Increased cardiac oxidative stress in a mouse transgenic model of non-alcoholic fatty liver disease and insulin resistance

Introduction: Diabetic cardiomyopathy is characterized by impaired myocardial function and ischemic tolerance in patients with type 2 diabetes. This has been related to reduced oxidative capacity and increased oxidative stress in cardiomyocytes. Non-alcoholic fatty liver (NAFL) and insulin resistance are associated with increased cardiovascular risk and cardiac mortality. Here, we aimed to investigate how NAFL and insulin resistance relate to cardiac oxidative stress and mitochondrial oxidative capacity in mice.

Methods: Female mice, aged 18 and 36 weeks (w), with adipose tissue-specific overexpression of the sterol regulatory-element binding protein-1c (aP2-SREBP-1c: AP2), which serves as a model of NAFL, and wild-type controls (CON) underwent hyperinsulinemic-euglycemic clamps to assess insulin sensitivity (n = 5 – 7). Mitochondria were isolated from the whole heart by differential centrifugation. Mitochondrial respiration and reactive oxygen species production were assessed ex vivo from isolated mitochondria by high-resolution respirometry and Amplex Red method, respectively (n = 4). Cardiac morphology and function were measured in vivo by NMR imaging in 36-weeks old mice (n = 8).

Results: Whole body insulin sensitivity was 71% and 70% lower in both 18- and 36-weeks old AP2 mice than in aged-matched CON (p < 0.05). Ex vivo cardiac mitochondrial oxidative capacity on tricarboxylic acid cycle-derived substrates was unchanged in 18 w old, but 93% higher in 36 w old AP2 mice (state u respiration: 2.86 ± 0.06, CON: 1.47 ± 0.43 nmol/mg protein/s; p < 0.05). Oxidative capacity on β-oxidation-derived substrates was 60% and 125% greater in 18 w old AP2 mice (2.18 ± 0.22, CON: 1.36 ± 0.19; p < 0.05) and in 36 w old AP2 mice (2.45 ± 0.66, CON: 1.09 ± 0.22 nmol/mg protein/s; p < 0.05). H₂O₂ production by mitochondrial complex III was 51% higher (p < 0.05) only in 36 w old mice, than in age-matched CON. Furthermore, there was a 34% increase in left ventricular mass and the 21% increase in wall thickness (p < 0.001) suggesting myocardial hypertrophy in the 36 w old AP2 mice. Finally, these older AP2 mice had 24% greater stroke volume and 29% higher cardiac output (for both p < 0.05 vs. CON). Data were analyzed for statistically significant differences applying two-tailed unpaired t-tests, p < 0.05 was considered significant.

Conclusions: Peripheral insulin resistance in a mouse model of hepatic steatosis associates with increased cardiac mitochondrial respiration and oxidative stress, which develops progressively with rising age. These changes are associated with left ventricle hypertrophy and increased cardiac output, which might reflect adaptation to increased blood pressure. Insulin resistance and steatosis may therefore lead to higher myocardial energy turnover and oxidative stress rendering the hearts vulnerable for ischemic intolerance and impaired myocardial function.

Acknowledgment: DFG feasibility grant, German Diabetes Association Menarini