PPARβ/δ Agonism with GW501516 Increases Myotube PGC-1α Content and Reduces BCAA Media Content Independent of Changes in BCAA Catabolic Enzyme Expression

Figure 5

Effect of GW501516 on mitochondrial metabolism and content. (a) Time trial of mitochondrial metabolism following treatment with and without GW501516 (GW) at 1 μM for 24 hours. (b and c) Basal and peak mitochondrial metabolism following treatment described in “a” presented as (b) raw values or (c) normalized to nuclei content, which did not differ between groups (see Figure 3). (d) Effect of GW at 1 μM for 24 hours on mitochondrial staining both without and with normalization to nuclei content, with representative images at right. (e) Nuclei-normalized basal and peak mitochondrial metabolism following treatment described in “c” normalized to mitochondrial staining described in “d”. (f) Relationship between peak mitochondrial function and content as analyzed above presented as peak mitochondrial function normalized to nuclei content versus mitochondrial staining normalization to nuclei content. (g and h) Effect of GW at 1 μM for 24 hours on mitochondrial proton leak (uncoupling), ATP production, mitochondrial spare respiratory capacity, and non-mitochondrial (non-mt) respiration, both without and with normalization to DAPI, respectively. Notes: * indicates between groups. States of mitochondrial metabolism, as well as mitochondrial staining, were analyzed using student’s t-test. States of mitochondrial metabolism were calculated by subtracting non-mitochondrial respiration from basal and FCCP-induced peak mitochondrial oxygen consumption (OCR). Time course of mitochondrial function was analyzed using two-way ANOVA with Bonferroni’s correction for multiple comparisons. Metabolic analyses were performed using individual replicates per treatment condition and repeated across two independent experiments with per group in the final analyses. Mitochondrial staining was performed using individual replicates per treatment condition and was repeated across two independent experiments with per group in the final analyses using the average of three measurements per experiment. Images in “d” of representative individual myotubes were taken using the 20× objective with red line indicating 150 μm. Relationship between peak mitochondrial metabolism and mitochondrial content was analyzed using linear regression. Metabolic calculations were performed as follows: Basal Respiration = Measurement #3 − Measurement #12; Peak Respiration = Measurement #7 − Measurement #12; Proton Leak = Measurement #average of 4–6 − Measurement #12; Spare Respiratory Capacity = Measurement #7 − Measurement #3; ATP-Linked Respiration = Measurement #3 − Measurement #4–6; Non-Mitochondrial Respiration = Measurement #12. Other Abbreviations: Oligo: oligomycin; FCCP: carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone; and Rot: rotenone.

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