Exploring the role of sex in the association of late chronotype on cardiorespiratory fitness.

Circadian rhythms differ between young adult males and females. For example, males tend to be later chronotypes, preferring later timing of sleep and activity, than females. Likewise, there are sex differences in body composition and cardiorespiratory fitness. Few studies have investigated the association between circadian rhythms, cardiorespiratory fitness, and body composition. We sought to determine whether chronotype and circadian phase were associated with cardiorespiratory fitness, body composition, and anthropometric measures in sedentary males and females. Fifty-nine adults participated in the study. Circadian phase and chronotype were measured using dim light melatonin onset (DLMO) and the Morningness-Eveningness Questionnaire (MEQ) score. We used peak oxygen uptake (VO2peak ) results from a maximal graded exercise test to assess cardiorespiratory fitness. Body composition, BMI, and circumferences were collected as markers of adiposity. We observed a sex difference in the association between DLMO and VO2peak . For males, a later DLMO was associated with a lower VO2peak . VO2peak did not vary based on DLMO in females. Later circadian phase was also associated with increased body fat percentage, fat mass index, and abdominal circumference in males, but not females. Collectively, these results suggest that males who are later chronotypes may be at risk of obesity and low cardiorespiratory fitness.

Long days restore regular estrous cyclicity in mice lacking circadian rhythms.

Many female mammals have recurring cycles of ovulation and sexual behaviors that are regulated by reproductive hormones and confer reproductive success. In addition to sexual behaviors, circadian behavioral rhythms of locomotor activity also fluctuate across the estrous cycle in rodents. Moreover, there is a bidirectional relationship between circadian rhythms and estrous cyclicity since mice with disrupted circadian rhythms also have compromised estrous cycles resulting in fewer pregnancies. In the present study, we assessed whether extending day length, which alters circadian rhythms, normalizes estrous cyclicity in mice. We found that Period (Per) 1/2/3 triple knockout (KO) mice, that have disabled canonical molecular circadian clocks, have markedly disrupted estrous cycles. Surprisingly, extending the day length by only 2 h per day restored regular 4- or 5-day estrous cycles to Per1/2/3 KO mice. Longer days also induced consistent 4-day, rather than 5-day, estrous cycles in wild-type C57BL/6J mice. These data demonstrate that extending daytime light exposure could be used for enhancing reproductive success.

Chronic environmental circadian disruption increases atherosclerosis and dyslipidemia in female, but not male, ApolipoproteinE-deficient mice.

Shift work chronically disrupts circadian rhythms and increases the risk of developing cardiovascular disease. However, the mechanisms linking shift work and cardiovascular disease are largely unknown. The goal of this study was to investigate the effects of chronically shifting the light-dark (LD) cycle, which models the disordered exposure to light that may occur during shift work, on atherosclerosis. Atherosclerosis is the progressive accumulation of lipid-filled lesions within the artery wall and is the leading cause of cardiovascular disease. We studied ApolipoproteinE-deficient (ApoE -/- ) mice that are a well-established model of atherosclerosis. Male and female ApoE -/- mice were housed in control 12L:12D or chronic LD shift conditions for 12 weeks and fed low-fat diet. In the chronic LD shift condition, the light-dark cycle was advanced by 6 h every week. We found that chronic LD shifts exacerbated atherosclerosis in female, but not male, ApoE -/- mice. In females, chronic LD shifts increased total serum cholesterol concentrations with increased atherogenic VLDL/LDL particles. Chronic LD shifts did not affect food intake, activity, or body weight in male or female ApoE -/- mice. We also examined eating behavior in female ApoE -/- mice since aberrant meal timing has been linked to atherosclerosis. The phases of eating behavior rhythms, like locomotor activity rhythms, gradually shifted to the new LD cycle each week in the chronic LD shift group, but there was no effect of the LD shift on the amplitudes of the eating rhythms. Moreover, the duration of fasting intervals was not different in control 12L:12D compared to chronic LD shift conditions. Together these data demonstrate that female ApoE -/- mice have increased atherosclerosis when exposed to chronic LD shifts due to increased VLDL/LDL cholesterol, independent of changes in energy balance or feeding-fasting cycles.

Heart rate recovery as an assessment of cardiorespiratory fitness in young adults.

Background: Cardiorespiratory fitness, typically measured as peak oxygen uptake (VO2peak) during maximal graded exercise testing (GXTmax), is a predictor of morbidity, mortality, and cardiovascular disease. However, measuring VO2peak is costly and inconvenient and thus not widely used in clinical settings. Alternatively, postexercise heart rate recovery (HRRec), which is an index of vagal reactivation, is a valuable assessment of VO2peak in older adults and athletes. However, the validity of HRRec as a clinical indicator of cardiorespiratory fitness in young, sedentary adults, who are a rapidly growing population at risk for developing obesity and cardiovascular disease, has not been fully elucidated. Methods: We investigated the association between cardiorespiratory fitness, measured by VO2peak (mL·kg-1·min-1), and HRRec measures after a GXTmax in 61 young (25.2 ± 6.1 years), sedentary adults (40 females) using 3 methods. We examined the relationship between VO2peak and absolute (b·min-1) and relative (%) HRRec measures at 1, 2, and 3 min post GXTmax, as well as a measure of the slow component HRRec (HRRec 1 min minus HRR 2 min), using Pearson's correlation analysis. Results: VO2peak (36.5 ± 7.9 mL·kg-1·min-1) was not significantly correlated with absolute HRRec at 1 min (r = 0.18), 2 min (r = 0.04) or 3 min (r = 0.01). We also found no significant correlations between VO2peak and relative HRRec at 1 min (r = 0.09), 2 min (r = -0.06) or 3 min (r = -0.10). Lastly, we found no correlation between the measure of the slow component HRRec and VO2peak (r = -0.14). Conclusions: Our results indicate that HRRec measures are not a valid indicator of cardiorespiratory fitness in young, sedentary adults.

Genetics and functional significance of the understudied methamphetamine sensitive circadian oscillator (MASCO).

The last 50 years have witnessed extraordinary discoveries in the field of circadian rhythms. However, there are still several mysteries that remain. One of these chronobiological mysteries is the circadian rhythm that is revealed by administration of stimulant drugs to rodents. Herein we describe the discovery of this circadian rhythm and its underlying oscillator, which is frequently called the methamphetamine-sensitive circadian oscillator, or MASCO. This oscillator is distinct from canonical circadian oscillators because it controls robust activity rhythms independently of the suprachiasmatic nucleus and circadian genes are not essential for its timekeeping. We discuss these fundamental properties of MASCO and integrate studies of strain, sex, and circadian gene mutations on MASCO. The anatomical loci of MASCO are not known, so it has not been possible thus far to discover its novel molecular timekeeping mechanism or its functional significance. However, studies in mutant mice suggest that genetic approaches can be used to identify the neural network involved in the rhythm generation of MASCO. We also discuss parallels between human and rodent studies that support our working hypothesis that a function of MASCO may be to regulate sleep-wake cycles.

Estrogens and the circadian system.

Circadian rhythms are ~24 h cycles of behavior and physiology that are generated by a network of molecular clocks located in nearly every tissue in the body. In mammals, the circadian system is organized hierarchically such that the suprachiasmatic nucleus (SCN) is the main circadian clock that receives light information from the eye and entrains to the light-dark cycle. The SCN then coordinates the timing of tissue clocks so internal rhythms are aligned with environmental cycles. Estrogens interact with the circadian system to regulate biological processes. At the molecular level, estrogens and circadian genes interact to regulate gene expression and cell biology. Estrogens also regulate circadian behavior across the estrous cycle. The timing of ovulation during the estrous cycle requires coincident estrogen and SCN signals. Studies using circadian gene reporter mice have also elucidated estrogen regulation of peripheral tissue clocks and metabolic rhythms. This review synthesizes current understanding of the interplay between estrogens and the circadian system, with a focus on female rodents, in regulating molecular, physiological, and behavioral processes.

High-fat feeding disrupts daily eating behavior rhythms in obesity-prone but not in obesity-resistant male inbred mouse strains.

Abnormal meal timing, like skipping breakfast and late-night snacking, is associated with obesity in humans. Disruption of daily eating rhythms also contributes to obesity in mice. When fed a high-fat diet, male C57BL/6J mice have disrupted eating behavior rhythms and they become obese. In contrast to obesity-prone C57BL/6J mice, some inbred strains of mice are resistant to high-fat diet-induced obesity. In this study, we sought to determine whether there are distinct effects of high-fat feeding on daily eating behavior rhythms in obesity-prone and obesity-resistant male mice. Male obesity-prone (C57BL/6J and 129X1/SvJ) and obesity-resistant (SWR/J and BALB/cJ) mice were fed low-fat diet or high-fat diet for 6 wk. Consistent with previous studies, obesity-prone male mice gained more weight and adiposity during high-fat diet feeding than obesity-resistant male mice. The amplitude of the daily rhythm of eating behavior was markedly attenuated in male obesity-prone mice fed high-fat diet, but not in obesity-resistant males. In contrast, high-fat feeding did not differentially affect locomotor activity rhythms in obesity-prone and obesity-resistant male mice. Together, these data suggest that regulation of the daily rhythm of eating may underlie the propensity to develop diet-induced obesity in male mice.

Circadian disruption with constant light exposure exacerbates atherosclerosis in male ApolipoproteinE-deficient mice.

Disruption of the circadian system caused by disordered exposure to light is pervasive in modern society and increases the risk of cardiovascular disease. The mechanisms by which this happens are largely unknown. ApolipoproteinE-deficient (ApoE-/-) mice are studied commonly to elucidate mechanisms of atherosclerosis. In this study, we determined the effects of light-induced circadian disruption on atherosclerosis in ApoE-/- mice. We first characterized circadian rhythms of behavior, light responsiveness, and molecular timekeeping in tissues from ApoE-/- mice that were indistinguishable from rhythms in ApoE+/+ mice. These data showed that ApoE-/- mice had no inherent circadian disruption and therefore were an appropriate model for our study. We next induced severe disruption of circadian rhythms by exposing ApoE-/- mice to constant light for 12 weeks. Constant light exposure exacerbated atherosclerosis in male, but not female, ApoE-/- mice. Male ApoE-/- mice exposed to constant light had increased serum cholesterol concentrations due to increased VLDL/LDL fractions. Taken together, these data suggest that ApoE-/- mice are an appropriate model for studying light-induced circadian disruption and that exacerbated dyslipidemia may mediate atherosclerotic lesion formation caused by constant light exposure.

Circadian rhythm phase shifts caused by timed exercise vary with chronotype.

BACKGROUNDThe circadian system entrains behavioral and physiological rhythms to environmental cycles, and modern lifestyles disrupt this entrainment. We investigated a timed exercise intervention to phase shift the internal circadian rhythm.METHODSIn 52 young, sedentary adults, dim light melatonin onset (DLMO) was measured before and after 5 days of morning (10 hours after DLMO; n = 26) or evening (20 hours after DLMO; n = 26) exercise. Phase shifts were calculated as the difference in DLMO before and after exercise.RESULTSMorning exercise induced phase advance shifts (0.62 ± 0.18 hours) that were significantly greater than phase shifts from evening exercise (-0.02 ± 0.18 hours; P = 0.01). Chronotype also influenced the effect of timed exercise. For later chronotypes, both morning and evening exercise induced phase advances (0.54 ± 0.29 hours and 0.46 ±0.25 hours, respectively). In contrast, earlier chronotypes had phase advances from morning exercise (0.49 ± 0.25 hours) but had phase delays from evening exercise (-0.41 ± 0.29 hours).CONCLUSIONLate chronotypes - those who experience the most severe circadian misalignment - may benefit from phase advances induced by exercise in the morning or evening, but evening exercise may exacerbate circadian misalignment in early chronotypes. Thus, personalized exercise timing prescription, based on chronotype, could alleviate circadian misalignment in young adults.TRIAL REGISTRATIONTrial registration can be found at www.clinicaltrials.gov (NCT04097886).FUNDINGFunding was supplied by NIH grants UL1TR001998 and TL1TR001997, the Barnstable Brown Diabetes and Obesity Center, the Pediatric Exercise Physiology Laboratory Endowment, the Arvle and Ellen Turner Thacker Research Fund, and the University of Kentucky.

Estradiol regulates daily rhythms underlying diet-induced obesity in female mice.

The circadian system is a critical regulator of metabolism and obesity in males, but its role in regulating obesity in females is poorly understood. Because there are sex differences in the development of obesity and susceptibility to obesity-related disorders, we sought to determine the role of estrogens in regulating the circadian mechanisms underlying diet-induced obesity. When fed high-fat diet, C57BL/6J male mice gain weight, whereas females are resistant to diet-induced obesity. Here, we demonstrate that estradiol regulates circadian rhythms in females to confer resistance to diet-induced obesity. We found that ovariectomized females with undetectable circulating estrogens became obese and had disrupted daily rhythms of eating behavior and locomotor activity when fed a high-fat diet. The phase of the liver molecular circadian rhythm was also altered by high-fat diet feeding in ovariectomized mice. Estradiol replacement in ovariectomized females a fed high-fat diet rescued these behavioral and tissue rhythms. Additionally, restoring the daily rhythm of eating behavior in ovariectomized females with time-restricted feeding inhibited diet-induced obesity and insulin resistance. Together, these data suggest that the circadian system is a target for treating obesity and its comorbidities in women after menopause, when circulating levels of estrogens are too low to protect their circadian rhythms.

Peripheral Circadian Oscillators.

Circadian rhythms are ~24-hour cycles of physiology and behavior that are synchronized to environmental cycles, such as the light-dark cycle. During the 20th century, most research focused on establishing the fundamental properties of circadian rhythms and discovering circadian pacemakers that were believed to reside in the nervous system of animals. During this time, studies that suggested the existence of circadian oscillators in peripheral organs in mammals were largely dismissed. The discovery of a single-locus circadian pacemaker in the nervous system of several animals affirmed the single-oscillator model of the circadian system. However, the discovery of the genes that constituted the molecular timekeeping system provided the tools for demonstrating the existence of bona fide circadian oscillators in nearly every peripheral tissue in animals, including rodents, in the late 1990s and early 2000s. These studies led to our current understanding that the circadian system in animals is a hierarchical multi-oscillatory network, composed of master pacemaker(s) in the brain and oscillators in peripheral organs. Further studies showed that altering the temporal relationship between these oscillators by simulating jet-lag and metabolic challenges in rodents caused adverse physiological outcomes. Herein we review the studies that led to our current understanding of the function and pathology of the hierarchical multi-oscillator circadian system.

The Mysterious Food-Entrainable Oscillator: Insights from Mutant and Engineered Mouse Models.

The food-entrainable oscillator (FEO) is a mysterious circadian clock because its anatomical location(s) and molecular timekeeping mechanism are unknown. Food anticipatory activity (FAA), which is defined as the output of the FEO, emerges during temporally restricted feeding. FAA disappears immediately during ad libitum feeding and reappears during subsequent fasting. A free-running FAA rhythm has been observed only in rare circumstances when food was provided with a period outside the range of entrainment. Therefore, it is difficult to study the circadian properties of the FEO. Numerous studies have attempted to identify the critical molecular components of the FEO using mutant and genetically engineered mouse models. Herein we critically review the experimental protocols and findings of these studies in mouse models. Several themes emerge from these studies. First, there is little consistency in restricted feeding protocols between studies. Moreover, the protocols were sometimes not optimal, resulting in erroneous conclusions that FAA was absent in some mouse models. Second, circadian genes are not necessary for FEO timekeeping. Thus, another noncanonical timekeeping mechanism must exist in the FEO. Third, studies of mouse models have shown that signaling pathways involved in circadian timekeeping, reward (dopaminergic), and feeding and energy homeostasis can modulate, but are not necessary for, the expression of FAA. In sum, the approaches to date have been largely unsuccessful in discovering the timekeeping mechanism of the FEO. Moving forward, we propose the use of standardized and optimized experimental protocols that focus on identifying genes that alter the period of FAA in mutant and engineered mouse models. This approach is likely to permit discovery of molecular components of the FEO timekeeping mechanism.

mPeriod2 Brdm1 and other single Period mutant mice have normal food anticipatory activity.

Animals anticipate the timing of food availability via the food-entrainable oscillator (FEO). The anatomical location and timekeeping mechanism of the FEO are unknown. Several studies showed the circadian gene, Period 2, is critical for FEO timekeeping. However, other studies concluded that canonical circadian genes are not essential for FEO timekeeping. In this study, we re-examined the effects of the Per2 Brdm1 mutation on food entrainment using methods that have revealed robust food anticipatory activity in other mutant lines. We examined food anticipatory activity, which is the output of the FEO, in single Period mutant mice. Single Per1, Per2, and Per3 mutant mice had robust food anticipatory activity during restricted feeding. In addition, we found that two different lines of Per2 mutant mice (ldc and Brdm1) anticipated restricted food availability. To determine if FEO timekeeping persisted in the absence of the food cue, we assessed activity during fasting. Food anticipatory (wheel-running) activity in all Period mutant mice was also robust during food deprivation. Together, our studies demonstrate that the Period genes are not necessary for the expression of food anticipatory activity.

High-Fat Feeding Does Not Disrupt Daily Rhythms in Female Mice because of Protection by Ovarian Hormones.

Obesity in women is increased by the loss of circulating estrogen after menopause. Shift work, which disrupts circadian rhythms, also increases the risk for obesity. It is not known whether ovarian hormones interact with the circadian system to protect females from obesity. During high-fat feeding, male C57BL/6J mice develop profound obesity and disruption of daily rhythms. Since C57BL/6J female mice did not develop diet-induced obesity (during 8 weeks of high-fat feeding), we first determined if daily rhythms in female mice were resistant to disruption from high-fat diet. We fed female PERIOD2:LUCIFERASE mice 45% high-fat diet for 1 week and measured daily rhythms. Female mice retained robust rhythms of eating behavior and locomotor activity during high-fat feeding that were similar to chow-fed females. In addition, the phase of the liver molecular timekeeping (PER2:LUC) rhythm was not altered by high-fat feeding in females. To determine if ovarian hormones protected daily rhythms in female mice from high-fat feeding, we analyzed rhythms in ovariectomized mice. During high-fat feeding, the amplitudes of the eating behavior and locomotor activity rhythms were reduced in ovariectomized females. Liver PER2:LUC rhythms were also advanced by ~4 h by high-fat feeding, but not chow, in ovariectomized females. Together these data show circulating ovarian hormones protect the integrity of daily rhythms in female mice during high-fat feeding.

Disruption of Daily Rhythms by High-Fat Diet Is Reversible.

In mammals a network of circadian clocks coordinates behavior and physiology with 24-h environmental cycles. Consumption of high-fat diet disrupts this temporal coordination by advancing the phase of the liver molecular clock and altering daily rhythms of eating behavior and locomotor activity. In this study we sought to determine whether these effects of high-fat diet on circadian rhythms were reversible. We chronically fed mice high-fat diet and then returned them to low-fat chow diet. We found that the phase of the liver PERIOD2::LUCIFERASE rhythm was advanced (by 4h) and the daily rhythms of eating behavior and locomotor activity were altered for the duration of chronic high-fat diet feeding. Upon diet reversal, the eating behavior rhythm was rapidly reversed (within 2 days) and the phase of the liver clock was restored by 7 days of diet reversal. In contrast, the daily pattern of locomotor activity was not restored even after 2 weeks of diet reversal. Thus, while the circadian system is sensitive to changes in the macronutrient composition of food, the eating behavior rhythm and liver circadian clock are specifically tuned to respond to changes in diet.

Genetic variation in melatonin pathway enzymes in children with autism spectrum disorder and comorbid sleep onset delay.

Sleep disruption is common in individuals with autism spectrum disorder (ASD). Genes whose products regulate endogenous melatonin modify sleep patterns and have been implicated in ASD. Genetic factors likely contribute to comorbid expression of sleep disorders in ASD. We studied a clinically unique ASD subgroup, consisting solely of children with comorbid expression of sleep onset delay. We evaluated variation in two melatonin pathway genes, acetylserotonin O-methyltransferase (ASMT) and cytochrome P450 1A2 (CYP1A2). We observed higher frequencies than currently reported (p < 0.04) for variants evidenced to decrease ASMT expression and related to decreased CYP1A2 enzyme activity (p ≤ 0.0007). We detected a relationship between genotypes in ASMT and CYP1A2 (r(2) = 0.63). Our results indicate that expression of sleep onset delay relates to melatonin pathway genes.

Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity.

Metabolic disorders, including obesity, diabetes, and cardiovascular disease, are widespread in Westernized nations. Gut microbiota composition is a contributing factor to the susceptibility of an individual to the development of these disorders; therefore, altering a person's microbiota may ameliorate disease. One potential microbiome-altering strategy is the incorporation of modified bacteria that express therapeutic factors into the gut microbiota. For example, N-acylphosphatidylethanolamines (NAPEs) are precursors to the N-acylethanolamide (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reduce food intake and obesity. Here, we demonstrated that administration of engineered NAPE-expressing E. coli Nissle 1917 bacteria in drinking water for 8 weeks reduced the levels of obesity in mice fed a high-fat diet. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis compared with mice receiving standard water or control bacteria. The protective effects conferred by NAPE-expressing bacteria persisted for at least 4 weeks after their removal from the drinking water. Moreover, administration of NAPE-expressing bacteria to TallyHo mice, a polygenic mouse model of obesity, inhibited weight gain. Our results demonstrate that incorporation of appropriately modified bacteria into the gut microbiota has potential as an effective strategy to inhibit the development of metabolic disorders.

Wheel-running activity modulates circadian organization and the daily rhythm of eating behavior.

Consumption of high-fat diet acutely alters the daily rhythm of eating behavior and circadian organization (the phase relationship between oscillators in central and peripheral tissues) in mice. Voluntary wheel-running activity counteracts the obesogenic effects of high-fat diet and also modulates circadian rhythms in mice. In this study, we sought to determine whether voluntary wheel-running activity could prevent the proximate effects of high-fat diet consumption on circadian organization and behavioral rhythms in mice. Mice were housed with locked or freely rotating running wheels and fed chow or high-fat diet for 1 week and rhythms of locomotor activity, eating behavior, and molecular timekeeping (PERIOD2::LUCIFERASE luminescence rhythms) in ex vivo tissues were measured. Wheel-running activity delayed the phase of the liver rhythm by 4 h in both chow- and high-fat diet-fed mice. The delayed liver phase was specific to wheel-running activity since an enriched environment without the running wheel did not alter the phase of the liver rhythm. In addition, wheel-running activity modulated the effect of high-fat diet consumption on the daily rhythm of eating behavior. While high-fat diet consumption caused eating events to be more evenly dispersed across the 24 h-day in both locked-wheel and wheel-running mice, the effect of high-fat diet was much less pronounced in wheel-running mice. Together these data demonstrate that wheel-running activity is a salient factor that modulates liver phase and eating behavior rhythms in both chow- and high-fat-diet fed mice. Wheel-running activity in mice is both a source of exercise and a self-motivating, rewarding behavior. Understanding the putative reward-related mechanisms whereby wheel-running activity alters circadian rhythms could have implications for human obesity since palatable food and exercise may modulate similar reward circuits.

Effects of light, food, and methamphetamine on the circadian activity rhythm in mice.

The circadian rhythm of locomotor activity in mice is synchronized to environmental factors such as light and food availability. It is well-known that entrainment of the activity rhythm to the light-dark cycle is attained by the circadian pacemaker in the suprachiasmatic nucleus (SCN). Locomotor activity is also controlled by two extra-SCN oscillators; periodic food availability entrains the food-entrainable oscillator (FEO) and constant consumption of low-dose methamphetamine reveals the output of the methamphetamine-sensitive circadian oscillator (MASCO). In this study, we sought to investigate the relationship between the SCN, FEO, and MASCO by examining the combinatorial effects of light, food restriction, and/or methamphetamine on locomotor activity. To investigate coupling between the SCN and FEO, we tested whether food anticipatory activity, which is the output of the FEO, shifted coordinately with phase shifts of the light-dark cycle. We found that the phase of food anticipatory activity was phase-delayed or phase-advanced symmetrically with the respective shift of the light-dark cycle, suggesting that the FEO is strongly coupled to the SCN and the phase angle between the SCN and FEO is maintained during ad libitum feeding. To examine the effect of methamphetamine on the output of the FEO, we administered methamphetamine to mice undergoing restricted feeding and found that food-entrained activity was delayed by methamphetamine treatment. In addition, restricted feeding induced dissociation of the MASCO and SCN activity rhythms during short-term methamphetamine treatment, when these rhythms are typically integrated. In conclusion, our data suggest that the outputs of the SCN, FEO and MASCO collectively drive locomotor activity.