Adrenergic regulation of clock gene expression in mouse liver

A main oscillator in the suprachiasmatic nucleus (SCN) conveys circadian information to the peripheral clock systems for the regulation of fundamental physiological functions. Although polysynaptic autonomic neural pathways between the SCN and the liver were observed in rats, whether activation of the sympathetic nervous system entrains clock gene expression in the liver has yet to be understood. To assess sympathetic innervation from the SCN to liver tissue, we investigated whether injection of adrenaline/noradrenaline (epinephrine/norepinephrine) or sympathetic nerve stimulation could induce mPer gene expression in mouse liver. Acute administration of adrenaline or noradrenaline increased mPer1 but not mPer2 expression in the liver of mice in vivo and in hepatic slices in vitro. Electrical stimulation of the sympathetic nerves or adrenaline injection caused an elevation of bioluminescence in the liver area of transgenic mice carrying mPer1 promoter-luciferase. Under a light-dark cycle, destruction of the SCN flattened the daily rhythms of not only mPer1, mPer2, and mBmal1 genes but also noradrenaline content in the liver. Daily injection of adrenaline, administered at a fixed time for 6 days, recovered oscillations of mPer2 and mBmal1 gene expression in the liver of mice with SCN lesion on day 7. Sympathetic nerve denervation by 6-hydroxydopamine flattened the daily rhythm of mPer1 and mPer2 gene expression. Thus, on the basis of the present results, activation of the sympathetic nerves through noradrenaline and/or adrenaline release was a factor controlling the peripheral clock.     

Glucocorticoid regulation of G-protein subunits in neonatal liver

The effect of dexamethasone administration in vivo on the steady-state levels of G-protein subunits in liver of neonatal rabbits was investigated using specific antibodies to each subunit as well as bacterial toxin-mediated ADP-ribosylation assays. Parallel measurements were also made of the activity of adenylyl cyclase, as influenced by a variety of activators. Dexamethasone administration modulated the levels of G-protein subunits in liver in an age-dependent and subunit-specific manner but not in 24-h-old newborns. The inductive effect of dexamethasone was observed in animals older than 24 h, the greatest effect being on 2- to 3-day-old neonates. In 48-h-old animals the -subunits G_s-1, G_s-2, G_i and the β-subunit Gβ increased 2.0-, 2.1-, 4.3- and 2.8-fold, respectively, compared to the control. The increases were much less for older animals. Dexamethasone treatment also modulated effector-mediated stimulation of adenylyl cyclase activity in vitro and mimicked its effects on G-protein levels; the greatest increase (˜ 2-fold) in the activation of adenylyl cyclase occurred in membranes isolated from 2- to 3-day-old animals. In older animals there was either no effect of dexamethasone or a decrease in activity. The degree of change in enzyme activity paralleled the change in the amount of G_s rather than of G_i or Gβ. These results suggest development-dependent regulation of hepatic G-proteins by glucocorticoids.     

Glucocorticoid receptor gene expression in rat pituitary gland intermediate lobe following ovariectomy

Using hybridization techniques and Northern blots we have identified a approximately 6.5 kb glucocorticoid receptor mRNA species in rat pituitary intermediate lobe. Glucocorticoid receptor mRNA concentrations, which are barely detectable or undetectable in normal animals, were greatly increased following ovariectomy. This ovariectomy-induced increase in glucocorticoid receptor mRNA content of the intermediate lobe, which was confirmed by in situ hybridization experiments, could be reversed by 17 beta-estradiol administration.     

Structure and regulation of expression of the mouse GH receptor

GH-binding protein (GHBP) in the mouse consists of a ligand-binding domain, which is identical to the extracellular portion of the GH receptor (GHR), and a hydrophilic C-terminal domain, in place of the transmembrane and intracellular domains of the GHR. The two proteins are encoded by separate mRNAs which are derived from a single gene by alternative splicing. Determination of the gestational profiles of GHR and GHBP mRNA expression in mouse liver and placenta shows that in the liver, the 1.4 kb mRNA corresponding to the mouse GHBP increases approximately 20-fold between non-pregnant and late pregnant mice, whereas the relative increase in the expression of the 4.2 kb mouse GHR was 8-fold. The rise in the steady-state levels of both mRNAs began on day 9 of gestation. Mouse GHBP mRNA levels continue to rise until day 15 of pregnancy, while GHR mRNA abundance reaches a plateau by day 13. By elucidating the temporal changes in GHR and GHBP mRNA abundance during pregnancy and lactation in multiple maternal tissues and by assessing the ontogeny of these mRNAs in fetal and early postnatal mouse liver, our studies have demonstrated that the alternative splicing of mouse GHR/GHBP mRNA precursor is regulated in a tissue-, developmental stage- and physiological state-specific manner. In vitro studies using hepatocytes in culture have begun to elucidate the hormonal factor(s) involved in the gestation control of the expression of GHR and GHBP. Treatment of hepatocytes with GH or estradiol (E2) alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR mRNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2- and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. Our studies in the mouse suggest that GHBP is an important cell-surface receptor for GH in the liver. These studies postulate that an arginine-glycine-aspartic acid sequence found on mouse GHBP but absent in other species is responsible for the association of GHBP with the plasma membrane by binding to one or more integrins on the surface of liver cells.     

Gene expression profiling identifies liver X receptor alpha as an estrogen-regulated gene in mouse adipose tissue

Estrogens reduce adipose tissue mass in both humans and animals. The molecular mechanisms for this effect are, however, not well characterized. We took a gene expression profiling approach to study the direct effects of estrogen on mouse white adipose tissue (WAT). Female ovariectomized mice were treated for 10, 24 and 48 h with 17beta-estradiol or vehicle. RNA was extracted from gonadal fat and hybridized to Affymetrix MG-U74Av2 arrays. 17beta-Estradiol was shown to decrease mRNA expression of liver X receptor (LXR) alpha after 10 h of treatment compared with the vehicle control. The expression of several LXRalpha target genes, such as sterol regulatory element-binding protein 1c, apolipoprotein E, phospholipid transfer protein, ATP-binding cassette A1 and ATP-binding cassette G1, was similarly decreased. We furthermore identified a 1.5 kb LXRalpha promoter fragment that is negatively regulated by estrogen. Several genes involved in lipogenesis and lipolysis were identified as novel targets that could mediate estrogenic effects on adipose tissue. Finally, we show that ERalpha is the main estrogen receptor expressed in mouse white adipose tissue (WAT) with mRNA levels several hundred times higher than those of ERbeta mRNA.     

Estrogen regulation of neurokinin B gene expression in the mouse arcuate nucleus is mediated by estrogen receptor alpha

Neurokinin B (NKB) gene expression is elevated in the infundibular (arcuate) nucleus of the hypothalamus in postmenopausal women. Estrogen replacement decreases both the number of NKB mRNA-expressing neurons and the level of expression within individual cells. Similarly, NKB gene expression is elevated in ovariectomized rats and reduced after estrogen treatment. The actions of estrogen in the brain can be mediated via either estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). In the rodent arcuate nucleus (ARC), more ERalpha- than ERbeta-containing cells are present, suggesting that ERalpha might be directly responsible for estrogen regulation of NKB gene expression. However, an indirect effect via ERbeta could not be ruled out. Here we used ERalpha knockout and ERbeta knockout mice to identify the type of ER responsible for mediating estrogen action on NKB gene expression in the ARC. Using in situ hybridization histochemistry, we have found that estrogen treatment significantly reduced NKB gene expression in the ARC of ovariectomized ERbeta knockout mice, but had no effect on NKB mRNA levels in ERalpha knockout mice. These data indicate that ERalpha mediates the increase in NKB gene expression associated with ovariectomy in rodents and might also be responsible for the increase in NKB in postmenopausal women.     

Glucocorticoid function of the adrenal cortex in the course of a 24-hour day in pathological states]

A study was made of blood hydrocortisone content for a period of 24 hours in the patients with congenital diseases of the heart before and after the operation, in patients with acute respiratory disease, severe pneumonia, and also with acute laryngeal stenosis of influenzal etiology. It was revealed that under pathological conditions the adrenal cortex took part in the compensatory reactions of the organism at any time of the day. As a result of this hydrocortisone concentration is steadily maintained at a definite level in the course of the 24 hours. These changes depended on the intensity of the stressor and the severity of metabolic disturbances in the organism.     

Characteristics of 24-hour ECG and the regulation of cardiac rhythm in adolescents

Holter 24-hour monitoring was performed in 50 healthy subjects aged 11-15 years. Daily changes and standard values of heart rate (HR), P-R and Q-T intervals, P, R, T wave amplitudes, ST segment displacement were determined. All the patients under study exhibited sinus arrhythmia, pacemaker migration periods, short cardiac rhythm pauses (up to 1.5 s). Three adolescents were recorded to have Degree I atrioventricular block at night, 3 presented with rare (up to 2 per min) supraventricular contraction beats, 1 had a nocturnal transient Wolff-Parkinson-White syndrome. Despite HR at night, there were periods of Q-T interval prolongation. Daily changes in the neurohumoral regulation of the heart and its relation to the occurrence of various electrocardiographic events were determined.     

Stereological measurement of porto-central gradients in gene expression in mouse liver

The liver is thought to consist of lobules, numerous repeating, randomly oriented units. Within these lobules, genes are expressed in gradients along the porto-central axis, which spans the distance between portal and central veins. We have developed a robust stereological method to map all points in an image to their position on this porto-central axis. This approach is based on the distribution of well-characterized periportal and pericentral enzymes, which are visualized on sections preceding and following the section of interest. Because expression of the model genes phosphoenolpyruvate carboxykinase and ornithine aminotransferase declines gradually with increasing distance from the portal vein and central vein, respectively, these genes can be used to prepare images with topographical information without any assumption about the shape of the hepatic unit, or about the direction or shape of the gradient to be determined. The "relative distance" image is a 2-dimensional image that accurately maps the relative position of hepatocytes on the porto-central axis in 3-dimensional space. It is superimposed on the serial section under investigation to relate local staining density to position on the porto-central axis and obtain the gene expression gradient. The method was used to determine the expression gradient of 2 periportal and 2 pericentral enzymes and their response to fasting. The "total distance" image was used to measure the length of the porto-central axis, which was approximately 210 microm in mice and found to decrease 13% after 1 day of starvation. The method can be applied to any tissue component that can be stained quantitatively.     

Murine alanine aminotransferase: cDNA cloning, functional expression, and differential gene regulation in mouse fatty liver

Alanine aminotransferase (ALT) is a widely used index of liver integrity or hepatocellular damage in clinics as well as a key enzyme in intermediatary metabolism. In this study, we have cloned the complementary DNAs of murine homologues of human alanine aminotransferase 1 and 2 (ALT1 and ALT2). The deduced peptides of murine ALT1 (mALT1) and ALT2 (mALT2) share 87% and 93% identity, respectively, with their human counterparts at the amino acid level. Murine ALT genes localize to separate chromosomes, with mALT1 gene (gpt1) on chromosome 15 and mALT2 gene (gpt2) on chromosome 8. The murine gpt1 and gpt2 differ in messenger RNA expression: gpt1 is mainly expressed in liver, bowel, and white adipose tissue and gpt2 is highly expressed in muscle, liver, and white adipose tissue. Expression of recombinant mALT1 and mALT2 proteins in Escherichia coli (E. coli) produced functional enzymes that catalyze alanine transamination. The potential diagnostic value of ALT isoenzymes in liver disease was evaluated in an obese animal model. In fatty livers of obese mice, ALT2 gene expression is induced 2-fold, but ALT1 remains the same. Furthermore, in fatty liver, total hepatic ALT activity is elevated significantly by 30% whereas aspartate aminotransferase (AST) activity remains unchanged. In conclusion, these results indicate that ALT2 may be responsible for the increased ALT activity in hepatic steatosis and provide evidence that an ALT isoenzyme-specific assay may have more diagnostic value than the total ALT activity assay currently in clinical use.     

Glucocorticoid regulation of osteoclast differentiation and expression of receptor activator of nuclear factor-kappaB (NF-kappaB) ligand, osteoprotegerin, and receptor activator of NF-kappaB in mouse calvarial bones

In the present study, dexamethasone treatment of neonatal mouse calvarial bones increased mRNA expression of tartrate-resistant acid phosphatase, calcitonin receptor (CTR), cathepsin K, carbonic anhydrase II, osteoprotegerin (OPG), and receptor activator of nuclear factor-kappaB (RANK) as well as mRNA and protein expression of RANK ligand (RANKL). The increase in OPG mRNA noted with dexamethasone was in contrast to 1,25(OH)(2)-vitamin D3 (D3) treatment, which decreased OPG expression. Stimulation of (45)Ca release by dexamethasone and hydrocortisone in calvariae was blocked by OPG. Stimulation of RANKL, RANK, OPG, and CTR mRNA expression by dexamethasone in calvariae was blocked by the glucocorticoid receptor antagonist RU 38,486. Greater than additive potentiations of CTR mRNA and RANKL mRNA and protein were observed when D3 and dexamethasone were combined. Vitamin D receptor mRNA was increased by dexamethasone and D3, whereas glucocorticoid receptor (GR) mRNA was decreased by dexamethasone and unaffected by D3. No synergistic interaction between dexamethasone and D3 on either vitamin D receptor or GR mRNA expression was noted. The data demonstrate that dexamethasone-induced bone resorption in calvarial bones is associated with increased differentiation of osteoclasts and regulation of the RANKL-RANK-OPG system. The increase in OPG expression and the decrease of GR expression noted with dexamethasone offer an explanation for why bone breakdown in mouse calvariae treated with glucocorticoids is less than that caused by resorptive agents like D3. The synergistic stimulation of RANKL by dexamethasone and D3 offers an explanation of how glucocorticoids and D3 interact to potentiate bone resorption.     

Glucocorticoid receptor gene and risk of cardiovascular disease

BACKGROUND: Genetic variants in immunomodulating genes have been suggested to contribute to the risk of cardiovascular disease. Glucocorticoids are important regulators of inflammatory processes and the immune system. Our aim was to determine the contribution of genetic glucocorticoid receptor variants, with different cortisol sensitivities, to the risk of cardiovascular disease. METHODS: The study was conducted in a large (n=7983) population-based, prospective cohort of the Rotterdam Study. The mean duration of follow-up was 8.9 years. Measures of cardiovascular disease were incident myocardial infarction, coronary heart disease, high-sensitivity C-reactive protein level, interleukin 6 level, and arteria carotis intima-media thickness. RESULTS: Persons homozygous for haplotype 3, which is a common variant of the glucocorticoid receptor gene, had a more than 2-fold increased risk of myocardial infarction (hazard ratio, 2.1; 95% confidence interval, 1.13-4.07) and an almost 3-fold increased risk of coronary heart disease (hazard ratio, 2.6; 95% confidence interval, 1.40-4.81) compared with nonhomozygous persons. In addition, their C-reactive protein and interleukin 6 levels were higher, and carotis intima-media thickness was greater. No associations were found for the other haplotypes. CONCLUSIONS: The glucocorticoid receptor gene haplotype 3 is a common genetic variant and is related to a more active proinflammatory system. This haplotype is associated with the risk of cardiovascular disease and its parameters. These results should be regarded as hypothesis generating until they have been replicated in other studies. Our findings suggest that genetically determined cortisol sensitivity is involved in the pathogenesis of cardiovascular disease and might identify a subgroup at risk.     

High-throughput microarray detection of olfactory receptor gene expression in the mouse

The large number of olfactory receptor genes necessitates high throughput methods to analyze their expression patterns. We have therefore designed a high-density oligonucleotide array containing all known mouse olfactory receptor (OR) and V1R vomeronasal receptor genes. This custom array detected a large number of receptor genes, demonstrating specific expression in the olfactory sensory epithelium for approximately 800 OR genes previously designated as ORs based solely on genomic sequences. The array also enabled us to monitor the spatial and temporal distribution of gene expression for the entire OR family. Interestingly, OR genes showing spatially segregated expression patterns were also segregated on the chromosomes. This correlation between genomic location and spatial expression provides unique insights about the regulation of this large family of genes.     

Glucocorticoid and mineralocorticoid receptor mRNA expression in rat brain

In the rat brain, the binding of corticosterone is mediated through two receptor types, the type I receptor and the type II receptor, which are presumed to be encoded by genes designated as MR and GR, respectively. We have studied the regulation of these receptors by glucocorticoids, utilizing a cytosol receptor binding assay. In addition, we have employed molecular probes for the GR and the MR to measure receptor mRNAs. The level of type II receptor binding is uniform across several brain regions, as is the expression of GR (type II) mRNA. In contrast, type I receptor binding is concentrated in the hippocampus, and the MR (type I) mRNA similarly shows a higher level of expression in hippocampus than in the other brain regions studied. Removal of endogenous glucocorticoids by adrenalectomy (ADX) induces an increase, and corticosterone administration results in a decrease, in the level of type I and type II binding in the hippocampus; however, no significant changes in the MR (type I) or GR (type II) mRNA levels are seen with these treatments. The diurnal variation of serum corticosterone in intact rats is correlated with a circadian regulation of type I receptor binding in the hippocampus, while MR (type I) mRNA expression is unaffected. Thus, the changes in type I and type II receptor binding capacity elicited by differing steroid conditions cannot be attributed to modulation of the steady state levels of MR (type I) or GR (type II) mRNA.