The P4 study: Subsequent pregnancy maternal physiology after hypertensive and normotensive pregnancies.

OBJECTIVES: Hypertensive disorders of pregnancy are associated with subsequent increased risk of cardiometabolic disease. Adverse cardiometabolic measures are noted soon after hypertensive versus normotensive pregnancy (NP); to what degree these persist into a subsequent pregnancy (SP) is unknown. This study aimed to assess women's physiology early in SP after hypertensive pregnancy (HP: preeclampsia or gestational hypertension) or NP and compare SP to 6 months postpartum findings from the index pregnancy. STUDY DESIGN: Prospective sub-study of the P4 (Postpartum, Physiology, Psychology and Paediatric) observational cohort. Measurements six months after NP versus HP, and the SP at 11-13 weeks gestation. MAIN OUTCOME MEASURES: Blood pressure (BP), blood and urine tests (urine ACR, HOMA-IR, LDL cholesterol), body composition, and contribution of maternal characteristics and inter-pregnancy factors to BP and body fat (FM%) in SP. RESULTS: 49 women (34 NP, 15 HP). In the SP, post-HP women had higher BP (112/70 mmHg HP vs 102/64 mmHg NP; p < .001), with no significant drop from six months postpartum to early SP. On regression analysis, systolic and diastolic BP at 6-months were the major predictors for SP systolic (p < 0.001) and diastolic (p = 0.009) BP respectively in the SP. Longer interpregnancy interval and increased FM% 6-months postpartum were associated with higher SP FM% (p < 0.001). CONCLUSIONS: BP and body fat six months postpartum were similar early in the SP for HP group, and postpartum BP and FM% were major predictors of their corresponding SP measurements. Postpartum/inter-pregnancy intervention programs to improve these cardiometabolic risk markers might help improve women's long-term health and require investigation.

Maternal macro- and micronutrient intake six months after hypertensive versus normotensive pregnancy: is poor diet quality contributing to future cardiometabolic disease risk?

BACKGROUND: Hypertensive pregnancy is associated with increased long-term cardiometabolic disease risk. Assessing dietary intake patterns after hypertensive (HP) versus normotensive pregnancy (NP) may provide insights into the mechanism of this risk. METHODS: This study was a prospective sub-study of the P4 (Postpartum, Physiology, Psychology and Paediatrics) cohort. Women were studied six months after NP versus HP (preeclampsia or gestational hypertension). Dietary energy, macronutrient and micronutrient intake were measured using a three-day food diary (FoodWorks™) and assessed against Australian and New Zealand Nutrient Reference Values to determine nutritional adequacy. Comparisons between breastfeeding and non-breastfeeding women were assessed, and linear regression modelling (using hypertensive status, breastfeeding status, and demographic/pregnancy variables) performed to assess predictors of energy intake. RESULTS: Seventy-four women (60 NP, 14 HP) were included. HP women had higher mean body mass index (p = 0.02) and lower breastfeeding rates (29% HP versus 83% NP, p < 0.001) compared to NP women. Twenty-four-hour energy intake and total fat intake were 17% and 20% lower after HP respectively. Nutrient deficiencies were prevalent across all participants, however more HP women had inadequate magnesium, calcium and phosphorus intake. Breastfeeding women had significantly increased energy (17%), carbohydrate (15%) and total fat intake (21%), and increased vitamin A, vitamin E, riboflavin, magnesium and iron intake compared to non-breastfeeding women. HP and breastfeeding status were independent predictors of energy intake. CONCLUSIONS: HP women had lower micronutrient intake and greater prevalence of nutritional inadequacy compared to NP women, reflecting poorer diet quality and potentially contributing to future increased cardiometabolic disease risk.

The relationship between adiponectin, progesterone, and temperature across the menstrual cycle.

BACKGROUND: The mechanisms regulating adiponectin, a highly abundant adipokine produced by adipocytes, have not been fully elucidated. Adiponectin levels are significantly higher in women when compared to men, suggesting sex-hormone involvement in its regulation. Previously, we have observed an inverse association between adiponectin and basal body temperature in pregnant women. These findings suggest that states where progesterone and temperature fluctuate, such as the menstrual cycle, could be associated with fluctuating adiponectin levels. AIM: The aim of this study was to examine the relationship between adiponectin, progesterone, and temperature across the menstrual cycle. SUBJECTS AND METHODS: A prospective study was performed. Fifteen non-obese pre-menopausal female subjects, all with regular cycles, and on no medication recorded a daily temperature and underwent blood sampling, indirect calorimetry, and bio-impendence studies in both the follicular and luteal phases of the menstrual cycle. RESULTS: Serum adiponectin levels did not vary significantly across the menstrual cycle or between those who did and did not ovulate. No correlation was found between adiponectin levels and sex steroids, insulin and glucose levels or basal energy expenditure and body composition. CONCLUSIONS: Our results indicate that adiponectin is not related to sex steroids or body composition in healthy premenstrual women.

Predictors of inotropic and chronotropic effects of NG-monomethyl-L-arginine.

BACKGROUND: The haemodynamic effects of intravenous infusion of the non-selective nitric oxide synthase (NOS) L-omega monomethyl arginine (L-NMMA) have previously been characterized in humans. Its effect of reducing cardiac index (CI) is an important reason for the increase in mortality in patients with septic shock receiving L-NMMA in a pivotal outcome trial for this indication. The mechanism for the reduction in CI however, is uncertain. METHODS: In this study, we investigated the haemodynamic and arterial stiffness response to a bolus intravenous infusion of L-NMMA (3 mg kg(-1) over 5 min) in 26 healthy human volunteers to clarify the likely cause of L-NMMA induced negative inotropic and chronotropic effects. Digital photoplethysmography (MicroMedical Pulse Trace) was used to derive two measures of arterial stiffness: stiffness index, a measure of large arterial stiffness, and reflection index (RI), a measure of small- to medium-sized arterial stiffness. Haemodynamic measurements of systolic blood pressure, diastolic blood pressure, heart rate, systemic vascular resistance index (SVRI), stroke index and CI were made using a bioimpedance monitor (BioZ Cardiodynamics). RESULTS: We found that changes in CI during L-NMMA are closely related to changes in RI and SVRI. CONCLUSION: The negative inotropic effect of L-NMMA may be a result of an increase in coronary vascular resistance and a resultant decrease in myocardial perfusion. The reduction in CI may also result from a direct reduction of the normal positive inotropic effect of NO by L-NMMA which is closely correlated with its effects on SVRI.

Does oestrogen allow women to store fat more efficiently? A biological advantage for fertility and gestation.

In normal healthy-weight humans, women have a higher percentage body fat than men, a difference that commences at puberty and continues throughout adult life, suggesting that the mechanism is related to sex steroids. The first half of pregnancy is also a stage of body fat gain in women. From an energy balance point, there is no explanation why women should be fatter than men, as the latter consume more calories proportionately. Moreover, women store fat in early pregnancy when caloric intake does not significantly change. The aim of this review is to focus on evidence supporting one mechanism that may account for these findings. That is, oestrogen reduces postprandial fatty acid oxidation leading to an increase in body fat which may account for the greater fat mass observed in women compared with men and the fat gain in early pregnancy. Therefore, female puberty and early pregnancy could be seen as states of efficient fat storage of energy in preparation for fertility, foetal development and lactation providing an obvious biological advantage. Further research into this mechanism of fat storage may provide further insights into the regulation of body fat.

Evidence for functional expression of vascular angiotensin II type 2 receptors in patients with insulin resistance.

AIM: Angiotensin II type 2 (AT2) receptors are believed to become over-expressed in response to cardiovascular damage and to mediate beneficial effects (e.g. vasodilation). It is unknown whether AT2 receptors are functionally expressed in patients with insulin resistance (INSR). In this study, we investigated the role of the highly selective AT2 receptor antagonist, PD123319, on arterial stiffness and haemodynamic parameters in patients with INSR, compared with an age- and gender-matched control (N) group to determine whether there is functional expression of vascular AT2 receptors in patients with INSR. METHODS: We studied 10 subjects with INSR [mean age 28 +/- 5 years, body mass index (BMI) 30.4 +/- 5.4 kg/m(2), mean cholesterol level 4.7 +/- 0.7 mmol/l, mean homeostasis model assessment 2.78 +/- 0.84] and 10 age- and gender-matched normal subjects (mean age 27 +/- 7 years, BMI 23.6 +/- 2.5 kg/m(2), mean cholesterol level 3.9 +/- 0.6 mmol/l). All were normotensive, non-smokers and on no medications. Subjects received a 3-min infusion of PD123319 (10 microg/min). At the end of the infusion, arterial stiffness indices [stiffness index (SI) and reflective index (RI)] and haemodynamic parameters [cardiac index, systemic vascular resistance index (SVRI) and stroke index (ZI)] were measured. RESULTS: RI (mean % change: INSR 13.8 +/- 15.5%, N -0.2 +/- 4.6, p = 0.04) and SVRI (mean % change: INSR 13.5 +/- 9.7%, N -1.5 +/- 5.7, p = 0.005) increased significantly in response to PD123319 infusion in patients with INSR compared with controls. There were no significant changes in SI, systolic blood pressure, diastolic blood pressure and ZI. CONCLUSION: The results suggest the functional expression of AT2 receptors in small vessels that determine the inflection of the digital volume pulse wave in patients with INSR, possibly as an indicator of early vascular damage.

Insulin resistance and protein catabolism in non-diabetic hemodialysis patients.

Chronic kidney disease (CKD) is associated with complex metabolic changes including insulin resistance. Siew et al. have highlighted an important relationship between insulin resistance and skeletal muscle protein turnover. If insulin resistance is implicated in sarcopenia of CKD, further research will be required to determine whether interventions that improve insulin sensitivity improve clinical outcomes and cardiovascular risk in CKD.

Toxicity of cholesterol oxidation products to Caco-2 and HepG2 cells: modulatory effects of alpha- and gamma-tocopherol.

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g ml(-1)) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (ic(50) value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.

Metabolic effects of oestrogens: impact of the route of administration.

To investigate whether oestrogen modulates GH secretion and action in adult life, we studied the impact of oestrogen replacement on circulating GH and IGF-I levels in post-menopausal women. Since the liver is the major source of circulating IGF-I and the oral route of oestrogen delivery causes non-physiologic effects on hepatic proteins, we compared the effects of oral and transdermal route of delivery. Oral ethinyl oestradiol administration resulted in a significant fall in mean IGF-I levels and a 3-fold increase in mean 24h GH. Transdermal administration of 17beta oestradiol resulted in a slight increase in serum IGF-I but no change in mean 24h GH levels. To determine whether differences in oestrogen type rather than in the route of delivery caused the different effects on the GH/IGF-I axis, we compared the effects of three oral oestrogen formulations. Ethinyl oestradiol, conjugated equine oestrogen and oestradiol valerate each induced a fall in IGF-I and a rise of mean 24h GH levels in post-menopausal women. To determine the metabolic significance of oestrogen-induced changes on GH/ IGF-I, we compared the effects of 24 weeks each of oral and transdermal oestrogen on energy metabolism and body composition in 18 post menopausal women in an open-label randomised cross-over study. When compared to the transdermal route, oral oestrogen reduced lipid oxidation, increased fat mass and reduced lean body mass. Oestrogen causes distinct, route dependent effects on the somatotrophic axis. The dissociation of the GH/IGF-I axis by the oral route is likely to arise from impaired hepatic IGF-I production which causes increased GH secretion through reduced feedback inhibition. The route of oestrogen therapy confers divergent effects on substrate oxidation and body composition. The suppression of lipid oxidation during oral oestrogen therapy may increase fat mass while the fall in IGF-I may lead to a loss of lean body mass. The route dependent changes in body composition observed during oestrogen replacement therapy may have important implications for post-menopausal health and oestrogen use in general.

Efficient fat storage in premenopausal women and in early pregnancy: a role for estrogen.

There is a sexual dimorphism in body fat in humans. Adipose tissue increases with puberty and early pregnancy in women, suggesting gonadal steroids can influence body fat. Previously, we have observed that oral estrogen, compared with transdermal estrogen, reduced postprandial lipid oxidation and increased body fat, possibly due to suppressed hepatic lipid oxidation. If estrogen effects lipid oxidation, we predicted that subjects with significantly different endogenous estrogen production would oxidize lipids at different rates. The aim of this study was to compare energy metabolism in 12 pregnant (19 wk gestation, 29 +/- 1 yr, 1.66 +/- 0.02 m, 73.5 +/- 2.4 kg), 11 nonpregnant premenopausal (29 +/- 2 yr, 1.68 +/- 0.02 m, 63.1 +/- 1.8 kg), and 28 postmenopausal (58 +/- 1 yr, 1.62 +/- 0.01 m, 69.9 +/- 1.0 kg) women who were not receiving estrogen, and to relate these findings to endogenous estrogen concentrations. All women underwent indirect calorimetry under identical situations in the basal and postprandial state following a standard mixed meal. Basal (5998 +/- 184 vs. 5712 +/- 184 vs. 5800 +/- 121 kJ.24 h, respectively) and postprandial energy expenditure (7172 +/- 239 vs. 6964 +/- 210 vs. 6955 +/- 147 kJ.24 h) was similar among groups. However, basal lipid oxidation was reduced in pregnant (45.3 +/- 6.1 mg/min, P < 0.05) and nonpregnant women (44.5 +/- 6.3 mg/min, P < 0.05) compared with postmenopausal women (58.4 +/- 2.9 mg/min). Postprandial lipid oxidation differed among groups, being least in pregnant women (8.8 +/- 6.2 mg/min) compared with nonpregnant (28.9 +/- 6.4 mg/min, P < 0.04) and postmenopausal (48.1 +/- 4.0 mg/min, P = 0.0001) women. There was a significant reciprocal increase in postprandial carbohydrate oxidation. Mean postprandial glucose levels were slightly but nonsignificantly higher in pregnant women. Insulin levels were significantly higher in postmenopausal compared nonpregnant, but not pregnant, women. In a multiple regression analysis, serum estradiol (log transformed) correlated negatively with postprandial lipid oxidation (r = -0.66, P = 0.0001) and positively with postprandial nonesterified free fatty acid levels, whereas no correlation was found with postprandial insulin, glucose, fat free mass, and fat mass. In summary, postprandial lipid oxidation is reduced in pregnancy compared with that in healthy nonpregnant women, who in turn have lower postprandial lipid oxidation than postmenopausal women. This implies that the premenopausal years and early pregnancy are states of efficient fat storage, possibly mediated through reduced lipid oxidation due to estrogen, therefore increasing body fat for reproduction, thus supporting the notion that fat mass can be regulated.

Body composition and energy metabolism in pregnancy.

The objective of the study was to measure energy metabolism and body composition during pregnancy and postpartum, compared to non-pregnant women, using non-invasive techniques. A longitudinal study of eight normotensive pregnant women was carried out at 19 +/- 1 and 36+/-1 weeks gestation, and postpartum. A cross-sectional study was also performed comparing postpartum to 12 non-pregnant women. Indirect calorimetry was performed while fasting to measure basal metabolic rate (BMR) and postprandially to measure diet-induced thermogenesis (DIT). Body composition consists of fat mass, lean body mass (LBM), and total body water (TBW) and was measured by bio-electrical impedance. Insulin resistance was indirectly assessed by glucose and insulin concentration and DIT.

Body composition and energy metabolism in normotensive and hypertensive pregnancy.

OBJECTIVE: To determine whether the insulin resistance syndrome and altered body composition are features of hypertensive pregnancy. DESIGN: Women were recruited in the third trimester of pregnancy from the antenatal clinic, day assessment unit, and maternity ward of St George Hospital, Sydney. POPULATION: Women with pre-eclampsia (n = 12), gestational hypertension (n = 12), essential hypertension in pregnancy (n = 11), and normotensive pregnancy (n = 10). METHODS: Energy metabolism was assessed by indirect calorimetry to measure basal metabolic rate and diet-induced thermogenesis. Body composition was measured as lean body mass, total body water and fat mass by bio-electrical impedance. Blood was collected for measurement of glucose, insulin and lipid profiles. Insulin resistance was indirectly assessed by the insulin and glucose concentrations and diet-induced thermogenesis. RESULTS: Women with essential hypertension and gestational hypertension were heavier than women with normotensive pregnancies both pre-pregnancy and in the third trimester, whereas women with pre-eclampsia were similar to those with normotensive pregnancy. Women with essential hypertension were otherwise similar to normotensive pregnancy but women with gestational hypertension had a reduced diet-induced thermogenesis and almost double insulin levels. Women with pre-eclampsia had a similar body composition and insulin levels but reduced basal metabolic rate, diet-induced thermogenesis and glucose levels compared with normotensive pregnancy. CONCLUSIONS: Women who develop gestational hypertension, but not pre-eclampsia, are more likely to be overweight. Women with essential hypertension are similar to women with normotensive pregnancy throughout pregnancy. Both gestational hypertension and pre-eclampsia appear to be associated with some degree of insulin resistance, greater than that occurring in normal pregnancy.

Anabolic-androgenic steroids: medical assessment of present, past and potential users.

OBJECTIVE: To document adverse effects of anabolic-androgenic steroid (AAS) use in community-based users attending a medical clinic. DESIGN AND SETTING: Prospective recruitment, questionnaire-based interview, physical examination and investigations, with follow-up, of people who attended, anonymously, an inner-city hospital clinic established specifically to examine AAS use. PARTICIPANTS: 58 men, comprising 27 past AAS users, 14 present users and 17 potential users (who formed the control group). MAIN OUTCOME MEASURE: Clinical adverse effects and abnormal laboratory findings. RESULTS: Cyclical use of oral and intramuscular, human and veterinary AASs were reported. The most commonly reported source of AASs was friends (59%), gymnasiums (25%) and doctors (14%). The most common reported adverse effects were alterations in libido (61%), changes in mood (48%), reduced testis volume (46%) and acne (43%). Although mean systolic and diastolic blood pressure was not significantly different between groups, five present (29%), 10 past (37%) and one potential user (8%) were hypertensive. Gynaecomastia was found in 10 past users (37%; P<0.01 v. potential users), two present users (12%) and no potential users. Mean testis volume was significantly smaller in present users (18 mL; P<0.02) than in the other groups. Twenty past users (83%), eight present users (62%) and five potential users (71%) had abnormal liver function test results (P=0.5). After discussion of test results, only 11 participants (19%) reported they would not use AASs in the future. CONCLUSIONS: Adverse effects were reported by or detected in most of the AAS users who attended the clinic. Despite awareness of adverse consequences, most participants planned future use of AASs.

Effects of nicotinic acid on insulin sensitivity and blood pressure in healthy subjects.

Insulin resistance and hyperinsulinaemia are associated with hypertension although a causative relationship has not been established. The aim of this study was to determine whether a short term reduction in insulin sensitivity induced by nicotinic acid treatment (NA) would alter blood pressure. The study was a double-blind randomised placebo-controlled cross-over study. Seven healthy volunteers, three males and four females were randomised to placebo or NA 500 mg daily for 7 days then 1 g daily for a further 7 days. Hyperinsulinaemic euglycaemic clamp, indirect calorimetry, 24-h ambulatory blood pressure monitoring (ABPM) and forearm blood flow measurement (FABF) were performed at day 14 of each treatment phase. NA significantly reduced the glucose infusion rate required to maintain euglycaemia in all subjects (placebo vs NA; 31.5+/-4.2 vs. 26.2+/-4.6 micromol/kg/min, P = 0.002) associated with a decrease in non-oxidative glucose disposal. NA did not significantly alter 24-h mean systolic or diastolic blood pressure. Fasting glucose, insulin and non-esterified free fatty acid (NEFA) levels remained unchanged, energy expenditure and substrate oxidation were not altered by NA. These results suggest a short term reduction in insulin sensitivity with NA is not accompanied by a change in blood pressure. This may relate to the short duration of treatment, to a dissociation between insulin resistance and hypertension or to other homeostatic mechanisms which prevent blood pressure rising in subjects not predisposed to hypertension.

Cell cycle-dependent repetitive Ca(2+ )waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization.

Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+ )waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.

Thirty years of stimulus-secretion coupling: from Ca(2+) toGTP in the regulation of exocytosis.

Calcium, initially considered as the universal link between receptor stimulation and the onset of exocytosis in secretory cells, is now recognised as only one of a number of intracellular activators. In cells of haematopoietic origin (including mast cells), the key activator is one or more GTPases. Cells of this class, stimulated with GTPgammaS can undergo exocytosis in the effective absence of Ca(2+). A number of GTP-binding proteins that mediate exocytosis (G(E)) have been proposed but the best evidence supports roles for members of the Rho family of monomeric GTPases and for betagamma-subunits derived from G(i3). While preactivated Rac and Cdc42 can induce secretion from permeabilised mast cells in the absence of a guanine nucleotide betagamma-subunits only act to enhance the secretion induced by other GTP-binding proteins (likely to be members of the Rho family of monomeric GTPases). Further work is required to identify downstream effectors activated by these GTP-binding proteins and to show how they interact with the SNAP and SNARE isoforms known to be present in these cells.

Route-dependent endocrine and metabolic effects of estrogen replacement therapy.

The route of estrogen replacement therapy has a major impact on the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis. Estrogen administration by the oral, but not the transdermal, route reduces IGF-I and increases GH levels in postmenopausal women. This perturbation of the GH-IGF-I axis occurs with different forms of estrogen treatment, indicating that the dissociation of the somatotropic axis and concomitant increase in GH-binding protein levels are intrinsic effects of the oral route of estrogen administration. In clinical studies, oral estrogen reduced postprandial lipid oxidation, compared with transdermal estrogen. Oral estrogen was also associated with a reduction in lean body mass and an increase in fat mass, compared with transdermal estrogen. In contrast, the route of estrogen therapy had no impact on carbohydrate metabolism or the estrogen-induced increase in bone mineral density. The findings of route-dependent changes in body composition add a new dimension to health considerations concerning estrogen therapy in postmenopausal women and may have significant implications for estrogen replacement therapy in young hypogonadal females.

Examination of the role of ADP-ribosylation factor and phospholipase D activation in regulated exocytosis in chromaffin and PC12 cells.

The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.

Regulation of exocytosis from rat peritoneal mast cells by G protein beta gamma-subunits.

We applied G protein-derived beta gamma-subunits to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and GTPgammaS over approximately 20-30 min. beta gamma-Subunits applied to the permeabilized cells retard this loss of sensitivity to stimulation (run-down) and it can be inferred that they interact with the regulatory mechanism for secretion. While alpha-subunits are without effect, beta gamma-subunits at concentrations >10(-8 )M enhance the secretion due to Ca2+ and GTPgammaS. Unlike the small GTPases Rac and Cdc42, beta gamma-subunits cannot induce secretion in the absence of an activating guanine nucleotide, and thus further GTP-binding proteins (likely to be Rho-related GTPases) must be involved. The enhancement due to beta gamma-subunits is mediated largely through interaction with pleckstrin homology (PH) domains. It remains manifest in the face of maximum activation by PMA and inhibition of PKC with the pseudosubstrate inhibitory peptide. Soluble peptides mimicking PH domains inhibit the secretion due to GTPgammaS and block the enhancement due to beta gamma-subunits. Our data suggest that beta gamma-subunits are components of the pathway of activation of secretion due to receptor-mimetic ligands such as mastoparan and compound 48/80.