OBESITY AND THE CIRCADIAN CLOCK
Oren Froy ∗
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
ABSTRACT
Mammals have developed an endogenous circadian clock located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus that responds to the environmental light-dark cycle. The SCN clock receives light information from the retina and transmits synchronization cues to peripheral clocks in the liver, heart, etc., regulating cellular and physiological functions. The circadian clock also regulates metabolism and energy homeostasis in peripheral tissues by mediating the expression and/or activity of certain metabolic enzymes, hormones, and transport systems. Pronounced biological rhythms extend life span, as longevity was increased in older hamsters given fetal suprachiasmatic nuclei implants that restored higher amplitude rhythms. Destruction of the SCN results eventually in the absence of bodily rhythms. Disruption of circadian rhythms leads to hormone imbalance, psychological and sleep disorders, cancer proneness, and malignant growth. Recent data suggest that disruption of circadian rhythms in the SCN and peripheral tissues leads to manifestations of the metabolic syndrome and hyperphagia. Indeed, shift work and sleep deprivation in humans have been shown to be associated with increased adiposity. Similarly, homozygous Clock (a key gene of the biological clock) mutant mice have a greatly attenuated diurnal feeding rhythm, are hyperphagic and obese, and develop a metabolic syndrome. Bmal1-/- (a key
∗ Correspondence concerning this article should be addressed to: Oren Froy, Phone: 972-8-948-9746; Fax: 972-8936-3208; E-mail: froy@agri.huji.ac.il.
and Cancer Research, edited by Pauline R. Ramonde, and Fochas, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,
gene of the biological clock) knockout mice, similarly to Clock mutant mice, exhibited suppressed diurnal variations in glucose and triglycerides as well as abolished gluconeogenesis. In addition, high-fat diet disrupts circadian rhythms. Interestingly, Bmal1-/- knockout mice have reduced life span. Thus, disruption in circadian rhythms leads to obesity and reduced life expectancy, whereas resetting of circadian rhythms leads to well-being and increased longevity.
Keywords: clock; obesity, feeding, nutrition, metabolism, circadian rhythms
1. INTRODUCTION
Obesity has become a serious and growing public health problem. Previous ways to combat obesity have failed and new approaches need to be taken. The biological clock regulates the expression and/or activity of enzymes and hormones involved in metabolism. However, recently, there is a growing body of evidence that metabolism, food consumption, timed meals, and some nutrients feed back to entrain the clock. Moreover, disruption of circadian rhythms leads to obesity and the metabolic syndrome. This chapter will summarize recent findings concerning the relationship between feeding regimens, obesity, metabolism, and circadian rhythms.
2. CIRCADIAN RHYTHMS
The rotation of earth around its axis imparts light and dark cycles of 24 hours. Organisms on earth developed the ability to predict these cycles and evolved to restrict their activity to the night or day. By developing an endogenous circadian (circa - about and dies - day) clock, which is entrained to external time cues, animals and plants ensure that physiological processes are carried out at the appropriate, optimal time of day or night (Panda et al., 2002). In mammals, the circadian clock influences nearly all aspects of physiology and behavior, including sleep-wake cycles, cardiovascular activity, endocrine system, body temperature, renal activity, physiology of the gastrointestinal tract, hepatic metabolism, etc. (Panda et al., 2002; Reppert and Weaver, 2002). The fraction of cyclically expressed transcripts in each peripheral tissue ranges between 5-10% of the total population and the vast majority of these genes are tissue-specific (Kornmann et al., 2001; Akhtar et al., 2002; Duffield et al., 2002; Panda et al., 2002; Storch et al., 2002; Schibler et al., 2003).
3. CIRCADIAN RHYTHMS, WELL-BEING, AND LIFE SPAN
Disruption of biological rhythms has a negative effect in the short and long terms. Clinical epidemiology in humans indicates that myocardial infarction, pulmonary edema, hypertensive crises, and asthma and allergic rhinitis attacks all peak at certain times during the day (Maron et al., 1994; Staels, 2006; Burioka et al., 2007). These findings reveal the
prominent influence of the circadian clock on human physiology and pathophysiology (Reppert and Weaver, 2002). In our modern age, there is a need to extend wakefulness or repeatedly invert the normal sleep-wake cycle. As a result, travelers experience the condition known as jet lag, with its associated symptoms of fatigue, disorientation, and insomnia. Likewise, shift workers exhibit altered nighttime melatonin levels and reproductive hormone profiles that could increase the risk of hormone-related diseases (Davis and Mirick, 2006). A number of other disorders, e.g., psychological and sleep disorders, are associated with irregular or pathological functioning of the central biological clock (Reppert and Weaver, 2002). Disruption of circadian coordination has also been found to accelerate cancer proneness and malignant growth, suggesting that the circadian clock controls tumor progression (Fu et al., 2002; Filipski et al., 2003; Davis and Mirick, 2006). Circadian rhythms change with normal aging, including a shift in the phase and decrease in amplitude (Scarbrough et al., 1997; Yamazaki et al., 2002; Hofman and Swaab, 2006). The life span of Drosophila fly mutants whose circadian period differs from that of the wild type was significantly reduced by up to 15% (Klarsfeld and Rouyer, 1998). Chronic reversal of the external light-dark cycle at weekly intervals resulted in a significant decrease in the survival time of cardiomyopathic hamsters (Penev et al., 1998). Similarly, longevity in hamsters is decreased with a disruption of rhythmicity and is increased in older animals given fetal suprachiasmatic implants that restore higher amplitude rhythms (Hurd and Ralph, 1998). Thus, disruption of circadian coordination may be manifested by hormone imbalance, psychological and sleep disorders, cancer proneness, and reduced life span (Penev et al., 1998; Reppert and Weaver, 2002; Fu et al., 2002; Filipski et al., 2003; Davis and Mirick, 2006; Kondratov et al., 2006). In contrast, resetting of circadian rhythms has led to wellbeing and increased longevity (Hurd and Ralph, 1998; Klarsfeld and Rouyer, 1998; Karasek, 2004).
4. THE LOCATION OF THE MAMMALIAN BIOLOGICAL CLOCK
In mammals, the central circadian clock is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus in the brain. The SCN clock is composed of multiple, single-cell circadian oscillators, which, when synchronized, generate coordinated circadian outputs that regulate overt rhythms (Welsh et al., 1995; Liu et al., 1997; Herzog et al., 1998; Reppert and Weaver, 2001). SCN oscillation is not exactly 24 h, therefore, it is necessary to entrain the circadian pacemaker each day to the external light-dark cycle to prevent drifting (or freerunning) out of phase. Light is the most potent synchronizer for the SCN (Quintero et al., 2003). Light is perceived by the retina and the signal is transmitted via the retinohypothalamic tract (RHT) leading to the SCN (Gooley et al., 2001; Lucas et al., 2001; Reppert and Weaver, 2002). Similar clock oscillators have been found in peripheral tissues, such as the liver, intestine, and retina (Lee et al., 2001; Froy and Chapnik, 2007, Reppert and Weaver, 2002). Thus, the central circadian clock (often termed the master clock) is located within the SCN (Matsumoto et al., 1996; LeSauter et al., 1996), whereas peripheral clocks are found within non-SCN cells of the organism, including other regions of the central nervous system (CNS) (Lee et al., 2001; Reppert and Weaver, 2002; Froy et al., 2006; Young, 2006;
Froy and Chapnik, 2007). The SCN receives the light information, interprets it, and transmits it further to peripheral oscillators located outside the SCN, apparently via neuronal connections or circulating humoral factors. Complete destruction of the SCN abolishes circadian rhythmicity in the periphery, as it leads to loss of synchrony among individual cells and damping of the rhythm at the population level (Yoo et al., 2004; Welsh et al., 2004). The SCN must periodically send signals to peripheral oscillators in order to prevent the dampening of circadian rhythms in these tissues. The mechanisms by which the SCN accomplishes this task are not well-understood (Le Minh et al., 2001). However, several humoral factors expressed cyclically by the SCN, transforming growth factor α (TGFα) (Kramer et al., 2001), prokineticin 2 (PK2) (Cheng et al., 2002), and cardiotrophin-like cytokine (CLC) (Kraves and Weitz, 2006) have been shown to inhibit nocturnal locomotor activity when injected into the cerebral ventricle. In turn, SCN rhythms can be altered by neuronal and endocrine inputs (Saeb-Parsy et al., 2000).
5. SCN CONNECTION TO ENERGY HOMEOSTASIS BRAIN CENTERS
The SCN provides its most intense output to the subparaventricular zone (SPZ) and dorsomedial nucleus of the hypothalamus (DMH) (Saper et al., 2005a; Saper et al., 2005b). Furthermore, the DMH has many outputs to other regions of the brain, including the paraventricular nucleus (PVN), the lateral hypothalamus (LH), and ventrolateral preoptic nucleus (VLPO) that regulate corticosteroid release, wakefulness/feeding, and sleep, respectively. The role of the SPZ and DMH in regulating circadian rhythms was determined using lesion studies (Chou et al., 2003; Lu et al., 2001). Destruction of the ventral SPZ (vSPZ) reduced circadian rhythms of sleep-wakefulness and locomotor activity but had little effect on circadian regulation of body temperature (Lu et al., 2001). Conversely, degeneration of the dorsal SPZ (dSPZ) disrupted circadian regulation of body temperature with minimal effect on sleep-wakefulness and locomotor activity (Lu et al., 2001). Thus, vSPZ regulates sleep-wakefulness, whereas dSPZ regulates body temperature (Saper et al., 2005a). Ablation of DMH cell bodies, which receive inputs from both the SCN and the SPZ, resulted in severe impairment of circadian regulated sleep-wakefulness, locomotor activity, corticosteroid secretion, and feeding (Chou et al., 2003). Thus, the DMH constitutes a gateway between the master pacemaker neurons of the SCN and cell bodies located within brain centers important in energy homeostasis (Ramsey et al., 2007).
6. THE BIOLOGICAL CLOCK AT THE MOLECULAR LEVEL
Transcriptional-translational feedback loops lie at the very heart of the core clock mechanism. Generation of circadian rhythms is dependent on the concerted co-expression of specific clock genes. In mammals, the clock is an intracellular, transcriptional-translational mechanism sharing the same molecular components in SCN neurons and peripheral cells
(Schibler et al., 2003). Many clock gene products function as transcription factors, which possess PAS (PER, ARNT, SIM) and basic helix-loop-helix (bHLH) domains involved, respectively, in protein-protein and protein-DNA interactions. These factors ultimately activate or repress their own expression and, thus, constitute a self-sustained transcriptional feedback loop. Changes in concentration, subcellular localization, posttranslational modifications, and delays between transcription and translation lead to the achieved 24-h cycle (Dunlap, 1999; Panda et al., 2002; Reppert and Weaver, 2002).
Figure 1. The core mechanism of the mammalian circadian clock and its link to energy metabolism. The cellular oscillator is composed of a positive limb (CLOCK and BMAL1) and a negative limb (CRYs and PERs). CLOCK and BMAL1 dimerize in the cytoplasm and translocate to the nucleus. The CLOCK:BMAL1 heterodimer then binds to enhancer (E-box) sequences located in the promoter region of Per and Cry genes, activating their transcription. After translation, PERs and CRYs undergo nuclear translocation and inhibit CLOCK:BMAL1, resulting in decreased transcription of their own genes. The autoregulatory transcription–translation loop comprising CLOCK:BMAL1 and PER–CRY constitutes the core clock and generates 24-h rhythms of gene expression. CLOCK:BMAL1 heterodimer also induces the transcription of Rev-erbα and Rorα RORα and REV-ERBα regulate lipid metabolism and adipogenesis, and also participate in the regulation of Bmal1 expression. RORα stimulates and REVERBα inhibits Bmal1 transcription, acting through RORE. CLOCK:BMAL1 heterodimer also mediates the transcription of Pparα, a nuclear receptor involved in glucose and lipid metabolism. PPARα activates transcription of Rev-erba by binding to a peroxisome proliferator-response element (PPRE). PPARα also induces Bmal1 expression, acting through PPRE located in its promoter.
The core clock mechanism involves Clock, brain-muscle-Arnt-like 1 (Bmal1), Period1 (Per1), Period2 (Per2), Period3 (Per3), Cryptochrome1 (Cry1), and Cryptochrome2 (Cry2)
(Figure 1). In the mouse, the first clock gene identified, encodes the transcription factor CLOCK (Vitaterna et al., 1994), which dimerizes with BMAL1 to activate transcription. CLOCK and BMAL1, two PAS-bHLH transcription factors, are capable of activating transcription upon binding to E-box (5’- CACGTG -3’) and E-box-like promoter elements (Reppert and Weaver, 2002). BMAL1 can also dimerize with other CLOCK homologs, such as neuronal PAS domain protein 2 (NPAS2), to activate transcription and sustain rhythmicity (Asher and Schibler, 2006; Debruyne et al., 2006). The PERIOD proteins (PER1, PER2, and PER3) and the two CRYPTOCHROMEs (CRY1 and CRY2) operate as negative regulators (Zylka et al., 1998; Reppert and Weaver, 2001; Froy et al., 2002). Thus, CLOCK:BMAL1 heterodimers bind to E-box sequences and mediate transcription of a large number of genes including those of the negative feedback loop Pers and Crys. When PERs and CRYs are produced in the cytoplasm, they dimerize and translocate to the nucleus to inhibit CLOCK:BMAL1-mediated transcription (Figure 1). Pers and Bmal1 have robust oscillation in opposite phases correlating with their opposing functions (Froy et al., 2006). All the aforementioned clock genes exhibit a 24-h rhythm in cells. In addition, casein kinase I epsilon (CKIε) is thought to phosphorylate the PER proteins and, thereby, enhance their instability and degradation (Dunlap, 1999; Whitmore et al., 2000; Eide and Virshup, 2001; Eide et al., 2005a). CKIε also phosphorylates and partially activates the transcription factor BMAL1 (Eide et al., 2005b). Bmal1 expression is negatively regulated by the transcription factor reverse erythroblastosis virus α (REV-ERBα) (Preitner et al., 2002) and positively regulated by retinoic acid receptor-related orphan receptor α (RORα) (Sato et al., 2004) via the RORα response element (RORE) (Ueda et al., 2005).
7. EFFECT OF THE BIOLOGICAL CLOCK ON METABOLISM
Many hormones involved in metabolism, such as insulin (La Fleur et al., 1999), glucagon (Ruiter et al., 2003), adiponectin (Ando et al., 2005), corticosterone (De Boer and Van der Gugten, 1987), leptin, and ghrelin (Bodosi et al., 2004), have been shown to exhibit circadian oscillation. Leptin, an adipocyte-derived circulating hormone that acts at specific receptors in the hypothalamus to suppress appetite and increase metabolism, is extremely important in obesity. Leptin exhibits striking circadian patterns in both gene expression and protein secretion, with peaks during the sleep phase in humans (Kalra et al., 2003). Neither feeding time nor adrenalectomy affected the rhythmicity of leptin release. However, ablation of the SCN has been shown to eliminate leptin circadian rhythmicity in rodents, suggesting that the central circadian clock regulates leptin expression (Kalsbeek et al., 2001).
In addition to the endocrine control, the circadian clock has been reported to regulate metabolism and energy homeostasis in peripheral tissues (Froy, 2007). This is achieved by mediating the expression and/or activity of certain metabolic enzymes and transport systems (Hirota and Fukada, 2004; Kohsaka and Bass, 2006) involved in cholesterol metabolism, amino acid regulation, drug and toxin metabolism, the citric acid cycle, and glycogen and glucose metabolism (La Fleur et al., 1999; La Fleur, 2003; Davidson et al., 2004). Some examples are glycogen phosphorylase (Frederiks et al., 1987), cytochrome oxidase (Ximenes da Silva et al., 2000), lactate dehydrogenase (Rivera-Coll et al., 1993), acetyl-CoA
carboxylase (Fukuda and Iritani, 1991; Davies et al., 1992), malic enzyme, fatty acid synthase, glucose-6-phosphate dehydrogenase (Fukuda and Iritani, 1991) and many more. Moreover, lesion of rat SCN abolishes diurnal variations in whole body glucose homeostasis (Cailotto et al., 2005), altering not only rhythms in glucose utilization rates but also endogenous hepatic glucose production. In addition, a large number of nuclear receptors involved in lipid and glucose metabolism has been found to exhibit circadian expression (Yang et al., 2006). Similarly, glucose uptake and the concentration of the primary cellular metabolic currency adenosine triphosphate (ATP) in the brain and peripheral tissues have been found to fluctuate around the circadian cycle (Yamazaki et al., 1994; La Fleur, 2003; Kalsbeek et al., 2006).
8. ENERGY CENTERS AND CIRCADIAN RHYTHMS
Several brain regions have been associated with energy homeostasis. These regions are the ventromedial hypothalamic (VMH), paraventricular hypothalamus (PVH), dorsomedial hypothalamus (DMH), and arcuate nucleus (ARC) located at the mediobasal hypothalamus. Destruction of the ventromedial hypothalamic (VMH), paraventricular hypothalamus (PVH), and dorsomedial hypothalamus (DMH) regions result in obesity, whereas ablation of the lateral hypothalamus (LH) result in anorexia (Anand and Brobeck, 1951). Additional insight into the neural control of energy homeostasis comes from the melanocortin system (Fan et al., 1997; Huszar et al., 1997). Leptin, a satiety signal, stimulates pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART)-expressing neurons within the ARC to produce α-melanocyte-stimulating hormone (α-MSH), which subsequently activates the melanocortin 4 receptor (MC4R) and results in decreased food intake and increased energy expenditure (Adan et al., 1994; Cone, 2005). In humans, mutations in the proopiomelanocortin (POMC) and MC4R genes are associated with morbid obesity (Krude et al., 1998; Yeo et al., 1998; Vaisse et al., 1998; Hinney et al., 1999; Farooqi et al., 2000; Vaisse et al., 2000). In parallel, leptin suppresses a distinct set of neuropeptide Y (NPY) and agoutirelated protein (AgRP)-expressing neurons within the ARC. With no suppression of leptin, NPY and AgRP-expressing neurons release of AgRP, an antagonist of the MC4R (Ollmann et al., 1997; Quillan et al., 1998; Rossi et al., 1998). In the absence of leptin, such as during the fasted state, the orexigenic NPY/AgRP neuropeptides cause decreased energy expenditure and increased appetite (Baskin et al., 1999; Elias et al., 1999; Elias et al., 2000; Marsh et al., 1999; Stephens et al., 1995). Thus, agonists (α-MSH) and antagonists (AgRP) of the MC4R determine the weight-regulating effects of leptin in the central nervous system. Leptin can be the bridge between energy homeostasis and circadian control, due to its circadian oscillation and expression of its receptor in hypothalamic regions.
ARC neurons project to multiple nuclei involved in feeding behavior (Cowley et al., 2001; Elias et al., 1999; Elmquist et al., 1998; Elmquist et al., 1999), such as the lateral hypothalamus, which produces the hunger-stimulating neuropeptides melanin-concentrating hormone (MCH), orexin A, and orexin B (Flier and Maratos-Flier, 1998; Friedman and Halaas, 1998, Schwartz et al., 2000). Targeted deletion studies of MCH resulted in hypophagic lean mice with a high metabolic rate and demonstrated that MCH acts
downstream of leptin and the melanocortin system (Shimada et al., 1998). Orexins A and B are two neuropeptides generated from a single transcript that display a circadian rhythm of expression and are strongly induced by fasting (Sutcliffe and de Lecea, 2000; Willie et al., 2001). Indeed, mutant mice, in which Clock function is impaired, exhibit significantly higher energy intake and almost complete ablation of rhythmic expression in Cart and Orexin (Turek et al., 2005). Intracerebroventricular injection of orexin A stimulates food intake acutely in rats, in part through excitation of NPY in the ARC (Samson and Resch, 2000; Sutcliffe and de Lecea, 2000). Orexins also play a role in the regulation of sleep-wake rhythms, as mutations in the orexin B receptor (Lin et al., 1999; Willie et al., 2001) and deletion of the orexin gene (Hara et al., 2001a) caused narcolepsy and obesity (Nishino and Mignot, 2002; Fujiki et al., 2006).
9. CIRCADIAN RHYTHMS AND BODY WEIGHT
Fluctuations in body weight have been associated with changes in day length in various species, suggesting a central role for the circadian clock in regulating body weight. For example, in Siberian hamsters, modulation of body weight depends on photoperiod acting via the temporal pattern of melatonin secretion from the pineal gland (Gorman, 2003; Morgan et al., 2003). In studies performed on sheep, adipose tissue leptin levels were modulated by day length independently of food intake, body fatness and gonadal activity. In addition, increasing the length of the photoperiod resulted in increased activity of the lipogenesispromoting proteins lipoprotein lipase and malic enzyme, independent of nutritional status (Bocquier et al., 1998; Faulconnier et al., 2001). In humans, studies have demonstrated an increased incidence of obesity among shift workers (Di Lorenzo et al., 2003; Karlsson et al., 2001; Karlsson et al., 2003) (see Sleep and Obesity below).
In obese subjects, leptin, secreted from adipose tissue, retains diurnal variation in release, but with lower amplitude (Licinio, 1998). Leptin 24-h levels were lower in obese vs. nonobese adolescent girls, suggesting that blunted circadian variation may play a role in leptin resistance and obesity (Heptulla et al., 2001). Circadian patterns of leptin concentration were distinctly different between adult women with upper-body or lower-body obesity, with a delay in peak values of leptin of approximately 3 h in women with upper-body obesity (Perfetto et al., 2004). Indeed, leptin and the leptin receptor knockouts in animals or mutations in humans have been demonstrated to produce morbid, early onset obesity, hypoleptinemia, hyperphagia, hyperinsulinemia, and hyperglycemia (Zhang et al., 1994; Montague et al., 1997; Strobel et al., 1998; Clement et al., 1998). Similarly to leptin, the rhythmic expression of resistin and adiponectin was greatly blunted in obese (KK) and obese, diabetic (KK-Ay) mice (Ando et al., 2005). In humans, circulating adiponectin levels exhibit both ultradian pulsatility and a diurnal variation. In the latter case, the pattern of adiponectin release is out of phase with leptin with a significant decline at night, reaching a nadir in the early morning (Gavrila et al., 2003). In obese subjects, adiponectin levels were significantly lower than lean controls (Yildiz et al., 2004).
10. SLEEP AND OBESITY
Sleep is one of the output control systems of the biological clock. A large body of evidence accumulated thus far suggesting that short sleep duration is associated with increased body mass index (BMI - weight in kilograms divided by the square of height in meters) and elevated incidence of type 2 diabetes (Hasler et al., 2004; Megirian et al., 1998; Nilsson et al., 2004; Taheri et al., 2004; Gottlieb et al., 2005). Clinical studies have also identified changes in many aspects of energy metabolism following even just a few days of partial sleep restriction. Furthermore, self-reported short sleepers had significantly reduced circulating levels of the anorectic hormone leptin and increased levels of the orexigenic hormone ghrelin (Taheri et al., 2004). These neuroendocrine changes could explain, in part, reports of increased appetite following sleep loss (Spiegel et al., 1999). The relationship between BMI and reported total sleep time (TST) per 24 h showed that overweight (BMI=2529.9 kg*m-2) and obese (BMI=30-39.9 kg*m-2) patients slept less than patients with normal BMI. Interestingly, extremely obese subjects (BMI>40 kg*m-2) averaged greater TST than the obese subjects (Vorona et al., 2005). Indeed, previous studies have reported that obese patients were sleepier during the day and more likely to experience disturbed sleep at night compared with normal weight controls (Vgontzas et al., 1998). Daytime sleepiness could not wholly be explained by disturbed nighttime sleep, suggesting that a circadian abnormality likely underlies the daytime sleepiness observed in the obese patients (Vgontzas et al., 1998). Morning levels of cytokines associated with obesity, e.g., tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6), were significantly elevated in patients with sleep apnea compared with controls and also significantly correlated with excessive daytime sleepiness (Vgontzas et al., 1997).
Shift work is another example in which the normal synchrony between the light-dark cycle, sleeping, and eating is disturbed. Shift work has been associated with cardiovascular disease, obesity, diabetes and other metabolic disturbances. Night-shift workers, whose activity period is reversed in relation to the day/night cycle, are much more likely to develop the metabolic syndrome (Holmback et al., 2003). People who habitually sleep <6 or >9 hours per night have increased risk of developing type 2 diabetes and impaired glucose tolerance (Gottlieb et al., 2005). It has been reported that obesity, high triglycerides, and low concentrations of high-density lipoprotein cholesterol seem to cluster together more often in shift workers than in day workers (Karlsson et al., 2001; Karlsson et al., 2003). Similarly, duration of shift work was directly related to body mass index (BMI) and waist to hip ratio independent of age, sex, smoking status, physical activity, and educational level (van Amelsvoort et al., 1999; Parkes et al., 2002; Di Lorenzo et al., 2003). Recently it has been reported that subjects who experienced 38 h of continued wakefulness still exhibit significant endogenous circadian rhythms in leptin, glucose, and insulin with peaks around the usual time of waking (Shea et al., 2005). Feeding during the period of wakefulness was associated with systematic increases in leptin levels, while fasting during recovery sleep was associated with systematic decreases in leptin levels, glucose, and insulin (Shea et al., 2005). Shea et al. (2005) suggested that alterations in the sleep/wake schedule would lead to an increased daily range in circulating leptin, with lowest leptin upon awakening, which, by influencing food
intake and energy balance, could be implicated in the increased prevalence of obesity in the shift work population. These findings point to the adipocyte as an important factor in the development of obesity associated with shift work. In addition, sleep deprivation also leads to obesity (Sekine et al., 2002) and affects plasma leptin levels (Mullington et al., 2003). Sleep deprivation for 88 h followed by three nights of recovery sleep for 7 h or 14 h per night induced decreased amplitude of leptin levels during the sleep-deprived period that returned to normal after one night of sleep recovery (Mullington et al., 2003). Thus, shift work and sleep deprivation have been shown to be associated with increased adiposity, findings that have been linked to the sleep-associated peak in leptin secretion.
High fat diet and obesity also affect sleep itself. Mice fed a high-fat diet have increased sleep time, particularly in the non-rapid eye movement (NREM) stage, but decreased sleep consolidation (Jenkins et al., 2005). Similarly, the obese leptin-deficient ob/ob mice and rats harboring mutations in the leptin receptor exhibit increased NREM sleep time, decreased sleep consolidation, decreased locomotor activity, and a smaller compensatory rebound response to acute sleep deprivation (Danguir, 1989; Megirian et al., 1998; Laposky et al., 2006). On the other hand, food deprivation, which increases leptin levels, results in decreased sleep time (Danguir and Nicolaidis, 1979) and a more fragmented sleep pattern in rats (Borbely, 1977).
11. EFFECT OF CIRCADIAN RHYTHMS ON LIPID METABOLISM
Circadian clocks have been shown to be present in inguinal white adipose tissue, epididymal white adipose tissue, and brown adipose tissue (Zvonic et al., 2006; Zvonic et al., 2007). Diurnal variations in the sensitivity of adipose to adrenaline-induced lipolysis persist ex vivo, suggesting that the intrinsic nature of the adipocyte exhibits a diurnal variation (Suzuki et al., 1983). Adipose tissue secretes metabolic mediators, such as adiponectin, resistin, visfatin, and leptin that are circadianly controlled and can be entrained by meal timing. Novel transcriptomics studies revealed rhythmic expression of clock and adipokine genes, such as resistin, adiponectin, and visfatin, in visceral fat tissue (Ando et al., 2005). The expression of these mediators is blunted in obese patients (Saad et al., 1998; Kalsbeek et al., 2001; Gavrila et al., 2003). Fatty acid transport protein 1 (Fatp1), fatty acyl-CoA synthetase 1 (Acs1), and adipocyte differentiation-related protein (Adrp) exhibit diurnal variations in expression, suggesting that nocturnal expression of FATP1, ACS1, and ADRP will promote higher rates of fatty acid uptake and storage of triglyceride in rodents (Bray and Young, 2007).
Recent molecular studies established BMAL1 involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. Bmal1 knockout mice embryonic fibroblast cells failed to be differentiated into adipocytes. Loss of BMAL1 expression led to a significant decrease in adipogenesis and the gene expression of several key adipogenic/lipogenic factors (PPARγ2, aP2, C/EBPα, C/EBPδ, SREBP-1a, PEPCK, FAS). Furthermore, over-expression of BMAL1 in adipocytes increased lipid synthesis activity. These results indicate that BMAL1, a master regulator of circadian rhythm, also plays important roles in the regulation of adipose differentiation and lipogenesis in mature
edited by Pauline R. Ramonde, and Fochas,
adipocytes (Shimba et al., 2005). These findings may explain molecularly why clock disruption leads to obesity.
The circadian rhythmicity of a nuclear receptor family member, peroxisome proliferatoractivated receptor α (PPARα), provides a further example of a reciprocal link between circadian and metabolic processes. The CLOCK:BMAL heterodimer mediated transcription of PPARα, which subsequently binds to the peroxisome-proliferator response element and activates transcription of Bmal1 (Oishi et al., 2005; Inoue et al., 2005; Canaple et al., 2006) (Figure 1). PPARα regulates the transcription of genes involved in lipid and glucose metabolism upon binding of endogenous free fatty acids. These data are in concert with the finding that BMAL1-deficient embryonic fibroblasts fail to differentiate into adipocytes (Shimba et al., 2005) and demonstrate that PPARα may play a unique role at the intersection of circadian and metabolic pathways.
Another important candidate to link between the circadian clock and lipid metabolism is REV-ERBα. This pro-adipogenic transcription factor, whose levels increase dramatically during adipocyte differentiation (Chawla and Lazar, 1993), exhibits striking diurnal variations in expression in murine adipose tissue (Bray and Young, 2007) and rat liver (Torra et al., 2000). During adipocyte differentiation, REV-ERBα has been shown to act downstream of the differentiation factor peroxisome proliferator receptor-γ (PPARγ) by facilitating gene expression of PPAR target genes, including Ap2 and C/ebpα, but it has no effect on c/EBPb or SREBP-1 gene expression (Fontaine et al., 2003; Duez and Staels, 2008). Ectopic REV-ERBα expression in 3T3L1 pre-adipocytes promotes their differentiation into mature adipocytes (Fontaine et al., 2003). In addition to its role in lipid metabolism and adipocyte differentiation, REV-ERBα is a negative regulator of Bmal1 expression (Preitner et al., 2002). In contrast, retinoic acid–related orphan receptor α (RORα), which regulates lipogenesis and lipid storage in skeletal muscle, is a positive regulator of Bmal1 expression (Sato et al., 2004; Lau et al., 2004). Interestingly, CLOCK:BMAL1 heterodimer regulates the expression of both Rev-erbα and Rorα (Preitner et al., 2002; Ueda et al., 2002; Sato et al., 2004) (Figure 1).
12. CIRCADIAN RHYTHMS AND METABOLIC DISORDERS
Recent studies have suggested that disruption of circadian rhythms in the SCN and peripheral tissues may lead to manifestations of the metabolic syndrome (Broberger, 2005; Buijs and Kreier, 2006; Staels, 2006). In mice, a high-fat diet led to a mild metabolic syndrome of obesity, hyperlipidemia, and hyperglycemia, but had minimal effects on the rhythmic expression of the clock genes Clock, Bmal1, Per1, Per2, Cry1, and Cry2 in visceral adipose tissue and liver (Yanagihara et al., 2006; Satoh et al., 2006). In contrast, other reports showed disruption of circadian rhythms of clock gene and clock-controlled output gene expression (Kohsaka et al., 2007; Barnea et al., 2008) and even a phase delay in gene expression (Barnea et al., 2008). Similarly, daily rhythmicity in 7α-hydroxylase mRNA, encoding the hepatic enzyme controlling circadian cholesterol homeostasis, disappeared in mice fed high-fat diet. Interestingly, the oscillations of clock (Bmal1, Per1, Per2, Cry1, Cry2 and Dbp) and adipokine genes were mildly suppressed in the adipose tissue of obese KK
mice and greatly suppressed in the adipose of obese, diabetic (KK-Ay) mice compared with wild type mice (Ando et al., 2005). Similarly, obese diabetic mice exhibited circadian oscillation of most genes in the liver, but some genes had attenuated, but still rhythmic, expression (Ando et al., 2006).
The most compelling linkage between metabolic disorders and the circadian clock is demonstrated by the phenotypes of clock gene mutants and knockouts. Homozygous Clock mutant mice have a greatly attenuated diurnal feeding rhythm, are hyperphagic and obese, and develop a metabolic syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis, and hyperglycemia (Turek et al., 2005). However, some studies found that in Clock mutant mice, serum levels of triglyceride and free fatty acids were significantly lower than in wild-type control mice, whereas total cholesterol and glucose levels did not differ (Oishi et al., 2006a). Combination of the Clock mutation with the leptin knockout (ob/ob) resulted in significantly heavier mice than the Clock or ob/ob phenotype separately (Oishi et al., 2006b). Loss of circadian rhythms in Clock mutant mice exhibited attenuated expression of hypothalamic peptides associated with energy balance, such as ghrelin and orexin (Turek et al., 2005) and suppressed gluconeogenesis (Rudic et al., 2004). Surprisingly, in Clock mutant mice, high fat diet amplified the diurnal variation in glucose tolerance and insulin sensitivity and obesity was attenuated through impaired dietary fat absorption (Rudic et al., 2004; Oishi et al., 2006a). Bmal1-/- knockout mice, similarly to Clock mutant mice, exhibited suppressed diurnal variations in glucose and triglycerides as well as abolished gluconeogenesis. Although recovery from insulin-induced hypoglycemia was impaired in Clock mutant and Bmal1-/knockout mice, the counter-regulatory response of corticosterone and glucagon was retained (Rudic et al., 2004). Thus, CLOCK and BMAL1 regulate the recovery from insulin-induced hypoglycemia, glucose tolerance, insulin sensitivity, and fat absorption. Circadian control of glucose metabolism is implicated by the variation in glucose tolerance and insulin action across the day (Gagliardino et al., 1984; Van Cauter et al., 1997). Evidence suggests that loss of circadian rhythmicity of glucose metabolism may contribute to the development of metabolic disorders, such as type 2 diabetes, in both rodents (Velasco et al., 1988; Oster et al., 1988; Shimomura et al., 1990) and humans (Spallone et al., 1993; Van Cauter et al., 1997). For example, daily cycles of insulin secretion and glucose tolerance are lost in patients with type 2 diabetes (Boden et al., 1999; Van Cauter et al., 1997), as are daily variations in plasma corticosterone levels and locomotor activity in streptozotocin-induced diabetic rats (Oster et al., 1988; Velasco et al., 1988). These findings indicate that a critical relationship exists between endogenous circadian rhythms and diabetes. The findings also suggest that time of day may be an important consideration for the diagnosis and treatment of metabolic disorders such as type 2 diabetes (Schmidt et al., 1981; Troisi et al., 2000). In addition, in type 1 diabetes patients, lipolysis increased earlier in the evening than in the healthy controls, and remained elevated throughout the night, indicating that lipolysis shows a distinct circadian rhythm that is altered in type 1 diabetes patients (Hagstrom-Toft et al., 1997). These findings point to the tight relationship between disruption of circadian rhythms and metabolic disorders.
13. TIMED MEALS AND CIRCADIAN RHYTHMS
Similarly to the control of the circadian clock on metabolism, feeding is a very potent synchronizer (zeitgeber) for peripheral clocks. Recent evidence indicates that clock gene expression in the liver and other peripheral tissues is entrained to periodic meals (Stephan, 2002) (Figure 2). Limiting the time and duration of food availability with no calorie reduction is termed restricted feeding (RF) (Cassone and Stephan, 2002; Schibler et al., 2003; Hirota and Fukada, 2004). Animals, which receive food ad libitum everyday at the same time for only a few hours, adjust to the feeding period within a few days and consume their daily food intake during that limited time (Honma et al., 1983; Grasl-Kraupp et al., 1994; Froy et al., 2006). Restricting food to a particular time of day has profound effects on the behavior and physiology of animals. 2-4 h before the meal, the animals display an anticipatory behavior, which is demonstrated by an increase in locomotor activity, body temperature, corticosterone secretion, gastrointestinal motility, and activity of digestive enzymes (Saito et al., 1976; Honma et al., 1983; Comperatore and Stephan, 1987; Stephan, 2002), all are known output systems of the biological clock. RF drives rhythms in arrhythmic clock mutant mice and animals with lesioned SCN, independently of the light-dark cycle, and in constant darkness (Stephan et al., 1979; Mistlberger, 1994; Hara et al., 2001b; Stephan, 2002; Oishi et al., 2002). In most incidents, RF affects circadian oscillators in peripheral tissues, such as liver, kidney, heart, and pancreas, with no effect on the central pacemaker in the SCN (Damiola et al., 2000; Hara et al., 2001b; Stokkan et al., 2001; Cassone and Stephan, 2002; Oishi et al., 2002; Schibler et al., 2003; Hirota and Fukada, 2004). Thus, RF uncouples the SCN from the periphery. Many physiological activities that are normally dictated by the SCN master clock, such as hepatic P450 activity, body temperature, locomotor activity, heart rate, etc., are phase-shifted by RF to the time of food availability (Hara et al., 2001b; Mistlberger, 1994; Boulamery-Velly et al., 2005; Hirao et al., 2006). As soon as food availability returns to normal, the SCN clock, whose phase remains unaffected, resets the peripheral oscillators (Damiola et al., 2000). The location of this food-entrainable oscillator (FEO) has been elusive. Lesions in the dorsomedial hypothalamic nucleus (DMH) (Mieda et al., 2006; Gooley et al., 2006; Landry et al., 2006; Landry et al., 2007), the brain stem parabrachial nuclei (PBN) (Davidson et al., 2000; Gooley et al., 2006), and the core and shell regions of nucleus accumbens (Mistlberger and Mumby, 1992; Mendoza et al., 2005a) revealed that these brain regions may be involved in FEO output, but they cannot fully account for the oscillation (Davidson, 2006). Neither vagal signals nor leptin are critical for the entrainment (Comperatore and Stephan, 1990; Mistlberger and Marchant, 1999). However, the role of the biological clock in the anticipatory behavior has been recently demonstrated, as mPer2 mutant mice did not exhibit wheel-running food anticipation (Feillet et al., 2006; Mistlberger, 2006).
Figure 2. Resetting signals of the central and peripheral clocks. Light is absorbed through the retina and is transmitted to the suprachiasmatic nuclei (SCN) via the retinohypothalamic tract (RHT). The SCN then dictates the entrainment of peripheral oscillators via humoral or neuronal cues. Food and feeding affect either peripheral clocks or the central clock in the SCN (see text).
Although it has been well established that RF does not entrain the SCN, several studies have shown otherwise: 1) Male Wistar rats given diurnal total parenteral nutrition (TPN) exhibited a phase shift in Per2 and Dbp (D-site binding protein) expression in the SCN and liver (Miki et al., 2003). 2) The peak time of Per2 expression in the SCN was significantly affected by glucose and amino acids in rats (Iwanaga et al., 2005). 3) CS mice exhibited a fixed phase-relationship of Per1, Per2 and Bmal1 rhythms in the SCN with feeding time in total darkness (DD) under RF conditions (Abe et al., 2007). 4) Mus domesticus mouse lines receiving scheduled feeding and kept in total darkness exhibited a phase-shift in Per2 expression in the SCN (Castillo et al., 2004).
Recent reports suggest that disruption of circadian rhythmicity affects tumor growth and mortality in rodents with cancer (Fu and Lee, 2003). Arrhythmicity induced by SCN lesions (Filipski et al., 2002; Filipski et al., 2003) or chronic jet lag (Filipski et al., 2004; Filipski et al., 2005) in mice or continuous bright light in rats (van den Heiligenberg et al., 1999) increases the rate of tumor growth and hastens death. Conversely, enhancement of host rhythms can inhibit tumor progression. Molecularly, disruption of the clock gene Per2 increases both spontaneous and radiation-induced tumor frequency (Fu et al., 2002). However, prolonged survival was achieved when mice inoculated with osteosarcoma were treated for RF during the light period. The internal desynchronization produced by meal timing during the light period attenuated tumor progression, an effect possibly resulting from improved host-mediated tumor control (Wu et al., 2004; Filipski et al., 2005; Davidson et al., 2006).
Although feeding time is a dominant cue for circadian rhythms in mammalian peripheral tissue, the effect of feeding or fasting on circadian gene expression and behavior is unclear (Kobayashi et al., 2004). The expression level and phase of Per1, Per2 and of the clock controlled gene, Dbp are significantly altered by feeding (Kobayashi et al., 2004). Although Kobayashi et al. (2004) showed no effect of fasting on the expression level and phase of Per1, Per2, and Dbp, another report showed that fasting induced a phase advance in clock gene and clock-controlled output gene expression (Barnea et al., 2008). In an effort to
understand the effect of feeding on synchronization of peripheral clocks, it was found that taste (Mistlberger and Rusak, 1987; Abe and Rusak, 1992), but not smell (Coleman and Hay, 1990; Davidson et al., 2001), appears to play some role because rats and hamsters will entrain to the presence of palatable foods and express anticipatory behavior even if regular rodent chow is available ad libitum (Mistlberger and Rusak, 1987; Abe and Rusak, 1992). This effect appears to be independent of meal size because rats fed large volumes of non-nutritive mash do not entrain to this regimen (Mistlberger and Rusak, 1987). In contrast, the nutritional value of the meal affects anticipation, as rats will anticipate the timed presentation when two of three major nutrient groups, protein, carbohydrate, or fat, are presented (Mistlberger et al., 1990).
Similarly to feeding, the glucocorticoid hormone analog dexamethasone was shown to induce circadian gene expression in cultured rat-1 fibroblasts and transiently change the phase of circadian gene expression in liver, kidney, and heart. This effect of dexamethasone did not affect cyclic gene expression in neurons of the SCN (Balsalobre et al., 2000). As daytime feeding phase-shifts peripheral oscillators faster in adrenalectomized mice, it is plausible that glucocorticoids inhibit the RF-induced uncoupling of peripheral clocks from the SCN (Le Minh et al., 2001). Analysis of whether insulin could also shift the clock in peripheral tissues revealed that insulin-dependent diabetic mice responded to RF. These results ruled out insulin as a necessary component for phase shifting of peripheral clocks (Davidson et al., 2002; Oishi et al., 2004).
14. CIRCADIAN RHYTHMS AND CALORIC RESTRICTION
Calorie restriction (CR) refers to a dietary regimen low in calories without malnutrition. CR restricts the amount of calories derived from carbohydrates, fats, or proteins to 25%-60% below that of control animals fed ad libitum (Masoro et al., 1995). It has been documented that calorie restriction significantly extends the life span of rodents by up to 50% (Koubova and Guarente, 2003; Masoro, 2005). In addition to the increase in life span, CR also delays the occurrence of age-associated pathophysiological changes, such as cancer, diabetes, kidney disease, cataracts, etc. (Weindruch and Sohal, 1997; Roth et al., 2002; Roth et al., 2004; Masoro, 2005). Theories on how CR modulates aging and longevity abound, but the exact mechanism is still unknown (Masoro, 2005). The most prevalent is the “free radical/oxidative stress theory of aging” that attributes the aging-associated deterioration to the continuous accumulation of oxidative damage generated by reactive oxygen species produced in the mitochondria (Harman, 1956; Sohal and Weindruch, 1996; Spindler, 2005).
As opposed to RF, CR entrains the clock in the SCN (Challet et al., 1998; Challet et al., 2003; Mendoza et al., 2005b; Resuehr and Olcese, 2005), indicating that calorie reduction could affect the central oscillator. CR during the daytime affects the temporal organization of the SCN clockwork and circadian outputs in mice under light/dark cycle. In addition, CR affects photic responses of the circadian system, indicating that energy metabolism modulates gating of photic inputs in mammals (Mendoza et al., 2005b). These findings suggest that synchronization of peripheral oscillators during CR could be achieved directly due to the temporal eating, as has been reported for RF (Damiola et al., 2000; Hara et al., 2001b;
Stokkan et al., 2001), or by synchronizing the SCN (Challet et al., 1998; Challet et al., 2003; Mendoza et al., 2005b), which, in turn, sends humoral or neuronal signals to entrain the peripheral tissues (Froy et al., 2006; Froy and Miskin, 2007).
15. EFFECT OF METABOLISM AND FOOD COMPONENTS ON CIRCADIAN RHYTHMS
Recent experiments have suggested a direct route through which food may influence peripheral clocks (Rutter et al., 2001). CLOCK and its homolog NPAS2 can bind efficiently to BMAL1 and consequently to their E-box sequences in the presence of reduced nicotinamide adenine dinucleotides (NADH and NADPH). On the other hand, the oxidized forms of the nicotinamide adenine dinucleotides (NAD+ and NADP+) inhibit DNA binding of CLOCK:BMAL1 or NPAS2:BMAL1 (Rutter et al., 2001; Rutter et al., 2002). The NAD(P) redox equilibrium depends on the metabolic state of the cell. The ratio between NAD(P)H and NAD(P)+ dictates the binding of CLOCK/NPAS2:BMAL1 to E-boxes and could result in phase-shifting of cyclic clock gene expression and, as a result, of output gene expression (Rutter et al., 2001; Rutter et al., 2002; Hirota and Fukada, 2004). However, the effect of redox on circadian rhythms still needs to be substantiated by in vivo experiments. In addition to the general metabolic state of the cell, several studies have identified single nutrients capable of resetting or phase-shifting circadian rhythms, such as glucose (Stephan and Davidson, 1998; Young et al., 2002; Hirota et al., 2002; Iwanaga et al., 2005), amino acids (Iwanaga et al., 2005), sodium (Mohri et al., 2003; Lamont et al., 2007), ethanol (Chen et al., 2004; Spanagel et al., 2005), caffeine (Antle et al., 2001), thiamine (Langlais and Hall, 1998; Bennett and Schwartz, 1999), and retinoic acid (McNamara et al., 2001; Shirai et al., 2006).
16. CONCLUSION
The prominent influence of the circadian clock on human physiology is demonstrated by the temporal and pronounced activity of a plethora of systems, such as sleep and wake cycles, feeding behavior, metabolism, physiological and endocrine activity. Disrupted biological rhythms lead to attenuated circadian feeding rhythms, hyperphagia, obesity, cancer proneness, and reduced life expectancy. As food components and feeding time have the ability to reset bodily rhythms, it is of extreme importance to understand the relationship between food, feeding, and the biological clock at the molecular level. Resetting the biological clock by food or feeding time may lead to better functionality of physiological systems, preventing obesity, promoting well-being, and extending life span.
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"I am a naval officer."
"You are a spy. Come with us!"
The usual spy mania spread throughout the restaurant. Blows were threatened, chairs were brandished, and there were shouts of "Kill the spy, kill him!" on all sides. If the officers hadn't fought the crowd off, I would have been badly beaten.
At headquarters I was shown a description and even a picture of myself. So there was no doubt but what I was their man.
"Under what name does this spy travel?" I demanded.
"Under the name of Marine Inspector von Eckmann.
"Why, I am he."
"But you just said you were Count von Luckner."
I was compelled, with great injunctions of secrecy, to take them into my confidence, and had them telephone the Admiralty for confirmation.
The prying old captain at Geestemunde soon took himself to other parts —by request!
As I explained, my plan was to slip through the British blockade as a neutral and if possible disguised as some other ship that actually existed. There happened to be a Norwegian vessel that was almost a dead ringer for the Pass of Balmaha. She was scheduled to sail from Copenhagen. I decided that we would take her name, and sail the day before she sailed, so that if the British caught us and wirelessed to Copenhagen to confirm our story they would receive word that such a craft had left port at the time we claimed. This other ship was named the Maleta. For some time she had been discharging grain from the Argentine. From Denmark she was to proceed to Christiania and there pick up a cargo. Why not a cargo of lumber for Melbourne?
I went to Copenhagen, donned old clothes, and got a job as a dock walloper on the pier where the real Maleta was moored. That enabled me to study her. There was one thing that promised to be difficult to counterfeit. That was the log book. This precious volume contained the life history of the Maleta, when she left the Argentine, what kind of cargo she carried, what course she steered, the wind, the weather, observations of sun and stars, etc., etc. That log book must be in the captain's cabin and I must have it. But a watchman was stationed aft, so how could it be done?
I discovered that the captain and both mates were still in Norway with their families. So it would be some days before the loss of the book would be noticed—if I got it.
So one night, in the uniform of a customs inspector, I stole aboard the Maleta. The watchman, as usual, was sitting near the captain's cabin. The ship was moored to the pier with ropes fore and aft. Stealthily I tiptoed to the bow and cut the ropes, not quite through but almost. A stiff wind was blowing. The ropes cracked and broke. The ship swung around. The watchman ran forward shouting, and at the same moment I ran aft. Fumbling around the captain's cabin I at first failed to locate the log. Finally, I discovered it under the skipper's mattress. Shoving it beneath my belt, I slipped out.
On board now, and also on the pier, half a dozen men were shouting and throwing ropes to haul her back so she wouldn't side-swipe a near-by ship. I joined in the shouting, pretended to help them for a minute, then clambered on to the dock and hurried off in the dark.
We now put on the final touches that were to turn the Pass of Balmaha into the Maleta. We painted her the same colour as the Maleta, arranged her deck the same, and decorated the cabins with the same ornaments. In my captain's cabin, I hung pictures of the King and Queen of Norway and also of their jovial relative, King Edward VII of England. The barometer, thermometer, and chronometer, and all the other instruments were of Norwegian make. I had a Norwegian library and a Norwegian phonograph and records. We had enough provisions from Norwegian firms to last us through the blockade. It would hardly do to have any Bismarck herring, sauerkraut, and pretzels in sight if the British boarded us, would it?
The names of the tailors sewn inside my suits and my officers' suits were replaced with labels from Norwegian tailors. On my underclothing we embroidered the name of the captain of the Maleta—Knudsen.
I had learned in Copenhagen that a donkey engine was being installed on the Maleta. Very well, we got a donkey engine of the same make from Copenhagen and installed it on our ship. The log book of the Maleta was solemnly put in place, and the first entry was made, "To-day put in a new donkey engine."
We got up our cargo papers in regular form, signed and sealed by both the Norwegian port authorities and British consul. We also had a letter signed by His Majesty's consul at Copenhagen stating that the Maleta was carrying lumber for the use of the Government of the Commonwealth of Australia. The letter requested all British ships to help us if any emergency arose. To prove that this document was genuine, it was even stamped with the British Imperial Seal (made in Germany!).
I also had a letter which a British officer had supposedly written to my shipowner and which my ship-owner had forwarded to me, warning us against German search officers, but advising us to place our trust in the British!
A sailor with the loneliness of the sea upon him nearly always takes with him on his voyages photographs of his people. Now the crews on British warships know sailor ways, so I inquired all about the procedure from captains of neutral ships who had had their ships searched. They told me that the British always inspected the fo'c'sle to see that everything looked right there. I immediately got together a lot of photographs to pass as those of Norwegian sailors' parents, brothers and sisters, uncles and aunts, sweethearts, wives and mothers-in-law. What did it matter whether the sweethearts were good-looking or not? Sailors' sweethearts are not always prize beauties. We sent a man to Norway for the pictures in order to have the names of Norwegian photographers stamped on them.
The British are smart people, by Joe, and they know how to search a ship. They attach special importance to sailors' letters. The sailor eagerly looks forward to the letter he will receive at the next port. He never throws
the letters away either, but always keeps a stack of them in his sea chest. Sometimes you will see him reading a letter that his mother sent him eight years before. So we had to get up a whole set of letters for our "Norwegian" sailors, each set totally different from the other.
Of course, the stolen log of the Maleta gave us a lot of useful information about her crew, and our fake letters were made to tally with this information. Women in the Admiralty and Foreign offices who knew Norwegian wrote them for us. We got old Norwegian stamps and Norwegian postmarks and postmarks of various ports the letters were supposed to have been sent to. Then we aged the letters in chemicals, and tore and smudged some of them.
I picked as my officers men who like myself had spent long years before the mast, who knew Norwegian, and were of the right spirit. First Officer Kling had been a member of the Filchner Expedition, in which he had distinguished himself. The officer whom I selected to go aboard captured ships was a former comrade of mine, a fellow of six feet four, whom I met by chance on a dock. In response to my question whether he wanted to accompany me, he asked:
"Is it one of those trips that is likely to send you to heaven?"
"Yes."
"Then I'm with you. My name is Preiss, and you are after prizes. So I'll bring you luck."
My artillery and navigation officer, Lieutenant Kircheiss, was a wizard navigator. Engineer Krauss was our motor expert. The boatswain, the carpenter, and the cook, the three mainstays of a voyage in a sailing vessel, I picked with like care. Of the men who were to go with me I only needed twenty-seven with a knowledge of Norwegian. There were just twentyseven aboard the real Maleta. In selecting my men, I interviewed each candidate personally but gave him no hint of why I wanted him. I tried to read these men's souls in order to discover in them the qualities of courage and endurance that would be needed.
Without giving them any clue concerning the adventure on which they were soon to engage, I sent them home on furlough to prevent them from meeting one another and talking over the questions I had put to them. Not until the hour of departure did I send for them.
Now we needed a name for our raider. We needed one that she could take for her official name as an auxiliary cruiser after running the blockade. I wanted to call her the Albatross out of gratitude to the albatross that saved me from drowning when I was a lad. But I discovered that there was already a vessel with that name, a mine-layer. Then I wanted to call the ship the Sea Devil, the name by which I personally was afterward to be called. My officers favoured some name that would suggest the white wings of our sailship. So we compromised on Seeadler, or Sea Eagle.
On a pitch-dark November night, the Seeadler, with a small emergency crew, raises anchor and sails out of the mouth of the Weser into the North Sea. There, some distance offshore, we drop anchor.
At a remote place along the docks at Wilhelmshaven, men appear one by one. By the light of a dimly burning lantern I gather my crew. None of them has any inkling of what is afoot. I hear them ask:
"Where are we off to? What is it?"
We piled them into a little steamer, and off. Soon they saw an imposing ship riding through the night.
"Hello, what sort of craft is this, a sailship?"
Aboard everything is ready, and everything is Norwegian. Their bunks are all prepared. Photographs are on the walls. Norwegian landscapes, photographs of Norwegian girls, Norwegian flags hang draped. A fully equipped Norwegian ship awaiting the arrival of its crew.
"Do you speak Norwegian, Karl?"
"Yes. Do you?"
"Yes."
"Strange business this!"
Some of the men do not speak Norwegian. The ones that do, have their bunks above deck. The ones that don't, have their bunks below. Germany below decks. Norway above. Strange!
We were away from all communication with land now. There was no longer need for secrecy.
"Boys, the British say not even a mouse can get through their blockade. But we will show them, by Joe, and under full pressure of sail. Then, once we reach the high seas, we will sink their ships, by Joe. Can we do it?"
"Sure, Count, we can do it! By Joe, you bet we can do it!" Not a man quailed, and I was happy to be in command of such a crew.
Next morning a scow of lumber lay alongside, and we stacked timber to a height of six feet over all the deck, and fastened it down with wire and chains.
Every man had his rôle. Every man must now prove his mettle as an actor. Officers and sailors were given the names of officers and sailors aboard the Maleta. They had to get used to their new names. Fritz Meyer was now Ole Johnsen, Miller became Bjornsen, Hans Lehman became Lars Carlsen, and they knew me only as Captain Knudsen. We had long practice drills until the new names slid off our tongues without getting stuck.
Each man also had to learn a lot about his native town that he never knew before! I had already assembled as much information as I could about the towns listed in the stolen log book, and the rest we invented. Each man had to learn the names of the main streets of his town, the principal hotels, taverns, and drug stores, as well as the names of the mayor and other officials. Much of this sort of material had already been woven into the letters we had prepared for the sailors. Each man had to familiarize himself with the set of photographs that had been allotted to him, and the names of them all, the contents of his letters, and fix in his mind a whole new past life, according to the life of the sailor of the real Maleta whose rôle he was to play.
One of the mechanics' helpers, Schmidt by name, I had taken for a principal rôle in our strategy. He was slender, beardless, and of delicate appearance, and could pass well enough in woman's clothes. Norwegian skippers often take their wives with them on their voyages. The captain's wife aboard the false Maleta would seem natural and tend to disarm suspicion, and, besides, British naval officers are always courteous and considerate toward women. In the presence of the captain's wife, a prize officer who might board us would be more obliging toward us all. We had a blonde wig for Schmidt and an outfit of women's clothes. We took great pains in schooling him to play the part of the captain's wife correctly. One difficulty was his big feet. Not even a Norwegian skipper's wife had such feet. There was, unfortunately, no way to make them smaller, so we arranged that the captain's wife should be slightly ill and remain seated during any possible search and have a rug thrown over her feet to keep them warm. The other difficulty was Schmidt's voice. It was too deep, and he knew no Norwegian. Well, the captain's wife can't talk because she has an awful toothache. A wad of cotton stuffed into Schmidt's cheek, and there was the swelling. He did know enough English to say "all right." We trained him to say a high-pitched "all right" something like a woman with a toothache. Except for that phrase, he was to keep his mouth shut. We had a large photograph made of Schmidt in his costume, signed it "thy loving Josephine," and hung it in my cabin. Now the Britishers could compare the photograph of the captain's wife with the lady in person. So from now on poor Schmidt's name was "Josefeena" as the Norwegians pronounce it.
We were ready to sail when, by Joe, what comes but a telegram from the Kaiser's aide. I am to report immediately direct to His Majesty. I guessed what was up. I had gone into the navy from the mercantile marine instead of through the usual cadet route. I had been a common ordinary sailor, and this had aroused a lot of antagonism in naval circles. There had been jealousy about my getting an independent command—highest of all naval honours. So attempts were being made to have my assignment annulled.
And now they had gone to the Emperor! Maybe I would lose this fine sailship of mine. Already it had given me a new lease on life, just by getting back into the old life, the life that had been so difficult to survive and so delightful to recall. Maybe I would have to go back to the navy, to the modern war of hissing steel, and deafening guns of superdreadnaughts. I had an affection for them too, but it was the enthusiasm of the mind. Here on the sailship was my heart. Well, I would fight them.
"Luckner," I thought, "you always have to fight, or you sink. That's life."
The Emperor had been very kind to the man who had risen from a common sailor to a naval officer. He had paid for my naval training out of his own private purse, and had taken a personal interest in my promotions. Many a time on board ship he had commanded me to tell stories of my adventures. I could talk to him. I could talk to him more boldly than other officers dared. I knew that he understood me.
Even to appear in the Imperial presence was a trying ordeal for most officers. Many took refuge in rigid "attention." Well, I had never quite got used to high class manners at sea, and the ramrod "attention" left me more embarrassed than otherwise. Even in the Emperor's presence, I kept the same free, brusque manner of an old-time seaman that was natural to me.
The Kaiser spoke bluntly.
"Well, Luckner, at the Admiralty they now tell me it is madness to attempt the blockade with a sailing ship. What do you think?"
"Well, Your Majesty, if our Admiralty says it's impossible and ridiculous, then I'm sure it can be done," I replied. "For the British Admiralty will think it impossible also. They won't be on the lookout for anything so absurd as a raider disguised as a harmless old sailing ship."
The Emperor looked at me with a frown, and then his face relaxed into a smile.
"You are right, Luckner. Go ahead! And may the hand of the Almighty be at your helm."
I knew now that there would be no more official interference. The true Maleta was now due to sail in a day, so we made ready to pull up anchor. Then a wireless came from the Admiralty:
Wait till the Deutschland makes port.
Our giant merchant submarine, the Deutschland, was on her way home from her famous transatlantic cruise to America. In an attempt to cut her off, the British had set a double watch. So the Seeadler would have to slip past twice as many cruisers and destroyers as otherwise. I still hoped that, if only detained a day or so, we might yet be able to slip across the North Sea ahead of the Maleta. But we lay there for three and a half weeks, and the sad news came that the real Maleta had sailed and passed through the blockade. If we now attempted to use her name and a search party boarded us, the jig would be up.
So we hurriedly examined Lloyd's Register in the hope of finding another Norwegian ship that might correspond to us. We picked out one called the Carmoe. We had no idea where she was, but hoped she might be in some distant port unbeknown to the wary British. It was a long chance, but we could think of nothing better. Now we had to change our ship from the Maleta to the Carmoe. Painting out one name and substituting another was easy enough, but changing all our ship's papers was far more difficult.
But with much use of chemical eraser we finally accomplished it, and we had papers that would pass if the visibility was not too bright during the search. Then, when we were all set again, we picked up a copy of a Norwegian commercial paper and found that the real Carmoe had just been seized by the British and taken to Kirkwall for examination.
"By Joe, and they said this Pass of Balmaha was a lucky ship! We must have a Jonah on board!"
Now, if you haven't any luck, you must go and get some! All you have to do is know how to do that, and you will be a great success at sea, or anywhere!
So away with Lloyd's Register! Let's take life's register and name our sea eagle after the girl of my heart. Surely she will bring us luck. So, out with the paint and on with another new name—the name of my sweetheart, Irma.
In that name was concentrated most of the beauty that I had found in life. It symbolized strange moments of beauty that had crossed my path during the most trying days I had so far known. It seemed to be a lovely silken thread that had run through the years since that first voyage, when as a miserable cabin boy I sailed to Australia on that Russian tramp.
Of course, there was no such name as Irma listed with Lloyd's, and all any British officer would have to do would be to consult his Register and the jig would be up. But somehow I had a premonition that the name Irma would bring us through.
When we applied eraser and ink to our shipping papers and wrote in the name of Irma—disaster. Two erasures were too much. The ink blotted. If we should be stupid enough to take the British for fools, then we ourselves would be the real fools. Where was our luck now? Fate seemed to be against us, but I had no intention of giving up. Calling the carpenter I said:
"Come on, Chips, I am going to make you admiral of the day. Get the ax and smash all the bull's-eyes, windows, portholes, and everything."
