1

Activation of lactate receptor positively regulates skeletal muscle mass in mice

Yoshitaka Ohno1,2, Masashi Nakatani1, Takafumi Ito2, Yuki Matsui2, Koki Ando2, Yohei
Suda2, Kazuya Ohashi2, Shingo Yokoyama2, Katsumasa Goto2,3

1

Faculty of Rehabilitation and Care, Seijoh University, Tokai, Japan, 2 Laboratory ofPhysiology, School of Health Sciences, Toyohashi SOZO University, Toyohashi, Japan, 3
Department of Physiology, Graduate School of Health Sciences, Toyohashi SOZO
University, Toyohashi, Japan* Corresponding author: Katsumasa Goto, Ph.D, Department of Physiology, Graduate
School of Health Sciences, Toyohashi SOZO University, 20-1 Matsushita, Ushikawa,Toyohashi, Aichi 440-8511, Japan. E-mail: gotok@sepia.ocn.ne.jp

Short title: Activation of GPR81 increases skeletal muscle mass in mice2

Summary
G protein-coupled receptor 81 (GPR81), a selective receptor for lactate, expresses inskeletal muscle cells, but the physiological role of GPR81 in skeletal muscle has not been
fully elucidated. As it has been reported that the lactate administration induces muscle
hypertrophy, the stimulation of GPR81 has been suggested to mediate muscle hypertrophy.
To clarify the contribution of GPR81 activation in skeletal muscle hypertrophy, in thepresent study, we investigated the effect of GPR81 agonist administration on skeletal muscle
mass in mice. Male C57BL/6J mice were randomly divided into control group and GPR81
agonist-administered group that received oral administration of the specific GPR81 agonist
3-Chloro-5-hydroxybenzoic acid (CHBA).

In both fast-twitch plantaris and slow-twitch

soleus muscles of mice, the protein expression of GPR81 was observed.

Oraladministration of CHBA to mice significantly increased absolute muscle weight and muscle
weight relative to body weight in the two muscles.

Moreover, both absolute and relativemuscle protein content in the two muscles were significantly increased by CHBA
administration. CHBA administration also significantly upregulated the phosphorylationlevel of p42/44 extracellular signal-regulated kinase-1/2 (ERK1/2) and p90 ribosomal S6
kinase (p90RSK). These observations suggest that activation of GRP81 stimulates
increased the mass of two types of skeletal muscle in mice in vivo.

Lactate receptor GPR81

may positively affect skeletal muscle mass through activation of ERK pathway.

Keywords: skeletal muscle, G protein-coupled receptor 81, muscle mass, p42/44
extracellular signal-regulated kinase-1/2, p90 ribosomal S6 kinase

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Introduction
Skeletal muscle exhibits high plasticity and changes its mass in response to external
stimuli. It is well known that mechanical loading induced by resistance training is a typicalstimulus for skeletal muscle hypertrophy [1].

It is generally accepted that skeletal muscle

mass is highly regulated by the balance between protein synthesis and degradation [2].

The

protein synthesis in skeletal muscle has been suggested to be mediated by anabolic signals,
such as p42/44 extracellular signal-regulated kinase-1/2 (ERK1/2) and its downstream target
p90 ribosomal S6 kinase (p90RSK) [3]. In skeletal muscle cells, the ERK/p90RSK
signaling pathway is activated by mechanical loading [4] and has been suggested to be
involved in mechanical loading-induced skeletal muscle hypertrophy [5].

Additionally, theprotein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway and its
downstream targets p70 S6 kinase (p70S6K) are also included in anabolic signals andinvolved in muscle hypertrophy [6-8]. However, the detailed mechanism of muscle
hypertrophic response to resistance training has not been elucidated.
flow-restriction training is a training under vascular occlusion.

For instance, blood-

This training increases thesize of human skeletal muscle [9,10] and the level of blood circulating metabolites, such as
interleukin-6 (IL-6), growth hormone (GH), and lactate [10,11].

In particular, IL-6 isthought to stimulate muscle satellite cells and play important roles in muscle hypertrophy[12]. IL-6 may also directly mediate protein synthesis through activation of anabolic
signaling pathways [13]. Furthermore, GH has been shown to play an anabolic role inregulating skeletal muscle mass [14]. Therefore, there is a possibility that the metabolites
contribute to muscle hypertrophy following resistance training.
Lactate is one of the secretions released from skeletal muscle cells.

It is generally

4

accepted that lactate synthesis in skeletal muscle cells is stimulated by high-intensity
exercise, and cytosolic lactate is oxidized to pyruvate in mitochondria. Especially, lactate
synthesized in fast-twitch muscle, which has few mitochondria, is transported by circulation
to slow-twitch muscle for use as an energy source [15-17]. To our knowledge, the effect of
intracellular lactate on not only protein synthesis but also muscle mass is unknown, but it has
been suggested that extracellular lactate contributes to increased muscle mass [18-20].
Administration of the lactate-containing compound to mice has been reported to enhance theeffects of low-intensity exercise, increasing skeletal muscle mass and muscle satellite cell
regulatory factors [18]. Our previous study also demonstrated that extracellular lactatecauses C2C12 myotube hypertrophy by activating the anabolic intracellular ERK pathway[19], which stimulates proliferation and differentiation of muscle cells [21,22].

In addition,

we have recently shown that oral administration of lactate stimulates not only skeletal
muscle hypertrophy but also regeneration of injured skeletal muscle via the activation ofmuscle satellite cells [20]. However, the molecular mechanisms of lactate-induced
hypertrophic responses remain unclear.
It has been reported that G protein-coupled receptor 81(GPR81), which is a selective
receptor for lactate, is expressed in rodent skeletal muscle cells [19,23]. Moreover, theexpression of GPR81 in human skeletal muscle has been confirmed at both mRNA [23] andprotein [24] levels. GPR81-mediated activation of the anabolic ERK-intracellular signals
triggered by both lactate and GPR81 agonist has been reported in cultured rat L6 skeletal
muscle cells [25]. However, the physiological role of GPR81 in skeletal muscle cells has
not been fully elucidated.
mediated by GPR81.

We hypothesize that the hypertrophic effects of lactate are

In fact, in our previous study using cultured skeletal muscle cells, the5administration of a GPR81 agonist led to hypertrophy of C2C12 myotubes throughactivation of the ERK/p90RSK pathway [19].

In contrast, GPR81 agonist administration

did not significantly change the Akt/mTOR/p70S6K pathway [19].

However, there is noevidence showing that GPR81 has a hypertrophic effect on skeletal muscle in vivo.
Therefore, in the present study, we focused on ERK pathway and investigated the effects of
GPR81-agonist administration on the ERK pathway and skeletal muscle mass in mice.
Mammalian skeletal muscles are classified into fast- and slow-twitch muscle based on
their contractile properties. The responses to extracellular stimuli depend on these muscle
types [26-28].

It is well known that monocarboxylate transporters (MCTs) are present in

the plasma membrane of skeletal muscle, and lactate is transported across the plasma
membrane by MCTs [29]. MCT1 and MCT4 are predominantly expressed in slow- andfast-twitch fibers, respectively. MCTs play a key role of not only export of lactate frommuscle cells but also import of lactate into muscle cells. The uptake of extracellular lactate
into muscle cells is stimulated by MCT1, while the export of intracellular lactate frommuscle cells is stimulated by MCT4 [30]. On the other hand, a selective receptor for lactate
GPR81 is expressed in skeletal muscle cells [19,23], and activation of ERK pathway
downstream of GPR81 is triggered by lactate as well as GPR81 agonist [19,25]. However,
it is unclear whether there is a difference in GPR81 expression between slow- and fast-twitch
skeletal muscles.
Thus, MCTs and GPR81 may be involved in skeletal muscle responses to lactate,either separately or in relation to each other. Although our previous study demonstrated
that administration of lactate caused muscle hypertrophy in mice [20], it is not clear whether
MCTs or GPR81 are involved in lactate-induced muscle hypertrophy in animals.

6

Therefore, we tried to investigate the potential role of GPR81 in the lactate-induced muscle
hypertrophy by evaluating the expression of GPR81 in fast- and slow-twitch muscles.

7

Methods
Animals and grouping
All animal experimental procedures were conducted in accordance with the Guide for
the Care and Use of Laboratory Animals, as adopted and promulgated by the National
Institutes of Health (Bethesda, MD, USA). The Animal Use Committee of Toyohashi
SOZO University approved the procedures of animal experiments in this study (A2016003,
A2017002, A2018002). Male 8-week-old C57BL/6J mice (n = 52) were used.

To

investigate the effect of GPR81 activation on skeletal muscle mass (muscle weight andprotein content), mice were randomly divided into control (Con; n = 26) and GPR81 agonistadministered (AG; n = 26) groups. All mice were housed in a clean room maintained at
approximately 23 °C with a 12/12 hours light-dark cycle. Solid diet and water were
provided ad libitum.

Administration of GPR81 agonist
In the present study, a 3-chloro-5-hydroxybenzoic acid (CHBA, Tokyo Chemical
Industry, Tokyo, Japan), was used as the GPR81 agonist [31,32].

In the AG group, CHBA

(50 mg/kg body weight) was administered to the mice with an oral sonde 5 days a week for 3
weeks, according to the previously reported method [20]. To avoid the side effects ofCHBA on mice, its dose was selected with consideration for the study in mice [31].

A

stock solution of CHBA (100 mg/ml) was prepared in dimethylsulfoxide (DMSO) anddiluted with water to the final DMSO concentration of 5%. The same amount of 5%
DMSO in water was administered to the Con group.

8

Sample preparation and evaluation of muscle massMice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50mg/kg) and then sacrificed by cervical dislocation 1, 2, and 3 weeks after the initiation of
CHBA administration (n = 8-9 mice per group in each time point).

The left plantaris andsoleus muscles were dissected from the hindlimb of mice immediately after sacrifice.
Isolated muscle tissues were rapidly weighed, frozen in liquid nitrogen, and stored at −80 °C
until later analysis.
Frozen muscles were homogenized with a tissue lysis reagent (CelLyticTM-MT, 100
μl/mg muscle weight, Sigma-Aldrich, St. Louis, MO, USA) containing 1% (v/v)
Protease/Phosphatase Inhibitor Cocktail (5872, Cell Signaling Technology, Danvers, MA,
USA) on ice. To measure the muscle protein content, part of each tissue lysate (50 μl) was
mixed with the equal amount of 2 N NaOH and completely solubilized at 37 °C for 1 h [33].
The protein content of the lysate was determined using the Bradford technique (protein
Assay kit, Bio-Rad, Hercules, CA, USA) and bovine serum albumin (Sigma-Aldrich) as thestandard protein. And then the absolute muscle protein content in each muscle sample wascalculated.

Western blot analysis
Western blot analysis was performed with some modifications to previously reported
methods [19,33]. The rest of each tissue lysate was sonicated to extract proteins,
centrifuged at 20,000 g at 4 °C for 10 min, and the supernatant was collected. Aftermeasuring the protein content in the supernatant using the Bradford technique, thesupernatant solutions were mixed with sodium-dodecylsulfate (SDS) sample buffer (62.59mM Tris-HCl (pH 6.8), 2.3% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 30% (v/v) glycerol,
0.05% (w/v) bromophenol blue) and heated at 95 °C for 5 min.

The SDS-polyacrylamide

gel electrophoresis (PAGE) was performed in a Mini-PROTEAN Tetra Vertical
Electrophoresis Cell (Bio-Rad) at 200 V.

Equal amounts of protein (3 μg) were loaded into

the wells on each gel (Mini-PROTEAN TGX precast gels, Bio-Rad). Molecular weightmarkers (Prestained Protein Standards, Bio-Rad) were applied to the gels to check theelectrophoresis and transfer process.
Following SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF)
membranes (Trans-Blot Turbo Mini 0.2 µm PVDF Transfer Packs, Bio-Rad) using TransBlot Turbo Transfer System (Bio-Rad) at 1.3 A and 25 V for 7 min.

After the transfer, themembranes were blocked for 20 min using Blocking One-P (Nacalai Tesque, Kyoto, Japan).
The membranes were then incubated with a primary antibody: phosphorylated ERK1/2
Thr202/Tyr204 (p-ERK1/2: 9101, Cell Signaling), total ERK1/2 (t-ERK1/2: 9102, Cell
Signaling), phosphorylated p90RSK Ser380 (p-p90RSK: 11989, Cell Signaling), total
p90RSK (t-p90RSK: 9355, Cell Signaling) overnight at 4 °C, followed by a secondary
antibody: anti-rabbit immunoglobulin G conjugate to horseradish peroxidase (Cell
Signaling) for 2 h at room temperature.

After the final wash, protein bands were visualizedwith chemiluminescence (ImmunoStar LD, Fujifilm Wako Pure Chemical Corporation,
Osaka, Japan), were captured using LumiCube (Liponics, Tokyo, Japan), and were analyzed
using JustTLC software (Sweday, Sodra Sandby, Sweden).
The protein expression level of GPR81 in mouse skeletal muscles (n=7) was also
evaluated by western blotting using a primary antibody: GPR81 (sc-32647, Santa Cruz
Biotechnology, Santa Cruz, CA, USA), β-actin (4967, Cell Signaling Technology) [19].

10

Statistical analysis
All values were expressed as means ± SEM. The statistically significant level of
GPR81 expression was assessed using F-test followed by unpaired Student's t-test.

Othersignificant levels in animal experiments were tested using two-way (agonist administrationand time) ANOVA. The significance level was set at 0.05.

11

Results
GPR81 expression in mouse skeletal muscle
Protein expression of GPR81 was observed in both fast-twitch plantaris and slowtwitch soleus muscles of mice. There was no significant difference in the expression levelof GPR81 between the two types of muscles (Figure 1).

Effects of lactate receptor agonist on muscle mass and intracellular signals in mouse skeletal
muscleFigures 2 and 3 show the body weight, plantaris and soleus muscle mass (muscle
weight and protein content) of mice in response to oral administration of CHBA, a GPR81
agonist.
There was no significant difference in mouse body weight between Con and AG
groups during the experimental period (Figure 2A).

In the plantaris and soleus muscles, asignificant main effect of CHBA administration and time was observed on the muscle weight(Figure 2B).

In the AG group, the absolute muscle weight and the muscle weight relative tobody weight was significantly higher than that in the Con group (p < 0.05).
A significant main effect of CHBA administration and time was also found on theprotein content (Figure 3). Administration of CHBA significantly increased absolute andrelative muscle protein content in both plantaris and soleus muscles (p < 0.05).
The changes in phosphorylation levels of ERK1/2 and p90RSK in response to CHBA
administration were shown in Figure 4. There was a significant main effect of CHBA
administration on the relative expression level of phosphorylated ERK1/2 (p-ERK1/2) and pp90RSK in mouse plantaris and soleus muscles.

CHBA administration significantly

12

upregulated the relative expression level of p-ERK1/2 and p-p90RSK (p < 0.05).

Asignificant main effect of time was observed on the relative expression level of p-p90RSK.

13

Discussion
The present study demonstrated that no significant difference in the expression level of
GPR81 was observed between the fast-twitch plantaris and slow-twitch soleus muscles ofmice. Oral administration of CHBA to mice increased the muscle weight and protein
content in both plantaris and soleus muscles. Furthermore, in both muscles, there was also
CHBA-associated increase in the phosphorylation level of ERK1/2 and p90RSK.

Effect of GPR81 agonist on skeletal muscle mass
The present study confirmed GPR81 expression in mouse skeletal muscles, such as
fast-twitch plantaris and slow-twitch soleus muscles (Figure 1).

This result is consistent

with the report using mice [23]. In the present study, the oral administration of CHBA
increased the absolute and relative muscle weight and protein content in both plantaris andsoleus muscles of mice. This is the first report showing administration of GPR81 agonist
increased skeletal muscle mass in mice in vivo.

Additionally, there was no significant

difference in the expression level of GPR81 between fast-twitch plantaris and slow-twitch
soleus muscles, suggesting that GPR81 agonist increased muscle mass similarly in bothtypes. Taken together with the previous observation that GPR81 agonists increased thediameter of C2C12 myotubes [19], the findings from the present study demonstrate that
activation of GPR81 stimulates increases in muscle mass at both cellular and tissue levels ofskeletal muscle.

GPR81-associated intracellular signals
The present study demonstrated that phosphorylation level of ERK1/2 and p90RSK14

was significantly increased following administration of CHBA to mice. This result is
supported by the studies showing that ERK pathway was activated by the GPR81 agonist
3,5-DHBA in rat L6 [25] and mouse C2C12 skeletal muscle cells [19].

Therefore, it wassuggested that the ERK/p90RSK pathway exists downstream of GPR81 in mouse skeletal
muscle tissue.

It is well known that the ERK/p90RSK pathway is an anabolic signal

involved in protein synthesis, leading to muscle hypertrophy [3-5].

Moreover, the previous

study showed that inhibition of the ERK/p90RSK pathway attenuated myotube hypertrophy
induced by the GPR81 ligand lactate in C2C12 cells [19]. There is a possibility that
GPR81-mediated activation of the ERK/p90RSK pathway contribute to the increase inskeletal muscle mass.

GPR81 stimulation and skeletal muscle hypertrophy
It has been suggested that extracellular lactate is one of the regulators of skeletal
muscle plasticity [19,20,34,35].

In skeletal muscle cells, extracellular lactate positively

affected the myotube hypertrophy and myotube formation [19,20,34].

The previous study

reported that oral lactate administration to mice increased blood lactate concentrations andenhanced skeletal muscle hypertrophy and regeneration [20]. Similarly, the study in micehas demonstrated that lactate promotes glycerol-induced muscle regeneration [34].
Recently, lactate has been shown to inhibit the loss of muscle mass due to caloric restriction[35]. On the other hand, it has been reported that lactate administration has no additional
hypertrophic effect in synergist ablation-induced mechanical overload in mouse skeletal
muscle [36]. Judging from the gain of muscle mass by synergist ablation and lactate
administration [36], the hypertrophic effect of lactate is not necessarily strong, but rather

15

small, compared to exercise-associated mechanical loading.
Although the mechanisms of lactate-induced increase in skeletal muscle mass have notbeen fully elucidated, as shown in the present study, lactate receptor GPR81 in skeletal
muscle may play a role in the mechanism of increasing skeletal muscle mass.

If GPR81

activation induces skeletal muscle hypertrophy in pathological as well as healthy humanskeletal muscle, CHBA and lactate might be a possible countermeasure to muscle atrophy
due to reduced physical activity. Further studies including human experiments are needed
to elucidate this issue.
Numerous studies have been performed using young mice on the effects of various
external stimuli on skeletal muscle hypertrophy and atrophy [7,8,37,38].

Furthermore, theage-related suppression of muscle hypertrophy in response to various stimuli is also reported[39-42]. In the present study, therefore, young mice were used to investigate the effects of
GPR81 agonist on skeletal muscle mass. Time-related changes in skeletal muscle mass andintracellular signal were observed in the present study, but the reasons for these changes
cannot be explained at present.

The mice used in this study were in the growth phase,

which may have influenced the time-related changes.
In conclusion, the present study demonstrated GPR81 agonist-associated increase inmuscle mass and activation of ERK pathway in mouse skeletal muscles.

Stimulation of

lactate receptor GPR81 may have a positive impact on skeletal muscle mass.

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Conflict of interest
The authors declare no conflict of interest.

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Acknowledgments
This study was partially supported by JSPS KAKENHI (18K10796, Y.O.; 18H03160,
K.G.; 19K22825, K.G.; 19KK0254, K.G.; 21K11276, Y.O.; 22H03474, K.G.; 22K19722,
K.G.; 22H03319, K.G.; 22K18413, K.G.), the Uehara Memorial Foundation (Y.O.), theMeiji Yasuda Life Foundation of Health and Welfare (Y.O.), a grant from Graduate Schoolof Health Science, Toyohashi SOZO University (K.G.), and a research grant from Toyohashi
SOZO university (K.G.).

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Figure captions
Figure 1. Representative expression patterns and mean expression of GPR81 in mouseplantaris and soleus muscles. GPR81: G-protein-coupled receptor 81.

Values are

expressed relative to the value of plantaris muscle. Values are means ± SEM.

n=7

per group.

Figure 2. Effects of oral CHBA administration on mouse body weight (A), absolute andrelative muscle weight of plantaris and soleus muscles (B). Relative muscle weight:
relative muscle weight to body weight; Con: control group; AG: 3-Chloro-5-hydroxybenzoic
acid (CHBA)-administered group; 1W, 2W, and 3W: CHBA administration for 1, 2, and 3
weeks, respectively. Values are means ± SEM.

n = 8-9 per group in each time point.

Significant main effect of CHBA administration, p < 0.05.

§:||: Significant main effect oftime, p < 0.05.

Figure 3. Effects of oral CHBA administration on absolute and relative protein content ofplantaris and soleus muscles. Relative protein content: relative protein content tobody weight. See Figure 2 for other abbreviations, statistics and symbols.
are means ± SEM.

Valuesn = 8-9 per group in each time point.

Figure 4. Representative expression patterns and mean expressions of p-ERK1/2/t-ERK1/2
and p-p90RSK/t-p90RSK in plantaris (A) and soleus (B) muscles in response to
CHBA. p-ERK1/2: phosphorylated p42/44 extracellular signal-regulated kinase-1/2;
t-ERK1/2: total ERK1/2; p-p90RSK: p90 ribosomal S6 kinase; t-p90RSK: total

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p90RSK. See Figure 2 for other abbreviations, statistics and symbols.

Values are

expressed relative to the value of Con at 1W. Values are means ± SEM.

n = 8-9

per group in each time point.

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