Acetic acid stimulates G-protein-coupled receptor GPR43 and induces intracellular calcium influx in L6 myotube cells

Short chain fatty acids (SCFAs) produced endogenously in the gut by bacterial fermentation of dietary fiber have been studied as nutrients that act as signaling molecules to activate G-protein coupled receptors (GPCRs) such as GPR41 and GPR43. GPR43 functioning involves the suppression of lipid accumulation and maintaining body energy homeostasis, and is activated by acetic acid or propionic acid. Previously, we reported that the orally administered acetic acid improves lipid metabolism in liver and skeletal muscles and suppresses obesity, thus improving glucose tolerance. Acetic acid stimulates AMP-activated protein kinase (AMPK) through its metabolic pathway in skeletal muscle cells. We hypothesized that acetic acid would stimulate GPR43 in skeletal muscle cells and has function in modulating gene expression related to muscle characteristics through its signal pathway. The objective of the current study was to clarify this effect of acetic acid. The GPR43 expression, observed in the differentiated myotube cells, was increased upon acetic acid treatment. Acetic acid induced the intracellular calcium influx in the cells and this induction was significantly inhibited by the GPR43-specific siRNA treatment. The calcineurin molecule is activated by calcium/calmodulin and is associated with proliferation of slow-twitch fibers. Calcineurin was activated by acetic acid treatment and inhibited by the concomitant treatment with GPR43-siRNA. Acetic acid induced nuclear localization of myocyte enhancer factor 2A (MEF2A), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and nuclear factor of activated t cells c1 (NFATc1). However, these localizations were abolished by the treatment with GPR43-siRNA. It was concluded that acetic acid plays a role in the activation of GPR43 and involves the proliferation of slow-twitch fibers in L6 skeletal muscles through the calcium-signaling pathway caused by induction of intracellular calcium influx.


Introduction
Prevalence of obesity and obesity-linked type 2 diabetes are worldwide health concerns that need urgent attention and solution. According to the International Diabetes Federation Diabetes Atlas, there were 463 million people living with diabetes globally in 2019. This number is expected to rise to 700 million by 2045 [1]. Dietary supplementation with SCFAs such as acetic acid focuses on preventing obesity and life-style related diseases [2][3][4]. Acetic acid significantly reduces body weight gain, suppresses liver lipogenesis and lipid accumulation in white adipose tissues, improves glucose tolerance, and protects against fatty liver [5][6][7][8].
It was demonstrated in our previous study that acetic acid stimulates the AMPK via an increase in the AMP/ATP ratio through its metabolic pathway, induces myoglobin and glucose transporter 4 (GLUT4) expressions, stimulates glucose incorporation, and suppresses lipid accumulation in the L6 skeletal muscle cells [6,9].
SCFAs administered in clinical trials show a positive effect on the ulcerative colitis Crohn's disease, diarrhea, and obesity [10,11]. SCFAs are produced in gut by bacterial fermentation and act as signaling molecules to activate G-protein coupled receptors GPR41 and GPR43. As compared to propionic acid and butyric acid, acetic acid activates GPR43 with a higher potency than that seen in GPR41 [12,13]. GPR43 is reported to couple to Gi/o and Gq/ 11 of G proteins [12][13][14], which interact with several downstream molecules, and activates the pathways. It is expressed in adipose tissues, intestine, and immune cells [2,4,[13][14][15]. GPR43 promotes the secretion of glucagon-like peptide 1 (GLP-1) in mixed colonic primary cultures and STC-1 cells [3,16]. GPR43 expressed in white adipose tissues is involved in the inhibition of fat accumulation and plays a key role in regulation of life-style related diseases [2]. However, the physiological functions of GPR43 in skeletal muscle cells have not been reported so far. We hypothesized that acetic acid would stimulate GPR43 in skeletal muscle cells and has function in modulating gene expression related to muscle characteristics through its signal pathway.
In this study, we emphasize on the function of acetic acid as an activator of GPR43 in L6 skeletal muscle cells. We reveal that acetic acid functions in the induction of calcium influx in L6 cells and increases expressions of several genes associated with slow-twitch fibers through the activation of GPR43.

L6 cell culture and siRNA transfection
L6 cells were cultured as previously described [9]. L6 myoblasts were grown in DMEM containing 10% (v/v) FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO 2 at 37˚C. For myotube differentiation, the medium was changed to DMEM containing 2% (v/v) horse serum when myoblasts were 80% confluent. Myotubes were harvested 8-11 days after differentiation, and experimental procedures were initiated. Differentiated myotubes were incubated with acetic acid and/or other reagents. L6 cells cultured in growth medium were transfected with 50 nM control siRNA or rat GPR43-siRNA (sense, 5'-CUGCUAUUGGCGCU UUGUATT-3' and antisense, 5'-UACAAAGCGCCAAUAGCAGTT-3') using Lipofectamine RNAiMAX for 24-48 h according to the manufacturer's instructions. The siRNA oligonucleotides were designed to interact with GPR43 mRNA. The down-regulation of the GPR43 targeted siRNA was confirmed by measuring the levels of its expression using the StepOnePlus detection system (Applied Biosystems, CA, USA).

Animal models
Eight and 20 weeks old SD rats were used for experimental procedures related to skeletal muscles. Rats were fed on a standard laboratory rodent diet (CE-2) which was purchased from Clea (Tokyo, Japan). The care and use of the animals in this study followed the guidelines of Okayama Prefectural University and the laws and notifications of the Japanese government. All animal experiments were approved by the Animal Care and Use Committee of Okayama Prefectural University (protocol number 30-4).

Nuclear extraction and western blotting
Nuclear extraction and western blotting were performed as described previously [9]. After separation of the cytosolic and nuclear fractions, protein concentrations of each of the extracts from nuclear, cytosolic, and total fraction were determined by Bradford assay. An aliquot (15-30 μg of protein) of each extract was used for western blotting to determine the protein content of AMPKα, phosphorylated Thr-172 AMPKα, myoglobin, GLUT4, MEF2A, PGC-1α, NFATc1, phosphorylated Ser259 NFATc1, CaMKKβ, Sp1, and α-tubulin proteins. Samples were applied to 10-15% SDS-PAGE, and immuno-blotted with the corresponding antibodies. Chemiluminescent signals were visualized and quantified with ImageQuant LAS-4000 and Multi Gauge V3.2 analyzing software (Fujifilm, Tokyo, Japan).

Quantitative RT-PCR analysis
Total RNA was isolated with Sepasol RNA I super G and genomic DNA was isolated with extraction buffer (4 M guanidine thiocinnate, 50 mM sodium citrate, 1 M Tris). The cDNA was synthesized using PrimeScript RT Reagent Kit with gDNA Eraser according to the manufacturer's instructions. Quantitative real-time PCR analyses were performed using the StepO-nePlus with KAPA SYBR 1 FAST qPCR Kit (Kapa Biosystems, Wilmington, MA) for quantification of the specific mRNA content. Data were normalized to β-actin mRNA and expressed relative to the untreated control cells. The oligonucleotide primers sequences used in this study are listed in Table 1.

Intracellular calcium measurements
The intracellular calcium concentrations were measured by detecting the fluorescence of cells treated with a calcium-sensitive indicator, Fluo-4 AM [17]. L6 cells harvested 10 days after differentiation were re-plated in the 96-well plates (Iwaki, Tokyo, Japan) at 1.5×10 4 cells/well for 24 h. Subsequently, the Ca 2+ levels were determined with the use of Calcium Kit II-Fluo 4 (Dojindo, Kumamoto, Japan) using Powerscan HT (BioTek, VT, US). Briefly, cells were washed twice with non-serum medium containing 2.5 mM probenecid in 24 h after re-plating. Then cells were incubated with 4 μg/ mL Fluo-4 AM and 0.025% (w/v) Pluronic F-127 for 30 min in dark at 37˚C. After washings twice with non-serum medium, cells measurement was performed on a Powerscan HT instrument with an excitation band of 485/20 nm and fluorence was measured at 528/20 nm. Baseline signals (F 0 ) were recorded 5 min before the addition of each stimulus. Subsequently, continuous fluorescence measurements were performed for 20 min. Results are shown as F/F 0 ratios after background subtraction, where F was the fluorescence signal intensity and F 0 was the baseline intensity, as calculated by the average of 5 frames before stimulus application [17].

SDH activity
SDH activity was measured using spectrophotometer (U-2900, Hitachi High-Tech Corporation., Tokyo, Japan) [18]. L6 cells were detached, washed in PBS, and centrifuged at 1,000g for 5 min at 4˚C. The supernatant was discarded and the cells were resuspended in PBS. After twice wash, the cell pellet was suspended in 20 mM hypotonic potassium phosphate buffer (pH 7.5). The cell lysate was freezed in liquid nitrogen and thawed at 37˚C three times. The cell lysate was added to the reaction solution (25 mM potassium phosphate buffer (pH 7.5), 1 mg/ml bovine serum albumin, 300 μM potassium cyanide, 20 mM succinate and 80 μM DCPIP), the reaction mixture was incubated at 37˚C for 8 min, and the baseline was measured at 600nm for 2 min. Then, the reaction was started by adding 50 μM DUB and the decrease of absorbance at 600 nm was measured for 3 min. The activity of SDH was compared by quantifying how much the added DCPIP was consumed.

Statistical analyses
The data values have been presented as mean ± SE. The data were regarded as normally distributed and were analyzed with one-way or two-way ANOVA, followed by the Tukey-Kramer post hoc test for multiple comparisons or analyzed with unpaired Student's t-test for direct comparison between two groups. For analysis of [Ca 2+ ] i measurements, peaks (maximum F/F 0 ratio within 20 min after stimulus) were compared using one-way ANOVA, followed by the Tukey-Kramer post hoc test for multiple comparisons [17]. P values <0.05 were considered significant. All statistical analyses were performed using Statcel 4 (OMS, Tokyo, Japan).

Expression of GPR43 mRNA in skeletal muscles and L6 myotube cells
It has been reported that GPR41 (ffar3) and GPR43 (ffar2) are activated by SCFAs and expressed in a large variety of tissues, including adipose tissue and gastrointestinal tract [19,20]. As shown in Fig 1, GPR43 gene was expressed in differentiated myotube cells. The mRNA transcription was quantified in L6 myotube cells during the course of myotube differentiation until 11 days of the induction of differentiation ( Fig 1A). Expression of GPR43 was negligible in the myoblasts at day 0 though it significantly increased during differentiation ( Fig 1A). On the other hand, expression of GPR41 did not change significantly. Upon treatment with acetic acid, expression of the GPR43 was significantly elevated whereas that of GPR41 remained unaltered ( Fig 1B). In the skeletal muscles of SD rats at 8 weeks and 20 weeks, GPR43 was expressed in gastrocnemius and soleus muscles and the extent of expression was higher in skeletal muscles of 20 weeks old rats than that of 8 weeks old rats (Fig 1C). A time-course experiment revealed that GPR43 was significantly increased 30 min after the treatment of acetic acid in L6 cells (Fig 1D).

Induction of intracellular calcium influx by acetic acid in L6 cells
The intracellular calcium influx was seen in L6 cells after stimulation with acetic acid as well as 25 μM ATP. The ATP molecules induce calcium influx through the P2X receptors on the cell membrane surface [21][22][23]. The calcium influx was monitored for 20 min (Fig 2A). In comparison to the non-stimulated condition (NC), acetic acid and ATP stimulation significantly stimulated an increase in the intracellular calcium influx within 8 min of acetic acid treatment. Calcium influx was increased in a dose dependent manner up to the acetic acid concentration of 0.5 mM. However, addition of acetic acid in a concentration higher than 0.5 mM led to a reduction in the calcium influx. Thus, the calcium influx reached its peak at 0.5 mM acetic acid concentration ( Fig 2B).

Silencing of GPR43 inhibits the expression of skeletal muscle related genes and proteins
In order to investigate the physiological role of GPR43 in L6 myoblasts, GPR43 was knocked down by transfection of GPR43-specific in siRNA in the L6 cells. The inducing effect of acetic acid on the expression of GPR43 was completely abolished by the GPR43-siRNA. The treatment of araA, which is an AMPK inhibitor, also showed a decline in the effect of acetic acid, but the change was not significant (Fig 3A). The expression levels of the muscle genes (mef2a, mb, ppargc1a, cycs, and sdha) and proteins (pAMPK, myoglobin, MEF2A, PGC-1α, NFATc1, and CaMKKβ) were elevated significantly in the acetic acid-treated cells (Fig 3B and 3C). However, there was a significant drop in the induction upon silencing of GPR43. On the contrary, phosphorylated NFATc1, which is inactive form, tended to decrese in the acetic acidtreated cells. In addition, araA showed a declining trend in the expressions of these genes and proteins (Fig 3B and 3C). Nuclear MEF2A, PGC-1α, and NFATc1 proteins are associated with the proliferation and generation of slow-twitch muscle fiber proteins [24][25][26]. We examined the impact of GPR43 on the nuclear localization of MEF2, PGC-1α, and NFATc1 upon the treatment with acetic acid. The MEF2A, PGC-1α, and NFATc1 proteins were localized in the nucleus post acetic acid treatment (Fig 3D). In contrast, their nuclear localization was drastically reduced in the cells transfected with GPR43-specific siRNA (Fig 3D).

Effect of acetic acid on the induction of [Ca 2+ ] i influx in L6 cells upon GPR43 silencing
In order to examine the physiological functions of GPR43, induction of [Ca 2+ ] i influx was analyzed in the GPR43-silenced L6 cells upon the treatment with acetic acid and GPR43 agonist [24]. Intracellular [Ca 2+ ] i influx induced by ATP was not changed in cells transfected GPR43-siRNA. However, the [Ca 2+ ] i influx induced both by acetic acid and the GPR43 agonist was completely inhibited by siRNA mediated silencing of GPR43 (Fig 5A and 5B). Induction of [Ca 2+ ] i influx via GPR activation is caused by the stimulation of PLC activity and production of inositol-1,4,5-triphosphates (IP 3 ) [28,29]. The PLC inhibitor, u73122, suppressed the intracellular [Ca 2+ ] i influx induced by acetic acid and GPR43 agonist (Fig 5C).

Inhibition of PLC suppresses the phosphorylation of AMPK
Acetic acid stimulates phosphorylation of AMPK. However, treatment with PLC inhibitor abolished the phosphorylation. Agonist of GPR43 also induced the phosphorylation of AMPK, which was diminished by the treatment of the PLC inhibitor (Fig 6).

Association of calmodulin (CaM) and calcineurin with the increase of intracellular calcium levels upon acetic acid treatment
Calcium/ CaM enhances the activation of calcineurin and stimulates de-phosphorylation of NFATc1, following an increase in the transactivation of target slow-twitch muscle fiber genes [26,30,31]. Calcineurin inhibitor, cyclosporine A (CsA), and CaM inhibitor, W7, completely suppressed the genes mef2a, mb, ppargc1a, cycs, sdha, and GPR43; and proteins such as myoglobin, MEF2A, PGC-1α, and NFATc1 that increased with the acetic acid treatment (Fig 7A  and 7B). The expression levels of MEF2A, PGC-1α, and NFATc1 proteins in the nuclear fraction were also decreased because of the treatment with those inhibitors (Fig 7C). Contrastingly, MEF2A and PGC-1α proteins in the cytosolic fraction remained unaffected, though the NFATc1 expression was significantly decreased upon the treatment with CsA and W7 in cytosolic as well as nuclear fractions (Fig 7D).

GPR43 is involved with mitochondrial proliferation
Relative quantity of mitochondrial DNA (mtDNA) and SDH activity, which is a mitochondrial respiratory enzyme, were significantly increased by the treatment of acetic acid and GPR43 agonist compared with the untreated control cells. However, transfection of GPR43-siRNA completely suppressed both the mtDNA levels and SDH activity in the cells (Fig 8A and 8B).

Discussion
The GPR43 receptor is coupled to the Gq/ 11 protein that induces the calcium influx upon activation [12][13][14]. And it is activated by acetic acid and induces Ca 2+ influx in transfected cells [12]. It has been reported that GPR43 is expressed in the adipose tissue, intestinal, and immune cells, and activation of GPR43 leads to the suppression of fat accumulation, increase of GLP-1 secretion, and improves insulin signaling, however, the expression and the function of GPR43 in skeletal muscles have not been fully investigated [2,32,33]. SCFAs generated in the gut by microbes work as endogenous ligands for the GPR43 and GPR41 and act as signaling molecules in the tissues [12,13,[34][35][36]. Acetic acid has much more affinity with GPR43 than that with GPR41 [13]. It was shown in our previous study that administered acetic acid functions as an activator of AMPK and accelerates mitochondrial lipid oxidation in skeletal muscles via AMP generation [6]. Dietary acetic acid may play a role as a GPR43 ligand. In this study, we showed that GPR43 is expressed in L6 skeletal muscle cells and is activated by the treatment of acetic acid (Fig 1B). GPR43 has been reported to be coupled to the Gq and Gi/o protein families; and through the intracellular signaling pathway, it elevates the generation of signaling molecules such as IP 3 and intracellular Ca 2+ , and inhibits cAMP accumulation [37,38]. In skeletal muscles, a rise in the intracellular calcium influx resulting from motor activation plays key role in contractile activity-dependent expression as well as fiber type-specific gene expression [30]. A number of Ca 2+ sensitive target genes have been identified in the skeletal muscles [30,39,40]. The key signaling pathway downstream to the elevation in intracellular calcium that translates this signal into a transcriptional response includes the Ca 2+ / CaM-dependent phosphatase calcineurin pathway [26,30]. Calcineurin is a serine/threonine phosphatase and is activated by the binding of Ca 2+ /CaM. The best-known transcriptional targets for calcineurin are members of the NFAT family. The NFAT transcription factor translocates to the nucleus upon de-phosphorylation by calcineurin [24,26], promotes binding to the nucleotide recognition sequence, and enhances the transcription of the target genes [41]. In the nucleus, NFAT protein interacts with target genes in conjunction with other transcriptional regulator, MEF2, binding sites for which are clustered in the promoter/enhancer regions controlling the transcription of genes encoding proteins of the slow-fiber program [42,43]. Upstream of GPR43 gene contains NFAT recognition site [44]. In this study, it indicates that NFAT controls gene expression of GPR43 as well as other muscle oxidative fiber genes.
MEF2 proteins are the transcription factors involved in skeletal muscle differentiation [26]. The Ca 2+ /CaM -dependent protein kinase (CaMK) is a potent activator of MEF2A activity [26,45,46]. Studies report that NFAT associates with MEF2 and recruits the p300 co-activator to the MEF2 target genes and serves to stabilize the NFAT-MEF2 complex. It was shown that the Ca 2+ /CaM-mediated activation of CaMK phosphorylates histone deacetylase 5 (HDAC5), which binds directly to the MEF2 proteins and represses the transcriptional activity, and the direct interaction of the Ca 2+ /CaM with the repressor core of HDAC5 releases HDAC5 from MEF2 proteins [26,47,48]. After this, the phosphorylated HDAC5 masks the nuclear localization sequence, exposes the nuclear export signal by binding of the 14-3-3 chaperone protein, and is subsequently sequestered in the cytosol [47,49]. Subsequently, the transcriptional adapter factor p300 can bind to MEF2 and acetylate the histone protein tails. It has been reported that PGC-1α gene expression is also regulated by calcium-signaling components in the skeletal muscle cells [50]. PGC-1α plays a key role in the regulation of mitochondrial biogenesis and oxidative metabolism. Promoter sequence of PGC-1α contains a MEF2 binding site and those expressions are regulated by mutual interactions [51,52].
Furthermore, the activities of PGC-1α and MEF2A are regulated by AMPK [53]. AMPK is known as the master regulator of metabolic homeostasis. Activation of AMPK also leads to an increased mitochondrial biogenesis in the skeletal muscles [25,52]. AMPK is involved in the long-term metabolic changes involving mitochondrial biogenesis and promotes an oxidative muscle phenotype [54]. AMPK is also activated by CaMKK that is activated by Ca 2+ /CaM [25,55,56]. In this study, it appears that acetic acid functions to activate AMPK through stimulation of calcium release via activation of GPR43. Our results show that MEF2A and PGC-1α genes' and proteins' expression levels are suppressed by the treatment of CsA and W7 (Fig 7). Acetic acid generated by microbial fermentation of the dietary fiber in gut could be an activator of GPR43 in skeletal muscles. The ingested acetic acid would also have the effect on the activation of GPR43 in skeletal muscles and may improve the muscle functioning of slowtwitch fiber [6].
In the previous study, it was presented that chronic intake of acetic acid has effects on lower lipid accumulation in adipose tissue, on suppression of lipogenesis in liver and of plasma triglyceride, and on improvement of glucose tolerance in type 2 diabetic rats [5,6]. Lu et al. showed that dietary supplementation of acetic acid protects high-fat diet induced obesity and increased expressions of GPR43 and 41 in the adipose tissue [57]. Kimura et al. found that SCFAs, including acetic acid, activate GPR43 that expressed in the adipose tissue of mice and contribute to the regulation of energy expenditure and the suppression of fat accumulation [2]. Treatment of acetic acid stimulates fatty acid metabolism and glucose incorporation in L6 skeletal muscle cells through the mechanism of AMPK activation [9]. In this study, it appears that acetic acid activates GPR43 and its activation leads to the stimulation of AMPK and the increase of genes expression relating to slow-twitch fiber in L6 skeletal muscle cells. These effects of acetic acid may lead to postprandial glucose uptake and lipid-lowering effect by vinegar ingestion. Actually in human study, it was shown that daily intake of vinegar, which contains 750-1500mg acetic acid, has effects on suppressions of body weight, BMI, visceral fat area, waist circumference, and serum triglyceride [8]. Furthermore, acute ingestion of vinegar, which is containing 6% acetic acid, has effects on postprandial glucose uptake in forearm muscle and on lipid-lowering effect, suggesting improvement of insulin sensitivity [58]. In conclusion, our data indicate that acetic acid activates GPR43 in L6 skeletal muscles and stimulates gene expressions associated with slow-twitch fiber by regulating the functions of NFATc1, MEF2A, PGC-1α, and CaMKK through calcium-signaling. Daily intake of acetic acid may prevent obesity and improve skeletal muscle functioning during ageing process via the function of GPR43 and AMPK. It would be needed further investigation in human skeletal muscle cells.