Stilbenes and resveratrol metabolites improve mitochondrial fatty acid oxidation defects in human fibroblasts

CPT2-deficient, VLCAD-deficient, or control human skin fibroblasts used in this study have been described previously [14, 25]. Mutations and genotypes are given in Table 1. Under standard conditions, fibroblasts were grown in complete Ham’s F10 medium (Invitrogen) with glutamine, 12% fetal calf serum (FCS), 100 U/ml penicillin, and 0,1 mg/ml streptomycin. In some experiments the FCS was reduced to 3%. This low serum value was chosen because it allowed normal fibroblasts growth, in contrast to serum-free media that results in blocking cell proliferation. For treatment, the medium was removed and replaced with fresh medium containing the various compounds to be tested. These included RSV (Cayman Chemical company, USA), or one of the following compounds: cis-RSV (Cayman Chemical company, USA), piceid (Art Molecule, France), RSV-3-O-D-glucuronide (Art Molecule, France), RSV-4-O-D-glucuronide (Bertin Pharma, France), RSV-3-O-sulfate (Art Molecule, France), dihydroRSV (Art Molecule, France). Stock solutions of these compounds were prepared in DMSO and protected from light, and controls received equivalent amounts of DMSO (≤0.05%).

Fatty acid oxidation (FAO) was measured by the production of ³H₂O from (9,10-³H) palmitate, as described previously [25]. Briefly, after removal of cell culture medium, fibroblasts seeded in 24-well plates were incubated in 200 μl/well of PBS buffer containing 125 μM tritiated palmitate + 1 mM carnitine, for 2 hours at 37°C. After incubation, the mixture was removed and de-proteinized, ³H₂O was collected on ion-exchange resin, and the eluate was counted by liquid scintillation. The FAO values were expressed relative to protein content determined by the Lowry method.

The method used has been described previously [26]. Briefly, fibroblasts were cultured in complete Ham’s F10 medium containing 200 μM palmitate and 400 μM carnitine for 72 hours at 37°C. The culture medium was collected, extracted, and analyzed for acylcarnitine content by electrospray MS-MS, using an API3000 triple quadrupole mass spectrometer (Sciex, Applied Biosystems, USA) detecting the precursors of an m/z ratio of 85, by reference to added internal deuterated standards. C16:0 acylcarnitines were quantified by reference to standard curves.

Fibroblasts (0.7 × 10⁵) were seeded on 24-well plates in Ham’s F10 medium containing 12% FBS 24 h prior to treatment with [³H]RSV (specific activity: 74 GBq/mmol, Ge Health Care Life Science, Velizy-Villacoublay, France) at various concentrations and for several time points at 37°C. At the end of the incubation periods, the labeled medium was removed, and the cells were washed with PBS and lysed with NaOH 1 M. Cell-associated radioactivity was counted in a liquid scintillation analyzer (Perkin Elmer, Life Sciences Inc., Boston, MA). In parallel, cells treated with unlabeled RSV for the same kinetic time points, were used to measure protein content by the Lowry method.

For efflux experiments, cells were loaded with radiolabeled RSV (75 μM) for 1 h at 37°C (early maximal incorporation peak of RSV). After incubation, the labeled medium was removed and replaced by standard culture medium. Following this, the amount of RSV remaining in the cells was determined at various time points by counting cell-associated radioactivity.

For analysis of RSV metabolism, 10⁶ fibroblasts were treated by 75 μM RSV for 1 h and the medium was replaced by standard RSV-free medium. The presence of RSV aglycone and RSV metabolites was then analyzed in media and cell pellets at 0, 6 h, or 48 h after providing RSV-free medium, by LC-MS, (Agilent Technologies), as previously described [27].

Western blots were performed as described previously [26]. The following antibodies were used: anti VLCAD (kindly provided by Dr S. Yamagushi, Japan), anti CPT2 (kindly provided by Dr C. Prip-Buus, France), anti porin (Abcam, Cambridge, UK), and anti ß-actin (Chemicon International, Temecula, USA). The bands were scanned by computerized densitometry (NIH Image J) and the results were expressed as arbitrary units, normalized to the amount of ß-actin.

Data are the means ± SEM. Differences between groups were analyzed by one-way analysis of variance (ANOVA) and the Fisher test, or by the paired t test. P < 0.05 was considered significant.

As shown in Figure 1A, fibroblasts from patients with CPT2 or VLCAD deficiency typically exhibited markedly reduced palmitate oxidation capacities (from 1.3 to 3.3 nmol/h/mg), compared to control fibroblasts (5.2 ± 0.2 nmol/h/mg). Under standard culture conditions, treatment of fibroblasts by 75 μM RSV for two days led to maximal increases in palmitate oxidation in control (+45%) and patient cells (from +83 to +252%). This resulted in the restoration of normal FAO rates in all the CPT2- or VLCAD-deficient treated cells. The beneficial effect of RSV on FAO was further investigated by analysis of acylcarnitine profiles. As seen in Figure 1B, abnormal accumulation of long-chain acylcarnitines was observed in untreated CPT2 or VLCAD deficient fibroblasts, and a 3-day treatment with 75 μM RSV normalized the C16:0 acylcarnitine production.

Under standard culture conditions, RSV (75 μM) was rapidly incorporated into control, CPT2- and VLCAD-deficient fibroblasts at 37°C, and its uptake was only discretely modified by low temperature (4°C; data not shown). In short-term experiments, uptake values in control cells reached about 90% of their maximal value at 12 minutes and remained stable up to 120 minutes, around 7000 pmol/mg proteins (Figure 2A). Interestingly, similar uptake levels were still observed after a 48 or 72 h exposure to RSV (Figure 2B). Patient 1 and patient 3 uptake values were in the range of 3000 to 6000 pmol/mg proteins between 12 and 120 minutes. In long-term experiments, values of RSV uptake in patient 1 generally remained lower than in control fibroblasts, In contrast, RSV accumulation in patient 3 fibroblasts transiently rose between 6 and 24 hours, and went back to control values at 48 and 72 hours. Thus, CPT2-deficient cells tended to incorporate less RSV than control or VLCAD-deficient cells (Figure 2B). However, RSV incorporation in long-term experiments remained at high levels in the three fibroblasts cell lines, representing more than 50% of their maximal uptake value (Figure 2B), and indicating a steady-state between RSV influx and efflux rates.

After exposure to 75 μM RSV for one hour, measurements of efflux showed that RSV was rapidly released in the extracellular medium, and 60% to 80% of intracellular RSV content was lost after 2 h (Figure 2C). In parallel, no detectable production of RSV glucuro- conjugates, and a very low level of RSV-3-O-sulfate, were measured (Table 2 and Table 3).

As shown in Figure 3, increasing fetal calf serum (FCS) content from 0.5 to 12% in the culture medium resulted in a marked decrease of RSV uptake values, both in control and in patient fibroblasts. This clearly suggests that some FCS components might reduce RSV bioavailability in the cell culture system.

We then compared the responses of control or patient cells to a 48 h treatment with 20 or 75 μM RSV in culture medium containing either 12% or 3% FCS. In the absence of RSV, western-blot experiments (Figure 4A and B) revealed a marked CPT2 or VLCAD protein deficiency in patient 1 and patient 4, respectively compared to control fibroblasts. Treatment with 75 μM RSV in 12% FCS medium resulted in a significant induction of CPT2 (Figure 4A) and VLCAD (Figure 4B) proteins in control and in patients’ cells. By contrast, treatment with 20 μM RSV in 12% FCS induced either modest increases or no changes in CPT2 (Figure 4A) and VLCAD (Figure 4B) proteins levels. Noticeably, for both RSV concentrations tested, the levels of CPT2 and VLCAD proteins in control and patient fibroblasts were significantly augmented when treatment was performed in 3% FCS medium.We then measured FAO capacities in control, and in patient fibroblasts exposed to 10, 20, 37.5, or 75 μM RSV in culture medium containing 12% or 3% FCS (Figure 5). Control cells treated in 12% FCS exhibited modest increases in FAO unless RSV reached 37.5 μM, when half-maximal FAO stimulation was observed (p < 0.01) (Figure 5A). When control fibroblasts were treated in 3% FCS medium, there was an increase in the slope of FAO dose–response curve, indicating an amplified response to RSV. Vehicle-treated patient cells exhibited more pronounced FAO deficiencies when grown in 3% FCS compared to 12% FCS medium (Figure 5B and C). After treatment with RSV in 3% FCS medium, patient cells exhibited a shift of dose–response curves towards higher values, compared to those observed in 12% FCS medium. Noticeably, in patient cells grown in 3% FCS medium, robust responses were already observed at 10 μM RSV, which triggered a +228% and +115% increase in FAO in VLCAD and CPT2-deficient fibroblasts, respectively, whereas, no response was observed in 12% FCS.We then compared incorporation levels of 20 μM RSV in 12% and 3% FCS medium (Figure 6A and B). Between 0 and 2 hours, control and patient cells accumulated larger amounts of RSV when treated in culture medium containing 3% (about 1000 pmoles/mg proteins), compared to 12% (about 750 pmol/mg proteins) FCS. At 24 and 48 hours, cellular uptake values reached 3000–4000 pmol/mg proteins in 3% FCS, i.e. more than twofold those observed in 12% FCS.

We evaluated the effects of compounds structurally related to RSV, and of RSV metabolites. Two of them are stilbene compounds naturally found in association with RSV in foods, namely cis-RSV and trans-piceid [28]. The four others are major human metabolites i.e. RSV-3-O- and −4-O-glucuronide, RSV-3-O-sulfate, and dihydro-RSV. Cells were treated with 75 μM of each compound in 12% FCS medium for 48 h. Initial screening was performed in one control, one CPT2-deficient and one VLCAD-deficient cell line (Figure 7A). In control fibroblasts, exposure to cis-RSV or to piceid induced similar changes (+25%) in FAO. Treatment with these compounds resulted in a more pronounced increase in FAO in CPT2- (+121 to +125%, respectively) and in VLCAD-deficient fibroblasts (+30 to +50%, respectively). Treatment with each of four RSV metabolites tested generally resulted in modest changes (<15%) in FAO capacities in control fibroblasts. In patient cells, exposure to 3- or 4-glucuro-RSV led to moderate or no increase in FAO. In contrast, treatment with dihydro-RSV augmented FAO by 130 or 52% in CPT2 or VLCAD-deficient fibroblasts, respectively. The effects of these compounds were further analyzed in additional control, and deficient fibroblasts (Figure 7B). Altogether, these experiments established that exposure to cis-RSV, to piceid, or to dihydro-RSV triggered significant increases in FAO in control and patient cells. Consistent with this, cis-RSV, piceid, or dihydro-RSV induced a significant up-regulation of CPT2 (Figure 8A) and VLCAD (Figure 8B) proteins levels, accounting for the stimulatory effects of these compounds on FAO capacities.

The effects of RSV, cis-RSV, piceid and dihydro-RSV on mitochondrial biogenesis was assessed by western blot analysis of two mitochondrial proteins generally admitted to reflect mitochondrial mass. Porin is a voltage dependent anion selective channel located in the outer mitochondrial membrane and Tfam, is the mitochondrial transcription factor A, which co-localizes with mtDNA in the mitochondrial nucleoids [29]. Western-blot analysis revealed a general stimulation of porin and Tfam protein expression in response to RSV, cis-RSV, piceid and dihydro-RSV, i.e. to all the compounds that were found to induce increases of FAO capacities (Figure 9).