Type 1 diabetic akita mouse hearts are insulin sensitive but manifest structurally abnormal mitochondria that remain coupled despite increased uncoupling protein 3

Abstract

Objective: Fatty acid-induced mitochondrial uncoupling and oxidative stress have been proposed to reduce cardiac efficiency and contribute to cardiac dysfunction in type 2 diabetes. We hypothesized that mitochondrial uncoupling may also contribute to reduced cardiac efficiency and contractile dysfunction in the type 1 diabetic Akita mouse model (Akita).

Research design and methods: Cardiac function and substrate utilization were determined in isolated working hearts and in vivo function by echocardiography. Mitochondrial function and coupling were determined in saponin-permeabilized fibers, and proton leak kinetics was determined in isolated mitochondria. Hydrogen peroxide production and aconitase activity were measured in isolated mitochondria, and total reactive oxygen species (ROS) were measured in heart homogenates.

Results: Resting cardiac function was normal in Akita mice, and myocardial insulin sensitivity was preserved. Although Akita hearts oxidized more fatty acids, myocardial O(2) consumption was not increased, and cardiac efficiency was not reduced. ADP-stimulated mitochondrial oxygen consumption and ATP synthesis were decreased, and mitochondria showed grossly abnormal morphology in Akita. There was no evidence of oxidative stress, and despite a twofold increase in uncoupling protein 3 (UCP3) content, ATP-to-O ratios and proton leak kinetics were unchanged, even after perfusion of Akita hearts with 1 mmol/l palmitate.

Conclusions: Insulin-deficient Akita hearts do not exhibit fatty acid-induced mitochondrial uncoupling, indicating important differences in the basis for mitochondrial dysfunction between insulin-responsive type 1 versus insulin-resistant type 2 diabetic hearts. Increased UCP3 levels do not automatically increase mitochondrial uncoupling in the heart, which supports the hypothesis that fatty acid-induced mitochondrial uncoupling as exists in type 2 diabetic hearts requires a concomitant increase in ROS generation.

FIG. 1.

Substrate oxidation and contractile performance of Akita hearts. 24-week-old wild-type and Akita mouse hearts were perfused in the isolated working mode without insulin (basal) or in the presence of 1 nmol/l insulin (n = 4–5). A: Left ventricular–developed pressure (LVDevP). B: Cardiac output. C: Cardiac power. D: Palmitate oxidation. E: Glucose oxidation. F: Glycolysis. G: Oxygen consumption. H: Cardiac efficiency. *P < 0.05 vs. wild type, ΨP < 0.05 vs. without insulin.

FIG. 2.

Intact insulin signaling in Akita hearts. Representative Western blot images showing cardiac protein levels of phosphorylated Akt (p-Akt) on Ser⁴⁷³ and total levels of Akt (t-Akt) and densitometric quantification of the ratio of p-Akt to t-Akt (p-Akt/t-Akt) after isolated working heart perfusions in the absence (basal) or presence of 1 nmol/l insulin at the age of 10 weeks (A) and 24 weeks (B). *P < 0.05 vs. basal, ΨP < 0.05 vs. wild-type (WT).

FIG. 3.

Preserved mitochondrial coupling and impaired mitochondrial oxidative capacity in Akita hearts. Mitochondrial respiratory rates (A–C) and ATP synthesis and ATP-to-O ratios (E–G) of saponin-permeabilized cardiac fibers from 24-week-old wild-type (WT) and Akita mice using palmitoyl carnitine (A and E), pyruvate (B and F), or glutamate (C and G) as a substrate (n = 6). Similar data were also obtained in 10-week-old mice after incubation with glutamate (D and H), n = 6. *P < 0.05 vs. wild type.

FIG. 4.

Fatty acid perfusion does not induce mitochondrial uncoupling. Mitochondrial respiratory rates (A) and ATP synthesis and ATP-to-O ratios (B) of saponin-permeabilized cardiac fibers generated from 24-week-old wild-type (WT) and Akita mouse hearts that were perfused with KHB containing 11 mmol/l glucose and 1 mmol/l palmitate. Palmitoyl carnitine was used as respiratory substrate (n = 6). C: Proton leak kinetics of mitochondria isolated from 24-week-old wild-type and Akita mouse hearts preperfused with KHB containing 11 mmol/l glucose and 1 mmol/l palmitate. Palmitoyl carnitine was used as respiratory substrate (n = 6).

FIG. 5.

Reduced OXPHOS expression and increased UCP expression. A: Myocardial gene expression in 24-week-old wild-type (WT) and Akita mice normalized to 16S RNA transcript levels (n = 8). Values represent fold change in mRNA transcript levels relative to wild type, which was assigned as one (dashed line). B: Representative Western blot images and densitometric quantification of UCP3 protein levels in isolated mitochondria from 24-week-old wild-type and Akita mice (n = 4). Coomassie blue staining was used as a loading control. *P < 0.05 vs. wild type.

FIG. 6.

Altered mitochondrial morphology in Akita hearts. Representative longitudinal electron microscopy images of left ventricular wall of 24-week-old wild-type (WT) and Akita mice (A), stereological quantification of mitochondrial volume density (B), and mitochondrial number (C) (n = 4). Magnifications (×10,000 and ×40,000) are shown on each image, and black arrows on the images indicate lipid droplets. *P < 0.05 vs. wild type.

FIG. 7.

Oxidative stress is absent in Akita hearts. H₂O₂ production in isolated mitochondria of wild-type (WT) and Akita mice at 24 weeks (A) and 10 weeks (D) of age (n = 4). Mitochondrial aconitase activity of wild-type and Akita mice at 24 weeks (B) and 10 weeks (E) of age (n = 4). C: Oxidation of DCFDA measured in cardiac whole-tissue extracts from 24-week-old wild-type and Akita mice (n = 5). Representative Western blot images and densitometric quantification of MnSOD (F) and PRDX3 (G) in isolated mitochondria of 24-week-old wild-type and Akita mice (n = 4). Coomassie blue staining was used as a loading control. *P < 0.05 vs. wild type.