Giulia Donadel

Early vascular and metabolic effects of rosuvastatin compared with simvastatin in patients with type 2 diabetes

OBJECTIVE To compare the short-term effects of rosuvastatin and simvastatin on insulin-resistance and endothelial dysfunction in middle-aged patients with type 2 diabetes and mild untreated dyslipidemia. METHODS AND DESIGN 29 Subjects randomly assigned to rosuvastatin 20mg/daily or simvastatin 20mg/daily for 4 weeks. Following data collected both pre- and post-treatment: fasting glucose, lipids, hs CRP, TNF-alpha, insulin sensitivity measured with euglycemic-hyperinsulinemic clamp and flow-mediated dilation with brachial artery reactivity technique. RESULTS Both treatments markedly reduced LDL cholesterol (p<0.001 for both). Insulin sensitivity did not change from relative baseline values in both groups, as well as fasting glucose and adiponectin. Simvastatin significantly improved flow-mediated dilation (p<0.01), to a greater extent than in patients taking rosuvastatin (p=0.09). We found no association between flow-mediated dilation improvement, LDL reduction and changes in hs CRP levels. CONCLUSION In type 2 diabetic individuals rosuvastatin was less effective than simvastatin at improving endothelium-dependent vasodilation within one month, without affecting insulin-resistance, adiponectin levels and inflammation.

Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells

Background The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro. Methodology/Principal Findings Human umbilical vein endothelial cells (HUVECs) were treated with insulin in presence of glucose 30 mM (HG) and glucosamine 10 mM (Gluc-N) with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine. Conclusions/Significance These data indicate that sildenafil might improve NOS activity of endothelial cells in insulin resistance conditions and suggest the potential therapeutic use of sildenafil for improving vascular function in diabetic patients.

Pharmacogenomics and pharmacogenetics of thiazolidinediones: role in diabetes and cardiovascular risk factors

The most important goal in the treatment of patients with diabetes is to prevent the risk of cardiovascular disease (CVD), the first cause of mortality in these subjects. Thiazolidinediones (TZDs), a class of antidiabetic drugs, act as insulin sensitizers increasing insulin-dependent glucose disposal and reducing hepatic glucose output. TZDs including pioglitazone, rosiglitazone and troglitazone, by activating PPAR-γ have shown pleiotropic effects in reducing vascular risk factors and atherosclerosis. However, troglitazone was removed from the market due to its hepatoxicity, and rosiglitazone and pioglitazone both have particular warnings due to being associated with heart diseases. Specific genetic variations in genes involved in the pathways regulated by TDZs have demonstrated to modify the variability in treatment with these drugs, especially in their side effects. Therefore, pharmacogenomics and pharmacogenetics are an important tool in further understand intersubject variability per se but also to assess the therapeutic potential of such variability in drug individualization and therapeutic optimization.

Peroxiredoxin 6, a novel player in the pathogenesis of diabetes

Enhanced oxidative stress contributes to the pathogenesis of diabetes and its complications. Peroxiredoxin 6 (PRDX6) is a key regulator of cellular redox balance, with the peculiar ability to neutralize peroxides, peroxynitrite, and phospholipid hydroperoxides. In the current study, we aimed to define the role of PRDX6 in the pathophysiology of type 2 diabetes (T2D) using PRDX6 knockout (−/−) mice. Glucose and insulin responses were evaluated respectively by intraperitoneal glucose and insulin tolerance tests. Peripheral insulin sensitivity was analyzed by euglycemic-hyperinsulinemic clamp, and molecular tools were used to investigate insulin signaling. Moreover, inflammatory and lipid parameters were evaluated. We demonstrated that PRDX6−/− mice developed a phenotype similar to early-stage T2D caused by both reduced glucose-dependent insulin secretion and increased insulin resistance. Impaired insulin signaling was present in PRDX6−/− mice, leading to reduction of muscle glucose uptake. Morphological and ultrastructural changes were observed in islets of Langerhans and livers of mutant animals, as well as altered plasma lipid profiles and inflammatory parameters. In conclusion, we demonstrated that PRDX6 is a key mediator of overt hyperglycemia in T2D glucose metabolism, opening new perspectives for targeted therapeutic strategies in diabetes care.

Parathyroid Hormone and Insulin Resistance in Distinct Phenotypes of Severe Obesity: A Cross-Sectional Analysis in Middle-Aged Men and Premenopausal Women

CONTEXT High levels of PTH are reported in obese individuals and related to increased cardiometabolic risk. OBJECTIVE Our objective was to evaluate whether the relationship between PTH, insulin resistance, and related metabolic parameters differ between metabolically healthy obese (MHO) and insulin-resistant obese (IRO) subjects. DESIGN AND SETTING We conducted a cross-sectional study among patients evaluated for bariatric surgery in our University Hospital. PATIENTS Patients initially included were 174 severely obese subjects (114 women, aged 40 ± 5 yr, body mass index of 45 ± 6 kg/m(2)) without diabetes, chronic kidney disease, or hyperparathyroidism. MHO (n = 43) and IRO (n = 86) subjects were identified according to quartiles of insulin resistance. MAIN OUTCOME MEASURES Fasting and postload glucose, insulin, total and high-density lipoprotein cholesterol, triglycerides, creatinine, calcium, phosphorus, PTH, 25-hydroxyvitamin D (25OHD), fibrinogen, and high-sensitivity C-reactive protein were assessed. Insulin sensitivity index was derived from a 75-g oral glucose tolerance test. Fat distribution and bone mineral density were assessed with dual-energy x-ray absorptiometry. RESULTS Although 25OHD levels were higher in MHO than in IRO subjects [72.23 (59.41-80.36) vs. 52.36 (41.98-62.57) nmol/liter, P = 0.002], PTH levels were comparable between groups (74.4 ± 13.2 vs. 72.1 ± 15.1 ng/liter, P = 0.34). No differences in serum calcium, phosphorus, bone mineral density, and renal function were detected. An independent inverse association between 25OHD and insulin resistance was seen in both groups. In contrast to IRO subjects, after adjusting for covariates, PTH levels were unrelated to insulin sensitivity index, fasting and postload glucose, insulin, and high-sensitivity C-reactive protein in MHO subjects. CONCLUSIONS MHO and IRO subjects show comparably high levels of circulating PTH, which are not associated with insulin resistance and related metabolic parameters in MHO subjects. Most of the associations observed in IRO subjects appear to be mediated by greater truncal fat mass.

Human placental lactogen (hPL-A) activates signaling pathways linked to cell survival and improves insulin secretion in human pancreatic islets

The search for factors either promoting islets proliferation or survival during adult life is a major issue for both type 1 and 2 diabetes mellitus. Among factors with mitogenic activity on pancreatic β-cells, human placental lactogen (hPL) showed stronger activity when compared to the other lactogen hormones: growth hormone (GH) and prolactin (PRL). The aim of the present work is to elucidate the biological and molecular events of hPL isoform A (hPL-A) activity on human cultured islets. We used pure human pancreatic islets and insulinoma cell lines (βTC-1 and RIN, murine and rat respectively) stimulated with hPL-A recombinant protein and we compared hPL-A activity with that of hGH. We showed that hPL-A inhibits apoptosis, both in insulinoma and human islets, by the phosphorylation of AKT protein. Indeed, the antiapoptotic role of hPL-A was mediated by PI3K, p38 and it was independent by PKA, Erk1/2. Compared with hGH, hPL-A modulated at different intervals and/or intensity by the phosphorylation of JAKs/STATs and MAPKinases. Moreover, hPL-A induced PDX-1 intracellular expression, improving beta cell activity and ameliorating insulin secretion in response to high glucose stimulation. Our data support the idea that hPL-A is involved in the regulation of beta cells activity. Importantly, we found that hPL-A can preserve and improve the ability of purified human pancreatic islets cultured to secrete insulin in vitro.

Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia

Diabetic hyperglycaemia causes endothelial dysfunction mainly by impairing endothelial nitric oxide (NO) production. Moreover, hyperglycaemia activates several noxious cellular pathways including apoptosis, increase in reactive oxygen species (ROS) levels and diminishing Na+–K+ ATPase activity which exacerbate vascular damage. Serum glucocorticoid kinase (SGK)-1, a member of the serine/threonine kinases, plays a pivotal role in regulating NO production through inducible NO synthase activation and other cellular mechanisms. Therefore, in this study, we aimed to investigate the protective role of SGK-1 against hyperglycaemia in human umbilical endothelial cells (HUVECs). We used retrovirus to infect HUVECs with either SGK-1, SGK-1Δ60 (lacking of the N-60 amino acids—increase SGK-1 activity) or SGK-1Δ60KD (kinase-dead constructs). We tested our hypothesis in vitro after high glucose and glucosamine incubation. Increase in SGK-1 expression and activity (SGK-1Δ60) resulted in higher production of NO, inhibition of ROS synthesis and lower apoptosis in endothelial cell after either hyperglycaemia or glucosamine treatments. Moreover, in this study, we showed increased GLUT-1 membrane translocation and Na+−K+ ATPase activity in cell infected with SGK-1Δ60 construct. These results suggest that as in endothelial cells, an increased SGK-1 activity and expression reduces oxidative stress, improves cell survival and restores insulin-mediated NO production after different noxae stimuli. Therefore, SGK-1 may represent a specific target to further develop novel therapeutic options against diabetic vascular disease.

SGK-1 protects kidney cells against apoptosis induced by ceramide and TNF-α

Ceramide regulates several different cellular responses including mechanisms leading to apoptosis. Serum- and glucocorticoid-inducible protein kinase (SGK)-1 is a serine threonine kinase, which activates survival pathways in response to stress stimuli. Recently, we demonstrated an anti-apoptotic role of SGK-1 in human umbilical endothelial cells treated with high glucose. In the present study, since ceramide induces apoptosis by multiple mechanisms in diabetes and its complication such as nephropathy, we aimed to investigate whether SGK-1 may protect even against apoptosis induced by ceramide in kidney cells. Human embryonic kidney (HEK)-293 cells stable transfected with SGK-1 wild type (SGK-1wt) and its dominant negative gene (SGK-1dn) have been used in this study. Apoptotic stimuli were induced by C2-ceramide and TNF-α to increase endogenous synthesis of ceramide. Upon activation with these stimuli, SGK-1wt transfected cells have a statistically significant reduction of apoptosis compared with SGK-1dn cells (P<0.001). This protection was dependent on activation of caspase-3 and Poly-ADP-ribose-polymerase-1 (PARP-1) cleavage. SGK-1 and AKT-1 two highly homologous kinases differently reacted to ceramide treatment, since SGK-1 increases in response to apoptotic stimulus while AKT-1 decreases. This enhancement of SGK-1 was dependent on p38-mitogen-activated-protein kinases (p38MAPK), cyclic-adenosine-monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide-3-kinase (PI3K) pathways. Especially, by using selective LY294002 inhibitor, we demonstrated that the most involved pathway in the SGK-1 mediated process of protection was PI3K. Treatment with inhibitor of SGK-1 (GSK650394) significantly enhanced TNF-α-dependent apoptosis in HEK-293 cells overexpressing SGK-1wt. Caspase-3, -8 and -9 selective inhibitors confirmed that SGK-1 reduced the activation of caspase-dependent apoptosis, probably by both intrinsic and extrinsic pathways. In conclusion, we demonstrated that in kidney cells, overexpression of SGK-1 is protective against ceramide-induced apoptosis and the role of SGK-1 can be potentially explored as a therapeutic target in conditions like diabetes, where ceramide levels are increased.

The role of epsilon PKC in acute and chronic diseases: Possible pharmacological implications of its modulators.

Epsilon Protein kinase C (εPCK) is a particular kinase that, when activated, is able to protect against different stress injuries and therefore has been proposed to be a potential molecular target against acute and chronic diseases. Particular attention has been focused on εPCK for its involvement in the protective mechanism of Ischemic Preconditioning (IPC), a powerful endogenous mechanism characterized by subthreshold ischemic insults able to protect organs against ischemic injury. Therefore, in the past decades several εPCK modulators have been tested with the object to emulate εPCK mediate protection. Among these the most promising, so far, has been the ΨεRACK peptide, a homologous of RACK receptor for εPKC, that when administrated can mimic its effect in the cells. However, results from studies on εPCK indicate controversial role of this kinase in different organs and diseases, such as myocardial infarct, stroke, diabetes and cancer. Therefore, in this review we provide a discussion on the function of εPCK in acute and chronic diseases and how the different activators and inhibitors have been used to modulate its activity. A better understanding of its function is still needed to definitively target εPCK as novel therapeutic strategy.

FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes

Background: Diabetes mellitus (DM) is a multifactorial disease orphan of a cure. Regenerative medicine has been proposed as novel strategy for DM therapy. Human fibroblast growth factor (FGF)-2b controls β-cell clusters via autocrine action, and human placental lactogen (hPL)-A increases functional β-cells. We hypothesized whether FGF-2b/hPL-A treatment induces β-cell differentiation from ductal/non-endocrine precursor(s) by modulating specific genes expression. Methods: Human pancreatic ductal-cells (PANC-1) and non-endocrine pancreatic cells were treated with FGF-2b plus hPL-A at 500 ng/mL. Cytofluorimetry and Immunofluorescence have been performed to detect expression of endocrine, ductal and acinar markers. Bromodeoxyuridine incorporation and annexin-V quantified cells proliferation and apoptosis. Insulin secretion was assessed by RIA kit, and electron microscopy analyzed islet-like clusters. Results: Increase in PANC-1 duct cells de-differentiation into islet-like aggregates was observed after FGF-2b/hPL-A treatment showing ultrastructure typical of islets-aggregates. These clusters, after stimulation with FGF-2b/hPL-A, had significant (p < 0.05) increase in insulin, C-peptide, pancreatic and duodenal homeobox 1 (PDX-1), Nkx2.2, Nkx6.1, somatostatin, glucagon, and glucose transporter 2 (Glut-2), compared with control cells. Markers of PANC-1 (Cytokeratin-19, MUC-1, CA19-9) were decreased (p < 0.05). These aggregates after treatment with FGF-2b/hPL-A significantly reduced levels of apoptosis. Conclusions: FGF-2b and hPL-A are promising candidates for regenerative therapy in DM by inducing de-differentiation of stem cells modulating pivotal endocrine genes.