Lactate administration induces skeletal muscle synthesis by influencing Akt/mTOR and MuRF1 in non-trained mice but not in trained mice

doi:10.21203/rs.3.rs-1986461/v1

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Lactate administration induces skeletal muscle synthesis by influencing Akt/mTOR and MuRF1 in non-trained mice but not in trained mice

https://doi.org/10.21203/rs.3.rs-1986461/v1

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Background: The perception regarding lactate has changed over the past decades, with some of its physiological roles having been revealed. However, the effects of exogenous lactate on skeletal muscle synthesis remain unclear. Therefore, this study aimed to confirm the effects of long-term and post-exercise lactate administration on skeletal muscle synthesis.

Methods: 32 ICR mice were randomly assigned to non-trained + placebo (Non/Pla), non-trained + lactate (Non/Lac), tranied + placebo (Tr/Pla), or trained + lactate (Tr/Lac) groups. Lactate (3 g/kg) or same volume of saline was immediately administered after exercise training (70% VO_2max). Lactate administration and/or exercise training was performed 5 days/week for 5 weeks.

Results: After the experimental period, lactate administration tended to increase skeletal muscle weight and significantly enhanced the gastrocnemius muscle weight. Furthermore, lactate administration increased the mRNA levels of Akt (p = 0.000) and mTOR (p = 0.009), decreased protein expression of MuRF1 (p = 0.001), and reduced blood IGF1 levels (p = 0.039). However, lactate administration after exercise training had no additional effects on almost factors.

Conclusions: These results suggest that long-term lactate administration have a potential to stimulate skeletal muscle synthesis, but lactate administration after exercise training may not be extra beneficial for increasing skeletal muscle because of the lactate administration timing.

Lactate administration

Exercise training

Skeletal muscle

Protein synthesis

Protein degradation

Skeletal muscles can be quantitatively altered by the balance between protein synthesis and degradation, which is affected by various factors [1, 2]. Exercises involving muscle contraction and relaxation are widely accepted strategies to increase the amount of skeletal muscle by upregulating protein synthesis [3, 4]. Previous research showed that long-term exercise training induces muscle hypertrophy by increasing protein synthesis signaling such as protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway [5, 6]. Furthermore, this signaling can block E3 ubiquitin ligases ((muscle ring-finger protein-1 [MuRF1] and muscle-specific F-box protein [MAFbx1]), which induce atrophy and protein degradation [7–9]. Thus, exercise training has been confirmed to induce skeletal muscle synthesis, but the trigger for exercise-induced muscle synthesis has not been fully elucidated.

During exercise, various metabolic intermediate and bioactive molecules are secreted from skeletal muscles [10, 11]. Among them, lactate had been recognized as a “fatigue-inducing molecule” because early studies reported that lactate induces acidosis and contributes to a decreased exercise capacity [12, 13]. However, recent studies confirmed that acidosis associated with exercise is induced by increased H⁺ production from ATP hydrolysis [14]. In addition, lactate is used as an energy resource that can transit between body compartments not only through the Cori cycle between the muscles and liver but also from cell to cell or between intracellular sites by the lactate shuttle (monocarboxylate transporters [MCTs]) [15, 16]. The perception of lactate has been changed by recent research, leading to new possibilities for the role of lactate.

High-intensity exercise, which produces more lactate than low intensity-exercise [17], is acknowledged as a strategy to further increase skeletal muscle synthesis [18, 19]. Similarly, previous studies have demonstrated that exercise training with hypoxia or blood flow restriction, in which lactate production is artificially upregulated [20, 21], increases muscle synthesis and strength over a similar low-intensity exercise in a normal state [22–24]. Thus, there is a possibility that secreted lactate during exercise may affect to increase skeletal muscle synthesis. Moreover, several cell studies on lactate have confirmed the effects of increased lactate concentrations on muscle synthesis. Guo et al. [25] showed that increased lactate concentration in CHO-K1 cells decreased cAMP accumulation, which is an inhibitor of mTOR, and Ohno et al. [26] reported that the higher the concentration of lactate (0 to 20 mM), the larger the myotube diameter in C2C12 cells. Accordingly, blood lactate may partially affect the mechanism of exercise-induced muscle synthesis.

Motivated by the relatively novel physiological roles of lactate, recent research has investigated the effects of exogenous lactate on skeletal muscles. Cerda-Kohler et al. [27] reported that an injection of 3 g/kg of lactate in mice increased blood lactate concentration (approximately 20 mM) after 5 to 15 min and upregulated the phosphorylation of Akt (Thr308 and Ser473) and P70S6K in type2 muscles after 40 min. In addition, our previous study demonstrated that oral administration of 2 g/kg of sodium lactate increased mRNA levels of IGF receptor, Akt and mTOR in the plantaris muscle after 30 to 60 min, but did not affect the degradation factors (MuRF1 and MAFbx) [28]. Previous studies have shown that exogenous lactate administration has positive effects on skeletal muscle synthesis.

These findings suggest that long-term lactate administration induces skeletal muscle synthesis and that lactate administration with exercise training can have additional effects on skeletal muscle synthesis by increasing blood lactate [29]. Nevertheless, only a few studies have investigated the effects of long-term lactate treatment and oral administration, and previous studies have not directly investigated the effect of lactate administration with exercise training on skeletal muscle synthesis. Therefore, the present study had two purposes: (a) to determine the effects of oral lactate administration for 5 weeks on skeletal muscle synthesis and protein synthesis and degradation factors; and (b) to confirm whether lactate administration after exercise training has additional effects on skeletal muscle synthesis.

Animal care

Ethical approval for this study was obtained from the Konkuk University Institutional Animal Care and Use Committee (No. KU19149). Six-week-old male ICR mice (n = 32) were obtained from Orient Bio (Seongnam, Korea) and assigned to standard cages (4 mice/cage) under controlled conditions (12:12-h light-dark cycle, 50% humidity, 23 ± 1°C). A standard commercial diet (Orient Bio, Seongnam, Korea) and water were provided ad libitum. Food intake and body weight were measured daily between 9 and 10 a.m.

Study design

Prior to the experiment, the blood lactate concentration of mice (n = 6) after oral lactate administration was measured from the blood obtained from the tail using Lactate Pro2 (Arkray Inc., Kyoto, Japan). Lactate (sodium lactate, 195–05965, Wako Chemical, Osaka, Japan) was orally administered at 3 g/kg [27, 29]. Blood lactate levels significantly increased from 3.03 ± 0.31 mmol/L to 8.22 ± 2.38 mmol/L after 15 min of lactate administration and returned to basal levels (approximately 3.67 ± 1.17 mmol/L) after 3 hours (Table 1). Therefore, exogenous lactate administration increases the blood lactate concentration.

Table 1

Blood lactate concentration after oral administration of lactate.
	Time after lactate administration (min)
	0	15	30	45	60	90	120	150	180
Lactate (mmol/L)	3.0 ± 0.3	8.2 ± 2.4^$	6.7 ± 0.8^$	6.5 ± 1.2^$	5.7 ± 0.3^$	5.7 ± 1.2^$	4.9 ± 0.7^$	5.2 ± 0.8^$	3.7 ± 1.1

Lactate concentration parameters during the resting period after oral administration of lactate. 0 min means before lactate administration and 15, 30, 45, 60, 90, 120, 150, and 180 min means after lactate administration. Results are presented as the mean ± standard deviation. $ p < 0.05 vs. 0 min.

In the study, the mice were randomly divided into four groups as follows: non-trained + placebo (Non/Pla; n = 8), non-trained + lactate (Non/Lac; n = 8), trained + placebo (Tr/Pla; n = 8), and Trained + lactate (Tr/Lac; n = 8). Exercise training intensity was set at the “lactate threshold” (approximately 70% maximal oxygen uptake [VO_2max]) based on a previous study and was gradually increased to avoid exercise adaptation [30]. Lactate and placebo groups were administered 3 g/kg of sodium lactate and saline solution, respectively. Lactate or saline was administered immediately after each exercise training. Exercise training and lactate administration were conducted 5 days/week from 10 to 11 a.m. Mice were sacrificed by intraperitoneal anesthesia with 10 µL/g of 1.25% avertin 48 h after the last exercise training and lactate administration. Blood and skeletal muscles (soleus, plantaris, gastrocnemius, extensor digitorum longus, and tibialis [EDL]) were collected immediately after sacrifice. The study design is illustrated in Fig. 1.

Blood analysis

Blood samples were immediately obtained and centrifuged at 1,500 × g for 15 min at 4℃ before collecting the supernatant (serum). After blood sample preparation, serum concentrations of lactate (KTB1100, Abbkine, Wuhan, China) and glucose (K039-H1, ARBOR ASSAYS, Ann Arbor, MI, US) were measured using a colorimetric kit according to the manufacturer’s protocol and assessed by colorimetric detection at 450 and 560 nm, respectively. Serum concentrations of IGF1 (ab100695, Abcam, Cambridge, UK) were analyzed using an ELISA kit. Serum IGF1 was detected using a microplate reader at 450 nm absorbance, according to the manufacturer’s instructions.

mRNA analysis

Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the mRNA levels of protein synthesis and degradation factors in the plantaris muscle. Total RNA was extracted using 1 ml TRIzol reagent (79306; Qiagen, Hilden, Germany), and 200 µL chloroform (038-02601, Wako Chemical, Osaka, Japan) was added. After centrifuging the sample at 12,000 × g for 15 min at 4°C, the supernatant was separated in another tube, and 200 µL isopropanol (d2377, DUKSAN, Seoul, Korea) was added before mixing and reacting. To collect RNA pellets, samples were centrifuged at 12,000 × g for 15 min at 4°C. RNA pellets were diluted in 30 µL of DEPC water and heated at 55°C. To synthesize complementary DNA (cDNA) from the extracted total RNA, amfiRivert cDNA Synthesis Platinum Master Mix (R5600; GenDEPOT, Katy, TX, USA) was used following the manufacturer’s instructions. cDNA was amplified using amfiEco Taq DNA polymerase (P0701; GenDEPOT, Katy, TX, USA) following the manufacturer’s instructions: 1 µL of cDNA with 24 µL of Taq DNA polymerase, 2 × reaction buffer, PCR water, and forward and reverse primers. Detailed primer sequences and qRT-PCR cycling conditions for each gene are presented in Additional file 1. The PCR products were separated using 1% agarose gels composed of 1 × TAE buffer 50 mL, 0.5 g LE agarose, and 1.25 µL Safe-Pinky DNA gel staining solution (S1001-025; GenDEPOT, Katy, TX, USA). PCR product bands were analyzed using Print Graph 2M (ATTO, Sungnam, Korea) and normalized to GAPDH.

Protein analysis

To confirm the protein expression of muscle synthesis and degradation factors, the EDL muscle was analyzed by western blotting. The EDL muscles were homogenized in 250 µL of protein extraction buffer (EzRIPA Lysis kit, ATTO, Tokyo, Japan). Whole lysates were centrifuged at 14,000 × g at 4℃ for 15 min, and the supernatants that were transferred to new tubes underwent protein concentration determination by a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). After samples were denatured by heating at 100°C for 5 min, that total protein was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 1 h with 5% skim milk (Difco, Franklin Lakes, NJ, USA) and washed four times for 5 min using phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBS-T). Membranes were incubated overnight (16–24 h) at 4℃ with 3% skim milk and primary antibodies against the following: GAPDH (1:1000, sc-35062, Santa Cruze Biotechnology, USA), IGF receptor (1:1000, #3027, Cell signaling technology, USA), Akt (1:1000, #4691, Cell signaling technology, USA), p-Akt (1:1000, #4058, Cell signaling technology, USA), mTOR (1:1000, #2983, Cell signaling technology, USA), p-mTOR (1:1000, #5536, Cell signaling technology, USA), P70S6K (1:1000, #2708, Cell signaling technology, USA), p-P70S6K (1:1000, #9205, Cell signaling technology, USA), MuRF1 (1:1000, sc-3998608, Santa Cruze Biotechnology, USA), MAFbx (1:1000, sc-166806, Santa Cruze Biotechnology, USA), and FoxO1 (1:1000, #9461, Cell signaling technology, USA). After washing with PBS-T, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (anti-mouse, sc-516102; Santa Cruz Biotechnology, USA; anti-rabbit, sc-2357, Santa Cruz Biotechnology, USA) with 3% skim milk for 1 h at room temperature (24–25°C). Immunodetection was performed using an ECL reagent (Amersham Biosciences, Uppsala, Sweden).

Statistical Analysis

All data were analyzed using the IBM SPSS Statistics 25 software. The significant main effects (supplement, Placebo vs. Lactate; and exercise, Non-trained vs. Trained) and interactions were analyzed using a two-way analysis of variance followed by the LSD post hoc test. Significant differences were considered at p < 0.05, and all results are shown as mean ± standard deviations.

Effects of long-term and post-exercise lactate administration on body weight, food intake, and skeletal muscle weight

Body weight, food intake, and skeletal muscle weight after 5 weeks are shown in Table 2. After the experiment, body weight did not differ between the groups. However, food intake had main effects (supplement, p = 0.000; and exercise, p = 0.014) and interactions (p = 0.002). Furthermore, body weight of the Non/Lac was significantly lower than that of the Non/Pla (p = 0.000), while that of the trained groups was significantly higher than that of the non-trained groups (Non/Pla vs. Tr/Pla, p = 0.000; Non/Lac vs. Tr/Lac, p = 0.000).

Muscle weight of five different skeletal muscle was measured and individually normalized to body weight. Lactate administration and exercise training tended to increase muscle weight, but the main effects and interactions were observed only in a few skeletal muscles. The soleus muscle only achieved the main effect after exercise (p = 0.031). The gastrocnemius muscle achieved the main effect of supplementation (p = 0.028) and interaction (p = 0.000), and that of Non/Lac and Tr/Pla were significantly higher than that of Non/Pla (p = 0.000; and p = 0.001 respectively). The plantaris, EDL, and tibialis muscles were not significantly different among the groups.

Table 2

Body weight and skeletal muscle weight after 5 weeks.
	Non-trained
	Placebo		Lactate		Placebo		Lactate
Body weights (g/mouse)	35.93 ± 0.78	34.64 ± 0.93		35.15 ± 0.64		35.47 ± 0.72		Supplement 0.966 Exercise 0.387 Interactions 0.152
Food intake (g/5 week)	121.22 ± 2.1	109.08 ± 2.0*		147.28 ± 3.4^#		148.87 ± 2.3^#		Supplement 0.000 Exercise 0.014 Interactions 0.002
Soleus (mg/g body weight)	0.44 ± 0.06	0.47 ± 0.06		0.49 ± 0.02^#		0.45 ± 0.07		Supplement 0.849 Exercise 0.031 Interactions 0.096
Plantaris (mg/g body weight)	0.99 ± 0.08	1.06 ± 0.07		1.06 ± 0.06		1.07 ± 0.11		Supplement 0.172 Exercise 0. 145 Interactions 0.369
Gastrocnemius (mg/g body weight)	8.40 ± 0.65	9.14 ± 0.35^*		8.98 ± 0.34^#		8.96 ± 0.9		Supplement 0.028 Exercise 0. 253 Interactions 0.000
Extensor digitorum longus (mg/g body weight)	0.64 ± 0.13	0.71 ± 0.11		0.68 ± 0.07		0.68 ± 0.11		Supplement 0.161 Exercise 0. 506 Interactions 0.532
Tibialis (mg/g body weight)	3.51 ± 0.24	3.58 ± 0.19		3.63 ± 0.19		3.54 ± 0.37		Supplement 0.913 Exercise 0. 689 Interactions 0.411

Muscle weight of five different skeletal muscle was normalized to body weight. Results are presented as the mean ± standard deviation. ^* p < 0.05, placebo vs. lactate; ^# p < 0.05, non-trained vs. trained.

Effects of long-term and post-exercise lactate administration on blood lactate, glucose, and IGF1 levels

The serum levels of lactate, glucose, and IGF1 were measured to determine whether these were affected by long-term and post-exercise lactate administration (Table 3). The serum lactate concentration did not significantly differ between the groups, the serum glucose concentration only had an exercise effect (p = 0.004), and the serum IGF1 concentration had main effects (supplement, p = 0.021; and exercise, p = 0.01) and interactions (p = 0.043); those of Non/Lac were significantly lower than those of Non/Pla (p = 0.039) and Tr/Lac (p = 0.007).

Table 3

Blood concentration of lactate, glucose, and IGF1.
	Non-trained		Trained
	Placebo	Lactate	Placebo	Lactate
Lactate (mmol/L)	3.71 ± 0.60	3.29 ± 0.72	3.18 ± 0.29	3.15 ± 0.55	Supplement 0.265 Exercise 0.101 Interaction 0.332
Glucose (mg/dL)	166.8 ± 20.6	155.4 ± 23.5	131.1 ± 32.0^#	135.5 ± 21.3	Supplement 0.691 Exercise 0.004 Interaction 0.375
IGF1 (ng/mL)	29.02 ± 4.03	25.17 ± 2.31*	31.34 ± 3.84	29.38 ± 2.94^#	Supplement 0.021 Exercise 0. 01 Interaction 0.043
Results are presented as the mean ± standard deviation. ^* p < 0.05, placebo vs. lactate; ^# p < 0.05, non-trained vs. trained.

Effects of long-term and post-exercise lactate administration on mRNA of protein synthesis and degradation factors in mice plantaris muscles

RT-PCR analysis was conducted to investigate the mRNA expression of protein synthesis and degradation factors due to long-term and post-exercise lactate administration. The mRNA levels of IGF receptor, Akt, mTOR, and P70S6K were measured to confirm protein synthesis (Figs. 2B-E). IGF receptor only achieved the main effect of exercise (p = 0.000). Meanwhile, the mRNA of Akt had the main effect of supplementation (p = 0.001) and interactions (p = 0.025); that in Non/Lac were significantly upregulated compared with that in Non/Pla (p = 0.000). Furthermore, mTOR only achieved the main effect of supplementation (p = 0.009), and that of Non/Lac was significantly upregulated compared with that of Non/Pla (p = 0.043). Although the mRNA level of mTOR was increased in Non/Pla, the P70S6K of mTOR downregulation was not significantly different between the groups.

The current study measured the mRNA levels of MuRF1, MAFbx, and FoxO1 to confirm changes in protein degradation factors after long-term and post-exercise lactate administration (Figs. 2F-H), which revealed that the mRNA levels of protein degradation factors were not affected.

Effects of long-term and post-exercise lactate administration on protein expression of protein synthesis and degradation factors in mice EDL muscle

To elucidate the effects of long-term and post-exercise lactate administration on protein expression of protein synthesis (Figs. 3B-E) and degradation (Figs. 3G-I) factors, western blotting was conducted. Unexpectedly, long-term and post-exercise lactate administration did not significantly affect protein expression of the synthesis factors of the IGF receptor, Akt phosphorylation, and mTOR phosphorylation. In addition, P70S6K phosphorylation was the only main effect of exercise (p = 0.025).

Regarding the protein expression of skeletal muscle degradation factors, MAFbx and FoxO1 were not affected by long-term and post-exercise lactate administration. However, the MuRF1 expression achieved the main effects (supplementation, p = 0.003; and exercise, p = 0.000) and interactions (p = 0.035), and that in Non/Lac and Tr/Pla were significantly downregulated compared with that in Non/Pla (p = 0.001 and p = 0.000, respectively).

While the effects of exogenous lactate administration have been investigated, those of oral lactate administration on muscle synthesis have not been elucidated. The present study investigated the effects of long-term lactate administration on skeletal muscle synthesis and the additional effects of lactate administration after exercise training. The main findings were that long-term lactate administration increased skeletal muscle weight and influenced the expression of protein synthesis and degradation factors. However, lactate administration after exercise training did not have any additional effects on skeletal muscle synthesis.

This study showed that lactate administration decreased the amount of food intake by 11.1% and tended to decrease body weight. Appetite is regulated by hormones, such as leptin and ghrelin [31]. Previous studies have reported that lactate inhibits ghrelin secretion by binding ghrelin-producing cells [32] and that central administration of lactate decreases energy intake in rodents [33]. Therefore, the study suggests that oral lactate administration can also reduce food intake by inhibiting ghrelin secretion.

An important feature of long-term exercise training is that it induces skeletal muscle synthesis [3]. Specifically, high-intensity exercise increases muscle synthesis and lactate concentration to a greater extent than low-intensity exercise [17]. This suggests that an increase in blood lactate concentration may be linked to exercise-induced muscle synthesis. Previous studies have reported that lactate activates cell proliferation and increases the myotube diameter and length, myonuclei number, and protein content in cells [26, 34, 35]. Moreover, administration of 1 g/kg of lactate increased the blood lactate concentration by 4.1 ± 0.3 mmol/L, and that lactate administration 5 days/week increased the weight of the tibialis anterior muscle after 2 weeks in mice [26]. Thus, we hypothesized that long-term lactate administration would increase skeletal muscle weight. As expected, the pilot results showed that acute lactate administration increased the blood lactate concentration. Additionally, the current study demonstrated that skeletal muscle weight tended to increase in the Non/Lac when normalized to body weight and that the weight of the gastrocnemius muscle significantly increased when normalized to body weight. These findings suggest that increasing blood lactate levels may positively affect skeletal muscle synthesis as part of exercise-induced muscle synthesis.

The Akt/mTOR pathway and muscle-specific E3 ubiquitin ligases regulate muscle synthesis and degradation and play important roles in exercise-induced muscle synthesis [36, 37]. Previous studies primarily focused on the effects of acute lactate intake on protein synthesis and showed that exogenous lactate intake can alter the protein balance by stimulating the Akt/mTOR pathway in type 2 skeletal muscles [27, 28]. The present study investigated the effects of oral administration of lactate on protein synthesis and confirmed that the mRNA levels of Akt and mTOR in the plantaris muscle were increased. However, long-term lactate administration did not affect protein expression in the Akt/mTOR pathway in the EDL muscle. Nevertheless, the current results showed that the weight of skeletal muscles increased after long-term lactate administration, which was expected as the concentration of muscle specific E3 ubiquitin ligases decreased. MuRF1, an E3 ubiquitin ligase expressed in skeletal muscle, regulates proteolysis [38]; long-term exercise training attenuates protein degradation by downregulating MuRF1 expression in skeletal muscle [39, 40]. Interestingly, this study showed that MuRF1 expression was downregulated by long-term lactate administration, resulting in increased skeletal muscle weight. These findings are different from those of previous studies and suggest that long-term lactate administration may have a different effect on protein balance when compared with acute administration. The present study confirms that long-term lactate administration can positively affect skeletal muscle weight and the expression of protein synthesis and degradation factors.

IGF1 is generally acknowledged as somatomedin C, which activates cell growth and proliferation [36, 41]. However, several studies have recommended that the IGF1 concentration does not simply influence muscle synthesis; rather, it requires interaction with IGF receptors, physical activity, bio parameters, or other factors [42, 43]. This study confirmed that long-term lactate administration tended to increase skeletal muscle mass despite decreasing levels of circulating IGF1. Similarly, our previous study in rats confirmed that the level of factors for skeletal muscle synthesis increased even though lactate levels were reduced by serum IGF1 [28]. Thus, we suggest that the potential for increasing skeletal muscle synthesis caused by lactate does not depend on IGF1, and that lactate may affect other muscle synthesis pathways. Tsukamoto et al. [44] proposed that lactate inhibits Sir2, which is a negative regulator of MyoD, by decreasing the NAD⁺/NADH ratio. Oishi et al. [35] argued that calcium satellite anabolism is mediated by calcium signals and that lactate may stimulate the calcium/calmodulin-activated serine-threonine phosphatases calcineurin and myogenin. However, since this study did not elucidate the mechanisms of skeletal muscle synthesis caused by lactate, further molecular mechanistic studies on the effects of lactate administration on skeletal muscle are needed.

Oishi et al. [35] confirmed that administration of lactate and caffeine compounds combined with exercise training for 4 weeks increased the weight of the gastrocnemius and tibialis anterior muscles, enhanced the total DNA content, and upregulated anabolic signals in rats. Hashimoto et al. [45] showed that lactate-based compounds increased skeletal muscle weight of the plantaris and gastrocnemius muscles in obese rats when combined with voluntary running exercises. However, although the present study showed that lactate affects skeletal muscle weight and protein synthesis and degradation factors, its administration after exercise training did not result in additional effects.

Several studies reported that most (75–80%) of the lactate produced from exercise is preferentially used and stored as an energy source by skeletal muscles during exercise, post-exercise, and rest under aerobic conditions [46, 47]. In addition, Brooks et al. [48] reported that the lactate shuttle, which is activated by exercise training, transports lactate to the skeletal muscles, heart, and liver for energy production and gluconeogenesis. The lactate shuttle, also known as MCT isoforms 1 to 4, transports lactate to “cell-cell” and “intracellular shuttles” and is activated by the external environment, specifically exercise [49]. Coles et al. [50] showed that the MCT1 and 4 proteins and mRNA expression were immediately enhanced after acute exercise (2 hours, 21 m/min, 15% grade) in rat skeletal muscles. Enoki et al. [51] also reported that long-term exercise training (20–25 m/min, 30 min/day, 7 days/week, 3 weeks) increased levels of MCT1 and 4 in rat skeletal muscles. In summary, lactate is immediately transported and eliminated by exercise activated MCTs. In our experiment, lactate was administered immediately after every episode of exercise training. Hence, we supposed that MCTs were activated by exercise training, and that lactate administered immediately post-exercise was preferentially used as an energy source for post-exercise recovery rather than for skeletal muscle synthesis. However, a limitation of this study was that we did not confirm MCT activation in the skeletal muscles. Therefore, a more detailed study investigating the activation of MCTs is needed to confirm the effects of lactate on molecular signaling in skeletal muscle synthesis. Nevertheless, since lactate tended to increase skeletal muscle weight, positive results of skeletal muscle synthesis may be observed if lactate is administered before exercise training to reduce the effect of MCTs.

In summary, the present study confirmed that (a) lactate can increase skeletal muscle mass and has positive effects on protein synthesis (mRNA levels of Akt and mTOR) and degradation (MuRF1 protein expression); however, (b) the effects of lactate on skeletal muscles tend to disappear after exercise training. Although the study did not elucidate the exact mechanisms of long-term and post-exercise lactate administration, a notable finding was that lactate has the potential to positively regulate skeletal muscle synthesis. Therefore, this study provides new insights into the effects of oral administration of exogenous lactate on skeletal muscle synthesis.

Ethics approval and consent to participate

Ethical approval for this study was obtained from the Konkuk University Institutional Animal Care and Use Committee (No. KU19149).

Consent for publication

Not applicable.

Availability of data and materials

All availability of data and materials included in this article.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF-2019S1A5B8099542 and NRF-2021R1G1A1011987).

Authors' contributions

S.K., J.K., H.P., and K.L. conceived and designed the experiment. S.K., and I.J. performed the experiment. S.K., J.K., D.H., and H.P. analyzed data. S.K., D.H., and I.J. prepared figures and tables. S.K. wrote original draft. All authors contributed to manuscript revision.

Acknowledgments

We thank all members of the Physical Activity and Performance Institute, Konkuk University, and members of the Department of Sports Medicine and Science at the Graduate School of Konkuk University.

Atherton PJ, Smith K. Muscle protein synthesis in response to nutrition and exercise. J Physiol. 2012;590(5):1049–57. doi: 10.1113/jphysiol.2011.225003.
Anthony TG. Mechanisms of protein balance in skeletal muscle. Domest Anim Endocrinol. 2016;56:S23–32. doi: 10.1016/j.domaniend.
Tipton KD, Wolfe RR. Exercise, Protein Metabolism, and Muscle Growth. Int J Sport Nutr Exerc Metab. 2001;11(1):109–32. doi: 10.1123/ijsnem.11.1.109.
Kumar V, Atherton P, Smith K, Rennie MJ. Human muscle protein synthesis and breakdown during and after exercise. J Appl Physiol. 2009;106(6):2026–39. doi: 10.1152/japplphysiol.91481.2008.
Bodine SC. mTOR signaling and the molecular adaptation to resistance exercise. Med Sci Sports Exerc. 2006;38(11):1950–7. doi: 10.1249/01.mss.0000233797.24035.35.
Devin JL, Bolam KA, Jenkins DG, Skinner TL. The influence of exercise on the insulin-like growth factor axis in oncology: Physiological basis, current, and future perspectives. Cancer Epidemiol Biomarkers Prev. 2016;25(2):239–49. doi: 10.1158/1055-9965.EPI-15-0406.
Latres E, Amini AR, Amini AA, Griffiths J, Martin FJ, Wei Y, et al. Insulin-like growth factor-1 (IGF-1) inversely regulates atrophy-induced genes via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. J Biol Chem. 2005;280(4):2737–44. doi: 10.1074/jbc.M407517200.
Zeng Z, Liang J, Wu L, Zhang H, Lv J, Chen N. Exercise-Induced Autophagy Suppresses Sarcopenia Through Akt/mTOR and Akt/FoxO3a Signal Pathways and AMPK-Mediated Mitochondrial Quality Control. Front Physiol. 2020;11:583478. doi: 10.3389/fphys.2020.583478.
Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, et al. The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell. 2004;14(3):395–403. doi: 10.1016/s1097-2765(04)00211-4.
Leal LG, Lopes MA, Batista ML. Physical exercise-induced myokines and muscle-adipose tissue crosstalk: A review of current knowledge and the implications for health and metabolic diseases. Front Physiol. 2018;9:1307. doi: /10.3389/fphys.2018.01307.
Hargreaves M, Spriet LL. Skeletal muscle energy metabolism during exercise. Nat Metab. 2020;2(9):817–28. doi: 10.1038/s42255-020-0251-4.
Hollidge-Horvat MG, Parolin ML, Wong D, Jones NL, Heigenhauser GJF. Effect of induced metabolic alkalosis on human skeletal muscle metabolism during exercise. Am J Physiol - Endocrinol Metab. 2000;278(2):E316–29. doi: 10.1152/ajpendo.1999.277.4.E647.
Sutton JR, Jones NL, Toews CJ. Effect of pH on muscle glycolysis during exercise. Clin Sci. 1981;61(3):331–8. doi: 10.1042/cs0610331.
Robergs RA, Ghiasvand F, Parker D, Davies RC, Eston RG, Fulford J, et al. Biochemistry of exercise-induced metabolic acidosis association. Am J Physiol. 2004. doi: 10.1152/ajpregu.00114.2004.
Brooks GA. Lactate shuttles in nature. Biochem Soc Trans. 2002;30(2):258–64. doi: 10.1042/bst0300258.
Hashimoto T, Brooks GA. Mitochondrial lactate oxidation complex and an adaptive role for lactate production. Med Sci Sports Exerc. 2008;40(3):486–94. doi: 10.1249/MSS.0b013e31815fcb04.
Gollnick PD, Bayly WM, Hodgson DR. Exercise intensity, training, diet, and lactate concentration in muscle and blood. Medicine and Science in Sports and Exercise. 1986;18(3)334–40. doi: 10.1249/00005768-198606000-00015.
Fry AC. The role of resistance exercise intensity on muscle fibre adaptations. Sport Med. 2004;34(10):663–79. doi: 10.2165/00007256-200434100-00004.
Widegren U, Wretman C, Lionikas A, Hedin G, Henriksson J. Influence of exercise intensity on ERK/MAP kinase signalling in human skeletal muscle. Pflugers Arch Eur J Physiol. 2000;441(2–3):317–22. doi: 10.1007/s004240000417.
Teixeira EL, Barroso R, Silva-Batista C, Laurentino GC, Loenneke JP, Roschel H, et al. Blood flow restriction increases metabolic stress but decreases muscle activation during high-load resistance exercise. Muscle and Nerve. 2018;57(1):107–11. doi: 10.1002/mus.25616.
Wang D, Wang Q, Yan G, Qiao Y, Zhu B, Liu B, et al. Hypoxia induces lactate secretion and glycolytic efflux by downregulating mitochondrial pyruvate carrier levels in human umbilical vein endothelial cells. Mol Med Rep. 2018;18(2):1710–7. doi: 10.3892/mmr.2018.9079.
Sudo M, Ando S, Kano Y. Repeated blood flow restriction induces muscle fiber hypertrophy. Muscle and Nerve. 2017;55(2):274–6. doi: 10.1002/mus.25415.
Karabulut M, Abe T, Sato Y, Bemben MG. The effects of low-intensity resistance training with vascular restriction on leg muscle strength in older men. Eur J Appl Physiol. 2010;108(1):147–55. doi: 10.1007/s00421-009-1204-5.
Feriche B, García-Ramos A, Morales-Artacho AJ, Padial P. Resistance Training Using Different Hypoxic Training Strategies: a Basis for Hypertrophy and Muscle Power Development. Sport Med - Open. 2017;3(1):12. doi: 10.1186/s40798-017-0078-z.
Li G, Wang HQ, Wang LH, Chen RP, Liu JP. Distinct pathways of ERK1/2 activation by hydroxy-carboxylic acid receptor-1. PLoS One. 2014;9(3):e93041. doi: 10.1371/journal.pone.0093041.
Ohno Y, Ando K, Ito T, Suda Y, Matsui Y, Oyama A, et al. Lactate stimulates a potential for hypertrophy and regeneration of mouse skeletal muscle. Nutrients. 2019;11(4):869. doi: 10.3390/nu11040869.
Cerda-Kohler H, Henríquez-Olguín C, Casas M, Jensen TE, Llanos P, Jaimovich E. Lactate administration activates the ERK1/2, mTORC1, and AMPK pathways differentially according to skeletal muscle type in mouse. Physiol Rep. 2018;6(18):e13800. doi: 10.14814/phy2.13800.
Kyun S, Yoo C, Park HY, Kim J, Lim K. The effects of exogenous lactate administration on the igf1/akt/mtor pathway in rat skeletal muscle. Int J Environ Res Public Health. 2020;17(21):7805. doi: 10.3390/ijerph17217805.
Jang I, Kim J, Kyun S, Hwang D, Lim K. Acute Administration of Exogenous Lactate Increases Carbohydrate Metabolism during Exercise in Mice. Metabolites. 2021;11(8):553. doi: 10.3390/metabo11080553.
Hwang H, Kim J, Lim K. The effect of a 2-week red ginseng supplementation on food efficiency and energy metabolism in mice. Nutrients. 2020;12(6):1726. doi: 10.3390/nu12061726.
Austin J, Marks D. Hormonal Regulators of Appetite. Int J Pediatr Endocrinol. 2009;2009:141753. doi: 10.1155/2009/141753.
Engelstoft MS, Park W mee, Sakata I, Kristensen L V., Husted AS, Osborne-Lawrence S, et al. Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells. Mol Metab. 2013;2(4):376–92. doi: 0.1016/j.molmet.2013.08.006
Lam CKL, Chari M, Wang PYT, Lam TKT. Central lactate metabolism regulates food intake. Am J Physiol - Endocrinol Metab. 2008;295(2):E491–6. doi: 10.1152/ajpendo.90481.
Ohno Y, Oyama A, Kaneko H, Egawa T, Yokoyama S, Sugiura T, et al. Lactate increases myotube diameter via activation of MEK/ERK pathway in C2C12 cells. Acta Physiol. 2018;223(2):e13042. doi: 10.1111/apha.13042.
Oishi Y, Tsukamoto H, Yokokawa T, Hirotsu K, Shimazu M, Uchida K, et al. Mixed lactate and caffeine compound increases satellite cell activity and anabolic signals for muscle hypertrophy. J Appl Physiol. 2015;118(6):742–9. doi: 10.1152/japplphysiol.00054.2014.
Schiaffino S, Mammucari C. Regulation of skeletal muscle growth by the IGF1-Akt/PKB pathway: Insights from genetic models. Skelet Muscle. 2011;1(1):1–4. doi: 10.1186/2044-5040-1-4.
Schiaffino S, Dyar KA, Ciciliot S, Blaauw B, Sandri M. Mechanisms regulating skeletal muscle growth and atrophy. FEBS J. 2013;280(17):4294–314. doi: 10.1111/febs.12253.
Bodine SC, Baehr LM. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1. Am J Physiol - Endocrinol Metab. 2014;307(6):E469–84. doi: 10.1152/ajpendo.00204.2014.
Zanchi NE, de Siqueira Filho MA, Lira FS, Rosa JC, Yamashita AS, de Oliveira Carvalho CR, et al. Chronic resistance training decreases MuRF-1 and Atrogin-1 gene expression but does not modify Akt, GSK-3β and p70S6K levels in rats. Eur J Appl Physiol. 2009;106(3):415–23. doi: 10.1007/s00421-009-1033-6.
Gielen S, Sandri M, Kozarez I, Kratzsch J, Teupser D, Thiery J, et al. Exercise training attenuates MuRF-1 expression in the skeletal muscle of patients with chronic heart failure independent of age: The randomized leipzig exercise intervention in chronic heart failure and aging catabolism study. Circulation. 2012;125(22):2716–27. doi: 10.1161/CIRCULATIONAHA.111.047381.
Huat TJ, Khan AA, Pati S, Mustafa Z, Abdullah JM, Jaafar H. IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells. BMC Neurosci. 2014;15:91. doi: 10.1186/1471-2202-15-91.
Nindl BC, Urso ML, Pierce JR, Scofield DE, Barnes BR, Kraemer WJ, et al. IGF-I measurement across blood, interstitial fluid, and muscle biocompartments following explosive, high-power exercise. Am J Physiol - Regul Integr Comp Physiol. 2012;303(10):R1080-9. doi: 10.1152/ajpregu.00275.2012.
Kraemer WJ, Ratamess NA, Nindl BC. Recovery responses of testosterone, growth hormone, and IGF-1 after resistance exercise. J Appl Physiol. 2017;122(3):549–58. doi: 10.1152/japplphysiol.00599.2016.
Tsukamoto S, Shibasaki A, Naka A, Saito H, Iida K. Lactate promotes myoblast differentiation and myotube hypertrophy via a pathway involving MyoD in vitro and enhances muscle regeneration in vivo. Int J Mol Sci. 2018;19(11);3649. doi: 10.3390/ijms19113649.
Hashimoto T, Yokokawa T, Narusawa R, Okada Y, Kawaguchi R, Higashida K. A lactate-based compound containing caffeine in addition to voluntary running exercise decreases subcutaneous fat mass and improves glucose metabolism in obese rats. J Funct Foods. 2019;56:84–91. doi: 10.1016/j.jff.2019.03.007.
Nalbandian M, Takeda M. Lactate as a signaling molecule that regulates exercise-induced adaptations. Biology (Basel). 2016;5(4):38. doi: 10.3390/biology5040038.
Brooks GA. Lactate as a fulcrum of metabolism. Redox Biol. 2020;35:101454. doi: 10.1016/j.redox.2020.101454.
Brooks GA. The Science and Translation of Lactate Shuttle Theory. Cell Metab. 2018;27(4):757–85. doi: 10.1016/j.cmet.2018.03.008.
Kitaoka Y, Takahashi Y, Machida M, Takeda K, Takemasa T, Hatta H. Effect of AMPK activation on monocarboxylate transporter (MCT)1 and MCT4 in denervated muscle. J Physiol Sci. 2014;64(1):59–64. doi: 10.1007/s12576-013-0290-7.
Coles L, Litt J, Hatta H, Bonen A. Exercise rapidly increases expression of the monocarboxylate transporters MCT1 and MCT4 in rat muscle. J Physiol. 2004;561(1):253–61. doi: 10.1113/jphysiol.2004.073478.
Enoki T, Yoshida Y, Hatta H, Bonen A. Exercise training alleviates MCT1 and MCT4 reductions in heart and skeletal muscles of STZ-induced diabetic rats. J Appl Physiol. 2003;94(6):2433–8. doi: 10.1152/japplphysiol.01155.2002.

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Lactate administration induces skeletal muscle synthesis by influencing Akt/mTOR and MuRF1 in non-trained mice but not in trained mice

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Effects of long-term and post-exercise lactate administration on blood lactate, glucose, and IGF1 levels

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