Pyrroloquinoline Quinone, a Redox-Active o-Quinone, Stimulates

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Pyrroloquinoline Quinone, a Redox-active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating SIRT1/PGC-1# Signaling Pathway Kazuhiro Saihara, Ryosuke Kamikubo, Kazuto Ikemoto, Koji Uchida, and Mitsugu Akagawa Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01185 • Publication Date (Web): 29 Nov 2017 Downloaded from http://pubs.acs.org on December 2, 2017

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Biochemistry

Pyrroloquinoline

Quinone,

a

Redox-active

o-Quinone, Stimulates Mitochondrial Biogenesis by Activating SIRT1/PGC-1α Signaling Pathway

Kazuhiro Saihara, † Ryosuke Kamikubo, † ,§ Kazuto Ikemoto,‡ Koji Uchida,§* Mitsugu Akagawa†* †

Department of Biological Chemistry, Division of Applied Life Science, Graduate

School of Life and Environmental Sciences, Osaka Prefecture University, Sakai 599-8531, Japan §

Department of Applied Biological Chemistry, Graduate School of Agricultural and

Life Sciences, University of Tokyo, Tokyo 113-8657, Japan ‡

Niigata Research Laboratory, Mitsubishi Gas Chemical Company, Inc., Niigata

950-3112, Japan

* Corresponding author E-mail: [email protected] (KU); [email protected] (MA)

1 ACS Paragon Plus Environment

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ABBREVIATIONS ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; CREB, cAMP

response

element-binding

protein;

MTCO1,

mitochondrially-encoded

cytochrome c oxidase subunit 1; mtDNA, mitochondrial DNA; LKB1, liver kinase B1; NMN,

β-nicotinamide

mononucleotide;

NAMPT,

nicotinamide

phosphoribosyltransferase; NMNAT, nicotinamide/nicotinic acid mononucleotide adenylyltransferase; NMNT, nicotinamide methyl transferase; NR, nicotinamide riboside; NRFs, nuclear respiratory factors; PGC-1α, peroxisome proliferator-activated receptor-gamma-coactivator-1α; nuclear respiratory factors; PQQ, pyrroloquinoline quinone; 5’-PRPP, 5’-phospho-ribosyl-1-pyrophosphate; SIRT1, sirtuin 1; SIRT3, sirtuin 3; Tfam, mitochondrial transcription factor A.


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Biochemistry

Abstract Pyrroloquinoline quinone (PQQ), a redox-active o-quinone found in various foods and mammalian tissues, has received increasing attention in a number of health benefits due to its ability to enhance mitochondrial biogenesis. However, its underlying molecular mechanism remains incompletely understood. We have now established that the exposure of mouse NIH/3T3 fibroblasts to a physiologically relevant concentration of PQQ significantly stimulates the mitochondrial biogenesis. The exposure of NIH/3T3 cells to 10–100 nM PQQ for 48 h resulted in increased Mitotracker staining, the mitochondrial DNA content, and the mitochondrially-encoded cytochrome c oxidase subunit 1 (MTCO1) protein. Moreover, we observed that PQQ treatment induces deacetylation of the peroxisome proliferator-activated receptor-gamma-coactivator-1α (PGC-1α), and facilitates its nuclear translocation and target gene expression, but does not affect its protein levels, implying increased activity of the NAD+-dependent protein deacetylase sirtuin 1 (SIRT1). Indeed, treatment with a SIRT1 selective inhibitor, EX-527, prevented the ability of PQQ to stimulate the PGC-1α-mediated mitochondrial biogenesis. We also found that the PQQ treatment caused a concentration-dependent increase in the cellular NAD+ levels, but not the total NAD+ and NADH levels. Our results suggest that the PQQ-inducible mitochondrial biogenesis can be attributed to activation of the SIRT1/PGC-1α signaling pathway by enhancing the cellular NAD+ formation.

Keywords: Pyrroloquinoline quinone, mitochondrial biogenesis, PGC-1α, sirtuin, NAD+


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Pyrroloquinoline quinone (PQQ, Figure 1) is a redox cofactor of bacterial dehydrogenases such as alcohol and aldose dehydrogenases

1,2

, and stably acts as an

efficient electron transfer catalyst from various organic substrates including NADH and thiol compounds

3–6

. Although PQQ is not biosynthesized in mammals, a trace amount

of PQQ has been detected in human and rat organs or tissues because of its presence in numerous dietary sources at pM to nM levels

7,8

. It has been

demonstrated that PQQ is an important nutrient involved

in

numerous

physiological

and

biochemical processes in both bacteria and higher organisms

Figure 1. Chemical structure of PQQ.

3,4,9

. Most importantly, nutritional studies of rodent models have established

that a PQQ deficiency shows divergent systemic responses, such as growth impairment, friable skin, compromised immune responsiveness, and abnormal reproductive 10–12

performance, whereas PQQ administration reverses these responses

. Moreover, a

PQQ deficiency in mice and rats reduces the number and size of the mitochondria by 20–30%, and suppresses the mitochondrial function and respiration

13,14

. Previous

studies 13–15 have also revealed that mitochondrial biogenesis in vivo is responsive to the dietary PQQ status, and PQQ supplementation with the addition of only milligram quantities of PQQ per kg of diet stimulates mitochondrial biogenesis with a concomitant increase in the mitochondria-related functions in vivo. PQQ has also been reported to elicit mitochondrial biogenesis in mouse Hepa1-6 hepatoma cells through activation of the

cAMP

response

element-binding

protein

(CREB)

proliferator-activated receptor-gamma-coactivator-1α (PGC-1α)

and

peroxisome

16

. PGC-1α is the

master transcription regulator that stimulates mitochondrial biogenesis by upregulating the nuclear respiratory factors (NRFs) and mitochondrial transcription factor A (Tfam), leading to an increased mitochondrial DNA (mtDNA) replication and gene transcription


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Biochemistry

17,18

. The transcription factor CREB increases transcription of the PGC-1α via a

conserved CREB-binding site in the proximal promoter and is activated by exercise or fasting

19

. On the other hand, the exposure of human HepG2 hepatoma cells to PQQ

significantly elevates the expression and activity of the NAD+-dependent protein deacetylases, sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3)20, which play a key role in mitochondrial biogenesis and enhanced mitochondrial-related functions, respectively 21,22

. However, the concentrations of PQQ, shown to have an effect (10−30 µM) in these

previous in vitro studies, are much higher than those observed in the blood or tissues in humans, rats, and mice, ranging approximately from 3 to 40 nM (or pmol/g) 23–25. Also, the primary target for PQQ to act upon in order to elicit mitochondrial biogenesis remains incompletely understood26, thus more research is required to define the exact molecular basis. Recently, a growing body of evidence is accumulating to show that defects in the mitochondrial function, dynamics, and biogenesis have been closely linked to a variety of

age-related

and

metabolic 17,27,28

neurodegenerative disease

disorders,

including

obesity,

diabetes,

and

Now, stimulation of the mitochondrial biogenesis has

been considered as a potential approach to preserve the mitochondrial function and to improve these disorders

29

. Hence, the induction of mitochondrial biogenesis by PQQ

has a number of health implications, and elucidating the molecular mechanism underlying the PQQ-inducible mitochondrial biogenesis could provide more specific preventive and therapeutic strategies against age-related metabolic disorders. In the present study, we demonstrated that the exposure of mouse NIH/3T3 fibroblasts and HepG2 cells to a physiologically-relevant concentration of PQQ significantly stimulates the mitochondrial biogenesis. We also investigated the molecular mechanism of the PQQ-induced

mitochondrial

biogenesis

and

found

that

PQQ

activates

the

SIRT1/PGC-1α pathway, with a concomitant increase in the cellular NAD+ formation.


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Materials and Methods

Materials The PQQ disodium salt was provided by Mitsubishi Gas Chemical Company, Inc. (Tokyo, Japan). Bovine calf serum (CS) was obtained from Equitech-Bio (Kerrville, TX, USA). β-Nicotinamide mononucleotide (NMN) was obtained from Sigma-Aldrich (St. Louis, MO, USA). EX527 and sirtinol were purchased from Cayman Chemical (Ann Arbor, MI, USA). Dithiothreitol (DTT), the protease inhibitor cocktail, Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, Triton X-100, Nonidet P-40 (NP-40), nicotinamide, NADH, and NAD+ were purchased from Nacalai Tesque (Kyoto, Japan). All of the other reagents used in the study were of analytical grade and obtained from commercial sources.

Cell culture The mouse embryo fibroblast cell line, NIH/3T3, was obtained from the Cell Resource Center for Biomedical Research, Tohoku University, Japan, and maintained in a 5% CO2 humidified atmosphere at 37 °C in DMEM supplemented with 10% CS, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were cultured until they reached 70–80% confluence. After overnight serum starvation, the cells were incubated in serum-free DMEM with or without PQQ for 0–48 h.

SDS-PAGE and immunoblot analysis After treatment, the cells were washed twice with ice-cold PBS and lysed with


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Biochemistry

radio-immunoprecipitation assay buffer containing 1% Triton X-100, the phosphatase inhibitor cocktail, and protease inhibitor cocktail. The protein concentration of each lysate was determined using the BCA Protein Assay Reagent Kit (Nacalai Tesque). SDS-PAGE and immunoblot analysis were performed as previously reported

30

.

Chemiluminescence was detected using EzWestLumiOne and Luminograph (ATTO, Tokyo, Japan). Quantification of the band intensity was performed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Antibodies for the acetylated-lysine, SIRT1, histone H3, LKB1, phosphor-LKB1 (p-Ser428), AMPKα, phospho-AMPKα (p-Thr172), ACC, phospho-ACC (p-Ser79), goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for the β-actin, GAPDH, and PGC-1α were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and MTCO1 was from Boster Biological Technology (Pleasanton, CA, USA). The antibody for the NAMPT was purchased from Flarebio Biotech LLC (Baltimore, MD, USA).

Preparation of cytoplasmic and nuclear extracts The cytoplasmic and nuclear extracts for immunoblotting were prepared as previously described with some modifications

31

. Briefly, the cells were washed with

cold PBS and scraped into PBS. The cells were collected by centrifugation at 1,500 × g for 5 min and resuspended in 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 1 × protease inhibitor cocktail (buffer A). After 15 min on ice, NP-40 was added to a final concentration of 10%, and the tubes were vortexed for 10 s. The lysates were centrifuged at 1,000 × g for 10 min to obtain the soluble cytoplasmic fraction (supernatant) and the nuclear pellet, which was resuspended in ice-cold buffer B (20 mM HEPES at pH 7.9, 0.4 M NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1% NP-40, 10% glycerol, 1 mM DTT, and 1 × protease inhibitor cocktail) and


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agitated at 4 °C for 15 min. The nuclear lysate was centrifuged for 5 min at 4 °C to obtain the soluble nuclear fraction (supernatant).

Mitotracker assay The cells were plated in 24-well plates for plate assay or 8-well chamber slides for fluorescence microscopic imaging. After overnight serum starvation, the cells were incubated in serum-free DMEM with or without PQQ for 48 h. The mitochondria were determined by staining with Mitotracker Green FM (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. For the plate assay, the fluorescence intensity of the cells was monitored at excitation and emission wavelengths of 485 and 520 nm, respectively, using an MTP-900Lab plate reader (Hitachi High-Technologies, Tokyo, Japan), and the values corrected for the cellular protein content determined by a BCA protein assay. For fluorescence microscopic imaging, the cells were imaged using a fluorescence microscope, BZ-9000 (Keyence, Osaka, Japan).

Quantitative real-time PCR (qPCR) for mtDNA The cells were cultured in 12-well plates until they reached 70–80% confluence. After overnight serum starvation, the cells were incubated in serum-free DMEM with or without PQQ for 0–48 h. The genomic DNA (containing both mitochondrial and nuclear DNA) was extracted according to standard procedures. Primers for the mouse mitochondrial 12S sRNA (Forward 5'-ACCGCGGTCATACGATTAAC, reverse 3'-CCCAGTTTGGGTCTTAGCTG)

and

nuclear

18S

sRNA

(forward

5'-CATTCGAACGTCTGCCCTATC, reverse 3'-CCTGCTGCCTTCCTTGGA) were purchased from Thermo Fisher Scientific. qPCR was performed on the Thermal Cycler Dice Real Time System Single (Takara Bio, Shiga, Japan) using the THUNDERBIRD


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Biochemistry

SYBR qPCR Mix (Toyobo). The mtDNA per nuclear DNA was calculated as the ratio of mitochondrial 12S rRNA DNA to nuclear 18S rRNA DNA. Quantification was performed using the ∆∆Ct method.

RNA isolation and quantitative reverse transcription PCR (qRT-PCR) The total RNA was isolated from NIH/3T3 fibroblasts using the SuperPrep Cell Lysis Kit for qPCR (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. The total RNA was converted to cDNA using the SuperPrep RT Kit for qPCR (Toyobo). Primers for the mouse Nrf1 (Forward 5'-AGCACGGAGTGACCCAAAC, reverse 3'-TGTACGTGGCTACATGGACCT),

Tfam

(forward

5'-ATTCCGAAGTGTTTTTCCAGCA, reverse 3'-TCTGAAAGTTTTGCATCTGGGT), and

18S

rRNA

(forward

5'-CATTCGAACGTCTGCCCTATC,

reverse

3'-CCTGCTGCCTTCCTTGGA) were purchased from Thermo Fisher Scientific. The quantitative real-time PCR was performed as already described. The Ct value was normalized with the housekeeping gene 18S rRNA and the relative fold change was computed by the ∆∆Ct method.

Measurement of cellular NAD+ and NADH The cells were cultured in 12-well plates until they reached 70–80% confluence. After overnight serum starvation, the cells were incubated in serum-free DMEM with or without PQQ for 0–48 h. After incubation, the cellular levels of NAD+ and total NAD+ + NADH were measured using an Amplite colorimetric total NAD and NADH assay kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instructions, and normalized to the cellular protein content determined by the BCA protein assay.

Determination of ATP levels


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The NIH/3T3 fibroblasts were cultured in a collagen-coated 96-well culture plate until they reached 70–80% confluence. After overnight serum starvation, the cells were incubated in serum-free DMEM with or without PQQ for 48 h. After incubation, the cellular ATP levels were determined using an ATP luminescence kit (TOYO B-Net, Tokyo, Japan) according to the manufacturer’s instructions. The luminescence intensity was measured using an MTP-900Lab plate reader and the values corrected for the cellular protein content determined by the BCA protein assay.

Immunocytochemistry The NIH/3T/3 cells were seeded on collagen-coated 8-well chamber slides. After treatment, the cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100, followed by blocking with Blocking One (Nacalai Tesque) for 1 h. The cells were incubated overnight with the anti-PGC-1α antibody (Santa Cruz Biotechnology) at 4 ˚C. The cells were washed three times with PBS, then incubated with the Cy3 Conjugated anti-rabbit IgG antibody (GE Healthcare UK Ltd., Buckinghamshire, UK) for 3 h under shaded conditions. After rinsing, the nuclei were stained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) (Nacalai Tesque), and the cells were imaged using a BZ-9000 fluorescence microscope.

Immunoprecipitation assays The PGC-1α acetylation was analyzed by immunoprecipitation of PGC-1α from the cell lysates with the anti-PGC-1α antibody as previously described with some modifications

32,33

. Briefly, the cells were lysed in ice-cold lysis buffer containing 20

mM Tris-HCl (pH 7.4), 137 mM NaCl, 5 mM nicotinamide, 1% NP-40, 1 mM sodium butyrate, and the 1 × protease inhibitor cocktail by sonication. After removal of the debris and preclearing, the cell lysates were incubated overnight with 1 µL of the


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Biochemistry

anti-PGC-1α antibody (Santa Cruz Biotechnology) at 4 ˚C. Twenty µL of PureProteome Protein G magnetic beads (Millipore, Billerica, MA) was then added. After 60 min of rotation, the beads were collected using a magnetic separation rack, then washed four times with lysis buffer. The bound proteins were resolved by SDS-PAGE, then subjected to an immunoblot analysis.

Mitochondrial dehydrogenase assay The NIH/3T3 fibroblasts were cultured in a collagen-coated 96-well culture plate until they reached 70–80% confluence. After overnight serum starvation, the cells were incubated in serum-free DMEM with or without PQQ for 48 h. The activity of the mitochondrial dehydrogenase in the cells was determined using the WST-8 assay kit (Nacalai Tesque) as previously described 34. All values were normalized to the cellular protein content determined by the BCA protein assay.

Results PQQ

Stimulates

Mitochondrial

Biogenesis

at

Physiologically-Relevant

Concentrations We first ascertained whether PQQ induces mitochondrial biogenesis at physiologically-relevant concentrations. The NIH/3T3 fibroblasts were incubated with 10–100 nM of PQQ for 48 h, then the mitochondria was selectively stained using Mitotracker Green FM dye

35

. Fluorescent microscopic imaging shown in Figure 2A

(upper) revealed that the dye significantly accumulated in the PQQ-treated cells as compared to the vehicle-treated cells. Using a fluorescent plate reader, we also evaluated the mitochondrial contents. As shown in Figure 2A (lower), we demonstrated


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

that the PQQ treatment caused a significant increase in the mitochondrial contents. Moreover, we observed that the mitochondrial DNA relative to the nuclear DNA (mtDNA/nDNA) was dose-dependently increased in the PQQ-treated cells (Figure 2B).

Figure 2. PQQ stimulates mitochondrial biogenesis, and enhances mitochondrial dehydrogenase activity and ATP production. (A–E) NIH/3T3 fibroblasts were incubated with the indicated concentrations of PQQ for 48 h. (A) Mitochondria were stained with Mitotracker Green FM. After rinsing with PBS, the cells were immediately imaged using a fluorescence microscope (upper) or the mitochondrial intensity was determined by a plate fluorometer (lower). The mitochondrial content was normalized to the cellular protein content. (B) The mtDNA content was determined by qPCR, calculated as the ratio of mtDNA/nuclear (n) DNA, and expressed as fold change versus vehicle-treated control. (C) The protein levels of MTCO1 were determined by immunoblotting, normalized for β-actin levels, and expressed as the fold change versus vehicle-treated control. (D) The mitochondrial dehydrogenase activity was determined using a WST-8 assay kit and normalized to the cellular protein content. (E) The cellular ATP levels were determined using an ATP luminescence kit and normalized to the cellular protein content. (A–E) The results are shown as mean ± S.E.M. (n ≧ 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle-treated control. (ANOVA, Dunnett’s multiple comparison test).


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Biochemistry

As additional validation, we determined the protein levels of the mtDNA-encoded cytochrome c oxidase subunit 1 (MTCO1), a mitochondrial marker. Consistent with an increase in the mitochondrial content, the treatment of cells with PQQ significantly up-regulated the MTCO1 proteins in a concentration-dependent manner (Figure 2C). Accordingly, we anticipated that the increase in the mitochondrial biogenesis would contribute to the enhanced mitochondrial activity and the elevated ATP production. Thus, we measured the mitochondrial dehydrogenase activity and cellular ATP content in the PQQ-treated cells. As shown in Figures 2D and E, PQQ significantly enhanced the mitochondrial dehydrogenase activity and increased the cellular ATP content.

PQQ Stimulates Nuclear Translocation of PGC-1α and Promotes Expression of the Nrf1 and Tfam Genes Mechanistically, the mitochondrial biogenesis and function are augmented by the transcriptional coactivator, PGC-1α, through activation of the NRFs

17,18

. A previous

study 16 has shown that exposure of 30 µM PQQ to the mouse hepatoma cells activates the promoter of PGC-1α, and increases the PGC-1α mRNA and protein expression, leading to mitochondrial biogenesis. To establish the mechanism underlying the PQQ-stimulated mitochondrial biogenesis at physiologically-relevant concentrations, we assessed the protein levels of PGC-1α in the NIH/3T3 cells exposed to PQQ. However, immunoblot analysis showed that the protein levels of PGC-1α were not affected by the treatment with PQQ (10–100 nM) during the 48-h incubation (Figures

3A and B). Meanwhile, the transcriptional activity of PGC-1α is critically modulated through deacetylation by SIRT1, which allows for its nuclear translocation to promote the transcription

21

. Hence, we next probed for acetylation of PGC-1α in the

immunoprecipitates from cells exposed to PQQ or the SIRT1 activator resveratrol 36. As shown in Figure 3C, the PGC-1α acetylation decreased after exposure of 100 nM PQQ


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

to the cells in a time-dependent fashion. Moreover, we evaluated the influence of the PQQ treatment on the subcellular localization of PGC-1α in the NIH/3T3 cells.

Figure 3. PQQ induces PGC-1α activation. (A and B) The NIH/3T3 fibroblasts were incubated with the indicated concentrations of PQQ for 48 h (A) or for times ranging from 6 to 48 h (B). After incubation, the protein levels of PGC-1α were determined by immunoblotting, normalized for the β-actin levels, and expressed as the fold change versus vehicle-treated control. (C) The NIH/3T3 fibroblasts were incubated with 100 nM PQQ for 0–9 h or with 50 µM resveratrol (Res) for 9 h. After incubation, the protein was immunoprecipitated from each cell lysate with the anti-PGC-1α antibody. The immunoprecipitates were analyzed by immunoblotting using antibodies against the acetylated-lysine and PGC-1α. The relative acetylation levels of PGC-1α were normalized for the PGC-1α levels, and expressed as the fold change versus the control. (D) The NIH/3T3 fibroblasts were incubated with 100 nM PQQ or 50 µM resveratrol (Res) for 6 h. After incubation, PGC-1α was immunostained by using the anti-PGC-1α antibody as red, the nuclei were stained with DAPI as blue, and the merged image of PGC-1α and nuclei as purple. (E and F) The NIH/3T3 fibroblasts were incubated with the indicated concentrations of PQQ for times ranging from 0 to 9 h (E) or for 6 h (F). After incubation, the nuclear levels of PGC-1α were determined by immunoblotting. The antibody against histone H3 was used as the loading controls for the nuclear protein. (G and H) The NIH/3T3 fibroblasts were incubated with the indicated concentrations of PQQ for 15 h. The mRNA levels of Nrf1 (G) and Tfam (H) were then analyzed by qRT-PCR. (A–C, G, and H) The results are shown as mean ± S.E.M. (n ≧ 3). (A, C, G, and H) N.S., not significant. **p < 0.01, ***p < 0.001 versus vehicle-treated control (ANOVA, Dunnett’s multiple comparison test). (B) N.S., not significant (unpaired two-tailed t-test).


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Biochemistry

As demonstrated by an immunofluorescence microscopic analysis (Figure 3D), PGC-1α was mainly observed in the cytoplasm of the untreated cells, whereas the PQQ treatment apparently promoted nuclear translocation of PGC-1α similar to the treatment of the PGC-1α activator resveratrol, implying the enhanced transcriptional activity of PGC-1α. This observation was further confirmed by determination of the PGC-1α levels in the nuclear fraction with immunoblotting. As shown in Figures 3E and F, the nuclear levels of PGC-1α significantly increased after incubation with 100 nM PQQ for 6 h, and the exposure to PQQ at concentrations from 10 to 1,000 nM for 6 h gave rise to a dose-dependent nuclear accumulation of PGC-1α. The activated PGC-1α stimulates a powerful induction of the NRF1 and NRF2 gene expressions, and furthermore, binds to and coactivates the transcriptional function of NRF1 on the promoter for Tfam, a direct regulator of the mtDNA replication/transcription 29. Hence, we assessed the expression levels of Nrf1 and Tfam mRNA in the NIH/3T3 cells exposed to PQQ. As expected, the qRT-PCR analysis revealed that the mRNA levels of Nrf1 and Tfam were significantly increased by the treatment with PQQ for 15 h (Figures 3G and H). These data strongly suggest that PQQ might provoke the SIRT1-mediated PGC-1α activation and enhance its target gene expression, leading to mitochondrial biogenesis.

PQQ Induces Mitochondrial Biogenesis Through SIRT1-mediated PGC-1α Activation Although a previous study20 demonstrated that exposure of 10–30 µM PQQ to human hepatoma cells increased the expression levels of SIRT1, we could not observe a significantly increased expression of the SIRT1 protein in NIH/3T3 cells exposed to 10– 100 nM PQQ during the 48-h incubation (Figures 4A and B). To further verify that the PGC-1α-mediated mitochondrial biogenesis by PQQ is attributed to the SIRT1 activity, we incubated NIH/3T3 cells with PQQ in the presence of a selective SIRT1 inhibitor,


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

EX-527

Page 16 of 36

37

attenuated

. As shown in Figures 4C and D, the treatment with EX-527 effectively the

PQQ-elicited

nuclear

entry

of

PGC-1α.

Figure 4. SIRT1 inhibition abrogates PQQ-stimulated mitochondrial biogenesis. (A and B) The NIH/3T3 fibroblasts were incubated with the indicated concentrations of PQQ for 48 h (A) or for times ranging from 6 to 48 h (B). After incubation, the protein levels of SIRT1 were determined by immunoblotting, normalized for the β-actin levels, and expressed as fold change versus the vehicle-treated control. (C and D) The NIH/3T3 fibroblasts were incubated with or without 100 nM PQQ in the presence or absence of the SIRT1 inhibitor, EX527 (10 µM), for 6 h. (C) After incubation, PGC-1α was immunostained using the anti-PGC-1α antibody as red, the nuclei were stained with DAPI as blue, and the merged image of PGC-1α and nuclei as purple. (D) After incubation, the nuclear levels of PGC-1α were determined by immunoblotting. The antibody against histone H3 was used as the loading controls for the nuclear protein. (E–I) The NIH/3T3 fibroblasts were incubated with or without 100 nM PQQ in the presence or absence of the SIRT1 inhibitor, EX527 (10 µM), for 48 h. (E) The Mitochondria were stained with Mitotracker Green FM, then the mitochondria intensity was determined by a plate fluorometer. The Mitochondrial content was normalized to the cellular protein content. (F) The mtDNA content was determined by qPCR, calculated as a ratio of the mtDNA/nuclear (n) DNA, and expressed as fold change versus the vehicle-treated control. (G) The protein levels of MTCO1 and β-actin were determined by immunoblotting. (H) The Mitochondrial dehydrogenase activity was determined using a WST-8 assay kit and normalized to the cellular protein content. (I) The cellular ATP levels were determined using an ATP luminescence kit and normalized to the cellular protein content. (A, B, E, F, H, and I) The results are shown as mean ± S.E.M. (n ≧ 3). (A) N.S., not significant (ANOVA, Dunnett’s multiple comparison test). (B) N.S., not significant (unpaired two-tailed t-test). (B, E, F, H, and I) The bars with different letters are significantly different (p < 0.05, ANOVA, Tukey-Kramer test).


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Biochemistry

Furthermore, the PQQ-stimulated increments in the mitochondrial contents, mtDNA, and MTCO1 expression were fully reversed by the incubation with EX-527 (Figures

4E–G). In accord with these observations, EX-527 treatment also abolished the PQQ-inducible facilitation of the mitochondrial dehydrogenase activity and ATP generation (Figures 4H and I). The increased SIRT1 activity is highly associated with activation of the AMP-activated protein kinase (AMPK), which functions as another important regulator of mitochondrial biogenesis

38,39

, because SIRT1 elicits activation of liver kinase B1

(LKB1), a primary AMPK kinase, via deacetylation and subsequent phosphorylation leading to AMPK phosphorylation

39,40

. Hence, we also sought to explore the

LKB1/AMPK signaling in response to the PQQ treatment. As shown in Figures 5A–D, we observed that treatment of NIH/3T3 cells with PQQ synchronously enhanced the phosphorylation of both LKB1 and AMPK in a time- and dose-dependent manner. In good agreement with this observation, the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, was also elevated by the PQQ treatment (Figures 5E and F). On the other hand, we also examined the influence of the SIRT1 inhibitors, EX-527, sirtinol, and nicotinamide, on the PQQ-inducible phosphorylation of LKB1 and AMPK. As shown in Figures 5G–I, we observed that the treatments with EX-527, sirtinol, and nicotinamide abrogated the phosphorylation of both LKB1 and AMPK in PQQ-exposed cells, indicating the necessity of SIRT1 for the PQQ-inducible AMPK activation. These results suggest that SIRT1 activation can represent a key mechanism

for

the

PQQ-stimulated

mitochondrial


biogenesis.

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Page 18 of 36

Figure 5. PQQ activates LKB1/AMPK signaling. (A–F) The NIH/3T3 fibroblasts were incubated with the indicated concentrations of PQQ or with 50 µM resveratrol (Res) for times ranging from 1 to 9 h (A, C, and E) or for 6 h (B, D, and F). After incubation, the levels of LKB1 and phospho-LKB1 (A and B), AMPKα and phospho-AMPKα (C and D), ACC and phospho-ACC (E and F) were determined by immunoblotting. The antibody against GAPDH was used as the loading controls. (G, H, and I) The NIH/3T3 fibroblasts were pretreated with 1 µM EX-527, 40 µM sirtinol, or 10 mM nicotinamide for 1 h, and then exposed to 100 nM PQQ for an additional 6 h. The phosphorylation levels of LKB1 and AMPK were determined by immunetting.

PQQ Elicits Enhanced Cellular NAD+ Levels In general, the enzymatic activity of SIRT1 is directly regulated by the cellular availability of its substrate NAD+ or the NAD+/NADH ratio, which reflects the cellular metabolic status and redox state, and is closely linked to physiological or pathological states

41,42

. The cellular NAD+/NADH ratio is intricately controlled by multiple

processes including glycolysis, the TCA cycle, and oxidative phosphorylation by the electron transport chain

42,43

. Simultaneously, in mammals, NAD+ is biosynthesized

from three major precursors, i.e., nicotinic acid, nicotinamide, and nicotinamide riboside (NR)

41

. Additionally, PQQ is known to non-enzymatically catalyze the

oxidation of NADH to generate NAD+ through its continuous redox cycling


3–5,44

.

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Biochemistry

Hence, to gain further insight into the molecular mechanism underlying the PQQ-stimulated mitochondrial biogenesis, we attempted to assess the time-dependent NAD+ levels in NIH/3T3 cells treated with PQQ in serum-free DMEM. As shown in

Figures 6A and B, we found that the intracellular NAD+ level was significantly increased after a 6-h incubation with 100 nM PQQ, but not the total NAD+ and NADH levels. Moreover, the exposure of the cells to 10–100 nM PQQ for 6 h caused a concentration-dependent increase in the NAD+ levels, even though the total NAD+ and NADH levels were not significantly altered by the PQQ exposure (Figures 6C and D).

Figure 6. PQQ increases in cellular NAD+ concentration. (A–D) The NIH/3T3 fibroblasts were incubated with the indicated concentrations of PQQ for times ranging from 1 to 15 h (A and B) or for 6 h (C and D). (A–D After the incubation, the cellular levels of NAD+ (A and C) and total NAD+ and NADH (B and D) were measured and normalized to the cellular protein content. (A–D) The results are shown as mean ± S.E.M. (n ≧ 3). N.S., not significant. **p < 0.01, ***p < 0.001 versus vehicle-treated control (ANOVA, Dunnett’s multiple comparison test).

Similarly, we observed that the treatment of HepG2 cells with 100 nM PQQ significantly elevated the cellular NAD+ level and mitochondrial content (Figure S1A–

C). These data suggest that the SIRT1-mediated PGC-1α activation by PQQ might be attributable to the elevated cellular NAD+ levels.


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Page 20 of 36

Discussion PQQ has now been increasingly recognized as an important biofactor for numerous physiological functions including the promotion of growth and reproduction neural and cardiovascular protection

11,12,45,46

,

47,48

, and enhancing the learning and memory

functions 49,50 in animals. In particular, the PQQ-inducible mitochondrial biogenesis has been receiving much attention due to its physiological importance and pharmacological effects. Notably, the PQQ administration reverses the mitochondrial changes, and significantly improves the lipid profile in a rat model of type 2 diabetes mellitus

15

.

Moreover, PQQ exerts cytoprotective effects on the oxidative stress-induced cardiomyocytes, concomitant with a preservative effect on the mitochondrial function 51, and also protects the cardiac function and mitochondria from ischemia/reperfusion oxidative damage in rats

48

. Thus, based on these findings, the PQQ-inducible

mitochondrial biogenesis may be directly attributed to its multiple physiological functions. However, many aspects of its molecular and physiological modes of action remain unclear. To better understand the molecular basis of the PQQ-inducible mitochondrial biogenesis, we used the NIH/3T3 mouse embryo fibroblast cell line because skin friability was the most striking feature in mice fed PQQ-deficient diets 10,12. Furthermore, PQQ has been noted to be prominently retained in the skin and kidney 24 h after oral administration to mice as compared to other tissues 52. In the present study, we indeed proved that the exposure of NIH/3T3 cells to a physiologically-relevant concentration of PQQ significantly increases the mitochondrial content, mtDNA content, and mitochondrially-encoded MTCO1 protein (Figure 2). In addition, we also characterized the molecular mechanism underlying the PQQ-induced mitochondrial biogenesis. Based on our data, we propose that the PQQ-stimulated mitochondrial biogenesis can be attributed to the activation of the SIRT1/PGC-1α


Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

signaling pathway via enhancing the cellular NAD+ formation. As shown in Figure 3, we observed that the PQQ treatment promotes deacetylation of PGC-1α and induces its nuclear translocation and target gene expression, but does not affect the protein levels of PGC-1α, suggesting activation of the SIRT1-dependent pathway. Accordingly, our result that inhibition of SIRT1 by EX-527 (SIRT1 selective inhibitor) prevented the ability of PQQ to stimulate the PGC-1α-mediated mitochondrial biogenesis strongly supports the key role of the SIRT1-dependent pathway in our proposed mechanism (Figure 4). Recently, some studies have demonstrated that pharmacological-treatment targeting for stimulating SIRT1, such as resveratrol tetramethylpyrazine

53

, metformin

54

, and

55

, increased the mitochondrial biogenesis, slowing senescence. In

addition, growing evidence suggests that increased mitochondrial biogenesis mediated by SIRT1 plays a central role in improving the life span and reducing aging-related diseases

56

. SIRT1 can be regulated by many mechanisms including post-translational

modifications and NAD+ levels

21

. Of these mechanisms, the SIRT1 activity is

significantly dependent on the cellular NAD+, since the protein deacetylation by SIRT1 requires NAD+ as a co-substrate to transfer an acetyl group from a protein substrate to the ADP-ribose moiety of NAD+. Intriguingly, we found that PQQ exposures to NIH/3T3 cells and HepG2 cells at physiologically-relevant concentrations significantly increase the cellular NAD+ levels without alterations in the total NAD+ and NADH levels (Figures 6A–D and Figure S1A and B). Recent studies have demonstrated that the NAD+ levels are limiting, making the availability of NAD+ pivotal for the mitochondrial function and biogenesis

43

. Indeed, we confirmed that the elevated

cellular NAD+ levels elicited by the exposure of NIH/3T3 cells to the NAD+ precursor NMN effectively stimulated mitochondrial biogenesis (Figure S2). In mammals, the salvage pathway starting from nicotinamide is considered as a principal NAD+ biosynthetic pathway, although the main precursor actually originated


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

is still under debate

Page 22 of 36

57

. In this pathway, the nicotinamide phosphoribosyltransferase

(NAMPT) converts nicotinamide and 5’-phospho-ribosyl-1-pyrophosphate (5’-PRPP) to NMN, which is the rate-limiting step in this NAD+ biosynthesis 58. NMN, together with ATP, is then reversibly converted into NAD+ by the nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT). All of the NAD+ biosynthetic pathways merge at the last step of the dinucleotide formation, catalyzed by NMNAT. Humans have three NMNAT genes that produce three NMNAT protein isoforms with distinct tissue expression patterns and subcellular localizations

59

. Overexpression of NAMPT

markedly increases NAD+ levels in neurons and fibroblasts, whereas overexpression of the cytoplasmic form of NMNAT does not alter cellular NAD+ levels

60,61

. Therefore,

intracellular NAMPT is the key element of cellular NAD+ biosynthesis, and can significantly influence the SIRT1 activity. A previous study reported that treatment of HepG2 cells with more than 10 µM PQQ leads to a significant increase in the intracellular

NAMPT

protein

20

.

Nevertheless,

at

physiologically-relevant

concentrations, the PQQ treatment did not completely induce NAMPT up-regulation in both HepG2 cells and NIH/3T3 cells (Figure S1D and Figures S3A and B). On the other hand, nicotinamide methyl transferase (NMNT) is the only enzyme that catabolizes nicotinamide by transferring a methyl group from S-adenosylmethionine to nicotinamide

57

. More recently, it has been demonstrated that NNMT knockdown

increases adipose NAD+ levels 62. Further studies are needed to elucidate whether PQQ modulates the NAD+ biosynthetic pathways to enhance cellular NAD+ levels. PQQ can stably catalyze the oxidation of NADH through its continuous and repeated redox cycling 3,44,63. A redox reaction between PQQ and NADH readily occurs to produce pyrroloquinoline quinol (PQQH2) and NAD+. Subsequently, PQQH2 can be reoxidized to PQQ through aerobic auto-oxidation or the reaction with radical species such as singlet oxygen, the aroxyl radical, and the peroxyl radical 5,64–66. Of note, PQQ


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Biochemistry

is 100–1,000 times more efficient than other quinone biofactors in assays designed to assess redox cycling, and can undergo thousands of redox catalytic cycles at neutral pH and moderate temperatures without degradation or polymerization 3. More recently, we have identified the

L-lactate

dehydrogenase (LDH) A chain as one of the major

PQQ-binding proteins from NIH/3T3 cells, and demonstrated that the binding of PQQ to a purified rabbit muscle LDH augments the enzymatic activity to convert L-lactate to pyruvate via the oxidation of NADH to NAD+ by acting as a redox catalyst 5. Furthermore, we have found that PQQ exposure attenuates lactate release from the NIH/3T3 cells, implying that PQQ may oxidize NADH to generate NAD+ due to its redox activity and potentiate the NAD+-dependent reaction in the cells. Also, the cationic redox active dye, methylene blue, is known to act as an effective NADH-oxidation catalyst via its redox cycling

67

. Interestingly, recent in vivo and in

vitro studies have revealed that methylene blue treatment significantly increases the cellular NAD+/NADH ratio and NAD+ content 68,69. Methylene blue has been shown to accumulate in the liver and heart with a high affinity after oral administration to mice, and increases the hepatic SIRT1 activity 68. Consistently, methylene blue treatment has enhanced the hepatic mtDNA contents and oxygen consumption rates. Moreover, treatment of streptozotocin-induced diabetic rats with methylene blue has been found to increase NAD+, the activity of SIRT3, and reduce the protein lysine acetylation in cardiac mitochondria

69

. A previous study has also shown that β-lapachone, a

redox-active o-quinone isolated from the lapacho tree (Tabebuia sp.), induces an increase in the cellular NAD+/NADH ratio through NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent NADH oxidation in mouse L6 myoblasts

70

. NQO1, a cytosolic

antioxidant flavoprotein, catalyzes the reduction of a wide range of quinones to their corresponding hydroquinones by utilizing NADH as an electron donor, which consequently increases intracellular NAD+ levels

71

. Notably, β-lapachone is a high


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 36

72

affinity substrate for NQO1

, and its pharmacological treatment to mice highly

increased the NAD+/NADH ratio and stimulates the AMPK signaling pathway and mitochondrial fatty acid oxidation in the liver

70

. Based on these facts, the NADH

oxidation through the redox-cycling reaction and/or NQO1-dependent reaction by PQQ could be involved in the mitochondrial biogenesis. In the present study, we also found that treatment of NIH/3T3 cells with PQQ at physiologically-relevant concentrations activates the LKB1/AMPK pathway (Figure 5). Furthermore, we demonstrated that the treatment with SIRT1 inhibitors prevented the phosphorylation of both LKB1 and AMPK in PQQ-exposed cells, indicating that SIRT1 acts upstream of the AMPK activation (Figures 5G–I). Similar to SIRT1, AMPK is an evolutionary conserved enzyme and acts as an energy status sensor through the intracellular AMP or AMP/ATP ratio in eukaryotes

33

. Currently available evidence

suggests that SIRT1 and AMPK exhibit mutual interactions with each other in both in 21,39

vivo and in vitro studies

. The activation of SIRT1 leads to deacetylation of LKB1,

which enhances the LKB1 binding to the STE20-related adaptor protein (STRAD) and mouse embryo scaffold protein (MO25), increasing its phosphorylation at Ser-428 and the kinase activity 39,40. LKB1 then promotes AMPK activation by phosphorylating the AMPK α catalytic subunit at Thr-172. In the meantime, it has been suggested that AMPK can activate SIRT1 by increasing the cellular NAD+ level by promoting the expression of NAMPT

33,39

, although the intracellular NAMPT levels remained

unchanged in PQQ-exposed cells (Figure S1, and Figures S3A and B). Moreover, the activated AMPK directly phosphorylates PGC-1α and makes it more susceptible to deacetylation by SIRT1

33,39

. Taken together, these facts indicate that AMPK activation

is critically involved in the mitochondrial biogenesis via the SIRT1/PGC-1α pathway. Thus, our finding that the LKB1/AMPK pathway is activated in response to the PQQ treatment further supports our proposed mechanism that the PQQ-dependent


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Biochemistry

mitochondrial biogenesis can be attributed to activation of the SIRT1/PGC-1α pathway. NAD+ is an essential electron transporter during mitochondrial respiration and oxidative phosphorylation, and the ratios of NAD+/NADH, which changes in diverse physiological conditions, can affect numerous enzymatic activities. Accumulating evidence has also indicated that NAD+ plays crucial roles in not only mitochondrial functions and the energy metabolism, but also calcium homeostasis and inflammation 73. The pivotal NAD+-dependent enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) and sirtuins, have also been shown to play important roles in cell death and aging

42,74,75

.

Recently, a number of studies in rodents and humans has shown that the NAD+ content and NAMPT level decline with age in multiple organs such as the pancreas, adipose tissue, skeletal muscle, liver, skin, and brain

75–79

. Additionally, accumulating evidence

has suggested that the NAD+ availability decreases in age-associated metabolic complications, such as type 2 diabetes 42,80,81. Importantly, the NMN treatment has been proved to enhance the NAD+ biosynthesis and ameliorate various pathologies in mouse disease models

80,82–84

. Recent studies have also shown that supplementation of NR at

pharmacological levels can provide physiological or metabolic benefits by boosting NAD+ levels

85–87

. Thus, enhancing the NAD+ levels is expected to have significant

preventive effects on various pathophysiological changes during the natural process of aging. Considering these facts, our results offer a novel hypothesis that PQQ can enhance the cellular NAD+ levels, thereby contributing to health and disease prevention via NAD+-dependent regulation. Further studies are need to establish the contribution of the PQQ-dependent increase in the cellular NAD+ level to its important nutritional and physiological functions.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ASSOCIATED CONTENT Supporting Information Figure S1. PQQ stimulates mitochondrial biogenesis and increases NAD+ level in HepG2 cells. Figure S2. NMN stimulates mitochondrial biogenesis through increased NAD+ level. Figure S3. PQQ does not affect the protein levels of NAMPT.

AUTHOR INFORMATION * Corresponding Author E-mail: [email protected] (KU); [email protected] (MA)

Funding This work was supported in part by a Grant-in-Aid for Scientific Research (A) (No. 22780124) (M.A.) and Grant-in-Aid for Scientific Research on Innovative Areas "Oxygen Biology: a new criterion for integrated understanding of life" (No. 26111011) (K.U.) of the Ministry of Education, Sciences, Sports, Technology (MEXT), Japan.

Notes The authors have declared that no competing interests exist.


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For table of content use only.

Pyrroloquinoline Quinone, a Redox-active o-Quinone, Stimulates Mitochondrial Biogenesis by Increasing Cellular NAD+ Level

Kazuhiro Saihara, Ryosuke Kamikubo, Kazuto Ikemoto, Koji Uchida, Mitsugu Akagawa


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Pyrroloquinoline Quinone, a Redox-Active o-Quinone, Stimulates

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