Antioxidant treatment normalizes mitochondrial energetics and myocardial insulin sensitivity independently of changes in systemic metabolic homeostasis in a mouse model of the metabolic syndrome

Abstract

Cardiac dysfunction in obesity is associated with mitochondrial dysfunction, oxidative stress and altered insulin sensitivity. Whether oxidative stress directly contributes to myocardial insulin resistance remains to be determined. This study tested the hypothesis that ROS scavenging will improve mitochondrial function and insulin sensitivity in the hearts of rodent models with varying degrees of insulin resistance and hyperglycemia. The catalytic antioxidant MnTBAP was administered to the uncoupling protein-diphtheria toxin A (UCP-DTA) mouse model of insulin resistance (IR) and obesity, at early and late time points in the evolution of IR, and to db/db mice with severe obesity and type-two diabetes. Mitochondrial function was measured in saponin-permeabilized cardiac fibers. Aconitase activity and hydrogen peroxide emission were measured in isolated mitochondria. Insulin-stimulated glucose oxidation, glycolysis and fatty acid oxidation rates were measured in isolated working hearts, and 2-deoxyglucose uptake was measured in isolated cardiomyocytes. Four weeks of MnTBAP attenuated glucose intolerance in 13-week-old UCP-DTA mice but was without effect in 24-week-old UCP-DTA mice and in db/db mice. Despite the absence of improvement in the systemic metabolic milieu, MnTBAP reversed cardiac mitochondrial oxidative stress and improved mitochondrial bioenergetics by increasing ATP generation and reducing mitochondrial uncoupling in all models. MnTBAP also improved myocardial insulin mediated glucose metabolism in 13 and 24-week-old UCP-DTA mice. Pharmacological ROS scavenging improves myocardial energy metabolism and insulin responsiveness in obesity and type 2 diabetes via direct effects that might be independent of changes in systemic metabolism.

Figure 1. Impact of MnTBAP on body composition and glucose tolerance in UCP-DTA and db/db mice

A, B. Relative distribution of fat and lean mass as measured by DEXA in 13 and 24 week-old UCP-DTA mice vs. controls, respectively. Lean and fat mass is expressed as % of total body mass (n=3–9). C–E. Glucose tolerance tests in 13 week-old UCP-DTA, 24 week-old UCP-DTA, and 9 week-old db/db mice, respectively (n=6–15). Mice were treated with MnTBAP or saline, as indicated. Maximum glucose concentration detectable by the glucometer is 600mg/dl. *p<0.05 vs. other genotypes; ^$ p<0.05 vs. WT saline and WT MnTBAP.

Figure 2. Evidence for mitochondrial oxidative stress and reversal by MnTBAP

A, B. No change in H₂O₂ emission and aconitase activity in 13 week-old UCP-DTA mice (n=3–4). C. The rate of H₂O₂ emission is increased in hearts of 24 week-old UCP-DTA mice treated with saline but not MnTBAP (n=6–7). D. Mitochondrial aconitase activity in hearts of 24 week-old UCP-DTA mice compared to WT littermates (n =4–6). *p<0.05 vs. all other or indicated groups. Saline-treated control wildtype (WT) mice, MnTBAP-treated WT mice; UCP-DTA treated with saline, UCP-DTA treated with MnTBAP.

Figure 3. Impact of MnTBAP on mitochondrial function and myocardial substrate metabolism in 13 week-old UCP-DTA mice

A. Oxygen consumption, ATP synthesis, and ATP/O ratios measured using saponin-permeabilized LV fibers with palmitoyl-carnitine and malate as substrates (n =9–13). B. Oxygen consumption, ATP synthesis, and ATP/O ratios using saponin-permeabilized fibers with glutamate and malate as substrates ( n=6–7). *p<0.05 vs. all or indicated groups. C. Electron microscopy (1:10,000) of left ventricular section in 13 week-old UCP-DTA mice, and quantification of mitochondrial volume density and number. n=4. D–F. Palmitate oxidation, cardiac power and mVO₂ measured ex vivo in isolated working hearts (n=6). *p<0.05 vs. all other or indicated groups. Saline-treated control wildtype (WT) mice, MnTBAP-treated WT mice, UCP-DTA treated with saline, UCP-DTA treated with MnTBAP. Dw=dry weight.

Figure 4. Impact of MnTBAP on mitochondrial function and myocardial substrate metabolism in 24 week-old UCP-DTA mice

A. Oxygen consumption, ATP synthesis, and ATP/O ratios measured using saponin-permeabilized LV fibers with palmitoyl-carnitine and malate as substrates (n=6–8). B. Abundance and densitometric analysis of selected OxPhos subunits in the whole heart homogenates as measured by western blotting (n=4–6). C. Electron microscopy images (1:10,000) and quantification of mitochondrial number and volume density in left ventricular sections of 24 week-old UCP-DTA mouse hearts (n=4). D–F. Palmitate oxidation, cardiac power and mVO₂ measured ex vivo in isolated working hearts (n=6–8). *p<0.05 vs. all other or indicated groups. Dashed line or Saline-treated control wildtype (WT) mice, MnTBAP-treated WT mice, UCP-DTA treated with saline, UCP-DTA treated with MnTBAP. Dw=dry weight.

Figure 5. Impact of MnTBAP on mitochondrial function and myocardial substrate metabolism in 9 week-old db/db mice

A–C. Oxygen consumption, ATP synthesis, and ATP/O ratios measured using saponin-permeabilized LV fibers with palmitoyl-carnitine and malate as substrates (n=6–8). D–F. Palmitate oxidation, cardiac power and mVO₂ measured ex vivo in isolated working hearts (n=4–5). *p<0.05 vs. all other or indicated groups. Saline-treated control wildtype (WT) mice, MnTBAP-treated WT mice, db/db treated with saline, db/db treated with MnTBAP. Dw=dry weight.

Figure 6. Impact of MnTBAP on insulin-stimulated cardiomyocyte glucose uptake and myocardial glucose utilization in 13 and 24-week-old UCP-DTA mice

A. Insulin-stimulated 2-deoxy-³H-glucose uptake in isolated cardiomyocytes from 13 week-old and control hearts treated with saline or MnTBAP (n=4–5). B–D. Glycolysis, glucose oxidation, and cardiac power measured in isolated working heart preparations with or without 1nM insulin in 13 week-old UCP-DTA and control mice (n=5–7). E. Insulin-stimulated 2-deoxy-³H-glucose uptake in isolated cardiomyocytes from 24 week-old UCP-DTA and control hearts treated with saline or MnTBAP (n=5–7). F–H. Glycolysis, glucose oxidation, and cardiac power measured in isolated working heart preparations with or without 1nM insulin in 24 week-old UCP-DTA or control mice (n=5–7). I–K. Glycolysis, glucose oxidation, and cardiac power in db/db hearts measured in isolated working heart preparations without insulin (n=3–4). *p<0.05 vs. other genotypes in the same treatment group; # p<0.05 for insulin- stimulated vs. basal state for same genotype. Saline-treated control wildtype (WT) mice, MnTBAP-treated WT mice, UCP-DTA or db/db mice treated with saline, UCP-DTA or db/db mice, treated with MnTBAP. Dw=dry weight.

Figure 7. Insulin signaling in hearts of 13 or 24 week-old UCP-DTA and control mice treated with saline or MnTBAP

A, B. Western blots and densitometric analysis representing phosphorylation of Akt in 13 week-old UCP-DTA and control hearts perfused with or without 1nM insulin (n=4). C–E. Western blots and densitometric analysis representing phosphorylation of Akt and GSK3 in 24 week-old UCP-DTA and control hearts with or without 1nM insulin. E, F. Fold increase of insulin stimulated phospho-Akt and GSK3 over basal (n=5). *p<0.05 vs. other genotypes in the same groups; # p<0.05 for insulin- stimulated vs. basal state for same genotype. Saline-treated control wildtype (WT) mice, MnTBAP-treated WT mice, UCP-DTA treated with saline, UCP-DTA treated with MnTBAP.