Identification of novel GH-regulated genes in C2C12 cells.

Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.

Musculoskeletal complications of acromegaly: what radiologists should know about early manifestations.

The purpose of this article is to summarise the early musculoskeletal complications of acromegaly. Some of the early signs of acromegaly may be evaluated by the musculoskeletal radiologist. In the early stage of disease, peripheral nerve enlargement associated with carpal tunnel syndrome or cubital tunnel syndrome and thickening of retinacula, such as A1 pulley in trigger finger, represent the features that may be seen by radiologists and are worthy of an endocrinological evaluation. Due to the insidious nature of the disease, the diagnosis of acromegaly is significantly delayed. Few and nonspecific symptoms characterise the initial phases of the disease, and therefore, most patients will have generally consulted many specialists (most frequently musculoskeletal radiologists) before an adequate endocrinological assessment is performed. For this reason, initial clinical signs are much more important than symptoms for an early diagnosis of acromegaly. The first and most important therapeutic approach to acromegaly is early diagnosis, whereas the therapeutic goals are to eliminate morbidity and reduce mortality to the expected age- and sex-adjusted rates and prevent the development of systemic complications. Musculoskeletal radiologists should be aware that these features may be early manifestations of acromegaly. When both radiological and clinical abnormalities are present, an endocrinological workup is useful to diagnose the disease in an early phase.

Body composition after endogenous (Cushing's syndrome) and exogenous (rheumatoid arthritis) exposure to glucocorticoids.

Exposure to chronic glucocorticoid (GC) excess determines changes in body composition. The aim of the study was to compare body composition in women exposed to endogenous hypercortisolism (Cushing's syndrome, CS), exogenous glucocorticoid treatment (rheumatoid arthritis, RA) and controls. Fifty-one CS women, 26 RA women treated with low-dose prednisone (5 mg/day or 10 mg/2 days), and 78 female controls were included. Fourteen CS patients were hypercortisolemic, 37 in remission (10 required hydrocortisone substitution after surgery). Body composition parameters were measured by dual-energy X-ray absorptiometry scanning (DEXA). RA patients had a greater waist-hip ratio (WHR) (p<0.01), less lean body mass (LBM) (p<0.01), and lumbar bone mineral density (BMD) (p<0.01) than controls. CS patients, globally and those with cured disease, had more total fat (both percentage and kg) and trunk fat percentage, and less whole body-BMD than RA patients (p<0.05, p<0.01, p<0.05, respectively). Active CS patients had less whole body-BMD and more LBM than RA patients (p<0.05, p=0.01, respectively). Cured CS patients not taking hydrocortisone had more total fat [both percentage (p<0.05) and kg (p<0.05)], trunk fat percentage (p<0.05), lumbar BMD (p<0.01) than RA patients. Cured CS patients requiring hydrocortisone only differed from RA patients by smaller WHR (p<0.01). All the differences in BMD disappeared when the data were reanalyzed including only the estrogen-deficient groups. Hypercortisoliof CS determines an irreversible increase in body fat, greater than in RA. Endogenous and exogenous exposure to GC negatively affects body composition by increasing the WHR. There appears to be no additional effect on BMD in estrogen-deficient women.

Diagnosis of cure in Cushing's syndrome: lessons from long-term follow-up.

It is generally assumed that endocrine 'cure' of hypercortisolism after successful treatment for Cushing's syndrome (CS) is associated with reversal of increased morbidity and mortality, typical of the active disease. However, recent data do not support this idea; increased cardiovascular risk is still present 5 years after endocrine cure, and health-related quality of life (HRQoL), although improved when compared to the active phase of hypercortisolism, is still impaired when compared to normal population. Abnormal body composition typical of hypercortisolism (i.e., increased total and trunk fat, reduced bone mass and lean body mass) is not completely normalized, even years after controlling hypercortisolism. Thus, control of hypercortisolism in CS does not normalize HRQoL, long-term cardiovascular risk and morbidity, body composition nor some metabolic parameters. Whether the same occurs in patients exposed to pharmacological doses of exogenous glucocorticoids, and whether the body composition abnormalities associated with the exposure to exogenous glucocorticoids are reversible or not, are worth considering.

Sympathovagal imbalance in transsexual subjects.

CONTEXT: Autonomic nervous system imbalance is related to cardiovascular risk. Heart rate variability (HRV) indexes are associated with age, race, and sex, but the role of sex hormones is still unknown. OBJECTIVE: To evaluate sympathovagal balance (SB) in transsexuals. PATIENTS: Eighteen transsexual subjects, 12 male-to-female (group 1) and 6 female- to-male (group 2), compared with 34 age-matched controls: 17 males (group 3) and 17 females (group 4). Autonomic testing of SB was performed by Power Spectral Analysis (PSA) of HRV in clinostatism (c) and orthostatism (o). PSA identifies power peaks: high frequency (HF) expresses vagal activity, while low frequency (LF) expresses sympathetic activity. RESULTS: Group 1 showed lower LFc than groups 2, 3, and 4 (p<0.001, p=0.05, p<0.001, respectively), and lower LFo than groups 3 and 4 (p=0.01); HFc was lower than in groups 2, 3, and 4 (p=0.02, p=0.02, p<0.001, respectively), and HFo was lower than in groups 3 and 4 (p<0.001). LFo/HFo ratio was higher in group 1 than in group 4 (p<0.001). No differences emerged between groups 2 and 3. Group 2 showed lower HFo than group 4 (p=0.03), and a higher LFo/HFo ratio (p=0.01). Group 3 showed lower HFo and HFc than group 4 (p=0.02, p=0.05, respectively), and a higher LFo/HFo ratio (p=0.03). CONCLUSION: In this study we found a sympathovagal imbalance due to a reduced sympathetic and parasympathetic influence on heart rate. Sex hormone therapy per se may play a role in this imbalance, and HRV measurement could be useful in detecting cardiovascular risk in transsexuals.

Somatostatin receptor scintigraphy in thoracic diseases.

Somatostatin (SS) receptor scintigraphy is useful for the diagnosis of lesions with high density of SS receptors, and above all neuroendocrine tumors. For several years, only indium-labeled octreotide has been applied to visualise in vivo tissues with SS receptor overexpression. Radiolabeled octreotide became the gold standard for the detection of neuroendocrine tumors. More recently, however, several new SS analogues with varying affinity for SS receptor subtypes have been developed, and different radionuclides as radiolabels have been introduced. Moreover, significant improvements have been made by the introduction of hybrid machines, such as single photon emission computed tomography/ computed tomography (SPECT/CT) or positron emission tomography (PET)/CT that enable to perform whole-body imaging quickly and with high anatomical resolution in several body areas, including the chest. The development of more specific radiopharmaceuticals, together with the modern technique of imaging, may provide excellent quality images with high contrast, allowing to depict very small lesions and making them easy to interpret. Indeed, in the management of SS receptor-positive lesions, the contribution of nuclear medicine is essential in several clinical settings, such as initial diagnosis, disease staging, follow-up, treatment planning, and treatment monitoring. In addition, the tracer uptake might be used as a prognostic parameter and as a predictor of treatment response. In the chest, apart in (neuro)endocrine tumors, SS receptors have been demonstrated in granulomatous diseases, like sarcoidosis and other immune-mediated disorders, such as anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis. In this paper we review and discuss the role of SS receptor scintigraphy in diagnosis, staging or follow- up of thoracic SS receptor-positive lesions.

The rational use of fine needle aspiration biopsy (FNAB) in diagnosing thyroid nodules.

AIM: Fine needle aspiration biopsy (FNAB) plays a crucial role in the diagnosis of thyroid nodules and enables the number of surgical operations to be reduced. Theoretically, FNAB should be carried out on all nodules, though currently only those displaying certain characteristics are biopsied. Indeed, to perform FNAB on all nodules may be regarded as an excess of zeal. Therefore, it seems advisable that the endocrinologist should be able to confirm on the spot the necessity and utility of FNAB. METHODS: We evaluated on a sample of 263 consecutive requests (209 female, 57 male; age 56.7+/-13.7 years) for FNAB in 2004: 1) the appropriateness of the investigation, 2) expected efficacy, 3) practical efficacy, 4) efficiency. FNAB was performed under echo-guidance in accordance with the standard technique. In 50%, 36%, 6%, 3%, 2% and 1% of cases, the echographic diagnosis was of MNG, UNG, pseudo-nodular lesion in ATD, lymph-node, neck cyst, suspected parathyroid lesion and tumefaction of the salivary glands, respectively. A pre-FNAB clinical risk score was assigned to each case on the basis of clinical and echographic data, with a maximum possible score of 11. The results of FNAB were subdivided into 5 categories according to the criteria of the BTA (Thy1-Thy5). After FNAB, a decisional category was assigned, ranging from ''observation'' to ''surgery''; this was subsequently (7-18 months) compared with the management strategy adopted by the attending physician. Information was gathered by means of telephone enquiry. RESULTS: 1) Appropriateness: on the basis of clinical and echographic findings, FNAB was not judged appropriate in 24% of cases because of either the lack of confirmation of a significant target (34%) or a low pre-FNAB risk score (range 0-2) (66%). The decisional category was ''observation'' in 87% of cases and ''further investigation'' in 13%. 2) Expected efficacy: FNAB was performed in 76% of cases. The biopsies (3%) performed on swollen lymph-nodes and extra-thyroid neck tumefactions, in which biochemical evaluation was positive, proved to be diagnostic but not classifiable according to the BTA. In 82% of the remaining cases, the result was Thy2 (observation) or Thy 4-5 (surgery). Thy3 results (surgery) were rare (1%). Thy1 results (16%) were yielded by the aspiration of colloid cysts (29%), solid lesions (10%) characterised by means of PTH-FNAB and Tg-FNAB, nodules (9%) no longer detectable on repetition of FNAB, nodules (16%) in which FNAB was already a repetition of a non-diagnostic investigation (2003), and nodules (9%) in which the presence of normal thyrocytes, ''hot'' scintigraphic image and prior decision of the surgeon advised against repeating FNAB. Of the patients with Thy1 results, 26% refused to repeat FNAB. In all, 95% of FNAB supported by biochemical evaluation yielded results that usefully contributed to patient management. The correlation between pre-FNAB clinical risk and cytological score according to the BTA proved significant (P<0.001). No difference in diameter was recorded between nodules with adequate cytology (23.3+/-0.9 mm) and those with inadequate cytology (25.2+/-1.6 mm). 3) Practical efficacy: 75% of patients were reached by telephone. In most cases, observation was the most frequent clinical choice, after echography and/or FNAB. The decisional category assigned after FNAB correlated significantly (P<0.001) with the approach adopted by the attending physician. d) Efficiency: following FNAB, 11 patients were assigned to surgery. DTC was detected in 100% of these cases (1 follicular carcinoma, 1 insular carcinoma, 9 papillary carcinoma). The success of FNAB (9/11) in detecting lesions that proved malignant on histological examination (11/11) was significant (P<0.05). Of the 2 Thy 3 cases, 1 was follicular carcinoma and 1 was follicular adenoma with adjacent papillary carcinoma. The incidence of thyroid carcinomas in the population studied was 5.5%. CONCLUSIONS: 1) Together with clinical-biochemical evaluation, echo-guided FNAB re-mains the first-line diagnostic test in the management of thyroid nodules; 2) a pre-FNAB clinical risk score is useful in limiting the number of probably inappropriate investigations; 3) efficacy, in terms of cytology results that are useful for patient management after FNAB (and after biochemical evaluation, when indicated) is high, enabling patients to be stratified in classes with different subsequent pathways; 4) in the vast majority of cases, FNAB influences subsequent clinical decisions; 5) false negatives cannot be excluded, while false positives are practically nil; 6) further indications may be yielded by studies on larger populations, and new prospects may emerge from the application of other techniques associated to FNAB.

White matter alterations in the brains of patients with active, remitted, and cured cushing syndrome: a DTI study.

BACKGROUND AND PURPOSE: Cushing syndrome appears after chronic exposure to elevated glucocorticoid levels. Cortisol excess may alter white matter microstructure. Our purpose was to study WM changes in patients with Cushing syndrome compared with controls by using DTI and the influence of hypercortisolism. MATERIALS AND METHODS: Thirty-five patients with Cushing syndrome and 35 healthy controls, matched for age, education, and sex, were analyzed through DTI (tract-based spatial statistics) for fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity (general linear model, family-wise error, and threshold-free cluster enhancement corrections, P < .05). Furthermore, the influence of hypercortisolism on WM DTI changes was studied by comparing 4 subgroups: 8 patients with Cushing syndrome with active hypercortisolism, 7 with Cushing syndrome with medication-remitted cortisol, 20 surgically cured, and 35 controls. Cardiovascular risk factors were used as covariates. In addition, correlations were analyzed among DTI values, concomitant 24-hour urinary free cortisol levels, and disease duration. RESULTS: There were widespread alterations (reduced fractional anisotropy, and increased mean diffusivity, axial diffusivity, and radial diffusivity values; P < .05) in patients with Cushing syndrome compared with controls, independent of the cardiovascular risk factors present. Both active and cured Cushing syndrome subgroups showed similar changes compared with controls. Patients with medically remitted Cushing syndrome also had reduced fractional anisotropy and increased mean diffusivity and radial diffusivity values, compared with controls. No correlations were found between DTI maps and 24-hour urinary free cortisol levels or with disease duration. CONCLUSIONS: Diffuse WM alterations in patients with Cushing syndrome suggest underlying loss of WM integrity and demyelination. Once present, they seem to be independent of concomitant hypercortisolism, persisting after remission/cure.

Current diagnostic guidelines for biochemical diagnosis of acromegaly.

Acromegaly is a rare and chronic disease that, in the majority of cases, is due to the presence of a benign growth hormone (GH)-producing tumor of the pituitary. In the past, the diagnosis of acromegaly was established basically on physical changes, and only the patients with a severe clinical picture were brought to medical attention. The development of a radioimmunoassay for detecting GH allowed for the first time to confirm the diagnosis biochemically. Subsequently, methods for measuring insulin-like growth factor 1 (IGF-I) became available and added another important biochemical marker for the diagnosis and follow-up of these patients. Progressive improvements in assay methods have allowed for progressively better definitions of normality and, as a result, have permitted the diagnosis to be biochemically established in patients with only mild forms of the disease. Moreover, new potential markers of disease activity, such as other GH-dependent IGF system parameters, have been investigated and proposed in the diagnostic work-up and for monitoring the therapeutic outcome. Optimal assessment of disease activity, for both diagnostic and follow-up purposes, is mandatory. This subject has been strongly debated regarding proper cut-off values using highly sensitive GH assays as well as the problems linked to IGF system components measurement. Consequently, several consensus reports, as well as original studies, have been issued giving special attention to diagnostic procedures, cut-off revisions and definition of disease activity. The present review discuss principally the biochemical diagnosis of acromegaly based on these articles and on the experience collected in an endocrinological unit considered as reference center for pituitary diseases.

Gender dimorphism in body composition abnormalities in acromegaly: males are more affected than females.

BACKGROUND: Acromegaly changes body composition (BC), but long-term gender differences have not been reported. OBJECTIVE: To evaluate BC in active and controlled acromegalic patients. DESIGN AND METHODS: Clinical and biochemical variables and BC (by dual-energy X-ray absorptiometry) were evaluated in 60 acromegalic patients (19 active, 41 controlled) and 105 controls, matched for age and gender. RESULTS: Acromegalic males (n=24) had more total mass (89+/-13 vs 76.5+/-15.3 kg, P<0.001), lean body mass (LBM; 64.6+/-8.7 vs 56.4+/-5.8 kg, P<0.001), and bone mineral content (BMC; 2.9+/-0.5 vs 2.6+/-0.3 kg, P<0.05) than controls (n=33). Controlled male patients (n=14) had more total mass (89+/-14.7 vs 76.5+/-15.3 kg, P<0.05) and a trend to have more LBM (61.8+/-9.4 vs 56.4+/-5.8 kg, P=0.065) than controls. Only in active disease was a decrease in fat mass (FM) observed, compared with controlled patients and controls (males: 19.5+/-5.3 vs 27+/-6.2 and 25.9+/-4%, P<0.001; females: 30.3+/-6.7 vs 37.1+/-5.8 and 36.5+/-6.6%, P<0.01). In females, no further differences were observed. No differences in BMC were found between eugonadal and hypogonadal acromegalic patients, but in hypogonadal females, acromegaly appeared to prevent the BMC loss seen in hypogonadal postmenopausal controls. GH and IGF1 levels were negatively correlated with FM (males, P<0.05; females, P<0.001), but in the regression analysis GH was a predictor of FM only in women. CONCLUSIONS: Control of acromegaly reverts decreased FM in both genders; only in males more total mass and a trend for more LBM persist. The anabolic effect of GH on bone reverted in cured males, but persisted in females and appeared to override the bone loss of menopause.

Pituitary tumor disappearance in a patient with newly diagnosed acromegaly primarily treated with octreotide LAR.

We describe the case of an acromegalic patient primarily treated with octreotide LAR in whom the pituitary tumor disappeared after 18 months of treatment. A 68-yr-old woman, with clinical suspicion of acromegaly, was admitted to our Unit with the ultrasonographical evidence of cardiac hypertrophy, arrhythmias, right branch block and interatrial septum aneurism. She referred hands and feet enlargement since the age of 30 and facial disfigurements since the age of 50. At the age of 45 she underwent surgery for carpal tunnel syndrome and at the age of 61 an euthyroid nodular goiter was diagnosed. Hormonal evaluation showed elevated circulating GH levels (25+/-3.2 ng/ml), not suppressible after oral glucose load, and elevated IGF-I levels (646 ng/ml), whereas the remaining pituitary function was normal. Visual perimetry was normal, whereas magnetic resonance imaging (MRI) showed an intrasellar pituitary adenoma with maximal diameter of 9 mm. In order to improve cardiovascular function before surgery, the patient started octreotide LAR 20 mg every 4 weeks for 3 months. Then based on IGF-I values, the dose was adjusted to 30 mg. After 6 months a second MRI showed significant tumor reduction (>50% of baseline maximal diameter), GH and IGF-I were within the normal range and the patient continued the treatment. After one-year therapy, an improvement of cardiac alterations was recorded and the patient was referred to the neurosurgeon. However, she refused the operation. At 18-month follow-up, MRI showed the complete disappearance of direct and indirect signs of pituitary adenoma. To our knowledge, this is the first case of complete radiological remission of pituitary tumor during octreotide LAR treatment in acromegaly.

Potential indications for somatostatin analogues: immune system and limphoproliferative disorders.

Among hormones and neuropeptides influencing the immune system, somatostatin seems to play a key role not only in inhibiting specific immune cell activities, but also in promoting selected functions of particular immune cell subsets. Indeed, controversial effects have been observed in experimental conditions where somatostatin seems to stimulate certain cell functions, such as secretion of specific products (immunoglobulin, cytokines), cell migration and adhesion to extracellular matrix components. However, interestingly, cortistatin (CST), a neuropeptide that strongly resembles somatostatin, from both the structural and functional points of view, seems to have potential roles in regulating immune responses, as well as other lymphoid cell functions. The unexpected wide distribution of CST in a number of human organs, but particularly in immune cells, points to a broader physiological role of CST than previously presumed. The actions of somatostatin and its synthetic analogs (SSA) are mediated by five membrane G protein-coupled receptors subtypes (SSTR1-5), displaying a tissue specific distribution. The majority of somatostatin-target tissues, including lymphoid tissues, may co-express multiple somatostatin receptor (SSTR). The number of SSTRs in lymphoid cells is significantly lower compared to neuroendocrine tissues. However, the presence of receptors allowed the localization by in vivo SSTR scintigraphy of lymphoproliferative disorders, as well as granulomatous and autoimmune diseases. In specific cases, this technique may contribute to establishing the diagnosis and staging the disease. Recent studies evaluating the specific and quantitative SSTR distribution in lymphoid organs and cells, in both normal conditions and immune disorders, have largely contributed to better understand the phenomenology of in vivo receptor imaging and also the involvement of the different SSTR in determining the uptake of radiolabeled SSAs. Moreover, since lymphomas are highly radiosensitive malignancies, a promising approach in refractory patients with malignant lymphomas may be represented by radionuclide-targeted therapy with radioactive-coupled SSAs combined with gene therapy. This latter technique seems effective in inducing the expression or increasing the number of given SSTR in order to ameliorate the impact of radionuclide-targeted therapy. Medical treatment of lymphoproliferative diseases with currently available synthetic analogs have produced unsatisfactory and conflicting results. This might be due to the affinity of the current available SSAs for specific SSTR. However, the synthesis of new compounds with distinct properties has reopened a challenge in this field. The application of receptor-based localization and anti-tumor strategies should also be taking into account the new knowledge recently emerged on the physiopathology of neuropeptide receptors: firstly, neuropeptide receptor homo- and heterodimerization, which may involve different subtypes of SSTRs, as well as other neuropetide receptors, and secondly, the role of endogenous SSTR ligands, such as CST.