Direct Relationship Between Metabolic Flexibility Measured During Glucose Clamp and Prolonged Fast in Men

Figures

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  Figure A1. (1) After acute t-BHP treatment (50 µM) for 30 min, and (2) 24 h after acute t-BHP treatment.

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  Figure A2. IP3R flexibility index (Iflex) in (1) human epidermal keratinocytes in basal conditions and following treatment with 0.05% orchid extract (Gastrodia elata) (passage 5); (2) young human dermal fibroblasts (passage 6, P6) and replicatively senescent human dermal fibroblasts (passage 31, P31). Statistical difference was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test, and is presented with *: p < 0.05, ** p < 0.01, ***: p < 0.001 and ****: p < 0.0001. (3) Effect of palmitate-induced lipotoxicity for 3 h on the VDAC1/IP3R flexibility index (Iflex) in primary human fibroblasts (passage 6), showing that 500 µM and 800 µM palmitate for 3 h remain lipotoxic 24 h after returning to a control medium. Statistical difference was analyzed using the Kruskal–Wallis test, and is presented with *: p < 0.05, and ****: p < 0.0001. Measurements were performed on a single set, with 6 technical replicates.

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  Figure 1. Effects of replicative senescence on (A) the proteasome activity assessed through the rate of hydrolysis of succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methylcoumarin (LLVY-AMC), (B) gene expression of p21, (C) the number of VDAC1/IP3R complexes per nucleus quantified using in situ proximity ligation assay, and (D) the cellular level of reactive oxygen species (ROS) assessed following CellROXTM staining (n = 3 to 4 series). Exploration of the effects of replicative senescence on (E) the generalized polarization (GP) index of membrane fluidity, (F) the global elastic modulus of the cell cytoskeleton resistance, and (G) the elastic modulus of the cytoskeleton resistance in the vicinity of the VDAC1/IP3R complexes. Panels (E–G) show data obtained from a single experimental set. Results are presented for young human dermal fibroblasts (HDFs, P5 or P6) and HDFs aged through replicative senescence (P30 or P31) from two patients (HFDs1 and HFDs2). (H) Representative images of in situ proximity ligation assay between VDAC1 and IP3R (red dots). (I) Representative images of in-CellROXTM staining (original magnification ×63 and scale bar = 20 µm). Statistical differences were analyzed using…

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  Figure 2. (A) Dose–response analysis of VDAC1/IP3R complexes quantified using in situ proximity ligation assays, after acute 10 and 30 min exposure to tert-butyl hydroperoxide (t-BHP) in human dermal fibroblasts (HDFs, passage 6). (B) Persistence of VDAC1/IP3R complex modulation at 24 and 48 h after a single acute 30 min exposure to t-BHP (50 µM) in HDFs (passage 6) (n = 2 series). Validation study: HDFs (passage 6 or 7) were exposed to t-BHP (50 µM) for 30 min. (C) VDAC1–IP3R complex formation (n = 6 series) and (D) intra-mitochondrial calcium levels assessed by Rhod-2 AM labelling (n = 3 series) were measured immediately after exposure (30 min) and 24 h after the acute oxidative stress. (E) Representative images of in situ proximity ligation assay between VDAC1 and IP3R (red dots) (original magnification ×63 and scale bar = 20 µm). (F) Representative images of Rhod2 AM staining (original magnification ×63 and scale bar = 20 µm). Statistical differences were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. They are presented with, *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

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  Figure 3. Correlations between the number of VDAC1/IP3R complexes per nucleus and (A) the generalized polarization (GP) index of membrane fluidity, (B) the global elastic modulus of the cell cytoskeleton resistance, and (C) the elastic modulus of the cytoskeleton resistance in the vicinity of the VDAC1/IP3R complexes. Measurements were performed on a single experimental set after 24 h in untreated and treated (50 µM t-BHP for 30 min with or without pre- and post-treatment with orchid extract) HDFs (passage 6). Correlations were explored by simple linear regression and centred second-order polynomial (quadratic) regression.

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  Figure 4. Effects of acute oxidative stress on (A) the VDAC1/IP3R flexibility index (Iflex) and (B) the intra-mitochondrial calcium content assessed following Rhod2 AM staining, in human dermal fibroblasts (HDFs, passage 6 or 7) (n = 3 series). (C) Representative images of Rhod2 AM staining (original magnification ×63 and scale bar = 20 µm). The HFDs were treated with 0.05% orchid extract (Gastrodia elata) for 40 h, 16 h before and 24 h after t-BHP treatment (50 µM, 30 min). Statistical difference was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. It is presented with *: p < 0.05, ***: p < 0.001, and ****: p < 0.0001. (D) Linear regression between Iflex and BHI measured at 24 h (BHI 24 h) in HDFs (passage 6 or 7). (E) Matrix of correlation (Pearson r) between Iflex, the number of VDAC1/IP3R complexes measured at 30 min (VDAC1/IP3R 30 min) and 24 h (VDAC1/IP3R 24 h), BHI measured at 24 h (BHI 24 h), and mitochondrial respiration parameters measured at 24 h in HFDs (passage 6 or 7).