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. 2023 Jun 19;13(6):766. doi: 10.3390/metabo13060766

Figure 2.

Figure 2

Effect of JPH both with and without insulin resistance on mitochondrial biogenic signaling, content, and function. (a) Time course of the effect of JPH-203 (JPH) at 1 µM for 24 h with and without insulin resistance (IR) on mitochondrial metabolism. (b,c) Effect of JPH with and without IR on basal (left) and peak (right) mitochondrial metabolism both without (b) and with (c) normalization to cell nuclei content (presented in Figure S3). (d) Mitochondrial content of cells described in “a” indicated by NAO staining without (left) and with normalization to cell nuclei content (presented in Figure S3). (e) Effect of JPH with and without IR on mRNA expression of mitochondrial biogenesis including peroxisome proliferator-activated receptor-alpha (Ppara), peroxisome proliferator-activated receptor-delta (Ppard), peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc1a), nuclear respiratory factor 1 (Nrf1), and mitochondrial transcription factor A (Tfam). (f) Effect of JPH with and without IR on mRNA expression of mitochondrial content including citrate synthase (Cs), cytochrome c oxidase 1 (Cox1), NADH dehydrogenase 1 (Nd1), NADH dehydrogenase 5 (Nd5), cytochrome b (Cytb), cytochrome c oxidase subunit 5a (Cox5a), and ATP synthase subunit 5b (Atp5b). (g) Effect of JPH with and without IR on mRNA expression of mitochondrial dynamics including mitofusin 1 (Mtfn1), mitofusin 2 (Mfn2), mitochondrial fission protein 1 (Fis1), and Dynamin-related protein 1 (Drp1). Notes: Two-way ANOVA and one-way ANOVA with Bonferroni’s correction for multiple comparisons were used to assess differences in metabolism, mitochondrial content, and gene expression following 24 h treatment. Dissimilar letters within each graph and above each group indicate p ≤ 0.05 between groups, while J, IR, and I represent main and interaction effects for two-way ANOVA (with NS indicating no significant effect). States of mitochondrial metabolism were calculated by subtracting non-mitochondrial respiration from basal or FCCP-induced peak oxygen consumption. Metabolic measurements were performed using n = 23 individual replicates per treatment condition and were repeated across 2 independent experiments with n = 46 per group in the final analyses. No wells responded with negative raw values. Mitochondrial staining was performed using n = 23 individual replicates per treatment condition and were repeated across 2 independent experiments with n = 46 per group in the final analyses using the average of 3 measurements per experiment (less background). Images in “d” of representative individual myotubes were taken using the 10× objective. Target gene expression was normalized to average TATA-binding protein (Tbp) using 3 replicates per group across 2 independent experiments with n = 6 for the final analysis.