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Summary: A healthy heart adapts to changes in nutrient availability and energy demands. In metabolic diseases like type 2 diabetes (T2D), increased reliance on fatty acids for energy production contributes to mitochondrial dysfunction and cardiomyopathy. A principal regulator of cardiac metabolism is 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2), which is a central driver of glycolysis. We hypothesized that increasing PFK-2 activity could mitigate cardiac dysfunction induced by high-fat diet (HFD). Wild type (WT) and cardiac-specific transgenic mice expressing PFK-2 (GlycoHi) were fed a low fat or HFD for 16 weeks to induce metabolic dysfunction. Metabolic phenotypes were determined by measuring mitochondrial bioenergetics and performing targeted quantitative proteomic and metabolomic analysis. Increasing cardiac PFK-2 had beneficial effects on cardiac and mitochondrial function. Unexpectedly, GlycoHi mice also exhibited sex-dependent systemic protection from HFD, including increased glucose homeostasis. These findings support improving glycolysis via PFK-2 activity can mitigate mitochondrial and functional changes that occur with metabolic syndrome.

Diabetic cardiomyopathy is associated with an increase in oxidative stress. However, antioxidant therapy has shown a limited capacity to mitigate disease pathology. The molecular mechanisms responsible for the modulation of reactive oxygen species (ROS) production and clearance must be better defined. The objective of this study was to determine how insulin affects superoxide radical (O2•–) levels. O2•– production was evaluated in adult cardiomyocytes isolated from control and Akita (type 1 diabetic) mice by spin-trapping electron paramagnetic resonance spectroscopy. We found that the basal rates of O2•– production were comparable in control and Akita cardiomyocytes. However, culturing cardiomyocytes without insulin resulted in a significant increase in O2•– production only in the Akita group. In contrast, O2•– production was unaffected by high glucose and/or fatty acid supplementation. The increase in O2•– was due in part to a decrease in superoxide dismutase (SOD) activity. The PI3K inhibitor, LY294002, decreased Akita SOD activity when insulin was present, indicating that the modulation of antioxidant activity is through insulin signaling. The effect of insulin on mitochondrial O2•– production was evaluated in Akita mice that underwent a 1-week treatment of insulin. Mitochondria isolated from insulin-treated Akita mice produced less O2•– than vehicle-treated diabetic mice. Quantitative proteomics was performed on whole heart homogenates to determine how insulin affects antioxidant protein expression. Of 29 antioxidant enzymes quantified, thioredoxin 1 was the only one that was significantly enhanced by insulin treatment. In vitro analysis of thioredoxin 1 revealed a previously undescribed capacity of the enzyme to directly scavenge O2•–. These findings demonstrate that insulin has a role in mitigating cardiac oxidative stress in diabetes via regulation of endogenous antioxidant activity.

Many Amyotrophic Lateral Sclerosis (ALS) patients experience hypermetabolism, or an increase in measured vs. calculated metabolic rate. The cause of hypermetabolism and the effects on neuronal metabolism in ALS are currently unknown, but the efficacy of dietary interventions shows promise for metabolism as an ALS therapeutic target. The goal of this study is to measure changes in metabolic pathways as a function of disease progression in spinal cords of the SOD1G93A mouse model of ALS. We conducted a comprehensive assessment of protein expression for metabolic pathways, antioxidants, chaperones, and proteases in lumbar spinal cord from male SOD1G93A mice at pre-onset, onset, and end-stages of the disease using targeted proteomic analysis. These results reveal that protein content of metabolic proteins including proteins involved in glycolysis, β-oxidation, and mitochondrial metabolism is altered in SOD1G93A mouse spinal cord well before disease onset. The changes in mitochondrial metabolism proteins are associated with decreased maximal respiration and glycolytic flux in SOD1G93A dermal fibroblasts and increased hydrogen peroxide and lipid hydroperoxide production in mitochondria from sciatic nerve and gastrocnemius muscle fibers at end stage of disease. Consistent with redox dysregulation, expression of the glutathione antioxidant system is decreased, and peroxiredoxins and catalase expression are increased. In addition, stress response proteases and chaperones, including those involved in the mitochondrial unfolded protein response (UPRmt), are induced before disease onset. In summary, we report that metabolic and stress response changes occur in SOD1G93A lumbar spinal cord before motor symptom onset, and are primarily caused by SOD1G93A expression and do not vary greatly as a function of disease course.