The involvement of sympathetic nervous system in ...

Life Sciences 201 (2018) 54–62

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The involvement of sympathetic nervous system in essence of chickenfacilitated physiological adaption and circadian resetting

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Yinhua Nia, Lingyan Maa, Tao Wua, Ai Lin Limb, Wanjing Zhanga, Luna Yanga, ⁎ ⁎⁎ Yoshihiro Nakaob, , Zhengwei Fua, a b

College of Biotechnology and Bioengineering, Zhejiang University of Technology, China Scientific Research and Applications, Brands Suntory, Singapore

A R T I C LE I N FO

A B S T R A C T

Keywords: Sympathetic nervous system Superior cervical ganglionectomy Essence of chicken Circadian clock Physiological function

Aim: The impact of the sympathetic nervous system (SNS) on the regulation of circadian rhythm and physiological functions is still not clear. Previous studies have found that essence of chicken (EC) supplementation facilitated the physiological adaption and circadian resetting in rats subjected to jet lag. Herein, the effects of SNS on the circadian clock and the hypothesis that EC-induced acceleration of circadian resetting is dependent on the SNS are investigated. Main methods: Male Wistar rats with superior cervical ganglionectomy (SCGx) were used to investigate the role of the SNS in circadian rhythm and physiological functions. SCGx rats were further fed with or without ECcontaining diet for 2 weeks and subjected to artificial jet lag. Key findings: Loss of SNS did not affect the circadian rhythm both in the hypothalamic suprachiasmatic nuclei (SCN) and peripheral clocks, including the liver and heart. The serum lipid levels were increased significantly in SCGx rats, together with the up-regulation of lipogenic gene expression in the liver and slight effect on serum hormones. The quicker resetting process of the clock genes in peripheral tissues of EC-fed rats was abolished after SCGx. In contrast, the phase shift of serum melatonin and corticosterone were faster in EC-fed rats, compared to that of control rats. Significance: The SNS controls different aspects of physiological functions, and it has little effect on circadian system under normal light/dark condition. The effects EC on peripheral circadian synchrony and physiological functions were dependent on, at least partly, through the regulation of sympathetic nerve function.

1. Introduction The mammalian master clock, which located in the hypothalamic suprachiasmatic nuclei (SCN), resets and synchronizes the circadian rhythm of peripheral tissues by entraining signals, including the neuroendocrine axis and autonomic nervous system [1,2]. Melatonin is one of the key hormonal signals that controls and adjusts the circadian time of the master clocks [3]. Exogenous melatonin administration could result in the alteration and entrainment of the free-running circadian rhythm [4]. In addition, glucocorticoid (GC) is another hormonal signal that acts as internal synchronizer of the peripheral circadian oscillators [5]. The exogenous administration of glucocorticoid affects the circadian phase of peripheral clocks differently, depending on the time of administration and endogenous rhythm of glucocorticoid itself [5,6]. Chronic administration of glucocorticoid would cause the dysregulation of lipid metabolism and behavioral changes in addition to the alteration ⁎

of the peripheral clock gene expression [7]. It has been known that autonomic nervous system (ANS) activity is involved in regulation of sleep and wakefulness [8]. Studies have also found that parasympathetic nervous system (PNS) is responsive to the sleep–wake process [9,10]. However, the impact of ANS in the circadian regulation of the master or peripheral clock is still unknown. Recent study has indicated that the loss of vagus nerve by vagotomy did not affect the circadian phase and resetting process of clock genes in the heart of rats [11]. Similarly, the expression of clock genes was not affected after the denervation of the sympathetic nervous system (SNS) in the liver, although the normal circadian changes in blood glucose level were abolished [12,13]. Interestingly, complete denervation of the autonomic inputs to the liver did not impair the 24-h profile in plasma glucose or the daily rhythms in the gene expression of liver enzymes [14], implicating the importance of ANS balance to control daily variations of blood glucose concentration. In addition, acute

Correspondence to: Y. Nakao, Scientific Research and Applications, Brands Suntory, 3 Biopolis Drive, Synpase, 138623, Singapore. Correspondence to: Z. Fu, College of Biological Engineering, Zhejiang University of Technology, No.6 District, Zhaohui, Hangzhou, Zhejiang 310032, China. E-mail addresses: [email protected] (Y. Nakao), [email protected] (Z. Fu).

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https://doi.org/10.1016/j.lfs.2018.03.047 Received 30 January 2018; Received in revised form 19 March 2018; Accepted 24 March 2018 Available online 27 March 2018 0024-3205/ © 2018 Published by Elsevier Inc.


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2. Materials and methods

time point and each group). The experimental design was shown in Fig. S1. To determine whether the effects of EC on the circadian system and physiological changes during artificial jet lag were depending on SNS, total of 150 rats were divided into three groups as follows: (1) SCGx control group (n = 30), fed with standard rodent chow and kept in normal light/dark cycle, (2) SCGx-Normal resetting group (n = 60), fed with standard rodent chow and subjected to artificial jet lag, (3) SCGxEC resetting group (n = 60), fed with EC containing diet and subjected to jet lag. SCGx was conducted after 2 weeks of feeding, and jet lag was induced by extending the light period for 24 h, together with the reversal feeding schedule after 3 days of recovery of SCGx. Control rats were sacrificed before jet lag, and jet lagged rat were sacrificed at 4-h intervals of the daily cycle on the 3rd day (Time 48–72 h) and 5th day (Time 96–120 h) after reversal (n = 5). The detailed experimental design is shown in Fig. S2. All rats were sacrificed after deep anesthesia by intraperitoneal injection of sodium pentobarbital (45 mg/kg body weight). SCN, liver and heart were removed quickly, snap-frozen in liquid nitrogen and stored at −80 °C until use. Blood was also collected and then centrifuged at 6000 g for 5 min at 4 °C, and serum was stored at −80 °C. All experiments were performed according to institutional guidelines. The study was approved by the Research Committee of Zhejiang University of Technology.

2.1. Materials

2.4. SCN collection

Essence of chicken (EC, 70 mL/bottle,) was supplied by Brands Suntory, Singapore. The specific composition of EC is described as previously [20]. Normal commercial diet containing 7.5% w/w freezedried EC powder (about 6 g/bottle) was fed to each rat at first 2 h with a dosage of 2.5 g/rat/day, and the normal commercial diet was fed to the mice after they had completely consumed the EC containing diet every day.

Brain tissue was removed and carefully placed on dry ice to freeze and keep the sample intact, then keep at −80 °C until use. To collect SCN, the brain tissue was trimmed and glued to a stage, and 1 mm coronal section encompassing the SCN was prepared using a Motorized Advance Tissue Slicer (Vibroslice, Campden Instruments, London, UK). The section was then placed on a frozen glass slide, and both SCN were then punched out with a 0.4 mm diameter needle. SCN punches were frozen at −80 °C until total RNA extraction.

administration of adrenaline or noradrenaline or electrical stimulation of the sympathetic nerves could induce the expression of Per1 in the liver of mice [15]. However, the effect of the SNS on the master clock or other peripheral clocks still needs to be clarified. Essence of chicken (EC) supplementation has proved to be effective in alleviating physical and mental stress and promoting metabolism [16]. L-carnosine, one of the main active components in EC, was found to be able to reduce the neural activities of sympathetic nerves and facilitate the activities of parasympathetic nerves [17,18]. In our previous studies, we have found that EC supplementation facilitates the reentrainment of peripheral clock in rats subjected to experimental jet lag [19]. However, the exact mechanism and the involvement of SNS in the effects of EC on circadian synchrony are still unknown. In the present study, the impact of SNS on circadian clock and physiological functions was assessed by the removal of superior cervical ganglion (SCG). Next, the hypothesis that the acceleration of the circadian clock resetting by EC was partly through the sympathetic nerve activities was tested. Superior cervical ganglionectomy (SCGx) rats were fed with or without EC-containing diet for 2 weeks and then subjected to artificial jet lag by the reversal of both light/dark cycle and feeding schedule. The circadian resetting processes and physiological functions were compared.

2.2. Superior cervical ganglionectomy (SCGx) 2.5. Immunohistochemistry SCGx was conducted as previously described [21]. Briefly, the ventral neck region was shaved and disinfected after anesthesia. The salivary glands were exposed by a 2 cm vertical incision and retracted to expose the underlying muscles. After sectioning the omohyoid muscles and dissecting the common carotid artery, the SCG which behind the carotid bifurcations were gently pulled and then removed. Sham surgery was also conducted using the same technique without removing the SCG.

The removed SCG were fixed in 4% paraformaldehyde in PBS and Paraffin-embedded. Immunohistochemical detection of the enzyme tyrosine hydroxylase was performed by staining with anti-tyrosine hydroxylase antibody (Millipore, Billerica, MA), followed by secondary antibody (AlexaFluor 594, Jackson Immunoresearch Laboratories, Baltimore, PA, USA) using standard techniques. 2.6. Hormone and biochemical analyses

2.3. Animals and experimental design Serum levels of corticosterone (CORT), melatonin and insulin were determined by corticosterone ELISA kit (Enzo life Sciences, Lausen, Switzerland), melatonin ELISA kit (IBL, Hamburg, Germany) and insulin ELISA kit (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) according to the manufacturer's instruction. Serum levels of triglyceride (TG), total cholesterol (TC) and free fatty acid (FFA) were determined by commercial kits purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). The levels of serum highdensity lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were assessed by Hangzhou Chain Medical labs (Hangzhou, China).

Seven-week-old male Wistar rats (200–220 g) were purchased from the China National Laboratory Animal Resource Center (Shanghai, China). They were fed and kept in a temperature-controlled (22 ± 1 °C) and light-controlled (200 lx, 12/12-h light/dark cycle) animal facility. The onset of light was defined as Zeitgeber time 0 (ZT0), and the onset of darkness defined as ZT12. Water was available ad libitum, while standard rodent chow was restricted to only during the dark period from the beginning of each experiment. After 1 week of adaptation, rats were divided into different groups for further experiments. To investigate the role of the SNS in the regulation of circadian rhythm and physiological changes in vivo, a total of 90 rats were kept for 2 weeks and then divided into three groups as follows: (1) Control group (n = 30), (2) Superior cervical ganglionectomy group (SCGx, n = 30), (3) Sham operation group, (Sham, n = 30), which received the same surgical anatomy without removing the superior cervical ganglia. After 3 days of recovery, rats under normal feeding schedule were sacrificed at 4-h intervals of the daily cycle starting at ZT0 (n = 5 for each

2.7. RNA extraction and RT-qPCR Total RNA of SCN, liver and heart were extracted by using TRIzol reagent (Takara Biochemicals, Dalian, China) according to the manufacturer's instructions. cDNA was synthesized using the a reverse transcriptase kit (Toyobo, Osaka, Japan). Quantitative real-time PCR (qPCR) was performed on an Eppendorf MasterCycler ep RealPlex4 55


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rhythm, SCG was surgically removed and the expression profiles of clock genes in the SCN, liver and heart, including Bmal1, Cry1, Per1 and Rev-erbα, were examined. Blepharoptosis induced by SCGx was found in rats after surgery (Fig. S3A), and the sympathetic nature of the SCG was further confirmed by immunohistochemical staining of tyrosine hydroxylase (TH, Fig. S3B). The expression of Bmal1 and Rev-erbα in the SCN of control rats displayed rhythmic patterns with the peak time at ZT2 and ZT8, respectively (Fig. 1A, Table S2). Though the expression of Cry1 and Per1 vibrated during the day, cosine wave analysis showed that the rhythmicity of these two genes was lost in the SCN of control rats (Table S2). Both sham surgery and SCGx had little effect on the expression pattern of clock genes in the SCN, and no difference was found between sham and SCGx rats as well (Fig. 1A, Table S2). On the other hand, expression profiles of all four clock genes exhibited a robust daily rhythmicity both in the liver and heart of control rats (Fig. 1B, C, Table S3, S4). The phases of all clock genes in the liver changed slightly with peak time shifted by 1–3 h after sham operation, and SCGx had little effect on the phase of these genes comparted to sham operated group (Fig. 1B, Table S3). The rhythmicity of clock genes in the heart was not affected by either sham operation or SCGx, and no difference was found between sham and SCGx rats, similar to those in the SCN (Fig. 1C, Table S4).

(Wesseling-Berzdorf, Germany) using the SYBR Green Realtime PCR Master Mix (Toyobo) as described previously [20,22]. Primers used for real-time PCR are shown in Table S1. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as a housekeeping gene for normalization. 2.8. Analysis of locomotor activity To assess the impact of EC on circadian rhythm of locomotor activity in SCGx rats, nine rats (n = 3 per group) were housed in individual cages and their behavior activity was monitored using an online PC computer equipped with CompACTAMS (Activity Monitoring System, Muromachi, Tokyo, Japan) by collecting data every 5 min. 2.9. Statistical analyses All data are presented as means ± SEM. Differences in mean values were assessed using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test. P values < 0.05 were considered to indicate statistical significance. Significant differences in circadian phases of the genes and hormones between groups were determined by two-way ANOVA followed by the Dunnett's post hoc test. The acrophase time of each cycling gene or serum hormone was estimated from the acrophase of the most highly correlated cosine wave as described previously [20]. The cosine fit is considered to be statistically significant when F is greater than F0.05 (*F > F0.05).

3.2. Loss of the SNS on serum hormones and lipid levels To examine the effect of SCGx on physiological changes, serum levels of hormone and lipid were evaluated. Serum corticosterone levels in control rats displayed robust daily rhythmicity with the highest level at ZT11, the peak coinciding with the time of light/dark period transition (Fig. 2A, Table S5). Sham operation and SCGx did not affect the phase of corticosterone in serum. SCGx caused a significant reduction of corticosterone level at ZT12 compared to that of control group, and no

3. Results 3.1. The role of the SNS in the regulation of circadian rhythm To investigate the involvement of SNS in the regulation of circadian

Fig. 1. Circadian genes expression pattern in the SCN, liver and heart of rats after SCGx. (A) mRNA expression of circadian clock genes in the SCN. (B) mRNA expression of circadian clock genes in the liver. (C) mRNA expression of circadian clock genes in the heart. Data represent a percentage of the maximal gene expression level referring to each photoperiodic group. The white/black bar on the bottom represents the duration of the light/dark cycle. n = 5 for each time point and each group. 56


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Fig. 2. The impact of loss of the sympathetic nervous system on serum hormone and lipid levels. (A) Daily rhythm of serum corticosterone, melatonin and insulin levels. (B) Serum total cholesterol, triglyceride and free fatty acid levels after SCGx at indicated time points. n = 5. *P < 0.05, **P < 0.01 vs. Control group, # P < 0.05, ##P < 0.01vs. Sham group. (C) mRNA expression of lipogenic genes in the liver of rats. n = 5. *P < 0.05, **P < 0.01 vs. Control group, #P < 0.05, ## P < 0.01 vs. Sham group.

3.3. Effect of EC feeding on the resetting process of clock genes in the SCN of SCGx rats

difference was found between SCGx and sham group (Fig. 2A, Table S5). Similar results were found in serum melatonin levels which peaked at ZT22-ZT0, with SCGx showing a marked decrease of melatonin levels at ZT 12 and ZT16 compared to control group, and ZT16 compared to sham group (Fig. 2A, Table S5). The serum insulin levels fluctuated without rhythmicity in all three groups, with a significant increase at ZT20 in Sham and SCGx rats (Fig. 2A, Table S5). No differences were found either the phase or the level of insulin between sham and SCGx rats (Fig. 2A). Next, serum lipid levels were assessed at ZT8 and ZT20 as lipid levels are easily affected by exogenous stimulations at these two time points, according to our previous study [20]. The levels of serum TC and TG were increased significantly both at ZT8 and ZT20 in the SCGx rats compared with that of either control group or sham group, but FFA levels only increased at ZT8. Sham operation did not affect serum lipid levels at both time points (Fig. 2B). To further investigate the mechanism underlying the alteration of serum lipid levels, mRNA expressions of lipogenic genes in the liver were evaluated by qPCR. The expressions of sterol regulatory element binding protein-1c (Srebp-1c), the master transcriptional regulator of lipogenesis and its downstream regulatory targets, including fatty acid synthase (Fas) and acetyl-coAcarboxylase (Acc), were all up-regulated or tended to be increased in the liver of SCGx rats at ZT8 compared with that of either control group or sham group. As for ZT20, there were no significant difference in the mRNA expression levels between SCGx and sham group. (Fig. 2C).

We have previously found that the resetting process of experimental jet lag was facilitated by EC consumption [19]. To further determine whether the SNS is involved in the EC-induced rapid resetting of clock genes, SCGx rats were used and fed with an EC-containing diet. The expression pattern of clock genes in the SCN between the normal resetting and EC resetting groups is shown in Fig. 3. In the normal resetting group, there was a significant daily alteration in the expression of four examined clock genes on the third day after reversal of the light/ dark cycle (Fig. 3A). Cosine wave analyses revealed that the peak time of these genes were shifted by 0–4 h in both normal resetting and EC resetting group. No significant difference was found between normal resetting and EC resetting group (Table S6). As for the fifth day, the peak phase of Bmal1 was shifted by 10 h in the SCN of normal resetting rats, while only 6 h phase shift was found in EC resetting rats. In contrast, the phase shift of Rev-erbα was 7 h in normal resetting group and 12 h in EC resetting group. No difference was found in Cry1 and Per1 expression patterns between the two groups (Fig. 3B, Table S6). 3.4. Effect of EC feeding on the resetting process of clock genes in the liver of SCGx rats Since the resetting process was not accelerated in the master clock of SCGx rats by EC diet feeding, we next examined the role of EC on the 57


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Fig. 3. The effect of EC feeding on the resetting process of the clock genes in the SCN of SCGx rats. The jet lag was conducted by reversing the light/dark cycle and feed schedule. Circadian clock genes expression patterns in the SCN were assessed by qPCR at (A) the 3rd day (Time 48–72 h) and (B) 5th day (Time 96–120 h) after reversal (n = 5). The black/white bar on the top of column represents the duration of the dark/light cycle in SCGx control group. The white/black bar on the bottom of column represents the duration of the light/dark cycle after reversal in SCGx-Normal resetting and SCGx-EC resetting group.

Fig. 4. The effect of EC feeding on the resetting process of the clock genes in the liver of SCGx rats. The jet lag was conducted by reversing the light/dark cycle and feed schedule. Circadian clock genes expression patterns in the liver were assessed by qPCR at (A) the 3rd day (Time 48–72 h) and (B) 5th day (Time 96–120 h) after reversal (n = 5). The black/white bar on the top of column represents the duration of the dark/light cycle in SCGx control group. The white/black bar on the bottom of column represents the duration of the light/dark cycle after reversal in SCGx-Normal resetting and SCGx-EC resetting group.

showed in Fig. S4. The phase of Bmal1, Cry1 and Per1 was shifted 4 h, compared to that of control rats, which were shifted 8 h in the heart of EC fed rats on the third day after the reversal of light and feeding schedule (Fig. S4A). The resetting processes of Bmal1 and Cry1 were similar in both the normal resetting and EC resetting group on day 5, with the peak shifted either 12 h or 8 h. Additionally, the resetting process of Per1 was finished in the heart of EC fed rats, and only 8 h of peak shift was found in normal resetting group (Fig. S4B). These results confirmed the EC-induced acceleration of phase shift of clock gene expressions in the heart. Next, the resetting processes of clock genes in the heart of SCGx rats were also evaluated. The peak time of four clock genes were all shifted by 3–6 h on the third day of jet lag, and there was no indication of an effect of EC feeding on the heart clock compared with that of normal resetting group (Fig. 5A, Table S8). The resetting processes were completed on the fifth day for these clock genes, with phases shifted 11–12 h and the expression pattern of the four genes almost identical

resetting of peripheral clock. Interestingly, even though the robust rhythmicity was all preserved in all examined clock genes in the liver of both groups, the resetting processes of Bmal1, Cry1 and Rev-erbα in the liver were completed on the third day of jet lag and kept 9–12 h phase shift on the fifth day in both resetting groups (Fig. 4, Table S7). No significant difference was found between normal resetting and EC resetting group (Fig. 4, Table S7). On the other hand, the peak phases of Per1 in normal resetting group or EC resetting group were shifted by 8–9 h on day 3 and finished resetting process on day 5 after the reversal of light/dark cycle (Fig. 4).

3.5. Effect of EC feeding on the resetting process of clock genes in the heart of SCGx rats The acceleration effects circadian resetting by EC found in our previous study were focused on the pineal gland and liver, and the effect of EC on the resetting process of heart clock in normal rats was 58


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Fig. 5. The effect of EC feeding on the resetting process of the clock genes in the heart of SCGx rats. The jet lag was conducted by reversing the light/dark cycle and feed schedule. Circadian clock genes expression patterns in the heart were assessed by qPCR at (A) the 3rd day (Time 48–72 h) and (B) 5th day (Time 96–120 h) after reversal (n = 5). The black/white bar on the top of column represents the duration of the dark/light cycle in SCGx control group. The white/black bar on the bottom of column represents the duration of the light/dark cycle after reversal in SCGx-Normal resetting and SCGx-EC resetting group.

No significant differences of the resetting speed were found in three examined genes between resetting groups (Fig. 6, Table S9).

between the two resetting groups (Fig. 5B, Table S8).

3.6. Effect of EC feeding on the resetting process of lipogenic genes in the liver of SCGx rats

3.7. Effect of EC feeding on the resetting process of serum hormones of SCGx rats

Since loss of the SNS also influenced the serum lipid levels and hepatic ligogenic gene expressions, the resetting process of these genes were examined. The rhythmic expression phases of Srebp-1c and Fas were shifted significantly after jet lag and the resetting processes of both groups were almost completed on day 5 (Fig. 6, Table S9). Though no robust rhythmicity was found in the expression of Acc, the overall phase resetting also seem to be completed in the two groups on day 5.

To further investigate the potential hormone signals that may involve in the EC-induced rapid resetting of circadian clock, the rhythm of serum corticosterone, melatonin and insulin levels were determined. Serum glucocorticoid is a hormone signal act as an internal timing signal that synchronizes the peripheral clocks, and is rhythmically secreted after the reversal of light/dark cycle. The resetting process of

Fig. 6. The effect of EC feeding on the resetting process of the lipogenic genes in the liver of SCGx rats. The jet lag was conducted by reversing the light/dark cycle and feed schedule. Lipogenic genes expression patterns in the liver were assessed by qPCR at (A) the 3rd day (Time 48–72 h) and (B) 5th day (Time 96–120 h) after reversal (n = 5). The black/white bar on the top of column represents the duration of the dark/light cycle in SCGx control group. The white/black bar on the bottom of column represents the duration of the light/dark cycle after reversal in SCGx-Normal resetting and SCGx-EC resetting group. 59


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Fig. 7. The effect of EC feeding on the resetting process of the serum hormones of SCGx rats. The jet lag was conducted by reversing the light/dark cycle and feed schedule. Serum corticosterone, melatonin and insulin levels were assessed at (A) the 3rd day (Time 48–72 h) and (B) 5th day (Time 96–120 h) after reversal (n = 5). The black/white bar on the top of column represents the duration of the dark/light cycle in SCGx control group. The white/black bar on the bottom of column represents the duration of the light/dark cycle after reversal in SCGx-Normal resetting and SCGx-EC resetting group.

affect the circadian rhythm in the SCN and peripheral clocks, including the liver and heart. Consistently, studies have found that the clock genes were unaffected after the denervation of the SNS in the liver [12]. These results suggested that the SNS had little effect on circadian system under normal light/dark condition. SCGx affected the serum hormones slightly, but changed the serum lipid levels more severely, indicating that the SNS controls different aspects of physiological functions. Interestingly, the accelerating effect of the peripheral clock resetting process induced by EC was not preserved after SCGx. In contrast, the phase shift of serum melatonin and corticosterone were faster in EC fed rats, compared to that of control rats. Taken together, this suggests that the regulation of EC on circadian system under jet lag conditions might be executed partly through the SNS. The SCG is the uppermost ganglia of the paravertebral sympathetic chain, which contains a mixture of neuronal phenotypes that selectively innervate different target tissues located in the brain [23,24]. Removal of the SCG by SCGx is now representing a valuable microsurgical model to study the role of the SNS in different physiological and pathological processes, including homeostatic regulation, neuronal functions, and circadian biology [21]. Irreversible blepharoptosis is used as an indicator of the successful removal of the SCG, which could be easily identified after SCGx [21]. In addition, there are a number of neuron types in the SCG, including TH containing neurons [25]. Therefore, the sympathetic nature of SCG could also be corroborated via the identification of TH containing neurons. Our results revealed both blepharoptosis and immunohistochemical staining of TH neuron in removed tissues (Fig. S1), suggesting the surgery of SCGx was performed successfully. The sympathetic innervation to the pineal gland via the SCG determines the melatonin secretion, indicating the important role of SNS in the regulation of pineal function [26]. The loss of neural pathways that mediate the rhythmic pineal melatonin secretion would lead to decreased melatonin secretion [27]. Studies have also found that bilateral SCGx abolished the circadian rhythm of melatonin [28]. In accordance with that, the serum level of melatonin displayed significant rhythmicity in control rats, and it was decreased markedly after SCGx (Fig. 2A). Growing evidence has shown that the robust daily variation of GC is

corticosterone was completed in EC-fed rats on day 3, 2–3 h faster than that of normal resetting rats (Fig. 7, Table S10). Moreover, the concentration of corticosterone in EC resetting group around the peak time was much higher than that of control and normal resetting group on day 5 (Fig. 7B). On the other hand, the serum melatonin level was increased slightly in the EC resetting group in the dark phase, while it was higher in the light phase in the normal resetting group on the third day (Fig. 7A). The peak time of both resetting groups was shifted by about 4 h (Fig. 7A, Table S10). The overall levels of melatonin were also higher in both resetting group, while the phase shift was 2 h faster in EC resetting group than that of normal resetting group (Fig. 7B, Table S10). Though the serum insulin levels remained arrhythmic in both two groups, the peak time was shifted clearly after jet lag, and the resetting process tended to be faster in EC fed rats than that of normal resetting group on day 5 (Fig. 7, Table S10). 3.8. Effect of EC feeding on the resetting process of locomotor activities of SCGx rats Locomotor activity, as an output signal of the master clock, was also monitored during the jet lag. In the control condition, the nighttime activity was about 80% and daytime activity was 20% of total activity. After the reversal of light/dark cycle, this activity pattern took 3 days to be re-entrained in both resetting groups (Fig. S4). In addition, we have found this night/day activity ratio was stable even among different experiments. To further analyze the impact of EC on locomotor activity, we compared the recovery rates of nighttime activity in normal resetting or EC resetting groups in rats with or without SCGx (Fig. S6). It took about 5 days to recover to 80% of total activity in rats without SCGx, while it took only 3 days in SCGx rats. However, EC feeding did not affect the recovery rates significantly in control or SCGx rats. 4. Discussion This study first investigated the impact of SNS on the regulation of circadian clock and physiological changes, which was based on the hypothesis that EC-induced acceleration of circadian resetting may be partly carried out through the SNS. We found that loss of SNS did not 60


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regulation. On the other hand, the resetting processes of serum corticosterone and melatonin in EC fed SCGx rats were 2–3 h faster than those of SCGx-Normal resetting rats (Fig. 7), indicating that EC-induced accelerations of corticosterone and melatonin resynchronization, which contribute to the quick resetting of circadian clock, were preserved in SCGx rats. Thus, the sympathetic regulation is dominant and independent with hormonal regulation during the resetting process of circadian clock. Chronic circadian disruption would cause metabolic disorder of liver, leading to the development of metabolic diseases, such as obesity, dyslipidemia, fatty liver disease, and even liver cancer [39,40]. Studies have found that EC supplementation promoted the physiological adaptation to the prolonged circadian disruption by coupling both the circadian oscillators and metabolic regulators [20]. In the present study, the synchrony of lipogenic regulators were not different from the SCGx rats fed with or without EC (Fig. 6), suggesting that the amelioration of lipid metabolism disorder was effected at least partly, by the regulation of sympathetic nerve function. In summary, this study demonstrates that SNS plays an important role in the homeostasis of both the circadian clock system and physiological functions. Importantly, EC-induced physiological adaptation and circadian resynchronization were abolished after the removal of SCG, indicating the involvement of SNS in the effect of EC in ameliorating these physiological alterations. Further studies focusing on the detailed components of EC in the regulation of circadian clock and lipid metabolism and the impact of SNS during these processes would provide more mechanistic insights into the effect of EC on physiological functions. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lfs.2018.03.047.

mediated by multimodal forms of regulation, including the control of SCN via the neuroendocrine axis and autonomic nervous system, as well as intrinsic mechanisms governed by the resident adrenal cortical clock [29–31]. The central circadian system regulates GC release in a HPA axis-independent manner by modulating the adrenal sensitivity to ACTH through SCN-mediated activation of the autonomic nervous system [32]. Though the circadian phase of GC was slightly affected (1 h of phase shift) by the removal of SCG, the peak level of GC was decreased significantly (Fig. 2A), suggesting that the main impact of loss of SNS on GC was reducing the overall GC level in circulation. The secretion and sensitivity of insulin is also under the control of circadian clock. Evidence has shown that diurnal variation of insulin levels was found in vivo and in vitro [2,33,34]. On the other hand, insulin is a critical mediator of dietary-induced changes in sympathetic activity, and in contrast to the role PNS, stress-induced sympathetic neural activity to the pancreas inhibits insulin secretion. In accordance with previous studies, though the daily rhythmicity was not as robust as other hormones, the levels of insulin displayed a series of peaks and troughs (Fig. 2A). The SCGx did not affect the diurnal variation of insulin levels significantly, and the increased levels at ZT20 in Sham and SCGx rat was mostly likely a result of recovery from the surgery. Hepatic lipid metabolism is regulated by both the sympathetic and the parasympathetic nerve systems. Specifically, sympathetic stimulation regulates the secretion of TG and apolipoprotein B (ApoB) and the release of very low density lipoprotein (VLDL) [13]. Recent study has found that denervation of the hepatic sympathetic nerves caused an increase of VLDL secretion, resulting in elevated plasma level of cholesterol and TG [35]. Consistently, our results also found the increase of serum lipid levels in both time points, including TC, TG and FFA (Fig. 2B). Previous evidence for the role of SNS in regulating hepatic lipid metabolism was mainly focused on hepatic VLDL-TG secretion [36,37], few studies investigated the involvement of SNS in lipogenisis. Therefore, we further investigated the mRNA expressions of lipogenic genes in the liver, and found the significant up-regulation of the main lipogenic regulators in the liver of SCGx rats (Fig. 2C). These results suggested the increased serum lipid levels might be resulted from both the increase of lipogensis and VLDL-TG secretion. EC has been widely used as a functional supplement, and our previous results found that the resetting rate of locomotor activity was similar between normal resetting and EC resetting groups, suggesting that EC did not affect the behavioral circadian resetting. Therefore, the main facilitating effects of EC are on peripheral clocks, not SCN or the output signal of SCN. Consistent with our previous study, we also found that there was no significant difference in the resetting rate of locomotor activity between normal resetting and EC resetting group in SCGx rats in the current study (Fig. S5). On the other hand, the recover speed of nighttime activity to 80% of total activity in SCGx rats was faster than normal rats, suggesting that loss of the SNS might accelerate the resetting process of locomotor activity. Further studies are required to analyze the detailed effect and mechanism of SCGx on the SCN clock or output signals. In addition, although the rhythmic clock gene expression in the SCN were found, the peak phases of these genes were slightly different from the study by Pembroke et al. or others [38]. The possible reason for this inconsistence might be the techniques used, since the circadian clock gene profiles were measured by in situ hybridization or laser-capture microdissection (LCM) and RNA-seq in the other studies. These techniques are more advanced and therefore might cause the differences compared with our study. However, the detailed role of sympathetic nervous system in regulation of SCN clock is needed to be clarified in our further study. The mechanism of EC on peripheral circadian resetting process may involve different aspects, including the secretion and rhythm glucocorticoids and melatonin [19], as well as the involvement of vagus nerve [11]. In the present study, the accelerating effects of EC on circadian system were abolished after the removal of SCG (Fig. 3–5), suggesting the critical role of SNS in the EC-mediated circadian

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