Michele Longo

Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells

Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.

Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes

Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.

Prostate health index (phi) and prostate cancer antigen 3 (PCA3) significantly improve diagnostic accuracy in patients undergoing prostate biopsy.

Prostate health index (phi) and prostate cancer antigen 3 (PCA3) have been recently proposed as novel biomarkers for prostate cancer (PCa). We assessed the diagnostic performance of these biomarkers, alone or in combination, in men undergoing first prostate biopsy for suspicion of PCa.

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.

Understanding type 2 diabetes: from genetics to epigenetics

The known genetic variability (common DNA polymorphisms) does not account either for the current epidemics of type 2 diabetes or for the family transmission of this disorder. However, clinical, epidemiological and, more recently, experimental evidence indicates that environmental factors have an extraordinary impact on the natural history of type 2 diabetes. Some of these environmental hits are often shared in family groups and proved to be capable to induce epigenetic changes which alter the function of genes affecting major diabetes traits. Thus, epigenetic mechanisms may explain the environmental origin as well as the familial aggregation of type 2 diabetes much easier than common polymorphisms. In the murine model, exposure of parents to environmental hits known to cause epigenetic changes reprograms insulin sensitivity as well as beta-cell function in the progeny, indicating that certain epigenetic changes can be transgenerationally transmitted. Studies from different laboratories revealed that, in humans, lifestyle intervention modulates the epigenome and reverts environmentally induced epigenetic modifications at specific target genes. Finally, specific human epigenotypes have been identified which predict adiposity and type 2 diabetes with much greater power than any polymorphism so far identified. These epigenotypes can be recognized in easily accessible white cells from peripheral blood, indicating that, in the future, epigenetic profiling may enable effective type 2 diabetes prediction. This review discusses recent evidence from the literature supporting the immediate need for further investigation to uncover the power of epigenetics in the prediction, prevention and treatment of type 2 diabetes.

Productive Infection of Bovine Papillomavirus Type 2 in the Urothelial Cells of Naturally Occurring Urinary Bladder Tumors in Cattle and Water Buffaloes

Background Papillomaviruses (PVs) are highly epitheliotropic as they usually establish productive infections within squamous epithelia of the skin, the anogenital tract and the oral cavity. In this study, early (E) and late (L) protein expression of bovine papillomavirus type 2 (BPV-2) in the urothelium of the urinary bladder is described in cows and water buffaloes suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. Methods and Findings E5 protein, the major oncoprotein of the BPV-2, was detected in all tumors. L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection was detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium. Finally, the early protein E2, required for viral DNA replication and known to be a pivotal factor for both productive and persistent infection, was detected by Western blot and immunohistochemically. Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium. Conclusion This study shows that both active and productive infections by BPV-2 in the urothelium of the bovine and bubaline urinary bladder can occur in vivo.

IL-3 synergises with basophil-derived IL-4 and IL-13 to promote the alternative activation of human monocytes

Basophil‐derived IL‐4 is involved in the alternative activation of mouse monocytes, as recently shown in vivo. Whether this applies to human basophils and monocytes has not been established yet. Here, we sought to characterise the interaction between basophils and monocytes and identify the molecular determinants. A basophil‐monocyte co‐culture model revealed that IL‐3 and basophil‐derived IL‐4 and IL‐13 induced monocyte production of CCL17, a marker of alternative activation. Critically, IL‐3 and IL‐4 acted directly on monocytes to induce CCL17 production through histone H3 acetylation, but did not increase the recruitment of STAT5 or STAT6. Although freshly isolated monocytes did not express the IL‐3 receptor α chain (CD123), and did not respond to IL‐3 (as assessed by STAT5 phosphorylation), the overnight incubation with IL‐4 (especially if associated with IL‐3) upregulated CD123 expression. IL‐3‐activated JAK2‐STAT5 pathway inhibitors reduced the CCL17 production in response to IL‐3 and IL‐4, but not to IL‐4 alone. Interestingly, monocytes isolated from allergen‐sensitised asthmatic patients exhibited a higher expression of CD123. Taken together, our data show that the JAK2‐STAT5 pathway modulates both basophil and monocyte effector responses. The coordinated activation of STAT5 and STAT6 may have a major impact on monocyte alternative activation in vitro and in vivo.

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.

PREP1 deficiency downregulates hepatic lipogenesis and attenuates steatohepatitis in mice

Aims/hypothesisThe aim of this study was to investigate the function of Prep1 (also known as Pknox1) in hepatic lipogenesis.MethodsThe hepatic lipogenesis pathway was evaluated by real-time RT-PCR and Western blot. Biochemical variables were assessed using a clinical chemistry analyser.ResultsSerum triacylglycerols and liver expression of fatty acid synthase (FAS) were significantly decreased in Prep1 hypomorphic heterozygous (Prep1i/+) mice compared with their non-hypomorphic littermates. Upstream FAS expression, phosphorylation of protein kinase C (PKC)ζ, liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) increased in Prep1i/+ mice, while protein and mRNA levels of the lipid phosphatase inhibitor of PKCζ, SH2-containing inositol 5′-phosphatase 2 (SHIP2), was more than 60% reduced. Consistent with these findings, HepG2 cells transfected with Prep1 cDNA exhibited increased triacylglycerol accumulation and FAS expression, with strongly reduced PKCζ, LKB1, AMPK and ACC phosphorylation. Further experiments revealed the presence of both Prep1 and its major partner Pbx1 at the Ship2 (also known as Inppl1) promoter. PBX-regulating protein 1 (PREP1) and pre-B cell leukaemia transcription factor 1 (PBX1) enhanced Ship2 transcription. The PREP1HR mutant, which is unable to bind PBX1, exhibited no effect on Ship2 function, indicating transcriptional activation of Ship2 by the PREP1/PBX1 complex. Treatment with a methionine- and choline-deficient diet (MCDD) induced steatosis in both Prep1i/+ and non-hypomorphic control mice. However, alanine aminotransferase increase, intracellular triacylglycerol content and histological evidence of liver steatosis, inflammation and necrosis were significantly less evident in Prep1i/+ mice, indicating that Prep1 silencing protects mice from MCDD-induced steatohepatitis.Conclusions/interpretationOur results indicate that Prep1 silencing reduces lipotoxicity by increasing PKCζ/LKB1/AMPK/ACC signalling, while levels of PREP1 expression may determine the risk of steatohepatitis and its progression.

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.