ABSTRACT Objective: Pyrroloquinoline quinone (PQQ) is a novel supplement involved in processes such as mitochondrial biogenesis and cellular energy metabolism. Since endurance exercise and PQQ exhibit similar mechanisms for mitochondrial biogenesis, it is plausible that PQQ may have ergogenic value. Therefore, the purpose of this study was to examine the effects of a six-week endurance exercise training program on mitochondrial biogenesis and aerobic performance in nonendurance-trained males.
Methods: Twenty-three males were randomized to consume 20 mg/day of PQQ or placebo (PLC). Both groups followed a supervised six-week endurance exercise training program. Body composition was assessed by dual-energy-x-ray-absorptiometry (DEXA). Aerobic exercise performance and peroxisome proliferator-activated receptor c coactivator-1a (PGC-1a), a biochemical marker for mitochondrial biogenesis, were assessed before and after the six-week endurance training/supplementation program.
Results: There were no significant differences between groups in aerobic performance after endurance-training (p > 0.05). However, there were significant improvements in peak oxygen consumption (VO2peak) and total exercise test duration after endurance-training, irrespective of group (p < 0.05). The PQQ group had a significant increase in PGC-1a protein levels from baseline to post endurance training compared to PLC (p < 0.05). Furthermore, the PQQ group had higher PGC-1a protein levels after 6 weeks of endurance training compared to PLC (p < 0.05). Conclusions: Supplementation of PQQ does not appear to elicit any ergogenic effects regarding aerobic performance or body composition but appears to impact mitochondrial biogenesis by way of significant elevations in PGC-1a protein content.
Abbreviations: ATP: Adenosine triphosphate; CS: Citrate synthase; CREB: cAMP response elementbinding protein, ETC: Electron transport chain; LDH: Lactate dehydrogenase; mRNA: messenger ribonucleic acid; mtDNA: Mitochondrial DNA; NADþ: Oxidized Nicotinamide adenine dinucleotide; NADH: Nicotinamide adenine dinucleotide; NRF-1: Nuclear receptor factor 1; NRF-2: Nuclear receptor factor 2; PGC-1a: Peroxisome proliferator-activated receptor c coactivator-1a; PLC: Placebo; PQQ: Pyrroloquinoline quinone; RER: Respiratory exchange ratio; TFAM: Mitochondrial transcription factor A; VO2peak: Peak oxygen uptake
Introduction
Aerobic exercise training is widely regarded as a potent modality through which there is an enhancement in physical performance and the conferring of health benefits that can instigate intramuscular adaptations to improve oxidative capacity, mitochondrial density, capillary density, resistance to fatigue, greater oxygen utilization and phosphorylation to occur for ATP generation (1). This continual generation of ATP is essential to skeletal muscle bioenergetics, especially in response to cellular stress such as exercise. Therefore, strategic approaches in exercise training to instigate mitochondrial biogenesis can be relevant toward the metabolic plasticity of skeletal muscle (3). transportation, and subsequent elevations in mitochondrial function (1,2). Moreover, exercise-induced enhancements in mitochondrial function correspond to the efficiency at which it can regulate skeletal muscle function to improve muscular performance via the utilization of oxygen and the mediated delivery of electrons from reducing equivalents to allow for oxidative.
Aerobic exercise training is known to induce the up-regulation of mitochondrial biogenesis through which the synthesis and incorporation of new proteins and mitochondrial DNA (mtDNA) transcription occurs (1,4–6). Mitochondrial biogenesis is initiated with the increased transcription of both nuclear DNA and the 13 genes comprising mtDNA (7,8). Peroxisome proliferator-activated receptor c coactivator-1a (PGC-1a) is identified as a transcriptional co activator that is a master regulator for mitochondrial biogenesis (9–11). This transcriptional co-activator stimulates mitochondrial biogenesis through the activation of nuclear respiratory factors 1 and 2 (NRF-1, NRF-2). Moreover, NRF-1/2 functions to promote mitochondrial biogenesis through the regulation and activation of nuclear gene mitochondrial transcription factor A (TFAM) (9,10). This multifunctional protein plays an essential role in mtDNA replication and packaging, as well as in the transcription of mtDNA (9). Overall, the process of expanding preexisting mitochondria via mitochondrial biogenesis allows for increases in mitochondrial mRNA expression, protein content, number, mitochondrial size, and function in response to changes within energy status, and improvements in oxidative ability (3). Up-regulations in PGC-1a activity could enhance oxidative capacity, increase fatty acid b-oxidation, attenuate muscle glycogenolysis, delay the onset of muscle fatigue, and improve aerobic exercise performance (3–5).
Pyrroloquinoline quinone (PQQ) has been identified as a novel supplement that appears to promote benefits related to cognitive, immune, anti-diabetic, and anti-oxidative properties (12,13). Furthermore, there is data suggesting its role in continuous redox-cycling activity to oxidize nicotinamide adenine dinucleotide (NADH) to modulate lactate dehydrogenase (LDH) activity in an animal model (14). Moreover, there has been research examining the mechanisms of action behind PQQ supplementation on mitochondrial biogenesis (13,15–18). In fact, there are data in rodents to show that PQQ treatment enabled elevations in markers of mitochondrial biogenesis such as PGC-1a, NRF-1/2, cAMP response element-binding protein (CREB), as well as TFAM (15,16). Furthermore, the overexpression of PGC-1a activity in skeletal muscle has been attributed to the remodeling of muscle tissue to a fiber-type composition that is metabolically more oxidative and less glycolytic (6,9,19).
PQQ cannot be synthesized by the human body, so it must be obtained through a diet of nutrient-rich, plantbased foods or exogenous supplementation (12,13). To this respect, the supplemental use of PQQ has been examined based on its properties as an antioxidant as well as its role impacting mitochondrial function and biogenesis (12,15,18,20,21). However, most of the research has solely explored the effects of PQQ upon redox modulation and mitochondrial biogenesis within rodents (15,16). Therefore, there is a paucity of research investigating the effectiveness of PQQ within humans (22). There is strong evidence supporting that aerobic exercise can upregulate PGC-1a expression and instigate adaptations to improve mitochondrial function and oxidative capacity (3,4,9,10). Therefore, it is plausible that supplemental PQQ in conjunction with aerobic exercise training in humans could provide a differential additive effect toward being an ergogenic aid, but the literature is non-existent.
Therefore, the purpose of this study was to investigate the effects of PQQ supplementation following a six-week endurance exercise training program on aerobic performance and indices of mitochondrial biogenesis (PGC-1a content) in non-endurance-trained young males.
Materials and methods
Experimental approach
In a randomized, double-blind, placebo-controlled, parallel experimental design, participants visited the laboratory on three separate occasions in the following manner: visit 1 ¼ entry/familiarization session, visit 2 ¼ baseline testing prior to six weeks of aerobic exercise training/supplement distribution, visit 3 ¼ follow-up testing after six weeks of aerobic exercise training/supplementation. Prior to the testing sessions at visit 2 and 3, participants had their body composition assessed, provided their 48-hour dietary intake, and performed an aerobic exercise performance test. Muscle biopsies were taken prior to each exercise testing session. Blood pressure was assessed prior to and immediately following exercise and heart rate was assessed continually during exercise. Figure 1 presents an illustration of the experimental protocol for testing sessions in the lab during visits 2 and 3.
Participants
Twenty-three apparently healthy, non-endurance trained males who had not been involved in a habitual endurance training program for more than one hour per week for at least 6 months prior to the onset of the study and were between the ages of 18–30 years volunteered to participate in the proposed study. Only participants considered as either low or moderate risk and with no contraindications to exercise as outlined by the American College of Sports Medicine (ACSM) and who have not consumed any nutritional supplements (excluding multi-vitamins) 3 months prior to the study participated. All eligible participants, in a double-blind manner, were randomly assigned to one of two treatment groups involving placebo (PLC) and PQQ using a random number generator (www.random.com). There were 12 participants in the PQQ group and 11 within the PLC group. All participants signed university-approved informed consent documents and approval was granted by the Institutional Review Board for the Protection of Human Subjects in Research of Baylor University. Additionally, all experimental procedures involved in the study conformed to the ethical consideration of the Declaration of Helsinki.
Body composition testing
At each testing session for visit 2 and 3, total body mass (kg) was determined on a standard dual beam balance scale (Detecto Bridgeview, IL). Percent body fat, fat mass, and fatfree mass were determined using DEXA (Hologic Discovery Series W, Waltham, MA, USA). Quality control calibration procedures were performed on a spine phantom (Hologic X-CALIBER Model DPA/QDR-1 anthropometric spine phantom) and a density step calibration phantom prior to each testing session. Total body water (total, intracellular, and extracellular) was determined with bioelectrical impedance (Tanita Inc., Arlington Heights, IL, USA) using a low energy, high frequency current (500 micro amps at a frequency of 50 kHz).
Peak oxygen consumption exercise sessions
Before and after six weeks of aerobic training and supplementation (visit 2 and 3), participants performed a graded exercise test on a bicycle ergometer (Medgraphics, Monark, Ergomedic, Model No. 828E, St Paul, MN) based on an established protocol (23). The participants warmed up on the bike for four minutes with a workload of 15 Watts. Following the warm up, the cycle ergometer work rate began to increase by 15 Watts per minute increments. The participants maintained a pedaling speed of 60 revolutions per minute (rpm) throughout the entire test. This incremental work rate continued until the participants could no longer maintain 60 rpm or volitionally decided to stop the test. Subjects were verbally encouraged by study personnel to provide a true maximal effort in order to achieve peak oxygen consumption (VO2peak). Additional variables measured included the respiratory exchange ratio (RER), ventilation, maximal heart rate and the exercise test duration. Oxygen consumption was measured every 15 seconds via an opencircuit sampling system (Parvo Medics, Provo, UT, USA). Blood pressure was determined before and after the exercise session utilizing a mercurial sphygmomanometer with standard procedures and heart rate was monitored continuously for each participant throughout the testing session using a heart rate monitor (Polar RS400, Polar Inc, Lake Success, NY).
It is important to note that participants did not perform the aerobic exercise test in a fasted state. However, dietary conditions prior to both exercise testing sessions as visit 2 and 3 were controlled with participants being instructed to consume the identical meal prior to both testing sessions.
Furthermore, both exercise testing sessions were conducted at the same time of day with respect to each participant.
Aerobic exercise training protocol
Participants participated in an aerobic exercise training program five days weekly for a total duration of six weeks. Participants signed up for designated training times and sessions were supervised by study personnel. For the first two weeks, participants rode a stationary bicycle ergometer at an intensity based on the heart rate that coincided with 65% of the VO2peak achieved during the baseline bicycle exercise test. Their training zone was defined as this heart rate (±6 beats per minute). Heart rate was monitored continuously for each participant throughout each training session using a heart rate monitor (Polar RS400, Polar Inc, Lake Success, NY). The objective was to accumulate 30 minutes at each exercise session of continuous aerobic exercise in the prescribed heart rate zone. During the first two weeks, the flywheel load on the bicycle ergometer was adjusted by study personnel to maintain the target intensity. For the remaining four weeks of training, intensity was increased to a level equivalent to a heart rate (±6 beats per minute) corresponding to 75% of the VO2peak. Flywheel load was adjusted by study personnel to maintain the prescribed intensity.
Supplementation protocol
In a double-blind fashion, participants were randomly assigned to orally ingest 20 mg of cellulose PLC (n ¼ 11) or 20 mg of PQQ (n ¼ 12) (Nascent Health Sciences, LLC, New York, NY, USA) daily during the six-week exercise training period. Based on independent analysis (Weifang Shengyu Pharma Co, Ltd.), the PQQ product was shown to be 99% pure. Both supplements were in capsule form and identical in size, shape and color. Upon reporting to the laboratory, 30 minutes prior to exercise training, study personnel provided each participant with their respective supplement. On non-exercise days, participants were instructed to take their respective supplements in the morning upon waking. Supplement compliance was evaluated through the use of a standardized weekly supplement compliance form throughout the training period.
Dietary records and analyses
The participants’ diets were not standardized, and they were asked not to change their dietary habits throughout the study. However, participants were required to keep dietary records for 48 hours prior to the testing session at Visit 2 and 3. The 48-hour dietary records were evaluated with the Food Processor dietary assessment software program (ESHA Research, Salem OR, USA) to determine the average daily macronutrient consumption of fat, carbohydrate, and protein in the diet prior to supplementation and exercise.
Muscle biopsies
Using a 14-gauge fine-needle aspiration procedure, percutaneous muscle biopsies (30–40 mg) were obtained from the middle portion of the vastus lateralis muscle of the leg at the midpoint between the patella and the greater trochanter of the femur at a depth between 1 and 2 cm under local anesthesia with 1% Lidocaine based on our standard laboratory protocol (24). The leg used for the baseline initial biopsy at Visit 2 was randomly selected using a random number generator (www.random.org). The next biopsy at Visit 3 involved the same leg. For both biopsies, attempts were made to extract the tissue from the same location by using the pre-biopsy scar, depth markings on the needle, and a successive puncture that was made approximately 0.5 cm to the former from medial to lateral. Following removal, muscle samples were immediately frozen in liquid nitrogen and stored at -80 C for later analysis. Three muscle samples were obtained at visit 2 at baseline, post exercise testing at 30 minutes and 2 hours. Moreover, 3 additional muscle samples were obtained at visit 3 in the same time-oriented manner. This summated to 6 total muscle biopsies performed during the study.
Skeletal muscle cellular extraction
Based on our previous approach (25,26), approximately 20 mg of each muscle sample was weighed and subsequently homogenized using a commercial cell extraction buffer (Biosource, Camarillo, CA) and a tissue homogenizer. The cell extraction buffer was supplemented with 1 mM phenylmethanesulphonylfluoride (PMSF) and a protease inhibitor cocktail (Sigma Chemical Company, St. Louis, MO) with broad specificity for the inhibition of serine, cysteine, and metallo-proteases.
Muscle protein quantification
From the muscle tissue sample obtained at the testing sessions during Visit 2 and 3, total protein was quantified from the skeletal muscle cellular extracts spectrophotometrically based on the DC colorimetric protein assay method at a wavelength of 750 nm (Bio-Rad Hercules, CA, USA) using bovine serum albumin as the protein standard.
Skeletal muscle marker of mitochondrial biogenesis
Muscle samples were analyzed in duplicate for protein levels of PGC-1a (Biomatik, Cambridge ON, Canada) using an enzyme-linked immunoabsorbent assay (ELISA) Kit based on manufacturer’s guidelines. The absorbances, which was directly proportional to the concentration of analyte in the sample, was measured at a wavelength of 450 nm using a microplate reader (iMark, Bio-Rad, Hercules, CA). A set of standards of known concentrations for PGC-1a protein was utilized to construct a standard curve by plotting the net absorbance values of the standards against the respective peptide concentrations. Using data reduction software (Microplate Manager, Bio-Rad, Hercules, CA), PGC-1a concentrations were determined and normalized to muscle total protein content. The coefficient of variation for the analysis of PGC-1a was 5.91%.
Statistical analyses
Independent t-test was calculated on each dependent variable separately to determine if significant differences existed at baseline between the two groups. If a significant difference existed at baseline in any of the dependent variables, the one-way analysis of covariance (ANCOVA) test was analyzed for the affected dependent variable while accounting for the baseline/pre-exercise time point value as a covariate. If the analyzed dependent variable was not significantly different between groups at baseline, Levenes test of homogeneity of variance was then implemented. If this statistical test failed for homogeneity of variance, nonparametric analyses such as the Mann-Whitney U and Wilcoxon Signed-rank Tests were conducted. All variables were tested for normality through the utilization of the Shapiro Wilk Test as well as visually analyzing the distribution via the Q-Q plots. If these tests were successively passed, separate 2 x 2 [Group (PQQ, PLC] x Time [Pre, Post] mixed methods factorial analyses of variance (ANOVA) were carried out for all criterion variables of interest pertaining to the 6 weeks aerobic training adaptations. If a significant interaction effect was present between group and time, simple effects analyses were conducted. However, if no significant interaction effects were present, main effects for group and time were analyzed via pair-wise comparisons from the statistical output. To control for alpha inflation of the ANOVA, the Bonferroni correction test was utilized.
An a-priori power calculation showed that 10 participants per group was adequate to detect a significant difference between groups in the marker of PGC-1a mRNA expression in response to aerobic exercise training, given a type I error rate of 0.05 and a power of 0.80. In addition to reporting probability values, an index of effect size was reported to reflect the magnitude of the observed effect. The index of effect size utilized was partial Eta squared (g2 ), which estimates the proportion of variance in the dependent variable that can be explained by the independent variable. Partial Eta squared effect sizes were determined to be: weak ¼ 0.17, medium ¼ 0.24, strong ¼ 0.51, very strong ¼ 0.70 (27). All statistical procedures were performed using SPSS 25.0 software and a probability level of < 0.05 was adopted throughout the study. In addition, for all statistical analyses not meeting the sphericity assumption for the within-subjects’ analyses, a Huynh-Feldt correction factor was applied to the degrees of freedom in order to adjust (increase) the critical F-value to a level that would prevent the likelihood of committing a type I error. All data was presented as mean ± standard deviation (SD).
Results
Compliance and anthropometric baseline data
The group-specific baseline anthropometric, hemodynamic, and VO2peak data describing the 23 participants who completed the study are expressed in Table 1. A total of 55 individuals who reported interest in participating in this training study were initially screened. However, following the screening process, 24 participants were eligible and began participation in the study. However, one participant failed to finish the study due to compliance conflicts with the training program. Therefore, the 23 remaining participants completed the requirements for the study and were 100% compliant by attending every exercise training session.
Dietary intake
All 23 participants recorded their food intake for two consecutive days prior to each laboratory visit at the beginning and end of the six-week endurance training study. Moreover, independent t-test statistical analyses were completed on the dietary food logs corresponding to visit 2 and 3 for each participant. There were no significant differences for total kilocalories, fat, carbohydrate, or protein intake between PQQ and PLC groups (p > 0.05). The data for kilocalorie, fat, carbohydrate, and protein intake for both groups are presented in Table 2. All 23 participants had 100% supplement compliance and successively completed every training session throughout the six weeks.
Body composition
The changes in total body mass, lean mass, fat mass and total body water due to six weeks of aerobic training are presented in Table 3. There were no statistically significant interactions between group and time for changes in lean mass (p ¼ 0.665; partial η2 ¼ 0.009), fat mass (p ¼ 0.260; partial η2 ¼ 0.060), and total body water (p ¼ 0.811; partial η2 ¼ 0.001). Furthermore, the main effect of group revealed no statistically significant changes throughout the study in lean mass (p ¼ 0.739; partial η2 ¼0.005), fat mass (p ¼ 0.488; partial η2 ¼ 0.023), and total body water (p ¼ 0.611; partial η2 ¼ 0.013). Similarly, the main effect of time also revealed no statistically significant differences in fat mass (p ¼ 0.113; partial η2 ¼ 0.115) and total body water (p ¼ 0.988; partial η2 ¼
Conclusions
Our data add to the ongoing investigation behind the functional significance of this vitamin-like compound. There is a scarcity of research to affirm the physiological functions of PQQ within humans. Nevertheless, this is the first exercise training study investigating the effects of PQQ supplementation in humans. There is a need for future studies to both replicate and examine if variations in endurance training modalities may incur similar or greater benefits. Furthermore, based upon the results herein, PQQ supplementation does not appear to elicit any ergogenic effects regarding aerobic performance or body composition but appears to impact mitochondrial biogenesis by way of significant elevations in PGC-1a protein content.
