Cecilia Nigro

Increased hexosamine biosynthetic pathway flux dedifferentiates INS-1E cells and murine islets by an extracellular signal-regulated kinase (ERK)1/2-mediated signal transmission pathway

Aims/hypothesisBeta cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (decline of glucose-stimulated insulin secretion, downregulation of specific gene expression). Apoptosis and dysfunction are caused, at least in part, by lipoglucotoxicity. The mechanisms implicated are oxidative stress, increase in the hexosamine biosynthetic pathway (HBP) flux and endoplasmic reticulum (ER) stress. Oxidative stress plays a role in glucotoxicity-induced beta cell dedifferentiation, while glucotoxicity-induced ER stress has been mostly linked to beta cell apoptosis. We sought to clarify whether ER stress caused by increased HBP flux participates in a dedifferentiating response of beta cells, in the absence of relevant apoptosis.MethodsWe used INS-1E cells and murine islets. We analysed the unfolded protein response and the expression profile of beta cells by real-time RT-PCR and western blot. The signal transmission pathway elicited by ER stress was investigated by real-time RT-PCR and immunofluorescence.ResultsGlucosamine and high glucose induced ER stress, but did not decrease cell viability in INS-1E cells. ER stress caused dedifferentiation of beta cells, as shown by downregulation of beta cell markers and of the transcription factor, pancreatic and duodenal homeobox 1. Glucose-stimulated insulin secretion was inhibited. These effects were prevented by the chemical chaperone, 4-phenyl butyric acid. The extracellular signal-regulated kinase (ERK) signal transmission pathway was implicated, since its inhibition prevented the effects induced by glucosamine and high glucose.Conclusions/interpretationGlucotoxic ER stress dedifferentiates beta cells, in the absence of apoptosis, through a transcriptional response. These effects are mediated by the activation of ERK1/2.

Personalized medicine and type 2 diabetes: lesson from epigenetics.

Similarly to genetic polymorphisms, epigenetic modifications may alter transcriptional activity and contribute to different traits of the Type 2 diabetes phenotype. The establishment of these epigenetic marks may precede diabetes onset and predict the disease. Current evidence now indicates that epigenetic differences represent markers of diabetes risk. Studies on epigenome plasticity revealed that cytokines and other metabolites, by affecting DNA methylation, may acutely reprogram gene expression and contribute to the Type 2 diabetes phenotype even in the adult life. The available evidence further indicates that epigenetic marks across the genome are subject to dynamic variations in response to environmental cues. Finally, different genes responsible for the interindividual variability in antidiabetic drug response are subjected to epigenetic regulation. Determining how specific epigenetic profiles determine diabetes is a challenging task. In the near future, the identification of epigenetic marks predictive of diabetes risk or response to treatment may offer unanticipated opportunities to personalize Type 2 diabetes management.

Platelet‐Rich Plasma Increases Growth and Motility of Adipose Tissue‐Derived Mesenchymal Stem Cells and Controls Adipocyte Secretory Function

Adipose tissue‐derived mesenchymal stem cells (Ad‐MSC) and platelet derivatives have been used alone or in combination to achieve regeneration of injured tissues. We have tested the effect of platelet‐rich plasma (PRP) on Ad‐MSC and adipocyte function. PRP increased Ad‐MSC viability, proliferation rate and G1‐S cell cycle progression, by at least 7‐, 2‐, and 2.2‐fold, respectively, and reduced caspase 3 cleavage. Higher PRP concentrations or PRPs derived from individuals with higher platelet counts were more effective in increasing Ad‐MSC growth. PRP also accelerated cell migration by at least 1.5‐fold. However, PRP did not significantly affect mature adipocyte viability, differentiation and expression levels of PPAR‐γ and AP‐2 mRNAs, while it increased leptin production by 3.5‐fold. Interestingly, PRP treatment of mature adipocytes also enhanced the release of Interleukin (IL)‐6, IL‐8, IL‐10, Interferon‐γ, and Vascular Endothelial Growth Factor. Thus, data are consistent with a stimulatory effect of platelet derivatives on Ad‐MSC growth and motility. Moreover, PRP did not reduce mature adipocyte survival and increased the release of pro‐angiogenic factors, which may facilitate tissue regeneration processes.

Circulating miRNAs as intercellular messengers, potential biomarkers and therapeutic targets for Type 2 diabetes

miRNAs have emerged as key epigenetic regulators of metabolism. Their deregulation contributes to metabolic abnormalities, proposing their potential role as therapeutic targets for Type 2 diabetes. The exciting finding that miRNAs exist in the bloodstream suggests that circulating miRNAs may act in a hormone-like fashion. Despite the fact that significant progress has been made in understanding circulating miRNAs, this topic is full of complexities and many questions remain unanswered. The goal of this review is to bring together up-to-date knowledge about circulating miRNAs and their role as intercellular communicators as well as potential biomarkers and therapeutic targets in metabolic diseases, providing examples of possible clinical applications for circulating miRNAs in diabetes and cardiovascular complications.

Hepatocyte nuclear factor (HNF)-4α-driven epigenetic silencing of the human PED gene

Aims/hypothesisOverexpression of PED (also known as PEA15) determines insulin resistance and impaired insulin secretion and may contribute to progression toward type 2 diabetes. Recently, we found that the transcription factor hepatocyte nuclear factor (HNF)-4α binds to PED promoter and represses its transcription. However, the molecular details responsible for regulation of PED gene remain unclear.MethodsHere we used gain and loss of function approaches to investigate the hypothesis that HNF-4α controls chromatin remodelling at the PED promoter in human cell lines.ResultsHNF-4α production and binding induce chromatin remodelling at the −250 to 50 region of PED, indicating that remodelling is limited to two nucleosomes located at the proximal promoter. Chromatin immunoprecipitation assays also revealed concomitant HNF-4α-induced deacetylation of histone H3 at Lys9 and Lys14, and increased dimethylation of histone H3 at Lys9. The latter was followed by reduction of histone H3 Lys4 dimethylation. HNF-4α was also shown to target the histone deacetylase complex associated with silencing mediator of retinoic acid and thyroid hormone receptor, both at the PED promoter, and at GRB14 and USP21 regulatory regions, leading to a reduction of mRNA levels. Moreover, HNF-4α silencing and PED overexpression were accompanied by a significant reduction of hepatic glycogen content.Conclusions/interpretationThese results show that HNF-4α serves as a scaffold protein for histone deacetylase activities, thereby inhibiting liver expression of genes including PED. Dysregulation of these mechanisms may lead to upregulation of the PED gene in type 2 diabetes.

Peroxisome proliferator-activated receptor-γ activation enhances insulin-stimulated glucose disposal by reducing ped/pea-15 gene expression in skeletal muscle cells: evidence for involvement of activator protein-1.

Background: PPARγ modulation of glucoregulatory response in skeletal muscle has been only partially elucidated. Results: PPARγ inhibits the transcription of the diabetes-associated gene ped/pea-15 via AP-1. Conclusion: ped/pea-15 is downstream of a PPARγ-regulated inflammatory network. Significance: These studies further elucidate the gene network responsible for inflammation-induced insulin resistance. The gene network responsible for inflammation-induced insulin resistance remains enigmatic. In this study, we show that, in L6 cells, rosiglitazone- as well as pioglitazone-dependent activation of peroxisome proliferator-activated receptor-γ (PPARγ) represses transcription of the ped/pea-15 gene, whose increased activity impairs glucose tolerance in mice and humans. Rosiglitazone enhanced insulin-induced glucose uptake in L6 cells expressing the endogenous ped/pea-15 gene but not in cells expressing ped/pea-15 under the control of an exogenous promoter. The ability of PPARγ to affect ped/pea-15 expression was also lost in cells and in C57BL/6J transgenic mice expressing ped/pea-15 under the control of an exogenous promoter, suggesting that ped/pea-15 repression may contribute to rosiglitazone action on glucose disposal. Indeed, high fat diet mice showed insulin resistance and increased ped/pea-15 levels, although these effects were reduced by rosiglitazone treatment. Both supershift and ChIP assays revealed the presence of the AP-1 component c-JUN at the PED/PEA-15 promoter upon 12-O-tetradecanoylphorbol-13-acetate stimulation of the cells. In these experiments, rosiglitazone treatment reduced c-JUN presence at the PED/PEA-15 promoter. This effect was not associated with a decrease in c-JUN expression. In addition, c-jun silencing in L6 cells lowered ped/pea-15 expression and caused nonresponsiveness to rosiglitazone, although c-jun overexpression enhanced the binding to the ped/pea-15 promoter and blocked the rosiglitazone effect. These results indicate that PPARγ regulates ped/pea-15 transcription by inhibiting c-JUN binding at the ped/pea-15 promoter. Thus, ped/pea-15 is downstream of a major PPARγ-regulated inflammatory network. Repression of ped/pea-15 transcription might contribute to the PPARγ regulation of muscle sensitivity to insulin.

Epigenetics: spotlight on type 2 diabetes and obesity

Type 2 diabetes (T2D) and obesity are the major public health problems. Substantial efforts have been made to define loci and variants contributing to the individual risk of these disorders. However, the overall risk explained by genetic variation is very modest. Epigenetics is one of the fastest growing research areas in biomedicine as changes in the epigenome are involved in many biological processes, impact on the risk for several complex diseases including diabetes and may explain susceptibility. In this review, we focus on the role of DNA methylation in contributing to the risk of T2D and obesity.

Adenoviral gene transfer of PLD1-D4 enhances insulin sensitivity in mice by disrupting phospholipase D1 interaction with PED/PEA-15.

Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.

PED/PEA-15 inhibits hydrogen peroxide-induced apoptosis in Ins-1E pancreatic beta-cells via PLD-1.

The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from TgPED/PEA-15 mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1EPED/PEA-15). In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1EPED/PEA-15 cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1EPED/PEA-15. These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

Methylglyoxal-Glyoxalase 1 Balance: The Root of Vascular Damage

The highly reactive dicarbonyl methylglyoxal (MGO) is mainly formed as byproduct of glycolysis. Therefore, high blood glucose levels determine increased MGO accumulation. Nonetheless, MGO levels are also increased as consequence of the ineffective action of its main detoxification pathway, the glyoxalase system, of which glyoxalase 1 (Glo1) is the rate-limiting enzyme. Indeed, a physiological decrease of Glo1 transcription and activity occurs not only in chronic hyperglycaemia but also with ageing, during which MGO accumulation occurs. MGO and its advanced glycated end products (AGEs) are associated with age-related diseases including diabetes, vascular dysfunction and neurodegeneration. Endothelial dysfunction is the first step in the initiation, progression and clinical outcome of vascular complications, such as retinopathy, nephropathy, impaired wound healing and macroangiopathy. Because of these considerations, studies have been centered on understanding the molecular basis of endothelial dysfunction in diabetes, unveiling a central role of MGO-Glo1 imbalance in the onset of vascular complications. This review focuses on the current understanding of MGO accumulation and Glo1 activity in diabetes, and their contribution on the impairment of endothelial function leading to diabetes-associated vascular damage.