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Dietary fish oil differentially ameliorates high-fructose diet-induced hepatic steatosis and hyperlipidemia in mice depending on time of feeding
Dietary fish oil differentially ameliorates high-fructose diet-induced hepatic steatosis and hyperlipidemia in mice depending on time of feeding Katsutaka Oishi, Tatsuya Konishi, Chiaki Hashimoto, Saori Yamamoto, Yoshinori Takahashi, Yasuhiko Shiina PII: DOI: Reference:
S0955-2863(17)30511-9 doi: 10.1016/j.jnutbio.2017.09.024 JNB 7860
To appear in:
The Journal of Nutritional Biochemistry
Received date: Revised date: Accepted date:
13 June 2017 22 August 2017 30 September 2017
Please cite this article as: Oishi Katsutaka, Konishi Tatsuya, Hashimoto Chiaki, Yamamoto Saori, Takahashi Yoshinori, Shiina Yasuhiko, Dietary fish oil differentially ameliorates high-fructose diet-induced hepatic steatosis and hyperlipidemia in mice depending on time of feeding, The Journal of Nutritional Biochemistry (2017), doi: 10.1016/j.jnutbio.2017.09.024
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Ref. JNB_2017_426
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Dietary fish oil differentially ameliorates high-fructose diet-induced hepatic
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steatosis and hyperlipidemia in mice depending on time of feeding
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Katsutaka Oishi1,2,3,*, Tatsuya Konishi4, Chiaki Hashimoto1,2, Saori Yamamoto1,
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Yoshinori Takahashi4, Yasuhiko Shiina4
1. Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
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2. Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.
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3. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan.
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4. Maruha Nichiro Corporation, Tsukuba, Ibaraki, Japan.
*Corresponding author: Katsutaka OISHI, PhD Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Tel/Fax: +81-29-861-6053 E-mail: [email protected]
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Abstract
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Chrononutrition is the science of nutrition based on chronobiology. Numerous epidemiological studies have shown that fish oil (FO) reduces the risk of cardiovascular
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events through various actions such as lowering triglycerides. The present study aimed to determine the time of day when the hypertriglyceridemia-decreasing ability of FO is
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optimal in mice. A high-fructose diet (HFrD) that induces hyperlipidemia in mice was replaced with the same diet containing 4% FO (HFrD-4%FO) at different times of the day for two weeks as described below. Mice were fed with HFrD alone (CTRL) or with
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HFrD containing 4%FO for 12 hours around the time of activity onset (breakfast (BF)-FO), or offset (dinner (DN)-FO). Plasma and liver concentrations of triglycerides
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and total cholesterol were reduced in BF-FO but not in DN-FO mice compared with CTRL mice. The temporal expression of genes associated with fatty acid (FA) synthesis
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such as Fasn, Acaca, Scd1 and Acly in the liver was significantly suppressed in both
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BF-FO and DN-FO mice. Expression levels of Scd1 in epididymal adipose tissue were significantly suppressed only in the BF-FO mice. Plasma concentrations of docosahexaenoic acid and eicosapentaenoic acid were far more increased in BF-FO, than in DN-FO mice. Significantly more of these n-3 polyunsaturated fatty acids (PUFA) were excreted in the feces of DN-FO than of BF-FO mice. These findings suggest that dietary FO exerts more hypolipidemic activity at the time of breakfast than dinner, because the intestinal absorption of n-3 PUFA is more effective at that time.
Keywords: circadian rhythm; chrononutrition; fish oil; chronopharmacology; docosahexaenoic acid; eicosapentaenoic acid. 2
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1. Introduction
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Hypertriglyceridemia is independently associated with an increased risk of cardiovascular diseases [1]. Epidemiological studies have shown that fish oil (FO) rich
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in n-3 polyunsaturated fatty acids (PUFA) reduces the risk of cardiovascular events such as cardiac death and myocardial infarction via anti-inflammatory, anti-atherosclerotic
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suppression and antithrombotic pathways [2]. The ability of FO to lower triglycerides (TG) has been recognized in experimental animals and humans [3], and the critical components of FO responsible for the hypotriglyceridemic effects are thought to be
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docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Although the TG-lowering ability of FO is established, the precise mechanisms of action are not
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completely understood. Mechanisms such as reducing hepatic and adipose lipogenesis de novo by suppressing lipogenic gene expression, inhibiting key enzymes involved in
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hepatic TG synthesis and increasing the β-oxidation of fatty acids (FA) as well as the
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expression of lipoprotein lipase (LPL), result in increased amounts of TG being removed from circulating very low density lipoprotein (VLDL) and chylomicron particles and seem to be involved in the ability of FO to decrease TG [4]. The remarkable increase in dietary fructose consumption among western countries during the past 40 years is implicated in the increasing prevalence of metabolic and cardiovascular disorders such as non-alcoholic fatty liver disease (NAFLD) and hyperlipidemia [5-7]. Fructose is both a potent substrate for and stimulator of lipogenesis de novo, and a high-fructose diet (HFrD) induces extreme hyperlipidemia in humans [8] and experimental animals including rodents and rhesus monkeys [9-12]. Several studies of experimental animals have shown that FO prevents HFrD-induced 4
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hypertriglyceridemia [10-12].
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The circadian clock system entrains various daily rhythms of behavior and physiology
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such as the sleep-wake cycle, core body temperature, blood pressure, immune functions, hormonal secretion and metabolism to environmental cues such as light-dark cycles in
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mammals. The master circadian oscillator in the suprachiasmatic nucleus (SCN) is driven by self-sustained transcription-translation-based feedback loops consisting of the
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periodic expression of clock genes [13]. Studies of clock genes have shown that oscillatory mechanisms function in various peripheral tissues such as the heart, lungs, liver, kidneys and adipose tissues and that these peripheral oscillators play important
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roles in regulating various physiological functions [14, 15]. In fact, hundreds of circadian clock-controlled genes that regulate an enormous diversity of biological
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processes including lipid absorption, synthesis and metabolism have been identified in peripheral tissues using DNA microarray technology [16-18]. The circadian clock
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governs changes in mitochondrial FA oxidation that peak at the end of the rest period
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[19, 20]. Dietary fat and molecular clocks both regulate intestinal digestion and dietary lipid absorption [21, 22]. Chrononutrition is the science of nutrition based on chronobiology, namely the circadian rhythms of the absorption, distribution, metabolism and elimination of food and food components. The principle of consuming foodstuffs at the time of the day when they would be maximally beneficial to health has been established. The present study investigated the effects of FO consumed at different times of the day on TG levels in mice fed with an HFrD.
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2. Materials and methods
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2.1. Animals and diets
Five-week-old male ddY mice (Japan SLC Inc., Hamamatsu, Japan) were fed with a
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normal non-purified diet (CE-2; Clea Japan Inc., Tokyo, Japan) ad libitum for three weeks under a 12 h light-12 h dark cycle (LD 12:12); lights on at Zeitgeber Time (ZT) 0
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and lights off at ZT12. After acclimatization, the mice were fed with the HFrD (F2HFrD; Oriental Yeast Co. Ltd., Tokyo, Japan) without or with FO (DHA-22K; Maruha Nichiro Corporation) ad libitum or time-restricted for two weeks as described
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below. Supplemental Table 1 shows the FA composition of DHA-22K and the composition of the experimental diets.
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Fish oil was administered ad libitum for two weeks using HFrD without or with 2% FO (HFrD-2%FO; Supplemental Table 1). Thereafter, the mice were sacrificed at ZT18 to
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obtain and whole blood was withdrawn under inhalational anesthesia with sevoflurane.
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The HFrD was replaced with the same diet containing 4% FO (HFrD-4%FO; Supplemental Table 1) for 12 hours a day (time-imposed restricted feeding) as described below. After acclimatization, the mice were given HFrD-4%FO from ZT18 to ZT6 (breakfast (BF)-FO), from ZT6 to ZT18 (dinner (DN)-FO), or given HFrD alone (CTRL). After nine days on the replaced or CTRL diets, feces were collected for 24 hours and stored at -80ºC and two weeks of these diets, the mice were sacrificed at ZT2, 8, 14, and 20 after 14 days to obtain blood and tissue samples, because many metabolic parameters and gene expression levels fluctuate at various times of the day [16, 17]. Whole blood was withdrawn under inhalational anesthesia with sevoflurane, and then the liver and the epididymal white adipose tissue (WAT) were dissected, rapidly frozen 6
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and stored in liquid nitrogen.
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Animal care and all experiments proceeded under the approval of the Animal Care and
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Use Committee at the National Institute of Advanced Industrial Science and Technology
2.2. Measurement of blood metabolic parameters
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(Permission No.: 2015-020).
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Mouse blood collected into EDTA-coated tubes was immediately separated by centrifugation for 15 min at 5,800 g. Platelet-poor plasma was collected and stored at -80ºC. Plasma glucose (Glc), free fatty acids (FFA), TG, and total cholesterol (T-Cho)
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concentrations were measured using LabAssayTM Glucose, NEFA, Triglyceride and
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Cholesterol kits (Wako Pure Chemical Industries Ltd., Osaka, Japan).
2.3. Measurements of hepatic lipids
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Hepatic lipids were assayed as described [23]. In brief, frozen liver fragments (0.3 g)
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were disrupted in 1 mL of 50 mmol/L NaCl using a Polytron homogenizer. Three volumes of chloroform:methanol (2:1, vol/vol) were added to homogenates (0.3 mL) and then 0.5 mL of the organic phase was evaporated and resolved in 0.2 mL of isopropanol containing 20% Triton X-100. Hepatic lipid concentrations were then measured using kits as described above.
2.4. Real-time reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted using RNAiso Plus (Takara Bio Inc., Otsu, Japan). Single-stranded cDNA was synthesized using PrimeScriptTM RT reagent kits with gDNA Eraser (Takara Bio Inc.). Real-time RT-PCR proceeded using SYBR® Premix Ex 7
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TaqTM II (Takara Bio Inc.) and a LightCyclerTM (Roche Diagnostics, Mannheim,
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Germany) with the primer sequences shown in Supplementary Table 2. The
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amplification conditions were 95C for 10 s followed by 45 cycles of 95C for 5 s, 57C for 10 s and 72C for 10 s. The amount of target mRNA was normalized relative to that
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of Actb.
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2.5. Fatty acid analysis
Lipids were extracted from 100 μL of plasma or 100 mg of feces as described by Bligh and Dyer [24] and then 1.5 mL of 0.5 M sodium hydroxide/methanol was added to the
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extract. The mixture was incubated at 100ºC for 9 min, cooled, and then incubated at 100ºC for 7 min with BF3/methanol (2 mL). The reactant was partitioned into hexane (3
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mL) and distilled water (3 mL) layers and the upper layer was analyzed using a GC-2010 PLUS gas chromatograph (GC) (Shimadzu, Kyoto, Japan) with a flame
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ionization detector (FID) and a DB-WAX capillary column (0.25 mm i.d. × 50 m; J&W
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Scientific, Folsom, CA, USA). The column temperature was increased from 170 ºC to 210ºC in 1.5ºC/min increments. The injector and detector temperatures were set at 250ºC. The carrier gas was He at a flow rate of 1.1 mL/min. The split ratio was 100:1. H2 and air were supplied to the FID. Fatty acids were identified by comparing retention times with the Supelco 37 Component FAME Mix lipid standard (Sigma-Aldrich, St. Louis, MO, USA). Tricosanoic acid methyl ester served as the internal standard for quantitative FA determination.
2.6. Statistical analysis All data are expressed as means ± standard error of the means (SEM) and were analyzed 8
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using Excel-Toukei 2010 software (Social Survey Research Information Co. Ltd.,
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Tokyo, Japan). Data were statistically evaluated using two-way analyses of variance
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(ANOVA) followed by the Tukey-Kramer post-hoc test. Differences between two groups were analyzed using unpaired Student or Welch t-tests. P < 0.05 was taken to
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indicate statistical significance.
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3. Results
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We initially assessed the ability of FO to decrease plasma lipid concentrations in mice fed with the HFrD. The consumption of the HFrD-2%FO ad libitum significantly
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reduced plasma of T-Cho, TG, and FFA, although body weight was slightly increased (Fig. 1).
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We replaced the HFrD with HFrD-4%FO between ZT18 and ZT6 (BF-FO) and between ZT6 and ZT18 (DN-FO) for two weeks (Fig. 2) to determine the effects of time-imposed restricted FO intake on hyperlipidemia. Total daily food consumption was
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similar among the CTRL, BF-FO and DN-FO groups (Fig. 3A), and estimated-FO consumption was also similar between the BF-FO and DN-FO groups (Fig. 3B). Body
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weight gain (Fig. 3C) and the relative weight of the liver (Fig. 3D) and WAT (Fig. 3E) were similar among all groups.
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Plasma Glc concentrations were identical among CTRL, BF-FO, and DN-FO groups
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(Fig. 4A). Plasma T-Cho concentrations were significantly decreased in BF-FO mice compared with those of CTRL mice during the active phase (Fig. 4B). Plasma TG and FFA concentrations fluctuated in a circadian manner that peaked at the end of the active phase in all groups, and FO-induced lipid lowering effects were statistically significant only in the BF-FO mice compared with CTRL mice (Fig. 4C and D). Hepatic contents of T-Cho, TG, and FFA were significantly suppressed depending on the time of day in BF-FO, compared with CTRL mice (Fig. 5A-C), but not in DN-FO mice. Figure 6 shows temporal expression profiles of genes related to cholesterol metabolism in the liver. Supplementation with FO significantly suppressed the mRNA expression of 10
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cholesterol synthetic genes such as Hmgcr and Hmgcs1 and upregulated that of biliary
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cholesterol transporter genes such as Abcg5 and Abcg8 (Fig. 6A-D). These effects were
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similar between BF-FO and DN-FO mice. Fish oil supplementation did not affect the mRNA expression levels of Cyp7a1 that encodes the rate-limiting enzyme for bile acid
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synthesis (Fig. 6E).
Supplementation with FO significantly reduced the hepatic mRNA expression of genes
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related to FA synthesis such as Fasn, Acaca, Scd1 and Acly, especially at the end of the active phase in both BF-FO and DN-FO mice (Fig. 7A-D), and this effect appeared to be slightly more obvious in the BF-FO, than in the DN-FO group. Expression levels of
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β-oxidation-related genes such as Cpt1a and Acox1 were little affected by FO supplementation (Fig. 7E and F).
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Adipose mRNA expression levels of Scd1 were significantly suppressed in BF-FO mice but were not statistically significant in DN-FO, compared with CTRL mice (Fig. 8C).
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Supplementation with FO did not affect the mRNA expression of any other examined
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genes related to FA synthesis (Fig. 8A, B and D). We assessed the effects of FO supplementation on temporal fluctuations in plasma FA concentrations. We found that plasma concentrations of palmitic, stearic, oleic and linoleic acids fluctuated in a circadian manner, and increased during the active phase in CTRL mice (Fig. 9A-D). Daily averaged concentrations of these FA were significantly reduced in BF-FO, compared with CTRL mice, although stearic, oleic and linoleic acid concentrations were reduced in DN-FO mice (Suppl. Fig. 1). The concentrations of oleic acid as well as linoleic acid, an essential FA that is abundant in lard, were significantly lower in BF-FO, than DN-FO mice (Suppl. Fig. 1). The circadian phase of linoleic acid fluctuations that increased during the dark period in CTRL and DN-FO 11
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mice were obviously different in BF-FO mice. Plasma concentrations of arachidonic
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acid were essentially constant during the day in all three groups and significantly
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decreased throughout the day in both BF-FO and DN-FO, compared with CTRL mice (Fig. 9E, Suppl. Fig. 1). Plasma concentrations of DHA and EPA were remarkably
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increased in both BF-FO and DN-FO, compared with CTRL mice throughout the day (Fig. 9F and G, Suppl. Fig. 1). Plasma concentrations of these n-3 PUFAs slightly
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fluctuated and peaked at the night-to-day and day-to-night transitions in BF-FO and DN-FO mice, respectively, but remained essentially constant in CTRL mice (Fig. 9F and G). Notably, the daily averaged concentrations of these n-3 PUFA were significantly
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higher in BF-FO, than in DN-FO mice (Suppl. Fig. 1). Figure 10 shows total daily fecal contents of FA. The fecal excretion of palmitic, stearic,
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oleic, linoleic and arachidonic acids were little affected in DN-FO, compared with CTRL mice (Fig. 10A-E). Fecal excretion of these FA was slightly but not significantly
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reduced in BF-FO, compared with DN-FO mice, whereas FO supplementation
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remarkably increased the fecal excretion of DHA and EPA (Fig. 10F and G). The amounts of these n-3 PUFA excreted into feces of was two-fold higher in DN-FO, than in BF-FO mice. .
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4. Discussion
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Fish oil supplementation has been recognized to improve the hyperlipidemia by decreasing lipogenesis in experimental animals and humans [3]. The present study
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evaluated the time-of-day effects of FO supplementation on HFrD-induced hyperlipidemia in mice. The results revealed that FO supplementation at the
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beginning of the active period (BF-FO) reduced liver and plasma concentrations of TG and T-Cho in mice. The present findings indicate that the circadian rhythms of various functions such as bile secretion, intestinal lipid absorption, FA synthesis de
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novo and β-oxidation are involved in the feeding time-dependent hypolipidemic effects of FO.
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Plasma concentrations of both DHA and EPA were significantly higher in BF-FO than DN-FO mice, although these groups consumed similar amounts of these n-3
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PUFA. These findings were in line with the significant decrease in the fecal
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excretion of these n-3 PUFA in BF-FO, compared with DN-FO mice. On the other hand, the fecal excretion of other FA were similar between BF-FO and DN-FO mice, except for oleic acid. These findings suggest that the intestinal absorption mechanisms of DHA and EPA, which are subject to circadian fluctuation, differ from those of other dietary FA, probably due to their chain length and bond saturation. In fact, the intestinal absorption and lymphatic transport is less effective for DHA and EPA than for other saturated and unsaturated FA [25, 26], because their low hydrolysis rate at the 1 and 3 positions of TG render them resistant to hydrolysis by pancreatic lipase [27, 28]. Therefore, intestinal absorption appears to be a critical determinant of plasma DHA and EPA concentrations, and the efficiency 13
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of this process is under circadian regulation.
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Lipid digestion before absorption includes several steps such as partial gastric
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digestion, emulsification by bile, hydrolysis by pancreatic lipase and micelle formation in the intestinal lumen. Thereafter, FA are taken up from the intestinal
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lumen into enterocytes via protein-independent diffusion and protein-dependent mechanisms such as CD36 and FABP2 that are expressed in the apical membranes
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of enterocytes. Most of these processes are enhanced during the active period and decreased during the resting period [29]. For example, bile acid synthesis is regulated in a circadian manner via the rhythmic expression of Cyp7a1, which is
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directly transcribed by clock molecules [30, 31]. Circadian changes in the efficiency of the intestine to absorb DHA and EPA seem to be regulated by several
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processes as described above.
The gut microbiota has emerged as an important contributor to the development of
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obesity and metabolic disorders through interactions with dietary factors [32, 33].
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Kishino et al. revealed that PUFA metabolism by gut bacteria affects host lipid composition [34]. Caesar et al. revealed that FO supplementation can affect the composition of the gut microbiota, which improves lipid metabolism by suppressing adipose inflammation [35]. The prebiotic effects of FO might have been involved in the FO-induced changes in the plasma FA composition and metabolic improvements identified herein. Several studies have found that the composition of gut microbiota conspicuously changes in a circadian fashion, and this is governed by the host circadian clock [36, 37]. The microbiota appears to play a critical role in circadian clock-nutrition interaction [38]. Therefore, circadian changes in intestinal microbiota composition might be involved in the 14
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time-of-day-dependent intestinal FO absorption.
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Plasma concentrations of linoleic acid, an essential FA, were significantly lower in
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BF-FO, than DN-FO mice, although its daily fecal excretion rather tended to be lower in BF-FO mice. Furthermore, both the plasma concentrations and fecal
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excretion of palmitic acid, which is abundant in FO, were similar between the BF-FO and DN-FO mice, even though the efficiency of intestinal DHA and EPA
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absorption seems to be regulated in a circadian manner. These observations suggest that the regulatory mechanisms of plasma FA concentrations differs as noted below. We found here that FO supplementation reduced plasma concentrations of several
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FA such as palmitic, stearic and oleic acids, especially during the dark period and that these effects were more significant in BF-FO, than DN-FO mice. These effects
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seemed, at least in part, to result from a reduction in lipogenesis de novo via the suppressed expression of lipogenic genes such as Fasn, Acaca, Scd1, and Acly,
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since lipogenesis de novo follows a circadian rhythm that peaks with nocturnal
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feeding in mice [39]. The mRNA expression of these lipogenic genes was significantly suppressed in BF-FO mice, especially during the dark period. During the dark-to-light transition, plasma concentrations of both DHA and EPA, which are both responsible for suppressing lipogenic gene expression, were significantly higher in BF-FO, than in DN-FO mice. These time-of-day-dependent increases in plasma n-3 PUFA might be involved in the suppression of lipogenesis de novo in BF-FO mice. Linoleic acid is an essential n-6 PUFA, which is metabolized to arachidonic acid. The circadian
phase of plasma linoleic acid obviously differed between BF-FO and
DN-FO mice, peaking at ZT2 and ZT20, respectively. Furthermore, plasma linoleic 15
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acid concentrations were significantly lower in BF-FO than DN-FO mice,
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suggesting that the time of FO feeding affects not only plasma DHA and EPA
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concentrations but also the circadian regulation of linoleic acid metabolism. On the other hand, plasma concentrations of arachidonic acid, which were also
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significantly reduced by FO supplementation, were identical throughout the day between BF-FO and DN-FO mice.
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Multiple mechanisms such as a reduction in lipogenesis de novo via the suppressed expression of lipogenic genes, inhibition of the key enzymes associated with hepatic TG synthesis, and increased β- FA oxidation are involved in the ability of
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n-3 PUFA to lower TG [4]. Although the ability of FO to lower TG is established, the precise mechanisms of this action are not completely understood. Several
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studies have indicated that other factors besides DHA and EPA such as palmitoleate induce the lipid-lowering effects of FO [40]. In addition to liver and adipose
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tissues, heart and skeletal muscle can facilitate the TG-lowering effects of FO by
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upregulating FA uptake and oxidation [3]. Lipogenesis de novo follows a circadian rhythm in mice that peaks with nocturnal feeding, whereas the hepatic β-oxidation of FA is enhanced during the light period [39]. We found here that FO supplementation at the time of activity onset obviously reduced hepatic and plasma lipid accumulation by suppressing lipogenic gene expression compared with supplementation at the time of activity offset. The fecal excretion of DHA and EPA derived from FO was significantly higher in DN-FO, than in BF-FO mice, although plasma concentrations of these were significantly higher in the BF-FO mice, suggesting that the intestinal absorptions of n-3 PUFA is most effective during the onset of activity. Chronobiological studies on the long-term supplementation of FO 16
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would be more beneficial, since hyperlipidemia and NAFLD are chronic diseases.
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functional mechanism of FO-induced TG reduction.
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Further studies from a chronobiological aspect should reveal new insight into the
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Acknowledgements
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This study was supported by the Japan Society for the Promotion of Science (JSPS)
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KAKENHI Grant Number JP16K00940 (to K. Oishi).
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Figure legends
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Fig. 1. Body weight (A), food consumption (B), and blood Glc (C), T-Cho (D), TG (E) and FFA (F) concentrations in mice fed with high-fructose diet without (CTRL) or with
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2% fish oil (FO) for two weeks. Values are shown as means ± SEM; n = 24 - 25 (A), n = 12-14 (B), n = 4 per group (C-F). Significant differences between groups: *P < 0.05,
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**P < 0.01. FFA, free fatty acid; Glc, glucose; T-Cho, total cholesterol; TG, triglyceride.
Fig. 2. Experimental design of time-restricted administration of fish oil to mice fed with
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high-fructose diet. Fish oil (FO) supplementation was administered in a restricted manner by replacing high-fructose diet (HFrD) with HFrD containing 4% FO for 12
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hours each day for two weeks. Mice were housed under 12 h light-12 h dark cycles (LD 12:12), with lights on at Zeitgeber Time (ZT) 0 and lights off at ZT12. CTRL, mice fed
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with HFrD ad libitum throughout the day; BF-FO, mice fed with HFrD between ZT6
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and ZT18 and with HFrD containing 4% fish oil between ZT18 and ZT6. DN-FO, mice fed with HFrD between ZT18 and ZT6, and with HFrD containing 4% fish oil between ZT6 and ZT18. White and black bars, light and dark periods, respectively.
Fig. 3. Food consumption (A), estimated fish oil consumption (B), body weight changes (C), as well as relative liver (D) and epididymal adipose tissue (E) weight in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Values are shown as means ± SEM; n = 8 - 9 (A), n = 8 (B), n = 24 - 25 (C) and n = 5 - 6 (D, E) per group Significant main effects or 19
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interactions are shown. A: Significant differences between before (Pre) and after (Post)
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experiment in each group, *P < 0.01. WAT, epididymal white adipose tissues.
Fig. 4. Temporal fluctuations of plasma Glc (A), T-Cho (B), TG (C) and FFA (D)
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concentrations in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet
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containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Gray shading, dark period. Values are shown as means ± SEM; n = 6 - 7 per time point and group. Significant main effects or interactions are shown. Significant main effects or interactions are shown.
*
P < 0.05 and
**
P < 0.01, significant differences at
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corresponding ZT between CTRL and BF-FO. §P < 0.05, significant differences at
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corresponding ZT between BF-FO and DN-FO.
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Fig. 5. Temporal fluctuations of hepatic T-Cho (A), TG (B), and FFA (C) contents in
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mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks day (See Fig. 2). Gray shading, dark period. Values are shown as means ± SEM, n = 6 - 7 per time point and group. Significant main effects or interactions are shown. *P < 0.05, significant differences at corresponding ZT between CTRL and BF-FO. §P < 0.05 and §§P < 0.01, significant differences at corresponding ZT between BF-FO and DN-FO.
Fig. 6. Temporal expression profiles of hepatic Hmgcr (A), Hmgcs1 (B), Abcg5 (C), Abcg8 (D) and Cyp7a1 (E) mRNA in mice supplemented with fish oil in time-restricted 20
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fashion. Administration of fish oil was restricted by replacing high-fructose diet with
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same diet containing 4% fish oil for 12 hours each day for two weeks day (See Fig. 2).
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Gray shading indicates dark period. Maximal value of CTRL mice in each gene is expressed as 100%. Values are means ± SEM, n = 6-7 per time point and per group.
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Significant main effects or interactions are shown. *P < 0.05 and **P < 0.01, significant †
differences at corresponding ZT between CTRL and BF-FO. P < 0.05 and
††
P < 0.01,
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significant differences at corresponding ZT between CTRL and DN-FO.
Fig. 7. Temporal expression profiles of hepatic Fasn (A), Acaca (B), Scd1 (C), Acly (D),
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Cpt1a (E) and Acox1 (F) mRNAs in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with
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same diet containing 4% fish oil for 12 hours each day for two weeks day (See Fig. 2). Gray shading indicates dark period. Maximal value of CTRL mice in each gene is
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expressed as 100%. Values are means ± SEM, n = 6-7 per time point and per group. Significant main effects or interactions are shown. *P < 0.05 and **P < 0.01, significant
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†
differences at corresponding ZT between CTRL and BF-FO. P < 0.05 and
††
P < 0.01,
significant differences at corresponding ZT between CTRL and DN-FO. §P < 0.01, significant differences at corresponding ZT between BF-FO and DN-FO.
Fig. 8. Temporal expression profiles of adipose Fasn (A), Acaca (B), Scd1 (C) and Acly (D) mRNA in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Gray shading 21
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indicates dark period. Maximal value of CTRL mice in each gene is expressed as 100%.
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Values are means ± SEM, n = 6-7 per time point and per group. Significant main effects
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or interactions are shown. Significant differences at corresponding ZT between CTRL and BF-FO, *P < 0.01.
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Fig. 9. Temporal fluctuations of plasma palmitic acid (A), stearic acid (B), oleic acid (C), linoleic acid (D), arachidonic acid (E), EPA (F) and DHA (G) concentrations in
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mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Gray shading indicates dark period. Values are means ± SEM, n = 6-7 per time point and per group. Significant main effects
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or interactions are shown. *P < 0.05 and
**
P < 0.01, significant differences at
†
††
P < 0.01, significant
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corresponding ZT between CTRL and BF-FO. P < 0.05 and
differences at corresponding ZT between CTRL and DN-FO. §P < 0.01, significant
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differences at corresponding ZT between BF-FO and DN-FO.
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Fig. 10. Total daily fecal excretion of palmitic acid (A), stearic (B), oleic (C), linoleic (D), arachidonic (E) acids, EPA (F) and DHA (G) in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Values are means ± SEM, n = 5-6 per group. Significant main †
effects are shown. *P < 0.05, significant differences between CTRL and BF-FO. P < 0.01, significant differences between CTRL and DN-FO. §P < 0.05, significant differences between BF-FO and DN-FO.
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References
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[1] Cullen P. Evidence that triglycerides are an independent coronary heart disease risk factor. Am J Cardiol. 2000;86:943-9.
SC
[2] Saravanan P, Davidson NC, Schmidt EB, Calder PC. Cardiovascular effects of marine omega-3 fatty acids. Lancet. 2010;376:540-50.
MA NU
[3] Shearer GC, Savinova OV, Harris WS. Fish oil -- how does it reduce plasma triglycerides? Biochim Biophys Acta. 2012;1821:843-51. [4] Backes J, Anzalone D, Hilleman D, Catini J. The clinical relevance of omega-3 fatty
ED
acids in the management of hypertriglyceridemia. Lipids Health Dis. 2016;15:118. [5] Bray GA, Nielsen SJ, Popkin BM. Consumption of high-fructose corn syrup in
PT
beverages may play a role in the epidemic of obesity. Am J Clin Nutr. 2004;79:537-43. [6] Miller A, Adeli K. Dietary fructose and the metabolic syndrome. Curr Opin
CE
Gastroenterol. 2008;24:204-9.
AC
[7] Samuel VT. Fructose induced lipogenesis: from sugar to fat to insulin resistance. Trends Endocrinol Metab. 2011;22:60-5. [8] Hudgins LC, Parker TS, Levine DM, Hellerstein MK. A dual sugar challenge test for lipogenic sensitivity to dietary fructose. J Clin Endocrinol Metab. 2011;96:861-8. [9] Nagai Y, Nishio Y, Nakamura T, Maegawa H, Kikkawa R, Kashiwagi A. Amelioration of high fructose-induced metabolic derangements by activation of PPARalpha. Am J Physiol Endocrinol Metab. 2002;282:E1180-90. [10] Bremer AA, Stanhope KL, Graham JL, Cummings BP, Ampah SB, Saville BR, et al. Fish oil supplementation ameliorates fructose-induced hypertriglyceridemia and insulin resistance in adult male rhesus macaques. J Nutr. 2014;144:5-11. 23
ACCEPTED MANUSCRIPT
[11] Erion DM, Popov V, Hsiao JJ, Vatner D, Mitchell K, Yonemitsu S, et al. The role of
T
the carbohydrate response element-binding protein in male fructose-fed rats.
RI P
Endocrinology. 2013;154:36-44.
[12] Karsenty J, Landrier JF, Rousseau-Ralliard D, Robbez-Masson V, Margotat A,
SC
Deprez P, et al. Beneficial effects of omega-3 fatty acids on the consequences of a fructose diet are not mediated by PPAR delta or PGC1 alpha. Eur J Nutr.
MA NU
2013;52:1865-74.
[13] Weaver DR. Introduction to Circadian Rhythms and Mechanisms of Circadian Oscillations. In: Gumz LM, editor. Circadian Clocks: Role in Health and Disease. New
ED
York, NY: Springer New York; 2016. p. 1-55.
[14] Froy O. Metabolism and circadian rhythms--implications for obesity. Endocr Rev.
PT
2010;31:1-24.
[15] Mazzoccoli G, Pazienza V, Vinciguerra M. Clock genes and clock-controlled genes
CE
in the regulation of metabolic rhythms. Chronobiol Int. 2012;29:227-51.
AC
[16] Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, et al. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell. 2002;109:307-20.
[17] Ueda HR, Chen W, Adachi A, Wakamatsu H, Hayashi S, Takasugi T, et al. A transcription factor response element for gene expression during circadian night. Nature. 2002;418:534-9. [18] Oishi K, Miyazaki K, Kadota K, Kikuno R, Nagase T, Atsumi G, et al. Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes. J Biol Chem. 2003;278:41519-27. [19] Peek CB, Affinati AH, Ramsey KM, Kuo HY, Yu W, Sena LA, et al. Circadian 24
ACCEPTED MANUSCRIPT
clock NAD+ cycle drives mitochondrial oxidative metabolism in mice. Science.
T
2013;342:1243417.
RI P
[20] Bray MS, Young ME. Regulation of fatty acid metabolism by cell autonomous circadian clocks: time to fatten up on information? J Biol Chem. 2011;286:11883-9.
SC
[21] Scheving LA. Biological clocks and the digestive system. Gastroenterology. 2000;119:536-49.
MA NU
[22] Bron R, Furness JB. Rhythm of digestion: keeping time in the gastrointestinal tract. Clin Exp Pharmacol Physiol. 2009;36:1041-8.
[23] Oishi K, Yamamoto S, Itoh N, Nakao R, Yasumoto Y, Tanaka K, et al. Wheat
ED
alkylresorcinols suppress high-fat, high-sucrose diet-induced obesity and glucose intolerance by increasing insulin sensitivity and cholesterol excretion in male mice. J
PT
Nutr. 2015;145:199-206.
[24] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J
CE
Biochem Physiol. 1959;37:911-7.
AC
[25] Christensen MS, Hoy CE, Becker CC, Redgrave TG. Intestinal absorption and lymphatic transport of eicosapentaenoic (EPA), docosahexaenoic (DHA), and decanoic acids: dependence on intramolecular triacylglycerol structure. Am J Clin Nutr. 1995;61:56-61. [26] Ikeda I, Sasaki E, Yasunami H, Nomiyama S, Nakayama M, Sugano M, et al. Digestion and lymphatic transport of eicosapentaenoic and docosahexaenoic acids given in the form of triacylglycerol, free acid and ethyl ester in rats. Biochim Biophys Acta. 1995;1259:297-304. [27] Bottino NR, Vandenburg GA, Reiser R. Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis. Lipids. 25
ACCEPTED MANUSCRIPT
1967;2:489-93.
T
[28] Yang LY, Kuksis A, Myher JJ. Lipolysis of menhaden oil triacylglycerols and the
RI P
corresponding fatty acid alkyl esters by pancreatic lipase in vitro: a reexamination. J Lipid Res. 1990;31:137-47.
SC
[29] Gnocchi D, Pedrelli M, Hurt-Camejo E, Parini P. Lipids around the Clock: Focus on Circadian Rhythms and Lipid Metabolism. Biology (Basel). 2015;4:104-32.
Regulation
of
bile
acid
MA NU
[30] Duez H, van der Veen JN, Duhem C, Pourcet B, Touvier T, Fontaine C, et al. synthesis
by
the
nuclear
receptor
Rev-erbalpha.
Gastroenterology. 2008;135:689-98.
ED
[31] Noshiro M, Usui E, Kawamoto T, Kubo H, Fujimoto K, Furukawa M, et al. Multiple mechanisms regulate circadian expression of the gene for cholesterol
PT
7alpha-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis. J Biol Rhythms. 2007;22:299-311.
CE
[32] Cox LM, Blaser MJ. Pathways in microbe-induced obesity. Cell Metab.
AC
2013;17:883-94.
[33] Kovatcheva-Datchary P, Arora T. Nutrition, the gut microbiome and the metabolic syndrome. Best Pract Res Clin Gastroenterol. 2013;27:59-72. [34] Kishino S, Takeuchi M, Park SB, Hirata A, Kitamura N, Kunisawa J, et al. Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition. Proc Natl Acad Sci U S A. 2013;110:17808-13. [35] Caesar R, Tremaroli V, Kovatcheva-Datchary P, Cani Patrice D, Bäckhed F. Crosstalk between gut microbiota and dietary lipids aggravates WAT inflammation through TLR signaling. Cell Metab. 2015;22:658-68. [36] Thaiss CA, Zeevi D, Levy M, Zilberman-Schapira G, Suez J, Tengeler AC, et al. 26
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Transkingdom control of microbiota diurnal oscillations promotes metabolic
T
homeostasis. Cell. 2014;159:514-29.
RI P
[37] Voigt RM, Forsyth CB, Green SJ, Mutlu E, Engen P, Vitaterna MH, et al. Circadian disorganization alters intestinal microbiota. PLoS One. 2014;9:e97500.
SC
[38] Asher G, Sassone-Corsi P. Time for food: the intimate interplay between nutrition, metabolism, and the circadian clock. Cell. 2015;161:84-92.
MA NU
[39] Liu S, Brown JD, Stanya KJ, Homan E, Leidl M, Inouye K, et al. A diurnal serum lipid integrates hepatic lipogenesis and peripheral fatty acid use. Nature. 2013;502:550-4.
ED
[40] Shiba S, Tsunoda N, Wakutsu M, Muraki E, Sonoda M, Tam PS, et al. Regulation of lipid metabolism by palmitoleate and eicosapentaenoic acid (EPA) in mice fed a
AC
CE
PT
high-fat diet. Biosci Biotechnol Biochem. 2011;75:2401-3.
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S0955-2863(17)30511-9 doi: 10.1016/j.jnutbio.2017.09.024 JNB 7860
To appear in:
The Journal of Nutritional Biochemistry
Received date: Revised date: Accepted date:
13 June 2017 22 August 2017 30 September 2017
Please cite this article as: Oishi Katsutaka, Konishi Tatsuya, Hashimoto Chiaki, Yamamoto Saori, Takahashi Yoshinori, Shiina Yasuhiko, Dietary fish oil differentially ameliorates high-fructose diet-induced hepatic steatosis and hyperlipidemia in mice depending on time of feeding, The Journal of Nutritional Biochemistry (2017), doi: 10.1016/j.jnutbio.2017.09.024
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Ref. JNB_2017_426
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Dietary fish oil differentially ameliorates high-fructose diet-induced hepatic
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steatosis and hyperlipidemia in mice depending on time of feeding
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Katsutaka Oishi1,2,3,*, Tatsuya Konishi4, Chiaki Hashimoto1,2, Saori Yamamoto1,
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Yoshinori Takahashi4, Yasuhiko Shiina4
1. Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
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2. Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.
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3. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan.
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4. Maruha Nichiro Corporation, Tsukuba, Ibaraki, Japan.
*Corresponding author: Katsutaka OISHI, PhD Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Tel/Fax: +81-29-861-6053 E-mail: [email protected]
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Abstract
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Chrononutrition is the science of nutrition based on chronobiology. Numerous epidemiological studies have shown that fish oil (FO) reduces the risk of cardiovascular
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events through various actions such as lowering triglycerides. The present study aimed to determine the time of day when the hypertriglyceridemia-decreasing ability of FO is
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optimal in mice. A high-fructose diet (HFrD) that induces hyperlipidemia in mice was replaced with the same diet containing 4% FO (HFrD-4%FO) at different times of the day for two weeks as described below. Mice were fed with HFrD alone (CTRL) or with
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HFrD containing 4%FO for 12 hours around the time of activity onset (breakfast (BF)-FO), or offset (dinner (DN)-FO). Plasma and liver concentrations of triglycerides
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and total cholesterol were reduced in BF-FO but not in DN-FO mice compared with CTRL mice. The temporal expression of genes associated with fatty acid (FA) synthesis
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such as Fasn, Acaca, Scd1 and Acly in the liver was significantly suppressed in both
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BF-FO and DN-FO mice. Expression levels of Scd1 in epididymal adipose tissue were significantly suppressed only in the BF-FO mice. Plasma concentrations of docosahexaenoic acid and eicosapentaenoic acid were far more increased in BF-FO, than in DN-FO mice. Significantly more of these n-3 polyunsaturated fatty acids (PUFA) were excreted in the feces of DN-FO than of BF-FO mice. These findings suggest that dietary FO exerts more hypolipidemic activity at the time of breakfast than dinner, because the intestinal absorption of n-3 PUFA is more effective at that time.
Keywords: circadian rhythm; chrononutrition; fish oil; chronopharmacology; docosahexaenoic acid; eicosapentaenoic acid. 2
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1. Introduction
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Hypertriglyceridemia is independently associated with an increased risk of cardiovascular diseases [1]. Epidemiological studies have shown that fish oil (FO) rich
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in n-3 polyunsaturated fatty acids (PUFA) reduces the risk of cardiovascular events such as cardiac death and myocardial infarction via anti-inflammatory, anti-atherosclerotic
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suppression and antithrombotic pathways [2]. The ability of FO to lower triglycerides (TG) has been recognized in experimental animals and humans [3], and the critical components of FO responsible for the hypotriglyceridemic effects are thought to be
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docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Although the TG-lowering ability of FO is established, the precise mechanisms of action are not
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completely understood. Mechanisms such as reducing hepatic and adipose lipogenesis de novo by suppressing lipogenic gene expression, inhibiting key enzymes involved in
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hepatic TG synthesis and increasing the β-oxidation of fatty acids (FA) as well as the
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expression of lipoprotein lipase (LPL), result in increased amounts of TG being removed from circulating very low density lipoprotein (VLDL) and chylomicron particles and seem to be involved in the ability of FO to decrease TG [4]. The remarkable increase in dietary fructose consumption among western countries during the past 40 years is implicated in the increasing prevalence of metabolic and cardiovascular disorders such as non-alcoholic fatty liver disease (NAFLD) and hyperlipidemia [5-7]. Fructose is both a potent substrate for and stimulator of lipogenesis de novo, and a high-fructose diet (HFrD) induces extreme hyperlipidemia in humans [8] and experimental animals including rodents and rhesus monkeys [9-12]. Several studies of experimental animals have shown that FO prevents HFrD-induced 4
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hypertriglyceridemia [10-12].
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The circadian clock system entrains various daily rhythms of behavior and physiology
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such as the sleep-wake cycle, core body temperature, blood pressure, immune functions, hormonal secretion and metabolism to environmental cues such as light-dark cycles in
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mammals. The master circadian oscillator in the suprachiasmatic nucleus (SCN) is driven by self-sustained transcription-translation-based feedback loops consisting of the
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periodic expression of clock genes [13]. Studies of clock genes have shown that oscillatory mechanisms function in various peripheral tissues such as the heart, lungs, liver, kidneys and adipose tissues and that these peripheral oscillators play important
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roles in regulating various physiological functions [14, 15]. In fact, hundreds of circadian clock-controlled genes that regulate an enormous diversity of biological
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processes including lipid absorption, synthesis and metabolism have been identified in peripheral tissues using DNA microarray technology [16-18]. The circadian clock
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governs changes in mitochondrial FA oxidation that peak at the end of the rest period
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[19, 20]. Dietary fat and molecular clocks both regulate intestinal digestion and dietary lipid absorption [21, 22]. Chrononutrition is the science of nutrition based on chronobiology, namely the circadian rhythms of the absorption, distribution, metabolism and elimination of food and food components. The principle of consuming foodstuffs at the time of the day when they would be maximally beneficial to health has been established. The present study investigated the effects of FO consumed at different times of the day on TG levels in mice fed with an HFrD.
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2. Materials and methods
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2.1. Animals and diets
Five-week-old male ddY mice (Japan SLC Inc., Hamamatsu, Japan) were fed with a
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normal non-purified diet (CE-2; Clea Japan Inc., Tokyo, Japan) ad libitum for three weeks under a 12 h light-12 h dark cycle (LD 12:12); lights on at Zeitgeber Time (ZT) 0
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and lights off at ZT12. After acclimatization, the mice were fed with the HFrD (F2HFrD; Oriental Yeast Co. Ltd., Tokyo, Japan) without or with FO (DHA-22K; Maruha Nichiro Corporation) ad libitum or time-restricted for two weeks as described
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below. Supplemental Table 1 shows the FA composition of DHA-22K and the composition of the experimental diets.
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Fish oil was administered ad libitum for two weeks using HFrD without or with 2% FO (HFrD-2%FO; Supplemental Table 1). Thereafter, the mice were sacrificed at ZT18 to
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obtain and whole blood was withdrawn under inhalational anesthesia with sevoflurane.
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The HFrD was replaced with the same diet containing 4% FO (HFrD-4%FO; Supplemental Table 1) for 12 hours a day (time-imposed restricted feeding) as described below. After acclimatization, the mice were given HFrD-4%FO from ZT18 to ZT6 (breakfast (BF)-FO), from ZT6 to ZT18 (dinner (DN)-FO), or given HFrD alone (CTRL). After nine days on the replaced or CTRL diets, feces were collected for 24 hours and stored at -80ºC and two weeks of these diets, the mice were sacrificed at ZT2, 8, 14, and 20 after 14 days to obtain blood and tissue samples, because many metabolic parameters and gene expression levels fluctuate at various times of the day [16, 17]. Whole blood was withdrawn under inhalational anesthesia with sevoflurane, and then the liver and the epididymal white adipose tissue (WAT) were dissected, rapidly frozen 6
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and stored in liquid nitrogen.
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Animal care and all experiments proceeded under the approval of the Animal Care and
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Use Committee at the National Institute of Advanced Industrial Science and Technology
2.2. Measurement of blood metabolic parameters
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(Permission No.: 2015-020).
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Mouse blood collected into EDTA-coated tubes was immediately separated by centrifugation for 15 min at 5,800 g. Platelet-poor plasma was collected and stored at -80ºC. Plasma glucose (Glc), free fatty acids (FFA), TG, and total cholesterol (T-Cho)
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concentrations were measured using LabAssayTM Glucose, NEFA, Triglyceride and
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Cholesterol kits (Wako Pure Chemical Industries Ltd., Osaka, Japan).
2.3. Measurements of hepatic lipids
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Hepatic lipids were assayed as described [23]. In brief, frozen liver fragments (0.3 g)
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were disrupted in 1 mL of 50 mmol/L NaCl using a Polytron homogenizer. Three volumes of chloroform:methanol (2:1, vol/vol) were added to homogenates (0.3 mL) and then 0.5 mL of the organic phase was evaporated and resolved in 0.2 mL of isopropanol containing 20% Triton X-100. Hepatic lipid concentrations were then measured using kits as described above.
2.4. Real-time reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted using RNAiso Plus (Takara Bio Inc., Otsu, Japan). Single-stranded cDNA was synthesized using PrimeScriptTM RT reagent kits with gDNA Eraser (Takara Bio Inc.). Real-time RT-PCR proceeded using SYBR® Premix Ex 7
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TaqTM II (Takara Bio Inc.) and a LightCyclerTM (Roche Diagnostics, Mannheim,
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Germany) with the primer sequences shown in Supplementary Table 2. The
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amplification conditions were 95C for 10 s followed by 45 cycles of 95C for 5 s, 57C for 10 s and 72C for 10 s. The amount of target mRNA was normalized relative to that
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of Actb.
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2.5. Fatty acid analysis
Lipids were extracted from 100 μL of plasma or 100 mg of feces as described by Bligh and Dyer [24] and then 1.5 mL of 0.5 M sodium hydroxide/methanol was added to the
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extract. The mixture was incubated at 100ºC for 9 min, cooled, and then incubated at 100ºC for 7 min with BF3/methanol (2 mL). The reactant was partitioned into hexane (3
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mL) and distilled water (3 mL) layers and the upper layer was analyzed using a GC-2010 PLUS gas chromatograph (GC) (Shimadzu, Kyoto, Japan) with a flame
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ionization detector (FID) and a DB-WAX capillary column (0.25 mm i.d. × 50 m; J&W
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Scientific, Folsom, CA, USA). The column temperature was increased from 170 ºC to 210ºC in 1.5ºC/min increments. The injector and detector temperatures were set at 250ºC. The carrier gas was He at a flow rate of 1.1 mL/min. The split ratio was 100:1. H2 and air were supplied to the FID. Fatty acids were identified by comparing retention times with the Supelco 37 Component FAME Mix lipid standard (Sigma-Aldrich, St. Louis, MO, USA). Tricosanoic acid methyl ester served as the internal standard for quantitative FA determination.
2.6. Statistical analysis All data are expressed as means ± standard error of the means (SEM) and were analyzed 8
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using Excel-Toukei 2010 software (Social Survey Research Information Co. Ltd.,
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Tokyo, Japan). Data were statistically evaluated using two-way analyses of variance
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(ANOVA) followed by the Tukey-Kramer post-hoc test. Differences between two groups were analyzed using unpaired Student or Welch t-tests. P < 0.05 was taken to
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indicate statistical significance.
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3. Results
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We initially assessed the ability of FO to decrease plasma lipid concentrations in mice fed with the HFrD. The consumption of the HFrD-2%FO ad libitum significantly
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reduced plasma of T-Cho, TG, and FFA, although body weight was slightly increased (Fig. 1).
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We replaced the HFrD with HFrD-4%FO between ZT18 and ZT6 (BF-FO) and between ZT6 and ZT18 (DN-FO) for two weeks (Fig. 2) to determine the effects of time-imposed restricted FO intake on hyperlipidemia. Total daily food consumption was
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similar among the CTRL, BF-FO and DN-FO groups (Fig. 3A), and estimated-FO consumption was also similar between the BF-FO and DN-FO groups (Fig. 3B). Body
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weight gain (Fig. 3C) and the relative weight of the liver (Fig. 3D) and WAT (Fig. 3E) were similar among all groups.
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Plasma Glc concentrations were identical among CTRL, BF-FO, and DN-FO groups
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(Fig. 4A). Plasma T-Cho concentrations were significantly decreased in BF-FO mice compared with those of CTRL mice during the active phase (Fig. 4B). Plasma TG and FFA concentrations fluctuated in a circadian manner that peaked at the end of the active phase in all groups, and FO-induced lipid lowering effects were statistically significant only in the BF-FO mice compared with CTRL mice (Fig. 4C and D). Hepatic contents of T-Cho, TG, and FFA were significantly suppressed depending on the time of day in BF-FO, compared with CTRL mice (Fig. 5A-C), but not in DN-FO mice. Figure 6 shows temporal expression profiles of genes related to cholesterol metabolism in the liver. Supplementation with FO significantly suppressed the mRNA expression of 10
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cholesterol synthetic genes such as Hmgcr and Hmgcs1 and upregulated that of biliary
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cholesterol transporter genes such as Abcg5 and Abcg8 (Fig. 6A-D). These effects were
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similar between BF-FO and DN-FO mice. Fish oil supplementation did not affect the mRNA expression levels of Cyp7a1 that encodes the rate-limiting enzyme for bile acid
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synthesis (Fig. 6E).
Supplementation with FO significantly reduced the hepatic mRNA expression of genes
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related to FA synthesis such as Fasn, Acaca, Scd1 and Acly, especially at the end of the active phase in both BF-FO and DN-FO mice (Fig. 7A-D), and this effect appeared to be slightly more obvious in the BF-FO, than in the DN-FO group. Expression levels of
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β-oxidation-related genes such as Cpt1a and Acox1 were little affected by FO supplementation (Fig. 7E and F).
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Adipose mRNA expression levels of Scd1 were significantly suppressed in BF-FO mice but were not statistically significant in DN-FO, compared with CTRL mice (Fig. 8C).
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Supplementation with FO did not affect the mRNA expression of any other examined
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genes related to FA synthesis (Fig. 8A, B and D). We assessed the effects of FO supplementation on temporal fluctuations in plasma FA concentrations. We found that plasma concentrations of palmitic, stearic, oleic and linoleic acids fluctuated in a circadian manner, and increased during the active phase in CTRL mice (Fig. 9A-D). Daily averaged concentrations of these FA were significantly reduced in BF-FO, compared with CTRL mice, although stearic, oleic and linoleic acid concentrations were reduced in DN-FO mice (Suppl. Fig. 1). The concentrations of oleic acid as well as linoleic acid, an essential FA that is abundant in lard, were significantly lower in BF-FO, than DN-FO mice (Suppl. Fig. 1). The circadian phase of linoleic acid fluctuations that increased during the dark period in CTRL and DN-FO 11
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mice were obviously different in BF-FO mice. Plasma concentrations of arachidonic
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acid were essentially constant during the day in all three groups and significantly
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decreased throughout the day in both BF-FO and DN-FO, compared with CTRL mice (Fig. 9E, Suppl. Fig. 1). Plasma concentrations of DHA and EPA were remarkably
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increased in both BF-FO and DN-FO, compared with CTRL mice throughout the day (Fig. 9F and G, Suppl. Fig. 1). Plasma concentrations of these n-3 PUFAs slightly
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fluctuated and peaked at the night-to-day and day-to-night transitions in BF-FO and DN-FO mice, respectively, but remained essentially constant in CTRL mice (Fig. 9F and G). Notably, the daily averaged concentrations of these n-3 PUFA were significantly
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higher in BF-FO, than in DN-FO mice (Suppl. Fig. 1). Figure 10 shows total daily fecal contents of FA. The fecal excretion of palmitic, stearic,
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oleic, linoleic and arachidonic acids were little affected in DN-FO, compared with CTRL mice (Fig. 10A-E). Fecal excretion of these FA was slightly but not significantly
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reduced in BF-FO, compared with DN-FO mice, whereas FO supplementation
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remarkably increased the fecal excretion of DHA and EPA (Fig. 10F and G). The amounts of these n-3 PUFA excreted into feces of was two-fold higher in DN-FO, than in BF-FO mice. .
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4. Discussion
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Fish oil supplementation has been recognized to improve the hyperlipidemia by decreasing lipogenesis in experimental animals and humans [3]. The present study
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evaluated the time-of-day effects of FO supplementation on HFrD-induced hyperlipidemia in mice. The results revealed that FO supplementation at the
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beginning of the active period (BF-FO) reduced liver and plasma concentrations of TG and T-Cho in mice. The present findings indicate that the circadian rhythms of various functions such as bile secretion, intestinal lipid absorption, FA synthesis de
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novo and β-oxidation are involved in the feeding time-dependent hypolipidemic effects of FO.
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Plasma concentrations of both DHA and EPA were significantly higher in BF-FO than DN-FO mice, although these groups consumed similar amounts of these n-3
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PUFA. These findings were in line with the significant decrease in the fecal
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excretion of these n-3 PUFA in BF-FO, compared with DN-FO mice. On the other hand, the fecal excretion of other FA were similar between BF-FO and DN-FO mice, except for oleic acid. These findings suggest that the intestinal absorption mechanisms of DHA and EPA, which are subject to circadian fluctuation, differ from those of other dietary FA, probably due to their chain length and bond saturation. In fact, the intestinal absorption and lymphatic transport is less effective for DHA and EPA than for other saturated and unsaturated FA [25, 26], because their low hydrolysis rate at the 1 and 3 positions of TG render them resistant to hydrolysis by pancreatic lipase [27, 28]. Therefore, intestinal absorption appears to be a critical determinant of plasma DHA and EPA concentrations, and the efficiency 13
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of this process is under circadian regulation.
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Lipid digestion before absorption includes several steps such as partial gastric
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digestion, emulsification by bile, hydrolysis by pancreatic lipase and micelle formation in the intestinal lumen. Thereafter, FA are taken up from the intestinal
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lumen into enterocytes via protein-independent diffusion and protein-dependent mechanisms such as CD36 and FABP2 that are expressed in the apical membranes
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of enterocytes. Most of these processes are enhanced during the active period and decreased during the resting period [29]. For example, bile acid synthesis is regulated in a circadian manner via the rhythmic expression of Cyp7a1, which is
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directly transcribed by clock molecules [30, 31]. Circadian changes in the efficiency of the intestine to absorb DHA and EPA seem to be regulated by several
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processes as described above.
The gut microbiota has emerged as an important contributor to the development of
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obesity and metabolic disorders through interactions with dietary factors [32, 33].
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Kishino et al. revealed that PUFA metabolism by gut bacteria affects host lipid composition [34]. Caesar et al. revealed that FO supplementation can affect the composition of the gut microbiota, which improves lipid metabolism by suppressing adipose inflammation [35]. The prebiotic effects of FO might have been involved in the FO-induced changes in the plasma FA composition and metabolic improvements identified herein. Several studies have found that the composition of gut microbiota conspicuously changes in a circadian fashion, and this is governed by the host circadian clock [36, 37]. The microbiota appears to play a critical role in circadian clock-nutrition interaction [38]. Therefore, circadian changes in intestinal microbiota composition might be involved in the 14
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time-of-day-dependent intestinal FO absorption.
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Plasma concentrations of linoleic acid, an essential FA, were significantly lower in
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BF-FO, than DN-FO mice, although its daily fecal excretion rather tended to be lower in BF-FO mice. Furthermore, both the plasma concentrations and fecal
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excretion of palmitic acid, which is abundant in FO, were similar between the BF-FO and DN-FO mice, even though the efficiency of intestinal DHA and EPA
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absorption seems to be regulated in a circadian manner. These observations suggest that the regulatory mechanisms of plasma FA concentrations differs as noted below. We found here that FO supplementation reduced plasma concentrations of several
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FA such as palmitic, stearic and oleic acids, especially during the dark period and that these effects were more significant in BF-FO, than DN-FO mice. These effects
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seemed, at least in part, to result from a reduction in lipogenesis de novo via the suppressed expression of lipogenic genes such as Fasn, Acaca, Scd1, and Acly,
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since lipogenesis de novo follows a circadian rhythm that peaks with nocturnal
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feeding in mice [39]. The mRNA expression of these lipogenic genes was significantly suppressed in BF-FO mice, especially during the dark period. During the dark-to-light transition, plasma concentrations of both DHA and EPA, which are both responsible for suppressing lipogenic gene expression, were significantly higher in BF-FO, than in DN-FO mice. These time-of-day-dependent increases in plasma n-3 PUFA might be involved in the suppression of lipogenesis de novo in BF-FO mice. Linoleic acid is an essential n-6 PUFA, which is metabolized to arachidonic acid. The circadian
phase of plasma linoleic acid obviously differed between BF-FO and
DN-FO mice, peaking at ZT2 and ZT20, respectively. Furthermore, plasma linoleic 15
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acid concentrations were significantly lower in BF-FO than DN-FO mice,
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suggesting that the time of FO feeding affects not only plasma DHA and EPA
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concentrations but also the circadian regulation of linoleic acid metabolism. On the other hand, plasma concentrations of arachidonic acid, which were also
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significantly reduced by FO supplementation, were identical throughout the day between BF-FO and DN-FO mice.
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Multiple mechanisms such as a reduction in lipogenesis de novo via the suppressed expression of lipogenic genes, inhibition of the key enzymes associated with hepatic TG synthesis, and increased β- FA oxidation are involved in the ability of
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n-3 PUFA to lower TG [4]. Although the ability of FO to lower TG is established, the precise mechanisms of this action are not completely understood. Several
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studies have indicated that other factors besides DHA and EPA such as palmitoleate induce the lipid-lowering effects of FO [40]. In addition to liver and adipose
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tissues, heart and skeletal muscle can facilitate the TG-lowering effects of FO by
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upregulating FA uptake and oxidation [3]. Lipogenesis de novo follows a circadian rhythm in mice that peaks with nocturnal feeding, whereas the hepatic β-oxidation of FA is enhanced during the light period [39]. We found here that FO supplementation at the time of activity onset obviously reduced hepatic and plasma lipid accumulation by suppressing lipogenic gene expression compared with supplementation at the time of activity offset. The fecal excretion of DHA and EPA derived from FO was significantly higher in DN-FO, than in BF-FO mice, although plasma concentrations of these were significantly higher in the BF-FO mice, suggesting that the intestinal absorptions of n-3 PUFA is most effective during the onset of activity. Chronobiological studies on the long-term supplementation of FO 16
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would be more beneficial, since hyperlipidemia and NAFLD are chronic diseases.
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functional mechanism of FO-induced TG reduction.
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Further studies from a chronobiological aspect should reveal new insight into the
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Acknowledgements
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This study was supported by the Japan Society for the Promotion of Science (JSPS)
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KAKENHI Grant Number JP16K00940 (to K. Oishi).
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Figure legends
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Fig. 1. Body weight (A), food consumption (B), and blood Glc (C), T-Cho (D), TG (E) and FFA (F) concentrations in mice fed with high-fructose diet without (CTRL) or with
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2% fish oil (FO) for two weeks. Values are shown as means ± SEM; n = 24 - 25 (A), n = 12-14 (B), n = 4 per group (C-F). Significant differences between groups: *P < 0.05,
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**P < 0.01. FFA, free fatty acid; Glc, glucose; T-Cho, total cholesterol; TG, triglyceride.
Fig. 2. Experimental design of time-restricted administration of fish oil to mice fed with
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high-fructose diet. Fish oil (FO) supplementation was administered in a restricted manner by replacing high-fructose diet (HFrD) with HFrD containing 4% FO for 12
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hours each day for two weeks. Mice were housed under 12 h light-12 h dark cycles (LD 12:12), with lights on at Zeitgeber Time (ZT) 0 and lights off at ZT12. CTRL, mice fed
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with HFrD ad libitum throughout the day; BF-FO, mice fed with HFrD between ZT6
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and ZT18 and with HFrD containing 4% fish oil between ZT18 and ZT6. DN-FO, mice fed with HFrD between ZT18 and ZT6, and with HFrD containing 4% fish oil between ZT6 and ZT18. White and black bars, light and dark periods, respectively.
Fig. 3. Food consumption (A), estimated fish oil consumption (B), body weight changes (C), as well as relative liver (D) and epididymal adipose tissue (E) weight in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Values are shown as means ± SEM; n = 8 - 9 (A), n = 8 (B), n = 24 - 25 (C) and n = 5 - 6 (D, E) per group Significant main effects or 19
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interactions are shown. A: Significant differences between before (Pre) and after (Post)
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experiment in each group, *P < 0.01. WAT, epididymal white adipose tissues.
Fig. 4. Temporal fluctuations of plasma Glc (A), T-Cho (B), TG (C) and FFA (D)
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concentrations in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet
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containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Gray shading, dark period. Values are shown as means ± SEM; n = 6 - 7 per time point and group. Significant main effects or interactions are shown. Significant main effects or interactions are shown.
*
P < 0.05 and
**
P < 0.01, significant differences at
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corresponding ZT between CTRL and BF-FO. §P < 0.05, significant differences at
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corresponding ZT between BF-FO and DN-FO.
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Fig. 5. Temporal fluctuations of hepatic T-Cho (A), TG (B), and FFA (C) contents in
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mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks day (See Fig. 2). Gray shading, dark period. Values are shown as means ± SEM, n = 6 - 7 per time point and group. Significant main effects or interactions are shown. *P < 0.05, significant differences at corresponding ZT between CTRL and BF-FO. §P < 0.05 and §§P < 0.01, significant differences at corresponding ZT between BF-FO and DN-FO.
Fig. 6. Temporal expression profiles of hepatic Hmgcr (A), Hmgcs1 (B), Abcg5 (C), Abcg8 (D) and Cyp7a1 (E) mRNA in mice supplemented with fish oil in time-restricted 20
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fashion. Administration of fish oil was restricted by replacing high-fructose diet with
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same diet containing 4% fish oil for 12 hours each day for two weeks day (See Fig. 2).
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Gray shading indicates dark period. Maximal value of CTRL mice in each gene is expressed as 100%. Values are means ± SEM, n = 6-7 per time point and per group.
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Significant main effects or interactions are shown. *P < 0.05 and **P < 0.01, significant †
differences at corresponding ZT between CTRL and BF-FO. P < 0.05 and
††
P < 0.01,
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significant differences at corresponding ZT between CTRL and DN-FO.
Fig. 7. Temporal expression profiles of hepatic Fasn (A), Acaca (B), Scd1 (C), Acly (D),
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Cpt1a (E) and Acox1 (F) mRNAs in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with
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same diet containing 4% fish oil for 12 hours each day for two weeks day (See Fig. 2). Gray shading indicates dark period. Maximal value of CTRL mice in each gene is
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expressed as 100%. Values are means ± SEM, n = 6-7 per time point and per group. Significant main effects or interactions are shown. *P < 0.05 and **P < 0.01, significant
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†
differences at corresponding ZT between CTRL and BF-FO. P < 0.05 and
††
P < 0.01,
significant differences at corresponding ZT between CTRL and DN-FO. §P < 0.01, significant differences at corresponding ZT between BF-FO and DN-FO.
Fig. 8. Temporal expression profiles of adipose Fasn (A), Acaca (B), Scd1 (C) and Acly (D) mRNA in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Gray shading 21
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indicates dark period. Maximal value of CTRL mice in each gene is expressed as 100%.
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Values are means ± SEM, n = 6-7 per time point and per group. Significant main effects
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or interactions are shown. Significant differences at corresponding ZT between CTRL and BF-FO, *P < 0.01.
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Fig. 9. Temporal fluctuations of plasma palmitic acid (A), stearic acid (B), oleic acid (C), linoleic acid (D), arachidonic acid (E), EPA (F) and DHA (G) concentrations in
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mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Gray shading indicates dark period. Values are means ± SEM, n = 6-7 per time point and per group. Significant main effects
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or interactions are shown. *P < 0.05 and
**
P < 0.01, significant differences at
†
††
P < 0.01, significant
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corresponding ZT between CTRL and BF-FO. P < 0.05 and
differences at corresponding ZT between CTRL and DN-FO. §P < 0.01, significant
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differences at corresponding ZT between BF-FO and DN-FO.
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Fig. 10. Total daily fecal excretion of palmitic acid (A), stearic (B), oleic (C), linoleic (D), arachidonic (E) acids, EPA (F) and DHA (G) in mice supplemented with fish oil in time-restricted fashion. Administration of fish oil was restricted by replacing high-fructose diet with same diet containing 4% fish oil for 12 hours each day for two weeks (See Fig. 2). Values are means ± SEM, n = 5-6 per group. Significant main †
effects are shown. *P < 0.05, significant differences between CTRL and BF-FO. P < 0.01, significant differences between CTRL and DN-FO. §P < 0.05, significant differences between BF-FO and DN-FO.
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References
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[1] Cullen P. Evidence that triglycerides are an independent coronary heart disease risk factor. Am J Cardiol. 2000;86:943-9.
SC
[2] Saravanan P, Davidson NC, Schmidt EB, Calder PC. Cardiovascular effects of marine omega-3 fatty acids. Lancet. 2010;376:540-50.
MA NU
[3] Shearer GC, Savinova OV, Harris WS. Fish oil -- how does it reduce plasma triglycerides? Biochim Biophys Acta. 2012;1821:843-51. [4] Backes J, Anzalone D, Hilleman D, Catini J. The clinical relevance of omega-3 fatty
ED
acids in the management of hypertriglyceridemia. Lipids Health Dis. 2016;15:118. [5] Bray GA, Nielsen SJ, Popkin BM. Consumption of high-fructose corn syrup in
PT
beverages may play a role in the epidemic of obesity. Am J Clin Nutr. 2004;79:537-43. [6] Miller A, Adeli K. Dietary fructose and the metabolic syndrome. Curr Opin
CE
Gastroenterol. 2008;24:204-9.
AC
[7] Samuel VT. Fructose induced lipogenesis: from sugar to fat to insulin resistance. Trends Endocrinol Metab. 2011;22:60-5. [8] Hudgins LC, Parker TS, Levine DM, Hellerstein MK. A dual sugar challenge test for lipogenic sensitivity to dietary fructose. J Clin Endocrinol Metab. 2011;96:861-8. [9] Nagai Y, Nishio Y, Nakamura T, Maegawa H, Kikkawa R, Kashiwagi A. Amelioration of high fructose-induced metabolic derangements by activation of PPARalpha. Am J Physiol Endocrinol Metab. 2002;282:E1180-90. [10] Bremer AA, Stanhope KL, Graham JL, Cummings BP, Ampah SB, Saville BR, et al. Fish oil supplementation ameliorates fructose-induced hypertriglyceridemia and insulin resistance in adult male rhesus macaques. J Nutr. 2014;144:5-11. 23
ACCEPTED MANUSCRIPT
[11] Erion DM, Popov V, Hsiao JJ, Vatner D, Mitchell K, Yonemitsu S, et al. The role of
T
the carbohydrate response element-binding protein in male fructose-fed rats.
RI P
Endocrinology. 2013;154:36-44.
[12] Karsenty J, Landrier JF, Rousseau-Ralliard D, Robbez-Masson V, Margotat A,
SC
Deprez P, et al. Beneficial effects of omega-3 fatty acids on the consequences of a fructose diet are not mediated by PPAR delta or PGC1 alpha. Eur J Nutr.
MA NU
2013;52:1865-74.
[13] Weaver DR. Introduction to Circadian Rhythms and Mechanisms of Circadian Oscillations. In: Gumz LM, editor. Circadian Clocks: Role in Health and Disease. New
ED
York, NY: Springer New York; 2016. p. 1-55.
[14] Froy O. Metabolism and circadian rhythms--implications for obesity. Endocr Rev.
PT
2010;31:1-24.
[15] Mazzoccoli G, Pazienza V, Vinciguerra M. Clock genes and clock-controlled genes
CE
in the regulation of metabolic rhythms. Chronobiol Int. 2012;29:227-51.
AC
[16] Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, et al. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell. 2002;109:307-20.
[17] Ueda HR, Chen W, Adachi A, Wakamatsu H, Hayashi S, Takasugi T, et al. A transcription factor response element for gene expression during circadian night. Nature. 2002;418:534-9. [18] Oishi K, Miyazaki K, Kadota K, Kikuno R, Nagase T, Atsumi G, et al. Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes. J Biol Chem. 2003;278:41519-27. [19] Peek CB, Affinati AH, Ramsey KM, Kuo HY, Yu W, Sena LA, et al. Circadian 24
ACCEPTED MANUSCRIPT
clock NAD+ cycle drives mitochondrial oxidative metabolism in mice. Science.
T
2013;342:1243417.
RI P
[20] Bray MS, Young ME. Regulation of fatty acid metabolism by cell autonomous circadian clocks: time to fatten up on information? J Biol Chem. 2011;286:11883-9.
SC
[21] Scheving LA. Biological clocks and the digestive system. Gastroenterology. 2000;119:536-49.
MA NU
[22] Bron R, Furness JB. Rhythm of digestion: keeping time in the gastrointestinal tract. Clin Exp Pharmacol Physiol. 2009;36:1041-8.
[23] Oishi K, Yamamoto S, Itoh N, Nakao R, Yasumoto Y, Tanaka K, et al. Wheat
ED
alkylresorcinols suppress high-fat, high-sucrose diet-induced obesity and glucose intolerance by increasing insulin sensitivity and cholesterol excretion in male mice. J
PT
Nutr. 2015;145:199-206.
[24] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J
CE
Biochem Physiol. 1959;37:911-7.
AC
[25] Christensen MS, Hoy CE, Becker CC, Redgrave TG. Intestinal absorption and lymphatic transport of eicosapentaenoic (EPA), docosahexaenoic (DHA), and decanoic acids: dependence on intramolecular triacylglycerol structure. Am J Clin Nutr. 1995;61:56-61. [26] Ikeda I, Sasaki E, Yasunami H, Nomiyama S, Nakayama M, Sugano M, et al. Digestion and lymphatic transport of eicosapentaenoic and docosahexaenoic acids given in the form of triacylglycerol, free acid and ethyl ester in rats. Biochim Biophys Acta. 1995;1259:297-304. [27] Bottino NR, Vandenburg GA, Reiser R. Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis. Lipids. 25
ACCEPTED MANUSCRIPT
1967;2:489-93.
T
[28] Yang LY, Kuksis A, Myher JJ. Lipolysis of menhaden oil triacylglycerols and the
RI P
corresponding fatty acid alkyl esters by pancreatic lipase in vitro: a reexamination. J Lipid Res. 1990;31:137-47.
SC
[29] Gnocchi D, Pedrelli M, Hurt-Camejo E, Parini P. Lipids around the Clock: Focus on Circadian Rhythms and Lipid Metabolism. Biology (Basel). 2015;4:104-32.
Regulation
of
bile
acid
MA NU
[30] Duez H, van der Veen JN, Duhem C, Pourcet B, Touvier T, Fontaine C, et al. synthesis
by
the
nuclear
receptor
Rev-erbalpha.
Gastroenterology. 2008;135:689-98.
ED
[31] Noshiro M, Usui E, Kawamoto T, Kubo H, Fujimoto K, Furukawa M, et al. Multiple mechanisms regulate circadian expression of the gene for cholesterol
PT
7alpha-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis. J Biol Rhythms. 2007;22:299-311.
CE
[32] Cox LM, Blaser MJ. Pathways in microbe-induced obesity. Cell Metab.
AC
2013;17:883-94.
[33] Kovatcheva-Datchary P, Arora T. Nutrition, the gut microbiome and the metabolic syndrome. Best Pract Res Clin Gastroenterol. 2013;27:59-72. [34] Kishino S, Takeuchi M, Park SB, Hirata A, Kitamura N, Kunisawa J, et al. Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition. Proc Natl Acad Sci U S A. 2013;110:17808-13. [35] Caesar R, Tremaroli V, Kovatcheva-Datchary P, Cani Patrice D, Bäckhed F. Crosstalk between gut microbiota and dietary lipids aggravates WAT inflammation through TLR signaling. Cell Metab. 2015;22:658-68. [36] Thaiss CA, Zeevi D, Levy M, Zilberman-Schapira G, Suez J, Tengeler AC, et al. 26
ACCEPTED MANUSCRIPT
Transkingdom control of microbiota diurnal oscillations promotes metabolic
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homeostasis. Cell. 2014;159:514-29.
RI P
[37] Voigt RM, Forsyth CB, Green SJ, Mutlu E, Engen P, Vitaterna MH, et al. Circadian disorganization alters intestinal microbiota. PLoS One. 2014;9:e97500.
SC
[38] Asher G, Sassone-Corsi P. Time for food: the intimate interplay between nutrition, metabolism, and the circadian clock. Cell. 2015;161:84-92.
MA NU
[39] Liu S, Brown JD, Stanya KJ, Homan E, Leidl M, Inouye K, et al. A diurnal serum lipid integrates hepatic lipogenesis and peripheral fatty acid use. Nature. 2013;502:550-4.
ED
[40] Shiba S, Tsunoda N, Wakutsu M, Muraki E, Sonoda M, Tam PS, et al. Regulation of lipid metabolism by palmitoleate and eicosapentaenoic acid (EPA) in mice fed a
AC
CE
PT
high-fat diet. Biosci Biotechnol Biochem. 2011;75:2401-3.
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