Perinatal loss of insulin signaling in cardiomyocytes impairs mitochondrial function. There is an early defect in pyruvate utilization and progressive reduction in mitochondrial oxidative capacity and ATP synthesis with glutamate and palmitoyl carnitine substrates. There is a modest decline in cardiac contractility at an early age, which does not progress to heart failure, however cardiac function was significantly impaired in vitro
following calcium-induced inotropic stress. Two mechanisms for mitochondrial dysfunction were identified. First, there was a coordinate reduction in TCA and FA enzymes that presumably impaired the delivery of reducing equivalents to the electron transport chain, which remained functionally competent. Second, mitochondria from insulin receptor deficient myocytes were more susceptible to FA- induced oxidative stress and mitochondrial uncoupling. These changes promote a mitochondrial biogenic response that occurs in the absence of increased PGC-1α expression. These data identify an important role for insulin signaling pathways in modulating mitochondrial bioenergetics and integrity. We recently reported that PI3K signaling regulates mitochondrial function in the heart 32
. The present study now shows that insulin signaling per se
also regulates myocardial mitochondrial oxygen consumption and ATP synthesis rates. The blunted activation of PI3K targets such as Akt and its downstream substrate GSK3β in insulin-perfused CIRKO hearts is consistent with the hypothesis that reduced PI3K signaling could contribute to mitochondrial dysfunction in CIRKO hearts. However, given that basal levels of phosphorylation of these kinases were not reduced, it is also likely that PI3K or Akt –independent signals downstream of the insulin receptor also play an important role.
The mitochondrial defect appears initially to be specific for pyruvate utilization and subsequently for FA utilization. These observations provide a mechanistic basis for our previously published observations that in isolated perfused working hearts from 16–20- week-old CIRKO mice, rates of glucose and FA oxidation were both reduced 23
. An important mechanism for reduced pyruvate flux appears to be reduced content of two key subunits of the pyruvate dehydrogenase complex. The E1-alpha subunit is a key regulator of PDH flux and is the substrate of the regulatory kinase PDK4 33, 34
. We provide novel evidence that the E1α1 subunit of PDH may be an insulin-regulated transcript in the heart, and that reduced protein levels of this subunit in mitochondrial matrix might occur on the basis of transcriptional repression. Increased phosphorylation of the E1-alpha subunit by PDK4, decreases its stability, while blocking degradation of the E1-alpha subunit of PDH increases the activity of and flux through the enzyme complex 35
. However PDHE1 phosphorylation was not increased in CIRKO hearts and the expression of its kinase (PDK4) was actually reduced. Mitochondrial dysfunction in CIRKO mice is associated with modest reduction in cardiac function. We previously reported that cardiac function was reduced in isolated working hearts that were perfused with glucose and FA as substrates under normal workload 23
. In the present study we chose to study glucose perfused hearts because of the defect in pyruvate utilization in mitochondria. We reasoned that in the presence of glucose alone, contractile dysfunction following calcium-induced inotropic stress would be amplified.
We speculate that an early defect in glucose/pyruvate metabolism could initially lead to the increase in FA utilization that was observed in the mitochondria of 8-week-old CIRKO mice. However, this cannot be sustained over time because of the coordinate reduction in levels of mitochondrial beta-oxidation enzymes. This hypothesis is also supported by the observation of increased rates of FA oxidation (FAO) in isolated working hearts obtained from 8-week-old CIRKO mice (see supplementary Figure S8
) but reduced FAO in 16–20-week-old mice 23
. The reduction in gene and protein expression levels of a broad array of regulators of FA metabolism was striking, and extends our previously reported findings that demonstrated reduced mRNA for acyl CoA dehydrogenases. The likely mechanism for these changes is insulin-mediated regulation of expression levels of the PPAR-α gene in the heart.
The second major mechanism that contributed to mitochondrial dysfunction is oxidative stress and ROS-mediated mitochondrial dysfunction. Increased ROS production was evident in the hearts of 8-week-old CIRKO mice and was sufficient to reduce the activity levels of the redox sensitive enzyme aconitase. This increase in ROS, also likely reduced mitochondrial energetics by promoting mitochondrial uncoupling as evidenced by reduced ATP/O ratios in palmitoyl carnitine treated mitochondria and increased oligomycin-insensitive respiration rates that were normalized by treating animals with the antioxidant MnTBAP. MnTBAP reduces ROS but could increase H2O2 generation, underscoring that mitochondrial superoxide likely mediates the changes observed. Increased ROS could reflect changes in superoxide generation or detoxification. Although, increased FA flux could contribute to increased ROS production in 8-week-old CIRKO hearts it is unlikely to represent the mechanism in older hearts in which FA oxidation is reduced. Proteomic analysis revealed changes in stoichiometry of ETC subunits, which could potentially contribute to increased superoxide generation. A major role for a reduction in ROS degradation pathways as a contributor to increased H2O2 production in CIRKO mitochondria appears unlikely, as neither MnSOD levels nor catalase content were changed in 8-week-old mice and in 24-week-old animals, catalase content was marginally lower. However it is possible that reduced TCA flux could limit the supply of reducing equivalents to replenish NADPH pools that maintain antioxidants such as glutathione in the reduced state. Taken together, deficient insulin signaling in the heart likely promotes mitochondrial oxidative stress by multiple mechanisms.
Mitochondrial biogenesis has been described in the hearts of insulin resistant mice and has been attributed to activation of PGC-1α-mediated signaling 36
. Here we show that mitochondrial number and volume density increased in CIRKO mice despite the lack of coordinate changes in mRNA levels of key regulators of the mitochondrial biogenesis pathway such as PGC-1α. Thus, the possibility exists that this proliferative response in CIRKO hearts is a consequence of reduced ATP generation or increased oxidative stress that promote mitochondrial biogenesis. In this regard it is important to discuss the discrepancy between citrate synthase (CS) activities and increased mitochondrial volume density in aging CIRKO mice. CS activity is widely used as an indirect estimate of mitochondrial mass. However, in CIRKO mice, our proteomic analyses indicate CS protein content in mitochondrial matrix was already significantly lower in 8-week-old CIRKO mice. This observation therefore supports the notion that the morphological “biogenic” response that we observed represents an adaptation to pre-existing mitochondrial dysfunction in this model.
In conclusion, we demonstrate that insulin signaling is a regulator of mitochondrial oxidative capacity via mechanisms that may determine TCA cycle flux and the mitochondrial metabolism of pyruvate and fatty acids. Moreover, impaired insulin signaling predisposes cardiac mitochondria to oxidative stress, which not only might damage mitochondria, but also impairs energetics by activating mitochondrial uncoupling. Thus insulin signaling plays an essential role in the maintenance of mitochondrial homeostasis in the heart. Given the perinatal timing of insulin receptor deletion in CIRKO hearts, it is important to note that metabolic maturation of the heart continues to occur throughout the neonatal period, thus we cannot rule out that the phenotypes that we have observed might reflect unique effects of insulin resistance during this important developmental window. Future studies in mice with inducible KO of insulin receptors in adult hearts will be required to clarify this.
Diabetes and obesity are independent risk factors for the development of heart failure 37
. There is a growing body of evidence that acquired defects in insulin signaling, which may impair cardiac metabolism and are associated with LV dysfunction, develop in the heart in diabetes and obesity 38
. The present study provides new insights into potential mechanisms linking impaired post-natal insulin signaling with the development of mitochondrial dysfunction in the heart.