atypical protein kinases C, p70 S6 kinase and SGK-1. These effects are insulin-independent. These data are in accordance with a work of Ayala, who found that in mice with an high fat diet sildenafil improved insulin sensitivity measured with hyperinsulin- emic-euglycemic clamp with no increased levels of Akt-1 phosphorylation [21]. Hyperglycaemic impairment of nitric oxide (NO) production by endothelial cells is implicated in the effect of diabetes to increase cardiovascular disease risk. As already reported [38,39], we found that in insulin resistance conditions, insulin treatment does not induce NO production; chronic treatment with sildenafil improves NO production in basal conditions as well as in insulin resistance conditions in e-NOS-dependent manner. This effect was reduced when insulin resistance was induced by Gluc-N. In these conditions, the positive effect of sildenafil on eNOS phosphory- lation was completely inhibited by Gluc-N. However eNOS mediated NO production was partly restored even in presence of Gluc-N. These results could be explained by the different mechanism of glucotoxicity. In fact, it is known that Gluc-N, by activating hexosamine pathway, causes O-GlcNac modifications at protein sites which are involved in insulin signalling [40]. Interestingly, under insulin resistance conditions we observed that insulin blunted the increase in NO generation induced by sildenafil. We demonstrated that insulin treatment was able to up-regulate PDE5 mRNA expression in HUVEC. It is possible that insulin increased PDE-5 mRNA expression through the transcription factors Sp1, AP-1 and AP-2. Indeed, AP-1 consensus sequence has been located at – 485 from the starting PDE-5 gene expression sequence (http://variome.kobic.re.kr/SNPatPromoter/ snpatpromoter.jsp?id = NM_001083). References 1. Fleming I, Bauersachs J, Schafer A, Scholz D, Aldershvile J, et al. 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These findings indicate that sildenafil improves insulin signalling and NO production in endothelial cells, probably by reducing oxidative stress induced by hyperglycemia and by increasing intracellular Ca2+ levels. The novel finding that insulin stimulates PDE-5 expression can explain the reduced response to sildenafil therapy in diabetic patients with elevated insulin resistance and hyperinsulinemia. Our data clarify the mechanism through which sildenafil improves insulin action in endothelial cells. Further investigation are necessary to discover all the substrates involved in this process and to understand if this novel action of sildenafil is specific for this molecule or it is a class effect of PDE-5i. In conclusion, we hypothesize that sildenafil can be potentially used to improve insulin action in type 2 diabetic patients leading to new therapeutic strategies for type 2 diabetes and other related cardiovascular disease. 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Chou SH, Chai CY, Wu JR, Tan MS, Chiu CC, et al. (2006) The effects of debanding on the lung expression of ET-1, eNOS, and cGMP in rats with left ventricular pressure overload. Exp Biol Med (Maywood ) 231: 954–959. PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e14542