J. Pineal Res. 2015; 59:267–275

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/jpi.12259

Journal of Pineal Research

Melatonin promotes adipogenesis and mitochondrial biogenesis in 3T3-L1 preadipocytes Abstract: Melatonin is synthesized in the pineal gland, but elicits a wide range of physiological responses in peripheral target tissues. Recent advances suggest that melatonin controls adiposity, resulting in changes in body weight. The aim of this study was to investigate the effect of melatonin on adipogenesis and mitochondrial biogenesis in 3T3-L1 mouse embryo fibroblasts. Melatonin significantly increased the expression of peroxisome proliferator-activated receptor-c (PPAR-c), a master regulator of adipogenesis, and promoted differentiation into adipocytes. Melatonin-treated cells also formed smaller lipid droplets and abundantly expressed several molecules associated with lipolysis, including adipose triglyceride lipase, perilipin, and comparative gene identification-58. Moreover, the hormone promoted biogenesis of mitochondria, as indicated by fluorescent staining, elevated the citrate synthase activity, and upregulated the expression of PPAR-c coactivator 1a, nuclear respiratory factor-1, and transcription factor A. The expression of uncoupling protein 1 was also observable both at mRNA and at protein level in melatonin-treated cells. Finally, adiponectin secretion and the expression of adiponectin receptors were enhanced. These results suggest that melatonin promotes adipogenesis, lipolysis, mitochondrial biogenesis, and adiponectin secretion. Thus, melatonin has potential as an anti-obesity agent that may reverse obesity-related disorders.

Introduction Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, is synthesized in the mammalian pineal gland and also in many other tissues and organs during the dark phase of the circadian cycle [1]. The rhythmic nature of melatonin synthesis is due to an endogenous circadian clock in the suprachiasmatic nucleus of the hypothalamus, which is entrained by the light/dark cycle of the 24-hr day. In turn, melatonin confers circadian rhythmicity to its target organs [2]. Besides, there is growing evidence that melatonin also has an antioxidant properties [3] and regulates a number of neuroendocrine and other physiological processes such as immune defense and tumor suppression [4,5]. More recently, the possible role of melatonin in obesity or adiposity has attracted attention, as well as its potential to prevent obesity-related metabolic disorders. Indeed, chronic supplementation with melatonin suppresses body weight and reduces adiposity in laboratory animals [6–9]. In sharp contrast, rats from which the pineal gland has been surgically removed accumulate white adipose tissue (WAT) in subcutaneous, retroperitoneal, and epididymal areas as a result of reduced levels of circulating melatonin [10, 11]. Moreover, melatonin peaks at a lower concentration during the night in patients with metabolic syndrome and type 2 diabetes than in healthy subjects [12–14]. However, the mechanism underlying melatonin-induced reduction in adiposity remains unclear.

Hisashi Kato1, Goki Tanaka1, Shinya Masuda2, Junetsu Ogasawara3, Takuya Sakurai3, Takako Kizaki3, Hideki Ohno3 and Tetsuya Izawa1,4 1

Graduate School of Health and Sports Science, Doshisha University, Kyotanabe, Kyoto, Japan; 2Division of Diabetic Research, Clinical Research Institute, National Hospital Organization, Kyoto Medical Center, Fushimi, Kyoto, Japan; 3Department of Molecular Predictive Medicine and Sports Science, Kyorin University, Mitaka, Tokyo, Japan; 4Faculty of Health and Sports Science, Doshisha University, Kyotanabe, Kyoto, Japan Key words: 3T3-L1, adipogenesis, adiponectin, melatonin, mitochondrial biogenesis Address reprint requests to Tetsuya Izawa, Graduate School of Health and Sports Science, Doshisha University, 1-3 Tatara-Miyakodani, Kyotanabe, Kyoto 610-0321, Japan. E-mail: [email protected] Received May 6, 2015; Accepted June 26, 2015.

Adiposity is usually linked to dysfunction in WAT, such as inflammation and abnormal secretion of adipokines. Furthermore, adiposity is regulated mainly either by adipogenesis, by a balance between lipolysis and lipogenesis, or by both. Indeed, in rats with low melatonin due to pinealectomy, lipolysis diminished, while lipogenesis increased [15]. However, the mechanism underlying these effects has not been elucidated. Moreover, investigation of the link between melatonin and adipogenesis has yielded contradictory results. Some studies demonstrate that melatonin stimulates adipogenesis in 3T3-L1 fibroblasts by upregulating peroxisome proliferator-activated receptor-c (PPAR-c) and CCAAT/enhancer-binding protein (C/ EBP)-a [16, 17]. In contrast, other studies show that melatonin suppresses adipocyte differentiation by downregulating C/EBP-b, which regulates the expression of PPAR-c and C/EBP-a [18]. Thus, the contribution of melatonin to adipogenesis is, at present, debatable. Recent advances indicate that adipocyte differentiation is associated with increased mitochondrial biogenesis and mitochondrial content [19]. In addition, mitochondrial content is reduced in adipocytes of obese db/db mice [20], suggesting that a change in mitochondrial biogenesis is a hallmark of adipogenesis. Therefore, changes in mitochondrial content should be observed during melatonin-induced adipogenesis. Indeed, several studies show that melatonin improves mitochondrial function in liver [21], skeletal muscle [22], and WAT in Zucker diabetic fatty 267

Kato et al. rats [23]. However, there is no direct evidence that demonstrates melatonin to increase mitochondria content and activity in adipocytes by themselves. If melatonin truly increases mitochondria content, it may explain why the hormone boosts levels of circulating adiponectin [24, 25], as it has been shown that adiponectin synthesis depends on the quantity and quality of mitochondria [26]. To characterize the effects of melatonin on adiposity, as well as its potential to prevent obesity-related metabolic abnormalities, we investigated its activity in 3T3-L1 cells, one of the best-characterized and reliable models to study differentiation of preadipocytes into adipocytes [27]. We found melatonin to promote differentiation of 3T3-L1 cells into adipocytes with small lipid droplets by inducing the expression of adipogenic and lipolytic molecules. In addition, the hormone enhanced the secretion of adiponectin and the expression of adiponectin receptor 1 and 2 (adipoR1-2). Finally, we demonstrate for the first time that melatonin increases mitochondrial content and activity.

Materials and methods Cell culture 3T3-L1 mouse embryo fibroblasts were generously provided by Dr. Takeshi Hashimoto of Ritsumeikan University, Shiga, Japan. Cells were cultured according to Gonzalez et al. [16] and Alonso-Vale et al. [18]. Briefly, 3T3-L1 preadipocytes were seeded at 5000 cells/cm2, grown until confluence in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), and maintained in the same medium for 2–3 days postconfluency at 37°C and 5% CO2. Differentiation was then induced by treatment for 48 hr with 1 lM dexamethasone, 0.5 mM isobutylmethylxanthine, 1.67 lM insulin, and 10% FBS. Cells were cultured for six additional days thereafter in DMEM containing 0.41 mM insulin and 10% FBS [28]. The medium was refreshed every 2 days. With this regime, fully differentiated adipocytes were obtained by day 8. To determine the effects of melatonin on adipocyte differentiation, cells were cultured with or without 1 mM melatonin throughout experiments. Cell viability Cell viability was measured using Cell Counting Kit-8, following the manufacturer’s instructions (Dojindo Molecular Technologies, Kumamoto, Japan). Briefly, after 8 days in culture, cells were incubated for 2 hr at 37°C in fresh medium containing CCK-8 reagent. Absorbance at 450 nm was then measured using Model 680 microplate reader (Bio-Rad, Hercules, CA, USA) to determine the amount of formazan generated, which is proportional to the number of viable cells. Results are expressed in arbitrary units, with control cells set to 1.0. Fluorescent staining and size of lipid droplet Lipid droplets and nuclei were stained using Adipocyte Fluorescent Staining kit (PMC, Hokkaido, Japan) according to the manufacturer’s protocol. Briefly, cells were 268

washed once with washing buffer and fixed overnight at room temperature with 10% formalin. Thereafter, formalin was removed, and cells were incubated for 30 min at room temperature with BODIPY. BODIPY was then washed off, and cells were stained for 30 min at room temperature with Hoechst 33258. Finally, cells were washed once with washing buffer and treated with mounting agent. Images were obtained with BZ-8100 fluorescent microscope (KEYENCE, Osaka, Japan). The average size of 200 lipid droplets per sample was measured on Image J software (version 1.47v; NIH, Bethesda, MD, USA), using images of cells at 809 magnification. Analysis of gene expression by quantitative realtime PCR Total RNA was extracted using ISOGEN II (Nippon gene, Tokyo, Japan). First-strand cDNA was synthesized using PrimScriptTM II first standard cDNA Synthesis Kit (TKR, Shiga, Japan), following the manufacturer’s protocol. For real-time PCR, RNA was reverse-transcribed using KAPA SYBRÒ FAST qPCR Kit Master Mix ABI PrismTM (KAPA BIO, Wilmington, MA, USA) and then amplified on Applied Biosystems StepOneÒ Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The amplification protocol included an initial denaturation step for 10 min at 95°C followed by 40 cycles consisting of denaturation for 15 s at 95°C, annealing for 1 min at 60°C, and extension for 1 min at 72°C. Relative expression was normalized to 18S ribosomal RNA using the DDCt method. Amplification of specific transcripts was confirmed by obtaining melting curves between 68 and 95°C at the end of PCR. The sequences of all primers are listed in Table 1. Western blotting Cells were washed twice with phosphate-buffered saline (PBS) and homogenized in EzRIPA lysis buffer (20 mM HEPES pH 7.5, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, 150 mM sodium chloride), supplemented with protease and phosphatase inhibitors (ATTO, Tokyo, Japan). The homogenate was incubated on ice for 15 min and centrifuged for 15 min at 14 000 g at 4°C. The supernatant was recovered and further cleared by a second round of centrifugation. Samples were frozen at 80°C until analyzed. Samples did not contain significantly different amounts of total protein (data not shown). Therefore, identical volumes from each sample were mixed with Laemmli sample buffer and heated for 2 min at 95°C. After separation on 8–12.5% SDS-PAGE gels, proteins were transferred to PVDF membranes (ATTO), which were blocked for 60 min with TBS containing 0.1% Tween-20 (TBS-T) and 5% skim milk, or with Block AceÒ Powder (DSP, Osaka, Japan) dissolved in purified water. Membranes were then probed at 4°C overnight in TBS-T containing 0.4% NaN3 and 1:1000 dilutions of specific antibodies against PPAR-c (Santa Cruz Biotechnology, Dallas, TX, USA), hormonesensitive lipase (HSL) (Cell Signaling Technology, Danvers, MA, USA), adipocyte triglyceride lipase (ATGL) (Cell Signaling Technology), perilipin A (Abcam, Cambridge,

Melatonin-induced responses in adipocytes Table 1. Primers for quantitative real-time PCR Gene

Sense primer (50 –30 )

Antisense primer (50 –30 )

c/ebpb pparc c/ebpa aP2 hsl atgl pgc-1a tfam nrf1 cytC ucp2 ucp1 adiponectin leptin adipoR1 adipoR2 18S

ACCGGGTTTCGGGACTTGA GGAGCCTAAGTTTGAGTTTGCTGTG ACATCAGCGCCTACATCGACC ACCGCAGACGACAGGAA GGCAGTGGTGTGTAACTAGGATTG CACTTTAGCTCCAAGGATGA CACCGTAAATCTGCGGGAATG CGCAGCACCTTTGGAGAA GCCGTCGGAGCACTTACT ATAGGGGCATGTCACCTCAAAC AGATACATGAACTCTGCCTTGGG TGCGATGTCCATGTACACCAA AACTTGTGCAGGTTGGATGG TCAACTCCCTGTTTCCAAAT ACGTTGGAGAGTCATCCCGTAT TCCCAGGAAGATGAAGGGTTTAT TTCTGGCCAACGGTCTAGACAAC

CCCGCAGGAACATCTTTAAGTGA TGCAGCAGGTTGTCTTGGATG TTGGCCTTCTCCTGCTGTCG CTCATGCCCTTTCATAAAC ATCCATGCTGTGTGAGAACGC TGGTTCAGTAGGCCATTCCT TATCCATTCTCAAGAGCAGCGAAAG CCCGACCTGTGGAATACTT CTGTTCCAATGTCACCACC GTGGTTAGCCATGACCTGAAAG GGACCGCATCTCAAAATAGC CTTCCTCCAAGTTGCTTATGTGG GCGATACACATAAGCGGCTT TCTTCACGAATGTCCCACGA CTCTGTGTGGATGCGGAAGAT TTCCATTCGTTCGATAGCATGA CCAGTGGTCTTGGTGTGCTGA

Abbreviations are defined in the text.

UK), comparative gene identification-58 (CGI-58) (Santa Cruz Biotechnology), PPAR-c coactivator-1a (PGC-1a) (Merck Millipore, MA, USA), cytochrome c oxidase-IV (COX IV) (Abcam, Cambridge, UK), uncoupling protein 1 (UCP1) (Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam). Subsequently, membranes were labeled for 60 min with 1:2500 dilutions of anti-rabbit or anti-mouse immunoglobulin G (GE Healthcare, Buckinghamshire, UK). Bands were visualized using ECL Prime system (GE Healthcare) and quantified on ChemiDocTM MP system (Bio-Rad). Protein abundance was normalized to GAPDH.

buffer containing 25 mM Tris–HCl pH 7.4 and 1 mM EDTA. The homogenate was centrifuged to obtain the infranatant, which was then diluted 50-fold with purified water, and mixed with assay buffer consisting of 50 mM Tris–HCl pH 8.1, 0.2 mM DTNB, 0.1 mM acetyl-CoA, and 0.5 mM oxaloacetate. Absorbance at 412 nm was measured over 15 min in a U-1900 spectrophotometer (HITACHI, Tokyo, Japan). Background was determined using assay buffer without 0.5 mM oxaloacetate and subtracted from all samples. Citrate synthase activity was calculated as nmol/min/mg total protein. Total protein was measured by Bradford assay [29].

Lipolysis

Adiponectin secretion

To measure lipolysis, differentiated cells were incubated for 2 hr in fresh medium with or without 10 lM isoproterenol. Aliquots of the medium were collected and stored at 80°C until analysis. Glycerol release, a measure of lipolysis, was determined using Adipolysis Assay Kit (Cayman, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Lipolysis was calculated in nmol per mg total protein per hr. Total protein in samples was measured by Bradford’s method [29], using a commercially available kit.

Aliquots of the culture medium at the eighth day of cell culture were collected and stored at 80°C until analysis. The concentration of adiponectin in these aliquots was measured in duplicate using the Adiponectin ELISA kit (Otsuka Pharmaceutical, Tokyo, Japan), with sensitivity range of 0.25–8.0 ng/mL. Coefficient of variation (CV) was

Melatonin promotes adipogenesis and mitochondrial biogenesis in 3T3-L1 preadipocytes.

Melatonin is synthesized in the pineal gland, but elicits a wide range of physiological responses in peripheral target tissues. Recent advances sugges...
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