Mineral-Enriched Deep-Sea Water Modulates Lactate Metabolism via PGC-1α-Mediated Metabolic Reprogramming

Metabolic disorders such as diabetes and obesity are serious global health issues. These diseases are accelerated by mineral deficiencies, emphasizing the importance of addressing these deficiencies in disease management plans. Lactate metabolism is fundamentally linked to glucose metabolism, and several clinical studies have reported that blood lactate levels are higher in obese and diabetic patients than in healthy subjects. Balanced deep-sea water contains various minerals and exhibits antiobesity and antidiabetic activities in mice; however, the impact of balanced deep-sea water on lactate metabolism is unclear. Thus, we evaluated the effects of balanced deep-sea water on lactate metabolism in C2C12 myotubes, and found that balanced deep-sea water mediated lactate metabolism by regulating the gene expression levels of lactate dehydrogenases A and B, a monocarboxylate transporter, and a mitochondrial pyruvate carrier. The activities of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and signaling molecules involved in PGC-1α activation were also upregulated by treatment with balanced deep-sea water. These results suggest that balanced deep-sea water, which can mediate lactate metabolism, may be used to prevent or treat obesity and diabetes mellitus.


BDSW Cytotoxicity
The cytotoxicity of BDSW was evaluated by measuring the viability of mouse C2C12 myotube cells. BDSW did not show significant cytotoxic effects for C2C12 cells at hardness levels of up to 3000 ( Figure 1). Because of the lack of a measurable effect of water hardness on cell viability, no further morphological analyses of the cells were conducted.

BDSW Modulated Expression of Lactate Production-Related Genes
To evaluate the effects of BDSW on lactate production, we examined the mRNA expression of LDH A and LDH B in C2C12 myotubes. BDSW decreased the LDH A mRNA expression in a hardnessdependent manner (Figure 2A), whereas the LDH B mRNA expression gradually increased as the BDSW hardness increased ( Figure 2B). These results suggest that BDSW can modulate lactate production.

BDSW Modulated Expression of Lactate Production-Related Genes
To evaluate the effects of BDSW on lactate production, we examined the mRNA expression of LDH A and LDH B in C2C12 myotubes. BDSW decreased the LDH A mRNA expression in a hardness-dependent manner (Figure 2A), whereas the LDH B mRNA expression gradually increased as the BDSW hardness increased ( Figure 2B). These results suggest that BDSW can modulate lactate production.  Effects of BDSW on the expression of genes related to lactate production in C2C12 myotubes. Cells were incubated with BDSW of various hardness levels (0, 500, 1000, or 2000) or 10 μM SRT1720 (SRT) for 12 h. The mRNA levels of (A) LDH A and (B) LDH B represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. untreated control.

BDSW Enhanced PGC-1α Expression and Activity in C2C12 Myotubes
To study the effects of BDSW on lactate homeostasis, we measured PGC-1a expression and activity in C2C12 myotubes. BDSW treatment of C2C12 myotubes increased the PGC-1α mRNA levels in a hardness-dependent manner ( Figure 3A) and decreased PGC-1α acetylation ( Figure 3B). These results suggest that BDSW treatment can upregulate the PGC-1α gene expression and protein deacetylation, which significantly promotes the PGC1-α activity.

BDSW Enhanced PGC-1α Expression and Activity in C2C12 Myotubes
To study the effects of BDSW on lactate homeostasis, we measured PGC-1a expression and activity in C2C12 myotubes. BDSW treatment of C2C12 myotubes increased the PGC-1α mRNA levels in a hardness-dependent manner ( Figure 3A) and decreased PGC-1α acetylation ( Figure 3B). These results suggest that BDSW treatment can upregulate the PGC-1α gene expression and protein deacetylation, which significantly promotes the PGC1-α activity.   Relative PGC-1α mRNA levels in C2C12 myotubes incubated with BDSW of various hardness levels (0, 500, 1000, or 2000) or 10 μM SRT1720 (SRT) for 12 h. The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01 vs. untreated control. (B) Representative western blots and relative quantification of the acetylated versus total PGC-1α protein. Cells were incubated with BDSW of various hardness levels (0, 500, 1000, or 2000), 0.5 mM AICAR (AC), or 10 μM SRT1720 (SRT) for 1 h. PGC-1α was immunoprecipitated from nuclear extracts and immunoblotted using an anti-acetylated lysine (Ac-Lys) antibody to measure the acetylated protein or an anti-PGC-1α antibody to determine the total protein. IP, immunoprecipitation. The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. untreated control.

Potential Mechanisms of BDSW Effects on Lactate Metabolism
To clarify the molecular mechanism involved in the mediation of lactate production by BDSW, we used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the expression of genes encoding regulators of LDH B and LDH A in C2C12 myotubes. We found that BDSW treatment increased the ERR-α and RXR-α expression ( Figure 4A,B) and decreased that of Myc ( Figure 4C). These results suggested that BDSW could modulate lactate production by reprogramming genes that regulate LDH A and LDH B expression. To assess the effects of BDSW on the transport of lactate and pyruvate, we examined MCT1, MPC1, and MPC2 mRNA levels in C2C12 myotubes and observed an increase in MCT1 ( Figure 5A) and MPC1 ( Figure 5B) expression upon an increase in BDSW hardness. (A) Relative PGC-1α mRNA levels in C2C12 myotubes incubated with BDSW of various hardness levels (0, 500, 1000, or 2000) or 10 µM SRT1720 (SRT) for 12 h. The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01 vs. untreated control. (B) Representative western blots and relative quantification of the acetylated versus total PGC-1α protein. Cells were incubated with BDSW of various hardness levels (0, 500, 1000, or 2000), 0.5 mM AICAR (AC), or 10 µM SRT1720 (SRT) for 1 h. PGC-1α was immunoprecipitated from nuclear extracts and immunoblotted using an anti-acetylated lysine (Ac-Lys) antibody to measure the acetylated protein or an anti-PGC-1α antibody to determine the total protein. IP, immunoprecipitation. The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. untreated control.

Potential Mechanisms of BDSW Effects on Lactate Metabolism
To clarify the molecular mechanism involved in the mediation of lactate production by BDSW, we used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the expression of genes encoding regulators of LDH B and LDH A in C2C12 myotubes. We found that BDSW treatment increased the ERR-α and RXR-α expression ( Figure 4A,B) and decreased that of Myc ( Figure 4C). These results suggested that BDSW could modulate lactate production by reprogramming genes that regulate LDH A and LDH B expression. To assess the effects of BDSW on the transport of lactate and pyruvate, we examined MCT1, MPC1, and MPC2 mRNA levels in C2C12 myotubes and observed an increase in MCT1 ( Figure 5A) and MPC1 ( Figure 5B) expression upon an increase in BDSW hardness.

Signaling Molecules Involved in PGC-1α Activation
The AMPK-mediated increase in SIRT1 activity regulates the activity of downstream SIRT1 targets (e.g., PGC-1α) [11]. Through these pathways, PGC-1α induces transcription programs that enhance lactate metabolism and mediate skeletal muscle adaptation to improve metabolic health [6,11]. AMPK is activated through its phosphorylation by AMPK kinases, liver kinase B1, and Ca 2+ /calmodulin-dependent protein kinase II in response to an increase in the AMP:ATP ratio. Figure 5. Effects of BDSW on the expression of genes related to lactate (MCT1) and pyruvate (MPC1 and MPC2) transport in C2C12 myotubes. Cells were incubated with BDSW of various hardness levels (0, 500, 1000, or 2000) or 10 µM SRT1720 (SRT) for 12 h. The relative expression levels of (A) MCT1 and (B) MPC1 and MPC2 represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. untreated control.

Signaling Molecules Involved in PGC-1α Activation
The AMPK-mediated increase in SIRT1 activity regulates the activity of downstream SIRT1 targets (e.g., PGC-1α) [11]. Through these pathways, PGC-1α induces transcription programs that enhance lactate metabolism and mediate skeletal muscle adaptation to improve metabolic health [6,11]. AMPK is activated through its phosphorylation by AMPK kinases, liver kinase B1, and Ca 2+ /calmodulin-dependent protein kinase II in response to an increase in the AMP:ATP ratio. Phosphorylated AMPK is critical in skeletal tissues.
In light of these findings, we next studied the effects of BDSW treatment on signaling molecules involved in PGC-1α activation in C2C12 myotubes. We found that BDSW treatment augmented the AMPK and SIRT1 phosphorylation ( Figure 6A). These results suggested that BDSW could control lactate homeostasis via PGC-1α activation by enhancing AMPK and SIRT1 activity. of AMPK), nicotinamide (NA; a SIRT1 repressor), and rapamycin [RP; a mammalian target of rapamycin (mTOR) repressor], on the phosphorylation of AMPK and SIRT1. BDSW-induced AMPK and SIRT1 phosphorylation was reduced following treatment with CC and NA, respectively, but not with RP ( Figure 6B).  Representative western blots and quantitative analysis of phospho-AMPK (p-AMPK), total AMPK, p-SIRT1, and total SIRT1 levels in C2C12 myotube lysates following treatment with BDSW of various hardness levels (0, 500, 1000, or 2000), 0.5 mM AICAR (AC), or 10 µ M SRT1720 (SRT) for 1 h. β-Actin was used as a loading control. The data represent protein levels relative to those of β-actin and are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. untreated control. (B) Representative western blots and quantitative analysis of p-AMPK, total AMPK, p-SIRT1, and total SIRT1 levels in C2C12 myotubes treated with 0.5 μM compound C (CC), 1 mM nicotinamide (NA), or 100 nM rapamycin (RP) for 0.5 h, followed by incubation with BDSW with a hardness of 2000 for 1 h. β-Actin was used as a loading control. CON, control. The data represent protein levels relative to those of β-actin and are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 vs. CON.

Discussion
Lactic acid metabolism is fundamentally linked to glucose metabolism; therefore, metabolic disorders that affect glucose metabolism, such as obesity and diabetes, alter lactic acid homeostasis. Additionally, fasting plasma lactate levels are higher in obese and diabetic patients compared to those in healthy individuals [20][21][22], and the release of lactate by muscle preparation from obese patients with and without diabetes mellitus is significantly higher than that observed in the control [23]. Figure 6. Effects of BDSW, with and without kinase inhibitors, on the activities of signaling molecules involved in lactate metabolism in C2C12 myotubes. (A) Representative western blots and quantitative analysis of phospho-AMPK (p-AMPK), total AMPK, p-SIRT1, and total SIRT1 levels in C2C12 myotube lysates following treatment with BDSW of various hardness levels (0, 500, 1000, or 2000), 0.5 mM AICAR (AC), or 10 µM SRT1720 (SRT) for 1 h. β-Actin was used as a loading control. The data represent protein levels relative to those of β-actin and are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. untreated control. (B) Representative western blots and quantitative analysis of p-AMPK, total AMPK, p-SIRT1, and total SIRT1 levels in C2C12 myotubes treated with 0.5 µM compound C (CC), 1 mM nicotinamide (NA), or 100 nM rapamycin (RP) for 0.5 h, followed by incubation with BDSW with a hardness of 2000 for 1 h. β-Actin was used as a loading control. CON, control. The data represent protein levels relative to those of β-actin and are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 vs. CON.
To confirm the effect of BDSW treatment on signaling molecules involved in PGC-1α activation, we investigated the effects of inhibitory substances, Compound C (CC; an ATP-competitive repressor of AMPK), nicotinamide (NA; a SIRT1 repressor), and rapamycin [RP; a mammalian target of rapamycin (mTOR) repressor], on the phosphorylation of AMPK and SIRT1. BDSW-induced AMPK and SIRT1 phosphorylation was reduced following treatment with CC and NA, respectively, but not with RP ( Figure 6B).

Discussion
Lactic acid metabolism is fundamentally linked to glucose metabolism; therefore, metabolic disorders that affect glucose metabolism, such as obesity and diabetes, alter lactic acid homeostasis. Additionally, fasting plasma lactate levels are higher in obese and diabetic patients compared to those in healthy individuals [20][21][22], and the release of lactate by muscle preparation from obese patients with and without diabetes mellitus is significantly higher than that observed in the control [23]. However, there have been no in vitro studies investigating the mechanism of regulation of lactate metabolism in diabetes and obesity.
In this study, we evaluated the effects of BDSW, which shows antidiabetic and antilipidemic activities, on lactate metabolism in myotubes. Our results showed that BDSW mediated lactate metabolism in C2C12 myotubes by regulating the expression of LDH A, LDH B, MCT1, and MPC1, and also increased the activities of PGC-1α and its associated signaling molecules (Figure 7). We have previously shown that BDSW inhibits adipocyte hypertrophy and hepatic steatosis in a high-fat diet (HFD)-induced obesity mouse model [17]. BDSW ameliorated glycometabolism in HFD-induced type-2 diabetic mice and streptozotocin-induced type-1 diabetic mice [15,16]. Additionally, BDSW upregulated mitochondrial biogenesis and function in muscle tissues from obese mice, 3T3-L1 preadipocytes, and myotube cells [18,19]. Our findings, showing that BDSW improved lactate metabolism in C2C12 myotubes, suggest that BDSW can beneficially affect the onset and progression of obesity and diabetes, although this hypothesis requires further study. However, there have been no in vitro studies investigating the mechanism of regulation of lactate metabolism in diabetes and obesity.
In this study, we evaluated the effects of BDSW, which shows antidiabetic and antilipidemic activities, on lactate metabolism in myotubes. Our results showed that BDSW mediated lactate metabolism in C2C12 myotubes by regulating the expression of LDH A, LDH B, MCT1, and MPC1, and also increased the activities of PGC-1α and its associated signaling molecules (Figure 7). We have previously shown that BDSW inhibits adipocyte hypertrophy and hepatic steatosis in a high-fat diet (HFD)-induced obesity mouse model [17]. BDSW ameliorated glycometabolism in HFD-induced type-2 diabetic mice and streptozotocin-induced type-1 diabetic mice [15,16]. Additionally, BDSW upregulated mitochondrial biogenesis and function in muscle tissues from obese mice, 3T3-L1 preadipocytes, and myotube cells [18,19]. Our findings, showing that BDSW improved lactate metabolism in C2C12 myotubes, suggest that BDSW can beneficially affect the onset and progression of obesity and diabetes, although this hypothesis requires further study.
We hypothesized that BDSW could promote metabolic adaptation in skeletal muscles through PGC-1α activation. Our study showed that PGC-1α induced the LDH B expression in C2C12 myotubes following BDSW treatment. Furthermore, PGC-1α activity reduced the expression of LDH A, as well as that of one of its regulators (Myc). These results indicated that changes in PGC-1α expression could modulate the composition of the LDH complex, suggesting that BDSW can affect lactate production via PGC-1α activation in C2C12 myotubes. Our findings suggest that PGC-1α activity regulates lactate homeostasis in C2C12 myotubes in response to BDSW treatment, thus improving the metabolic health. Although we did not measure lactate and ATP in C2C12 myotubes or in the culture medium, we expect that the lactate content would decrease and that of ATP would increase in BDSW-treated C2C12 myotubes (Figure 7).
In conclusion, the described regulatory effects of BDSW on the lactate metabolism provide an insight into the mechanism of the antidiabetic and antiobesity activities of BDSW. However, further studies in animal models and through clinical trials are required to fully elucidate how minerals in the form of BDSW can be used in the prevention and treatment of diabetes mellitus and obesity.

BDSW Preparation
DSW was pumped at a depth of 0.5 km from a region located 6.7 km from the shoreline of Oho-Ri, Goseong (38.200 • N and 128.340 • E, East Sea, Korea), as previously described [13]. To eliminate marine microorganisms, DSW was filtered through a microfiltration membrane (Synopex, Pohang, Korea). After filtration, reverse osmosis was used to separate DSW minerals from desalted water (Vontron Technology Co., Ltd., Beijing, China). Extracted minerals (Mg and Ca) were combined with desalted DSW at a 3:1 ratio (Mg to Ca) to obtain BDSW. Mg and Ca contents of the BDSW were measured using a Dionex ICS-1100 basic integrated ion chromatography system (Thermo Fisher Scientific, Sunnyvale, CA, USA). The hardness of BDSW was determined based on the Mg and Ca concentrations as follows: hardness = Mg (mg/L) × 4.1 + Ca (mg/L) × 2.5. The mineral content of the BDSW at a hardness of 2000 is shown in Table 1.

BDSW Treatment
BDSW or desalted water was used to dissolve the DMEM powder. DMEM containing BDSW was diluted with DMEM containing desalted water to prepare BDSW of various hardness levels (500, 1000, 1500, 2000, 2500, or 3000). DMEM prepared with desalted water was used as a control. C2C12 myotubes were exposed to BDSW of various hardness levels or to AICAR (0.5 mM) or SRT1720 (10 µM) as positive controls for 1 or 12 h.

Inhibitor Treatments
C2C12 myotubes were incubated for 30 min with or without 0.5 µM CC (AMPK phosphorylation inhibitor), 1 mM NA (SIRT1 phosphorylation inhibitor), or 100 nM RP (mTOR phosphorylation inhibitor), followed by incubation with BDSW at a hardness of 2000 for 1 h.

Cell Toxicity
C2C12 myotubes were plated into a 96-well culture plate and exposed to BDSW of various hardness levels (0, 500, 1000, 1500, 2000, 2500, or 3000), AICAR (0.25 mM or 0.5 mM), or SRT1720 (5 µM or 10 µM) for 12 h. The culture media were replaced with a medium containing MTT (5 mg/mL), and the plate was incubated in the dark for 4 h. DMSO was used to dissolve the formazan crystals formed. Absorbance was measured at 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

qRT-PCR
Total RNA was isolated from C2C12 myotubes using TRIzol (Invitrogen), as described previously [19]. The PrimeScript kit was used to synthesize cDNA from RNA following the manufacturer's protocol. qRT-PCR was performed in triplicate using the FastStart SYBR Green master mix and a CFX96 Touch RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Gene expression levels were normalized to those of β-actin (set to 1.0), and relative expression was calculated using the 2 −∆∆Ct method. Table 2 shows the sequences of the primers that were used for these experiments. Table 2. Sequences of the primers used for qRT-PCR analysis in this study.

Western Blot Analysis
Cells were washed with Tris-buffered saline and lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany), which were incubated with 5% skim milk at 25 • C for 1 h. Subsequently, the membranes were incubated with primary antibodies (1:1000) at 4 • C overnight, followed by incubation with secondary antibodies (1:3000) for 1 h on a shaker. Bands were visualized using the ECL western blot substrate, and band intensities were quantified using the ImageJ software v1.51j (National Institutes of Health, Bethesda, MD, USA).

Immunoprecipitation
Immunoprecipitation was performed using a nuclear complex Co-IP kit (Active Motif, Carlsbad, CA, USA) to analyze the PGC-1α activity. For co-immunoprecipitation, nuclear protein extracts were incubated with agitation at 4 • C overnight with diluted anti-PGC-1α and then absorbed onto protein G agarose beads at 4 • C for 2 h. The immunoprecipitates used for immunoblotting were obtained with either anti-PGC-1α or anti-Ac-Lys. Immunoreactive bands were detected by ECL, and band intensities were measured using the ImageJ software.

Statistical Analysis
Data were analyzed by one-way analysis of variance, followed by Dunnett's test, using the SPSS Statistics software v.11.0 (SPSS, Inc., Chicago, IL, USA). Statistical significance was considered at a p-value < 0.05.