Glucocorticoids Affect 24 h Clock Genes Expression in Human Adipose Tissue Explant Cultures

Aims

to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock genes expression.

Subjects and Methods

VAT and SAT biopsies were obtained from morbid obese women (body mass index≥40 kg/m²) (n = 6). In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX) and AT explants treated with DEX (2 hours) were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR.

Results

CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element) was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements) in the SAT (situation not present in VAT). A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues.

Conclusions

24 h patterns in CLOCK and BMAL1 (positive clock elements) and PER2 (negative element) mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.     

Comparison of β-Adrenergic and Glucocorticoid Signaling on Clock Gene and Osteoblast-Related Gene Expressions in Human Osteoblast

Most living organisms exhibit circadian rhythms that are generated by endogenous circadian clocks, the master one being present in the suprachiasmatic nuclei (SCN). Output signals from the SCN are believed to transmit standard circadian time to peripheral tissue through sympathetic nervous system and humoral routes. Therefore, the authors examined the expression of clock genes following treatment with the β-adrenergic receptor agonist, isoprenaline, or the synthetic glucocorticoid, dexamethasone, in cultured human osteoblast SaM-1 cells. Cells were treated with 10^?6 M isoprenaline or 10^?7 M dexamethasone for 2?h and gene expressions were determined using real-time polymerase chain reaction (PCR) analysis. Treatment with isoprenaline or dexamethasone induced the circadian expression of clock genes human period 1 (hPer1), hPer2, hPer3, and human brain and muscle Arnt-like protein 1 (hBMAL1). Isoprenaline or dexamethasone treatment immediately increased hPer1 and hPer2 and caused circadian oscillation of hPer1 and hPer2 with three peaks within 48?h. hPer3 expression had one peak after isoprenaline or dexamethasone treatment. hBMAL expression had two peaks after isoprenaline or dexamethasone treatment, the temporal pattern being in antiphase to that of the other clock genes. Dexamethasone treatment delayed the oscillation of all clock genes for 2–6?h compared with isoprenaline treatment. The authors also examined the expression of osteoblast-related genes hα-1 type I collagen (hCol1a1), halkaline phosphatase (hALP), and hosteocalcin (hOC). Isoprenaline induced oscillation of hCol1a1, but not hALP and hOC. On the other hand, dexamethasone induced oscillation of hCol1a1 and hALP, but not hOC. Isoprenaline up-regulated hCol1a1 expression, but dexamethasone down-regulated hCol1a1 and hALP expression in the first phase. (Author correspondence: togariaf@dpc.agu.ac.jp)     

Comparison of β-adrenergic and glucocorticoid signaling on clock gene and osteoblast-related gene expressions in human osteoblast

Most living organisms exhibit circadian rhythms that are generated by endogenous circadian clocks, the master one being present in the suprachiasmatic nuclei (SCN). Output signals from the SCN are believed to transmit standard circadian time to peripheral tissue through sympathetic nervous system and humoral routes. Therefore, the authors examined the expression of clock genes following treatment with the β-adrenergic receptor agonist, isoprenaline, or the synthetic glucocorticoid, dexamethasone, in cultured human osteoblast SaM-1 cells. Cells were treated with 10(-6) M isoprenaline or 10(-7) M dexamethasone for 2?h and gene expressions were determined using real-time polymerase chain reaction (PCR) analysis. Treatment with isoprenaline or dexamethasone induced the circadian expression of clock genes human period 1 (hPer1), hPer2, hPer3, and human brain and muscle Arnt-like protein 1 (hBMAL1). Isoprenaline or dexamethasone treatment immediately increased hPer1 and hPer2 and caused circadian oscillation of hPer1 and hPer2 with three peaks within 48?h. hPer3 expression had one peak after isoprenaline or dexamethasone treatment. hBMAL expression had two peaks after isoprenaline or dexamethasone treatment, the temporal pattern being in antiphase to that of the other clock genes. Dexamethasone treatment delayed the oscillation of all clock genes for 2-6?h compared with isoprenaline treatment. The authors also examined the expression of osteoblast-related genes hα-1 type I collagen (hCol1a1), halkaline phosphatase (hALP), and hosteocalcin (hOC). Isoprenaline induced oscillation of hCol1a1, but not hALP and hOC. On the other hand, dexamethasone induced oscillation of hCol1a1 and hALP, but not hOC. Isoprenaline up-regulated hCol1a1 expression, but dexamethasone down-regulated hCol1a1 and hALP expression in the first phase.     

Expression of Clock Genes in Human Subcutaneous and Visceral Adipose Tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41?±?11 yrs of age) presenting a wide range of BMI (21.4 to 48.6?kg/m²) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p?<?.0001) in SAT in comparison to VAT was PER1 of female (372%) and male (326%) subjects. Different patterns of expression between the AM and PM periods were observed, in particular REV-ERBα, which was reduced (p?<?.05) at the PM period in SAT and VAT of both women and men (women: ～53% lower; men: ～78% lower), whereas CLOCK expression was not altered. Relationships between clock genes were different in SAT vs. VAT. BMI was negatively correlated with SATPER1 (r?=??.549; p?=?.001) and SATPER2 (r?=??.613; p?=?.0001) and positively with VATCLOCK (r?=?.541; p?=?.001) and VATBMAL1 (r?=?.468; p?=?.007) only in women. These data suggest that the circadian clock machinery of adipose tissue depots differs between female and male subjects, with a sex-specific effect observed for some genes. BMI correlated with clock genes, but at this moment it is not possible to establish the cause-effect relationship. (Author correspondence: mzanquetta@gmail.com)     

Circadian variations in clock gene expression of human bone marrow CD34+ cells

Time-dependent variations in clock gene expression have recently been observed in mouse hematopoietic cells, but the activity of these genes in human bone marrow (BM) has so far not been investigated. Since such data can be of considerable clinical interest for monitoring the dynamics in stem/progenitor cells, the authors have studied mRNA expression of the clock genes hPer1 , hPer2, hCry1, hCry2, hBmal1, hRev-erb alpha, and hClock in human hematopoietic CD34-positive (CD34( +)) cells. CD34(+) cells were isolated from the BM samples obtained from 10 healthy men at 6 times over 24 h. In addition, clock gene mRNA expression was analyzed in the whole BM in 3 subjects. Rhythms in serum cortisol, growth hormone, testosterone, and leukocyte counts documented that subjects exhibited standardized circadian patterns. All 7 clock genes were expressed both in CD34(+) cells and the whole BM, with some differences in magnitude between the 2 cell populations. A clear circadian rhythm was shown for hPer1, hPer2, and hCry2 expression in CD34(+) cells and for hPer1 in the whole BM, with maxima from early morning to midday. Similar to mouse hematopoietic cells, h Bmal1 was not oscillating rhythmically. The study demonstrates that clock gene expression in human BM stem/progenitor cells may be developmentally regulated, with strong or weaker circadian profiles as compared to those reported in other mature tissues.     

Expression of clock genes in human subcutaneous and visceral adipose tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41 ± 11 yrs of age) presenting a wide range of BMI (21.4 to 48.6 kg/m(2)) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p 相似文献   

Obesity Is Accompanied by Disturbances in Peripheral Glucocorticoid Metabolism and Changes in FA Recycling

The glucocorticoid activating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) is of major interest in obesity‐related morbidity. Alterations in tissue‐specific cortisol levels may influence lipogenetic and gluco/glyceroneogenetic pathways in fat and liver. We analyzed the expression and activity of 11βHSD1 as well as the expression of phosphoenolpyruvate carboxykinase (PEPCK), sterol regulatory element binding protein (SREBP), and fatty acid synthase (FAS) in adipose and liver and investigated putative associations between 11βHSD1 and energy metabolism genes. A total of 33 obese women (mean BMI 44.6) undergoing gastric bypass surgery were enrolled. Subcutaneous adipose tissue (SAT), omental fat (omental adipose tissue (OmAT)), and liver biopsies were collected during the surgery. 11βHSD1 gene expression was higher in SAT vs. OmAT (P = 0.013), whereas the activity was higher in OmAT (P = 0.009). The SAT 11βHSD1 correlated with waist circumference (P = 0.045) and was an independent predictor for the OmAT area in a linear regression model. Energy metabolism genes had AT depot–specific expression; higher leptin and SREBP in SAT than OmAT, but higher PEPCK in OmAT than SAT. The expression of 11βHSD1 correlated with PEPCK in both AT depots (P = 0.05 for SAT and P = 0.0001 for OmAT). Hepatic 11βHSD1 activity correlated negatively with abdominal adipose area (P = 0.002) and expression positively with PEPCK (P = 0.003). In human obesity, glucocorticoid regeneration in the SAT is associated with central fat accumulation indicating that the importance of this specific fat depot is underestimated. Central fat accumulation is negatively associated with hepatic 11βHSD1 activity. A disturbance in peripheral glucocorticoid metabolism is associated with changes in genes involved in fatty acid (FA) recycling in adipose tissue (AT).     

Peripheral circadian clocks are diversely affected by adrenalectomy

Glucocorticoids are considered to synchronize the rhythmicity of clock genes in peripheral tissues; however, the role of circadian variations of endogenous glucocorticoids is not well defined. In the present study, we examined whether peripheral circadian clocks were impaired by adrenalectomy. To achieve this, we tested the circadian rhythmicity of core clock genes (Bmal1, Per1-3, Cry1, RevErbα, Rora), clock-output genes (Dbp, E4bp4) and a glucocorticoid- and clock-controlled gene (Gilz) in liver, jejunum, kidney cortex, splenocytes and visceral adipose tissue (VAT). Adrenalectomy did not affect the phase of clock gene rhythms but distinctly modulated clock gene mRNA levels, and this effect was partially tissue-dependent. Adrenalectomy had a significant inhibitory effect on the level of Per1 mRNA in VAT, liver and jejunum, but not in kidney and splenocytes. Similarly, adrenalectomy down-regulated mRNA levels of Per2 in splenocytes and VAT, Per3 in jejunum, RevErbα in VAT and Dbp in VAT, kidney and splenocytes, whereas the mRNA amounts of Per1 and Per2 in kidney and Per3 in VAT and splenocytes were up-regulated. On the other hand, adrenalectomy had minimal effects on Rora and E4bp4 mRNAs. Adrenalectomy also resulted in decreased level of Gilz mRNA but did not alter the phase of its diurnal rhythm. Collectively, these findings suggest that adrenalectomy alters the mRNA levels of core clock genes and clock-output genes in peripheral organs and may cause tissue-specific modulations of their circadian profiles, which are reflected in changes of the amplitudes but not phases. Thus, the circulating corticosteroids are necessary for maintaining the high-amplitude rhythmicity of the peripheral clocks in a tissue-specific manner.     

Expression of Obesity Markers and Persistent Organic Pollutants Levels in Adipose Tissue of Obese Patients: Reinforcing the Obesogen Hypothesis?

Introduction

Persistent Organic Pollutants (POPs) accumulate in adipose tissue and some are described to possess endocrine disrupting capacities. Therefore, it is important to evaluate their effects on key endocrine pathways in adipose tissue (AT), to further evaluate their potential role in metabolic pathologies such as obesity.

Objectives

The aim is twofold: (i) evaluate gene expression levels of obesity marker genes, i.e. the adipokines leptin (LEP), adiponectin (ADIPOQ) and Tumor Necrosis Factor α (TNFα) and the nuclear receptor, Peroxisome Proliferator Activated Receptor γ (PPARγ) in paired subcutaneous (SAT) and visceral (VAT) AT of obese subjects (n = 50) and to relate these values to serum concentrations of LEP and ADIPOQ (ii) evaluate the association of expression levels of marker genes in AT and serum with POP concentrations in AT.

Results and Conclusions

Leptin and adiponectin levels in serum were positively correlated to respectively expression levels of leptin in SAT and adiponectin in VAT. Our study shows more significant correlations between gene expression of obesity marker genes and POP concentrations in VAT compared to SAT. Since VAT is more important than SAT in pathologies associated with obesity, this suggests that POPs are able to influence the association between obesity and the development of associated pathologies. Moreover, this finding reveals the importance of VAT when investigating the obesogen hypothesis. Concerning PPARγ expression in VAT, negative correlations with polychlorinated biphenyls (PCBs) concentrations were found in non T2D patients. LEP serum concentrations correlated with several PCBs in women whereas in men no correlations were found. This strengthens the potential importance of gender differences in obesity and within the obesogen hypothesis.     

Short-term propofol anaesthesia down-regulates clock genes expression in the master clock

ABSTRACT

Background: Propofol anesthesia triggers phase-advances of circadian rhythms controlled by the suprachiasmatic nuclei (SCN), the master clock. Besides, inhalational anesthesia has been associated with a subsequent reduction of Per2 mRNA levels in the whole brain of rodents. The acute effects of propofol anesthesia per se on the SCN molecular clockwork remain unclear. Here we aim to study the expression of Per1 and Per2 clock genes in the SCN of rats exposed to constant darkness after a single dose of propofol. Methods: Thirty 2-months old rats were randomly divided into 2 groups receiving a single dose of either 120 mg/kg propofol 1% (n=15), or intralipid® 10% (n=15) in late day (projected circadian time (CT) 10, i.e., 10h after the expected time of lights on). Thereafter, rat brains were sampled in darkness 1h, 2h or 3h after the treatment (projected CT11, CT12 or CT13). Expression of Per1 and Per2 mRNA was analyzed by in situ hybridization in SCN coronal sections. Results: Per1 expression was affected by time and treatment. Per1 expression in the SCN after propofol treatment decreased at CT11 and CT12 when compared to the vehicle group. For Per2 expression, we observed only a treatment effect. Observed in dark conditions without hypothermia or/and concomitant surgery, such down-regulation of clock genes Per is only correlated to propofol treatment. This may explain “jet-lag-like” symptoms described by patients after anesthesia. Conclusion: We show here for the first time that short-term propofol anesthesia leads to a transient down-regulation of Per1 and Per2 expression in the SCN.     

The cell adhesion molecule EphA4 is involved in circadian clock functions

Circadian (～24 h) rhythms of cellular network plasticity in the central circadian clock, the suprachiasmatic nucleus (SCN), have been described. The neuronal network in the SCN regulates photic resetting of the circadian clock as well as stability of the circadian system during both entrained and constant conditions. EphA4, a cell adhesion molecule regulating synaptic plasticity by controlling connections of neurons and astrocytes, is expressed in the SCN. To address whether EphA4 plays a role in circadian photoreception and influences the neuronal network of the SCN, we have analyzed circadian wheel‐running behavior of EphA4 knockout (EphA4^?/?) mice under different light conditions and upon photic resetting, as well as their light‐induced protein response in the SCN. EphA4^?/? mice exhibited reduced wheel‐running activity, longer endogenous periods under constant darkness and shorter periods under constant light conditions, suggesting an effect of EphA4 on SCN function. Moreover, EphA4^?/? mice exhibited suppressed phase delays of their wheel‐running activity following a light pulse during the beginning of the subjective night (CT15). Accordingly, light‐induced c‐FOS (FBJ murine osteosarcoma viral oncogene homolog) expression was diminished. Our results suggest a circadian role for EphA4 in the SCN neuronal network, affecting the circadian system and contributing to the circadian response to light.     

Circadian oscillators in the mouse brain: molecular clock components in the neocortex and cerebellar cortex

The circadian timekeeper of the mammalian brain resides in the suprachiasmatic nucleus of the hypothalamus (SCN), and is characterized by rhythmic expression of a set of clock genes with specific 24-h daily profiles. An increasing amount of data suggests that additional circadian oscillators residing outside the SCN have the capacity to generate peripheral circadian rhythms. We have recently shown the presence of SCN-controlled oscillators in the neocortex and cerebellum of the rat. The function of these peripheral brain clocks is unknown, and elucidating this could involve mice with conditional cell-specific clock gene deletions. This prompted us to analyze the molecular clockwork of the mouse neocortex and cerebellum in detail. Here, by use of in situ hybridization and quantitative RT-PCR, we show that clock genes are expressed in all six layers of the neocortex and the Purkinje and granular cell layers of the cerebellar cortex of the mouse brain. Among these, Per1, Per2, Cry1, Arntl, and Nr1d1 exhibit circadian rhythms suggesting that local running circadian oscillators reside within neurons of the mouse neocortex and cerebellar cortex. The temporal expression profiles of clock genes are similar in the neocortex and cerebellum, but they are delayed by 5 h as compared to the SCN, suggestively reflecting a master–slave relationship between the SCN and extra-hypothalamic oscillators. Furthermore, ARNTL protein products are detectable in neurons of the mouse neocortex and cerebellum, as revealed by immunohistochemistry. These findings give reason to further pursue the physiological significance of circadian oscillators in the mouse neocortex and cerebellum.     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.     

Impacts of Visceral Adipose Tissue and Subcutaneous Adipose Tissue on Metabolic Risk Factors in Middle‐aged Japanese

Regional fat distribution rather than overall fat volume has been considered to be important to understanding the link between obesity and metabolic disorders. We aimed to evaluate the independent associations of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) with metabolic risk factors in apparently healthy middle‐aged Japanese. Participants were 1,119 men and 854 women aged 38–60 years who were not taking medications for diabetes, hypertension, or dyslipidemia. VAT and SAT were measured by use of computed tomography (CT) scanning. VAT and SAT were significantly and positively correlated with each other in men (r = 0.531, P < 0.001) and women (r = 0.589, P < 0.001). In multiple regression analyses, either measure of abdominal adiposity (VAT or SAT) was positively associated with blood pressure, fasting plasma glucose, and log triglyceride (P < 0.001) and inversely with high‐density lipoprotein (HDL)‐cholesterol (P < 0.001). When VAT and SAT were simultaneously included in the model, the association of VAT with triglycerides was maintained (P < 0.001) but that of SAT was lost. The same was true for HDL‐cholesterol in women. For fasting plasma glucose, the association with VAT was strong (P < 0.001) and the borderline association with SAT was maintained (P = 0.060 in men and P = 0.020 in women). Both VAT and SAT were independently associated with blood pressure (P < 0.001). Further adjustment for anthropometric indices resulted in the independent association only with VAT for all risk factors. In conclusion, impacts of VAT and SAT differed among risk factors. VAT showed dominant impacts on triglyceride concentrations in both genders and on HDL‐cholesterol in women, while SAT also had an independent association with blood pressure.     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)