins are a novel class of modulators for the transient receptor potential vanilloid type 1 (TRPV1) channel.

Klotho up-regulates renal calcium channel transient receptor potential vanilloid 5 (TRPV5) by intra- and extracellular N-glycosylation-dependent mechanisms.

The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel.

Low-cytotoxic synthetic bromorutaecarpine exhibits anti-inflammation and activation of transient receptor potential vanilloid type 1 activities.

Transient receptor potential vanilloid 1 activation by dietary capsaicin promotes urinary sodium excretion by inhibiting epithelial sodium channel α subunit-mediated sodium reabsorption.

Transient Receptor Potential Vanilloid 1 Regulates Mitochondrial Membrane Potential and Myocardial Reperfusion Injury.

Silencing Transient Receptor Potential Vanilloid Receptor Subtype I-containing Sensory Neurons to Treat Bone Cancer Pain.

The essential role of transient receptor potential vanilloid 1 in simvastatin-induced activation of endothelial nitric oxide synthase and angiogenesis.

Activation of mu opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1) via β-arrestin-2-mediated cross-talk.

Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis.

A critical role for the transient receptor potential channel type 6 in human platelet activation.