The Acute Effects of Swimming Exercise on PGC-1-FNDC5/Irisin

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Article The Acute Effects of Swimming Exercise on PGC-1α-FNDC5/Irisin-UCP1 Expression in Male C57BL/6J Mice

Eunhee Cho 1, Da Yeon Jeong 2, Jae Geun Kim 2,3 and Sewon Lee 4,5,6,*

1 Department of Human Movement Science, Graduate School, Incheon National University, Incheon 22012, Korea; [email protected] 2 Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Korea; [email protected] (D.Y.J.); [email protected] (J.G.K.) 3 Institute for New Drug Development, Division of Life Sciences, Incheon National University, Incheon 22012, Korea 4 Division of Sport Science, College of Arts & Physical Education, Incheon National University, Incheon 22012, Korea 5 Sport Science Institute, College of Arts & Physical Education, Incheon National University, Incheon 22012, Korea 6 Health Promotion Center, College of Arts & Physical Education, Incheon National University, Incheon 22012, Korea * Correspondence: [email protected]; Tel.:+82-32-835-8572

Abstract: Irisin is a myokine primarily secreted by skeletal muscles and is known as an exercise- induced hormone. The purpose of this study was to determine whether the PGC-1α -FNDC5 /Irisin- UCP1 expression which is an irisin-related signaling pathway, is activated by an acute swimming exercise. Fourteen to sixteen weeks old male C57BL/6J mice (n = 20) were divided into control (CON, n n = 10) and swimming exercise groups (SEG, = 10). The SEG mice performed 90 min of acute swimming exercise, while control (non-exercised) mice were exposed to shallow water (2 cm of depth) for 90 min. The mRNA and protein expression of PGC-1α, FNDC5 and browning markers including Citation: Cho, E.; Jeong, D.Y.; Kim, J.G.; Lee, S. The Acute Effects UCP1 were evaluated by quantitative real-time PCR and western blotting. Serum irisin concentration of Swimming Exercise on was measured by enzyme-linked immunosorbent assay. An acute swimming exercise did not lead PGC-1α-FNDC5/Irisin-UCP1 to alterations in the mRNA and protein expression of PGC-1α in both soleus and gastrocnemius Expression in Male C57BL/6J Mice. muscles, the mRNA and protein expression of UCP1 in brown adipose tissue, mRNA browning Metabolites 2021, 11, 111. https:// markers in visceral adipose tissue and circulating irisin when compared with the control group. On doi.org/10.3390/metabo11020111 the other hand, an acute swimming exercise led to increases in the mRNA and protein expressions of FNDC5 in the soleus muscle, the protein expression of FNDC5 in the gastrocnemius muscles and the Academic Editor: Vassilis Mougios protein expression of UCP1 in subcutaneous adipose tissue. Received: 24 December 2020 Accepted: 14 February 2021 Keywords: irisin; swimming exercise; UCP1; PGC-1α; FNDC5 Published: 16 February 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional afﬁl- iations. Irisin is a myokine secreted primarily by skeletal muscles and is known as an exercise- induced hormone [1]. Exercise-induced muscle contraction stimulates the gene expression for ﬁbronectin type III domain-containing protein 5 (FNDC5), and irisin is cleaved from FNDC5 and released in circulation [1]. FNDC5 is controlled by peroxisome proliferator- activated receptor-gamma coactivator-1α (PGC-1α) in response to chronic exercise and is Copyright: © 2021 by the authors. α Licensee MDPI, Basel, Switzerland. positively related to PGC-1 in human skeletal muscle [2]. α This article is an open access article PGC-1 and FNDC5 expression and increment in circulating level of irisin are known distributed under the terms and to enhance energy consumption and stimulate the browning of white adipose tissue (WAT), conditions of the Creative Commons thereby ameliorating insulin resistance [3]. In addition, recent studies have reported that Attribution (CC BY) license (https:// irisin had a protective effect on the dysfunction of pancreatic β cell [4,5] and vascular creativecommons.org/licenses/by/ dysfunction [6]. In this regard, irisin is attracting attention as a signaling mechanism that 4.0/). can be used to treat metabolic diseases such as obesity, insulin resistance, and metabolic

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syndrome because it increases the expression of uncoupling protein 1 (UCP1) present in the inner membrane of mitochondria, thereby inducing the browning of WAT. It is well known that aerobic exercise improves metabolic health including weight management, glucose and cholesterol metabolism for the prevention and treatment of metabolic abnormalities [7]. In particular, increased blood levels of irisin with regular exercise improved energy metabolism and glucose homeostasis, suggesting that it may improve obesity, insulin resistance and its complications [8]. However, the effects of exercise on irisin and irisin-related signaling substances expression are still under discussion and controversial because different results were reported depending on the type of exercise (swimming vs. running, differences of recruited muscles), intensity, frequency and duration. Previous studies indicated that the circulating level of irisin was increased with acute endurance exercise in experimental animal models and humans [9–11]. Increased level of irisin is suggested to mediate the alteration of WAT browning by increasing energy consumption through the UCP1 expression, and it is evi- dent that browning of WAT is related to the beneﬁcial effect of exercise [1,12–15]. On the other hand, other studies suggested no effect of acute endurance exercise on circulating irisin [16,17]. Interestingly, Tsuchiya et al. indicated an acute bout of resistance exercise caused greater irisin responses compared with endurance exercise alone or combined resistance and endurance exercise [16]. Although previous studies reported that exercise activates those factors, studies on whether acute swimming exercise promotes this signaling pathway are still unclear. In addition, it is also uncertain whether the activation patterns appear differently according to muscle ﬁber types (Type I vs. Type II) and fat types (brown adipose tissue (BAT) vs. white adipose tissue). Therefore, the purpose of this study was to determine whether the PGC-1α-FNDC5/Irisin-UCP1 expression, which is an irisin-related signaling pathway, is activated by an acute swimming exercise.

2. Results 2.1. An Acute Swimming Exercise Did Not Increase mRNA and Protein Expressions of PGC-1 α in 2 Different Types of Skeletal Muscle Fibers A previous report suggested that chronic voluntary wheel running exercise activated PGC-1α expression in skeletal muscle, which promoted FNDC5 expression in skeletal muscle and irisin concentration in circulation [1]. In order to verify whether a 90 min acute swimming exercise activates PGC-1α in skeletal muscles, we evaluated mRNA and protein expression in gastrocnemius and soleus muscles which were mixed muscle ﬁber (fast and slow) and slow-twitch muscle ﬁber, respectively, between control (CON) and swimming exercise group (SEG). The mRNA expression of PGC-1α was comparable between groups in gastrocnemius (Figure1a) and soleus (Figure1b) muscles. The protein expression of PGC-1α was also examined in both soleus and gastrocnemius muscles in CON and SEG and the levels of PGC-1α protein were also comparable between groups in gastrocnemius Metabolites 2021, 11, x FOR PEER REVIEW 3 of 11 (Figure1c) and soleus (Figure1d) muscles. There was a tendency toward an increase in the mRNA and protein expressions of PGC-1α in soleus muscle but it did not reach to the statistical difference (p = 0.1448, p = 0.1473, respectively, Figure1b,d).

Figure 1. Cont.

Figure 1. An acute swimming exercise did not increase mRNA and protein expressions of PGC-1α in skeletal muscles. Male C57BL/6J mice were exposed to an acute swimming exercise for 90 min (SEG) or to shallow water for 90 min (CON). The levels of PGC-1α mRNA were comparable between groups in gastrocnemius (a) and soleus (b) muscles. In addition, the levels of PGC-1α protein were also comparable between groups in gastrocnemius (c) and soleus (d) muscles. Results are presented as mean ± SEM. n = 6–10 mice per group. CON, control group, SEG; swimming exercise group.

2.2. An Acute Swimming Exercise Activates mRNA and Protein Expressions of FNCD5 in 2 Different Types of Skeletal Muscle Fibers To determine whether an acute swimming exercise activates FNDC5 in skeletal muscles, we identified mRNA and protein expression of FNDC5 in two different types of skeletal muscle fibers. The mRNA expression of FNDC5 was comparable between groups in gastrocnemius (p = 0.4444, Figure 2a), while the mRNA FNDC5 was significantly increased in SEG compared to CON in soleus (p = 0.0497, Figure 2b) muscle. The protein expressions of FNDC5 were also examined in both soleus and gastrocnemius muscles in CON and SEG. The levels of FNDC5 protein were significantly increased after the acute exercise in both gastrocnemius (4.657 folds increase, Figure 2c) and soleus (2.435 folds increase, Fig- ure 2d) muscles.

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Figure 1. An acute swimming exercise did not increase mRNA and protein expressions of PGC-1α Figurein skeletal 1. An muscles. acute swimming Male C57BL/6J exercise mice did not were increa exposedse mRNA to an acuteand protein swimming expressions exercise of for PGC-1 90 minα in(SEG) skeletal or to muscles. shallow Male water C57BL/6J for 90 min mice (CON). were Theexposed levels to of anPGC-1 acuteα mRNAswimming were exercise comparable for 90 between min (SEG) or to shallow water for 90 min (CON). The levels of PGC-1α mRNA were comparable be- groups in gastrocnemius (a) and soleus (b) muscles. In addition, the levels of PGC-1α protein were tween groups in gastrocnemius (a) and soleus (b) muscles. In addition, the levels of PGC-1α pro- also comparable between groups in gastrocnemius (c) and soleus (d) muscles. Results are presented tein were also comparable between groups in gastrocnemius (c) and soleus (d) muscles. Results ± areas meanpresentedSEM. as meann = 6–10 ± SEM. mice n per= 6–10 group. mice CON, per group. control CON, group, control SEG; group, swimming SEG; exercise swimming group. exer- cise2.2. group. An Acute Swimming Exercise Activates mRNA and Protein Expressions of FNCD5 in 2 Different Types of Skeletal Muscle Fibers 2.2. An Acute Swimming Exercise Activates mRNA and Protein Expressions of FNCD5 in 2 To determine whether an acute swimming exercise activates FNDC5 in skeletal mus- Differentcles, we Types identified of Skeletal mRNA Muscle and Fibers protein expression of FNDC5 in two different types of skeletalTo determine muscle fibers. whether The an mRNA acute expression swimming of exFNDC5ercise activateswas comparable FNDC5 in between skeletal groups muscles,in gastrocnemius we identified mRNA (p = 0.4444, and protein Figure 2expressia), whileon the of FNDC5 mRNA inFNDC5 two differentwas significantly types of skel- in- etalcreased muscle in SEGfibers. compared The mRNA to CONexpression in soleus of FNDC5 (p = 0.0497, was comparable Figure2b) muscle. between The groups protein in gastrocnemiusexpressions of ( FNDC5p = 0.4444, were Figure also 2a), examined while the in mRNA both soleusFNDC5 and was gastrocnemius significantlymuscles increased in CON and SEG. The levels of FNDC5 protein were significantly increased after the acute in SEG compared to CON in soleus (p = 0.0497, Figure 2b) muscle. The protein expressions exercise in both gastrocnemius (4.657 folds increase, Figure2c) and soleus (2.435 folds of FNDC5 were also examined in both soleus and gastrocnemius muscles in CON and increase, Figure2d) muscles. SEG. The levels of FNDC5 protein were significantly increased after the acute exercise in both2.3. Angastrocnemius Acute Swimming (4.657 Exercise folds increase, Did Not IncreaseFigure 2c) Circulating and soleus Irisin (2.435 in Serum folds increase, Fig- ure 2d)The muscles. activation of FNDC5 in skeletal muscles resulted in an elevation in the level of circulating irisin which causes the browning of WAT by UCP1 [18]. Therefore, to verify Metabolites 2021, 11, x FOR PEER REVIEW 4 of 11 the acute effect of swimming exercise on circulating irisin, we used an enzyme-linked immunosorbent assay (ELISA) with an antibody specific to irisin. An acute swimming exercise for 90 min did not lead to an increase in circulating irisin (p = 0.3247, Figure3).

Figure 2. Cont.

Figure 2. An acute swimming exercise increased mRNA and protein expressions of FNDC5 in skeletal muscles. Male C57BL/6J mice were exposed to an acute swimming exercise for 90 min (SEG) or to shallow water for 90 min (CON). The level of FNDC5 mRNA was comparable between groups in gastrocnemius (a), whereas the level of that was significantly increased in SEG compared to CON in soleus muscle (b). In addition, the levels of FNDC5 protein were also significantly increased in SEG compared to CON in both gastrocnemius (c) and soleus muscles (d). Re- sults are presented as mean ± SEM. n = 7–10 mice per group. * p < 0.05 vs. CON. CON, control group; SEG, swimming exercise group.

Figure 3. An acute swimming exercise did not increase circulating irisin concentration in serum. Male C57BL/6J mice were exposed to an acute swimming exercise for 90 min (SEG) or to shallow

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Figure2.3. An 2. AcuteAn acute Swimming swimming Exercise exercise Did increased Not Increase mRNA Circulating and protein Irisin expressions in Serum of FNDC5 in Figureskeletal 2. An muscles. acute swimming Male C57BL/6J exercise mice increased were exposed mR toNA an and acute protein swimming expressions exercise for of 90FNDC5 min (SEG) in The activation of FNDC5 in skeletal muscles resulted in an elevation in the level of skeletalor to muscles. shallow waterMale forC57BL/6J 90 min (CON).mice were The exposed level of FNDC5 to an mRNAacute swimming was comparable exercise between for 90 groups min circulating irisin which causes the browning of WAT by UCP1 [18]. Therefore, to verify (SEG)in or gastrocnemius to shallow water (a), whereas for 90 themin level (CON). of that The was level signiﬁcantly of FNDC5 increased mRNA in was SEG comparable compared to CONbetween groupsthe in gastrocnemiusacute effect of swimming(a), whereas exercise the level on of circ thatulating was significantly irisin, we used increased an enzyme-linked in SEG com- im- in soleus muscle (b). In addition, the levels of FNDC5 protein were also signiﬁcantly increased in pared to CON in soleus muscle (b). In addition, the levels of FNDC5 protein were also signifi- SEGmunosorbent compared to assay CON in(ELISA) both gastrocnemius with an antibody (c) and sp soleusecific muscles to irisin. (d ).An Results acute areswimming presented exer- as cantly increased in SEG compared to CON in both gastrocnemius (c) and soleus muscles (d). Re- meancise± forSEM. 90 minn = 7–10did not mice lead per group.to an increase * p < 0.05 in vs. circulating CON. CON, irisin control (p = group;0.3247, SEG, Figure swimming 3). sults are presented as mean ± SEM. n = 7–10 mice per group. * p < 0.05 vs. CON. CON, control exercise group. group; SEG, swimming exercise group.

2.3. An Acute Swimming Exercise Did Not Increase Circulating Irisin in Serum The activation of FNDC5 in skeletal muscles resulted in an elevation in the level of circulating irisin which causes the browning of WAT by UCP1 [18]. Therefore, to verify the acute effect of swimming exercise on circulating irisin, we used an enzyme-linked immunosorbent assay (ELISA) with an antibody specific to irisin. An acute swimming exercise for 90 min did not lead to an increase in circulating irisin (p = 0.3247, Figure 3). Figure 3. An acute swimming exercise did not increase circulating irisin concentration in serum. MaleFigure C57BL/6J 3. An acute mice swimming were exposed exercise to an did acute not swimming increase circulating exercise for irisin 90 minconcentration (SEG) or toin shallowserum. waterMale for C57BL/6J 90 min (CON).mice were Results exposed are presented to an acute as swimming mean ± SEM. exercisen = 9 for mice 90 permin group. (SEG) CON,or to shallow control group; SEG, swimming exercise group.

2.4. An Acute Swimming Exercise Increases Protein Expression of UCP1 in Subcutenous White Adipose Tissue To test whether an acute swimming exercise increased UCP1 in adipose tissues, we examined mRNA and protein expression in 2 different types of adipocytes which were subcutaneous adipocytes and brown adipocytes. The UCP1 mRNA in subcutaneous WAT and BAT after an acute swimming exercise was not different from that of CON (Figure4a,b ), whereas the protein expressionof UCP1 was signiﬁcantly increased in SEG compared to CON (Figure4c). The protein expression in BAT did not alter by an acute swimming Figureexercise 3. An acute (p = 0.1341, swimming Figure exercise4d). did not increase circulating irisin concentration in serum. Male C57BL/6J mice were exposed to an acute swimming exercise for 90 min (SEG) or to shallow 2.5. Change in mRNA Expression of Browning Markers in Visceral WAT after Acute Swimming Exercise A previous study showed acute treadmill exercise did not change mRNA UPC1 expression in visceral WAT [19]. To test whether an acute swimming exercise increased browning in visceral WAT, we examined browning marker mRNA in visceral WAT. There was a tendency toward an increase in the mRNA of acetyl-CoA carboxylase (ACC)α and FNDC5 in visceral WAT, but it did not reach to the statistical difference (p = 0.0519, p = 0.0583, respectively. Figure5a,b). In addition, the mRNA expression of PRDM16 and Metabolites 2021, 11, x FOR PEER REVIEW 5 of 11

water for 90 min (CON). Results are presented as mean ± SEM. n = 9 mice per group. CON, control group, SEG; swimming exercise group.

Metabolites 2021, 11, 111 subcutaneous adipocytes and brown adipocytes. The UCP1 mRNA in subcutaneous WAT5 of 11 and BAT after an acute swimming exercise was not different from that of CON (Figure 4a,b), whereas the protein expression of UCP1 was significantly increased in SEG compared to CON (Figure 4c). The protein expression in BAT did not alter by an acute swim- peroxisome-proliferator-activated receptor-gamma (PPARγ) in visceral WAT did not alter ming exercise (p = 0.1341, Figure 4d). by an acute swimming exercise (p = 0.5517, p = 0.9142, respectively. Figure5c,d).

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Figure 4. An acute swimming exercise increase UCP1 protein expression in subcutaneous WAT, Figure 4. An acute swimming exercise increase UCP1 protein expression in subcutaneous WAT, whereaswhereas it did itnot did change not change UCP1UCP1 mRNAmRNA expression expression in insubcut subcutaneousaneous WAT WAT and BATBAT and and UCP1 UCP1 protein expression in BAT. Male C57BL/6J mice were exposed to an acute swimming exercise for 90 min proteinand expression FNDC5 inin BAT.visceral Male WAT, C57BL/6J but mice it did were not exposed reach to to an the acute statistical swimming difference exercise for ( p = 0.0519, p 90 min(SEG) (SEG) or or to to shallow shallow water water for for 90 90 min min (CON). (CON). The The level level of UCP1of UCP1mRNA mRNA was was comparable comparable between between= 0.0583,groups groups in respectively. bothin both subcutaneous subcutaneous Figure WAT WAT 5a,b). (a) and(a) andIn BAT addition, BAT (b). (b In). addition,In the addition, mRNA the the level levelexpression of UCP1of UCP1 protein pro-of PRDM16 was and teinperoxisome-proliferator-activated wassigniﬁcantly significantly increased increased in SEG in SEG compared compared receptor-gamma to CON to CON in subcutaneous in subcutaneous (PPAR WATγ WAT) (inc), visceral whereas (c), whereas the WAT level the did of not alter levelby ofUCP1 anUCP1 acute protein protein swimming was was comparable comparable exercise in BATin BAT(p (d =) between(0.5517,d) between groups. p = groups. 0.9142, Results Results respectively. are presentedare presented as Figure mean as mean± 5c,d).SEM. ± SEM.n n= = 5–9 5–9 mice mice per per group. group. * p *

2.5. Change in mRNA Expression of Browning Markers in Visceral WAT after Acute Swimming Exercise A previous study showed acute treadmill exercise did not change mRNA UPC1 expression in visceral WAT [19]. To test whether an acute swimming exercise increased browning in visceral WAT, we examined browning marker mRNA in visceral WAT. There was a tendency toward an increase in the mRNA of acetyl-CoA carboxylase (ACC)α

Figure 5. Cont.

Figure 5. Change in browning markers mRNA expression in visceral WAT after acute swimming exercise. Male C57BL/6J mice were exposed to an acute swimming exercise for 90 min (SEG) or to shallow water for 90 min (CON). The level of ACCα mRNA (a), and FNDC5 mRNA (b) was not significantly increased in SEG compared to CON in visceral WAT. The mRNA expression of PRDM16 (c) and PPARγ mRNA (d) was comparable between groups. Results are presented as mean ± SEM. n = 4 mice per group. * p < 0.05 vs. CON. CON, control group; SEG, swimming exercise group; WAT, white adipose tissue.

3. Discussion The purpose of this study was to determine whether the PGC-1α-FNDC5/Irisin- UCP1 expression, which is an irisin-related signaling pathway, is activated by an acute swimming exercise. One previous study found that when mice were treated with voluntary wheel running exercise for three weeks, the expressions of PGC-1α and FNDC5 genes were increased in skeletal muscles, and the expression of the UCP1 gene was increased in adipose tissues [1]. However, studies on whether even acute swimming exercise activates the PGC-1α-FNDC5 /Irisin-UCP1 signaling mechanism and whether the activation patterns appear differently according to muscle fiber types (Type I vs. Type I and II mixed) and fat types (brown fat vs. white fat) are insufficient. Our hypothesis was that irisin- related signaling substances would increase after 90 min of acute swimming exercise. However, unlike the expectation, there were no differences in the mRNA and protein expressions of PGC-1α in skeletal muscles, the mRNA and protein expression of UCP1 in brown adipose tissue, circulating irisin and browning markers in visceral WAT in the acute swimming group when compared with the control group. On the other hand, an acute swimming exercise led to increases in the expression of FNDC5 protein in both the soleus muscle (2–3 folds increase) that has many type I muscle fibers and the gastrocnemius muscle (4–5 folds increase) that has both type I and II muscle fibers. In this study, only the expression of FNDC5 in skeletal muscle increased, while the expression of PGC-1α

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and FNDC5 in visceral WAT, but it did not reach to the statistical difference (p = 0.0519, p = 0.0583, respectively. Figure 5a,b). In addition, the mRNA expression of PRDM16 and peroxisome-proliferator-activated receptor-gamma (PPARγ) in visceral WAT did not alter by an acute swimming exercise (p = 0.5517, p = 0.9142, respectively. Figure 5c,d).

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Figure 5. Change in browning markers mRNA expression in visceral WAT after acute swimming Figureexercise. 5. Change Male C57BL/6J in browning mice weremarkers exposed mRNA to an expressi acute swimmingon in visceral exercise WAT for 90 after min (SEG)acute orswimming exercise.to shallow Male water C57BL/6J for 90 min mice (CON). were The exposed level of toACC anα acutemRNA swimming (a), and FNDC5 exercisemRNA for ( b90) was min not (SEG) or to shallowsignificantly water increased for 90 min in SEG (CON). compared The tolevel CON of in ACC visceralα mRNA WAT. The (a), mRNA and FNDC5 expression mRNA of PRDM16 (b) was not significantly(c) and PPAR increasedγ mRNA (din) wasSEG comparable compared between to CON groups. in visceral Results WAT. are presented The mRNA as mean expression± SEM. of PRDM16n = 4 mice (c)per and group. PPAR * pγ< mRNA 0.05 vs. CON.(d) was CON, comparable control group; between SEG, swimming groups. exerciseResults group;are presented WAT, as meanwhite ± SEM. adipose n tissue.= 4 mice per group. * p < 0.05 vs. CON. CON, control group; SEG, swimming exer- cise3. group; Discussion WAT, white adipose tissue. The purpose of this study was to determine whether the PGC-1α-FNDC5/Irisin- 3. DiscussionUCP1 expression, which is an irisin-related signaling pathway, is activated by an acute swimmingThe purpose exercise. of One this previous study study was found to determine that when mice whether were treated the PGC-1 with voluntaryα-FNDC5/Irisin- wheel running exercise for three weeks, the expressions of PGC-1α and FNDC5 genes were UCP1 expression, which is an irisin-related signaling pathway, is activated by an acute increased in skeletal muscles, and the expression of the UCP1 gene was increased in adipose swimmingtissues [1]. exercise. However, One studies previous on whether study even found acute swimming that when exercise mice activateswere treated the PGC- with volun- tary1α wheel-FNDC5 running /Irisin-UCP1 exercise signaling for three mechanism weeks, and the whether expressions the activation of PGC-1 patternsα and appear FNDC5 genes weredifferently increased according in skeletal to muscle muscles, fiber types and the (Type expression I vs. Type Iof and the II UCP1 mixed) gene and fat was types increased in adipose(brown tissues fat vs. white[1]. However, fat) are insufficient. studies on Our whet hypothesisher even was acute that irisin-related swimming signaling exercise activates thesubstances PGC-1α-FNDC5 would increase /Irisin-UCP1 after 90 min signaling of acute swimmingmechanism exercise. and whether However, unlikethe activation the pat- expectation, there were no differences in the mRNA and protein expressions of PGC-1α ternsin skeletal appear muscles, differently the mRNA according and protein to muscle expression fiber oftypes UCP1 (Type in brown I vs. adipose Type tissue,I and II mixed) andcirculating fat types irisin (brown and browning fat vs. white markers fat) in are visceral insufficient. WAT in the Our acute hypothesis swimming was group that irisin- relatedwhen comparedsignaling with substances the control would group. increase On the other after hand, 90 anmin acute of swimmingacute swimming exercise exercise. However,led to increases unlike in the the expectation, expression of FNDC5there were protein no indifferences both the soleus in the muscle mRNA (2–3 and folds protein ex- pressionsincrease) of that PGC-1 has manyα in typeskeletal I muscle muscles, fibers the and mRNA the gastrocnemius and protein muscle expression (4–5 folds of UCP1 in increase) that has both type I and II muscle fibers. In this study, only the expression of brownFNDC5 adipose in skeletal tissue, muscle circulating increased, irisin while theand expression browning of PGC-1markersα was in notvisceral increased WAT in the acuteafter swimming the exercise. group In a previous when study compared conducted with with the human, control alteration group. of mRNAOn thePGC-1 otherα hand, an acuteexpression swimming in skeletal exercise muscle led orto serumincreases irisin in was the notexpression accompanied of FNDC5 by corresponding protein in both the soleuschanges muscle in FNDC5 (2–3 folds [20]. Moreover, increase) theythat suggested has many that type large I muscle intra- and fibers inter-individual and the gastrocnem- iusvariations muscle (4–5 in PGC-1 foldsα, FNDC5increase)gene that expression has both and type circulating I and II irisin muscle in response fibers.to In a this single study, only resistance exercise and heavy-intensity endurance training [20]. In addition, a mouse the expression of FNDC5 in skeletal muscle increased, while the expression of PGC-1α study conducted by Pang et al. has reported that PGC-1α expression increased during 30 min of treadmill exercise but decreased during 1 h of treadmill exercise, suggesting that the regulation of gene and protein expression may differ according to the duration of exercise [11]. A few studies suggested 6-h prolonged acute bout of swimming exercise- induced an elevated mRNA expression of PGC-1 immediately after [21] and elevation of PGC-1α protein was maintained 18 and 24 h after the exercise in rat epitrochlearis muscle [22]. Given these previous findings, the expression of PGC-1α is considered to occur when some time has passed after exercise. Therefore, the duration of exercise is considered to play an important role in the regulation of the PGC-1α-FNDC5/Irisin- UCP1 signaling mechanism. Furthermore, the changes in FNDC5 expression were not accompanied by changes in PGC-1α, suggesting that a factor other than PGC-1α may be Metabolites 2021, 11, 111 7 of 11

involved in the regulation of FNDC5 expression. Although FNDC5 is known as PGC-1α dependent myokine, the expression of FNDC5 increased without PGC-1α activation in our study. Indeed, the increase in FNDC5 expression is not necessarily caused by PGC- 1α activation and further study is needed on the irisin-related mechanisms involved in FNDC5 expression. For example, Natalicchio et al. shown that higher irisin concentration by palmitate occurred without an increase in FNDC5 mRNA levels in human myotubes [5]. Furthermore, in our study the increases in FNDC5 expression were not accompanied by an increase in serum irisin, indicating that different signaling processes may be involved in irisin release from other tissues in addition to transcriptional regulation in skeletal muscles. For instance, Roca-Rivada et al. shown that FNDC5 protein was produced and released from both visceral and subcutaneous adipose tissue in a rat model [23]. Thus, it seems that various upstream mechanisms induced by exercise can affect the expression of PGC-1α-FNDC5/Irisin-UCP1 expression. The results of previous studies that examined the irisin response after exercise are controversial. Contrary to previous studies which showed increased circulating irisin concentration after exercise [2,24,25], inconsistent studies were also reported [2,20,26]. A previous study showed prolonged, moderate-intensity aerobic exercise increased circulating irisin concentration at 54 min and then decreased at 90 min during an acute treadmill exercise [27]. In the previous study, circulating irisin level was further declined by 20 min of the recovery period in humans [27]. In addition, Nygaard et al. have reported that the concentration of irisin was transiently increased after aerobic exercise and then decreased over time and returned to the pre-exercise concentration after 6 h [9]. These two previous studies have indicated that acute exercise may lead to a transient increase in irisin and the elevated irisin concentration was maintained for a short time. In the present study, to create an environment similar to the swimming exercise group, the control group mice were exposed to shallow water for 90 min in a water tank ﬁlled with water to approximately 2 cm heights. Although we did not measure the body temperature of CON mice, it is possible that the body temperature of the CON mice might be declined during this period of time and led to increases in the irisin-related signaling substances such as PGC-1α and irisin in the control group. For this reason, we may not see the differences between CON and SEG in PGC-1α and irisin. As a precedent, it was found that in the white adipose of rat exposed to cold, the level of UCP expression almost reached the level of mitochondria presented in brown fat [28] and Lee et al. also suggest that exercise- induced irisin secretion could have evolved from shivering-related muscle contraction to increase brown fat thermogenesis [29], indicating cold-induced thermogenesis may affect the irisin-related signaling pathway. More detailed studies are required to further investigate the effect of shallow water exposure on the irisin-related signaling pathway. In addition, swimming exercise may be different from treadmill exercise or voluntary wheel running exercise because it leads to a change in body temperature during exercise, which results in an alteration in the irisin-related signaling pathway. In addition, a study suggested that 6-h prolonged swimming exercise reduced muscle glycogen concentrations in the epitrochlearis, triceps and red region of the gastrocnemius muscles, whereas it did not change in soleus, plantaris and white region of the gastrocnemius muscle in the experimental rat model [30]. On the other hand, the 6-h prolonged treadmill running caused a reduction in glycogen concentration in soleus, plantaris and red region of gastrocnemius muscle [30]. Furthermore, the protein expression of PGC-1α was signiﬁcantly elevated by treadmill exercise, not a swimming exercise in rat soleus muscle, suggesting different parts of muscles may be stimulated according to exercise type (treadmill vs. swimming) [30]. In this study, we did not separate the white and red regions of the gastrocnemius muscle. Because the red region of the gastrocnemius muscle was highly recruited both by swimming and running exercise, it is necessary to investigate the signaling pathways using the red part of the gastrocnemius muscle in the future. Therefore, well-controlled additional studies are needed to verify whether acute or chronic swimming exercises activate the PGC-1α-FNDC5/Irisin-UCP1 signaling pathway. Metabolites 2021, 11, 111 8 of 11

4. Materials and Methods 4.1. Experimental Animal Model Fourteen to sixteen weeks old male C57BL/6J mice (n = 20) were purchased from the Shizuoka Laboratory Center (Shizuoka, Japan). The mice were housed on a 12-h light/dark cycle (7:00 a.m., lights on and 7:00 p.m., lights off) and were given access to a standard chow diet and water ad libitum. The animal facility was controlled under speciﬁc pathogen-free conditions at 22 ◦C. All of the animal care and lab experimental procedures were performed in accordance with the Animal Care and Use Committee of the Incheon National University (Incheon, South Korea, permission# INU-ANIM-2018-17).

4.2. Procedure for an Acute Swimming Exercise Before control and exercise treatment, all mice were acclimatized to swim in a glass water tank for 5 min/day for a week. After acclimation for swimming exercise, control and swimming exercise mice were randomly assigned to control (CON, n = 10) and swimming exercise group (SEG, n = 10). The SEG mice performed 90 min of acute swimming exercise in an environment of more than 15 cm deep in a glass water tank (45 × 45 × 45 cm), and 31 ± 1 ◦C water temperature according to protocols based on the previous studies [31,32]. Control (non-exercised) mice were exposed to shallow water (2 cm of depth) for 90 min. The CON mice could stand normally with their head out of the water and did not have to swim. After exercise and control treatment, all mice were gently dried with a cloth towel and then immediately anesthetized.

4.3. Preparations of Serum and Tissues Following the completion of the treatment, all mice were anesthetized by an intraperi- toneal injection of 2.5% tribromoethanol (0.01 mL/g of body weight). Under anesthesia, whole blood was obtained from the vena cava and held for 30 min at room temperature. Then, the blood sample was centrifuged at 12,000 rpm at 4 ◦C for 10 min and sera were transferred in separate tubes without disturbing blood clots. After that, soleus and gastrocnemius muscles, anterior subcutaneous WAT, epididymal WAT and interscapular BAT from mouse were rapidly excised respectively and transferred in separate tubes. All collected samples were stored at −80 ◦C refrigerator until the analysis.

4.4. Quantitative Real-Time PCR Total RNA was isolated from the skeletal muscles, BAT, subcutaneous and visceral WAT, and reverse-transcribed to obtain cDNA using a Maxime RT PreMix kit (Intron Biotechnology, Seoul, South Korea). Real-time PCR ampliﬁcation of the cDNA was analyzed with SYBR Green Real-time PCR Master Mix (Toyobo Co. Ltd., Osaka, Japan) in a Bio-Rad CFX 96 Real-Time Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The results were analyzed using the CFX Manager software and normalized by a house- keeping gene, β-actin. The primers used were as follows: For β-actin: forward: 5’-TAA AAC GCA GCT CAG TAA CAG TCC G-3’ Reverse: 5’-TGG AAT CCT GTG GCA TCC ATG AAA C-3’. For FNDC5: forward: 5’AGA AGA AGG ATG TGC GGA TG-3’ reverse: 5’-TCT TGA AGA GCA CAG GCT CA-3’. For PGC-1α: forward: 5’-AAT GCA GCG GTC TTA GCA CT-3’ reverse: 5’-GTG TGA GGA GGG TCA TCG TT3’. For UCP-1: forward: 5’-GCG TTC TGG GTA CCA TCC TA-3’ reverse: 5’-GCT CTG AGC CCT TGG TGT AG-3’. For ACCα: forward: 5’-GAA GTC AGA GCC ACG GCA CA-3’ reverse: 5’-GGC AAT CTC AGT TCA AGC CAG TC-3’. For PRDM16: forward: 5’-AGC ACG GTG AAG CCA TTC-3’ reverse: 5’- GCG TGC ATC CGC TTG TG-3’. For PPAR-γ: 5’-TGT CGG TTT CAG AAG TGC CTT G-3’ reverse: 5’-TTC AGC TGG TCG ATA TCA CTG GAG-3’.

4.5. Procedure for Western Blotting In this experiment, a conventional western blot system (Bio-Rad Laboratories, Her- cules, CA, USA). Proteins in skeletal muscles and adipose tissues were analyzed by electrophoresis USA) was used to analyze speciﬁc protein levels using a typical type of elec- Metabolites 2021, 11, 111 9 of 11

trophoresis. The skeletal muscles and adipose tissues collected were homogenized with CelLytic MT lysis buffer (Sigma-Aldrich, St Louis, MO, USA) mixed with protease in- hibitors cocktail (Sigma-Aldrich, St Louis, MO, USA). The total protein concentration was measured by a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). An equal amount of proteins was separated by electrophoresis using sodium dodecyl sulphate (SDS)-polyacrylamide gel and then transferred to PVDF membrane. After the membranes were blocked with 5% skim milk, the proteins were incubated with the use of primary antibodies at the given dilutions: β-actin (Santa Cruz Biotechnology, catalog# sc-47778, 1:1000), PGC-1α, (Abcam, catalog# ab-54481, 1:500), FNDC5 (Abcam, catalog# ab-174833, 1:500) and UCP1 (Abcam, catalog# ab-10983, 1:1000). After incubation, wash steps were performed using TBST mixed with tris-buffered saline (TBS) and tween 20 twice for 5 min and twice for 10 min. The membranes were incubated with a secondary antibody (Abcam, 1:2000) for 1 h at room temperature. Thereafter, the wash procedure was performed twice for 5 min and 10 min twice for 30 min. The final band intensity was quantified using a Chemidoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to that of the corresponding internal reference, β-actin.

4.6. Procedure for ELISA Serum irisin concentration was measured according to the manufacturer’s experimental procedure using a commercial ELISA kit (AdipoGen Life Sciences, San Diego, CA, USA, AG-45A-0046YEK-KI01). To produce a standard curve of optical density (OD) versus irisin concentration, we added specimens, standard samples and HRP-labeled antibodies to micro-pores pre-coated with the irisin antibody, and the OD values of the standard samples and specimens were then detected with a microplate spectrophotometer at a wavelength of 450 nm. The concentration of irisin in the samples was subsequently determined by comparing the OD value of the samples to the standard curve.

4.7. Statistical Analysis All values were presented as means ± SEM. All statistical data analysis was performed using GraphPad Prism 6.05 version software (La Jolla, CA, USA). The means of independent two groups in mRNA and protein expression and circulating irisin concentration were assessed using unpaired t-tests. The signiﬁcance level was set as p < 0.05.

5. Conclusions An acute swimming exercise did not lead to elevations in the mRNA and protein expressions of PGC-1α in skeletal muscles, the mRNA and protein expression of UCP1 in BAT, circulating irisin and browning markers in visceral WAT when compared with the control group. On the other hand, an acute swimming exercise led to increases in the mRNA and protein expressions of FNDC5 in the soleus muscle, the protein expression of FNDC5 in the gastrocnemius muscles and the protein expression of UCP1 in subcutaneous WAT. Our results demonstrated that an acute swimming exercise for 90 min appears to activate the parts of PGC-1α-FNDC5/Irisin-UCP1 signaling components.

Author Contributions: Conceptualization, E.C., J.G.K. and S.L.; formal analysis, E.C., D.Y.J.; investi- gation, E.C., D.Y.J., J.G.K. and S.L.; methodology, E.C., D.Y.J.; writing—original draft preparation, E.C. and S.L.; writing—review and editing, J.G.K. and S.L.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Incheon National University Research grant in 2018 and a grant of NRF-2019R1F1A1040823 for Sewon Lee. Institutional Review Board Statement: All of the animal care and lab experimental procedures were performed in accordance with the Animal Care and Use Committee of the Incheon National University (Incheon, South Korea, permission# INU-ANIM-2018-17). Informed Consent Statement: Not applicable. Metabolites 2021, 11, 111 10 of 11

Data Availability Statement: The data presented in this study are available in article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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