Can lifelong endurance exercise improve ageing through beneficial effects on circadian timing function, muscular performance and health status in men? Protocol for a comparative cross-sectional study.

A well-synchronized circadian system is a manifestation of an individual's health. A gradual weakening of the circadian timing function characterizes aging. Regular exercise has been suggested as a modality to improve many detrimental changes associated with aging. Therefore, we aim to examine the benefits and risks of lifelong endurance exercise on age-dependent changes in the circadian time-keeping function, the performance of the muscular system and health status. The study protocol has a comparative cross-sectional design, including groups of senior (65 to 75 years old, n=16) and young (20-30 years old, n=16) endurance runners and triathletes. Age-matched groups of young and elderly sedentary men are included as controls. The circadian function is evaluated mainly by measurement of urinary 6-sulphatoxymelatonin, a metabolite of the hormone melatonin shown to participate in the modulation of sleep cycles. The 6-sulphatoxymelatonin will be assessed in urine samples collected upon awakening in the morning and in the late evening, as a marker of melatonin production. In addition, sleep/activity rhythms and sleep quality will be measured by wrist actigraphy. Performance of the muscular system will be assessed by examination of muscular strength and quantifying of gene expression in the skeletal muscle tissue samples. Health status and age-induced reduction in immune function are to be analysed via the balance of pro- and anti-inflammatory immune markers in the plasma and skeletal muscle, body composition, bone density and physical fitness.

Artificial Dim Light at Night during Pregnancy Can Affect Hormonal and Metabolic Rhythms in Rat Offspring.

Artificial light at night (ALAN) is considered an environmental risk factor that can interfere with the circadian control of the endocrine system and metabolism. We studied the impact of ALAN during pregnancy on the hormonal and biochemical parameters in rat pups at postnatal (P) days P3, P10, and P20. Control dams (CTRL) were kept in a standard light-dark regime, and ALAN dams were exposed to dim ALAN (<2 lx) during the whole pregnancy. A plasma melatonin rhythm was found in all CTRL groups, whereas in ALAN pups, melatonin was not rhythmic at P3, and its amplitude was lowered at P10; no differences were found between groups at P20. Plasma corticosterone was rhythmic at P20 in both groups, with decreased mesor in ALAN pups. Plasma thyroid hormones exhibited an inconsistent developmental pattern, and vasopressin levels were suppressed at the beginning of the dark phase at P20 in ALAN compared to CTRL. Glucose and cholesterol showed significant daily rhythms in CTRL but not in ALAN offspring at P3. Exposure to ALAN during pregnancy disturbed the development of daily rhythms in measured hormones and metabolites, suggesting that ALAN during pregnancy can act as an endocrine disruptor that can interfere with the normal development of the progeny.

Sleep Problems and 6-Sulfatoxymelatonin as Possible Predictors of Symptom Severity, Adaptive and Maladaptive Behavior in Children with Autism Spectrum Disorder.

In children with autism spectrum disorder (ASD), sleep disturbances are a frequent comorbidity with an adverse effect on their behavior and functioning. It was suggested that melatonin deficit is at least partly responsible for the sleep problems. The study aimed to investigate, in a sample of 56 children with ASD aged 2.8-13.3 years, if the sleep problems and melatonin secretion can serve as predictors of adaptive functioning and severity of the ASD core symptoms. We demonstrated that, after adjustment for age, the Sleep score assessed by the Children's Sleep Habits Questionnaire predicts the Adaptive behavior composite score only in children younger than 6 years, and the preferred predictive model is for the domain Socialization. The age-adjusted Sleep score predicted Externalizing and Internalizing maladaptive behavior, with a near-zero contribution of age to the relationship between the Internalizing maladaptive behavior and Sleep score. After adjustment for age, the reduced night-time melatonin secretion predicted a higher severity of ASD symptoms in the domain Social affect and the Calibrated Severity Score, but not the sleep problems. Our results emphasize the importance of assessing sleep problems as a modifiable predictor of behavior in children with ASD and support the hypothesis about the role of melatonin in pathophysiology of ASD.

Effect of 17ß-estradiol on the daily pattern of ACE2, ADAM17, TMPRSS2 and estradiol receptor transcription in the lungs and colon of male rats.

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17ß-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 µg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19.

Sleep Characteristics According to Gender and Age Measured by Wrist Actigraphy.

The sleep/wake rhythm is one of the most important biological rhythms. Quality and duration of sleep change during lifetime. The aim of our study was to determine differences in sleep efficiency, movement, and fragmentation during sleep period between genders and according to age. Sleep period was monitored by wrist actigraphy under home-based conditions. Seventy-four healthy participants-47 women and 27 men participated in the study. The participants were divided by age into groups younger than 40 years and 40 years and older. Women showed lower sleep fragmentation and mobility during sleep compared to men. Younger women showed a higher actual sleep and sleep efficiency compared to older women and younger men. Younger men compared to older men had a significantly lower actual sleep, lower sleep efficiency and significantly more sleep and wake bouts. Our results confirmed differences in sleep parameters between genders and according to age. The best sleep quality was detected in young women, but gender differences were not apparent in elderly participants, suggesting the impact of sex hormones on sleep.

Impact of Dim Light at Night on Urinary 6-Sulphatoxymelatonin Concentrations and Sleep in Healthy Humans.

Artificial light at night can have negative effects on human wellbeing and health. It can disrupt circadian rhythms, interfere with sleep, and participate in the progress of civilisation diseases. The aim of the present study was to explore if dim artificial light during the entire night (ALAN) can affect melatonin production and sleep quality in young volunteers. We performed two experiments in real-life home-based conditions. Young volunteers (n = 33) were exposed to four nights of one lux ALAN or two nights of five lux ALAN. Melatonin production, based on 6-sulphatoxymelatonin/creatinine concentrations in urine, and sleep quality, based on actimetry, were evaluated. Exposure to ALAN one lux during the entire night did not suppress aMT6s/creatinine concentrations but did aggravate sleep quality by increasing sleep fragmentation and one-minute immobility. ALAN up to five lux reduced melatonin biosynthesis significantly and interfered with sleep quality, as evidenced by an increased percentage of one-minute immobility and a tendency of increased fragmentation index. Our results show that people are more sensitive to low illuminance during the entire night, as previously expected. ALAN can interfere with melatonin production and sleep quality in young, healthy individuals, and both processes have different sensitivities to light.

Dim Light at Night Disturbs Molecular Pathways of Lipid Metabolism.

Dim light at night (dLAN) is associated with metabolic risk but the specific effects on lipid metabolism have only been evaluated to a limited extent. Therefore, to explore whether dLAN can compromise lipid metabolic homeostasis in healthy individuals, we exposed Wistar rats to dLAN (~2 lx) for 2 and 5 weeks and analyzed the main lipogenic pathways in the liver and epididymal fat pad, including the control mechanisms at the hormonal and molecular level. We found that dLAN promoted hepatic triacylglycerol accumulation, upregulated hepatic genes involved in de novo synthesis of fatty acids, and elevated glucose and fatty acid uptake. These observations were paralleled with suppressed fatty acid synthesis in the adipose tissue and altered plasma adipokine levels, indicating disturbed adipocyte metabolic function with a potential negative impact on liver metabolism. Moreover, dLAN-exposed rats displayed an elevated expression of two peroxisome proliferator-activated receptor family members (Pparα and Pparγ) in the liver and adipose tissue, suggesting the deregulation of important metabolic transcription factors. Together, our results demonstrate that an impaired balance of lipid biosynthetic pathways caused by dLAN can increase lipid storage in the liver, thereby accounting for a potential linking mechanism between dLAN and metabolic diseases.

Inhibition of VEGF mediated post receptor signalling pathways by recently developed tyrosine kinase inhibitor in comparison with sunitinib.

Inhibition of angiogenesis involves blocking of tyrosine kinases (TK) implicated in signalling of vascular endothelial growth factor receptors (VEFGR). The inhibition of TK results in a disruption of Ras/Raf/MEK/ERK1/2 and PI3K/Akt signalling pathways. We evaluated recently developed TK inhibitor 22SYM and compared its anti-angiogenic effects with an approved multitargeted TK inhibitor sunitinib L-malate (sunitinib). Both compounds significantly inhibited migration and proliferation of human umbilical vein endothelial cells and ERK1/2 and Akt phosphorylation induced by VEGF. The lower inhibitory activity of 22SYM probably reflects its lower bioavailability and higher specific binding to VEGFR2 TK, which may decrease its potential side effects and toxicity in comparison with sunitinib.

Prenatal hypoxia in rats increased blood pressure and sympathetic drive of the adult offspring.

Decreased oxygenation during pregnancy and early periods of ontogeny can affect normal body development and result in diseases in adulthood. The aim of this study was to use the model of prenatal intermittent hypoxia (PIH) and evaluate the effects of short-term hypoxia at the end of gestation on blood pressure (BP) control in adulthood. Wistar rats were exposed daily to PIH for 4 h during gestational day 19 and 20. In adult male rats, heart rate (HR), systolic BP and pulse pressure (PP) were acquired by radiotelemetry during 1 week. On the basis of HR variability and BP variability, sympathovagal balance (LF/HF) and spontaneous baroreflex sensitivity (sBRS) were evaluated. Systolic BP and PP were significantly elevated in PIH rats in comparison with control rats during the light and dark phase of the day, while LF/HF increased only during the light phase of the day. In contrast, sBRS tended to decrease only during the dark phase in PIH rats. In all measured and calculated parameters, significant circadian rhythms were present and were not affected by PIH. In conclusion, our data suggest that short intermittent hypoxia at the end of gestation can increase BP and PP via significant changes in LF/HF, which occur especially during the passive phase of the day. Results suggest that minor changes in the autonomous nervous system activity induced by environmental conditions during the perinatal period may contribute to development of hypertension in adulthood.

Decreased melatonin levels and increased levels of advanced oxidation protein products in the seminal plasma are related to male infertility.

Melatonin, an indolamine secreted by the pineal gland, is known as a powerful free-radical scavenger and wide-spectrum antioxidant. Therefore, the aim of this study was to correlate markers of oxidative protein damage (advanced oxidation protein products, AOPPs) and the total antioxidant capacity (TAC) with melatonin levels in the seminal plasma of men with azoospermia (n=37), theratozoospermia (n=29) and fertile controls (normozoospermia, n=37). Melatonin concentration was measured by radioimmunoassay. The levels of AOPP as well as TAC efficiency (determined by the ferric reducing antioxidant power, FRAP) were estimated by spectrophotometric methods. The concentration of melatonin and AOPP significantly differed in azoospermic (P<0.0001) and theratozoospermic (P<0.0001) patients versus fertile men, and correlated negatively (r=-0.33, P=0.0016). The TAC levels were significantly higher in azoospermia than in theratozoospermia (P=0.0022) and the control group (P=0.00016). In azoospermia, the AOPP concentration was also significantly higher than that observed in theratozoospermia (P=0.00029). Decreased levels of melatonin together with elevated AOPP altered the oxidative-antioxidative balance in the ejaculate, thereby reducing fertility. Therefore, melatonin and AOPP levels may serve as additional diagnostic markers of semen quality and male reproductive potential.

Immunohistochemical definition of MT(2) receptors and melatonin in the gastrointestinal tissues of rat.

The gastrointestinal tract of vertebrate species contains melatonin, which participates in several physiological functions. Some of these effects are mediated via specific membrane receptors (MT(1) and MT(2)). In the present study, the distribution of the MT(2) receptor protein in the gastrointestinal tract was localized, and changes in MT(2) receptor density were observed in relation to the expected circadian changes in melatonin concentrations. Immunohistochemistry was performed in the rat stomach, duodenum, colon and pancreas. The amount of MT(2) was determined by Western blot. Melatonin concentrations were measured by radioimmunoassay (RIA). In the stomach, duodenum and colon, the most intense immunoreactivity was observed in the Muscularis mucosae and in the Muscularis externa. In the pancreas, the immunolabelling was less intense. There was a clear daily rhythm of melatonin concentrations in the stomach, duodenum and pancreas, with higher levels during the dark period. The density of MT(2) receptors did not exhibit circadian variation. Moreover, circadian changes in melatonin concentrations were not found in the colon. The density of MT(2) was the highest in the colon. Our results provide evidence for the heterogeneous distribution of MT(2) receptors in different parts and layers of the gastrointestinal tract, which could indicate a physiological role of melatonin in the gastrointestinal tract.

Effect of rhythmic melatonin administration on clock gene expression in the suprachiasmatic nucleus and the heart of hypertensive TGR(mRen2)27 rats.

OBJECTIVES: Plasma melatonin concentrations in non-dipping patients show a blunted daily rhythm. Melatonin has a capacity to improve disturbances in biological rhythms. Hypertensive TGR(mRen2)27 (TGR) rats with an upregulated renin-angiotensin system and inverted blood pressure profile were used to elucidate whether melatonin is able to influence the control of blood pressure. DESIGN: Melatonin was administered in drinking water to normotensive Sprague-Dawley (SD) and hypertensive TGR rats during the dark phase of the light: dark cycle 12: 12 for 4 weeks. METHODS: The effect of melatonin on blood pressure was monitored, and the expression of clock genes per2 and bmal1 and melatonin receptor MT1 in the suprachiasmatic nucleus (SCN) and the heart was measured by real time polymerase chain reaction during a 24-h cycle. RESULTS AND CONCLUSION: The administration of melatonin did not influence clock gene expression in the SCN but its effect on clock gene expression in the heart was phase dependent in both SD and TGR rats. Melatonin administration did not decrease the expression of melatonin receptors in the SCN and the heart. Melatonin did not decrease blood pressure in TGR rats but influenced the peripheral oscillator in the heart independently of the SCN. A modified function of molecular circadian oscillators in the heart can interfere with anticipation and disturb the adaptation of this organ to pressure overload.

Effect of L-NAME-induced hypertension on melatonin receptors and melatonin levels in the pineal gland and the peripheral organs of rats.

Melatonin plays a role in blood pressure (BP) control. The aim of this study was to determine whether melatonin concentrations and melatonin receptor levels are altered in L-NAME-treated, NO-deficient hypertensive rats. Two groups of male adult Wistar rats were investigated: rats (n=36) treated with NO-synthase inhibitor L-NAME (40 mg kg(-1)) and age-matched controls (n=36). BP was measured weekly by tail-cuff plethysmography. After 4 weeks, L-NAME administration increased BP (178+/-1 vs. control 118+/-1 mm Hg). At the end of treatment, rats were killed in regular 4 h intervals over a 24-h period. Melatonin concentrations in the plasma, pineal gland, heart and kidney and melatonin receptor (MT(1)) density in the aorta were determined. A significant daily rhythm of melatonin concentrations was found in the blood, pineal gland, kidney and heart of both control and hypertensive rats. Peak nighttime pineal melatonin concentrations were higher in L-NAME-treated rats than in controls (3.38+/-0.48 vs. 1.75+/-0.33 ng per pineal gland). No differences between both groups were found in melatonin concentrations in blood, kidney and heart or in the MT(1) receptor density in the aorta. Our results suggest that L-NAME treatment enhances melatonin production in the pineal gland, potentially by decreasing an inhibitory effect of NO on melatonin production in the pineal gland. However, the enhanced pineal melatonin formation was insufficient to increase melatonin concentrations in circulation, heart and kidney of L-NAME-treated rats, indicating an increased use of melatonin in hypertensive animals.

Endocrine rhythms and expression of selected genes in the brain, stellate ganglia, and adrenals of hypertensive TGR rats.

Hypertensive TGR(mRen2)27 (TGR) rats represent a strain with genetically upregulated renin-angiotensin-aldosterone system. Simultaneously with development of hypertension, a daily profile in blood pressure (BP) inverts and in mature TGR rats BP is higher during the lighttime (L) than the darktime (D). Physiological mechanisms of inverted BP rhythm generation are not understood. In our study we determined circadian profiles of plasma hormones related to BP control (aldosterone, corticosterone, melatonin, prolactin) in TGR and control Sprague-Dawley (SD) rats over 24 h and expression of genes encoding catecholamine synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) in adrenals and stellate ganglia. Plasma levels of corticosterone and aldosterone were higher in TGR than SD rats but acrophases of their rhythms were not changed. Darktime peak of prolactin in TGR rats was decreased in comparison with SD animals and pineal melatonin levels started to rise earlier in TGR than in SD rats. In adrenals we found upregulated expression of TH, DBH, and PNMT mRNA at the beginning of the lighttime in TGR compared to SD rats. Expression of TH and DBH in stellate ganglia was not different in TGR rats in comparison with SD, but PNMT expression was higher during L compared to D in TGR rats. We hypothesize that upregulated adrenal medulla functioning in the morning and disturbed communication between circadian oscillators and mechanisms involved in BP control can explain the reversed BP profile in TGR rats.

Diabetes induces changes in melatonin concentrations in peripheral tissues of rat.

OBJECTIVE: Exogenous melatonin was found to protect target organs under conditions of diabetes mellitus, however, concentrations of the hormone in peripheral tissues have not been determined. Therefore the aim of the present study was to measure the daily profile of melatonin levels in the pineal gland, plasma, pancreas, kidney, spleen, duodenum and colon of control and diabetic rats. MATERIAL & METHODS: Diabetes was induced by a single injection of streptozotocin (STZ, 65 mg/kg of body weight) and samples were collected over a 24 hr cycle on day 17 after STZ treatment. Melatonin and corticosterone levels were measured directly in plasma and after extraction in the pineal gland and peripheral organs (pancreas, kidney, spleen, duodenum and colon). RESULTS: A significant daily rhythm of melatonin concentrations was found not only in the pineal gland and plasma but also in the pancreas, kidney, spleen and duodenum. The daily pattern of melatonin levels in the colon was arrhythmic without a characteristic night-time increase of hormone concentration. Experimentally induced diabetes resulted in lower melatonin levels in the pancreas, kidney and duodenum as compared to control. No differences between STZ-treated and control rats were found in the spleen and colon. Plasma corticosterone levels were enhanced in diabetic rats in comparison with controls and the daily profile was not rhythmic. CONCLUSION: Our data suggest that the lower amplitude of melatonin rhythm in target organs induced by experimental diabetes can contribute to desynchronization of daily rhythms and can lower the antioxidative capacity of tissues.

Rhythmic clock gene expression in heart, kidney and some brain nuclei involved in blood pressure control in hypertensive TGR(mREN-2)27 rats.

Hypertensive TGR(mREN-2)27 rats exerting inverted blood pressure (BP) profile were used to study clock gene expression in structures responsible for BP control. TGR and control Sprague Dawley male rats were synchronized to the light:dark cycle 12:12 with food and water ad libitum. Daily rhythm in per2, bmal1, clock and dbp expression in the suprachiasmatic nucleus (SCN), rostral ventrolateral medulla (RVLM), nucleus of the solitary tract (NTS), heart and kidney was determined in both groups. Sampling occurred in regular 4 h intervals when rats of both strains were 11-weeks-old. Blood pressure and relative heart weight were significantly elevated in TGR rats in comparison with control. Expression of bmal1 and clock was up regulated in SCN of TGR rats but daily rhythm in per2 and dbp expression was similar in both groups. Mesor of per2 expression in RVLM was significantly higher in TGR than in control rats. In NTS of TGR rats expression of per2 was phase delayed by 3.5 h in comparison with control and bmal1 did not exert rhythmic pattern. Our study provided the first evidence about modified function of central and peripheral circadian oscillators in TGR rats at the level of clock gene expression. Expression of clock genes exerted up regulation in SCN and RVLM and down regulation in NTS. Circadian oscillators in selected brain structures were influenced more than oscillators in the heart and kidney by additional renin gene. Interactions of RAS and circadian system probably contribute to the development of inverted BP profile in TGR rats.

Effect of streptozotocin-induced diabetes on daily expression of per2 and dbp in the heart and liver and melatonin rhythm in the pineal gland of Wistar rat.

The circadian system is a flexible framework allowing a proper adjustment of physiological functions to the regularly changing environment. Pathways that are used to synchronize components of the circadian system have been shown to be susceptible to pathophysiological conditions. In our study, we investigated effects of streptozotocin (STZ)-induced diabetes mellitus on function of the circadian system at the level of melatonin synthesis and expression of per2 and dbp in the heart and liver in 8-week-old Wistar rats. Rhythmic pattern of clock gene per2 and transcription factor dbp in controls and STZ-treated animals was determined. Streptozotocin administration had a more substantial effect on per2 expression in the liver than in the heart. Pronounced phase advance in the rhythm of dbp expression in both the liver and the heart was observed. The melatonin rhythm reflecting the phase of the master clock was not affected by STZ application. Changes in per2 and dbp expression in the heart and liver imply alterations in input pathway or peripheral oscillators with possible consequences on function of analysed organs.