Animal Science Journal (2016) 87, 1371–1378
doi: 10.1111/asj.12559
ORIGINAL ARTICLE Oleuropein induces mitochondrial biogenesis and decreases reactive oxygen species generation in cultured avian muscle cells, possibly via an up-regulation of peroxisome proliferator-activated receptor γ coactivator-1α Motoi KIKUSATO, Hikaru MUROI, Yuichiro UWABE, Kyohei FURUKAWA and Masaaki TOYOMIZU Animal Nutrition, Life Sciences, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan
ABSTRACT It has been shown that oleuropein, a phenolic compound in the fruit and leaves of the olive tree (Olea europaea) induces mammalian uncoupling protein 1 (UCP1) expression via an increased secretion of noradrenaline and adrenaline. This study investigated the effects of oleuropein on avian UCP (avUCP) expression as well as genes related to mitochondrial oxidative phosphorylation and biogenesis in cultured avian muscle cells, together with reactive oxygen species generation. Oleuropein induced avUCP as well as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor-1 (NRF1), mitochondrial transcription factor A (TFAM) and ATP5a1 (a component of mitochondrial adenosine triphosphate synthase) gene expression and cytochrome c oxidase activity, indicating the induction of mitochondrial biogenesis. Sirtuin-1 (SIRT1) gene expression was also up-regulated by this compound, which could contribute to an increase in PGC-1α activity. Oleuropein suppressed the level of superoxide generation per mitochondrion, possibly via the up-regulation of avUCP and manganese superoxide dismutase (MnSOD) expression. Based on these findings, this study is the first to show that oleuropein may induce avUCP expression in avian muscle cells independent of the catecholamines, in which PGC-1α may be involved.
Key words: avUCP, mitochondrial superoxide, NRF1, SIRT1.
INTRODUCTION Oleuropein is a major phenolic compound in olive oil and leaves of the olive tree (Olea europaea) (Omar 2010). It has been reported that oleuropein has antioxidant activity (Briante et al. 2002; Kruk et al. 2005) as well as anti-obesity effects that take place through the up-regulation of uncoupling protein (UCP) expression (Oi-Kano et al. 2008). UCP is well known to promote energy expenditure by driving proton leak activity, which dissipates the mitochondrial membrane potential without adenosine triphosphate (ATP) synthesis. This activity also plays an important role in suppressing mitochondrial reactive oxygen species (ROS) generation (Keipert et al. 2010; Toime & Brand 2010; Basu Ball et al. 2011). Thus, UCP up-regulation may provide beneficial effects, not only in relation to energy substrate utilization, but also with respect to the cellular oxidative balance. Previous studies have shown that, in rats receiving oleuropein, the stimulation of UCP1 expression in brown adipose tissue was associated with increased noradrenaline and adrenaline secretion (Oi-Kano et al. 2008). Despite this finding, the precise mechanism underlying the induction of UCP1 expression in the presence of © 2016 Japanese Society of Animal Science
oleuropein is yet to be elucidated. It is well known that UCP expression is induced through the β-adrenergic system, wherein peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays an important role in inducing UCP expression. Moreover, this coactivator functions as a master regulator of mitochondrial biogenesis (Ventura-Clapier et al. 2008) as well as redox balance (St-Pierre et al. 2003; Lin et al. 2005; Scarpulla et al. 2012). We hypothesized that PGC-1α may be involved in the induction of UCP expression due to oleuropein supplementation, and also postulated that oleuropein may exert further beneficial effects on cellular metabolism if PGC-1α is up-regulated under these conditions. Regulation of UCP expression via the PGC-1α pathway has also been observed in birds receiving the β-adrenergic agonist isoproterenol (Joubert et al. 2011), or in birds exposed to a cold environment (Ueda et al. 2005). Correspondence: Motoi Kikusato, Animal Nutrition, Life Sciences, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Miyagi, Sendai 981-8555, Japan. (Email:
[email protected]) Received 8 June 2015; accepted for publication 26 August 2015.
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Moreover, we previously showed that expression of avian UCP (avUCP), which shares a 71–73% amino acid identity with mammalian UCP2 and UCP3 (Raimbault et al. 2001; Toyomizu et al. 2002), was up-regulated in the skeletal muscles of birds fed a diet containing olive oil (Mujahid et al. 2009). Based on these observations, we evaluated here the effects of oleuropein on mitochondrial oxidative metabolism in cultured avian muscle cells, and found that avUCP expression was up-regulated in oleuropein-exposed cells independently of the action of catecholamines; this effect was possibly mediated by the up-regulation of PGC-1α expression. In addition, we also found that oleuropein induced mitochondrial biogenesis and decreased the level of superoxide generation by mitochondria in avian muscle cells.
MATERIALS AND METHODS Ethics statement The Animal Care and Use Committee of the Graduate School of Agricultural Science, Tohoku University, approved all procedures, and every effort was made to minimize pain or discomfort to the animals.
Primary culture of avian skeletal muscle cells Five 0-day-old male chicks (Ross strain, Gallus gallus domesticus) were obtained from a commercial hatchery (Matsumoto-Keien, Zao, Miyagi, Japan). The chicks were killed by decapitation, and skeletal muscle cells were isolated from the Superficial pectoralis muscles of the chicks as previously described (Furukawa et al. 2015; Kikusato et al. 2015). The muscles were dissected and digested with a mixture of collagenase (1 mg/mL) and dispase (1000 U/mL) for 20 min at 37 °C. Cells were collected by centrifugation, washed and resuspended in basal medium (80% Dulbecco’s modified Eagle’s medium (DMEM), 20% M199) supplemented with 10% fetal bovine serum (FBS), 1.5 × 105 U/L penicillin and 0.15 g/L streptomycin. The cell suspension was transferred to ϕ90 mm-dishes to allow fibroblast attachment, and unattached cells were then collected. Thereafter, the cells were seeded onto collagen Type I-coated φ35 mm-dishes or 24-well microplates at a density of 4.5 × 104 cells/cm2 in each case, and were incubated for 48 h at 37 °C under 95% air/5% CO2 until the cells reached sub-confluence. Oleuropein (12247, Sigma-Aldrich, St Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) and this was added to the culture medium at 0.1% volume (v/v), following which the cells were incubated for 1-4 h and then used for subsequent experiments. Oleuropein non-treated (0 μmol/L) cells were incubated with the medium containing 0.1% (v/v) DMSO. © 2016 Japanese Society of Animal Science
Quantification of messenger RNA (mRNA) expression using real-time reverse transcription polymerase chain reaction (RT-PCR) The effects of oleuropein on the mRNA levels of avUCP and PGC-1α as well as the transcription factors (peroxisome proliferator-activated receptor (PPAR)α, PPARβ/δ), mitochondrial biogenesis-related factors (nuclear respiratory factor (NRF) and mitochondrial transcription factor A (TFAM)), sirtuin-1 (SIRT1), manganese superoxide dismutase (MnSOD) and ATP5a1 (a component of mitochondrial ATP synthase) were determined by realtime RT-PCR analysis using a CFX Connect™ system (Bio-Rad Laboratories, Hercules, CA, USA). Isolation of tissue RNA and synthesis of complementary DNA (cDNA) were conducted as previously described (Kikusato et al. 2015). Reverse transcription was carried out in the presence and absence of DNA polymerase to check for the unintended amplification of genes from DNA retained in the isolation process. Primer sets used to amplify each gene are listed in Table 1. To correct for differences in the amounts of template cDNA used, results are presented as ratios of the target mRNA to 18S ribosomal RNA (18S) levels. All data are shown as fold changes relative to control values at each incubation time.
Western blot analysis avUCP protein content was quantified as previously described (Toyomizu et al. 2011), with minor modifications. The cells were collected and solubilized with radioimmunoprecipitation assay (RIPA) buffer, and then incubated in boiling water for 5 min. The samples were loaded onto 12% polyacrylamide gels (MiniPROTEAN® TGX™ Gels, Bio-Rad, Hercules, CA, USA) and electrophoresis was carried out. Thereafter, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (0.2 μm) using a semi-dry transfer apparatus (Trans-Blot® Turbo™ Bio-Rad) according to the manufacturer’s instructions. The membranes were treated with Tris-buffered saline (TBS) blocking buffer containing 5% (w/v) skimmed milk and 0.1% (v/v) Tween-20 for 1 h at room temperature. Membranes were incubated overnight at 4 °C with a polyclonal recognizing avUCP (see (Toyomizu et al. 2011) for detailed information) at a dilution of 1:500. After rinsing with 1 × TBS buffer containing 0.1% (v/v) Tween-20 for 30 min (three times), membranes were incubated in blocking buffer at room temperature for 1 h with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (7074S, Cell Signaling Technology, Beverly, MA, USA) at a dilution of 1:1000. After washing with 1 × TBS buffer with 0.1% (v/v) Tween-20 for 30 min (three times), the membranes were incubated with chemiluminescent substrate solution (Chemi-Lumi One Super, Nacalai Tesque Inc., Kyoto, Japan) for 1 min. Immuno-reactive avUCP protein on the membranes Animal Science Journal (2016) 87, 1371–1378
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Table 1
Primer sequences
Gene avUCP PGC-1α PPARα† PPARβ/δ† NRF1 TFAM† ATP5a1 SIRT1 MnSOD 18S
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Primer set Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
ACT CTg TgA AgC AgC TCT ACA CC ATg TAC CgC gTC TTC ACC ACA TC ATC AgA ACA AgC CCT gTg gT gAC TCA ggT gTC AAT ggA AgT g CAA ACC AAC CAT CCT gAC gAT ggA ggT CAg CCA TTT TTT ggA CAT ggA gCC CAA gTT TgA gT Cgg Agg ATg TTg TCT Tgg AT CAT ggC ACT TAA CAg TgA AgC Ag gTg ACC ACg GTC TGG TAC AT AgC Agg TTT ACg Agg AAg CA TTg AAg CCA CTT CgA ggT CT CTT ggT gCC gCA TTT gTT gCT gAT ACC TCA gCA gTg CCA gTT TT gAT CAg CAA AAg gCT ggA Tgg T ACg AgC CgC TTT CgC TAC TAC CAC TCT TCC TgA CCT gCC TTA C TAg ACg TCC CTg CTC CTT ATT A TAg ATA ACC TCg AgC CgA TCg gAC TTg CCC TCC AAT ggA TCC
Annealing temp. (°C)
GenBank Accession No.
66
NP_989438.1
60
NP_001006457.1
60
NP_001001464.1
60
NP_990059.1
62
NP_001025817.1
59
NM_204100.1
57
NP_989617.1
60
NP_001004767.1
58
NP_989542.1
63
M59389.1
avUCP, avian uncoupling protein; MnSOD, manganese superoxide dismutase; NRF1, nuclear respiratory factor-1; PGC-1α, peroxisome proliferatoractivated receptor γ coactivator-1α; PPAR, peroxisome proliferator-activated receptor; SIRT1, sirtuin-1; TFAM, mitochondrial transcription factor A; 18S, 18 s-rRNA. † The primer sets for these genes were used as previously described (PPARα and PPARβ/δ, (Joubert et al. 2011); TFAM, (Gan et al. 2015).
was imaged using a VersaDoc Model 5000 (Bio-Rad). Protein sizes were then estimated by using a pre-stained molecular-mass standard (Bio-Rad), and protein contents were densitometrically quantified by Quantity One® software (Bio-Rad). The membrane was washed with Tris-buffered saline (TBS) (20 mmol/L Tris/HCl and 139 mmol/L NaCl, pH7.6) with 0.1% (v/v) Tween-20 to remove the residue of chemiluminescent solution, and subsequently washed with TBS including 15% (v/v) hydrogen peroxide to inactivate HRP activity of the secondary antibody. Immunoblotting was then carried out with anti-β-actin antibody (1:10 000 dilution, anti-monoclonal antibody prepared from mouse, G043, Applied Biological Materials Inc., Richmond, BC, Canada). The subsequent procedures for estimating protein sizes were conducted in the same manner as for the detection of avUCP, and detected by using a HRP-conjugated anti-mouse secondary antibody (Cell Signaling Technology, 7076S). avUCP levels were corrected for β-actin levels, with values shown as fold changes relative to control values.
Measurement of mitochondrial superoxide production in cultured avian muscle cells Mitochondrial superoxide production was detected using MitoSOX™ Red Mitochondrial Superoxide Indicator (Life Technologies, San Diego, CA, USA) as previously described (Furukawa et al. 2015; Kikusato et al. 2015). The dye was dissolved in DMSO, and this was then added to Hanks’ balanced salt solution (HBSS) to make a 0.1% solution. After the incubation of cells with oleuropein, the medium was replaced with pre-warmed HBSS Animal Science Journal (2016) 87, 1371–1378
supplemented with 5 μmol/L MitoSOX™ Red. Levels of superoxide generation were then measured as the change in fluorescence at excitation and emission wavelengths (λex/λem) of 510 nm/590 nm for 30 min. The assay was carried out on a computer-controlled fluorescence microplate reader at 37 °C. Thereafter, the cellular protein content was determined using micro bicinchoninic acid (microBCA) assay, with bovine serum albumin (BSA) as the standard, and superoxide levels corrected for protein content. All data are shown as fold changes relative to control values.
Measurement of mitochondrial cytochrome c oxidase activity Mitochondrial cytochrome c oxidase (COX) activity, which is indicative of respiratory complex IV activity, in cell homogenates was measured polarographically using a Clark-type oxygen electrode (Rank Brothers, Cambridge, UK), as previously described (Wiedemann et al. 2000) with minor modifications. Cells were collected in HBSS solutions and gently homogenized with a Polytron PT-3100 (Kinematica, Littau, Switzerland) at 1000 rpm for 5 sec. The homogenates were then mixed with 100 mmol/L Tris/HCl and (pH7.0) transferred into a 30 °C-warmed O2-measurement chamber, to which 50 mmol/L N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and 0.1 mmol/L L-ascorbate (pH 7.0), and 2 μmol/L oxidized bovine heart cytochrome c (C3131, Sigma-Aldrich) were added. After monitoring oxygen consumption, 50 μmol/L potassium cyanide (KCN) were added to inhibit mitochondria-dependent COX © 2016 Japanese Society of Animal Science
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oxygen consumption. Mitochondrial COX activity was calculated by subtracting the KCN-inhibited oxygen consumption rate from total oxygen consumption rate, which was corrected for the protein levels.
Measurement of mitochondrial superoxide production in isolated avian muscle mitochondria Mitochondria were isolated from the Superficial pectoralis muscles of three 1-week-old chickens, as previously described (Kikusato & Toyomizu 2013). They were fed a standard diet for broiler chickens and reared in electrically heated batteries until used in experiments. The levels of superoxide generated from isolated mitochondria were determined fluorometrically by measurement of the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red, Invitrogen, Carlsbad, CA, USA) coupled to enzymatic reduction by HRP as previously described (Kikusato & Toyomizu 2013). Mitochondria (0.1 mg protein/mL) were suspended in a 37 °C-warmed assay medium (80 mmol/L KCl, 50 mmol/L HEPES (pH 7.2), 1 mmol/L ethyleneglycoltetraacetic acid, 5 mmol/L K2HPO4, 5 mmol/L MgCl2, and 0.3% BSA (w/v)). Four mmol/L succinate was added to the mitochondrial suspension to induce the generation of superoxide. Thereafter, oleuropein (50 μmol/L injection × 4 times) or the same amount of DMSO, and 1 μmol/L carbonyl cyanide p-triflouromethoxyphenyl hydrazone (FCCP) were added into the mitochondrial suspension. The measurement was conducted in triplicate.
Statistical analysis All data are expressed as the mean ± standard error (SE) of 4-6 individual samples. Statistical differences between the groups were identified using non-parametric KruskalWallis test followed by Steel-Dwass multiple comparison test or Student’s t-test. Values of P < 0.05 were considered to indicate statistical significance in each test.
RESULTS Time course of the effect of oleuropein on avUCP expression The effect of oleuropein on avUCP gene expression in avian muscle cells was evaluated. As illustrated in Figure 1A, there was no change in avUCP mRNA levels after 1 h of incubation of cells with oleuropein, while after 2 h the mRNA level was significantly increased. Following a longer incubation with oleuropein (4 h), avUCP mRNA levels returned to near-normal values. The level of avUCP protein in the 2 h-incubation cells tended to be increased compared to that of non-treated cells (Fig. 1B) (P = 0.09 vs. 50 μmol/L, P = 0.08 vs. 200 μmol/L), although no significant differences in mRNA or protein levels were observed for the different oleuropein concentrations tested (Fig. 1A, B). © 2016 Japanese Society of Animal Science
Figure 1 Time course of avian uncoupling protein (avUCP) messenger RNA (mRNA) level (A) and the protein level at 2 h-incubation (B) in avian muscle cells treated (or not) with oleuropein (50 and 200 μmol/L). Real-time RT-PCR was used to quantify the mRNA levels and the results were normalized to 18S rRNA levels. The amount of avUCP protein was quantified by western blot analysis using anti-avUCP antibody, with the results corrected for β-actin levels. All data are shown as fold changes relative to control values at each incubation time. Values are a,b means ± SE, n = 4-6. P < 0.05, with different letters signifying values that are statistically different.
Effects of oleuropein on PGC-1α, PPARs and SIRT1 gene expression To clarify the mechanism underlying the up-regulation of avUCP gene expression with oleuropein, the effects of oleuropein on PGC-1α, PPARα and PPARβ/δ gene expression were evaluated. As illustrated in Figure 2A, PGC-1α mRNA levels in oleuropein-treated cells showed a pattern similar to avUCP mRNA levels, whereas no effect was seen on PPARα (Fig. 2B) or PPARβ/δ (Fig. 2C) mRNA levels at any given incubation concentration or time. A strong positive correlation was observed between avUCP and PGC-1α mRNA levels in cells incubated with oleuropein for 2 h (Fig. 2D). As SIRT1 deacetylates PGC-1α and thereby induces the co-transcriptional activity, the level of SIRT1 mRNA was also measured and found to be significantly increased (Fig. 2E). Overall, these results suggest that oleuropein up-regulates avUCP expression in avian muscle cells, probably through the expression of PGC-1α following its activation by SIRT1.
Effects of oleuropein on mitochondrial biogenesis As illustrated in Figure 3A, oleuropein-treated cells showed a significant increase in NRF1 mRNA expression compared with non-treated cells. mRNA levels of TFAM and ATP5a1 as well as mitochondrial COX activity were also increased in oleuropein-treated cells (Fig. 3B). Overall, the results suggest that oleuropein may induce mitochondrial biogenesis, possibly via the up-regulation of PGC-1α activity in avian muscle cells.
Effects of oleuropein on mitochondrial superoxide generation While an objective of this study was to determine if mitochondrial superoxide generation is suppressed in cells Animal Science Journal (2016) 87, 1371–1378
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Figure 2 Time course of change in messenger RNA (mRNA) levels of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) (A), peroxisome proliferator-activated receptor-α (PPARα) (B) and PPARβ/δ (C) in avian muscle cells incubated with oleuropein (50 and 200 μmol/L), and relationship between avUCP and PGC-1α mRNA levels (D), and sirtuin-1 (SIRT1) mRNA level (E) following a 2 h-incubation of avian muscle cells with oleuropein. Real-time RT-PCR was used to quantify the mRNA levels, with results normalized to 18S rRNA levels. All data are shown as fold changes relative to control values at each incubation time. The values graphed in panel D represent Pearson correlation coefficients for the relationship between avUCP and PGC-1α gene levels. Values are means ± SE, a,b n = 4-6. *P < 0.05 compared with control group. P < 0.05, with different letters signifying values that are statistically different.
Figure 3 Levels of nuclear respiratory factor-1 (NRF1), mitochondrial transcription factor A (TFAM) and adenosine triphosphate (ATP)5a1 messenger RNA (mRNA) (A), and mitochondrial cytochrome c oxidase (COX) activity (B) following a 2 h-incubation of avian muscle cells with 50 μmol/L oleuropein. Real-time RT-PCR was used to quantify the mRNA levels and results were normalized to 18S rRNA levels. The COX activity was measured polarographically. Data are represented as fold change relative to control values. Values are means ± SE, n = 5-6. *P < 0.05 compared to non-treated cells.
exposed to oleuropein, no difference in superoxide levels was observed between control and treated cells (Fig. 4A). However, superoxide production was significantly lowered by oleuropein when levels were normalized to mitochondrial COX activity (Fig. 4B). These results suggest that the superoxide generation per mitochondrion might be suppressed by oleuropein. To further explore the mechanism whereby oleuropein lowers mitochondrial superoxide generation, the possible involvement Animal Science Journal (2016) 87, 1371–1378
of MnSOD was evaluated. As a result, MnSOD mRNA levels were significantly increased in oleuropein-treated cells (Fig. 4C). Furthermore, an influence of anti-oxidant activity due to oleuropein on the lowering of mitochondrial superoxide generation was also evaluated. Whereas the superoxide generated from isolated avian muscle mitochondria was suppressed by FCCP that was injected as a positive control agent, oleuropein supplementation had little effect on the superoxide generation rate at the concentrations injected up to 200 μmol/L (Fig. 4D). Overall results suggested that oleuropein suppresses mitochondrial superoxide generation, via the up-regulation of avUCP and MnSOD expression in avian muscle cells.
DISCUSSION The present study demonstrates that oleuropein added to the culture medium increases avUCP gene expression in cultured muscle cells in a manner that occurs independently of noradrenaline and adrenaline secretion. It was also observed that oleuropein could induce mitochondrial biogenesis. While the enhancement of oxidative metabolism through mitochondrial biogenesis could serve as a therapeutic target in various metabolic diseases, mitochondria is considered as a generator of ROS. In this regard, the present study demonstrates that the superoxide generation per mitochondrion in oleuropein-treated cells was suppressed, possibly as a consequence of the © 2016 Japanese Society of Animal Science
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Figure 4 Mitochondrial superoxide levels corrected for protein values (A) and for mitochondrial cytochrome c oxidase (COX) activity (B), and manganese superoxide dismutase (MnSOD) messenger RNA (mRNA) levels (C) following a 2 h-incubation of avian muscle cells with 50 μmol/L oleuropein. Values are means ± SE, n = 5-6. *P < 0.05 compared to non-treated cells. The effect of oleuropein (50-200 μmol/L) and carbonyl cyanide p-triflouromethoxyphenyl hydrazone (FCCP) (1 μmol/L) on the superoxide generation rate in isolated avian muscle mitochondria (D). The measurement was conducted in triplicate and the representative result is shown.
up-regulation of avUCP and MnSOD expression. It could therefore be postulated that oleuropein administration may have a beneficial effect on cellular oxidative metabolism. It should be noted that the up-regulating effect of oleuropein on avUCP and PGC-1α expression was only observed for a limited period of time. Given the fact that oleuropein undergoes hydrolysis to yield compounds such as hydroxytyrosol and elenoic acid (Briante et al. 2001; Visioli & Galli 2002; Yuan et al. 2015), it can be assumed that, following 4 h of incubation, the oleuropein introduced into the culture medium might be metabolized and consequently lose its efficacy to up-regulate expression of those genes. With regard to this possibility, we obtained data showing that hydroxytyrosol had no effect on avUCP or PGC1α mRNA levels in avian muscle cells (data not shown). Thus, the continuous administration of oleuropein or structural modifications preventing its biotransformation is required to maintain its effects. The present study revealed that PGC-1α may play an important role in up-regulating avUCP expression in oleuropein-treated cells. PGC-1α binds to several transcription factors (Lin et al. 2005), of which PPARs and thyroid hormone receptor are known to induce the transcription of UCP3 expression in mammalian skeletal muscle (Brun et al. 1999; Solanes et al. 2005). A previous study reported that, in skeletal muscle of birds receiving isoproterenol, the stimulation of avUCP mRNA expression was associated with increases in PGC-1α as well as © 2016 Japanese Society of Animal Science
PPARα and PPARβ/δ mRNA expression (Joubert et al. 2011). In contrast, the present study showed that PPAR mRNA expression levels were not altered in response to oleuropein treatment. From these outcomes, it is conceivable that the up-regulating mechanism of avUCP due to oleuropein might be different from that due to the β-adrenergic system in avian muscle cells, and that oleuropein might induce the transcription of PGC-1α by binding to other transcriptional factors such as thyroid hormone receptor (TR) (Puigserver 2005; Joubert et al. 2011). To identify the transcriptional factor that induces avUCP gene expression in concert with PGC-1α in oleuropein-treated cells, the protein levels or nuclear binding state of the factors need to be analyzed. In the signaling process governing mitochondrial biogenesis, PGC-1α interacts with NRF and induces TFAM transcription, which drives the transcription and replication of mitochondrial DNA and components of the mitochondrial electron transport chain (Scarpulla 2011). Moreover, PGC-1α is known to control NRF (Wu et al. 1999) as well as MnSOD gene expression (St-Pierre et al. 2003; Pfluger et al. 2008). Based on these findings, it can be postulated that, in avian muscle cells exposed to oleuropein, PGC-1α may play a pivotal role in not only up-regulating avUCP expression but also by inducing mitochondrial biogenesis and activating anti-oxidant defense systems. To further define the role of this coactivator in oleuropein-treated cells, the possible interaction of SIRT1 with PGC-1α should also be considered. PGC-1α undergoes deacetylation by SIRT1 and thereby Animal Science Journal (2016) 87, 1371–1378
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drives its co-transcriptional activity (Nemoto et al. 2005). One could therefore postulate that oleuropein might increase PGC-1α activity through a SIRT1-dependent modification, thereby augmenting avUCP as well as oxidative metabolism in avian muscle cells. The molecular mechanism underlying SIRT1 gene expression in cells exposed to oleuropein nevertheless remains unclear. A few studies showed that SIRT1 expression is induced by forkhead box O3 (FoxO3) or epithelial NO synthase (eNOS) in response to fasting or calorie restriction (Nemoto et al. 2004; Nisoli et al. 2005). Given that eNOS expression in avian muscle remains to be identified, it is possible that oleuropein might affect FoxO3 activity or its nuclear content in avian muscle cells. To clarify this point, further investigation is required to verify this possibility. Another polyphenol, resveratrol, has attracted scientific attention in recent years given the variety of health benefits associated with it in terms of improvements in metabolic diseases (Lagouge et al. 2006; Ramadori et al. 2009), extension of life span (Baur et al. 2006) and calorie restriction-like effects (Pearson et al. 2008). The precise mechanism by which resveratrol functions in vivo and in vitro has been extensively investigated (Price et al. 2012). Apart from this polyphenol, several isoflavones and their derivatives were recently identified as compounds that promote mitochondrial biogenesis (Rasbach & Schnellmann 2008). We suggest that oleuropein may have beneficial effects on mitochondrial biogenesis and oxidative metabolism, and that further investigation is warranted to more fully elucidate its biological properties.
ACKNOWLEDGMENT This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 25850182 (M.K.) and 24380147 (M.T.).
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Animal Science Journal (2016) 87, 1371–1378
