a b s t r a c tCatecholamines are an important regulator of lipolysis in adipose tissue. Here we show that rat adipocytes, isolated from mesenteric adipose tissue, express genes of catecholamine biosynthetic enzymes and produce catecholamines de novo. Administration of tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, in vitro significantly reduced concentration of catecholamines in isolated adipocytes. We hypothesize that the sympathetic innervation of adipose tissues is not the only source of catecholamines, since adipocytes also have the capacity to produce both norepinephrine and epinephrine.
Interactions between the hypothalamic-pituitary-adrenocortical (HPA) system and melatonin secretion have been demonstrated, but only the effects of melatonin on the activity of the HPA system have been studied in man. Alterations of melatonin secretion described as low-melatonin syndrome have been demonstrated in patients suffering from a major depressive episode, and an inhibitory factor on melatonin secretion has been postulated. We investigated whether corticotropin-releasing hormone (CRH), which is thought to be involved in HPA abnormalities in depressed patients, can also suppress melatonin secretion in healthy volunteers. Ten healthy male human volunteers in a double-blind study design received randomized hourly intravenous injections from 08.00 to 18.00 h that contained 10 µg human CRH, 1 µg adrenocorticotropic hormone (ACTH), or placebo to simulate pulsatile hormone secretion. Plasma melatonin and cortisol responses during the treatment and nocturnal sleep electroencephalograms after the treatment were recorded. Administration of CRH reduced melatonin secretion significantly below values obtained after administration of placebo and ACTH. Cortisol secretion was significantly enhanced by ACTH in comparison to both placebo and CRH. Electroencephalographic sleep parameters revealed no treatment effects. Our findings suggest that CRH has an inhibitory effect on the pineal secretion of melatonin in normal man. A mechanism via a release of cortisol was not supported by our results. Secondary hormonal effects from changes in nocturnal sleep architecture were excluded. Further investigation of the action of CRH on melatonin secretion as well as the mutual feedback between the HPA system and the pineal gland may extend our knowledge of neuroendocrine alterations mediating the adaptive response to stress and the eventual involvement in the pathogenesis of depression.
The sympathoadrenal system is the main source of catecholamines (CAs) in adipose tissues and therefore plays the key role in the regulation of adipose tissue metabolism. We recently reported existence of an alternative catecholamine producing system directly in adipose tissue cells, and here we investigated effect of various stressors - physical (cold) and emotional stress (immobilization) on dynamics of this system. Acute or chronic cold exposure increased intracellular NE and EPI concentration in isolated rat mesenteric adipocytes. Gene expression of catecholamine biosynthetic enzymes did not change in adipocytes but was increased in stromal vascular fraction (SVF) after 28-day cold. Exposure of rats to a single immobilization stress caused increases in NE and EPI levels, and also gene expression of catecholamine biosynthetic enzymes in adipocytes. In SVF changes were similar but more pronounced. Animals adapted to a long-term cold exposure (28 days, 4°C) did not show those responses found after a single immobilization stress either in adipocytes or SVF. Our data indicate that gene machinery accommodated in adipocytes, which is able to synthesize NE and EPI de novo, is significantly activated by stress. Cold-adapted animals keep their adaptation even after an exposure to a novel stressor (immobilization). These findings suggest the functionality of CAs produced endogenously in adipocytes. Taken together, the newly discovered catecholamine synthesizing system in adipocytes is activated in stress situations and might significantly contribute to regulation of lipolysis and other metabolic or thermogenetic processes.
The 90 000-M, glucocorticoid receptor was purified to homogeneity according to sodium dodecylsulfate/polyacrylamide gel electrophoresis. An affinity column containing either dexamethasone-17P-carboxylic acid or dexamethasone-21 -methanesulfonate bound to the matrix with the help of a disulfide bond is used in this study. Using this affinity matrix, in a single step, 8700-fold purification of glucocorticoid receptor from rat liver cytosol could be achieved. Following the method of activation and DNA-cellulose chromatography the 90000-M, receptor subunit was purified to homogeneity.The existence of steroid binding proteins in the target organ has been investigated in many laboratories [1 -71. The glucocorticoid resceptor protein from rat liver cytosol has been characterised in crude or partially purified receptor preparations [8 -191. The partial purification of the glucocorticoid receptor by a two-step chromatographic procedure on either phosphocellulose [20,21] or DNA-cellulose [19] with an intermediate activation step has been reported by several groups. We report here the purification of glucocorticoid receptor subunit of molecular weight 90 000 to homogeneity by hormone-affinity chromatography, activation and DNA-affinity chromatography. MATERIALS AND METHODSCytaminium dihydrochloride (Merck, Darmstadt), dexamethasone from Sigma Chemical Co, [3H]dexamethasone (2.5 Ci/mmol) from Amersham Buchler (Braunschweig). All other chemicals were of analytical grade obtained commercially. BuffersBuffer A: 1 mM EDTA, 20 mM sodium phosphate, 10 glycerine, 50 mM KC1, pH 7.0; buffer B: 1 mM EDTA, 20 mM sodium phosphate, 10 % glycerine, 2 mM 2-niercaptoethanol, 90 mM KCl, pH 7.8.Oxidution of 9a-Fluoro-16sr-methylprednisolone u d h Periodic Acid (221 1 g dexamethasone (spec.act. 1 mCi/g) was dissolved in 200 ml ethyl alcohol. 120 ml (0.025 M) periodic acid and 3.8 ml (2.5 M) sulfuric acid in 75 ml distilled water were added to this solution. After stirring for 24 h at room temperature, alcohol was removed under reduced pressure, the precipitated crude product was collected by filtration and dried over phosphorous pentoxide. The crude acid thus obtained was dissolved in 1 1 ethyl acetate and washed 3 times with 100-ml portions of 0.01 M NaOH. The wash was brought to pH 2 with 1 M HCl; kept for 2 h at room temperature and stored at 4 "C overnight. The acid was collected by filtration and recrystallised from methanol/water to yield pure product (yield 945 mg, 98%). m.p. > 250°C; mass spectrum (high resolution) m/e found 378.1838, calculated 378.1842. NMR 6 = 115 ppm, 1.6ppm (C-18 and C-19), 0.92ppm doublet) 16a-methyl; RF = 0.048 (CHC13/MeOH, 9 : l), RF = 0.58 (ethyl acetate/ethanol/NH3, 5 : 5 : 1). Synthesis o j Dex-I7~-curhoxy-his-~-aminoetkanethiol0.5 mmol (189 mg, spec.act. 1 pCi/mg) dex-17P-carboxylic acid was added to a solution of N-hydroxysuccinimide (57.5 mg, 0.5 mmol) in dry ethyl acetate (8 ml). A solution of dicyclohexylcarbodiimide (103 mg, 0.5 mmol) in 2 ml dry ethyl acetate was added and the reaction mixture was le...
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