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Originally published In Press as doi:10.1074/jbc.M412385200 on March 17, 2005

J. Biol. Chem., Vol. 280, Issue 20, 19507-19515, May 20, 2005
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Free Fatty Acids Inhibit Serum Deprivation-induced Apoptosis through GPR120 in a Murine Enteroendocrine Cell Line STC-1*

Susumu Katsuma{ddagger}§, Noriyuki Hatae§, Takeaki Yano§, Yoshinao Ruike§, Mai Kimura§, Akira Hirasawa§, and Gozoh Tsujimoto§||

From the {ddagger}Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 and the §Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

Received for publication, November 2, 2004 , and in revised form, March 16, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Free fatty acids (FFAs)1 play important physiological roles in many tissues. For example, impairment of insulin-mediated glucose uptake and glycogen synthesis in muscle (1) and potentiation of glucose-stimulated insulin secretion in pancreatic islets (2) have been considered as a result of intracellular metabolism of FFAs to long chain acyl-CoA esters. However, it is also suggested that FFAs may act as ligands for cell-surface receptors because fatty acid derivatives, such as prostaglandins and leukotrienes, have their specific cell-surface G protein-coupled receptors (GPCRs) (3, 4).

Recently, several groups reported that three orphan receptors, GPR40, GPR41, and GPR43, can be activated by FFAs (5-9). GPR40 can be activated by medium and long chain FFAs, but not by short chain FFAs (5, 6), and GPR41 and GPR43 can be activated by short chain FFAs, but not by medium and long chain FFAs (7-9). Pharmacological analysis of GPR40 and its tissue distribution suggested that GPR40 is abundantly expressed in the pancreas as a functional receptor for long chain FFAs and that FFAs amplify glucose-stimulated insulin secretion from pancreatic beta cells by activating GPR40 (5).

We have demonstrated very recently (10) that an orphan GPCR, GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long chain FFAs. Furthermore, we showed that the stimulation of GPR120 by FFAs promotes the secretion of glucagon-like peptide-1 (GLP-1) and activates the extracellular signal-regulated kinase (ERK) cascade (10). However, other physiological functions of GPR120 triggered by FFA stimulation are uncertain. In the present study, we show that unsaturated long chain FFAs, such as linolenic acid, inhibit apoptosis of a murine enteroendocrine cell line STC-1 under serum-free conditions. Also, we reveal that inhibition of apoptosis by the FFAs of STC-1 cells is mediated through GPR120. This is the first real evidence that FFA-induced apoptosis inhibition is mediated through GPCR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Fatty acids and 3-isobutyl-1-methylxanthine were obtained from Sigma. U0126, PD98059, SB203580, U73122 [GenBank] , U73343 [GenBank] , LY294002, wortmannin, and forskolin were purchased form Calbiochem. SP600125 was from Biomol. Pertussis toxin (PT) was obtained from Seikagaku Corp. Antibodies were purchased from Cell Signaling Technology.

Cell Culture and Treatment—We used a murine enteroendocrine cell line STC-1, an immortalized mouse cell line derived from small bowel endocrine tumors, that arose as a consequence of the mating of two independent transgenic mouse lines, rat insulin promoter simian virus 40 Tag x rat insulin promoter-polyoma small Tag (11). STC-1 cells were maintained in Dulbecco's modified Eagle's medium containing 15% (v/v) horse serum (HS) and 5% (v/v) fetal bovine serum (FBS). The human breast cancer cell line MDA-MB-231 was maintained in Dulbecco's modified Eagle's medium containing 10% (v/v) FBS. STC-1 cells were serum-starved for 2 h and treated with or without fatty acids. After 24 h of treatment, we used the WST-1 assay kit (Roche Applied Science) to assess viable cell numbers. Total RNA was isolated using Isogen (Nippon Gene) and subjected to RT-PCR. Each FFA was dissolved in dimethyl sulfoxide (Me2SO). The final concentration of Me2SO was adjusted to 0.1% (v/v). FFAs were added to serum-free medium and then sonicated just before use, and the resulting medium was used as serum-free, FFA-containing medium. The medium containing the same amount of Me2SO was used as the control.



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  		FIG. 1.
FFAs induce cell survival of serum-starved STC-1 cells. A, STC-1 cells were serum-starved for 2 h and then stimulated for 24 h with the indicated FFAs (100 µM) or 15% (v/v) HS and 5% (v/v) FBS (HS/FBS). Cell variability was assessed using the WST-1 method and expressed as a percentage compared with Me2SO-treated cells. Data show mean ± S.E. of three independent experiments. *, p < 0.001; **, p < 0.01; #, p < 0.05 versus control. B, STC-1 cells were serum-starved for 2 h and then stimulated for 24 h with the indicated concentrations of linolenic acid, palmitoleic acid, and docosahexaenoic acid. Cell variability was assessed using the WST-1 method and expressed as a percentage compared with Me2SO-treated cells. Data show mean ± S.E. of three independent experiments. *, p < 0.001; #, p < 0.01 versus control.

 



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  		FIG. 2.
Inhibition of caspase-3 activity by FFAs. A, caspase-3 activity was measured in serum-starved STC-1 cells treated with or without fatty acids (100 µM) and HS/FBS for 5 h. Caspase-3 activity was expressed as a percentage compared with Me2SO-treated cells. Data show mean ± S.E. of three independent experiments. *, p < 0.001; **, p < 0.01; #, p < 0.05 versus control. B, caspase-3 activity was measured in serum-starved STC-1 cells treated with or without linolenic acid, palmitoleic acid, and docosahexaenoic acid (1, 3, 10, 30, and 100 µM). Caspase-3 activity was expressed as a percentage compared with Me2SO-treated cells. Data show mean ± S.E. of four independent experiments. *, p < 0.001; #, p < 0.05 versus control.

 



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  		FIG. 3.
Inhibition of DNA fragmentation by FFAs. A, DNA fragmentation was assessed quantitatively by an ELISA-based method using anti-histone and anti-DNA antibodies in serum-starved STC-1 cells treated with or without fatty acids (100 µM) and HS/FBS for 24 h. Data show mean ± S.E. of three independent experiments. *, p < 0.01; **, p < 0.05 versus control. B, DNA fragmentation was measured quantitatively in serum-starved STC-1 cells treated with or without linolenic acid (3, 10, 30, and 100 µM). Data show mean ± S.E. of three independent experiments. *, p < 0.01; **, p < 0.05 versus control.

 
Caspase-3 Activity Assay—The activity of caspase-3 was determined by the EnzChek Caspase-3 Assay Kit (Molecular Probes) following the manufacturer's instructions (12). Briefly, 3 x 106 cells were serum-starved for 2 h and treated with the indicated reagents. Five hours later, the cells were collected and lysed, and caspase-3 activity in the extracts was measured.

DNA Fragmentation Assay—DNA fragmentation was measured using the Cell Death Detection ELISAPLUS kit (Roche Applied Science) following the manufacturer's instructions (12). Briefly, STC-1 cells, cultured in 24-well culture plates, were treated with the indicated reagents. After 24 h, the cells were lysed, and enzyme immunoassay using mouse monoclonal antibodies directed against DNA and histones was carried out to detect mono- and oligonucleosomes in the cytoplasmatic fraction of cell lysates. The absorbance was normalized by the total cell number after 24 h of treatment.

Plasmids and Transfection—Plasmids were transfected into STC-1 cells using Lipofectamine PLUS or Lipofectamine 2000 reagents (Invitrogen) as described previously (10). One or 2 days after transfection, cells were serum-starved for 2 h, treated with the indicated reagents, and caspase-3 activity measured as described above. Expression plasmids containing murine GPR120 and GPR40 genes were constructed as described previously (10). pCMV-G{alpha}q, pCMV-G{alpha}qQ209L, pCMV-{beta}ARK1nt, and pCMV-{beta}ARK1ct were kindly provided by J. Yamauchi (Nara Institute of Science and Technology). Plasmids containing the dominant-negative (DN) JNK and p38 were kindly provided by Y. Izumi (Osaka City Medical School) and S. Mitsuyama (Kumamoto University) (13, 14). The truncated form of Akt (DN-Akt, amino acids 1-147) was isolated from STC-1 cells and cloned into pIRES-neo expression vector (Clontech).

Transfection of STC-1 Cells with Short Hairpin RNA Expression Vector—DNA oligonucleotides targeting GPR120 and GPR40 were synthesized and inserted into the short interfering RNA expression vector pSilencer1.0 (Ambion) as described previously (10). Plasmids were transfected into STC-1 cells with Lipofectamine 2000 (Invitrogen). Transfected cells were examined for GPR120 and GPR40 mRNA levels by real time RT-PCR.

Real Time PCR Analysis—Total RNA was reverse-transcribed and used for real time PCR analysis by using the DNA Engine Opticon2 System (MJ Research) according to the manufacturer's instructions. PCR primers were as follows: for mouse GPR120, mGPR120-1, 5'-GCATAGGAGAAATCTCATGG-3'; mGPR120-2, 5'-GAGTTGGCAAACGTGAAGGC-3'; for mouse GPR40, GPR40-2, 5'-AGTCCTCGTCACACATATTG-3'; GPR40-3, 5'-AATGCCTCCAATGTGGATAG-3'; for mouse early growth response gene-1 (Egr-1), mEgr-1, 5'-ACGACAGCAGTCCCATCTAC-3'; mEgr-2, 5'-GTTACGCATGCAGATTCGAC-3'; for mouse GAPDH, mGAPDH-1, 5'-TGACCACAGTCCATGCCATC-3'; mGAPD-H-4, 5'-TTGAAGTCGCAGGAGACAAC-3'; for human GPR120, hGPR-120-1, 5'-TGGTCATTGTGATCAGTTAC-3'; hGPR120-4, 5'-CATTCCTGCACAGTGTCATG-3'; for human GPR40, hGPR40-1, 5'-TCAGCCTCTCTCTCCTGCTC-3'; hGPR40-4, 5'-CGCACACACTGTCTTCAGGC-3'; for human GAPDH, hGAPDH-1, 5'-TCAAGATCATCAGCAATGCC-3'; and hGAPDH-4, 5'-TTGAAGTCAGAGGAGACCAC-3'.



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  		FIG. 4.
Effects of linolenic acid on ERK1/2, JNK, p38, and Akt activation in STC-1 cells. A, Western blotting. Serum-starved STC-1 cells were stimulated with linolenic acid (100 µM) for 0, 2, 5, and 10 min. Western blotting was performed using anti-phospho- and anti-total kinase antibodies. B, effects of protein kinase inhibitors. Serum-starved STC-1 cells were preincubated for 30 min with the following inhibitors: U0126 (10 µM), SB203580 (10 µM), SP600125 (10 µM), wortmannin (500 nM), or LY294002 (10 µM). Cells were then stimulated with or without linolenic acid (100 µM) for 10 min. Western blotting was performed using anti-phospho- and anti-total kinase antibodies. DMSO, Me2SO.

 



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  		FIG. 5.
Effects of protein kinase inhibitors on linolenic acid- or palmitoleic acid-induced inhibition of caspase-3 activity. A, effects of protein kinase inhibitors. Serum-starved STC-1 cells were preincubated for 30 min with the following inhibitors: PD98059 (PD, 30 µM), U0126 (10 µM), SB203580 (SB, 10 µM), SP600125 (SP, 10 µM), wortmannin (Wort, 500 nM) or LY294002 (LY, 10 µM). Cells were then stimulated with or without linolenic acid or palmitoleic acid (100 µM) for 5 h. Caspase-3 activity was measured and expressed as a percentage of caspase-3 activity inhibition compared with cells without linolenic acid or palmitoleic acid treatment. Data show mean ± S.E. of four independent experiments. Asterisk, p < 0.05 versus Me2SO (DMSO) treatment. B-D, effects of dominant-negative mutants of p38 (B), JNK (C), and Akt (D) on linolenic acid-induced inhibition of caspase-3 activity. STC-1 cells were transfected with dominant-negative mutants of p38 (DN-p38), JNK (DN-JNK), Akt (DN-Akt), or empty vector. After 24 h, cells were serum-starved, and caspase-3 activity was measured. Data show mean ± S.E. of three independent experiments. Asterisk, p < 0.05.

 
Western Blotting—Activation of ERK1/2, JNK, p38, and Akt in STC-1 cells was assayed by Western blotting as described previously (10). Briefly, cells were serum-starved for 2 h and treated with 100 µM of each FFA. After 10 min of stimulation, total cell extracts were prepared and subjected to Western blotting using anti-phospho- and anti-total kinase antibodies (Cell Signaling Technology).

Measurement of cAMP Formation—cAMP levels in STC-1 cells were determined as reported previously (15). Reactions were started by the addition of test agents along with 500 µM 3-isobutyl-1-methylxanthine. After incubation for 10 min at 37 °C, reactions were terminated by the addition of 10% trichloroacetic acid. The level of cAMP was measured by radioimmunoassay with a cAMP assay system (Amersham Biosciences).

Statistical Analysis—One-way analysis of variance was used to evaluate treatment effects. If the analysis of variance value was significant, comparisons between the control and treatment group were performed using analysis of variance followed by Dunnett's test to localize the significant difference. A p value of less than 0.05 was considered significant. All statistics were run with InStat 2.00 (GraphPad Software).



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  		FIG. 6.
FFAs promote ERK activation and immediate early gene expression in STC-1 cells. A, ERK activation. STC-1 cells were serum-starved for 2 h and treated with 100 µM of each FFA. After 2 min of stimulation, total cell extracts were prepared and subjected to Western blotting using anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies. C18:3 ME, linolenic acid methyl ester. B, Egr-1 expression. STC-1 cells were serum-starved for 2 h with or without pretreatment with U0126 and treated with 100 µM of each FFA. After 30 min of stimulation, total RNA were prepared and subjected to real time RT-PCR analysis of Egr-1 and GAPDH genes. The GAPDH gene was used as a control. Data show mean ± S.E. of three independent experiments. #, p < 0.05 versus control; *, p < 0.05 versus FFA treatment. DMSO, Me2SO.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Free Fatty Acids Enhance Cell Survival of Serum-starved STC-1 Cells—We first examined the effects of saturated and unsaturated FFAs with a chain length of C12 to C22 on cell survival of murine enteroendocrine STC-1 cells. As shown in Fig. 1A, linolenic acid (C18:3), palmitoleic acid (C16:1), and docosahexaenoic acid (C22:6) markedly promoted cell survival of serum-starved STC-1 cells at 100 µM. Arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5) had a moderate effect. Myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1), linoleic acid (C18:2), and eicosatrienoic acid (C20:3) exhibited a small positive effect on cell survival. Medium chain fatty acids, octanoic acid (C8) and lauric acid (C12), and saturated long chain fatty acids, arachidic acid (C20) and docosanoic acid (C22), did not show any positive effects upon cell survival. In this condition, we did not observe changes in the total number of STC-1 cells by treatment with linolenic acid or palmitoleic acid (data not shown). In addition, we examined this cell survival effect using various doses of linolenic acid, palmitoleic acid, and docosahexaenoic acid (1-100 µM), and we found that stimulation of these unsaturated FFAs increased viable cell number in a dose-dependent manner (Fig. 1B). These results indicate that saturated FFAs with a chain length of C14 to C18 and mono- and polyunsaturated FFAs of C16 to C22 enhance cell survival of serum-starved STC-1 cells.



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  		FIG. 7.
Transient transfection of GPR120, but not GPR40, enhances linolenic acid-induced inhibition of caspase-3 activity. STC-1 cells were transfected with GPR120-EGFP and GPR40-EGFP expression vectors. Two days after transfection, cells were serum-starved, and caspase-3 activity was measured in serum-starved STC-1 cells treated with or without linolenic acid (100 µM). Caspase-3 activity was measured and expressed as a percentage of caspase-3 activity inhibition compared with Me2SO-treated empty vector-transfected cells. Data show mean ± S.E. of four independent experiments. *, p < 0.05; **, p < 0.01.

 
Free Fatty Acids Inhibit Caspase-3 Activity of Serum-starved STC-1 Cells—The enhanced cell survival of serum-starved STC-1 cells by FFAs could be explained by anti-apoptotic effects. We therefore measured caspase-3 activity that is responsible for apoptosis induction in most cell types. As shown in Fig. 2A,at100 µM, linolenic acid, palmitoleic acid, and docosahexaenoic acid markedly promoted the inhibition of caspase-3 activity in serum-starved STC-1 cells (about 30-40% inhibition of caspase-3 activity). Oleic acid, linoleic acid, eicosatrienoic acid, arachidonic acid, and eicosapentaenoic acid had little effect (about 10-20% inhibition). Other fatty acids did not show any positive effects on caspase-3 activity after 5 h of stimulation. In addition, we examined this inhibition using various doses of linolenic acid, palmitoleic acid, and docosahexaenoic acid (1-100 µM), and we demonstrated that the anti-apoptotic effect increased in a dose-dependent manner (Fig. 2B). These results indicate that mono- and polyunsaturated FFAs of C16 to C22 significantly enhance the inhibition of caspase-3 activity in serum-starved STC-1 cells.

Free Fatty Acids Inhibit DNA Fragmentation of Serum-starved STC-1 Cells—We next examined the effects of FFAs on serum deprivation-induced DNA fragmentation, which constitutes the final cellular event during apoptosis. DNA fragmentation was assessed quantitatively by an ELISA-based method using anti-histone and anti-DNA antibodies. The results of this analysis showed that at 100 µM, linolenic acid and palmitoleic acid markedly promoted inhibition of DNA fragmentation of serum-starved STC-1 cells (Fig. 3A). Oleic acid, eicosatrienoic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid had a little effect. Other fatty acids did not show any effects upon DNA fragmentation. In addition, we examined this inhibition with various doses of linolenic acid (3-100 µM), and we showed that the effect increased in a dose-dependent manner (Fig. 3B). These results indicate that mono- and polyunsaturated FFAs of C16 to C22 significantly enhanced inhibition of serum deprivation-induced DNA fragmentation in STC-1 cells.



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  		FIG. 8.
Knock-down of GPR120, but not GPR40, decreases the effect on linolenic acid-induced inhibition of caspase-3 activity. A, short hairpin RNA-mediated knock-down of GPR120 and GPR40 in STC-1 cells. Plasmids were transfected into STC-1 cells, and transfected cells were examined for the levels of GPR120 and GPR40 mRNA by real time RT-PCR. mRNA levels were expressed as a percentage compared with those of control vector-transfected cells. Data show mean ± S.E. of three independent experiments. Asterisk, p < 0.05. B, ERK activation. Cells were serum-starved for 2 h and treated with 100 µM of linolenic acid or palmitoleic acid. After 2 min of stimulation, total cell extracts were prepared and subjected to Western blotting using anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies. C, measurement of caspase-3 activity. Caspase-3 activity in transfected cells was measured and expressed as a percentage of caspase-3 activity inhibition compared with Me2SO-treated cells (DMSO). Data show mean ± S.E. of three independent experiments. Asterisk, p < 0.05.

 
Inhibition of Caspase-3 Activity by Unsaturated FFAs Involves Activation of ERK and Akt in STC-1 Cells—To delineate the signaling pathways involved in unsaturated FFA-promoted inhibition of apoptosis, we examined the activation of ERK, c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and Akt/protein kinase B (Akt). As shown in Fig. 4A, these four protein kinases were markedly activated by treatment with linolenic acid. Next, we assessed the effects of specific inhibitors for ERK kinase (U0126 and PD98059), p38 (SB203580), and JNK (SP600125). In addition, we used two inhibitors (wortmannin and LY294002) for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt (16, 17). Western blot analysis showed that an ERK kinase inhibitor, U0126, and PI3K inhibitors, wortmannin and LY294002, completely inhibited the activation of ERK and Akt, respectively (Fig. 4B). Also, we measured caspase-3 activity in linolenic acid- or palmitoleic acid-stimulated STC-1 cells with or without pretreatment with these inhibitors. As shown in Fig. 5A, pretreatment with ERK kinase inhibitors and PI3K inhibitors significantly reduced the linolenic acid- or palmitoleic acid-induced inhibition of caspase-3 activity, whereas SB203580 and SP600125 did not have any effects. In addition, we found that the transfection of STC-1 cells with the DN mutant of p38 or JNK did not affect the anti-apoptotic effect of linolenic acid (Fig. 5, B and C). Furthermore, transfection of STC-1 cells with DN-Akt significantly inhibited the anti-apoptotic effect of linolenic acid (Fig. 5D). Taken together, these results suggest that the activation of ERK and PI3K-Akt pathways, but not JNK and p38 pathways, leads to linolenic acid-induced inhibition of caspase-3 activity in serum-deprived STC-1 cells.

FFAs Promote ERK Activation and Immediate Early Gene Expression in STC-1 Cells—We next investigated the effects of various FFAs on ERK activation in STC-1 cells. As shown in Fig. 6A, at 100 µM, linolenic acid and palmitoleic acid strongly promoted ERK activation in serum-starved STC-1 cells, whereas oleic acid, palmitic acid, stearic acid, linolenic acid methyl ester, and octanoic acid showed lesser effects on ERK activation. Because the induction of immediate early response genes is frequently observed concomitant with ERK activation (12, 18, 19), we next examined the induction of one of the immediate early genes, early growth response gene-1 (Egr-1), by FFA stimulation in STC-1 cells. After 0.5 h of stimulation, Egr-1 was markedly up-regulated by linolenic acid and palmitoleic acid, whereas oleic acid, octanoic acid, palmitic acid, stearic acid, and linolenic acid methyl ester had a little effect (Fig. 6B). This induction was significantly inhibited by pretreatment with an ERK kinase inhibitor, U0126 (Fig. 6B), suggesting that Egr-1 induction by FFAs is mainly mediated by ERK activation. Fig. 6 demonstrates that the patterns of ERK activation and early gene induction by FFAs were in accordance with those of FFA-promoted cell survival as shown in Figs. 1, 2, 3.



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  		FIG. 9.
Linolenic acid-mediated inhibition of caspase-3 activity is mediated through the Gq pathway. A, effect of PT on linolenic acid-induced inhibition of caspase-3 activation. STC-1 cells were treated with PT (100 ng/ml) for 16 h, serum-starved for 2 h, and treated with 100 µM of linolenic acid. Cells were then stimulated with or without linolenic acid (LA, 100 µM) for 5 h, and caspase-3 activity was measured. B, effect of PT on linolenic acid-induced ERK activation. STC-1 cells were treated with PT (100 ng/ml) for 16 h, serum-starved for 2 h, and treated with 100 µM of linolenic acid. After 2 min of stimulation, total cell extracts were prepared and subjected to Western blotting using anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies. C, measurement of cAMP formation. STC-1 cells were incubated for 10 min with or without linolenic acid (LA, 100 µM) or with forskolin (FSK,5 µM). The level of cAMP was measured by radioimmunoassay as described in the "Experimental Procedures." DMSO,Me2SO. D, effect of G{alpha}q inhibition on linolenic acid-induced caspase-3 inhibition in serum-deprived STC-1 cells. STC-1 cells were transfected with pCMV-{beta}ARK1nt (nt), pCMV-{beta}ARK1ct (ct), or empty vector pCMV. After 24 h, cells were serum-starved for 7 h, and caspase-3 activity was measured. Data show mean ± S.E. of three independent experiments. Asterisk, p < 0.05. E, involvement of G{alpha}q on caspase-3 inhibition in serum-deprived STC-1 cells. STC-1 cells were transfected with GqWT, GqQ209L, or empty vector pCMV. After 24 h, cells were serum-starved for 7 h, and caspase-3 activity was measured. Data show mean ± S.E. of three independent experiments. Asterisk, p < 0.05.

 
Linolenic Acid-mediated Inhibition of Caspase-3 Activity in Serum-starved STC-1 Cells Is Mediated through GPR120 — STC-1 cells have been shown to express two cell-surface FFA receptors, GPR40 and GPR120, at the same transcriptional level (10). To examine which receptor is involved in the anti-apoptotic effects of FFAs on STC-1 cells, we transiently transfected expression vectors containing each FFA receptor and measured the caspase-3 activity of linolenic acid-stimulated transfected cells. As shown in Fig. 7, transfection with an expression plasmid containing GPR120-enhanced green fluorescent protein (EGFP), but not GPR40-EGFP, enhanced the inhibition of caspase-3 activity of linolenic acid-stimulated cells. Next, we transfected short hairpin RNA vectors targeting GPR40 and GPR120 into STC-1 cells, and we examined mRNA levels of GPR120 and GPR40 by RT-PCR. As shown in Fig. 8A, GPR40 or GPR120 mRNA expression was significantly reduced compared with that of cells transfected with the control vector. Then we measured linolenic acid- and palmitoleic acid-induced ERK activation in these cells. Western blotting of phospho-ERK1/2 indicated that reduced expression of GPR120, but not GPR40, decreased the level of linolenic acid- and palmitoleic acid-induced ERK1/2 phosphorylation (Fig. 8B). Finally, we examined caspase-3 activity in linolenic acid- and palmitoleic acid-stimulated cells. As shown in Fig. 8C, reduced expression of GPR120, but not GPR40, significantly reduced the effects of linolenic acid and palmitoleic acid on caspase-3 activity inhibition. Collectively, we conclude that our data demonstrate that the linolenic acid- and palmitoleic acid-promoted inhibition of caspase-3 activity in serum-starved STC-1 cells is mainly mediated through GPR120.

Linolenic Acid-mediated Inhibition of Caspase-3 Activity Is Mediated through the Gq Pathway but Not the Gi nor the Gs Pathway—We investigated which G proteins are involved in the linolenic acid-induced inhibition of caspase-3 activity downstream of GPR120-mediated signaling. As shown in Fig. 9A, pretreatment of STC-1 cells with PT, an endotoxin that catalyzes ADP-ribosylation of G{alpha}i/o (20), did not reduce the anti-apoptotic effect of linolenic acid. Also, linolenic acid-induced ERK activation was not affected by PT pretreatment (Fig. 9B), suggesting that the anti-apoptotic effect of linolenic acid was not mediated through the Gi/o pathway. We next examined the effect of linolenic acid on changes in cAMP. We did not detect a significant increase in cAMP production, although forskolin, an adenylate cyclase activator, elicited a marked increase in cAMP production (Fig. 9C), suggesting that the Gs pathway was not involved in linolenic acid-induced signaling in STC-1 cells. Previously, we showed that linolenic acid induces an increase in [Ca2+]i via GPR120 in STC-1 cells (10), suggesting that FFA-promoted signaling in STC-1 cells may be coupled with the Gq pathway. To examine whether the anti-apoptotic effect of linolenic acid is mediated through the Gq pathway, we examined caspase-3 activity in linolenic acid-stimulated STC-1 cells transfected with plasmids expressing a plasmid encoding the regulator of G-protein signaling domain of {beta}-adrenergic receptor kinase 1 ({beta}ARK1nt), which is known to bind to G{alpha}q/11 and inhibit these cellular functions (21, 22). As shown in Fig. 9D, transfection of {beta}ARK1nt caused an inhibition of linolenic acid-promoted caspase-3 inhibition. In contrast, transfection of the COOH terminus of {beta}ARK1 ({beta}ARK1ct), which is known to bind to G{beta}{gamma} and inhibit these cellular functions (23, 24), did not affect its inhibition (Fig. 9D). In addition, we measured caspase-3 activity in STC-1 cells transfected with plasmids expressing a wild-type G{alpha}q (GqWT) or a constitutive active form of G{alpha}q (GqQ209L). As shown in Fig. 9E, transfection of GqQ209L, but not GqWT, induced the inhibition of caspase-3 activity in serum-deprived STC-1 cells (Fig. 9E). Phospholipase C (PLC) is well known to be activated by G{alpha}q (25). We found that U73122 [GenBank] , a specific inhibitor of PLC, but not U73343 [GenBank] , an inactive analog of U73122 [GenBank] , significantly reduced the anti-apoptotic effect of linolenic acid on STC-1 cells (Fig. 10A) as well as linolenic acid-promoted ERK activation (Fig. 10B). These results suggest that linolenic acid-mediated apoptosis inhibition is mediated through the Gq-PLC-ERK pathway.



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  		FIG. 10.
Involvement of PLC on linolenic acid-induced caspase-3 inhibition in serum-deprived STC-1 cells. A, effect of PLC inhibitor on linolenic acid-induced caspase-3 inhibition. Serum-starved STC-1 cells were preincubated for 30 min with U73122 [GenBank] (1 µM) or U73343 [GenBank] (1 µM). Cells were then stimulated with or without linolenic acid (100 µM) for 5 h. Caspase-3 activity was measured and expressed as a percentage of caspase-3 activity inhibition compared with cells without linolenic acid treatment. Data show mean ± S.E. of four independent experiments. Asterisk, p < 0.05. B, effect of PLC inhibitor on linolenic acid-induced ERK activation. Serum-starved STC-1 cells were preincubated for 30 min with U73122 [GenBank] (1 µM) or U73343 [GenBank] (1 µM). Cells were then stimulated with or without linolenic acid (100 µM) for 2 min. Total cell extracts were prepared and subjected to Western blotting using anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies. Asterisk, p < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
GPCRs of the GPR40 family have been shown to be activated by FFAs as follows: GPR40 by medium and long chain FFAs and GPR41 and GPR43 by short chain carboxylic acids, respectively (5-9). More recently, we identified a novel GPCR, GPR120, as another GPCR for long chain FFAs (10). GPR120 and GPR40 are activated by similar properties of FFAs, and GPR40 directly and GPR120 indirectly promote glucose-stimulated insulin secretion (5, 10). Also, we showed that FFAs promote the secretion of GLP-1 and the activation of ERK through GPR120 (10). The secretion of GLP-1 was not affected by pretreatment with an ERK kinase inhibitor, PD98059 (10), suggesting that its secretion is not involved in the ERK cascade. As the ERK pathway is closely linked to growth and differentiation signals (26), the stimulatory effects of FFAs through GPR120 might evoke other cellular events aside from hormone secretion. In the present study, we found that saturated FFAs with a chain length of C14 to C18 and unsaturated FFAs of C16 to C22 enhance cell survival of serum-starved STC-1 cells, with linolenic acid, palmitoleic acid, and docosahexaenoic acid having the most potency. This property is in accordance with that of the [Ca2+]i response evoked by FFAs in GPR120-expressing HEK293 cells (10), leading to the idea that FFAs promote cell survival of serum-starved STC-1 cells through GPR120. RNA interference experiments clearly showed that the inhibition of apoptosis by the FFAs of STC-1 cells is mainly mediated through GPR120 (Fig. 8).

Based on the results described in this study, we propose the mechanism of FFA-induced cell survival in STC-1 cells as follows. FFAs stimulate cell-surface GPR120 and activate the following two independent pathways: a pathway mediated by PLC-ERK and a further pathway mediated by PI3K. Stimulation of STC-1 cells by linolenic acid promoted Akt activation (Fig. 4A), which was almost completely inhibited by PI3K inhibitors (Fig. 4B). Because Akt is a major target of PI3K involved in cell survival or proliferation (16, 17), it follows that the PI3K-mediated cell survival pathway is clearly involved in Akt activation. In addition, we found that other MAPKs, JNK and p38, were also activated in STC-1 cells by linolenic acid. Neither chemical inhibitors nor dominant-negative mutants of these MAPKs affected the anti-apoptotic activity induced by linolenic acid (Fig. 5), suggesting that these MAPKs might involve other cellular events aside from cell survival. Further studies are required to clarify the roles of these MAPKs in linolenic acid-stimulated STC-1 cells.

Numerous studies have documented that long chain FFAs influence the cell survival and proliferation. Saturated FFAs have been reported to have toxicity on various types of cells, such as breast cancer MDA-MB-231 cells (27, 28), pheochromocytoma PC12 cells (29), Chinese hamster ovary cells (30), pancreatic beta cells (31), rat testicular Leydig cells (32), and rat neonatal cardiomyocytes (33). However, studies on FFA-induced effects are limited, and the mechanism of such effects remains largely unknown. Recently, it was reported that the unsaturated FFA, oleic acid, exhibited the positive effects upon the cell survival of four breast cancer cell lines, MDA-MB-231, ZR-75-1, MCF-7, and T47D, whereas the saturated FFA, palmitic acid, promoted apoptosis (27, 28). A possible explanation for the opposite effects of these two FFAs came from the observation that oleic acid and palmitic acid caused an increase or decrease in PI3K activity in these cells, respectively. Also, mitochondria and cardiolipin were shown to play a critical role in saturated FFA-induced apoptosis in breast cancer cells (21). We showed independently that the unsaturated FFA-promoted cell survival of MDA-MB-231 cells involves the activation of ERK and PI3K, which is in accordance with existing literature (Refs. 27 and 28 and data not shown). In addition, we found that this cell line expresses two cell-surface FFA receptors, GPR120 and GPR40 (data not shown). Taken together with the recent report (34) that another human breast cancer cell line, MCF-7, also expresses GPR40 mRNA, the present results allow us to propose a model to describe how unsaturated FFAs promote cell survival of breast cancer cells via GPR120 or GPR40. It will be of great value to next determine the precise mechanisms involved in the action of FFA upon breast cancer cells through GPR120 and GPR40.

In conclusion, we demonstrate here, for the first time, that FFA-induced inhibition of apoptosis is mediated through GPCR. Further studies will be needed to advance our understanding of the precise mechanisms and pathways involved with GPR120 and its role in anti-apoptosis under various physiological conditions and in different cell types.


   FOOTNOTES
 
* This work was supported in part by a research grant from the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by the 21st Century Centers of Excellence Program "Knowledge Information Infrastructure for Genome Science." Back

|| To whom correspondence should be addressed: Dept. of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan. Fax: 81-75-753-4523; Tel.: 81-75-753-4544; E-mail: gtsuji{at}pharm.kyoto-u.ac.jp.

1 The abbreviations used are: FFAs, free fatty acids; DN, dominant-negative; GPCR, G-protein-coupled receptor; GLP-1, glucagon-like peptide-1; PLC, phospholipase C; PI3K, phosphatidylinositol 3-kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PT, pertussis toxin; FBS, fetal bovine serum; HS, horse serum; RT, reverse transcription; h, human; m, mouse; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay. Back


   ACKNOWLEDGMENTS
 
We thank J. Yamauchi (Nara Institute of Science and Technology), Y. Izumi (Osaka City Medical School), and S. Mitsuyama (Kumamoto University) for providing plasmids.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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