Figure 2
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Perturbations in Skm OXPHOS alter lipid metabolism and storage
Data for wild‐type (wt, black bars and traces) and LowOXPHOS (orange bars and traces) mice are shown.
A Representative images of mice and body weight graph following the expression of mitochondrial ATPIF1H49K (days of doxycycline) (wt, n = 12; LowOXPHOS, n = 12).
B Representative images of hindlimb muscles. Arrows point to the soleus.
C, D Transversal slices of red fibers from soleus stained with hematoxylin/eosin (C). Higher im‐WAT infiltrations in LowOXPHOS mice are shown. Quantifications in (D) (wt, n = 6; LowOXPHOS, n = 6; 10 fields/mouse).
E, F iTRAQ ratio (E) and representative WB expression (F) of Skm proteins from de novo lipid synthesis. The expression of ATP citrate lyase (ACLY), acetyl‐CoA carboxylase (ACAC), fatty acid synthase (FASN) and their phosphorylation (p) are shown. Two samples per condition; each sample contains protein extracts from 3 mice. GAPDH is shown as a loading control. Quantifications in lateral histograms (wt, n = 6; LowOXPHOS, n = 6). In (E), a higher intensity of red color represents a higher LowOXPHOS/wt expression ratio.
G Immunocapture (IP) of Skm acetylated proteins blotted with anti‐ACLY antibody.
H Skm acetyl‐CoA amounts (wt, n = 8; LowOXPHOS, n = 8).
I Schematic representation of de novo FFA synthesis and the increase in LD. Elevated acetyl‐CoA levels may be derived from dysregulation of the FFA β‐oxidation, glycolysis, or BCAA metabolism.
J Myocyte rates of aerobic glycolysis to lactate production. Bars are the mean ± SEM of n = 3 experiments, 9 replicas/condition.
K, L 14C(u)‐glucose uptake (K) and oxidation to CO2 (L) in myocytes expressing or not the ATP synthase inhibitor ATPIF1H49K. Bars are the mean ± SEM of n = 3 experiments, 6 replicas/condition.
M Representative WB expression of Skm PDH and its phosphorylation. Two samples per condition; each sample contains extracts from 3 mice. Quantification in histograms (wt, n = 6; LowOXPHOS, n = 6).
N Skm levels of lactate (wt, n = 6; LowOXPHOS, n = 6).
O FFA β‐oxidation in primary cultures of myocytes derived from wt or LowOXPHOS mouse hindlimbs. Bars are the mean ± SEM of n = 3 experiments, 9 replicas/condition.
P FFA amounts in Skm extracts (wt, n = 8; LowOXPHOS, n = 8).
Q, R Transversal slices of soleus stained with hematoxylin/eosin (Q). Arrows indicate LDs in LowOXPHOS mice. Oil Red O staining and enzymatic activity of respiratory CI in transverse contiguous slices (R). Images are representative of n = 4 mice/genotype, 10 images/mouse. Note the colocalization of oxidative fibers with LDs (R).
S Electron microscopy of transversal slices of the soleus. Intrafiber LDs surrounded by mitochondria in LowOXPHOS mice are shown. Histogram shows the quantifications of n = 4 animals/genotype, 10 images/animal.
T LD formation upon ATP synthase inhibition (ATPIF1H49K) in starved myocytes after 24 h of palmitate supplementation in the presence or absence of BCAA. Blue: DAPI, nuclei; green: BODIPY‐positive LDs. Histograms show the quantification expressed as the number of LDs/nuclei. Bars are the mean ± SEM of n = 3 experiments, 12 fields/condition. See Fig EV3E.
U LD formation upon ATP synthase inhibition (5 μM oligomycin) in myocytes after 24 h of palmitate supplementation. Histograms show the quantification expressed as fold of control of the BODIPY/DAPI fluorescence intensity. Seven fields/condition. See Fig EV3F.
Data information: Bars are the mean ± SEM of the indicated (n) mice/genotype *P < 0.05 when compared to wt by ANOVA and Student's t‐test. See also Fig EV3.