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(Circulation Research. 2007;100:1063.)
© 2007 American Heart Association, Inc.

Integrative Physiology

Activation of Transient Receptor Potential Vanilloid Type-1 Channel Prevents Adipogenesis and Obesity

Li Li Zhang^*, Dao Yan Liu^*, Li Qun Ma, Zhi Dan Luo, Ting Bing Cao, Jian Zhong, Zhen Cheng Yan, Li Juan Wang, Zhi Gang Zhao, Shan Jun Zhu, Mark Schrader, Florian Thilo, Zhi Ming Zhu, Martin Tepel

From the Center for Hypertension and Metabolic Diseases, Department of Hypertension and Endocrinology, Daping Hospital, Third Military Medical University (L.L.Z., D.Y.L., L.Q.M., Z.D.L., T.B.C., J.Z., Z.C.Y., L.J.W., Z.G.Z., S.J.Z., Z.M.Z.), Chongqing, PR China; and Charité Campus Benjamin Franklin (M.S., F.T., M.T.), Berlin, Germany.

Correspondence to Dr Zhi Ming Zhu, Center for Hypertension and Metabolic Diseases, Department of Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, Chongqing 400042, PR China. E-mail zhuzm{at}yahoo.com

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We tested the hypothesis that activation of transient receptor potential vanilloid type-1 (TRPV1) by capsaicin prevents adipogenesis. TRPV1 channels in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans were detected by immunoblotting and quantitative real-time RT-PCR. The effect of TRPV1 on cytosolic calcium was determined fluorometrically in 3T3-L1-preadipocytes and in human visceral fat tissue. Adipogenesis in stimulated 3T3-L1-preadipocytes was determined by oil red O-staining of intracellular lipid droplets, triglyceride levels, expression of peroxisome proliferator-activated receptor-, and expression of fatty acid synthase. Long-term feeding experiments were undertaken in wild-type mice and TRPV1 knockout mice.

We detected TRPV1 channels in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans. In vitro, the TRPV1 agonist capsaicin dose-dependently induced calcium influx and prevented the adipogenesis in stimulated 3T3-L1-preadipocytes. RNA interference knockdown of TRPV1 in 3T3-L1-preadipocytes attenuated capsaicin-induced calcium influx, and adipogenesis in stimulated 3T3-L1-preadipocytes was no longer prevented. During regular adipogenesis TRPV1 channels were downregulated which was accompanied by a significant and time-dependent reduction of calcium influx. Compared with lean counterparts in visceral adipose tissue from obese db/db and ob/ob mice, and from obese human male subjects we observed a reduced TRVP1 expression. The reduced TRPV1 expression in visceral adipose tissue from obese humans was accompanied by reduced capsaicin-induced calcium influx. The oral administration of capsaicin for 120 days prevented obesity in male wild type mice but not in TRPV1 knockout mice assigned to high fat diet. We conclude that the activation of TRPV1 channels by capsaicin prevented adipogenesis and obesity.

Key Words: transient receptor potential vanilloid type-1 • RNAi • TRPV1 knockout adipogenesis • obesity

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Visceral obesity as clinically assessed by waist circumference depends on the proliferation and growth of preadipocytes which is closely regulated by several genes and extracellular factors.^1–3 Among these factors capsaicin ((E)-N-[(4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide) has been shown to affect lipid metabolism and obesity.^4,5 However, the underlying mechanisms by which capsaicin affect visceral adipose tissue have not been completely clarified yet. Recent studies indicated that capsaicin activates the transient receptor potential vanilloid type-1 (TRPV1) channel.^6–8 The capsaicin receptor TRPV1 belongs to the family of nonselective cation channels with high calcium permeability.⁹

Now, we tested the hypothesis that capsaicin-induced activation of TRPV1 in preadipocytes prevents adipogenesis and obesity. We showed that the activation of TRPV1 channels by capsaicin increased cytosolic calcium and prevented adipogenesis of preadipocytes in vitro. The effects of capsaicin on adipogenesis were attenuated after TRPV1 knockdown. Furthermore, capsaicin prevented TRPV1 downregulation during adipogenesis. Finally the administration of capsaicin prevented obesity in male wild-type mice but not in TRPV1 knockout mice assigned to high fat diet in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detailed methods, sequences, and reagents used can be found in the online data supplement available at http://circres.ahajournals.org.

Culture of murine 3T3-L1 preadipocytes,^10–15 RNA interference technique,¹⁶ measurements of cytosolic calcium,^17,18 immunoblottings, immunohistochemistry, determination of adipocyte size, PCR, and standardized techniques^17–19 were performed as described previously.

All mice (db/db mice, ob/ob mice, C57BL/6 wild-type mice, and TRPV1 knock-out mice) were purchased from the Jackson Laboratory (Bar Harbor, Maine). Procedures were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee.

Subjects were classified obese if waist circumference was >90 cm.^20,21 The protocol was approved by the local Ethics Committee. All patients gave written informed consent.

All values reported are mean±SEM. Comparisons between groups were analyzed using Student t test or one-way ANOVA with Bonferroni’s multiple comparison post hoc test as appropriate (GraphPad Prism, La Jolla, Calif). Two-sided probability values <0.05 were considered to indicate statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
First, we detected TRPV1 in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans by immunoblotting (Figure 1a) and immunohistochemistry (Figure 1b). The immunoblots confirmed the molecular mass of TRPV1 of 95 kDa and showed that the antibodies identified TRPV1 in both 3T3-L1-preadipocytes and visceral adipose tissue. Immunohistochemistry demonstrated specific staining for TRPV1 in the cell membrane of 3T3-L1-preadipocytes and adipocytes from mice and humans.

View larger version (46K): [in this window] [in a new window]   Figure 1. The TRPV1 channel agonist capsaicin increases calcium, prevents adipogenesis, and prevents downregulation of TRPV1 channels in 3T3–L1-preadipocytes. a, Immunoblot of TRPV1 obtained from 3T3–L1-preadipocytes (3T3–L1), from visceral adipose tissue (vis fat). The predicted molecular weight of TRPV1 is 95 kDa. M denotes marker. Immunoblots are representative of 3 separate experiments showing similar results. b, Immunohistochemical staining of 3T3–L1-preadipocytes (3T3–L1), visceral adipose tissue (vis fat) from mouse and from humans with anti-TRPV1 antibody showing TRPV1 on the membrane surface. A negative control (only second antibody added) is shown in upper left panel. For 3T3–L1-preadipocytes an overlay with the nucleus stained using PI (red color) and TRPV1 stained in green is shown. Panels are representative of 3 separate experiments showing similar results. Bar denotes 10 µm. c, Concentration-dependent increase of cytosolic free calcium concentration in 3T3–L1-preadipocytes after addition of capsaicin. Fura2-loaded 3T3–L1-preadipocytes were stimulated with increasing capsaicin concentrations (in nmol/L) as indicated beside each tracing and the fluorescence was measured at 510 nm emission with excitation wavelengths of 340 nm and 380 nm. Data are mean±SEM from 6 separate experiments. d, Capsaicin prevents adipogenesis in 3T3–L1-preadipocytes. Identification of adipogenesis in 3T3–L1-preadipocytes in the absence (control) and presence of TRPV1 agonist capsaicin or TRPV1 antagonist capsazepine. Lipid droplets were visualized by oil red O-staining. Upper panels were obtained without induction of adipogenesis of 3T3–L1-preadipocytes (unstimulated), whereas lower panels were obtained after induction of adipogenesis of 3T3–L1-preadipocytes for several days (stimulated) as indicated. Scale bar indicates 10 µm; magnification, x400. Representative pictures of 3 separate experiments are shown. e, Capsaicin prevents the downregulation of TRPV1 expression during adipogenesis of 3T3–L1-preadipocytes. Adipogenesis was induced in the absence (control) and presence of TRPV1 agonist capsaicin or TRPV1 antagonist capsazepine for 8 days. TRPV1 expression in 3T3–L1-preadipocytes was measured at the indicated days. Data are mean±SEM. **P<0.01 compared with control (absence of capsaicin, day 1). f, Reduced capsaicin-induced calcium influx into adipocytes compared with 3T3–L1-preadipocytes. 3T3–L1-preadipocytes were cultured without induction of adipogenesis (unstimulated) and with induction of adipogenesis (stimulated) to generate adipocytes for 8 days. Fluorescence tracings after addition of 1 µmol/L capsaicin to fura2-loaded 3T3–L1-preadipocytes or fura2-loaded adipocytes are shown. Data are mean±SEM from 6 separate experiments. g, Time-dependent reduction of capsaicin (1 µmol/L)-induced calcium influx during adipogenesis. Calcium influx into 3T3-L1-preadipocytes which were cultured with induction of adipogenesis in the absence (control) and presence of capsaicin was measured at different time points as indicated. Data are mean±SEM from 6 separate experiments. *P<0.05 compared with control (absence of capsaicin, day 3). +P<0.05 compared with capsaicin (day 8). h, RNAi knockdown of TRPV1 reduces capsaicin-induced calcium influx into 3T3-L1-preadipocytes. Fluorescence tracings after addition of 1µmol/L capsaicin to fura2-loaded 3T3-L1-preadipocytes after control transfection (Control) and after transfection with RNAi against TRPV1 (RNAi). Data are mean±SEM from 6 separate experiments. i, Capsaicin did not prevent adipogenesis in TRPV1 knockdown 3T3-L1-preadipocytes. 3T3-L1-preadipocytes after control transfection (3T3-L1) and after transfection with RNAi against TRPV1 (TRPV1-RNAi-3T3-L1) were stimulated with by 3-isobutyl-1-methylxanthine, dexamethasone, and insulin in the absence (control) and presence of TRPV1 agonist capsaicin. Scale bar indicates 10 µm; magnification, x200. Representative pictures of 3 separate experiments are shown.

Caterina et al⁶ and Savidge et al²² showed that the specific agonist capsaicin induces calcium influx through TRPV1 channels. To confirm the functional properties of TRPV1 channels in 3T3-L1-preadipocytes intracellular calcium concentrations were measured using the fluorescent dye technique. We used 3T3-L1-preadipocytes because they are an established model for characterizing the events responsible for adipocyte differentiation.^10,12 As shown in Figure 1c, the TRPV1 agonist capsaicin dose-dependently increased intracellular calcium in fura-2 loaded 3T3-L1-preadipocytes. The capsaicin-induced calcium increase could be observed at a concentration of 10 nmol/L, whereas the maximum effect was obtained at 1 µmol/L.

Based on previous findings by Miller et al¹⁰ and Shi et al¹¹ that a calcium increase inhibits differentiation of preadipocytes we now evaluated the effect of capsaicin on adipogenesis of 3T3-L1-preadipocytes. Adipogenesis was induced in 3T3-L1-preadipocytes using 3-isobutyl-1-methylxanthine, dexamethasone, and insulin as recommended.^12,13 The differentiation of preadipocytes was examined by four different functional tests, including oil red O-staining of intracellular lipid droplets,¹³ determination of triglyceride levels, expression of peroxisome proliferator-activated receptor- (PPAR-), a key transcriptional regulator of adipogenesis,¹⁴ and the expression of fatty acid synthase, a key enzyme in de novo lipogenesis.¹⁵ Increased oil red O-staining of lipid droplets in cells indicated a time-dependent adipocyte differentiation after induction of adipogenesis with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (Figure 1d). After induction of adipogenesis cells differentiated into morphologically distinct, fat-laden adipocytes with accumulated cytoplasmic triglycerides that stained with oil red O. Under control conditions increased lipid droplets could be observed approximately 3 days after induction of adipogenesis. In the presence of the TRPV1 agonist capsaicin (final concentration, 1µmol/L) we observed a reduced oil red O-staining of cells after the induction of adipogenesis. In contrast, administration of the TRPV1 antagonist capsazepine did not prevent adipogenesis in 3T3-L1-preadipocytes.

Determination of cellular triglyceride levels confirmed that capsaicin prevented adipogenesis. Compared with control conditions the induction of adipogenesis for 8 days significantly increased triglyceride levels from 3.4±0.2 mmol/L to 10.8±0.8 mmol/L in 3T3-L1-preadipocytes (each n=5; P<0.01). The triglycerides levels after induction of adipogenesis for 8 days in the presence of capsaicin were significantly lower compared with the values in the absence of capsaicin (6.2±0.4 mmol/L versus 10.8±0.8 mmol/L; each n=5; P<0.01). In contrast, administration of the TRPV1 antagonist capsazepine did not prevent the increase of triglyceride levels in 3T3-L1-preadipocytes during adipogenesis (12.3±0.6 mmol/L; n=5). Compared with control conditions the induction of adipogenesis for 8 days significantly increased the expression of PPAR- from 1.00±0.06 to 2.02±0.01 in 3T3-L1-preadipocytes (each n=5; P<0.01). The expression of PPAR- after induction of adipogenesis for 8 days in the presence of capsaicin was significantly lower compared with the values in the absence of capsaicin (1.18±0.16 versus 2.02±0.01; each n=5; P<0.01). In contrast, administration of the TRPV1 antagonist capsazepine did not prevent the increased expression of PPAR- in 3T3-L1-preadipocytes during adipogenesis (1.75±0.12; n=5). Finally, compared with control conditions the induction of adipogenesis for 8 days significantly increased the expression of fatty acid synthase from 1.01±0.08 to 2.99±0.09 in 3T3-L1-preadipocytes (each n=4; P<0.01). The expression of fatty acid synthase after induction of adipogenesis for 8 days in the presence of capsaicin was significantly lower compared with the values in the absence of capsaicin (1.17±0.10 versus 2.99±0.09; each n=4; P<0.01). In contrast, administration of the TRPV1 antagonist capsazepine did not prevent the increased expression of fatty acid synthase in 3T3-L1-preadipocytes during adipogenesis (2.86±0.15; n=4).

As negative controls we showed that without the induction of adipogenesis by 3-isobutyl-1-methylxanthine, dexamethasone, and insulin neither capsaicin nor capsazepine had a significant effect on the production of lipid droplets in 3T3-L1-preadipocytes (Figure 1d). Without induction of adipogenesis triglyceride levels in 3T3-L1-preadipocytes were similar at day 8 under control conditions (3.4±0.2mmol/L), in the presence of capsaicin (3.3±0.2mmol/L) or capsazepine (3.7±0.3 mmol/L; each n=5; p=n.s.). Without induction of adipogenesis expression of PPAR- in 3T3-L1-preadipocytes was similar at day 8 under control conditions (1.00±0.06), in the presence of capsaicin (1.23±0.03) or capsazepine (1.00±0.04; each n=5; p=n.s.). Finally, without induction of adipogenesis expression of fatty acid synthase in 3T3-L1-preadipocytes was similar at day 8 under control conditions (1.01±0.08), in the presence or capsaicin (1.10±0.09) or capsazepine (0.99±0.10; each n=4; p=n.s.). From these experiments it is concluded that the capsaicin-induced calcium increase through TRPV1 channels prevented adipogenesis.

During adipogenesis of 3T3-L1-preadipocytes induced by 3-isobutyl-1-methylxanthine, dexamethasone, and insulin a time-dependent downregulation of TRPV1 expression could be observed by immunoblotting (Figure 1e). The downregulation of TRPV1 expression during adipogenesis was prevented in the presence of the TRPV1 agonist capsaicin. Eight days after induction of adipogenesis in 3T3-L1-preadipocytes, TRPV1 channel expression was significantly reduced from control value of 1.00±0.08 to 0.46±0.03 (each n=4; P<0.01), whereas it was 1.35±0.03 (n=4; P<0.01 compared with control) when the induction of adipogenesis was performed in the presence of capsaicin. The administration of the TRPV1 antagonist capsazepine did not prevent the downregulation of TRPV1 channel after induction of adipogenesis in 3T3-L1-preadipocytes.

The capsaicin-induced calcium increase was significantly lower in mature adipocytes compared with 3T3-L1-preadipocytes (32±4%, versus 100±11%; each n=10; P<0.01; Figure 1f). In accordance with the observed downregulation of TRPV1 channel expression during adipogenesis, we observed a significant and time-dependent reduction of calcium influx during adipogenesis. On the other hand, the calcium influx was unchanged when adipogenesis was blocked in the presence of capsaicin (Figure 1g). Resting cytosolic calcium concentration was not significantly different in mature adipocytes compared with 3T3-L1-preadipocytes or stimulated 3T3-L1-preadipocytes in the presence of capsaicin (104±2 nmol/L; 101±4 nmol/L; 105±3 nmol/L; each n=12; p=n.s.), probably indicating long-lasting increased calcium extrusion after transient rise in intracellular calcium.

To verify that TRPV1 channels are involved in capsaicin-induced calcium increase in 3T3-L1-preadipocytes and capsaicin-dependent prevention of adipogenesis, TRPV1 knockdown was performed by gene silencing with RNA interference using specific RNA interference (RNAi).¹⁶ Compared with empty-control-transfected 3T3-L1-preadipocytes the TRPV1 expression in RNAi-transfected 3T3-L1-preadipocytes was significantly reduced to 38±3% (n=3; P<0.05). The thereby reduced TRPV1 channel expression caused a significant reduction of capsaicin-induced calcium increase from 100±16% in empty-control-transfected 3T3-L1-preadipocytes to 16±3% in TRPV1 RNAi-transfected 3T3-L1-preadipocytes (n=10; P<0.01; Figure 1h). Adipogenesis was induced in TRPV1 knockdown 3T3-L1-preadipocytes and control-transfected 3T3-L1-preadipocytes by 3-isobutyl-1-methylxanthine, dexamethasone, and insulin and adipogenesis was evaluated by oil red O-staining of lipid droplets (Figure 1i). As expected, in TRPV1 knockdown 3T3-L1-preadipocytes the administration of capsaicin did not prevent adipogenesis, whereas in control-transfected 3T3-L1-preadipocytes the TRPV1 agonist capsaicin prevented adipogenesis. These experiments confirmed the necessity of TRPV1 channel activation in capsaicin-dependent prevention of adipogenesis.

Now the question arises, whether downregulation of TRPV1 channels is a common finding in several animal models of obesity as well as in human obesity. We therefore investigated TRPV1 channel expression using immunoblotting and mRNA detection in visceral adipose tissue from genetically obese db/db mice, which have a nonfunctional leptin receptor, and from obese ob/ob mice, which have a naturally occurring leptin deletion.²³ As shown in Figure 2a through 2d, genetically obese db/db mice (body weight, 27±1g; abdominal circumference, 8.1±0.1 cm; visceral fat, 1.6±0.1 g) had significantly lower TRPV1 channel expression (Figure 2b) and TRPV1 mRNA (Figure 2c; 0.50±0.01 versus 0.71±0.05; each n=6; P<0.01) in visceral adipose tissue compared with their lean littermates (body weight, 16±1g; abdominal circumference, 6.1±0.3 cm; visceral fat, 0.3±0.1 g). As shown in Figure 2e through 2h, leptin-deficient obese ob/ob mice (body weight, 50±4 g; abdominal circumference, 11.6±0.1 cm; visceral fat, 5.0±0.4 g) had significantly lower TRPV1 channel expression (Figure 2f) and TRPV1 mRNA (Figure 2g; 0.73±0.10 versus 1.41±0.13; each n=6; P<0.01) in visceral adipose tissue compared with lean C57BL/6 counterparts (body weight, 25±1 g; abdominal circumference, 7.9±0.2 cm; visceral fat, 0.6±0.2 g).

View larger version (36K): [in this window] [in a new window]   Figure 2. Reduced TRPV1 expression in visceral adipose tissue from obese db/db mice (upper panel), obese ob/ob mice (middle panel) and obese human male subjects (lower panel). Picture of body shape and abdominal situs showing visceral fat (a and e); expression of TRPV1 in visceral adipose tissue (b and f); representative agarose gel electrophoresis of PCR products from mRNA of TRPV1 and GAPDH genes in visceral adipose tissue (c and g), M denotes bp marker; and summary data (d and h); each n=4. **P<0.01 between groups. Lower panels show data from age-matched human male control subjects and obese human male subjects. Waist circumference (i); each n=8. ***P<0.001 between groups; expression of TRPV1 in visceral adipose tissue and subcutaneous fat (j); representative quantitative real-time RT-PCR from visceral adipose tissue (k) from 1 human male control subject (black line) and from 1 obese human male subject (red line); y-axis denotes fluorescence at 530 nm in arbitrary units, and x-axis denotes number of cycles of the quantitative real-time RT-PCR; The shift of the red line to the right compared with the black line indicates reduced TRPV1 transcripts in obese subjects; summary data of quantitative real-time RT-PCR are given in (l); n=4. *P<0.05 between groups. Quantitative in-cell Western assay of TRPV1 expression in visceral adipose tissue (m), representative capsaicin-induced calcium influx into visceral adipose tissue from 1 human control subject (open circles) and 1 obese human subject (filled circles) (n); and summary data (o); each n=4; *P<0.05).

We further investigated TRPV1 channel expression in adipose tissue from human male subjects. Visceral adipose tissue and subcutaneous fat was obtained during cholecystectomy. Subjects were classified obese if waist circumference was more than 90 cm according to the Asian WHO criteria.^20,21 Eight lean male control subjects (age, 43±1 years; body mass index, 24.4±1.4 kg/m², serum triglycerides, 4.6±0.4 mmol/L) had a waist circumference of 82.8±1.5 cm, whereas 8 age-matched obese male subjects (age, 44±2 years, body mass index, 26.3±0.4 kg/m², serum triglycerides, 4.9±0.6 mmol/L) had a significantly higher waist circumference of 92.0±0.8 cm (P<0.001 for the waist circumference between the groups; Figure 2i). Western blot shows that obese human male subjects had significantly lower TRPV1 channel expression in visceral adipose tissue (0.70±0.15 versus 4.93±1.07; each n=8; P<0.01) and subcutaneous fat (1.22±0.08 versus 1.70±0.13; each n=8; P<0.01) compared with age-matched lean human male control subjects (Figure 2j). Using quantitative real-time RT-PCR from visceral adipose tissue (Figure 2k and 2l) we showed that obese human male subjects had significantly reduced TRPV1 mRNA compared with age-matched lean human male control subjects (normalized ratio, 0.43±0.16 versus 1.05±0.19, n=4; P<0.05).

Next we evaluated whether reduced TRPV1 expression is also linked to reduced calcium influx in visceral adipose tissue from 4 control subjects (body mass index, 23.5±0.3 kg/m²) and 4 age-matched obese subjects (body mass index, 32.4±2.0 kg/m²). Normalized TRPV1 protein expression by quantitative in-cell Western assay in visceral adipose tissue was significantly lower in obese subjects compared with control subjects (normalized TRPV1 expression, 1.02±007 versus 5.05±0.49; each n=4, P<0.05; Figure 2m). The capsaicin-induced calcium influx was significantly lower in obese subjects compared with control subjects (0.2±0.1 versus 1.3±0.2; each n=4; P<0.05; Figure 2n and 2o).

These findings indicated that downregulation of TRPV1 channel in visceral adipose tissue is a common finding for obesity. Hence a putative intervention which activates TRPV1 channels to prevent obesity should start early during adipogenesis.

As proof of principle we now evaluated whether continuous activation of TRPV1 channels by capsaicin can prevent obesity, hypertrophy of adipocytes, and prevent downregulation of TRPV1 channels in male mice assigned to high fat diet. We first analyzed the biometrical characteristics of mice on control diet and high fat diet, both in the absence and presence of TRPV1 channel agonist capsaicin (Figure 3). After 120 days mice on high fat diet had significantly higher body weight compared with control mice (44±1 g versus 29±1 g; each n=10; P<0.01). Mice on high fat diet plus capsaicin remained lean (31±1 g; n=10; P<0.01 compared with mice on high fat diet). Body weight of mice on control diet plus capsaicin was not significantly different to mice on control diet (27±1 g versus 29±1 g; each n=10; p=n.s.). As shown in Figure 3c we observed a decrease in food intake within the first days after starting capsaicin administration. However, 10 days after starting capsaicin administration the food intake was not significantly different between the groups (food intake per mouse per day in control mice, 4.6±0.1 g; mice on high fat diet, 4.8±0.1 g; control mice with capsaicin, 4.8±0.1; mice on high fat diet with capsaicin, 4.7±0.1 g; p=n.s. by ANOVA). These data indicated that long term feeding with capsaicin did not change food intake. Mice on high fat diet showed obesity with significantly increased waist circumference and increased plasma triglyceride levels compared with control mice or mice on high fat diet plus capsaicin. Mice on high fat diet had significantly more visceral adipose tissue, subcutaneous fat and brown fat compared with control mice or mice on high fat diet plus capsaicin (Figure 3d through 3i). These data indicated that the administration of the TRPV1 agonist capsaicin prevented obesity in mice on high fat diet.

View larger version (36K): [in this window] [in a new window]   Figure 3. The TRPV1 channel agonist capsaicin prevents obesity in mice on high fat diet. a, Time course of body weight from control mice (open circles), mice on high fat diet (filled circles), control mice with capsaicin (open squares), and mice on high fat diet with capsaicin (filled squares; each n=10); Body weight (b), daily food intake per mouse during the first 10 days after the start of capsaicin administration in control mice (open circles), mice on high fat diet (filled circles), control mice with capsaicin (open squares), and mice on high fat diet with capsaicin (filled squares; each n=10) (c), picture of body shape of mice (d), waist circumference (e), serum triglycerides (f), visceral adipose tissue (g), subcutaneous fat (h), brown fat (i) at the age of 120 days of control mice (n=10), mice on high fat diet (n=10), control mice plus capsaicin (n=10), and mice on high fat diet plus capsaicin (n=10). **P<0.01 for the comparison high fat vs control; ++P<0.01 for the comparison high fat plus capsaicin vs high fat.

Figure 4 shows adipocyte size and TRPV1 channel expression in adipose tissue in mice. Mice on high fat diet had significantly increased adipocyte size in visceral adipose tissue compared with control mice (3708±199 µm², n=78; versus 1062±37 µm², n=101 P<0.001). Mice on high fat diet plus capsaicin had small adipocytes size (1255±64 µm², n=90; P<0.001 compared with mice on high fat diet). Similar findings could be obtained in subcutaneous fat and brown fat (Figure 4b through 4d). Mice on high fat diet had significantly reduced TRPV1 channel expression in visceral adipose tissue compared with control mice (0.44±0.06 versus 1.00±0.05; each n=10; P<0.001). Furthermore, mice on high fat diet plus capsaicin had significantly increased TRPV1 channel expression in visceral adipose tissue (1.18±0.08, n=10; P<0.001) compared with mice on high fat diet (Figure 4e). The administration of capsaicin increased TRPV1 channel expression in visceral adipose tissue by 169%. As shown in Figure 4f mice on high fat diet had reduced TRPV1 mRNA in visceral adipose tissue compared with control mice. Furthermore, mice on high fat diet plus capsaicin had increased TRPV1 mRNA in visceral adipose tissue compared with mice on high fat diet, supporting the protein expression data.

View larger version (59K): [in this window] [in a new window]   Figure 4. The TRPV1 channel agonist capsaicin prevents adipocyte hypertrophy and the reduction of TRPV1 expression in visceral adipose tissue from mice on high fat diet. a, Representative cross sections of visceral adipose tissue (vis fat), subcutaneous fat (sc fat), and brown fat (br fat) from control mice, mice on high fat diet, control mice plus capsaicin, and mice on high fat diet plus capsaicin. Scale bar indicates 10 µm; magnification, x400. Summary data of adipocyte size of visceral adipose tissue (b), subcutaneous fat (c), and brown fat (d) in mice. ***P<0.001 for the comparison high fat vs control; +++P<0.001 for the comparison high fat plus capsaicin vs high fat. e, Expression of TRPV1 in visceral adipose tissue (vis fat) from mice. ***P<0.001 for the comparison high fat vs control; +++P<0.001 for the comparison high fat plus capsaicin vs high fat. f, Representative agarose gel electrophoresis of PCR products from mRNA of TRPV1 and GAPDH genes in visceral adipose tissue mice M denotes bp marker. Panels are representative of 3 separate experiments showing similar results.

As appropriate controls TRPV1 knockout mice were exposed to high fat diet in the presence and absence of capsaicin. As could be predicted from the findings presented above, in TRPV1 knockout mice on high fat diet for 60 days the body weight was not significantly different in the absence and presence of the TRPV1 agonist capsaicin (30.3±0.7 g versus 29.8±0.4 g; each n=3; p=n.s.). In contrast wild-type mice on high fat diet for 60 days had a body weight of 32.0±0.4 g, whereas wild-type mice on high fat diet plus capsaicin a significantly reduced body weight of 27.2±1.3 g (each n=5; P<0.05 compared with mice on high fat diet). These data strongly support the hypothesis that the preventive effects of capsaicin are mediated by the TRPV1 channel.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
TRPV1 channels have been described in several tissues.²⁴ This study for the first time detected TRPV1 transcripts and channel protein in 3T3-L1-preadipocytes and adipose tissue both in mice and humans. A limitation of the present study is that although we observed reduced TRPV1 expression both on transcript level and on protein level in two obese animal models and in obese humans compared with lean counterparts it is yet unknown whether there is a linear association between body mass index or waist circumference and TRPV1 expression levels. This would need larger cohort studies which may be difficult because of the limited availability of human visceral adipose tissue.

The TRPV1 agonist capsaicin dose-dependently increased intracellular calcium in 3T3-L1-preadipocytes. After TRPV1 knockdown by gene silencing with RNA interference using specific RNAi the capsaicin-induced calcium increase was significantly attenuated in 3T3-L1-preadipocytes. The capsaicin-induced calcium influx through TRPV1 channels has already been reported in HEK293 cells transfected with TRPV1 channels, neurons, and hepatoblastoma cells.^6,22,25 Furthermore, capsaicin had several effects on nonneuronal tissues.²⁶ Capsaicin activates migration of human polymorphonuclear cells and elicited a substantial constriction in isolated arterioles which may be related to capsaicin-induced transplasmamembrane calcium influx.^26,27 As detailed in a review by Szallasi and Blumberg, capsaicin is a known specific agonist of TRPV1 channels.²⁶ The potency of capsaicin-induced calcium influx was 320 nmol/L in neurons, 660 nmol/L in mast cells and 380 nmol/L in glioma cells.²⁶ In our study the capsaicin-induced calcium influx was dose-dependent and thus confirmed the functional properties of TRPV1 channels in preadipocytes. It should be noted that capsaicin concentrations as low as 10nmol/L showed calcium influx in preadipocytes. The characteristics of capsaicin-induced calcium entry through TRPV1 channels was similar to that reported by Caterina et al⁶ and Savidge et al²² and matches the characteristics of the TRPV1 channel.

Previous studies indicated that adipogenesis is regulated by calcium.^10,11 Shi et al reported that elevated calcium markedly suppressed adipogenesis with a decrease in triglyceride accumulation and a substantial inhibition in PPAR- expression.¹¹ In the present study adipogenesis was induced in 3T3-L1-preadipocytes using 3-isobutyl-1-methylxanthine, dexamethasone, and insulin.^12,13 We showed that capsaicin-induced elevation of cytosolic calcium reduced intracellular lipid droplets, reduced triglyceride levels, reduced expression of PPAR- and reduced fatty acid synthase in stimulated preadipocytes, whereas administration of the TRPV1 antagonist capsazepine did not prevent adipogenesis. Furthermore, after TRPV1 knockdown the administration of capsaicin did not prevent adipogenesis in TRPV1 knockdown 3T3-L1-preadipocytes. Hence we confirmed that increased calcium after administration of capsaicin significantly blocked adipogenesis in stimulated 3T3-L1-preadipocytes.^11,28,29

As proof of principle the effect of capsaicin on adipogenesis and obesity was then investigated in vivo, both in control mice and in TRPV1 knockout mice. Our long term feeding experiments showed that the administration of capsaicin prevented obesity in male wild-type mice assigned to high fat diet but not in TRPV1 knockout mice assigned to high fat diet, indicating that TRPV1 is directly involved in adipogenesis and obesity in vivo.

Capsaicin has been reported to reduce adiposity in rats and mice, which was partly explained by its effect on energy and lipid metabolism via catecholamine secretion from the adrenal medulla through sympathetic activation of the central nervous system. However, these effects on catecholamine secretion were observed after intravenous administration of capsaicin in anesthetized rats.³⁰ It is unclear whether these mechanisms may also explain the long term effects of oral administration of capsaicin on obesity. Furthermore, sympathetic activity has shown consistent elevation in obesity, whereas catecholamine levels in obesity have been conflicting, with high, normal, and low levels reported.³¹

In the present long term feeding study changes of food intake in the presence of capsaicin have been ruled out by measurements of food intake. We showed a decrease in food intake within the first few days after starting the administration of capsaicin confirming a recent study by Wang et al who reported reduced short term food intake after TRPV1 activation of sensory neurons.³² We showed that 10 days after starting capsaicin administration the food intake was similar in the presence and absence of capsaicin, which is in accordance with findings in obese Zucker rats.³³ An effect of red pepper on thermogenesis has been described.⁴ However, several evidences indicated that afferent reflexes are mainly responsible for diet-induced thermogenesis.^34–36

Using normal diet, Rong et al did not report a significant difference of feeding behavior, growth rate and body weight between TRPV1 knockout mice and wild-type mice.³⁷ However, no long term feeding studies comparing high fat diet with normal diet have yet been reported in TRPV1 knockout mice. In the present study we showed for the first time that capsaicin did not prevent adipogenesis and obesity in TRPV1 knockout mice on high fat diet.

In conclusion we present in vitro and in vivo evidences that capsaicin-induced calcium influx through TRPV1 channels prevents adipogenesis, prevents downregulation of TRPV1 expression and finally prevents obesity.

	Acknowledgments

 
Sources of Funding

This study was supported by grants for Natural Science Foundation of China (No. 30470830) and 973 program (grant No. 2006CB503804 and 2006CB503905).

Disclosures

None.

	Footnotes

 
*Both authors contributed equally to this article.

Original received October 9, 2006; resubmission received October 30, 2006; revised resubmission received February 12, 2007; accepted February 22, 2007.

	References

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