Effect of Quercetin Treatment on Mitochondrial Biogenesis

nutrients

Article Eﬀect of Quercetin Treatment on Mitochondrial Biogenesis and Exercise-Induced AMP-Activated Protein Kinase Activation in Rat Skeletal Muscle

Keiichi Koshinaka 1,*, Asuka Honda 1, Hiroyuki Masuda 2 and Akiko Sato 1 1 Department of Health and Sports, Niigata University of Health and Welfare, 1398 Shimami-cho, Kita-ku, Niigata 950-3198, Japan; [email protected] (A.H.); [email protected] (A.S.) 2 Department of Health and Nutrition, Niigata University of Health and Welfare, 1398 Shimami-cho, Kita-ku, Niigata 950-3198, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-25-257-4761

Received: 16 February 2020; Accepted: 7 March 2020; Published: 10 March 2020

Abstract: The purpose of this study was to evaluate the effect of chronic quercetin treatment on mitochondrial biogenesis, endurance exercise performance and activation levels of AMP-activated protein kinase (AMPK) in rat skeletal muscle. Rats were assigned to a control or quercetin group and were fed for 7 days. Rats treated with quercetin showed no changes in the protein levels of citrate synthase or cytochrome C oxidase IV or those of sirtuin 1, peroxisome proliferator-activated receptor gamma coactivator-1α or phosphorylated AMPK. After endurance swimming exercise, quercetin-treated rats demonstrated no differences in blood and muscle lactate levels or glycogen utilization speed compared to control rats. These results indicate that quercetin treatment does not stimulate mitochondrial biogenesis in skeletal muscle and does not influence metabolism in a way that might enhance endurance exercise capacity. On the other hand, the AMPK phosphorylation level immediately after exercise was significantly lower in quercetin-treated muscles, suggesting that quercetin treatment might provide a disadvantage to muscle adaptation when administered with exercise training. The molecular results of this study indicate that quercetin treatment may not be advantageous for improving endurance exercise performance, at least after high-dose and short-term therapy.

Keywords: AMPK; rat; skeletal muscle; quercetin; exercise

1. Introduction Quercetin is one of the most common flavonoids found in many foods, including onions, red wines, blueberries and leeks [1]. Quercetin is a powerful antioxidant and has been shown to have cardioprotective and anti-inflammatory properties and to reduce the risk of metabolic syndrome and muscle atrophy [2–5]. In addition to its pharmacological effects, quercetin is thought to have possible ergogenic effects, especially in endurance exercise. Several human and rodent studies [6–9] demonstrated that quercetin treatment increased VO2 max and time to fatigue and improved performance in the 30-km time trial and maximal 12-min test. It is well known that mitochondrial biogenesis in skeletal muscle plays a substantial role in determining endurance exercise performance. Exercise training improves performance and increases mitochondrial content. From this perspective, a landmark study conducted by Davis et al. [7] has received the most attention in the field of quercetin-related research. The authors demonstrated that relatively short-term (7 days) quercetin supplementation increased mitochondrial biogenesis in mouse skeletal muscle in the absence of exercise training and resulted in a remarkable improvement in endurance exercise capacity. However, a subsequent study in humans did not replicate this exercise-like

Nutrients 2020, 12, 729; doi:10.3390/nu12030729 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 729 2 of 13 effect on mitochondrial biogenesis [9], while another human study found only a weak effect [10]. Although the exact cause of this discrepancy is unclear, authors of previous studies hypothesized that quercetin’s effect on mitochondrial biogenesis may differ between human and rodents [10–12]. If this is true, it will have a major impact because rodent-derived cells and tissues are extremely beneficial for identifying bioactive substances that stimulate mitochondrial biogenesis and for analyzing the molecular mechanisms of plant-derived substances such as polyphenols. Thus far there is very little information regarding quercetin’s effect on mitochondria in skeletal muscle, despite the experimental importance of this issue. We hypothesized that quercetin treatment might not be a stimulus for inducing mitochondrial biogenesis even in rodent skeletal muscle. Mitochondrial biogenesis is a complicated process in which peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α has been shown to play a major role [13]. Increased activity of PGC-1α induced by PGC-1α deacetylation and phosphorylation results in stimulation of mitochondrial DNA (mtDNA) replication and gene expression. It is well documented that AMP-activated protein kinase (AMPK) plays a role in both deacetylation and phosphorylation [13]. AMPK activation increases PGC-1α deacetylase sirtuin (SIRT) 1 activity via nicotinamide adenine dinucleotide stimulation and also increases PGC-1α phosphorylation as a direct effect of AMPK kinase activity. Furthermore, AMPK activation increases both PGC-1α and SIRT1 protein expression levels, contributing to the activities of these molecules. In this context, AMPK is considered to be a master switch that induces mitochondrial biogenesis and it is expected to be activated by quercetin treatment if quercetin has an impact on mitochondria. Indeed, in cultured L6 [14] and C2C12 cells [15,16], AMPK activation levels were successfully increased after incubation with quercetin. However, to the best of our knowledge, no studies thus far have examined the in vivo effects of acute and chronic oral quercetin treatment on muscle AMPK activation levels. Importantly, a previous study reported that resveratrol, another polyphenol that promotes mitochondrial biogenesis, stimulated muscle AMPK and increased mitochondrial biogenesis only in cultured cells and not following in vivo treatment [17]. Therefore, we consider it important to identify quercetin’s effect on muscle AMPK in vivo to determine whether quercetin impacts mitochondrial biogenesis in living organisms. In contrast to the findings discussed above, accumulating studies have suggested that antioxidant supplementation hampers several acute exercise-induced cell signaling responses as well as training-induced adaptations, including mitochondrial biogenesis [18,19]. Since quercetin is a powerful antioxidant, when administered along with exercise it might have a detrimental effect on exercised skeletal muscle. Several studies have demonstrated that exercise stimulates AMPK activation [20,21]. Therefore, we hypothesized that quercetin treatment would result in reduced AMPK activation in response to exercise. In the present study, in addition to focusing on the effects of quercetin treatment on the basal (resting) level of AMPK in skeletal muscle, we also investigated the impact of quercetin on exercise-induced AMPK activation. The primary purpose of this study was to evaluate the effect of chronic quercetin treatment on mitochondrial biogenesis in rat skeletal muscle and to compare our results with those of the previous landmark study [7]. Quercetin’s possible ergogenic effect was also assessed by measuring the rate of glycogen expenditure during submaximal endurance exercise. The second goal of this study was to investigate the effect of chronic quercetin treatment on AMPK activation both at baseline and immediately after acute exercise. To further characterize the possible effect of quercetin on skeletal muscle, AMPK activation levels were measured both after acute administration of quercetin in vivo and acute treatment in vitro.

2. Materials and Methods

2.1. Animals This research was approved by the Animal Studies Committee of Niigata University of Health and Welfare (29015-01013). Male Wistar rats (4 weeks, n = 65) were obtained from CLEA Japan Nutrients 2020, 12, 729 3 of 13

(Tokyo, Japan). Rats were housed individually at constant room temperature (23 C 1) in a 12-h ◦ ± light (06:00 18:00 h)/12-h dark cycle and were provided standard laboratory chow (MF: Oriental Yeast, − Tokyo, Japan) and water ad libitum till experimental day (1 week later).

2.2. Quercetin Stimulation in Vitro After 3-h fasting (09:00), rats were anesthetized by pentobarbital sodium (50 mg/body weight (BW)), and epitrochlearis muscles, located in the upper arm, were dissected out. The muscles were then incubated with shaking for 30 min at 35 ◦C in 4 mL of oxygenated Krebs-Henseleit buﬀer containing 8 mM glucose, 32 mM mannitol and 0.1% radioimmunoassay-grade bovine serum albumin, in the absence or presence of 100 µM quercetin (ChromaDex, Los Angeles, CA, USA), 100 µM resveratrol (Fujiﬁlm Wako Pure Chemical, Osaka, Japan) or 0.5 mM 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) (Tronto Research Chemical, North York, Canada) in 0.1% DMSO. Flasks were gassed continuously with 95% O2/5% CO2 during incubation. After incubation, muscles were clamp-frozen in liquid nitrogen for western blot analysis.

2.3. Acute Effect of Quercetin Administration After 3-h fasting, rats were orally administrated either orange-flavored Tang (Kraft Foods, Northfield, IL, USA) [7] as control or quercetin (25 mg/kg BW) mixed with Tang. Then, rats were maintained in a fasting and resting condition for 4 h. After that, epitrochlearis muscles were dissected out under anesthesia and clamp-frozen in liquid nitrogen for western blot analysis.

2.4. Chronic (7 Days) Eﬀect of Quercetin Treatment Rats were randomly assigned to either a control or quercetin group and were maintained under ad libitum feeding for 7 days. During this period, rats were given once-daily oral supplementation of either Tang or quercetin as described above, with daily measurement of food intake. On day 7, the last supplementation was administered at 17:00. On the next morning (06:00), food was removed and rats were maintained in the fasting state for 3 h. After that, soleus and epitrochlearis muscles from some rats were dissected out under anesthesia and clamp-frozen in liquid nitrogen for western blot analysis and evaluation of muscle metabolites. Also, epididymal and retroperitoneal fat pads were removed and weighed. The remaining rats were used for endurance exercise testing before sacriﬁce.

2.5. Endurance Exercise Testing On subsequent 2 days before testing, all rats have become accustomed to swimming for 10 min/day without a weight. On the testing day, after 3-h fasting, rats performed a swimming exercise for 1 h with a weight equal to ~2.5% of body weight. Immediately after exercise, blood was taken by tail-tip cut and then rats were rapidly anesthetized with isoﬂurane. Epitrochlearis muscles were dissected out under anesthesia and clamp-frozen in liquid nitrogen for measurement of muscle metabolites and western blot analysis.

2.6. Blood Parameters Blood glucose and lactic acid levels were measured using product Glutest Every (Sanwa Kagaku, Nagoya, Japan) and Lactate pro (Arkray, Kyoto, Japan), respectively.

2.7. Western Blot Analysis Epitrochlearis and soleus muscles were homogenized as previously described [21]. Primary antibodies were purchased as follows. From Cell Signaling Technology (Beverly, MA, USA); anti-AMPK-α, phospho-(p-)AMPK (Thr172), citrate synthase (CS), cytochrome C oxidase (COX) IV and SIRT1. From Merck (Temecula, CA, USA); anti-PGC-1α, monocarboxylate transporter (MCT) 1, MCT4 and MCT4. Anti-carnitine palmitoyltransferase (CPT) 1B, glucose transporter (GLUT) 4 and Nutrients 2020, 12, 729 4 of 13

Nutrients 2020, 12, x FOR PEER REVIEW 4 of 13 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Abcam (Tokyo, Japan), Biogenesis (Poole, United Kingdom) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Equal Biogenesis (Poole, United Kingdom) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Equal protein concentrations were loaded in each lane and also visually confirmed by coomassie brilliant protein concentrations were loaded in each lane and also visually conﬁrmed by coomassie brilliant blue blue staining of the blot membrane. The phosphorylated AMPK abundance was normalized by staining of the blot membrane. The phosphorylated AMPK abundance was normalized by AMPK-α AMPK-α abundance and other proteins were normalized by GAPDH abundance. abundance and other proteins were normalized by GAPDH abundance.

2.8.2.8. Muscle Muscle metabolites Metabolites EpitrochlearisEpitrochlearis muscles muscles were were homogenized homogenized in in0.3 0.3 M Mperchloric perchloric acid acid (PCA) (PCA) and and the the extracts extracts were were usedused to tomeasure measure glycogen glycogen by by the the amyloglucosidase amyloglucosidase method method [22]. [22 The]. The remaining remaining PCA PCA extracts extracts were were centrifuged at 1,000 g for 10 min at 4°C. After centrifugation, the supernatant was collected and centrifuged at 1000 g for 10 min at 4 ◦C. After centrifugation, the supernatant was collected and neutralizedneutralized by by the the addition addition of of2M 2M KOH, KOH, followe followedd by by fluorometric ﬂuorometric measurem measurementsents of oflactate lactate [23]. [23 ].

2.9.2.9. Statistics Statistics ValuesValues are are expressed expressed as asmeans means ± SE.SE. Differences Diﬀerences between between groups groups were were determined determined using using an an ± unpairedunpaired t-test.t-test. p

3. Results3. Results

3.1.3.1. Quercetin Quercetin stimulation Stimulation in vitro In Vitro WeWe first ﬁrst examined examined whetherwhether quercetin quercetin aﬀ affectedected AMPK AM inPK mature, in mature, intact intact skeletal skeletal muscle bymuscle measuring by measuringAMPK phosphorylation AMPK phosphorylation at Thr 172, at aThr critical 172, site a critical for AMPK site activationfor AMPK (Figure activation1A). Following(Figure 1A). the Followingshort, 30-min the short, incubation 30-min period incubation with epitrochlearisperiod with epitrochlearis muscle, AICAR, muscle, a pharmacological AICAR, a pharmacological AMPK agonist, AMPKsuccessfully agonist, increasedsuccessfully the increased AMPK phosphorylation the AMPK phosphorylation level, whereas level, quercetin whereas did quercetin not. In did contrast, not. In resveratrol,contrast, resveratrol, which is anotherwhich ispolyphenol another polyphenol that is known that is to known be an to AMPK be an stimulantAMPK stimulant [17,24], increased[17,24], increasedAMPK phosphorylationAMPK phosphorylation levels after levels incubation after incubation for the same for the duration same duration (Figure1 (FigureB). 1B).

FigureFigure 1. Effect 1. Eﬀect of ofquercetin quercetin stimulation stimulation on onAMPK AMPK phosphorylation phosphorylation in invitro. vitro Isolated. Isolated epitrochlearis epitrochlearis µ musclesmuscles were were incubated incubated for 30 for min 30 mineither either in the in absence the absence (-) or presence (-) or presence (+) of 100 (+ )μM of quercetin 100 M quercetin (A) or (A) or 100 µM resveratrol (B). Values are means SE (n = 4–5). Sample stimulated with 0.5 mM 100 μM resveratrol (B). Values are means ± SE (n± = 4–5). Sample stimulated with 0.5 mM 5- aminoimidazole-4-carboxamide-1-beta-d-ribofuranosi5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranosidede (AICAR) (AICAR) was used was as used a positive as a positive control control (n = (n = 1). A representative blot is shown above each ﬁgures. The obtained values of p-AMPK were 1). A representative blot is shown above each figures. The obtained values of p-AMPK were normalized with the value for AMPK. #p < 0.05 vs. resveratrol (-). C: control (absence of stimulant), Q: normalized with the value for AMPK. #p < 0.05 vs. resveratrol (-). C: control (absence of stimulant), quercetin, R: resveratrol, A: AICAR. Q: quercetin, R: resveratrol, A: AICAR.

3.2. Acute effect of quercetin administration

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3.2. Acute Effect of Quercetin Administration Nutrients 2020, 12, x FOR PEER REVIEW 5 of 13 No previous studies have examined the effect of a single quercetin application to AMPK in skeletal muscleNo previousin vivo .studies A positive have e ffexaminedect on AMPK the effect would of suggesta single that quercetin quercetin application facilitates to mitochondrial AMPK in skeletalbiogenesis. muscle However,in vivo. weA positive found that effect AMPK on AMPK phosphorylation would suggest levels werethat quercetin unaffected facilitates by a single mitochondrialquercetinNutrients 2020 application biogenesis., 12, x FOR PEER to However, the REVIEW epitrochlearis we found muscle that AMPK (Figure phosphorylation2). levels were unaffected5 of 13 by a single quercetin application to the epitrochlearis muscle (Figure 2). No previous studies have examined the effect of a single quercetin application to AMPK in skeletal muscle in vivo. A positive effect on AMPK would suggest that quercetin facilitates mitochondrial biogenesis. However, we found that AMPK phosphorylation levels were unaffected by a single quercetin application to the epitrochlearis muscle (Figure 2).

FigureFigure 2. Acute 2. Acute effect eﬀ ofect quercetin of quercetin administration administration on AMPK on AMPK phosphorylation. phosphorylation. Epitrochlearis Epitrochlearis muscles muscles

werewere dissected dissected out out6 hours 6 h after after oral oral quercetin quercetin or or vehicle vehicle (control) (control) administration. administration. A A representative representative blot blotis is shownFigure shown 2. above Acuteabove figure.effect figure. of The quercetin The obtained obtained administration values values of p-AMPKonof AMPKp-AMPK phosphorylation. were were normalized normalized Epitrochlearis with with the the value muscles value for AMPK.for were dissected out 6 hours after oral quercetin or vehicle (control) administration. A representative AMPK.Values Values are means are meansSE ± (n SE= (6).n = C: 6). control, C: control, Q: quercetin. Q: quercetin. blot is shown above± figure. The obtained values of p-AMPK were normalized with the value for 3.3. ChronicAMPK. (7 Values days) are Eff ectmeans of Quercetin± SE (n = 6). Treatment C: control, Q: (with quercetin. Epitrochlearis Muscle) 3.3. Chronic (7 days) effect of quercetin treatment (with epitrochlearis muscle) To investigate whether repeated quercetin administration affected mitochondrial biogenesis, rats To3.3. investigate Chronic (7 days) whether effect ofrepeated quercetin treatmentquercetin (with administration epitrochlearis muscle) affected mitochondrial biogenesis, were supplemented with quercetin for 7 days. Body weight increased gradually during the treatment rats were Tosupplemented investigate whether with quercetin repeated quercetinfor 7 days administration. Body weight affected increased mitochondrial gradually biogenesis, during the period but the rate of body weight gain was identical between the control and quercetin groups treatmentrats wereperiod supplemented but the rate with of body quercetin weight for ga7 indays was. Body identical weight between increased the gradually control andduring quercetin the (Figure3A). The rats in both groups ate the same amount of food throughout the treatment period groupstreatment (Figure period3A). The but ratsthe ratein both of body groups weight ate ga thein wassame identical amount between of food the throughout control and the quercetin treatment (Figure3B). As shown in Figure4A, quercetin supplementation did not a ffect epididymal fat mass. periodgroups (Figure (Figure 3B). As3A). shown The rats in inFigure both groups4A, querce ate thetin same supplementation amount of food did throughout not affect the epididymal treatment fat The retroperitoneal fat mass was slightly lower in the quercetin-treated group than the control group mass. periodThe retroperitoneal (Figure 3B). As fatshown mass in was Figure slightly 4A, querce lowetinr in supplementation the quercetin-treated did not affectgroup epididymal than the control fat butmass. the diThefference retroperitoneal was not fat statistically mass was slightly significant lowe (rp in= the0.09, quercetin-treated Figure4B). group than the control group but the difference was not statistically significant (p = 0.09, Figure 4B). group but the difference was not statistically significant (p = 0.09, Figure 4B).

FigureFigure 3. 3.Effects Effects ofof 7-day7-day quercetin quercetin treatment treatment on on body body weight weight and andfood foodintake. intake. Rats were Rats assigned were assigned to to Figureeithereither 3. Effects a controla control of or 7-day or quercetin quercetin quercetin groups groups treatment andand werewere on maintained body weight under under and ad ad libitumfood libitum intake. feeding feeding Rats for for 7were days. 7 days. assigned During During to this period,this period, dailybody daily weightbody weight (A) and (A) total and foodtotal food intake intake (B) were (B) were measured. measured. Values Values are are means means SE± SE (n (n= 13–14). either a control or quercetin groups and were maintained under ad libitum feeding for 7 days.± During this period,= 13–14). daily body weight (A) and total food intake (B) were measured. Values are means ± SE (n = 13–14).

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FigureFigure 4.4. EEffectsffects ofof 7-day7-day quercetin quercetin treatment treatment on on fat-pad fat-pad mass. mass. Rats Rats were were assigned assigned to to either either a control a control or quercetinor quercetin groups groups and and were were maintained maintained under under ad libitumad libitum feeding feeding for 7for days. 7 days. After After that, that, epididymal epididymal (A) and retroperitoneal (B) fat-pads were removed and weighed. Values are means SE (n = 6–7). (A) and retroperitoneal (B) fat-pads were removed and weighed. Values are means± ± SE (n = 6–7). Next, weFigure measured 4. Effects of mitochondrial7-day quercetin treatment content on fat-pad in epitrochlearis mass. Rats were assigned muscle to either obtained a control at the end of the Next, weor quercetin measured groups mitochondrialand were maintained content under ad libitumin epitro feedingchlearis for 7 days. muscle After that, obtained epididymal at the end of the treatment period. The citrate synthase (CS) and cytochrome C oxidase (COX) IV enzymes are treatment period.(A) and retroperitoneal The citrate (B) synthase fat-pads were (CS) removed and andcytochrome weighed. Values C oxidase are means (COX)± SE (n = 6–7).IV enzymes are major major indicators of mitochondrial content. Western blot analysis revealed that the levels of these indicators of mitochondrial content. Western blot analysis revealed that the levels of these proteins proteins wereNext, not we a ffmeasuredected by mitochondrial quercetin treatmentcontent in epitro (Figurechlearis5A muscle and B). obtained As supporting at the end of evidence, the there were not affected by quercetin treatment (Figure 5A and B). As supporting evidence, there were no were notreatment effects ofperiod. quercetin The citrate treatment synthase on(CS) several and cytochrome proteins C oxidase associated (COX) with IV enzymes mitochondrial are major biogenesis, effects ofindicators quercetin of mitochondrial treatment on content. several Western proteins blot an associatedalysis revealed with that mitochondr the levels of ialthese biogenesis, proteins including including PGC-1α (Figure5C) and SIRT1 (Figure5D). To better understand the metabolic impact PGC-1αwere (Figure not affected 5C) and by quercetin SIRT1 (Figure treatment 5D). (Figure To 5A better and B). understand As supporting the evidence, metabolic there impactwere no of quercetin of quercetineffects onof quercetin skeletal treatment muscle, on we several quantified proteins associated the protein with mitochondr expressionial levelsbiogenesis, of criticalincluding molecules in on skeletal muscle, we quantified the protein expression levels of critical molecules in glycolyticPGC-1/glycogenolyticα (Figure 5C) and and SIRT1 lipid (Figure metabolisms, 5D). To better namely understand glucose the metabolic transporter impact of (GLUT) quercetin 4 (Figure5E), glycolytic/glycogenolyticon skeletal muscle, andwe quantified lipid metabolisms, the protein namelyexpression glucose levels transporterof critical molecules (GLUT) in4 (Figure 5E), carnitine palmitoyltransferase (CPT) 1B (Figure5F), monocarboxylate transporter (MCT) 1 (Figure5G) carnitineglycolytic/glycogenolytic palmitoyltransferase and (CPT) lipid metabolisms, 1B (Figure namely 5F), monocarboxylateglucose transporter (GLUT) transporter 4 (Figure (MCT) 5E), 1 (Figure and MCT4 (Figure5H). The two groups did not di ffer in the expression levels of any of these molecules. 5G) andcarnitine MCT4 palmitoyltransferase (Figure 5H). The (CPT) two 1Bgroups (Figure did5F), monocarboxylatenot differ in the transporter expression (MCT) levels 1 (Figure of any of these Level of5G) glyceraldehyde-3-phosphate and MCT4 (Figure 5H). The two dehydrogenase groups did not di (GAPDH)ffer in the expression was identical levels of between any of these the two groups molecules. Level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identical between (control,molecules. 1.00 0.03; Level quercetin, of glyceraldehyde-3-phosphat 0.99 0.04). e dehydrogenase (GAPDH) was identical between the twothe groups two± groups (control, (control, 1.00 1.00 ± ±0.03; 0.03;± quercetin, 0.99 0.99 ± 0.04). ± 0.04).

Figure 5.FigureEﬀects 5. Effects of 7-day of 7-day quercetin quercetin treatmenttreatment on on metabolic metabolic molecules molecules in epitrochlearis in epitrochlearis muscles. Rats muscles. Rats were assignedwere assigned to either to either a control a control or or quercetinquercetin groups groups and andwere weremaintained maintained under ad underlibitum feeding ad libitum feeding for 7 days. After that, epitrochlearis muscles were dissected out as Western blot samples for CS (A), for 7 days. After that, epitrochlearis muscles were dissected out as Western blot samples for CS (A), COX IV (B), PGC-1 α (C), SIRT1 (D), GLUT4 (E), CPT1B (F), MCT1 (G) and MCT4 (H). A representative blot is shown above each ﬁgures. Values are means SE (n = 6–13). All values were normalized by Figure 5. Effects of 7-day quercetin treatment on metabolic± molecules in epitrochlearis muscles. Rats GAPDH abundance. C: control, Q: quercetin. were assigned to either a control or quercetin groups and were maintained under ad libitum feeding for 7 days. After that, epitrochlearis muscles were dissected out as Western blot samples for CS (A),

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NutrientsNutrients 2020 2020, 12, 12, x, xFOR FOR PEER PEER REVIEW REVIEW 7 of7 of 13 13 We next examined whether chronic quercetin treatment affected energy metabolism during exercise.COXCOX IV IV After(B), (B), PGC-1 thePGC-1 treatment α α(C), (C), SIRT1 period,SIRT1 (D), (D), the GLUT4 bloodGLUT4 lactate(E), (E), CPT1 CPT1 levelB B(F), did(F), MCT1 notMCT1 di ff(G) er(G) betweenand and MCT4 MCT4 the (H). two(H). A groupsA at restrepresentativerepresentative (basal level) blot blot (Figure is is shown shown6A). above Immediatelyabove ea eachch figures. figures. after Values Values endurance are are means means exercise, ± ±SE SE ( then ( n= =6–13). blood 6–13). Alllactate All values values level were were increased approximatelynormalizednormalized by by GAPDH two-foldGAPDH abundance. abundance. but this changeC: C: control, control, was Q: Q: identicalquercetin. quercetin. in both groups. Similarly, the lactate level in epitrochlearis muscle was identical in both groups at rest and immediately after exercise (Figure6B). WeWe Thenext next same examined examined magnitude whether whether of the chronic chronic lactate quercetin levels quercetin shown tr treatment ineatment Figure affected 6affected are associated energy energy metabolism withmetabolism glycogen during during levels at exercise.exercise.rest and After After during the the treatment exercisetreatment (Figure period, period,7 ).the the At blood rest,blood thelactat lactat glycogene elevel level leveldid did not innot epitrochlearisdiffer differ between between muscle the the two two was groups groups una ff atected at restrest by(basal (basal quercetin level) level) (Figure supplementation. (Figure 6A). 6A). Immediately Immediately One-hour after after endurance en endurancedurance exercise exercise, exercise, resulted the the blood blood in substantial lactate lactate level level consumption increased increased of approximatelyapproximatelymuscle glycogen two-fold two-fold in both but but groupsthis this change change but thewas was magnitudes identical identical in in both theboth twogroups. groups. groups Similarly, Similarly, were comparable. the the lactate lactate level level in in epitrochlearisepitrochlearisFigure muscle 8muscleA shows was was the identical identical e ffect ofin in exerciseboth both groups groups on AMPK at at rest rest phosphorylationand and immediately immediately levels after after exercise in exercise epitrochlearis (Figure (Figure 6B). muscle. 6B). BeforeTheThe same same exercise, magnitude magnitude quercetin of of the the had lactate lactate no e fflevels ect.levels Immediatelyshown shown in in Figure Figure after 6 exercise,6are are associated associated there with was with aglycogen glycogen two-fold levels increaselevels atat rest inrest theand and AMPK during during phosphorylation exercise exercise (Figure (Figure level 7). 7). inAt At the rest, rest, control the the glycogen groupglycogen and level level a significantly in in epitrochlearis epitrochlearis smaller muscle increasemuscle was was in the unaffectedunaffectedquercetin by by group quercetin quercetin (p < 0.05).supplementation. supplementation. This reduced One-ho responseOne-hourur toendurance endurance exercise wasexercise exercise not due resulted resulted to a di ffin erencein substantial substantial in AMPK consumptionconsumptioncontent (Figure of of muscle muscle8B). glycogen glycogen in in both both groups groups but but the the magnitudes magnitudes in in the the two two groups groups were were comparable.comparable.

Figure 6. Eﬀects of 7-day quercetin treatment on exercise-induced lactate metabolism. Rats were FigureFigure 6. 6. Effects Effects of of 7-day 7-day quercetin quercetin treatment treatment on on exercise-induced exercise-induced lactate lactate metabolism. metabolism. Rats Rats were were assigned to either a control or quercetin groups and were maintained under ad libitum feeding for 7 assignedassigned to to either either a acontrol control or or quercetin quercetin gr groupsoups and and were were maintained maintained under under ad ad libitum libitum feeding feeding for for 7 7 days. After that, rats underwent swimming exercise for 1 h. Immediately after exercise, blood was days.days. After After that, that, rats rats underwent underwent swimming swimming exercise exercise for for 1 1hour. hour. Immediately Immediately after after exercise, exercise, blood blood was was taken for blood lactate (A) and epitrochlearis muscles were dissected out as samples for muscle lactate takentaken for for blood blood lactate lactate (A) (A) and and epitrochlearis epitrochlearis muscles muscles were were dissected dissected out out as as samples samples for for muscle muscle lactate lactate (B). Values are means SE (n = 6–7). (B).(B). Values Values are are means means ± ±SE SE± (n ( n= =6–7). 6–7).

Figure 7. Eﬀects of 7-day quercetin treatment on exercise-induced glycogen metabolism. Rats were FigureFigure 7. 7. Effects Effects of of 7-day 7-day quercetin quercetin treatment treatment on on exerci exercise-inducedse-induced glycogen glycogen metabolism. metabolism. Rats Rats were were assigned to either a control or quercetin groups and were maintained under ad libitum feeding for 7 assigned to either a control or quercetin groups and were maintained under ad libitum feeding for 7 assigneddays. to After either that, a control rats underwent or quercetin swimming groups exerciseand were for maintained 1 h. Immediately under ad after libitum exercise, feeding epitrochlearis for 7 days. After that, rats underwent swimming exercise for 1 hour. Immediately after exercise, days.muscles After werethat, dissectedrats underwent out as samples. swimming Values exer arecise means for 1 SEhour. (n = 6–7).Immediately after exercise, epitrochlearisepitrochlearis muscles muscles were were dissected dissected out out as as samples. samples. Values Values are± are means means ± ±SE SE (n ( n= =6–7). 6–7).

FigureFigure 8A 8A shows shows the the effect effect of of exercise exercise on on AMPK AMPK phosphorylation phosphorylation levels levels in in epitrochlearis epitrochlearis muscle. muscle. BeforeBefore exercise, exercise, quercetin quercetin had had no no effect. effect. Immediately Immediately after after exercise, exercise, there there was was a atwo-fold two-fold increase increase in in thethe AMPK AMPK phosphorylation phosphorylation level level in in the the control control group group and and a asignificantly significantly smaller smaller increase increase in in the the

NutrientsNutrients 2020 2020, 12, 12, x, xFOR FOR PEER PEER REVIEW REVIEW 8 8of of 13 13 quercetinNutrientsquercetin2020 group group, 12, 729 ( p( p< <0.05). 0.05). This This reduced reduced response response to to exerci exercisese was was not not due due to to a adifference difference in in AMPK AMPK8 of 13 contentcontent (Figure (Figure 8B). 8B).

Figure 8. Effects of 7-day quercetin treatment on exercise-induced AMPK phosphorylation. Rats were FigureFigure 8. 8. Effects Effects of of 7-day 7-day quercetin quercetin treatment treatment on on exerci exercise-inducedse-induced AMPK AMPK phosph phosphorylation.orylation. Rats Rats were were assigned to either a control or quercetin groups and were maintained under ad libitum feeding for 7 assignedassigned to to either either a acontrol control or or quercetin quercetin gr groupsoups and and were were maintained maintained under under ad ad libitum libitum feeding feeding for for 7 7 days. After that, rats underwent swimming exercise for 1 h. Immediately after exercise, epitrochlearis days.days. AfterAfter that,that, ratsrats underwentunderwent swimmingswimming exerexercisecise forfor 1 1 hour.hour. ImmediatelyImmediately afterafter exercise,exercise, muscles were dissected out as Western blot samples for p-AMPK (A) and AMPK (B). A representative epitrochlearisepitrochlearis muscles muscles were were dissected dissected out out as as Western Western blot blot samples samples for for p-AMPK p-AMPK (A) (A) and and AMPK AMPK (B). (B). blot is shown above each figures. The obtained values of p-AMPK were normalized with the value AA representative representative blot blot is is shown shown above above each each figures. figures. The The obtained obtained values values of of p-AMPK p-AMPK were were normalized normalized for AMPK. Values are means SE (n = 6–7). #p < 0.05 vs. control in exercised group. C: control, Q: withwith the the value value for for AMPK. AMPK. Values Values± are are means means ± ±SE SE ( n( n= =6–7). 6–7). # p# p< <0.05 0.05 vs. vs. control control in in exercised exercised group. group. C: C: quercetin, EX: exercise, EX (+): exercised, EX (-): rest. control,control, Q: Q: quercetin, quercetin, EX: EX: exercise, exercise, EX EX (+): (+): exercised, exercised, EX EX (-): (-): rest. rest. 3.4. Chronic (7 Days) Effect of Quercetin Treatment (with Soleus Muscle) 3.4.3.4. Chronic Chronic (7 (7 days) days) effect effect of of querc quercetinetin treatment treatment (with (with soleus soleus muscle) muscle) The aforementioned results were obtained using epitrochlearis muscle, which consists primarily of fast-twitchTheThe aforementioned aforementioned glycolytic results fibers. results were Thewere obtained previous obtained using reportusing ep ep byitrochlearisitrochlearis Davis et muscle, al. muscle, [7], showing which which consists consists a positive primarily primarily effect ofofof fast-twitch quercetinfast-twitch onglycolytic glycolytic mitochondrial fibers. fibers. The biogenesis,The previous previous was report report obtained by by Davis Davis using et et al. soleusal. [7], [7], showing muscle,showing whicha apositive positive is composed effect effect of of quercetinmainlyquercetin of on slow-twitchon mitochondrial mitochondrial oxidative biogenesis, biogenesis, fibers. was Wewas thereforeobtained obtained using performed using soleus soleus additional muscle, muscle, which which analyses is is composed composed of mitochondrial mainly mainly ofbiogenesis-associatedof slow-twitchslow-twitch oxidativeoxidative proteins fibers.fibers. in soleus WeWe thereforetherefore muscle following peperformedrformed 7-day additionaladditional quercetin analyses supplementationanalyses ofof mitochondrialmitochondrial (Figure9). biogenesis-associatedAsbiogenesis-associated was the case in the proteins proteins experiments in in soleus soleus using muscle muscle epitrochlearis following following muscle, 7-day 7-day quercetin thequercetin protein supplementation supplementation expression levels (Figure (Figure of CS 9).(Figure9). As As was was9A), the the COX case case IV in in (Figure the the experiments experiments9B), PGC-1 using usingα (Figure epitro epitro9C)chlearischlearis and SIRT1 muscle, muscle, (Figure the the protein 9proteinD) were expression expression not increased levels levels by of of CSquercetinCS (Figure (Figure treatment.9A), 9A), COX COX IV Levels IV (Figure (Figure of p-AMPK 9B), 9B), PGC-1 PGC-1 (control,αα (Figure (Figure 1.00 9C) 9C)0.02; and and quercetin,SIRT1 SIRT1 (Figure (Figure 0.99 9D) 9D)0.04), were were AMPK not not increased increased (control, ± ± by1.00by quercetin quercetin0.18; treatment. quercetin; treatment. Levels 0.79 Levels of0.11) of p-AMPK p-AMPK and GAPDH (control, (control, (control, 1. 1.0000 ± ±0.02; 0.02; 1.00 quercetin, quercetin,0.05; quercetin,0.99 0.99 ± ±0.04), 0.04), 1.03 AMPK AMPK0.06) (control, (control, were ± ± ± ± 1.00identical1.00 ± ±0.18; 0.18; between quercetin; quercetin; the two0.79 0.79 groups. ± ±0.11) 0.11) and and GAPDH GAPDH (control, (control, 1.00 1.00 ± ±0.05; 0.05; quercetin, quercetin, 1.03 1.03 ± ±0.06) 0.06) were were identicalidentical between between the the two two groups. groups.

FigureFigureFigure 9. 9.9. Effects EEffectsﬀects of ofof 7-day 7-day7-day quercetin quercetinquercetin treatment treatmenttreatment on onon metabolic metabolicmetabolic molecules moleculesmolecules in inin soleus soleussoleus muscles. muscles.muscles. Rats RatsRats were werewere assignedassignedassigned to toto either eithereither a a acontrol controlcontrol or oror quercetin quercetinquercetin gr groupsgroupsoups and andand were werewere maintained maintainedmaintained under underunder ad adad libitum libitumlibitum feeding feedingfeeding for forfor 7 7 7 A days.days.days. After AfterAfter that, that,that, epitrochlearis epitrochlearisepitrochlearis muscles muscles were were dissecteddissected dissected outout out asas as WesternWestern Western blot blot blot samples samples samples for for for CS CS CS ( (A),), (A), COX COX COX IV (B), PGC-1α (C) and SIRT1 (D) A representative blot is shown above each ﬁgures. Values are means IVIV (B), (B), PGC-1 PGC-1αα (C) (C) and and SIRT1 SIRT1 (D) (D) A A representative representative blot blot is is shown shown above above each each figures. figures. Values Values are are means means± SE (n = 7). All values were normalized by GAPDH abundance. C: control, Q: quercetin. ± ±SE SE ( n( n= =7). 7). All All values values were were normalized normalized by by GAPD GAPDHH abundance. abundance. C: C: control, control, Q: Q: quercetin. quercetin.

Nutrients 2020, 12, 729 9 of 13

4. Discussion A previous rodent study conducted by Henagan et al. [25] demonstrated the effect of 3- and 8-week quercetin treatment on mouse muscle PGC-1α mRNA under a high-fat diet and found that low-dose treatment with 50 µg/day for 8 weeks but not 3 weeks, significantly increased PGC-1α mRNA, whereas high-dose treatment with 600 µg/day did not increase mRNA levels with either treatment duration. The study by Henagan et al. [25] did not measure the protein levels of PGC-1α or mitochondrial enzymes. More recently, the same group also reported that mice treated with low-dose quercetin for 9 weeks under a high-fat diet exhibited greater mitochondrial content in mouse muscle than control mice fed the same diet [26]. The authors assessed mitochondrial content by measuring mtDNA but the validity of mtDNA as a marker of mitochondrial content has been debated because, in some cases, mitochondrial enzyme activity and protein content do not correlate with mtDNA levels [7,27,28]. In the present study, we conducted protein-level analysis of mitochondrial enzymes (CS and COX IV). Also, we analyzed two different muscles, one rich in slow-twitch oxidative fibers (soleus) and the other composed mainly of first-twitch glycolytic fibers (epitrochlearis). This was done because these fiber types differ in several ways, for instance the relative abundance of basal mitochondrial content and SIRT1 protein [29], which could result in different capacities for mitochondrial adaptation. Our results clearly demonstrated that quercetin treatment did not stimulate mitochondrial biogenesis in skeletal muscle regardless of muscle fiber type. In the present study, we used a high-dose quercetin treatment (25 mg/kg BW/day). Although quercetin treatment might have properties related to hormesis effect in mitochondrial biogenesis, this dose used in the present study was reported to induce dramatic increase in mitochondrial proteins [7]. We cannot explain this discrepancy. However, the fact that quercetin failed to influence mitochondrial biogenesis in vivo was supported by our finding that it also did not affect SIRT1 or PGC-1α protein levels. To further characterize why quercetin did not impact skeletal muscle, we focused on AMPK, the primary molecule responsible for stimulating mitochondrial biogenesis. In previous studies, quercetin induced AMPK activation when C2C12 and L6 myotubes were directly incubated with quercetin in vitro [14–16]. Unlike experiments using ~100 µM resveratrol [17,24] and studies that evaluated ~100 µM quercetin treatment of cultured cells [14–16], we demonstrated that 100 µM quercetin did not directly stimulate AMPK activation in mature, intact skeletal muscle. Quercetin aglycone is used frequently in such experiments but this form of quercetin is known to be rare or undetectable in the blood of humans and rats [30–32]. In the case of oral ingestion of quercetin, several quercetin metabolites were previously shown to appear in the blood [30–32]. Although the blood concentrations of these metabolites and their direct action on AMPK activity in skeletal muscle were not examined in the present study, these metabolites may affect muscle AMPK directly or indirectly after oral ingestion of quercetin. To examine this possibility, we measured AMPK activation in muscles 4 h after a single (acute) oral dose of quercetin and also 16 h after the last oral ingestion during chronic treatment. Again, our results demonstrated that quercetin did not affect muscle AMPK activation after oral challenge. This is a plausible reason why quercetin treatment does not induce muscle mitochondrial biogenesis in vivo. It is agreed that mitochondrial content is important for maximal endurance performance but it is not the sole determinant. We examined whether quercetin had ergogenic effects other than mitochondrial biogenesis. Another factor is glycogen metabolism, which is associated with the availability of lactate and free fatty acid (FFA). Lactate levels are controlled by the rates of lactate production and utilization. Lactate is an easily utilized substrate that is proactively transported during endurance exercise by MCT1 and MCT4 proteins, which are responsible for lactate uptake and extrusion, respectively, across the muscle cell membrane [33]. Given that exercise training induces MCT1 and MCT4 proteins [33], upregulation of these molecules has been implicated as a positive adaptation for enhancing endurance performance. So far, no studies have tested the effect of quercetin on these important proteins. In the present study, we showed for the first time that quercetin treatment had no impact on either MCT1 or MCT4 and that lactate metabolism during exercise, as measured by Nutrients 2020, 12, 729 10 of 13 blood and muscle lactate levels, was unchanged. Since AMPK activation is thought to upregulate MCT proteins [33,34], our results regarding MCT1 and MCT4 are supported by present evidence showing that quercetin did not affect AMPK activation levels. The increase in blood FFA levels during endurance exercise also contributes to improved endurance capacity, specifically by coordinating the regulation of glycogen metabolism. Greater oxidation of FFAs, as observed in trained subjects, reduces glycogen use and increases endurance capacity. Maintaining higher glycogen levels before exercise also delays glycogen depletion during exercise. Only one previous study measured glycogen levels at rest and during exercise in quercetin-treated subjects. Dumke et al. demonstrated [35] that 3-week treatment with quercetin in trained athletes did not alter glycogen utilization speed during endurance exercise. Consistent with this human study, we showed that quercetin treatment of rodents did not influence glycogen levels either at rest or immediately after endurance exercise. Furthermore, the metabolic molecules GLUT4 and CPT1B, which may alter glycogen utilization speed by facilitating glucose and FFA uptake into muscle cells, were not influenced by quercetin treatment. We did not identify any adaptations in response to quercetin treatment that would exert an ergogenic effect on endurance performance. While many studies have reported quercetin’s ergogenic effect on endurance exercise [6–9], other studies have shown no effect of chronic quercetin treatment [11,36,37]. Importantly, Casuso et al. [27] demonstrated that chronic quercetin treatment and exercise training impaired training-induced citrate synthase adaptation in skeletal muscle. This detrimental effect of quercetin on mitochondria might mask some of quercetin’s ergogenic effects, especially when quercetin is administered to subjects engaged in exercise training. In the present study, quercetin treatment blunted exercise-induced AMPK activation. Although precise role of AMPK activation in exercise-induced muscle adaptation is still under debate [20], this might contribute to the decreased training-induced mitochondrial adaptation [27]. Identifying the mechanisms underlying the reduced AMPK response to exercise is beyond the scope of this study; however, given that production of reactive oxygen species and reactive nitrogen species are increased during muscle contractile activity and that these small molecules are capable to stimulate AMPK activation [18,19], cancellation of these molecules during exercise by quercetin’s antioxidant function might play a role in this phenomenon. In contrast, the present study showed that the level of glycogen, a negative AMPK regulator, apparently did not contribute to the reduced AMPK response because the glycogen level was the same at baseline and immediately after exercise. We examined the exercise-induced AMPK activation level only in the epitrochlearis muscle, located in the upper arm, because this muscle is a predominant working muscle in the swimming exercise model [38]. As a result, we could not extrapolate the evidence obtained in this glycolytic muscle to oxidative muscles such as the soleus. However, our results indicate that repetitive quercetin treatment might worsen AMPK-induced muscle adaptation, including mitochondrial biogenesis. As experimental limitations in this study, we cannot mention more about quercetin’s hormesis effect and ineffectual term because we did experiments with one dose and one term for the treatment. The longer-term effect of quercetin treatment needs to be examined in the future. Also, we could not examine whether the blunted exercise-induced AMPK activation actually might result in any detrimental effect in exercise training.

5. Conclusions The following results of this study are of special interest to athletes: 1) quercetin treatment did not stimulate mitochondrial biogenesis in skeletal muscle, 2) quercetin treatment did not inﬂuence metabolism in a way that might enhance endurance exercise capacity and 3) quercetin treatment might provide a disadvantage to muscle adaptation when administered with exercise training. The molecular results of this study indicate that quercetin treatment may not be advantageous for improving endurance exercise performance, at least after high-dose and short-term therapy. Nutrients 2020, 12, 729 11 of 13

Author Contributions: K.K. designed all of the experiments, summarized the data and wrote the manuscript. K.K. and A.S. performed the animal experiments and K.K., A.H. and H.M. performed the biochemical analyses. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a research fund from the Bourbon Corporation. Acknowledgments: We are grateful to the Bourbon Corporation for providing the quercetin. Conﬂicts of Interest: The Bourbon Corporation had no control over the interpretation, writing, or publication of this work. The authors have no further relationships with the Bourbon Corporation, and did not obtain additional ﬁnancial support for this study.

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