e the synthesis of NO in the liver cells, efforts were made to determine the possible effect of OPC 8212 site glucose induced NO production in the liver cells in the synthesis of Glut-4 itself, a protein. It was serendipitously found that the glucose induced NO production also resulted in the synthesis of Glut-4 in the liver cells as quantitated by ELISA by using Glut-4 antibody. It was found that the incubation of GLS with different amounts of glucose that resulted in the increased synthesis of Glut-4 was maximally increased at 0.02M glucose in the incubation mixture. Further increase of the sugar concentrations in the reaction mixture, however not only resulted in the decreased synthesis of NO as described in the 7 Glucose Transporter-4 in Mice Hepatocytes doi: 10.1371/journal.pone.0081935.g005 5). The addition of 0.1mM NAME to the reaction mixture containing NO had no effect on Glut-4 synthesis but the addition of NAME to the reaction mixture containing glucose completely inhibited the synthesis of the transporter, indicating NAME caused the inhibition of Glut-4 synthesis stimulated by glucose due to the inhibition of NO synthesis induced by glucose. The addition of NAME to the reaction mixture had no effect of the added NO induced Glut-4 synthesis in the reaction mixture indicating NAME itself is not the inhibitor of Glut-4 synthesis. 
To determine whether the actual synthesis of Glut-4 occurred in the GLS was due to the increased synthesis of NO, or merely due to the release of preformed Glut-4 from the cells by NO, in parallel experiments the synthesis of Glut-4 in the GLS in the presence of glucose was determined by in vitro translation of Glut-4 mRNA as described in the Materials and Methods, and the quantitation of Glut-4 was made by immunoblot analysis. It was found that the integrated area of the Glut-4 band in the immunoblot in the absence of added glucose in 15601771 the incubation mixture was 0.399 mm2 which was increased to 1.2850.064 mm2 in the presence of 0.02M glucose in the incubation mixture. In contrast, increase of the glucose concentration from 0.02M to 0.03M in the incubation mixture actually resulted in the decreased synthesis of Glut-4, as estimated by the integrated area of the band which was 0.6940.034mm2. The role of glucose induced NO synthesis in the expression of proinsulin genes I and II in the mice liver cells As glucose has been reported to have an essential role in the synthesis and secretion of insulin both in the pancreatic cells and in the adult mice hepatocytes, the role of glucose induced NO synthesis by GANOS in the liver cells was investigated to determine the role of NO, if any, in the actual synthesis of insulin in the mice liver cells in the presence of glucose. It was found that the treatment of GLS with different amounts of glucose resulted in the synthesis of insulin that was quantitated by ELISA. It was found that at 0.02M glucose, the synthesis of insulin was maximally stimulated over the control . The increased synthesis of insulin in the presence of glucose was related to the increase of glucose induced NO synthesis in the hepatocytes as described above. Addition of 0.1mM L-NAME to the reaction mixture was found to result in the inhibition of both insulin and NO syntheses at all concentrations 15102954 of glucose used in the reaction mixture. The incubation of GLS with glucose showed the expression of both proinsulin genes I and II as determined by cDNA analysis which was expressed due to NO synthesis compared to con
