Melinjo (Gnetum gnemon L.) seed extract induces uncoupling protein 1 expression in brown fat and protects mice against diet-induced obesity, inflammation, and insulin resistance

Accepted Manuscript Melinjo (Gnetum gnemon L.) seed extract induces uncoupling protein 1 expression in brown fat and protects mice against dietinduced obesity, inflammation, and insulin resistance

Takeshi Yoneshiro, Ryuji Kaede, Kazuki Nagaya, Manami Saito, Julia Aoyama, Mohamed Elfeky, Yuko Okamatsu-Ogura, Kazuhiro Kimura, Akira Terao PII: DOI: Reference:

S0271-5317(17)31091-6 doi:10.1016/j.nutres.2018.06.012 NTR 7914

To appear in:

Nutrition Research

Received date: Revised date: Accepted date:

28 November 2017 25 May 2018 28 June 2018

Please cite this article as: Takeshi Yoneshiro, Ryuji Kaede, Kazuki Nagaya, Manami Saito, Julia Aoyama, Mohamed Elfeky, Yuko Okamatsu-Ogura, Kazuhiro Kimura, Akira Terao , Melinjo (Gnetum gnemon L.) seed extract induces uncoupling protein 1 expression in brown fat and protects mice against diet-induced obesity, inflammation, and insulin resistance. Ntr (2018), doi:10.1016/j.nutres.2018.06.012

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Melinjo (Gnetum gnemon L.) seed extract induces uncoupling protein 1 expression in brown fat and protects mice against diet-induced obesity, inflammation, and insulin resistance

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Authors:

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Takeshi Yoneshiroa, Ryuji Kaedea, Kazuki Nagayaa, Manami Saitoa, Julia Aoyamaa,

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Mohamed Elfekya,c, Yuko Okamatsu-Oguraa, Kazuhiro Kimuraa, Akira Teraoa,b

a

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Affiliations:

Laboratory of Biochemistry, Department of Biomedical Sciences, Graduate School of

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Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan School of Biological Sciences, Tokai University, Sapporo, Hokkaido 005-8601, Japan

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Department of Biochemistry, Faculty of Veterinary Medicine, Alexandria University,

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Behera 22785, Egypt

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Address correspondence to: Akira Terao, DVM, PhD

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School of Biological Sciences, Tokai University 5-1-1-1 Minamisawa, Minami-ku, Sapporo, Hokkaido 005-8601, Japan Tel.: +81-11-571-5111 ext. 2913, Fax: +81-11-571-6904, E-mail: [email protected]

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Abbreviations: melinjo (Gnetum gnemon L.) seed extract (MSE)

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Brown adipose tissue (BAT) white adipose tissue (WAT)

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uncoupling protein 1 (UCP1)

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temperature-sensitive transient receptor potential (TRP) channels

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Sirtuin 1 (SIRT1)

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Abstract Dietary supplementation with melinjo (Gnetum gnemon L.) seed extract (MSE) has been proposed as an anti-obesity strategy. However, it remains unclear how MSE

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modulates energy balance. We tested the hypothesis that dietary MSE reduces energy intake and/or increases physical activity and metabolic thermogenesis in brown and

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white adipose tissue (BAT and WAT) in mice. Twenty-four C57BL/6J mice were

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provided with normal diet, high-fat diet (HFD), or HFD with 1% MSE added, for 17 weeks. Food intake, spontaneous locomotor activity, hepatic triglyceride (TG) content,

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and blood parameters were examined. Mitochondrial thermogenesis-associated molecule and inflammatory marker expression levels in BAT and WAT were examined

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by quantitative PCR and western blotting. Dietary MSE did not affect energy intake or spontaneous locomotor activity, but significantly suppressed HFD-induced fat

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accumulation, hyperglycemia, and hyperinsulinemia. Homeostasis model assessment of insulin resistance score and hepatic TG content were both lower in the MSE-supplemented HFD-fed group than in the HFD-fed group, indicating reduced

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insulin resistance and a less fatty liver. Dietary MSE upregulated thermogenic

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uncoupling protein 1 (UCP1) and mitochondrial marker cytochrome c oxidase subunit IV protein expression in BAT; this was closely associated with sirtuin 1 mRNA induction. mRNAs of adipose inflammatory markers, such as monocyte chemotactic 1 and interleukin-1, were induced by HFD but suppressed by MSE. Considering that UCP1 protein expression is the most physiologically relevant parameter to assess the thermogenic capacities of BAT, our results indicate that dietary MSE supplementation induces BAT thermogenesis and reduces obesity-associated adipose tissue inflammation, hepatic steatosis, and insulin resistance. 3

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Keywords: Melinjo seed extract; brown adipose tissue; uncoupling protein 1; obesity;

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diabetes; sirtuin 1

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1. Introduction Obesity is a major global health concern and there is an urgent need for effective

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treatments. Brown adipose tissue (BAT) is a major site of sympathetically-activated non-shivering thermogenesis during cold exposure or overfeeding; thus, it controls body

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temperature and energy balance [1]. The contribution of BAT to energy expenditure is

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increasingly appreciated in mice and humans, and there is a possibility that increasing

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BAT-mediated thermogenesis may serve as a novel approach to combat obesity [1].

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Indeed, activation of BAT thermogenesis by cold exposure or by using a β3-adrenergic receptor agonist increases whole-body energy expenditure and reduces body fat content

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[2,3]. Although chronic use of a cold regimen or pharmacological agents is likely to be limited in patients with obesity because of possible side effects [3], the thermogenic

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effects of these approaches can be mimicked by the ingestion of food ingredients with

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agonist activity for temperature-sensitive transient receptor potential (TRP) channels. For example, capsaicin analogs or menthol can potentiate energy expenditure and prevent body fat accumulation through the activation of uncoupling protein 1 (UCP1), a key molecule involved in BAT thermogenesis [1,4-6], by a TRP channel-mediated pathway [1]. In addition to TRP-activating substances, there are several food ingredients with

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anti-obesity effects that potentially stimulate energy expenditure. One of these is melinjo (Gnetum gnemon L.) seed extract (MSE). A previous study in mice with

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diet-induced obesity revealed that dietary supplementation with MSE reduced body fat and led to improved systemic glucose homeostasis and survival [7]. MSE contains

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resveratrol and its-related compounds (resveratrols), such as trans-resveratrol, gnetin C,

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gnemonoside A, and gnemonoside D. Since trans-resveratrol is a caloric restriction

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mimetic [8,9], the beneficial effects of MSE could be attributed to the effects of

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resveratrol on energy metabolism [7]. However, the impacts of MSE on energy expenditure or thermogenesis have not yet been determined; thus, the mechanism by

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which this food ingredient protects mice against obesity remains elusive. Sirtuin 1 (SIRT1) is a critical regulator of cellular energy homeostasis,

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mitochondrial biogenesis, and UCP1 transcription in BAT and white adipose tissue

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(WAT) [10-13]. SIRT1 deacetylates peroxisome proliferator-activated receptor γ, thus allowing recruitment of PR-domain-containing 16 [10], a master regulator of brown adipocyte differentiation. As resveratrols can activate SIRT1 [10], we hypothesized that UCP1 expression in BAT and WAT may be upregulated by dietary supplementation with MSE. Therefore, we examined the impacts of dietary supplementation of MSE on thermogenic UCP1 expression in BAT and WAT, as well as on food intake and

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spontaneous locomotor activity, to examine the involvement of potential thermogenic activation in the anti-obesity and anti-diabetic effects of MSE in high-fat diet (HFD)-fed

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mice.

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2. Methods and materials

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2.1. Animals.

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Experimental procedures were performed in accordance with the Guidelines for

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Animal Care and Use of Hokkaido University and approved by the University Committee for the Care and Use of Laboratory Animals. All efforts were made to

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minimize the number of animals used and any discomfort experienced by the mice. The number of mice per group was selected based on the average of number of mice used in

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previous studies performed to analyze the anti-obesity effects of thermogenic food

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ingredients [5-7]. Male C57BL/6J mice, 3 weeks of age (CLEA Japan Inc., Hamamatsu, Japan), were purchased and housed in plastic cages within an air-conditioned room in an animal facility, approved by the Association for Assessment and Accreditation of Laboratory Animal Care International, at 22ºC with a 12 h light:12 h dark cycle [14].

2.2. Diets.

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Control diet (CD) and HFD were purchased from Oriental Yeast Co. Ltd. (Tokyo, Japan). The CD was composed of 60% carbohydrate, 28% protein, and 12% fat;

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it provided a total of 3.45 kcal/g (Table 1) [14]. The HFD was composed of 19% carbohydrate, 18% protein, and 62% fat; it provided a total of 5.06 kcal/g [14].

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Lyophilized MSE powder, containing 0.1% trans-resveratrol, 1.6% gnetin C, 18.7%

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gnemonoside A, 3.8% gnemonoside D, and 9.0 % dextrin, was obtained from Yamada

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Bee Company, Inc. (Okayama, Japan) and prepared as described previously [15]. The

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powder was standardized to contain > 20.0% resveratrol derivatives. The MSE-supplemented diet was prepared by adding 1% w/w lyophilized MSE powder to

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the HFD.

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2.3. Dietary treatments.

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The mice were allowed to acclimatize to the laboratory environment with free access to normal lab chow and tap water for 1 week. Then, the mice were divided into three groups by stratified randomization to balance baseline body weight, and provided with the following diets for 17 ± 0.3 weeks: CD (n = 8); HFD (n = 8); or HFD supplemented with 1% MSE (n = 8). Body weight was monitored once per week. Food intake was measured once per week, from week 13 to week 15, and daily energy intake

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was calculated, as reported previously [14]. Spontaneous locomotor activity was measured with an infrared sensor (Biotex, Kyoto, Japan) at 10 to 12 weeks after

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randomization (n = 6) [14]. Mice were fasted for 6 h before sacrifice, then anesthetized with isoflurane; arterial blood was collected from the abdominal aorta into heparinized

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tubes [14]. The blood collected was immediately centrifuged (5 min, 2500 × g, 4ºC) and

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the plasma was frozen at −80ºC until biochemical analysis. After the mice were

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sacrificed by cervical dislocation, the liver, gastrocnemius skeletal muscle, and

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interscapular BAT, inguinal WAT (iWAT), and gonadal WAT (gWAT) were removed and weighed. Fat specimens from one side of the body were transferred into RNAlater

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Stabilization Solution (Life Technologies Inc., Carlsbad, CA, USA) for gene expression analysis. Fat specimens from the other side of the body were transferred into liquid

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nitrogen for western blot analysis.

2.4. Biochemical analysis. Plasma glucose, non-esterified fatty acids (NEFA) and triglycerides (TG), and

hepatic TG were measured with commercial kits (Glucose CII-test, NEFA C-test, and Triglyceride E-test; Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin was analyzed with an ELISA assay kit (Mouse Insulin ELISA Kit; Morinaga Institute of

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Biological Science, Yokohama, Japan). Insulin sensitivity and islet β cell function were appraised by the homeostasis model assessment of insulin resistance (HOMA-IR) and

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× fasted insulin / (fasted glucose − 3.5)), respectively [16,17].

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β-cell function (HOMA-β) calculated as (fasted insulin × fasted glucose / 22.4) and (20

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2.5. RNA analysis.

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Total RNA was extracted using RNAiso reagent (Takara Bio, Shiga, Japan).

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The mRNA expression levels of Ucp1 and Sirt1 were quantified by real-time RT-PCR using the respective cDNA fragment as a standard and were normalized to β-actin

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(Actb) mRNA levels [14]. The mRNA expression levels of the inflammatory markers monocyte chemoattractant protein 1 (Mcp1), tumor necrosis factor α (Tnfa), interleukin

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1β (Il-1b), and interleukin 6 (Il-6) were also quantified. Briefly, 2 μg of total RNA were

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reverse transcribed with an oligo(dT) 15-adaptor primer and Moloney murine leukemia virus reverse transcriptase (Life Technologies). Real-time quantitative PCR was performed on a fluorescence thermal cycler (LightCycler system; Roche Diagnostics, Mannheim, Germany) by using SYBR Green PCR kits (Roche Diagnostics). Primer sequences used are provided in Table 2.

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2.6. Western blot analysis. BAT, iWAT, and gWAT specimens were homogenized in RIPA buffer (Nacalai

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Tesque, Kyoto, Japan). After centrifugation at 800 × g for 10 min at 4ºC, the supernatant was analyzed for UCP1 and mitochondrial cytochrome c oxidase IV (COX-IV) content

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by western blotting. Equal amounts of protein (5 μg for BAT, 30 μg for iWAT/gWAT)

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were separated by 13.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis

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and transferred onto polyvinylidene fluoride membranes (Immobilon; Millipore,

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Bedford, MA, USA). After being blocked with 5% skim milk (Morinaga Milk Industry Co., Tokyo, Japan), the membrane was incubated with a primary anti-rat UCP1 antibody,

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kindly provided by Dr. Teruo Kawada (Kyoto University, Kyoto, Japan), or with a primary anti-mouse COX-IV antibody (Molecular Probes, Eugene, OR, USA),

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overnight at 4ºC. The bound antibody was visualized with an enhanced

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chemiluminescence system (Amersham, Little Chalfont, Bucks, UK) by using horseradish peroxidase-linked goat anti-rabbit immunoglobulin (Zymed Laboratories, San Francisco, CA, USA) for UCP1 or goat anti-mouse immunoglobulin (Jackson Immunoresearch Laboratories, West Grove, PA, USA) for COX-IV.

2.7. Statistical analyses.

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Data are presented as means ± SD and were analyzed with IBM SPSS Statistics 17.0 (IBM Japan, Tokyo, Japan). Body weight was analyzed by two-way repeated

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measures analysis of variance (ANOVA). Differences in variables among the three experimental groups were assessed by one-way factorial ANOVA or Kruskal-Wallis test

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with Fisher's least significant difference or Games-Howell post hoc tests, as appropriate.

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Correlation between Ucp1 and Sirt1 mRNA expression levels was assessed by using the

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Pearson correlation coefficient. A P value ≤ 0.05 was considered statistically significant.

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3. Results

We first examined the anti-obesity effect of MSE in HFD-fed mice. Initial body

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weight was similar among the three groups; however, final body weight and body

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weight gain were significantly higher in the HFD group than in the CD and MSE groups (Figure 1A). HFD markedly increased BAT, iWAT, and gWAT weights and adiposity index compared with CD (Table 2, Figure 1B); however, HFD-induced gains in fat pad weight were decreased by dietary MSE (P < 0.05). In contrast, skeletal muscle mass and liver weight did not differ among the experimental groups (Table 2). Thus, body weight gain seemed to result largely from increased WAT mass (Figure 1B). Consistent with

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this explanation, hepatic TG content in the MSE group was significantly lower than that in the HFD group (P < 0.05) and comparable to that in the CD group (Figure 1C). These

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results suggest that dietary MSE protects against HFD-induced fat accumulation and hepatic steatosis.

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We next examined whether the MSE-induced reduction in body weight gain

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improved glucose and lipid homeostasis. Compared with in the CD group (6.17 ± 0.46

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mmol/l), plasma fasting glucose concentration was significantly higher in the HFD and

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MSE groups by +182% and +82%, respectively (P < 0.01, Table 2). MSE supplementation, however, markedly reduced HFD-induced hyperglycemia (P < 0.001).

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The HFD group exhibited markedly higher plasma insulin concentration than the CD group (1.68 ± 1.08 vs 0.13 ± 0.13 ng/ml, P < 0.01), but dietary MSE relieved this

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HFD-induced hyperinsulinemia (0.46 ± 0.25 ng/ml, P < 0.01 vs. HFD). HOMA-IR

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values, which correlate closely with insulin sensitivity assessed by intraperitoneal glucose tolerance tests [16,17], were markedly elevated in the HFD (1.39 ± 0.94, P < 0.01) and MSE groups (0.24 ± 0.12, P < 0.01) compared with the CD group (0.03 ± 0.03; Figure 1D). It should be noted that the increase in HOMA-IR values was markedly smaller in the MSE group (6.8-fold increase) than in the HFD group (40.0-fold increase, P < 0.01). Moreover, HOMA-β, an index of β-cell function, did not

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differ significantly between the three experimental groups (Figure 1E). Thus, the observed hyperglycemia was attributable to decreased insulin sensitivity, rather than

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blunted insulin secretion. Plasma TG (P < 0.05) and NEFA (P < 0.05) concentrations were significantly lower in the MSE group than in the HFD group (Table 2). Despite

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these body fat-reducing effects of MSE, food intake and caloric intake did not differ

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significantly between the HFD and MSE groups (Figure 2A). Neither HFD feeding nor

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MSE supplementation affected spontaneous locomotor activity (Figure 2B, 2C).

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Because MSE suppressed diet-induced weight gain and insulin resistance without affecting energy intake and physical activity, we reasoned that dietary MSE might

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stimulate metabolic thermogenesis. To explore this possibility, we examined the expression of thermogenesis-related molecules in adipose tissue. In interscapular BAT,

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UCP1 protein expression was significantly increased in the MSE group compared with

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the CD (P < 0.001) and HFD groups (P < 0.05; Figure 3A). A similar trend was observed in Ucp1 mRNA expression (ANOVA P = 0.095; Figure 3D). The MSE group also exhibited the highest COX-IV protein content in BAT among the three experimental groups (P < 0.05; Figure 3A), which was positively correlated with UCP1 protein expression (r = 0.610, P < 0.001). Compared with in the CD group, Sirt1 expression in BAT was significantly elevated in the MSE group, but not in the HFD

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group (Figure 3E). Moreover, the Sirt1 expression level was closely and positively correlated with the Ucp1 expression level (P < 0.01, Figure 3F). It has been established

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that cold acclimation induces UCP1-positive beige/brite adipocyte formation in certain white fat depots such as iWAT and contributes to energy homeostasis control [19]. We

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therefore examined UCP1 and COX-IV gene and protein expression in WAT. Dietary

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MSE resulted in no significant change in UCP1 expression in iWAT or gWAT, while

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COX-IV expression in iWAT was increased in the MSE group compared with the CD

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and HFD groups (Figures 3B-3D). This indicates a marginal effect of MSE on browning of white fat, at least in our experimental condition.

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Obesity is closely associated with chronic inflammation in adipose tissue, accompanied by increased proinflammatory and decreased anti-inflammatory cytokine

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levels. This state is associated with the development of insulin resistance. We therefore

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investigated inflammatory marker gene expression in adipose tissue. Consistent with the increased body fat accumulation, Mcp1 and Il-1b levels in WAT were notably increased in the HFD mice compared with the CD mice (P < 0.05; Figure 4A, 4C); however, dietary MSE significantly suppressed HFD-induced increases in the inflammatory marker gene expression levels (P < 0.05). Similarly, Tnfa and Il-6 mRNA expression in WAT also tended to be increased in the HFD group, but not in the MSE group,

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compared with the CD group (Figure 4B, 4D). These results clearly suggest that MSE effectively inhibits HFD-induced adipose inflammation. In contrast, in BAT, HFD

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feeding did not significantly increase expression levels of inflammatory markers as compared with CD feeding, with the exception of Mcp1 (Figures 4A-4D), which

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implies negligible inflammation in BAT. Despite the apparent lack of inflammation in

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BAT, Il-6 mRNA was notably augmented in the MSE group compared with the CD

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group (Figure 4D), which suggests inflammation-independent upregulation of Il-6 in

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BAT by dietary MSE.

4. Discussion

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MSE has been reported to prevent diet-induced obesity and improve survival in

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mice [7]. To clarify the mechanisms of action of MSE, we examined the effects of dietary MSE on food intake and energy expenditure, focusing particularly on BAT thermogenesis. Although the ingested dose of MSE used in the present study (10 g/kg diet) was half of that used in a previous study (20 g/kg diet) [7], we observed significant anti-obesity effects similar to those seen in [7]: dietary MSE markedly suppressed body fat accumulation and hepatic TG content in HFD-fed mice. No notable difference in

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energy intake, spontaneous locomotor activity, or skeletal muscle mass was found between the HFD and MSE groups; therefore, the observed body fat reduction is

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unlikely to have resulted from changes in food intake or physical activity. Consistent with previous findings [14], HFD feeding increased UCP1 protein expression levels in

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BAT compared with CD feeding. It should be emphasized that UCP1 protein expression

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levels in BAT were significantly higher in the MSE group than in the HFD group,

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despite the fact that these two groups consumed comparable amounts of the HFD.

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Given that UCP1 protein abundance is the most relevant indicator of BAT thermogenic capacity [20], our results imply that dietary MSE induces BAT recruitment and

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thermogenesis. Moreover, dietary MSE significantly upregulated mitochondrial COX-IV protein expression, which was positively related to UCP1 induction. UCP1

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localizes in the mitochondrial inner membranes, and mitochondrial biogenesis and

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content are crucial factors in UCP1-mediated thermogenesis [20,21]. Thus, it seems likely that dietary MSE increases BAT thermogenesis by inducing UCP1 expression and mitochondrial biogenesis. Although UCP1 induction was not observed in WAT, at least under our experimental conditions, COX-IV protein expression was significantly and selectively upregulated by MSE in iWAT, which is liable to browning, but not in gWAT, which is resistant to browning. It is thus possible that increasing the MSE dose or

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duration of treatment may induce UCP1 expression in iWAT, which could be verified by comparative analysis to determine the dose- and duration-dependency of the MSE

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effects. Collectively, these results support our hypothesis and suggest that dietary MSE significantly increases thermogenic UCP1 expression in BAT, thus providing a potential

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mechanism for the anti-obesity effects of MSE.

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The specific bioactive ingredients in MSE responsible for BAT recruitment are

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not yet known. However, resveratrols, including trans-resveratrol, could be among the

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substances responsible because dietary trans-resveratrol is known to increase energy expenditure and cellular mitochondrial biogenesis via SIRT1 activation [10]. It has also

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been documented that dietary resveratrol supplementation activates BAT thermogenesis and enriches mitochondrial content [12]. Consistent with these earlier findings, we

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observed significant dietary-MSE-induced upregulation of UCP1 in BAT, although the

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calculated dose of resveratrols in our study (MSE, 10 g/kg diet; resveratrols, 20% of MSE; hence, resveratrols comprised approximately 2 g/kg of diet) was half of that used in a previous study (approximately 4 g/kg of diet) [12]. Additionally, brown fat activation by resveratrol is reportedly mediated by Sirt1 activation [12]. Indeed, in the present study, Sirt1 expression in BAT was increased by dietary MSE supplementation. We also observed a significant positive correlation between Sirt1 and Ucp1 gene

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expression levels, again supporting resveratrols as the potentially responsible compounds. Therefore, it is highly possible that Sirt1 activation is involved in the BAT

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recruitment observed in the MSE group, and that resveratrols are involved to some extent in thermogenic effects of dietary MSE.

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Obesity-related adipose tissue inflammation with macrophage infiltration

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contributes substantially to glucose intolerance and insulin resistance [22]. The

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macrophage infiltration is triggered by the release of proinflammatory adipokines,

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including MCP1. Subsequently, macrophages interact with adipocytes through TNFα release to increase proinflammatory adipokine secretion and decrease anti-inflammatory

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adipokine secretion, thus leading to the development of insulin resistance [22]. In the present study, glucose homeostasis and insulin sensitivity, assessed via fasting blood

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glucose, insulin, and HOMA-IR, were significantly improved in the MSE group

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compared with the HFD group. This improvement coincided with significant suppression of Mcp1, Tnfa, Il-1b, and Il-6 mRNA expression in WAT. Therefore, our results suggest that dietary MSE supplementation prevents adipose tissue inflammation by suppressing fat accumulation via activation of BAT thermogenesis, and that this might be involved in reducing hyperglycemia. Among the inflammatory markers, Il-6 was selectively induced in BAT, but not in

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WAT, in the MSE group. This Il-6 induction seems not to be a consequence of adipose tissue inflammation because adipose tissue inflammation was largely suppressed in the

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MSE group, as described above. Although IL-6 was originally thought to be a proinflammatory cytokine, current understanding suggests that the role of IL-6 is more Central

IL-6

delivery

paradoxically

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body-fat-reducing

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complex.

and

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insulin-sensitizing effects, and upregulates UCP1 expression in BAT via sympathetic

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nervous system stimulation [23-25]. A study using Il-6 knockout mice documented an

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indispensable role of IL-6 in UCP1 induction after cold exposure in iWAT [26]. Furthermore, IL-6 is expressed and released by brown adipocytes themselves in

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response to thermogenic stimuli [25], indicating the occurrence of autocrine effects [23,25]. Finally, BAT-derived IL-6 is required for the beneficial effects of BAT

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transplantation on glucose homeostasis [27]. It is thus tempting to speculate that dietary

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MSE induces IL-6 production and secretion in BAT, thus contributing to further BAT activation and exerting anti-diabetic effects. This hypothesis could be explored in future studies using Il-6 knockout mice. Although we observed a significant induction of UCP1 protein expression in BAT upon MSE consumption, this does not define a causal relationship between increased BAT thermogenic capacities and observed body fat reduction: this is a notable limitation

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of the present study. Additionally, possible effects of dietary MSE on macronutrient absorption in the intestine were not investigated in this study. Thus, further studies

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employing Ucp1-knockout mice, where comparisons of whole-body energy expenditure and macronutrient absorption are made between wild type and Ucp1-knockout mice, are

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important. It should be emphasized that previous studies with Ucp1-KO mice have

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shown an indispensable role for UCP1 in the anti-obesity effects of thermogenic food

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ingredients [5,6]. Moreover, the extents of UCP1 induction shown in these previous

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studies are comparable to the UCP1 induction observed in the current study (~200% by capsinoids [5]; ~250% by menthol [6]; 254% by MSE). Therefore, it seems that

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induction of UCP1 is, at least in part, involved in the body fat reduction by dietary MSE.

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In conclusion, our results suggest that dietary MSE supplementation induces BAT

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thermogenesis, consequently suppressing body fat accumulation and adipose inflammation and thus preventing HFD-induced fatty liver and insulin resistance. Our data provide compelling evidence that the food ingredient MSE is a novel promising natural ingredient that recruits BAT. Additional studies are needed to assess the beneficial effects of MSE on BAT thermogenesis and energy homeostasis in humans.

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Acknowledgment

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This work was supported in part by a Yamada Research Grant and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports,

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Science and Technology of Japan [grant number 15K07140]. AT has received funding

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from the Yamada Bee Farm. The other authors declare no conflict of interest. The

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funding sources had no involvement in the collection, analysis, or interpretation of data.

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We thank Ruth Tunn and Ryan Chastain-Gross from Edanz Group (Fukuoka, Japan) for editing a draft of this manuscript. TY and AT: designed the research; TY, RK, KN, MS,

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JA, ME, and AT: conducted the research; YOO and KK: contributed to the interpretation

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of data; TY and AT: analyzed the data and wrote the manuscript.

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FIGURE LEGENDS Figure 1. Anti-obesity effects of dietary melinjo seed extract (MSE)

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(A) Body weight. (B) Tissue weight relative to body weight. (C) Hepatic triglyceride (TG) content. (D) Homeostatic model assessment of insulin resistance (HOMA-IR). (E)

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HOMA-β cell function (HOMA-β). Data are presented as means with SD. * P < 0.05.

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Values are means ± SD (n = 8/group). Body weight was analyzed by two-way repeated

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measures ANOVA. One-way factorial ANOVA, with Fisher's least significant difference

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or Games-Howell post hoc tests, was used to compare among the three experimental

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groups. * P < 0.05 vs. control diet (CD), † P < 0.05 vs. high-fat diet (HFD).

Figure 2. Spontaneous locomotor activity and energy intake

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(A) Dietary energy intake from protein, fat, and carbohydrate (n = 8/group). (B)

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Spontaneous locomotor activity per cage during the light and dark phases was measured at 10–12 weeks after randomization (n = 6/group). (C) Cumulative locomotor activity (24 h) (n = 6/group). Values are means ± SD and were analyzed by one-way ANOVA with Fisher's least significant difference post hoc tests. * P < 0.05.

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Figure 3. Dietary melinjo seed extract (MSE) enhances the thermogenic capacity of brown adipose tissue (BAT)

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(A–C) Uncoupling protein 1 (UCP1) and mitochondrial cytochrome c oxidase IV (COX-IV) protein expression in BAT (A), inguinal white adipose tissue (iWAT; B), and

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gonadal (g)WAT (C). (D) Ucp1 mRNA expression normalized to β-actin (Actb) mRNA

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levels. (E) Sirtuin 1 (Sirt1) mRNA expression in BAT. Values are means ± SD (n =

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8/group). Comparisons among the three experimental groups were performed by using

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one-way factorial ANOVA or Kruskal-Wallis test, with Fisher's least significant difference or Games-Howell post hoc tests, as appropriate. (F) Correlation between

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Ucp1 and Sirt1 transcript levels in BAT assessed by using the Pearson correlation

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coefficient. * P < 0.05.

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Figure 4. Dietary melinjo seed extract (MSE) prevents high-fat diet (HFD)-induced inflammation in adipose tissue

(A–D) Monocyte chemoattractant protein 1 (Mcp1; A), tumor necrosis factor α (Tnfa; B), interleukin 1β (Il-1b; C), and interleukin 6 (Il-6; D) mRNA expression in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) normalized to β-actin (Actb) mRNA levels. Values are means ± SD (n = 8/group). Values were 29

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log-transformed and analyzed by one-way factorial ANOVA with Fisher's least

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significant difference post hoc test. * P ≤ 0.05.

Figure 5. Dietary melinjo seed extract (MSE) induces uncoupling protein 1

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(UCP1)-mediated thermogenesis in brown adipose tissue (BAT) through induction

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of sirtuin 1 (Sirt1), leading to the improvement of adipose tissue inflammation and

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systemic insulin resistance

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Table 1. Ingredient composition of the diets fed to mice (g/kg) HFD + CD

HFD 1% MSE

397.0

60.0

59.4

α-Corn starch

132.0

160.0

158.4

Sucrose

100.0

55.0

54.5

Lard

70.0

330.0

326.7

20.0

19.8

Casein

200.0

Cellulose

50.0

Vitamin mix

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-

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Soy oil

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Maltodextrin

253.4

66.1

65.4

10.0

10.0

9.9

Mineral mix

35.0

35.0

34.7

L-Cysteine

3.0

3.0

3.0

2.5

2.5

2.5

0.0

0.0

10.0

999.5

997.6

997.7

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256.0

Choline bitartrate

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MSE

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Total

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CD and HFD were obtained from Oriental Yeast Co. Ltd. (Tokyo, Japan).

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Total calculated digestible energy: 345, 506, and 505 kcal/100 g for CD, HFD, and HFD + MSE, respectively. PFC ratio: 28: 12: 60 for CD, 18: 62: 19 for HFD, and 18: 62: 20 for HFD + MSE. Fiber content: 4.0, 0.7, and 0.7 g/100 g for CD, HFD, and HFD + MSE, respectively. Abbreviations: HFD, high-fat diet; CD, control diet; MSE, melinjo seed extract; PFC ratio, protein/fat/carbohydrate ratio. 31

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Table 2. Primer sequences Reverse

Ucp1

5′-GGCCTCTACGACTCAGTCCA-3′

5′-TAAGCCGGCTGAGATCTTGT-3′

Actb

5′-AAGTGTGACGTTGACATCCG-3′

5′-GATCCACACAGAGTACTTGC-3′

Il-6

5′-AGTTGCCTTCTTGGGACTGA-3′

5′-TCCACGATTTCCCAGAGAAC-3′

Il-1b

5′-TGCCACCTTTTGACAGTGATG-3′

5′-GAGTGATACTGCCTGCCTGA-3′

Tnfa

5′-TCGAGTGACAAGCCTGTAGC-3′

5′-GGGAGTAGACAAGGTACAAC-3′

Mcp1

5′-AGGTGTCCCAAAGAAGCTGT-3′

5′-ACAGAAGTGCTTGAGGTGGT-3′

Sirt1

5′-AGGGAACCTTTGCCTCATCT-3′

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Forward

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5′-GAGGTGTTGGTGGCAACTCT-3′

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Abbreviations: Actb, β-actin; Il-1b, interleukin 1β; Il-6, interleukin 6; Mcp1, monocyte

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uncoupling protein 1

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chemoattractant protein 1; Sirt1, sirtuin 1; Tnfa, tumor necrosis factor α; Ucp1,

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Table 3. Tissue weights and blood parameters a HFD

HFD + MSE

47.5 ± 9.5

89.6 ± 35.3 *

49.8 ± 9.3 †

Inguinal WAT (mg)

113.5 ± 50.6

891.6 ± 616.0 *

217.4 ± 102.6 †

Gonadal WAT (mg)

251.1 ± 76.1

1345.2 ± 548.1 *

Gastrocnemius muscle (mg)

128.0 ± 33.6

140.0 ± 32.8

127.1 ± 17.6

Liver (mg)

1033 ± 141

1203 ± 435

900 ±95

Glucose (mmol/l)

6.17 ± 0.46

17.42 ± 4.19 *

11.43 ± 2.08 * †

Insulin (ng/ml)

0.13 ± 0.13

1.68 ± 1.08 *

0.46 ± 0.25 †

TG (mg/ml)

79.1 ± 8.0

92.4 ± 22.0

57.1 ± 13.2 †

NEFA (μEq/l)

1191 ± 196

1517 ± 314 *

942 ± 128 †

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384.7 ± 189.1 †

Values are means ± SD (n = 8/group). The data were analyzed by one-way ANOVA

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a

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Interscapular BAT (mg)

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CD

with Fisher's least significant difference or Games-Howell post hoc tests, as appropriate.

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* P < 0.05 vs. CD, † P < 0.05 vs. HFD.

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Abbreviations: BAT, brown adipose tissue; HFD, high-fat diet; MSE, melinjo (Gnetum gnemon L.) seed extract; NEFA, non-esterified fatty acid; CD, control diet; TG, triglyceride; WAT, white adipose tissue.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5