Superoxide and Respiratory Coupling in Mitochondria of Insulin-Deficient Diabetic Rats

Abstract

Mitochondrial reactive oxygen species have been implicated in both diabetic complications and the progression of the underlying diabetic state. However, it is not clear whether mitochondria of diabetic origin are intrinsically altered to generate excess reactive oxygen species independent of the surrounding diabetic milieu. Mitochondria were isolated from gastrocnemius, heart, and liver of 2-wk and 2-month streptozotocin diabetic rats and controls. We rigidly quantified mitochondrial superoxide, respiration and ATP production, respiratory coupling, the expression of several proteins with antioxidant properties, and the redox state of glutathione. Both fluorescent assessment and electron paramagnetic spectroscopy revealed that superoxide production was unchanged or reduced in the 2-month diabetic mitochondria compared with controls. Kinetic analysis of the proton leak showed that diabetic heart and muscle mitochondria were actually more coupled compared with control despite an approximate 2- to 4-fold increase in uncoupling protein-3 content. Adenine nucleotide translocator type 1 expression was reduced by approximately 50% in diabetic muscle mitochondria. Catalase was significantly up-regulated in muscle and heart tissue and in heart mitochondria, whereas glutathione peroxidase expression was increased in liver mitochondria of diabetic rats. We conclude that gastrocnemius, heart, and liver mitochondria of streptozotocin diabetic rats are not irrevocably altered toward excess superoxide production either by complex I or complex III. Moreover, gastrocnemius and heart mitochondria demonstrate increased, not decreased, respiratory coupling. Mitochondria of insulin-deficient diabetic rats do show signs of adaptation to antecedent oxidative stress manifested as tissue-specific enzyme and uncoupling protein expression but remain remarkably robust with respect to superoxide production.

Mitochondrial superoxide and coenzyme Q in insulin-deficient rats: increased electron leak

“…Data for ROS production, proton conductance, and oxygen consumption from 34 rats (17 control and 17 STZ-diabetic), comprising groups II and III of a previous publication (15), has previously been reported in that manuscript. Here, we combine these results with 57 newly studied animals (26 control and 31 STZ-diabetic), increasing the numbers for data comparison and enabling adequate numbers to assess ROS in relation to multiple mitochondrial functional parameters assessed under different substrate and inhibitor conditions.…”

“…Muscle and heart tissues were minced for 1 min prior to homogenization. Mitochondria were then isolated and washed three times, as previously described (11,15,17). Mitochondria prepared in this fashion were highly pure, as indicated by the distribution of glyceraldehyde-3-phosphate dehydrogenase and porin in whole tissue and mitochondrial extracts (15).…”

“…However, existing data do not support that expectation. We recently found that skeletal muscle mitochondria from insulin-deficient streptozotocin diabetic rats generated less H 2 O 2 and unchanged amounts of superoxide [directly assessed by electron paramagnetic spectroscopy (EPR)] compared with control mitochondria (15). Moreover, a recent study (7) showed that heart mitochondria of an insulin-deficient type 1 model, the Akita mouse, manifest no greater ROS production than nondiabetic controls.…”

Rodent models of diabetic cardiomyopathy

“…However, recent studies have revealed important differences between models of type 1 and type 2 diabetes. Mitochondrial reactive oxygen species (ROS) production is increased in the hearts of type 2 diabetic models, whereas type 1 diabetic models show no increase or even reduced production of ROS that originate from mitochondria Herlein et al, 2009). Fatty acid-induced mitochondrial uncoupling is another trait of type 2 diabetic hearts that does not seem to be present in type 1 diabetic models .…”

“…Studies investigating oxidative stress in STZ-diabetic hearts revealed increased cellular ROS levels, enhanced superoxide production, increased NADPH oxidase expression (subunit p47) and decreased GSSG/GSH ratios (ratio of oxidized to reduced glutathione) (Ghosh et al, 2004;Ghosh et al, 2005;Ceylan-Isik et al, 2006;Lashin et al, 2006;Wold et al, 2006;Singh et al, 2008). The mitochondrial origin of increased superoxide remains controversial because direct measurements of mitochondrial superoxide production showed no increase in STZ hearts (Herlein et al, 2009 (Lopaschuk et al, 1983;Flarsheim et al, 1996;Hattori et al, 2000;Choi et al, 2002;Zhao et al, 2006;Suarez et al, 2008). Furthermore, several studies have demonstrated increased connective tissue content in STZ-diabetic hearts, which can be attenuated by treatment of mice with the aldosterone antagonist spironolactone, suggesting that increased aldosterone action may contribute to cardiac fibrosis (Miric et al, 2001;Westermann et al, 2007;Singh et al, 2008;Ueno et al, 2008;Van Linthout et al, 2008).…”