Palmitic Acid Methyl Ester Enhances Adipogenic Differentiation in Rat Adipose Tissue-Derived Mesenchymal Stem Cells through a G Protein-Coupled Receptor-Mediated Pathway

Adipogenic differentiation from stem cells has become a research target due to the increasing interest in obesity. It has been indicated that adipocytes can secrete palmitic acid methyl ester (PAME), which is able to regulate stem cell proliferation. However, the effects of PAME on adipogenic differentiation in stem cell remain unclear. Here, we present that the adipogenic differentiation medium supplemented with PAME induced the differentiation of rat adipose tissue-derived mesenchymal stem cells (rAD-MSCs) into adipocyte. rAD-MSCs were treated with PAME for 12 days and then subjected to various analyses. The results from the present study show that PAME significantly increased the levels of adipogenic differentiation markers, PPARγ and Gpd1, and enhanced adipogenic differentiation in rAD-MSCs. Furthermore, the level of GPR40/120 protein increased during induction of adipocyte differentiation in rAD-MSCs. Cotreatment with PAME and a GPR40/120 antagonist together inhibited the PAME-enhanced adipogenic differentiation. Moreover, PAME significantly increased phosphorylation of extracellular signal-regulated kinases (ERK), but not AKT and mTOR. Cotreatment with PAME and a GPR40/120 antagonist together inhibited the PAME-enhanced ERK phosphorylation and adipogenic differentiation. PAME also increased the intracellular Ca2+ levels. Cotreatment with PAME and a Ca2+ chelator or a phospholipase C (PLC) inhibitor prevented the PAME-enhanced ERK phosphorylation and adipogenic differentiation. Our data suggest that PAME activated the GPR40/120/PLC-mediated pathway, which in turn increased the intracellular Ca2+ levels, thereby activating the ERK, and eventually enhanced adipogenic differentiation in rAD-MSCs. The findings from the present study might help get insight into the physiological roles and molecular mechanism of PAME in regulating stem cell differentiation.


Introduction
Obesity is a tremendous health problem worldwide. Excess adiposity is an established risk factor for metabolic diseases, heart diseases, hypertension, stroke, and several types of cancer [1]. Obesity is defined as an excessive accumulation of adipose tissue and can be divided into two main types: hyperplasia (adipocyte number increase) and hypertrophy (adipocyte size increase). However, several studies have demonstrated that the adipocyte number increases when body fat reaches 25% of the total body weight in children and adults [2,3]. Interestingly, adipocyte precursors from obese subjects proliferate more rapidly in culture than the cells from lean individuals [4,5]. New adipocytes constantly arise from a preexisting population of undifferentiated progenitor cells. On the other hand, the mesenchymal stem cells (MSCs), a major source of adipocyte generation in adipose tissue, can increase adipocyte number [1,6].
A recent study reported that perivascular adipose tissue releases PAME, causing vasorelaxation [17]. PAME, an endogenous fatty acid methyl ester (FAME), has been reported to possess potent antifibrotic [18][19][20] and antiinflammatory activities [21,22] and regulates cell proliferation in MSCs [23][24][25]. PAME is detected in adipogenic differentiated mesenchymal stromal cells, but not in the undifferentiated mesenchymal stromal cells [26]. Taken together, these data suggest that PAME plays a critical role in the stem cell differentiation. However, the effects of PAME on the differentiation of stem cell into the adipocyte lineage remain unclear. PAME has a structure similar to fatty acid, but its target molecule is still unclear. Several orphan G protein-coupled receptors (GPCRs), including GPR40, GPR43, GPR41, and GPR120, have been identified as receptors for free fatty acid and can be activated by free fatty acid and their derivatives. GPR43 and GPR41 are activated by short-chain fatty acids, whereas GPR40 and GPR120 are activated by medium-to long-chain fatty acids [27]. Ichimura et al. demonstrated in a human study that the level of GPR120 is significantly higher in obese individuals than in lean control [28]. Furthermore, the level of GPR120 mRNA increases during induction of adipocyte differentiation in 3T3-L1 cells [29]. Knockdown of GPR120 by siRNA or GPR120 gene deficiency suppresses the expression of adipogenic genes and lipid accumulation in 3T3-L1 cells and mouse embryonic fibroblast, respectively [28,29]. Gao et al. also demonstrated that low concentrations of GPR120 agonists can increase the ability of adipogenic differentiation in MSCs [30]. However, the mechanism underlying PAME enhancing the GPR40/120-mediated adipocyte differentiation in rat AD-MSCs (rAD-MSCs) has not been elucidated. In the present investigation, we delineated the signaling pathway involved in the PAME-enhanced rAD-MSC adipogenic differentiation.

Preparation and Expansion of rAD-MSCs.
Adult male Sprague-Dawley rats (350-450 g) were used as adipose tissue donors. The rAD-MSCs were obtained from the adipose tissues of rats that were anesthetized using urethane (1.5 g/kg, i.p.) and sacrificed. The protocol was approved by the Institution of Animal Care and Use Committee of the Tzu Chi University (IACUC Approval No. 107003). Adipose tissues were washed using phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin (GIBCO; Grand Island, NY). The tissues were minced and digested with 0.1% collagenase type I (GIBCO) in PBS for 60 min at 37°C, and collagenase was then inactivated with an equal amount of culture medium (DMEM-high glucose supplemented with 10% FBS and 1% penicillin/streptomycin) (GIBCO). After centrifugation for 10 min at 2,000 rpm, the rAD-MSCs were incubated at 37°C in a fully humidified atmosphere with 5% CO 2 , and the culture medium was changed every 48 h.

Oil
Red O Staining. rAD-MSCs were seeded in 24 mm poly-l-lysine-coated coverslips. After differentiation into adipocytes, adipogenic induction medium was removed and the cells were washed thrice with PBS. Cells were fixed in 10% formaldehyde for 30 min at room temperature and washed thrice with distilled water. Distilled water was then removed from the coverslips, and 500 μL of Oil Red O working solution (from 3 mL of stock solution, which contains 250 mg Oil Red O in 50 mL 60% triethyl phosphate, in 2 mL distilled water, and filtered through Whatman filter paper No. 1.) was added to each coverslip. Cells were stained for 10 min at room temperature and then washed thrice with distilled water. Cells were examined under the microscope, and images were captured at 10x magnifications using an Olympus CX31 microscope. For quantitation of lipid droplets, cells were extracted with 200 μL of 60% triethyl phosphate for 10 min, and absorbance was measured using 2 Stem Cells International Thermo Scientific Multiskan Spectrum ELISA plate reader (Thermo, Waltham, MA) at 490 nm.
The membrane was treated with 5% BSA in Tween-Trisbuffered saline (T-TBS) buffer (0.05% Tween 20; 200 mM Tris-HCl, pH 7.4; and 1.5 M NaCl) for 1 h at room temperature to block the nonspecific IgGs and then incubated with primary antibodies diluted in T-TBS buffer overnight at 4°C. The information of primary and secondary antibodies used in this study is shown as follows: all primary antibodies including anti-PPARγ, anti-p-ERK1/2, anti-ERK1/2, anti-p-AKT, anti-AKT, anti-p-mTOR, and anti-mTOR purchased from Cell Signaling (Danvers, MA); anti-GPR40 purchased from Boster Bio (Pleasanton, CA); and anti-GPR120 purchased from Novus Biologicals (Centennial, CO) were in 1,000 dilution; anti-β-actin purchased from Millipore was in 10,000 dilution. Secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG) were diluted in T-TBS buffer (1 : 5,000), and the immunoreactive proteins were visualized with an ECL detection system (GE Healthcare; Uppsala, Sweden). The resulting bands were analyzed using Image-Pro Plus software and normalized using β-actin.
2.6. Quantitative Real-Time PCR. Total RNAs were extracted from rAD-MSCs using the Trizol reagent (Ambion; Carlsbad, CA), and the cDNA was synthesized with Verso™ cDNA Kit (Thermo) using 3 μg of total RNAs. The quantitation of cDNA amount was performed with quantitative real time-PCR using a Maxima SYBR Green qPCR Master Mix (2x) and ROX Solution (Thermo) and then detected using the LightCycler 480 System (Roche; Basel, Switzerland). The following PCR primers were used: Pparγ, forward: 5′-TTG AGT GCC GAG All gene expression was analyzed using the comparative C t method (2 -ΔΔCt ), where ΔΔC t = ΔC t ðsampleÞ − ΔC t ðreferenceÞ relative to Gapdh levels. 2.8. Statistical Analyses. Data from at least three independent experiments were presented as means ± SEM and compared with unpaired t-test. Data obtained from three or more groups were subjected to one-way ANOVA followed by Fisher's least significant difference test, and statistical value of * p < 0:05 was considered significant.

Effects of PAME on rAD-MSC Adipogenic
Differentiation. Initially, we examined the effect of PAME on rAD-MSC adipogenic differentiation. Treatment with PAME (30, 50, or 100 μM) significantly enhanced rAD-MSC adipogenic differentiation (Figures 1(a) and 1(b)). The enhancement of PAME on the rAD-MSC adipogenic differentiation was confirmed by examining the mRNA and protein levels of adipogenic differentiation markers, peroxisome proliferator-activated receptor gamma (Pparγ), and glycerol-3-phosphate dehydrogenase 1 (Gpd1), in rAD-MSCs treated with or without PAME (50 μM). Quantitative RT-PCR assay demonstrated that the mRNA levels of both Pparγ (Figure 1(c)) and Gpd1 (Figure 1(d)) were significantly increased in the PAME-treated group as compared with the adipogenic induction medium-treated control group. The protein levels of PPARγ detected by Western blot analyses also increased in the PAME-treated group (Figure 1(e)). PAME (50 μM) significantly enhanced rAD-MSC adipogenic differentiation, and this enhancement was blocked by 0.5% BSA (Figure 1(f)). To further examine the specificity of PAME on rAD-MSC adipogenic differentiation, the adipogenic differentiation effect of SAME, a structural analog of FAME, and PA, a precursor of PAME, was tested to serve as negative controls. As shown in (Figure 1(g)), treatment with SAME (50 μM) or PA (50 μM) for 12 days did not significantly affect the adipogenic differentiation of rAD-MSCs.
3.2. Involvement of GPR40 and GPR120 in the PAME-Induced rAD-MSC Adipogenic Differentiation. The G protein-coupled receptors GPR40 and GPR120, also known as FFAR1 and FFAR4, respectively, are known to be activated by medium-to long-chain fatty acids [27]. Moreover, GPR40 has been shown to be the sensor of C10 methyl ester [31]. Accordingly, we examined whether GPR40/120 are involved in the PAME-enhanced rAD-MSC adipogenic differentiation. rAD-MSCs were induced to differentiate into adipocytes under the adipogenic induction medium. After induction of adipogenic differentiation with adipogenic induction medium for 12 days, the protein levels of both GPR40 (Figure 2(a)) and GPR120 (Figure 2(b)) were significantly increased in rAD-MSCs treated with or without PAME (50 μM    Stem Cells International (50 μM) enhanced the rAD-MSC adipogenic differentiation, and this effect was abolished by cotreatment with a GPR40 antagonist (DC260126 or GW1100) or a GPR120 antagonist (AH7614). To confirm the enhancement effect of PAME on the rAD-MSC adipogenic differentiation, the mRNA levels of the adipogenic differentiation markers, Pparγ and Gpd1, in rAD-MSCs were examined. Quantitative RT-PCR assay demonstrated that the mRNA levels of Pparγ (Figure 2(e)) and Gpd1 (Figure 2(f)) were significantly decreased in the cells cotreated with PAME (50 μM) and GW1100 or AH7614 as compared with the PAME-treated group.

Discussion
PAME, a fatty acid ester of palmitic acid in mammalian cells, represents an endogenous naturally occurring FAME [34]. It has been reported that endogenous PAME is released from superior cervical ganglion [35], retinal [36], and perivascular adipose tissue [17]. Liu et al. showed that PAME was synthesized through PA methylation via the AdoMet-dependent catechol-O-methyltransferase catalytic pathway in adipocytes [37]. Recently, we detected PAME in the rat bone marrow [25]. Lee et al. reported that the culture medium of differentiated adipocytes of NIH 3T3 and 3T3-L1 cells  Figure 5: PAME increases adipogenic differentiation through activating the GPR40/120/PLC/ERK pathway. Treatment of rAD-MSCs with PAME (50 μM) in adipogenic induction medium for 12 days enhanced the adipogenic differentiation, and these effects were abolished by cotreatment with U73122, U0126, or BAPTA-AM (a, b). PAME (50 μM) significantly increased the mRNA levels of Pparγ (c) and Gpd1 (d) in rAD-MSCs, and these effects were abolished by cotreatment with U73122, BAPTA-AM, or U0126. These antagonists were dissolved in 0.1% DMSO (vehicle). Values shown in parentheses represent the number in each group. PAME (50 μM) increased the protein levels of PPARγ in rAD-MSCs, and these effects were abolished by cotreatment with GW1100, AH7614, U73122, BAPTA-AM, or U0126 (e) (n = 3). All data represent mean ± SEM. # p < 0:05, versus the AIM group; $ p < 0:05, versus the AIM+PAME group. AIM: adipogenic induction medium; Veh: vehicle. 9 Stem Cells International contained a significant concentration of PAME [17]. PAME has been shown to inhibit phagocytosis [38,39], fibrotic effects [18][19][20], inflammation [21,22], and oxidative stress [40]; to induce vasodilation [17,35,36,41,42]; and to prevent nonalcoholic steatohepatitis [43]. In stem cells, PAME is able to induce cell cycle arrest at the G 2 /M phase in hBM-MSCs [25]. However, the molecular mechanisms underlying PAME-induced stem cell differentiation are still unclear. In the present study, we demonstrated that PAME activated the GPR40/120/PLC/ERK signaling pathway, leading to the enhancement of adipogenic differentiation in rAD-MSCs. p53, also known as a tumour suppressor protein, can regulate cell cycle and promote differentiation of human embryonic stem cells and 3T3-L1 cells [44][45][46][47][48]. We previously showed that PAME was able to induce cell cycle arrest at the G 2 /M phase in hBM-MSCs via the p53/p21 pathway [25]. p53 can regulate the MAPK signaling pathways [49] and lead to accelerated differentiation of PC12 cells mediated by activation of the MAPK cascade [50]. In the present study, we demonstrated that PAME activated the PLC/ERK signaling pathway, leading to the enhancement of adipogenic differentiation in rAD-MSCs. It has been demonstrated that activation of the MEK/ERK signaling promotes adipogenesis by enhancing Pparγ and C/ebpα gene expression during the differentiation of 3T3-L1 preadipocytes [51]. ERK1 and ERK2 are involved in recruitment and maturation of hBM-MSCs induced to adipocyte differentiation [52,53].
Calcium performs the physiological functions in cells by activating various intracellular signaling pathways. It is involved in several cell functions including apoptosis, exocytosis, and gene activation during the differentiation and/or proliferation processes. It has been demonstrated that increasing calcium in the early stages of differentiation inhibits human adipocyte differentiation, whereas increasing calcium in the late stage promotes human adipocyte differentiation and increases expression of PPARγ, an adipogenic differentiation transcription factor [54]. In the present study, we demonstrated that PAME significantly increased the intracellular calcium levels, and cotreatment with BAPTA-AM, a Ca 2+ chelator, abolished the PAME-enhanced adipogenic differentiation of rAD-MSCs and decreased the levels of Pparγ and Gpd1 mRNA. It has been demonstrated that exogenous calcium treatment enhanced the adipogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells via negatively regulating the Wnt5a/β-catenin signaling pathway [55]. In the present study, we demonstrated that PAME activated the PLC/ERK signaling pathway, and cotreatment with U73122 (a PLC inhibitor) or U0126 (an ERK inhibitor) abolished the PAME-enhanced adipogenic differentiation of rAD-MSCs and decreased the levels of Pparγ and Gpd1 mRNA. Interaction between ERK and Wnt/βcatenin pathway can regulate proliferation and differentiation in embryonic stem cells [56,57]. It has been demonstrated that stimulating the MEK-ERK pathway results in the activation of the Wnt/β-catenin pathway, leading to the differentiation of human embryonic stem cell towards the endoderm lineage [58]. Tian et al. showed that blockade of the ERK1/2 activity inactivates the Wnt/β-catenin signaling in the BM-MSCs and suggested a novel SPRY4-ERK1/2-Wnt/β-catenin regulatory loop, which plays a key role in cell adipogenic determination and in balancing of bone structure and homeostasis of bone marrow microenvironment [59].
GPR40/120 are receptors for both saturated and unsaturated fatty acids. It has been reported that a carboxylic group of the FFAs is required for GPR40 receptor activation. If the carboxylic group of FFAs is replaced by the methyl ester group, then the GPR40 receptor cannot be activated [60] Figure 6: Schematic representation of the PAME-enhanced adipogenic differentiation in rAD-MSCs. PAME stimulated the GPR40 and GPR120, subsequently activating PLC, which in turn caused IP 3 generation, thereby leading to ERK1/2 activation through increases of intracellular Ca 2+ levels, and eventually increased the expression of adipogenic differentiation genes, Pparγ and Gpd1.

10
Stem Cells International and GPR40 receptor activation has not been confirmed. Some studies showed that replacement of the carboxylic group with other different functional groups still can activate the GPR40/120 receptors [31,61,62]. In the present study, we demonstrated that PAME, which does not have carboxylic group, can still enhance adipogenic differentiation through activating GPR40/120. The α-linolenic acid has been shown to stimulate the release of glucagon-like peptide-1 in cultured STC-1 intestinal endocrine cells and in mice and rats [63,64]. However, α-linolenic acid is an agonist of both GPR120 and GPR40, which are both expressed in the intestine, and GPR40 has also been shown to mediate the glucagon-like peptide-1 release [65]. Although GPR40 and GPR120 share only 10% homology between their amino acid sequences [60,63], the binding activity of FAs to GPR120 and GPR40 is similar. Many of the natural agonists, such as EPA, DHA, and α-linolenic acid, activate both receptors, making the difficulty to distinguish which of these two receptors contribute a specific biologic process [66][67][68].
In the present study, we demonstrated that PAME can promote MSC adipogeneic differentiation. MSCs play an important role in adipogenesis, which can lead to obesity [69]. It has been reported that adipose tissue secretes numerous adipokines and FFAs, thereby promoting MSC differentiation into adipocytes. Obese patients have significantly elevated PA levels in the blood [70]. A recent study reported that PAME is biosynthesized from PA via catechol-Omethyltransferase catalysis [37]. Moreover, a study has suggested that catechol-O-methyltransferase may participate in the development of obesity [71]. These findings suggest that more adipose tissue will synthesize and release more PAME, which in turn causes more MSCs to differentiate into adipocytes, forming a vicious circle.

Conclusions
The experimental findings presented in this paper introduce our basic observation that PAME can induce adipogenic differentiation through activating the GPR40/120-mediated signaling pathway. We propose a model of the molecular mechanism underlying PAME-enhanced adipogenic differentiation in rAD-MSCs as shown in Figure 6. Although in vivo studies of adipocyte-derived PAME promoting the adipogenic differentiation of MSC and leading to obesity are still ongoing, the findings from the present in vitro studies suggest that inhibition of the PAME activities may be a new direction for consideration in animal models of obesity to increase the possibility of future clinical applications.

Data Availability
All materials are available by the corresponding author.

Conflicts of Interest
The authors declare no conflicts of interest.