Insights into the molecular function of the inactivating mutations of B-Raf involving the DFG motif.

BRAF gene mutations have been associated with human cancers. Among the naturally occurring mutations, two that involve amino acids of the conserved DFG motif in the activation loop (D594V and G596R), appear to be inactivating. Aim of this study was to analyze the molecular mechanisms involved in the loss of function of B-Raf inactivating mutation G596R. Furthermore, the ability of the B-Raf DFG motif mutants to generate heterodimers with C-Raf and the possible functional consequences of the B-Raf/C-Raf heterodimer formation was examined. Wet molecular experiments in HEK293T cells demonstrate that B-Raf(G596R) is a kinase-impaired mutant. Molecular dynamics simulations show that the loss of function of B-Raf(G596R) depends on a restraining effect of Arg596 on the catalytic residue Asp594, which results in the loss of the appropriate spatial localization and/or conformation of the latter necessary for anchoring ATP to the enzyme. Exploration of B-Raf/C-Raf heterodimer formation indicates the occurrence of functioning heterodimers in the case of all the DFG B-Raf mutants, independently from the expected differences in spatial conformation of the activation loop, although the transforming activity of the mutants appear negligible. In conclusion, this study delivers novel information on the functional properties of the B-Raf DFG motif inactivating mutants and on the mechanisms driving B-Raf/C-Raf heterodimerization and consequent C-Raf transactivation.

Recent advances in adrenal autoimmunity.

Autoimmune Addison's disease (AAD) results from the immune-mediated destruction of adrenocortical cells. AAD is a major component of the autoimmune polyendocrine syndromes type 1 (APS 1) and type 2. The adrenal autoimmune process is made evident by the apperance of circulating autoantibodies against the steroidogenic enzyme 21-hydroxylase. Detection of 21-hydroxylase in patients with endocrine autoimmune diseases enables the identification of subjects with preclinical AAD. An impaired response to a corticotrophin stimulation test marks the irreversible stage of preclinical AAD and predicts progression towards clinical AAD in over 80% of cases. APS 1 is caused by mutations of the autoimmune regulator (AIRE) gene, which encodes an activator of transcription, Aire, that induces the expression of autoantigens in thymic medullary epithelial cells and promotes immunological tolerance. Isolated and APS 2-related AAD is an autoimmune disease with evidence for complex genetic susceptibility caused by T-cell-mediated destruction of adrenocortical cells, with a major contribution of HLA genes. The target cells in the adrenal cortex participate in the immune reaction by releasing chemokines, such as CXCL-10, that attract Th1 cells.

Portal vein glucose sensors do not play a major role in modulating physiological responses to insulin-induced hypoglycemia in humans.

OBJECTIVE: Experimental data from animal studies indicate that portal vein glucose sensors play a key role in the responses to slow-fall hypoglycemia. However, their role in modulating these responses in humans is not well understood. The aim of the present study was to examine in humans the potential role of portal vein glucose sensors in physiological responses to insulin-induced hypoglycemia mimicking the slow fall of insulin-treated diabetic subjects. RESEARCH DESIGN AND METHODS: Ten nondiabetic subjects were studied on two different occasions during intravenous insulin (2 mU . kg(-1) . min(-1)) plus variable glucose for 160 minutes. In both studies, after 60 min of normal plasma glucose concentrations, hypoglycemia (47 mg/dl) was induced slowly (60 min) and maintained for 60 min. Hypoglycemia was preceded by the ingestion of either oral placebo or glucose (28 g) given at 30 min. RESULTS: Plasma glucose and insulin were not different with either placebo or glucose (P > 0.2). Similarly, counterregulatory hormones, substrates, and symptoms were not different with either placebo or glucose. The Stroop color and colored words subtest of the Stroop test deteriorated less (P < 0.05) with glucose than placebo. CONCLUSIONS: In contrast to animals, in humans, prevention of portal hypoglycemia with oral glucose from the beginning of insulin-induced slow-fall hypoglycemia has no effect on sympathoadrenal and symptomatic responses to hypoglycemia.

Effect of oral amino acids on counterregulatory responses and cognitive function during insulin-induced hypoglycemia in nondiabetic and type 1 diabetic people.

OBJECTIVE: Amino acids stimulate glucagon responses to hypoglycemia and may be utilized by the brain. The aim of this study was to assess the responses to hypoglycemia in nondiabetic and type 1 diabetic subjects after ingestion of an amino acid mixture. RESEARCH DESIGN AND METHODS: Ten nondiabetic and 10 diabetic type 1 subjects were studied on three different occasions during intravenous insulin (2 mU . kg(-1) . min(-1)) plus variable glucose for 160 min. In two studies, clamped hypoglycemia (47 mg/dl plasma glucose for 40 min) was induced and either oral placebo or an amino acid mixture (42 g) was given at 30 min. In the third study, amino acids were given, but euglycemia was maintained. RESULTS: Plasma glucose and insulin were no different in the hypoglycemia studies with both placebo and amino acids (P > 0.2). After the amino acid mixture, plasma amino acid concentrations increased to levels observed after a mixed meal (2.4 +/- 0.13 vs. placebo study 1.7 +/- 0.1 mmol/l, P = 0.02). During clamped euglycemia, ingestion of amino acids resulted in transient increases in glucagon concentrations, which returned to basal by the end of the study. During clamped hypoglycemia, glucagon response was sustained and increased more in amino acid studies versus placebo in nondiabetic and diabetic subjects (P < 0.05), but other counter-regulatory hormones and total symptom score were not different. Beta-OH-butyrate was less suppressed after amino acids (200 +/- 15 vs. 93 +/- 9 micromol/l, P = 0.01). Among the cognitive tests administered, the following indicated less deterioration after amino acids than placebo: Trail-Making part B, PASAT (Paced Auditory Serial Addition Test) (2 s), digit span forward, Stroop colored words, and verbal memory tests for nondiabetic subjects; and Trail-Making part B, digit span backward, and Stroop color tests for diabetic subjects. CONCLUSIONS: Oral amino acids improve cognitive function in response to hypoglycemia and enhance the response of glucagon in nondiabetic and diabetic subjects.

Mapping of human autoantibody epitopes on aromatic L-amino acid decarboxylase.

CONTEXT: Aromatic l-amino acid decarboxylase (AADC) is target of autoantibodies in autoimmune polyendocrine syndrome I (APS I), especially in patients with autoimmune hepatitis. Little information is currently available on AADC autoantibody epitopes and on the interrelation between autoantibody-mediated inhibition of enzymatic activity and epitope specificity. DESIGN: We tested the immunoreactivity of full-length porcine AADC and of eight fragments of the enzyme with human serum from 18 patients with APS I, 199 with non-APS I autoimmune Addison's disease, 124 with type 1 diabetes mellitus, 36 with Graves' disease, and 141 healthy control subjects, and we evaluated the autoantibody-mediated enzymatic inhibition. RESULTS: AADC antibodies (Ab) were detected in 12 of 18 (67%) APS I patients and in six of 199 (3%) autoimmune Addison's disease patients. Four patients with autoimmune hepatitis were all positive for AADCAb. None of the 141 healthy control subjects, 82 patients with nonautoimmune adrenal insufficiency, 124 with type 1 diabetes mellitus, and 36 with Graves' disease were found positive. Two epitope regions, corresponding to amino acids 274-299 (E1) and 380-471 (E2) were identified. Localization of E1 was confirmed by displacement studies with synthetic peptides corresponding to peptides of porcine AADC. All 12 AADCAb-positive APS I sera reacted with E1, and seven of 12 (58%) reacted also with E2. E2-specific, but not E1-specific, autoantibodies were associated with a significant inhibition of in vitro AADC enzymatic activity. CONCLUSIONS: We mapped the human AADCAb epitopes to the middle and COOH-terminal regions of the enzyme. Autoantibodies to the COOH-terminal region induce a significant inhibition of enzymatic activity.

Identification of differentially expressed proteins in papillary thyroid carcinomas with V600E mutation of BRAF.

BRAF, a serine/threonine kinase of the RAF family, is a downstream transducer of the RAS-regulated MAPK pathway. V600E mutation of BRAF protein is the most common genetic alteration occurring in papillary thyroid carcinomas and is prognostic of poor clinicopathological outcomes. Protein expression in the subclass of PTC bearing the BRAF(V600E) mutation was investigated by using 2-DE and MS/MS techniques and compared to that of matched normal thyroid tissues from seven patients. 2-D gel image analysis revealed that the expression of eight polypeptide spots, corresponding to five proteins, were significantly underexpressed in PTC bearing BRAF(V600E) mutation whereas 25 polypeptides, representing 19 distinct proteins, were significantly upregulated in tumour tissue, as compared to normal thyroid. Among the differentially expressed polypeptides, mitochondrial proteins, ROS-scavenger enzymes, apoptosis-related proteins as well as proteins involved in tumour cell proliferation were identified. Although dissimilarities between the present results and those previously reported can be ascribed to the use of different 2-DE techniques, the possibility that BRAF(V600E) mutation is responsible for changes in protein expression distinct from those induced by other oncogenes cannot be ruled out.

Exercise and diabetes.

Physical activity activates has acute and chronic effects on glucose, lipid and protein metabolism. In type 1 diabetic subjects, the lack of the physiological inhibition of insulin secretion during exercise results in a potential risk of hypoglycemia. On the other hand, exercise-induced activation of counterregulatory hormones might trigger an acute metabolic derangement in severe insulin-deficient subjects. Thus, diabetic patients, before starting exercise sessions, must be carefully educated about the consequences of physical activity on their blood glucose and the appropriate modifications of diet and insulin therapy. Long-term effects of regular exercise are particularly advantageous for type 2 diabetic patients. Regular aerobic exercise reduces of visceral fat mass and body weight without decreasing lean body mass, ameliorates insulin sensitivity, glucose and blood pressure control, lipid profile and reduces the cardiovascular risk. For these reasons, regular aerobic physical activity must be considered an essential component of the cure of type 2 diabetes mellitus. In this regard, individual behavioral strategies have been documented to be effective in motivating sedentary type 2 diabetic subjects to the adoption and the maintenance of regular physical activity.

Genotyping of an Italian papillary thyroid carcinoma cohort revealed high prevalence of BRAF mutations, absence of RAS mutations and allowed the detection of a new mutation of BRAF oncoprotein (BRAF(V599lns)).

OBJECTIVES: The genes RET and RAS, and more recently BRAF, have been shown to be frequently mutated in human papillary thyroid carcinomas (PTC). The aim of this study was to genotype for these mutations a cohort of thyroid tumours collected at our institutions. DESIGN AND PATIENTS: Thyroid tumours removed from 51 subjects were analysed, including 43 PTC and 8 non-PTC tumours [3 follicular adenomas (FA), 4 follicular carcinomas (FTC) and 1 anaplastic carcinoma (AC)]. MEASUREMENTS: RET/PTC1 and RET/PTC3 expression was evaluated by reverse transcriptase-polymerase chain reaction, whereas screening of BRAF (exon 15) and RAS (HRAS, KRAS2 and NRAS) mutations were performed, respectively, by single strand conformation polymorphism and denaturing high-pressure liquid chromatography. RESULTS: RET/PTC expressions was positive in 5/43 (11.6%) PTC and in none of the non-PTC tumour. Similarly, BRAF mutations were positive only in PTC, but with a higher prevalence (24/43 positives, 55.8%). All but one BRAF mutation resulted in the prototypic substitution of valine 600 with a glutamic acid. In one case, a somatic in-frame insertion of three bases at codon 599 resulted in the insertion of an additional valine. RET/PTC expression and BRAF mutations were mutually exclusive. Screening of the RAS gene allowed identification of oncogenic mutations in 1/3 (33.3%) FA and 3/4 (75%) FTC. None of the PTCs was positive for RAS. CONCLUSIONS: These data indicate that BRAF mutations are the most frequent genetic event in PTC and that RAS mutations, besides being a genetic hallmark of follicular tumours, are rare or completely absent in PTC from our area. Together, BRAF mutations and rarer RET rearrangements accounted for a genetic event in two-thirds of PTCs. This study showed a novel and presumably oncogenic mutation of BRAF, which is BRAF(V599Ins).

Metabolic and endocrine effects of physiological increments in plasma ghrelin concentrations.

BACKGROUND: Growing evidence indicates that the administration of large amounts of ghrelin to humans increases circulating concentrations of several pituitary and adrenal hormones, induces hyperglycemia and reduces serum insulin concentrations. At present, it is not known whether physiological increments in plasma ghrelin concentrations affect glucose kinetics or hormone concentrations in humans. METHODS AND RESULTS: We compared the effects of two- and three-fold increments in plasma ghrelin concentrations in eight healthy subjects during a 2 h intravenous infusion of 7.5 (GHRE7.5), 15 (GHRE15) pmol kg(-1) min(-1) acylated human ghrelin or placebo (PL), in a randomized double-blind study. Compared with PL (146 +/- 24 pM) plasma ghrelin concentrations increased at 120 min (p<0.001) about two-fold after GHRE7.5 (300 +/- 35 pM) and three-fold after GHRE15 (494 +/- 30 pM). GHRE15 significantly increased circulating concentrations of NEFA, GH, ACTH, epinephrine, and prolactin (p<0.01). GHRELIN7.5 significantly (p<0.01) increased only serum GH concentrations. Neither ghrelin infusions changed glucose flux or circulating concentrations of glucose, insulin, C-peptide, glucagon, IGF-1, cortisol and norepinephrine. CONCLUSIONS: GH secretion is the only response that is stimulated by physiological increments in plasma ghrelin concentrations; about three-fold increases in plasma ghrelin concentrations are required to elicit the responses of epinephrine, prolactin, ACTH and NEFA.

Make your diabetic patients walk: long-term impact of different amounts of physical activity on type 2 diabetes.

OBJECTIVE: To establish the impact of different amounts of increased energy expenditure on type 2 diabetes care. RESEARCH DESIGN AND METHODS: Post hoc analysis of long-term effects of different amounts of increased energy expenditure (metabolic equivalents [METS] per hour per week) through voluntary aerobic physical activity was performed in 179 type 2 diabetic subjects (age 62 +/- 1 years [mean +/- SE]) randomized to a physical activity counseling intervention. Subjects were followed for 2 years and divided into six groups based on their increments in METs per hour per week: group 0 (no activity, n = 28), group 1-10 (6.8 +/- 0.3, n = 27), group 11-20 (17.1 +/- 0.4, n = 31), group 21-30 (27.0 +/- 0.5, n = 27), group 31-40 (37.5 +/- 0.5, n = 32), and group >40 (58.3 +/- 1.8, n = 34). RESULTS: At baseline, the six groups did not differ for energy expenditure, age, sex, diabetes duration, and all parameters measured. After 2 years, in group 0 and in group 1-10, no parameter changed; in groups 11-20, 21-30, 31-40, and >40, HbA(1c), blood pressure, total serum cholesterol, triglycerides, and estimated percent of 10-year coronary heart disease risk improved (P < 0.05). In group 21-30, 31-40, and >40, body weight, waist circumference, heart rate, fasting plasma glucose, serum LDL and HDL cholesterol also improved (P < 0.05). METs per hour per week correlated positively with changes of HDL cholesterol and negatively with those of other parameters (P < 0.001). After 2 years, per capita yearly costs of medications increased (P = 0.008) by USD393 in group 0, did not significantly change in group 1-10 (USD 206, P = 0.09), and decreased in group 11-20 (USD -196, P = 0.01), group 21-30 (USD -593, P = 0.009), group 31-40 (USD -660, P = 0.003), and group >40 (USD -579, P = 0.001). CONCLUSIONS: Energy expenditure >10 METs . h(-1) . week(-1) obtained through aerobic leisure time physical activity is sufficient to achieve health and financial advantages, but full benefits are achieved with energy expenditure >20 METs . h(-1) . week(-1).

Elevated serum interferon-gamma-inducible chemokine-10/CXC chemokine ligand-10 in autoimmune primary adrenal insufficiency and in vitro expression in human adrenal cells primary cultures after stimulation with proinflammatory cytokines.

Chemokines are a large family of cytokines involved in the pathogenesis of inflammatory and autoimmune diseases. Among CXC chemokines, CXC chemokine ligand 10 (CXCL10) has been identified to play an important role in several endocrinological autoimmune diseases, such as Hashimoto's thyroiditis, Graves' disease, and type 1 diabetes mellitus. Although the mechanisms leading to glandular autoimmune process may be at least in part shared by different endocrine organs, the role of CXCL10 in autoimmune adrenal insufficiency is unknown. The aim of this study was to evaluate the role of CXCL10 in Addison's disease (AD). Serum CXCL10 levels were assayed in 64 patients with clinically evident autoimmune AD, 20 patients with autoimmune subclinical AD, nine patients with nonautoimmune AD, and 48 healthy volunteers. Clinically evident and subclinical AD, but not nonautoimmune AD patients, showed a significant increase in serum CXCL10 levels compared with healthy subjects: 119.9 pg/ml (range, 39.8-427.6) and 124.0 pg/ml (range, 37.0-384.7) vs. 75.6 pg/ml (range, 22.4-164.0; P < 0.001 for both groups). Comparable serum CXCL10 levels were found between patients with an isolated form of AD and patients with other autoimmune conditions associated with AD, suggesting a specific influence of the adrenal autoimmune process in determining elevated CXCL10 concentrations in such patients. No relationship was found between serum CXCL10 levels and anti-21-hydroxylase or adrenal cortex autoantibody titers or between CXCL10 levels and duration of disease. The role of CXCL10 in the adrenal gland was also evaluated in vitro in human zona fasciculata cells (hZFC). CXCL10, although not basally detected in cultured hZFC, was strongly induced by interferon-gamma and synergistically increased by TNF-alpha addition. Hydrocortisone or ACTH alone had no effect on CXCL10 secretion in hZFC, but they both significantly inhibited cytokine-induced CXCL10 secretion. Taken together, these data suggest a potential role of hZFC, through the production of CXCL10, in regulating the recruitment of specific subsets of activated lymphocytes in autoimmune AD.

BRAF(V599E) mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas.

Activating mutations of BRAF have been identified in a variety of human cancers, most notably melanomas and papillary thyroid carcinomas (PTCs). The aim of the present study was to disclose the role of BRAF mutations in thyroid carcinoma development. Seventy-two thyroid tumors, including 60 PTCs, six follicular adenomas, five follicular carcinomas, and one anaplastic carcinoma, were studied. BRAF mutation screening focused on exon 15 and exon 11 of the gene by single-stranded conformational polymorphism and sequence analysis. Search of RET/PTC expression was conducted with the RT-PCR technique. The molecular genetic study of the BRAF gene showed the presence of a missense thymine to adenine transversion at nucleotide 1796, resulting in the V599E substitution, in 24 of 60 PTCs (40%), none of six follicular adenomas, and none of five follicular carcinomas or one anaplastic carcinoma. Moreover, nine of 60 PTCs (15%) presented RET/PTC expression. A genetico-clinical association analysis showed a statistically significant correlation between BRAF mutation and development of PTCs of the classic papillary histotype (P = 0.038). On the contrary, no link could be detected between expression of BRAF(V599E) and age at diagnosis, gender, dimension, and local invasiveness of the primary cancer, presence of lymph node metastases, tumor stage, and multifocality of the disease. These data clearly confirm that BRAF(V599E) is the more common genetic alteration found to date in adult sporadic PTCs, that it is unique for this thyroid cancer histotype, and that it might drive the development of PTCs of the classic papillary subtype.

Italian addison network study: update of diagnostic criteria for the etiological classification of primary adrenal insufficiency.

Primary adrenal insufficiency (PAI) is clinically evident in one in 8000 individuals. A correct etiological classification is critical for correct disease management. To update the diagnostic criteria for the etiological classification of PAI, a multicentric network was established in Italy, and 222 patients with PAI were studied. Both 21-hydroxylase and adrenal cortex autoantibodies (21OHAb and ACA, respectively) were tested in two independent laboratories on coded samples and found in 65-66% and 58-61% of cases, respectively. Autoimmune polyendocrine syndrome I was diagnosed in 11 of the 222 patients. Of the remaining 211 patients, 38 (18%) had a nonautoimmune form of PAI. In 145 subjects (65%), the presence of adrenal autoantibodies, without signs of other forms of PAI, led to a diagnosis of autoimmune Addison's disease. In six cases (3%), PAI remained idiopathic. Logistic regression analysis showed a 92.2-92.7% probability of correct reclassification for the two 21OHAb assays and 84.5-85.9% for the ACA assays. We conclude that the simultaneous presence of both 21OHAb and ACA permits unambiguous diagnosis of autoimmune Addison's, whereas subjects with low antibody titers should undergo both instrumental and biochemical tests to exclude other causes of PAI. Lastly, we developed a comprehensive flowchart for the classification of PAI for use in routine clinical practice.

Insulin is required for prandial ghrelin suppression in humans.

Accumulating evidence indicates that ghrelin plays a role in regulating food intake and energy homeostasis. In normal subjects, circulating ghrelin concentrations decrease after meal ingestion and increase progressively before meals. At present, it is not clear whether nutrients suppress the plasma ghrelin concentration directly or indirectly by stimulating insulin secretion. To test the hypothesis that insulin regulates postprandial plasma ghrelin concentrations in humans, we compared the effects of meal ingestion on plasma ghrelin levels in six C-peptide-negative subjects with type 1 diabetes and in six healthy subjects matched for age, sex, and BMI. Diabetic subjects were studied during absence of insulin (insulin withdrawal study), with intravenous infusion of basal insulin (basal insulin study) and subcutaneous administration of a prandial insulin dose (prandial insulin study). Meal intake suppressed plasma ghrelin concentrations (nadir at 105 min) by 32 +/- 4% in normal control subjects, 57 +/- 3% in diabetic patients during the prandial insulin study (P < 0.002 vs. control subjects), and 38 +/- 8% during basal insulin study (P = 0.0016 vs. hyperinsulinemia; P = NS vs. control subjects) but did not have any effect in the insulin withdrawal study (P < 0.001 vs. other studies). In conclusion, 1). insulin is essential for meal-induced plasma ghrelin suppression, 2). basal insulin availability is sufficient for postprandial ghrelin suppression in type 1 diabetic subjects, and 3). lack of meal-induced ghrelin suppression caused by severe insulin deficiency may explain hyperphagia of uncontrolled type 1 diabetic subjects.

Ret/PTC activation does not influence clinical and pathological features of adult papillary thyroid carcinomas.

OBJECTIVE: RET proto-oncogene rearrangements (ret/PTCs) represent the most common genetic alterations found in papillary thyroid carcinomas (PTCs). Correlation of ret/PTC expression with clinical outcome is controversial. The aim of the present study was to analyze the frequency of RET rearrangements in adult PTCs, and to investigate if ret/PTCs influence biological behavior and clinical features of the cancers. DESIGN: Ret/PTC rearrangements were looked for in tIssue samples of 48 PTCs collected at our institution. Data about clinical and pathological features of the tumors were also reviewed. Three separate association analyses were carried out on the cohort evaluating the effects of, respectively, ret/PTC positivity, preferential RET tyrosine kinase domain (RET-TK) expression, and ret/PTC plus RET-TK positivity, on age, sex, tumor size, staging, number of neoplastic foci, and histological subtype. METHODS: The genetic study was conducted with the RT-PCR-Southern blot technique. Standard Student's t-test and Fisher exact test were applied for the association analyses. RESULTS: The molecular genetic study demonstrated the positivity of ret/PTC1 and ret/PTC3 in 13 of 48 tumors (27.1%), and an exclusive or preferential RET-TK expression in 17 cases (35.4%). None of the three genetico-clinical analyses showed any significant association between ret/PTC expression and the clinical and pathological features of the cancers. CONCLUSIONS: These data indicate that RET rearrangements may not play any distinctive role in driving histotype development and cancer progression in these neoplasms. Moreover, they weaken the possibility of using ret/PTC as a prognostic marker for papillary thyroid carcinomas.

Validation of a counseling strategy to promote the adoption and the maintenance of physical activity by type 2 diabetic subjects.

OBJECTIVE: There is enough evidence that physical activity is an effective therapeutic tool in the management of type 2 diabetes. The present study was designed to validate a counseling strategy that could be used by physicians in their daily outpatient practice to promote the adoption and maintenance of physical activity by type 2 diabetic subjects. RESEARCH DESIGN AND METHODS: The long-term (2-year) efficacy of the behavioral approach (n = 182) was compared with usual care treatment (n = 158) in two matched, randomized groups of patients with type 2 diabetes who had been referred to our Outpatient Diabetes Center. The outcome of the intervention was consistent patient achievement of an energy expenditure of >10 metabolic equivalents (METs)-h/week through voluntary physical activity. RESULTS: After 2 years, 69% of the patients in the intervention group (27.1 +/- 2.0 METs x h/week) and 18% of the control group (4.1 +/- 0.8 METs x h/week) achieved the target (P < 0.001) with significant (P < 0.001) improvements in BMI (intervention group 28.9 +/- 0.2 versus control group 30.4 +/- 0.3 kg/m(2)) and HbA(1c) (intervention group 7.0 +/- 0.1 versus control group 7.6 +/- 0.1%). CONCLUSIONS: This randomized, controlled study shows that physicians can motivate most patients with type 2 diabetes to exercise long-term and emphasizes the value of individual behavioral approaches in daily practice.

Dehydroepiandrosterone, 17alpha-hydroxyprogesterone and aldosterone responses to the low-dose (1 micro g) ACTH test in subjects with preclinical adrenal autoimmunity.

OBJECTIVE: The appearance of 21-hydroxylase autoantibodies (21OHAbs) identifies subjects with preclinical adrenal insufficiency. In 21OHAb-positive subjects, the adrenocortical function is best evaluated by peak cortisol (F) levels after the low-dose (1 micro g) ACTH stimulation test (LDT). No information is currently available on the correlation between F and other adrenocortical hormone responses to the LDT in subjects with an ongoing autoimmune adrenal process. In this study, we tested the hypothesis that the dehydroepiandrosterone (DHEA), 17alpha-hydroxyprogesterone (17OHP) and aldosterone (A) responses to the LDT are consensual to that of F during the preclinical phase of autoimmune adrenal insufficiency. DESIGN AND PATIENTS: We studied 12 subjects positive for 21OHAb, in the absence of clinical signs of adrenal insufficiency. On the basis of peak F levels after the LDT, and according to the lower level of normal observed in 15 healthy volunteers (510.4 nmol/l), patients were subdivided into two groups: group A, n = 6 subjects with normal F response; and group B, n = 6 subjects with impaired F response. Results were expressed as absolute delta increase (Delta) between peak and basal levels. RESULTS: DeltaF was significantly higher in group A (314.5 +/- 115.8 nmol/l) than in group B (151.7 +/- 88.2 nmol/l) (P = 0.041). DeltaDHEA and Delta17OHP were also significantly higher in group A (17.0 +/- 13.5 nmol/l and 6.1 +/- 4.4 nmol/l, respectively) than in group B (0.69 +/- 2.25 nmol/l and 1.9 +/- 1.7 nmol/l, respectively) (P = 0.002 and P = 0.041). The difference in DeltaA between the two groups did not reach statistical significance (group A 321.8 +/- 272.0 pmol/l vs. group B 157.0 +/- 154.0 pmol/l). DeltaDHEA, Delta17OHP and DeltaA tended to correlate positively with DeltaF (P = 0.039, P = 0.039 and P = 0.044, respectively), but the correlations did not reach significance after correction of the P-value. CONCLUSIONS: Our study demonstrates a high concordance between F and DHEA, 17OHP and A responses to the LDT in subjects with preclinical adrenal autoimmunity, thus strengthening the concept that the LDT is an accurate test to identify early adrenal dysfunction.

Ghrelin is not necessary for adequate hormonal counterregulation of insulin-induced hypoglycemia.

Ghrelin is a novel enteric hormone that stimulates growth hormone (GH), ACTH, and epinephrine; augments plasma glucose; and increases food intake by inducing the feeling of hunger. These characteristics make ghrelin a potential counterregulatory hormone. At present, it is not known whether ghrelin increases in response to insulin-induced hypoglycemia. To answer this question, we compared plasma ghrelin concentrations after a short-term insulin infusion that was allowed or not (euglycemic clamp) to cause hypoglycemia (2.7 +/- 0.2 mmol/l at 30 min) in five healthy volunteers. In both studies, plasma ghrelin concentrations decreased (P < 0.01) after insulin infusion (hypoglycemia by 14%, euglycemia by 22%), reached a nadir at 30 min, and returned to baseline at 60 min, without differences between the hypoglycemia and the euglycemia studies. Glucagon, cortisol, and GH increased in response to hypoglycemia despite the decreased ghrelin. There was a strong correlation (R(2) = 0.91, P < 0.002) between the insulin sensitivity of the subjects and the percentage suppression of ghrelin from baseline. These data demonstrate that ghrelin is not required for the hormonal defenses against insulin-induced hypoglycemia and that insulin can suppress ghrelin levels in healthy humans. These results raise the possibility that postprandial hyperinsulinemia is responsible for the reduction of plasma ghrelin that occurs during meal intake.

[Prevalence of the neuroendocrine phenotype in thymus neoplasms]. / Prevalenza del fenotipo neuroendocrino nelle neoplasie del timo.

To determine the prevalence of neuroendocrine differentiation in human thymic neoplasms, which are unusual tumours that may range from well-differentiated to overtly malignant, poorly differentiated lesions, an immunohistochemical study was conducted in 23 thymic neoplasms re-classified on the basis of the new 1999 WHO classification. Immunohistochemical evidence of neuroendocrine differentiation in the form of reactivity to the markers synaptophysin, neuron-specific enolase and chromogranin A was found in 6 of 23 tissues (26%). Two of 3 patients with thymic carcinoids (or well-differentiated thymic neuroendocrine carcinoma) were affected by multiple endocrine neoplasia type 1 (MEN-1). Myasthenia gravis was present in 2/6 patients with neuroendocrine differentiation. This study demonstrates the high prevalence of neuroendocrine markers in human thymic neoplasms. Whether and in what percentage of cases immunohistochemical reactivity may be correlated with clinical behaviour and outcome remains a controversial issue. Finally, the association between thymic carcinoids and MEN-1 is a strong indication for clinical and possibly genetic screening of all patients presenting this feature, just as all MEN-1 patients have to undergo thoracic imaging and prophylactic thymectomy in selected cases.