Emanuela Faloia

Overexpression of the Renin-Angiotensin System in Human Visceral Adipose Tissue in Normal and Overweight Subjects*

To evaluate the expression of the renin-angiotensin system (RAS) genes in visceral (VAT) and subcutaneous adipose tissue (SAT) in normotensive subjects with different body mass index (BMI). Adipose tissue was obtained from 22 normotensive (12 normal weight and 10 overweight) patients during surgery for colecystectomy. Angiotensinogen (AGT), angiotensin II receptor type 1 (AT1), angiotensin converting enzyme (ACE) mRNA, and protein levels were measured by reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. The AGT mRNA and AT1 receptor mRNA levels were significantly higher in VAT than in SAT; AGT mRNA levels were higher, although not significantly, in overweight subjects in both SAT and VAT. There was no significant difference in ACE gene expression in the two tissues, and no expression of angiotensin II receptor type 2 (AT2). Finally, we failed to find mRNA for the renin gene in adipose tissue. The presence of AGT and ATI receptor in SAT and VAT was confirmed by Western blot analysis. Our study demonstrates the presence--and different levels of expression--of the various components of the RAS system (AGT, ATI, and ACE) in human SAT and VAT, and highlights the different role and regulation of the system in the two tissues. Its high expression in VAT suggests that its regulation and function are involved in all conditions where visceral adiposity is present.

Altered Cellular Ca2+ and Na+ Transport in Diabetes Mellitus

Platelet intracellular Ca2+ concentration ([Ca2+]i) and its response to stimuli (ADP and thrombin) were studied in 15 insulin-dependent and 22 non-insulin-dependent diabetes mellitus patients with the fluorescent probe Fura 2. The activity of Ca2+-ATPase and Na+-K+-ATPase, membrane fluidity, and cholesterol and phospholipid content were also determined in platelet membranes. Compared with control subjects, diabetic patients showed 1) increased platelet [Ca2+]1 in the resting state, 2) higher Ca2+ levels after stimulation with thrombin and ADP, due entirely to increased resting concentrations, 3)reduced activity of Na+-K+-ATPase, 4)increased activity of Ca2+-ATPase, 5) higher fluidity of the platelet membrane, and 6)increased membrane concentration of total phospholipids. Na+-K+-ATPase activity was inversely related to platelet [Ca2+]1 in each group studied, whereas Ca2+-ATPase activity was positively correlated with intracellular Ca2+ levels. The data obtained in diabetic subjects suggest an abnormality in Ca2+ and Na+ transport across the platelet membrane that might be responsible for the reported platelet hyperreactivity to stimuli in diabetes.

Decreased nitric oxide levels and increased calcium content in platelets of hypertensive patients

Decreased levels of nitric oxide (NO) may contribute to impaired endothelium-dependent vasodilatation in essential hypertension. Moreover, in hypertension, major platelets aggregation and endothelial adhesion, and increased atherogenetic risks are also present. Nitric oxide produced by platelet NO synthase, which is similar to endothelial NO synthase, inhibits platelets aggregation by increasing cytoplasmic cyclic GMP levels and contributes in a major way to the antithrombogenic properties of endothelium. The aim of this study was to investigate platelet NO production and cytosolic Ca2+ levels in patients with essential hypertension and in healthy subjects. We studied NO production in 36 subjects (21 patients had essential hypertension and 15 subjects were normotensive); NO synthase activity was evaluated by measuring nitrite levels by the Griess reaction in the supernatant of sonicated platelets. Cytosolic Ca2+ levels were measured in intact platelets using the fluorescent probe Fura 2-AM. Nitric oxide levels in platelets were found higher in normotensive than in hypertensive patients (P < .0001). Nitric oxide levels in hypertensive women were significantly higher than in hypertensive men (P < .001). Hypertensive women and men had lower levels of nitrite than normotensive women and men (P < .001 and P < .002, respectively). Platelet cytosolic Ca2+ levels were higher in hypertensive patients than in normotensive subjects (P < .001). An inverse correlation was found between platelet cytosolic Ca2+ and NO levels (r = 0.74, P < .002). These data confirm the link between hypertension and altered platelets function and suggest a role for NO in cardiovascular events. Moreover, the higher levels of nitric oxide in child-bearing age women than in men further support the protective effect of estrogens on cardiovascular diseases.

Body composition, fat distribution and metabolic characteristics in lean and obese women with polycystic ovary syndrome

The polycystic ovary syndrome (PCOS), characterized by chronic anovulation and hyperandrogenism, has many features of metabolic syndrome and can be considered a metabolic disease. Approximately 50% of patients with PCOS are overweight or obese with abdominal fat accumulation. Some metabolic alterations and abdominal fat distribution have also been reported in lean women with PCOS. The aim of this study was to evaluate the effect, if any, of obesity on metabolic features, body composition and fat distribution in patients with PCOS. Body composition and abdominal fat distribution (evaluated by DEXA), waist circumference, blood pressure, lipid profile, glucose tolerance and homeostasis model assessment index were determined in 23 lean [mean age 23±5 yr, mean body mass index (BMI) 22±2 kg/m2] and 27 overweight-obese (mean age 21±5 yr, mean BMI 32±5 kg/m2) patients with PCOS and in 20 age- and weight-matched eumenorrhoic women. Patients exhibited slight but non-significant differences in metabolic parameters, waist circumference, blood pressure and total and abdominal fat content compared with weight-matched controls. None of the lean subjects suffered from metabolic syndrome according to the National Cholesterol Education Program — Adult Treatment Panel III (NCEP-ATPIII) criteria as opposed to 10 overweightobese patients and three overweight-obese control subjects (37% and 33.3% of each subgroup, respectively). Our data do not show significant metabolic alterations in lean PCOS women. Results indicate that obesity seems to underpin the metabolic alterations exhibited by the overweightobese patients. However, since women with PCOS are at increased cardiovascular risk, further studies are needed to evaluate metabolic alterations and body composition in these patients.

Comparison of circulating and local adipose tissue renin-angiotensin system in normotensive and hypertensive obese subjects

The renin-angiotensin-aldosterone system (RAAS) plays a well-recognized role in the regulation of BP and in salt and water balance. Since hypertension affects a considerable proportion of obese patients, circulating RAAS has been studied in obese subjects with and without hypertension, albeit with conflicting results. Furthermore, attention has recently focused on the expression of the components of the Renin-angiotensin system (RAS) in some organs, including adipose tissue where it seems to be involved in the regulation of growth and differentiation. The aim of our study was to investigate circulating RAAS and adipose tissue RAS in obese patients with and without hypertension and in matched controls. PRA, and plasma and urinary aldosterone levels were measured in 35 obese, 30 hypertensive obese patients and in 20 controls. In addition, the expression of angiotensinogen (AGT) and angiotensin II type 1 receptor (AT1) genes was studied in sc adipose tissue from 8 obese, 6 hypertensive obese and 6 healthy subjects. As previously demonstrated in other studies, there were no significant differences in the levels of circulating RAAS components in the 3 groups. As regards local RAS, interestingly, we found that AT1 gene was significantly more expressed in sc adipose tissue from obese patients with hypertension than in those without hypertension and controls. By contrast, AGT levels were similar in the 3 groups. Our data do not support the hypothesis of an involvement of circulating RAAS in the development of obesity-related hypertension. On the other hand, local RAS seems to be differently regulated in sc adipose tissue from obese patients with hypertension with respect to normotensive obese patients and controls.

Use of the desmopressin test in the differential diagnosis of pseudo-Cushing state from Cushing's disease.

CONTEXT The desmopressin (DDAVP) test has been proposed to discriminate Cushings disease (CD) from pseudo-Cushing states (PC); however, current information on its value is scarce and contradictory. OBJECTIVE The aim of the study was to assess the ability of the DDAVP test in distinguishing between these conditions, with emphasis on subjects with mild hypercortisolism. DESIGN AND SETTING We conducted a retrospective/prospective study at the Division of Endocrinology, Polytechnic University of Marche, Ancona, Italy. PATIENTS The study included 52 subjects with CD, 28 with PC, and 31 control subjects (CT). INTERVENTION(S) We performed the DDAVP test and standard diagnostic procedures for the diagnosis of Cushings syndrome. MAIN OUTCOME MEASURE(S) The diagnosis/exclusion of CD was measured. RESULTS Interpretation of the DDAVP test based on percentage and absolute increment of cortisol and ACTH did not afford acceptable values of both sensitivity (SE) and specificity (SP). CD diagnosis based on simultaneous positivity for basal serum cortisol greater than 331 nmol/liter and absolute ACTH increment greater than 4 pmol/liter and its exclusion in subjects negative for one or both measures yielded an SE of 90.3% and an SP of 91.5%. The approach was also highly effective in distinguishing PC from: 1) CD with moderate values of urinary free cortisol (SE, 86.9%; SP, 92.8%); 2) CD with moderate values of serum cortisol after dexamethasone suppression (SE, 86.6%; SP, 92.8%); and 3) CD with moderate values of midnight serum cortisol (SE, 100%; SP, 92.8%). CONCLUSIONS Interpretation of the DDAVP test through a combination of parameters allowed effective discrimination of CD from PC, even in subjects with mild hypercortisolism.

Diabetes mellitus induces red blood cell plasma membrane alterations possibly affecting the aging process

Various alterations of red blood cell (RBC) plasma membrane appear both in diabetes mellitus and during the physiological aging process. Diabetes mellitus decreases RBC life-span; therefore, it may change the plasma membrane by acting through its effect on the aging process. In order to clarify the issue, RBCs from normal subjects and insulin-dependent diabetic patients were fractionated in five subpopulations of different mean age (fraction 1: early young RBC, fraction 5: mature RBC). Thereafter, plasma membranes were prepared and enzymatic activities, membrane fluidity and lipid peroxidation were evaluated. NA+, K(+)-ATPase activity decreased during aging and it was higher in all RBC subpopulations from normal subjects in comparison to diabetic patients. Next, lipid peroxidation and fluidity increased during aging in both the study groups; in this case, however, in all subpopulations, except for that from fraction 1, RBCs from diabetic patients showed higher membrane fluidity and lipid peroxidation in comparison to normal subjects. Data herein reported suggest that diabetes mellitus affects the plasma membrane independently of (lipid peroxidation and fluidity) or dependently on (Na+, K(+)-ATPase) its effect on aging. In the case of lipid peroxidation and fluidity diabetes mellitus seems to affect the membrane by decreasing RBC life span, whereas in the case of Na+K(+)-ATPase it seems to alter this enzymatic activity which in turn might affect RBC aging. Acetylcholinesterase activity decreased during aging in RBCs from normal subjects, but it increased in RBCs from diabetic patients; RBC subpopulation from fraction 1, on the other hand, showed similar values in normal subjects and diabetic patients. In this case the effect of diabetes mellitus appears only during aging.

Adipose Tissue as an Endocrine Organ? A Review of Recent Data Related to Cardiovascular Complications of Endocrine Dysfunctions

Clinical and experimental data obtained in the last few years have modified the concept of adipose tissue as one solely directed at energy storage and release. The adipose tissue is a target organ for glucocorticoids and several studies have been carried out on the function of hypothalamic‐pituitary‐adrenal axis in obese subjects without conclusive results. A recent and innovative finding is that adipose tissue can produce cortisol from its inactive precursor, cortisone. The identification of leptin, a hormone synthesised by fat tissue, has ushered in the modern view of this tissue as a true endocrine organ. Leptin is produced primarily by subcutaneous and to a lesser extent by visceral adipose tissue, and has a central role in controlling body weight and, especially in regulating fat stores. Leptin is also involved in several complex functions, including physiological processes associated with puberty. Another hormone of fat tissue is angiotensinogen, which is produced in larger amounts by visceral than subcutaneous fat. Human and animals adipose tissue express a whole renin‐angiotensin system (RAS). Angiotensin II, the final effector of this system is probably produced locally by adipose tissue. The function of adipose RAS is not well known. RAS can participate together with other hormones and substances, in adipocyte differentiation and fat tissue growth, but could be also involved in the pathogenesis of complications of obesity including arterial hypertension.

Corticotrophin-releasing hormone and desmopressin tests in the differential diagnosis between Cushing's disease and pseudo- Cushing state: a comparative study

Background  We recently proposed a new and effective way of interpreting human corticotrophin‐releasing hormone (hCRH) and desmopressin (DDAVP) tests, for the differential diagnosis between Cushing’s disease (CD) and pseudo‐Cushing state (PC), based on the simultaneous analysis of ACTH and cortisol.

Human corticotropin releasing hormone test performance in the differential diagnosis between Cushing's disease and pseudo-Cushing state is enhanced by combined ACTH and cortisol analysis

OBJECTIVE Corticotropin releasing hormone (CRH) test does not reliably distinguish Cushings disease (CD) from normality or pseudo-Cushing state (PC). We assessed whether this could be achieved with a novel approach while preserving the ability of the test to distinguish CD from ectopic ACTH syndrome (EAS). Design Retrospective/prospective study. SUBJECTS AND METHODS We studied 51 subjects with CD, 7 with EAS, 26 with PC, and 31 controls (CT). Human CRH (hCRH) test was performed at 0830 h by measuring plasma ACTH and serum cortisol at -15, 0, 15, 30, 45, 60, 90, and 120 min. RESULTS The area under the curve-ACTH exhibited a significant negative correlation with baseline serum cortisol in CT and PC, but not in CD or EAS patients. ACTH response to hCRH was blunted in PC compared with CT, whereas peak serum cortisol was higher in PC than in CT subjects. These findings suggested that ACTH-dependent Cushings syndrome can be diagnosed by the presence of two hCRH test parameters and excluded if either or both are absent. Application of i) basal serum cortisol >12 microg/dl and peak plasma ACTH >54 pg/ml, or ii) peak serum cortisol >21 microg/dl and peak plasma ACTH >45 pg/ml, had 91.3% (95% confidence intervals (CI) 81-97.1) and 94.8% (CI 85.6-98.9) sensitivity and 98.2% (CI 90.6-99.9) and 91.2% (CI 80.7-97) specificity respectively, in diagnosing ACTH-dependent Cushings syndrome. The >14% serum cortisol increase from mean baseline values to the mean of 15 and 30 min values in patients who were positive for the test completely discriminated between CD and EAS. CONCLUSIONS Simultaneous plasma ACTH and serum cortisol analysis enables the hCRH test to distinguish CD from PC and from normality, while preserving its ability to discriminate CD from EAS.