UCP1 and the regulation of mitochondrial respiration in brown adipose tissue by oxaloacetate

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PROJECT SUMMARY Control of metabolic flux through the TCA cycle has been investigated dating back many decades. However, there are issues that still require clarification. One important, but largely neglected, issue regards the role of oxaloacetate (OAA) inhibition of succinate dehydrogenase (SDH) in regulating mitochondrial function. Recent findings in my laboratory focused on skeletal muscle mitochondria show that while low membrane potential (ΔΨ) drives respiration, low ΔΨ also initiates a sequence of events leading to mitochondrial OAA accumulation, inhibition of SDH, and consequent inhibition or metabolic “braking” of respiration. Given that low ΔΨ appears to be the initiating factor, the current proposal is focused on the role of OAA in regulating respiration in brown adipose tissue (BAT) wherein ΔΨ is intrinsically low due to the action of uncoupling protein 1 (UCP1). Our overall hypothesis is that mitochondrial OAA metabolism and inhibition of SDH is regulated by definable interactions between UCP1 controlled ΔΨ and downstream events and that there are physiological consequences. Our aims address three issues. Aim 1 will delineate molecular events initiated by UCP1-regulated ΔΨ and culminating in OAA inhibition of O2 flux. Aim 2 examines UCP1 perturbations in live mice to determine if this translates to OAA effects on mitochondrial and cellular function. Aim 3 attempts to modulate OAA inhibition of SDH in a way that may prove beneficial towards mitochondrial, cellular, and whole-body metabolism. To accomplish our objectives, we will use a novel NMR assay to assess mitochondrial OAA content as well as other metabolites. A major reason why our knowledge of OAA effects on respiration is lacking is that OAA is difficult to assay since the metabolite is not stable. In fact, metabolomics services, whether commercial or in university cores, do not offer quantification of OAA. Our research will also use a novel adaptation of existing technology that we developed to assess mitochondrial respiration at clamped concentrations of ADP. In this way, we can assess mitochodnrial respiration beyond the often-determined state 3 and state 4, which are not physiological states. Finally, we will use up-to-date mass spectroscopy methods for targeted metabolite expression and for [13C]isotopomer tracer studies directed at metabolic flux. Our project is applicable to the clinical issue of obesity and its complications since understanding BAT physiology and how it is regulated will add to our overall knowledge of whole-body energetics.