A new index to simplify the screening of hypertension in overweight or obese youth.

BACKGROUND AND AIMS: Hypertension (HTH) is a frequent complication in pediatric obesity. To simplify the screening of HTH in overweight/obese (Ow/Ob) youth, we compared the performance of a new index (High Blood Pressure index, HBPi) with respect to the standard criteria of the IV Report [systolic BP (SBP) and/or diastolic BP (DBP) ≥95th percentile for age, gender and height]. We also compared the performance of HBPi with other simplified indices such as the BP/height ratio and the absolute height-specific BP thresholds. Ten pediatrics' outpatient centers participating in the "CARdiometabolic risk factors in ITALY study" provided medical records of 4225 Ow/Ob children and adolescents (age 6-16 years). METHODS AND RESULTS: Centers were divided into two groups: training set (TS) (n = 2204 participants) and validation set (VS) (n = 2021 participants). The simplified HBPi (mmHg) was: (SBP/2 + DBP/10) - age + (1 × female gender). In the TS, a HBPi value ≥57 mmHg in both children and adolescents had high sensitivity (0.89), specificity (0.97), positive (0.89) and negative (0.97) predictive values in classifying youth at high risk of HTN compared with the IV Report. In the VS, the HBPi showed a better performance than high levels of BP/height ratio and height-specific BP thresholds in classifying individuals at risk of HTN: area under curves 0.95 (0.93-0.96), 0.80 (0.78-0.82), 0.76 (0.74-0.79), respectively; specificities 0.95 (0.94-0.96), 0.69 (0.67-0.72), 0.60 (0.57-0.62), respectively. CONCLUSIONS: HBPi, combining SBP and DBP, gender and age, may help pediatricians to implement HTN screening in Ow/Ob youth.

Impaired fasting glucose and impaired glucose tolerance in children and adolescents with overweight/obesity.

OBJECTIVE: To investigate in a large sample of overweight/obese (OW/OB) children and adolescents the prevalence of prediabetic phenotypes such as impaired fasting glucose (IFG) and impaired glucose tolerance (IGT), and to assess their association with cardiometabolic risk (CMR) factors including hepatic steatosis (HS). METHODS: Population data were obtained from the CARdiometabolic risk factors in children and adolescents in ITALY study. Between 2003 and 2013, 3088 youths (972 children and 2116 adolescents) received oral glucose tolerance test (OGTT) and were included in the study. In 798 individuals, abdominal ultrasound for identification of HS was available. RESULTS: The prevalence of IFG (3.2 vs. 3.3%) and IGT (4.6 vs. 5.0%) was similar between children and adolescents. Children with isolated IGT had a 2-11 fold increased risk of high LDL-C, non-HDL-C, Tg/HDL-C ratio, and low insulin sensitivity, when compared to those with normal glucose tolerance (NGT). No significant association of IFG with any CMR factor was found in children. Among adolescents, IGT subjects, and to a lesser extent those with IFG, showed a worse CMR profile compared to NGT subgroup. In the overall sample, IGT phenotype showed a twofold increased risk of HS compared to NGT subgroup. CONCLUSIONS: Our study shows an unexpected similar prevalence of IFG and IGT between children and adolescents with overweight/obesity. The IGT phenotype was associated with a worse CMR profile in both children and adolescents. Phenotyping prediabetes conditions by OGTT should be done as part of prediction and prevention of cardiometabolic diseases in OW/OB youth since early childhood.

Adipose tissue microbiota in humans: an open issue.

BACKGROUND: A specific 'adipose tissue' microbiota has been recently identified in mice and hypothesized in humans. The purpose of this study was to verify the presence of microbiota of human whole adipose tissue and isolated adipocytes by combining culture-dependent and independent methods. METHODS: Standard microbiological cultural techniques and 16S ribosomal RNA (16S rRNA) gene sequencing (Illumina technology) on DNA and RNA were employed to study (a) whole abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) from 14 obese and five normal-weight subjects and (b) mature adipocytes isolated from SAT and VAT after collagenase digestion or mechanical separation. To optimize the 16S rRNA gene detection, we used different DNA extraction methods (lysis with proteinase K, proteinase K+lysozyme and microbeads) and amplification procedures (semi-quantitative standard PCR and real-time quantitative PCR). RESULTS: Microbiological cultures were negative in all analyzed samples. In enzymatically isolated adipocytes, 90% of the sequenced bacterial DNA belonged to Clostridium histolyticum, the bacterium from which the collagenase enzyme was isolated. Bacterial 16S rRNA gene was not detected from DNA and RNA of whole SAT and VAT, as well as of mechanically isolated mature adipocytes, even after blocking with a specific primer the nonspecific amplification of human mitochondrial 12S rRNA. CONCLUSIONS: Our results do not support the presence of a human adipose tissue microbiota. In addition, they emphasized the technical problems encountered when applying metagenomic studies to human tissues with very low or absent bacterial load.

White blood cell count may identify abnormal cardiometabolic phenotype and preclinical organ damage in overweight/obese children.

BACKGROUND AND AIMS: Subclinical inflammation is a central component of cardiometabolic disease risk in obese subjects. The aim of the study was to evaluate whether the white blood cell count (WBCc) may help to identify an abnormal cardiometabolic phenotype in overweight (Ow) or obese (Ob) children. METHODS AND RESULTS: A cross-sectional sample of 2835 Ow/Ob children and adolescents (age 6-18 years) was recruited from 10 Italian centers for the care of obesity. Anthropometric and biochemical variables were assessed in the overall sample. Waist to height ratio (WhtR), alanine aminotransferase (ALT), lipids, 2 h post-load plasma glucose (2hPG), left ventricular (LV) geometry and carotid intima-media thickness (cIMT) were assessed in 2128, 2300, 1834, 535 and 315 children, respectively. Insulin resistance and whole body insulin sensitivity index (WBISI) were analyzed using homeostatic model assessment (HOMA-IR) and Matsuda's test. Groups divided in quartiles of WBCc significantly differed for body mass index, WhtR, 2hPG, HOMA-IR, WBISI, lipids, ALT, cIMT, LV mass and relative wall thickness. Children with high WBCc (≥8700 cell/mm(3)) showed a 1.3-2.5 fold increased probability of having high normal 2hPG, high ALT, high cIMT, or LV remodeling/concentric LV hypertrophy, after adjustment for age, gender, pubertal status, BMI and centers. CONCLUSIONS: This study shows that WBCc is associated with early derangements of glucose metabolism and preclinical signs of liver, vascular and cardiac damage. The WBCc may be an effective and low-cost tool for identifying Ow and Ob children at the greatest risk of potential complications.

Permanence of molecular features of obesity in subcutaneous adipose tissue of ex-obese subjects.

OBJECTIVE: Bariatric surgery represents a powerful tool for morbid obesity treatment. However, after stabilization of weight loss that follows surgical interventions, ex-obese patients face the problem of residual tissues removal. Actually, it is unknown whether the characteristics of this residual subcutaneous adipose tissue (SAT) are 'restored' with regard to molecular and morphological features. DESIGN: To clarify this issue, we compared the SAT gene expression profile of ex-obese patients (ExOB-SAT, mean body mass index (BMI): 27.2±1.3 kg m(-2)) with that of lean (normal weight, NW-SAT, mean BMI: 22.6±1.1 kg m(-2)), overweight (OW-SAT, BMI: 27.65±0.2 kg m(-2)) and obese patients, according to BMI classes (OB1-SAT: 30 > or = BMI < or = 34.9, OB2-SAT: 35 > or = BMI < or = 39.9, OB3-SAT: BMI > or = 40). SUBJECTS AND METHODS: A total of 58 samples of SAT were collected during surgical interventions. Gene expression levels were assessed by microarrays and significant genes were validated by RT-qPCR. Adipocyte hypertrophy, inflammatory infiltration and fibrosis were assessed by morphological techniques. RESULTS: Global gene expression in ExOB-SAT was closely related to gene expression of OB3-SAT by hierarchical clustering procedures, in spite of different BMI. Metallothioneins (MT1A and MT2A) were the key over-expressed genes in both groups. At morphologic level, adipocyte hypertrophy and inflammatory infiltration improved after weight loss in ExOB-SAT, despite a persistence of fibrosis. CONCLUSIONS: Taken together, these results demonstrate that SAT gene expression is not fully restored, even after an extensive and stable weight loss. The persistence of 'obesity molecular features' in ExOB-SAT suggests that the molecular signature of adipose tissue is not solely dependent on weight loss and may need longer time period to completely disappear.

The nicotinic acetylcholine receptor α7 in subcutaneous mature adipocytes: downregulation in human obesity and modulation by diet-induced weight loss.

BACKGROUND: It is known that cholinergic anti-inflammatory reflex regulates inflammation in peripheral tissues. Nicotinic acetylcholine receptors (nAChRs) are mediators of this anti-inflammatory pathway and also non-neuronal cells express functional nAChrs. A role for α7-subtype acetylcholine cholinergic receptor (α7nAChR) in insulin sensitivity improvement has already been shown in rodents both in vivo and in vitro. However, no data are available on α7nAChR expression in human adipocytes. OBJECTIVE: To investigate the expression and protein content of α7nAChR in human subcutaneous adipose tissue (SAT) and in isolated mature adipocytes. DESIGN: A total of 39 SAT biopsy specimens obtained from obese and normal-weight subjects were used to assess α7nAChR messenger RNA levels and to stimulate α7nAChR with a specific agonist and antagonist in vitro. Additional SATs from eight non-diabetic obese subjects were also studied, before and after a 3-month lifestyle intervention. RESULTS: α7nAChR expression was significantly lower in the SAT of obese subjects compared with that of normal-weight subjects. In mature adipocytes isolated from morbidly obese subjects (body mass index > 40 kg m(-2)), α7nAChR expression was 75% lower compared with adipocytes from normal-weight subjects. In adipocytes of obese subjects, α7nAChR was downregulated also at protein level. In eight non-diabetic obese subjects, a lifestyle intervention (3 months of diet and physical activity) induced a significant weight loss and an increase in α7nAChR SAT expression. In vitro stimulation of adipocytes with the specific α7nAChR agonist PNU282987 induced a significant anti-inflammatory effect. Furthermore, a similar downregulation of the inflammatory profile, associated with a significant increase in α7nAChR protein level, was observed after genistein stimulation. CONCLUSIONS: These results provide evidence that α7nAChR expression levels are significantly decreased in obese subjects, and that this receptor modulates inflammatory gene expression in human adipocytes. The upregulation of α7nAChR by genistein stimulation opens new insights for the management of low-grade inflammation linked to human obesity.

Percutaneous transluminal angioplasty improves glucose control and quality of life in patients with critical limb ischemia.

AIM: To evaluate the benefit of endovascular peripheral revascularization on glucose control in patients with chronic limb ischemia. METHODS AND RESULTS: Over a 12 month period, 61 patients (41 male, range 49-88 years of age) presenting with critical limb ischemia (CLI) were treated according to the Trans Atlantic Inter Society Consensus (TASC II) guidelines. After discharge, all patients were asked to measure their glucose level three times daily, and glycated hemoglobin was checked monthly up to 12 months, as well as to fill a questionnaire to assess their Quality of Life (QoL). The revascularization procedure was successful in 90% of cases. Glycemic control and glycated hemoglobin in 22 diabetic patients subgroup were significantly improved after the treatment and remained stable over the follow-up period. There was a significant improvement in QoL that increased steadily from the operation and to reach a plateau after six months. CONCLUSIONS: Peripheral percutaneous angioplasty in subjects with CLI significantly improves glycemic control and ameliorates QoL. Revascularization positively effects also long-term diabetes control as well as QoL.

Predictors of the early impairment of renal disease in human obesity.

OBJECTIVE: The mechanisms underlying the association of the increased albumin excretion rate (AER) with adiposity have yet to be clarified. We therefore investigated (1) the predictors of AER after 3 months of lifestyle intervention in a large cohort of nondiabetic obese women and (2) the relationships between AER and the adipose tissue gene expression of adipokines linked to inflammation and insulin resistance. SUBJECTS: A total of 269 obese nondiabetic women (age 49.9+/-13.1 years, body mass index (BMI) 36.8+/-4.6 kg m(-2)) participated in this program. Measurements used were anthropometrics parameters, blood pressure, oral glucose tolerance test, lipids, creatinine, AER, homeostasis model assessment of insulin resistance (HOMA-IR) and glomerular filtration rate at baseline and after 3 months of lifestyle intervention. At baseline, in a subgroup of 34 women, subcutaneous adipose tissue biopsy was carried out for the analysis of mRNA expression levels of adiponectin, suppressor of cytokine signaling 3 (SOCS-3), tumor necrosis factor alpha (TNF-alpha), pentraxine 3 (PTX-3), angiotensinogen and angiotensin-converting enzyme, and a blood sample was also taken from this group for the measurement of circulating adiponectin, interleukin-6, TNF-alpha and PTX-3. Microalbuminuria was defined as albumin/creatinine ratio >or=3.5 mg mmol(-1). Real-time PCR was used to quantify mRNA. RESULTS: Six percent of obese women had microalbuminuria. When dividing the whole cohort into three groups according to AER changes (decrease, stability and increase), we noted that 2 h glucose, insulin and HOMA-IR significantly decreased (P<0.05 for all) only in women who had a decrease in AER, whereas BMI and waist circumference significantly decreased in all the three groups (P<0.05). At baseline, higher AER was associated to significantly higher adipose tissue mRNA expression levels of SOCS-3 and PTX-3 (P<0.05) and to higher TNF-alpha and angiotensinogen expression. CONCLUSIONS: In obese women, weight loss alone is not sufficient to induce the AER decrease that occurs only with a concomitant improvement in glucose homeostasis. The adipose tissue gene expression profile seems to favor the early renal impairment often seen in obese subjects.

Sympathoadrenergic and metabolic factors are involved in ambulatory blood pressure rise in childhood obesity.

We investigated in a young Italian obese population, the relationship between ambulatory BP (ABP) and several pathophysiological factors linking obesity to hypertension. A total of 89 obese children and adolescents underwent a 24-h ambulatory BP monitoring (ABPM) and an oral glucose tolerance test. The circulating levels of insulin, lipids, uric acid, C-reactive protein, interleukin-6, renin and aldosterone and the 24-h urinary levels of epinephrine, norepinephrine and albumin excretion rate were measured. Nine percent of subjects had daytime sustained hypertension (SH), 26% night-time hypertension and 11% a non-dipping pattern. SH subjects compared to those with sustained normotension (SN) were more obese (P<0.05), with a more frequent family history of hypertension (P<0.05), higher urinary catecholamine (P<0.05) and heart rate values (P<0.05) after adjustment for standard deviation score (SDS) of body mass index (BMI) and sex. Subjects with night-time hypertension compared to those with night-time normotension were more obese (P<0.0001), with a higher prevalence of impaired glucose tolerance (P<0.05) and metabolic syndrome (P<0.05) and higher 2-h glucose (P<0.05), uric acid (P<0.05) and triglycerides (P<0.05). In multivariate regression analysis, daytime systolic BP (SBP) remained independently correlated with urinary norepinephrine and SDS-BMI (P<0.05 for both), daytime diastolic BP (DBP) with waist circumference (P<0.05) and night-time SBP and DBP with SDS-BMI (P<0.01 for both). The risk of having systolic and diastolic hypertension increased with the increase in SDS-BMI and waist circumference, respectively. In conclusion, in our cohort of obese children and adolescents, daytime and night-time hypertension were associated with activation of the sympathoadrenal system and worst metabolic conditions, respectively.

Diagnosis and management of Cushing's syndrome: results of an Italian multicentre study. Study Group of the Italian Society of Endocrinology on the Pathophysiology of the Hypothalamic-Pituitary-Adrenal Axis.

The past 45 yr' experience with Cushing's syndrome (CS) has led to the awareness of its complex nature and, by the same token, brought about an increase in the diagnostic and therapeutic dilemmas. We carried out a retrospective multicentre study on the diagnostic work-up and treatment in 426 patients with CS, subdivided as follows: 288 with Cushing's disease (CD), 80 with an adrenal adenoma, 24 with an adrenal carcinoma, 25 with ectopic ACTH and/or CRH secretion, and 9 with ACTH-independent nodular adrenal hyperplasia. Normal urinary free cortisol (UFC) values among multiple collections were recorded in about 10% of patients with CS. In 28% of patients with ACTH-independent CS, basal ACTH concentrations were within the normal range but did not respond to CRH stimulation. Measurement of ACTH levels by immunoradiometric assay, rather than by RIA, offered a greater chance of recognizing patients with ACTH-independent CS or ectopic secretion. A 50% increase in ACTH or cortisol levels after CRH yielded a diagnostic accuracy of 86% and 61%, respectively, in the differential diagnosis of ACTH-dependent CS. An 80% decrease in cortisol levels after 8 mg dexamethasone overnight, or in UFC values after the classical 2-day administration, excluded an ectopic secretion but carried a low negative predictive value given the high number of nonsuppressors among patients with CD. Pituitary imaging identified an adenoma in 61% of patients with CD. At inferior petrosal sinus sampling, an ACTH centre: periphery gradient after CRH less than 3, correctly classified all patients with ectopic secretion but misdiagnosed 15% of 76 patients with CD. Transsphenoidal pituitary surgery, the standard therapy for CD, resulted in complete remission (appearance of clinical signs of adrenal insufficiency associated with low/normal UFC excretion and, when available, low/normal morning plasma ACTH and cortisol levels) in 69% of patients. The overall relapse rate after pituitary surgery was 17%. The probability of relapse-free survival, as assessed by Kaplan-Meier analysis, was 95% at 12 months, 84% at 2 yr, and 80% at 3 yr. Risk of relapse was significantly correlated with postoperative baseline plasma ACTH and cortisol peak after CRH. No relapses were observed among patients who did not respond to CRH. Other therapeutic approaches for CD, such as pituitary irradiation and medical therapy, resulted in normalization of cortisol secretion in about half of treated cases. In summary, an accurate selection of the available diagnostic tools leads to the correct diagnosis in the majority of patients with CS. The therapeutic options for CD, adrenal carcinoma, and ectopic secretion are, as yet, not fully satisfactory. The high incidence of relapse after pituitary surgery calls for a prolonged follow-up.

Effect of an antiserotoninergic drug, metergoline, on the ACTH and cortisol response to insulin hypoglycemia and lysine-vasopressin in man.

The effect of metergoline, a specific antiserotoninergic drug, on ACTH secretion was investigated in 29 normal volunteers and in 4 patients with increased ACTH production (3 with Addison's disease, 1 with Cushing's disease). In 15 normal subjects, a 4-day treatment with 10 mg daily of metergoline significantly blunted the ACTH response to insulin hypoglycemia. Mean peak ACTH values before and after treatment were, respectively, 333 +/- 39.2 (SE) and 235 +/- 38.8 pg/ml (P less than 0.05). The corresponding values of plasma cortisol were 29.6 +/- 2.96 and 20.5 +/- 2.67 mug/100 ml (P less than 0.05). In contrast, metergoline failed to affect the ACTH response to lysine-vasopressin (LVP) administered iv (8 subjects studied) and im (6 subjects studied). In 3 patients suffering from Addison's disease, an appreciable although not statistically significant lowering of the plasma ACTH levels was noted during metergoline administration. The mean pre- and post-treatment values of plasma ACTH in these patients were, respectively, 1116 +/- 192.2 and 666 +/- 100.8 pg/ml, 4240 +/- 50.0 and 3398 +/- 368.0 pg/ml, and 431 +/- 44.0 and 352 +/- 23.9 pg/ml. In one patient with Cushing's disease caused by a pituitary adenoma, metergoline did not appreciably modify plasma ACTH levels. Taken together, these results lend support to the concept of a physiological stimulating effect of serotonin on ACTH secretion. Moreover, they are compatible with the view that serotonin exerts its action chiefly at the hypothalamic level while LVP promotes ACTH release by a primary action on the pituitary.

Galanin is released by adrenocorticotropin-secreting pituitary adenomas in vivo and in vitro.

Galanin, a brain-gut peptide, is also synthesized and released by the pituitary. In man, galanin-like immunoreactivity and galanin messenger RNA have been detected specifically in normal and tumoral corticotropes, but little is known about the production and release of galanin by the human pituitary. We evaluated galanin release by 5 ACTH-secreting pituitary adenomas in culture and plasma galanin concentrations in the inferior petrosal sinuses (IPSs) of 15 patients with Cushing's disease before and after CRH administration. For comparison, the galanin response to CRH was evaluated in 8 normal controls. Galanin secretion by pituitary tumor cultures ranged from 30-230 pmol/4 h. Incubation with CRH induced an increase in galanin concentrations (100 pM CRH: 151 +/- 32%; 1 nM CRH: 232 +/- 43%; 10 nM CRH: 246 +/- 35%; and 100 nM CRH: 270 +/- 44% unstimulated levels at 24 h, P < 0.05). The stimulatory effect of CRH seemed to be dose-dependent. Basal and CRH-stimulated ACTH and galanin concentrations also exhibited a strong positive correlation in single tumor cultures. At IPS sampling, mean basal plasma galanin concentrations in the dominant IPS were somewhat higher than those registered at the periphery (18.6 +/- 1.94 vs. 15.8 +/- 1.60 pmol/L, P = 0.05). Administration of CRH induced a modest but significant increase in galanin concentrations at all three sampling sites. No correlations were found between ACTH and galanin levels in the IPSs and at the periphery. Different from what was observed in patients with Cushing's disease, CRH did not modify plasma galanin concentrations in normal subjects. In conclusion, this study demonstrates that galanin is released by human tumoral corticotropes and responds to CRH. The role of locally produced galanin is, as yet, unknown but may possibly be that of a autocrine/paracrine modulator.

The desmopressin test in the differential diagnosis between Cushing's disease and pseudo-Cushing states.

Differentiating Cushing's disease (CD) from pseudo-Cushing (PC) states may still be difficult in current practice. Because desmopressin (1-deamino-8D-arginine vasopressin, DDAVP), a vasopressin analogue, stimulates ACTH release in patients with CD but not in the majority of normal, obese, and depressed subjects, we investigated its ability to discriminate CD from PC states. One hundred seventy-three subjects (76 with active CD, 30 with PC, 36 with simple obesity, and 31 healthy volunteers) were tested with an iv bolus of 10 microg DDAVP. Sixty-one of these subjects also underwent a control study with saline. DDAVP induced marked ACTH and cortisol rises in CD (P < 0.005 vs. saline, for both ACTH and cortisol) but not in PC. A significant ACTH elevation occurred upon DDAVP administration also in normal and obese subjects, but it was much smaller than that observed in patients with CD (P < 0.0001). A peak absolute ACTH increase (> or =6 pmol/L), after DDAVP, allowed us to recognize 66 of 76 patients with CD and 88 of 97 subjects of the other groups. The same criterion correctly identified 18 of 20 patients with mild CD (24-h urinary free cortisol < or = 690 nmol/day) and 29 of 30 PC, resulting in a diagnostic accuracy of 94%, which was definitely higher than that displayed by urinary free cortisol, overnight 1-mg dexamethasone suppression test, and midnight plasma cortisol. In conclusion, the DDAVP test seems to be a useful adjunctive tool for the evaluation of hypercortisolemic patients chiefly because of its ability to differentiate mild CD from PC states.

Effect of gamma-aminobutyric acid on growth hormone and prolactin secretion in man: influence of pimozide and domperidone.

To investigate the possibility that GH release induced by gamma-aminobutyric acid (GABA) in man is mediated by a dopaminergic mechanism(s), we evaluated the effect of two antidopaminergic compounds, pimozide and domperidone, on the plasma GH response to acute GABA administration (5 g, orally). Only the former compound can freely cross the blood-brain barrier. In 9 normal volunteers, GABA caused a significant increase of plasma GH levels (P < 0.0001 vs. GH levels in a group of 14 controls). In these subjects, pimozide (6 mg/day, orally, for 4 days) significantly blunted the GH elevation induced by GABA (P < 0.01). Unlike pimozide, in 8 additional subjects, domperidone (4 mg injected iv immediately before GABA administration) did not influence the GABA-induced GH response. GABA did not alter either baseline or pimozide-stimulated plasma PRL levels. Likewise, it did not significantly modify the brisk PRL rise after domperidone injection. These findings are consistent with the hypothesis that GABA-stimulated GH secretion is mediated via dopamine release at a suprapituitary level. With regard to PRL secretion, no GABA-dopamine interactions are readily apparent.

Glucocorticoid receptors in anorexia nervosa and Cushing's disease.

BACKGROUND: Patients with anorexia nervosa do not display cushingoid features in spite of elevated cortisol plasma levels. Whether a cortisol resistance or a reduced availability of the metabolic substrates necessary to develop the effect of glucocorticoids is responsible for this has not been established. METHODS: Twenty-two patients with severe restrictive anorexia nervosa, 10 patients with active Cushing's disease, and 24 healthy volunteers without psychiatric disorders or mood alterations were investigated. Glucocorticoid receptor characteristics were examined on mononuclear leukocytes by measuring [3H]dexamethasone binding and the effect of dexamethasone on [3H]thymidine incorporation, which represents an index of DNA synthesis. RESULTS: The number of glucocorticoid receptors on mononuclear leukocytes (MNL) was comparable in patients with anorexia nervosa, patients with active Cushing's disease, and normal subjects (binding capacity 3.3 +/- 0.23 vs. 3.7 +/- 0.30 and 3.5 +/- 0.20 fmol/10(6) cells). Conversely, glucocorticoid receptor affinity was significantly decreased in anorexia nervosa as well as in Cushing's patients compared to control subjects (dissociation constant 4.0 +/- 0.31 and 4.1 +/- 0.34 vs. 2.9 +/- 0.29 nmol/L, p < .001) and inversely correlated with the levels of urinary free cortisol in both groups of patients. Basal [3H]thymidine incorporation in MNL was significantly reduced in anorexia nervosa as well as in Cushing's patients compared to control subjects (p < .001) and was diminished by dexamethasone to an extent similar to control subjects in patients with anorexia nervosa, but significantly (p < .001) less in those with Cushing's disease. In patients with anorexia nervosa, the incorporation of [3H]thymidine into the MNL was inversely correlated with urinary free cortisol levels. CONCLUSIONS: These data indicate that the lack of cushingoid features in patients with anorexia nervosa is not ascribable to a reduced sensitivity to glucocorticoids but is more likely due to the paucity of metabolic substrates.

Alpha 2-adrenoceptor sensitivity in anorexia nervosa: GH response to clonidine or GHRH stimulation.

Growth hormone (GH) response to clonidine and growth hormone-releasing hormone (GHRH) stimulation, together with baseline somatomedin C (SmC) levels, were examined in parallel in a group of 21 patients with anorexia nervosa (AN) and in 10 controls. In addition, the Hamilton Rating Scale for Depression (HRS) was administered to the patients. Clonidine (2.5 micrograms/kg body weight, iv) induced GH elevations that were not significantly different between patients and controls. In contrast, GHRH (1 microgram/kg body weight, iv) produced a significantly higher GH response in anorectics than in controls. The ratio between GH responses (area under the curve, or AUC) to GHRH and to clonidine was significantly higher in patients than in controls. Baseline SmC levels (6 patients) were significantly lower in anorectics than in controls. Minor depressive symptomatology was present in all patients. When viewed in relation to the GH hyperresponsiveness to GHRH, the apparent normality of the response to clonidine in anorectics reflects the existence of an actual alpha 2-adrenoceptor subsensitivity. As clonidine reportedly acts via release of endogenous GHRH, an excessive, rather than a normal, GH response to clonidine was to be anticipated.

Lack of growth hormone response to acute administration of dexamethasone in anorexia nervosa.

High plasma growth hormone (GH) levels, associated with abnormal hormone responses to provocative stimuli, point to an altered GH secretion in anorexia nervosa. The GH-releasing effect of acutely administered glucocorticoids, firmly established in normal subjects, has not been reported in these patients. In this study, acute iv administration of 4 mg of dexamethasone, compared with saline, increased plasma GH in nine normal-weight women (AUC 848.2 +/- 127.95 vs 242.8 +/- 55.35 micrograms.l-1.min-1, p < 0.05, respectively) but was ineffective in 11 anorectic patients (AUC 3271.8 +/- 1407.11 vs 2780.0 +/- 1162.04 micrograms.l-1.min-1, NS). After dexamethasone, a significant lowering of plasma cortisol was observed in normal women (AUC 25367.0 +/- 3128.43 vs 47347.1 +/- 4456.61 nmol.l-1.min-1, after dexamethasone and saline, respectively, p < 0.05), but not in anorectic patients (AUC 77809.3 +/- 8499.92 vs 78454.9 +/- 7603.62 nmol.l-1.min-1, NS). In both groups, plasma adrenocorticotrophin (ACTH) displayed a significant decrease after dexamethasone (AUC 523.6 +/- 92.08 vs 874.2 +/- 115.03 pmol.l-1.min-1, p < 0.05, after dexamethasone and saline, respectively, in anorectic patients and 377.5 +/- 38.41 vs 1004.9 +/- 200.51 pmol.l-1.min-1, p < 0.05, in controls). However, when considering the hormonal decremental areas, a significant dexamethasone-induced ACTH inhibition, compared to saline, was evidenced in normal (delta AUC -414.4 +/- 65.75 vs 222.9 +/- 42.40 pmol.l-1.min-1, p < 0.05) but not in anorectic women (delta AUC -254.2 +/- 96.92 vs 2.9 +/- 132.32 pmol.l-1.min-1, NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Salivary cortisol measurement in normal-weight, obese and anorexic women: comparison with plasma cortisol.

OBJECTIVE: To compare salivary, plasma and urinary free cortisol (UFC) measurements in patients with anorexia nervosa, in whom an overdrive of the hypothalamic-pituitary-adrenal (HPA) axis is well established but information on salivary cortisol is lacking, in viscerally obese patients in whom subtle abnormalities of cortisol secretion and metabolism are postulated, and in normal-weight healthy women. PARTICIPANTS AND EXPERIMENTAL DESIGN: Measurement of salivary cortisol offers a convenient way to assess the concentrations of free, biologically active cortisol in plasma in different physiopathological settings. Forty-seven drug-free, newly diagnosed women with active restrictive anorexia nervosa, 30 restrictive anorexic women undergoing chronic psychopharmacological treatment, 47 women with mild-to-moderate visceral obesity, 103 women with severe central obesity and 63 normal-weight healthy women entered the study. Salivary and blood samples were collected at 0800 h, 1700 h and 2400 h, together with three consecutive 24-h urine specimens for UFC determination. In controls and patients with anorexia nervosa (n=83), salivary and plasma cortisol were also measured after a 1-mg overnight dexamethasone suppression test (DST). In patients with anorexia nervosa, mood was rated by the Hamilton scale for anxiety and depression. RESULTS: Untreated patients with anorexia nervosa showed increased plasma and salivary cortisol and UFC concentrations (all P<0.001 compared with controls), and decreased cortisol suppression after DST in plasma and saliva (P<0.0001 and P<0.005 respectively compared with controls). These alterations were less pronounced, although still statistically significant, in treated patients with anorexia nervosa. Salivary cortisol was highly correlated with paired plasma cortisol in the whole population and after splitting the participants by group (P<0.0001). However, for plasma cortisol values greater than 500 nmol/l (the corticosteroid-binding globulin saturation point), this parallelism was lost. Taking plasma cortisol as a reference, the level of agreement for post-dexamethasone salivary and plasma cortisol was 58.9% among suppressors and 77.8% among non-suppressors (chi2 test: P<0.01). Decreased 0800 h/2400 h cortisol ratios were observed in plasma and saliva in drug-free patients with anorexia nervosa (P<0.005 and P<0.05 respectively compared with controls), and in saliva in severely obese patients (P<0.05 compared with controls). Depression and anxiety scores were unrelated to cortisol concentrations in any compartment. CONCLUSIONS: Salivary cortisol measurement is a valuable and convenient alternative to plasma cortisol measurement. It enables demonstration of the overdrive of the HPA axis in anorexia nervosa and subtle perturbations of the cortisol diurnal rhythm in women with visceral obesity. With the establishment of more specific and widely acceptable cut-off values for dynamic testing, measurement of salivary cortisol could largely replace plasma cortisol measurement.

Altered morning and nighttime pulsatile corticotropin and cortisol release in polycystic ovary syndrome.

An overdrive of the hypothalamic-pituitary-adrenal (HPA) axis has been postulated in patients with polycystic ovary syndrome (PCOS). However, little is known concerning the pulsatile modes of corticotropin (ACTH) and cortisol secretion in these patients. To further investigate this issue, spontaneous ACTH and cortisol release were evaluated in 16 normal-weight patients with PCOS and 16 control women. Nine PCOS patients and eight controls were studied between 8 AM and 12 AM (noon), and seven PCOS patients and eight controls between 11 PM and 3 AM. Venous blood samples were taken at 10-minute intervals. Cluster analysis was used to assess ACTH and cortisol pulse frequency and amplitude, deconvolution to calculate mean hormone secretion rates, and approximative entropy (ApEn) to measure the orderliness of ACTH and cortisol time-series data. PCOS patients compared with controls displayed increased ACTH and cortisol release (area under the curve [AUC] and mean plasma concentration) both in the morning and at night. This was not due to increased hormonal secretory burst frequency, but to higher hormonal interpeak valley concentrations and, in the case of ACTH, nighttime pulse amplitudes. Mean ACTH and cortisol secretion rates also were increased in PCOS patients. Further, both controls and PCOS patients exhibited significant (0 to 20 minutes lagged) concordance between individual daytime pulsatile ACTH and cortisol release episodes. As shown by increased ApEn values, PCOS patients had more disorderly daytime cortisol release. In addition, the normal daytime correlation between the amount of pulsatile ACTH and cortisol release as observed in the controls was lost in PCOS patients. Finally, cross-correlation analysis showed a more prominent negative correlation in PCOS patients versus controls between plasma cortisol and 40- to 120-minute delayed ACTH concentrations in the morning, indicating a more sustained negative feedback of cortisol on ACTH release in PCOS at this time. Taken together, these findings demonstrate the existence of multifaceted dysregulation of the HPA axis in PCOS.

Effects of gamma aminobutyric acid (GABA) and muscimol on endocrine pancreatic function in man.

The high concentrations of gamma aminobutyric acid (GABA) in the pancreatic islets and the neurotransmitter role played by this amino acid in the central nervous system, make it plausible that GABA also intervenes in the control of endocrine pancreatic function. In 12 normal subjects, a single oral dose of 5 or 10 g GABA, as compared to placebo, caused a significant (p less than 0.01) and dose-dependent (p less than 0.01) increase of plasma levels of immunoreactive insulin, C peptide and glucagon, without affecting plasma glucose concentration. By contrast, in 15 additional subjects, a single oral dose of 5 mg muscimol, a specific GABA receptor agonist, did not consistently influence the above parameters. Although the lack of effects of muscimol might indicate that the action of GABA is not mediated through specific receptors, the results with GABA suggest that this amino acid plays a specific role in the regulation of endocrine pancreatic function.