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δ-Tocopherol promotes thermogenic gene expression via PGC-1α upregulation in 3T3-L1 cells
Biochemical and Biophysical Research Communications xxx (2018) 1e7
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d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3-L1 cells Rieko Tanaka-Yachi a, *, Masato Shirasaki a, Rena Otsu a, Chie Takahashi-Muto b, Hideki Inoue a, Yoshinori Aoki c, Taisuke Koike c, Chikako Kiyose d a
Department of Applied Bioscience, Kanagawa Institute of Technology, Kanagawa, Japan Department of Clinical Nutrition, Kitasato Junior College of Health and Hygienic Sciences, Niigata, Japan Mitsubishi-Chemical Foods Corporation, Tokyo, Japan d Department of Nutrition and Life Science, Kanagawa Institute of Technology, Kanagawa, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2018 Accepted 4 October 2018 Available online xxx
Activation of thermogenic adipocytes (brown and beige) has been considered an attractive target for weight loss and treatment of metabolic disease. Peroxisome proliferator-activated receptor g coactivator-1 a (PGC1-a) is a master regulator of thermogenic gene expression in thermogenic adipocytes. We previously reported that a-tocopherol upregulated PGC-1a gene expression and promoted thermogenic adipocyte differentiation in mammalian adipocytes. In this study, we investigated the effects of the vitamin E analogs (a-, g- and d-tocopherol) on PGC-1a and uncoupling protein 1 (UCP1) gene expression in 3T3-L1 cells. The expression of PGC-1a and UCP1 increased significantly with the addition of d-tocopherol. In d-tocopherol-treated cells, nuclear translocation of PGC-1a increased, as did p38 mitogen-activated protein kinase (MAPK) expression and phosphorylation. Our results suggest that p38 MAPK activation by d-tocopherol contributes to PGC-1a activation and UCP1 induction. © 2018 Elsevier Inc. All rights reserved.
Keywords: Tocopherol Thermogenic adipocytes PGC-1a UCP1 p38 MAPK
1. Introduction Adipose tissue is characterized as either white adipose tissue (WAT) or brown adipose tissue (BAT). White adipocytes have large lipid droplets and few mitochondria, whereas beige and brown adipocytes have multilocular lipid droplets and a large number of mitochondria containing uncoupling protein 1 (UCP1). UCP1 uncouples the respiratory chain, allowing for fast substrate oxidation and functions as a radiator for thermogenesis to maintain body temperature. Human BAT is localized in the supraclavicular and neck region [1,2], and there is a negative correlation between BAT mass and body mass index [3]. Classical brown adipocytes are derived from a myf-5-positive, muscle-like cellular lineage. Beige adipocytes, identified in 2012, are novel thermogenic adipocytes [4]. Beige adipocytes, which are differentiated from white adipose depots, are derived from a myf-5 -negative, white adipocyte lineage. It has been reported that human BAT located in the supraclavicular depots consisted mainly of beige adipocytes [4,5].
Peroxisome proliferator-activated receptor g co-activator-1 a (PGC1-a) plays an important role in the regulation of mitochondrial biogenesis and thermogenic gene expression. PGC1-a is expressed mainly in skeletal muscle cells and brown and beige adipocytes, and its expression is regulated by fasting, exercise, and cold exposure [6e8]. Several factors control PGC-1a activation, including protein kinases, such as p38 mitogen-activated protein kinase (MAPK), 50 AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and ribosomal protein S6 kinase (S6K) [9e13]. Activated PGC1-a regulates hermogenic gene expression through its interaction with PPARg and other transcription factors. In addition, it has been reported that a PPARg agonist, rosiglitazone, promotes thermogenic adipocyte differentiation in mouse WAT [14]. Tocopherols (toc) are present naturally as four analogs (a-, b-, g-, d-), each differing in the number and position of methyl groups on the chroman ring. Landrier et al. demonstrated that a-toc influenced PPARg expression and target gene expression [15]. In this study, we investigated the function these tocopherols in vitro.
* Corresponding author. Department of Applied Bioscience, Kanagawa Institute of Technology, 1030, Shimo-ogino, Atsugi-shi, Kanagawa, 243-0292, Japan. E-mail address: [email protected] (R. Tanaka-Yachi). https://doi.org/10.1016/j.bbrc.2018.10.021 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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2. Materials and methods 2.1. Materials Tocopherol analogs (a-toc, g-toc, and d-toc) were purchased from Wako Pure Chem. Ind. Ltd. (Osaka, Japan). Rosiglitazone was purchased from Wako Pure Chem. Ind. Ltd. (Osaka, Japan). Rabbit anti-PGC-1a antibody (AB3242) was purchased from Merck & Co., Inc., and rabbit anti-UCP1 antibody (ab10983) was purchased from Abcam. Rabbit anti-phospho-p38-MAPK antibody (#9211) and rabbit anti-phospho-AMPK antibody (#2535) were purchased from CST Japan K.K. (Tokyo, Japan). An inhibitor of p38 MAPK (SB203580) was purchased from Invivogen. 4,6-Diamidino-2-phenylindole Dihydrochloride (DAPI) stain was purchased from AAT Bioquest, Inc. 2.2. Cell culture and differentiation 3T3-L1 cells were cultured at 37 C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and Lglutamine (292 mg/ml). Cells were seeded in 6-well plates at a density of 4 105 cells per well and cultured for three days postconfluency. Cells were maintained in DMEM supplemented with 10% fatal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine (292 mg/ml), insulin (10 mg/ml), dexamethasone (2.5 mM) and 3-isobutyl-1-methylxanthine (0.5 mM). Each vitamin E analog was added at the induction of differentiation, and cells were cultured for 10 days. Rosiglitazone, a PPARg agonist, was added at a final concentration of 10 mM as a positive control. Control cells were treated with equivalent DMSO. SB203580, the specific inhibitor of p38 MAPK was treated during the last 24 h. 2.3. RNA interference A siRNA duplex oligomer (Stealth RNAi) was purchased from Invitrogen; the sequence was 50 -GCG GAC AGA AUU GAG AGA CCG CUU U-30 . Control siRNA (Silencer Negative Control siRNA) was obtained from Nippon EGT. Cells were transfected with siRNA at a final concentration of 20 nM using Lipofectamine RNAi MAX (Invitrogen), according to the manufacturer's instructions, and during the last 72 h of the culture period.
The membranes were blocked for 5 min using Bullet Blocking One (Nacalai Tesque, Inc.). Membranes were incubated with primary antibodies for 2 h, and then incubated with secondary antibodies conjugated to peroxidase (Bio-Rad Laboratories, Inc.) for 1 h. The proteins were visualized by enhanced chemiluminescence using a Universal Hood II (Bio-Rad Laboratories, Inc.). 2.6. Immunofluorescence staining Cells were washed with PBS briefly and fixed in 3.7% formaldehyde in PBS at room temperature for 15 min. Cells were permeabilized for 5 min with 50 mg/ml digitonin and quenched with 50 mM NH4Cl in PBS for 10 min. The cells then were washed with PBS three times and treated with blocking buffer (3% BSA in PBS) for 45 min. Cells were incubated with primary antibodies for 2 h and incubated with secondary antibodies and DAPI for 1 h, before being washed with PBS three times and mounting. Images were obtained using a laser-scanning microscope with a 63 1.4 PlanApochromat oil immersion lens. 2.7. Statistical analysis All data are expressed as mean ± SD. Statistical analyses were performed by one-way ANOVA, followed by Bonferroni's post hoc test. Data were visualized using Kaleida Graph ver.4.5 (Hulinks Inc., Tokyo, Japan). Differences were considered significant at p < 0.05. 3. Results 3.1. Effects of vitamin E analogs on gene expressions of PPARg, PGC1a, and UCP1 Gene expression of PPARg, PGC-1a, and UCP1 in 3T3-L1 cells increased significantly with the addition of rosiglitazone (PPARg agonist). These findings confirmed our previous report that a-toc induced the expressions of PPARg, PGC-1a, and UCP1. Conversely, g-toc did not affect expression of these genes. Surprisingly, d-toc significantly increased gene expression of all three genes to a similar magnitude as adding rosiglitazone (Fig. 1A). Next, we investigated the changes in expression of PGC-1a and UCP1 genes after adding different concentrations of d-toc. As a result, expression of PGC-1a and UCP1 genes increased in a concentrationdependent manner across a range of d-toc from 1 to 50 mM (Fig. 1B).
2.4. Measurement of mRNA expression using real-time PCR Total RNA was extracted using Sepasol RNA II (Nacalai Tesque Inc.). The quantity and purity of the RNA were determined by measuring absorbance at 258/280 nm. Total RNA was reverse-transcribed into cDNA using a high-capacity RNA-to-cDNA kit, according to the manufacturer's protocol. A 7500 Fast Real-Time PCR system and real-time PCR kit (TaqMan® Gene Expression Assays) were employed, according to the manufacturer's instructions. Beta-actin was used as an internal control. The assay IDs of the primer/probe mixtures in the TaqMan gene expression assays were as follows: PPARg (Pparg); Mm01184322_m1, PGC-1a (Ppargc1a); Mm01208835_m1, UCP1 (Ucp1); Mm01244861_m1, b-actin (Actb); Mm00607939_m1, ERK1 (Mapk3); Mm01973540_m1, ERK2 (Mapk1); Mm00442479_m1, p38 MAPK(Mapk14); Mm01301009_m1, AMPK (Prkaa1); Mm01296700_m1, S6K (Rps6kb2); Mm00445440_m1, SIRT1 (Sirt1); Mm0116521_m1. 2.5. Measurement of protein expression by western blotting Total protein from cells was separated in a 10% polyacrylamide gel and then transferred to polyvinylidene difluoride membranes.
3.2. Effects of d-tocopherol addition on size of lipid droplet and UCP1 staining We observed cell morphology with optical microscopy. The size of lipid droplets was smaller than the control and resembled a “multilocular lipid droplet,” which is characteristic of brown and beige adipocytes (Fig. 2A). Next, we determined the intracellular expression pattern of UCP1 protein by immunofluorescent staining. As shown in Fig. 2B, staining of UCP1 was enhanced in cells treated with d-toc. 3.3. Effects of d-tocopherol on PGC-1a activation and the validation by PGC-1a knockdown Protein expression of PGC-1a was measured by western blotting. Unlike the results from gene expression analysis, a-tocopherol did not affect protein expression of PGC-1a. Conversely, d-toc significantly increased expression of PGC-1a (Fig. 3A). We examined the intracellular distribution of PGC-1a by immunofluorescence staining. Nuclear translocation of PGC-1a was enhanced by dtoc addition (Fig. 3B). Next, we examined gene expression of UCP1
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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Fig. 1. Effects of tocopherols on gene expression of PPARg, PGC-1a, and UCP1. (A) Gene expression of PPARg, PGC-1a, and UCP1 in 3T3-L1 cells. Cells were cultured with 10 mM tocopherol or 10 mM rosiglitazone. Control cells were treated with DMSO. (B) Concentration dependency of d-toc on PGC-1a and UCP1 gene expression. Gene expression was measured by real-time PCR, which was normalized with b-actin activity. The values presented are mean ± SD for three samples. Statistical analysis was performed by one-way ANOVA, followed by Bonferroni's post hoc test (*p < 0.05, **p < 0.01).
in PGC-1a knockdown cells to verify whether the increase of UCP1 expression by d-toc was due to PGC-1a induction. Gene expression of PGC-1a was suppressed to approximately 20% following siRNA treatment, and the staining of PGC-1a decreased significantly following PGC-1a siRNA treatment (Fig. 3C and D). In these cells, any increase of UCP1 mRNA expression by d-toc was eliminated (Fig. 3E). Similarly, the increase of UCP1 staining by d-toc was suppressed by PGC-1a knockdown (Fig. 3F).
Next, we examined expression of PGC-1a and UCP1 in p38 MAPK inhibitor (SB203580) treated cells to verify whether PGC-1a became induced and activated due to p38 MAPK activation. The increase of PGC-1a and UCP1 expression by d-toc tended to be suppressed by SB203580 treatment, but it was not completely eliminated (Fig. 4D).
3.4. Effects of d-tocopherol on kinases and deacetylases that regulate PGC-1a activation
In this study, we compared the effects of tocopherol analogs on the transcription factors and UCP1 gene expression in 3T3-L1 adipocytes. We previously reported that a-toc upregulated PGC-1a gene expression in vivo and in vitro [16]. We confirmed that a-toc induced the expression of PPARg, PGC-1a, and UCP1 mRNA in 3T3L1 cells. Consistent with previous reports, g-tocopherol did not affect expression of these genes. Surprisingly, however, d-toc induced gene expression more markedly than a-toc. The effects of d-toc were comparable with that of rosiglitazone, which was used as a positive control. In particular, the induction of PGC-1a and UCP1 expression was larger than expected; therefore, the influence of PGC-1a expression should be considered further, rather than PPARg-dependent regulation. In addition, our results showed that
PGC-1a activation can be regulated by several protein kinases and deacetylases. Therefore, we examined gene expression in ERK1, ERK2, p38 MAPK, AMPK, S6K, and SIRT1 in 3T3-L1 cells. Gene expression of p38 MAPK increased significantly by d-toc addition. On the other hand, d-toc did not affect the expression of other kinases and SIRT1 (Fig. 4A). Moreover, we detected the activated p38 MAPK by immunofluorescence staining. As a result, phosphorylated p38 MAPK (p-p38 MAPK) increased in d-toc treated cells (Fig. 4B). Protein expression of p38 MAPK and p-p38 MAPK increased significantly in cells treated with 50 mM d-toc (Fig. 4C).
4. Discussion
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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Fig. 2. Effects of d-tocopherol on cell morphology and intracellular expression of UCP1. (A) Effects of d-tocopherol on cell morphology. Cells were observed by optical microscopy. Scale bar, 20 mm. (B) Immunofluorescence staining of UCP1. Cells were fixed, permeabilized, stained for UCP1 (in green) and nuclei with DAPI (in blue), and visualized with confocal microscopy. Scale bar, 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
antioxidant activity is not proportional to changes in gene expression. At final concentrations between 1 and 50 mM, expression of UCP1 and PGC-1a increased in a concentration-dependent manner with the addition of d-toc. However, cell death was observed in cells treated with 100 mM d-toc, and gene expression tended to decrease compared to cells treated with 50 mM. Conversely, cell death was not observed in a-toc treated cells. In adipocytes, a high concentration of d-toc may induce cell death. Delta-toc treatment also increased PGC-1a and UCP1 protein expression. Conversely, a-toc did not affect PGC-1a protein expression when the final concentration was 10 mM. UCP1 protein expression tended to increase in a-toc treated cells, but the increase was not significant. We examined protein expression levels in cells treated with higher concentrations of a-toc. When comparing them at the same concentrations, the data suggest that d-toc was more effective than a-toc at inducing the expression of PGC-1a and UCP1. We examined the influence of the PGC-1a knockdown on UCP1
induction with d-toc. As a result, PGC-1a siRNA canceled the induction of UCP1 gene expression with d-toc treatment. These results showed that PGC-1a is indispensable for UCP1 induction with d-toc. Nuclear translocation of PGC-1a occurs when it is phosphorylated and activated. Therefore, we suggest that d-toc promote activation of PGC-1a. Protein kinases, such as AMPK and p38 MAPK, control PGC-1a function by phosphorylating and activating the protein [9e13]. Delta-tocotrienol, an isoform of vitamin E, has been reported to have a more potent anticancer activity compared to other tocotrienols [17e19]. It has been reported that d-tocotrienol promoted p38 MAPK activation in cancer cells [17]. In this study, we suggest that d-toc could activate p38 MAPK in 3T3-L1 cells. The cell death observed in d-toc treatments may be related to p38 MAPK activation. However, p38 MAPK inhibition did not completely eliminate the effects of d-toc, suggesting that other factors also contribute to this effect. In conclusion, we demonstrated that d-tocopherol promoted PGC-1a activation and UCP1 expression in mammalian adipocytes.
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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Fig. 3. The effect of d-tocopherol on PGC-1a protein expression and the influence of PGC-1a knockdown. (A) Protein expression of PGC-1a. Cells were cultured with 10 mM tocopherols. Control cells were treated with DMSO. Protein expression was measured with western blotting, normalized with GAPDH. The values are mean ± SD for three samples. Statistical analysis was performed by one-way ANOVA, followed by Bonferroni's post hoc test (*p < 0.05). (B) Immunofluorescence staining of PGC-1a. Cells were cultured with 10 mM d-tocopherol. Control cells were treated with DMSO. Cells were fixed, permeabilized, stained for PGC-1a (in red) and nuclei with DAPI (in blue), and visualized with confocal microscopy. Scale bar, 20 mm. (C), (D) Confirmation of knockdown efficiency by real-time PCR and immunofluorescence staining. siControl; Control siRNA treated cells, siPGC-1a; PGC-1a siRNA treated cells. Cells were treated with the indicated siRNAs for 72 h. The values are mean ± SD for three samples. Statistical analysis was performed with one-way ANOVA, followed by a t-test (*p < 0.05). (E), (F) The influence of PGC-1a knockdown in the UCP1 gene expression and UCP1 staining. Cells were cultured with 10 mM d-tocopherol. Control cells were treated with DMSO. Gene expression was measured with real-time PCR and normalized with b-actin. The values are mean ± SD for three samples. Statistical analysis was performed with one-way ANOVA, followed by Bonferroni's post hoc test (*p < 0.05, **p < 0.01). For immunofluorescence staining, cells were fixed, permeabilized, stained for UCP1 (green) and nuclei with DAPI (in blue), and visualized with confocal microscopy. Scale bar, 20 mm.
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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Fig. 4. Effects of d-tocopherol on expression of PGC-1a activators. (A) Gene expression of activators for PGC-1a in cells. Cells were cultured with 10 mM or 50 mM d-tocopherol. Control cells were treated with DMSO. Gene expression of ERK1 (Mapk3), ERK2 (Mapk1), p38 MAPK (Mapk14), AMPK (Prkaa1), S6K (Rps6kb2), and SIRT1 (Sirt1) was measured with real-time PCR and normalized with b-actin. (B) Immunofluorescence staining of phosphorylated p38 MAPK (p-p38 MAPK). Cells were cultured with 10 mM d-tocopherol. Control cells were treated with DMSO. Cells were fixed, permeabilized, stained for p-p38 MAPK (in red) and nuclei with DAPI (in blue), and visualized by confocal microscopy. Scale bar, 20 mm. (C) Protein expression of p38-MAPK and p-p38 MAPK. Expression was measured by western blotting and normalized with GAPDH. (D) Protein expression of PGC-1a and UCP1 in p38 MAPK inhibitor (SB203580) treated cells. Expression was measured by western blotting and normalized with GAPDH. The values are mean ± SD for three samples. Statistical analysis was performed with one-way ANOVA, followed by Bonferroni's post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Our present findings suggest that d-tocopherol has the potential ability to prevent obesity and obesity-related disease by enhancing energy consumption in adipocytes.
Funding This work was supported by JSPS KAKENHI Grant Number 15K16220.
Acknowledgments The authors would like to thank Enago (www.enago.jp) for the English language review. References [1] A.M. Cypess, S. Lehman, G. Williams, I. Tal, D. Rodman, A.B. Goldfine, F.C. Kuo, E.L. Palmer, Y.H. Tseng, A. Doria, G.M. Kolodny, C.R. Kahn, Identification and
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d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3-L1 cells Rieko Tanaka-Yachi a, *, Masato Shirasaki a, Rena Otsu a, Chie Takahashi-Muto b, Hideki Inoue a, Yoshinori Aoki c, Taisuke Koike c, Chikako Kiyose d a
Department of Applied Bioscience, Kanagawa Institute of Technology, Kanagawa, Japan Department of Clinical Nutrition, Kitasato Junior College of Health and Hygienic Sciences, Niigata, Japan Mitsubishi-Chemical Foods Corporation, Tokyo, Japan d Department of Nutrition and Life Science, Kanagawa Institute of Technology, Kanagawa, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2018 Accepted 4 October 2018 Available online xxx
Activation of thermogenic adipocytes (brown and beige) has been considered an attractive target for weight loss and treatment of metabolic disease. Peroxisome proliferator-activated receptor g coactivator-1 a (PGC1-a) is a master regulator of thermogenic gene expression in thermogenic adipocytes. We previously reported that a-tocopherol upregulated PGC-1a gene expression and promoted thermogenic adipocyte differentiation in mammalian adipocytes. In this study, we investigated the effects of the vitamin E analogs (a-, g- and d-tocopherol) on PGC-1a and uncoupling protein 1 (UCP1) gene expression in 3T3-L1 cells. The expression of PGC-1a and UCP1 increased significantly with the addition of d-tocopherol. In d-tocopherol-treated cells, nuclear translocation of PGC-1a increased, as did p38 mitogen-activated protein kinase (MAPK) expression and phosphorylation. Our results suggest that p38 MAPK activation by d-tocopherol contributes to PGC-1a activation and UCP1 induction. © 2018 Elsevier Inc. All rights reserved.
Keywords: Tocopherol Thermogenic adipocytes PGC-1a UCP1 p38 MAPK
1. Introduction Adipose tissue is characterized as either white adipose tissue (WAT) or brown adipose tissue (BAT). White adipocytes have large lipid droplets and few mitochondria, whereas beige and brown adipocytes have multilocular lipid droplets and a large number of mitochondria containing uncoupling protein 1 (UCP1). UCP1 uncouples the respiratory chain, allowing for fast substrate oxidation and functions as a radiator for thermogenesis to maintain body temperature. Human BAT is localized in the supraclavicular and neck region [1,2], and there is a negative correlation between BAT mass and body mass index [3]. Classical brown adipocytes are derived from a myf-5-positive, muscle-like cellular lineage. Beige adipocytes, identified in 2012, are novel thermogenic adipocytes [4]. Beige adipocytes, which are differentiated from white adipose depots, are derived from a myf-5 -negative, white adipocyte lineage. It has been reported that human BAT located in the supraclavicular depots consisted mainly of beige adipocytes [4,5].
Peroxisome proliferator-activated receptor g co-activator-1 a (PGC1-a) plays an important role in the regulation of mitochondrial biogenesis and thermogenic gene expression. PGC1-a is expressed mainly in skeletal muscle cells and brown and beige adipocytes, and its expression is regulated by fasting, exercise, and cold exposure [6e8]. Several factors control PGC-1a activation, including protein kinases, such as p38 mitogen-activated protein kinase (MAPK), 50 AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and ribosomal protein S6 kinase (S6K) [9e13]. Activated PGC1-a regulates hermogenic gene expression through its interaction with PPARg and other transcription factors. In addition, it has been reported that a PPARg agonist, rosiglitazone, promotes thermogenic adipocyte differentiation in mouse WAT [14]. Tocopherols (toc) are present naturally as four analogs (a-, b-, g-, d-), each differing in the number and position of methyl groups on the chroman ring. Landrier et al. demonstrated that a-toc influenced PPARg expression and target gene expression [15]. In this study, we investigated the function these tocopherols in vitro.
* Corresponding author. Department of Applied Bioscience, Kanagawa Institute of Technology, 1030, Shimo-ogino, Atsugi-shi, Kanagawa, 243-0292, Japan. E-mail address: [email protected] (R. Tanaka-Yachi). https://doi.org/10.1016/j.bbrc.2018.10.021 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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2. Materials and methods 2.1. Materials Tocopherol analogs (a-toc, g-toc, and d-toc) were purchased from Wako Pure Chem. Ind. Ltd. (Osaka, Japan). Rosiglitazone was purchased from Wako Pure Chem. Ind. Ltd. (Osaka, Japan). Rabbit anti-PGC-1a antibody (AB3242) was purchased from Merck & Co., Inc., and rabbit anti-UCP1 antibody (ab10983) was purchased from Abcam. Rabbit anti-phospho-p38-MAPK antibody (#9211) and rabbit anti-phospho-AMPK antibody (#2535) were purchased from CST Japan K.K. (Tokyo, Japan). An inhibitor of p38 MAPK (SB203580) was purchased from Invivogen. 4,6-Diamidino-2-phenylindole Dihydrochloride (DAPI) stain was purchased from AAT Bioquest, Inc. 2.2. Cell culture and differentiation 3T3-L1 cells were cultured at 37 C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and Lglutamine (292 mg/ml). Cells were seeded in 6-well plates at a density of 4 105 cells per well and cultured for three days postconfluency. Cells were maintained in DMEM supplemented with 10% fatal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine (292 mg/ml), insulin (10 mg/ml), dexamethasone (2.5 mM) and 3-isobutyl-1-methylxanthine (0.5 mM). Each vitamin E analog was added at the induction of differentiation, and cells were cultured for 10 days. Rosiglitazone, a PPARg agonist, was added at a final concentration of 10 mM as a positive control. Control cells were treated with equivalent DMSO. SB203580, the specific inhibitor of p38 MAPK was treated during the last 24 h. 2.3. RNA interference A siRNA duplex oligomer (Stealth RNAi) was purchased from Invitrogen; the sequence was 50 -GCG GAC AGA AUU GAG AGA CCG CUU U-30 . Control siRNA (Silencer Negative Control siRNA) was obtained from Nippon EGT. Cells were transfected with siRNA at a final concentration of 20 nM using Lipofectamine RNAi MAX (Invitrogen), according to the manufacturer's instructions, and during the last 72 h of the culture period.
The membranes were blocked for 5 min using Bullet Blocking One (Nacalai Tesque, Inc.). Membranes were incubated with primary antibodies for 2 h, and then incubated with secondary antibodies conjugated to peroxidase (Bio-Rad Laboratories, Inc.) for 1 h. The proteins were visualized by enhanced chemiluminescence using a Universal Hood II (Bio-Rad Laboratories, Inc.). 2.6. Immunofluorescence staining Cells were washed with PBS briefly and fixed in 3.7% formaldehyde in PBS at room temperature for 15 min. Cells were permeabilized for 5 min with 50 mg/ml digitonin and quenched with 50 mM NH4Cl in PBS for 10 min. The cells then were washed with PBS three times and treated with blocking buffer (3% BSA in PBS) for 45 min. Cells were incubated with primary antibodies for 2 h and incubated with secondary antibodies and DAPI for 1 h, before being washed with PBS three times and mounting. Images were obtained using a laser-scanning microscope with a 63 1.4 PlanApochromat oil immersion lens. 2.7. Statistical analysis All data are expressed as mean ± SD. Statistical analyses were performed by one-way ANOVA, followed by Bonferroni's post hoc test. Data were visualized using Kaleida Graph ver.4.5 (Hulinks Inc., Tokyo, Japan). Differences were considered significant at p < 0.05. 3. Results 3.1. Effects of vitamin E analogs on gene expressions of PPARg, PGC1a, and UCP1 Gene expression of PPARg, PGC-1a, and UCP1 in 3T3-L1 cells increased significantly with the addition of rosiglitazone (PPARg agonist). These findings confirmed our previous report that a-toc induced the expressions of PPARg, PGC-1a, and UCP1. Conversely, g-toc did not affect expression of these genes. Surprisingly, d-toc significantly increased gene expression of all three genes to a similar magnitude as adding rosiglitazone (Fig. 1A). Next, we investigated the changes in expression of PGC-1a and UCP1 genes after adding different concentrations of d-toc. As a result, expression of PGC-1a and UCP1 genes increased in a concentrationdependent manner across a range of d-toc from 1 to 50 mM (Fig. 1B).
2.4. Measurement of mRNA expression using real-time PCR Total RNA was extracted using Sepasol RNA II (Nacalai Tesque Inc.). The quantity and purity of the RNA were determined by measuring absorbance at 258/280 nm. Total RNA was reverse-transcribed into cDNA using a high-capacity RNA-to-cDNA kit, according to the manufacturer's protocol. A 7500 Fast Real-Time PCR system and real-time PCR kit (TaqMan® Gene Expression Assays) were employed, according to the manufacturer's instructions. Beta-actin was used as an internal control. The assay IDs of the primer/probe mixtures in the TaqMan gene expression assays were as follows: PPARg (Pparg); Mm01184322_m1, PGC-1a (Ppargc1a); Mm01208835_m1, UCP1 (Ucp1); Mm01244861_m1, b-actin (Actb); Mm00607939_m1, ERK1 (Mapk3); Mm01973540_m1, ERK2 (Mapk1); Mm00442479_m1, p38 MAPK(Mapk14); Mm01301009_m1, AMPK (Prkaa1); Mm01296700_m1, S6K (Rps6kb2); Mm00445440_m1, SIRT1 (Sirt1); Mm0116521_m1. 2.5. Measurement of protein expression by western blotting Total protein from cells was separated in a 10% polyacrylamide gel and then transferred to polyvinylidene difluoride membranes.
3.2. Effects of d-tocopherol addition on size of lipid droplet and UCP1 staining We observed cell morphology with optical microscopy. The size of lipid droplets was smaller than the control and resembled a “multilocular lipid droplet,” which is characteristic of brown and beige adipocytes (Fig. 2A). Next, we determined the intracellular expression pattern of UCP1 protein by immunofluorescent staining. As shown in Fig. 2B, staining of UCP1 was enhanced in cells treated with d-toc. 3.3. Effects of d-tocopherol on PGC-1a activation and the validation by PGC-1a knockdown Protein expression of PGC-1a was measured by western blotting. Unlike the results from gene expression analysis, a-tocopherol did not affect protein expression of PGC-1a. Conversely, d-toc significantly increased expression of PGC-1a (Fig. 3A). We examined the intracellular distribution of PGC-1a by immunofluorescence staining. Nuclear translocation of PGC-1a was enhanced by dtoc addition (Fig. 3B). Next, we examined gene expression of UCP1
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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Fig. 1. Effects of tocopherols on gene expression of PPARg, PGC-1a, and UCP1. (A) Gene expression of PPARg, PGC-1a, and UCP1 in 3T3-L1 cells. Cells were cultured with 10 mM tocopherol or 10 mM rosiglitazone. Control cells were treated with DMSO. (B) Concentration dependency of d-toc on PGC-1a and UCP1 gene expression. Gene expression was measured by real-time PCR, which was normalized with b-actin activity. The values presented are mean ± SD for three samples. Statistical analysis was performed by one-way ANOVA, followed by Bonferroni's post hoc test (*p < 0.05, **p < 0.01).
in PGC-1a knockdown cells to verify whether the increase of UCP1 expression by d-toc was due to PGC-1a induction. Gene expression of PGC-1a was suppressed to approximately 20% following siRNA treatment, and the staining of PGC-1a decreased significantly following PGC-1a siRNA treatment (Fig. 3C and D). In these cells, any increase of UCP1 mRNA expression by d-toc was eliminated (Fig. 3E). Similarly, the increase of UCP1 staining by d-toc was suppressed by PGC-1a knockdown (Fig. 3F).
Next, we examined expression of PGC-1a and UCP1 in p38 MAPK inhibitor (SB203580) treated cells to verify whether PGC-1a became induced and activated due to p38 MAPK activation. The increase of PGC-1a and UCP1 expression by d-toc tended to be suppressed by SB203580 treatment, but it was not completely eliminated (Fig. 4D).
3.4. Effects of d-tocopherol on kinases and deacetylases that regulate PGC-1a activation
In this study, we compared the effects of tocopherol analogs on the transcription factors and UCP1 gene expression in 3T3-L1 adipocytes. We previously reported that a-toc upregulated PGC-1a gene expression in vivo and in vitro [16]. We confirmed that a-toc induced the expression of PPARg, PGC-1a, and UCP1 mRNA in 3T3L1 cells. Consistent with previous reports, g-tocopherol did not affect expression of these genes. Surprisingly, however, d-toc induced gene expression more markedly than a-toc. The effects of d-toc were comparable with that of rosiglitazone, which was used as a positive control. In particular, the induction of PGC-1a and UCP1 expression was larger than expected; therefore, the influence of PGC-1a expression should be considered further, rather than PPARg-dependent regulation. In addition, our results showed that
PGC-1a activation can be regulated by several protein kinases and deacetylases. Therefore, we examined gene expression in ERK1, ERK2, p38 MAPK, AMPK, S6K, and SIRT1 in 3T3-L1 cells. Gene expression of p38 MAPK increased significantly by d-toc addition. On the other hand, d-toc did not affect the expression of other kinases and SIRT1 (Fig. 4A). Moreover, we detected the activated p38 MAPK by immunofluorescence staining. As a result, phosphorylated p38 MAPK (p-p38 MAPK) increased in d-toc treated cells (Fig. 4B). Protein expression of p38 MAPK and p-p38 MAPK increased significantly in cells treated with 50 mM d-toc (Fig. 4C).
4. Discussion
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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Fig. 2. Effects of d-tocopherol on cell morphology and intracellular expression of UCP1. (A) Effects of d-tocopherol on cell morphology. Cells were observed by optical microscopy. Scale bar, 20 mm. (B) Immunofluorescence staining of UCP1. Cells were fixed, permeabilized, stained for UCP1 (in green) and nuclei with DAPI (in blue), and visualized with confocal microscopy. Scale bar, 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
antioxidant activity is not proportional to changes in gene expression. At final concentrations between 1 and 50 mM, expression of UCP1 and PGC-1a increased in a concentration-dependent manner with the addition of d-toc. However, cell death was observed in cells treated with 100 mM d-toc, and gene expression tended to decrease compared to cells treated with 50 mM. Conversely, cell death was not observed in a-toc treated cells. In adipocytes, a high concentration of d-toc may induce cell death. Delta-toc treatment also increased PGC-1a and UCP1 protein expression. Conversely, a-toc did not affect PGC-1a protein expression when the final concentration was 10 mM. UCP1 protein expression tended to increase in a-toc treated cells, but the increase was not significant. We examined protein expression levels in cells treated with higher concentrations of a-toc. When comparing them at the same concentrations, the data suggest that d-toc was more effective than a-toc at inducing the expression of PGC-1a and UCP1. We examined the influence of the PGC-1a knockdown on UCP1
induction with d-toc. As a result, PGC-1a siRNA canceled the induction of UCP1 gene expression with d-toc treatment. These results showed that PGC-1a is indispensable for UCP1 induction with d-toc. Nuclear translocation of PGC-1a occurs when it is phosphorylated and activated. Therefore, we suggest that d-toc promote activation of PGC-1a. Protein kinases, such as AMPK and p38 MAPK, control PGC-1a function by phosphorylating and activating the protein [9e13]. Delta-tocotrienol, an isoform of vitamin E, has been reported to have a more potent anticancer activity compared to other tocotrienols [17e19]. It has been reported that d-tocotrienol promoted p38 MAPK activation in cancer cells [17]. In this study, we suggest that d-toc could activate p38 MAPK in 3T3-L1 cells. The cell death observed in d-toc treatments may be related to p38 MAPK activation. However, p38 MAPK inhibition did not completely eliminate the effects of d-toc, suggesting that other factors also contribute to this effect. In conclusion, we demonstrated that d-tocopherol promoted PGC-1a activation and UCP1 expression in mammalian adipocytes.
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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Fig. 3. The effect of d-tocopherol on PGC-1a protein expression and the influence of PGC-1a knockdown. (A) Protein expression of PGC-1a. Cells were cultured with 10 mM tocopherols. Control cells were treated with DMSO. Protein expression was measured with western blotting, normalized with GAPDH. The values are mean ± SD for three samples. Statistical analysis was performed by one-way ANOVA, followed by Bonferroni's post hoc test (*p < 0.05). (B) Immunofluorescence staining of PGC-1a. Cells were cultured with 10 mM d-tocopherol. Control cells were treated with DMSO. Cells were fixed, permeabilized, stained for PGC-1a (in red) and nuclei with DAPI (in blue), and visualized with confocal microscopy. Scale bar, 20 mm. (C), (D) Confirmation of knockdown efficiency by real-time PCR and immunofluorescence staining. siControl; Control siRNA treated cells, siPGC-1a; PGC-1a siRNA treated cells. Cells were treated with the indicated siRNAs for 72 h. The values are mean ± SD for three samples. Statistical analysis was performed with one-way ANOVA, followed by a t-test (*p < 0.05). (E), (F) The influence of PGC-1a knockdown in the UCP1 gene expression and UCP1 staining. Cells were cultured with 10 mM d-tocopherol. Control cells were treated with DMSO. Gene expression was measured with real-time PCR and normalized with b-actin. The values are mean ± SD for three samples. Statistical analysis was performed with one-way ANOVA, followed by Bonferroni's post hoc test (*p < 0.05, **p < 0.01). For immunofluorescence staining, cells were fixed, permeabilized, stained for UCP1 (green) and nuclei with DAPI (in blue), and visualized with confocal microscopy. Scale bar, 20 mm.
Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021
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Fig. 4. Effects of d-tocopherol on expression of PGC-1a activators. (A) Gene expression of activators for PGC-1a in cells. Cells were cultured with 10 mM or 50 mM d-tocopherol. Control cells were treated with DMSO. Gene expression of ERK1 (Mapk3), ERK2 (Mapk1), p38 MAPK (Mapk14), AMPK (Prkaa1), S6K (Rps6kb2), and SIRT1 (Sirt1) was measured with real-time PCR and normalized with b-actin. (B) Immunofluorescence staining of phosphorylated p38 MAPK (p-p38 MAPK). Cells were cultured with 10 mM d-tocopherol. Control cells were treated with DMSO. Cells were fixed, permeabilized, stained for p-p38 MAPK (in red) and nuclei with DAPI (in blue), and visualized by confocal microscopy. Scale bar, 20 mm. (C) Protein expression of p38-MAPK and p-p38 MAPK. Expression was measured by western blotting and normalized with GAPDH. (D) Protein expression of PGC-1a and UCP1 in p38 MAPK inhibitor (SB203580) treated cells. Expression was measured by western blotting and normalized with GAPDH. The values are mean ± SD for three samples. Statistical analysis was performed with one-way ANOVA, followed by Bonferroni's post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Our present findings suggest that d-tocopherol has the potential ability to prevent obesity and obesity-related disease by enhancing energy consumption in adipocytes.
Funding This work was supported by JSPS KAKENHI Grant Number 15K16220.
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Please cite this article in press as: R. Tanaka-Yachi, et al., d-Tocopherol promotes thermogenic gene expression via PGC-1a upregulation in 3T3L1 cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.021