Estrogen and leptin have differential effects on FSH and LH release in female rats

Prior experiments have shown that the adipocyte hormone leptin can advance puberty in mice. We hypothesized that it would also stimulate gonadotrophin secretion in adults. Since the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) is drastically affected by estrogen, we hypothesized that leptin might have different actions dependent on the dose of estrogen. Consequently in these experiments, we tested the effect of injection of leptin into the third cerebral ventricle of ovariectomized animals injected with either the oil diluent, 10 microg or 50 microg of estradiol benzoate 72 hr prior to the experiment. The animals were ovariectomized 3-4 weeks prior to implantation of a cannula into the third ventricle 1 week before the experiments. The day after implantation of an external jugular catheter, blood samples (0. 3 ml) were collected just before and every 10 min for 2 hr after 3V injection of 5 microl of diluent or 10 microg of leptin. Both doses of estradiol benzoate equally decreased plasma LH concentrations and pulse amplitude, but there was a graded decrease in pulse frequency. In contrast, only the 50-microg dose of estradiol benzoate significantly decreased mean plasma FSH concentrations without significantly changing other parameters of FSH release. The number of LH pulses alone and pulses of both hormones together decreased as the dose of estrogen was increased, whereas the number of pulses of FSH alone significantly increased with the higher dose of estradiol benzoate, demonstrating differential control of LH and FSH secretion by estrogen, consistent with alterations in release of luteinizing hormone releasing hormone (LHRH) and the putative FSH-releasing factor (FSHRF), respectively. The effects of intraventricularly injected leptin were drastically altered by increasing doses of estradiol benzoate. There was no significant effect of intraventricular injection of leptin (10 microg) on the various parameters of either FSH or LH secretion in ovariectomized, oil-injected rats, whereas in those injected with 10 microg of estradiol benzoate there was an increase in the first hr in mean plasma concentration, area under the curve, pulse amplitude, and maximum increase of LH above the starting value (Deltamax) on comparison with the results in the diluent-injected animals in which there was no alteration of these parameters during the 2 hr following injection. The pattern of FSH release was opposite to that of LH and had a different time-course. In the diluent-injected animals, probably because of the stress of injection and frequent blood sampling, there was an initial significant decline in plasma FSH at 20 min after injection, followed by a progressive increase with a significant elevation above the control values at 110 and 120 min. In the leptin-injected animals, mean plasma FSH was nearly constant during the entire experiment, coupled with a significant decrease below values in diluent-injected rats, beginning at 30 min after injection and progressing to a maximal difference at 120 min. Area under the curve, pulse amplitude, and Deltamax of FSH was also decreased in the second hour compared to values in diluent-injected rats. In contrast to the stimulatory effects of intraventricular injection of leptin on pulsatile LH release manifest during the first hour after injection, there was a diametrically opposite, delayed significant decrease in pulsatile FSH release. This differential effect of leptin on FSH and LH release was consistent with differential effects of leptin on LHRH and FSHRF release. Finally, the higher dose of E2 (50 microg) suppressed release of both FSH and LH, but there was little effect of leptin under these conditions, the only effect being a slight (P < 0.04) increase in pulse amplitude of LH in this group of rats. The results indicate that the central effects of leptin on gonadotropin release are strongly dependent on plasma estradiol levels. These effects are consistent w     

Effect of TNF-alpha on prolactin secretion from rat anterior pituitary and dopamine release from the hypothalamus: comparison with the effect of interleukin-1 beta.

The effects of human recombinant interleukin-1 beta and -6 and tumor necrosis factor-alpha (TNF-alpha) on the releases of PRL and dopamine were examined using monolayer cultures of rat pituitary cells and hypothalamic cells. The release of PRL from rat pituitary cells in 30 min was increased about 2-fold (p less than 0.05) by 10(5) U/l interleukin-1 beta, 10(5) U/l interleukin-6 or 100 micrograms/l TNF-alpha. TNF-alpha at 100 micrograms/l significantly increased PRL release within 5 min incubation and this effect continued throughout the next 30 min of incubation. Incubation for 5 min with TNF-alpha caused dose-dependent stimulation of PRL release. These cytokines did not modulate [3H]-dopamine release from primary cultures of hypothalamic cells. These results suggest that these cytokines stimulate PRL release directly at the pituitary gland, without modifying the release of dopamine from the hypothalamus.     

The possible role of prolactin in the circadian rhythm of leptin secretion in male rats

In humans there is a circadian rhythm of leptin concentrations in plasma with a minimum in the early morning and a maximum in the middle of the night. By taking blood samples from adult male rats every 3 hr for 24 hr, we determined that a circadian rhythm of plasma leptin concentrations also occurs in the rat with a peak at 0130h and a minimum at 0730h. To determine if this rhythm is controlled by nocturnally released hormones, we evaluated the effect of hormones known to be released at night in humans, some of which are also known to be released at night in rats. In humans, prolactin (PRL), growth hormone (GH), and melatonin are known to be released at night, and adrenocorticotropic hormone (ACTH) release is inhibited. In these experiments, conscious rats were injected intravenously with 0.5 ml diluent or the substance to be evaluated just after removal of the first blood sample (0.3 ml), and additional blood samples (0.3 ml) were drawn every 10 min thereafter for 2 hr. The injection of highly purified sheep PRL (500 microg) produced a rapid increase in plasma leptin that persisted for the duration of the experiment. Lower doses were ineffective. To determine the effect of blockade of PRL secretion on leptin secretion, alpha bromoergocryptine (1.5 mg), a dopamine-2-receptor agonist that rapidly inhibits PRL release, was injected. It produced a rapid decline in plasma leptin within 10 min, and the decline persisted for 120 min. The minimal effective dose of GH to lower plasma leptin was 1 mg/rat. Insulin-like growth factor (IGF-1) (10 microg), but not IGF-2 (10 microg), also significantly decreased plasma leptin. Melatonin, known to be nocturnally released in humans and rats, was injected at a dose of 1 mg/rat during daytime (1100h) or nighttime (2300h). It did not alter leptin release significantly. Dexamethasone (DEX), a potent glucocorticoid, was ineffective at a 0. 1-mg dose but produced a delayed, significant increase in leptin, manifest 100-120 min after injection of a 1 mg dose. Since glucocorticoids decrease at night in humans at the time of the maximum plasma concentrations of leptin, we hypothesize that this increase in leptin from a relatively high dose of DEX would mimic the response to the release of corticosterone following stress in the rat and that glucocorticoids are not responsible for the circadian rhythm of leptin concentration. Therefore, we conclude that an increase in PRL secretion during the night may be responsible, at least in part, for the nocturnal elevation of leptin concentrations observed in rats and humans.     

Chromium-induced excretion of urinary lipid metabolites,DNA damage,nitric oxide production,and generation of reactive oxygen species in Sprague-Dawley rats

Chromium and its salts induce cytotoxicity and mutagenesis, and vitamin E has been reported to attenuate chromate-induced cytotoxicity. These observations suggest that chromium produces reactive oxygen species which may mediate many of the untoward effects of chromium. We have therefore examined and compared the effects of Cr(III) (chromium chloride hexahydrate) and Cr(VI) (sodium dichromate) following single oral doses (0.50 ld₅₀) on the production of reactive oxygen species by peritoneal macrophages, and hepatic mitochondria and microsomes in rats. The effects of Cr(III) and Cr(VI) on hepatic mitochondrial and microsomal lipid peroxidation and enhanced excretion of urinary lipid metabolites as well as the incidence of hepatic nuclear DNA damage and nitric oxide (NO) production were also examined. Increases in lipid peroxidation of 1.8- and 2.2-fold occurred in hepatic mitochondria and microsomes, respectively, 48 hr after the oral administration of 25 mg Cr(VI)/kg, while increases of 1.2- and 1.4-fold, respectively, were observed after 895 mg Cr(III)/kg. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) were determined at 0–96 hr after Cr administration. Between 48 and 72 hr post-treatment, maximal excretion of the four urinary lipid metabolites was observed with increases of 1.5- to 5.4-fold in Cr(VI) treated rats. Peritoneal macrophages from Cr(VI) treated animals 48 hr after treatment resulted in 1.4- and 3.6-fold increases in chemiluminescence and iodonitrotetrazolium reduction, indicating enhanced production of Superoxide anion, while macrophages from Cr(III) treated animals showed negligible increases. Increases in DNA single strand breaks of 1.7-fold and 1.5-fold were observed following administration of Cr(VI) and Cr(III), respectively, at 48 hr post-treatment. Enhanced production of NO by peritoneal exudate cells (primarily macrophages) was monitored following Cr(VI) administration at both 24 and 48 hr post-treatment with enhanced production of NO being observed at both timepoints. The results indicate that both Cr(VI) and Cr(III) induce an oxidative stress at equitoxic doses, while Cr(VI) induces greater oxidative stress in rats as compared with Cr(III) treated animals.     

The anti-inflammatory effect of leptin on experimental colitis: involvement of endogenous glucocorticoids

The present study was designed to compare the effect of leptin on acute colonic inflammation with that of acute stress exposure, which acts via the hypothalamic-pituitary-adrenal (HPA) axis. Sprague-Dawley rats of both sexes were administered intrarectally with acetic acid. Either leptin (10 microg/kg; i.p.) or saline was injected immediately before and 6 h after the induction of colitis. A group of rats was exposed to water avoidance stress (WAS) for 30 min at the 6th h of colitis induction. RU-486 (2 mg/kg; i.p.), a glucocorticoid receptor antagonist, was injected intraperitoneally, at 12 and 1 h before the initial leptin injection, and at 1 h before the second leptin injection or exposure to WAS. Rats were decapitated at 24 h and the distal 8 cm of the colon were removed for macroscopic and microscopic scoring, determination of tissue wet weight index (WI) and tissue myeloperoxidase activity (MPO). Acetic acid-induced colitis significantly increased macroscopic and microscopic damage scores, WI and MPO, compared to control group. Exposure to acute WAS or treatment with leptin reduced the elevations in damage scores, WI and MPO induced by colitis, but no additive inhibitory effect was observed when WAS and leptin were applied together. RU-486 treatment reversed the inhibitory effects of leptin or WAS on colonic inflammation. Our results demonstrate that exogenous leptin mimics the effects of HPA axis activation on colitis-induced inflammatory process. The results also suggest that the anti-inflammatory effect of leptin involves a tissue neutrophil-dependent mechanism and is dependent on the release of glucocorticoids.     

Inducible nitric oxide synthase mediates cytokine release: the time course in conscious and septic rats

Nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-beta (IL-1beta) are postulated to play a key pathophysiologic role during sepsis. In this study, we examined the time course of inducible NO synthase (iNOS) mRNA expression and the plasma TNF-alpha and IL-1beta in lipopolysaccharide (LPS)-treated conscious rats. The hemodynamic pattern in septic shock is more similar to clinical conditions without anesthesia. The data showed that a significant increase in iNOS mRNA levels was found in the spleen, lung, liver, with slight elevation in the heart and kidney at 3 h after LPS administration. However, iNOS mRNA levels were not elevated significantly in all tissues examined at 24 h. In the plasma, TNF-alpha and IL-1beta culminated within 1 h, and reduced gradually to baseline levels in a relatively short period (within 9 h). The results suggest that local NO production by activation of iNOS mRNA expression and cytokine release may contribute to LPS-induced organ dysfunction at various time points.     

High-dose leptin activates human leukocytes via receptor expression on monocytes

Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 +/- 5% for monocytes, 12 +/- 4% for neutrophils, and 5 +/- 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-alpha and IL-6 mRNA and a dose-dependent production of TNF-alpha and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3-5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-alpha (5-fold), IL-6 (19-fold), and IFN-gamma (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.     

Tumor necrosis factors alpha and beta differ in their capacities to generate interleukin 1 release from human endothelial cells

The capacity of the tumor necrosis factors, TNF-alpha and TNF-beta, products of activated macrophages and lymphocytes, respectively, to stimulate interleukin 1 (IL-1) release from endothelial cells derived from human umbilical veins was examined in vitro. Recombinant TNF-alpha caused IL-1 release by 4 hr with maximal levels of 17 U/ml by 24 hr; half-maximal stimulation occurred at approximately 80 pM. In contrast, recombinant TNF-beta was a relatively poor stimulus for IL-1 release. Even at concentrations as high as 600 pM, only 3 U of IL-1/ml were recovered; maximal IL-1 release (10 to 12 U/ml) required up to 5 nM TNF-beta. Natural, glycosated human TNF-beta was comparable in activity to recombinant TNF-beta. TNF-beta did not directly inhibit the IL-1 comitogenesis assay, nor was there evidence that TNF-beta induced the release of an IL-1 inhibitor, in that supernatants generated in the presence of TNF-beta did not inhibit thymocyte proliferation to a recombinant IL-1 standard. Binding of the recombinant TNF to endothelial monolayers was assessed by using [125I]TNF-alpha in competition studies with cold TNF-alpha and TNF-beta. Binding of TNF-alpha was half-maximal at 80 pM with an average of 664 receptors/cell and Kd = 0.043 nM. Although TNF-beta was capable of fully competing for [125I]TNF-alpha binding, half-maximal binding occurred at 800 pM TNF-beta. These data suggest that the TNF receptors on human endothelial cells may reflect the structural differences between these two homologous cytokines.     

Dissociation between increased growth hormone and prolactin secretion during the morning hours of early pregnancy in the rat

We investigated whether serum growth hormone (GH) concentration changes in association with the rise in serum prolactin (PRL) concentration known to occur during the early morning hours in the pregnant rat. Animals were kept in a room with the lights on from 0500 to 1900 hours (hr) daily and decapitated for the collection of trunk blood at 2200 or 2400 hr on Day 6 of pregnancy or at 0200, 0400, 0800 or 1000 hr on Day 6 of pregnancy. Serum GH concentration rose more than 4-fold from low levels at 2200 and 2400 hr to higher levels at 0400 and 0800 hr and then declined by 1000 hr. Serum prolactin (PRL) concentration followed a similar pattern except that it returned to low levels earlier, by 0800 hr. Serum luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone concentrations showed no significant changes. Serum GH levels at 0800 hr in pregnant rats were higher than those observed in cyclic rats (13 time periods sampled). The results demonstrate that serum GH concentration is elevated during a circumscribed period in the 6- to 7-day pregnant rat. The time of onset of the rise is similar to that for serum PRL but the elevation in GH levels persists longer than that for PRL.     

In vitro metabolism of [14C]4-chlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl by hepatic microsomes from rats and pigeons. Evidence against an obligatory arene oxide in aromatic hydroxylation reactions.

1. The catalytic activities of cytochromes P-450IA1 and P-450IIB1 in control and Aroclor 1254 treated rats and pigeons (1 mmol/kg) were assessed using [14C]4-chloro- and [14C]2,2',5,5'-tetrachlorobiphenyl as substrates. Treatment of rats resulted in increases of the total amount of chloroform-extractable metabolites of [14C]4-chlorobiphenyl from 37.2 (control) to 199.4 and 221.6 nmol/hr per mg microsomal protein at 48 and 120 hr post treatment. The portion of [14C]4-chloro-3',4'-dihydroxybiphenyl (M4) and of a second unidentified dihydroxylated metabolite (M3) increased during these incubations from 13.7% for controls to 53.5% at 48 hr and 69.12% at 120 hr post treatment. 2. [14C]4-chloro-3'-hydroxybiphenyl (M1) and [14C]4-chloro-4'-hydroxybiphenyl (M2) were the major metabolites formed by pigeon hepatic microsomes; however, the amounts formed were 38.7- and 29.3-fold less, respectively, than in untreated rats. Treatment of pigeons with Aroclor 1254 increased the metabolite formation from 1.0 (control) to 13.6 and 22.4 nmol/hr per mg microsomal protein at 48 hr and 120 hr post treatment respectively; however, only small amounts of metabolites M3 (0.5 nmol/hr per mg protein) and M4 (2.0 nmol/hr per mg protein) were detected. 3. Treatment of rats with Aroclor 1254 resulted in an approximately two-fold increase in the rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl, and the ratio of 3- to 4-hydroxylation increased from 0.45 (control) to 0.6 and 0.8 at 48 hr and 120 hr post treatment respectively. The rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl by control and Aroclor 1254 treated pigeons was up to 23-fold lower than in rats and there was no evidence for the formation of the diol metabolite M3. However, as with rats, the ratio of meta- to para-carbon atom hydroxylation increased from 0.58 (controls) to 0.72 at 120 hr post treatment. 4. From the evidence presented, it is suggested that cytochromes P-450IA1 and P-450IIB1 may not metabolize PCB-congeneric substrates via an obligatory arene oxide intermediate.     

Evidence of nitric oxide, a flow-dependent factor, being a trigger of liver regeneration in rats.

The hypothesis tested was that the hemodynamic consequence of partial hepatectomy (PHX) triggers the cascade of events that leads to liver regeneration. After PHX, all the portal flow must go through the remaining vascular bed, thus producing increased shear stress and release of nitric oxide (NO), which then initiates the next stages of the regeneration process. As an index of triggering of the regeneration cascade, we used an in vitro bioassay detecting the appearance of proliferating factors (PFs; various growth factors, cytokines, and hormones) in plasma 4 h after two-thirds PHX in rats. PF levels, assessed using proliferation of cultured hepatocytes, were elevated in two-thirds PHX rats, fully blocked by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), and restored by L-arginine. L-NAME inhibited liver weight restoration at 48 h but resulted in high mortality. L-NAME lacked toxic effects in non-PHX rats. NO was directly antiproliferative on cultured cells, suggesting that the proliferative effect of NO in vivo was secondary to the activation of other proliferative stimuli. The data support the hypothesis that vascular shear stress induced release of NO following PHX serves as a primary trigger to initiate the regeneration process.     

Oxidative stress, nitric oxide production, and renal sodium handling in leptin-induced hypertension

Chronic hyperleptinemia induces arterial hypertension in experimental animals and may contribute to the development of hypertension in obese humans; however, the mechanism of hypertensive effect of leptin is not completely elucidated. We investigated the effect of leptin on whole-body oxidative stress, nitric oxide production, and renal sodium handling. The study was performed on male Wistar rats divided into 3 groups: 1) control, fed standard chow ad libitum, 2) leptin-treated group, receiving leptin injections (0.25 mg/kg twice daily s.c. for 7 days), 3) pair-fed group, in which food intake was adjusted to the leptin group. Leptin caused 30.5% increase in systolic blood pressure. Plasma concentration and urinary excretion of 8-isoprostanes in animals receiving leptin was 46.4% and 49.2% higher, respectively. The level of lipid peroxidation products, malonyldialdehyde + 4-hydroxyalkenals, increased by 52.5% in the renal cortex and by 48.4% in the renal medulla following leptin treatment, whereas aconitase activity decreased in these regions of the kidney by 45.3% and 39.2%, respectively. Urinary excretion of nitric oxide metabolites (NOx) was 55.0% lower, and fractional excretion of NOx was 55.8% lower in the leptin-treated group. Urinary excretion of cGMP decreased in leptin-treated rats by 26.3%. Following leptin treatment, absolute and fractional sodium excretion decreased by 35.0% and 41.2%, respectively. These results indicate that hyperleptinemia induces systemic and intrarenal oxidative stress, decreases the amount of bioactive NO possibly due to its degradation by reactive oxygen species, and causes renal sodium retention by stimulating tubular sodium reabsorption. NO deficiency and abnormal renal Na+ handling may contribute to leptin-induced hypertension.     

Effects of estrogen on serum leptin levels and leptin mRNA expression in adipose tissue in rats

OBJECTIVE: In this study, we examined changes in serum leptin levels during the estrus cycle and the role of estrogen in these changes. METHODS: We measured serum leptin levels during normal estrus cycles in intact rats and estradiol-17beta (E2)-induced artificial estrus cycles in ovariectomized rats. RESULTS: Serum leptin levels increased 1.6-fold from 4.2 +/- 0.2 ng/ml during diestrus stage 2 to 6.7 +/- 0.9 ng/ml during proestrus stage during the 4-day estrus cycle. During the E2-induced estrus cycle, serum leptin levels increased 2.3-fold from 2.3 +/- 0.1 ng/ml at estrus to 5.4 +/- 1.2 ng/ml at proestrus. E2 also increased serum leptin concentrations and leptin mRNA expression in adipose tissue of immature rats. DISCUSSION: These findings suggest that increased serum leptin induced by estrogen during proestrus may trigger the preovulatory release of luteinizing hormone. Furthermore, our findings indicate that estrogen has a positive effect on leptin production in adipose tissue.     

In vivo effects of the controlled NO donor/scavenger ruthenium cyclam complexes on blood pressure

Ruthenium(II/III) complexes able to bind and release NO* were tested in vivo, in conscious Wistar rats instrumented for continuous blood pressure (BP) measurement and administration of in bolus injections (5 to 100 nmol/Kg i.v.) of trans-[Ru(II)Cl(NO+)(cyclam)](PF6)2 (cyclam-NO) or sodium nitroprusside (SNP). For normotensive rats, cyclam-NO produced a sustained 10% BP reduction of basal MAP during 7 +/- 0.4 to 11 +/- 0.3 min. In acute hypertensive rats, cyclam-NO produced BP reduction 3-fold larger than in normotensive rats and similar to that of SNP (maximal effect: 41 +/- 1.3 vs. 45 +/- 2.2 mmHg, respectively). However, the duration of the effect of cyclam-NO was 13 to 21-fold longer than that of SNP. The hypotensive effect of cyclam-NO was fully blocked in presence of continuous infusion of a NO* scavenger, carboxy-PTIO (6 mmol/Kg/min), or of the inhibitor of cGMP activation, methylene blue (83 nmol/Kg/min), or of the cyclam-NO precursor, trans-[RuCl(tfins)(cyclam)](tfms) (cyclam-tfms) (500 mmol/Kg/min). The long lasting BP reduction of cyclam-NO can be interpreted in terms of a slower rate of NO* release (k-NO = 2.2 x 10(-3) S(-1) at 35 degrees C) following chemical reduction (E(0') = 0.10 V vs NHE). In summary, cyclam-NO showed an hypotensive effect around 20 times longer than SNP in either normotensive or hypertensive rats, which was completely inhibited by methylene blue or carboxy-PTIO. Continuous infusion of cyclam-tfms completely blocked the hypotensive effect of cyclam-NO by scavenging the NO* released by the reduced cyclam-NO.     

Extra-adipocyte leptin release in human obesity and its relation to sympathoadrenal function

The link between the human sympathoadrenalmedullary system and the adipocyte hormone leptin is controversial. We measured total and regional norepinephrine spillover, epinephrine secretion rate, and extra-adipocyte leptin release in 22 lean [body mass index (BMI) < 26] and 20 obese (BMI > 28) normotensive men who underwent arterial and central venous catheterization. Because plasma clearance of leptin is primarily by renal removal, for men at steady state we could estimate whole body leptin release to plasma from renal plasma leptin extraction. Whole body leptin release was 1,950 +/- 643 (means +/- SE) ng/min in obese men and 382 +/- 124 ng/min in lean men (P < 0.05). Total and renal norepinephrine spillover rates correlated directly with whole body leptin secretion rate. Leptin is released from multiple nonadipocyte sites, which we tested by use of simultaneous arteriovenous blood sampling. We found a surprisingly large contribution of brain leptin release to the plasma leptin pool, 529 +/- 175 ng/min (> 40% whole body leptin release), with greater leptin release in obese than in lean men, 935 +/- 321 vs. 160 +/- 59 ng/min (P = 0.045). In parallel with leptin measurements, we also quantified brain serotonin turnover and jugular overflow of neuropeptide Y (NPY). Brain serotonin turnover was higher in obese than in lean men, 227 +/- 112 vs. 21 +/- 14 ng/min (P = 0.019), as was overflow of NPY from the brain, 12.9 +/- 1.4 vs. 5.3 +/- 2.2 ng/min (P = 0.042). These results suggest that leptin is released within the brain and at an increased rate in obese humans, in whom activation of brain serotonergic and NPY mechanisms also exists.     

TNF-alpha enhances contraction and inhibits endothelial NO-cGMP relaxation in systemic vessels of pregnant rats

Tumor necrosis factor-alpha (TNF-alpha) is elevated in the plasma of preeclamptic women and may have a role in pregnancy-induced hypertension. However, whether the hemodynamic effects of TNF-alpha reflect the direct effects on vascular reactivity is unclear. We tested the hypothesis that TNF-alpha impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. We measured isometric contraction in aortic strips isolated from virgin and pregnant Sprague-Dawley rats (nontreated vs. treated for 2 h with 10-1,000 pg/ml TNF-alpha). In endothelium-intact vascular strips, TNF-alpha caused greater enhancement of phenylephrine (Phe) contraction in pregnant than virgin rats. TNF-alpha caused significant inhibition of ACh- and bradykinin-induced vascular relaxation and nitrite/nitrate production that were more prominent in pregnant than virgin rats. N(G)-nitro-L-arginine methyl ester [L-NAME, 100 microM, an inhibitor of nitric oxide (NO) synthase] or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 1 microM, an inhibitor of cGMP production in smooth muscle) inhibited ACh relaxation and enhanced Phe contraction in nontreated but to a lesser extent in TNF-alpha-treated vessels, particularly those of pregnant rats. Endothelium removal enhanced Phe contraction in nontreated but not TNF-alpha-treated vessels, especially those of pregnant rats. Relaxation of Phe contraction with the NO donor sodium nitroprusside was not different between nontreated and TNF-alpha-treated vessels. Thus TNF-alpha enhances vascular contraction and inhibits endothelium-dependent NO-cGMP-mediated vascular relaxation in systemic vessels, particularly those of pregnant rats. The results support a direct role for TNF-alpha as a possible mediator of increased vascular resistance associated with pregnancy-induced hypertension.     

Role of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) in the regulation of blood pressure by leptin in lean and obese rats

We investigated the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in hemodynamic action of leptin. The effect of leptin (1 mg/kg i.p.) on systolic blood pressure (SBP) was examined in lean rats and in rats made obese by feeding highly palatable diet for either 1 or 3 months. Separate groups received NO synthase inhibitor, L-NAME, or EDHF inhibitors, the mixture of apamin+charybdotoxin or sulfaphenazole, before leptin administration. Leptin increased NO production, as evidenced by increase in plasma and urinary NO metabolites and cyclic GMP. This effect was impaired in both obese groups. In lean rats either leptin or EDHF inhibitors had no effect on blood pressure. L-NAME increased blood pressure in lean animals and this effect was prevented by leptin. However, when leptin was administered to animals pretreated with both L-NAME and EDHF inhibitors, blood pressure increased even more than after L-NAME alone. In the 1-month obese group leptin had no effect on SBP, however, pressor effect of leptin was observed in animals pretreated with EDHF inhibitors. In the 3-month obese group leptin alone increased SBP, and EDHF inhibitors did not augment its pressor effect. The results suggest that leptin may stimulate EDHF when NO becomes deficient, e.g. after NOS blockade or in short-term obesity. Although the effect of leptin on NO production is impaired in the 1-month obese group, BP does not increase, probably because EDHF compensates for NO deficiency. In contrast, leptin increases BP in 3-month obesity because its effect on EDHF is also attenuated.     

Pinealectomy and melatonin administration in rats: their effects on plasma leptin levels and relationship with zinc

The aim of this study was to examine effects of pinealectomy and melatonin administration plasma leptin levels and its relationship with zinc in rats. The study was conducted on 40 adult male Sprague-Dawley rats. They were divided into four groups each containing 10 animals. Group 1 served as control. Group 2 was pinealectomized group. Animals in Group 3 were pinealectomized and injected with melatonin (3 mg/kg/day, ip). Group 4 received melatonin alone (3 mg/kg/day, ip). At the end of the experiments, all animals were decapitated and trunk blood collected. Plasma leptin and zinc levels were determined by radioimmunoassay and Atomic Absorption Spectrophotometer methods, respectively. Although mean weights of the animals at the beginning were not significantly different among the groups, the mean weight of the pinealectomized group was found to be significantly lower than all other groups at the end of a six-month period (p < 0.01). Plasma leptin and zinc levels were the highest in melatonin-administered group (group 4; p < 0.01). The lowest plasma leptin and zinc levels were obtained in the pinealectomized group (group 2; p < 0.01). Changes in these two parameters were not statistically significant in groups 1 and 3. Our findings indicate that pinealectomy results in a decrease in leptin and zinc levels in rats, and that melatonin administration to pinealectomized rats prevents the decrease in the these parameters. In addition, long-term administration of melatonin to rats leads to an increase in both leptin and zinc concentrations.