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.
