NF-kappaB-induced oxidative stress contributes to mitochondrial and cardiac dysfunction in type II diabetes

Abstract

Aims: Inflammatory molecules and their transcription factor, nuclear factor kappa-B (NF-kappaB), are thought to play important roles in diabetes-induced cardiac dysfunction. Here, we investigated the effects of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, in diabetic mice.

Methods and results: Obese db/db mice and heterozygous lean mice (n = 8) were allowed free access to drinking water (control) or water containing PDTC (100 mg/kg) for 20 weeks. Left ventricular (LV) function was measured using echocardiography at baseline and at study end. Mice were sacrificed and LV removed for gene expression, biochemical, immunofluorescence, and mitochondrial assays. LV and mitochondrial reactive oxygen species (ROS), superoxide and peroxynitrite were measured using electron spin resonance spectroscopy. Enhanced NF-kappaB activity in db/db mice was associated with increased oxidative stress as demonstrated by increased ROS, superoxide, and peroxynitrite production, and increased NF-kappaB, gp91phox, and Nox1 expression; PDTC ameliorated these effects. Mitochondrial free radical production and structural damage were higher in the db/db group than in the control, db/db PDTC, and PDTC-treated heterozygous animal groups.

Conclusion: This study demonstrates that NF-kappaB blockade with PDTC mitigates oxidative stress and improves mitochondrial structural integrity directly, through down-regulation of increased oxygen-free radicals, thereby increasing ATP synthesis and thus restoring cardiac function in type II diabetes.

Figure 1

(A) Mean GSH levels for each experimental group as determined by a kit method. (B) Mean GSH/GSSG ratios for each experimental group. Note the marked decrease in GSH levels and GSH/GSSG ratio for the db/db group and the normalization of these parameters by PDTC treatment. *P < 0.05 vs. control; ^#P < 0.05 vs. db/db; ^$P < 0.05 vs. db/db PDTC; ^@P < 0.05 vs. hz PDTC.

Figure 2

Gene expression levels of (A) gp91phox and (B) Nox1 as determined by real-time RT–PCR and western blotting. Both of these genes were significantly upregulated in db/db LV tissue, and PDTC treatment attenuated expression of these genes. Production levels of (C) superoxide, (D) total ROS, and (E) peroxynitrite as determined by EPR spectroscopy. Levels of superoxide, total ROS, and peroxynitrite production were all increased significantly in db/db LV tissue; these levels were normalized with PDTC treatment. (F) Protein expression levels of 3-nitrotyrosine in LV tissues from all experimental groups. *P < 0.05 vs. control; ^#P < 0.05 vs. db/db; ^$P < 0.05 vs. db/db PDTC; ^@P < 0.05 vs. hz PDTC.

Figure 3

(A and B) Mitochondrial purity as determined by transmission electron microscopy (TEM) and western blot for the mitochondrial markers ANT and VDAC. (C) Results from swelling assay presented in graphical form. (D) Ultrastructural changes of LV tissue and isolated LV mitochondria as examined with TEM; (×40 magnification). Note the disordered appearance of the mitochondria and lack of cristae in db/db animals, and the preservation of appearance with PDTC treatment.

Figure 4

Mitochondrial production rates of (A) ROS, (B) superoxide, and (C) hydrogen peroxide, as determined by EPR, were significantly higher in db/db mice than in any other group. PDTC treatment normalized production rates of ROS, superoxide, and hydrogen peroxide in isolated LV mitochondria. *P < 0.05 vs. control; ^#P < 0.05 vs. db/db; ^$P < 0.05 vs. db/db PDTC; ^@P < 0.05 vs. hz PDTC.

Figure 5

(A) Mitochondrial complex III activity as determined by EPR, (B) ATP production, and (C) ATP/ADP ratios were all decreased in db/db animals when compared with other groups. Treatment with PDTC restored mitochondrial complex III activity and ATP production and improved ATP/ADP ratio. *P < 0.05 vs. control; ^#P < 0.05 vs. db/db; ^$P < 0.05 vs. db/db PDTC; ^@P < 0.05 vs. hz PDTC.

Figure 6

(A) NF-κB p65 activity as determined by ELISA was significantly higher in db/db mice and was attenuated with PDTC treatment. (B) Tissue gene expression and (C) mitochondrial protein expression of NF-κB p50 (as determined by real-time RT–PCR and western blot, respectively) were significantly higher in db/db animals; this expression was attenuated, but not normalized, with PDTC treatment. *P < 0.05 vs. control; ^#P < 0.05 vs. db/db; ^$P < 0.05 vs. db/db PDTC; ^@ < 0.05 vs. hz PDTC.