Circadian synchronization of liver regeneration in adult rats: the role played by adrenal hormones

The role played by the adrenal hormones in the regulation of liver proliferation in adult rats was investigated under various experimental conditions. In untreated control groups, cell growth was very low and endogenous corticosterone levels showed a clearly-defined circadian rhythm with a peak in the evening. Adrenalectomy depressed the level of endogenous corticosterone immediately and the growth rate of the liver increased significantly. We were able to prevent this effect by repeated injections of corticosterone at physiological doses. After a 1/3 hepatectomy and a sham-operation, the corticosterone blood level maintained its normal circadian pattern with the exception of a transient increase during the first two post-operative hours. After a hepatectomy of this kind, a negative correlation was found to exist between the adrenal hormone level and the waves of DNA synthesis; the subsequent mitoses appeared in two successive circadian waves of decreasing amplitude, a maximum value being reached in the morning. In rats submitted to a 1/3 hepatectomy and an adrenalectomy simultaneously, the endogenous corticosterone level fell significantly after a post-operative peak. The regenerating pattern was completely different from that induced by 1/3 hepatectomy alone. The rise in the labelling index began earlier and rose to significantly higher values; it was then followed by a single large mitotic wave without any circadian rhythm. These results favour the hypothesis that adrenal hormones have a significant effect on the negative control of liver regeneration. Circadian changes in the corticosterone level were responsible for the nycthemeral pattern observed in the regenerating liver after a partial hepatectomy. The results show a marked inhibition of the G1-S transition, particularly in the evening, when the endogenous corticosterone concentration was at its highest. Also discussed is the relationship between corticoids and 'chalones', which synergetically inhibit the passage from G0 into the cell cycle.     

高原鼠兔肾上腺皮质功能的每日节律及急性低氧效应

机体对各种有害刺激如冷、热、毒、损伤、缺氧等均产生应激反应,表现出肾上腺皮质功能增强,皮质激素分泌增加,从而提高了机体对外界恶劣环境的适应能力与耐受能力。本文试图探讨高原上的土生动物--高原鼠兔(Ochotona curzoniae)对低氧应激反应的特点,以及高原鼠兔肾上腺皮质酮分泌的每日节律(Circadian rhythm);并与实验大鼠进行比较。     

Effect of chronic intracerebroventricular infusion of corticotropin-releasing factor on circadian corticosterone rhythm in the rat

The effects of chronic (14 day) intracerebroventricular infusion of various amounts of ovine corticotropin-releasing factor (oCRF) on the circadian blood corticosterone rhythm in male rats were examined. Control (saline-infused) rats showed distinct blood corticosterone rhythms over 48 h with nadirs at 0900 h and peaks at 2100 h on days 6-7 and 13-14. oCRF at 3 pmol/h did not affect the circadian corticosterone rhythm on these days. When oCRF was infused at a rate of 12 pmol/h, blood corticosterone was increased throughout the 48 h periods. A significant circadian rhythm remained at days 6-7, but continuous infusion for an additional 7 days disrupted the rhythm. Higher doses of oCRF (48 and 240 pmol/h) obliterated the rhythm during both periods; the disruption was characterized by an increase in corticosterone during the lights-on period without a substantial change in the evening maximum. Thus, the blood corticosterone concentration was eventually confined within a narrow range, not exceeding the normal circadian peak, over a wide dose range of centrally administered CRF. Significant effects of oCRF on body and adrenal weight were observed only at the two highest doses used. These findings may provide some insight into the state of the hypothalamic-pituitary-adrenal axis in animals exposed to chronic stress and in patients with depression.     

Altered Entrainment to the Day/Night Cycle Attenuates the Daily Rise in Circulating Corticosterone in the Mouse

The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT_1B receptors, decreasing responsiveness to light. Consequently, 5-HT_1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light∶dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT_1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h) conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH) stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8-10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8–10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Four waves of hepatocyte proliferation linked with three waves of hepatic fat accumulation during partial hepatectomy-induced liver regeneration

Partial hepatectomy (PH) triggers hepatocyte proliferation-mediated liver repair and is widely used to study the mechanisms governing liver regeneration in mice. However, the dynamics of the hepatocyte proliferative response to PH remain unclear. We found that PH-induced mouse liver regrowth was driven by four consecutive waves of hepatocyte replication. The first wave exhibited the highest magnitude followed by two moderate waves and one minor wave. Underlying this continuous hepatocyte replication was persistent activation of cell cycle components throughout the period of liver regeneration. Hepatocyte mitotic activity in the first three proliferative cycles showed a circadian rhythm manifested by three corresponding mitosis peaks, which were always observed at Zeitgeber time 0. The Bmal1-Clock/Wee1/Cdc2 pathway has been proposed by others to govern the circadian rhythm of hepatocyte mitosis during liver regeneration. However, we did not observe the correlations in the expression or phosphorylation of these proteins in regenerating livers. Notably, Bmal1 protein displayed frequent changes in hepatic distribution and cellular localization as the liver regrowth progressed. Further, three waves of hepatic fat accumulation occurred during hepatic regeneration. The first started before and lasted through the first round of hepatocyte proliferation, whereas the second and third occurred concomitantly with the second and third mitotic peaks, respectively. CONCLUSION: PH-induced liver regeneration consists of four continuous waves of hepatocyte proliferation coupled with three waves of hepatic fat accumulation. Bmal1, Wee1, and Cdc2 may not form a pathway regulating the circadian rhythm of hepatocyte mitosis during liver regeneration.     

Serum corticosteroids at the high and low points of the circadian rhythm in rats with regenerating adrenals.

Serum levels of 11-deoxycorticosterone (DOC), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) and corticosterone (B) were determined at the high (1800 h) and low (0800 h) points of the circadian rhythm in control rats and in rats with regenerating adrenals. The levels of DOC at 0800 h in quiescent rats with regenerating adrenals were 6.5 times greater than in the control group. The levels of 18-OH-DOC and B, however, were not significantly different between these groups. A circadian rhythm for B, 18-OH-DOC and DOC was evident in control rats with a 12,20 and 3.5 fold increase, respectively, at 1800 h as compared to 0800 h. In animals with regenerating adrenals there was only a minimal change in the levels of B and 18-OH-DOC at 1800 h. There was, however, a 2 fold further increase in the levels of DOC at 1800 h as compared with the elevated levels at 0800 h. These findings show that the decrease in 11β and 18-hydroxylase activity of the regenerating adrenal is most clearly evident at the high point of the circadian rhythm. Furthermore, only by taking into account physiological variations in adrenal activity can an accurate assessment of DOC secretion in the adrenal regeneration model of hypertension be obtained.     

Timed maternal melatonin treatment reverses circadian disruption of the fetal adrenal clock imposed by exposure to constant light

Surprisingly, in our modern 24/7 society, there is scant information on the impact of developmental chronodisruption like the one experienced by shift worker pregnant women on fetal and postnatal physiology. There are important differences between the maternal and fetal circadian systems; for instance, the suprachiasmatic nucleus is the master clock in the mother but not in the fetus. Despite this, several tissues/organs display circadian oscillations in the fetus. Our hypothesis is that the maternal plasma melatonin rhythm drives the fetal circadian system, which in turn relies this information to other fetal tissues through corticosterone rhythmic signaling. The present data show that suppression of the maternal plasma melatonin circadian rhythm, secondary to exposure of pregnant rats to constant light along the second half of gestation, had several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affected the mRNA expression level of clock genes and clock-controlled genes as well as it lowered the content and precluded the rhythm of corticosterone. Third, an altered in vitro fetal adrenal response to ACTH of both, corticosterone production and relative expression of clock genes and steroidogenic genes was observed. All these changes were reversed when the mother received a daily dose of melatonin during the subjective night; supporting a role of melatonin on overall fetal development and pointing to it as a 'time giver' for the fetal adrenal gland. Thus, the present results collectively support that the maternal circadian rhythm of melatonin is a key signal for the generation and/or synchronization of the circadian rhythms in the fetal adrenal gland. In turn, low levels and lack of a circadian rhythm of fetal corticosterone may be responsible of fetal growth restriction; potentially inducing long term effects in the offspring, possibility that warrants further research.     

Circadian rhythm of circulating corticosterone and prolactin levels in spontaneously hypertensive rats

Circulating levels of corticosterone and prolactin were measured by radioimmunoassay at hourly intervals during a 24 h period to establish the diurnal rhythm of these hormones in spontaneously hypertensive rats (SHR). Corticosterone levels exhibited a distinct circadian variation with concentrations reaching a zenith at 2000 h and a nadir at 1200 h in male and female SHR. Corticosterone levels in females were consistently greater than males. Circulating prolactin levels were greater during the light h than dark in the female; the opposite occurred in males. Measurement of pituitary prolactin content tended to be low when serum prolactin levels were high and vice versa. The circadian rhythm of circulating corticosterone and prolactin in the hypertensive SHR was found to be similar to the day-night patterns established for normotensive rats. However, these measurements were made under quiescent conditions. It is suggested that because SHR are hyper-responsive to stress and because corticosterone and prolactin have synchronous effects on stress-induced adrenal steroidogenesis, further investigation of prolactin and corticosterone may reveal a participatory role of these hormones in the pathogenesis of the genetically-programmed hypertension of SHR.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36–40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G₂/M phase of the cell cycle was significantly higher than that in G₀/G₁ phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G₂/M phase.     

Diurnal variations of serum testosterone levels in intact and gonadectomized male and female rhesus monkeys.

A radioimmunoassay for serum testosterone which does not require chromatographic separation was used to measure the diurnal variations in intact and orchidecomized males and intact and ovariectomized females. The intact male rhesus monkey shows a distinctive diurnal variation in serum levels of testosterone characterized by lower values during the day and a marked increase in the early evening (1900-2200 hr). The testosterone levels remain high throughout most of the lights-off period in the intact male. In contrast to the intact male, the markedly lowered serum levels of testosterone in the orchidectomized male were higher during the day and consistently showed a nadir during the early evening (2000-2200 hr). The evening nadir of testosterone levels was 51.0% lower than the 24-hr mean whereas the maximum serum level was 46.4% higher. A similar circadian pattern of testosterone was seen in both the intact and ovariectomized females. The testosterone values were higher during the day and consistently showed a nadir during the early evening. These results suggest that the adrenal secretion of testosterone varies in a diurnal pattern characterized by an early evening nadir. This adrenal pattern is overshadowed by a much larger gonadal rhythm in the intact male.     

Opiate receptor blockade and diurnal pituitary and adrenal hormone levels

Peripheral pituitary hormone levels exhibit circadian variations though the mechanism of these changes is unknown. In order to investigate the possible role of endogenous opiates in such changes we have studied the influence of opiate receptor blockade with naloxone (6.8 mg) on pituitary hormones in the morning and again in the evening in six normal male volunteers. Basal ACTH, cortisol, aldosterone and prolactin were higher in the morning than in the evening. Following naloxone at 0700h both ACTH and cortisol rose indicating a tonic inhibition of ACTH by endogenous opiates at that time. At 2230h cortisol rose following naloxone but ACTH did not, suggesting that endogenous opiates do not play an important role in the diurnal rhythm of this hormone and consistent with the suggestion that endogenous opiates can effect cortisol levels independently of their action on ACTH. Neither aldosterone nor prolactin were influenced by naloxone. In contrast TSH was unaffected by naloxone in the morning but fell in the evening (mean + SE decrement over 120 min -0.6 +/- 0.3 mU/l as compared with the control +0.6 +/- 0.4 mU/l; p less than 0.01). Thus, endogenous opiates probably tonically stimulates TSH levels in the evening when TSH may increase and possibly play a role in the circadian rhythm of TSH.     

Circadian rhythm of hypothalamo-hypophyseal-adrenal activity in the chicken.

Corticosteroid production, ACTH content in the adenohypophysis and CRF content in the median eminence were studied in vitro and in vivo in the 3-weeks-old chicken according to SAFFRAN and SCHALLY in four different parts of the day: at 6 and 12 a.m., and 6 and 12 p.m. The chicken adrenals in vitro produced only corticosterone in fluorimetrically measurable amounts, with the maximum during the day and the minimum in the evening and at night. Sensitivity to ACTH of the adrenals in vitro was higher during the day than in the evening or at night. After paper-chromatographic separation, steroid hormones equivalent to corticosterone and cortisol could be detected fluorimetrically in the plasma. The "cortisol" level was about 2-2.5 times higher than the corticosterone level. Plasma "cortisol" level displayed the peak at 6 a.m. and the minimum at 12 p.m., while corticosterone at 12 a.m. and 12 p.m., respectively. ACTH content in the adenohypophysis was lowest at 6 a.m. then increased gradually until 12 p.m. CRF level in the median eminence fell during the forenoon, was lowest at noon, to rise up to 6 p.m. and persist at a high level between 6 p.m. and 6 a.m. Thus, in the 3-week-old chicken the functioning of the hypothalamo-hypophyseal-adrenal system displays a circadian rhythm characteristic of animals showing diurnal activity.     

AUTOSYNCHRONIZATION OF RAT LIVER CELLS WITH ENDOGENOUS CORTICOSTERONE AFTER PARTIAL HEPATECTOMY

After adrenalectomy in adult male rats ³H-TdR incorporation into the liver parenchymal cells is increased 4–8 times and the mitotic index rises from 0–31 % to 1–3%; this is inhibited by corticosterone. After hepatectomy the serum corticosterone level increases from 18 μg/100 ml to 57 μg/100 ml. The corticosterone binding capacity of the serum declines from 2–06 to 0–17. The activity of tyrosine transaminase doubles, whereas the incorporation of ³H-TdR into the liver cells is decreased by a factor of 5–7. Thereafter the binding capacity increases again and reaches, 24 hr after operation, a value of 3–82. The tyrosine transaminase activity and the serum corticosterone content return to normal. ³H-TdR incorporation, however, increases by a factor of 7-7 of the initial value. We concluded that in the first few hours after partial hepatectomy corticosterone blocks the liver cells in G₁ and an accumulation of the cells occurs at this cell cycle phase. Folio wing the binding of the corticosterone by serum proteins a little later the liver cells enter the S-phase synchronously.     

Effects of galanin on the secretion and proliferative activity of the immature and regenerating adrenal glands of rats

The effects of galanin and the galanin-receptor antagonist (galanin-A) [D-Thr(6),D-Trp(8,9),15-ol]-galanin(1-15) on the immature and regenerating rat adrenal glands have been investigated in vivo. Adult female rats with adrenal regeneration and their offpring (20-day-old) were given three subcutaneous injections (28, 16, and 4 h before being killed) of 2 nmol/100 g galanin and/or galanin-A, and 0.1 mg/100 g vincristin 3 h before being killed. Plasma corticosterone concentration was measured by radioimmunoassay, and the mitotic index ( per thousand of metaphase-arrested cells) was evaluated. In immature rats, galanin increased plasma corticosterone concentration, without affecting mitotic index; the secretagogue effect was reversed by galanin-A, which alone was ineffective. In rats with regenerating adrenal, galanin-A increased both blood level of corticosterone and mitotic index; galanin was ineffective, but blocked the effects of galanin-A. These findings allowed us to draw the following conclusions: 1) galanin exerts a moderate glucocorticoid secretagogue action on immature rat adrenals, but endogenous galanin does not play a major physiological role in the functional control of the gland; and 2) endogenous galanin exerts a maximal tonic inhibitory control on both glucocorticoid secretion and proliferative activity of regenerating rat adrenals, whose physiological relevance remains to be investigated.     

Evaluation of the inhibitory effect of circulating corticosteroids on circadian stimulated and stress induced secretion of ACTH in rats

Male rats were bilaterally adrenalectomized in order to measure the extent of inhibition exerted by endogenous corticosteroids on both basal ACTH secretion along its circadian rhythm and ether-stress induced ACTH secretion. In intact controls, plasma ACTH levels at the circadian maximum exceeded by 4 times the circadian minimum, and ACTH response 15 min after ether-stress surpassed the circadian minimum by 20 times. In adrenalectomized rats, the daily minimum was 8 times that of the controls. Nevertheless the circadian maximum was 3 times above the rhythm's minimum, while the maximal stress response (15 min) surpassed the circadian minimum by 8 times. In adrenalectomized rats supplemented with a solid source of corticosterone inducing a stable plasma corticosterone level equivalent to the controls' circadian minimum (3 micrograms/100 ml), the ACTH rhythm still fluctuated twice as high as in intact controls. The tonic feed-back inhibition exerted by endogenous corticosteroids on ACTH secretion appeared thus significantly stronger than the GABAergic inhibition to the corticotropic system which was previously studied under similar standard conditions.     

Influence of melatonin on rat thyroid, adrenals and testis secretion during the day.

The effect of pinealectomy and exogenous melatonin on circadian rhythm of triiodothyronine (T3), thyroxin (T4), corticosterone and testosterone in sham-operated and pinealectomized rats were investigated. The hormones concentration were RIA-measured and the circadian rhythm secretion were analysed by cosinor method. The findings suggest that pinealectomy abolishes the rhythmical character of corticosterone secretion and disturbs the circadian rhythm of T3, T4 and testosterone. Exogenous melatonin has the suppressive effect of diurnal secretion of T3, T4 and testosterone in pinealectomized rats but stimulates the rhythmical corticosterone secretion.