Role of Circadian Clock on the Pathogenesis and Lifestyle Management in Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a rising epidemic affecting around 32.4% of the global population [1]. The mechanism underlying the development and progression of NAFLD, as well as the prediction of hepatic and extrahepatic risk, is a complex area and is not yet fully understood [2]. Several risk factors, such as ethnicity, gender, age, body composition, weight, and the presence of metabolic syndrome can increase the risk of this liver disease [3]. In fact, the progression of chronic liver diseases, including NAFLD, occurs differently in men and women [4]. However, it seems that multiple etiopathogenic factors, such as obesity, genetics, environmental factors, insulin resistance, and changes in the gut microbiota, act in parallel or sequentially, causing NAFLD [5]. In this sense, following pioneer attempts, and in order to overcome the negatives attributed to NAFLD, it has been proposed to change the term NAFLD to MAFLD (metabolic dysfunction associated fatty liver disease) [6].

The epidemiological evidence in this field has suggested a close relationship between unhealthy lifestyle and NAFLD, making lifestyle correction the first-line approach in these patients [7]. In this regard, a link has been reported between the pathogenesis of chronic liver disease and some behaviors and activities, such as shift work, inadequate sleep, energy-dense fatty foods, or the exposure to artificial light [8]. The phenomenon of absence of synchrony between sleep and the circadian rhythm is called circadian misalignment and it has been associated with an increased incidence of metabolic and cardiovascular disorders [9]. Interestingly, sex differences in sleep appear in infancy and persist into childhood [4]. These underlying differences may be exacerbated at puberty and lead to objective sleep disturbances, which contribute to the higher prevalence of insomnia symptoms in young adolescents [10].

Circadian rhythms are controlled by the clock’s proteins and regulate the body’s functions [11], determining, for example, the most appropriate eating time. Concretely, the circadian clock contributes to the pathogenesis of NAFLD and non-alcoholic steatohepatitis because it enhances lipid dysregulation, oxidative stress, and inflammation [12,13]. Animal models have shown that the composition and timing of a diet influences the circadian clock and hepatic fat accumulation [14], leading to the awareness that for metabolic health it is not only important ‘what we eat’, but also ‘when we eat’ [15]. Since many genes controlled by the circadian clock are essential in the body’s metabolic processes, behavioral approaches, such as restricting the time of food intake, may have benefits in NAFLD independent of the weight loss effects [16]. Therefore, recent data recommend a lifestyle intervention to improve sleep duration and quality, along with diet and physical activity, for better metabolic benefits [17].

The purpose of this narrative review focuses on (i) the influence of circadian rhythms on the pathogenesis of NAFLD and (ii) on the role of sleep habits in the development of this liver disease. Finally, emerging evidence on chrononutrition and the use of fasting strategies to ‘synchronize’ lifestyle habits with the circadian clock in the management of patients with NAFLD will be reviewed.

The rotation of the earth on its axis and around the sun determines regular changes in the environment with regards to light–dark cycles [18]. Many organisms have developed an endogenous “time-keeping” system in order to synchronize with external time signals (zeitgebers) [19]. The periodicity of this clock is approximately 24 h and is, hence, also called a circadian rhythm [20]. In mammals, the circadian pacemaker resides in the suprachiasmatic nuclei (SCN), and is controlled by specialized retinal ganglion cells that measure ambient light [8]. Clock proteins are the endogenous timing mechanisms that control these circadian rhythms and help to maintain circadian homeostasis [18]. Importantly, circadian clock proteins are also active in other organs (peripheral clocks), such as the lung or liver [21]. At the heart of the molecular complex are the core transcription factors Circadian Locomotor Output Cycles Kaput (CLOCK) and Brain and Muscle Arnt-like protein-1 (BMAL1) [22]. CLOCK and BMAL1 drive the transcription of their own repressors, period (PER) and cryptochrome (CRY), resulting in a strongly self-regulated feedback loop [23] (Figure 1). During the daylight hours, increased transcription of PER and CRY genes results in the accumulation of the circadian repressors PER and CRY [24]. This process results in CLOCK:BMAL1-driven inhibition of PER, CRY, and clock-controlled genes. The highly controlled degradation of PER and CRY relieves transcriptional repression and allows CLOCK:BMAL1-mediated transcription to proceed again, establishing cycles in circadian gene expression [25].

The central clock in the SCN synchronizes with the peripheral clock to regulate liver metabolism [26]. The SCN is not sensitive to feeding patterns, whereas the liver peripheral clock depends on the feeding pattern for the amplitude and phase oscillation of its transcripts [27]. Concretely, hepatic metabolism processes, including glucose, lipid, and cholesterol/bile acid metabolism are highly dynamic, influenced by feeding/fasting and circadian rhythms [28]. For example, the release of the GLP-1, a peptide hormone produced in the intestinal epithelium which stimulates insulin and inhibits glucagon secretion, is circadian in both rodents and humans and this rhythmicity is lost with the consumption of a high fat/palmitate diet and with sleep deprivation [29,30]. Lastly, the production of hormones, such as melatonin or cortisol, also depends on the SCN rhythmic activity in response to light/darkness, whereas others hormones, such as FGF21, adiponectin, leptin, or insulin oscillate, following a circadian basis but are also regulated by feeding/fasting cycles [31]. In this regard, it has been demonstrated in a study of 110 individuals that the food intake timing relative to melatonin onset was significantly associated with the percentage of body fat (p = 0.02) and body mass index (p = 0.04), while controlling for sex [32].

Thus, in the absence of a robust hepatic circadian clock the body becomes more susceptible to metabolic disturbances, including insulin resistance or increased adiposity, which contribute to the pathogenesis of fatty liver diseases, diabetes, and obesity, as well as fibrosis and hepatocellular carcinoma [8,33]. In an animal model of male Wistar rats kept on a 12 h light/dark cycle, it was shown that forced activity during the sleep phase decoupled daily activity from the biological clock [34]. On the other hand, human studies have shown that dyssynchrony between feeding/breakfasting time and the circadian clock can lead to obesity and dysmetabolism [35]. Furthermore, it has recently been suggested that circadian rhythm alterations may have metabolic consequences in subjects carrying specific single nucleotide polymorphisms in other genes related to the circadian clock, such as PNPLA3, PPARY, STAT3, and PPARGCα, which, in turn, are related to NAFLD development and progression [18]. The circadian clock therefore plays an integral role in the coordination of metabolism, which has led to a closer examination of its contribution to a range of diseases, including NAFLD.

Additionally, it has also been described that disruption of circadian clock function can affect the gut microbiome [36,37]. Circadian rhythms have been shown to modulate the composition and function of commensal bacteria, as well as host susceptibility to bacterial and viral infections [38]. In this regard, a study of 14 male and female mice showed that disruption of the circadian clock by deletion of BMAL1 abolished the rhythmicity of fecal microbiota composition in both sexes [39]. In the same study, BMAL1 deletion also induced alterations in fecal bacterial abundance with different effects depending on sex. Moreover, some bacteria, such as cyanobacteria (Synechococcus elongatus), have their own circadian rhythms, providing a survival advantage to them [40]. Manipulating the circadian rhythms of gut microbial abundance and activity may therefore be a promising future approach.

Increasing evidence suggests that our circadian clock interacts with metabolic functions and that meal timing is an important factor in metabolic regulation [32,97]. Chrononutrition is a new discipline that investigates the relationship between circadian rhythms, nutrition, and metabolism, and has developed rapidly in recent years [98]. It has clearly been demonstrated that when we eat is as important as what and how much we eat for the progression of chronic diseases [99]. The eating behavior of our modern society is often characterized by prolonged and irregular daily eating patterns which, together with the Western-style diet, sedentary lifestyle, and chronic sleep deprivation, may contribute to an increased risk of metabolic diseases, including NAFLD [100,101]. Concretely, late-night eating (defined as eating dinner within 2 h of bedtime) is associated with increased risk of metabolic diseases [102].

The chronotype (with chronos meaning time in Greek) is defined as an individual’s circadian preference for activity being determined by the expression of at least a dozen core circadian genes [103]. Individuals’ chronotype can be categorized as “early” or “morning”, when people go to bed and wake up early, whereas “late” or “evening” will define people who go to bed and wake up late [104,105]. A recent study has shown that individuals with a late chronotype presented a significantly higher visceral adiposity index, liver fat equation, hepatic steatosis, and non-alcoholic steatohepatitis indexes [106], and a lower adherence to a healthy diet, calculated with the Chrononutrition Profile Questionnaire [107]. Moreover, people with late chronotypes reported 0.25 less servings of fruit and 0.13 less servings of vegetables per day compared to people with early chronotypes [108]. Similarly, in 194 patients with type-2 diabetes mellitus who do not work shift work, a late chronotype was associated with skipping breakfast (p = 0.014) [109]. In fact, it has been demonstrated that frequent breakfast skipping was independently associated with higher cardiovascular mortality risk (p < 0.001) in 3004 participants with metabolic associated fatty liver disease (MAFLD), but not in MAFLD-free individuals (p = 0.280) [110].

Current management of NAFLD includes diet and lifestyle changes for achieving weight loss [111]. However, the difficulty of adhering to diets, as well as the current “obesogenic” environment, characterized by physical, social, and cultural factors that encourage sedentary lifestyles and obesity, lead to the alteration of the biological clock [112]. In this regard, some studies have suggested alternative approaches to reducing the risks associated with metabolic diseases, such as intermittent fasting (IF) or time-restricted feeding (TRF) [113,114], which have been recently reviewed in detail in NAFLD [115]. In fact, it has been demonstrated that these nutritional strategies are useful for realigning lifestyle behaviors with circadian biological rhythms [116] (Figure 2).

Firstly, it should be noted that TRF is not synonymous with IF. Intermittent fasting usually refers to a period of water-only or very low-calorie fasting lasting of less than 24 h, followed by a period of normal eating for one or two days to induce changes in metabolism [15]. However, it may also include fasting on alternate days, eating little or no food for two consecutive days (5:2) or periodic fasting (2–21 days of minimal calories), followed by normal eating on non-restricted days [15,113]. Caloric intake in this type of diet is strictly regulated on fasting days. In this regard, an umbrella review of 11 meta-analyses of 130 randomized clinical trials has described beneficial associations of IF with anthropometric and cardiometabolic outcomes, supporting the use of this approach in overweight or obese adults [114]. Importantly, both IF and calorie-restricted diets have been shown to be equally effective in managing weight loss and improving cardiometabolic outcomes [117]. However, some benefits of IF, such as promoting ketogenesis, being linked to circadian biology and showing pronounced weight loss outcomes in individuals with elevated body mass index, have made it emerge as a promising strategy in patients with cardiovascular diseases [118]. In addition, a meta-analysis to estimate the effects of IF in adults with NAFLD confirmed an improvement in ALT (p ≤ 0.00) and AST (p ≤ 0.00) compared to a non-fasting group [101]. These results appear to be related to a decrease in body weight and a reduction in body mass index. In fact, previous meta-analyses have shown that subjects following IF had greater reductions in both body weight and body mass index compared to the control group [119,120].

In contrast, TRF is a dietary approach defined as feeding within a period of ≤10 h and fasting for at least 14 h a day, where feeding is consolidated to the active period, aligning peripheral and central circadian rhythms [121]. This strategy could be divided into early (8 a.m. to 4 p.m.) and late (1 p.m. to 9 p.m.) [122]. When applied to human populations, TRF does not restrict the number of calories consumed or require a dietary profile of macronutrients, making it easier for patients to adopt. Indeed, the use of this strategy has been reported to improve weight loss, cardiometabolic health, and overall wellbeing [123]. A study in mice showed that TRF is able to alleviate metabolic diseases with benefits proportional to the duration of fasting, due to its pleiotropic beneficial effects on metabolism, such as reducing insulin signaling and switching energy utilization from glucose to fat during fasting, or reducing biomarkers of inflammation [124]. In humans, a 12-week RCT demonstrated that fasting for 16 h (16/8 TRF), combined with a low-sugar diet, was associated with a significant reduction (p < 0. 05) in body fat and body weight, as well as circulating levels of fasting blood glucose and liver biomarkers (ALT: 34 ± 13.9 to 21.2 ± 5.4 U/L; AST: 26.3 ± 6.2 to 20.50 ± 4 U/L; γ-glutamyl transpeptidase: 33 ± 15 to 23.2 ± 11.1 U/L) compared to the control group [125]. In this study, a significant decrease (p < 0.05) was also observed in the fibrosis score (6.3 ± 1 to 5.2 ± 1.2 kPa) and in the controlled attenuation parameter (322.9 ± 34.9 to 270.9 ± 36.2 dB/m). However, to date only one study conducted on 15 men compared the effects of early TRF and late TRF, finding only a difference in mean fasting blood glucose, which was lower in early TRF (p = 0.02) [126].

In summary, both IF and TRF could be easily adoptable behavioral interventions to prevent/act on outcomes in patients with metabolic syndrome and NAFLD [127]. However, it is important to keep in mind some limitations of these nutritional strategies, such as the difficulty of maintaining the daily feeding window of 10–6 h while maintaining family, social, or work life [15]. Another major limitation is the lack of standardization of protocols for both diets and the scarce research in NAFLD patients, so no clear conclusions can be established at present.

Sleep disturbances have been implicated in the pathogenesis of NAFLD, influencing patients’ health and quality of life. The misalignment with the circadian rhythm thus adds to the metabolic burden of this complex disease. Understanding the molecular mechanisms involved in the control of the circadian clock is therefore essential not only to advance scientific knowledge, but also to improve public health by identifying new therapeutic targets and lifestyle modifications.

Indeed, public strategies aimed at optimizing shift work schedules and respecting biological clocks may be useful to reduce social jetlag. Along with these strategies, the message of the importance of taking care of the details of sleep should be reinforced, starting with reducing artificial lighting and ending with observing social schedules respecting the hormonal rhythm. Therefore, integrative analyses of precision variables should include age, sex, body phenotype, and lifestyle variables, together with personalized issues (genetics, epigenetics, microbiota composition, etc.), in order to contribute to the prescription of more personalized treatments. However, as research in this field is limited, future studies should be conducted to propose safe and effective feeding strategies to improve the management of these patients.

Conceptualization, N.P.-D.-d.-C., G.C. and E.B.; methodology, N.P.-D.-d.-C., G.C, C.R. and G.P.C.; investigation, N.P.-D.-d.-C. and G.C; data curation, N.P.-D.-d.-C. and G.C; writing—original draft preparation, N.P.-D.-d.-C. and G.C; writing—review and editing, G.P.C., C.R. and E.B.; visualization, N.P.-D.-d.-C., G.C., G.P.C., A.A., C.R. and E.B.; supervision, E.B.; project administration, E.B.; funding acquisition, E.B. All authors have read and agreed to the published version of the manuscript.

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All data are publicly available.

The authors declare no conflict of interest.

Italian Ministry for Education, University and Research (Ministero dell’Istruzione, dell’Università e della Ricerca—MIUR) under the program “Dipartimenti di Eccellenza 2018–2022”, project code D15D18000410001.