Peripheral Clock Gene Research Articles

Lack of Association Between Prokineticin 2 Gene and Japanese Methamphetamine Dependence

Disruption of circadian rhythms may be involved in the pathophysiology of psychiatric disorders, including drug addiction. Recently, we detected the significant association between prokineticin 2 receptor gene (PROKR2) and Japanese methamphetamine dependence patients. Also, prokineticin 2 (PK2) gene deficient mice showed reduced physiological and behavioral parameters, including circadian locomotor activity, circulating glucocorticoid, glucose levels and the expression of peripheral clock genes compared with WT mice. These evidences indicate that PK2 gene (PROK2) is a good candidate gene for the pathogenesis of methamphetamine dependence. To evaluate the association between PROK2 and methamphetamine dependence, we conducted a case-control study of Japanese samples (215 methamphetamine dependence and 232 controls) with four tagging SNPs selected by HapMap database. The age and sex of the control subjects did not differ from those of the methamphetamine dependence patients. Written informed consent was obtained from each subject. This study was approved by the ethics committees at Fujita Health University, Nagoya University Graduate School of Medicine and each participating member of the Institute of the Japanese Genetics Initiative for Drug Abuse (JGIDA). We did not detect an association between PROK2 and Japanese methamphetamine dependence patients in allele/genotype-wise analysis, or the haplotype analysis. Our findings suggest that PROK2 does not play a major role in the pathophysiology of methamphetamine dependence in the Japanese population.

Spectral modulation attenuates molecular, endocrine, and neurobehavioral disruption induced by nocturnal light exposure

The human eye serves distinctly dual roles in image forming (IF) and non-image-forming (NIF) responses when exposed to light. Whereas IF responses mediate vision, the NIF responses affect various molecular, neuroendocrine, and neurobehavioral variables. NIF responses can have acute and circadian phase-shifting effects on physiological variables. Both the acute and phase-shifting effects induced by photic stimuli demonstrate short-wavelength sensitivity peaking ≈450-480 nm. In the current study, we examined the molecular, neuroendocrine, and neurobehavioral effects of completely filtering (0% transmission) all short wavelengths <480 nm and all short wavelengths <460 nm or partially filtering (~30% transmission) <480 nm from polychromatic white light exposure between 2000 and 0800 in healthy individuals. Filtering short wavelengths <480 nm prevented nocturnal light-induced suppression of melatonin secretion, increased cortisol secretion, and disrupted peripheral clock gene expression. Furthermore, subjective alertness, mood, and errors on an objective vigilance task were significantly less impaired at 0800 by filtering wavelengths <480 nm compared with unfiltered nocturnal light exposure. These changes were not associated with significantly increased sleepiness or fatigue compared with unfiltered light exposure. The changes in molecular, endocrine, and neurobehavioral processes were not significantly improved by completely filtering <460 nm or partially filtering <480 nm compared with unfiltered nocturnal light exposure. Repeated light-dark cycle alterations as in rotating nightshifts can disrupt circadian rhythms and induce health disorders. The current data suggest that spectral modulation may provide an effective method of regulating the effects of light on physiological processes.

Nocturnin: a circadian target of Pparg‐induced adipogenesis

Nuclear receptors (NRs) control cell fate and regulate tissue function. Some of the NRs are expressed in a circadian and tissue-specific manner. Clock genes are part of the circadian network and fine-tune gene expression in adipose and skeletal tissues. Pparg, a master transcription factor that determines adipogenesis, exhibits a circadian expression pattern in white adipose tissue and liver. Here we report the finding that the message and protein for a peripheral clock gene, nocturnin, is markedly upregulated with Pparg activation in adipocytes and bone marrow stromal cells. Nocturnin is also expressed in relatively high amounts in other tissues that may have physiologic relevance for bone, including the brain and hypothalamus. Of importance, we found polymorphic strain differences in bone marrow nocturnin expression that relate to phenotypic determinants of skeletal acquisition. Defining the function of nocturnin in peripheral tissues should provide new insights into lineage allocation and the intimate relationship between nuclear receptors and physiologic timekeeping.

Effects of acute and chronic STZ-induced diabetes on clock gene expression and feeding in the gastrointestinal tract

Diabetes may shift clock gene expression within peripheral organs. However, little is known about the effect of diabetes on the gastrointestinal molecular clock. We therefore investigated the effect of diabetes on gastrointestinal clock gene expression. As peripheral clock gene expression is strongly driven by food intake, we also determined the effect of STZ-induced diabetes on patterns of food intake. The effects of acute (1 week) and chronic (12 weeks) STZ-induced diabetes on period (per) genes in the stomach body, proximal and distal colon, liver, kidney, and lung of C57BL/6J mice were assessed using real-time polymerase chain reaction. Food intake studies were completed using automated feeding equipment. Rhythmicity in expression of per2 and per3 persisted in all organs. However, per2 and per3 expression of STZ-injected mice was generally phase delayed within the gastrointestinal tract but not within the kidney or lung as compared with vehicle-injected mice. The phase delay was most pronounced for per2 in the proximal colon at 12 weeks. Food intake was rhythmic with larger circadian amplitude for diabetic mice than for control mice. Thus, STZ-induced diabetes differentially alters peripheral per expression. STZ-induced diabetes does not alter the circadian phase of food intake. Alterations in clock gene expression in a mouse model of diabetes are most pronounced in those organs that are intimately associated with food processing and metabolism.

Effects of Nocturnal Light on (Clock) Gene Expression in Peripheral Organs: A Role for the Autonomic Innervation of the Liver

BackgroundThe biological clock, located in the hypothalamic suprachiasmatic nucleus (SCN), controls the daily rhythms in physiology and behavior. Early studies demonstrated that light exposure not only affects the phase of the SCN but also the functional activity of peripheral organs. More recently it was shown that the same light stimulus induces immediate changes in clock gene expression in the pineal and adrenal, suggesting a role of peripheral clocks in the organ-specific output. In the present study, we further investigated the immediate effect of nocturnal light exposure on clock genes and metabolism-related genes in different organs of the rat. In addition, we investigated the role of the autonomic nervous system as a possible output pathway of the SCN to modify the activity of the liver after light exposure.Methodology and Principal FindingsFirst, we demonstrated that light, applied at different circadian times, affects clock gene expression in a different manner, depending on the time of day and the organ. However, the changes in clock gene expression did not correlate in a consistent manner with those of the output genes (i.e., genes involved in the functional output of an organ). Then, by selectively removing the autonomic innervation to the liver, we demonstrated that light affects liver gene expression not only via the hormonal pathway but also via the autonomic input.ConclusionNocturnal light immediately affects peripheral clock gene expression but without a clear correlation with organ-specific output genes, raising the question whether the peripheral clock plays a “decisive” role in the immediate (functional) response of an organ to nocturnal light exposure. Interestingly, the autonomic innervation of the liver is essential to transmit the light information from the SCN, indicating that the autonomic nervous system is an important gateway for the SCN to cause an immediate resetting of peripheral physiology after phase-shift inducing light exposures.

A two-clock model of circadian timing in the immune system of mammals

It has been confirmed that clock genes, as well as the pineal hormone, have a role in the hypothalamic suprachiasmatic nucleus, the circadian endogenous pacemaker. It seems that the peripheral clock genes in the cells of the immune system subtly control biorhythms; their seeming lack of impact only showing that they work well. Some biorhythms even seem to be independent of a light/dark circadian regime. This apparent conflict in the mammalian time structure can be resolved by a two-clocks control model involving: (a) the endogenous gene clock, which is dominant in the neural system and (b) the exogenous clock of the immune system. Interactions between these two clocks can explain both the frequently observed individual differences in circadian rhythms and the subtle role of the peripheral clock genes. The endogenous clock facilitates an alternation in the immune system which counters external attacks in daytime and induces repair and advancement by night.

Endocrine-dependent expression of circadian clock genes in insects.

Current models state that insect peripheral oscillators are directly responsive to light, while mammalian peripheral clock genes are coordinated by a master clock in the brain via intermediate factors, possibly hormonal. We show that the expression levels of two circadian clock genes, period (per) and Par Domain Protein 1 (Pdp1) in the peripheral tissue of an insect model species, the linden bug Pyrrhocoris apterus, are inversely affected by contrasting photoperiods. The effect of photoperiod on per and Pdp1 mRNA levels was found to be mediated by the corpus allatum, an endocrine gland producing juvenile hormone. Our results provide the first experimental evidence for the effect of an endocrine gland on circadian clock gene expression in insects.

Circadian Traffic of Hematopoietic Stem Cells Is Orchestrated by the Molecular Clock and Mediated by β3 Adrenergic Signals from the Sympathetic Nervous System.

Under homeostasis, low numbers of haematopoietic stem cells (HSPC) are detectable in the bloodstream of mammals. Here, we show the mechanisms behind the robust circadian oscillations of circulating HSPC in mice. In normal 12h light-12h darkness cycles (LD) there were 2–4-fold fluctuations in Lin-Sca-1+c-kit+ cells and competitive repopulating stem cell units (CRU; 16 weeks) between the peak (5h after the initiation of light) and trough (12h later). Circadian fluctuations of HSPC were entrained by photic cues since the pattern was dramatically altered when mice were subjected to continuous light or a “jet lag” of 12h. Because CXCL12 mediates HSPC migration, we evaluated whether it is subjected to circadian control. Indeed, CXCL12 protein and mRNA levels in the bone marrow (BM) fluctuate under all light conditions in antiphase with circulating HSPC. Since the sympathetic nervous system (SNS) affects G-CSF-induced HSPC mobilization (Cell 2006; 124:407–21), we analyzed its role in circadian HSPC release. SNS disruption by injection of 6-hydroxydopamine significantly hampered HSPC egress from the BM, as revealed by the marked reductions in the number of CRU in the blood obtained from sympathectomized animals compared to control mice. Moreover, unilateral section of the sciatic and femoral nerves revealed that the circadian oscillations of Cxcl12 were severely altered in the denervated tibiae but not in the contralateral sham-operated limbs, suggesting the requirement of adrenergic signals locally delivered in the BM. To dissect further the underlying mechanism, we treated BM stromal cells (MS-5) with adrenergic agonists and antagonists. Norepinephrine and isoproterenol, a non-selective β-adrenergic agonist, reduced CXCL12 production in a dose-dependent manner. Unexpectedly, this effect was mediated by the β3- (Adrb3) but not the β2-adrenergic receptor (Adrb2) since it was induced by a β3-adrenergic agonist and inhibited by a specific β3 antagonist while Adrβ2 engagement or blockade had no effect. Similar results were obtained in primary myeloid BM cultures. Further, isoproterenol treatment significantly reduced Cxcl12 expression in stroma derived from Adrb2−/− but not Adrb3−/− mice. In addition, isoproterenol administration increased circulating HSPC in wild-type but not Adrb3−/− mice in which HSPC homing was blocked by inactivation of endothelial selectins and α4 integrins. Since SNS signals can modulate osteoblast proliferation via peripheral expression of clock genes, we profiled the expression of core clock genes in the BM, and found altered patterns of Clock, Bmal1, Per1, Per2, Cry1 and Rev-erb-α under continuous light or jet lag conditions. However, treatment of stromal cells obtained from the BM of Bmal1−/− or Per1−/−Per2m/mmice with isoproterenol reduced Cxcl12 to the same extent as in wild-type control stroma, suggesting that peripheral clock genes do not directly regulate Cxcl12. The physiological oscillations in circulating HSPC may impact the harvested stem cell yield since more HSPC were obtained at the acrophase in mice injected with G-CSF or AMD-3100. All together, these results demonstrate that the cyclical HSPC release and Cxcl12 BM expression are regulated by the central and not the peripheral molecular clock through signals from the SNS that are transduced locally by the β3-adrenergic receptor in BM stromal cells. Since osteoblasts are reported to express only Adrb2, these results also suggest the contribution of other stromal components in the physiological release of HSPC.

Entrainment to Feeding but Not to Light: Circadian Phenotype of VPAC2Receptor-Null Mice

The master clock driving mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and entrained by daily light/dark cycles. SCN lesions abolish circadian rhythms of behavior and result in a loss of synchronized circadian rhythms of clock gene expression in peripheral organs (e.g., the liver) and of hormone secretion (e.g., corticosterone). We examined rhythms of behavior, hepatic clock gene expression, and corticosterone secretion in VPAC2 receptor-null (Vipr2-/-) mice, which lack a functional SCN clock. Unexpectedly, although Vipr2-/- mice lacked robust circadian rhythms of wheel-running activity and corticosterone secretion, hepatic clock gene expression was strongly rhythmic, but advanced in phase compared with that in wild-type mice. The timing of food availability is thought to be an important entrainment signal for circadian clocks outside the SCN. Vipr2-/- mice consumed food significantly earlier in the 24 h cycle than wild-type mice, consistent with the observed timing of peripheral rhythms of circadian gene expression. When restricted to feeding only during the daytime (RF), mice develop rhythms of activity and of corticosterone secretion in anticipation of feeding time, thought to be driven by a food-entrainable circadian oscillator, located outside the SCN. Under RF, mice of both genotypes developed food-anticipatory rhythms of activity and corticosterone secretion, and hepatic gene expression rhythms also became synchronized to the RF stimulus. Thus, food intake is an effective zeitgeber capable of coordinating circadian rhythms of behavior, peripheral clock gene expression, and hormone secretion, even in the absence of a functional SCN clock.

Circadian Rhythms in the CNS and Peripheral Clock Disorders: Function of Clock Genes: Influence of Medication for Bronchial Asthma on Circadian Gene

Bronchial asthma is a chronic inflammatory disorder of the airways, in which inflammation causes bronchial hyper-responsiveness and flow limitation in the presence of various stimuli. Pulmonary function in asthmatic patients frequently deteriorates between midnight and early morning, which has suggested a role for chronotherapy. Although relationships between bronchial asthma and the function of clock genes remain unclear, some medications given for asthma such as glucocorticoids or β2-adrenoceptor agonists may influence clock genes in vivo. In our studies of clock gene mRNA expressions in human bronchial epithelial cells in vitro and peripheral blood cells in vivo, we demonstrated that glucocorticoid or β2-adrenoceptor agonist treatment strongly induced human Per1 mRNA expression both in vitro and in vivo. Human peripheral blood cells provide a useful indication of peripheral clock gene mRNA expression in vivo.

Attenuated Circadian Rhythms in Mice Lacking theProkineticin 2Gene

Circadian clocks drive daily rhythms in virtually all organisms. In mammals, the suprachiasmatic nucleus (SCN) is recognized as the master clock that synchronizes central and peripheral oscillators to evoke circadian rhythms of diverse physiology and behavior. How the timing information is transmitted from the SCN clock to generate overt circadian rhythms is essentially unknown. Prokineticin 2 (PK2), a clock-controlled gene that encodes a secreted protein, has been indicated as a candidate SCN clock output signal that regulates circadian locomotor rhythm. Here we report the generation and analysis of PK2-null mice. The reduction of locomotor rhythms in PK2-null mice was apparent in both hybrid and inbred genetic backgrounds. PK2-null mice also displayed significantly reduced rhythmicity for a variety of other physiological and behavioral parameters, including sleep-wake cycle, body temperature, circulating glucocorticoid and glucose levels, as well as the expression of peripheral clock genes. In addition, PK2-null mice showed accelerated acquisition of food anticipatory activity during a daytime food restriction. We conclude that PK2, acting as a SCN output factor, is important for the maintenance of robust circadian rhythms.

Peripheral clock gene expression in CS mice with bimodal locomotor rhythms

CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct bimodal rhythms (similar to rhythm splitting, but hereafter referred to as bimodal rhythms) under constant darkness. In the present study, we compared clock-related gene expression ( mPer1, mBmal1 and Dbp) in the SCN and peripheral tissues (liver, adrenal gland and heart) between CS and C57BL/6J mice. In spite of normal robust oscillation in the SCN of both mice, behavioral rhythms and peripheral rhythms of clock-related genes were significantly different between these mice. However, when daytime restricted feeding was given, no essential differences between the two strains were observed. These results indicate that unusual circadian behaviors and peripheral gene expression in CS mice do not depend on the SCN but rather mechanisms outside of the SCN.

Circadian Expression of Plasminogen Activator Inhibitor-1 in Angiotensin II Type 1a Receptor Knockout Mice

Both the peripheral biological clock and the renin-angiotensin system regulate mRNA expression of plasminogen activator inhibitor-1 (PAI-1). Our objective was to determine whether angiotensin II (Ang II) type 1 (AT1) receptor-mediated signaling contributes to the development of circadian expression of PAI-1 and clock genes in the heart, aorta, liver, and kidney. We sacrificed AT1a receptor knockout (AT1a-KO) and wild-type (WT) 12 week-old mice every 4 hr. We examined mRNA expression for PAI-1 and clock genes (Per2, Bmal1, and Clock) in heart, aorta, liver, and kidney by using the quantitative reverse transcription-polymerase chain reaction. PAI-1 mRNA showed circadian oscillation with a peak occurring during the light phase in the heart, liver, aorta, and kidney of WT mice. Peak expression of PAI-1 in the liver and aorta was decreased in AT1a-KO mice. On the other hand, cardiac PAI-1 expression in AT1a-KO mice was reduced in the dark phase, during which time its expression level was low. There were no significant differences between WT and AT1a-KO mice in renal PAI-1 expression. Clock genes oscillated synchronously in WT and AT1a-KO mice, and there were no significant differences between the WT and the AT1a-KO mice in their expression. Plasma angiotensin II showed little oscillation in the WT mice. We conclude that AT1a receptor-mediated Ang II signaling modulates the circadian expression of PAI-1 in an organ-specific manner. The effect of the renin-angiotensin system on PAI-1 expression appears to be independent of peripheral clock gene expression.

Human Intestinal Circadian Clock: Expression of Clock Genes in Colonocytes Lining the Crypt

Biological clock components have been detected in many epithelial tissues of the digestive tract of mammals (oral mucosa, pancreas, and liver), suggesting the existence of peripheral circadian clocks that may be entrainable by food. Our aim was to investigate the expression of main peripheral clock genes in colonocytes of healthy humans and in human colon carcinoma cell lines. The presence of clock components was investigated in single intact colonic crypts isolated by chelation from the biopsies of 25 patients (free of any sign of colonic lesions) undergoing routine colonoscopy and in cell lines of human colon carcinoma (Caco2 and HT29 clone 19A). Per‐1, per‐2, and clock mRNA were detected by real‐time RT‐PCR. The three‐dimensional distributions of PER‐1, PER‐2, CLOCK, and BMAL1 proteins were recorded along colonic crypts by immunofluorescent confocal imaging. We demonstrate the presence of per‐1, per‐2, and clock mRNA in samples prepared from colonic crypts of 5 patients and in all cell lines. We also demonstrate the presence of two circadian clock proteins, PER‐1 and CLOCK, in human colonocytes on crypts isolated from 20 patients (15 patients for PER‐1 and 6 for CLOCK) and in colon carcinoma cells. Establishing the presence of clock proteins in human colonic crypts is the first step toward the study of the regulation of the intestinal circadian clock by nutrients and feeding rhythms.

Circadian Expression of Plasminogen Activator Inhibitor-1 in Angiotensin II Type 1a Receptor Knockout Mice

Both the peripheral biological clock and the renin-angiotensin system regulate mRNA expression of plasminogen activator inhibitor-1 (PAI-1). Our objective was to determine whether angiotensin II (Ang II) type 1 (AT1) receptor-mediated signaling contributes to the development of circadian expression of PAI-1 and clock genes in the heart, aorta, liver, and kidney. We sacrificed AT1a receptor knockout (AT1a-KO) and wild-type (WT) 12 week-old mice every 4 hr. We examined mRNA expression for PAI-1 and clock genes (Per2, Bmal1, and Clock) in heart, aorta, liver, and kidney by using the quantitative reverse transcription-polymerase chain reaction. PAI-1 mRNA showed circadian oscillation with a peak occurring during the light phase in the heart, liver, aorta, and kidney of WT mice. Peak expression of PAI-1 in the liver and aorta was decreased in AT1a-KO mice. On the other hand, cardiac PAI-1 expression in AT1a-KO mice was reduced in the dark phase, during which time its expression level was low. There were no significant differences between WT and AT1a-KO mice in renal PAI-1 expression. Clock genes oscillated synchronously in WT and AT1a-KO mice, and there were no significant differences between the WT and the AT1a-KO mice in their expression. Plasma angiotensin II showed little oscillation in the WT mice. We conclude that AT1a receptor-mediated Ang II signaling modulates the circadian expression of PAI-1 in an organ-specific manner. The effect of the renin-angiotensin system on PAI-1 expression appears to be independent of peripheral clock gene expression.

Effect of feeding on peripheral circadian rhythms and behaviour in mammals.

Although feeding time is a dominant cue for circadian rhythms in mammalian peripheral tissue, the effect of feeding and fasting on circadian gene expression and behaviour is unknown. Here we report that fasting does not affect the phase of rhythmic mRNA expression levels of the clock genes, mPer1, mPer2 and of the clock controlled gene, mDBP. However, the levels of each of these genes were significantly altered in different ways and recovered by feeding. We also found that feeding enhances phase-shifting to a new light-dark cycle of rhythmic mPer2 mRNA expression in the heart. Furthermore, feeding enhances the phase-shifting to new light-dark cycle of behaviour more than fasting. Our data indicate that feeding is an important cue for circadian behaviour rhythms as well as for the photo-entrainment of peripheral clock gene expression.

Daily injection of insulin attenuated impairment of liver circadian clock oscillation in the streptozotocin-treated diabetic mouse

Recent studies have shown a dampened amplitude of clock gene rhythm in the heart and liver of streptozotocin (STZ)-treated rats and mice, however it is unknown whether impairment is due to dysfunction of the suprachiasmatic nucleus (SCN) or not. Rhythmic expression of mPER2 was dampened in the STZ-treated mouse liver but not SCN and cerebral cortex. Injection of insulin could normalize an impairment of mPer2 and mPER2 expression rhythm in the liver, when it was injected at nighttime, but not at daytime. In the present study, we demonstrated that insulin-dependent diabetes impaired oscillation of the peripheral clock gene and its product. Insulin injection can recover dampened oscillation of the peripheral clock depending on its injection time.

Alterations of circadian expressions of clock genes in Dahl salt-sensitive rats fed a high-salt diet.

In mammals, behavioral and physiologic processes display 24-hour rhythms that are regulated by a circadian system consisting of central and peripheral oscillators. Because various cardiovascular functions show diurnal variations and abnormal patterns of circadian blood pressure variation carry a high risk of cardiovascular complications, we investigated whether the expression of clock genes is altered in an animal model of hypertension. In Dahl salt-sensitive rats fed a high-salt (4% NaCl) diet for 6 weeks (DS-H), radiotelemetry monitoring showed increased amplitude of circadian variations in blood pressure. The ratio of heart weight to body weight and the ratio of kidney weight to body weight were higher in DS-H. Echocardiographic data showed that the wall thickness of the left ventricle was greater in DS-H. Northern blot analysis and single cosinor analysis revealed that the amplitudes of circadian expression changes of the clock genes (mPer2, Bmal1, and dbp) in the heart, liver, and kidney were significantly decreased in DS-H rats compared with a normal-salt-diet group, except for Bmal1 in the liver. The circadian expression changes of plasminogen activator inhibitor-1, a clock-regulated gene, were attenuated in the hearts of DS-H. The present results demonstrate that DS-H show altered circadian expression of peripheral clock genes. Detailed analyses of the relation between circadian expression of clock genes and blood pressure regulation might reveal a role for chronologic therapy of cardiovascular disease.

Neural regulation of circadian rhythms.

Neural regulation of circadian rhythms.