Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart.

SYNOPSIS Diabetic cardiomyopathy refers to the changes in contractility that occur to the diabetic heart that can arise in the absence of vascular disease. Mitochondrial bioenergetic deficits and increased free radical production are pathological hallmarks of diabetic cardiomyopathy but the mechanisms and causal relationships between mitochondrial deficits and the progression of disease are not understood. We evaluated cardiac mitochondrial function in a rodent model of chronic type 1 diabetes (OVE26 mice) prior to onset of contractility deficits. We found that the most pronounced change in OVE26 heart mitochondria is severe metabolic inflexibility. This inflexibility is characterized by large deficits in mitochondrial respiration measured in the presence of non-fatty acid substrates. Metabolic inflexibility occurred concomitantly with decreased activities of pyruvate dehydrogenase and complex II. Hyper-acetylation of protein lysine was also observed. Treatment of control heart mitochondria with acetic anhydride, an acetylating agent, preferentially inhibited respiration by non-fatty acid substrates and increased superoxide production. We have concluded that metabolic inflexibility, induced by discrete enzymatic and molecular changes, including hyper-acetylation of protein lysines, precedes mitochondrial defects in a chronic rodent model of type 1 diabetes.

BackgroundPeroxisome proliferator activated receptor-alpha (PPARα) is a ubiquitously expressed nuclear receptor. The role of endogenous PPARα in retinal neuronal homeostasis is unknown. Retinal photoreceptors are the highest energy-consuming cells in the body, requiring abundant energy substrates. PPARα is a known regulator of lipid metabolism, and we hypothesized that it may regulate lipid use for oxidative phosphorylation in energetically demanding retinal neurons.ResultsWe found that endogenous PPARα is essential for the maintenance and survival of retinal neurons, with Pparα -/- mice developing retinal degeneration first detected at 8 weeks of age. Using extracellular flux analysis, we identified that PPARα mediates retinal utilization of lipids as an energy substrate, and that ablation of PPARα ultimately results in retinal bioenergetic deficiency and neurodegeneration. This may be due to PPARα regulation of lipid transporters, which facilitate the internalization of fatty acids into cell membranes and mitochondria for oxidation and ATP production.ConclusionWe identify an endogenous role for PPARα in retinal neuronal survival and lipid metabolism, and furthermore underscore the importance of fatty acid oxidation in photoreceptor survival. We also suggest PPARα as a putative therapeutic target for age-related macular degeneration, which may be due in part to decreased mitochondrial efficiency and subsequent energetic deficits.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0451-x) contains supplementary material, which is available to authorized users.

BackgroundThe healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Diabetes mellitus disrupts this metabolic flexibility and promotes cardiomyopathy through mechanisms that are not completely understood. Phosphofructokinase 2 (PFK‐2) is a primary regulator of cardiac glycolysis and substrate selection, yet its regulation under normal and pathological conditions is unknown. This study was undertaken to determine how changes in insulin signaling affect PFK‐2 content, activity, and cardiac metabolism.Methods and ResultsStreptozotocin‐induced diabetes mellitus, high‐fat diet feeding, and fasted mice were used to identify how decreased insulin signaling affects PFK‐2 and cardiac metabolism. Primary adult cardiomyocytes were used to define the mechanisms that regulate PFK‐2 degradation. Both type 1 diabetes mellitus and a high‐fat diet induced a significant decrease in cardiac PFK‐2 protein content without affecting its transcript levels. Overnight fasting also induced a decrease in PFK‐2, suggesting it is rapidly degraded in the absence of insulin signaling. An unbiased metabolomic study demonstrated that decreased PFK‐2 in fasted animals is accompanied by an increase in glycolytic intermediates upstream of phosphofructokianse‐1, whereas those downstream are diminished. Mechanistic studies using cardiomyocytes showed that, in the absence of insulin signaling, PFK‐2 is rapidly degraded via both proteasomal‐ and chaperone‐mediated autophagy.ConclusionsThe loss of PFK‐2 content as a result of reduced insulin signaling impairs the capacity to dynamically regulate glycolysis and elevates the levels of early glycolytic intermediates. Although this may be beneficial in the fasted state to conserve systemic glucose, it represents a pathological impairment in diabetes mellitus.

Therapy for treatment-resistant breast cancer provides limited options and the response rates are low. Therefore, the development of therapies with alternative chemotherapeutic strategies is necessary. AG311 (5-[(4-methylphenyl)thio]-9H-pyrimido [4,5-b]indole-2,4-diamine), a small molecule, is being investigated in preclinical and mechanistic studies for treatment of resistant breast cancer through necrosis, an alternative cell death mechanism. In vitro, AG311 induces rapid necrosis in numerous cancer cell lines as evidenced by loss of membrane integrity, ATP depletion, HMGB1 (high-mobility group protein B1) translocation, nuclear swelling, and stable membrane blebbing in breast cancer cells. Within minutes, exposure to AG311 also results in mitochondrial depolarization, superoxide production, and increased intracellular calcium levels. Additionally, upregulation of mitochondrial oxidative phosphorylation results in sensitization to AG311. This AG311-induced cell death can be partially prevented by treatment with the mitochondrial calcium uniporter inhibitor, Ru360 [(m)[(HCO 2 )(NH 3 ) 4 Ru] 2 OCl 3 ], or an antioxidant, lipoic acid. Additionally, AG311 does not increase apoptotic markers such as cleavage of poly (ADP-ribose) polymerase (PARP) or caspase-3 and -7 activity. Importantly, in vivo studies in two orthotopic breast cancer mouse models (xenograft and allograft) demonstrate that AG311 retards tumor growth and reduces lung metastases better than clinically used agents and has no gross or histopathological toxicity. Together, these data suggest that AG311 is a first-in-class antitumor and antimetastatic agent inducing necrosis in breast cancer tumors, likely through the mitochondria.