Activity-Based Protein Profiling Reveals Mitochondrial Oxidative Enzyme Impairment and Restoration in Diet-Induced Obese Mice

<div><p>High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD, or if mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar, or elevated, relative to standard diet (SD) mice; thereby, IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, β-oxidation, and respiratory chain function at levels similar to or higher than SD mice.</p> </div

Ingenuity Pathway Analysis of 293 proteins labeled by <b>ATP-ABP</b> in mouse skeletal muscle mitochondria lysates from HFD and SD fed mice for 2-, 8-, and 16-weeks.

<p>The <i>p</i>-value indicates the significance of pathway enrichment. The ratio is the number of proteins identified by probe-labeling to the number of proteins assigned to that pathway by IPA.</p

Comparison of citrate synthase activity measured by an activity assay and by LC-MS based ABPP using ATP-ABP

<p>. (A) The citrate synthase activity was measured for each skeletal muscle mitochondrial lysates from individual mice. Results show the mean +/− standard error. (B) Protein abundance identified by <b>ATP-ABP</b> labeling of citrate synthase. Results show the mean of the abundance measured by MS +/− standard error. A single outlier at 2- and 16-week time points was removed from the HFD group. Each box in (A) and (B) contains all measured data for each point. The line within the box indicates the mean, and capped dashed lines represent the standard error from the mean.</p

Synthesis of ATP-ABP.

<p>Synthesis of ATP-ABP.</p

Activity-based protein profiling of skeletal muscle mitochondria with ATP-ABP.

<p>(A) Probe structure of <b>ATP-ABP</b>, and reactivity with ATPases and ATP-binding proteins. The ε-amino group of lysine reacts with the mixed anhydride of <b>ATP-ABP</b> to form a stable amide bond on the protein to a six-carbon moiety containing the click chemistry (CC) compatible alkyne. Subsequent CC reaction to biotin-azide permits enrichment of <b>ATP-ABP</b>-labeled proteins. (B) Sample preparation of <b>ATP-ABP</b> labeled skeletal muscle mitochondrial proteins. Mitochondria were extracted from skeletal muscle tissue from individual mice, labeled with the probe (pink square), captured on streptavidin agarose resin (yellow circle), digested on-resin with trypsin, and the subsequent peptides were measured by LC-MS/MS.</p

Comparison of cytochrome C oxidase activity between HFD- and SD-fed mice at two weeks, measured by an absorbance assay.

<p>The cytochrome C oxidase activity was measured for each skeletal muscle mitochondrial lysate from individual mice. Results show the mean +/− standard error. A single outlier for both HFD- and SD-fed mouse groups was removed. The fold change closely correlates with the fold change for COX41 measured by <b>ATP-ABP</b> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047996#pone-0047996-g003" target="_blank"><b>Figure 3</b></a>).</p