Panel (A) represents one blot of six such independent experiments. found a positive correlation between liver hypertrophy/increased G6PD activity (r2=0.77; P=0.0009) and liver hypertrophy/superoxide production (r2=0.51; P=0.0091) in fa/fa rats. Increased G6PD and NADPH oxidase expression and activity, in young hyper-glycemic and -insulinemic rats prior to the development of diabetes, appear to be contributing factors for the induction of oxidative stress. Since inhibition of G6PD activity decreases oxidative stress, we conclude that it behaves as a pro-oxidant in the fa/fa rat liver, in type 2 diabetes. (Transduction Laboratory, San Jose, CA, USA), goat polyclonal FIIN-2 anti-Nox-4 and anti- p47test for multiple comparisons. Differences were considered significant at p 0.05. Results Glucose and insulin levels are increased in young Zucker fa/fa rats Nine to Eleven weeks old Zucker fa/fa rats (3456 g; n=6) were obese (P 0.05) as compared to lean rats (26415 FIIN-2 g; n=6), and had high (P 0.05) non-fasting plasma glucose [12711 (lean) and 34112 (Zucker fa/fa) mg/dl] and insulin [0.670.07 (lean) and 3.040.22 (Zucker fa/fa) ng/ml] levels. Glucose-6-phosphate dehydrogenase activity is elevated in type 2 diabetes Since glucose metabolism is altered in type 2 diabetes, it is possible that G6PD expression and/or activity may also be modulated. We performed Western blot analysis to determine whether G6PD expression was changed in liver of Zucker fa/fa rats. The results indicated that the expression of G6PD increased (P 0.05) by 117 % in liver (Fig 1 A top panel and B) and hepatocytes (Fig 1 A bottom panel) of Zucker fa/fa as compared to the lean rats. In addition, we found that the activity of G6PD was significantly higher in the liver and hepatocytes of Zucker fa/fa animals by FIIN-2 400 % (Fig 1 C) and 160 % (P 0.05), respectively, as compared to age-matched lean rat. Additionally, NADP+, substrate, -dependent enzyme activity curve was also significantly shifted to the left in hepatic tissue from Zucker fa/fa rats (Fig 1 D). Consistently, the NADPH (Fig 1 E) and 6-phospho-gluconate (Fig 1 F), products of G6PD, levels were significantly increased in the liver of Zucker fa/fa rats as compared to lean rats. Open in a separate window Figure 1 Glucose-6-phosphate dehydrogenase activity is elevated in type 2 diabetic modelZucker lean and fa/fa rat liver homogenates (Panel A top) and hepatocyte lysates (Panel A bottom) were analyzed on 9% SDS-PAGE. Western blot analysis was performed using rabbit polyclonal Rabbit Polyclonal to UGDH anti-G6PD antibody (A). All input lanes contain 35 g of total protein content. Panel (A) represents one blot of six such independent experiments. (B) The graph represents G6PD protein expression in lean (n=6) and ZDF (n=6) rat liver as estimated by densitometric analysis (total: upper and lower band) and normalized with -tubulin values. The graph represents G6PD activity (C), effect of NADP+-dependent on activity (D), NADPH levels (E) and 6-phospho-gluconate levels (F) from lean (n=6) and Zucker fa/fa (n=6) rat liver, respectively. Statistical analysis for (BCF) was performed using Students t-test and (lean Zucker fa/fa) was considered statistically significant. Glucose-6-phosphate dehydrogenase activity is elevated by PI3 and Src kinases in type 2 diabetes EGF associated tyrosine kinases-, PKC- and Src-dependent pathways have been shown to activate G6PD in renal cortical cells [21] and in the failing human hearts [22]. To elucidate how G6PD is activated in fa/fa, we studied the effects of kinases involved in the insulin signaling pathway such as, PI3 kinase [23] and Src family of protein tyrosine kinases [24], on G6PD activity. We found that there was an increase in the amount of total-Src (Fig 2 A & B) and phospho-Src416 (Fig. FIIN-2 2 A & C) in fa/fa lean rats. Next, we treated liver with (i) PI3 kinase inhibitor-LY294002 (active form; 10 M) and “type”:”entrez-nucleotide”,”attrs”:”text”:”LY303511″,”term_id”:”1257646067″,”term_text”:”LY303511″LY303511 (non-active form; 10 M); and (ii) Src kinase inhibitor-PP2 (active form; 10 M) and PP3 (non-active form; 10 M), respectively, to determine the role of PI3 kinase and Src kinase in activating G6PD. The liver was perfused with the inhibitors for 30 minutes as described earlier (See Methods) and then the activity of G6PD was determined in liver homogenates in the presence of LY294002/”type”:”entrez-nucleotide”,”attrs”:”text”:”LY303511″,”term_id”:”1257646067″,”term_text”:”LY303511″LY303511 and PP2/PP3. Both active form of inhibitors attenuated (P 0.05) G6PD activity in fa/fa, but not in lean liver homogenate (Fig 2 D & E). Total-Src was increased in isolated hepatocytes (Fig 2 A), and treatment with PI3 kinase and Src kinase inhibitors decreased (P 0.05) G6PD activity (Control: 0.8920.159; LY294002: 0.3580.2893; PP2: 0.4060.3802 nmol/min/mg protein) in hepatocytes from fa/fa rats. Open in a separate window Figure 2 Glucose-6-phosphate dehydrogenase is activated by Src kinase in the.