Reduced cardiac efficiency is a well-described characteristic of type 2 diabetic hearts in animal models and humans (3
). In this study, we addressed the following question: Do impaired cardiac efficiency and fatty acid–induced mitochondrial uncoupling contribute to cardiac contractile dysfunction in type 1 diabetic Akita mice? Similar to type 2 diabetic hearts, substrate metabolism shifts toward increased fatty acid metabolism, and mitochondrial oxidative capacity is impaired. However, in contrast to type 2 diabetic mouse models, Akita mice show relatively preserved contractile function under ambient conditions, no impairment in cardiac efficiency, and no evidence of mitochondrial uncoupling (normal Voligo
, ATP-to-O ratios, and proton leak). Perfusion of hearts with high fatty acid concentrations was not sufficient to induce mitochondrial uncoupling despite the presence of increased UCP3 levels. Moreover, the absence of oxidative stress distinguishes the Akita mouse from type 2 diabetic models. Thus, the molecular mechanisms for mitochondrial dysfunction importantly differ between insulin-deficient type 1 and insulin-resistant type 2 diabetic mice. We also show that increased UCP3 does not invariably lead to increased mitochondrial uncoupling in the heart, supporting the hypothesis that fatty acid–induced mitochondrial uncoupling in diabetic hearts may require a concomitant increase in ROS generation.
The absence of mitochondrial uncoupling in Akita hearts was associated with the absence of oxidative stress. Mitochondrial superoxide production, measured as H2
generation, was actually decreased, and the activity of mitochondrial aconitase and oxidation of DCFDA, which are independent measures of oxidative damage, were unaltered in Akita hearts. The association of mitochondrial uncoupling with increased ROS and lipid peroxide generation in type 2 diabetic db/db
) and the lack of uncoupling and the absence of oxidative stress in type 1 diabetic Akita hearts suggest a causal interdependence of ROS production and mitochondrial uncoupling in diabetic hearts. Superoxide increases proton conductance by UCP3 in isolated rat skeletal muscle mitochondria, and lipid peroxidation products, such as hydroxynonenal, increase proton leak in isolated mitochondria from multiple tissues, including the heart, via UCPs and adenine nucleotide translocator (7
). Although fatty acids appear to be required for proton transport of UCP2 and UCP3 when reconstituted in liposomes, Echtay et al. (24
) showed that hydroxynonenal-induced proton conductance does not require the presence of fatty acids (29
). Thus, it appears that for fatty acid–induced mitochondrial uncoupling, as observed to occur in type 2 diabetic hearts, an increase in ROS may be required in addition to increased fatty acid availability and utilization.
The absence of oxidative stress or of any increase in ROS or H2
production in Akita hearts was unexpected. Inhibition of mitochondrial H2
production in the presence of rotenone suggested that complex I accounted for most of the ROS production in Akita and control hearts. This contrasts with similar studies in mitochondria isolated from db/db
hearts in which additional mitochondrial complexes (likely complex III) also contributed to ROS generation (6
). The reduction in ROS generation in Akita mice suggested that there might be a defect at the level of mitochondrial complex I. This was also supported by the observation that mitochondrial respirations in the presence of glutamate and pyruvate, which are complex I substrates, were also reduced, whereas this was not the case with palmitoyl carnitine that delivers reducing equivalents to complex I and complex II. Thus, a simple increase in substrate flux might not be sufficient to increase mitochondrial ROS generation in diabetic hearts in the absence of specific alterations in mitochondrial complex activities that amplify ROS generation.
Oxidative stress has been proposed to contribute to mitochondrial dysfunction in the hearts of type 1 diabetes mouse models (30
). In the present study, we demonstrated impaired mitochondrial function and perturbed mitochondrial morphology in the absence of any evidence of oxidative stress. Interestingly, impaired mitochondrial morphology in the type 1 diabetic OVE26 mouse can be normalized by overexpression of manganese superoxide dismutase (MnSOD) or catalase in the heart, but MnSOD-deficient mice die from early dilated cardiomyopathy and have normal cardiac mitochondrial morphology (30
). In other transgenic models, knockout of the mitochondrial transcription factor A leads to reduced mitochondrial DNA replication and transcription and enlarged mitochondria with abnormal cristae morphology, and combined deletion of the cardiac insulin and IGF1 receptor results in impaired oxidative phosphorylation (OXPHOS) gene transcription and decreased mitochondria that appeared to be less dense in electron micrographs (33
). Thus, in some contexts, changes in the content of electron transport chain subunits can be associated with changes in mitochondrial morphology. Because insulin is a positive regulator of OXPHOS gene expression and because OXPHOS gene expression is decreased in the Akita mouse heart, insulin deficiency as opposed to oxidative stress might be an important contributor to mitochondrial dysfunction and altered morphology in the Akita mouse model (36
). Defective insulin signaling has been reported to reduce expression levels of genes involved in β-oxidation (23
), yet fatty acid oxidation genes were unchanged or increased in Akita mice. We believe that this reflects the impact of PPARα activation in the face of increased fatty acid availability.
An important observation in the present study is that increased content of UCP3 in the heart should not be taken to indicate increased mitochondrial uncoupling. We previously reported that increased mitochondrial uncoupling activity can occur in the absence of any changes in UCP3 content in hearts from ob/ob
). It is important to discuss what might drive UCP3 levels in Akita mouse hearts. Besides detoxification of ROS, UCP3 has been postulated to play a role in the regulation of fatty acid metabolism. Conditions associated with increased fatty acid availability, such as fasting, high-fat feeding, and diabetes, result in increased UCP3 expression, likely due to increased PPARα signaling (12
). In general, muscle UCP3 protein content is negatively related to fatty acid oxidative capacity, and induction of UCP3 is most pronounced in glycolytic muscle upon fasting and high-fat feeding (41
). Thus, increased UCP3 expression may reflect an adaptive response to fatty acid overload (43
). Based on the fatty acid cycling model, it has been proposed that export of fatty acid anions from the matrix into the intermembrane space via UCPs may help to lower intramitochondrial fatty acid levels if increased fatty acid availability exceeds mitochondrial oxidative capacity (43
). Thereby, oxidation of intramitochondrial fatty acids into harmful lipid peroxides could be prevented. Expression of MTE1 often parallels that of UCP3, and this was also the case in the present study. MTE1 catalyzes conversion of acyl-CoAs back to fatty acid anions, which can then be translocated out of the matrix by UCPs (13
). In the cytosol, they may be reesterified and oxidized in the mitochondria, stored as triglycerides, or used for other pathways. Nonesterified fatty acids can be generated within mitochondria and exported from the matrix, and these events are markedly increased in cardiac mitochondria from streptozotocin-induced type 1 diabetic rats (13
). Despite increased basal fatty acid oxidation, Akita mice also revealed increased lipid droplets; thus, it is tempting to speculate that fatty acyl-CoAs would be converted to fatty acid anions by increased MTE1 activity, exported by UCP3, reesterified, and stored as triglycerides. Thus, UCPs would contribute to match fatty acid availability with oxidation to prevent intramitochondrial lipotoxic effects.
A number of important differences between Akita mice and previously evaluated models of type 2 diabetes were observed (3
). First, there was a relative preservation of cardiac function and preserved inotropic responses to a calcium-induced increase in workload. This likely reflects the more profound mitochondrial dysfunction in the type 2 models and a greater degree of mitochondrial uncoupling. Preliminary studies do suggest, though, that cardiac dysfunction may develop in Akita mice after a longer-term hemodynamic challenge, such as after isoproterenol infusion (supplementary results and supplementary Table 5), but the mechanisms for this remains to be elucidated. Second, insulin sensitivity was preserved in Akita hearts in contrast to the insulin resistance reported in models of type 2 diabetes (2
). Although insulin levels in diabetic Akita are 40% of insulin levels in nondiabetic controls (19
), these ambient insulin concentrations might be sufficient to maintain mitochondrial function to a greater extent than models in which insulin signaling is severely impaired. Finally, myocardial V
was not increased despite increased oxidation rates of exogenous palmitate. Although this may partly reflect the absence of mitochondrial uncoupling, it is also possible that reduced oxidation of glucose and endogenous triglycerides coupled with mitochondrial dysfunction reduced myocardial V
in intact perfused hearts.
In conclusion, despite a significant increase in UCP3 content, insulin-deficient Akita hearts do not develop fatty acid–induced mitochondrial uncoupling, suggesting that underlying mechanisms for mitochondrial dysfunction may importantly differ between insulin-responsive type 1 versus insulin-resistant type 2 diabetic hearts. Increased UCP3 levels do not invariably lead to increased mitochondrial uncoupling in the heart, thereby supporting the hypothesis that fatty acid–induced mitochondrial uncoupling in diabetic hearts requires a concomitant increase in ROS or lipid peroxide generation.