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Transglutaminase 2 (TG2 or TGM2) is a multi-functional enzyme which catalyzes transamidation reactions or acts as a G-protein in intracellular signalling. Tgm2-/- Mice lacking TG2 activity are glucose intolerant and show impairment of insulin secretion, suggesting an important physiological role for TG2 in the pancreatic beta cell. We have previously described a TGM2 heterozygous missense mutation ((c.998A>G, p.N333S) in a 14 year-old patient with insulin-treated diabetes and in his diabetic father. The aim of this study was to further investigate the role of TG2 in early-onset type 2 diabetes. We analysed the TGM2 gene in 205 patients with clinically defined Maturity Onset Diabetes of the Young (MODY) or early-onset type 2 diabetes. We found two novel heterozygous mutations (c.989T>G, p.M330R; c.992T>A, p.I331N), which were not detected in 300 normoglycemic controls. All mutations were in residues which are located close to the catalytic site and impaired transamidating activity in vitro. Gene expression of TGM family genes and localization of TG2 in normal human pancreas indicated that TG2 is the only transglutaminase significantly expressed in human pancreatic islet cells. We conclude that reduced TG2 activity can contribute to disorders of glucose metabolism possibly via an impairment of insulin secretion.

SummaryHypertension is a health problem affecting over 1 billion people worldwide. How the immune system gets activated under hypertensive stimuli to contribute to blood pressure elevation is a fascinating enigma. Here we showed a splenic role for placental growth factor (PlGF), which accounts for the onset of hypertension, through immune system modulation. PlGF repressed the expression of the protein Timp3 (tissue inhibitor of metalloproteinases 3), through the transcriptional Sirt1-p53 axis. Timp3 repression allowed costimulation of T cells and their deployment toward classical organs involved in hypertension. We showed that the spleen is an essential organ for the development of hypertension through a noradrenergic drive mediated by the celiac ganglion efferent. Overall, we demonstrate that PlGF mediates the neuroimmune interaction in the spleen, organizing a unique and nonredundant response that allows the onset of hypertension.

Recent evidence suggests that insulin signaling through the insulin receptor A type (Ex11-), regulates insulin gene transcription. Because chronic hyperglycemia negatively affects insulin receptor function and regulates alternative splicing of the insulin receptor, we inquired whether chronic exposure of pancreatic beta-cells to high glucose results in alterations in insulin signaling due to changes in insulin receptor expression and relative abundance of its spliced isoforms. Our results demonstrate that the insulin receptor is localized in insulin secretory vescicles in human pancreatic beta-cells. Furthermore, we find that alterations in insulin expression and secretion caused by chronic exposure to high glucose are paralleled by decreased insulin receptor expression and increased relative abundance of the Ex11+ isoform in both human islets and RIN beta-cells. PDX-1 and HMGI(Y) transcription factors are down-regulated by high glucose. These changes are associated with defects in insulin signaling involving insulin receptor-associated PI 3-kinase/Akt/PHAS-I pathway in RIN beta-cells. Re-expression in RIN beta-cells chronically exposed to high glucose of the Ex11-, but not the Ex11+, isoform restored insulin mRNA expression. These data suggest that changes in early steps of insulin receptor signaling may play a role in determining beta-cell dysfunction caused by chronic hyperglycemia.

Recent studies have identified several polymorphisms in the human insulin receptor substrate-1 (IRS-1) gene. The most prevalent IRS-1 variant, a Gly-->Arg change at the codon 972, has been reported to be increased in prevalence among patients with type 2 diabetes. Carriers of the Arg(972) substitution are characterized by lower fasting insulin and C-peptide levels compared with non-carriers, suggesting that the Arg(972) IRS-1 variant may contribute to impairment of insulin secretion. In this study, we stably overexpressed both wild-type IRS-1 (RIN-WT) and Arg(972) IRS-1 variant (RIN-Arg(972)) in RIN beta cells to investigate directly whether the polymorphism in codon 972 of IRS-1 impairs insulin secretion. The Arg(972) IRS-1 variant did not affect expression or function of endogenous IRS-2. RIN-WT showed a marked increase in both glucose- and insulin-stimulated tyrosine phosphorylation of IRS-1 compared with control RIN cells. The Arg(972) IRS-1 variant did not alter the extent of either glucose- or insulin-stimulated tyrosine phosphorylation of recombinant IRS-1. However, RIN-Arg(972) showed a significant decrease in binding of the p85 subunit of phosphatidylinositol-3-kinase (PI 3-kinase) with IRS-1, compared with RIN-WT. Compared with control RIN cells, insulin content was reduced to the same extent in RIN-WT or RIN-Arg(972) at both the protein and mRNA levels. Both glucose- and sulfonylurea-induced insulin secretion was increased in RIN-WT compared with control RIN cells. By contrast, RIN cells expressing Arg(972) IRS-1 exhibited a marked decrease in both glucose- and sulfonylurea-stimulated insulin secretion compared with RIN-WT. These data suggest that the insulin signaling pathway involving the IRS-1/PI 3-kinase may play an important role in the insulin secretory process in pancreatic beta cells. More importantly, the results suggest that the common Arg(972) IRS-1 polymorphism may impair glucose-stimulated insulin secretion, thus contributing to the relative insulin deficiency observed in carriers of this variant.

A polymorphism in the interleukin 12B gene was recently reported to be strongly associated with type 1 diabetes in 422 Australian and British families. We analyzed the same polymorphism in 470 Italian type 1 diabetic patients and 544 matched control subjects and found no evidence of association with the disease.

Senescence is a phenomenon characterized by a progressive decline of body homeostasis. Premature senescence acts when the cellular system is not able to adequately respond to noxious stimuli by synthesis of stressor molecules. Among those, serum-and-glucocorticoidinducible kinase-1 (SGK-1) dramatically increases under typical physiopathological conditions, such as glucocorticoid or mineralcorticoids exposure, inflammation, hyperglycemia, and ischemia. SGK-1 has been implicated in mechanism regulating oxidative stress, apoptosis, and DNA damage, which are all leading to a state of accelerating aging. Moreover, SGK-1-sensitive ion channels participate in the regulation of renal Na(+)/K(+) regulation, blood pressure, gastric acid secretion, cardiac action potential, and neuroexcitability. Recently, we demonstrated in endothelial cells as an increase in SGK-1 activity and expression reduces oxidative stress, improves cell survival and restores insulin-mediated nitric oxide production after hyperglycemia. Moreover, we showed as SGK-1 delays the onset of senescence by increasing telomerase activity, significantly decreasing reactive oxygen species (ROS) production, and by directly interacting with hTERT. Therefore, SGK-1 may represent a specific target to further develop novel therapeutic options against chronic diseases such as diabetes typical of aging. SGK-1 has been also associated with cancer, neurodegenerative diseases, and cardiovascular disease, among other age-related diseases. However, to date, the data available on SGK-1 and aging, are sparse, controversial, and only from C. elegans experimental models. In this review we sought to discuss the possible implication of SGK-1 in mechanisms regulating senescence and age-related diseases. Moreover, we aimed to discuss and identify the possible role of SGK-1 as possible molecular target to counteract and prevent aging.

Sulfonylureas may stimulate glucose metabolism by protein kinase C (PKC) activation. Because interaction of insulin receptors with PKC plays an important role in controlling the intracellular sorting of the insulin-receptor complex, we investigated the possibility that the sulfonylurea glimepiride may influence intracellular routing of insulin and its receptor through a mechanism involving PKC, and that changes in these processes may be associated with improved insulin action. Using human hepatoma Hep-G2 cells, we found that glimepiride did not affect insulin binding, insulin receptor isoform expression, and insulin-induced receptor internalization. By contrast, glimepiride significantly increased intracellular dissociation of the insulin-receptor complex, degradation of insulin, recycling of internalized insulin receptors, release of internalized radioactivity, and prevented insulin-induced receptor down-regulation. Association of PKC-betaII and -epsilon with insulin receptors was increased in glimepiride-treated cells. Selective depletion of cellular PKC-betaII and -epsilon by exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) or treatment of cells with PKC-betaII inhibitor G06976 reversed the effect of glimepiride on intracellular insulin-receptor processing. Glimepiride increased the effects of insulin on glucose incorporation into glycogen by enhancing both sensitivity and maximal efficacy of insulin. Exposing cells to TPA or G06976 inhibitor reversed these effects. Results indicate that glimepiride increases intracellular sorting of the insulin-receptor complex toward the degradative route, which is associated with both an increased association of the insulin receptor with PKCs and improved insulin action. These data suggest a novel mechanism of action of sulfonylurea, which may have a therapeutic impact on the treatment of type 2 diabetes.

Molecular scanning of human IRS-1 gene revealed a common polymorphism causing Gly--Arg972 change. Diabetic and pre-diabetic carriers of Arg972 IRS-1 are characterized by low fasting levels of insulin and C-peptide. To investigate directly whether the Arg 972 IRS-1 affects human islet cells survival, we took advantage of the unique opportunity to analyze pancreatic islets isolated from three donors heterozygous for the Arg972 and six donors carrying wild-type IRS-1. Islets from carriers of Arg972 IRS-1 showed a two-fold increase in the number of apoptotic cells as compared with wild-type. IRS-1-associated PI3-kinase activity was decreased in islets from carriers of Arg972 IRS-1. Same results were reproduced in RIN rat b-cell lines stably expressing wild-type IRS-1 or Arg972 IRS-1. Using these cells, we characterized the downstream pathway by which Arg972 IRS-1 impairs b-cell survival. RIN-Arg972 cells exhibited a marked impairment in the sequential activation of PI3-kinase, Akt, and BAD as compared with RI N-WT. Impaired BAD phosphorylation resulted in increased binding to Bcl-XL instead of 14-3-3 protein, thus sequestering the Bcl-XL antiapoptotic protein to promote survival. Both caspase-9 and caspase-3 activities were increased in RIN-Arg972 cells. The results show that the common Arg972 polymorphism in IRS-1 impairs human b-cell survival and causes resistance to antiapoptotic effects of insulin by affecting the PI3-kinase/Akt survival pathway. These findings establish an important role for the insulin signaling in human b-cell survival and suggest that genetic defects in early steps of insulin signaling may contribute to b-cell failure.

Type 2 (non-insulin-dependent) diabetes results from decreased insulin action in peripheral target tissues (insulin resistance) and impaired pancreatic beta-cell function. These defects reflect both genetic components and environmental risk factors. Recently, the common Gly(972)--Arg amino acid polymorphism of insulin receptor substrate 1 (Arg(972) IRS-1) has been associated with human type 2 diabetes. In this study, we report on some functional and morphological properties of isolated human islets carrying the Arg(972) IRS-1 polymorphism. Insulin content was lower in variant than control islets (94 +/- 47 vs. 133 +/- 56 microU/islet; P0.05). Stepwise glucose increase (1.7 to 16.7 mmol/l) significantly potentiated insulin secretion from control islets, but not Arg(972) IRS-1 islets, with the latter also showing a relatively lower response to glyburide and a significantly higher response to arginine. Proinsulin release mirrored insulin secretion, and the insulin-to-proinsulin ratio in response to arginine was significantly lower from Arg(972) IRS-1 islets than from control islets. Glucose utilization and oxidation did not differ in variant and wild-type islets at both low and high glucose levels. Electron microscopy showed that Arg(972) IRS-1 beta-cells had a severalfold greater number of immature secretory granules and a lower number of mature granules than control beta-cells. In conclusion, Arg(972) IRS-1 islets have reduced insulin content, impaired insulin secretion, and a lower amount of mature secretory granules. These alterations may account for the increased predisposition to type 2 diabetes in individuals carrying the Gly(972)--Arg amino acid polymorphism of IRS-1.

Background A significant proportion of individuals clinically diagnosed with familial hypercholesterolemia (FH), but without any disease-causing mutation, are likely to have polygenic hypercholesterolemia. We evaluated the distribution of a polygenic risk score, consisting of 12 low-density lipoprotein cholesterol (LDL-C)-raising variants (polygenic LDL-C risk score), in subjects with a clinical diagnosis of FH. Methods and Results Within the Lipid Transport Disorders Italian Genetic Network (LIPIGEN) study, 875 patients who were FH-mutation positive (women, 54.75%; mean age, 42.47±15.00 years) and 644 patients who were FH-mutation negative (women, 54.21%; mean age, 49.73±13.54 years) were evaluated. Patients who were FH-mutation negative had lower mean levels of pretreatment LDL-C than patients who were FH-mutation positive (217.14±55.49 versus 270.52±68.59 mg/dL, P