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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective:

Visceral obesity contributes to the development of obesity-related disorders such as diabetes, hyperlipidemia, and fatty liver disease, as well as cardiovascular diseases. In this study, we determined whether topical application of capsaicin can reduce fat accumulation in visceral adipose tissues.

Methods and Results:

We first observed that topical application of 0.075% capsaicin to male mice fed a high-fat diet significantly reduced weight gain and visceral fat. Fat cells were markedly smaller in the mesenteric and epididymal adipose tissues of mice treated with capsaicin cream. The capsaicin treatment also lowered serum levels of fasting glucose, total cholesterol, and triglycerides. Immunoblot analysis and RT-PCR revealed increased expression of adiponectin and other adipokines including peroxisome proliferator-activated receptor (PPAR) α, PPARγ, visfatin, and adipsin, but reduced expression of tumor necrosis factor-α and IL-6.

Conclusions:

These results indicate that topical application of capsaicin to obese mice limits fat accumulation in adipose tissues and may reduce inflammation and increase insulin sensitivity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Although obesity is associated with insulin resistance, a risk factor for both type 2 diabetes and cardiovascular disease (1), the extent of whole body fat accumulation does not necessarily determine the occurrence of these diseases. In fact, visceral obesity is the main risk factor for inappropriate storage of triglycerides in adipocytes, and fat accumulation in visceral adipose tissues plays crucial roles in the development of obesity-related disorders such as type 2 diabetes, hyperlipidemia, hypertension, and metabolic syndrome (2,3). Adipocytes are highly specialized cells that affect energy homeostasis by secreting a number of biologically active adipokines. Research into adipokines has strongly influenced our understanding of the relationship between increased adipose tissue and obesity (4). In fact, down-regulation of adiponectin is a mechanism whereby obesity can cause insulin resistance and diabetes (5). Targeting the adipokines associated with visceral obesity may therefore be a useful strategy for preventing obesity-induced metabolic pathologies.

Adiponectin is a 30-kDa adipokine that is highly expressed in adipocytes, and its concentration is inversely related to visceral fat accumulation. It has antidiabetic, antihypertensive, and antiatherogenic properties, as well as antiinflammatory and antioncogenic actions (2). Although the mechanism by which plasma levels of adiponectin are reduced in individuals with high levels of visceral fat is not clear, co-culture with visceral fat inhibits adiponectin secretion from subcutaneous adipocytes (6). This finding suggests that visceral adipose tissue releases some factor(s) inhibiting adiponectin synthesis or secretion. Adiponectin improves insulin sensitivity by increasing energy expenditure and fatty acid oxidation through activation of adenosine monophosphate-activated protein kinase (AMPK), and by increasing the expression of target genes of peroxisome proliferator-activated receptor α (PPARα) such as fatty acid transolocase/CD36, acyl-CoA oxidase, and uncoupling protein (UCP) 2 (5).

Capsaicin, a major pungent ingredient of hot chilies and other peppers, has direct effects on isolated adipocytes in vitro (7,8), and has antiobesity activity in animal model (9,10). Moreover there is epidemiological evidence for an association between consumption of capsaicinoid-containing foods and lower incidence of obesity (11,12). Shin and Moritani (13) have reported that consuming capsaicin 1 h before low intensity exercise improved lipolysis and may therefore be a valuable supplement in the treatment of individuals with hyperlipidemia and/or obesity. According to these animal and human studies, dietary capsaicin may be considered a functional agent that helps to prevent obesity. However, it is not ideal for controlling obesity in humans because the long-term consumption of capsaicin may be limited by its pungency. Capsaicin is commonly applied topically to reduce aches and burning feelings, the frequently reported symptoms of painful neuropathy (14). It is currently used in topical creams and high-dose dermal patches to relieve the pain of peripheral neuropathies such as postherpetic neuralgia caused by shingles, and can be directly applied to abdominal skin with no side effects in animal models and humans (15,16).

In this study, we used an in vivo mouse model fed a high-fat diet (HFD to examine the effect of topically applied capsaicin as a treatment for preventing visceral obesity.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animal and human experiments

Male C57BL/6 mice were used in this study. All experimental procedures were approved by the University of Ulsan Animal Care and Use Committee and conformed to National Institutes of Health guidelines. The mice were fed a 60% HFD (Research Diets, New Brunswick, NJ) and were split into two groups: a control group (n = 7), and an experimental group (n = 7) treated once a day with 0.075% capsaicin cream. The capsaicin cream was prepared by mixing capsaicin into a hydrophilic cream base (Sigma, St. Louis, MO), and 100 mg was applied to shaved abdominal skin on each occasion. For capsaicin posttreatment, obese mice were fed a 60% HFD for 7 weeks and then received either a control cream (n = 8) or the 0.075% capsaicin cream (n = 7) for a further 7 weeks while still on the HFD. Then mice were weighed and killed by cervical dislocation. The mesenteric fat epididymal fat of each mouse were pooled and weighed, and blood was collected from the heart.

Histological analysis

Visceral fat tissue was fixed in 10% neutral buffered formalin for 24 h, cut into small pieces, and embedded in paraffin for histological analysis. The samples were cut by microtome, mounted on D-polylysine-coated glass slides, deparaffinized in xylene, and stained with hematoxylin and eosin to evaluate adipocyte size. The area occupied by adipocytes was calculated using float morphology in the Image J program (http://rsb.info.nih.gov/ij/).

RNA isolation and analysis

Visceral fat tissues were homogenized with TRIzol (Invitrogen, Carlsbad, CA). RT-PCR was used to measure levels of gene expression, which were normalized to the amount of GAPDH mRNA. cDNAs were synthesized with Go Taq DNA polymerase (Promega, Madison, WI) in a MyCycler Thermal Cycler (Bio-Rad). Polymerase chain reaction products were analyzed by electrophoresis in agarose gels and staining with ethidium bromide to verify that single amplicons of the expected sizes had been obtained.

Western blot analysis

Visceral fat tissues were homogenized in HNTG lysis buffer (50-mmol/l 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid, 150-mmol/l NaCl, 1% triton X-100, and 10% glycerol) supplemented with protease inhibitor cocktail (Sigma). Lysates were clarified by centrifugation at 12000g for 20 min at 4 °C. Protein concentrations were determined by the Bradford assay (Pierce, Rockford, IL), and equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked in Tris-buffered saline with Tween 20 containing 5% nonfat dry milk for 2 h at room temperature. Bound primary antibodies were detected with peroxidase-coupled secondary antibody and an enhanced chemiluminescence detection system (Pierce). Band intensities were determined by densitometric analysis with the Gel Doc gel documentation system (Bio-Rad, Hercules, CA) and the Image J program (NIH), and were normalized to β-actin. All assays were performed in duplicate.

Plasma analysis

Blood was collected in haparinized tubes, and plasma was prepared immediately by centrifugation (890g, 4 °C, 15 min). Plasma concentrations of glucose, triglyceride, and total cholesterol were measured with a commercial enzymatic colorimetric kit (Asan Pharm, Seoul, Korea).

Statistical analysis

Data are presented as means ± s.e.m. Two-tailed t-tests were used to analyze differences in body weight gain and food intake, and plasma data were analyzed by two-way ANOVA. Statistics were calculated using GraphPad Prism version 5.0 software (GraphPad Software, La Jolla, CA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Capsaicin reduces body weight of obese mice

A previous study showed that dietary capsaicin reduced obesity-induced insulin resistance in obese mice fed an HFD (17). In this study, capsaicin cream was topically applied to the skins of mice fed an HFD for 8 weeks. The increase of body weight seen in the control HFD-fed mice was not observed in the 0.75% capsaicin-treated group (Figure 1a). To examine visceral fat, we measured the weights of livers, and mesenteric and epididymal adipose tissues. The mesenteric adipose tissue of the capsaicin-treated mice weighed less than that of the control obese mice (Figure 1b), and was much less enlarged than that of the control obese mice (Figure 1c).

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Figure 1. Topical application of capsaicin decreases adiposity. Capsaicin cream (0.075%) was applied to C57BL/6 mice fed a high-fat diet (HFD) for 7 weeks. (a) Total body weights (BW), (b) BW and adipose tissue weights of individual mice. (c) Representative anatomical photographs of control mice and mice treated with capsaicin cream. BW and tissue weights were measured with an analytical balance. Data are mean ± s.e.m. *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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To investigate the effect of capsaicin on the adipose tissues of mice that are already obese, we fed mice the HFD for 7 weeks and then treated half of them with capsaicin cream for another 7 weeks while continuing to provide the HFD. Figure 2a shows that body weight increased steadily in the control group whereas there was little or no increase in body weight in the capsaicin-treated group (P < 0.001). As in the experiment in Figure 1, the weights of mesenteric (0.33 ± 0.04) and epididymal adipose tissue (0.81 ± 0.06) were lower in the capsaicin-treated mice than in the control mice (0.51 ± 0.05 and 1.43 ± 0.17, respectively) (Figure 2b,c). These results indicate that topical application of capsaicin cream is equally effective in pre-obese and post-obese mice. It is noteworthy that food intake increased steadily in both groups (Figure 2d,e), indicating that the effect of capsaicin on weight gain could not be attributed to reduced food intake.

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Figure 2. Topical application of capsaicin to mice that are obese reduces further gain of BW and adipose tissue weight. Mice were fed an HFD continuously for 14 weeks. After 7 weeks the mice were divided into two groups, one of which received treatment with 0.075% capsaicin cream for the remaining 7 weeks, the other control cream. (a) Total BW gain. (b) BW and adipose tissue weights of individual mice. (c) Total weight of adipose tissues. (d) Total food intake. (e) daily food calories (kcal/day). Data are mean ± s.e.m. of mice. ***P < 0.001, *P < 0.05. BW, body weight; CAP, capsaicin; HFD, high-fat diet.

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Capsaicin reduces lipid droplets in visceral tissues and the insulin resistance of obese mice

We compared the lipid contents in visceral tissues of obese mice with and without capsaicin application in an experiment of the kind described in Figure 2. Cross-sections of mesenteric and epididymal adipose tissue, and liver, showed a marked decrease or complete absence of lipid droplets in the capsaicin-treated mice (Figure 3a). Capsaicin treatment also reduced the frequency of large and medium-sized (50-100 µm2 and 40-50 × 102 µm2) adipocytes in both mesenteric and epididymal adipose tissue (Figure 3b).

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Figure 3. Effect of topical application of capsaicin on adipose tissue, plasma glucose, and lipids. (a) Representative histological images of mesenteric and epididymal adipose tissues (H&E). (b) Cell size was determined by morphometry. (c) Glucose, total cholesterol, and triglyceride levels in plasma of killed mice. Data are mean ± s.e.m. of mice in each group in an experiment of the type described in the legend to Figure 2. **P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Plasma glucose, cholesterol, and triglyceride levels after capsaicin treatment measured as described previously (17) were also lower in the capsaicin-treated mice, in agreement with the histological data.

To better understand the molecular mechanism by which capsaicin application reduces lipid accumulation in mesenteric adipose tissue, we measured transcript levels of several genes involved in inflammation and lipid metabolism. As shown in Figure 4a, expression of tumor necrosis factor-α (TNF-α) and IL-6 was somewhat lower in the obese mice receiving topical application of capsaicin than in the obese control group. However, substantial increases were observed in the expression of a number of adipokines and genes related to lipid metabolism, such as adiponectin, adipsin, visfatin, lipoprotein lipase (LPL), PPAR(α, β/δ, γ), UCP2, fatty acid-binding protein 4 (FABP4), fatty acid transporter 1, and acyl-CoA synthetase long-chain family member 1 (ACSL1) (Figure 4b), and western blots showed that adiponectin and leptin expression also increased at the protein level (Figure 4c). These findings suggest that the topical application of capsaicin inhibits lipogenesis in mesenteric adipose tissues by upregulating adipokines, especially adiponectin and leptin.

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Figure 4. Effects of topical application of capsaicin on expression of obesity-related genes in mesenteric adipose tissues. (a) Expression of TNF-α and IL-6 genes. (b) Expression of adiponectin, adipsin, visfatin, LPL, ACSL1, FABP4, FATP1, UCP2, and PPARs genes. mRNA. ACSL1, acyl-CoA synthetase long-chain family member 1; FATP1, fatty acid transporter 1; LPL, lipoprotein lipase; PPAR, peroxisome proliferator-activated receptor; UCP2, uncoupling protein 2 (c) Protein levels of adiponectin and leptin were determined by western blot analysis with adiponectin and anti-leptin antibodies.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The effects of capsaicin on body weight and fat mass have been interpreted as due to enhancement of energy metabolism and thermogenesis, or inhibition of inflammation (7,18). Epidemiological data reveal an association between consumption of capsaicinoid-containing foods and reduced incidence of human obesity (12,19). We have shown above that topical application of capsaicin, as described previously (15), is capable of reducing visceral fat and suppressing tissue inflammation in HFD-induced obese mice. Capsaicin application reduced body weight and fat gain in the HFD-induced obese mice, and reduced fat mass in mesenteric and epididymal adipose tissues. Interestingly, we observed a similar effect of capsaicin on pre-obesity (Figure 1) and post-obesity mice (Figure 2); topical application of capsaicin cream prevented the increase of visceral fat mass induced by overfeeding in the preo-besity mice, and may have eliminated part of the visceral fat that had been already gained by post-obesity mice (Figure 2). As we were primarily interested in the therapeutic potential of capsaicin, we focused on post-obesity mice. Most of the effects of topical application of capsaicin were found to be as strong as, or stronger than, those obtained by dietary application (17). For example, topical application of capsaicin caused a much greater reduction in the ratio of adipose tissue weight to body weight than dietary supplementation. This strong effect is confirmed by the histological data in Figure 3a showing that capsaicin treatment reduced the number of large adipocytes in visceral tissues.

Although topical application of capsaicin reduces visceral adiposity, the molecular basis of its action is unknown. A possible explanation is increased energy expenditure. Indeed it has been reported that leptin receptor mRNA levels are reduced, whereas mRNA levels for UCP2 are elevated in visceral fat, which may point to increased energy expenditure (20). This idea prompted us to test the effect of topical application of capsaicin on genes related to lipid metabolism, and we observed that capsaicin treatment upregulated the expression of UCP2, ACSL1 and fatty acid transport protein 1 in mesenteric adipose tissues (Figure 4). ACSL1 has the function of directing the metabolic partitioning of fatty acids toward β-oxidation in adipocytes (21). The functions of ACSLs in adipose tissue are of particular interest because of the central role of white adipose tissue in energy storage. In addition, we found an enhanced level of LPL mRNA in the capsaicin-treated mice. LPL is important for whole body energy homeostasis and fat accumulation in adipose tissue; it is the rate-limiting enzyme in hydrolysis of triglyceride-rich lipoproteins and plays a critical role in lipoprotein and free fatty acid metabolism. Increased LPL activity also reduces fat accumulation and improves HFD-induced insulin resistance (22). Hence, it seems that topical application of capsaicin enhances energy expenditure and reduces fat accumulation by upregulating UCP2, fatty acid transporter 1, ACSL1, and LPL, which in turn protect against visceral obesity. How might capsaicin application mediate these effects? Several studies have indicated that the AMPK signaling pathway is involved in regulating gene expression related to lipid metabolism (23), and capsaicin causes activation of AMPK (24,25,26). AMPK activation by both adiponectin and insulin is associated with a change in cellular energy state dependent on the activities of fatty acid transporter 1 and ACSL1 (27). The former mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes (28). These findings suggest that capsaicin inhibits fat accumulation in adipocyte by regulating lipid metabolism via the AMPK signaling pathway. However, we found that the leptin level was not significantly changed by capsaicin application. Leptin is the most important adipocytokine involved in the inter-organ cross-talk controlling weight homeostasis, insulin sensitivity, and many other key biological parameters, and it is highly expressed in adipocytes. Obesity is characterized by elevated systemic levels of central and peripheral leptin resistance (29). Serum leptin level is elevated during the development of obesity and is decreased by diet-induced weight loss (30).

Adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ secreting a number of biologically active adipokines (5). We have previously shown that dietary intake or intraperitoneal injection of capsaicin reduces visceral fat and obesity-induced inflammation by stimulating the expression of adiponectin (7,17). Adiponectin, as an adipokine, has recently attracted much attention. In skeletal muscle, it increases the expression of molecules involved in fatty acid transport, fatty acid combustion, and energy dissipation. These changes lower tissue triacyglyceride content (31). We showed above that the expression of adiponectin was substantially upregulated by topical capsaicin treatment. Since plasma adiponectin level is reduced in obesity (5), our findings suggest that the reduced visceral fat accumulation resulting from topical application of capsaicin leads to adiponectin secretion from the visceral adipose tissue. It also remains possible that topical application of capsaicin increases energy metabolism via the sensory-central sympathetic nerve axis and suppresses the accumulation of visceral fat, as does oral administration of the transient receptor potential vanilloid I (32,33).

We also showed that the inflammatory cytokines, TNF-α and IL-6, were downregulated by topical capsaicin treatment. This suggests that capsaicin may inhibit the adipose tissue inflammatory response. Obese adipose tissue secretes IL-6 and TNF-α, and overproduction of these proinflammatory mediators relative to antiinflammatory adipokines (e.g., adiponectin) is thought to be the central mechanism underlying adverse metabolic effects (34). Increased production of TNF-α by adipose tissue is implicated in the insulin resistance associated with obesity (35). This inverse correlation between the levels of adiponectin and IL-6 or TNF-α was found in the adipose tissues of patients with metabolic syndrome (36). Several lines of evidence suggest that topical application of capsaicin suppresses harmful obesity-induced inflammatory responses by decreasing levels of TNF-α and IL-6 and enhancing levels of PPARs (37). We previously also demonstrated that the in vitro effect of capsaicin on adipocytokine release from adipose tissue/adipocytes results from activation of PPARγ and/or inactivation of NF-κB (7,38). A potent antiinflammatory action of capsaicin is supported by the upregulation of heme oxygenase 1, which results in increased adiponectin secretion and decreases in TNF-α and IL-6, and reduces weight gain in Zucker fat rats (39).

Taken together, our data suggest that topical application of capsaicin alters adipocyte function in such a way as to reduce visceral obesity and inflammation, and increase insulin sensitivity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0094050). D-J.Y. and A-R.K. are supported by the BK-21 fund.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References