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.

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.

Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells.

The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

The route of estrogen replacement therapy confers divergent effects on substrate oxidation and body composition in postmenopausal women.

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. To investigate whether these perturbations have metabolic consequences, we compared the effects of 24 wk each of oral (Premarin 1.25 mg) and transdermal (Estraderm 100TTS) estrogen on energy metabolism and body composition in 18 postmenopausal women in an open-label randomized crossover study. Energy expenditure, lipid oxidation (lipid(ox)), and carbohydrate oxidation (CHOox) were measured by indirect calorimetry in the fasted and fed state before and after 2 and 6 mon treatment. Lean body mass, fat mass, and total body bone mineral density were measured by dual X-ray absorptiometry before and after 6 mon treatment. Mean (+/-SE) Luteinizing hormone levels fell to comparable levels during oral and transdermal estrogen, and bone mineral density was significantly increased by both treatments. Mean IGF-I was significantly lower during oral estrogen (77+/-7 versus 97+/-7 microg/liter, P < 0.05) treatment. Lipid(ox) 30-60 min after a standardized meal was significantly lower (36+/-5 versus 54+/-5 mg/min, P < 0.01) and CHOox higher (147+/-13 versus 109+/-12 mg/min, P < 0.05) with oral compared with transdermal estrogen. Oral estrogen resulted in a 1.2+/-0.5 kg (P < 0.05) increase in fat mass and a 1.2+/-0.4 kg (P < 0.01) decrease in lean mass compared with transdermal estrogen. Lean body mass (0.4+/-0.2 kg) and fat mass (0. 1+/-0.4 kg) did not change significantly during transdermal estrogen. In summary, when compared with the transdermal route, oral estrogen reduces lipid(ox), increases fat mass, and reduces lean body mass. The route of estrogen therapy confers distinct and divergent effects on substrate oxidation and body composition. The suppression of lipidox during oral estrogen therapy may increase fat mass although the fall in IGF-I may lead to a loss of lean body mass. The route-dependent changes in body composition observed during estrogen replacement therapy may have important implications for postmenopausal health.

Induction of exocytosis from permeabilized mast cells by the guanosine triphosphatases Rac and Cdc42.

We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K 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 guanosine triphosphate (GTP)gammaS over about 20-30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTPgammaS, retard the loss of sensitivity (run-down) and, more importantly, enable secretion to be stimulated by Ca2+ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca2+ and GTPgammaS. Our data suggest that Rac2 and Cdc42 should be considered as candidates for GE, GTPases that mediate exocytosis in cells of hematopoeitic origin.

Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.

Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown.

The role of cytoplasmic pH in the inhibitory action of high osmolarity on secretion from bovine adrenal chromaffin cells.

Elevated osmolarity is known to inhibit secretion from a wide range of cells including bovine adrenal chromaffin cells. The mechanism of this inhibition is unclear but the elevated osmolarity has been proposed to oppose an osmotic driving force involved in exocytotic fusion. Using the fluorescent indicators quene 1 and fura2, we monitored the effect of elevated osmolarity on cytoplasmic pH (pHi) and cytoplasmic free Ca2+ [( Ca2+]i). Elevated osmolarity increased both pHi and [Ca2+]i, but had no effect on the [Ca2+]i rise elicited by either K+ or nicotine. Elevating pHi with NH4Cl was shown to inhibit secretion from chromaffin cells. The elevation of pHi by hyperosmolar solutions is proposed as one of the mechanisms by which elevated osmolarity inhibits secretion.

The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C.

The control of cytoskeletal actin and exocytosis was examined in intact and digitonin-permeabilized chromaffin cells. Cytoskeletal actin was assayed by determining the actin content of Triton-insoluble cytoskeletons. The secretagogues nicotine, high K+ and Ba2+ resulted in a rapid reduction in the amount of actin associated with the cytoskeleton. The effect of nicotine but not high K+ on cytoskeletal actin was independent of external Ca2+ and the reduction in cytoskeletal actin was mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate suggesting a role for protein kinase C. In digitonin-permeabilized cells micromolar calcium produced both catecholamine secretion and a reduction in cytoskeletal actin. The reduction in cytoskeletal actin was transient. Secretion was enhanced by the GTP analogue guanosine 5'-(3-O-thio)triphosphate and the analogue also reduced cytoskeletal actin at low calcium levels. The effects of guanosine 5'-(3-O-thio)triphosphate were inhibited by the phospholipase C inhibitor neomycin and were mimicked by 12-O-tetradecanoylphorbol-13-acetate. An additional GTP analogue, guanyl-5'-yl imidodiphosphate, had no effect on cytoskeletal actin. These results provide further evidence for a requirement for reorganisation of cortical actin in the secretory processes and suggest that the reduction in actin associated with the cytoskeleton may be mediated by protein kinase C and/or calcium in intact and permeabilized chromaffin cells.

A comparison of the effects of oral and transdermal estrogen replacement on insulin sensitivity in postmenopausal women.

Previous studies have shown that oral, but not transdermal, administration of estrogen stimulates GH secretion in postmenopausal women. Because GH impairs insulin action, the impact of estrogen replacement therapy on carbohydrate metabolism may be influenced by the route of administration. The aim of this study was to prospectively compare the effects of oral and transdermal estrogen replacement on glucose tolerance and insulin sensitivity in postmenopausal women. In an open label, randomized, cross-over study, nine postmenopausal women were randomized to transdermal estrogen patches (Estraderm-TTS 100) or oral conjugated estrogen (Premarin, 1.25 mg) daily for 12 weeks and then crossed over to receive the alternative treatment for a further 12 weeks. An oral glucose tolerance test and hyperinsulinemic euglycemic clamp (HEC) were performed before treatment and at the end of 10 weeks of treatment. Oral and transdermal estrogen both significantly reduced LH to the same degree. Mean GH did not significantly change with transdermal estrogen, but rose significantly during oral estrogen therapy. Peak and mean glucose and insulin levels during the oral glucose tolerance test were not altered by estrogen therapy and were not significantly different between treatments. Mean glucose and insulin levels were maintained at an identical level during the HEC performed at pretreatment and during estrogen therapy. The mean glucose infusion rate required to maintain euglycemia during the HEC (mean +/- SEM, pretreatment, 40.4 +/- 4.8 mumol/kg.min) was unaltered by oral (39.8 +/- 4.6 mumol/kg.min) or transdermal estrogen treatment (42.1 +/- 4.2 mumol/kg.min). However, during the transdermal estrogen phase (60 +/- 10 mumol/L), the mean nonesterified free fatty acid concentration was suppressed to a significantly lower level during the HEC than during the oral estrogen phase (120 +/- 20 mumol/L; P < 0.05). We conclude that compared to the oral route, transdermal estrogen therapy is associated with a slight, but significant, improvement of insulin action on lipid metabolism. However, in the short term, the route of estrogen replacement therapy does not have a major impact on glucose metabolism in postmenopausal women.

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 expenditure in acromegaly.

To investigate whether GH is a regulator of body composition and energy metabolism in adult life, we have compared body composition and resting energy expenditure (REE) in a cross-sectional study in 20 acromegalic and 20 normal subjects, pair-matched for sex, age, height, and weight. In a longitudinal study, 8 acromegalic patients were also studied before and after 12 weeks of treatment [n = 6 during octreotide (100 micrograms, 3 times/day); n = 2 after pituitary surgery], and 7 patients were studied 12 weeks after withdrawal of octreotide. REE was measured by indirect calorimetry and fat mass and fat-free soft tissue mass (FFSTM) by dual energy x-ray absorptiometry. A subgroup of 12 matched pairs of subjects and 7 treated patients had measurement of extracellular water (ECW) by 24Na dilution, which when subtracted from FFSTM provided an estimate of body cell mass (BCM). Fat mass was significantly reduced (25.4 +/- 2.2 vs. 29.7 +/- 2.7 kg; P = 0.007) and FFSTM increased (53.3 +/- 2.2 vs. 49.2 +/- 2.3 kg; P = 0.003) in acromegaly with ECW (25.6 +/- 1.6 vs. 21.1 +/- 0.9 L; P = 0.0003), but not BCM, significantly elevated. Treatment of acromegaly increased fat mass and reduced FFSTM [change (delta), -1.3 +/- 0.4 kg; P = 0.004]; the latter reflected a significant fall in ECW (delta, -2.2 +/- 0.4 L; P = 0.002), but not BCM. The opposite effect on body composition occurred after treatment withdrawal. REE was increased in acromegaly (1682 +/- 49 vs. 1540 +/- 45 Cal/24 h; P = 0.02) and significantly related to insulin-like growth factor-I (P = 0.02). REE was significantly reduced (delta, -154 +/- 17 Cal/24 h; P = 0.0001) with treatment and increased after treatment withdrawal (P = 0.003). In acromegaly, there is a reversible 1) reduction in fat mass; 2) increase in FFSTM, accounted for by an increase in ECW, but not BCM; and 3) increase in REE, which is dependent on disease activity. We conclude from these observations in acromegaly that GH is a regulator of energy metabolism and body composition.

Do androgens regulate growth hormone-binding protein in adult man?

To determine whether adult serum GH-binding protein (GHBP) is regulated by androgen, serum GHBP concentrations were compared between 20 normal and 18 hypogonadal men matched for age and body mass index, and the effect of im testosterone treatment (250 mg testosterone enanthate) on GHBP levels in the 18 hypogonadal men was studied. Nine of the hypogonadal subjects had coexistent GH deficiency. Serum GHBP concentration was measured by a ligand immunofunctional assay. The mean serum GHBP level in untreated hypogonadal men was not significantly different from that of normal men (0.98 +/- 0.15 vs. 1.17 +/- 0.16 nmol/L). The mean serum insulin-like growth factor I (IGF-I) level was significantly lower in the hypogonadal men (132 +/- 22 vs. 206 +/- 17 ng/mL; P < 0.01). Basal testosterone (3.7 +/- 0.7 nmol/L) in hypogonadal men increased during treatment to a mean level of 29.1 +/- 2.8 nmol/L, which was not significantly higher than that in normal men (22.6 +/- 1.9 nmol/L). The mean serum GHBP level in hypogonadal men fell significantly during treatment to 0.60 +/- 0.11 nmol/L (P = 0.0003), whereas the serum IGF-I level rose significantly to 151 +/- 26 ng/mL (P < 0.04). The decrease in GHBP level was significant in both the GH-sufficient and GH-deficient subjects (P < 0.02 in both instances), whereas the increase in IGF-I level was significant in the GH-sufficient group (199 +/- 22 to 235 +/- 29 ng/mL; P < 0.04) but not in the GH-deficient group (53 +/- 7 to 55 +/- 5 ng/mL; P > 0.8). Thus, serum GHBP is normal in hypogonadal men but is reduced by testosterone treatment irrespective of endogenous GH-secretory status. It was concluded that the effect of testosterone on GHBP is pharmacological and occurs independent of GH mediation.

Energy metabolism and substrate oxidation in acromegaly.

Short term GH administration increases lipid breakdown and oxidation (lipidox) and reduces glucose uptake and carbohydrate oxidation (CHOox). It is not clear whether similar shifts in substrate oxidation occur in acromegaly, and our aim was to investigate this. Using indirect calorimetry, we compared energy expenditure, CHOox, and lipidox in 20 acromegalic patients and 20 normal subjects pair-matched for sex, age, height, and weight. Investigations were performed in the basal state (12-h fast) and during a 75-g oral glucose tolerance test (OGTT). Acromegalic patients had significantly higher fasting glucose levels and greater glucose and insulin responses during an OGTT than normal subjects. Fasting nonesterified free fatty acid and insulin-like growth factor (IGF)-binding protein-1 levels were similar in the two groups, and both were acutely suppressed by oral glucose to the same degree. Basal energy expenditure was significantly greater in the acromegalic patients (1682 +/- 49 vs. 1540 +/- 45 Cal/24 h; P < 0.05), who showed a trend toward higher basal CHOox. Oral glucose resulted in a significantly higher rise in energy expenditure in the normal compared to the acromegalic subjects. During the OGTT, CHOox significantly increased in both groups, but rose to a higher level in the acromegalic patients (177 +/- 10 vs. 138 +/- 9 mg/min; P = 0.004). Oral glucose significantly reduced lipidox in both groups, but lipidox was reduced to a significantly lower level in the acromegalic patients (32 +/- 4 vs. 46 +/- 3 mg/min; P = 0.004). In acromegaly, basal CHOox (r = 0.56; P = 0.01) and postglucose CHOox (r = 0.79; P = 0.0001) were both positively correlated to IGF-I, but not to insulin and/or glucose. In normal subjects, postglucose CHOox was positively correlated to IGF-I. In summary, hyperinsulinemia in acromegaly was associated with higher glucose levels and a blunted thermogenic response to glucose, and displayed no relationship to the pattern of substrate oxidation. CHOox was increased, and lipidox was reduced in acromegaly, and the extent of IGF-I elevation was related to CHOox in the basal and postglucose states. We conclude that 1) the chronic effects of GH excess on substrate oxidation differ from the short term effects of GH administration; 2) impaired insulin action in acromegaly extends to effects on energy expenditure; and 3) IGF-I may be an important regulator of substrate oxidation in acromegaly.

Adults with growth hormone deficiency have abnormal body composition but normal energy metabolism.

We compared body composition and energy metabolism in a cross-sectional study involving 21 GH-deficient (GHD) and 29 normal adults. Fifteen subjects from each group were pair-matched for age, sex, height, and weight. Comparison of body composition by dual energy x-ray absorptiometry in the 15 pairs showed that GHD subjects had a significantly greater fat mass (23.9 +/- 1.8 vs. 19.8 +/- 2.6 kg; P = 0.02), lower bone mineral content (2.6 +/- 0.1 vs. 2.9 +/- 0.1 kg; P = 0.06), and lower fat-free soft tissue mass (FFSTM; 45.8 +/- 2.5 vs. 48.9 +/- 2.6 kg; P = 0.02). To determine which subcompartment changes were responsible for the reduced FFSTM in GH deficiency, extracellular water (ECW) was measured by 24Na dilution in 11 of the subject pairs, and body cell mass (BCM) was derived by subtracting ECW from FFSTM. The GHD subjects had lower ECW (19.4 +/- 1.0 vs. 21.0 +/- 1.0 kg; P = 0.05) and BCM (26.6 +/- 1.9 vs. 28.6 +/- 2.1 kg; P = 0.08) than their normal counterparts, with the difference approaching statistical significance. When the relationship between ECW and FFSTM was compared in the larger group of 18 GHD and 27 normal subjects, no statistically significant difference was found between the 2 regression lines, indicating that ECW and BCM were proportionately reduced in GH deficiency. Energy expenditure and fuel utilization before and after a standardized mixed meal were measured by indirect calorimetry in 12 matched pairs of GHD and normal subjects. Comparison of energy expenditure and fuel utilization showed no significant difference in fasting or postprandial energy expenditure, or carbohydrate or fat oxidation rates between the two groups. We conclude that GHD adults have an increased FM, a reduced FFSTM due to a proportionate reduction in ECW and BCM, and no detectable disturbance in energy metabolism.

A major role for protein kinase C in calcium-activated exocytosis in permeabilised adrenal chromaffin cells.

The role of endogenously activated protein kinase C in calcium-activated exocytosis was examined in digitonin-permeabilised bovine adrenal chromaffin cells. Protein kinase C activity was reduced by down-regulation following long-term treatment with PMA or by using the inhibitor sphingosine. Both treatments resulted in a substantial reduction in catecholamine secretion elicited by micromolar calcium, indicating that endogenous activation of protein kinase C is a major requirement for calcium-activated exocytosis in chromaffin cells.

The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis.

The effect of caffeine on catecholamine secretion and intracellular free Ca2+ concentration [( Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura-2-loaded cells and cell populations. In cell populations caffeine elicited a large (approximately 200 nM) transient rise in [Ca2+]i that was independent of external Ca2+. This rise in [Ca2+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (greater than 200 nM) rise in [Ca2+]i, whereas a minority failed to respond. The latter, whose caffeine-sensitive store was empty, buffered a Ca2+ load induced by a depolarizing stimulus more effectively than those whose store was full. The caffeine-sensitive store in bovine chromaffin cells may be involved in Ca2+ homeostasis rather than in triggering exocytosis.

Spatial localization of the stimulus-induced rise in cytosolic Ca2+ in bovine adrenal chromaffin cells. Distinct nicotinic and muscarinic patterns.

The spatial distribution of the intracellular free Ca2+ (Ca2+i) rise elicited by different stimuli in bovine adrenal chromaffin cells was examined in single fura-2-loaded cells. In response to the potent secretagogues nicotine and high K+, Ca2+i was initially localized exclusively to the entire subplasmalemmal area of the cell. In response to the ineffective secretagogues, methacholine and muscarine, the rise in Ca2+i originated only in one pole of the cell and even at the peak of the response Ca2+ was still generally restricted to this same area of the cell. These results suggest that the triggering of exocytosis from these cells requires a specific spatial distribution of Ca2+i.