Caffeine alters circadian rhythms and expression of disease and metabolic markers

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Abstract

The circadian clock regulates many aspects of physiology, energy metabolism, and sleep. Restricted feeding (RF), a regimen that restricts the duration of food availability entrains the circadian clock. Caffeine has been shown to affect both metabolism and sleep. However, its effect on clock gene and clock-controlled gene expression has not been studied. Here, we tested the effect of caffeine on circadian rhythms and the expression of disease and metabolic markers in the serum, liver, and jejunum of mice supplemented with caffeine under ad libitum (AL) feeding or RF for 16 weeks. Caffeine significantly affected circadian oscillation and the daily levels of disease and metabolic markers. Under AL, caffeine reduced the average daily mRNA levels of certain disease and inflammatory markers, such as liver alpha fetoprotein (Afp), C-reactive protein (Crp), jejunum alanine aminotransferase (Alt), growth arrest and DNA damage 45β (Gadd45β), Interleukin 1α (Il-1α), Il-1β mRNA and serum plasminogen activator inhibitor 1 (PAI-1). Under RF, caffeine reduced the average daily levels of Alt, Gadd45β, Il-1α and Il-1β mRNA in the jejunum, but not in the liver. In addition, caffeine supplementation led to decreased expression of catabolic factors under RF. In conclusion, caffeine affects circadian gene expression and metabolism possibly leading to beneficial effects mainly under AL feeding.

Introduction

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus is the central oscillator that controls the approximately 24-h cycle (circadian rhythms) of behavior and physiology in mammal (Reppert and Weaver, 2002). Circadian oscillators are also found in various peripheral tissues, such as the liver and digestive system (Lee et al., 2001, Froy and Chapnik, 2007, Pan and Hussain, 2009). The clockwork consists of a primary loop that is composed of the helix-loop-helix transcription factors CLOCK and BMAL1, which mediate transcription of the Period (Per1, Per2, Per3) and Cryptochrome (Cry1, Cry2) genes through E-box enhancer elements. In turn, PER and CRY proteins repress CLOCK/BMAL1-mediated gene trans-activation, thereby shutting down their own transcription (Reppert and Weaver, 2002). Light is the most potent synchronizer for the SCN, yet food consumption, timed meals, and some nutrients also entrain the circadian clock (Schibler et al., 2003, Froy, 2007).

Limiting the time and duration of food availability has been shown to affect the circadian clock and life span (Duffy et al., 1990a, Duffy et al., 1990b, Schibler et al., 2003, Froy, 2010). Interestingly, diurnal restricted feeding (RF) in nocturnal animals shifts many physiological activities in peripheral tissues normally dictated by the SCN. Recently, we found that long term RF led to robust circadian rhythms, altered metabolism, and to the lowering of the inflammatory state and disease proneness (Sherman et al., in press). Robust circadian rhythms have been previously associated with aging retardation and extended life span, as longevity was increased in older hamsters given fetal suprachiasmatic implants that restored higher amplitude rhythms (Hurd and Ralph, 1998, Hofman and Swaab, 2006). Conversely, disruption of circadian rhythms by shift work, sleep deprivation, or by mutations in clock genes can lead to manifestations of the metabolic syndrome, as well as certain types of cancer, coronary heart disease, depression, and overall reduced life expectancy (Davis and Mirick, 2006, Filipski et al., 2006, Froy, 2007).

In addition to RF, caffeine, the world's most popular psychoactive compound, has been shown to shift circadian rhythms. Caffeine administered systemically at the mid-sleep period induced arousal without shifts, and dose-dependently attenuated shifts to a 3-h sleep deprivation procedure in Syrian hamsters (Antle et al., 2001). It has been shown that when ingested at night caffeine suppressed melatonin levels in humans, causing reduction in sleep duration (Wright et al., 1997). Caffeine can also alter physiological rhythms as it delays the phase of circadian activity through ryanodine receptors in mouse brain slices containing the SCN (Ding et al., 1998). However, to the best of our knowledge, its effect on clock gene expression has not been tested.

The circadian clock regulates the expression and/or activity of enzymes and hormones involved in metabolism. In turn, key metabolic enzymes and transcription activators interact with and affect the core clock mechanism (La Fleur et al., 1999, Pan and Hussain, 2007, Pan and Hussain, 2009, Green et al., 2008, Froy, 2010). In addition, caffeine has been shown to affect metabolism (Magkos and Kavouras, 2005). As resetting the biological clock by timed meals or nutrients is of general interest, we set out to test the effect of caffeine on circadian gene expression. In addition, as RF leads to high amplitude circadian rhythms and decreased expression of disease markers, we investigated the combination of RF and caffeine, on the expression of the circadian clock, disease, inflammatory, and metabolic markers.

Section snippets

Animals, treatments, and tissues

12 week-old male C57BL/6 mice (n = 144) were entrained to a light–dark cycle (12 h light:12 h darkness, LD) for two weeks with food ad libitum (AL). Subsequently, mice were fed AL or restricted in feeding time (restricted feeding, RF) and each group (n = 72) was divided into 3 subgroups (n = 24 each): a control group and two groups supplemented with 3.5 mg/kg/day or 7 mg/kg/day caffeine for 16 weeks. The RF group was given food between ZT3 and ZT6 (ZT0 – lights on). Average body weight and food

Results

To examine the effect of caffeine on circadian rhythms, mice received food supplemented with caffeine (3.5 mg/kg/day or 7 mg/kg/day) for 16 weeks either ad libitum (AL) or restricted to 3 h (RF) as the mean daily caffeine intake for US consumers is 4 mg/kg/day (Barone and Roberts, 1996). A period of time of 16 weeks allows circadian rhythms and aging-related biomarkers to change and stabilize (Fu et al., 2006). The expression levels of several clock genes, metabolic, disease, and inflammatory markers

Discussion

In this study we investigated the effect of long-term caffeine treatment on several physiological and molecular parameters under the influence of the circadian clock. Although RF affected body weight and food consumption to some degree, caffeine itself did not have any additive or synergistic effect. However, major effects on circadian oscillation, disease, and metabolic marker levels were found with caffeine.

Conflict of interest statement

None of the authors has any conflict of interest to disclose.

Acknowledgements

All authors read and approved the final manuscript.

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