Taking biological rhythms into account: From study design to results reporting.

Numerous physiological and behavioral processes in living organisms exhibit strong rhythmicity and are regulated within a 24-hour cycle. These include locomotor activity and sleep patterns, feeding-fasting cycles, hormone synthesis, body temperature, and even mood and cognitive abilities, all of which are segregated into different phases throughout the day. These processes are governed by the internal timing system, a hierarchical multi-oscillator structure conserved across all organisms, from bacteria to humans. Circadian rhythms have been seen across multiple taxonomic kingdoms. In mammals, a hierarchical internal timing system is comprised of so-called central and periphereal clocks. Although these rhythms are intrinsic, they are under environmental influences, such as seasonal temperature changes, photoperiod variations, and day-night cycles. Recognizing the existence of biological rhythms and their primary external influences is crucial when designing and reporting experiments. Neglecting these physiological variations may result in inconsistent findings and misinterpretations. Thus, here we propose to incorporate biological rhythms into all stages of human and animal research, including experiment design, analysis, and reporting of findings. We also provide a flowchart to support decision-making during the design process, considering biological rhythmicity, along with a checklist outlining key factors that should be considered and documented throughout the study. This comprehensive approach not only benefits the field of chronobiology but also holds value for various other research disciplines. The insights gained from this study have the potential to enhance the validity, reproducibility, and overall quality of scientific investigations, providing valuable guidance for planning, developing, and communicating scientific studies.

Skin Temperature Circadian Rhythms and Dysautonomia in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: The Role of Endothelin-1 in the Vascular Tone Dysregulation.

There is accumulating evidence of autonomic dysfunction in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS); however, little is known about its association with circadian rhythms and endothelial dysfunction. This study aimed to explore the autonomic responses through an orthostatic test and analysis of the peripheral skin temperature variations and vascular endothelium state in ME/CFS patients. Sixty-seven adult female ME/CFS patients and 48 healthy controls were enrolled. Demographic and clinical characteristics were assessed using validated self-reported outcome measures. Postural changes in blood pressure, heart rate, and wrist temperature were recorded during the orthostatic test. Actigraphy during one week was used to determine the 24-h profile of peripheral temperature and activity. Circulating endothelial biomarkers were measured as indicators of endothelial functioning. Results showed that ME/CFS patients presented higher blood pressure and heart rate values than healthy controls in the supine and standing position (p < 0.05 for both), and also a higher amplitude of the activity rhythm (p < 0.01). Circulating levels of endothelin-1 (ET-1) and vascular cell adhesion molecule-1 (VCAM-1) were significantly higher in ME/CFS (p < 0.05). In ME/CFS, ET-1 levels were associated with the stability of the temperature rhythm (p < 0.01), and also with the self-reported questionnaires (p < 0.001). This suggests that ME/CFS patients exhibited modifications in circadian rhythm and hemodynamic measures, which are associated with endothelial biomarkers (ET-1 and VCAM-1). Future investigation in this area is needed to assess dysautonomia and vascular tone abnormalities, which may provide potential therapeutic targets for ME/CFS.

Coupling Between Subregional Oscillators Within the Suprachiasmatic Nucleus Determines Free-Running Period in the Rat.

Rats housed in a 22-h light-dark cycle (11:11, T22) exhibit 2 distinct circadian locomotor activity (LMA) bouts simultaneously: one is entrained to the LD cycle and a second dissociated bout maintains a period greater than 24 h. These 2 activity bouts are associated with independent clock gene oscillations in the ventrolateral (vl-) and dorsomedial (dm-) suprachiasmatic nucleus (SCN), respectively. Previous results in our laboratory have shown that the vl- and dm-SCN oscillators are weakly coupled under T22 and that the period of the dissociated bout depends on coupling between the 2 subdivisions. Here, we sought to study the behavior of the T22 SCN pacemaker upon release into free-running conditions and compare it to the behavior of the system upon release from typical 24-h (12:12, T24) entrainment. T22-desynchronized rats or T24-entrained rats were released into constant darkness (DD). Activity rhythms in T22 rats rapidly resynchronized upon release into DD, and the free-running period (FRP) of the fused rhythm was longer than the FRP of T24 rats. We then asked whether the in vivo period changes were also present in the ex vivo SCN. Per1-luc rats were desynchronized in T22 for assessment of SCN Per1-luc ex vivo. Similar to behavioral FRP, the period of ex vivo SCN explanted from T22 rats was longer than that for T24 animals. Mathematical models supported the observed behavior of the dual oscillator system as the result of mutual coupling between the vl- and dm-SCN oscillators. This bidirectionally coupled model predicted both the FRP of the T22 system and its phase-shifting response to light. Together, these data support a model of pacemaker organization in which a light-sensitive vl-SCN oscillator is mutually coupled with a light-insensitive dm-SCN oscillator, and together they determine the period of the coupled system as a whole and its response to light pulses.

Handling missing data in rest-activity time series measured by actimetry.

A handling procedure of off-wrist episodes in actimetry time series of motor activity is presented using two records (regular vs. irregular sleep-wake cycle and daytime activity) of 14 consecutive days sampled in 1-minute epochs. We generated single missing value (NA) intervals of 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h as well as random NA episodes following probabilistic rules to simulate real-life off-wrist episodes. Then, we replaced these episodes with "zeroes" (i.e., the default of immobility records), mean or median of the remaining 13 days corresponding to the missing bins. Single missing episodes of up to 12 h resulted in less than 5% variation from the original values. The irregular series showed higher variability in acrophase, MESOR, L5, M10 and RA compared to the regular series. Random missing allocation simulating real-life off-wrist episodes resulted in significant changes in most parameters, and the imputation of zeroes significantly increased the variance; however, replacing NA with mean or median resulted in patterns similar to those of NA. We recommend replacing 'zeroes' with NA whenever possible, given the risk of inflating invariance using zeroes. If the parameters cannot be computed in the presence of NA, we recommend using the weekly mean of corresponding timepoints.

Circadian Patterns of Patients with Type 2 Diabetes and Obstructive Sleep Apnea.

Sleep apnea, a condition that modifies sleep and circadian rhythms, is highly prevalent in patients with diabetes. However, it is not known if there is an association between sleep apnea, circadian alterations and glycemic regulation in this type of patient. Here, a polysomnographic study was carried out on 21 women and 25 men (mean age = 64.3 ± 1.46 years) with diagnoses of type 2 diabetes to detect the presence of sleep apnea. Moreover, patients wore an actigraph and a temperature sensor on the wrist for one week, to study the manifestation of the circadian rhythms. The correlations of circadian and polysomnographic variables with the severity of apnea, measured by the apnea-hypopnea index, and with glycemic dysregulation, measured by the percentage of glycated hemoglobin, were analyzed. The mean apnea-hypoapnea index of all the participants was 39.6 ± 4.3. Apnea-hypoapnea index correlated with % N1, negatively with % N3, and also the stability of the active circadian rhythm. However, no significant correlation was found between the apnea-hypopnea index and wrist temperature rhythm and glycated hemoglobin. Glycated hemoglobin levels were negatively associated with the percentage of variance explained by the wrist temperature circadian rhythm (calculated via 24 and 12 h rhythms). This association was independent of body mass index and was strongest in patients with severe apnea. In conclusion, patients with diabetes showed altered circadian rhythms associated with a poor glycemic control and this association could partially be related to the coexistence of sleep apnea.

Neonatal overfeeding during lactation rapidly and permanently misaligns the hepatic circadian rhythm and programmes adult NAFLD.

Childhood obesity is a strong risk factor for adult obesity, type 2 diabetes, and cardiovascular disease. The mechanisms that link early adiposity with late-onset chronic diseases are poorly characterised. We developed a mouse model of early adiposity through litter size reduction. Mice reared in small litters (SLs) developed obesity, insulin resistance, and hepatic steatosis during adulthood. The liver played a major role in the development of the disease. OBJECTIVE: To gain insight into the molecular mechanisms that link early development and childhood obesity with adult hepatic steatosis and insulin resistance. METHODS: We analysed the hepatic transcriptome (Affymetrix) of control and SL mice to uncover potential pathways involved in the long-term programming of disease in our model. RESULTS: The circadian rhythm was the most significantly deregulated Gene Ontology term in the liver of adult SL mice. Several core clock genes, such as period 1-3 and cryptochrome 1-2, were altered in two-week-old SL mice and remained altered throughout their life course until they reached 4-6 months of age. Defective circadian rhythm was restricted to the periphery since the expression of clock genes in the hypothalamus, the central pacemaker, was normal. The period-cryptochrome genes were primarily entrained by dietary signals. Hence, restricting food availability during the light cycle only uncoupled the central rhythm from the peripheral and completely normalised hepatic triglyceride content in adult SL mice. This effect was accompanied by better re-alignment of the hepatic period genes, suggesting that they might have played a causal role in mediating hepatic steatosis in the adult SL mice. Functional downregulation of Per2 in hepatocytes in vitro confirmed that the period genes regulated lipid-related genes in part through peroxisome proliferator-activated receptor alpha (Ppara). CONCLUSIONS: The hepatic circadian rhythm matures during early development, from birth to postnatal day 30. Hence, nutritional challenges during early life may misalign the hepatic circadian rhythm and secondarily lead to metabolic derangements. Specific time-restricted feeding interventions improve metabolic health in the context of childhood obesity by partially re-aligning the peripheral circadian rhythm.

Sleeve gastrectomy in patients with severe obesity restores circadian rhythms and their relationship with sleep pattern.

Advances in research have linked alterations of circadian rhythms with obesity. However, few studies have focused on the recovery of the circadian expression after a weight loss treatment. Our aim was to study the alterations of the circadian rhythmicity due to morbid obesity and the recovery of the circadian pattern after weight loss in a cohort of patients who underwent sleeve gastrectomy. For this purpose, 41 patients with severe obesity (75% women; age [mean (SD)] 49.7 ± 10 years; BMI 44.3 ± 6.2 kg/m2) were monitored before bariatric surgery and 9 months afterward. On both occasions, activity and wrist temperature were determined by actigraphy and were related with weight loss. Anthropometric, biochemical, and sleep-related variables, along with dietary intake and physical activity, were analyzed in relation with circadian rhythmicity. Results show significant differences in the circadian expression before and after 9 months of bariatric surgery, with more stable and less fragmented rhythms after weight loss. Moreover, only after surgery were the circadian variables associated to sleep timing and chronotype. The findings of this study indicate that weight loss treatment in patients with morbid obesity improves the circadian rhythm expression, and in such a way that it could be associated with better sleep quality. Moreover, it allows the recovery of the relationship between sleep patterns and circadian rhythm that was lost due to the obesity.

Higher eating frequency is associated with lower adiposity and robust circadian rhythms: a cross-sectional study.

BACKGROUND: Although overweight and obesity are assumed to arise from an energy imbalance, evidence has shown that the frequency and timing of meals are also potential risk factors for obesity. However, the lack of a consistent approach to define eating patterns relative to internal circadian rhythms limits the extent of these findings. OBJECTIVES: The objective of this study was to investigate the association of the circadian pattern of energy intake with adiposity and with internal circadian rhythms. METHODS: A total of 260 Spanish adults (aged 20-30 y; 78.1% women) were included in a 6-d cross-sectional study. Participants documented sleep and dietary intake within the study period. From these data, we evaluated the chronotype, eating patterns (meal timing, eating duration, and eating frequency), and we obtained the daily profile of energy intake. In addition, we evaluated the circadian pattern of wrist temperature (internal circadian rhythm marker). Circadian patterns of energy intake and wrist temperature were analyzed, and their association among them and with anthropometric variables and diet quality was studied. RESULTS: The greater fragmentation of the circadian pattern of energy intake was associated with lower BMI (in kg/m2; -10.55; 95% CI: -16.96, -4.13; P = 0.001). In addition, a greater eating frequency (≥5 eating occasions/d) was significantly associated with lower BMI (-1.88; 95% CI: -3.27, -0.48) and higher energy intake after 20:00 (4.14% of kcal; 95% CI: 1.67, 7.16). Furthermore, a greater eating frequency was associated with lower fragmentation (P = 0.042) and greater stability of the circadian pattern of wrist temperature (P = 0.016). CONCLUSIONS: The daily pattern of energy intake is associated with adiposity and robust circadian rhythms. Our results shed light on the relevance of eating frequency as a potential zeitgeber for the circadian system. Although more evidence is needed, eating frequency could be considered for future chrono-nutritional recommendations for the prevention of circadian misalignment and obesity.

ADHD subtypes are associated differently with circadian rhythms of motor activity, sleep disturbances, and body mass index in children and adolescents: a case-control study.

To date, few studies have examined the circadian pattern of motor activity in children and adolescents newly diagnosed with attention-deficit/hyperactivity disorder (ADHD). The objective was to study the circadian pattern of motor activity in subjects with ADHD (medication naïve) and to investigate the relationships between alterations in circadian patterns, the ADHD subtype (combined or inattentive), sleep disturbances and body mass index (BMI). One-hundred twenty children and adolescents (60 medication naïve ADHD and 60 controls) were included in a gender- and age-matched case-control study. ADHD was diagnosed according to the DSM-IV-TR, the Schedule for Affective Disorders and Schizophrenia-Present and Lifetime Version, and the Conner's Parents Rating Scale-Revised. Circadian rhythms of motor activity and sleep parameters were measured using actigraphy and the Sleep Disturbance Scale for Children. BMI and dietary intake were also evaluated. ADHD patients showed a trend towards eveningness and greater sleep disturbances than controls. Additionally, patients with ADHD-combined had significantly higher mean values of motor activity and showed a significant delay in bedtime. Furthermore, among ADHD-C patients hyperactivity symptoms were significantly associated with the least 5 h of activity. Regarding patients with ADHD-inattentive, increased fragmentation of the circadian pattern was associated with inattention symptoms, and they also showed a significant increase in BMI of 2.52 kg/m2 [95% CI 0.31, 4.73] in comparison with controls. Our findings highlight the potential use of actigraphy as a clinical tool to aid in the diagnosis of ADHD. It should be noted that evaluating motor activity variables could also allow the differentiation between ADHD subtypes.

Adiposity and body mass index of young women are associated with altered 24-hour profile of wrist temperature and sleep quality.

Sleep alterations are linked to circadian disturbances; however, these variables are also related with adiposity, which could impair the study of the previous relationship. Here, we investigated whether the association between adiposity and impaired circadian health is independent of sleep and circadian related variables in a sample of young women. One hundred ninety-two women (18-30 years of age) participated in this cross-sectional study. To evaluate circadian rhythmicity, subjects wore a temperature sensor for six consecutive days, and variables related with the amplitude and stability of the rhythm were calculated. We also assessed adiposity markers (body mass index [BMI], fat mass, waist, and hip circumference), sleep and circadian variables (sleep schedules and quality, social jet lag, and chronotype), energy intake, and physical activity. Partial correlations were conducted to test the associations between circadian and sleep-related variables with adiposity markers. Results showed the circadian pattern of temperature clearly depended on BMI and, to a lesser extent, on sleep variables. Specifically, we showed that higher adiposity was significantly associated with lower amplitude, percentage of variance explained by the rhythm, and intradaily variability and L10 values of the temperature 24 h rhythm. Furthermore, sleep quality was the only variable significantly related with both adiposity and circadian pattern of wrist temperature, while social jet lag and chronotype were only associated with the circadian pattern of temperature. Therefore, we consider BMI should be taken into account when sleep health or disturbances are studied by means of circadian rhythm analyses.

Entraining effects of variations in light spectral composition on the rest-activity rhythm of a nocturnal rodent.

The ability to predict and adjust physiology and behavior to recurring environmental events has been necessary for survival on Earth. Recent discoveries revealed that not only changes in irradiance but also light spectral composition can stimulate the suprachiasmatic nucleus (SCN), ensuring the body's synchronization to the environment. Therefore, using a lighting system that modulates spectral composition during the day using combined red-green-blue (RGB) lights, we evaluated the effect of variations in light spectral composition on the rest-activity rhythm of rodents. Male Wistar rats (n = 17) were gestated and raised under different lighting conditions and exposed to a long photoperiod (16 h light: 8 h dark). The difference between groups was the presence of variations in light spectral composition during the day (RGB-v) to simulate daily changes in natural light, or not (RGB-f). After weaning, spontaneous motor activity was recorded continuously for rhythm evaluation. Our results indicated that animals under RGB-v did not present a reactive peak of activity after the beginning of the light phase, suggesting that this group successfully detected the variations aimed at mimicking daily alterations of natural light. Furthermore, RGB-v animals exhibited an earlier activity acrophase in comparison to animals under RGB-f (RGB-v = 12:16 - "hh:mm", RGB-f = 13:02; p < 0.001), which might have been due to the capability to predict the beginning of the dark phase when exposed to variations in light spectrum. However, this earlier activity acrophase can be also explained by the blue-light peak that occurred in RGB-v. The spectral and waveform analysis of daily patterns of motor activity revealed that rats in the RGB-v group were better entrained to a circadian rhythm throughout the experiment. RGB-v showed higher interdaily stability (IS) values (29.75 ± 6.5, n = 9) than did RGB-f (t(15)  =  2.74, p  =  0.015). Besides, the highest power content (PC) on the first harmonic (circadian) was reached earlier in the RGB-v group. The circadianity index (CI) of the whole period was higher in the RGB-v group (t(15)  =  3.47, p = 0.003). Thus, we could consider that locomotor activity rhythm was entrained to the light-dark cycle in the RGB-v rats earlier compared to the RGB-f rats. Our results provide additional evidence for the effect of variations in light spectral composition on the rest-activity pattern of nocturnal rodents. This suggests that these animals might predict the arrival of the activity phase by its advanced acrophase when exposed to RGB-v, demonstrating a better synchronization to a 24-h rhythm.

Seasonal variation of body weight loss after bariatric surgery.

Seasonal variations have been described in humans in several variables such as sleep, mood, appetite, food preferences, or body weight. We hypothesized that these variations could also influence the decrease in body weight rate in patients submitted to body weight loss interventions. Thus, here we tested the variations of weight loss according to the time of the year the surgery took place in a group patients (n = 1322) submitted to bariatric surgery in the Hospital Universitari de la Vall d'Hebron in Barcelona (geographical coordinates: 41°25'41″N 2°8'32″E). For the analysis, the percentage of total body weight loss (%TWL), excess body weight loss (%EWL) and percentage of body mass index loss (%BMIL) were calculated at 3 (n = 1255), 6 (n = 1172), 9 (n = 1002), and 12 months (n = 1076) after surgery. For %EWL and %BMIL a statistically significant seasonal variation was detected when the variables were calculated at 3 months, but not at the other times, with more weight loss in summer-fall. However, seasonal variations were not detected for %TWL (p = 0.09). The mean amplitude of the seasonal rhythm for %EWL was of 1.8%, while for the rhythm of %BMIL was 0.7%. Moreover, a second peak was detected in January-February modulating the seasonal rhythm of the two variables. Results confirm seasonal variations in humans and indicate that short term responses to weight loss can be modulated by the time of year.

Seasonal variations of changes in lipid and glucidic variables after bariatric surgery.

Seasonality is a phenomenon that is characterized by changes over the year in sleep, mood, behaviour, appetite and body weight. In humans, seasonal variations have been found in certain variables, such as lipid variables and body mass index. We hypothesize that this rhythm could influence the expected variation of the levels of biochemical variables in cases of body weight loss. Thus, the goal of this study was to observe whether the time of year in which bariatric surgery (BS) took place modulated the changes in several variables related to glucidic and lipid metabolism. Blood samples were obtained from 24 women and 10 men before BS and 1 and 3 months after BS. We calculated the percentage of variation that occurred for each individual and for each variable as a function of the time of the year. Data were adjusted to a 12-month period sinusoidal curve, with significance being set at p < 0.05. The results showed that almost all of the studied variables changed due to the BS according to a seasonal rhythm. Most of the variables showed a decrease that was most prominent in winter. In the cases of body mass index (BMI), adrenocorticotropin hormone (ACTH), and cortisol, the highest variation occurred in winter. Insulin and cholesterol in high-density lipoproteins (cHLD) variations were higher in springtime. Glucose variation showed a decrease after surgery with acrophase in summer-fall and plasminogen activator inhibitor-1 (PAI-1) and homeostatic model assessment-insulin resistance (HOMA-IR) in spring-summer. Ghrelin levels showed increases with a rhythm of variation with an acrophase in summer-fall. The seasonal rhythm found in this study fits nearly with the inverse of the endogenous circannual rhythm of the variables studied. The time of the year when the highest variation takes place is related to the circannual rhythm of the variable. The results agree with the manifestation of seasonal rhythm in human biochemical variables, which are reflected in the responses to weight loss after BS.

Circadian rhythm abnormalities and autonomic dysfunction in patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis.

Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) patients frequently show autonomic symptoms which may be associated with a hypothalamic dysfunction. This study aimed to explore circadian rhythm patterns in rest and activity and distal skin temperature (DST) and their association with self-reported outcome measures, in CFS/ME patients and healthy controls at two different times of year. Ten women who met both the 1994 CDC/Fukuda definition and 2003 Canadian criteria for CFS/ME were included in the study, along with ten healthy controls matched for age, sex and body mass index. Self-reported measures were used to assess fatigue, sleep quality, anxiety and depression, autonomic function and health-related quality of life. The ActTrust actigraph was used to record activity, DST and light intensity, with data intervals of one minute over seven consecutive days. Sleep variables were obtained through actigraphic analysis and from subjective sleep diary. The circadian variables and the spectral analysis of the rhythms were calculated. Linear regression analysis was used to evaluate the relationship between the rhythmic variables and clinical features. Recordings were taken in the same subjects in winter and summer. Results showed no differences in rhythm stability, sleep latency or number of awakenings between groups as measured with the actigraph. However, daily activity, the relative amplitude and the stability of the activity rhythm were lower in CFS/ME patients than in controls. DST was sensitive to environmental temperature and showed lower nocturnal values in CFS/ME patients than controls only in winter. A spectral analysis showed no differences in phase or amplitude of the 24h rhythm, but the power of the second harmonic (12h), revealed differences between groups (controls showed a post-lunch dip in activity and peak in DST, while CFS/ME patients did not) and correlated with clinical features. These findings suggest that circadian regulation and skin vasodilator responses may play a role in CFS/ME.

Forced desynchronization model for a diurnal primate.

The circadian system is organized in a hierarchy of multiple oscillators, with the suprachiasmatic nucleus (SCN) as the master oscillator in mammals. The SCN is formed by a group of coupled cell oscillators. Knowledge of this coupling mechanism is essential to understanding entrainment and the expression of circadian rhythms. Some authors suggest that light-dark (LD) cycles with periods near the limit of entrainment may be good models for promoting internal desynchronization, providing knowledge about the coupling mechanism. As such, we evaluated the circadian activity rhythm (CAR) pattern of marmosets in LD cycles at lower limits of entrainment in order to study induced internal dissociation. To that end, two experiments were conducted: (1) 6 adult females were under symmetrical LD cycles T21, T22 and T21.5 for 60, 35 and 48 days, respectively; and (2) 4 male and 4 female adults were under T21 for 24 days followed by 18 days of LL, back to T21 for 24 days, followed by 14 days of LL. The CAR of each animal was continuously recorded. In experiment 1, vocalizations were also recorded. Under Ts shorter than 24 days, a dissociation pattern was observed for CAR and vocalizations. Two simultaneous circadian components emerged, one with the same period as the LD cycle, called the light-entrained component, and the other in free-running, denominated the non-light-entrained component. Both components were displayed in the CAR for all the animals in T21, five animals (83.3%) in T21.5 and two animals (33.3%) in T22. Our results are in accordance with the multioscillatory nature of the circadian system. Dissociation is partial synchronization to the LD cycle, with at least one group of oscillators synchronized by relative coordination and masking, while another group of oscillators free runs, but is also masked by the LD cycle. Since only T21 promoted the emergence of both circadian components in the circadian rhythms of all marmosets, it was considered the promoter period of circadian rhythm dissociation in this species, and is proposed as a good animal model for forced desynchronization in non-human diurnal primates.

Seasonal variation in plasma lipids and lipases in young healthy humans.

Although intermediate metabolism is known to follow circadian rhythms, little information is available on the variation in lipase activities (lipoprotein and hepatic lipase, LPL and HL, respectively) and lipids throughout the year. In a cross-sectional study, we collected and analysed blood from 245 healthy students (110 men and 135 women) between 18 and 25 years old from the University of Barcelona throughout the annual campaign (March, May, October and December) of the blood bank. All subjects gave their written informed consent to participate. All blood samples were taken after breakfast at 8:00 and 11:00 am. Plasma glucose, total plasma protein, triacylglycerides (TAG), free fatty acids (FFA), free cholesterol and esterified cholesterol (FC and TC, respectively), cholesterol in low-density lipoproteins (cLDL), cholesterol in high-density lipoproteins (cHDL), phospholipids (PL) and lipase activities (LPL and HL) were determined. Cosinor analysis was used to evaluate the presence (significance of fit cosine curve to data and variance explained by rhythm) and characteristics of possible 12-month rhythms (acrophase, MESOR and amplitude). Statistically significant seasonal rhythms were detected for all the variables studied except proteins, with most of them peaking in the winter season. The lowest value for cLDL and the HL occurs in summer, while for cHDL and the LPL it is in winter. These findings demonstrate for the first time that in physiological conditions, plasma LPL and HL activities and lipids follow seasonal rhythms. The metabolic significance of this pattern is discussed.

Daily Variation of UV-induced Erythema and the Action of Solar Filters.

UV rays may cause several degrees of skin damage, which makes sunscreen research necessary. In addition, skin sensitivity shows daily variations, which can interfere in the detection of the efficacy of the filters. Here, we studied the UV-induced erythema in hairless rats at two times of the day (light and darkness) using a colorimeter method. The effect of an emulsion with solar filters with or without melatonin was also assayed. Results indicate that the value of a* (from CIELAB color space values L* a* b) was the most useful variable to evaluate the erythema. However, at the UV intensity used, erythema was only detected when irradiation was carried out during the activity phase of the animal, enabling the detection of the protective action of the sunscreen at this time. Thus, daily variations in skin sensitivity have been demonstrated and should be taken into account in dermatological research.

Melatonin pharmacokinetics after transdermal administration changes according to the time of the day.

Melatonin is a neurohormone with multiple and different actions, such as chronobiotic or antioxidant. Melatonin is usually orally administered, but dermal administration is also useful in dermatological diseases or as adjuvant to certain skin treatments. Here, we studied the variability of the pharmacokinetics of melatonin and its metabolite AFMK, when melatonin is transdermally administered to Hairless rat at two different times of day (Zeitgeber Time 4 (ZT4) and ZT16). Moreover, in order to obtain the bioavailability, kinetics after intravenous administration was also studied. In addition, a permeation study was carried out, at both ZTs, to test the amount of melatonin retained in the skin after transdermal administration. Results showed that pharmacokinetic parameters of melatonin administered exogenously depended on the time of the day. When intravenous data were fitted to a compartmental model, the extrapolated plasma concentration at time 0 and the area under the curve were higher at ZT4, while clearance, volumes of central and peripheral compartments and volume of distribution at the steady state were higher at ZT16. Transdermal administration was best fitted to a one-compartment model and tmax, half-life of absorption and area under the curve showed higher values at ZT4, while the absorption rate and constant of absorption were higher at ZT16. AFMK was detected in all cases, but no differences between the two ZTs were observed. Transdermal administration showed better bioavailability also at ZT4. Results indicate that time of day is a variable that should be taken into account when melatonin is transdermally administered.

Human adipose tissue expresses intrinsic circadian rhythm in insulin sensitivity.

In humans, insulin sensitivity varies according to time of day, with decreased values in the evening and at night. Mechanisms responsible for the diurnal variation in insulin sensitivity are unclear. We investigated whether human adipose tissue (AT) expresses intrinsic circadian rhythms in insulin sensitivity that could contribute to this phenomenon. Subcutaneous and visceral AT biopsies were obtained from extremely obese participants (body mass index, 41.8 ± 6.3 kg/m(2); 46 ± 11 y) during gastric-bypass surgery. To assess the rhythm in insulin signaling, AKT phosphorylation was determined every 4 h over 24 h in vitro in response to different insulin concentrations (0, 1, 10, and 100 nM). Data revealed that subcutaneous AT exhibited robust circadian rhythms in insulin signaling (P < 0.00001). Insulin sensitivity reached its maximum (acrophase) around noon, being 54% higher than during midnight (P = 0.009). The amplitude of the rhythm was positively correlated with in vivo sleep duration (r = 0.53; P = 0.023) and negatively correlated with in vivo bedtime (r = -0.54; P = 0.020). No circadian rhythms were detected in visceral AT (P = 0.643). Here, we demonstrate the relevance of the time of the day for how sensitive AT is to the effects of insulin. Subcutaneous AT shows an endogenous circadian rhythm in insulin sensitivity that could provide an underlying mechanism for the daily rhythm in systemic insulin sensitivity.-Carrasco-Benso, M. P., Rivero-Gutierrez, B., Lopez-Minguez, J., Anzola, A., Diez-Noguera, A., Madrid, J. A., Lujan, J. A., Martínez-Augustin, O., Scheer, F. A. J. L., Garaulet, M. Human adipose tissue expresses intrinsic circadian rhythm in insulin sensitivity.

Melatonin administration modifies circadian motor activity under constant light depending on the lighting conditions during suckling.

Early lighting conditions have been described to produce long-term effects on circadian behavior, which may also influence the response to agents acting on the circadian system. It has been suggested that melatonin (MEL) may act on the circadian pacemaker and as a scavenger of reactive oxygen and nitrogen species. Here, we studied the oxidative and behavioral changes caused by prolonged exposure to constant light (LL) in groups of rats that differed in MEL administration and in lighting conditions during suckling. The rats were exposed to either a light-dark cycle (LD) or LL. At 40 days old, rats were treated for 2 weeks with a daily subcutaneous injection of MEL (10 mg/kg body weight) or a vehicle at activity onset. Blood samples were taken before and after treatment, to determine catalase (CAT) activity and nitrite level in plasma. As expected, LL-reared rats showed a more stable motor activity circadian rhythm than LD rats. MEL treatment produced more reactivity in LD- than in LL rats, and was also able to alter the phase of the rhythm in LD rats. There were no significant differences in nitrite levels or CAT activity between the groups, although both variables increased with time. Finally, we also tested depressive signs by means of sucrose consumption, and anhedonia was found in LD males treated with MEL. The results suggest that the lighting conditions in early infancy are important for the long-term functionality of the circadian system, including rhythm manifestation, responses to MEL and mood alterations.