The FASEB Journal article fj.14-268300. Published online April 17, 2015.
The FASEB Journal • Research Communication
Transient receptor potential vanilloid type-1 channel regulates diet-induced obesity, insulin resistance, and leptin resistance Eunjung Lee,*,†,‡ Dae Young Jung,* Jong Hun Kim,*,§ Payal R. Patel,* Xiaodi Hu,* Yongjin Lee,* Yoshihiro Azuma,* Hsun-Fan Wang,* Nicholas Tsitsilianos,* Umber Shafiq,* Jung Yeon Kwon,*,§ Hyong Joo Lee,† Ki Won Lee,†,§,1 and Jason K. Kim*,†,{,1 *Program in Molecular Medicine and {Department of Medicine, Division of Endocrinology, Metabolism, and Diabetes, University of Massachusetts Medical School, Worcester, Massachusetts, USA; †World Class University Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul, Republic of Korea; ‡Traditional Alcoholic Beverage Research Team, Korea Food Research Institute, Seongnam, Republic of Korea; and §Advanced Institutes of Convergence Technology, Seoul National University, Suwon, Republic of Korea Insulin resistance is a major characteristic of obesity and type 2 diabetes, but the underlying mechanism is unclear. Recent studies have shown a metabolic role of capsaicin that may be mediated via the transient receptor potential vanilloid type (TRPV)-1 channel. In this study, TRPV1 knockout (KO) and wild-type (WT) mice (as controls) were fed a high-fat diet (HFD), and metabolic studies were performed to measure insulin and leptin action. The TRPV1 KO mice became more obese than the WT mice after HFD, partly attributed to altered energy balance and leptin resistance in the KO mice. The hyperinsulinemic-euglycemic clamp experiment showed that the TRPV1 KO mice were more insulin resistant after HFD because of the ∼40% reduction in glucose metabolism in the white and brown adipose tissue, compared with that in the WT mice. Leptin treatment failed to suppress food intake, and leptin-mediated hypothalamic signal transducer and activator of transcription (STAT)-3 activity was blunted in the TRPV1 KO mice. We also found that the TRPV1 KO mice were more obese and insulin resistant than the WT mice at 9 mo of age. Taken together, these results indicate that lacking TRPV1 exacerbates the obesity and insulin resistance associated with an HFD and aging, and our findings further suggest that TRPV1 has a major role in regulating glucose metabolism and hypothalamic leptin’s effects in obesity.—Lee, E., Jung, D. Y., Kim, J. H., Patel, P. R., Hu, X., Lee, Y., Azuma, Y., Wang, H.-F., Tsitsilianos, N., Shafiq, U., Kwon, J. Y., Lee, H. J., Lee, K. W., Kim, J. K. Transient receptor potential vanilloid type-1 channel regulates dietinduced obesity, insulin resistance, and leptin resistance. FASEB J. 29, 000–000 (2015). www.fasebj.org ABSTRACT
Abbreviations: DG, deoxyglucose; DG-6-P, deoxyglucose 6phosphate; HFD, high-fat diet; HGP, hepatic glucose production; KO, knockout; MEF, mouse embryonic fibroblast; MRS, magnetic resonance spectroscopy; p-STAT, phosphorylated STAT; RNAi, RNA interference; SH, Src homology; siRNA, small interfering RNA; SOCS, suppressor of cytokine signaling; (continued on next page)
0892-6638/15/0029-0001 © FASEB
Key Words: capsaicin • hyperinsulinemic-euglycemic clamp glucose metabolism • aging
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THE INCREASING PREVALENCE IN OBESITY continues to be a global health problem and is having a major impact on the worldwide incidence of diabetes. Obesity is characterized by altered secretion of hormones, metabolites, and inflammatory cytokines from adipose tissue (1, 2). Among them, leptin regulates energy balance as a major satiety factor and through its effects on energy expenditure (3). Although leptin therapy has been shown to improve energy homeostasis in lipodystrophic subjects and obese diabetic rats, underlying leptin resistance in most obese humans has limited its therapeutic potential (4–8). Thus, understanding the mechanism of leptin resistance plays an important role in developing new targets to treat obesity and type 2 diabetes. The transient receptor potential vanilloid type (TRPV)1 channel is a member of the TRP protein superfamily, a large group of cation-permeable channels expressed in mammalian cells. TRPV1 is a nonselective cation channel that is highly permeable to Ca2+ and Na+ and is involved in thermogenesis and pain sensing (9–11). Capsaicin, a major component of chili pepper, is a natural and specific agonist for the TRPV1 channel and induces the pain and thermogenic responses collectively known as the spicy sensation. Several recent reports have shown that dietary consumption of chili pepper or capsaicin-containing foods results in an increased energy expenditure that is associated with a lower incidence of obesity (12–14). In 1 Correspondence: J.K.K., University of Massachusetts Medical School, Program in Molecular Medicine, 368 Plantation St., AS9.1041, Worcester, MA, 01605, USA. E-mail: jason.kim@ umassmed.edu; K.W.L., Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea. E-mail: [email protected] doi: 10.1096/fj.14-268300 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.
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addition, dietary capsaicin improves insulin sensitivity in obese mice that have been fed a high-fat diet (HFD) (15). The underlying mechanism by which capsaicin affects energy balance and glucose metabolism may involve its activation of the TRPV1 channel. Zhang et al. (12) recently found that capsaicin inhibits adipogenesis in 3T3-L1 preadipcoytes, and RNA interference (RNAi) knockdown of TRPV1 channel rescues adipocytes from capsaicin’s effects. Furthermore, long-term capsaicin intake prevents dietinduced obesity in mice, but such an antiobesity effect of capsaicin is lost in TRPV1 knockout (KO) mice. In contrast, Motter and Ahern (16) reported that TRPV1-null mice in fact gain less weight after long-term high-fat feeding than do wild-type (WT) mice. This effect was associated with increased thermogenic capacity in TRPV1-null mice. Although these findings regarding the role of the TRPV1 channel in obesity are contradictory, Riera et al. (17) found that mice lacking a TRPV1 channel show longevity. Taken together, these findings imply that capsaicin and the TRPV1 channel have an important role in energy homeostasis. In the current study, we examined the effects of TRPV1 deficiency in diet-induced obesity, insulin resistance, and leptin action in mice. MATERIALS AND METHODS Animals and diet Male TRPV1-deficient (TRPV1 KO; C57BL/6 background) mice and WT (C57BL/6 background) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed under controlled temperature (23°C) and lighting (12 h of light, 7 AM to 7 PM; 12 h of dark, 7 PM to 7 AM). Starting at 12 wk of age, mice were fed an HFD (Harlan Teklad TD 93075; 55% kcal from fat, 24% kcal from carbohydrate, and 21% kcal from protein; Harlan Laboratories, Indianapolis, IN, USA) ad libitum for 5 wk to induce obesity. The animal studies were approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. Body composition and energy balance Whole-body fat and lean mass were noninvasively measured by 1H magnetic resonance spectroscopy (MRS; Echo Medical Systems, Houston, TX, USA). Indirect calorimetry and energy balance parameters including food and water intake, energy expenditure, respiratory exchange ratio, and physical activity were noninvasively assessed for 3 or 6 days with metabolic cages (TSESystems, Inc., Chesterfield, MO, USA). We used the LabMaster platform (TSE-Systems, Inc.), with easy-to-use calorimetry and featuring fully automated monitoring for food and water and XYZ activity. The Labmaster cages that are most similar to the home cages at our facility were used, thereby allowing the use of bedding in the cage and minimizing any animal anxiety during the experimental period. The system provides intuitive software with flexibility for experimental setup and data use.
(continued from previous page) STAT, signal transducer and activator of transcription; TRP, transient receptor potential channels, M, melastatin, V, vanilloid, C, cation, P, polycystin; VO2, oxygen consumption; VCO2, carbon dioxide production; WT, wild-type
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Hyperinsulinemic-euglycemic clamp After the mice were fed chow or an HFD, they underwent survival surgery 5 or 6 days before the hyperinsulinemic-euglycemic clamp experiments, to establish an indwelling catheter in the jugular vein. On the day of the clamp experiment, the mice were withheld food overnight (;15 h), and a 2 h clamp was conducted in conscious mice with a primed and continuous infusion of human insulin (150 mU/kg body weight priming followed by 2.5 mU/kg per minute; Humulin; Eli Lilly, Indianapolis, IN, USA) (18). To maintain euglycemia, 20% glucose was infused at variable rates during the clamp. Whole-body glucose turnover was assessed with a continuous infusion of [3-3H]glucose (PerkinElmer, Waltham, MA, USA), and 2-deoxy-D-[1-14C]glucose (2-[14C]DG) was administered as a bolus (10 mCi) 75 min after the start of the clamp experiment, to measure insulin-stimulated glucose uptake in individual organs. At the end of the clamp, the mice were anesthetized, and tissues were taken for biochemical analysis (18). Biochemical analysis and calculation Glucose concentrations during the clamp experiments were analyzed in 10 ml plasma by a glucose oxidase method on a GM9 Analyzer (Analox Instruments, Ltd., Hammersmith, London, UK). Plasma concentrations of [3-3H]glucose, 2-[14C]deoxyglucose (DG), and 3H2O were determined after deproteinization of the plasma samples (18). For the determination of tissue 2[14C]deoxyglucose 6-phosphate (DG-6-P) content, the samples were homogenized, and the supernatants were placed in an ionexchange column to separate 2-[14C]DG-6-P from 2-[14C]DG. Rates of basal hepatic glucose production (HGP) and insulinstimulated whole-body glucose turnover were determined as previously described (18). The insulin-stimulated rate of HGP was determined by subtracting the glucose infusion rate from wholebody glucose turnover. Whole-body glycolysis and glycogen plus lipid synthesis from glucose were calculated (18). The insulinstimulated glucose uptake in individual tissues was assessed by determining the tissue (e.g., skeletal muscle) content of 2-[14C] DG-6-P and plasma 2-[14C]DG clearance profile. Plasma hormone measurement Plasma levels of leptin, insulin, and adiponectin (total and high molecular weight) were measured with ELISA kits (ALPCO Diagnostics, Salem, NH, USA). Plasma samples were collected in mice that were withheld food overnight (5 h). Blood was collected carefully into heparin-coated Eppendorf tubes (Eppendorf AG, Hamburg, Germany) and centrifuged at 12,000 rpm for 3 minutes at 4°C. For ELISA assays, the supernatant of centrifuged blood was used according to the manufacturer’s instructions. Leptin administration study For long-term administration of leptin, osmotic pumps (1007D; Alzet Corp., Cupertino, CA, USA) containing leptin (20 mg/d; R&D Systems, Minneapolis, MN, USA) or saline were implanted subcutaneously in anesthetized mice (19). After implantation of the osmotic pumps, the mice were returned to their cages to recover from anesthesia and then were placed in the TSE metabolic cages for 6 consecutive days, to measure daily food intake during long-term leptin administration. To measure neuroendocrine signaling of leptin, the mice were withheld food overnight and then injected with leptin (2 mg/kg) intraperitoneally. After 2 h, the mice were anesthetized with a ketamine (100 mg/ml) and xylazine (20 mg/ml) mixture, and tissues were rapidly removed from each mouse. After lysis of tissue samples, a total of 20 mg of protein was used for Western blot analysis.
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Cell culture
Immunoblot analysis
Mouse embryonic fibroblasts (MEFs) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.
Total cell and tissue lysates from mice were prepared and subjected to Western blot analysis (20). After blotting, a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) was incubated with the primary antibodies against phosphorylated (p)STAT3 (Tyr705), STAT3, and suppressor of cytokine signaling (SOCS)-3 (Cell Signaling Technology, Beverly, MA, USA), and b-actin (Sigma-Aldrich, St. Louis MO, USA) at 4°C overnight. Protein bands were visualized with a chemiluminescence detection kit after hybridization with a horseradish peroxidaseconjugated secondary antibody.
RNAi The MEFs were grown in 6-well plates and transfected with either a TRPV1-specific siRNA oligonucleotide (cat nos. 1441844 and 1441845) or scrambled oligonucleotide (cat no. SN-1001; both from Bioneer, Daejeon, South Korea) as controls, with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) used according to the manufacturer’s instructions. After 24 h, the cells were treated with leptin (100 ng/ml) or vehicle for 10 min, to determine the effect of RNAi-mediated knockdown of TRPV1 on leptin signaling.
Statistical analyses Data are expressed as means 6 SE, and differences between groups were examined for statistical significance by ANOVA with Fisher’s exact test. P , 0.05 was used as the criterion for statistical significance. All analyses were performed with Statistical Analysis Software (SAS, Inc., Cary, NC, USA).
Quantitative PCR in MEFs
RESULTS
The cellular RNA was extracted from WT and TRPV1 KO MEFs by the TRIzol method. After reverse transcription, PCR of cDNA synthesis from cellular RNA was performed. The expression of each gene was assessed by real-time PCR with the following primers: for mouse Socs3, 59-GGGTGGCAAAGAAAAGGAG-39 (forward), 59- GTTGAGCGTCAAGACCCAGT-39 (reverse); and for mouse b-actin, 59-TGTCCACCTTCCAGCAGATGT-39 (forward), 59-AGCTCAGTAACAGTCCGCCTAGA-39 (reverse).
Increased obesity in TRPV1 KO mice during highfat feeding When fed a chow diet, the TRPV1 KO mice and agematched WT mice showed similar body weight and body composition (whole-body fat and lean mass). In contrast, during high-fat feeding, TRPV1 KO mice gradually gained
Figure 1. TRPV1 deletion accelerates diet-induced obesity in mice. TRPV1 KO and WT mice (12 wk old) were fed an HFD (n = 10–12) or chow diet (n = 6) for 5 wk. A) Body weight was measured during 5 wk of HFD or chow. B, C) Whole-body fat (B) and lean mass (C) were measured weekly by 1H MRS. *P , 0.05 vs. HFD-fed WT mice.
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more body weight and were significantly heavier than WT mice after 4 wk of the HFD (Fig. 1A). Increased weight gain during consumption of the HFD was mostly attributed to the increases (;50%) in whole-body fat mass in the TRPV1 KO mice, compared with the WT mice, and increased adiposity was observed as early as after 1 wk of the HFD (Fig. 1B). Whole-body lean mass did not differ between the groups (Fig. 1C).
Increased obesity in HFD-fed TRPV1 KO mice is attributed to positive energy balance To determine the mechanism of increased obesity in the HFD-fed TRPV1 KO mice, we performed indirect calorimetry by using metabolic cages in both groups of mice. Daily food intake was measured for 3 consecutive days by the TSE metabolic cages. Food intake on days 1 and 2 tended to be higher in the TRPV1 KO mice, and food intake on day 3 was significantly increased, compared with that of WT mice (Fig. 2A). Oxygen consumption (VO2) and carbon dioxide production (VCO2) rates were measured by the metabolic cages at hourly intervals for 3 consecutive days. Our findings indicate that VO2 and VCO2 rates were significantly reduced, mostly during the night cycle in the TRPV1 KO mice, compared with that of the WT mice (Fig. 2B, C). Furthermore, physical activity was reduced significantly (;35%) in the TRPV1 KO mice compared with the activity in the WT mice after 5 wk of the HFD (Fig. 2D). Timeline analysis showed that this reduction in physical
activity in the TRPV1 KO mice occurred largely during the night cycle, which is consistent with reduced nightcycle energy expenditure rates in the TRPV1 KO mice. It is important to note that night-cycle activity was lower, despite higher food intake in the TRPV1 KO mice, suggesting nonfeeding behavior alterations in these KO mice. A metabolic cage study was performed in the chowfed mice for comparison, and physical activity did not differ between the chow- and the HFD-fed WT mice. In contrast, the TRPV1 KO mice showed a significant decrease in physical activity after 4 wk of HFD, compared with that in the chow-fed state (Fig. 2E). TRPV1 KO mice are more insulin resistant after high-fat feeding To determine the role of TRPV1 in glucose homeostasis, we fed male TRPV1 KO and WT mice chow or an HFD for 5 wk and performed a 2 h hyperinsulinemic-euglycemic clamp to measure insulin sensitivity in awake mice. Basal plasma insulin levels did not differ between the TRPV1 KO and WT mice fed the chow diet, and insulin levels were raised similarly in both groups during the clamp experiment (Fig. 3A). The HFD increased basal insulin levels in both groups of mice, and insulin increased to comparable levels during the clamp. While consuming the chow diet, the TRPV1 KO mice showed normal insulin sensitivity, with glucose metabolism rates comparable to those in the WT mice (Fig. 3B–D). After being fed the HFD, the WT mice developed insulin resistance, with 30 to 50% decreases in
Figure 2. Altered energy balance in TRPV1-deficient mice. Indirect calorimetry was performed in metabolic cages for 3 consecutive days in age-matched WT and TRPV1 KO mice (n = 6 for each group). A) Food intake was measured on days 1, 2, and 3 by the metabolic cages. A 24 h timeline is shown of the Vo2 consumption (B) and VCo2 production (C ) rates. D) A 24 h timeline of physical activity in HFD-fed mice. E) Averaged physical activity during the night cycle in WT and TRPV1 KO mice before (baseline) and after 5 wk of high-fat feeding. *P , 0.05 vs. WT mice or baseline.
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Figure 3. Increased insulin resistance in HFD-fed TRPV1 KO mice. All mice were 12 wk of age at the beginning of the experiment and were fed an HFD (n = 10–12) or chow diet (n = 6 for each group) for 5 wk. A) Plasma insulin levels were measured with ELISA. Samples were taken from the mice at the beginning and end of hyperinsulinemic-euglycemic clamp experiments. B) Steady-state glucose infusion rate during the experiments in awake mice. C ) Insulin-stimulated whole-body glucose turnover was calculated by infusion of [3H]glucose during the tests. D) Insulin-stimulated whole-body glycolysis rates. *P , 0.05 vs. WT mice or chow-fed mice.
glucose infusion rates and whole-body glucose turnover during clamp experiments, compared with the results in the chow-fed WT mice (Fig. 3B, C). HFD-induced insulin resistance was exacerbated in the TRPV1 KO mice with 50 to 70% reductions in glucose infusion rates and whole-body glucose turnover in HFD-fed TRPV1 KO mice, compared with the chow-fed TRPV1 KO mice. In addition, glucose infusion rates and whole-body glucose turnover were significantly lower in HFD-fed TRPV1 KO mice, compared with the HFD-fed WT mice. Insulin-stimulated whole-body glycolysis tended to be lower in the HFD-fed TRPV1 KO mice than in the HFD-fed WT mice (Fig. 3D). During the clamp experiments, a bolus injection of [14C]2-DG was used to measure the insulin-stimulated glucose uptake in individual organs. Consistent with normal insulin sensitivity in the chow-fed mice, there were no significant differences in tissue-specific glucose uptake in the TRPV1 KO and WT mice fed the chow diet (Fig. 4A–D). However, insulin-stimulated glucose uptake in heart tended to be lower in the TRPV1 KO mice (1176 6 98 vs. 1628 6 188 nM/g per minute in the WT mice; P = 0.059), but the difference did not reach statistical significance. After consuming the HFD, the TRPV1 KO mice showed significant reductions in insulin-stimulated glucose uptake in white and brown adipose tissue, compared with that in the WT mice (Fig. 4A, B). Myocardial glucose metabolism was also reduced significantly TRPV1 KO MICE DEVELOP INSULIN AND LEPTIN RESISTANCE
in TRPV1 KO mice, compared with that in the WT mice after HFD (Fig. 4C). Skeletal muscle glucose metabolism was not significantly different between the TRPV1 KO and WT mice fed chow or an HFD (Fig. 4D). Furthermore, basal HGP rates did not differ between the TRPV1 KO and WT mice on either diet, and after the HFD, the ability of insulin to suppress HGP was blunted similarly in both the TRPV1 KO and WT mice (Fig. 4E). Thus, TRPV1 KO mice developed more severe insulin resistance in the peripheral organs, compared with that in the WT mice after HFD. This effect was selective for reduced glucose metabolism in white and brown fat as well as the heart of HFD-fed TRPV1 KO mice. Our findings of increased insulin resistance in HFD-fed TRPV1 KO mice suggest that the effects of TRPV1 deficiency on insulin resistance is coupled to increased obesity after high-fat feeding in these mice. TRPV1 KO mice develop leptin resistance Recent studies have shown that leptin affects locomotor activity and energy expenditure in addition to its wellestablished effect on feeding behavior (21, 22). In the current study, we found that serum leptin levels were significantly elevated (.2-fold) in the TRPV1 KO mice, compared with those in the WT mice after HFD (Fig. 5A). 5
Figure 4. Reduced glucose metabolism in adipose tissue and heart of HFD-fed TRPV1 KO mice. Insulin-stimulated glucose uptake in individual organs was calculated by injecting 2-[14C]DG during hyperinsulinemic-euglycemic clamp experiments. A–D) Insulin-stimulated glucose uptake in white and brown adipose tissue, heart, and skeletal muscle from WT and TRPV1 KO mice fed chow (n = 6 for each group) or an HFD (n = 10–12). E) Basal and clamp HGP rates and hepatic insulin action were calculated as percentage of insulin-mediated suppression of HGP. *P , 0.05 vs. WT mice or chow-fed mice.
As a major adipokine, leptin is primarily secreted by adipocytes, and higher leptin levels in TRPV1 KO mice may be attributed to increased fat mass in these mice after HFD. Thus, we normalized circulating leptin levels to whole-body fat mass and found that HFD-fed TRPV1 KO mice still showed a significantly higher leptin level, compared with that in the HFD-fed WT mice (Fig. 5B). This difference was selective for leptin, as circulating levels of total and high-molecular-weight adiponectin were not altered in TRPV1 KO mice fed chow or HFD (Supplemental Fig. 1). We tested leptin’s effects on food intake in TRPV1 KO and WT mice by administering leptin long term with the osmotic pump (Alzet) and by measuring daily food intake with metabolic cages. Plasma leptin levels were similarly elevated after long-term leptin administration in both groups of mice (Fig. 5C). After 24 h of leptin administration, food intake was reduced by ;25% in WT mice, demonstrating potent inhibitory effects of leptin on feeding behavior (Fig. 5D, E). In contrast, leptin administration failed to suppress food intake in TRPV1 KO mice, thereby demonstrating leptin resistance in these mice. Next, we performed small interfering (si)RNA-mediated knockdown of TRPV1 in MEFs. Leptin activated STAT3 6
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phosphorylation in control MEFs, but leptin-stimulated p-STAT3 was profoundly blunted in TRPV1-deficient MEFs (Fig. 6A). Moreover, to determine the hypothalamic effect of TRPV1 deletion on leptin signaling in vivo, we injected leptin intraperitoneally into the TRPV1 KO and WT mice and obtained samples of the hypothalamus for molecular analysis. Hypothalamic STAT3 phosphorylation increased by more than 2-fold after leptin injection into the WT mice, but leptin failed to affect hypothalamic STAT3 phosphorylation in TRPV1 KO mice (Fig. 6B). SOCS3 is an Src homology (SH)-2 domain-containing protein that is expressed in response to leptin-stimulated STAT3 activation and provides a feedback inhibition of leptin receptor signaling (23). To that end, we found that SOCS3 expression at mRNA and protein levels increased significantly (2–4-fold) in primary cultured MEFs from TRPV1 KO mice, compared with levels in the WT mice (Fig. 6C, D). In addition, SOCS3 expression was measured in white adipose tissue samples and tended to increase in TRPV1 KO mice that did not show further elevations after leptin stimulation (Fig. 6E). Taken together, these results indicate that TRPV1 plays an important role in leptin-mediated STAT3 phosphorylation in hypothalamus and further suggest that TRPV1 deficiency
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Figure 5. Leptin resistance in TRPV1 KO mice. A) Plasma leptin levels were measured by ELISA in WT (n = 12) and TRPV1 KO (n = 10) mice, before and after 5 wk on an HFD. B) Plasma leptin levels were normalized to whole-body fat mass, measured with 1H MRS. C–E) Leptin (20 mg/d) was infused continuously for 6 d with implantable osmotic pumps in 12-wk-old WT (n = 5) and TRPV1 KO (n = 5) mice. Daily food intake was measured with metabolic cages; basal levels in each mouse were obtained by measuring average food intake for 3 d. Plasma leptin levels (C), daily food intake (D), and percentage change in food intake at baseline and after leptin treatment (E) are shown. *P , 0.05, baseline vs. leptin infused mice. WT vs. KO mice, chow diet vs. high-fat diet.
results in systemic leptin resistance, possibly associated with increased SOCS3 expression (24). TRPV1 KO mice develop obesity, insulin resistance, and leptin resistance with aging
lost in aging TRPV1 KO mice (Fig. 7F, G). These data indicate that TRPV1 deficiency causes leptin resistance and further increases obesity and insulin resistance in mice with aging. DISCUSSION
Both insulin resistance and leptin resistance are associated with aging, but the underlying mechanism remains poorly understood. To examine the potential role of TRPV1 in aging-associated changes in metabolism, we performed longitudinal metabolic studies in male TRPV1 KO and WT mice at 3 and 9 mo of age while feeding the mice a standard chow diet. At 3 mo of age, the TRPV1 KO and WT mice showed similar body weight and body composition (Fig. 7A–C). At 9 mo of age, the WT mice showed a modest increase in body weight resulting from increases in both fat and lean mass. Age-matched TRPV1 KO mice showed a significantly higher body weight, compared with that of the WT mice, largely because of a 2-fold increase in wholebody fat mass in the TRPV1 KO mice. A 2 h hyperinsulinemic-euglycemic clamp was performed in young (3 mo of age) and older (9 mo of age) TRPV1 KO and WT mice. Glucose infusion rates during clamp decreased by 50% in WT mice at 9 mo of age, compared with those at 3 mo of age, indicating agingassociated insulin resistance in the WT mice (Fig. 7D). Glucose infusion rates were further reduced in 9-mo-old TRPV1 KO mice, which showed severe insulin resistance with aging. Circulating leptin levels were increased by more than 3-fold in aging TRPV1 KO mice, compared with those in the aging WT mice (Fig. 7E). Whereas leptin administration significantly suppressed food intake in aging WT mice, this leptin effect was completely TRPV1 KO MICE DEVELOP INSULIN AND LEPTIN RESISTANCE
Previous evidence has implicated dietary calcium consumption in the modulation of adipocyte metabolism and obesity (25–28). Recent studies have further shown that calcium channel blockers attenuate obesity and related hypertension, suggesting that calcium channels may be directly involved in energy homeostasis and metabolic disease (29, 30). TRP channels as mediators of cation influx (e.g., Ca2+) have been examined for their roles in metabolic diseases, including obesity, type 2 diabetes, hypertension, and cardiovascular disease (31, 32). In obesity studies, the expression pattern for TRP canonical (C) 4, TRP melastatin (M)8, TRP polycystin (P)2, TRP mucolipin (ML), and TRPV6 channels were reported to correlate with obesity in genome scans (33). The TRPV4 channel was recently reported to control energy homeostasis and promote obesity (34, 35). We have also found that mice with homozygous deletion of TRPM2 are protected from dietinduced obesity and insulin resistance (36). Although all of these studies suggest a potential role of calcium channels in energy homeostasis, the underlying mechanism remains poorly understood. The role of the TRPV1 channel in obesity and glucose metabolism is controversial. Zhang et al. (12) and Yu et al. (13) have reported that the antiobesity effects of capsaicin are mediated by activation of TRPV1 channels. In contrast, Motter and Ahern (16) found that 7
Figure 6. Impaired leptin signaling in TRPV1-deficient cells. A) p-STAT3 at Tyr705, with or without leptin (100 ng/ml) for 10 minutes, as determined by Western blot analysis in MEFs with siRNA-mediated knockdown of TRPV1. The numbers above the Western blot lanes denote the relative normalized phosphorylation levels compared to the control at each time point. B) p-STAT at Tyr705 was examined by Western blot in the hypothalamus of the WT and TRPV1 KO mice after intraperitoneal injection of leptin (2 mg/kg body weight). Quantitative data are shown as means 6 SE (n = 3 mice/group). Quantitative analyses of p-STAT3 and STAT3 were performed with ImageJ software in 3 independent experiments. C, D) SOCS3 mRNA and protein levels in primary cultured MEFs from WT and TRPV1 KO mice. E) White adipose tissue expression of SOCS3 in the WT and TRPV1 KO mice, without or with leptin stimulation.
TRPV1-null mice are resistant to diet-induced obesity. Thus, it is unclear whether activation of the TRPV1 channel promotes or blocks diet-induced obesity. In our current study, we demonstrated that TRPV1 KO mice become more obese than WT mice while consuming an HFD, supporting the notion that TRPV1 activation may mediate capsaicin’s antiobesity effects. The discrepancy between our findings and those of Motter and Ahern may involve several factors, including different composition of HFD (55% fat from calories in our HFD vs. 26% fat from calories) and the pair feeding regimen used by Motter and Ahern. In addition, we examined the effects of intermediate-term high-fat feeding (i.e., 5 wk), whereas Motter and Ahern observed body weight after 10 to 20 wk of high-fat feeding. In addition to increased obesity during high-fat feeding, TRPV1 KO mice also gained more adiposity with aging than did the WT mice. At 9 mo of age, TRPV1 KO mice were also more insulin resistant than the age-matched WT mice, which may have been attributed to increased fat mass in TRPV1 KO mice. Contrary to our findings, 8
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Riera et al. (17) reported that mice lacking TRPV1 showed improved glucose tolerance, compared with WT mice at 22 mo of age. However, in their study, improved glucose tolerance was associated with increased pancreatic b-cell mass and higher insulin secretion, which may account for enhanced glucose clearance during a glucose tolerance test in TRPV1 KO mice. Consistent with our data, insulin tolerance tests found TRPV1 KO mice to be more insulin resistant than WT mice at 22 mo of age, and the researchers further concluded that an increased b-cell mass is a compensatory effect against insulin resistance in TRPV1 KO mice. Furthermore, our findings demonstrate that TRPV1 KO mice are more insulin resistant after consuming an HFD or with aging, because of the increased obesity in these mice. Obesity is a major determinant of insulin resistance, and in earlier work, we have shown that reduction in whole-body adiposity improves insulin sensitivity in mice (37). However, it is interesting to note that insulin resistance in HFD-fed TRPV1 KO mice was selective for white and brown adipose tissue and heart,
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Figure 7. Increased obesity, insulin resistance, and leptin resistance in aging TRPV1 KO mice. A) Body weights were measured at 3 and 9 mo of age in WT (n = 4) and TRPV1 KO (n = 4) mice fed a chow diet. B, C) Whole-body fat and lean mass were measured by 1H MRS at 3 and 9 mo of age. D) Steady-state glucose infusion rates during hyperinsulinemic-euglycemic clamp tests were measured in awake mice to assess insulin sensitivity at 9 mo of age (n = 4 for each group). E) Plasma leptin levels were measured with ELISA in 9-mo-old WT (n = 4) and TRPV1 KO (n = 4) mice. F) Daily food intake was measured in vehicle- and leptin-injected mice before and 24 h after leptin injection. G) Percentage change in food intake before and after leptin injection. *P , 0.05 vs. WT mice or 3-mo-old mice.
whereas skeletal muscle and liver insulin activity remained comparable to that in the HFD-fed WT mice. Since the current study examined mice with global deletion of TRPV1, local effects of TRPV1 in selective organs may further affect systemic glucose metabolism. To that end, TRPM2-deficient mice also showed increased glucose metabolism selectively in heart and white adipose tissue (36). In addition, we examined other TRP channels for potential compensatory effects and found that TRPM2, TRPC1, and TRPC3 mRNA levels were not altered in TRPV1 KO MEFs (Supplemental Fig. 2). However, TRPV4 expression was reduced in the KO MEFs, and the potential role of TRPV4 as well as the organ-specific nature of TRPV1’s effects should be explored in future studies. Leptin is a major adipokine that plays a critical role in energy intake and energy expenditure by stimulating a hypothalamic leptin signaling pathway that has not been fully elucidated. Leptin resistance is a major cause of the failure of leptin therapy to treat obese human subjects, and understanding the molecular mechanism of leptin resistance is important for the development of TRPV1 KO MICE DEVELOP INSULIN AND LEPTIN RESISTANCE
therapies to treat obesity. Our findings demonstrate that TRPV1 KO mice are more obese after an HFD, partly because of increased food intake and decreases in energy expenditure and physical activity, and these effects may be coupled to leptin resistance in these mice. Our data showing that siRNA-mediated TRPV1 knockdown in MEFs results in loss of leptin’s effects on STAT3 phosphorylation, a major downstream leptin signal, further demonstrate an important role of TRPV1 in leptin signaling. Consistent with these data, STAT3 phosphorylation at Tyr705 residue was significantly stimulated by capsaicin (10 mM) alone and as a cotreatment with leptin (100 ng/ml) for 10 minutes in WT MEFs, and oral treatment of capsaicin with HFD for 12 wk reduced food intake compared to that in vehicle-treated mice without alterations in plasma leptin levels in mice (data not shown). These data further support TRPV1 regulation of STAT3 phosphorylation, leptin signaling, and feeding behavior in mice. However, we found that TRPV1 does not directly interact with leptin receptor by stimulation of leptin treatment (data not shown), whereas tissue-specific gene expression profiles from 9
BioGPS (38) have shown a similar mRNA expression pattern of leptin receptor to TRPV1. To that end, increased SOCS3 expression in the context of TRPV1 deficiency may underlie impaired leptin signaling and leptin resistance. To our knowledge, this is the first report on the role of TRPV1 channels in leptin resistance. Further study is needed to determine how TRPV1 regulates the leptin signaling pathway. Taken together, we have shown that TRPV1 regulates energy balance, and loss of TRPV1 exacerbates HFD-induced and aging-associated obesity in mice. This detrimental effect of TRPV1 deficiency on obesity results from loss of sensitivity to leptin-controlled energy homeostasis. In addition, loss of TRPV1 caused diet-induced insulin resistance to deteriorate, especially in adipose tissue and heart, but not in skeletal muscle and liver. Thus, our findings identify TRPV1 as a novel regulator of leptin signaling and support the potential role of a TRPV1 agonist, such as capsaicin, in the treatment of obesity and insulin resistance. This work was supported by U.S. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK080756, U24-DK093000, and R24-DK090963 to J.K.K.; National Leap Research Program Grant 2010-0029233 to K.W.L.; and National Research Foundation of Korea Grant 2012M3A9C4048818 to K.W.L. funded by the Ministry of Science, Information and Communications Technology, and Future Planning.
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The FASEB Journal • Research Communication
Transient receptor potential vanilloid type-1 channel regulates diet-induced obesity, insulin resistance, and leptin resistance Eunjung Lee,*,†,‡ Dae Young Jung,* Jong Hun Kim,*,§ Payal R. Patel,* Xiaodi Hu,* Yongjin Lee,* Yoshihiro Azuma,* Hsun-Fan Wang,* Nicholas Tsitsilianos,* Umber Shafiq,* Jung Yeon Kwon,*,§ Hyong Joo Lee,† Ki Won Lee,†,§,1 and Jason K. Kim*,†,{,1 *Program in Molecular Medicine and {Department of Medicine, Division of Endocrinology, Metabolism, and Diabetes, University of Massachusetts Medical School, Worcester, Massachusetts, USA; †World Class University Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul, Republic of Korea; ‡Traditional Alcoholic Beverage Research Team, Korea Food Research Institute, Seongnam, Republic of Korea; and §Advanced Institutes of Convergence Technology, Seoul National University, Suwon, Republic of Korea Insulin resistance is a major characteristic of obesity and type 2 diabetes, but the underlying mechanism is unclear. Recent studies have shown a metabolic role of capsaicin that may be mediated via the transient receptor potential vanilloid type (TRPV)-1 channel. In this study, TRPV1 knockout (KO) and wild-type (WT) mice (as controls) were fed a high-fat diet (HFD), and metabolic studies were performed to measure insulin and leptin action. The TRPV1 KO mice became more obese than the WT mice after HFD, partly attributed to altered energy balance and leptin resistance in the KO mice. The hyperinsulinemic-euglycemic clamp experiment showed that the TRPV1 KO mice were more insulin resistant after HFD because of the ∼40% reduction in glucose metabolism in the white and brown adipose tissue, compared with that in the WT mice. Leptin treatment failed to suppress food intake, and leptin-mediated hypothalamic signal transducer and activator of transcription (STAT)-3 activity was blunted in the TRPV1 KO mice. We also found that the TRPV1 KO mice were more obese and insulin resistant than the WT mice at 9 mo of age. Taken together, these results indicate that lacking TRPV1 exacerbates the obesity and insulin resistance associated with an HFD and aging, and our findings further suggest that TRPV1 has a major role in regulating glucose metabolism and hypothalamic leptin’s effects in obesity.—Lee, E., Jung, D. Y., Kim, J. H., Patel, P. R., Hu, X., Lee, Y., Azuma, Y., Wang, H.-F., Tsitsilianos, N., Shafiq, U., Kwon, J. Y., Lee, H. J., Lee, K. W., Kim, J. K. Transient receptor potential vanilloid type-1 channel regulates dietinduced obesity, insulin resistance, and leptin resistance. FASEB J. 29, 000–000 (2015). www.fasebj.org ABSTRACT
Abbreviations: DG, deoxyglucose; DG-6-P, deoxyglucose 6phosphate; HFD, high-fat diet; HGP, hepatic glucose production; KO, knockout; MEF, mouse embryonic fibroblast; MRS, magnetic resonance spectroscopy; p-STAT, phosphorylated STAT; RNAi, RNA interference; SH, Src homology; siRNA, small interfering RNA; SOCS, suppressor of cytokine signaling; (continued on next page)
0892-6638/15/0029-0001 © FASEB
Key Words: capsaicin • hyperinsulinemic-euglycemic clamp glucose metabolism • aging
•
THE INCREASING PREVALENCE IN OBESITY continues to be a global health problem and is having a major impact on the worldwide incidence of diabetes. Obesity is characterized by altered secretion of hormones, metabolites, and inflammatory cytokines from adipose tissue (1, 2). Among them, leptin regulates energy balance as a major satiety factor and through its effects on energy expenditure (3). Although leptin therapy has been shown to improve energy homeostasis in lipodystrophic subjects and obese diabetic rats, underlying leptin resistance in most obese humans has limited its therapeutic potential (4–8). Thus, understanding the mechanism of leptin resistance plays an important role in developing new targets to treat obesity and type 2 diabetes. The transient receptor potential vanilloid type (TRPV)1 channel is a member of the TRP protein superfamily, a large group of cation-permeable channels expressed in mammalian cells. TRPV1 is a nonselective cation channel that is highly permeable to Ca2+ and Na+ and is involved in thermogenesis and pain sensing (9–11). Capsaicin, a major component of chili pepper, is a natural and specific agonist for the TRPV1 channel and induces the pain and thermogenic responses collectively known as the spicy sensation. Several recent reports have shown that dietary consumption of chili pepper or capsaicin-containing foods results in an increased energy expenditure that is associated with a lower incidence of obesity (12–14). In 1 Correspondence: J.K.K., University of Massachusetts Medical School, Program in Molecular Medicine, 368 Plantation St., AS9.1041, Worcester, MA, 01605, USA. E-mail: jason.kim@ umassmed.edu; K.W.L., Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea. E-mail: [email protected] doi: 10.1096/fj.14-268300 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.
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addition, dietary capsaicin improves insulin sensitivity in obese mice that have been fed a high-fat diet (HFD) (15). The underlying mechanism by which capsaicin affects energy balance and glucose metabolism may involve its activation of the TRPV1 channel. Zhang et al. (12) recently found that capsaicin inhibits adipogenesis in 3T3-L1 preadipcoytes, and RNA interference (RNAi) knockdown of TRPV1 channel rescues adipocytes from capsaicin’s effects. Furthermore, long-term capsaicin intake prevents dietinduced obesity in mice, but such an antiobesity effect of capsaicin is lost in TRPV1 knockout (KO) mice. In contrast, Motter and Ahern (16) reported that TRPV1-null mice in fact gain less weight after long-term high-fat feeding than do wild-type (WT) mice. This effect was associated with increased thermogenic capacity in TRPV1-null mice. Although these findings regarding the role of the TRPV1 channel in obesity are contradictory, Riera et al. (17) found that mice lacking a TRPV1 channel show longevity. Taken together, these findings imply that capsaicin and the TRPV1 channel have an important role in energy homeostasis. In the current study, we examined the effects of TRPV1 deficiency in diet-induced obesity, insulin resistance, and leptin action in mice. MATERIALS AND METHODS Animals and diet Male TRPV1-deficient (TRPV1 KO; C57BL/6 background) mice and WT (C57BL/6 background) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed under controlled temperature (23°C) and lighting (12 h of light, 7 AM to 7 PM; 12 h of dark, 7 PM to 7 AM). Starting at 12 wk of age, mice were fed an HFD (Harlan Teklad TD 93075; 55% kcal from fat, 24% kcal from carbohydrate, and 21% kcal from protein; Harlan Laboratories, Indianapolis, IN, USA) ad libitum for 5 wk to induce obesity. The animal studies were approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. Body composition and energy balance Whole-body fat and lean mass were noninvasively measured by 1H magnetic resonance spectroscopy (MRS; Echo Medical Systems, Houston, TX, USA). Indirect calorimetry and energy balance parameters including food and water intake, energy expenditure, respiratory exchange ratio, and physical activity were noninvasively assessed for 3 or 6 days with metabolic cages (TSESystems, Inc., Chesterfield, MO, USA). We used the LabMaster platform (TSE-Systems, Inc.), with easy-to-use calorimetry and featuring fully automated monitoring for food and water and XYZ activity. The Labmaster cages that are most similar to the home cages at our facility were used, thereby allowing the use of bedding in the cage and minimizing any animal anxiety during the experimental period. The system provides intuitive software with flexibility for experimental setup and data use.
(continued from previous page) STAT, signal transducer and activator of transcription; TRP, transient receptor potential channels, M, melastatin, V, vanilloid, C, cation, P, polycystin; VO2, oxygen consumption; VCO2, carbon dioxide production; WT, wild-type
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Hyperinsulinemic-euglycemic clamp After the mice were fed chow or an HFD, they underwent survival surgery 5 or 6 days before the hyperinsulinemic-euglycemic clamp experiments, to establish an indwelling catheter in the jugular vein. On the day of the clamp experiment, the mice were withheld food overnight (;15 h), and a 2 h clamp was conducted in conscious mice with a primed and continuous infusion of human insulin (150 mU/kg body weight priming followed by 2.5 mU/kg per minute; Humulin; Eli Lilly, Indianapolis, IN, USA) (18). To maintain euglycemia, 20% glucose was infused at variable rates during the clamp. Whole-body glucose turnover was assessed with a continuous infusion of [3-3H]glucose (PerkinElmer, Waltham, MA, USA), and 2-deoxy-D-[1-14C]glucose (2-[14C]DG) was administered as a bolus (10 mCi) 75 min after the start of the clamp experiment, to measure insulin-stimulated glucose uptake in individual organs. At the end of the clamp, the mice were anesthetized, and tissues were taken for biochemical analysis (18). Biochemical analysis and calculation Glucose concentrations during the clamp experiments were analyzed in 10 ml plasma by a glucose oxidase method on a GM9 Analyzer (Analox Instruments, Ltd., Hammersmith, London, UK). Plasma concentrations of [3-3H]glucose, 2-[14C]deoxyglucose (DG), and 3H2O were determined after deproteinization of the plasma samples (18). For the determination of tissue 2[14C]deoxyglucose 6-phosphate (DG-6-P) content, the samples were homogenized, and the supernatants were placed in an ionexchange column to separate 2-[14C]DG-6-P from 2-[14C]DG. Rates of basal hepatic glucose production (HGP) and insulinstimulated whole-body glucose turnover were determined as previously described (18). The insulin-stimulated rate of HGP was determined by subtracting the glucose infusion rate from wholebody glucose turnover. Whole-body glycolysis and glycogen plus lipid synthesis from glucose were calculated (18). The insulinstimulated glucose uptake in individual tissues was assessed by determining the tissue (e.g., skeletal muscle) content of 2-[14C] DG-6-P and plasma 2-[14C]DG clearance profile. Plasma hormone measurement Plasma levels of leptin, insulin, and adiponectin (total and high molecular weight) were measured with ELISA kits (ALPCO Diagnostics, Salem, NH, USA). Plasma samples were collected in mice that were withheld food overnight (5 h). Blood was collected carefully into heparin-coated Eppendorf tubes (Eppendorf AG, Hamburg, Germany) and centrifuged at 12,000 rpm for 3 minutes at 4°C. For ELISA assays, the supernatant of centrifuged blood was used according to the manufacturer’s instructions. Leptin administration study For long-term administration of leptin, osmotic pumps (1007D; Alzet Corp., Cupertino, CA, USA) containing leptin (20 mg/d; R&D Systems, Minneapolis, MN, USA) or saline were implanted subcutaneously in anesthetized mice (19). After implantation of the osmotic pumps, the mice were returned to their cages to recover from anesthesia and then were placed in the TSE metabolic cages for 6 consecutive days, to measure daily food intake during long-term leptin administration. To measure neuroendocrine signaling of leptin, the mice were withheld food overnight and then injected with leptin (2 mg/kg) intraperitoneally. After 2 h, the mice were anesthetized with a ketamine (100 mg/ml) and xylazine (20 mg/ml) mixture, and tissues were rapidly removed from each mouse. After lysis of tissue samples, a total of 20 mg of protein was used for Western blot analysis.
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Cell culture
Immunoblot analysis
Mouse embryonic fibroblasts (MEFs) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.
Total cell and tissue lysates from mice were prepared and subjected to Western blot analysis (20). After blotting, a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) was incubated with the primary antibodies against phosphorylated (p)STAT3 (Tyr705), STAT3, and suppressor of cytokine signaling (SOCS)-3 (Cell Signaling Technology, Beverly, MA, USA), and b-actin (Sigma-Aldrich, St. Louis MO, USA) at 4°C overnight. Protein bands were visualized with a chemiluminescence detection kit after hybridization with a horseradish peroxidaseconjugated secondary antibody.
RNAi The MEFs were grown in 6-well plates and transfected with either a TRPV1-specific siRNA oligonucleotide (cat nos. 1441844 and 1441845) or scrambled oligonucleotide (cat no. SN-1001; both from Bioneer, Daejeon, South Korea) as controls, with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) used according to the manufacturer’s instructions. After 24 h, the cells were treated with leptin (100 ng/ml) or vehicle for 10 min, to determine the effect of RNAi-mediated knockdown of TRPV1 on leptin signaling.
Statistical analyses Data are expressed as means 6 SE, and differences between groups were examined for statistical significance by ANOVA with Fisher’s exact test. P , 0.05 was used as the criterion for statistical significance. All analyses were performed with Statistical Analysis Software (SAS, Inc., Cary, NC, USA).
Quantitative PCR in MEFs
RESULTS
The cellular RNA was extracted from WT and TRPV1 KO MEFs by the TRIzol method. After reverse transcription, PCR of cDNA synthesis from cellular RNA was performed. The expression of each gene was assessed by real-time PCR with the following primers: for mouse Socs3, 59-GGGTGGCAAAGAAAAGGAG-39 (forward), 59- GTTGAGCGTCAAGACCCAGT-39 (reverse); and for mouse b-actin, 59-TGTCCACCTTCCAGCAGATGT-39 (forward), 59-AGCTCAGTAACAGTCCGCCTAGA-39 (reverse).
Increased obesity in TRPV1 KO mice during highfat feeding When fed a chow diet, the TRPV1 KO mice and agematched WT mice showed similar body weight and body composition (whole-body fat and lean mass). In contrast, during high-fat feeding, TRPV1 KO mice gradually gained
Figure 1. TRPV1 deletion accelerates diet-induced obesity in mice. TRPV1 KO and WT mice (12 wk old) were fed an HFD (n = 10–12) or chow diet (n = 6) for 5 wk. A) Body weight was measured during 5 wk of HFD or chow. B, C) Whole-body fat (B) and lean mass (C) were measured weekly by 1H MRS. *P , 0.05 vs. HFD-fed WT mice.
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more body weight and were significantly heavier than WT mice after 4 wk of the HFD (Fig. 1A). Increased weight gain during consumption of the HFD was mostly attributed to the increases (;50%) in whole-body fat mass in the TRPV1 KO mice, compared with the WT mice, and increased adiposity was observed as early as after 1 wk of the HFD (Fig. 1B). Whole-body lean mass did not differ between the groups (Fig. 1C).
Increased obesity in HFD-fed TRPV1 KO mice is attributed to positive energy balance To determine the mechanism of increased obesity in the HFD-fed TRPV1 KO mice, we performed indirect calorimetry by using metabolic cages in both groups of mice. Daily food intake was measured for 3 consecutive days by the TSE metabolic cages. Food intake on days 1 and 2 tended to be higher in the TRPV1 KO mice, and food intake on day 3 was significantly increased, compared with that of WT mice (Fig. 2A). Oxygen consumption (VO2) and carbon dioxide production (VCO2) rates were measured by the metabolic cages at hourly intervals for 3 consecutive days. Our findings indicate that VO2 and VCO2 rates were significantly reduced, mostly during the night cycle in the TRPV1 KO mice, compared with that of the WT mice (Fig. 2B, C). Furthermore, physical activity was reduced significantly (;35%) in the TRPV1 KO mice compared with the activity in the WT mice after 5 wk of the HFD (Fig. 2D). Timeline analysis showed that this reduction in physical
activity in the TRPV1 KO mice occurred largely during the night cycle, which is consistent with reduced nightcycle energy expenditure rates in the TRPV1 KO mice. It is important to note that night-cycle activity was lower, despite higher food intake in the TRPV1 KO mice, suggesting nonfeeding behavior alterations in these KO mice. A metabolic cage study was performed in the chowfed mice for comparison, and physical activity did not differ between the chow- and the HFD-fed WT mice. In contrast, the TRPV1 KO mice showed a significant decrease in physical activity after 4 wk of HFD, compared with that in the chow-fed state (Fig. 2E). TRPV1 KO mice are more insulin resistant after high-fat feeding To determine the role of TRPV1 in glucose homeostasis, we fed male TRPV1 KO and WT mice chow or an HFD for 5 wk and performed a 2 h hyperinsulinemic-euglycemic clamp to measure insulin sensitivity in awake mice. Basal plasma insulin levels did not differ between the TRPV1 KO and WT mice fed the chow diet, and insulin levels were raised similarly in both groups during the clamp experiment (Fig. 3A). The HFD increased basal insulin levels in both groups of mice, and insulin increased to comparable levels during the clamp. While consuming the chow diet, the TRPV1 KO mice showed normal insulin sensitivity, with glucose metabolism rates comparable to those in the WT mice (Fig. 3B–D). After being fed the HFD, the WT mice developed insulin resistance, with 30 to 50% decreases in
Figure 2. Altered energy balance in TRPV1-deficient mice. Indirect calorimetry was performed in metabolic cages for 3 consecutive days in age-matched WT and TRPV1 KO mice (n = 6 for each group). A) Food intake was measured on days 1, 2, and 3 by the metabolic cages. A 24 h timeline is shown of the Vo2 consumption (B) and VCo2 production (C ) rates. D) A 24 h timeline of physical activity in HFD-fed mice. E) Averaged physical activity during the night cycle in WT and TRPV1 KO mice before (baseline) and after 5 wk of high-fat feeding. *P , 0.05 vs. WT mice or baseline.
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Figure 3. Increased insulin resistance in HFD-fed TRPV1 KO mice. All mice were 12 wk of age at the beginning of the experiment and were fed an HFD (n = 10–12) or chow diet (n = 6 for each group) for 5 wk. A) Plasma insulin levels were measured with ELISA. Samples were taken from the mice at the beginning and end of hyperinsulinemic-euglycemic clamp experiments. B) Steady-state glucose infusion rate during the experiments in awake mice. C ) Insulin-stimulated whole-body glucose turnover was calculated by infusion of [3H]glucose during the tests. D) Insulin-stimulated whole-body glycolysis rates. *P , 0.05 vs. WT mice or chow-fed mice.
glucose infusion rates and whole-body glucose turnover during clamp experiments, compared with the results in the chow-fed WT mice (Fig. 3B, C). HFD-induced insulin resistance was exacerbated in the TRPV1 KO mice with 50 to 70% reductions in glucose infusion rates and whole-body glucose turnover in HFD-fed TRPV1 KO mice, compared with the chow-fed TRPV1 KO mice. In addition, glucose infusion rates and whole-body glucose turnover were significantly lower in HFD-fed TRPV1 KO mice, compared with the HFD-fed WT mice. Insulin-stimulated whole-body glycolysis tended to be lower in the HFD-fed TRPV1 KO mice than in the HFD-fed WT mice (Fig. 3D). During the clamp experiments, a bolus injection of [14C]2-DG was used to measure the insulin-stimulated glucose uptake in individual organs. Consistent with normal insulin sensitivity in the chow-fed mice, there were no significant differences in tissue-specific glucose uptake in the TRPV1 KO and WT mice fed the chow diet (Fig. 4A–D). However, insulin-stimulated glucose uptake in heart tended to be lower in the TRPV1 KO mice (1176 6 98 vs. 1628 6 188 nM/g per minute in the WT mice; P = 0.059), but the difference did not reach statistical significance. After consuming the HFD, the TRPV1 KO mice showed significant reductions in insulin-stimulated glucose uptake in white and brown adipose tissue, compared with that in the WT mice (Fig. 4A, B). Myocardial glucose metabolism was also reduced significantly TRPV1 KO MICE DEVELOP INSULIN AND LEPTIN RESISTANCE
in TRPV1 KO mice, compared with that in the WT mice after HFD (Fig. 4C). Skeletal muscle glucose metabolism was not significantly different between the TRPV1 KO and WT mice fed chow or an HFD (Fig. 4D). Furthermore, basal HGP rates did not differ between the TRPV1 KO and WT mice on either diet, and after the HFD, the ability of insulin to suppress HGP was blunted similarly in both the TRPV1 KO and WT mice (Fig. 4E). Thus, TRPV1 KO mice developed more severe insulin resistance in the peripheral organs, compared with that in the WT mice after HFD. This effect was selective for reduced glucose metabolism in white and brown fat as well as the heart of HFD-fed TRPV1 KO mice. Our findings of increased insulin resistance in HFD-fed TRPV1 KO mice suggest that the effects of TRPV1 deficiency on insulin resistance is coupled to increased obesity after high-fat feeding in these mice. TRPV1 KO mice develop leptin resistance Recent studies have shown that leptin affects locomotor activity and energy expenditure in addition to its wellestablished effect on feeding behavior (21, 22). In the current study, we found that serum leptin levels were significantly elevated (.2-fold) in the TRPV1 KO mice, compared with those in the WT mice after HFD (Fig. 5A). 5
Figure 4. Reduced glucose metabolism in adipose tissue and heart of HFD-fed TRPV1 KO mice. Insulin-stimulated glucose uptake in individual organs was calculated by injecting 2-[14C]DG during hyperinsulinemic-euglycemic clamp experiments. A–D) Insulin-stimulated glucose uptake in white and brown adipose tissue, heart, and skeletal muscle from WT and TRPV1 KO mice fed chow (n = 6 for each group) or an HFD (n = 10–12). E) Basal and clamp HGP rates and hepatic insulin action were calculated as percentage of insulin-mediated suppression of HGP. *P , 0.05 vs. WT mice or chow-fed mice.
As a major adipokine, leptin is primarily secreted by adipocytes, and higher leptin levels in TRPV1 KO mice may be attributed to increased fat mass in these mice after HFD. Thus, we normalized circulating leptin levels to whole-body fat mass and found that HFD-fed TRPV1 KO mice still showed a significantly higher leptin level, compared with that in the HFD-fed WT mice (Fig. 5B). This difference was selective for leptin, as circulating levels of total and high-molecular-weight adiponectin were not altered in TRPV1 KO mice fed chow or HFD (Supplemental Fig. 1). We tested leptin’s effects on food intake in TRPV1 KO and WT mice by administering leptin long term with the osmotic pump (Alzet) and by measuring daily food intake with metabolic cages. Plasma leptin levels were similarly elevated after long-term leptin administration in both groups of mice (Fig. 5C). After 24 h of leptin administration, food intake was reduced by ;25% in WT mice, demonstrating potent inhibitory effects of leptin on feeding behavior (Fig. 5D, E). In contrast, leptin administration failed to suppress food intake in TRPV1 KO mice, thereby demonstrating leptin resistance in these mice. Next, we performed small interfering (si)RNA-mediated knockdown of TRPV1 in MEFs. Leptin activated STAT3 6
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phosphorylation in control MEFs, but leptin-stimulated p-STAT3 was profoundly blunted in TRPV1-deficient MEFs (Fig. 6A). Moreover, to determine the hypothalamic effect of TRPV1 deletion on leptin signaling in vivo, we injected leptin intraperitoneally into the TRPV1 KO and WT mice and obtained samples of the hypothalamus for molecular analysis. Hypothalamic STAT3 phosphorylation increased by more than 2-fold after leptin injection into the WT mice, but leptin failed to affect hypothalamic STAT3 phosphorylation in TRPV1 KO mice (Fig. 6B). SOCS3 is an Src homology (SH)-2 domain-containing protein that is expressed in response to leptin-stimulated STAT3 activation and provides a feedback inhibition of leptin receptor signaling (23). To that end, we found that SOCS3 expression at mRNA and protein levels increased significantly (2–4-fold) in primary cultured MEFs from TRPV1 KO mice, compared with levels in the WT mice (Fig. 6C, D). In addition, SOCS3 expression was measured in white adipose tissue samples and tended to increase in TRPV1 KO mice that did not show further elevations after leptin stimulation (Fig. 6E). Taken together, these results indicate that TRPV1 plays an important role in leptin-mediated STAT3 phosphorylation in hypothalamus and further suggest that TRPV1 deficiency
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Figure 5. Leptin resistance in TRPV1 KO mice. A) Plasma leptin levels were measured by ELISA in WT (n = 12) and TRPV1 KO (n = 10) mice, before and after 5 wk on an HFD. B) Plasma leptin levels were normalized to whole-body fat mass, measured with 1H MRS. C–E) Leptin (20 mg/d) was infused continuously for 6 d with implantable osmotic pumps in 12-wk-old WT (n = 5) and TRPV1 KO (n = 5) mice. Daily food intake was measured with metabolic cages; basal levels in each mouse were obtained by measuring average food intake for 3 d. Plasma leptin levels (C), daily food intake (D), and percentage change in food intake at baseline and after leptin treatment (E) are shown. *P , 0.05, baseline vs. leptin infused mice. WT vs. KO mice, chow diet vs. high-fat diet.
results in systemic leptin resistance, possibly associated with increased SOCS3 expression (24). TRPV1 KO mice develop obesity, insulin resistance, and leptin resistance with aging
lost in aging TRPV1 KO mice (Fig. 7F, G). These data indicate that TRPV1 deficiency causes leptin resistance and further increases obesity and insulin resistance in mice with aging. DISCUSSION
Both insulin resistance and leptin resistance are associated with aging, but the underlying mechanism remains poorly understood. To examine the potential role of TRPV1 in aging-associated changes in metabolism, we performed longitudinal metabolic studies in male TRPV1 KO and WT mice at 3 and 9 mo of age while feeding the mice a standard chow diet. At 3 mo of age, the TRPV1 KO and WT mice showed similar body weight and body composition (Fig. 7A–C). At 9 mo of age, the WT mice showed a modest increase in body weight resulting from increases in both fat and lean mass. Age-matched TRPV1 KO mice showed a significantly higher body weight, compared with that of the WT mice, largely because of a 2-fold increase in wholebody fat mass in the TRPV1 KO mice. A 2 h hyperinsulinemic-euglycemic clamp was performed in young (3 mo of age) and older (9 mo of age) TRPV1 KO and WT mice. Glucose infusion rates during clamp decreased by 50% in WT mice at 9 mo of age, compared with those at 3 mo of age, indicating agingassociated insulin resistance in the WT mice (Fig. 7D). Glucose infusion rates were further reduced in 9-mo-old TRPV1 KO mice, which showed severe insulin resistance with aging. Circulating leptin levels were increased by more than 3-fold in aging TRPV1 KO mice, compared with those in the aging WT mice (Fig. 7E). Whereas leptin administration significantly suppressed food intake in aging WT mice, this leptin effect was completely TRPV1 KO MICE DEVELOP INSULIN AND LEPTIN RESISTANCE
Previous evidence has implicated dietary calcium consumption in the modulation of adipocyte metabolism and obesity (25–28). Recent studies have further shown that calcium channel blockers attenuate obesity and related hypertension, suggesting that calcium channels may be directly involved in energy homeostasis and metabolic disease (29, 30). TRP channels as mediators of cation influx (e.g., Ca2+) have been examined for their roles in metabolic diseases, including obesity, type 2 diabetes, hypertension, and cardiovascular disease (31, 32). In obesity studies, the expression pattern for TRP canonical (C) 4, TRP melastatin (M)8, TRP polycystin (P)2, TRP mucolipin (ML), and TRPV6 channels were reported to correlate with obesity in genome scans (33). The TRPV4 channel was recently reported to control energy homeostasis and promote obesity (34, 35). We have also found that mice with homozygous deletion of TRPM2 are protected from dietinduced obesity and insulin resistance (36). Although all of these studies suggest a potential role of calcium channels in energy homeostasis, the underlying mechanism remains poorly understood. The role of the TRPV1 channel in obesity and glucose metabolism is controversial. Zhang et al. (12) and Yu et al. (13) have reported that the antiobesity effects of capsaicin are mediated by activation of TRPV1 channels. In contrast, Motter and Ahern (16) found that 7
Figure 6. Impaired leptin signaling in TRPV1-deficient cells. A) p-STAT3 at Tyr705, with or without leptin (100 ng/ml) for 10 minutes, as determined by Western blot analysis in MEFs with siRNA-mediated knockdown of TRPV1. The numbers above the Western blot lanes denote the relative normalized phosphorylation levels compared to the control at each time point. B) p-STAT at Tyr705 was examined by Western blot in the hypothalamus of the WT and TRPV1 KO mice after intraperitoneal injection of leptin (2 mg/kg body weight). Quantitative data are shown as means 6 SE (n = 3 mice/group). Quantitative analyses of p-STAT3 and STAT3 were performed with ImageJ software in 3 independent experiments. C, D) SOCS3 mRNA and protein levels in primary cultured MEFs from WT and TRPV1 KO mice. E) White adipose tissue expression of SOCS3 in the WT and TRPV1 KO mice, without or with leptin stimulation.
TRPV1-null mice are resistant to diet-induced obesity. Thus, it is unclear whether activation of the TRPV1 channel promotes or blocks diet-induced obesity. In our current study, we demonstrated that TRPV1 KO mice become more obese than WT mice while consuming an HFD, supporting the notion that TRPV1 activation may mediate capsaicin’s antiobesity effects. The discrepancy between our findings and those of Motter and Ahern may involve several factors, including different composition of HFD (55% fat from calories in our HFD vs. 26% fat from calories) and the pair feeding regimen used by Motter and Ahern. In addition, we examined the effects of intermediate-term high-fat feeding (i.e., 5 wk), whereas Motter and Ahern observed body weight after 10 to 20 wk of high-fat feeding. In addition to increased obesity during high-fat feeding, TRPV1 KO mice also gained more adiposity with aging than did the WT mice. At 9 mo of age, TRPV1 KO mice were also more insulin resistant than the age-matched WT mice, which may have been attributed to increased fat mass in TRPV1 KO mice. Contrary to our findings, 8
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Riera et al. (17) reported that mice lacking TRPV1 showed improved glucose tolerance, compared with WT mice at 22 mo of age. However, in their study, improved glucose tolerance was associated with increased pancreatic b-cell mass and higher insulin secretion, which may account for enhanced glucose clearance during a glucose tolerance test in TRPV1 KO mice. Consistent with our data, insulin tolerance tests found TRPV1 KO mice to be more insulin resistant than WT mice at 22 mo of age, and the researchers further concluded that an increased b-cell mass is a compensatory effect against insulin resistance in TRPV1 KO mice. Furthermore, our findings demonstrate that TRPV1 KO mice are more insulin resistant after consuming an HFD or with aging, because of the increased obesity in these mice. Obesity is a major determinant of insulin resistance, and in earlier work, we have shown that reduction in whole-body adiposity improves insulin sensitivity in mice (37). However, it is interesting to note that insulin resistance in HFD-fed TRPV1 KO mice was selective for white and brown adipose tissue and heart,
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Figure 7. Increased obesity, insulin resistance, and leptin resistance in aging TRPV1 KO mice. A) Body weights were measured at 3 and 9 mo of age in WT (n = 4) and TRPV1 KO (n = 4) mice fed a chow diet. B, C) Whole-body fat and lean mass were measured by 1H MRS at 3 and 9 mo of age. D) Steady-state glucose infusion rates during hyperinsulinemic-euglycemic clamp tests were measured in awake mice to assess insulin sensitivity at 9 mo of age (n = 4 for each group). E) Plasma leptin levels were measured with ELISA in 9-mo-old WT (n = 4) and TRPV1 KO (n = 4) mice. F) Daily food intake was measured in vehicle- and leptin-injected mice before and 24 h after leptin injection. G) Percentage change in food intake before and after leptin injection. *P , 0.05 vs. WT mice or 3-mo-old mice.
whereas skeletal muscle and liver insulin activity remained comparable to that in the HFD-fed WT mice. Since the current study examined mice with global deletion of TRPV1, local effects of TRPV1 in selective organs may further affect systemic glucose metabolism. To that end, TRPM2-deficient mice also showed increased glucose metabolism selectively in heart and white adipose tissue (36). In addition, we examined other TRP channels for potential compensatory effects and found that TRPM2, TRPC1, and TRPC3 mRNA levels were not altered in TRPV1 KO MEFs (Supplemental Fig. 2). However, TRPV4 expression was reduced in the KO MEFs, and the potential role of TRPV4 as well as the organ-specific nature of TRPV1’s effects should be explored in future studies. Leptin is a major adipokine that plays a critical role in energy intake and energy expenditure by stimulating a hypothalamic leptin signaling pathway that has not been fully elucidated. Leptin resistance is a major cause of the failure of leptin therapy to treat obese human subjects, and understanding the molecular mechanism of leptin resistance is important for the development of TRPV1 KO MICE DEVELOP INSULIN AND LEPTIN RESISTANCE
therapies to treat obesity. Our findings demonstrate that TRPV1 KO mice are more obese after an HFD, partly because of increased food intake and decreases in energy expenditure and physical activity, and these effects may be coupled to leptin resistance in these mice. Our data showing that siRNA-mediated TRPV1 knockdown in MEFs results in loss of leptin’s effects on STAT3 phosphorylation, a major downstream leptin signal, further demonstrate an important role of TRPV1 in leptin signaling. Consistent with these data, STAT3 phosphorylation at Tyr705 residue was significantly stimulated by capsaicin (10 mM) alone and as a cotreatment with leptin (100 ng/ml) for 10 minutes in WT MEFs, and oral treatment of capsaicin with HFD for 12 wk reduced food intake compared to that in vehicle-treated mice without alterations in plasma leptin levels in mice (data not shown). These data further support TRPV1 regulation of STAT3 phosphorylation, leptin signaling, and feeding behavior in mice. However, we found that TRPV1 does not directly interact with leptin receptor by stimulation of leptin treatment (data not shown), whereas tissue-specific gene expression profiles from 9
BioGPS (38) have shown a similar mRNA expression pattern of leptin receptor to TRPV1. To that end, increased SOCS3 expression in the context of TRPV1 deficiency may underlie impaired leptin signaling and leptin resistance. To our knowledge, this is the first report on the role of TRPV1 channels in leptin resistance. Further study is needed to determine how TRPV1 regulates the leptin signaling pathway. Taken together, we have shown that TRPV1 regulates energy balance, and loss of TRPV1 exacerbates HFD-induced and aging-associated obesity in mice. This detrimental effect of TRPV1 deficiency on obesity results from loss of sensitivity to leptin-controlled energy homeostasis. In addition, loss of TRPV1 caused diet-induced insulin resistance to deteriorate, especially in adipose tissue and heart, but not in skeletal muscle and liver. Thus, our findings identify TRPV1 as a novel regulator of leptin signaling and support the potential role of a TRPV1 agonist, such as capsaicin, in the treatment of obesity and insulin resistance. This work was supported by U.S. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK080756, U24-DK093000, and R24-DK090963 to J.K.K.; National Leap Research Program Grant 2010-0029233 to K.W.L.; and National Research Foundation of Korea Grant 2012M3A9C4048818 to K.W.L. funded by the Ministry of Science, Information and Communications Technology, and Future Planning.
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