Assessing the interaction of the UCP system and fatty acids on epicardial adipose tissue mitochondrial respiration

Figures

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  FIGURE 1. Schematic representation of the respirometry traces following SUIT, for Protocol 1: NADH‐linked OXPHOS capacity (A) and for Protocol 2: FAO‐linked OXPHOS capacity (B), applied to permeabilized subcutaneous and epicardial adipose tissues. Blue line and left y axis: O2 concentration over time; red line and right y axis: Tissue mass specific flux O2. Respiratory States, by sequential order: L, non‐phosphorylation LEAK state (orange); P, OXPHOS state (green); E, uncoupled respiration state (blue) ROX, residual respiration state (grey); REN, residual endogenous substrates (lavender); LCI, LEAK state of complex I; PCI, complex I‐linked OXPHOS capacity; PCI+CII, complex I + II‐linked OXPHOS capacity; PFAO, fatty acid oxidation‐linked OXPHOS capacity; PFAO+CI, fatty acid oxidation + complex I‐linked OXPHOS capacity; PFAO+CI+CII, fatty acid oxidation +complex I+ II‐linked OXPHOS capacity; Substrates by sequential order: P, pyruvate; M, Malate; G, Glutamate; ADP, Adenosine diphosphate; Omy, Oligomycin; FCCP, Carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone; AA, Antimycin A; Mal.1, Malate; Oct, Octanoyl‐carnitine; CCP, Carbonyl cyanide phenylhydrazone.

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  FIGURE 2. Significant alteration in mitochondrial complex expression and increased UCP1 mRNA levels in EAT compared to SAT. The protein levels of mitochondrial complex I, subunit NDUFB8 (A and B), complex II, subunit SDHB (A and C), complex III, subunit UQCRC2 (A and D), complex IV, subunit MTCO1 (A and E) and complex V, subunit ATP5A (A and F) were analyzed by Western blot. The mRNA expression of mitochondrial complex V, subunit ATP5C1 (G) and UCP1 (H) was analyzed by qRT‐PCR. The number of patients is indicated for each assay. Gene expression was quantified by RT‐qPCR, and protein expression by Western blot, with normalization to the housekeeping gene or protein β‐Actin, respectively. The number of patients included in each assay is indicated. A p ≤ .05 was considered statistically different, *p ≤ .05; **p ≤ .01; ***p ≤ .001; ****p ≤ .0001. EAT, epicardial adipose tissue; UCP1, uncoupling protein 1; SAT, subcutaneous adipose tissue.

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  FIGURE 3. NADH‐linked OXPHOS in EAT versus SAT (A) and impact of the UCP1 inhibition by GDP (B). Oxygen fluxes are expressed per tissue mass. The number of patients included in each assay is indicated. A p ≤ .05 was considered statistically different, *p ≤ .05; **p ≤ .01; ***p ≤ .001; ****p ≤ .0001. EAT, epicardial adipose tissue; GDP, guanosine 5′‐diphosphate; UCP1, uncoupling protein 1; SAT, subcutaneous adipose tissue; NADH, nicotinamide adenine dinucleotide (N‐linked pathway); L, non‐phosphorylation LEAK state; P, OXPHOS state; E, uncoupled respiration state; LCI, LEAK state of complex I; PCI, complex I‐linked OXPHOS capacity; PCI+CII, complex I + II‐linked OXPHOS capacity.

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  FIGURE 4. FAO‐linked OXPHOS in EAT versus SAT (A) and the impact of the UCP1 inhibition by GDP (B). Oxygen fluxes are expressed per tissue mass. The number of patients included in each assay is indicated. A p ≤ .05 was considered statistically different, *p ≤ .05; **p ≤ .01; ***p ≤ .001; ****p ≤ .0001. EAT, epicardial adipose tissue; GDP, guanosine 5′‐diphosphate; UCP1, uncoupling protein 1; SAT, subcutaneous adipose tissue; FAO, fatty acid oxidation (F‐linked pathway); E, uncoupled respiration state; REN, residual endogenous substrates; PFAO, fatty acid oxidation‐linked OXPHOS capacity; PFAO+CI, fatty acid oxidation + complex I‐linked OXPHOS capacity; PFAO+CI+CII, fatty acid oxidation + complex I + II‐linked OXPHOS capacity.

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  FIGURE 5. Mitochondrial complex‐coupling and flux control ratios in the NADH‐linked OXPHOS (left panel, A–C) and impact of UCP1 inhibition by GDP (D–F, right panel, D–F). P/E, phosphorylation system control ratio (A and D), OXPHOS P‐L control efficiency, OXPHOS control efficiency (B and E), and N/NS, N‐linked pathway capacity over NS–pathway capacity (C and F). The number of patients included in each assay is indicated. A p ≤ .05 was considered statistically different, **p ≤ .01; ***p ≤ .001; ****p ≤ .0001. AT, epicardial adipose tissue; GDP, guanosine 5'‐diphosphate; UCP1, uncoupling protein 1; SAT, subcutaneous adipose tissue; FAO, fatty acid oxidation (F‐linked pathway); E, uncoupled respiration state; REN, residual endogenous substrates; PFAO, fatty acid oxidation‐linked OXPHOS capacity; PFAO+CI, fatty acid oxidation + complex I‐linked OXPHOS capacity; PFAO+CI+CII, fatty acid oxidation + complex I + II‐linked OXPHOS capacity.

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  FIGURE 6. NADH‐Linked OXPHOS in EAT with and without GDP. Overall impact of the UCP1 inhibition by GDP (A), and in the EAT from patients in the NDM (B), DM (C), NCAD (D) and CAD (E) groups. Oxygen fluxes are expressed per tissue mass. The number of patients included in each assay is indicated. A p ≤ .05 was considered statistically different, *p ≤ .05. EAT, epicardial adipose tissue; CAD, coronary artery disease group; DM, diabetes mellitus group; NCAD, non‐coronary artery disease group; NDM, non‐diabetes mellitus group. GDP, guanosine 5′‐diphosphate; NADH, nicotinamide adenine dinucleotide (N‐linked pathway); L, non‐phosphorylation LEAK state; P, OXPHOS state; E, uncoupled respiration state; LCI, LEAK state of complex I; PCI, complex I‐linked OXPHOS capacity; PCI+CII, complex I + II‐linked OXPHOS capacity.

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  FIGURE 7. NADH‐Linked OXPHOS in EAT with and without GDP. Overall impact of the UCP1 inhibition by GDP (A), and in the EAT from patients in the NDM (B), DM (C), NCAD (D) and CAD (E) groups. Oxygen fluxes are expressed per tissue mass. The number of patients included in each assay is indicated. A p ≤ .05 was considered statistically different, *p ≤ .05. EAT, epicardial adipose tissue; CAD, coronary artery disease group; DM, diabetes mellitus group; NCAD, non‐coronary artery disease group; NDM, non‐diabetes mellitus group; FAO, fatty acid oxidation (F‐linked pathway); E, uncoupled respiration state; REN, residual endogenous substrates; PFAO, fatty acid oxidation‐linked OXPHOS capacity; PFAO+CI, fatty acid oxidation + complex I‐linked OXPHOS capacity; PFAO+CI+CII, fatty acid oxidation + complex I + II‐linked OXPHOS capacity.