Effects of short-term fasting on the rhythmic expression of core circadian clock and functional genes in skeletal muscle of goldfish (Carassius auratus)

Abstract

Molecular oscillators exist in peripheral tissues like pacemaker cells. Food intake is a dominant zeitgeber for peripheral clocks in vertebrates. Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the effects of the rhythmic expression of clock components in skeletal muscle following short-term fasting in goldfish. Here, we characterized the molecular clock components and their daily transcription in COSINOR, and assessed the effect of 7-day fasting on the circadian patterns of the candidate genes expression in goldfish skeletal muscle. For the core clock genes, clock, bmal1a, cry1, cry2, cry3, per1, per2 and per3 showed circadian rhythmicity in fed goldfish, but not for bmal1a, cry2 and per1 in the fasted state. Of the 8 candidate functional genes analyzed, igf1, igf2 and igfbp2 showed circadian rhythmicity in the fed state, but circadian pattern was only observed for mRNA of myog, igfbp2 and mstn in fasted goldfish. Additionally, Spelman's correlation analysis showed the circadian expression of the myog and mstn presented positive and negative correlation with the transcription pattern of clock and per2 genes in fasted goldfish, respectively. Our results demonstrated that the peripheral clocks might be reset to respond rapidly to withholding of food in teleost skeletal muscle.

Introduction

Almost all organisms display diurnal rhythms in physiology, behavior and metabolism due to the existence of daily environmental cycles (Ferrell and Chiang, 2015; Hardin and Panda, 2013). In metazoans, the generation of circadian rhythm appears to be a consequence of specialised tissues known as “central clocks”, which help living organisms anticipate predictable changes in the environment (Velarde et al., 2009). Apart from the central clock, peripheral tissues like skeletal muscle, heart, liver, spleen, kidney and intestine possess local circadian clock, namely peripheral clocks (Liu et al., 2007) that can influence diverse internal biological processes including many aspects of gene expression, homeostasis and metabolism (Storch et al., 2002).

Central and peripheral circadian clocks synchronize diverse physiological processes to the approximate 24-hour light/dark cycles, and are sustained by the rhythmic expression of circadian clock genes through the interaction of negative and positive transcriptional-translational feedback loops (Shavlakadze et al., 2013). The molecular mechanism of the endogenous clocks is argued to be conserved along the phylogenetic scale, from unicellular organisms and insects to multicellular plants and mammals (Panda et al., 2002). In mammals, the positive feedback loop of the core clock is formed by two members of the pas-bhlh (Per-Arnt-Sim basic Helix-Loop-Helix) family of transcription factors clock (circadian locomotor output control kaput) and bmal1 (brain muscle arnt-like 1), which heterodimerize and drive transcription of additional core clock genes cryptochrome (cry1 and cry2) and period (per1, per2 and per3). The per and cry proteins, constituting the negative feedback loops of the core molecular clock by forming a multimeric complex, are translocated into the cell nucleus where they inhibit their own transcriptional activity by binding to the clock:bmal1 heterodimer and blocking its function (Amaral and Johnston, 2011; Velarde et al., 2009). The interlocked loop involving the rev-erba (NR1D1, transcriptional repressor of bmal1) and orphan nuclear receptors rorα genes (transcriptional activator of bmal1) act to reinforce the molecular clock (Takahashi et al., 2008). These genes are tightly linked to the feedback loops through their proteins that activate or repress bmal1 expression, which are part of the core molecular clock. This autoregulatory mechanism results in a self-sustained, cyclic expression of circadian clock genes with an approximately 24-h circadian periodicity (Vatine et al., 2011). On the other hand, several hundred noncircadian genes are rhythmically expressed in mouse skeletal muscle and liver, which are under regulation of the molecular clock and are responsible for the integration between many physiological pathways and the molecular clock (Amaral and Johnston, 2011; Miller et al., 2007). It has recently been shown in mice that myod, an important regulator of myogenesis, was directly regulated by bmal1 and clock in skeletal muscle (Andrews et al., 2010).

To date, our knowledge of the molecular mechanisms of the teleost circadian system is based mainly on model species such as Japanese medaka (Oryzias latipes), zebrafish (Danio rerio), spotted green pufferfish (Tetraodon nigrovirides), three-spined stickleback (Gasterosteus aculeatus) and tiger pufferfish (Takifugu rubripes) (Cuesta et al., 2014; Vatine et al., 2011). Importantly, the main differences with respect to the mammalian system may be the type of cry genes and the regulation of per2. For example, six cry genes have been found in zebrafish, which could be divided into two groups: similarity to mammalian cry genes and similarity to the Drosophila melanogaster cry genes (Kobayashi et al., 2000). The circadian oscillators may need a predominant signals to sustain their rhythmicity, such as temperature, light and food availability (Damiola et al., 2000; Mendoza, 2007). Surprisingly, changing of these signals may lead to disruption of circadian rhythms, which are associated with an increased risk of cardiovascular disease and metabolic syndrome in humans and animals (Karlsson et al., 2001; Staels, 2006). However, the central oscillator in the SCN is primarily entrained by cues from light. Peripheral oscillators are likely to have a high degree of independence from central circadian signals. The molecular clock in many peripheral tissues could be strongly affected by daily feeding signals, which have little effect on the phase of the central clock (Mendoza, 2007; Zhdanova and Reebs, 2006).

The feeding factors may influence the circadian clock network which consequently affects the metabolic outputs in central and peripheral tissues via transcriptional regulation (Cagampang and Bruce, 2012; Lazado et al., 2014). For instance, the dietary composition could induce a significant inverse effect on the circadian rhythms in sea bass fed on a commercial diet (Gutierrez et al., 1984). In addition, feeding schedule affects a daily rhythmicity of plasma insulin and muscle glycogen levels in fish (Perez et al., 1988). The feeding-fasting treatment on the daily rhythms can alter the rhythm of glucose and insulin (Paredes et al., 2014). Damiola suggested that restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the SCN (Damiola et al., 2000). Furthermore, 24 h of fasting dampened circadian rhythmicity for myog mRNA in skeletal muscle of adult mice (Shavlakadze et al., 2013). In goldfish, daily meals with different sizes and energy could affect rhythm activity and significantly shorten the time required for resynchronization (Sánchez-Vázquez et al., 2001).

As the most abundant tissue of fish body, skeletal muscle is the main edible part (Zhang et al., 2011). The post-embryonic muscle growth usually occurs during two growth processes, the hyperplasia and hypertrophy growth (Zimmerman and Lowery, 1999). The rates of these two processes are known to be regulated by several internal myogenic regulatory factors and external stimuli, e.g. diet treatment (Rescan, 2005; Watabe, 1999). Novel genes involved in muscle growth regulation have been identified using microarrays (Rescan et al., 2007; Salem et al., 2006). 23 nutritionally regulated genes were identified in the fast skeletal muscle of Atlantic salmon by using subtracted cDNA libraries (Bower and Johnston, 2010).

The clock system in skeletal muscle plays critical roles in skeletal muscle physiology ranging from structural maintenance to functional regulation (Chatterjee and Ma, 2016; Chatterjee et al., 2013; McCarthy et al., 2007; Woldt et al., 2013). The expression of specific genes driven by peripheral clocks are involved in muscle physiology (Andrews et al., 2010; McCarthy et al., 2007). For instance, myod, exhibiting a circadian rhythm in its mRNA and protein levels, is a direct target of the circadian transcriptional activators clock and bmal1 (Andrews et al., 2010). Currently, peripheral clocks receive scant attention in fish skeletal muscle, with the exception of some single study in a few fish.

Goldfish (Carassius auratus) is a well studied species, with a demonstrated capacity of entrainment by feeding cues and a robust circadian system in skeletal muscle (Velarde et al., 2009). Some peripheral tissues (e.g. skeletal muscle) are affected by food deprivation, so the study of peripheral oscillators has been of particular significance in fish (Blanco et al., 2016). Therefore, the objectives of this study were to investigate the effects of short-term fasting (7 d) on the rhythmic expression of the core circadian clock and functional genes in goldfish skeletal muscle.