Metabolism and disposition of [14C]-methylcyclosiloxanes in rats.

Octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) are low molecular weight cyclic volatile methyl siloxanes (cVMSs) primarily used as intermediates or monomers in the production of high molecular weight silicone polymers. The use of D4 as a direct ingredient in personal care products has declined significantly over the past 20 years, although it may be present as a residual impurity in a variety of consumer products. D5 is still used as an intentional ingredient in cosmetics, consumer products and in dry cleaning. Persons who may be exposed include occupational exposure for workers, and potential inhalation or dermal exposure for consumers and the general public. Because of the diverse use, especially of D5, and the potential for human exposure, a comprehensive program was undertaken to understand the kinetics, metabolism, enzyme induction and toxicity of D4 and D5 in rats following relevant routes of exposure. Physiologically based pharmacokinetic (PBPK) models utilizing these studies have been reported for D4 and D5 in the rat and human following dermal and inhalation exposures, with the oral uptake component of the model being limited in its description. Data from high dose oral studies in corn oil and simethicone vehicles and neat were used in the D4/D5 harmonized PBPK model development. It was uncertain if the inability to adequately describe the oral uptake was due to unrealistic high doses or unique aspects of the chemistry of D4/D5. Low dose studies were used to provide data to refine the description of oral uptake in the model by exploring the dose dependency and the impact of a more realistic food-like vehicle. Absorption, distribution, metabolism and elimination (ADME) of D4 and D5 was determined following a single low oral gavage dose of 14C-D4 and 14C-D5 at 30 and 100mg/kg body weight (bw), respectively, in a rodent liquid diet. Comparison of the low vs. high dose oral gavage administration of D4 and D5 demonstrated dose-dependent kinetic behavior. Data and modeling results suggest differences in metabolism between low and high dose administration indicating high dose administration results in or approaches non-linear saturated metabolism. These low dose data sets were used to refine the D4/D5 multi-route harmonized PBPK model to allow for a better description of the disposition and toxicokinetics of D4/D5 following oral exposure. With a refined oral uptake description, the model could be used in risk assessment to better define the internal dose of D4 and D5 following exposure to D4 and D5 via multiple routes.

VGF peptides upon osmotic stimuli: changes in neuroendocrine regulatory peptides 1 and 2 in the hypothalamic-pituitary-axis and plasma.

VGF or VGF nerve growth factor inducible is a protein that has been found to play a role in regulating energy homeostasis and metabolism. From VGF precursor derive two neuroendocrine regulatory peptides NERP-1 and NERP-2 that, intracerebroventricular (icv) injected, modulate the antidiuretic hormone (ADH) release. Thus, we investigated possible modulations of the NERPs and other VGF peptides (namely VGF C-terminus, TLQP and PGH) in the hypothalamic-pituitary-axis, adrenal gland and plasma upon osmotic stimuli. The latter tissues were studied using water deprived (WD), salt loaded (SL), rehydrated after salt cargo and control rats by immunohistochemistry and immunoenzymatic assays. The high-performance liquid chromatography ensured the endogenous presence of the two NERPs in both plasma and hypothalamus. Upon dehydration, NERP-1 levels increased in the median eminence (M.E.) only, while using SL rats, the values of both NERPs increased in the M.E. and even in the hypothalamus. Conversely, in the blood of WD and SL rats, the levels of NERP-1 and NERP-2 decreased while, using pituitary from both rat groups, levels of NERP-2 increased and those of NERP-1 decreased. Reduction in the VGF C-terminus peptide levels was observed exclusively in the M.E. (using WD rats) and pituitary (using WD and SL rats), while PGH and TLQP peptide levels never changed in all tissues tested. By immunohistochemistry, the VGF peptides studied (apart from the TLQP peptides) were present in the hypothalamic and pituitary ADH containing neurons of the control rats, while using WD and SL rats, an immunostaining increase was selectively revealed for VGF C-terminus peptides in the hypothalamic neurons that produce ADH. All VGF changes found using SL rats disappeared after only 1h of rehydration. In conclusion, we hypothesize that NERPs may be involved in both autocrine and endocrine mechanisms important for the fluid balance.

Trilostane-induced inhibition of cortisol secretion results in reduced negative feedback at the hypothalamic-pituitary axis.

Cushing's disease caused by pituitary corticotroph adenoma in dogs is usually treated by medical treatment, and the efficacy of this treatment has been reported. However, controversy remains as to whether reduced negative feedback through the inhibition of cortisol secretion, similar to Nelson's syndrome, may appear as an adverse effect. The purpose of this study was to investigate the effect of reduced negative feedback through the inhibition of cortisol secretion by daily trilostane administration on the pituitary-adrenal axis in clinically normal dogs. Dogs were administered 5mg/kg trilostane twice a day every day for 8 weeks (n=8) or 16 weeks (n=3). After the initiation of trilostane administration, plasma adrenocorticotropic hormone (ACTH) concentrations were increased remarkably. As assessed by magnetic resonance imaging (MRI) during administration, the pituitary became enlarged. After trilostane administration, the cytoplasmic areas of the pituitary corticotrophs were increased and the ratio of pituitary corticotrophs to all cells in the anterior lobe was greater in the trilostane-treated dogs than that in untreated animals. In addition, histological examinations revealed bilateral adrenal cortical hyperplasia. Using real-time PCR quantification, the expression of proopiomelanocortin (POMC) mRNA in the pituitary and ACTH receptor (ACTH-R) mRNA in the adrenal gland was greater in the dogs treated with trilostane than in untreated dogs. These results indicate that reduced negative feedback induced hyperfunction of the pituitary corticotrophs and pituitary enlargement in healthy dogs. These changes suggest that the inhibition of cortisol secretion by trilostane may increase the risk for accelerating the growth of corticotroph adenomas in dogs with Cushing's disease.

Vitamin A concentrations in piglet extrahepatic tissues respond differently ten days after vitamin A treatment.

Periodic supplementation to infants and young children is encouraged in developing countries by the WHO. We investigated vitamin A (VA) in extrahepatic tissues of piglets after supplementation with retinyl acetate to determine long-term storage. 3, 4-Didehydroretinyl acetate (DRA) as a tracer was used to evaluate uptake from chylomicra in 4 h. Sows were fed a VA-depleted diet throughout pregnancy and lactation. Male castrated piglets (n = 28, 11.6 +/- 0.5 d) from these sows were weaned onto a VA-free diet for 1 wk, assigned to 4 groups, and dosed orally with 0, 26.2, 52.4, or 105 micromol VA. After 10 d, 5.3 micromol DRA was administered to determine short-term uptake of 3, 4-didehydroretinol (DR). Four hours later, piglets were killed; adrenal glands, kidney, lung, and spleen were collected and analyzed for retinol and DR. Retinol concentrations of kidney and adrenal gland were higher than control, but treated groups did not differ. Retinol concentration was highest in kidney (1.70-2.52 nmol/g), followed by adrenal gland (0.30-0.48 nmol/g), lung (0.15-0.21 nmol/g), and spleen (0.11-0.15 nmol/g). Total retinol in kidney and spleen was different among the groups (P < 0.05). Unesterified retinol was the major VA form; the percent retinol of total VA was lowest in adrenal glands. DR did not differ among the groups. In 4 h, the minimum estimated chylomicron contribution to tissue DR was 63-280% higher than the maximum DR exposure from retinol-binding protein. Constant dietary intake may be important in maintaining VA concentrations in extrahepatic tissues.

Chronic consumption of a low-fat diet leads to increased hypothalamic agouti-related protein and reduced leptin.

OBJECTIVE: This study examined the hypothesis that dietary fat under ad libitum feeding conditions influences expression levels (mRNA) of the mouse agouti-related protein (AgRP), leptin, leptin receptor (OBRb), and neuropeptide Y (NPY) at early stages of development. METHODS: C57Bl/6J male mice were placed on a high-fat diet (HFD) or a low-fat diet (LFD) shortly after weaning. Groups of mice were euthanized at various ages and real-time one-step reverse transcriptase polymerase chain reaction was used to analyze gene expression in the hypothalamus (AgRP, NPY, OBRb), the adrenal gland (AgRP), the testis (AgRP), and epididymal fat (leptin). RESULTS: Leptin expression increased linearly with age but only under the HFD despite body weight gain under both diets. This pattern of expression coincided with reduced expression of hypothalamic AgRP under an HFD, whereas OBRb and NPY did not fluctuate in response to diet. By contrast, consumption of an LFD (i.e., high carbohydrate) increased hypothalamic AgRP and suppressed adipose leptin, which is consistent with the notion that leptin could regulate AgRP centrally. In contrast, AgRP expression in the adrenal gland initially decreased and then increased with age under both diets. CONCLUSIONS: Dietary fat can have a tissue-dependent effect on AgRP that may be unfettered by leptin under an HFD.

Effect of tibolone administration on central and peripheral levels of allopregnanolone and beta-endorphin in female rats.

OBJECTIVE: We evaluated the effects of tibolone oral administration on neuroendocrine function by investigating the modulation exerted by tibolone administration on allopregnanolone and central and peripheral beta-endorphin (beta-EP) levels in ovariectomized rats. DESIGN: Female Wistar rats (N = 64) were included: 48 rats were ovariectomized, 8 cycling rats were included as controls, and 8 cycling rats were treated with placebo. The ovariectomized animals were divided into six groups: untreated rats and those that received 14-day oral treatment with either placebo, estradiol valerate (E2V) 0.05 mg/kg/d, or tibolone (0.1, 0.5, or 2 mg/kg/d. beta-EP levels were assessed in the frontal lobe, parietal lobe, hippocampus, hypothalamus, anterior pituitary, neurointermediate pituitary, and plasma, whereas allopregnanolone levels were measured in the frontal lobe, parietal lobe, hippocampus, hypothalamus, anterior pituitary, adrenal glands, and serum. RESULTS: The administration of tibolone (0.5 and 2 mg/kg/d) in ovariectomized rats induces a significant increase of allopregnanolone in the frontal lobe, parietal lobe, hippocampus, hypothalamus, whereas in serum a significant increase of allopregnanolone occurs only with the dose of 2 mg/kg/d, a significant decrease in allopregnanolone levels occurs in the adrenal glands. No changes occurred in the anterior pituitary. Tibolone doses of 0.5 and 2 mg/kg/d induced a significant increase in beta-EP content in the frontal lobe, hypothalamus, and neurointermediate lobe; and, at doses of 2 mg/kg/d, in the parietal lobe, anterior pituitary, and plasma, without changes in the hippocampus. Compared with E2V, 0.5 mg/kg/d tibolone showed a similar effect on allopregnanolone and beta-EP in most brain regions. CONCLUSIONS: Tibolone administration affects beta-EP and allopregnanolone levels, playing a role as a neuroendocrine modulator.

Dopamine receptor expression and function in human normal adrenal gland and adrenal tumors.

Dopamine is known to play a role in the modulation of aldosterone and catecholamine secretion from the adrenal gland, where dopamine receptors (DR), in particular the DR type 2 (D(2)), have been found to be expressed. DR expression has also been demonstrated in some types of benign adrenal tumors. The aims of the current study were to evaluate DR expression and D(2) localization in the normal adrenal gland and in different types of benign and malignant adrenal tumors, as well as to evaluate the in vitro effects of the dopamine agonists bromocriptine and cabergoline on hormone secretion in nontumoral adrenal cells. Adrenal tissues from 25 patients, subjected to adrenal surgery for different diseases, were studied. These included three normal adrenals; five adrenal hyperplasias; four aldosterone-secreting, two cortisol-secreting, and two clinically nonfunctioning adrenal adenomas; two aldosterone-secreting, two cortisol-secreting, and two androgen-secreting adrenal carcinomas; and three pheochromocytomas. In all tissues, DR and D(2) isoform (D(2long) and D(2short)) expression was evaluated by RT-PCR. D(2) localization was also evaluated by immunohistochemistry using a specific polyclonal antibody, whereas D(2)-like receptor expression was evaluated by receptor-ligand binding study, using the radiolabeled D(2) analog (125)I-epidepride. The effects of bromocriptine and cabergoline on baseline and ACTH and/or angiotensin II-stimulated aldosterone, cortisol, and androstenedione secretion were evaluated in cell cultures derived from five different adrenal hyperplasia. At RT-PCR, both D(1)-like and D(2)-like receptors were expressed in all normal and hyperplastic adrenals. D(2) and D(4) were expressed in aldosterone- and cortisol-secreting adenomas, cortisol-secreting carcinomas, and clinically nonfunctioning adenomas, whereas no DR was expressed in aldosterone- and androgen-secreting carcinomas. D(2), D(4), and D(5) were expressed in pheochromocytomas. In all D(2)-positive tissues, both D(2) isoforms were expressed, with the exception of one case of aldosterone-secreting adenoma and the cortisol-secreting carcinomas, in which only the D(2long) isoform was expressed. D(2)-like receptor expression was confirmed at receptor-ligand binding study. At immunohistochemistry, D(2) was mainly localized in the zona glomerulosa and reticularis of the adrenal cortex and, to a lesser extent, in the zona fasciculata and medulla of normal and hyperplastic adrenal tissue. In the positive tumors, D(2) was localized in the tumoral cells. At the in vitro study, a significant inhibition of both baseline and ACTH-stimulated aldosterone secretion was found after high-dose cabergoline, but not bromocriptine, administration; and a significant inhibition of angiotensin-II-stimulated aldosterone secretion was found after both bromocriptine and cabergoline administration in the adrenal hyperplasias. In conclusion, the current study demonstrated that both D(1)-like and D(2)-like receptors are expressed in the normal adrenal gland and in a percentage of adrenal adenomas or carcinomas. Bromocriptine and cabergoline induce only a minor inhibition of the secretion of adrenal hormones in the nontumoral adrenal gland in vitro, not excluding, however, the possible effective use of dopamine agonists in vivo in the treatment of adrenal tumors.

Molecular cloning and expression of guinea pig cytochrome P450c21 cDNA (steroid 21-hydroxylase) isolated from the adrenals.

In mammals, the P450c21 enzyme mediates 21-hydroxylase activity by transforming progesterone and 17-hydroxyprogesterone into deoxycorticosterone (DOC) and 11-deoxycortisol (11-DOC), respectively. Previous studies have shown that among the adrenal steroid hydroxylase enzymes involved in C19 steroid and glucocorticoid syntheses, P450c21 plays an important role, because it is localized at the key branch between glucocorticoids and C19 steroid production. Its implication in congenital adrenal hyperplasia is also of great clinical interest. In this study, in addition to describing the isolation of the P450c21 cDNA from guinea pig (GP) adrenal and comparing it to those from other species, we report on its tissue-distribution and on the activity of the recombinant protein towards progesterone and 17-hydroxyprogesterone. The guinea pig P450c21 includes the full-length coding region (1464 nucleotide) that is translated to a protein of 488 amino acids. The clone shares highly conserved regions with other species. The guinea pig P450c21 cDNA hybridized with a major transcript of 2.1kb and with two minor related transcripts of 1.8 and 1.5 kb and was found to be adrenal-specific among the various tissues analyzed. Characterization of the enzymatic activity by transient transfection of the guinea pig P450c21 cDNA in human embryonic kidney 293 cells indicated a net preference for the 21-hydroxylation of 17-hydroxyprogesterone in comparison to the progesterone substrate. Assays showed a maximum conversion rate of 12.5% for the conversion of progesterone into deoxycorticosterone (mineralocorticoid pathway), whereas the guinea pig P450c21 demonstrated a higher activity with 17alpha-hydroxyprogesterone, with 55% of 11-deoxycortisol formation (glucocorticoid pathway) after 48 h. Adrenocorticotropin and an analogue of the second messenger cyclic adenosine monophosphate specifically increased the abundance of P450c21 mRNA levels in guinea pig adrenal cells.

Fetal hypoxemia on a molecular level: adaptive changes in the hypothalamic-pituitary-adrenal (HPA) axis and the lungs.

The development of diseases in later life, such as diabetes type II, hypertension and cardiovascular disease, is linked to abnormal intrauterine conditions that reduce birth weight. Obviously, fetal development can be disturbed so profoundly, that fetal programming is changed permanently. We have examined the effects of hypoxia, or more precisely hypoxemia, on the fetal hypothalamic-pituitary-adrenal (HPA) axis and lungs using molecular biology techniques in order to elucidate the underlying mechanisms. Chronically catheterized fetal sheep were subjected to a hypoxemia (48 h) without change in arterial pH or paCO2. Major changes occurred, although the degree of hypoxemia was just moderate. There was a transient increase in the fetal plasma ACTH-concentrations with an upregulation of the cortisol-concentrations, which was more pronounced in the older, hypoxemic fetuses (134-136 days of gestation) than in the younger, hypoxemic animals (126-130 days of gestation; term is 145 days). There was an unique, differential regulation for pro-opiomelanocortin messenger RNA (mRNA), the precursor molecule of e.g. ACTH, in the pars distalis and pars intermedia of the pituitary gland. This finding supported the increased bioactivity besides the increased concentrations for ACTH. Simultaneously, there was an increase in the mRNAs of the ACTH-receptor and of the steroid-synthesizing enzymes in the fetal adrenal gland of the older, hypoxemic fetuses. No changes in the fetal plasma androstenedione-concentrations were observed. Clearly, there was a selective increase of the cortisol-synthesis. Growth and maturation of the fetal lung might also have been affected, because of the increase in surfactant-protein A mRNA in the older, hypoxemic animals and the decrease in the insulin-like growth factor-I and its binding protein-5 mRNA in the younger, hypoxemic fetuses. In summary, even a moderate degree of hypoxemia was shown to affect the different levels of fetal organism profoundly, offering a pathophysiological basis for changes in fetal development.

A new peptidic ligand and its receptor regulating adrenal function in rats.

We searched for peptidic ligands for orphan G protein-coupled receptors utilizing a human genome data base and identified a new gene encoding a preproprotein that could generate a peptide. This peptide consisted of 43 amino acid residues starting from N-terminal pyroglutamic acid and ending at C-terminal arginine-phenylalanine-amide. We therefore named it QRFP after pyroglutamylated arginine-phenylalanine-amide peptide. We subsequently searched for its receptor and found that Chinese hamster ovary cells expressing an orphan G protein-coupled receptor, AQ27, specifically responded to QRFP. We analyzed tissue distributions of QRFP and its receptor mRNAs in rats utilizing quantitative reverse transcription-polymerase chain reaction and in situ hybridization. QRFP mRNA was highly expressed in the hypothalamus, whereas its receptor mRNA was highly expressed in the adrenal gland. The intravenous administration of QRFP caused the release of aldosterone, suggesting that QRFP and its receptor have a regulatory function in the rat adrenal gland.

Differential expression of AT1 receptors in the pituitary and adrenal gland of SHR and WKY.

The renin-angiotensin (ANG) system has been implicated in the development of hypertension in spontaneously hypertensive rats (SHR). Because SHR are more susceptible to stress than normotensive Wistar-Kyoto rats (WKY), we measured the mRNA expression of AT1A, AT1B, and AT2 receptors in the hypothalamo-pituitary-adrenal (stress) axis of male SHR in comparison to age-matched WKY at prehypertensive (3 to 4 weeks), developing (7 to 8 weeks), and established (12 to 13 weeks) stages of hypertension. AT1A receptor mRNA was mainly expressed in the hypothalamus and adrenal gland. AT1B receptor mRNA was detected in the pituitary and adrenal gland. AT2 receptor mRNA was prominent only in the adrenal gland. When compared with WKY, SHR showed increased AT1A receptor mRNA levels in the pituitary gland at all ages in contrast to reduced pituitary AT1B receptor mRNA levels. In the adrenal gland of SHR, AT1B receptor mRNA levels were decreased at the hypertensive stages when compared with WKY. The reduced expression of adrenal AT1B receptor mRNA was localized selectively in the zona glomerulosa by in situ hybridization. No differences were observed between WKY and SHR in the expression of hypothalamic ANG receptors. ANG significantly increased plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone in dexamethasone-treated SHR but not in WKY. The aldosterone response to ANG was similar in SHR and WKY. Our results suggest a differential gene expression of AT1A and AT1B receptors in the hypothalamo-pituitary-adrenal axis of SHR and normotensive WKY and imply the participation of AT1 receptors in an exaggerated endocrine stress response of SHR to ANG.

Molecular cloning and tissue distribution of the ovine somatostatin receptor subtype 5: osst5.

The sheep is a valuable model to study growth hormone (GH) neuroregulation since its GH secretion pattern is close to that in humans and an integrated physiological approach is possible in this species. Somatostatin receptor subtype 5 (sst5) appears to be important in GH regulation but the ovine sst5 gene (osst5) has not yet been cloned. We report here the cloning of sst5 in that species. We screened a cDNA sheep library and isolated a 1.24 kb cDNA, which includes the whole coding region of osst5. The predicted protein consists of 367 amino acids exhibiting a putative seven transmembrane domain topology typical of G protein-coupled receptors. Nucleotide sequence comparisons with that of other species sst5 showed that osst5 displays 83.8, 81 and 79.7% homology with human, rat, and mice sst5, respectively. Southern blot analysis of ovine cortex DNA demonstrated that osst5 is encoded by a single gene. Osst5 transiently expressed in Chinese Hamster ovary (CHO) cells exhibit a high affinity for somatostatin-14. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that osst5 mRNAs are present in pituitary, cortex, hypothalamus, hippocampus, colon and adrenal gland. The cloning of osst5 should provide a useful tool to study the mechanisms through which somatostatin inhibits hormone secretion in the sheep.

Aromatase overexpression enhances the stimulatory effects of adrenal androgens on MCF7 breast cancer cells.

The intratumoral conversion of adrenal androgens into estrogens by the aromatase enzyme complex may be an important mechanism of autocrine stimulation in hormone-dependent breast tumor. The effects of estrogens on tumor development are mediated by the activity of estrogen receptor alpha that induces gene expression and cell proliferation. Thus, estrogen biosynthesis 'in situ' and/or estrogen receptor action are the main targets of endocrine treatment in endocrine-dependent breast carcinoma. In the present study we demonstrate that three major adrenal androgens, dehydroepiandrosterone, 5-androstene-3beta, 17beta-diol and 4-androstene 3,17-dione, all acquire an estradiol-like biological efficacy in aromatase transfected MCF7 breast cancer cells. Our results suggest that in postmenopausal women aromatase inhibitors might be considered as an adjuvant approach to the treatment of hormone-dependent breast tumors that overexpress aromatase.

Cloning and expression of zebra finch (Taeniopygia guttata) steroidogenic factor 1: overlap with hypothalamic but not with telencephalic aromatase.

The zebra finch (Taeniopygia guttata) brain is highly sexually dimorphic. The organization and production of sex-specific song is considerably influenced by estrogens and androgens. Because the brain itself expresses several steroidogenic enzymes, the local production of sex steroids may contribute to sex differences in neural development. Sex steroid production in gonads is directed by a master regulatory factor, steroidogenic factor 1 (SF1). We have identified a cDNA encoding the homologue of SF1 in the zebra finch and utilized reverse transcription-polymerase chain reaction and in situ hybridization to examine early and late developmental expression of SF1 in brain and in early gonadal development. We found that SF1 is expressed early in embryonic development in the Rathke pouch, beginning at stage 15 and extending to at least stage 27 in both males and females. The earliest expression of SF1 in gonads was found at stage 17 for both males and females and extended to at least stage 27. In brain, we assessed SF1 mRNA expression in posthatch and adult telencephalon, and we compared SF1 and aromatase mRNA expression in adult hypothalamus. In the telencephalon and hippocampus, aromatase was expressed independently of SF1, whereas in the hypothalamus, aromatase and SF1 expression were more closely associated. Expression of SF1 and of aromatase overlapped in restricted regions of the hypothalamus, suggesting that SF1 may regulate aromatase expression in these regions. These findings suggest that steroidogenesis in the zebra finch brain may be regulated by both SF1-dependent and SF1-independent mechanisms. No sex differences were detected in SF1 expression in brain.

Increased body fat mass and suppression of circulating leptin levels in response to hypersecretion of epinephrine in phenylethanolamine-N-methyltransferase (PNMT)-overexpressing mice.

Epinephrine is a major stress hormone that plays a central role in the control of metabolic function and energy homeostasis. To evaluate the role of epinephrine and the physiological and pathophysiological consequences of sustained elevation of epinephrine on metabolic and endocrine function, we studied several metabolic parameters and circulating leptin levels in a newly developed transgenic mouse model of phenylethanolamine-N-methyltransferase (PNMT) overexpression. A 100-fold overexpression of PNMT and subsequent elevation of epinephrine levels resulted in a marked suppression of circulating leptin levels in the transgenic animals (1.14 +/- 0.05 vs. 2.17 +/- 0.35 ng/ml; P < 0.01), which correlated negatively with plasma epinephrine (r = -0.82; P < 0.05), thus providing evidence for an inhibitory action of epinephrine on leptin production in vivo. In parallel, we found a marked increase in the body fat content of the transgenic animals (12.54 +/- 1.5 vs. 6.22 +/- 0.2%; P < 0.01) that was accompanied by enlarged adipocytes, indicating an increased lipid storage in PNMT transgenic mice. Interestingly, however, transgenic animals had normal body weight and did not exhibit major alterations in carbohydrate metabolism, as evidenced by analysis of random and fasted blood glucose levels, plasma insulin and C peptide levels, and insulin tolerance test. The metabolic alterations observed were not secondary to changes in food intake or increased activity of the hypothalamic-pituitary-adrenal axis, as there were no differences in these parameters. In summary, sustained primary overproduction of epinephrine resulted in suppression of plasma leptin levels and increased lipid storage in the PNMT transgenic mice. The concerted action of the sympathoadrenal system and reduced leptin may contribute to defending energy reservoirs while maintaining a normal body weight, which may be of vital importance under conditions of stress and energy deficiency.

Agouti-related protein-like immunoreactivity: characterization of release from hypothalamic tissue and presence in serum.

A novel RIA was used to examine the release of agouti-related protein-like immunoreactivity (AGRP-LI) from perfused rat hypothalamic tissue slices and to characterize AGRP-LI in rat serum. A continuous low level basal AGRP-LI release was observed from hypothalami of rats fed ad libitum before the rats were killed. Basal AGRP-LI release was 3-fold greater in rats fasted 48 h. In fasted animals leptin dose-dependently suppressed basal AGRP-LI release. In fed animals no change in basal AGRP-LI release was detected in response to 10(-6) M alpha-MSH, orexin B, melanin-concentrating hormone, or serotonin. HPLC analysis of AGRP-LI in rat serum identified a single peak that eluted in close proximity to synthetic AGRP (87-132) and mouse [Leu127Pro]AGRP and that was identical to the peak seen in hypothalamic and adrenal tissue extracts. The serum concentration of AGRP-LI in rats fed ad libitum was 0.865+/-0.323 nmol/liter (mean +/- SE). Food deprivation resulted in a slow, but statistically significant rise in serum immunoreactivity at 48 h [1.174+/-0.118 nmol/liter (mean +/- SE)]. Bilateral adrenalectomy did not change serum levels of AGRP-LI. These studies demonstrate that in the rat there are different levels of basal hypothalamic AGRP-LI release in fed and fasted states and that in the fasted rat this release can be profoundly suppressed by leptin. These studies also suggest that AGRP is present in the systemic circulation of rats.

[The biogenic amine content of rat tissues in the postresuscitation period following hemorrhagic shock and the effect of the preparation semax]. / Soderzhanie biogennykh aminov v tkaniakh krys v postreanimatsionnom periode posle gemorragicheskogo shoka i vliianie preparata semaks.

LPO products were measured in plasma and biogenic amines (serotonin, adrenalin, noradrenalin) in tissues of rats in different periods after hemorrhagic shock provoked by taking blood and maintenance of arterial pressure at the level of 40 mm Hg for 1 hour. Resuscitation was conducted by administration of autoblood. It was found that splenic serotonin levels decreased on experiment day 7 and went up on day 28. On late experiment stages noradrenalin levels in the adrenals were high. Early after resuscitation the trend was noted to higher LPO products concentration in plasma and serotonin in the brain stem. Intravenous injection of semax prevented serotonin fall in the spleen on experiment day 7. It is suggested that biogenic amines, especially serotonin system, are involved in mechanisms of postresuscitation disorders, in cerebral defects in particular, through prolongation of secondary hypoxia early after hemorrhagic shock and activation of hypothalamo-hypophyso-adrenal system late after the shock.

CART peptide analysis by Western blotting.

CART peptides have been implicated in leptin-regulated feeding, reward and reinforcement, neurotropism, and other processes. In this Western blotting study, at least six different CART peptides varying from 4 to 14 kD were found in rat brain, pituitary, gut, and adrenal gland. The peptides may be processed differently in different tissues and one species found in the rat was not found in human hypothalamus. The higher molecular weight species are likely to include preproCART and proCART, while lower molecular weight peptides may be CART 55-102 and 62-102, physiologically active fragments.

Differential involvement of central and peripheral norepinephrine in the central lipopolysaccharide-induced interleukin-6 responses in mice.

Intracerebroventricular injection of lipopolysaccharide (LPS) induces a marked increase in circulating interleukin (IL)-6 levels and in IL-6 mRNA expression in brain and peripheral organs. Recently, it was reported that intraperitoneal administration of alpha-adrenoceptor antagonists inhibits centrally injected LPS-induced increases in plasma IL-6 levels, suggesting the involvement of the norepinephrine (NE) system in the central LPS-induced IL-6 response. However, the localization (either central or peripheral) of NE involvement in the central LPS-induced IL-6 response has not been characterized. In the present study, mice were pretreated with 6-hydroxydopamine (6-OHDA) administered intracerebroventricularly or intraperitoneally to deplete central or peripheral stores of NE, respectively. Intracerebroventricular LPS (50 ng/mouse) markedly increased plasma IL-6 levels and IL-6 mRNA expression in choroid plexus, hypothalamus, pituitary, adrenals, heart, liver, spleen, and lymph nodes, but with minimal effect in lung, kidney, and testis, as revealed by RT-PCR. Pretreatment with intracerebroventricular 6-OHDA (50 microg/mouse) decreased the LPS-induced plasma IL-6 levels by 39% and the LPS-induced IL-6 mRNA expression in liver, spleen, and lymph nodes, but not in choroid plexus, hypothalamus, pituitary, adrenals, and heart. Pretreatment with intraperitoneal 6-OHDA (100 mg/kg) decreased the LPS-induced plasma IL-6 levels by 36% and the LPS-induced IL-6 mRNA expression in all the peripheral organs displaying increased IL-6 mRNA. Central LPS-induced increase in plasma corticosterone levels was decreased slightly by central but not by peripheral NE depletion. These results suggest that central NE and peripheral NE are differentially involved in the central LPS-induced IL-6 mRNA expression in peripheral organs.

The effect of L-ascorbic acid on age-related changes of pyridinoline in cartilage collagen of guinea pigs.

In order to elucidate the effect of L-ascorbic acid (AsA) on the formation of pyridinoline, a mature crosslink of collagen, its content in cartilage collagen of guinea pigs supplemented with and without AsA in the growing process (4-8 weeks of age) and in the period of maturity (10-14 weeks of age) was examined. The AsA-deficient animals, for four weeks during the growing process, had a significantly higher content of pyridinoline in their collagen than the AsA-supplemented group, indicating that the depletion of AsA induced increasing contents of pyridinoline. On the other hand, in the period of maturity, the pyridinoline content in the collagen decreased with age, whereas no difference between AsA-deficient and -supplemented groups was observed. Based on these results, it is assumed that AsA affects the formation of pyridinoline, especially in the growing period.