Effects Of Short-term Fasting On The Rhythmic Expression Of Clock-related Genes In Skeletal Muscle And Liver Of Carassius Auratus

Biological rhythm refered to a cyclical phenomenon of organisms’ behaviour and physiological function, which was the mechanism that organisms survived in long-term natural selection and evolution process to adapt to the environment. Biological rhythm was controled by the biological clock genes. The central clock was believed to be located in suprachiasmatic nucleus (SCN) in the hypothalamus, mainly affected by the light, however secondary clock was discovered in peripheral tissues, mainly affected by feeding. The expression of circadian clock genes were not only ubiquitous but also tissue-specific, and the change of expression promoted a change of clock gene’s expression, then leaded to some physiological functions anomaly, and the results caused some corresponding diseases. Restricted feeding invariably affected physiological activity and behavior for organisms, for example, when food were restricted in some animals, which could strengthen clock output signal and caused their physiological and bio-chemical changes.The core machinery of the circadian clock consisted of intracellular transcriptional-translational feedback loops regulated by clock genes and corresponding proteins. This regulatory network was composed of two main arms:the transcriptional activation (positive feedback) and the repressor (negative feedback) arms. The positive arm of the core clock was formed by two members:circadian locomotor output cycles kaput (Clock) and Aryl hydrocarbon receptor nuclear translocator-like (Bmal1). In the negative feedback loop, Per and Cry proteins blocked the action of Clock:Arntl1 and thereby inhibited their transcription. This autoregulatory mechanism resulted in a cyclic, self-sustained expression of clock genes with an approximately 24-h period. Some important functional genes had daily rhythmicity besides clock genes in peripheral tissue in teleost fish. It was important significance that circadian clock genes and functional genes had a jarless daily rhythmicity for teleost metabolism and homeostasis. Starvation stress was a more powerful zeitgeber among food restriction factors, and had a great influence on growth and development, physiological and biochemical indexes, gene expression in teleost fish.In the present study, the expression of circadian clock genes and functional genes were determined using RT-qPCR in fast myotomal muscle and liver of Carassius auratus fasted for a week, cosinor analysis was carried out on daily rhythmicity of clock-related genes using MATLAB 7.0 software, and correlation of mRNA levels between genes was analyzed by Spearman’s correlation test in SPSS 16.0. The goal of this study was to investigate effects of short-term fasting on the rhythmic expression of clock-related genes in skeletal muscle and liver of Carassius auratus. The study was beneficial to understand daily rhythmicity of circadian clock genes and functional genes in peripheral tissues in teleost fish, and grasped impact of short-term fasting on teleost fish. The research results were advantageous for further studying regulatory mechanism of the circadian clock system in teleost fish. Results were as follows:(1) Circadian clock gene(Clock, Bmalla, Cry1, Cyr2, Cry3, Per1, Per2 and Per3) and four functional genes (IGF1, IGF2,IGFBP2 and EPOR) presented a circadian rhythm in muscle tissue significantly (p<0.30); Significant positive correlation was found between Clock, a positive regulatory element, and many genes; Negative regulatory elements (Cry1, Cry2, Per1, Per2 and Per3) presented a otical biasing feature; Interestingly, Cry3 presented a dark bias and had negative correlation with most genes significantly (r<-0.50). Circadian rhythm of IGFs alwals might depend on circadian rhythm of clock genes. These results further demonstrated clock system was more complex in teleosts because of the existence of interspecific difference and paralogous gene’s difference.(2) Circadian rhythm of circadian clock genes and functional genes was severely impacted in muscle tissue of Carassius auratus fasted for a week:(a) Circadian rhythm of Bmalla, Cry2, Per1, IGF1, IGF2 and EPOR was destroyed (p<0.30); (b) MyoG, MSTN, ucpl and EPO obtained significant circadian rhythmicity (p<0.30); (c) Starvation stress transform phase of Clock, Cry1, Cry3, Per2, Per3 and IGFBP2; (d) IGFBP2 presented a significant circadian rhythm before and after short-term fasting (p<0.30); (e) correlation (Clock to Cry1, Per2 and Per3;Cry3 to Pe2) was the same as the control groups (r> 0.50 or r<-0.50).(3) Clock, Bmalla, Cry2, Cry3 and Per3 presented a significant circadian rhythm in liver tissue of Carassius auratus (p<0.30). pnp5a, ppara, fas and LPL presented a significant circadian rhythm, and meanwhile presented a high level’s expression in the daytime and a low level’s expression at night (p<0.30). Correlation degree of Clock was closed to other clock genes compared with Bmalla in positive regulatory elements, but Bmalla was more familiar with functional genes. In addition, some negative control components (Cry2, Cry3 and Per3) had a more familiar with functional genes, which might be caused by the interaction between these regulatory elements and functional genes. Interestingly, Cry3 presented a significant negative correlation with genes related correlations all the time (r<-0.50).(4) Circadian rhythm of circadian clock genes and functional genes was severely impacted in liver tissue of Carassius auratus fasted for a week:(a) Clock, Bmalla, Cry3, Per3, Ampkla, Ampk1b, ppara, AANAT, ERK2, JNK1 and LPL presented a significant circadian rhythm before and after short-term fasting (p<0.30), and their amplitude, mesor and acrophase were moved for fasting; (b) Circadian rhythm of Cry2, pnp5a and fas was destroyed (p<0.30); (c) Cryl and pparg obtained significant circadian rhythmicity (p<0.30); (d) Significant positive correlations (Per3 to ppara; Bmalla to Ampk1b，AANAT、JNK1) were unaffected (r> 0.50); Correlations (Cry3 to Ampk1a, JNK1; Per3 to Ampk1a, Ampk1b) were transformed into significant positive correlations (r> 0.50), therefore, daily rhythmicity of functional genes might be administrated by other feedback loops in liver.In a conclusion, biological clock systems were more complex in teleosts because of the existence of interspecific difference and tissue specificity. What’s more, peripheral tissues had some independent biological clock systems. Daily rhythmicity, correlations, amplitude, mesor, acrophase of circadian clock genes and functional genes were severely impacted by starvation stress in peripheral tissues. Finally, A new equilibrium was established through altering the original clock system by short-term starvation stress.