AbstractWhite adipose tissue (WAT) is classically associated with energy storage in the form of triacylglycerol and is directly associated with metabolic disorders, including obesity. Mitochondria regulates cellular expenditure and are active in WAT. Although isolated mitochondria have been classically used to assess their functions, several artifacts can be introduced by this approach. Although methods to assess mitochondrial physiology in permeabilized WAT were proposed, important limitations that affect organelle function exist. Here, we established and validated a method for functional evaluation of mice mesenteric WAT (mWAT) mitochondria by using mechanical permeabilization in combination with lipid depletion and high-resolution respirometry. We observed that mild stirring of mWAT for 20 minutes at room temperature with 4% fatty acid-free albumin selectively permeabilized white adipocytes plasma membrane. In these conditions, mWAT mitochondria were intact and coupled, exhibiting succinate-induced respiratory rates that were sensitive to classical modulators of oxidative phosphorylation. Finally, the respiratory capacity of mWAT in females was significantly higher than in males, an observation that agrees with reported data using isolated mitochondria. The functional assessment of mWAT mitochondria through mild mechanical permeabilization, lipid depletion and high resolution respirometry proposed here will contribute to a better understanding of WAT biology in several pathophysiological contexts.
Timing Polymerase Pausing with TV-PRO-seq
