Effect of Aspirin on Mitochondrial Dysfunction and Stress in the Pancreas and Heart of Goto-Kakizaki Diabetic Rats

Figures

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  Figure 1. Effects of aspirin (ASA) treatment on mitochondrial ROS and membrane-bound NOX activity, lipid peroxidation and SOD levels in the pancreas and heart of control Wistar and GK rats (n = 5). Mitochondrial ROS production (A) in the pancreas and heart of control Wistar and GK rats was measured using DCFDA as a probe. Total homogenates from the tissues of these animals were analyzed for membrane-bound NOX production (B) using the lucigenin-enhanced chemiluminescence method, as described in the Materials and Methods. NADPH-dependent LPO (C) was measured in the pancreas and heart of control Wistar and GK rats using the LPO-586™ assay kit from Oxis Int. Inc. (Portland, OR, USA) and SOD levels (D) were measured using the SOD kit from Abcam (Cambridge, UK) as per the manufacturer’s protocol. Results are expressed as mean ± SD. from three independent experiments and asterisks indicate a significant difference. * p < 0.05 compared to control, ** p < 0.001 compared to control, ∆ p < 0.05 compared to GK, ∆∆ p < 0.001 compared to GK animals.

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  Figure 2. Glutathione levels and metabolism in the pancreas and heart of control Wistar and GK rats treated with aspirin (ASA) (n = 5). GSH concentrations in the pancreas and heart of control Wistar and GK rats were measured in the cytosol (A) and mitochondria (B) using the enzymatic recycling method of Griffith, as described in the Materials and Methods. GST activity, using CDNB as a substrate, was measured in the cytosol (C) and mitochondria (D). GSH-Px activity in the pancreas and heart of control Wistar and GK rats was measured in the cytosol (E) and mitochondria (F) using cumene hydroperoxide as a substrate and GSH-reductase activity was measured in the cytosol (G) and mitochondria (H) using GSSG/NADPH as a substrate. Values are means ± SD from three independent experiments. Asterisks indicate a significant difference. * p < 0.05 compared to control, ** p < 0.001 compared to control, ∆ p < 0.05 compared to GK, ∆∆ p < 0.001 compared to GK animals.

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  Figure 3. CYP 450 levels in the pancreas and heart of control Wistar and GK rats treated with aspirin (ASA) (n = 5). CYP 2E1 (A) and CYP 3A4 (B) activities in the pancreas and heart microsomal fractions were measured using standard substrates, as described in the Materials and Methods. Results are expressed as mean + SD from three independent experiments and asterisks indicate a significant difference. * p < 0.05 compared to control, ** p < 0.001 compared to control, ∆ p < 0.05 compared to GK rats.

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  Figure 4. Effect of aspirin (ASA) on the activities of mitochondrial complexes and ATP levels in the pancreas and heart of control Wistar and GK rats (n = 5). Mitochondrial respiratory enzyme Complexes I (A), II/III (B) and IV (C) activities were measured in freshly prepared mitochondrial fractions from the pancreas and heart of control Wistar and GK rats using enzyme-specific substrates, as described in the Materials and Methods. The ATP content (D) in the rat tissues was determined using the ATP Bioluminescent cell assay kit (Sigma, St. Louis, MO, USA). Results are expressed as mean + SD from three independent experiments and asterisks indicate a significant difference. * p < 0.05 compared to control, ** p < 0.001 compared to control, ∆ p < 0.05 compared to GK, ∆∆ p < 0.001 compared to GK rats.

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  Figure 5. Effect of aspirin (ASA) on glutamate dehydrogenase activities in the pancreas and heart of control Wistar and GK rats (n = 5). Glutamate dehydrogenase activity was measured in the pancreas and heart of control Wistar and GK rats using the kit protocol, as described in the Materials and Methods, and the NADH produced after 30 min of reaction was determined using a standard curve (A). A time course reaction with absorbance values at 450 nm over 30 min at 10 min intervals is shown in (B). Values are means ± SD from three independent experiments and asterisks indicate a significant difference. * p < 0.05 compared to control, ** p < 0.001 compared to control, ∆ p < 0.05 compared to GK rats.