Lack of TRPV 2 impairs thermogenesis in mouse brown adipose tissue
2016; Springer Nature; Volume: 17; Issue: 3 Linguagem: Inglês
10.15252/embr.201540819
ISSN1469-3178
AutoresWuping Sun, Kunitoshi Uchida, Yoshiro Suzuki, Yiming Zhou, Minji Kim, Yasunori Takayama, Nobuyuki Takahashi, Tsuyoshi Goto, Shigeo Wakabayashi, Teruo Kawada, Yuko Iwata, Makoto Tominaga,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoArticle11 February 2016free access Transparent process Lack of TRPV2 impairs thermogenesis in mouse brown adipose tissue Wuping Sun Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Kunitoshi Uchida Corresponding Author Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Yoshiro Suzuki Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Yiming Zhou Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Search for more papers by this author Minji Kim Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan Search for more papers by this author Yasunori Takayama Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Nobuyuki Takahashi Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan Search for more papers by this author Tsuyoshi Goto Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan Search for more papers by this author Shigeo Wakabayashi Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Teruo Kawada Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan Search for more papers by this author Yuko Iwata Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Makoto Tominaga Corresponding Author Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Wuping Sun Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Kunitoshi Uchida Corresponding Author Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Yoshiro Suzuki Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Yiming Zhou Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Search for more papers by this author Minji Kim Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan Search for more papers by this author Yasunori Takayama Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Nobuyuki Takahashi Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan Search for more papers by this author Tsuyoshi Goto Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan Search for more papers by this author Shigeo Wakabayashi Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Teruo Kawada Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan Search for more papers by this author Yuko Iwata Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Makoto Tominaga Corresponding Author Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan Search for more papers by this author Author Information Wuping Sun1,2, Kunitoshi Uchida 1,2, Yoshiro Suzuki1,2, Yiming Zhou1, Minji Kim3, Yasunori Takayama1,2, Nobuyuki Takahashi3, Tsuyoshi Goto3, Shigeo Wakabayashi4, Teruo Kawada3, Yuko Iwata4 and Makoto Tominaga 1,2 1Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki, Japan 2Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan 3Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan 4Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan *Corresponding author. Tel: +81 564 59 5287/5286; Fax: +81 564 59 5285; E-mail: [email protected] *Corresponding author. Tel: +81 564 59 5287/5286; Fax: +81 564 59 5285; E-mail: [email protected] EMBO Rep (2016)17:383-399https://doi.org/10.15252/embr.201540819 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Brown adipose tissue (BAT), a major site for mammalian non-shivering thermogenesis, could be a target for prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca2+-permeable non-selective cation channel, plays vital roles in the regulation of various cellular functions. Here, we show that TRPV2 is expressed in brown adipocytes and that mRNA levels of thermogenic genes are reduced in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice. The induction of thermogenic genes in response to β-adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca2+ concentrations in wild-type brown adipocytes. In addition, TRPV2KO mice have more white adipose tissue and larger brown adipocytes and show cold intolerance, and lower BAT temperature increases in response to β-adrenergic receptor stimulation. Furthermore, TRPV2KO mice have increased body weight and fat upon high-fat-diet treatment. Based on these findings, we conclude that TRPV2 has a role in BAT thermogenesis and could be a target for human obesity therapy. Synopsis Mice lacking TRPV2 show impaired BAT thermogenesis and are prone to obesity on a high-fat diet. TRPV2-mediated increases in thermogenic gene expression upon β-adrenergic receptor stimulation might involve intracellular calcium signals. TRPV2KO mice show cold intolerance and impaired BAT thermogenesis upon β-adrenergic receptor activation. TRPV2KO mice are prone to be obese and insulin resistant on a high-fat diet. TRPV2-mediated increases in intracellular calcium concentrations might promote thermogenic gene expression upon β-adrenergic receptor activation. Introduction The prevalence of obesity has increased worldwide, and it is believed to be the result of an imbalance between the intake and expenditure of energy 1. Obesity is also a serious health problem that is implicated in various diseases including type II diabetes, hypertension, coronary heart diseases, and cancer 2, and it is characterized by increased adipose tissue mass that results from increased fat cell size and number, suggesting that the main contributor to obesity is adipose tissue 3. Unlike white adipose tissue (WAT), brown adipose tissue (BAT) is specialized for the efficient dissipation of chemical energy in the form of heat, and BAT is a major site for mammalian non-shivering thermogenesis with mitochondrial uncoupling protein 1 (UCP1) 45. Although BAT was originally identified in infants and rodents, recent studies have reported that BAT also exists in adult humans as demonstrated using a combination of high-resolution imaging techniques 67. This novel finding highlights the crucial role for BAT in the regulation of energy metabolism and fat deposition 89. Upon cold exposure or β-adrenergic receptor stimulation, BAT functions are activated and "browning" of WAT occurs as well due to the emerged UCP1-positive "beige" cells 1011. When activated, UCP1 in mitochondria uncouples the respiratory chain and heat is generated 1213. Moreover, BAT activity in human and the amount of BAT tissue are inversely correlated with adiposity 14. Thus, BAT could be a promising target for human obesity prevention and treatment, and understanding the molecular mechanisms for thermogenesis in brown adipocytes is the subject of intense investigation. The concentrations of intracellular Ca2+ ([Ca2+]i) and the amplitude of its fluctuations have primary importance for survival and function in a plethora of cell types 15. For many cells, there have been extensive studies of [Ca2+]i signals, whereas relatively little knowledge about Ca2+ signaling in white and brown adipocytes is available despite its suggested importance 16. Transient receptor potential vanilloid 2 (TRPV2) is activated by noxious heat with an activation temperature threshold of higher than 52°C 17 and by a number of chemical ligands, for example, 2-aminoethoxydiphenyl borate (2APB) and lysophosphatidylcholine (LPC) in a species-specific manner 1819. SKF96365 (SKF) is a TRPV2-selective antagonist 1820. Importantly, TRPV2 is also reported to be a mechano-sensitive channel activated by mechanical stretch and cell swelling 2122. Several studies have reported the involvement of TRP channels in adipose tissue functions. For example, TRPV1 was reported to be involved in the regulation of food intake and glucose homeostasis in white fat during obesity 23. And the roles of TRPV1 and TRPV3 have been shown in the regulation of adipogenesis; their activation-mediated Ca2+ influx prevents white adipocyte differentiation and plays anti-adipogenic roles in vivo 2425. TRPM8 stimulation by its ligands increased UCP1 expression in brown adipocytes and BAT through PKA phosphorylation 26. Moreover, activation of TRPV1 by capsaicin or TRPM8 by cold temperature or menthol enhanced UCP1 expression 2728, whereas knockdown of TRPV4 facilitated UCP1 expression in white adipocytes 29. Although recent studies indicate that the involvement of [Ca2+]i increases in the functions of adipose tissues 2629, the roles of Ca2+ influx in BAT are still not well understood. In this study, we demonstrated that TRPV2 was functionally expressed in mouse brown adipocytes in culture. mRNA levels of thermogenic genes were significantly lower in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice, and increases in the genes in response to β-adrenergic receptor stimulation were significantly lower in TRPV2KO brown adipocytes and significantly suppressed by reduction in intracellular Ca2+ concentrations in wild-type (WT) brown adipocytes. More interestingly, TRPV2KO interscapular BAT (iBAT) showed impaired adaptive thermogenesis upon cold exposure and administration of a β3-adrenergic receptor agonist. And TRPV2KO mice showed significantly heavier body weight and fat upon high-fat-diet (HFD) treatment for 8 weeks continuously. These data suggest that regulation of Ca2+ influx by TRPV2 is critical for maintaining iBAT thermogenic functions under physiological conditions and stimulation. Results TRPV2 was functionally expressed in the differentiated mouse brown adipocytes in culture To explore the expression pattern of TRP channels in adipocytes, we first examined TRPV2 expression in primary cultured brown adipocytes and BAT. RT–PCR and real-time RT–PCR analyses revealed that the expression level of Trpv2 mRNA looked the highest in the differentiated mouse brown adipocytes among Trpv1, Trpv2, Trpv3, and Trpv4 (Fig 1A and B). The same result was obtained in the analysis of mouse iBAT (Fig 1C). The mRNA expression of Trpm8 was significantly lower than Trpv2 in iBAT (Fig EV1A). Although Trpv2 mRNA expression was not different among brown adipocytes, CD11b-negative (CD11b (−)) and CD11b-positive (CD11b (+), macrophages) cells isolated from WT iBAT (Fig EV1B-D), the CD11b (+) fractions were negligibly small compared with brown adipocytes, suggesting the little contribution of macrophages to the BAT functions. We next examined the expression of TRPV2 protein in differentiated brown adipocytes from WT and TRPV2KO mice by Western blotting, and lack of TRPV2 was confirmed in TRPV2KO mice (Fig 1D). Then, we examined the functional expression of TRPV2 in mouse brown adipocytes using Ca2+ imaging and whole-cell patch-clamp methods. A TRPV2 agonist, either 2APB or LPC, increased [Ca2+]i which was blocked by SKF (Fig EV1E and F), indicating that the observed 2APB- or LPC-evoked [Ca2+]i increases were mediated by TRPV2 activation. Adipocytes showed increases in [Ca2+]i upon activation of α1-adrenergic receptor by nor-epinephrine (NE) through Gq-coupled receptor signaling, indicating that they were differentiated adipocytes (Figs 1E and EV1E–G), and such 2APB- or LPC-evoked [Ca2+]i increases were drastically reduced in TRPV2KO brown adipocytes (Figs 1E and EV1G), further indicating the functional TRPV2 expression in WT differentiated brown adipocytes. Whole-cell patch-clamp recordings showed that 2APB activated currents with outward rectification that was blocked by SKF (Fig 1F). Mean densities of the 2APB-induced currents in mouse brown adipocytes at −60 mV and + 100 mV were significantly smaller in the cells given both 2APB and SKF compared with cells given 2APB alone (Fig 1G). These results demonstrated that TRPV2 is functionally expressed in the differentiated mouse brown adipocytes. Figure 1. TRPV2 was functionally expressed in mouse differentiated brown adipocytes RT–PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3, and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (−)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template. Results of real-time RT–PCR analysis of Trpv1, Trpv2, Trpv3, and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene unaffected by adipogenesis. Data are presented as mean ± SEM, n = 6. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3, and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms. Averaged traces of [Ca2+]i changes in response to 500 μmol/l 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One μmol/l NE was used to confirm differentiation. Five μmol/l ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mouse brown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; **P < 0.01. Unpaired Student's t-test. A representative trace of whole-cell current activated by 3 mmol/l of 2APB in the presence or absence of 10 μmol/l SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage-ramp pulse protocol. The right inset indicates the current–voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue), and current in the presence of 2APB alone (c, red) at the time points of a, b, and c, respectively. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red), and currents in the presence of 2APB+SKF (blue) at −60 mV and + 100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; **P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. mRNA expression of Trpv2 and Trpm8, and functional expression of TRPV2 in adipose tissues and differentiated brown adipocytes A. The mRNA expression levels of Trpv2 and Trpm8 in iBAT normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 10; **P < 0.01. Unpaired Student's t-test. B–D. The mRNA expression levels of Cd11b, Ucp1, and Trpv2 in brown adipocytes, CD11b-negative (Cd11b (-)) cells and CD11b-positive (Cd11b (+)) cells isolated from WT iBAT. Data are presented as mean ± SEM, n = 6; **P < 0.01 vs. brown adipocytes group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. E, F. Changes in intracellular Ca2+ concentration ([Ca2+]i) in mouse brown adipocytes. Responses to a TRPV2 agonist, 500 μmol/l 2APB (E) or 30 μmol/l LPC (F), were inhibited by a selective TRPV2 antagonist 10 μmol/l SKF96365 (SKF). One μmol/l norepinephrine (NE) was used to confirm differentiation. Five μmol/l ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mouse brown adipocytes. G. Averaged traces of [Ca2+]i changes in response to 30 μmol/l LPC in the differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. Data are presented as mean ± SEM, WT cells (n = 115), and TRPV2KO brown adipocytes (n = 89); **P < 0.01. Unpaired Student's t-test. Download figure Download PowerPoint Lack of TRPV2 facilitated brown adipocyte differentiation In order to examine the involvement of TRPV2 in adipocyte differentiation like TRPV1 and TRPV3 2425, we compared the mRNA expression of Trpv1, Trpv2, Trpv3, and Trpv4 in pre-adipocytes from mouse iBAT and 6-day differentiated brown adipocytes. Only Trpv2 mRNA level was significantly increased in 6-day differentiated brown adipocytes (Fig 2A). To further clarify the involvement of TRPV2 in the differentiation and the thermogenic function of mouse brown adipocytes, we analyzed the brown adipocytes from WT and TRPV2KO mice. Continuous treatment with either 2APB or LPC for 6 days significantly reduced the number of differentiated brown adipocytes (Fig 2B). Then, we compared the differentiated brown adipocyte number in WT and TRPV2KO cells. Although number of differentiated brown adipocytes and triglyceride levels were not different between WT and TRPV2KO cells with control differentiation medium, significantly more differentiated brown adipocytes and higher triglyceride levels were observed in TRPV2KO adipocytes when we used ten-time-diluted differentiation medium (Fig 2C and D). These results can be interpreted that differentiation was saturated in the condition with control medium and that the difference became significant in the condition with reduced differentiation efficiency. This TRPV2-dependent brown adipocyte differentiation is consistent with the data shown in Fig 2B. These results demonstrated that TRPV2 could be activated even in the in vitro condition and that this activation could prevent mouse brown adipocyte differentiation. Figure 2. Brown adipocytes from TRPV2KO mice exhibited facilitated differentiation A. Results of real-time RT–PCR analysis of Trpv1, Trpv2, Trpv3, and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. *P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test. B. The number of 6-day differentiated mouse brown adipocytes treated with TRPV2 agonists, 100 μmol/l 2APB or 10 μmol/l LPC. Mean ± SEM, n = 6, *P < 0.05 and **P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. C, D. Comparison of the numbers of 6-day differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, **P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. Download figure Download PowerPoint TRPV2-dependent increases in the expression of thermogenic genes could involve intracellular Ca2+ signaling in brown adipocytes Because expression of Trpv2 was increased in the differentiated brown adipocytes, we also compared mRNA levels of thermogenic genes between WT and TRPV2KO brown adipocytes. Ucp1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1a) mRNA levels were significantly lower in TRPV2KO brown adipocytes, while peroxisome proliferator-activated receptor γ (Pparg) and β3-adrenergic receptor (Adrb3) mRNA levels were not different (Fig 3A), suggesting that TRPV2 is involved in the thermogenic function of brown adipocytes. It is known that the mRNA levels of Ucp1 and Pgc1a are enhanced by sympathetic nerve activation after cold exposure 3031. Thus, we examined the effect of isoproterenol (ISO), a β-adrenergic receptor agonist, on brown adipocytes. Although WT brown adipocytes exhibited the increases in Ucp1 and Pgc1a mRNA expression levels after ISO treatment, these increases were almost abolished in the TRPV2KO cells (Fig 3B and C). Thus, modulation of the basal expression level of thermogenic genes and the response to β-adrenergic receptor stimuli may be impaired in TRPV2KO brown adipocytes. In order to investigate the pathways downstream of β-adrenergic receptor activation, we examined the effects of forskolin on brown adipocytes. mRNA expression level of Ucp1 (Fig 3D) and non-esterified fatty acid (NEFA) release (Fig 3E) were significantly enhanced in brown adipocytes from WT mice treated with forskolin. On the other hand, the enhancement was significantly reduced in TRPV2KO brown adipocyte. These results suggested that TRPV2-dependent induction in the expression of thermogenic genes in brown adipocytes involves a cyclic adenosine monophosphate pathway. To test the importance of [Ca2+]i for the thermogenic gene expression in brown adipocytes, we applied BAPTA-AM, a cell-permeant Ca2+ chelator. BAPTA-AM treatment significantly reduced the induction of Ucp1 and Pgc1a mRNA expression by ISO (Fig 3F and G), suggesting that [Ca2+]i changes possibly due to Ca2+ influx through TRPV2 are critical for the induction of thermogenic genes upon activation of sympathetic nervous system. Figure 3. Basal expression of genes related to BAT function and their changes A. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1a), peroxisome proliferator-activated receptor γ (Pparg), and β-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; **P < 0.01 vs. WT. Unpaired Student's t-test. B, C. Changes in Ucp1 (B) and Pgc1a (C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 μmol/l isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; **P < 0.01 vs. DMSO group; #P < 0.05; ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. D, E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 μmol/l forskolin for 4 h. Data are presented as mean ± SEM, n = 6; *P < 0.05; **P < 0.01 vs. DMSO group; ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. F, G. Changes in Ucp1 mRNA (F) and Pgc1a mRNA (G) in the differentiated brown adipocytes treated with 10 μmol/l ISO alone or 10 μmol/l ISO plus 10 μmol/l BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; *P < 0.05; **P < 0.01 vs. DMSO group; ##P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. Download figure Download PowerPoint TRPV2KO mice might have exhibited an energy imbalance We examined whether TRPV2KO mice exhibited impairments in energy metabolism. Table 1 depicts a comprehensive evaluation of the multiple metabolic parameters in WT and TRPV2KO mice. Under at libitum feeding conditions, blood glucose, plasma insulin, NEFA, and serum cholesterol levels were not different between WT and TRPV2KO mice. TRPV2KO mice exhibited significantly smaller body weights until 8 weeks of age (Fig 4A) as previously reported 32. On the other hand, food intake and water intake were not different between WT and TRPV2KO mice (Fig 4B and C). We then compared the weights of tissues related to energy metabolism upon normalization to their body weights to minimize the effects of body weight difference. The normalized weights of iBAT, iWAT, and epididymal WAT (eWAT) were significantly larger in TRPV2KO mice compared with WT mice at 8 weeks of age (Fig 4D). Moreover, the mRNA levels of Ucp1 and Pgc1a were significantly lower in TRPV2KO iBAT similar to the brown adipocytes (Fig 3A), while Pparg, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1) mRNA levels were not different (Fig 4E). PR domain containing 16 (Prdm16) expression was not different, either (Fig 4E), suggesting that differentiation of brown adipocytes is not different in the tissue level. On the other hand, the Ucp2 expression levels were not different between WT and TRPV2KO iBAT probably because expression levels were very low. Interestingly, the expression levels of genes associated with lipid metabolism (lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36)) were slightly higher in TRPV2KO iBAT although the differences were not significant (Fig 4E). These results suggested that an energy imbalance might have existed in TRPV2KO mice. Table 1. Blood biochemical parameters and oxygen consumption in WT and TRPV2KO mice Blood glucose (mg/dl) Plasma insulin (ng/ml) Serum NEFA (mEq/l) Serum cholesterol (mg/dl) Oxygen consumption (ml/kg/h) Dark Light WT 157.63 ± 9.79 0.94 ± 0.13 0.91 ± 0.16 58.09 ± 8.69 3804.74 ± 151.62 3044.55 ± 151.21 TRPV2KO 154.50 ± 6.22n.s. 1.39 ± 0.27n.s. 0.84 ± 0.13n.s. 44.52 ± 4.46n.s. 4063.26 ± 90.85n.s. 2984.90 ± 66.20n.s. NEFA: Non-esterified fatty acid. Data are represented as the mean ± SEM of 8 mice in each group. n.s. indicates that no significant differences were observed between WT and TRPV2KO mice. Unpaired Student's t-test. Figure 4. Energy imbalance in TRPV2-deficient mice A. Body weight changes from 3 weeks to 8 weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; *P < 0.05; **P < 0.01 vs. WT group. Unpaired Student's t-test. B, C. Changes in food intake (B) and water intake (C) from 3 weeks to 8 weeks of age between WT and TRPV2KO mice (n = 6). Data are presented as mean ± SEM. D. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15). Data are presented as mean ± SEM; *P < 0.05; **P < 0.01 vs. WT group. Unpaired Student's t-test. E. The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1a, Pparg, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl), and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; *P < 0.05 vs. WT group. Unpaired Student's t-test. Download figure Download PowerPoint Adipocytes from TRPV2KO iBAT exhibited an accumulation of lipid droplets and an increase in cell size To further explore the involvement of TRPV2 in mouse iBAT function, we performed a histological study by a hematoxylin and eosin staining. Surprisingly, we observed more and larger lipid droplets rather than multiple lipid droplets in adipocytes from 8-week-old TRPV2KO mice (Fig 5A). When comparing the droplet size distribution, mean values were significantly larger in TRPV2KO iBAT in 8-week-old mice compared with WT iBAT (Fig 5B). In addition, cell diameters were
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