Mitochondria Bioenergetic Functions and Cell Metabolism Are Modulated by the Bergamot Polyphenolic Fraction

Abstract

The bergamot polyphenolic fraction (BPF) was evaluated in the F₁F_O-ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F₁F_O-ATPase, dependent on the natural cofactor Mg²⁺, increased by 15%, whereas the enzyme activity in the presence of Ca²⁺ was inhibited by 10%. By considering this opposite BPF effect, the F₁F_O-ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters.

Keywords: F1FO-ATPase; bergamot polyphenolic fraction; cell metabolism; mitochondria; mitochondrial permeability transition pore; porcine aortic endothelial cells.

Figure 1

Dose–response curve of the BPF on F₁F_O-ATPase activity. F₁F_O-ATPase activated by Mg²⁺ (Mg²⁺-activated F₁F_O-ATPase) (●) and by Ca²⁺ (Ca²⁺-activated F₁F_O-ATPase) (○) activities in the presence of increasing BPF concentrations are expressed as percentages of the total mitochondrial F-ATPase activity sustained by Mg²⁺ or Ca²⁺, respectively. Data represent the mean ± SD from three independent experiments carried out on different mitochondrial preparations. * indicates significant differences with respect to the control (p  ≤  0.05).

Figure 2

BPF effects on selected oxidative phosphorylation parameters: state 3 and 4 respiration rates and their ratio as the RCR. Glutamate/malate- (A) and succinate- (B) stimulated mitochondrial respiration in the presence of 50 µg/mL (red) (█), 100 µg/mL (yellow) (█), and in the absence (green) (█) of the BPF, respectively. Data expressed as columns represent the mean ± SD from three independent experiments carried out on different mitochondrial preparations. Different letters indicate significant differences (p ≤ 0.05) among treatments within the same parameter (state 3; state 4, and state 3/state 4).

Figure 3

Evaluation of mPTP opening. Representative curves of at least three different experiments on the calcium retention capacity (CRC). The CRC was monitored in response to subsequent 10 μM CaCl₂ pulses (shown by the triangles), as detailed in Section 2.6, in the absence (control—black line) (−) and in the presence of the inhibitors 2 mM MgADP (red line) (−), 50 µg/mL BPF (green line) (−), and 100 µg/mL BPF (blue line) (−).

Figure 4

Effect of the BPF on the real-time ATP production rate in pAECs. (A) Evaluation of ATP production rate by mitochondrial OXPHOS (blue) (█) or by glycolysis (red) (█) in BPF-treated cells. (B) The ATP rate index, calculated as the ratio between the mitochondrial ATP production rate and the glycolytic ATP production rate, is shown on the y-axis (logarithmic scale) in pEACs treated without (control) and with the BPF. Data expressed as column charts ((A) plots) and points ((B) plots) represent the mean ± SD (vertical bars) from three experiments carried out on distinct cell preparations. Different lower-case letters indicate significantly different values (p ≤ 0.05) among BPF treatments (0, 50, 100, and 150 μg/mL) in the same metabolic pathway (OXPHOS or glycolysis) (A) and ratio value (B); different upper-case letters indicate different values (p ≤ 0.05) among treatments in ATP production rates due to sum of OXPHOS plus glycolysis (A).

Figure 5

Effect of the BPF on the cell metabolism of pAECs. (A) The mitochondrial respiration profile was obtained from the oxygen consumption rate (OCR) without (●, blue) and with 100 μg/mL BPF (■, orange) under basal respiration conditions and after the addition of 1.5 μM oligomycin (olig), 1.0 μM FCCP, and a mixture of 0.5 μM rotenone plus antimycin A (Rot + AA). Inhibitor injections are shown as dotted lines. (B) Mitochondrial parameters (basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and ATP turnover) in the absence (█, blue) or in the presence of 100 μg/mL BPF (█, orange). (C) The glycolysis profile was obtained from the extracellular acidification rate (ECAR) without (●, blue) and with 100 μg/mL BPF (■, orange) under basal glycolysis conditions and after the addition of 10 mM glucose (port A), 1 μM oligomycin (port B), and 50 mM 2-deoxyglucose (2DG). Compound injections are shown as dotted lines. (D) Glycolytic parameters (glycolysis, glycolytic capacity, and glycolytic reserve) in the absence (█, blue) or in the presence of 100 μg/mL BPF (█, orange). Data expressed as points (A,C) and column charts (B,D) represent the mean ± SD (vertical bars) from three experiments carried out on different cell preparations. * indicates significant differences (p ≤ 0.05) among treatments within the same parameter.

Figure 6

BPF effect on pAECs’ viability. Cells were treated with different doses of BPF for 5 h. * p < 0.05, one-way ANOVA, and post hoc Tukey’s test between each treatment vs. control (CTR) group.