Effect of exogenously administered glucagon versus spontaneous endogenous counter-regulation on glycaemic recovery from insulin-induced hypoglycaemia in patients with type 2 diabetes treated with a novel glucokinase activator, AZD1656, and metformin.

AIMS: To study the effect of exogenous i.m. glucagon on recovery from controlled insulin-induced hypoglycaemia in patients with type 2 diabetes treated with the novel glucokinase activator AZD1656, in combination with metformin. METHODS: This was a single-centre randomized, open, two-way crossover phase I, automated glucose clamp (Biostator(®); Life Science Instruments, Elkhart, MD, USA) study (NCT00817271) in eight patients (seven men and one woman, mean age 58.6 years, body mass index 28.1 kg/m(2)). All patients received a stable dose of metformin twice daily, ranging from 1000 to 2250 mg. A 2-day titration phase commenced with 40 mg AZD1656 twice daily, escalating to 80 mg twice daily if tolerated. This was followed by a single dose of 80 or 160 mg AZD1656, administered on days 5 and 8 when metabolic studies were performed. After an overnight fast on days 5 and 8, controlled hypoglycaemia was induced using an exogenous i.v. infusion of insulin. Plasma glucose was lowered in a stepwise fashion over 3 h to attain a target nadir of 2.7 mmol/l. This was sustained for 30 min, at the end of which the hypoglycaemic clamp was released. In random sequence, patients either received an i.m. injection of 1 mg glucagon or were allowed to recover from hypoglycaemia by endogenous counter-regulation. To avoid prolonged hypoglycaemia, a reverse glucose clamp was applied from 4 to 6 h post-dose. RESULTS: Three patients received 40 mg AZD1656 twice daily and five patients 80 mg twice daily. Mean plasma glucose at 20 min after release of the hypoglycaemic clamp was significantly lower (3.1 ± 0.3 mmol/l) for AZD1656 alone than for AZD1656 + glucagon (4.9 ± 0.8 mmol/l; p < 0.001 between the groups). Catecholamine and cortisol responses were similar on the AZD1656 + glucagon and AZD alone study days. Growth hormone response was 18% lower for AZD1656 alone (p = 0.01), consistent with the effect of a pharmacological dose of glucagon on growth hormone secretion. No safety or tolerability concerns were observed during treatment with AZ1656. CONCLUSIONS: Exogenous glucagon was effective as a rescue treatment for hypoglycaemia induced during treatment with AZD1656, given in combination with metformin in patients with type 2 diabetes.

Pharmacodynamic effects of the oral glucokinase activator AZD6370 after single doses in healthy volunteers assessed with euglycaemic clamp.

AIMS: This study evaluated the safety, tolerability, pharmacokinetics and pharmacodynamic effects of the glucokinase activator (GKA) AZD6370 in non-diabetic subjects, using the euglycaemic clamp to avoid the risk of hypoglycaemia. METHODS: Oral single ascending doses of AZD6370 10-650 mg or subcutaneous short-acting insulin 4 or 12 U were given to healthy fasting subjects. AZD6370 safety, tolerability and pharmacokinetics were assessed. Pharmacodynamic effects on serum (S)-insulin and glucose infusion rate (GIR) were investigated with euglycaemic clamp. AZD6370 10-20 mg was also assessed when taken with food without euglycaemic clamp. RESULTS: AZD6370 was well tolerated and no safety concerns were raised. AZD6370 was rapidly absorbed and eliminated, and plasma concentration was proportional to dose. Both S-insulin and GIR increased following AZD6370 administration. The observed increase in GIR correlated with increasing AZD6370 area under the plasma concentration vs. time curve, demonstrating a dose-concentration-dependent pharmacodynamic effect. AZD6370 at doses of 50 and 80 mg had similar effects to short-acting insulin 4 U on peripheral S-insulin levels but greater effects on GIR, suggesting an effect beyond the increase of peripheral S-insulin levels at lower doses. In the food interaction part of the study, performed without euglycaemic clamp, dose escalation was stopped at a low dose (20 mg) because of hypoglycaemia. CONCLUSION: The euglycaemic clamp was successfully used to avoid hypoglycaemia and to demonstrate pharmacodynamic effects, that is, markedly increased insulin secretion and glucose utilisation, following administration of AZD6370 in healthy fasting subjects. In addition to the effect on pancreatic insulin secretion, the data support an extra-pancreatic (hepatic) component of GKA action.

Safety, pharmacokinetics and pharmacodynamics of multiple-ascending doses of the novel glucokinase activator AZD1656 in patients with type 2 diabetes mellitus.

AIMS: To assess the safety, pharmacokinetics and pharmacodynamics of multiple-ascending doses of the novel glucokinase activator AZD1656 in patients with type 2 diabetes mellitus (T2DM). METHODS: This randomized, single-blind, placebo-controlled, monotherapy study was carried out in two parts. In part A, 32 patients received AZD1656 (7, 20, 40 or 80 mg) twice daily or placebo for 8 days in hospital. In part B, another 20 patients received, as outpatients, individually titrated AZD1656 15-45 mg twice daily or placebo for 28 days. Safety, pharmacokinetics and pharmacodynamic variables were evaluated. RESULTS: AZD1656 was generally well tolerated. Pharmacokinetics of AZD1656 were virtually dose- and time-independent. AZD1656 was rapidly absorbed and eliminated. An active metabolite was formed which had a longer half-life than AZD1656, but showed ∼15% of the area under the plasma concentration versus time curve from 0 to 24 h compared with that of AZD1656. Renal excretion of AZD1656 and the metabolite was low. In part A, fasting plasma glucose (FPG) was reduced by up to 21% and mean 24-h plasma glucose was reduced by up to 24% with AZD1656 versus placebo, depending on dose. No dose-related changes in serum insulin or C-peptide were observed with AZD1656 at the end of treatment. Results in part B confirmed the glucose-lowering effect of AZD1656 versus placebo. CONCLUSIONS: AZD1656 was well tolerated with predictable pharmacokinetics in patients with T2DM. Dose-dependent reductions in plasma glucose were observed.

Higher than expected estradiol levels in aromatase inhibitor-treated, postmenopausal breast cancer patients.

OBJECTIVE: Vaginal estradiol is considered contraindicated in aromatase inhibitor (AI)-treated patients because of the risk of elevated estrogen levels. This leaves limited treatment options for patients experiencing gynecological symptoms. However, in clinical practice, no precise estimation has been performed of circulating estrogens and aromatase index in postmenopausal breast cancer patients on long-lasting AI or tamoxifen treatment. METHODS: Steroid hormones were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) and extraction radioimmunoassay (RIA). Postmenopausal AI-treated patients (n =33) were compared with tamoxifen-treated patients (n =34) and controls without vaginal treatment (n =56), with vaginal estradiol (n =25), or with estriol (n =11) treatment. RESULTS: By use of LC-MS/MS, median (range) estradiol plasma concentrations were 16.7 (2.4-162.6), 31.0 (13.4-77.1), 27.2 (7.8-115.8) and 33.3 (20.3-340.1) pmol/l in AI-treated breast cancer patients, tamoxifen-treated breast cancer patients, postmenopausal controls and postmenopausal controls on vaginal estradiol, respectively. The AI-treated group and subgroups had significantly lower estradiol and estrone concentrations than all other groups (p <0.05). There was extensive interindividual variation in estradiol concentration within the AI-treated group, measured using both LC-MS/MS (2.3-182.0 pmol/l) and extraction RIA (2.4-162.6 pmol/l). The AI-treated group had lower aromatase index compared to all other groups (p <0.05-0.001). CONCLUSION: Circulating estrogen levels may have been underestimated in previous longitudinal studies of AI-treated breast cancer patients. Additional studies are required to further evaluate the role of circulating estrogens in breast cancer patients suffering from gynecological symptoms.

Redistribution of ovarian blood flow after injection of human chorionic gonadotropin and luteinizing hormone in the adult pseudopregnant rat.

It is well known that LH and human CG (hCG) induce an increase in total ovarian blood flow. The effect of LH/hCG on luteal blood flow, however, is unknown. This work studies the effect of hCG on both luteal and ovarian blood flows at different stages of pseudopregnancy in adult female rats. Pseudopregnancy was induced by mating with sterile male rats. The length of pseudopregnancy was 13 +/- 1 days and, during this time, blood flow was measured by the injection of radioactive microspheres during anesthesia. At autopsy, the corpora lutea were identified and extirpated under a stereomicroscope. These, and the remaining ovary, were then counted for radioactivity and the blood flow was calculated. Progesterone levels were determined in plasma and ovarian tissues. Furthermore, the responsiveness of adenylate cyclase was tested in ovarian tissues at day 6 of pseudopregnancy. An intraarterial injection of hCG (50 IU) or vehicle (saline) was given 20 min before the blood flow determinations in anesthetized rats. The luteal blood flow was not changed by hCG on days 2, 6, and 11 of pseudopregnancy, whereas in the remaining ovary the blood flow increased more than 2-fold, thereby resulting in redistribution of the blood flow. Ten micrograms of NIH-LH-B9, tested at day 6 of pseudopregnancy, mimicked the effect of hCG. At day 6 of pseudopregnancy, hCG (50 IU) was given ip to conscious rats 200 min and 24 h before blood flow determinations. At 200 min after hCG there was a more pronounced redistribution of ovarian blood flow with a 45% reduction in luteal blood flow and a 4-fold increase in flow through the remaining ovary. LH as well as hCG doubled the progesterone content of the remaining ovary. In the corpora lutea an increased progesterone content was seen after 200 min of hCG exposure. At 24 h after hCG injection, all parameters had returned to control levels except that adenylate cyclase was nonresponsive. The increase in the total ovarian blood flow coincides with the increased steroidogenesis and these effects are likely due to release of metabolites and/or vasoactive substances. Despite this increase, the blood flow of the corpus luteum was not increased rendering vascular mechanisms unlikely as a part of the acute LH/hCG effects on corpus luteum of pseudopregnancy.

Beta-adrenergic receptor concentration in corpora lutea of different ages obtained from pregnant mare serum gonadotropin-treated rats.

We have measured the beta-adrenergic receptor content in 1- to 10-day-old corpora lutea. Corpora lutea of defined ages were obtained by sc injection of 8 IU PMSG to 26-day-old rats, leading to ovulation and formation of corpora lutea in the early morning of day 29. Membrane fractions of isolated corpora lutea were incubated with various concentrations of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol [125I]iodo-HYP, 12.5-2500 pM) for 60 min at 22 C. Alprenolol (10(-5) M) was used to determine nonspecific binding. Bound and free [125I]iodo-HYP was separated by filtration and washing on Whatman GF/C filters under vacuum. There was a 3-fold increase in beta-adrenergic receptor concentration during the first 2-3 days of corpus luteum formation, followed by a decline in the beta-adrenergic receptor content with luteal age. The rat luteal beta-adrenergic receptor seems to be of the beta 2-subtype as determined from adenylate cyclase stimulation as well as from displacement of [125I]iodo-HYP binding by various beta-adrenergic agonists.

Disproportional bone growth and reduced weight gain in gonadectomized male bovine growth hormone transgenic and normal mice.

Sex steroids have been shown to influence longitudinal bone growth during sexual maturation, partially by increased GH secretion. Mice transgenic for metallothionein promoter bovine GH were developed by pronuclear injection as a model with sex steroid-independent GH secretion. Prepubertal normal and transgenic, male and female mice were either gonadectomized or sham operated. The growth was divided into two segments: peripubertal growth from 30-60 days of age and adult growth from 60-90 days of age. Orchidectomy resulted in a decreased growth rate of the lumbar spine and a decreased weight gain during the peripubertal growth, whereas tibia growth was unaffected. The alteration in proportions between the lumbar spine and the tibia was apparent for both normal and bovine GH transgenic mice, suggesting that the effect was not mediated via decreased GH secretion. Orchidectomy resulted in increased adult tibial growth, whereas weight gain and lumbar growth were unaffected. In female mice, gonadectomy did not influence these parameters during either time period studied. In summary, we present data indicating that the male gonads in a GH secretion-independent manner stimulate pubertal growth of the spine and inhibit the tibial growth of adult animals.

Elevated levels of growth hormone increase bone mineral content in normal young mice, but not in ovariectomized mice.

Both estrogens and GH are necessary for normal bone remodeling. This study investigates the effect of elevated GH levels on the amount and density of bone in young female mice and its dependence on intact ovarian function. Metallothionein promoter-GH-transgenic mice were either sham operated or ovariectomized at 25-29 days of age, and the bone measurements were made at about 90 days of age. A 6-mm high cylinder containing only cortical bone was cut from the right tibia, and lumbar vertebrae 6 was measured as a bone with predominantly cancellous bone. The amounts of tibial and vertebral bone, measured by dry weight, mineral weight, organic weight, bone mineral content (measured by dual energy x-ray analysis), and volume, were increased in GH-transgenic animals compared to those in normal littermates. This stimulatory effect of elevated GH levels was not seen in ovariectomized mice. The real density of the tibial bone were slightly decreased in GH-transgenic animals compared to normal littermates. In conclusion, elevated levels of GH increase the amounts of vertebral (predominantly cancellous) bone and tibial (cortical) bone in young mice. Intact ovaries are a prerequisite for the stimulatory effect of elevated levels of GH. The fact that ovariectomy decreases the stimulatory effect of elevated GH levels suggests that the effect of elevated GH levels in bone is dependent upon the presence of basic sex steroid secretion.

Diurnal rhythm of testosterone secretion before and throughout puberty in healthy girls: correlation with 17beta-estradiol and dehydroepiandrosterone sulfate.

The regulation of androgen synthesis during puberty in females is complicated, with changes in steroidogenic and peripheral interconversion capacity. In the present study we have investigated the diurnal rhythm of testosterone secretion in 56 healthy girls before and during puberty, up to 2 yr postmenarche. The girls' ages ranged between 4.6-16.5 yr, and their height SD scores ranged between -3.6 and +3.7. One to 5 serum profiles (seven samples per 24 h) were taken from each girl for steroid measurements, and a total of 84 serum profiles were obtained. Serum testosterone concentrations were determined using a RIA with a detection limit of 30 pmol/L. The results demonstrate that there is a diurnal rhythm of testosterone secretion during both prepuberty and puberty in girls. The pattern has its nadir in the late evening or just after midnight, with the highest levels in the morning (0600-1000 h). Serum testosterone concentrations in prepubertal girls were significantly lower than those in pubertal girls and were significantly lower in early puberty than in girls in mid- or late puberty. No differences were found in levels between girls in midpuberty or late puberty. Before puberty, serum testosterone concentrations correlated with serum dehydroepiandrosterone sulfate, consistent with the adrenals being the major source of testosterone. After the onset of puberty, a correlation between testosterone and 17beta-estradiol was seen, consistent with the ovaries being the major source of testosterone during puberty. Furthermore, the present study showed that there is a relative hyperandrogenicity in early puberty, with high levels of androgens relative to estrogens.

Diurnal rhythm of 17 beta-estradiol secretion throughout pubertal development in healthy girls: evaluation by a sensitive radioimmunoassay.

Puberty is initiated by a nocturnal rise in gonadotropin secretion, which, in boys, results in an increased nocturnal secretion of testosterone. To characterize any similar diurnal rhythm of 17 beta-estradiol in healthy girls, we determined the secretion of 17 beta-estradiol before and during puberty. The study group consisted of 45 healthy girls whose height SD scores ranged from -3.7 to +4.9 compared with Swedish growth reference values. One to 6 profiles of 17 beta-estradiol (7 samples/24 h) were obtained from each girl during puberty and from 21 of the girls before clinical signs of puberty (a total of 76 serum profiles). Serum 17 beta-estradiol concentrations were determined using a modified RIA. The detection limit for the RIA was 1.8 fmol/tube, which corresponded to a serum level of 7.8 pmol/L in extracted serum. It was considered that levels above 50 pmol/L could be determined accurately without extraction. The serum levels of 17 beta-estradiol in prepubertal girls were, in most cases, below the detection limit, except in the morning, when in 17 of the 21 prepubertal girls, serum 17 beta-estradiol levels were just above the detection limit. All girls in early puberty (Tanner breast stage 2) had measurable serum levels of 17 beta-estradiol in the morning, whereas 10 of these 15 girls had levels below the detection limit around midnight. Later in puberty (Tanner breast stages 3 and 4), but before menarche, the diurnal rhythm was more obvious, with high levels of 17 beta-estradiol during the latter part of the night and in the morning. This diurnal rhythm was lost by 1 yr after menarche. There was a high degree of correlation between serum concentrations of 17 beta-estradiol and bone age, whereas there was much less, if any, correlation between 17 beta-estradiol and levels of sex hormone-binding globulin or dehydroepiandrosterone sulfate during puberty. We conclude that the nocturnal rise in gonadotropin secretion during puberty in girls is accompanied by an increased secretion of 17 beta-estradiol in the morning. This diurnal rhythm is lost 1 yr after menarche. Determination of 17 beta-estradiol levels in the morning could be useful in determining the initiation of puberty, whereas determinations in the late evening could provide information on the tempo of puberty.

Nocturnal application of transdermal estradiol patches produces levels of estradiol that mimic those seen at the onset of spontaneous puberty in girls.

The objective of pubertal induction in children with hypogonadism is to mimic spontaneous puberty in terms of physical and psychological development. In a clinical observation study, we induced puberty in 15 girls with hyper- or hypogonadotropic hypogonadism using low doses of transdermal estradiol patches attached only during the night and compared the estradiol concentrations obtained with those in healthy girls. Pubertal induction was started between the ages of 12.3 and 18.1 yr. A transdermal matrix patch of 17beta-estradiol (25 microg/24 h; Evorel, Janssen Pharmaceuticals-Cilag) was cut into pieces corresponding to 3.1, 4.2, or 6.2 microg/24 h initially and attached to the buttock. After 4-14 months, the dose was increased gradually. Serum 17beta-estradiol concentrations were measured every 2 h by RIA (detection limit, 6.0 pmol/L; 1.6 pg/mL). The results show that it is possible to mimic the spontaneous levels as well as the diurnal pattern of serum 17beta-estradiol in early puberty, by cutting a transdermal 17beta-estradiol matrix patch and attaching a part of it, corresponding to 0.08-0.12 microg estradiol/kg BW, to the buttock nocturnally. In most of the girls, breast development occurred within 3-6 months of the start of treatment.

Twenty-four-hour profiles of luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol levels: a semilongitudinal study throughout puberty in healthy boys.

To follow and correlate gonadotropin and sex steroid changes throughout puberty, 24-h profiles of LH, FSH, testosterone, and estradiol were taken on several occasions for between 2-9.5 yr in 12 healthy boys, aged 8.7-18.2 yr. Serum concentrations of LH and FSH were measured every 20 min, whereas testosterone and estradiol were measured every 2-4 h during the 24-h period. The prepubertal boys (Tanner stage 1) were subdivided into two groups: Pre 1, with a testicular volume of 1-2 mL, and Pre 2, with a testicular volume of 3 mL. Pubertal stages were classified, according to testicular volume, as early puberty (pubertal stage 2; 4-9 mL), midpuberty (pubertal stages 3-4; 10-15 mL), and late puberty (pubertal stage 5; > or = 16 mL). Mean levels of LH and FSH increased with pubertal development, although the increase in LH was greater than that in FSH. These increases were due to elevated basal levels of LH and FSH as well as to increases in the number of peaks and the peak amplitudes of LH. No diurnal rhythm was found in boys at stage Pre 1. Thereafter, a clear diurnal rhythm appeared for LH, and later in puberty, an ultradian rhythm was superimposed, as shown by time-sequence analyses. A diurnal rhythm also existed for FSH, but was much less marked than that for LH despite a clear covariation between LH and FSH, as shown from cross-correlation studies. Testosterone also showed diurnal variations from the late prepubertal stage, followed by increasing levels during both day and night in puberty. We conclude that during puberty, gonadotropin levels rise differently for LH and FSH, which may be due to the development of differences in feedback mechanisms. Despite covariation between LH and FSH, only LH showed a clear diurnal variation. In parallel, nocturnal variations in testosterone and estradiol were found. Changes in mean levels of LH, testosterone, and estradiol as well as their mean daytime and nighttime levels follow each other from the prepubertal stages to late puberty.

Mechanism of action of prostaglandin F2 alpha-induced luteolysis: evidence for a rapid effect on the guanine nucleotide binding regulatory component of adenylate cyclase in rat luteal tissue.

The initial events in prostaglandin F2 alpha-(PGF2 alpha)-induced luteolysis were studied in pregnant mare serum gonadotropin/human chorionic gonadotropin-(PMSG/hCG)-treated rats with luteinized ovaries. Injection with a potent PGF2 alpha analog (cloprostenol, 5 micrograms/ml) induced functional luteolysis, as assessed by plasma levels of progesterone and 20 alpha-dihydroprogesterone. At 0.5 and 3 h after cloprostenol administration the luteolytic effect was also evident as a reduced response of luteal adenylate cyclase to all stimulatory agents tested, LH, isoproterenol, fluoride, guanylylimidodiphosphate and forskolin. 24 h after cloprostenol the response to all agents, except to LH, had returned to normal. This general and transient block of the luteal adenylate cyclase system indicates that a common factor, possibly the stimulatory guanine nucleotide binding protein (Ns), is involved in the mechanism of action of PGF2 alpha. To test this hypothesis, we measured the functional coupling of the Ns protein to the beta-adrenergic receptor in luteal membranes. Binding competition curves showed a marked shift to the right in membranes prepared from rats injected with cloprostenol 0.5 and 3 h before membrane preparation, while at 24 h after cloprostenol the shift had disappeared. The total number of beta-adrenergic receptors was, however, not affected by the cloprostenol treatment. Computer analysis of the data indicates that, at 0.5 and 3 h after cloprostenol treatment, there was a reduced number of high affinity binding sites, 38 and 41%, respectively, compared to 53% for control membranes. The cellular mechanism for this action of PGF2 alpha on the Ns protein remains to be elucidated.

Catecholamine content and adenylate cyclase activity in corpora lutea of different ages of the PMSG-treated immature rat.

The catecholamine content and adenylate cyclase response were studied in a well-characterized corpus luteum model, where ovulation was induced by treatment of prepubertal Sprague-Dawley rats with pregnant mare's serum gonadotropin. The luteal content of noradrenaline, determined with HPLC, was constant during the first 7 days of pseudopregnancy, followed by a 3-fold increase in older corpora lutea. No detectable amounts of dopamine were found, while trace amounts of adrenaline were found in a few cases. The increase in noradrenaline content was not associated with a changed sensitivity of luteal adenylate cyclase to catecholamines. The response to adrenaline was maximal in 3-day-old corpora lutea, whereafter a decrease was seen. The significance of the increased endogenous levels of noradrenaline at the end of pseudopregnancy is at present unknown. However, the fact that the increase in noradrenaline occurs a few days before spontaneous luteolysis is of special interest, since it has been suggested that an adrenergic innervation is a prerequisite for the antigonadotropic effect of prostaglandin F2 alpha in the human corpus luteum.

Leptin levels show diurnal variation throughout puberty in healthy children, and follow a gender-specific pattern.

OBJECTIVE: To investigate the levels and diurnal rhythm of serum leptin in healthy children, and to investigate the association between leptin levels and sex steroids. METHODS: Four girls and four boys, all healthy volunteers, were followed longitudinally throughout puberty. Their chronological ages ranged from 8.7 to 19.5 years, and body composition, expressed as weight-for-height standard deviation scores (SDS), ranged between -1.7 and +2.4. Serum leptin, oestradiol and testosterone concentrations were measured by radioimmunoassay at 1000, 1400, 1800, 2200, 0200 and 0600 h. RESULTS: In all girls and boys, both prepubertally and during pubertal development, serum leptin levels increased during the night, with no difference in relative peak amplitude. In boys, the leptin concentrations increased until the initiation of puberty and then declined, whereas in girls, the concentrations increased throughout puberty. The inter-individual variation in mean leptin levels among girls decreased to 11% at the time of menarche. A positive correlation was found for both oestradiol and testosterone versus leptin in girls throughout puberty (r=0.64 and r=0.71 respectively, P<0.001). A negative correlation was found between leptin and testosterone in boys in mid- and late puberty (r=-0.66, P<0.01). No correlation was found between oestradiol and leptin in boys or between testosterone and leptin in pre- and early pubertal boys. CONCLUSION: Serum leptin concentrations show diurnal variation throughout pubertal development in both girls and boys. The changes in leptin levels during puberty follow a gender-specific pattern, probably due to an influence of sex steroids on leptin production.

Synthesis of prostaglandin F2 alpha, E2 and prostacyclin in isolated corpora lutea of adult pseudopregnant rats throughout the luteal life-span.

The ability of de novo biosynthesis of prostaglandins (PGs) in individual whole corpora lutea (CL) obtained from sterile-mated adult pseudopregnant rats on different days of the luteal phase and the post-luteolytic period was evaluated. Production of PGs, progesterone and 20 alpha-dihydroprogesterone were determined after in vitro incubation of CL extirpated from Day 2 to Day 19 after mating. A time-relationship with increased accumulation of PGs in the medium was demonstrated from 18 s to 5 h, with large increments during the first 30 min. Basal accumulation of PGs in the incubation medium was highest for 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) greater than PGE2 greater than PGF2 alpha greater than thromboxane B2 (TXB2) and basal accumulation of PGF2 alpha and PGE2 measured in the medium was maximal on Day 10-11 of pseudopregnancy, concomitantly with a decline in secretion of progesterone. Addition of arachidonic acid (AA) dose-dependently increased synthesis of PGs, with absolute amounts of PGE2 greater than 6-keto-PGF1 alpha greater than PGF2 alpha greater than TXB2 and addition of 14 microM indomethacin markedly inhibited accumulation of all PGs measured. Luteinizing hormone (LH, 10 micrograms/ml) stimulated progesterone secretion on all days during pseudopregnancy, but not on the post-luteolytic Day 19. LH increased PGF2 alpha, PGE2 and 6-keto-PGF1 alpha secretion on Day 13 of pseudopregnancy by 76%, 91% and 28%, respectively, but not on the other days tested. Furthermore, stimulation of PG-synthesis by addition of AA abrogated the LH-induced progesterone accumulation markedly, but only on Day 13 of pseudopregnancy. Epinephrine (5 micrograms/ml) increased production of progesterone and also PGs, but only on Day 2 of pseudopregnancy, whereas oxytocin (100 mIU/ml) was found to be without effect on progesterone as well as PG secretion on all days tested. The results of the present study demonstrates the independent ability of the rat CL to synthesize PGG/PGH2-derived prostaglandins, including the putative luteolysin PGF2 alpha. Secondly, we demonstrate that LH and AA-induced increases in PGF2 alpha and PGE2 production during the luteolytic period, may be an autocrine or paracrine mechanism involved in luteolysis.

Multiple pituitary hormone deficiency: management of puberty for optimal auxological results.

The overview in this paper focuses on ways of achieving optimal auxological results in puberty, principally in idiopathic and congenital multiple pituitary hormone deficiency (MPHD), suggested by the co-authors. We agreed that diagnosing gonadotrophin insufficiency/deficiency is difficult in young children and should be repeated in late prepuberty, but a firm diagnosis of MPHD helps avoid endocrine re-testing at the end of growth. The hypothalamic-pituitary axis must be reassessed periodically in evolving endocrinopathies, though current practice varies widely. Optimum age to induce puberty is 11-12 years in girls and 13-14 boys, and sex steroids are the preferred agents. Short-course testosterone to increase micropenis size is advantageous, but inducing early testicular maturation is not known to improve later fertility. There is also little evidence for increasing the dose of GH during puberty, though therapy should continue to final height, and possibly until peak bone mass is achieved. Delaying puberty is an option in septo-optic dysplasia, and minimising the dose of hydrocortisone is crucial in treating ACTH/cortisol insufficiency. Many unresolved questions remain in this difficult area.

Management of puberty in constitutional delay of growth and puberty.

Constitutional delay of growth and puberty (CDGP) is the most common presenting form of short stature, but no single test can infallibly discriminate CDGP and isolated hypogonadotrophic hypogonadism. Management of puberty in CDGP aims to optimise not only growth maintaining body proportions and improving peak bone mass without impairing growth potential--but also well-being; for example, the distress boys often suffer because of their lack of growth and pubertal progression can affect their school performance and social relationships. Typical sex steroid treatments to induce puberty in boys with CDGP include testosterone (T) enanthate, T undecanoate, mixed T esters, T transdermal patches, and oxandrolone p.o. Compared with other regimens, short-course low-dose depot T i.m. is an effective, practical, safe, well tolerated, and inexpensive regimen. Some unresolved problems in management include optimal timing and dose of sex steroid treatment, the role of GH in CDGP, and the management of CDGP in girls.

Vascular resistance is decreased in the luteal rat ovary by a 20 minute continuous infusion of noradrenaline.

The effect of a 20 min continuous infusion of noradrenaline (2 nanomoles/min) on the blood flow and vascular resistance of 2-, 6- and 11-day-old corpora lutea from adult pseudopregnant rats was studied. Pseudopregnancy was induced by mating with vasectomized male rats. The blood flow of the corpus luteum and the remaining ovary was measured with the microsphere technique. The basal blood flow varied between the luteal ages studied and was highest at day 6 of pseudopregnancy. Noradrenaline induced a two-fold increase in the blood flow of the corpus luteum at the luteal ages studied. The vascular resistance (blood pressure/blood flow) decreased for all luteal ages, while the vascular resistance for kidney, spleen and diaphragm was unchanged. Antidiuretic hormone was found to markedly decrease the luteal blood flow and the vascular resistance remained increased. The effect of noradrenaline infusion on the luteal blood flow thus in contrast to other vasoactive substances is biphasic, with an initial vasoconstriction followed by vasodilatation.

Effects of hysterectomy and uterine decidualization on in vivo levels of prostaglandins in the corpus luteum of adult pseudopregnant rats.

A possible role of the uterus in regulating content of luteal prostaglandins (PGs) was investigated. Pseudopregnancy was induced in adult virgin female rats by mating them with vasectomized male rats. On Day 5 of pseudopregnancy, decidualization of the uterus was induced or hysterectomy was performed. As controls, intact pseudopregnant animals with a luteal phase of 13 +/- 1 days were used. Measurements of in vivo tissue levels of PGF2 alpha, PGE2, and 6-keto-PGF1 alpha were performed by RIA after homogenization and extraction procedures in CL of pseudopregnancy and remainder of ovaries on Days 5, 13, and 19. Serum levels of progesterone and 20 alpha-dihydroprogesterone were determined by RIA. In hysterectomized animals, PGF2 alpha levels increased 2.5-fold in corpora lutea on Day 13 compared with levels on Day 5 of pseudopregnancy, but were still lower than in control rats undergoing functional luteolysis on Day 13. Decidual-tissue-bearing rats exhibited low levels of PGF2 alpha on Day 13 of pseudopregnancy. On Day 19, when luteolysis had occurred in decidual-tissue-bearing and hysterectomized rats, as judged by plasma levels of progestins, luteal content of PGF2 alpha was elevated to a similar level as that in control animals undergoing functional luteolysis on Day 13. When data pooled from control, decidual-tissue-bearing and hysterectomized rats were analyzed, a highly significant inverse correlation (r = -0.72, n = 46, p less than 0.001) between luteal PGF2 alpha content and ratio of plasma progestins was found.(ABSTRACT TRUNCATED AT 250 WORDS)