Circadian variations in plasma concentrations of cholecystokinin and gastrin in man.

Cholecystokinin (CCK) is a gut hormone which regulates gallbladder contraction and pancreatic enzyme secretion. In addition, CCK is also a major intestinal satiety signal. The knowledge about CCK in circulation, however, has been limited by difficulties in accurate measurement of the concentrations in plasma. Thus, CCK circulates in low concentrations and furthermore, it is structurally homologous to the antral hormone, gastrin, which circulates in higher concentrations. Therefore, most antibodies raised against CCK cross-react in immunoassays with gastrin. However, using highly sensitive and entirely specific in-house radioimmunoassays, which meet these challenges, we have now measured the daily concentration-variations of CCK and gastrin in plasma from young healthy men (n = 24). Plasma was sampled every third hour from each person during 24 h. The results show that the gastrointestinal secretion of both CCK and gastrin in man display significant circadian variations.

Spatiotemporal expression pattern of PERIOD 1 and PERIOD 2 in the mouse SCN is dependent on VIP receptor 2 signaling.

Neurons of the hypothalamic suprachiasmatic nucleus (SCN) express clock genes, which regulate their own transcription and generate daily output signals driving circadian rhythmic behavior and physiology. The neuropeptide VIP and its specific receptor, the VPAC2 receptor, are important for synchronization of clock neurons. In the present study, we characterized PER1 and PER2 expressing neurons in wild-type and VPAC2-deficient mice. We found evidence for distinct spatiotemporal circadian oscillation in the expression of the PER genes in two separate clusters of SCN neurons. In wild-type mice corresponding to the SCN shell and ventral core, high expression of PER was found at lights-off most likely representing an evening clock (E-clock). In another smaller cluster of neurons located in the central core of the SCN, PER expression peaks in antiphase at lights-on and could represent a morning clock (M-clock). BMAL1 immunoreactivity was found to be expressed in antiphase to PER in M and E neurons, respectively. PER was found in 98% of neurons expressing vasopressin (AVP) and in 92% of VIP neurons. The chemotype of M neurons was not identified. M but not E cells were responsive to long but not short photoperiods. The expression of the VPAC2 receptor was found in both M and E cells, and VPAC2-deficient mice displayed markedly blunted PER expression in both cell clusters of the SCN. Conclusion: These observations support the existence of M and E clocks involved in circadian and seasonal adaptation, which seem dependent on intact VIP/VPAC2 signaling in the SCN.

Calcitonin-gene related peptide and disease activity in cluster headache.

OBJECTIVE: To investigate the role of calcitonin gene-related peptide, pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide in cluster headache, we measured these vasoactive peptides interictally and during experimentally induced cluster headache attacks. METHODS: We included patients with episodic cluster headache in an active phase (n = 9), episodic cluster headache patients in remission (n = 9) and patients with chronic cluster headache (n = 13). Cluster headache attacks were induced by infusion of calcitonin gene-related peptide (1.5 µg/min) in a randomized, double-blind, placebo controlled, two-way cross-over study. At baseline, we collected interictal blood samples from all patients and during 11 calcitonin gene-related peptide-induced cluster headache attacks. RESULTS: At baseline, episodic cluster headache patients in remission had higher plasma levels of calcitonin gene-related peptide, 100.6 ± 36.3 pmol/l, compared to chronic cluster headache patients, 65.9 ± 30.5 pmol/l, ( p = 0.011). Episodic cluster headache patients in active phase had higher PACAP38 levels, 4.0 ± 0.8 pmol/l, compared to chronic cluster headache patients, 3.3 ± 0.7 pmol/l, ( p = 0.033). Baseline levels of vasoactive intestinal polypeptide did not differ between cluster headache groups. We found no attack-related increase in calcitonin gene-related peptide, PACAP38 or vasoactive intestinal polypeptide levels during calcitonin gene-related peptide-induced cluster headache attacks. CONCLUSIONS: This study suggests that cluster headache disease activity is associated with alterations of calcitonin gene-related peptide expression. Future studies should investigate the potential of using calcitonin gene-related peptide measurements in monitoring of disease state and predicting response to preventive treatments, including response to anti-calcitonin gene-related peptide monoclonal antibodies.

Role of light and the circadian clock in the rhythmic oscillation of intraocular pressure: Studies in VPAC2 receptor and PACAP deficient mice.

The intraocular pressure of mice displays a daily rhythmicity being highest during the dark period. The present study was performed to elucidate the role of the circadian clock and light in the diurnal and the circadian variations in intraocular pressure in mice, by using animals with disrupted clock function (VPAC2 receptor knockout mice) or impaired light information to the clock (PACAP knockout mice). In wildtype mice, intraocular pressure measured under light/dark conditions showed a statistically significant 24 h sinusoidal rhythm with nadir during the light phase and peak during the dark phase. After transfer of the wildtype mice into constant darkness, the intraocular pressure increased, but the rhythmic changes in intraocular pressure continued with a pattern identical to that obtained during the light/dark cycle. The intraocular pressure in VPAC2 receptor deficient mice during light/dark conditions also showed a sinusoidal pattern with significant changes as a function of a 24 h cycle. However, transfer of the VPAC2 receptor knockout mice into constant darkness completely abolished the rhythmic changes in intraocular pressure. The intraocular pressure in PACAP deficient mice oscillated significantly during both 24 h light and darkness and during constant darkness. During LD conditions, the amplitude of PACAP deficient was significantly lower compared to wildtype mice, resulting in higher daytime and lower nighttime values. In conclusion, by studying the VPAC2 receptor knockout mouse which lacks circadian control and the PACAP knockout mouse which displays impaired light signaling, we provided evidence that the daily intraocular pressure rhythms are primarily generated by the circadian master clock and to a lesser extent by environmental light and darkness.

Cranial parasympathetic activation induces autonomic symptoms but no cluster headache attacks.

Background Low frequency (LF) stimulation of the sphenopalatine ganglion (SPG) may increase parasympathetic outflow and provoke cluster headache (CH) attacks in CH patients implanted with an SPG neurostimulator. Methods In a double-blind randomized sham-controlled crossover study, 20 CH patients received LF or sham stimulation for 30 min on two separate days. We recorded headache characteristics, cephalic autonomic symptoms (CAS), plasma levels of parasympathetic markers such as pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal peptide (VIP), and mechanical detection and pain thresholds as a marker of sensory modulation. Results In the immediate phase (0-60 min), 16 (80%) patients experienced CAS after LF stimulation, while nine patients (45%) reported CAS after sham ( p = 0.046). We found no difference in induction of cluster-like attacks between LF stimulation (n = 7) and sham stimulation (n = 5) ( p = 0.724). There was no difference in mechanical detection and pain thresholds, and in PACAP and VIP plasma concentrations between LF and sham stimulation ( p ≥ 0.162). Conclusion LF stimulation of the SPG induced autonomic symptoms, but no CH attacks. These data suggest that increased parasympathetic outflow is not sufficient to induce CH attacks in patients. Study protocol ClinicalTrials.gov registration number NCT02510729.

PACAP in hypothalamic regulation of sleep and circadian rhythm: importance for headache.

The interaction between sleep and primary headaches has gained considerable interest due to their strong, bidirectional, clinical relationship. Several primary headaches demonstrate either a circadian/circannual rhythmicity in attack onset or are directly associated with sleep itself. Migraine and cluster headache both show distinct attack patterns and while the underlying mechanisms of this circadian variation in attack onset remain to be fully explored, recent evidence points to clear physiological, anatomical and genetic points of convergence. The hypothalamus has emerged as a key brain area in several headache disorders including migraine and cluster headache. It is involved in homeostatic regulation, including pain processing and sleep regulation, enabling appropriate physiological responses to diverse stimuli. It is also a key integrator of circadian entrainment to light, in part regulated by pituitary adenylate cyclase-activating peptide (PACAP). With its established role in experimental headache research the peptide has been extensively studied in relation to headache in both humans and animals, however, there are only few studies investigating its effect on sleep in humans. Given its prominent role in circadian entrainment, established in preclinical research, and the ability of exogenous PACAP to trigger attacks experimentally, further research is very much warranted. The current review will focus on the role of the hypothalamus in the regulation of sleep-wake and circadian rhythms and provide suggestions for the future direction of such research, with a particular focus on PACAP.

Part II: Biochemical changes after pituitary adenylate cyclase-activating polypeptide-38 infusion in migraine patients.

Background Intravenous infusion of pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) provokes migraine attacks in 65-70% of migraine without aura (MO) patients. We investigated whether PACAP38 infusion causes changes in the endogenous production of PACAP38, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), tumour necrosis factor alpha (TNFα), S100 calcium binding protein B (S100B), neuron-specific enolase and pituitary hormones in migraine patients. Methods We allocated 32 previously genotyped MO patients to receive intravenous infusion PACAP38 (10 pmol/kg/minute) for 20 minutes and recorded migraine-like attacks. Sixteen of the patients were carriers of the risk allele rs2274316 ( MEF2D), which confers increased risk of MO and may regulate PACAP38 expression, and 16 were non-carriers. We collected blood samples at baseline and 20, 30, 40, 60 and 90 minutes after the start of the infusion. A control group of six healthy volunteers received intravenous saline. Results PACAP38 infusion caused significant changes in plasma concentrations of VIP ( p = 0.026), prolactin ( p = 0.011), S100B ( p < 0.001) and thyroid-stimulating hormone (TSH; p = 0.015), but not CGRP ( p = 0.642) and TNFα ( p = 0.535). We found no difference in measured biochemical variables after PACAP38 infusion in patients who later developed migraine-like attacks compared to those who did not ( p > 0.05). There was no difference in the changes of biochemical variables between patients with and without the MEF2D-associated gene variant ( p > 0.05). Conclusion PACAP38 infusion elevated the plasma levels of VIP, prolactin, S100B and TSH, but not CGRP and TNFα. Development of delayed migraine-like attacks or the presence of the MEF2D gene variant was not associated with pre-ictal changes in plasma levels of neuropeptides, TNFα and pituitary hormones.

Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus, sculpturing time-of-day-dependent memory formation. This molecular mechanism constitutes the functional link between circadian rhythms and learning efficiency. In hippocampal neurons of wild-type mice, pP90RSK translocates into the nucleus upon stimulation with forskolin (left), whereas in Period1-knockout (Per1(-/-) ) mice (right) the kinase is trapped at the nuclear periphery, unable to efficiently phosphorylate nuclear CREB. Consequently, the presence of PER1 in hippocampal neurons is a prerequisite for the time-of-day-dependent phosphorylation of CREB, as it regulates the shuttling of pP90RSK into the nucleus. Representative immunofluorescence images show a temporal difference in phosphorylated cAMP response element-binding protein (pCREB; green color) levels in all regions of the dorsal hippocampus between a wild-type C3H mouse (WT; left) and a Period1-knockout (Per1(-/-) ; right) mouse. Images were taken 2 h after lights on, thus, when fluctuating levels of pCREB peak in WT mouse hippocampus. Insets show a representative hippocampal neuron, in response to activating cAMP signaling, stained for the neuronal marker NeuN (red), the nuclear marker DAPI (blue) and the activated CREB kinase pP90RSK (green). The image was taken 2 h after light onset (at the peak of the endogenous CREB phosphorylation that fluctuates with time of day). Magnification: 100X, inset 400X. Read the Editorial Highlight for this article on page 650. Cover image for this issue: doi: 10.1111/jnc.13332.

Phosphorylation of rat melanopsin at Ser-381 and Ser-398 by light/dark and its importance for intrinsically photosensitive ganglion cells (ipRGCs) cellular Ca2+ signaling.

The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca(2+) response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca(2+) response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.

Investigation of the pathophysiological mechanisms of migraine attacks induced by pituitary adenylate cyclase-activating polypeptide-38.

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide are structurally and functionally closely related but show differences in migraine-inducing properties. Mechanisms responsible for the difference in migraine induction are unknown. Here, for the first time, we present a head-to-head comparison study of the immediate and long-lasting observations of the migraine-inducing, arterial, physiological and biochemical responses comparing PACAP38 and vasoactive intestinal polypeptide. In a double-blind crossover study 24 female migraine patients without aura were randomly allocated to intravenous infusion of PACAP38 (10 pmol/kg/min) or vasoactive intestinal polypeptide (8 pmol/kg/min) over 20 min. We recorded incidence of migraine during and after infusion (0-24 h). Magnetic resonance angiography of selected extra- and intracranial arteries, blood samples (plasma PACAP38 and vasoactive intestinal polypeptide and serum tryptase), and vital signs (blood pressure, heart rate, respiratory frequency, and end-tidal pressure of CO2) was recorded before and up to 5 h after infusion. Twenty-two patients [mean age 24 years (range 19-36)] completed the study on both days. Sixteen patients (73%) reported migraine-like attacks after PACAP38 and four after vasoactive intestinal polypeptide (18%) infusion (P = 0.002). Three of four patients, who reported migraine-like attacks after vasoactive intestinal polypeptide, also reported attacks after PACAP38. Both peptides induced marked dilatation of the extracranial (P < 0.05), but not intracranial arteries (P > 0.05). PACAP38-induced vasodilatation was longer lasting (>2 h), whereas vasoactive intestinal polypeptide-induced dilatation was normalized after 2 h. We recorded elevated plasma PACAP38 at 1 h after the start of PACAP38 infusion only in those patients who later reported migraine attacks. Blood levels of vasoactive intestinal polypeptide and tryptase were unchanged after PACAP38 infusion. In conclusion, PACAP38-induced migraine was associated with sustained dilatation of extracranial arteries and elevated plasma PACAP38 before onset of migraine-like attacks. PACAP38 has a much higher affinity for the PAC1 receptor and we therefore suggest that migraine induction by PACAP38 may be because of activation of the PAC1 receptor, which may be a future anti-migraine drug target.

Diurnal changes of biochemical metabolic markers in healthy young males - the Bispebjerg study of diurnal variations.

BACKGROUND: To examine whether time of the day has an effect on the circulating levels of metabolism parameters. METHODS: Venous blood samples were obtained under standardized conditions from 24 healthy young men every third hour through 24 hours. The metabolic markers and melatonin were examined at each time-point and data were analyzed by rhythmometric statistical methods. RESULTS: The normal 24-h rhythms of the participants were confirmed by significant oscillation of melatonin (p < 0.0001). Cosinor analysis revealed significant diurnal 24-h rhythms of five of the seven examined markers: Total cholesterol (p = 0.01, amplitude (amp) = 0.18 mmol/L) peaking in the early afternoon, Glucose (p < 0.0001, amp = 0.35 mmol/L) peaking around midnight, C-peptide (p < 0.001, amp = 360 pmol/L), triglyceride (p < 0.0001, amp = 0.37 mmol/L) peaking in the afternoon and low-density lipoprotein cholesterol (p = 0.003, amp = 0.16 mmol/L) peaking in the morning. C-peptide, triglyceride, and glucose had the highest 24-h oscillations in proportion to the reference ranges of the parameters for healthy young men. Glycated haemoglobin (HbA1c) (p = 0.07, amp = 0.57 mmol/L) and high-density lipoprotein (p = 0.09, amp = 0.06 mmol/L) did not show significant oscillations. CONCLUSIONS: When diagnosing and monitoring metabolic disorders compensation for the 24-h variation of the biochemical metabolic markers is needed especially C-peptide, triglyceride and glucose. Furthermore, the stable HbA1c level through 24 h makes it an accurate diagnostic test for diabetes mellitus.

Diurnal variation of von Willebrand factor in plasma: the Bispebjerg study of diurnal variations.

BACKGROUND: Quantitation of von Willebrand factor (VWF) in plasma is a central element in assessing von Willebrand disease (VWD). VWF activity is known to vary, which has partly been ascribed to biological and preanalytical variation. However, a possible diurnal expression of VWF has not been thoroughly tested. OBJECTIVES: We examined whether VWF antigen and VWF activity in plasma display a diurnal profile in healthy young males, and whether such variation is related to changes in release or elimination. METHODS: Plasma from 20 healthy young males was collected at 9 time-points over 24 h (15 h of light and 9 h of darkness); the plasma concentration of melatonin was used as an internal control to confirm the normal 24-h rhythms of the individual participants. RESULTS: The data, analyzed by rhythmometric statistics, revealed a significant variation (P = 0.02) and total amplitude of 22.6% in VWF antigen concentrations over the 24-h period. A pronounced variation in VWF activity was also observed, although not significant according to the 24-h statistical model. To examine whether the diurnal pattern was related to changes in elimination or secretion, the ratio between (i) coagulation factor VIII and VWF and (ii) VWF propeptide and VWF was determined. Taken together, the data suggest changes in release and not in clearance. CONCLUSIONS: Diurnal variation in von Willebrand antigen and activity in plasma represents an important aspect of the biological variation. Standardized time-of-day plasma sampling for quantitation of VWF in VWD patients seems warranted.

Differential expression of melanopsin mRNA and protein in Brown Norwegian rats.

Melanopsin is expressed in a subpopulation of retinal ganglion cells rendering these cells intrinsically photosensitive (ipRGCs). The ipRGCs are the primary RGCs mediating light entrainment of the circadian clock and control of the pupillary light reflex, light regulated melatonin secretion and negative masking behaviour. Previous studies have demonstrated that melanopsin expression in albino rats is regulated by light and darkness. The present study was undertaken to study the influence of light and darkness during the circadian day and after extended periods of constant light and darkness on melanopsin expression in the pigmented retina of the Brown Norwegian rat (Rattus norvegicus). The diurnal and circadian expressions were examined in retinal extracts from rats euthanized every 4 h during a 24 h light/dark (LD) and a 24 h dark cycle (DD) using quantitative real-time PCR and Western blotting. To study whether light regulates melanopsin expression, rats were sacrificed after being placed in either constant light (LL) or darkness for 3 or 21 d. Flat mount retinas from animals kept during either LL or DD were also examined by immunohistochemistry. Melanopsin mRNA expression displayed a significant rhythmic change during the LD cycle with peak expression around dusk and nadir at dawn. Melanopsin protein also changed over the LD cycle with peak expression at the end of the night and nadir at dusk. Rhythmic expression of melanopsin mRNA but not melanopsin protein was found in constant darkness. After 3 or 21 d in either LL or DD melanopsin mRNA expression was unaltered. Melanopsin protein was at the same high level after 3 and 21 d in DD, whereas a significant decrease was found after prolonging the light period for 3 or 21 d. The change in melanopsin protein was primarily due to change in immunoreactivity in the dendritic processes. In conclusion we found that light and darkness are important for regulation of melanopsin protein expression whereas input from a retinal networks regulates melanopsin mRNA expression. It is likely to speculate that altered level of melanopsin is one way in which the retina adapts to environmental light and darkness conditions ensuring optimal light sensitivity for the transmission to the brain.

Extensive monitoring through multiple blood samples in professional soccer players.

The aim of this study was to make a comprehensive gathering of consecutive detailed blood samples from professional soccer players and to analyze different blood parameters in relation to seasonal changes in training and match exposure. Blood samples were collected 5 times during a 6-month period and analyzed for 37 variables in 27 professional soccer players from the best Danish league. Additionally, the players were tested for body composition, V[Combining Dot Above]O2max and physical performance by the Yo-Yo intermittent endurance submax test (IE2). Multiple variations in blood parameters occurred during the observation period, including a decrease in hemoglobin and an increase in hematocrit as the competitive season progressed. Iron and transferrin were stable, whereas ferritin showed a decrease at the end of the season. The immunoglobulin A (IgA) and IgM increased in the period with basal physical training and at the end of the season. Leucocytes decreased with increased physical training. Lymphocytes decreased at the end of the season. The V[Combining Dot Above]O2max decreased toward the end of the season, whereas no significant changes were observed in the IE2 test. The regular blood samples from elite soccer players reveal significant changes that may be related to changes in training pattern, match exposure, or length of the match season. Especially the end of the preparation season and at the end of the competitive season seem to be time points were the blood-derived values indicate that the players are under excessive physical strain and might be more subjected to a possible overreaching-overtraining conditions. We suggest that regular analyses of blood samples could be an important initiative to optimize training adaptation, training load, and game participation, but sampling has to be regular, and a database has to be built for each individual player.

Circadian rhythms and food anticipatory behavior in Wfs1-deficient mice.

The dorsomedial hypothalamic nucleus (DMH) has been proposed as a candidate for the neural substrate of a food-entrainable oscillator. The existence of a food-entrainable oscillator in the mammalian nervous system was inferred previously from restricted feeding-induced behavioral rhythmicity in rodents with suprachiasmatic nucleus lesions. In the present study, we have characterized the circadian rhythmicity of behavior in Wfs1-deficient mice during ad libitum and restricted feeding. Based on the expression of Wfs1 protein in the DMH it was hypothesized that Wfs1-deficient mice will display reduced or otherwise altered food anticipatory activity. Wfs1 immunoreactivity in DMH was found almost exclusively in the compact part. Restricted feeding induced c-Fos immunoreactivity primarily in the ventral and lateral aspects of DMH and it was similar in both genotypes. Wfs1-deficiency resulted in significantly lower body weight and reduced wheel-running activity. Circadian rhythmicity of behavior was normal in Wfs1-deficient mice under ad libitum feeding apart from elongated free-running period in constant light. The amount of food anticipatory activity induced by restricted feeding was not significantly different between the genotypes. Present results indicate that the effects of Wfs1-deficiency on behavioral rhythmicity are subtle suggesting that Wfs1 is not a major player in the neural networks responsible for circadian rhythmicity of behavior.

Expression of the clock genes Per1 and Bmal1 during follicle development in the rat ovary. Effects of gonadotropin stimulation and hypophysectomy.

Daily oscillations of clock genes have recently been demonstrated in the ovaries of several species. Clock gene knockout or mutant mice demonstrate a variety of reproductive defects. Accumulating evidence suggests that these rhythms act to synchronise the expression of specific ovarian genes to hypothalamo-pituitary signals and that they are regulated by one or both of the gonadotropins. The aim of this study has been to examine the spatio-temporal expression of the clock genes Per1 and Bmal1 during gonadotropin-independent and gonadotropin-dependent follicle development in the rat ovary. We have examined the ovaries of prepubertal rats, of prepubertal rats stimulated with equine chorionic gonadotropin (eCG)/human chorionic gonadotropin (hCG) and of hypophysectomised adult animals. Using quantitative reverse transcription with the polymerase chain reaction, in situ hybridisation histochemistry and immunohistochemistry, we have demonstrated that the expression of the two clock genes is low and arrhythmic in ovarian cells during early gonadotropin-independent follicle development in prepubertal animals and in hypophysectomised animals. We have also demonstrated that the expression of the clock genes becomes rhythmic following eCG stimulation in the theca interna cells and the secondary interstitial cells and that, following additional hCG stimulation, the expression of the clock genes also becomes rhythmic in the granulosa cells of preovulatory follicles. These findings link the initiation of clock gene rhythms in the rat ovary to the luteinising hormone receptor and suggest a functional link to androgen and progesterone production. In hypophysectomised animals, rhythmic clock gene expression is also observed in the corpora lutea and in secondary interstitial cells demonstrating that, in these compartments, entrainment of clock gene rhythms is gonadotropin-independent.

Rhythmic 24-hour variations of frequently used clinical biochemical parameters in healthy young males--the Bispebjerg study of diurnal variations.

PURPOSE: To evaluate the influence of time of day on the circulating concentrations of 14 frequently used clinical biochemical parameters in the Bispebjerg study of diurnal variations. MATERIALS AND METHODS: Venous blood samples were obtained under controlled environmental, activities and food conditions from 24 healthy young men every third hour through 24 hours, nine time points in total. At each time point, the parameters' concentrations were measured. The data were analyzed by rhythmometric statistical methods and in addition the biological variations were calculated. RESULTS: Significant oscillation of melatonin with an amplitude (amp) of 19.84 pg/ml and a peak at 03:34 h confirmed the normal 24-hour rhythms of the participants. Potassium (p < 0.0001, amp = 0.18 mmol/L), sodium (p < 0.0001, amp = 1.10 mmol/L) creatine kinase (p = 0.01, amp = 17.18 U/L), bilirubin (p < 0.0001, amp = 2.36 µmol/L) and aspartate aminotransferase (p < 0.0001, amp = 1.66 U/L) oscillated with gradually falling mean concentrations through the day to nadir around midnight. Urea nitrogen (p = 0.01, amp = 0.22 mmol/L) oscillated with gradually increasing mean concentrations through the day peaking around midnight. Lactate dehydrogenase (p < 0.0001, amp = 9.76 U/L) oscillated with gradually increasing concentrations through the early day peaking in the afternoon. Uric acid (p = 0.03, amp = 0.013 mmol/L) oscillated with gradually increasing concentrations through the night peaking in the morning. Potassium and sodium had the highest 24-hour oscillations in proportion to the reference intervals of the parameters for healthy young men. CONCLUSIONS: In the clinical setting, diurnal variations of clinical biochemical parameters commonly used through the day and night must be considered when concentration changes in the parameters are evaluated especially potassium and sodium.

Circadian regulation of protein cargo in extracellular vesicles.

The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (EVs), including exosomes, involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24-hour rhythmic abundance of individual proteins in small EVs using liquid chromatography-mass spectrometry in circadian-synchronized tendon fibroblasts. Furthermore, the release of small EVs enriched in RNA binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Last, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast small EVs is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for small EVs released by tendon fibroblasts, which supports the view that the circadian clock regulates protein cargo in EVs involved in cell-cell cross-talk.

Temporal phasing of locomotor activity, heart rate rhythmicity, and core body temperature is disrupted in VIP receptor 2-deficient mice.

Neurons of the brain's biological clock located in the hypothalamic suprachiasmatic nucleus (SCN) generate circadian rhythms of physiology (core body temperature, hormone secretion, locomotor activity, sleep/wake, and heart rate) with distinct temporal phasing when entrained by the light/dark (LD) cycle. The neuropeptide vasoactive intestinal polypetide (VIP) and its receptor (VPAC2) are highly expressed in the SCN. Recent studies indicate that VIPergic signaling plays an essential role in the maintenance of ongoing circadian rhythmicity by synchronizing SCN cells and by maintaining rhythmicity within individual neurons. To further increase the understanding of the role of VPAC2 signaling in circadian regulation, we implanted telemetric devices and simultaneously measured core body temperature, spontaneous activity, and heart rate in a strain of VPAC2-deficient mice and compared these observations with observations made from mice examined by wheel-running activity. The study demonstrates that VPAC2 signaling is necessary for a functional circadian clock driving locomotor activity, core body temperature, and heart rate rhythmicity, since VPAC2-deficient mice lose the rhythms in all three parameters when placed under constant conditions (of either light or darkness). Furthermore, although 24-h rhythms for three parameters are retained in VPAC2-deficient mice during the LD cycle, the temperature rhythm displays markedly altered time course and profile, rising earlier and peaking ∼4-6 h prior to that of wild-type mice. The use of telemetric devices to measure circadian locomotor activity, temperature, and heart rate, together with the classical determination of circadian rhythms of wheel-running activity, raises questions about how representative wheel-running activity may be of other behavioral parameters, especially when animals have altered circadian phenotype.

Localisation of the neuropeptide PACAP and its receptors in the rat parathyroid and thyroid glands.

PACAP (pituitary adenylate cyclase activating polypeptide) is widely distributed neuropeptide acting via three subtypes of receptors, PAC(1), VPAC(1) and VPAC(2). Here we examined the localisation and nature of PACAP-immunoreactive nerves in the rat thyroid and parathyroid glands and defined the distribution of PAC(1), VPAC(1) and VPAC(2) receptor mRNA's. In the parathyroid gland a large number of nerve fibres displaying PACAP-immunoreactivity were distributed beneath the capsule, around blood vessels and close to glandular cells. Most of the PACAP-nerves were sensory, since they co-stored CGRP (calcitonin-gene-related peptide) and were sensitive to capsaicin-treatment. mRNA's for PAC(1) and VPAC(2) receptors occurred in the parathyroid gland, mainly located in the glandular cells. In the thyroid gland PACAP-immunoreactive nerve fibres were associated with blood vessels, thyroid follicles and parafollicular C-cells. A high degree of co-existence between PACAP and VIP (vasoactive intestinal polypeptide) was observed in the intrathyroid nerve fibres and cell bodies of the thyroid ganglion indicating a common origin for the two peptides. A minor population of PACAP-immunoreactive nerve fibres with relation to blood vessels co-stored NPY (neuropeptide Y), whereas only a few fibres co-stored CGRP. PAC(1) and VPAC(1) receptor mRNA's occurred in follicular cells and blood vessels, whereas the expression of the VPAC(2) receptor was low. The findings suggest that PACAP plays a role in the regulation of parathyroid and thyroid blood flow and hormone secretion.