The Master Course on "Methodologies for Bio-Medical Research in Diabetes" was organized jointly by the University of Bari Aldo Moro, Bari, Italy, and the Italian Society of Diabetology (SID, Società Italiana di Diabetologia). This Course Program has been active throughout the academic year 2011-2012, and engaged selected students in lectures on diabetes research, training in laboratory work, and personal study on selected review articles and original papers. Lecturing activities involved 26 Italian and international experts in the field of diabetes and metabolism and were highly interactive also involving discussion of experimental data sets. The lectures addressed several aspects of diabetes aetiology, pathophysiology, and management, as well methodological and biotechnology topics. At the end of the Course, students were requested to write a review article on a research topic of their choice.

OBJECTIVE: The objective of the study was to assess the effect of high aldosterone levels on insulin sensitivity of adipose tissue in humans. METHODS: Visceral adipose tissue (VAT) was obtained from patients with aldosterone-producing adenoma (APA; n=14) and, as controls, nonfunctioning adenoma (NFA; n=14) undergoing laparoscopic adrenalectomy. Homeostasis model assessment index was higher and potassium was lower in APA than NFA (P<0.05). Immunohistochemistry, Western blotting, and real-time PCR were used to detect and quantify mineralocorticoid receptor (MR) expression. Transcript levels of peroxisome proliferative-activated receptor-gamma, insulin receptor, glucose transporter 4, insulin receptor substrate-1 and -2, leptin, adiponectin, IL-6, monocyte chemoattractant protein-1, glucocorticoid receptor (GR)-alpha, 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1, and HSD11B2 were quantified. The effect of increasing aldosterone concentrations on 2-deoxy-[3H]d-glucose uptake was tested in human sc abdominal adipocytes. RESULTS: Expression of MR was demonstrated in VAT, with no difference between APA and NFA as to mRNA levels of MR, GRalpha, HSD11B1, and glucose metabolism and inflammation factors. In cultured adipocytes, basal and insulin-stimulated glucose uptake were unaffected by 1-100 nM (normal/hyperaldosteronism) and impaired only by much higher, up to 10 microM, aldosterone concentrations. The impairment was prevented by RU486 but not by eplerenone. CONCLUSIONS: Gene expression of insulin signaling/inflammatory molecules was similar in VAT of APA and NFA patients, not supporting an effect of aldosterone excess on insulin sensitivity of adipose tissues. Only at pharmacological concentrations and through GR activation, aldosterone reduced glucose uptake in adipocytes. Insulin resistance in primary aldosteronism might occur in compartments other than fat and/or depend on concurrent environmental factors.

Abstract In 2007 the Italian COM.E.T.A. (COMorbidities Evaluation and Treatment in Acromegaly) study group started to assess the application in a clinical setting of the Versailles criteria for management of acromegaly complications by a first questionnaire focusing on cardiovascular co-morbidities. A further questionnaire on sleep apnea syndrome (SAS) was delivered by the COM.E.T.A. study group to 107 endocrine centers in Italy. The results of our survey suggest that SAS is a well-known comorbidity even if its estimated prevalence is lower than in the literature. Polysomnography is the preferred tool for diagnosis. Control of SAS is considered relevant both for quality of life and co-morbidities. Continuous positive airway pressure is the cornerstone of therapy, but patients' acceptance may be critical. Control of GH/IGF-I secretion is important to improve SAS. Management of SAS requires cooperation between specialists.

Aims.We evaluated the links between leptin and visfatin levels and fertilization rates in nonoverweight (NOW) women with PCOS (NOW-PCOS) from Apulia undergoing in vitro fertilization/embryo transfer (IVF). Materials and Methodology. We recruited 16 NOW women with PCOS (NOW-PCOS) and 10 normally ovulating NOW women (control-NOW). All women underwent IVF. Androgens, 17--estradiol (17-E2), and insulin levels were measured in plasma and/or serum and leptin and visfatin levels were assayed in both serum and follicular fluid (FF-leptin, FF-visfatin). Results. In NOW-PCOS, both serum and FF-leptin were significantly lower than in control-NOW. In NOW-PCOS, significant correlations were found between BMI and serum leptin and insulinemia and FF-leptin. By contrast, in control-NOW, FF-leptin levels were not correlated with insulinemia. Serum visfatin levels were not significantly different in NOW-PCOS and control-NOW, but FF-visfatin levels were 1.6-fold higher, although not significantly, in NOW-PCOS than in control-NOW. Conclusions. Both serum leptin levels and FF-leptin are BMI- and insulin-related in Southern Italian NOW-PCOS from Apulia. In line with other reports showing that FF-leptin levels are predictive of fertilization rates, lower than normal FF-leptin levels in NOW-PCOS may explain their lower fertilization rate and this may be related to the level of insulin and/or insulin resistance.

AIMS: We previously demonstrated the presence of two different populations among adult-onset autoimmune diabetes (latent autoimmume diabetes of adults; LADA) having high or low titre of antibodies to glutamic acid decarboxylase (GADA). The transcription factor 7-like 2 (TCF7L2) gene has been recognized as the major gene associated with Type 2 diabetes. The aim of the present study was to evaluate whether the phenotypic heterogeneity of LADA based on GADA titre is associated with TCF7L2 polymorphisms. METHODS: Two hundred and fifty patients identified as LADA, divided into two subgroups with low (< or = 32 arbitrary units) or high (> 32 units) GADA titre, 620 subjects with Type 2 diabetes [from the Non-Insulin Requiring Autoimmune Diabetes (NIRAD) study cohort of 5330 subjects] in addition to 551 consecutive cases of Type 1 diabetes and 545 normoglycaemic subjects were analysed for the rs12255372 and rs7903146 polymorphisms of the TCF7L2 gene using Taqman. RESULTS: The genotype and allele distributions of the two polymorphisms revealed similar frequencies in subjects with low GADA titre and Type 2 diabetes. High GADA titre, Type 1 diabetes and controls also showed comparable frequencies. A significant increase of GT/TT genotypes of the rs12255372 single-nucleotide polymorphism (SNP) and CT/TT genotypes of the rs7903146 SNP was observed in low GADA titre and Type 2 diabetes compared with high GADA titre, Type 1 diabetes and controls (P < or = 0.04 for both comparisons). The risk alleles of both variants were increased in low GADA titre and Type 2 diabetes compared with high GADA titre, Type 1 diabetes and control subjects (P < 0.02 for all comparisons). CONCLUSIONS: TCF7L2 common genetic variants of susceptibility are associated only with low GADA antibody titre in LADA patients.

With the increasing use of thyroid ultrasound, the recognition of thyroid nodules in a large proportion of apparently healthy subjects has become common. Because also the papillary thyroid microcarcinomas (PTMC) are being increasingly discovered, it is important to ascertain whether PTMC may exhibit heterogenous clinical features, associated with different aggressiveness. AIM: We retrospectively examined 122 subjects [98 female (80.3%), and 24 male (19.7%)] with thyroid cancer to find potential clinical and pathological findings that could be predictive of clinically aggressive behavior. RESULTS: Twenty of the 31 patients with true incidental cancer (64.5%) in comparison to 20 of the 91 patients with non-incidental cancer (21.9%) had a diameter &lt;10 mm, and this difference was statistically significant (p&lt;0.0001). There was a statistically significant association between size and invasiveness because 19.3% of invasive cancers were &lt;10 mm whereas 44.6% of non-invasive cancers were &lt;10 mm (p=0.005). The relationship between incidental discovery and invasiveness was also evaluated, but the proportion of incidental invasive cancer (19.3%) was not significantly different from that of incidental non-invasive cancer (30.8%). In the multivariate analysis, only size &lt;10 mm (odds ratio=0.35, p=0.013) and papillary vs other histotypes (odds ratio=0.35, p=0.04) were statistically significant protective factors against invasiveness. CONCLUSIONS: a) Incidentally discovered thyroid cancers are more frequently microcarcinomas; b) there appears to be no difference in terms of invasive behavior between incidental and non-incidental thyroid cancer; c) smaller tumor size emerges as a protective factor.

OBJECTIVE: In type 2 diabetes, prevalence of nonalbuminuric renal impairment is increasing worldwide, though its clinical significance remains unclear. This large-cohort study aimed at evaluating the association of this phenotype with cardiovascular risk factors and other complications. METHODS: Type 2 diabetic patients from the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study (n = 15,773), visiting consecutively 19 hospital-based Diabetes Clinics in years 2007-2008, were examined. Serum creatinine was assessed by the Jaffe method; albuminuria was measured by immunonephelometry or immunoturbidimetry. RESULTS: Of patients with renal impairment, as identified by an estimated glomerular filtration rate (eGFR) less than 60 ml/min per 1.73 m², 56.6% were normoalbuminuric, 30.8% were microalbuminuric, and 12.6% were macroalbuminuric. Percentages were similar when GFR was estimated using the more accurate Chronic Kidney Disease Epidemiology Collaboration equation instead of the simplified Modification of Diet in Renal Disease formula, and were independent of age, thus indicating that the increasing prevalence of this phenotype does not reflects misclassification of elderly patients. Nonalbuminuric renal impairment was not associated with HbA(1c) and correlated less strongly with retinopathy and hypertension than albuminuria, either alone or associated with reduced eGFR. It was associated with a higher prevalence of cardiovascular disease (CVD) than albuminuria alone, but lower than albuminuric renal impairment. Female sex correlated with nonalbuminuric renal impairment and male sex with the albuminuric forms. CONCLUSIONS: These data show that type 2 diabetic patients with nonalbuminuric renal impairment exhibit distinct clinical features, suggesting predominance of macroangiopathy as underlying renal pathology, and that this phenotype is associated with significant CVD burden.

Background: Clusterin (CLU) is a ubiquitous multifunctional factor involved in neoplastic transformation. The CLU transcript variants and protein forms play a crucial role in balancing cells proliferation and death. Methods: We investigated the regulation of CLU transcript variants expression in an in vivo model system consisting of both neoplastic tissues and fine needle aspiration biopsy (FNAB) samples isolated from patients undergoing thyroidectomy. Results: The immunohistochemical analyses showed an overall CLU up-regulation in papillary carcinoma. A specific CLU2 transcript variant increase was registered using qPCR in papillary carcinomas while CLU1 decreased. In addition, the analysis of CLU transcripts expression level showed an increase of the CLU2 transcript in the TIR 3 patients with histologically confirmed thyroid cancer. Conclusions: Our results suggest the existence of a specific alteration of CLU2:CLU1 ratio towards CLU2, thus providing the first circumstantial evidence for the potential use of CLU transcript variants as effective biomarkers for a more accurate assessment of the so called “indeterminate” thyroid nodules.

In obese subjects with obstructive sleep apnea (OSA), chronic intermittent hypoxia (CIH) may be linked to systemic and adipose tissue inflammation.

Diabetes and cancer are worldwide chronic diseases with a major impact on the quality and expectancy of life. Metabolic abnormalities observed during the onset and progression of diabetes may have a critical role on the initiation and progression of carcinogenesis. To date, there are no conclusive data on the mechanisms underlying the relationship between diabetes and any type of human cancer. However, recent evidence suggests that both hyperglycemia and hyperinsulinemia in diabetes could elicit cell damage responses, such as glucotoxicity, lipotoxicity and oxidative stress, which participate in the cell transformation process raising the risk of cancer development. In addition, clinical trials have revealed that several anti-diabetes therapies may potentially affect the risk of cancer though largely undefined mechanisms. In this review, we highlight epidemiological and pathophysiological aspects of diabetes, which may influence cancer initiation and progression.

Differences in the inherent properties of adipose tissue-derived stem cells (ASC) may contribute to the biological specificity of the subcutaneous (Sc) and visceral (V) adipose tissue depots. In this study, three distinct subpopulations of ASC, i.e. ASCSVF, ASCBottom, and ASCCeiling, were isolated from Sc and V fat biopsies of non-obese subjects, and their gene expression and functional characteristics were investigated. Genome-wide mRNA expression profiles of ASCSVF, ASCBottom and ASCCeiling from Sc fat were significantly different as compared to their homologous subsets of V-ASCs. Furthermore, ASCSVF, ASCCeiling and ASCBottom from the same fat depot were also distinct from each other. In this respect, both principal component analysis and hierarchical clusters analysis showed that ASCCeiling and ASCSVF shared a similar pattern of closely related genes, which was highly different when compared to that of ASCBottom. However, larger variations in gene expression were found in inter-depot than in intra-depot comparisons. The analysis of connectivity of genes differently expressed in each ASC subset demonstrated that, although there was some overlap, there was also a clear distinction between each Sc-ASC and their corresponding V-ASC subsets, and among ASCSVF, ASCBottom, and ASCCeiling of Sc or V fat depots in regard to networks associated with regulation of cell cycle, cell organization and development, inflammation and metabolic responses. Finally, the release of several cytokines and growth factors in the ASC cultured medium also showed both inter- and intra-depot differences. Thus, ASCCeiling and ASCBottom can be identified as two genetically and functionally heterogeneous ASC populations in addition to the ASCSVF, with ASCBottom showing the highest degree of unmatched gene expression. On the other hand, inter-depot seem to prevail over intra-depot differences in the ASC gene expression assets and network functions, contributing to the high degree of specificity of Sc and V adipose tissue in humans.

The The aim of our study is to investigate the molecular mechanisms of diabetic cardiomyopathy through the identification of remarkable genes for the myocardial function that are expressed differently between diabetic and normal subjects. Moreover, we intend to characterize both in human myocardial tissue and in the related cardiac progenitor cells the pattern of gene expression and the levels of expression and protein activation of molecular effectors involved in the regulation of the myocardial function and differentiation to clarify whether in specific human pathological conditions (type 2 diabetes mellitus, cardiac failure, coronary artery disease) specific alterations of the aforementioned factors could take place. Thirty-five patients scheduled for coronary artery bypass grafting (CABG) or for aortic or mitral valve replacement were recruited into the study. There were 13 men and 22 women with a mean age of 64.8 +/- 13.4 years. A list of anamnestic, anthropometric, clinical, and instrumental data required for an optimal phenotypical characterization of the patients is reported. The small cardiac biopsy specimens were placed in the nourishing buffer, in a sterile tube provided the day of the procedure, to maintain the stability of the sample for several hours at room temperature. The cells were isolated by a dedicated protocol and then cultured in vitro. The sample was processed for total RNA extraction and levels of gene expression and protein activation of molecular effectors involved in the regulation of function and differentiation of human myocardium was analyzed. In particular, cardiac genes that modulate the oxidative stress response or the stress induced by pro-inflammatory cytokines (p66Shc, SOCS-1, SOCS-3) were analyzed. From a small sample of myocardium cardiac stem cells and cardiomyoblasts were also isolated and characterized. These cells showed a considerable proliferative capacity due to the fact that they demonstrate stability up to the eleventh passage. Analysis of gene expression in a subgroup of subjects showed the trend of a decrease in levels of expression of cardiac-specific transcription genes and oxidative stress-related proteins in tissues of diabetic patients compared with controls subjects. This trend is not confirmed in isolated cells. As for the coronary artery disease, diabetic cardiomyopathy could be associated with a reduction of the cardiac stem and progenitor cells pool. The expansion of the cardiac resident cells pool could be associated with a preservation of cardiac performance, suggesting that a preserved stamina compartment can counteract the impact of diabetes on the myocardium.

Diabetic nephropathy (DN) is the major cause of chronic kidney disease in developed countries and contributes significantly to increased morbidity and mortality among diabetic patients. Morphologically, DN is characterized by tubulo-interstitial fibrosis, thickening of the glomerular basement membrane and mesangial expansion mainly due to accumulation of extracellular matrix (ECM). ECM turnover is regulated by metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) activities. In diabetic conditions, TIMP3 expression in kidney is strongly reduced, but the causes of this reduction are still unknown. The aim of this study was to elucidate at least one of these mechanisms which relies on differential expression of TIMP3-targeting microRNAs (miRs) in a hyperglycemic environment either in vitro (MES13 cell line) or in vivo (mouse kidney and human biopsies). Among the TIMP3-targeting miRs, miR-21 and miR-221 were significantly upregulated in kidneys from diabetic mice compared to control littermates, and in a mesangial cell line grown in high glucose conditions. In human samples, only miR-21 expression was increased in kidney biopsies from diabetic patients compared to healthy controls. The expression of miR-217, which targets TIMP3 indirectly through downregulation of SirT1, was also increased in diabetic kidney and MES13 cell line. In agreement with these result, SirT1 expression was reduced in mouse and human diabetic kidneys as well as in MES13 mesangial cell line. TIMP3 deficiency has recently emerged as a hallmark of DN in mouse and human. In this study, we demonstrated that this reduction is due, at least in part, to increased expression of certain TIMP3-targeting miRs in diabetic kidneys compared to healthy controls. Unveiling the post-transcriptional mechanisms responsible for TIMP3 downregulation in hyperglycemic conditions may orient toward the use of this protein as a possible therapeutic target in DN.

Glucagon-like peptide-1 and its analogs may preserve pancreatic beta-cell mass by promoting resistance to cytokine-mediated apoptosis. The mechanisms of TNFalpha-induced apoptosis and of its inhibition by exendin-4 were investigated in insulin-secreting cells. INS-1 and MIN6 insulinoma cells were exposed to 20 ng/ml TNFalpha, with or without pretreatment with 10 nm exendin-4. Treatment with TNFalpha increased c-Jun N-terminal protein kinase (JNK) phosphorylation 2-fold, reduced inhibitor-kappaBalpha (IkappaBalpha) protein content by 50%, induced opposite changes in caspase-3 and Bcl-2 protein content, and increased cellular apoptosis. Moreover, exposure to TNFalpha resulted in increased serine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-2 and reduced basal and insulin-induced Akt phosphorylation. However, in the presence of a JNK inhibitor, TNFalpha-induced apoptosis was diminished and serine phosphorylation of IRS proteins was prevented. When cells were pretreated with exendin-4, TNFalpha-induced JNK and IRS-1/2 serine phosphorylation was markedly reduced, Akt phosphorylation was increased, caspase-3 and Bcl-2 protein levels were restored to normal, and TNFalpha-induced apoptosis was inhibited by 50%. This was associated with a 2-fold increase in IRS-2 expression levels. A similar ability of exendin-4 to prevent TNFalpha-induced JNK phosphorylation was found in isolated pancreatic human islets. The inhibitory effect of exendin-4 on TNFalpha-induced JNK phosphorylation was abrogated in the presence of the protein kinase A inhibitor H89. In conclusion, JNK activation mediates TNFalpha-induced apoptosis and impairment of the IRS/Akt signaling pathway in insulin-secreting cells. By inhibiting JNK phosphorylation in a PKA-dependent manner, exendin-4 counteracts TNFalpha-mediated apoptosis and reverses the inhibitory events in the IRS/Akt pathway, resulting in promotion of cell survival.

AIMS/HYPOTHESIS: The mechanisms of the protective effects of exendin-4 on NEFA-induced beta cell apoptosis were investigated. METHODS: The effects of exendin-4 and palmitate were evaluated in human and murine islets, rat insulin-secreting INS-1E cells and murine glucagon-secreting alpha-TC1-6 cells. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting or immunofluorescence, respectively. Small interfering (si)RNAs for Ib1 and Gpr40 were used. Cell apoptosis was quantified by two independent assays. Insulin release was assessed with an insulin ELISA. RESULTS: Exposure of human and murine primary islets and INS-1E cells, but not alpha-TC1-6 cells, to exendin-4 inhibited phosphorylation of the stress kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and prevented apoptosis in response to palmitate. Exendin-4 increased the protein content of islet-brain 1 (IB1), an endogenous JNK blocker; however, siRNA-mediated reduction of IB1 did not impair the ability of exendin-4 to inhibit JNK and prevent apoptosis. Exendin-4 reduced G-protein-coupled receptor 40 (GPR40) expression and inhibited palmitate-induced phosphorylation of mitogen-activated kinase kinase (MKK)4 and MKK7. The effects of exendin-4 were abrogated in the presence of the protein kinase A (PKA) inhibitors, H89 and KT5720. Knockdown of GPR40, as well as use of a specific GPR40 antagonist, resulted in diminished palmitate-induced JNK and p38 MAPK phosphorylation and apoptosis. Furthermore, inhibition of JNK and p38 MAPK activity prevented palmitate-induced apoptosis. CONCLUSIONS/INTERPRETATION: Exendin-4 counteracts the proapoptotic effects of palmitate in beta cells by reducing GPR40 expression and inhibiting MKK7- and MKK4-dependent phosphorylation of the stress kinases, JNK and p38 MAPK, in a PKA-dependent manner.

PURPOSE: Project Leonardo represented a feasibility study to evaluate the impact of a disease and care management (D&CM) model and of the introduction of "care manager" nurses, trained in this specialized role, into the primary health care system. PATIENTS AND METHODS: Thirty care managers were placed into the offices of 83 general practitioners and family physicians in the Apulia Region of Italy with the purpose of creating a strong cooperative and collaborative "team" consisting of physicians, care managers, specialists, and patients. The central aim of the health team collaboration was to empower 1,160 patients living with cardiovascular disease (CVD), diabetes, heart failure, and/or at risk of cardiovascular disease (CVD risk) to take a more active role in their health. With the support of dedicated software for data collection and care management decision making, Project Leonardo implemented guidelines and recommendations for each condition aimed to improve patient health outcomes and promote appropriate resource utilization. RESULTS: Results show that Leonardo was feasible and highly effective in increasing patient health knowledge, self-management skills, and readiness to make changes in health behaviors. Patient skill-building and ongoing monitoring by the health care team of diagnostic tests and services as well as treatment paths helped promote confidence and enhance safety of chronic patient management at home. CONCLUSION: Physicians, care managers, and patients showed unanimous agreement regarding the positive impact on patient health and self-management, and attributed the outcomes to the strong "partnership" between the care manager and the patient and the collaboration between the physician and the care manager. Future studies should consider the possibility of incorporating a patient empowerment model which considers the patient as the most important member of the health team and care managers as key health care collaborators able to enhance and support services to patients provided by physicians in the primary health care system.

OBJECTIVE: To evaluate the feasibility and effectiveness of an intensive, multifactorial cardiovascular risk reduction intervention in a clinic-based setting. RESEARCH DESIGN AND METHODS: The study was a pragmatic, cluster randomized trial, with the diabetes clinic as the unit of randomization. Clinics were randomly assigned to either continue their usual care (n = 5) or to apply an intensive intervention aimed at the optimal control of cardiovascular disease (CVD) risk factors and hyperglycemia (n = 4). To account for clustering, mixed model regression techniques were used to compare differences in CVD risk factors and HbA1c. Analyses were performed both by intent to treat and as treated per protocol. RESULTS: Nine clinics completed the study; 1,461 patients with type 2 diabetes and no previous cardiovascular events were enrolled. After 2 years, participants in the interventional group had significantly lower BMI, HbA1c, LDL cholesterol, and triglyceride levels and significantly higher HDL cholesterol level than did the usual care group. The proportion of patients reaching the treatment goals was systematically higher in the interventional clinics (35% vs. 24% for LDL cholesterol, P = 0.1299; 93% vs. 82% for HDL cholesterol, P = 0.0005; 80% vs. 64% for triglycerides, P = 0.0002; 39% vs. 22% for HbA1c, P = 0.0259; 13% vs. 5% for blood pressure, P = 0.1638). The analysis as treated per protocol confirmed these findings, showing larger and always significant differences between the study arms for all targets. CONCLUSIONS: A multifactorial intensive intervention in type 2 diabetes is feasible and effective in clinical practice and it is associated with significant and durable improvement in HbA1c and CVD risk profile.

Increased apoptosis of cardiac progenitor cells (CPCs) has been proposed as a mechanism of myocardial damage and dysfunction. Glucagon-like peptide-1 (GLP-1) has been shown to improve heart recovery and function after ischemia and to promote cell survival. The protective effects of GLP-1 on oxidative stress-induced apoptosis were investigated in human CPCs isolated from human heart biopsies. Mesenchymal-type cells were isolated from human heart biopsies, exhibited the marker profile of CPCs, differentiated toward the myocardiocyte, adipocyte, chondrocyte, and osteocyte lineages under appropriate culture conditions, and expressed functional GLP-1 receptors. CPCs were incubated with GLP-1 with or without hydrogen peroxide (H(2)O(2)). Phospho- and total proteins were detected by immunoblotting and immunofluorescence analysis. Gene expression was evaluated by quantitative RT-PCR. The role of the canonical GLP-1 receptor was assessed by using the receptor antagonist exendin(9-39) and receptor-specific silencer small interfering RNAs. Cell apoptosis was quantified by an ELISA assay and by flow cytometry-detected Annexin V. Exposure of CPCs to H(2)O(2) induced a 2-fold increase in cell apoptosis, mediated by activation of the c-Jun N-terminal protein kinase (JNK) pathway. Preincubation of CPCs with GLP-1 avoided H(2)O(2)-triggered JNK phosphorylation and nuclear localization, and protected CPCs from apoptosis. The GLP-1 effects were markedly reduced by coincubation with the receptor antagonist exendin(9-39), small interfering RNA-mediated silencing of the GLP-1 receptor, and pretreatment with the protein kinase A inhibitor H89. In conclusion, activation of GLP-1 receptors prevents oxidative stress-mediated apoptosis in human CPCs by interfering with JNK activation and may represent an important mechanism for the cardioprotective effects of GLP-1.

Several protocols are actually available for in Vitro Fertilization and Embryo Transfer. The review summarizes the main differences and the clinic characteristics of the protocols in use with GnRH agonists and GnRH antagonists by emphasizing the major outcomes and hormonal changes associated with each protocol. The majority of randomized clinical trials clearly shows that in "in Vitro" Fertilization and Embryo Transfer, the combination of exogenous Gonadotropin plus a Gonadotropin Releasing Hormone (GnRH) agonist, which is able to suppress pituitary FSH and LH secretion, is associated with increased pregnancy rate as compared with the use of gonadotropins without a GnRH agonist. Protocols with GnRH antagonists are effective in preventing a premature rise of LH and induce a shorter and more cost-effective ovarian stimulation compared to the long agonist protocol. However, a different synchronization of follicular recruitment and growth occurs with GnRH agonists than with GnRH antagonists. Future developments have to be focused on timing of the administration of GnRH antagonists, by giving a great attention to new strategies of stimulation in patients in which radio-chemotherapy cycles are needed.

The Italian Standards for the Treatment of Diabetes Mellitus represent a consensus document of the two Italian scientific diabetes societies (AMD and SID), providing specific recommendations for the diagnosis and treatment of diabetes and its complications. The level of scientific evidence behind every recommendation has been classified in accordance with the National Guidelines Plan. An original processing system was employed: the document prepared by the Editorial Team was published online for 20 days, and the suggestions and criticisms of about 30 persons were evaluated and integrated to those provided by a panel of diabetologists and members of other healthcare professions dedicated to diabetes care, as well as lay members. Lastly, the document was approved by AMD and SID National Steering Committees. In this version, some highlights of the full document (www.siditalia.it; www.aemmedi.it) concerning the main and most representative recommendations on diagnosis and treatment of diabetes and its complications as well as on diabetes care in some specific contexts, are reported.

Stem cells are unique cells exhibiting self-renewing properties and the potential to differentiate into multiple specialised cell types. Totipotent or pluripotent stem cells are generally abundant in embryonic or fetal tissues, but the use of discarded embryos as sources of these cells raises challenging ethical problems. Adult stem cells can also differentiate into a wide variety of cell types. In particular, adult adipose tissue contains a pool of abundant and accessible multipotent stem cells, designated as adipose-derived stem cells (ASCs), that are able to replicate as undifferentiated cells, to develop as mature adipocytes and to differentiate into multiple other cell types along the mesenchymal lineage, including chondrocytes, myocytes and osteocytes, and also into cells of endodermal and neuroectodermal origin, including beta-cells and neurons, respectively. An impairment in the differentiation potential and biological functions of ASCs may contribute to the development of obesity and related comorbidities. In this review, we summarise different aspects of the ASCs with special reference to the isolation and characterisation of these cell populations, their relation to the biochemical features of the adipose tissue depot of origin and to the metabolic characteristics of the donor subject and discuss some prospective therapeutic applications.

OBJECTIVE: We studied phosphorylation of insulin-receptors substrate downstream molecules: 1) in the ex-vivo visceral adipose tissue (VAT) of patients with aldosterone-producing adenoma (APA) (no.=7) and non-functioning adenoma (NFA) (no.=7) undergoing laparoscopic adrenalectomy; 2) in aldosterone-treated sc adipocytes of subjects (no.=5) who requested abdominoplasty. PATIENTS AND METHODS: Western blotting was used to detect phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in VAT from APA and NFA patients, and in subcutaneous adipocytes pre-treated with different aldosterone concentrations. Phosphorylation of Akt and ERK1/2 was similar in VAT of patients with APA and NFA. Pre-treatment in adipocytes with both physiological (1 nM) and pharmacological (10 μM) doses of aldosterone did not affect basal or insulin-induced phosphorylation of Akt and ERK1/2. CONCLUSIONS: Our data give further evidence that insulin signaling in human VAT is not affected by primary aldosterone overproduction.

Insulin is a major endocrine hormone also involved in the regulation of energy and lipid metabolism via the activation of an intracellular signaling cascade involving the insulin receptor (INSR), insulin receptor substrate (IRS) proteins, phosphoinositol 3-kinase (PI3K) and protein kinase B (AKT). Specifically, insulin regulates several aspects of the development and function of adipose tissue and stimulates the differentiation program of adipose cells. Insulin can activate its responses in adipose tissue through two INSR splicing variants: INSR-A, which is predominantly expressed in mesenchymal and less-differentiated cells and mainly linked to cell proliferation, and INSR-B, which is more expressed in terminally differentiated cells and coupled to metabolic effects. Recent findings have revealed that different distributions of INSR and an altered INSR-A:INSR-B ratio may contribute to metabolic abnormalities during the onset of insulin resistance and the progression to type 2 diabetes. In this review, we discuss the role of insulin and the INSR in the development and endocrine activity of adipose tissue and the pharmacological implications for the management of obesity and type 2 diabetes.

Diabetic foot is a common complication in diabetes mellitus course, able to increase the overall morbidity/mortality risk of such a disease. The aim was to investigate the outcomes, the incidence of clinical events, the number of recurrent ulcers in patients with diabetic foot during 1 year follow-up after angioplasty (PTA) revascularization. From January 2007 to August 2009, 103 diabetic patients with diabetic foot undergoing revascularization of a lower limb by PTA were recruited. At 1 year follow-up we assessed: "major" (death, stroke, Myocardial Infaction (MI) and "minor" (Deep Vein Thrombosis (DVT), renal failure, restenosis) events incidence; recurrent ulcers incidence; the predictive elements of all these events. At 1 year follow-up, "major"/"minor" events incidence was 15% (deaths:5, stroke: 1, MI: 9%) and 34% (renal failure: 11, DVT: 9, restenosis: 14%), respectively. Obesity, high low density level-cholesterol levels and distal arterial lesions (at posterior tibial artery in particular) were statistically significantly associated with major events (p<0.05); only obesity resulted statistically associated with minors (p = 0.043). High levels of C-reactive protein had a statistically significant relationship with the recurrence of ulcers (p = 0.006) while distal arterial obstructions showed a trend toward significance. To improve diabetic foot mortality and morbidity rate, our study underlines the importance of a prompt diagnosis and appropriate revascularization treatment. Other studies are needed to ascertain these. © 2012 Asian Network for Scientific Information.

BACKGROUND: Type 2 diabetes (T2D) might occur within metabolic syndrome (MbS). One of the complications of T2D is an impaired (imp) cardiovascular autonomic function (CAF). AIMS: In subjects with T2D and age ≤ 55 years, the prevalence of impCAF and its relationship with BMI, waist, HbA(1c) values, MbS, hypertension, and family history of T2D and/or hypertension were analysed. METHODS: 180 subjects consecutively undergoing a day hospital for T2D were studied. The IDF criteria were used to diagnose MbS. To detect impCAF, 5 tests for the evaluation of CAF were performed with Cardionomic (Meteda, Italy). Univariate and multivariate analyses were performed. RESULTS: The prevalence of impCAF and MbS were 33.9% and 67.8%, respectively. Among diabetics with impCAF, 86.9% had MbS. ImpCAF was significantly associated with MbS, overweight, and HbA(1c) > 7%. Both logistic (P = 0.0009) and Poisson (P = 0.0113) models showed a positive association between impCAF and MbS. The degree of ImpCAF showed a positive linear correlation with BMI and HbA(1c) values. CONCLUSIONS: The study demonstrates that glycaemic control and overweight influence CAF and that T2D + MbS is more strongly associated with impCAF than isolated T2D. We suggest that MbS not only increases the cardiovascular risk of relatively young subjects with T2D but is also associated with impCAF.

BACKGROUND AND AIM: We evaluated the relationship between insulin resistance (IR) and insulin secretion with the metabolic syndrome (MS) in 885 subjects (377 men/508 women, age 49±11 years, BMI 29±5.2kgm(-2)) at risk of diabetes enrolled in the genetics, pathophysiology and evolution of type 2 diabetes (GENFIEV) study. METHODS AND RESULTS: All subjects underwent a 75-g oral glucose tolerance test (OGTT) for the estimation of plasma levels of glucose and C-peptide, as well as fasting insulin and lipid profile. IR was arbitrarily defined as HOMA-IR value above the 75th centile of normal glucose tolerance (NGT) subjects. Overall MS prevalence (National Cholesterol Treatment Panel-Adult Treatment Panel (NCEP-ATPIII) criteria) was 33%, 19% in subjects with NGT, 42% in impaired fasting glucose (IFG), 34% in impaired glucose tolerance (IGT), 74% in IFG+IGT subjects, and 56% in newly diagnosed diabetic patients. Prevalence was slightly higher with IDF criteria. MS prevalence was >50% in subjects with 2h glucose >7.8mmoll(-1), independently of fasting plasma glucose. IR prevalence was higher in subjects with MS than in those without (63% vs. 23%; p<0.0001) and increased from 54% to 73% and 88% in the presence of three, four or five traits, respectively. IR occurred in 42% of subjects with non-diabetic alterations of glucose homeostasis, being the highest in those with IFG+IGT (IFG+IGT 53%, IFG 45%, IGT 38%; p<0.0001). Individuals with MS were more IR irrespective of glucose tolerance (p<0.0001) with no difference in insulinogenic index. Hypertriglyceridaemia (OR: 3.38; Confidence Interval, CI: 2.294.99), abdominal obesity (3.26; CI: 2.18-4.89), hyperglycaemia (3.02; CI: 1.80-5.07) and hypertension (1.69; CI: 1.12-2.55) were all associated with IR. CONCLUSIONS: These results show that in subjects with altered glucose tolerance (in particular IFG+IGT) MS prevalence is high and is generally associated to IR. Some combinations of traits of MS may significantly contribute to identify subjects with IR.

Type 2 diabetes mellitus is increasing in prevalence at alarming rates. Concurrent with its expanding prevalence is the increase in the related risk of morbidity and mortality. Because diabetic patients are prone to cardiovascular disease, treatment strategies should address the cardiovascular risk factors, including blood pressure, lipids, and body weight, in addition to the glycemic aspects of the disease. Newer agents, such as glucagon-like peptide-1 (GLP-1) analogs and dipeptidyl peptidase-4 (DPP-4) inhibitors, have varying degrees of evidence to support their effects on body weight, blood pressure, and lipid levels, beyond glycated hemoglobin reduction. While GLP-1 agonists produce a weight loss, the DPP-4 inhibitors, conversely, appear to have a weight-neutral effect. Substantial evidence demonstrates that both medications produce modest reductions in systolic blood pressure and, in some cases, diastolic blood pressure, and reduce several markers of cardiovascular risk, including C-reactive protein. Moreover, GLP-1 influences endothelial function. The effect of the incretin hormones on serum lipids are either neutral or beneficial, with small, non-significant decreases in LDL cholesterol, increases in HDL cholesterol, and occasionally significant decreases in fasting triglyceride levels. Also, they have positive effects on hepatic steatosis. Although GLP-1 agonists and DPP-4 inhibitors are at present not appropriate for primary treatment of cardiovascular risks factors, the reduction of these parameters is evidently beneficial.

OBJECTIVE: Multiple endocrine neoplasia type 2 (MEN 2) is a genetic disease characterized by medullary thyroid carcinoma (MTC) associated (MEN 2A and 2B) or not familial MTC (FMTC) with other endocrine neoplasia due to germline RET gene mutations. The prevalence of these rare genetic diseases and their corresponding RET mutations are unknown due to the small size of the study population. METHODS: We collected data on germline RET mutations of 250 families with hereditary MTC followed in 20 different Italian centres. RESULTS AND CONCLUSIONS: The most frequent RET amino acid substitution was Val804Met (19.6%) followed by Cys634Arg (13.6%). A total of 40 different germline RET mutations were present. Six families (2.4%) were negative for germline RET mutations. The comparison of the prevalence of RET germline mutations in the present study with those published by other European studies showed a higher prevalence of Val804Met and Ser891Ala mutations and a lower prevalence of Leu790Phe and Tyr791Phe (P<0.0001). A statistically significant higher prevalence of mutations affecting non-cysteine codons was also found (P<0.0001). Furthermore, the phenotype data collection showed an unexpected higher prevalence of FMTC (57.6%) with respect to other MEN 2 syndromes (34% MEN 2A and 6.8% of MEN 2B). In conclusion, we observed a statistically significant different pattern of RET mutations in Italian MEN 2 families with respect to other European studies and a higher prevalence of FMTC phenotype. The different ethnic origins of the patients and the particular attention given to analysing apparently sporadic MTC for RET germline mutations may explain these findings.

AIMS: Postprandial hyperglycaemia in patients with Type 2 diabetes mellitus has been linked to the development of cardiovascular disease. This study compared the effects of mealtime (thrice-daily) nateglinide with once-daily glyburide on postprandial glucose levels in patients with Type 2 diabetes and postprandial hyperglycaemia. METHODS: Patients with Type 2 diabetes aged ≥ 21 years with 2-h postprandial glucose levels ≥ 11.1 mmol/l, HbA(1c) of 6.5-8.5% (48-69 mmol/mol) and BMI of 22-30 kg/m(2) were randomized to 6 weeks' double-blind treatment with nateglinide 120 mg three times daily prior to meals, or glyburide 5 mg once daily before breakfast. The primary endpoint was the baseline-adjusted change in plasma glucose from preprandial (fasting plasma glucose) to 2-h postprandial glucose levels (2-h postprandial glucose excursion) at 6 weeks. RESULTS: Patients were randomized to nateglinide (n = 122) or glyburide (n = 110). The treatment groups were similar in terms of age, gender, BMI, fasting plasma glucose, 2-h postprandial glucose and HbA(1c). At endpoint, nateglinide recipients had significantly greater reductions than those receiving glyburide in both the 2-h (-2.4 vs. -1.6 mmol/l; P = 0.02) and 1-h (-1.7 vs. -0.9 mmol/l; P = 0.016) postprandial glucose excursions. Adverse events, most commonly symptomatic hypoglycaemia, were reported in 26% of recipients of glyburide and 22% of recipients of nateglinide. Episodes of suspected mild hypoglycaemia were reported in 24% of recipients of glyburide and 10% of recipients of nateglinide. CONCLUSIONS: Nateglinide leads to greater reductions in postprandial glucose excursions and is associated with a lower risk of hypoglycaemia than glyburide in this selected population of patients with Type 2 diabetes.

The effect of elevated body mass index (BMI) on the oocyte quality was investigated in women undergoing in vitro fertilization (IVF) cycles. A total of 268 patients classified on the basis of BMI subject to the first reproductive treatment were included in this study: the normal weight (NW) group consisted of 160 patients with BMI 19-24.9 kg/m(2) and the overweight (OW) group consisted of 108 patients with BMI ≥ 25 kg/m(2). All women were treated with a standard long luteal protocol. The oocyte features were classified as extracytoplasmic or cytoplasmic abnormalities. Outcomes were oocyte morphology, embryo quality, fertilization and implantation rates, and the ovarian response to stimulation. A higher percentage of oocytes with granular cytoplasm was found in women with BMI ≥ 25 (p = 0.04). However, percentages of mature, immature oocytes and germinal vesicle were similar in both groups. No differences were found in fertilization and cleavage rates and percentages of embryo quality. The implantation rate (p < 0.001) was significantly lower in the OW group than in the NW group. The amount of gonadotrophins was significantly higher in OW group (p = 0.003). These findings suggest that the poor reproductive outcome of obese women is influenced by the release of ova with reduced fertilization potential.

OBJECTIVE: To determine the functional health status and treatment satisfaction in patients with type 2 diabetes from the Evaluation of Lantus Effect ON Optimization of use of single dose Rapid insulin (ELEONOR) study that investigated whether a telecare program helps optimization of basal insulin glargine with one bolus injection of insulin glulisine. RESEARCH DESIGN AND METHODS: Functional health status and treatment satisfaction were investigated using the 36-Item Short-Form (SF-36) Health Survey, the World Health Organization Well-Being Questionnaire (WBQ), and the Diabetes Treatment Satisfaction Questionnaire. RESULTS: Of 291 randomized patients, 238 completed the study (telecare: 114; self-monitoring blood glucose: 124). Significant improvements were detected in most SF-36 domains, in WBQ depression and anxiety scores, and in treatment satisfaction, without differences between study groups. CONCLUSIONS: An insulin regimen that substantially improves metabolic control, while minimizing the risk of hypoglycemia, can positively affect physical and psychologic well-being and treatment satisfaction irrespective of the educational support system used.

p66Shc, a 66 kDa proto-oncogene Src collagen homologue (Shc) adaptor protein, is classically known as a signalling protein implicated in receptor tyrosine kinase signal transduction. The p66Shc isoform exerts a physiologically relevant, inhibitory signalling effect on the Erk pathway in skeletal muscle myoblasts, which is necessary for actin cytoskeleton polymerization and normal glucose transport responses. More recently, p66Shc has been also identified as a sensor of oxidative stress-induced apoptosis and as a longevity protein in mammals, actions which require Ser36 phosphorylation of the protein and consequent accumulation of intracellular reactive oxygen species. Oxidative stress plays a key role in dysfunction of several organs and tissues, and this is of interest in metabolic diseases such as type 2 diabetes. Thus changes in p66Shc expression and/or function may play an important role in the pathogenesis of type 2 diabetes and potentially serve as an effective target for its treatment.

OBJECTIVE - To ascertain to which extent the use of HbA1c and oral glucose tolerance test (OGTT) for diagnosis of glucose tolerance could identify individuals with different pathogenetic mechanisms and cardiovascular risk profile. RESEARCH DESIGNANDMETHODS - A total of 844 subjects (44%men; age 49.5 ± 11 years; BMI 29 ± 5 kg/m2) participated in this study. Parameters of b-cell functionwere derived from deconvolution of the plasma C-peptide concentration after a 75-g OGTT and insulin sensitivity assessed by homeostasismodel assessment of insulin resistance (IR). Cardiovascular risk profile was based on determination of plasma lipids and measurements of body weight, waist circumference, and blood pressure. Glucose regulation categories by OGTT and HbA1c were comparedwith respect to insulin action, insulin secretion, and cardiovascular risk profile. RESULTS - OGTT results showed 42% of the subjects had prediabetes and 15% had type 2 diabetes mellitus (T2DM), whereas the corresponding figures based on HbA 1c were 38 and 11%, with a respective concordance rate of 54 and 44%. Subjects meeting both diagnostic criteria for prediabetes presented greater IR and impairment of insulin secretion and had aworse cardiovascular risk profile than those with normal glucose tolerance at both diagnostic methods. In a logistic regression analyses adjusted for age, sex, and BMI, prediabetic subjects, and even more T2DM subjects by OGTT, had greater chance to have IR and impaired insulin secretion. CONCLUSIONS - HbA1c identifies a smaller proportion of prediabetic individuals and even a smaller proportion of T2DM individuals than OGTT, with no difference in IR, insulin secretion, and cardiovascular risk profile. Subjects fulfilling both diagnostic methods for prediabetes or T2DM are characterized by a worse metabolic profile.

Self-monitoring of blood glucose (SMBG) is a core component of diabetes management. However, the International Diabetes Federation recommends that SMBG be performed in a structured manner and that the data are accurately interpreted and used to take appropriate therapeutic actions. We designed a study to evaluate the impact of structured SMBG on glycemic control in non-insulin-treated type 2 diabetes (T2DM) patients. The Prospective, Randomized Trial on Intensive SMBG Management Added Value in Non-insulin-Treated T2DM Patients (PRISMA) is a 12-month, prospective, multicenter, open, parallel group, randomized, and controlled trial to evaluate the added value of an intensive, structured SMBG regimen in T2DM patients treated with oral agents and/or diet. One thousand patients (500 per arm) will be enrolled at 39 clinical sites in Italy. Eligible patients will be randomized to the intensive structured monitoring (ISM) group or the active control (AC) group, with a glycosylated hemoglobin (HbA1c) target of <7.0%. Intervention will comprise (1) structured SMBG (4-point daily glucose profiles on 3 days per week [ISM]; discretionary, unstructured SMBG [AC]); (2) comprehensive patient education (both groups); and (3) clinician's adjustment of diabetes medications using an algorithm targeting SMBG levels, HbA1c and hypoglycemia (ISM) or HbA1c and hypoglycemia (AC). The intervention and trial design build upon previous research by emphasizing appropriate and collaborative use of SMBG by both patients and physicians. Utilization of per protocol and intent-to-treat analyses facilitates assessment of the intervention. Inclusion of multiple dependent variables allows us to assess the broader impact of the intervention, including changes in patient and physician attitudes and behaviors. ClinicalTrials.gov (NCT00643474).

The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-δ, peroxisome proliferator-activated receptor-γ, and C/EBPα mRNA levels were increased severalfold 2-6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBPδ mRNA induction support a role for this protein during early adipogenesis.

CONTEXT AND OBJECTIVE: High-mobility group box-1 (HMGB1) is a pro-inflammatory cytokine that may contribute to the pathogenesis of micro- and macrovascular complications commonly observed in diabetes. We investigated whether HMGB1 is associated with: i) markers of low-grade inflammation (LGI) and endothelial dysfunction (ED) and pulse pressure (PP, a marker of arterial stiffness); ii) prevalent nephropathy, retinopathy and cardiovascular disease (CVD) in type 1 diabetes; and iii) the potential mediating roles of LGI, ED and PP therein. DESIGN AND METHODS: This was a cross-sectional nested case-control study of 463 patients (226 women; mean age 40±10 years) with type 1 diabetes from the EURODIAB Prospective Complications Study. We used linear and binary or multinomial logistic regression analyses adjusted for traditional risk factors. RESULTS: Serum Ln-HMGB1 levels were positively associated with LGI and ED (standardised β=0.07 (95% confidence interval (CI): 0.02-0.12) and β=0.08 (95% CI: 0.02-0.14) respectively), but not with PP. Higher Ln-HMGB1 (per unit) was associated with greater odds of micro- and macroalbuminuria: odds ratio (OR)=1.24 (95% CI: 0.90-1.71) and OR=1.61 (95% CI: 1.15-2.25) respectively, P for trend=0.004. Further adjustments for LGI or ED did not attenuate these associations. No such associations were found between Ln-HMGB1 and estimated glomerular filtration rate (eGFR), retinopathy or CVD, however. CONCLUSIONS: In type 1 diabetes, higher serum HMGB1 levels are associated with greater prevalence and severity of albuminuria, though not with eGFR, retinopathy and CVD. Prospective studies are needed to clarify the causal role of HMGB1, if any, in the pathogenesis of vascular complications in type 1 diabetes.

BACKGROUND: We compared telecare and conventional self-monitored blood glucose (SMBG) programs for titrating the addition of one bolus injection of insulin glulisine in patients with type 2 diabetes uncontrolled on oral hypoglycemic agents for ≥3 months who were first titrated with basal insulin glargine. METHODS: This randomized, multicenter, parallel-group study included 241 patients (mean screening glycosylated hemoglobin [HbA(1c)], 8.8% [73 mmol/mol]). In the run-in phase, any antidiabetes medication, except for metformin, was discontinued. Metformin was then up-titrated to 2 g/day (1 g twice daily) until study completion. Following run-in, all patients started glargine for 8-16 weeks, targeting fasting plasma glucose (FPG) ≤5.6 mmol/L using conventional SMBG. Patients with FPG ≤7 mmol/L added a glulisine dose at the meal with the highest postprandial plasma glucose excursion, titrated to target 2-h postprandial plasma glucose level <7.8 mmol/L using telecare or SMBG for 24 weeks. Patients with FPG >7 mmol/L at week 16 were withdrawn from the study. RESULTS: After glargine titration, 224 patients achieved FPG ≤7 mmol/L, without any difference between telecare and SBMG groups (mean±SD, 6.2±0.8 vs. 6.0±0. 9 mmol/L, respectively). HbA(1c) levels were lower following titration and were similar for telecare and SMBG (7.9±0.9% vs. 7.8±0.9% [63 vs. 62 mmol/mol], respectively). Adding glulisine further reduced HbA(1c) in both groups (-0.7% vs. -0.7%); 45.2% and 54.8% (P=0.14), respectively, of patients achieved HbA(1c) ≤7.0% (≤53 mmol/mol). Weight change and hypoglycemia were similar between groups. CONCLUSIONS: Patients adding one dose of glulisine at the meal with the highest postprandial plasma glucose excursion to titrated basal glargine achieved comparable improvements in glycemic control irrespective of traditional or telecare blood glucose monitoring.

The chronic kidney disease (CKD)-Epidemiology Collaboration (EPI) equation was shown to be more accurate than the Modification of Diet in Renal Disease (MDRD) Study formula for estimating glomerular filtration rate (GFR) in the general population. This study was aimed at assessing cardiovascular disease (CVD) burden associated with CKD in type 2 diabetes, using these two GFR estimating formulas for CKD definition. METHODS: This cohort study examined 15,773 Caucasian patients with type 2 diabetes participating in the Renal Insufficiency And Cardiovascular Events Italian Multicenter Study (NCT00715481) and attending the baseline visit in 19 diabetes clinics in years 2007-2008. Serum creatinine was assessed by the modified Jaffe method. Albuminuria was measured by immunonephelometry or immunoturbidimetry. CKD was defined as an estimated GFR (eGFR) <60 mL/min/1.73 m(2) and/or micro/macroalbuminuria. RESULTS: Prevalence of impaired eGFR and CKD decreased from 18.7% to 17.2% (P=0.0012) and from 37.5% to 36.3% (P=0.077), respectively, with the CKD-EPI, as compared with the MDRD Study equation. Subjects with impaired eGFR or CKD with the MDRD Study equation only showed lower CVD prevalence rates and coronary heart disease risk scores, mainly driven by prevailing female sex, younger age and shorter diabetes duration, as compared with those with both formulas, whereas opposite figures were observed in patients falling into these categories with the CKD-EPI equation only. CONCLUSIONS: Estimating GFR in patients with type 2 diabetes using the CKD-EPI equation provides a better definition of CVD burden associated with CKD not only in individuals reclassified upward, but also in those reclassified downward

The widespread increase in life expectancy is accompanied by an increased prevalence of features of physical frailty. Signs and symptoms may include sarcopenia and osteopenia, reduced exercise capacity, and diminished sense of well-being. The pathogenesis of age-associated sarcopenia and osteopenia is multifactorial, and hormonal decline may be a contributing factor. Aging is associated with a progressive decrease in GH secretion, and more than 30% of elderly people have circulating IGF1 levels below the normal range found in the young. GH acts directly on target tissues, including skeletal muscle and bone among many others, but many effects are mediated indirectly by circulating (liver-derived) or locally produced IGF1. Aging is also associated with reduced insulin sensitivity which, in turn, may contribute to the impairment of IGF1 action. Recent experimental evidence suggests that besides the age-dependent decline in GH and IGF1 serum levels, the dysregulation of GH and IGF1 actions due to impairment of the post-receptor signaling machinery may contribute to the loss of muscle mass and osteopenia. This article will focus on the molecular mechanisms of impaired GH and IGF1 signaling and action in aging, and their role in the pathogenesis of sarcopenia and osteoporosis.

The p66(Shc) protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66(Shc) in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66(Shc), with Ser(36) replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2 increased reactive oxygen species and phosphorylation of p66(Shc) on Ser(36) in HepG2 cells. Overexpression of p66(Shc) promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H2O2. p66(Shc) activation also resulted in increased Erk-1/2, Akt and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66(Shc) phosphorylation on Ser(36). Increased p66Shc expression was associated with reduced mRNA levels of anti-oxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66(Shc) Ala(36) mutant inhibited p66(Shc) signaling, enhanced anti-oxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66(Shc) protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared to control subjects.

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66(Shc) acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66(Shc) in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66(Shc) on Ser(36) and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66(Shc) in HUVEC resulted in enhanced p66(Shc) phosphorylation on Ser(36), increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66(Shc) protein, in which Ser(36) was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66(Shc) resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66(Shc) acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66(Shc) on Ser(36).

OBJECTIVE To evaluate safety and efficacy of DiaPep277 in preserving β-cell function in type 1 diabetic patients. RESEARCH DESIGN AND METHODS DIA-AID 1 is a multinational, phase 3, balanced-randomized, double-blind, placebo-controlled, parallel-group clinical study. Newly diagnosed patients (N = 457, aged 16-45 years) were randomized to subcutaneous injections of DiaPep277 or placebo quarterly for 2 years. The primary efficacy end point was the change from baseline in the area under the glucagon-stimulated C-peptide curve. Secondary end points were the change from baseline in mixed-meal stimulated C-peptide secretion and in fasting C-peptide and achieving target HbA1c ≤7% (≤53 mmol/mol). Partial remission (target HbA1c on insulin ≤0.5 units/kg/day) and hypoglycemic event rate were exploratory end points. RESULTS DiaPep277 was safe and well tolerated. Significant preservation of C-peptide secretion was observed in the DiaPep277-treated group compared with the placebo (relative treatment effects of 23.4%, P = 0.037, and 29.2%, P = 0.011, in the modified intent-to-treat [mITT] and per-protocol [PP] populations, respectively). The mixed-meal stimulation failed to distinguish between the groups. There was a trend toward efficacy in fasting C-peptide levels, though not statistically significant. Significantly more DiaPep277-treated than placebo-treated patients maintained target HbA1c (mITT 56% versus 44%, P = 0.03; PP 60% versus 45%, P = 0.0082) and entered partial remission (mITT 38% versus 29%, P = 0.08; PP 42% versus 30%, P = 0.035). DiaPep277 treatment reduced the relative hypoglycemic event risk (mITT by 20%; PP by 28%). CONCLUSIONS DiaPep277 safely contributes to preservation of β-cell function and to improved glycemic control in patients with type 1 diabetes.

Diabetes is associated with many hemorheological alterations. The decrease of RBC deformability, increase of aggregability, vasoconstriction, increase of blood viscosity and decrease of oxygen supply have a significant effect on wound healing, such as in foot ulcers. Basically, there is endothelial dysfunction and alteration of permeability; these impair wound healing in diabetic patients. Microcirculation still functions and there is blood flow, even when there is a decrease in vessel diameter, without anatomical lesions in vessel walls. It is necessary to maintain a good oxygen supply. Analyzing microcirculation and hemorheology in diabetes and considering methodologies to treat diabetic foot ulcers (e.g., hyperbaric oxygen therapy, laser, and vacuum) may help in the treatment of patient pathologies.

OBJECTIVE: Zinc transporter 8 (ZnT8) is an islet beta-cell secretory granule membrane protein recently identified as an autoantibody antigen in type 1 diabetes. The aim of this study was to determine the prevalence and role of antibodies to ZnT8 (ZnT8As) in adult-onset diabetes. RESEARCH DESIGN AND METHODS: ZnT8As were measured by a radioimmunoprecipitation assay using recombinant ZnT8 COOH-terminal or NH(2)-terminal proteins in 193 patients with adult-onset autoimmune diabetes having antibodies to either GAD (GADAs) or IA-2 (IA-2As) and in 1,056 antibody-negative patients with type 2 diabetes from the Non Insulin Requiring Autoimmune Diabetes (NIRAD) study. RESULTS: ZnT8As-COOH were detected in 18.6% patients with autoimmune diabetes and 1.4% with type 2 diabetes. ZnT8As-NH(2) were rare. ZnT8As were associated with younger age and a high GADA titer. The use of GADAs, IA-2As, and ZnT8As in combination allowed a stratification of clinical phenotype, with younger age of onset of diabetes and characteristics of more severe insulin deficiency (higher fasting glucose and A1C, lower BMI, total cholesterol, and triglycerides) in patients with all three markers, with progressive attenuation in patients with two, one, and no antibodies (all P(trend) < 0.001). Autoantibody titers, association with high-risk HLA genotypes, and prevalence of thyroid peroxidase antibodies followed the same trend (all P < 0.001). CONCLUSIONS: ZnT8As are detectable in a proportion of patients with adult-onset autoimmune diabetes and seem to be a valuable marker to differentiate clinical phenotypes.

La nostra invenzione riguarda il possibile impiego di una proteina nota, denominata irisina, derivante dal clivaggio di un precursore di membrana chiamato fibronectin type III domain-containing protein 5 (FNDC5), come farmaco per la preservazione della funzionalità e della sopravvivenza delle popolazioni cellulari che compongono l’isola pancreatica, deputata alla produzione di insulina. I risultati sperimentali, da noi ottenuti, dimostrano la capacità dell’irisina di indurre la sintesi e di stimolare la secrezione di insulina glucosio-indotta da parte delle beta-cellule pancreatiche. Inoltre, l’irisina promuove la proliferazione delle beta-cellule e ne riduce la morte indotta dall’esposizione cronica ad acidi grassi saturi. Queste proprietà fanno dell’irisina un valido ausilio nella terapia farmacologica del diabete mellito di tipo 2 e di tutte le condizioni patologiche caratterizzate da alterazioni della funzione secretoria, dalla riduzione del numero delle beta-cellule pancreatiche e da modificazioni della normale architettura/funzione dell’isola pancreatica.