Differences in Brain Region Mitochondrial Bioenergetics Detected Using CK Clamp

Abstract

The brain is a non-uniform organ comprised of discrete structures of varying function and cell composition. The differing function of brain regions ostensibly would necessitate disparate energetic demands, thereby suggesting brain bioenergetics would be different among brain regions. This study aimed to address whether brain regions indeed have different bioenergetics by evaluating substrate preference, fuel-specific mitochondrial function at physiologically relevant energy demands as well as associated membrane potential and electron conductance using high-resolution respirometry (Oroboros O2k) in the frontal cortex, hypothalamus, and hippocampus. We hypothesize there will be different bioenergetics among brain regions. Substrate preference was established using a substrate-uncoupler-inhibitor-titration (SUIT) with additions, in order: ADP, Pyruvate/Malate, UK5099, Octanoylcarnitine, Rotenone, Succinate, and Antimycin A. There was no statistically significant interaction between region and substrate (p=0.15) nor a main effect of region (p=0.91), however, there was a main effect of substrate in which pyruvate/malate (+275%) and succinate/rotenone (+170%) had significantly greater respiration than octanoylcarnitine, independent of region (p<0.001). Given the results of the SUIT test, pyruvate/malate was used for subsequent tests. The SUIT method relies upon saturating ADP levels and non-physiological ATP:ADP concentration ratios, the extent to which this test obscures the interpretation of brain region respiration is unclear. Instead, an alternative approach that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiological ATP:ADP ratios, “CK Clamp,” was used. Function of mitochondria with a pyruvate fuel was then evaluated in all three brain regions in which energetic demand was clamped at ΔGATP = -12.87 (high demand), -14.08, -14.5, and -14.85 (low demand). There was a statistically significant interaction between brain region and clamped ΔGATP values (p = 0.02). Frontal cortex displayed higher respiration than hippocampus at -12.87 ΔGATP (33%). There was no statistically significant interaction between region and clamp for mitochondrial membrane potential assessed during the CK clamp (p=0.16), however there was a significant main effect of clamped ΔGATP (p<0.001) in which membrane potential increased (i.e., hyperpolarized) as energetic demand decreased. This agrees with the bioenergetic model that a decrease in energetic demand increases the proton motive force (aka, hyperpolarization), thereby applying a backpressure on the electron transport chain, slowing respiration. Electron conductance was then assessed by calculating the slope of respiration versus clamped ΔGATP. Electron conductance was greater in the frontal cortex than hypothalamus (+70%, p = 0.015). The conclusion of this study is there are differences in mitochondrial bioenergetics among brain regions and the CK clamp was more sensitive at detecting these differences than the SUIT method. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.