Association of Expression Levels of Clock Genes and Autophagy-Related Genes under Abnormal Light/Dark Cycle Stimulation in NAFLD Mice

Background: Environmental disorders of the circadian rhythms can lead to metabolism-related diseases or exacerbate pathological conditions. Non-alcoholic fatty liver disease (NAFLD) has emerged with a growing occurrence. In the present study, we attempted to indicate whether circadian clock may inuence lipid deposition and the expression levels of autophagy-related genes in liver of mice. Methods: High-fat diet and abnormal light/dark cycles were employed to induce a mouse model of NAFLD with circadian rhythm sleep disorder. Herein, liver samples were obtained at ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20 time-point to detect the rhythmic expressions of circadian genes, autophagy-related genes, and Rev-erbα. Results: Abnormal exposure to light aggravated lipid deposition in liver of mice and exacerbated disorders related to 24-h expression levels of clock genes, autophagy-related genes, and Rev-erbα. Besides, Rev-erbα could transcriptionally control the expression levels of autophagy-related genes. Conclusions: The long-term high-fat diet combined with abnormal light/dark cycle stimulation aggravated the development of NAFLD and disturbed the expressions levels of autophagy-related genes. An abnormal circadian expression may lead to NAFLD aggression. Besides, the abnormal expression levels of clock genes may create an association between circadian rhythm sleep disorder and autophagy.

droplets are isolated in double membrane vesicles and transport to lysosomes for degradation [7]. It regulates intracellular lipid stores. Singh et al. demonstrated that autophagy could be repressed in cultured hepatocytes, promoting TG storage in lipid droplets [8]. Numerous scholars pointed out that defective hepatic autophagy is closely associated with NAFLD [8][9][10].
To date, NAFLD has affected within 30% of the global population, and it has emerged with a growing occurrence [11,12]. In order to explore the origin of rapid increase in the incidence of NAFLD, we concentrated on circadian clocks in the current study. Since the human life style has dramatically changed worldwide during the past decades, and modern life style is globally forcing more and more people to enter the asynchrony state, it is highly essential to nd out an association between the social time and the internal circadian clock. Notably, environmental disorders of the circadian rhythms can lead to metabolism-related diseases or exacerbate pathological conditions. Genome-wide gene expression studies demonstrated that the circadian clock plays a signi cant role in liver physiology [13,14]. The liver clock regulates hepatic metabolism, including fatty acid, lipid, glucose, etc. [15,16]. In addition, autophagy activity and autophagy-related genes showed daily rhythms in mice [17]. The ULK1, a critical positive regulator for autophagy initiation, is an autophagy-related gene. It maintains its rhythmicity both in zebra sh liver and mice liver [17,18], and it controls the lipid metabolism in adipocytes [19]. Besides, microtubule-associated protein light chain 3 (LC3) is a homologue of yeast Atg8p and is associated with the autophagosome membranes. Cytosolic LC3-I and membrane-bound LC3-II are two forms of LC3. LC3-II is associated with the number of autophagosomes, playing a signi cant circadian oscillatory role in liver and other tissues of mice, including heart, kidney, and skeletal muscle [17,20]. It is involved in autophagosome formation, and it is transcriptionally regulated Bmal1. In addition, Atg5 is an essential molecule for autophagosome formation, and its rhythmic expression levels can be found in wild-type ies [21]. Meanwhile, inhibition of the Atg5 in hepatocytes could increase TG level [8]. Lamp1, a lysosomalassociated membrane protein 1, is involves in intracellular lipid autophagic clearance [8]. Moreover, the rhythmic expression of lamp1 was found in the liver of mice and in human retinal pigment epithelial cells [22,23].
However, whether circadian rhythm disruption leads to disruption of autophagy and ultimately affects the occurrence and development of NAFLD. In the present study, we utilized abnormal light/dark cycle to induce mice circadian asynchrony. Additionally, we assessed the degree of lipid deposition in the liver of mice and detected 24-h expression levels of circadian clock genes and autophagy-related genes in the liver of mice. Besides, we preliminary evaluated how abnormal expressions of circadian clock genes could lead to changes in expressions of autophagy-related genes, and this may ultimately lead to the occurrence and development of NAFLD.

A mouse model of NAFLD
We adopted 72 8-week-old C57 BL/6J mice, which were provided by Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China). Since the high fat diet is more likely cause TG accumulation in liver [24], we used the Western-type diet (containing 0.15% cholesterol and 21% fat) for feeding mice for 6 weeks under light/dark cycle leading to circadian rhythm sleep disorder in mice [25]. All mice were randomly divided into three groups: (1) mice that were subjected to normal diet and regular light/dark cycle (C57 ND group); (2) mice that received high-fat diet under normal light/dark cycle (C57 WD group); (3) mice that received high-fat diet under abnormal light/dark cycle (C57 WD + DD/LD group).

Cultivation Of Primary Hepatocytes
Primary hepatocytes were isolated from C57 ND mice in our vitro experiments. The cells were collected according to the collagenase perfusion method [26]. Arterial blood of the mouse liver was blocked and replaced by 5 ml/min EDTA solution (including 5 mM glucose, 0.5 mM EDTA, 4.15 mM NaHCO3, 136 mM NaCl, 5.4 mM KCl, 0.65 mM NaH2PO4, 0.85 mM Na2HPO4, and 1 mM HEPES) for 6 min. Then, the liver was perfused with 1 mg/ml collagenase type IV. The liver was isolated and cut into pieces. Tissue fragments were ltered, centrifuged, and washed. The cells were collected and incubated in William's E medium, containing 10% fetal bovine serum (FBS). After 4 h, the old medium was replaced with a new William's E medium, containing 10% FBS to remove the dead cells. These cells at passage 0 were directly utilized herein.

Short Interfering RNA (SIRNA) Transfection
The Rev-erbα siRNA was synthesized at RiboBio Co., Ltd. (Guangzhou, China). Besides, 100 nM siRNA (Rev-erbα siRNA or Control siRNA) and 50 nM LipofectamineTM 3000 (Thermo Fisher Scienti c, Waltham, MA, USA) were incubated at room temperature for 5 min. The Rev-erbα siRNA or Control siRNA were mixed with LipofectamineTM 3000 for 15 min to promote the formation of Rev-erbα siRNA/Lipofectamine complex and Control siRNA/Lipofectamine complex. Then, these complexes were added into the serum-free William's E medium, incubated for 6 h. Replace with a fresh William's E medium for another 48 h. The interference was detected via quantitative reverse transcription polymerase chain reaction (RT-qPCR).

Histological Analysis
Histological examination was performed to assess fatty liver degeneration in mice. The liver samples were xed in 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Then, the samples were embedded into para n and processed routinely. Finally, the samples were stained with hematoxylin and eosin (H&E) as described previously [27].

RNA Extraction And Reverse Transcription
Total RNA was extracted from liver tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. RNA concentration was determined by UV spectrophotometry. RNA integrity was assessed using agarose gel electrophoresis. Then, 1 µg RNA was reversely transcribed into cDNA using ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan) according to manufacturer's protocol.

RT-QPCR
The cDNA was ampli ed by ABI QuantStudio5 (Applied Biosystems, Foster City, CA, USA). The qPCR reaction mixture included 2.5 µl cDNA, 1 µl of 100 nM upstream primers, 1 µl of 100 nM downstream primers, 10 µl SYBR Green Master mix (Bio-Rad Laboratories, Hercules, CA, USA), and 20 µl H2O. The relative expression levels of target genes were calculated using the 2ΔΔCt method and normalized to 18S. The qPCR primers are presented in Table 1.

Statistical Analysis
The data were presented as mean ± standard deviation (SD). Single cosinor method was employed to analyze circadian rhythm as previously described [28,29]. The following equation was formulated for cosine function: Y(t) = M + A*cos(x*t + µ). The daily rhythm characteristics included mesor (midline estimating statistic of rhythm corresponding to the mean level), amplitude (half of the peak-to-trough difference of the tted cosine function), and acrophase (the crest time of rhythm given in degrees (°C), where 360 °C is corresponding to a 24-h cycle) were estimated by the above-mentioned function. Differences between the values of each pair of parameters were compared by one-way analysis of variance (ANOVA), and P ≤ 0.05 was considered statistically signi cant.

Results
1. The abnormal lipid accumulation in hepatic cells after undergoing high-fat diet and abnormal light/dark cycle stimulation To assess whether high-fat diet-induced NAFLD in mice, hematoxylin and eosin (H&E) staining was performed to evaluate lipid accumulation in the liver cells. As displayed in Fig. 1, there was no abnormal lipid accumulation in control mice that received normal diet under regular light/dark cycle. However, for mice that received high-fat diet for 6 weeks, lipid accumulation was noted in several liver cells. According to entrainment of the circadian system under light/dark cycle [30], we herein conducted the experiments on the base of high-fat diet and abnormal light/dark cycle stimulation. Figure 1 illustrates under high-fat diet and abnormal light/dark cycle, more lipid droplets were accumulated in the liver cells. The abovementioned results suggested that high-fat diet and abnormal light/dark cycle could aggravate the fatty liver degeneration in mice.

The rhythmic expression levels of circadian genes in liver tissues
Circadian clock genes can modulate liver lipid metabolism. Therefore, the rhythmic expression levels of clock genes (Bmal1, Clock, Per2, and Cry2) were detected in C57 ND, C57 WD, and C57 WD + LD/DD groups at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 ( Table 2). As shown in Fig. 2, the circadian oscillation of Bmal1 in C57 WD + LD/DD group was attenuated. The expression level of Per2 was signi cantly increased in C57 WD + LD/DD group compared with that in C57 ND group. The mesors of Per2 were remarkably elevated in C57 WD + LD/DD group compared with those in control group. Moreover, the amplitudes of Per2 were notably increased in C57 WD + LD/DD group compared with those in C57 ND group. Furthermore, the peak periodic values of Clock, Per2, and Cry2 were altered in C57 WD + LD/DD group compared with those in C57 ND group.

Circadian rhythmic expression patterns of autophagy-related genes in liver tissues
Autophagy is a highly conserved intracellular degradation system, and recently was shown to display circadian rhythms in mice. In the present study, we detected the expression levels of autophagy-related genes (ULK1, LC3 , Atg5, and Lamp1) in liver tissues of mice ( Table 2). As shown in Fig. 3, ULK1 and LC3 in C57 WD + LD/DD mice were lost its circadian rhythms. The mesors of LC3 in C57WD group and Lamp1 in C57 WD + LD/DD group were decreased compared with those in C57 ND group. Additionally, the amplitudes of Atg5 in C57 WD group were signi cantly attenuated compared with those in C57 ND group.
Besides, the amplitudes of Lamp1 in both C57 WD and C57 WD + LD/DD groups were markedly reduced compared with those in control group. Moreover, the peak values of Atg5 and Lamp1 were dramatically decreased in C57 WD and C57 WD + LD/DD groups, respectively, compared with those in control group. Meanwhile, the peak periodic values of Atg5 and Lamp1 in C57 WD + LD/DD groups were different from those in control group.

Diurnal expression of Rev-erbα gene and its relationship with autophagy-related genes
The mechanism of the abnormal light/dark cycle stimulation causing changes in the circadian rhythm of autophagy-related genes have not been fully elucidated. The circadian clock gene Rev-erbα, known as nuclear receptor 1D1, plays a circadian oscillatory role in liver, heart, and skeletal muscle. Notably, Reverbα regulates the expression levels of several circadian target genes and plays regulatory roles in lipid metabolism [31]. Moreover, Rev-erbα de ciency resulted in deactivation of the Stk11-Ampk-Sirt1-Ppargc1-α signaling pathway, whereas autophagy was up-regulated, resulting in both impaired mitochondrial biogenesis and increased clearance [32]. Additionally, Huang et al. pointed out that the circadian clock can directly regulate the expression levels of autophagy-related genes through Rev-erbα in zebra sh liver [18]. In the current research, we attempted to indicate whether Rev-erbα could induce changes in rhythmic expression levels of autophagy-related genes in C57 WD + LD/DD group. We rst examined the expression level of Rev-erbα in C57ND, C57WD and C57 WD + LD/DD groups ( Table 2). As shown in Fig. 4, the mesors were signi cantly decreased in C57 WD + LD/DD group compared with those in C57ND group. The amplitudes of Rev-erbα were markedly inhibited in C57 WD + LD/DD group than those in C57 ND group. Moreover, the peak periodic values in C57 WD + LD/DD group were markedly altered compared with those in control group (Fig. 4A). The rhythmic expression level of Rev-erbα changed in C57WD + LD/DD group.
In order to explore the relationship between Rev-erbα and autophagy-related genes, the ChIP assay was performed to indicate whether Rev-erbα could transcriptionally regulate the expression levels of autophagy-related genes. However, Rev-erbα promoted the expression level of Bmal1 as high as ~ 9.3fold. Besides, Rev-erbα upregulated the expression levels of autophagy-related genes (ULK1 and Atg5) within ~ 3.45-and ~ 4.78-fold, respectively (Fig. 4B).
To further verify the effects of Rev-erbα on the expression levels of ULK1 and Atg5, siRNA was used to detect the expression levels of ULK1 and Atg5. In primary liver cells, when the expression level of Rev-erbα decreased to ~ 1.56-fold, the expression levels of ULK1 and Atg5 were reduced to ~ 0.96-and ~ 1.19-fold, respectively (Fig. 4C). The above-mentioned results indicated that the alteration of expression levels of autophagy-related genes induced by circadian rhythm sleep disorders may be accomplished through Reverbα.

Discussion
In the present study, we showed that long-term high-fat diet combined with abnormal light/dark cycle stimulation may lead to or aggaravate NAFLD and change circadian expression patterns of circadian clock genes and autophagy-related genes. The pathogenic mechanisms of NAFLD have not been well understood. However, circadian clock participates in lipid and glucose metabolism, and is associated with metabolic syndrome. The relationship between the pathogenesis of NAFLD and impairment of circadian has been previously reported by a number of scholars. For instance, shift workers with irregular sleep time are more susceptible to obesity and associated disorders such as NAFLD [33]. The circadian clock genes, such as Clock, Per2, and Rev-erbα regulate liver lipid metabolism, and circadian rhythm disorder leads to lipid accumulation in liver cells [34][35][36]. Moreover, a regular eating time and a proper sleep time can reduce the risk of metabolic syndrome and NAFLD [37]. In the present study, high-fat diet and abnormal light/dark cycle stimulation led to the accumulation of lipid and the abnormal expression levels of circadian clock genes in the liver of mice. However, the speci c relationship between circadian rhythm disorder and the pathogenesis of NAFLD has not yet been fully elucidated.
Circadian rhythms are closely related to metabolism, while how such daily rhythms organize lipid metabolism in liver cells has not been fully understood. Apart from canonical lipolysis, autophagy, a highly conserved biological degradation process, contributes to lipid drops degradation. Consistent with our study, autophagy-related genes displayed circadian rhythms in both mice and in zebra sh [18,38], and its periodic induction may provide a new association between daily rhythms and lipid metabolism.
Autophagy has shown robust circadian clocks in the liver of mice, and it is accompanied by cyclic induction of autophagy-related genes involved in various steps of autophagy [17]. In the zebra sh liver, circadian rhythms directly regulate autophagy-related genes via Rev-erbα [18]. The master regulators of autophagy, TFEB and TFE3, exhibited a circadian expression manner [39]. In the present research, Reverbα showed alteration of biological rhythm during 24 h that resulted from nutrient and light stimulation.
Furthermore, we demonstrated that Rev-erbα could transcriptionally regulate the expression levels of ULK1 and Atg5. Meanwhile the changes in mRNA levels of Rev-erbα, ULK1, and Atg5 were consistent with results of ChIP assay. Therefore, it can be concluded that Rev-erbα may create an association between biological rhythm and autophagy, however, further research needs to be conducted to con rm this nding.
Selective autophagy termed 'lipophagy' may create a novel association between circadian clock and development and progression of NAFLD. In recent years, the function of autophagy in NAFLD has attracted scholars' attention. Liver-speci c knockout of the autophagy gene Atg7 in mice has displayed a remarkable lipid accumulation, which could mimick the human NAFLD condition [8]. Treatment with activators of autophagy could lower TG level in the liver and blood [40]. Tanaka et al. demonstrated that rubicon, an autophagy-inhibiting protein, could repress autophagy and prompt lipid accumulation in hepatocyte in NAFLD [41]. The present research revealed that the greater the changes in rhythmic expression levels of autophagy-related genes, the more lipid accumulated in the liver cells.

Conclusions
In summary, the current study revealed that high-fat diet and light stimulation may result in lipid accumulation in liver cells of mice, and alter expression levels of circadian clock genes and autophagyrelated genes. Furthermore, Rev-erbα may create an association between circadian rhythms and autophagy. Availability of data and material Not applicable.

Con icts of interest
The authors have no competing interests to disclose. XT take responsibility for the integrity of the data analysis. The sponsors had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.