Metabolic effects of bariatric surgery in mouse models of circadian disruption.

BACKGROUND/OBJECTIVES: Mounting evidence supports a link between circadian disruption and metabolic disease. Humans with circadian disruption (for example, night-shift workers) have an increased risk of obesity and cardiometabolic diseases compared with the non-disrupted population. However, it is unclear whether the obesity and obesity-related disorders associated with circadian disruption respond to therapeutic treatments as well as individuals with other types of obesity. SUBJECTS/METHODS: Here, we test the effectiveness of the commonly used bariatric surgical procedure, Vertical Sleeve Gastrectomy (VSG), in mouse models of genetic and environmental circadian disruption. RESULTS: VSG led to a reduction in body weight and fat mass in both Clock(Δ19) mutant and constant-light mouse models (P<0.05), resulting in an overall metabolic improvement independent of circadian disruption. Interestingly, the decrease in body weight occurred without altering diurnal feeding or activity patterns (P>0.05). Within circadian-disrupted models, VSG also led to improved glucose tolerance and lipid handling (P<0.05). CONCLUSIONS: Together these data demonstrate that VSG is an effective treatment for the obesity associated with circadian disruption, and that the potent effects of bariatric surgery are orthogonal to circadian biology. However, as the effects of bariatric surgery are independent of circadian disruption, VSG cannot be considered a cure for circadian disruption. These data have important implications for circadian-disrupted obese patients. Moreover, these results reveal new information about the metabolic pathways governing the effects of bariatric surgery as well as of circadian disruption.

Disrupted sleep-wake regulation in the MCI-Park mouse model of Parkinson's disease.

Disrupted sleep has a profound adverse impact on lives of Parkinson's disease (PD) patients and their caregivers. Sleep disturbances are exceedingly common in PD, with substantial heterogeneity in type, timing, and severity. Among the most common sleep-related symptoms reported by PD patients are insomnia, excessive daytime sleepiness, and sleep fragmentation, characterized by interruptions and decreased continuity of sleep. Alterations in brain wave activity, as measured on the electroencephalogram (EEG), also occur in PD, with changes in the pattern and relative contributions of different frequency bands of the EEG spectrum to overall EEG activity in different vigilance states consistently observed. The mechanisms underlying these PD-associated sleep-wake abnormalities are poorly understood, and they are ineffectively treated by conventional PD therapies. To help fill this gap in knowledge, a new progressive model of PD - the MCI-Park mouse - was studied. Near the transition to the parkinsonian state, these mice exhibited significantly altered sleep-wake regulation, including increased wakefulness, decreased non-rapid eye movement (NREM) sleep, increased sleep fragmentation, reduced rapid eye movement (REM) sleep, and altered EEG activity patterns. These sleep-wake abnormalities resemble those identified in PD patients. Thus, this model may help elucidate the circuit mechanisms underlying sleep disruption in PD and identify targets for novel therapeutic approaches.

Exercise-induced promotion of hippocampal cell proliferation requires beta-endorphin.

Adult hippocampal neurogenesis is influenced by a variety of stimuli, including exercise, but the mechanisms by which running affects neurogenesis are not yet fully understood. Because beta-endorphin, which is released in response to exercise, increases cell proliferation in vitro, we hypothesized that it could exert a similar effect in vivo and mediate the stimulatory effects of running on neurogenesis. We thus analyzed the effects of voluntary wheel-running on adult neurogenesis (proliferation, differentiation, survival/death) in wild-type and beta-endorphin-deficient mice. In wild-type mice, exercise promoted cell proliferation evaluated by sacrificing animals 24 h after the last 5-bromo-2'-deoxyuridine (BrdU) pulse and by using endogenous cell cycle markers (Ki67 and pH(3)). This was accompanied by an increased survival of 4-wk-old BrdU-labeled cells, leading to a net increase of neurogenesis. Beta-endorphin deficiency had no effect in sedentary mice, but it completely blocked the running-induced increase in cell proliferation; this blockade was accompanied by an increased survival of 4-wk-old cells and a decreased cell death. Altogether, adult neurogenesis was increased in response to exercise in knockout mice. We conclude that beta-endorphin released during running is a key factor for exercise-induced cell proliferation and that a homeostatic balance may regulate the final number of new neurons.

Circadian involvement in termination of the refractory period in two sparrows.

Endogenous daily rhythmicity is involved in the short-day photoperiodic response of breaking the refractory period in both white-crowned and golden-crowned sparrows. Under a modified "coincidence model," short-day photoperiodic induction occurs when light is noncoincident with a specific phase of an internal rhythm.

Multiple circadian oscillators regulate the timing of behavioral and endocrine rhythms in female golden hamsters.

A single daily "surge" in pituitary luteinizing hormone release was observed in ovariectomized-estrogen-treated hamsters expressing an intact circadian rhythm of locomotor activity. In contrast, two luteinizing hormone surges occurred within a single 24-hour period in hamsters whose activity rhythm had dissociated or "split" into two distinct components. These observations indicate that both behavioral and endocrine circadian rhythms are regulated by the same multioscillator system, which seems to be composed of at least two distinct circadian oscillators.

Melatonin: effects on the circadian locomotor rhythm of sparrows.

The continuous administration of low levels of melatonin via intraperitoneally placed Silastic capsules either (i) shortened the free-running period of activity or (ii) induced continuous activity in house sparrows (Passer domesticus) maintained in constant darkness. After the melatonin-filled capsules were removed, the period of the circadian rhythm of activity lengthened in rhythmic birds and normal rhythmicity was restored in continuously active birds. The results suggest that melatonin is involved in the physiological control of circadian rhythmicity in sparrows.

Melatonin: antigonadal and progonadal effects in male golden hamsters.

Melatonin induced marked testicular regression in hamsters maintained on photostimulatory long days (light-dark 14 : 10). In animals maintained on nonstimulatory short days (light-dark 6 : 18), small amounts of melatonin (50 micrograms per day; 100 millimeters capsule length) prevented testicular regression; but testicular atrophy occurred in hamsters that received larger amounts of melatonin (75 to 100 micrograms per day; 150 to 200 millimeters capsule length) and in control hamsters that received none. The results demonstrate that melatonin can exert either pro- or antigonadal effects and emphasize that the effects of melatonin on the testis cannot be properly assessed unless account is taken of the dosage and mode of melatonin administration and the photoperiod on which experimental animals are maintained.

Role for acetylcholine in mediating effects of light on reproduction.

The length of day, or photoperiod, regulates the annual cycle of reproductive activity in the golden hamster. The inhibitory effects of a short-day photoperiod on testicular function were prevented by nighttime, but not daytime, intraventricular injections of carbachol, a cholinergic agonist. Short pulses of light during the night also block short-day induced testicular regression. The findings suggest that acetylcholine may play an important role in the mechanism through which information about the light-dark environment is transferred to the hypothalamic-pituitary-gonadal axis.

Splitting of the circadian rhythm of activity is abolished by unilateral lesions of the suprachiasmatic nuclei.

The circadian rhythm of activity in vertebrates often splits into two components after continuous exposure to constant light. This observation suggests that at least two circadian pacemakers underlie the activity rhythm. After unilateral ablation of the hypothalamic suprachiasmatic nuclei in hamsters, the splitting phenomenon was eliminated and a single rhythm of activity was established. The period of the new circadian activity rhythm different from the periods of the split rhythm and that preceding the split. These results suggest an interaction between the bilaterally paired suprachiasmatic nuclei in the generation of the circadian rhythm of activity.

Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior.

In a search for genes that regulate circadian rhythms in mammals, the progeny of mice treated with N-ethyl-N-nitrosourea (ENU) were screened for circadian clock mutations. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome 5, a region syntenic to human chromosome 4. The power of ENU mutagenesis combined with the ability to clone murine genes by map position provides a generally applicable approach to study complex behavior in mammals.

Development of Leydig cell tumors and onset of changes in the reproductive and endocrine systems of aging F344 rats.

The age-dependent onset of spontaneous testicular interstitial cell tumors was examined in F344 male rats. Light microscopy of testes established that nodular interstitial cell hyperplasia was evident in 3 of 5 12-month-old rats and in 5 of 5 rats at 15, 18, 21, and 24 months of age. Involution of the seminiferous epithelium was evident in all testes exhibiting extensive interstitial cell proliferation. Striking increments in serum prolactin and estradiol levels were noted with advancing age, whereas serum levels of follicle-stimulating hormone were unequivocally lower at 21 and 24 months than at 6 months of age. No measurable changes were detected in serum testosterone concentrations between 6 and 18 months of age, but marked increments in this androgen, without any measurable change in circulating luteinizing hormone titers, were apparent in 21- and 24-month-old rats. These findings point to a dynamic relationship between testicular interstitial cell tumorigenesis and age-related changes in the synthesis and/or secretion of gonadal and adenohypophyseal hormones.

High corticosterone levels in prenatally stressed rats predict persistent paradoxical sleep alterations.

Prenatal stress predisposes rats to long-lasting disturbances that persist throughout adulthood (e.g., high anxiety, dysfunction of the hypothalamo-pituitary-adrenal axis, and abnormal circadian timing). These disturbances parallel to a large extent those found in depressed patients, in which hypercortisolemia and sleep alterations may be related to stress-inducing events. We studied sleep-wake parameters in control and prenatally stressed adult rats (3-4 months old) and examined possible relationships with their corticosterone levels (determined at 2 months of age). Under baseline conditions, prenatally stressed rats showed increased amounts of paradoxical sleep, positively correlated to plasma corticosterone levels. Other changes include increased sleep fragmentation, total light slow-wave sleep time, and a slight decrease in the percentage of deep slow-wave sleep relative to total sleep time. During recovery sleep from acute restraint stress, all sleep changes persisted and were correlated with stress-induced corticosterone secretion. High corticosterone levels under baseline conditions as well as an acute stress challenge may thus predict long-term sleep-wake alterations in rats. Taken together with other behavioral and hormonal abnormalities in prenatally stressed animals, the pronounced changes in sleep-wake parameters that are similar to those found in depressed patients suggest that prenatal stress may be a useful animal model of depression.

The circadian clock mutation alters sleep homeostasis in the mouse.

The onset and duration of sleep are thought to be primarily under the control of a homeostatic mechanism affected by previous periods of wake and sleep and a circadian timing mechanism that partitions wake and sleep into different portions of the day and night. The mouse Clock mutation induces pronounced changes in overall circadian organization. We sought to determine whether this genetic disruption of circadian timing would affect sleep homeostasis. The Clock mutation affected a number of sleep parameters during entrainment to a 12 hr light/dark (LD 12:12) cycle, when animals were free-running in constant darkness (DD), and during recovery from 6 hr of sleep deprivation in LD 12:12. In particular, in LD 12:12, heterozygous and homozygous Clock mutants slept, respectively, approximately 1 and approximately 2 hr less than wild-type mice, and they had 25 and 51% smaller increases in rapid eye movement (REM) sleep during 24 hr recovery, respectively, than wild-type mice. The effects of the mutation on sleep are not readily attributable to differential entrainment to LD 12:12 because the baseline sleep differences between genotypes were also present when animals were free-running in DD. These results indicate that genetic alterations of the circadian clock system and/or its regulatory genes are likely to have widespread effects on a variety of sleep and wake parameters, including the homeostatic regulation of sleep.

The mouse Clock mutation behaves as an antimorph and maps within the W19H deletion, distal of Kit.

Clock is a semidominant mutation identified from an N-ethyl-N-nitrosourea mutagenesis screen in mice. Mice carrying the Clock mutation exhibit abnormalities of circadian behavior, including lengthening of endogenous period and loss of rhythmicity. To identify the gene affected by this mutation, we have generated a high-resolution genetic map (> 1800 meioses) of the Clock locus. We report that Clock is 0.7 cM distal of Kit on mouse chromosome 5. Mapping shows that Clock lies within the W19H deletion. Complementation analysis of different Clock and W19H compound genotypes indicates that the Clock mutation behaves as an antimorph. This antimorphic behavior of Clock strongly argues that Clock defines a gene centrally involved in the mammalian circadian system.

Effects of stimulated physical activity on the circadian pacemaker of vertebrates.

A dogma in the field of circadian rhythms is that in order to keep accurate time, pacemakers that generate such rhythms must be relatively independent of changes in the external and internal environment. While it is true that the period of circadian oscillators is conserved within a narrow range, regardless of alterations in the external and internal environment, numerous perturbations have now been found that can change the period and/or induce a phase shift in circadian pacemakers. Many of these perturbations also alter the overall level of activity and/or metabolic state of the organism. In 1960, Aschoff suggested that alterations in the "level of excitement" may induce changes in circadian clocks. Although little attention has been given to this hypothesis over the past three decades, recent findings support its validity and open new avenues for studying the function and organization of circadian clock systems.

Combined effects on the circadian clock of agents with different phase response curves: phase-shifting effects of triazolam and light.

Although light provides the primary signal for the entrainment of circadian pacemakers, a number of endogenous substances and pharmacological agents are also capable of resetting circadian pacemakers. Very little is known about the combined effects of photic and nonphotic agents on clock functions. We conducted a "double-pulse" experiment, in which two discrete stimuli are presented at different times within a single circadian cycle, to determine the combined effects of a 1-hr light pulse and injections of a benzodiazepine, triazolam, on the circadian rhythm of activity in the golden hamster. Our results suggest that, first, when given together as done in these experiments, the effects of triazolam and light are partially but not completely additive. Triazolam-induced phase advances appeared to make the effects of subsequent 1-hr light pulses more negative; phase delays were increased, and phase advances were decreased. Second, it appears that triazolam and light may alter the circadian pacemaker in very different ways, beyond the obvious difference in the shape of their phase response curves. The phase-shifting mechanics of the circadian system of the golden hamster appear to involve a longer response time to triazolam than to light pulses. Alternative possibilities to account for this difference are discussed.

Changes in the phase response curve of the circadian clock to a phase-shifting stimulus.

Experiments were conducted in hamsters to determine whether the phase response curve (PRC) to injections of the short-acting benzodiazepine triazolam is a fixed or a labile property of the circadian clock. The results indicated that (1) both the shape and the amplitude of the PRC to triazolam generated on the first day of transfer from a light-dark cycle (LD 14:10) to constant darkness (DD) (i.e., PRCLD) were different from those of the PRC generated after many days in DD (PRCDD); and (2) the phase-shifting effects of triazolam on the activity rhythms of hamsters transferred from LD 14:10 or 12:12 to DD changed dramatically within the first 8-9 days spent in DD. In an attempt to accelerate the resynchronization of the circadian clock of hamsters subjected to an 8-hr advance in the LD cycle, triazolam was given to the animals at a time selected on the basis of the characteristics of PRCLD. The activity rhythms of five of eight triazolam-treated animals were resynchronized to the new LD cycle within 2-4 days after the shift, whereas those of most of the control animals were resynchronized 21-29 days after the shift. These findings suggest that attempts to use pharmacological or nonpharmacological tools to phase-shift circadian clocks under entrained conditions should take into account information derived from PRCs generated at the time of transition from entrained to free-running conditions.

Entrainment of the circadian activity rhythm to the light-dark cycle can be altered by a short-acting benzodiazepine, triazolam.

The circadian rhythm of locomotor activity in hamsters maintained in either constant darkness or constant light can be phase-shifted by a single injection of the short-acting benzodiazepine, triazolam. These results suggest that treatment with triazolam may also alter the entrainment pattern of circadian rhythms in animals that are synchronized to a light-dark (LD) cycle. To test this hypothesis, hamsters maintained on an LD 6:18 light cycle received daily injections of triazolam (or vehicle) for 10-12 days, and any subsequent effects on the phase relationship between the onset of activity and the LD cycle were determined. Daily injections of triazolam (but not vehicle) induced pronounced advances or delays in the phase relationship between the entrained activity rhythm and the LD cycle; the direction of the shift was dependent on the time of the injection. Taken together with data from previous studies, these results suggest that triazolam, and perhaps other short-acting benzodiazepines, can be used to manipulate the mammalian circadian clock under a variety of experimental conditions.

Diurnal, photoperiodic, and age-related changes in plasma growth hormone levels in the golden hamster.

The golden hamster has been used extensively as an animal model for the study of both circadian and seasonal rhythms, and their regulation by the light-dark (LD) cycle. More recently, this species has been used to examine how the generation and entrainment of circadian rhythms are altered in advanced age. Recent studies in both humans and rodents indicate that age-related changes in the diurnal rhythm of pituitary growth hormone (GH) release may mediate some of the adverse effects of aging on a variety of physiological systems. As a first step in determining whether or not age-related changes in circulating GH levels are associated with changes in the regulation and/or expression of circadian rhythms, the effects of age on both the ultradian and diurnal patterns of plasma GH levels were determined in 3- to 22-month-old male hamsters that were bled every 15 min for a 24-hr period while entrained to an LD 14:10 light cycle. An additional study involving a similar blood collection protocol examined whether or not the length of the day is involved in the regulation of plasma GH levels. Although the frequency of pulsatile GH release did not change with advanced age, both the mean levels of GH per sample and the mean amplitude per pulse of GH were significantly elevated in 3- to 4-month-old animals, compared to animals that were 12-13, 15-16, or 21-22 months of age. In hamsters aged 3-4 and 12-13 months, there was an increase in both mean levels and the mean amplitude per pulse of GH, but not pulse frequency, during the night as compared to daytime values. No such diurnal rhythm was detected in the two groups of older animals. A clear diurnal rhythm in GH levels was also detected in animals maintained in a short-day (LD 6:18) cycle, and the mean levels of GH per sample were greater in hamsters maintained on short compared to long (LD 14:10) days. These results indicate that there are pronounced age-related changes in pituitary GH release in the hamster, and that both the time of day and the length of the day influence the pattern of GH release.