Synergistic activation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase by Src kinase elevates superoxide in type 2 diabetic, Zucker fa/fa, rat liver - PubMed (original) (raw)

Synergistic activation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase by Src kinase elevates superoxide in type 2 diabetic, Zucker fa/fa, rat liver

Rakhee S Gupte et al. Free Radic Biol Med. 2009.

Abstract

Glucose metabolism through the glycolysis and hexosamine pathway has been shown to be altered in type 2 diabetes. However, the fate of glucose through the pentose phosphate pathway (PPP) is currently unclear. In this study, we determined whether the activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the PPP, is modulated in the liver of Zucker obese fa/fa rats (9-11 weeks of age). We found that G6PD expression and activity, NADPH levels, and 6-phosphogluconate generation were significantly increased in the liver of fa/fa rats. Inhibition of PI3 kinase and Src kinases decreased (p < 0.05) G6PD activity in the fa/fa but not in the lean rat liver, suggesting that G6PD activity is regulated by PI3/Src kinase signaling pathways. G6PD-derived NADPH increased (p < 0.05) superoxide anion levels by 70-90% in fa/fa vs lean rat liver, which was inhibited by the NADPH oxidase inhibitor gp91(ds-tat) (50 microM) and G6PD inhibitors 6-aminonicotinamide (1 mM) and dehydroepiandrosterone (100 microM), therefore indicating that elevated G6PD activity may be responsible for mediating superoxide generation. Interestingly, we also 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 hyperglycemic and hyperinsulinemic rats before the development of diabetes, seems to be a contributing factor in the induction of oxidative stress. Because inhibition of G6PD activity decreases oxidative stress, we conclude that G6PD behaves as a pro-oxidant in the fa/fa rat liver in type 2 diabetes.

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Figures

Figure 1

Figure 1. Glucose-6-phosphate dehydrogenase activity is elevated in type 2 diabetic model

Zucker 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 anti-G6PD antibody (A). All input lanes contain 35 μg 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 α-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 (B–F) was performed using Student’s t-test and P<0.05 (lean versus Zucker fa/fa) was considered statistically significant.

Figure 2

Figure 2. Glucose-6-phosphate dehydrogenase is activated by Src kinase in the liver of Zucker fa/fa rats

(A) Zucker lean (lanes 1 & 3) and fa/fa (lanes 2 & 4) 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 anti-pSrc (first panel) and anti-total Src antibody (second panel). All input lanes contain 35 μg of total protein content. Panel (A) represents one blot of six such independent experiments. (B) & (C) Graphs represents total-Src (B) and phosphorylated-Src416 (C) in lean (n=6) and ZDF (n=6) rat liver as estimated by densitometric analysis and normalized with total-Src or α-tubulin values. (D) & (E) Lean (D) and Zucker fa/fa (E) liver tissues were incubated at 37°C for 10 minutes without and with PI3 kinase inhibitor, LY294002 (10 μM), Src kinase inhibitor, PP2 (10 μM), and alkaline phosphatase (200 U/ml), respectively. Graphs represents glucose-6-phosphate dehydrogenase activity in, Lean (D) and Zucker fa/fa (E) liver homogenates, respectively. Statistical analysis was performed using Student’s t-test and P<0.05 (lean versus Zucker fa/fa) was considered statistically significant.

Figure 3

Figure 3. Protein expression of NAD(P)H oxidase is altered in Zucker fa/fa rat liver tissue

Lean and Zucker 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 mouse monoclonal anti-Nox1 and -Nox2 (A; first & second panels), goat polyclonal anti-Nox4 (A; third panel), mouse monoclonal anti-p67_phox_ (A; fourth panel) and goat polyclonal anti-p47_phox_ (A; bottom panel) antibodies, respectively. All lanes contain 35 μg of total protein content. Panel (A) represents one blot of six such independent experiments. Panel B: Graph represents NADPH oxidase subunit expression in lean (n=6) and Zucker fa/fa (n=6) rat liver as estimated by densitometric analysis and normalized with α-tubulin values. Panel C: Lean and Zucker fa/fa rat liver tissue was fractionated by ultracentrifugation at 100,000×g to separate the membrane and cytoplasmic fractions. Graph represents levels of p47_phox_ found in membrane as compared to cytoplasmic fraction in Zucker fa/fa rat liver. Statistical analysis was performed using Student’s t-test and P<0.05 (lean versus ZDF) was considered statistically significant.

Figure 4

Figure 4. Superoxide generation is elevated in the liver of Zucker fa/fa rats

Liver homogenates obtained from lean (n=6) and Zucker fa/fa (n=6) rat were used to detected O2− production by lucigenin (5 μM) chemiluminescence (A); Superoxide dismutase activity (SOD; B). Statistical analysis was performed using Student’s t-test and P<0.05(lean versus Zucker fa/fa) was considered statistically significant.

Figure 5

Figure 5. Glutathione peroxidase and reductase activity is decreased in the liver of Zucker fa/fa rats

Liver homogenates obtained from lean (n=6) and Zucker fa/fa (n=6) rat were used to estimate glutathione peroxidase (GSH-Px; A) and glutathione reductase (GSH reductase; B) activities.

Figure 6

Figure 6. Superoxide generated in liver is NADPH-dependent

Liver homogenates obtained from lean (n=6) and Zucker fa/fa (n=6) animals were treated with: (A) either NADPH oxidase inhibitor, gp91_ds-tat_ (50 μM), mitochondrial respiratory chain inhibitors, antimycin (10 μM) and rotenone (50 μM), and superoxide levels were determined by lucigenin (5 μM) chemiluminescence. (B) NADPH (100 μM) or NADH (100 μM) for 10 minutes at 37°C, and superoxide levels were determined as described above. These experiments were performed in the absence of NADPH regenerating system. Statistical analysis was performed using Student’s t-test and P<0.05 was considered statistically significant.

Figure 7

Figure 7. NADPH oxidase and glucose-6-phosphate dehydrogenase synergistically increases superoxide production in diabetic tissues

Liver tissues obtained from lean (n=6) and ZDF (n=6) animals were incubated (A) with PI3 kinase inhibitor, LY294002 (10 μM), and Src kinase inhibitor, PP2 (10 μM), for 10 minutes at 37°C and (B) either with 6-aminonicotinamide (5 mM) or dehydroepiandrosterone (DHEA; 100 μM), inhibitors of glucose-6-phosphate dehydrogenase, for 10 minutes at 37°C in the presence of NADPH regenerating system and superoxide levels were determined by lucigenin (5 μM) chemiluminescence. Statistical analysis was performed using Student’s t-test and P<0.05 (lean versus ZDF) was considered statistically significant.

Figure 8

Figure 8. Liver hypertrophy occurs in young Zucker fa/fa rats

Liver weight (A) and liver-to-body weight (B) was also determined in these rats. Graph represents the liver protein-to-DNA ratio in lean and Zucker fa/fa rats (C). The expression of PCNA in lean (lanes 1, 3, and 5) and Zucker fa/fa (lanes 2, 4, and 6) liver was determined by western blot analysis using mouse monoclonal anti-PCNA antibody (D). Correlation between liver weight and G6PD activity (E) or superoxide levels (F) was determined by regression analysis. Statistical analysis was performed using Student’s t-test and P<0.05 was considered statistically significant.

Figure 9

Figure 9. A schematic illustration of G6PD and NADPH oxidase activation in Zucker fa/fa rats

In the fa/fa rats, a congenital model of obesity and type 2 diabetes, changes in adipose tissue hormones and elevation in either insulin or glucose increases expression and activity of glucose-6-phosphate dehydrogenase (G6PD) and NADPH oxidases (Nox-1 and Nox-4). Conversely, glutathione reductase (GSH reductase) activity is decreased. This leads to an increase in superoxide (O2−) levels, which consequently either evokes liver growth or liver dysfunction.

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