The obstetric, gynaecological and fertility implications of homozygous PAI-1 deficiency: single-centre experience.

Complete plasminogen activator inhibitor type 1 (PAI-1) deficiency is an exceedingly rare autosomal recessive bleeding disorder previously identified and reported in a large Old Order Amish (OOA) kindred in Indiana [Fay et al. Blood 1997; 90: 204]. Mouse models suggest that proteolysis via the plasminogen activator/plasmin system plays a crucial role in reproduction including degradation of the follicular wall during ovulation, fertilization, embryo implantation and embryogenesis [Leonardsson et al., Proc Natl Acad Sci USA 1995; 92: 12446]. We report the obstetric, gynaecological and fertility histories of OOA individuals with homozygous PAI-1 deficiency. In this family, there are 10 affected members identified to date ranging in age between 10 and 32 years, including seven female patients and three male patients. To date, two women have achieved pregnancies without difficulty; however, they experienced antenatal bleeding and preterm labour. The early initiation and continuation of antifibrinolytic agents, Epsilon-aminocaproic acid or tranexamic acid, during the pregnancy and in the postpartum period, was believed to be successful in preventing major bleeding complications in our patients with complete PAI-1 deficiency.

Physiology: does gut hormone PYY3-36 decrease food intake in rodents?

Batterham et al. report that the gut peptide hormone PYY3-36 decreases food intake and body-weight gain in rodents, a discovery that has been heralded as potentially offering a new therapy for obesity. However, we have been unable to replicate their results. Although the reasons for this discrepancy remain undetermined, an effective anti-obesity drug ultimately must produce its effects across a range of situations. The fact that the findings of Batterham et al. cannot easily be replicated calls into question the potential value of an anti-obesity approach that is based on administration of PYY3-36.

Prm1p, a pheromone-regulated multispanning membrane protein, facilitates plasma membrane fusion during yeast mating.

Cell fusion occurs throughout development, from fertilization to organogenesis. The molecular mechanisms driving plasma membrane fusion in these processes remain unknown. While yeast mating offers an excellent model system in which to study cell fusion, all genes previously shown to regulate the process act at or before cell wall breakdown; i.e., well before the two plasma membranes have come in contact. Using a new strategy in which genomic data is used to predict which genes may possess a given function, we identified PRM1, a gene that is selectively expressed during mating and that encodes a multispanning transmembrane protein. Prm1p localizes to sites of cell-cell contact where fusion occurs. In matings between Deltaprm1 mutants, a large fraction of cells initiate zygote formation and degrade the cell wall separating mating partners but then fail to fuse. Electron microscopic analysis reveals that the two plasma membranes in these mating pairs are tightly apposed, remaining separated only by a uniform gap of approximately 8 nm. Thus, the phenotype of Deltaprm1 mutants defines a new step in the mating reaction in which membranes are juxtaposed, possibly through a defined adherence junction, yet remain unfused. This phenotype suggests a role for Prm1p in plasma membrane fusion.

Variability in bleeding phenotype in Amish carriers of haemophilia B with the 31008 C-->T mutation.

The aim of this study was to characterize the variability of bleeding phenotype and its association with plasma factor IX coagulant activity (FIX:C) in haemophilia B carriers in a large Amish pedigree with a unifying genetic mutation, C-to-T transition at base 31008 of the factor IX gene (Xq27.1-27.2). A cross-sectional survey of haemophilia B carriers included a multiple choice questionnaire evaluating symptoms of mucocutaneous bleeding, joint bleeding and bleeding after haemostatic stress [menstruation, postpartum haemorrhage (PPH), dental extractions and invasive surgeries]. Severity of bleeding was graded as 0 to 4, 0 being no bleeding whereas 4 being severe bleeding. Association between total bleeding scores and the FIX:C was evaluated. Sixty-four haemophilia B carriers participated in this study. Median age: 18 years (range 1-70 years); median bleeding score: 1 (range 0-8). Besides PPH, isolated symptoms of bruising, epistaxis, menorrhagia and postsurgical bleeding including dental extraction were not associated with lower FIX:C. Bleeding score >/=3 was associated with involvement of at least two bleeding sites and a lower mean FIX:C of 42 +/- 10.3% (95% CI 36.4-47.7) while a score >3 had involvement of /=3. Phenotypic variability existed among the carriers of haemophilia B who belonged to a single pedigree carrying a single unifying mutation. The utility of bleeding scores to define bleeding phenotype precisely in haemophilia B carriers needs further evaluation.

Mathematical model of thrombin generation and bleeding phenotype in Amish carriers of Factor IX:C deficiency vs. controls.

INTRODUCTION: Factor IX:C (FIX:C) levels vary in hemophilia B carriers even in pedigrees with a unifying genetic defect. Analyzing the balance between pro-and anticoagulants might increase our understanding of carriers' bleeding potential. AIM: In this research study, we evaluated bleeding scores (BS) and a novel mathematical model of thrombin generation (TG) in Amish FIX:C deficient carriers and controls. METHODS: Blood samples and BS were obtained from post-menarchal females, including 59 carriers and 57 controls from the same extended pedigree. Factors II, V, VII, VIII, IX, X, antithrombin, tissue factor pathway inhibitor and protein C were assayed to generate mathematical models of TG in response to 5pM tissue factor (TF) and for TF + thrombomodulin. BS was based on a modification of the MCMDM-1VWD scoring system. RESULTS: Carriers had a lower mean FIX:C (68% vs. 119%), von Willebrand factor antigen (108 vs.133) and Tissue activatable fibrinolysis inhibitor (103 vs. 111) compared to controls; both groups had a similar mean BS. Carriers demonstrated significantly lower TG parameters on both mathematical models compared to controls. Carriers with FIX:C ≤ 50% had lower TG curves than those >50% but similar BS. CONCLUSION: Thrombin generation showed significant differences between carriers and controls, between low (≤50%) and high (>50%) FIX:C carriers, and specifically in the TF + thrombomodulin model, between high FIX:C carriers and controls, although the BS were not different.

Biologic response to subcutaneous and intranasal therapy with desmopressin in a large Amish kindred with Type 2M von Willebrand disease.

The aim of this study was to characterize the adequacy and longevity of biological response to desmopressin (DDAVP) in a large Amish kindred of Type 2M von Willebrand disease (VWD) possessing C-to-T transition at nucleotide 4120 in exon 28 of A1 domain of von Willebrand factor (VWF) gene. Response to both intranasal (Stimate) and subcutaneous DDAVP administration was assessed. Rise in ristocetin cofactor activity (VWF:RCo) > or = 40% at 90-min post-Stimate and 1-2 h after subcutaneous DDAVP was defined as initial response; response longevity was assessed only after subcutaneous dosing by measuring VWF:RCo levels at time-points 1, 2, 4 and 6 h. Eleven patients (five males, six females; age range: 20-56 years) participated in intranasal and 9/11 (four males, five females) in subcutaneous testing. Baseline haemostatic profiles included: VWF:RCo < 15%, VWF:Ag < 40% and normal VWF multimers. Initial response was comparable by both intranasal (6/11; 54.5%) and subcutaneous (4/9; 44%) routes; sustained response (VWF:RCo > 40% for 2 h) was observed in only one in nine (11%) patients tested. Median VWF:RCo peak levels after intranasal (40%) and subcutaneous (39%) routes were equivalent. Peak VWF:Ag levels were significantly higher after subcutaneous than intranasal DDAVP (94% vs. 54%; P = 0.03). Area under the curve for VWF:RCo was significantly decreased (170 microg h mL(-1)) compared with VWF:Ag (471 microg h mL(-1)) and FVIII:C (624.60 microg h mL(-1)). This study suggests that in this population: (i) intra-individual DDAVP response is consistent with subcutaneous and intranasal administration; and (ii) extending DDAVP challenge test up to at least 6 h is required to characterize adequacy and longevity of biologic response prior to using DDAVP as a sole haemostatic intervention.

Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting.

Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10% of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.

Nocturnal rise of leptin in lean, obese, and non-insulin-dependent diabetes mellitus subjects.

We studied 24-h profiles of circulating leptin levels using a sensitive and specific RIA in lean controls and obese subjects with or without non-insulin-dependent diabetes mellitus (NIDDM) during normal routine activity. Serum leptin levels were significantly higher in obese (41.7 +/- 9.0 ng/ml; n = 11) and obese NIDDM (30.8 +/- 6.7; n = 9) subjects compared with those in lean controls (12.0 +/- 4.4, n = 6). In all the three groups, serum leptin levels were highest between midnight and early morning hours and lowest around noon to midafternoon. The nocturnal rise in leptin levels was significant when data were analyzed by ANOVA (lean: F = 3.17, P < 0.0001, n = 4; obese: F = 2.02, P < 0.005, n = 11; and obese NIDDM: F = 4.9, P < 0.0001, n = 5). The average circadian amplitude between acrophase and nadir was 75.6% in lean, 51.7%, in obese and 60.7% in obese NIDDM groups, respectively. No significant correlations (P > 0.05) were observed between circulating levels of leptin and either insulin or glucose levels in any of the 20 subjects studied for 24-h profiles. The nocturnal rise in leptin observed in the present study resembles those reported for prolactin, thyroid-stimulating hormone, and free fatty acids. We speculate that the nocturnal rise in leptin could have an effect in suppressing appetite during the night while sleeping.

Comparative antitumor effects of hormonal ablation, estrogen agonist, estrogen cytotoxic derivative, and antiestrogen in the PAIII rat prostatic adenocarcinoma.

The effects of hormonal ablation, estrogen, estrogen-derived cytotoxic agent, and estrogen antagonist therapies used clinically were evaluated on in vitro colony formation, in vivo growth, and lymphatic and pulmonary metastasis of the PAIII tumor. Ventral prostatic and seminal vesicle weights were evaluated in the same animals to assess androgen-related responses. Estradiol, estramustine phosphate, and testosterone had no effects on PAIII colony formation in vitro. Castration, hypophysectomy, estradiol benzoate, and estramustine phosphate treatment of PAIII-bearing Lobund Wistar rats produced significant (P less than 0.05) regression of male accessory sex organs. Of these treatments, only hypophysectomy had significant (P less than 0.05) inhibitory effects on primary PAIII growth and lymphatic and pulmonary metastasis. LY117018 [6-hydroxy-2-(p-hydroxyphenyl)benzo(b)thien-3-yl-p-2-(l-pyrrolidin yl)ethoxy phenyl ketone] has antiestrogenic activity but produces no significant agonist responses. LY117018 had no effect upon PAIII colony formation in vitro. Following s.c. implantation of PAIII cells, LY117018 (2.0, 10.0, or 20.0 mg/kg s.c.) had no effect on primary tumor growth in the tail. In vitro LY117018 administration produced marked antimetastatic effects. In a dose-dependent manner, LY117018 inhibited PAIII metastasis to the gluteal (97%) and iliac lymph nodes (88%) (P less than 0.05 for both). LY117018 also maximally inhibited pulmonary metastasis by 86% (P less than 0.05). Maximal regression of 42% for ventral prostatic and 35% for seminal vesicle weights were also seen after LY117018 administration (P less than 0.05 for both). Co-administration of estradiol benzoate had no antagonistic effect upon the antitumor responses produced by LY117018. The mechanism of action of LY117018 is not known. The failure of estradiol benzoate to affect PAIII growth and metastasis supports the contention that the responses to LY117018 are not attributable to simple antagonism of estrogen action. LY117018 may be exerting its antitumor effects through autocrine, paracrine, or endocrine mechanisms. LY117018 represents a class of agents with potential utility in treating metastatic cancer of the prostate.

Circulating ghrelin levels are decreased in human obesity.

Ghrelin is a novel endogenous natural ligand for the growth hormone (GH) secretagogue receptor that has recently been isolated from the rat stomach. Ghrelin administration stimulates GH secretion but also causes weight gain by increasing food intake and reducing fat utilization in rodents. To investigate the possible involvement of ghrelin in the pathogenesis of human obesity, we measured body composition (by dual X-ray absorption) as well as fasting plasma ghrelin concentrations (radioimmunoassay) in 15 Caucasians (8 men and 7 women, 31+/-9 years of age, 92+/-24 kg body wt, and 29+/-10% body fat, mean +/- SD) and 15 Pima Indians (8 men and 7 women, 33+/-5 years of age, 97+/-29 kg body wt, and 30+/-8% body fat). Fasting plasma ghrelin was negatively correlated with percent body fat (r = -0.45; P = 0.01), fasting insulin (r = -0.45; P = 0.01) and leptin (r = -0.38; P = 0.03) concentrations. Plasma ghrelin concentration was decreased in obese Caucasians as compared with lean Caucasians (P < 0.01). Also, fasting plasma ghrelin was lower in Pima Indians, a population with a very high prevalence of obesity, compared with Caucasians (87+/-28 vs. 129+/-34 fmol/ml; P < 0.01). This result did not change after adjustment for fasting plasma insulin concentration. There was no correlation between fasting plasma ghrelin and height. Prospective clinical studies are now needed to establish the role of ghrelin in the pathogenesis of human obesity.

Differential brain responses to satiation in obese and lean men.

Knowledge of how the brain contributes to the regulation of food intake in humans is limited. We used positron emission tomography and measures of regional cerebral blood flow (rCBF) (a marker of neuronal activity) to describe the functional anatomy of satiation (i.e., the response to a liquid meal) in the context of extreme hunger (36-h fast) in 11 obese (BMI > or =35 kg/m2, age 27+/-5 years, weight 115+/-11 kg, 38+/-7% body fat; mean +/- SD) and 11 lean (BMI < or =25 kg/m2, age 35+/-8 years, weight 73+/-9 kg, 19+/-6% body fat) men. As in lean men, satiation in obese men produced significant increases in rCBF in the vicinity of the ventromedial and dorsolateral prefrontal cortex and significant decreases in rCBF in the vicinity of the limbic/paralimbic areas (i.e., hippocampal formation, temporal pole), striatum (i.e., caudate, putamen), precuneus, and cerebellum. However, rCBF increases in the prefrontal cortex were significantly greater in obese men than in lean men (P < 0.005). rCBF decreases in limbic/paralimbic areas, temporal and occipital cortex, and cerebellum were also significantly greater in obese men than in lean men (P < 0.005), whereas rCBF decreases in the hypothalamus and thalamus were attenuated in obese men compared with lean men (P < 0.05). This study raises the possibility that the brain responses to a meal in the prefrontal areas (which may be involved in the inhibition of inappropriate response tendencies) and limbic/paralimbic areas (commonly associated with the regulation of emotion) may be different in obese and lean men. Additional studies are required to investigate how these differential responses are related to the pathophysiology of obesity.

Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Dopaminergic receptors in the rat anterior pituitary change during the estrous cycle.

We investigated whether rat anterior pituitary dopaminergic receptors change in affinity and/or number of binding sites (Bmax) during the estrous cycle and whether they correlate with circulating PRL levels. Dopamine receptors were quantitated in partially purified pituitary membranes by equilibrium binding using [3H]spiperone. AT 0900 h of diestrous day 2 and 0900 h of proestrus, Bmax [316.5 +/- 7.7 and 297.9 +/- 7.3 fmol/mg protein (mean +/- SE), respectively] and plasma PRL levels (18.2 +/- 4.2 and 25.0 +/- 7.3 ng/ml, respectively) were low. Bmax remained low (305.6 +/- 22.1 fmol/mg) at 1300 h of proestrus, but PRL increased 5-fold. At 1700 h of proestrus, Bmax significantly increased to 453.3 +/- 27.2 fmol/mg, coincident with a preovulatory PRL surge (241.5 +/- 45.4 ng/ml). By 0900 h of estrus, Bmax decreased to 389.5 +/- 20.1 fmol/mg and did not return to basal levels until diestrous day 2. PRL also decreased by 0900 h of estrus to 106.6 +/- 13.0 ng/ml and progressively fell to basal level by 0900 h of diestrous day 1. Receptor affinity was unchanged throughout the cycle (range, 0.08-0.15 nM). These data indicate that the number of dopaminergic binding sides changes significantly during the estrous cycle, with the sharpest increase in the afternoon of proestrus. Since a complex relationship is apparent between alterations in dopaminergic receptor number and plasma PRL levels, we postulate that these receptors may be regulated by dopamine, ovarian steroids, and/or PRL itself. Such a change in the number of dopaminergic binding sites may e an important component in pituitary responsiveness to dopamine inhibition of PRL secretion throughout the estrous cycle.

Minireview: ghrelin and the regulation of energy balance--a hypothalamic perspective.

The recently discovered hormone, ghrelin, has been recognized as an important regulator of GH secretion and energy homeostasis. Orexigenic and adipogenic ghrelin is produced by the stomach, intestine, placenta, pituitary, and possibly in the hypothalamus. The concentration of circulating ghrelin, principally derived from the stomach, is influenced by acute and chronic changes in nutritional state. To date, most studies focused on the role of ghrelin in GH secretion or its function in complementing leptin action to prevent energy deficits. The potential significance of ghrelin in the etiology of obesity and cachexia as well as in the regulation of growth processes is the subject of ongoing discussions. A large quantity of information based on clinical trials and experimental studies with ghrelin and previously available synthetic ghrelin receptor agonists (GH secretagogues) must now be integrated with a rapidly increasing amount of data on the central regulation of metabolism and appetite. In this overview, we summarize recent findings and strategies on the integration of ghrelin into neuroendocrine networks that regulate energy homeostasis.

Leptin inhibits in vitro hypothalamic somatostatin secretion and somatostatin mRNA levels.

Leptin, the product of the ob gene, is a recently discovered hormone secreted by adipocytes that regulates food intake and energy expenditure. The site of action of leptin is likely to be the hypothalamus, since this area is important in the control of food intake and leptin receptor mRNA is particularly abundant in this area. In order to further unravel the mechanisms by which leptin acts, we have studied the effect of leptin on in vitro somatostatin synthesis and secretion. Leptin administration to fetal rat neurones in monolayer culture led to a time dependent decrease in basal somatostatin secretion and somatostatin mRNA levels, the maximal effect being observed with 6x10(-8) M leptin after 24 h incubation. Furthermore, leptin completely blunted 10(-7) M Neuropeptide Y-induced increase in somatostatin secretion and somatostatin mRNA levels as well as 10(-3) M (Bu)2-cAMP and 10(-6) M A23187-induced somatostatin secretion. Finally, leptin (3x10(-8) M M) also inhibited low glucose (1.1 mM) induced-somatostatin secretion in perifused adult hypothalami. This data indicates that leptin can influence the neuroendocrine system by regulating hypothalamic somatostatin gene expression.

Prolonged inhibition of growth hormone secretion by peripheral injection of bombesin is mediated by somatostatin in the rat.

Peripherally injected bombesin inhibits GH secretion in conscious, freely moving rats and in sodium pentobarbital-anesthetized rats. This inhibition of GH secretion is unusually prolonged, lasting up to 90 min after a single ip injection. The duration of inhibition of GH secretion by bombesin is greater than that observed for somatostatin (SRIF) in the same bioassay. The inhibition of GH release occurs concomitantly with stimulation of gastrin release and is independent of stimulatory effects of bombesin on plasma glucose. The structurally related mammalian gastrin-releasing peptide also inhibits GH secretion in the pentobarbital-anesthetized rat after peripheral injection. Peripherally administered bombesin blocks GH-releasing factor stimulation of GH secretion. Prior treatment of pentobarbital-anesthetized rats with SRIF-specific anti-serum blocks the inhibitory effect of bombesin on GH secretion. No effect of bombesin on GH secretion was observed in primary cultures of rat anterior pituitary cells. These data suggest that peripherally administered bombesin stimulates SRIF secretion, most probably of hypothalamic origin, which, in turn, inhibits pituitary secretion of GH. This sensitivity of the hypothalamus to a peripherally rather than centrally administered peptide has important mechanistic and therapeutic implications.

An extremely sensitive in vitro model for elucidating structure-activity relationships of growth hormone-releasing factor analogs.

An improved rat anterior pituitary primary cell culture technique for studying GH-releasing activity of human pancreatic GH-releasing factor (hpGRF) and its analogs is described. Male pituitaries, dispersed by a combination of trypsin digestion and mechanical agitation, were plated at a density of 200,000 cells per well and cultured for 4 days. The attached cells were then stimulated with synthetic hpGRF which was comprised of the first 29 residues of the larger, originally isolated forms and which was amidated at the C-terminal (hpGRF-29). Analogs of hpGRF-29 which were modified in positions 1, 2, 3, or 7, and other secretagogues were similarly tested. Medium was collected after 3 h, and secreted hormone was measured by RIA. The cells were extremely sensitive to hpGRF-29 stimulation, and this effect was specific. The minimal effective dose of hpGRF-29 was an unprecedented 0.4 X 10(-15)M. No stimulation of LH, FSH, or PRL by hpGRF-29 was observed. Bombesin and vasoactive intestinal peptide were ineffective in stimulating GH release. [D-Trp6]LHRH (a potent LHRH agonist), also did not release GH but did stimulate secretion of LH and FSH at doses ranging from 0.4 X 10(-10)M to 1.0 X 10(-9)M. Responses of the cells to hpGRF-29 analogs were characterized by distinct heterologous dose-response curves. [D-Ala2]hpGRF-29 was 50 times more active than its parent 29-amino-acid peptide. [D-Thr7]hpGRF-29, another analog that differed from hpGRF-29 by the insertion of a D-isomer for the naturally occurring L-residue, was about 10,000 times less effective in stimulating GH secretion than was hpGRF-29 itself. Potencies of these and other analogs with respect to GH release in vitro were similar to those estimated in vivo. Thus, this primary cell culture provides an extremely sensitive, selective, and reproducible system for studying hpGRF structure-activity relationships. Further, such tremendous sensitivity to hpGRF can provide a system to study changes in pituitary sensitivity to hpGRF during different physiological states.

Structural requirements for the activation of rat anterior pituitary adenylate cyclase by growth hormone-releasing factor (GRF): discovery of (N-Ac-Tyr1, D-Arg2)-GRF(1-29)-NH2 as a GRF antagonist on membranes.

The efficacy and potency of 14 GH-releasing factor (GRF) analogs, substituted in position 1 to 7, on adenylate cyclase activation in crude homogenates from rat anterior pituitary were related to those of human pancreatic GRF(1-29)-amide and vasoactive intestinal peptide. Among several D-amino acid substitutions, that in position 2 was the only one to yield a super-agonist [with a Kact (concentration required for half-maximal adenylate cyclase activation) 2 times lower than that of GRF(1-29)-NH2]. By contrast, D-isomer substitution in position 1 and 3 was without effect and D-isomer substitution in position 4, 6, or 7 decreased the affinity of the analog. The N-acetylated analog of GRF was as potent and active as the parent peptide, and the identity of the amino acid in position 2 of (N-Ac-Tyr1)-GRF(1-29)-NH2 proved to be determining for enzyme activation, with D-Phe2 and D-Trp2 derivatives acting as partial agonists and the (N-Ac-Tyr1,D-Arg2) analog being an efficient competitive antagonist of GRF(1-29)-NH2. With use of this antagonist, it was possible to demonstrate that GRF and vasoactive intestinal peptide receptors represent distinct entities in the rat anterior pituitary.

Leptin inhibition of the hypothalamic-pituitary-adrenal axis in response to stress.

Leptin is a newly identified protein hormone that is synthesized and secreted by adipose tissue. Absence of the mature hormone is responsible for the obese phenotype of ob/ob mice. The hypothalamic-pituitary-adrenal axis (HPAA) is activated in ob/ob mice, and chronic administration of leptin to ob/ob mice decreases plasma corticosterone levels, suggesting that the adipose hormone is capable of inhibiting the HPAA. The aim of this study was to determine whether leptin feeds back acutely to inhibit the HPAA of normal mice and rats. Male C57BL mice were injected ip with 100 microl saline and 2 or 4 microg/g BW mouse leptin in saline vehicle, and 4 h later they were subjected to 2 h of restraint stress by taping the hind limbs together or no stress. Hind leg restraint stimulated the HPAA as measured by significant (P < 0.05) elevation of both ACTH and corticosterone. Pretreatment with recombinant mouse leptin blocked the stress-mediated stimulation of both plasma hormones. To determine whether this inhibition was exerted at the hypothalamic level through inhibition of CRH, we studied leptin action on isolated rat hypothalami perifused with Krebs-Ringer buffer containing glucose (5.5 mM). CRH secretion was stimulated by decreasing the glucose concentration of the buffer to 1.1 mM. A surge of CRH was released over a 2-h period (basal integrated release was 14.4 +/- 1.6 pg/2 h, n = 5 and increased to 34.7 +/- 3.1 pg/2 h, n = 14). This response was blocked by mouse leptin in a dose-dependent manner (integrated stimulated CRH secretion was 30.6 +/- 2.5 pg/2 h, n = 5; 20.5 +/- 3.6 pg/2 h, n = 7; 15.3 +/- 4.3 pg/2 h, n = 3 for 1 nM, 3 nM and 30 nM, respectively). Leptin did not alter secretion of ACTH from rat primary cultured pituitary cells. These data demonstrate that leptin can inhibit hypothalamic CRH release, either directly or indirectly through another hypothalamic neuropeptide such as neuropeptide-Y. Dysfunctional leptin, insufficient leptin levels, or leptin resistance should each result in a partial open loop, thereby accounting for elevated glucocorticoid levels that accompany and contribute to many obese phenotypes. Leptin's ability to inhibit CRH release is the likely explanation for its ability to inhibit activation of the HPAA in response to stress.

Absence of cocaine- and amphetamine-regulated transcript results in obesity in mice fed a high caloric diet.

Cart (cocaine- and amphetamine-regulated transcript) was first identified to be a major brain mRNA up-regulated by cocaine and amphetamine. The CART protein has been established as a satiety factor closely associated with the action of leptin. To assess CART's role as an anorexigenic signal, we have generated CART-deficient mice by gene targeting. On a high fat diet, CART-deficient and female heterozygous mice, but not male heterozygous mice, showed statistically significant increases in weekly food consumption, body weight, and fat mass compared with their wild-type littermates. Furthermore, CART-deficient and female heterozygous mice were significantly heavier when fed a high fat diet than on a regular chow diet at 17 wk of age and at the 14th wk of the feeding studies. However, wild-type or male heterozygous mice showed no weight variations attributable to caloric contents of the diet at that age. Contrary to the obese phenotypes shown in MC4R-, proopiomelanocortin-, or leptin-deficient mice, our results showed that CART deficiency predisposed mice to become obese on a calorically dense diet. The results also show that CART may not be a major anorectic signal compared with proopiomelanocortin or leptin in the regulation of energy homeostasis.