Relevance of fatty acid handling in the adaptive response to mitochondrial dysfunction - Primary mitochondrial disease has an overall incidence of ~1:5000, and manifests as a decrease in the capacity for ATP production by oxidative phosphorylation (oxphos). Skeletal muscle (SkM) is highly affected, leading to mitochondrial myopathy (MM). A hallmark of SkM is that it can oxidize both glucose and fatty acids (FA) for ATP synthesis. Glucose uptake in SkM substantially depends on insulin signaling which is feedback-inhibited by FA derivatives, especially when FA supply exceeds oxidation, as likely occur when oxphos is deficient. Lipid overload can also cause endoplasmic reticulum stress. Yet the fate of circulating FA substrates has been largely ignored in MM, but has implications for the ability to sustain elevated glycolysis in MM and to trigger, or worsen, cell stress responses. The overarching questions of this project are, “How is FA substrate handled when oxphos is deficient, and how is that handling relevant in the adaptation to mitochondrial dysfunction including the increased reliance on glycolysis?”. We hypothesize that substrate metabolism in an organism with MM is rewired such that the liver and heart acquire greater roles to handle FA; this multi-organ compensation would mitigate lipid burden and reductive stress in oxphos-deficient SkM. We further hypothesize that FGF21, derived from oxphos-deficient SkM from activation of the integrated stress response (ISR), acts on the liver and heart to effect a tighter muscle-liver/heart metabolic coupling that favors better adaptation to defective oxphos. We will test these hypotheses using SkM-specific, and then combined liver/SM models of MM, namely mice with deficient oxphos in SkM due to SkM-specific loss of the mitochondrial phosphate carrier, PiC, or mice deficient in a mitochondrial quality control protein. Aim 1 will determine the fate of FA substrate in oxphos-deficient SkM and oxphos-competent liver and heart, using FA tracers and by measuring β-oxidation intermediates in SkM, liver, heart and serum. β-oxidation flux will be inferred using a novel approach that couples correlation analysis with metabolite abundance, complemented by ex vivo β-oxidation flux by isotope tracing. Variables to be considered: level of dietary fat; ATP demand (rest vs. exercise); need for FGF21; oxphos competence of liver. Aim 2 tests the hypothesis that insulin-mediated glucose uptake, which can be inhibited by cytosolic lipids, in oxphos- deficient SkM depends on the ability of the liver and heart for greater storage and oxidation of FA, and that, at later stages of MM, this ability depends on FGF21. This will be tested using hyperinsulinemic-euglycemic clamp along with radiolabeled glucose and non-metabolizable glucose analog to evaluate glucose disposal, glucose uptake into SkM, liver, heart and adipose tissues, and hepatic glucose production. Aim 3 tests the hypothesis that ISR activation is sensitive to lipids in oxphos-deficient SkM, thus serving as a driver of FGF21 production and secretion that mitigates lipid accumulation in oxphos-deficient SkM. We expect these studies to reveal how FA substrate is handled in the context of 1° mitochondrial dysfunction, which is a major unanswered question, and to provide insight into the implications of FA handling on major cellular responses to oxphos deficiency.
