Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase C␣ (PKC␣). Here we show that HGA-induced PKC␣ activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKC␣ in HGA-treated L6 cells. A direct interaction of PKC␣ with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKC␣ co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKC␣ activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKC␣. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKC␣.

The endocannabinoid system regulates food intake, energy, and glucose metabolism at both central and peripheral levels. We have investigated the mechanism by which it may control glucose uptake in skeletal muscle cells. Detectable levels of the cannabinoid receptor type 1 (CB1) were revealed in L6 cells. Exposure of differentiated L6 myotubes to the CB1 antagonist rimonabant (SR141716) selectively increased 2-deoxyglucose uptake (2-DG) in a time-and dose-dependent manner. A similar effect was induced by genetic silencing of CB1 by small interfering RNA. Protein expression profiling revealed that both the regulatory p85 and the catalytic p110 subunits of the phosphatidylinositol-3-kinase (PI3K) were increased by SR141716. No significant change in the cellular content of other known molecules regulating PI3K was observed. However, phosphoinositidedependent kinase-1, Akt/protein kinase B, and protein kinase C activities were rapidly induced after SR141716 treatment of L6 cells in a PI3K-dependent manner. The stimulatory effect of SR141716 on PI3K expression and activity was largely prevented by

Bovine papillomavirus type 2 (BPV-2) is an oncogenic virus infecting both epithelial and mesenchymal cells. Its life cycle, similar to other papillomaviruses (PVs), appears to be linked to epithelial differentiation. Human and bovine PVs have been known to reside in a latent, episomal form in PBMCs; therefore, it is believed that blood cells, like all mesenchymal cells, function as non-permissive carriers. Here, for the first time in veterinary and comparative medicine, the BPV-2 E5 oncoprotein and the major structural L1 capsid protein, known to be expressed only in productive infections, were shown to occur in defined subsets of PBMCs. E5 oncoprotein was detected in sorted T-and B-cells as well as in monocytes by flow cytometry and Western blot analysis. However, CD4 + and CD8 + lymphocytes appeared to be the main circulating targets of the virus, thus possibly representing the most important reservoir of active BPV-2 in blood. L1 protein was identified by flow cytometry in a population of blood cells recognized as lymphocytes by morphological scatter properties. Western blot analysis was performed on lysates obtained from the sorted subpopulations of PBMCs and detected L1 protein in CD4 + and CD8 + cells only. Thus, this study showed that CD4 + and CD8 + lymphocytes are permissive for BPV-2 and are new, hitherto unknown sites of productive PV infection. In light of these observations, the life cycle of PVs needs to be revisited to gain novel insights into the epidemiology of BPV infection and the pathogenesis of related diseases. INTRODUCTIONBovine papillomaviruses (BPVs) are a heterogeneous group of viruses responsible for tumours of the skin, genital and paragenital area, eye, upper gastrointestinal tract and urinary bladder (IARC, 2007). BPVs, like all other papillomaviruses (PVs), are usually strictly species specific. However, cases of cross-species infection are known to occur. BPV type 1 (BPV-1) and BPV-2, belonging to the genus Deltapapillomavirus (de Villiers et al., 2004), are responsible for infections in equids resulting in sarcoids (Chambers et al., 2003;Trenfield et al., 1985), as well as in bison and water buffaloes leading to warts (Literák et al., 2006;Silvestre et al., 2009). In addition, a variant of BPV-8, classified in the genus Epsilonpapillomavirus, also causes papillomas of the skin in bison (Tomita et al., 2007). More recently, it has been suggested that BPVs could be responsible for cutaneous sarcoids in cats and captive African lions (Munday & Knight, 2010;Orbell et al., 2010).BPV-1 and -2 are closely related serotypes (Shafti-Keramat et al., 2009) and can infect both epithelial and mesenchymal cells. Similar to other PVs, BPV-1/-2 replication and virion production are confined to the epithelial region of the lesions, whilst infection of mesenchymal cells appears to be non-productive (Campo, 2006;Shafti-Keramat et al., 2009).BPV-2 is known to play a central role in bladder carcinogenesis of adult cattle reared on pasturelands rich in bracken fern. In these animals, tumours of the u...

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.

Bovine papillomavirus type 2 (BPV-2) has been shown to infect and play a role in urinary bladder carcinogenesis of buffaloes grazed on pastures with ferns from the Marmara and Black Sea Regions of Turkey. BPV-2 DNA has been found in both neoplastic and non-neoplastic lesions of the urinary bladder. Furthermore, this virus may be a normal inhabitant of the urinary bladder since BPV-2 DNA has also been detected in clinically normal buffaloes. The viral activation by fern immunosuppressant or carcinogen may trigger the urothelial cell transformation. The E5 oncoprotein was solely detected in urothelial tumours and appeared to be co-localized with the overexpressed and phosphorylated platelet derived growth factor (PDGF) b receptor in a doublecolour immunofluorescence assay. Our results indicate that the E5-PDGF b receptor interaction also occurs in spontaneous tumours of the bubaline urinary bladder, revealing an additional role of BPV-2 in bladder carcinogenesis of buffaloes.

Soluble guanylate cyclase (sGC) is a receptor for nitric oxide (NO). Binding of NO to ferrous (Fe2+) heme increases its catalytic activity, leading to the production of cGMP from GTP. Hydrogen sulfide (H2S) is a signalling molecule that exerts both direct and indirect anti-oxidant effects. In the present, study we aimed to determine whether H2S could regulate sGC redox state and affect its responsiveness to NO-releasing agents and sGC activators. Using cultured rat aortic smooth muscle cells, we observed that treatment with H2S augmented the response to the NO donor DEA/NO, while attenuating the response to the heme-independent activator BAY58-2667 that targets oxidized sGC. Similarly, overexpression of H2S-synthesizing enzyme cystathionine-γ lyase reduced the ability of BAY58-2667 to promote cGMP accumulation. In experiments with phenylephrine-constricted mouse aortic rings, treatment with rotenone (a compound that increases ROS production), caused a rightward shift of the DEA/NO concentration-response curve, an effect partially restored by H2S. When rings were pre-treated with H2S, the concentration-response curve to BAY 58-2667 shifted to the right. Using purified recombinant human sGC, we observed that treatment with H2S converted ferric to ferrous sGC enhancing NO-donor-stimulated sGC activity and reducing BAY 58-2667-triggered cGMP formation. The study identified an additional mechanism of cross-talk between the NO and H2S pathways at the level of redox regulation of sGC. Our results provide evidence that H2S reduces sGC heme Fe, thus, facilitating NO-mediated cellular signaling events.

We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-(PKC ) expression and by overexpression of a dominant negative PKC mutant (DN-PKC ). Conversely, overexpression of PKC increased insulin internalization as a function of the PKC levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-␣ or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKC inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKC bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKC appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.Insulin binding is followed by tyrosine phosphorylation of insulin receptor (IR) 1 and of its intracellular substrates (1). Thereafter, insulin signaling impinges on at least three major enzymatic systems, the Ras/extracellular signal-regulated kinases, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB), and the protein kinase C (PKC) signal transduction pathways. These systems contribute to insulin regulation of cell metabolism, differentiation, and growth (2).In addition, hormone-bound IR is rapidly removed from the cell surface and internalized within clathrin-coated vesicles (3). Internalized insulin is then either degraded or released intact in the extracellular medium (4, 5). IR endocytosis is necessary for insulin clearance from the circulation (6, 7), down-regulation of surface binding sites (8), and transduction of specific biological effects (9 -11). Defective insulin internalization may also cause insulin resistance in animal models (7). It is now clear that endocytosis of insulin-IR complexes is triggered by receptor tyrosine kinase activation (12, 13). Receptor autophosphorylation on tyrosine residues allows the migration of IR toward coated pits (14), but it may not be sufficient for inducing internalization (15,16). Nevertheless, the molecular mechanisms, downstream receptor kinase activation, which control the internalization and the subsequent intracellular itinerary, have not been elucidated.PI3K is necessary for the internalization of several growth factor receptors (17-19). PI3K and its downst...

BACKGROUND AND PURPOSEHydrogen sulfide (H 2 S) is a gasotransmitter produced from L-cysteine through the enzymatic action of cystathionine-γ-lyase (CSE) and/or cystathionine-β-synthase. D-Penicillamine is the D isomer of a dimethylated cysteine and has been used for the treatment of rheumatoid arthritis. As D-penicillamine is structurally very similar to cysteine, we have investigated whether D-penicillamine, as a cysteine analogue, has an effect on the H 2 S pathway. EXPERIMENTAL APPROACHWe tested the effect of D-penicillamine (0.01-1 mM) in mouse aortic rings mounted in isolated organ baths and determined whether it could affect H 2 S biosynthesis. In particular, we investigated any possible inhibitor or donor behaviour by using recombinant enzyme-based assays and an in vivo approach. KEY RESULTSD-Penicillamine, per se, showed little or no vasodilator effect, and it cannot be metabolized as a substrate in place of L-cysteine. However, D-penicillamine significantly reduced L-cysteine-induced vasodilatation in a concentration-dependent manner through inhibition of H 2 S biosynthesis, and this effect occurred at concentrations 10 times lower than those needed to induce the release of H 2 S. In particular, D-penicillamine selectively inhibited CSE in a pyridoxal-5′-phospate-dependent manner. CONCLUSIONS AND IMPLICATIONSTaken together, our results suggest that D-penicillamine acts as a selective CSE inhibitor, leading to new perspectives in the design and use of specific pharmacological tools for H 2 S research. In addition, the inhibitory effect of D-penicillamine on CSE could account for its beneficial action in rheumatoid arthritis patients, where H 2 S has been shown to have a detrimental effect. AbbreviationsMPST, 3-mercaptopyruvate sulfurtransferase; CBS, cystathionine-β-synthase; CSE, cystathionine-γ-lyase; DPD, N,N-dimethylp-phenylenediamine sulfate; D-pen, D-penicillamine; L-pen, L-penicillamine; H 2 S, hydrogen sulfide; IVM, intravital microscopy; PAG, D,L-propargylglycine; PE, phenylephrine; PLP, pyridoxal-5′-phosphate; RA, rheumatoid arthritis; TCA, trichloroacetic acid; ZnAc, zinc acetate BJP IntroductionHydrogen sulfide (H 2 S) is a gaseous molecule endogenously synthesized by cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST). These enzymes are differently distributed throughout the human body and within cell compartments. MPST is mainly expressed in mitochondria (Stipanuk, 2004;Shibuya et al., 2009), while CSE and CBS show a wide distribution in diverse cell types. CSE and CBS can both metabolize the substrate L-cysteine to release H 2 S, and this reaction is strictly dependent upon the enzyme cofactor pyridoxal-5′-phosphate (PLP). The role of H 2 S has been widely investigated in several organs and tissues, and many studies have confirmed its crucial role in body physiology. For instance, CSE represents the prominent enzyme within the cardiovascular system, where H 2 S is a major player involved in regulating the function of heart and bloo...