Circadian Rhythm in Mammary Cytoplasmic Estrogen Receptor Content of Balb/C Female Mice with and Without Pituitary Isografts

Cytoplasmic estrogen receptors were determined by the dextran-coated charcoal method in inguinal breast tissue of three groups of Balb/C female mice 6–8 weeks following subcutaneous implantation into the intact animals of three pituitary glands and three pieces of skeletal muscle (group I), three pituitary glands and three segments of hypothalamic tissue (group II), or three pieces of skeletal muscle (group III) obtained from animals of the same inbred strain as control. A circadian rhythm in estrogen receptor content was statistically quantified by cosinor analysis in the muscle implanted control and the pituitary and hypothalamic implant groups. In the pituitary and muscle implant group the circadian rhythm is of borderline significance with a P-value between 0.05 and 0.10. The timing (acrophase) and extent of change (amplitude) are similar in all three treatment groups. The average receptor content (MESOR) in the two pituitary-implanted groups, which in previous studies were shown to have an increased breast cancer incidence is about twice that of the control group. The reduction in the pituitary induced breast cancer rate by hypothalamic tissue addition to a cancer incidence between the animals with pituitary and muscle isograft and the mice carrying no pituitary at all has also been shown previously in this strain of mice and is not reflected in receptor content.     

The effect of multi-frequency whole-body vibration on night-shifted mouse model

The circadian rhythm controls several biological activities; therefore, a disorganized circadian rhythm may cause fatal health problems. The aim of this study was to assess the effects of circadian rhythm disturbances induced by simulated night shift activities on the abdominal adipose tissue, bone microstructures and muscle volume in the tibiae of mice. Moreover, we evaluated the effects of multi-frequency whole-body vibration as a countermeasure against the consequences of circadian rhythm disturbances. Twenty-four 5-week-old C57BL/6J male mice were equally assigned to three groups: the normal group (Nor), night shift group (NS), and night shift with multi-frequency whole-body vibration group (NS + V). The NS and NS + V groups were exposed to circadian rhythm disturbances for 4 weeks with 3-day intervals by changing the day and night cycle based on 7 o’clock. After 4 weeks, morphological changes in the adipose tissue, bone microstructures and muscle volume in the tibiae were evaluated from three-dimensional images using in vivo micro-computed tomography. As a result, the volume of the abdominal adipose tissue was significantly higher in the NS than in the Nor and NS + V groups. Also, the microstructures of the tibia were more enhanced in the NS + V than the NS group. The volume of tibial muscle was increased in all groups, while there were no significant changes in muscle volume. From these results, we can conclude that circadian rhythm disturbances induced by night shift activities may reduce bone condition and increase the accumulation of abdominal adipose tissue and these negative effects may be prevented or improved through applying multi-frequency whole-body vibration.

     

河北省石家庄市第一医院

目的:检测日间和夜间Lewis肺癌小鼠血清中内血管内皮生长因子(VEGF)水平和肺癌组织中VEGF蛋白表达的差别,探讨肿瘤血管生成的昼夜节律。方法:选择C57BL小鼠30只,制备Lewis肺癌小鼠模型后随机分为日间组(D组)和夜间组(N组),在光照-黑暗条件下饲养建立统一同步化日夜节律。随着成瘤过程,应用ElISA方法测定两组小鼠模型第0、3、5、7天血清中VEGF浓度水平;成瘤后第9天处死小鼠,应用Westeon-blot法检测瘤体中VEGF蛋白的表达,并分别进行相关分析。结果:随着成瘤过程,D组小鼠血清中VEGF浓度均显著高于N组(P<0.05);D组小鼠瘤体组织中VEGF蛋白表达灰度值(8.87±1.20)均明显高于N组(6.43±1.35),有统计学意义(P<0.05)。结论:Lewis小鼠休息期(白天)血清中VEGF浓度及瘤体中VEGF蛋白表达水平均明显高于活动期(夜间),存在着明显的日夜差异,说明肺癌组织的血管生成可能具有一定日夜节律。     

Constant light exposure aggravates POMC-mediated muscle wasting associated with hypothalamic alteration of circadian clock and SIRT1 in endotoxemia rats

Constant light exposure is widespread in the intensive care unit (ICU) and could increase the rate of brain dysfunction as delirium and sleep disorders in critical patients. And the activation of hypothalamic neuropeptides is proved to play a crucial role in regulating hypercatabolism, especially skeletal muscle wasting in critical patients, which could lead to serious complications and poor prognosis. Here we investigated the hypothesis that constant light exposure could aggravate skeletal muscle wasting in endotoxemia rats and whether it was associated with alterations of circadian clock and hypothalamic proopiomelanocortin(POMC) expression. Fifty-four adult male Sprague-Dawley rats were intraperitoneally injected with lipopolysaccharide(LPS) or saline, subjected to constant light or a 12:12?h light-dark cycle for 7 days. On day 8, rats were sacrificed across six time points in 24?h and hypothalamus tissues and skeletal muscle were obtained. Rates of muscle wasting were measured by 3-methylhistidine(3-MH) and tyrosine release as well as expression of two muscle atrophic genes, muscle ring finger 1(MuRF-1) and muscle atrophy F-box(MAFbx). The expression of circadian clock genes, silent information regulator 1(SIRT1), POMC and hypothalamic inflammatory cytokines were also detected. Results showed that LPS administration significantly increased hypothalamic POMC expression, inflammatory cytokine levels and muscle wasting rates. Meanwhile constant light exposure disrupted the circadian rhythm, declined the expression of SIRT1 as well as aggravated hypothalamic POMC overexpression and skeletal muscle wasting in rats with endotoxemia. Taken together, the results demonstrated that constant light exposure could aggravate POMC-mediated skeletal muscle wasting in endotoxemia rats, which is associated with alteration of circadian clocks and SIRT1 in the hypothalamus.     

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ERA) gene also oscillates in a circadian fashion. In contrast, ERA-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ERA-positive breast cancer cells do not display circadian oscillation of ERA expression. Our findings suggest that estrogen signaling could be affected not only in ERA-negative breast cancer, but also in ERA-positive breast cancer due to lack of circadian availability of ERA. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.Key words: circadian rhythm, clock genes, estrogen receptor alpha (ERA), breast cancer cells, entrainment, serum shock     

Interaction of sesame seed and tamoxifen on tumor growth and bone health in athymic mice

Some premenopausal breast cancer patients use phytoestrogen-rich soy and flaxseed to alleviate side effects induced by drugs such as tamoxifen (TAM). Lignan-rich flaxseed protects against breast cancer and increases the effectiveness of TAM. This study determined the interactive effects of lignan-rich sesame seed (SS) and TAM on estrogen-responsive MCF-7 breast tumor growth and bone health in ovariectomized athymic mice under premenopausal-simulated conditions. Ovariectomized mice with an estrogen implant and established MCF-7 tumors were treated for 8 weeks as follows: (i) positive control fed basal diet (BD), (ii) SS group fed BD supplemented with 10% ground SS, (iii) TAM group with TAM implant fed BD, (iv) SS + TAM group with TAM implant fed BD supplemented with 10% SS, and (v) negative control fed BD with no estrogen implant. Palpable tumor data, adjusted for body weight, showed that SS does not inhibit MCF-7 tumor growth and tends to negate the tumor inhibitory effect of TAM by increasing cell proliferation and reducing apoptosis. SS alone and combined with TAM enhanced femur biomechanical strength but caused no differences in bone mineral content or bone mineral density in either the femur or lumbar vertebrae. SS is not protective and interacts adversely with TAM in MCF-7 breast tumors but induces beneficial effects on bone both alone and when combined with TAM.     

Circadian features of carbohydrate metabolism in domestic fowls exposed to weekly 120 degree-shifts of synchronizer schedule.

Effects of weekly 8 hr advance- or delay-shifts on the circadian rhythm of plasma glucose, liver glycogen and muscle glycogen in male domestic fowls, beginning at about 3 days of age, were examined. Circadian rhythm in the aforesaid indices of carbohydrate metabolism in control birds was also studied. Blood and tissue samples were collected from birds in all the three groups at 4 hr intervals over a single 24 hr time scale both at 6th and 12th week of age. Plasma glucose and glycogen content in the tissues were determined by employing standard techniques. Cosinor rhythmometry was used for analyzing time series data. In general, a statistically significant circadian rhythm was documented for all the three indices in control and advance-schedule birds, irrespective of age. In contrast, in delay-schedule birds, statistically significant circadian rhythm could not be detected, excluding in muscle glycogen at 12th week of age. The poor growth rate in the delay-schedule birds could be imputed to the disappearance of circadian rhythm in the indices of carbohydrate metabolism.     

Postnatal development of TRH and TSH rhythms in the rat

We previously observed that under a 12-hour light/12-hour dark schedule (lights off at 19.00 h), adult male Sprague-Dawley rats showed a circadian rhythm for serum thyroid-stimulating hormone (TSH) with a zenith near midday. In the present work, the ontogenesis of serum TSH rhythm was determined as well as pituitary TSH variations. In addition, hypothalamic and blood TRH were measured in these rats aged 15, 25, 40 and 70 days when sacrificed. As from the first age studied (15 days), a hypothalamic thyrotropin-releasing hormone (TRH) circadian rhythm was present. The mesor and the amplitude of this hypothalamic TRH rhythm increased while the rats were growing up, in contrast with the decrease observed for these parameters as far as blood TRH circadian rhythm is concerned. The time of the acrophase moved from 17.32 h in the 15-day-old rats to 13.57 h in the 70-day-old rats, being constantly in phase opposition with the blood TRH acrophase. The low amplitude pituitary TSH circadian rhythm detected in the young rat disappeared in the adult while, in contrast, the serum TSH rhythm became consistent to reach the well-characterized circadian midday peak in the 70-day-old rats.     

Pituitary and gonadal function in hypogonadotrophic hypogonadal (hpg) mice bearing hypothalamic implants

GnRH receptor values are 30-50% of normal in pituitaries of hpg male mice, and testicular LH receptors only 8% of normal (160.4 +/- 17.6 and 2013 +/- 208.1 fmol/testis respectively). In male hpg mice bearing fetal preoptic area (POA) hypothalamic implants for 10 days there was no change in pituitary GnRH receptors, pituitary gonadotrophin content, or seminal vesicle weight. However, testicular weights and LH receptors were doubled in 4/10 mice and 2 had increased serum FSH levels. Between 26 and 40 days after implantation pituitary GnRH receptors and pituitary LH increased to normal male levels, although at 40 days serum and pituitary FSH concentrations had reached only 50% of normal values. Testicular and seminal vesicle weights increased more than 10-fold by 40 days after implantation and LH receptors to 70% of normal. In hpg female mice bearing hypothalamic implants for 30-256 days pituitary gonadotrophin concentrations were normal, even though GnRH receptors reached only 60% of normal values (6.18 +/- 0.4 and 9.8 +/- 0.4 fmol/pituitary respectively). Serum FSH was substantially increased from values of less than 30 ng/ml in hpg mice to within the normal female range in hypothalamic implant recipients. Ovarian and uterine weights increased after hypothalamic grafting from only 4-5% to over 74% of normal values. LH receptors increased from 6.5 +/- 1.3 fmol/ovary for hpg mice to 566.9 +/- 39.2 fmol/ovary for implant recipients. Vaginal opening occurred about 23 days after implantation and these animals displayed prolonged periods of oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-dependent effects of starvation on serum, pituitary and hypothalamic leptin levels in rats

Leptin is produced by white adipose tissue and other cell types and is involved in both short- and long-term appetite control. Here we studied effects of starvation on serum, pituitary and hypothalamic levels of leptin during 72 h period. Each of the starved groups was sacrificed simultaneously with the group of ad libitum fed animals. The progression of the discrete starvation response phases was monitored by testing the blood glucose, free fatty acid, urea and corticosterone levels. Starvation caused biphasic increase in corticosterone and free fatty acid levels, and significant but transient decrease in urea and glucose levels. Starvation also abolished diurnal rhythm of changes in leptin concentrations in serum and hypothalamic and pituitary tissues. Only 6 h starving period was sufficient to lock serum leptin at low levels, whereas 12 h were needed to silence leptin production/secretion in hypothalamus for the whole examined period. In contrast, leptin production by pituitary tissues of starved animals required 24 h to reach minimum, followed by full recovery by the end of starvation period. These results indicate the tissue specific pattern of leptin release and suggest that the locally produced leptin could activate its receptor in pituitary cells independently of serum levels of this hormone.     

Effects of methamphetamine on daily rhythms of hypothalamic norepinephrine, serotonin and plasma corticosterone levels in the rat

The relationship between daily rhythms of hypothalamic norepinephrine (NE) and serotonin (5HT) contents and the circadian rhythm of plasma corticosterone (Comp. B) levels was studied in the rat. It was found that levels of hypothalamic NE, 5HT and plasma Comp. B exhibited distinct daily flucturations in the control condition. In animals treated with methamphetamine, the daily rhythms of hypothalamic 5HT contents and plasma Comp. B levels remained unchanged, whereas the daily rhythm of hypothalamic NE contents was completely abolished. As a result, it was suggested that the daily rhythm of hypothalamic NE is not functionally related to the circadian change of pituitary-adrenocortical activity.     

Effects of Age and Parity on Mammary Gland Lesions and Progenitor Cells in the FVB/N-RC Mice

The FVB/N mouse strain is extensively used in the development of animal models for breast cancer research. Recently it has been reported that the aging FVB/N mice develop spontaneous mammary lesions and tumors accompanied with abnormalities in the pituitary glands. These observations have a great impact on the mouse models of human breast cancer. We have developed a population of inbred FVB/N mice (designated FVB/N-RC) that have been genetically isolated for 20 years. To study the effects of age and parity on abnormalities of the mammary glands of FVB/N-RC mice, twenty-five nulliparous and multiparous (3-4 pregnancies) females were euthanized at 16-22 months of age. Examination of the mammary glands did not reveal macroscopic evidence of mammary gland tumors in either aged-nulliparous or multiparous FVB/N-RC mice (0/25). However, histological analysis of the mammary glands showed rare focal nodules of squamous changes in 2 of the aged multiparous mice. Mammary gland hyperplasia was detected in 8% and 71% of the aged-nulliparous and aged-multiparous mice, respectively. Epithelial contents and serum levels of triiodothyronine were significantly higher in the experimental groups than the 14-wk-old control mice. Immuno-histochemical staining of the pituitary gland pars distalis showed no difference in prolactin staining between the control and the aged mice. Tissue transplant and dilution studies showed no effect of age and/or parity on the ability of putative progenitor cells present among the injected mammary cells to repopulate a cleared fat pad and develop a full mammary gland outgrowth. This FVB/N-RC mouse substrain is suitable to develop mouse models for breast cancer.     

红景天提取物提高高强度跑台运动小鼠的抗氧化能力

为了考察红景天提取物对高强度跑台运动小鼠的抗氧化能力的影响,本研究将30只昆明小鼠随机分成对照组、模型组和红景天提取物组,每组10只。红景天提取物组小鼠按照500 mg/kg bw的剂量灌胃红景天提取液(2 m L)。对照组和模型组小鼠灌胃等体积的蒸馏水,共灌胃4周。采用硫酸蒽酮比色法检测小鼠肝脏和肌肉组织糖原的含量;采用硫代巴比妥酸比色法检测小鼠骨骼肌组织中的丙二醛(MDA)水平;采用RT-PCR和Western blotting检测骨骼肌组织中的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)的表达水平;采用苏木精和伊红(HE)染色评价骨骼肌病理改变。研究显示,模型组小鼠的跑台运动时间显著低于红景天提取物组(61.32 min vs 83.22 min,p<0.05);与模型组相比,红景天提取物组小鼠骨骼肌的炎性细胞浸润明显减轻,肌纤维排列明显改善;红景天提取物组的肝糖原和肌糖原含量均显著高于模型组;红景天提取物组小鼠骨骼肌组织中的MDA水平显著低于模型组;红景天提取物组的SOD、GSH-Px和CAT m RNA和蛋白表达水平均显著高于模型组;红景天提取物可通过上调抗氧化酶表达来增加抗氧化能力,减弱骨骼肌损伤,并增加机体的抗疲劳能力。     

Immunoreactive beta-endorphin in the plasma, pituitary and hypothalamus of young female rats on the day of estrus and intact and chronically castrated old constant estrous female rats

Immunoreactive beta-endorphin (IR-beta-ENDO) was compared in the plasma, pituitary and hypothalamus of young female rats on the day of estrus and old constant estrous (CE) female rats, and in intact and chronically castrated old CE female rats. The concentration of IR-beta-ENDO in the plasma and the content and concentration of IR-beta-ENDO in the neurointermediate lobe of the pituitary were significantly greater in the old CE female rats than in the young female rats on the day of estrus. The content and concentration of IR-beta-ENDO in the anterior pituitary and hypothalamus were similar in the two age groups. To determine if estrogen contributed to the increase in plasma and pituitary levels of IR-beta-ENDO observed in the old animals, a group of old CE female rats were castrated and compared to sham operated control CE rats. Thirty days after castration, levels of plasma, pituitary and hypothalamic IR-beta-ENDO were comparable in the intact and the chronically castrated old female rats. These data indicate that in old CE female rats, plasma and pituitary IR-beta-ENDO are significantly increased in comparison to young female rats on the day of estrus, and that these increased levels of IR-beta-ENDO observed in old female rats do not appear to be influenced by gonadal estrogen.     

A photosensitive circadian oscillator in an insect endocrine gland: photic induction of rhythmic steroidogenesis in vitro

The paired prothoracic glands of the insect Rhodnius prolixus each comprise a group of about 200 structurally identical cells. The synthesis (and release) of steroid moulting hormones (ecdysteroids) by these glands is under circadian control in vivo. We monitored ecdysteroid synthesis by single glands during long-term incubations in vitro. Synthesis is rhythmic in vitro and persists in continuous darkness. Glands which are arrhythmic (from prolonged continuous light) respond to transfer to darkness in vitro with the initiation of a free-running circadian rhythm of ecdysteroid synthesis. Therefore, the glands possess a light-sensitive circadian oscillator. These properties are conventionally associated with nervous tissue of animals. It is suggested that rhythmicity is synchronized within the gland by the known structural and electrical coupling between its component cells. The glands share properties with known pacemakers such as the avian pineal. However, the glands in vivo receive input from both light cues and the cerebral neuropeptide, prothoracicotropic hormone. Rhythmic release of this neuropeptide is controlled by a second oscillator located in the brain. We conclude that the pacemaker in the endocrine system of R. prolixus comprises at least three oscillators, one in each prothoracic gland and one in the brain, which are coupled hormonally. We conclude that the prothoracic gland is an important component of the circadian system controlling development in R. prolixus and that peripheral endocrine glands may play a more active role in the generation of animal circadian organization than has been thought. Accepted: 30 August 1997     

Effects of three feeding schedules on tumor and host of Lewis lung carcinoma-bearing mice

The effect of three feeding schedules on tumor and host were examined in Lewis Lung bearing (TB) and nontumor bearing (NTB) C57/B1 mice. Both NTB and TB animals were divided into three groups: the control groups which were fed ad libitum; the intermittent fed (IMF) groups were fed for 32 hr and fasted for 16 hr in each 48-hr cycle, and the alternate day fed (ADF) groups were fed for a 24-hr interval in each 48-hr cycle. The animals were killed at the end of the fifteenth day, following a fed day for all groups. In the NTB groups, only the ADF group showed decreased food intake and lower body weight gain as compared to their control group. In the TB mice, as compared to their control group, the IMF group showed a significant reduction in the mean tumor weight with no change in the mean host weight, even though the daily food intakes of these two groups were the same over the experimental interval. In contrast, the ADF group showed reductions in both host and tumor weights as compared to their control group. The tumor to host weight ratios were significantly reduced for both the IMF and ADF groups as compared to the ratios found for the control groups, which suggests a differential effect on the tumor and on the host due to the feeding schedule. As assessed by the protein, RNA, and DNA concentrations, no compositional differences were noted for the tumors obtained from the animals that were maintained on each of the three different feeding schedules. In the NTB mice, no differences in tissue leucine (Leu) oxidation occurred between the groups for liver and skeletal muscle, whereas in the TB animals in vitro Leu oxidation capability by skeletal muscle specimens was markedly enhanced in the ADF group, but no difference was noted for the IMF group of the TB mice when compared to the control group. Taken together, these results suggest that the 32-hr fed:16-hr fast schedule (IMF) was beneficial and the 24-hr fed:24-hr fast schedule was detrimental compared to the ad libitum feeding schedule with respect to tumor and host relationships.     

Estrogen status and skeletal muscle recovery from disuse atrophy.

Although estrogen loss can alter skeletal muscle recovery from disuse, the specific components of muscle regrowth that are estrogen sensitive have not been described. The primary purpose of this study was to determine the components of skeletal muscle mass recovery that are biological targets of estrogen. Intact, ovariectomized (OVX), and ovariectomized with 17beta-estradiol replacement (OVX+E2) female rats were subjected to hindlimb suspension for 10 days and then returned to normal cage ambulation for the duration of recovery. Soleus muscle mass returned to control levels by day 7 of recovery in the intact animals, whereas OVX soleus mass did not recover until day 14. Intact rats recovered soleus mean myofiber cross-sectional area (CSA) by day 14 of recovery, whereas the OVX soleus remained decreased (42%) at day 14. OVX mean fiber CSA did return to control levels by day 28 of recovery. The OVX+E2 treatment group recovered mean CSA at day 14, as in the intact animals. Myofibers demonstrating central nuclei were increased at day 14 in the OVX group, but not in intact or OVX+E2 animals. The percent noncontractile tissue was also increased 29% in OVX muscle at day 14, but not in either intact or OVX+E2 groups. In addition, collagen 1a mRNA was increased 45% in OVX muscle at day 14 of recovery. These results suggest that myofiber growth, myofiber regeneration, and extracellular matrix remodeling are estrogen-sensitive components of soleus muscle mass recovery from disuse atrophy.     

The effects of aromatase overexpression on mammary growth and gene expression in the aromatase x transforming growth factor alpha double transgenic mice

The transforming growth factor alpha (TGF) and its receptor (EGFR) are expressed in many breast cancers. Typically, the progression of estrogen dependent primary breast cancers into a hormone-independent state, due to the loss of the estrogen receptor, is associated with increased levels of TGF and EGFR, leading to aggressive breast carcinomas. The relationship between breast tumorigenesis and TGF is evident in the transgenic mice overexpressing TGF in the mammary glands. In the aromatase transgenic mice, the mammary glands exhibit preneoplastic developments but do not form frank tumors. To test the interactions between growth factor overexpression with tissue estrogen, we have crossed the aromatase transgenic mice with the TGF transgenic mice to produce a double transgenic strain. The histological data for the mammary glands of aromatase x TGF double transgenic mice show that these mice develop hyperplastic changes similar to the aromatase parental strain but no tumors are formed. Consistently, the expression of cyclin D1 and PCNA is diminished in the double transgenic strain as compared to the parental strains. In addition, the expression of TGF, EGF and EGFR are also decreased in the double transgenic strain, suggesting that continuous estrogen presence in the tissue due to aromatase overexpression downregulates the expression of EGFR and its ligands.