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Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1 Katsutoshi Nishino a, Haruka Uesugi a, Akira Hirasawa b, Anna Ohtera a, Yusaku Miyamae a, c, Mohamed Neffati d, Hiroko Isoda c, e, Taiho Kambe a, Seiji Masuda a, Kazuhiro Irie f, Masaya Nagao a, * a
Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 606-8501, Japan Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, 305-8572, Japan d Arid Zone Research Institute (IRA), M edenine, 4119, Tunisia e Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Ibaraki, 305-8572, Japan f Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2019 Accepted 5 November 2019 Available online xxx
We examined whether the acetylenic fatty acids 6-octadecynoic acid (6-ODA) and 9-octadecynoic acid (9-ODA) perform as ligands for free fatty acid receptors of medium- and long-chain fatty acids FFAR1 and FFAR4, previously called GPR40 and GPR120, respectively. Phosphorylation of extracellular signalregulated kinase (ERK)-1/2 was increased through FFAR1 but not through FFAR4 expressed in HEK 293 cells, suggesting that 6-ODA and 9-ODA function as an FFAR1 ligand, but not as an FFAR4 ligand. Activation of ERK in FFAR1-expressing HEK293 cells by 6-ODA and 9-ODA peaked at 10 min after stimulation followed by a slow decrease, similar to ERK activation by rosiglitazone, which peaked at 10 min after stimulation and lasted longer. Glucose-dependent production of insulin from MIN6 insulinoma cells was induced by 6-ODA and 9-ODA in an FFAR1-dependent manner. In this process, 6-ODA and 9-ODA stimulated the production of insulin not in the first phase that occurred within 10 min after stimulation but in the second phase. F-actin-remodeling that reflects insulin granule recruiting to the plasma membrane in the second phase of insulin secretion by 6-ODA and 9-ODA suggested that they have an FFAR1-dependent function in insulin secretion from MIN6 cells. © 2019 Elsevier Inc. All rights reserved.
Keywords: 6-Octadecynoid acid 9-Octadecynoic acid Fatty acid receptor Insulin secretion
1. Introduction Free fatty acids (FFAs) are indispensable nutrients some of which are synthesized in the body or produced by intestinal bacteria. Although FFAs are utilized as an important energy source, FFAs have both beneficial and detrimental effects. FFAs show these effects not only through an intracellular peroxisome proliferatoractivated receptor, but also through several G-protein coupled receptors (GPCRs), including a family of four fatty acid receptors, FFAR1, 2, 3 and 4 (previously known as GPR40, GPR43, GPR41 and
Abbreviations: 6-ODA, 6-octadecynoic acid; 9-ODA, 9-octadecynoic acid; Lin, linoleic acid; a-Lnn, a-linolenic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FFAR1, free fatty acid receptor 1; FFAR4, free fatty acid receptor 4; ERK, extra-signal-regulated kinase. * Corresponding author. E-mail address: [email protected] (M. Nagao).
GPR120, respectively). While FFAR1, 2 and 3 are classified as putative closely related GPCRs deorphanized as receptors for FFAs [1e5], GPR120, later reclassified as FFAR4 [6], shared no homology with other fatty acid receptors but was identified as a receptor for FFAs [7]. Long-chain fatty acids (LCFAs) can activate both FFAR1 and FFAR4, but the selectivity of LCFAs for FFAR1 over FFAR4 is not clear. FFAR1 is expressed mainly by b cells of pancreatic islets and gut enteroendocrine cells where FFAR1 is also involved in the secretion of insulin and glucagon-like peptide-1 (GLP-1) which regulate the secretion of insulin by pancreatic b-cells. However, FFAR4 is involved in the secretion of incretins that stimulate insulin secretion from pancreatic b-cells, including glucagon-like peptide-1 (GLP-1), from enteroendocrine cells [7]. FFAR4 knock-out mice fed a high fat diet had fatty liver, obesity and glucose intolerance, which suggested that FFAR4 is related to obesity and adipogenesis [8].
https://doi.org/10.1016/j.bbrc.2019.11.037 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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FFAR4 is also involved in the control of inflammation as a receptor of u3 fatty acids in macrophages and adipocytes [9]. 6-octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified as a peroxisome proliferator-activated receptor g (PPARg) agonist in the methanol extract of Marrubium vulgare L [10]. Thiazolidinediones, synthetic PPARg agonists such as pioglitazone and rosiglitazone, that are used in the treatment of diabetes, are known as FFAR1 agonists [11]. These results prompted us to examine whether 6-ODA and 9-ODA transduce signals through FFAR1 or FFAR4. First, we examined the activation of ERK by 6-ODA and 9ODA in HEK 293 cells that express FFAR1 [12] or FFAR4 in a doxycycline-dependent manner. Secondly, to verify the biological function of 6-ODA and 9-ODA, we examined the production of insulin from mouse insulinoma MIN6 cells induced by 6-ODA and 9ODA. Glucose stimulated a biphasic pattern of insulin secretion: the first phase occurs rapidly but continues only a few minutes and then a second phase that was sustained for a longer time is followed. Since the second phase of insulin secretion is regulated by actin polymerization that facilitates the recruitment and fusion of the insulin-containing granules to the membrane by stimulation [13]. Lastly, we examined actin polymerization by 6-ODA or 9-ODA in MIN6 cells. 2. Materials and methods 2.1. Chemicals 6-octadecynoic acid (6-ODA) was synthesized as described previously [10]. 9-octadecynoic acid (9-ODA) was purchased from Alfa Aesar and a-linolenic acid (a-Lnn) from Cayman chemical. GW1100, a selective antagonist of FFAR1, was purchased from Focus Biomolecules and digitonin from Tokyo Chemical Industry. Other chemicals were from Wako or Nakalai tesque. 2.2. Cell line T-REx FFAR1 (previously called T-REx GPR40) cells that express FFAR1 in a doxycycline (Dox)-dependent manner were constructed using Flp-In™ T-REx™-293 cells as described previously [12]. T-REx FFAR4 (T-REx GPR120) cells that express FFAR4 in a doxycycline (Dox)-dependent manner were constructed in the same way as TREx FFAR1 cells. These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Wako) containing 10% heatinactivated fetal-bovine-serum (FBS, Biosera), 100 mg/ml Hygromycin B (Wako) and 10 mg/ml Blasticidin S hydrochloride (Wako). Both cells were incubated at 37 C in a humidified 5% CO2 incubator. Expression of FFAR1 and FFAR4 were induced by 1 mg/ml Dox (Wako). Mouse insulinoma MIN6 cells were provided by Professor Junichi Miyazaki, Osaka University. MIN6 cells were maintained in DMEM supplemented with 15% heat-inactivated FBS, and 55 mM 2mercaptoethanol (Wako). Each medium contains 100 U/ml penicillin and 100 mg/ml streptomycin (Nakalai tesque). 2.3. Reagents Each free fatty acid was dissolved at 100 mM in 0.1 M NaOH. The solutions were heated for 30 min at 70 C and diluted at 5 mM in a solution of 10% fatty acid free-albumin, from bovine serum (BSA, Wako). These solutions were heated for 10 min at 55 C, and then sterilized by filtration using a 0.45 mM filter. The sterilized solutions were diluted with Krebs solution [14] containing glucose at different concentrations. Rosiglitazone and phorbol 12-myristate 13-acetate (PMA) were
dissolved in DMSO at 100 and 1 mM, respectively. 2.4. Preparation of cell lysates T-REx FFAR1 or T-REx FFAR4 cells were seeded in 12-well plates (1.0 105 cells per well) and incubated for 24 h. Then 1 mg/ml of Dox was added and the cells were incubated for another 24 h. The medium was replaced by serum-free DMEM containing Dox at 1 mg/ml and cells were cultured for 24 h and then each fatty acid, rosiglitazone or PMA was added. After 0e60 min, cells were washed with phosphate-buffered saline (PBS) and lysed in PBS containing 0.5% digitonin, 1 mM Na3VO4 and 10 mM NaF on ice for 15 min. The lysates were centrifuged at 15,000 rpm, 10 min, 4 C, and the supernatants were analyzed by western blotting. 2.5. Western blotting Each cell lysate in 1x SDS PAGE sample buffer (Nacalai) was heated 70 C for 10 min, and each lysate containing 10 mg proteins was deparated in 8% polyacrylamide gels and blotted onto PVDF membranes (Merck Millipore). Anti-Erk-1,2 (1:2000, 137F5, Cell Signaling), anti-phospho-Erk-1/2 (1:1000, D13.14.4E, Cell Signaling), and anti-GAPDH (1:3000, FL-335, Santa Cruz) were used as first-antibodies. Horseradish peroxidase-conjugated anti-rabbit IgG antibodies (1:3000, NA937, GE Healthcare) was used as a second antibody. ECL™ Western Blotting Detection System (GE Healthcare) and ImageQuant LAS 500 (GE Healthcare) were used for detection. 2.6. Insulin secretion assay MIN6 cells were incubated in 96-well plates (2.0 104 cells per well) for 48 h in maintenance medium. After washing twice with Krebs solution, MIN6 cells were subsequently incubated in Krebs solution for 1 h. The solution was replaced by Krebs solution with or without each fatty acid and cells were incubated at 37 C. Insulin concentrations in the supernatants were determined by the Insulin High Range Assay Kit (Cisbio). 2.7. Fluorescent microscopy of F-actin and insulin MIN6 cells on coverslips in 12-well plate (2.0 105 cells per well) in maintenance medium were incubated for 48 h. MIN6 cells were treated with or without fatty acid as described above and incubated for 30 min. The cells were fixed in 10% formaldehyde neutral buffer solution (Wako) for 30 min at 37 C, and permeabilized in 0.2% Triton X-100 (Nakalai tesque) in PBS for 5 min at room temperature. The fixed coverslips were treated by a blocking solution (5% fatty acid free-BSA in PBS) for 1 h. The coverslips were placed on solution of anti-insulin antibody (1:50, H-86, Santa cruz) and incubated for 1 h, and insulin and F-actin was detected by Alexa Fluor 488 conjugated Goat anti-Rabbit IgG (1:100, A-11034, Invitrogen) and Alexa Fluor™ 568 Phalloidin (2U, A-11034, Invitrogen), respectively, using a FLUOVIEW FV10 laser scanning microscope (Olympus). SlowFade™ Antifade kit (Life Technologies) was used to prevent fading. 2.8. Statistical analysis Statistical significance was tested by Dunnett’s test (multiplesample comparison) using R (ver. 1.1.383, RStudio). Differences were considered statistically significant if the p -value was less than 0.05.
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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3. Results 3.1. 6-ODA and 9-ODA activated ERK through FFAR1 but not through FFAR4 expressed in HEK293 cells T-REx FFAR1 [12] and T-REx FFAR4 cells expressed FFAR1 and FFAR4, respectively, in a doxycycline (Dox)-dependent manner in HEK293 cells,. Firstly, we examined ERK activation by 6-ODA or 9ODA using T-REx FFAR1 cells with induction of FFAR1 by Dox. Fig. 1A shows the ERK activation by these fatty acids or rosiglitazone. PMA was used as a positive control that activates ERK in an ~ E). As reported by FFAR1 or FFAR4 -independent manner (Fig. 1 A Smith et al. [15], a PPARg synthetic agonist rosiglitazone activated ERK after 10 min after treatment and activation slowly declined but was sustained at 60 min. ERK by 6-ODA and 9-ODA showed a similar activation pattern (Fig. 1A). Secondly, dose-dependent activation of ERK for 10 min by 6-ODA or 9-ODA was examined in T-REx FFAR1 or T-REx FFAR4 cells. ERK was phosphorylated only by stimulation with 6-ODA or 9-ODA but not by the vehicle control in T-REx-FFAR1 cells when expression of FFAR1 was induced by Dox (Fig. 1B). The activated ERK band was not detected by stimulation of 6-ODA or 9-ODA without Dox-induced FFAR1 expression (Fig. 1C), suggesting that activation of ERK by 6-ODA and 9-ODA was dependent on FFAR1 expressed in T-REx FFAR1 cells. However, after expression of FFAR4 was induced in T-REx FFAR4 cells, an FFAR4
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agonist linoleic acid (Lin) could stimulate phosphorylation of ERK but not that by 6-ODA or 9-ODA (Fig. 1D and E), suggesting that neither 6-ODA nor 9-ODA was an agonist for FFAR4. Lin activated ERK both in T-REx-FFAR1 and T-REx FFAR4 cells in the presence of Dox, while a-linolenic acid (a-Lnn) activated ERK in T-REx FFAR1 cells but weakly in T-REx FFAR4 cells (Fig. 1 B ~ E). Unlike Lin, 6-ODA and 9-ODA activated ERK in an FFAR1-dependent manner but not in an FFAR4-dependent manner in HEK293 cells that express FFAR1 or FFAR4 (Fig. 1B ~ E). 3.2. Dose- and FFAR1-dependent production of insulin in MIN6 cells by 6-ODA or 9-ODA Secretion of insulin from MIN 6 cells in the presence of 2 mM or 25 mM glucose induced by 6-ODA or 9-ODA at 60 min was measured by homogeneous time-resolved fluorescence (HTRF) insulin assay with an insulin high range assay kit. 6-ODA and 9-ODA induced secretion of insulin dose dependently at the dose of 20 or 60 mM, where production of insulin in the presence of 25 mM glucose was higher than that in the presence of 2 mM glucose, suggesting that glucose-dependent secretion of insulin by MIN6 cells was enhanced in this dose range of 6-ODA or 9-ODA (Fig. 2). The production of insulin by 6-ODA and 9-ODA was inhibited by the FFAR1 antagonist GW1100, suggesting that the production of insulin by 6-ODA and 9-ODA was FFAR1-dependent (Fig. 2).
Fig. 1. Phosphorylation of ERK by fatty acids. ERK phosphorylation in T-REx FFAR1 cells (A, B, C) or T-REx FFAR4 cells (D, E) with (A, B, D) or without (C, E) Dox treatment with fatty acids was analyzed by Western Blotting. (A) ERK phosphorylation in T-REx FFAR1cells. Rosiglitazone and PMA (Phorbol 12-myristate 13-acetate) were used as positive controls. (BeE) Cells were treated with fatty acids for 10 min. PMA was used as a positive control.
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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K. Nishino et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 2. Effect of fatty acids on insulin secretion from MIN6 cells. Values are means ± SD, n ¼ 3. Insulin secretion in the presence of 0 mM glucose (white), 2 mM glucose (gray), and 25 mM glucose (Black). *, p < 0.05; **, p < 0.01 vs vehicle control. ♯, p < 0.05; ♯♯, p < 0.01 vs without 1 mM GW1100.
3.3. Production of insulin in MIN6 cells by 6-ODA or 9-ODA The time of insulin secretion by 6-ODA or 9-ODA from MIN 6 cells in the presence of 2 mM or 25 mM glucose was analyzed. At the dose of 60 mM, 6-ODA and 9-ODA induced insulin secretion from MIN6 cells in a pattern similar to that of a well-known FFAR1 agonist, a-Lnn at 60 mM (Fig. 3). Two phases of insulin secretion by multiple stimuli are known. The first phase of insulin secretion occurs within 10 min, followed by a second phase of secretion. FFAR1 ligands of fatty acid, such as
oleic acid is known to stimulate second phase of insulin secretion from pancreatic b-cells [16]. Stimulation of insulin secretion by 6ODA or 9-ODA was observed significantly 30 min after addition of each fatty acid to the medium of MIN6 cells, suggesting that 6-ODA and 9-ODA increased insulin secretion in the second phase (Fig. 3). The constant difference in value of secreted insulin with and without 6-ODA, 9-ODA or a-Lnn after 120 min suggested that stimulation of insulin secretion by these fatty acids was supposed to cease around this time point. 3.4. F-actin remodeling supported production of insulin in the second-phase by 6-ODA or 9-ODA stimulation Although clear insulin granules could not be observed (Fig. 4) without using the total internal reflection fluorescence microscopy (TIRFM) system [17] or high-resolution microscopy, F-actin remodeling in the second phase of insulin secretion after stimulation by 6-ODA or 9-ODA was observed as an increase of F-actin beneath plasma membrane and this increase was inhibited by GW1100, suggesting that this phenomenon was dependent on FFAR1 (Fig. 4). 4. Discussion
Fig. 3. Insulin secretion from MIN6 cells stimulated by various fatty acids in the presence of 25 mM glucose. Vehicle control (open circle), or 60 mM of 6-ODA (Black circle), 9-ODA (open triangle) and a-Lnn (Black triangle). Values are means ± SD, n ¼ 3. *, p < 0.05; **, p < 0.01 vs vehicle control.
The biological functions of fatty acids with triple bond(s) remain to be determined. For example, the C10 acetylenic acid, masutakic acid A was cytotoxic against Kato III cells [18], and 6-nonadecynoic acid had strong anti-fungal activity against the human fungal pathogens [19]. 6-ODA, was known only for its anti-fungal activity [19]. Previously, we found that 6-ODA and its derivative 9-ODA acted as a PPARg agonist [10] like thiazolidinediones, such as rosiglitazone and pioglitazone, that are utilized as anti-diabetic medicine. The finding that the synthetic PPARg agonist, rosiglitazone, that is not a fatty acid, could also act as an FFAR1 fatty acid receptor agonist [15,20] prompted us to examine the biological function of 6-ODA or 9-ODA through FFAR1.
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
K. Nishino et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 4. Fluorescent image of F-actin and insulin in MIN6 cells. A) 25 mM glucose. (B) 60 mM fatty acids in 25 mM glucose. (C) 60 mM fatty acids þ 1 mM GW1100 in 25 mM glucose. Scale bar ¼ 20 mm.
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Fatty acid receptor FFAR1, also known as GPR40 or FFA1, was deorphanized as a medium and long-fatty acid receptor [1,3,20] expressed in brain and pancreas [1]. FFAR1 is known to transduce signals through Gaq subunit of G-protein since intracellular calcium is increased by oleic acid, Lin, g-linolenic acid (g-Lnn), arachidonic acid and docosahexaenoic acid (DHA) in insulinoma MIN6 cells and stimulated insulin secretion [1]. FFA-dependent ERK activation was induced in human FFAR1 expressing Chinese hamster ovary cells by Lin, oleic acid and DHA [3]. FFAR1-deficient mice had essentially normal glucose tolerance and glucose-responding insulin secretion, but intralipid-responding insulin secretion was reduced by ~50% [21]. FFAR4, also known as GPR120, with no homology to FFAR1, is a fatty acid receptor; stimulated saturated FFAs with a chain length for C14 to C18, and by unsaturated FFAs with a chain length of C16 to C22 [7]. Stimulation of FFAR4 by long-chain fatty acids also resulted in elevation of intracellular calcium and activation of ERK cascade which suggested interaction with Gaq family of G proteins [7]. Oh et al. reported that FFAR4-mediated expression of serum response element-luciferase reporter was induced not only by FFAR4 agonist GW9508 (that is usually known as an FFAR1 agonist) but also by DHA and eicosapentaenoic acid (EPA), but not by saturated fatty acid palmitate [22]. In RAW264.7 cells, DHA and EPA, but not saturated fatty acid palmitate activated ERK via FFAR4 [22]. In this study, 6-ODA and 9-ODA, both fatty acids with a triple bond, could activate ERK through FFAR1 (Fig. 1B and C) but not through FFAR4 (Fig. 1D and E). As compared with Lin which could activate ERK through both FFAR1 (Fig. 1B and C) and FFAR4 (Fig. 1D and E), 6-ODA and 9-ODA activates ERK selectively through FFAR1 but not FFAR4 (Fig. 1B ~ E). Since FFAR4 expressed in HEK293 cells did not respond to g-Lnn in the SRE-luc reporter system [22], while g-Lnn could increase intracellular calcium in colon epithelial cell lines, HCT116 and HT29 that express FFAR4 endogenously [23], our results that a-Lnn weakly activated ERK in T-REx-FFAR4 cells expressing FFAR4 (Fig. 1D and E), but not 6-ODA and 9-ODA, might be a phenomenon specific to this cell type. Thiazolidinedione “glitazone” drugs, synthetic agonists for PPARg, are also known to function as FFAR1 ligands. Smith et al. described that activation of ERK by rosiglitazone was dependent on FFAR1 since an FFAR1 antagonist GW1100 inhibited ERK phosphorylation by rosiglitazone [15]. Previously, we showed that acetylenic fatty acids 6-ODA and 9-ODA function as a PPARg agonist [10]. The present study revealed that 6-ODA and 9-ODA also function as a ligand for fatty acid receptor FFAR1 since 6-ODA and 9-ODA activated ERK only when expression of FFAR1 was induced by Dox in T-Rex-FFAR1 cells (Fig. 1B and C). Smith et al. also reported that the patterns of ERK activation, whether sustainable or transient, was dependent on each thiazilidinedione. This study revealed that 6-ODA and 9-ODA could phosphorylate ERK through FFAR1 10 min after stimulation and phosphorylated ERK decayed but still could clearly be observed 60 min after stimulation by these fatty acids (Fig. 1A). The mechanism for prolonged sustainable activation of ERK by 6-ODA and 9-ODA through FFAR1 should be revealed. Although the position of a triple bond was important for antifungal activity of 6-ODA compared to 9-ODA against some fungi [19], 6-ODA and -ODA were almost equally effective for induction of FFAR1-dependent insulin secretion for MIN6 cells (Fig. 3). The biphasic pattern of glucose stimulated insulin secretion (GSIS) consists of the first phase that occurs rapidly and continues for only a few minutes and the second phase [13]. 6-ODA and 9-ODA induced not the first phase but the second phase of insulin secretion (Fig. 3). Seino et al. proposed a model that the first phase of insulin secretion occurs from glanules near plasma membrane and the second phase of insulin secretion occurs from granules
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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associated with the cortical actin network [13]. F-actin remodeling that mobilizes insulin granules inside of the F-actin barrier is believed to be important for the second phase of insulin secretion. Actin remodeling associated with the second phase of insulin secretion after stimulation of 6-ODA or 9-ODA to FFAR1 should be important, since this remodeling and secretion of insulin was inhibited by the FFAR1 antagonist GW1100 (Figs. 2 and 4). ERK activation through GPCRs is known to be regulated by two pathways [24], G protein-dependent and G-protein-independent, arrestin-dependent pathways. The pattern of spatiotemporal activation of ERK by the two pathways may be important for determining the functions of the ligands of GPCRs [25]. In this study 6ODA and 9-ODA stimulated ERK activation and insulin secretion through one of the GPCRs, FFAR1. Further studies are needed to reveal the function of 6-ODA and 9-ODA through FFAR1.
[9]
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Acknowledgment [13]
This work was supported by the JST-JICA’s Science and Technology Research Partnership for Sustainable Development (SATREPS) of Japan (JPMJSA1506) and by KAKENHI (25660294). We thank Professor Jun-ichi Miyazaki, Osaka University, for providing MIN6 cells.
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Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1 Katsutoshi Nishino a, Haruka Uesugi a, Akira Hirasawa b, Anna Ohtera a, Yusaku Miyamae a, c, Mohamed Neffati d, Hiroko Isoda c, e, Taiho Kambe a, Seiji Masuda a, Kazuhiro Irie f, Masaya Nagao a, * a
Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 606-8501, Japan Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, 305-8572, Japan d Arid Zone Research Institute (IRA), M edenine, 4119, Tunisia e Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Ibaraki, 305-8572, Japan f Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2019 Accepted 5 November 2019 Available online xxx
We examined whether the acetylenic fatty acids 6-octadecynoic acid (6-ODA) and 9-octadecynoic acid (9-ODA) perform as ligands for free fatty acid receptors of medium- and long-chain fatty acids FFAR1 and FFAR4, previously called GPR40 and GPR120, respectively. Phosphorylation of extracellular signalregulated kinase (ERK)-1/2 was increased through FFAR1 but not through FFAR4 expressed in HEK 293 cells, suggesting that 6-ODA and 9-ODA function as an FFAR1 ligand, but not as an FFAR4 ligand. Activation of ERK in FFAR1-expressing HEK293 cells by 6-ODA and 9-ODA peaked at 10 min after stimulation followed by a slow decrease, similar to ERK activation by rosiglitazone, which peaked at 10 min after stimulation and lasted longer. Glucose-dependent production of insulin from MIN6 insulinoma cells was induced by 6-ODA and 9-ODA in an FFAR1-dependent manner. In this process, 6-ODA and 9-ODA stimulated the production of insulin not in the first phase that occurred within 10 min after stimulation but in the second phase. F-actin-remodeling that reflects insulin granule recruiting to the plasma membrane in the second phase of insulin secretion by 6-ODA and 9-ODA suggested that they have an FFAR1-dependent function in insulin secretion from MIN6 cells. © 2019 Elsevier Inc. All rights reserved.
Keywords: 6-Octadecynoid acid 9-Octadecynoic acid Fatty acid receptor Insulin secretion
1. Introduction Free fatty acids (FFAs) are indispensable nutrients some of which are synthesized in the body or produced by intestinal bacteria. Although FFAs are utilized as an important energy source, FFAs have both beneficial and detrimental effects. FFAs show these effects not only through an intracellular peroxisome proliferatoractivated receptor, but also through several G-protein coupled receptors (GPCRs), including a family of four fatty acid receptors, FFAR1, 2, 3 and 4 (previously known as GPR40, GPR43, GPR41 and
Abbreviations: 6-ODA, 6-octadecynoic acid; 9-ODA, 9-octadecynoic acid; Lin, linoleic acid; a-Lnn, a-linolenic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FFAR1, free fatty acid receptor 1; FFAR4, free fatty acid receptor 4; ERK, extra-signal-regulated kinase. * Corresponding author. E-mail address: [email protected] (M. Nagao).
GPR120, respectively). While FFAR1, 2 and 3 are classified as putative closely related GPCRs deorphanized as receptors for FFAs [1e5], GPR120, later reclassified as FFAR4 [6], shared no homology with other fatty acid receptors but was identified as a receptor for FFAs [7]. Long-chain fatty acids (LCFAs) can activate both FFAR1 and FFAR4, but the selectivity of LCFAs for FFAR1 over FFAR4 is not clear. FFAR1 is expressed mainly by b cells of pancreatic islets and gut enteroendocrine cells where FFAR1 is also involved in the secretion of insulin and glucagon-like peptide-1 (GLP-1) which regulate the secretion of insulin by pancreatic b-cells. However, FFAR4 is involved in the secretion of incretins that stimulate insulin secretion from pancreatic b-cells, including glucagon-like peptide-1 (GLP-1), from enteroendocrine cells [7]. FFAR4 knock-out mice fed a high fat diet had fatty liver, obesity and glucose intolerance, which suggested that FFAR4 is related to obesity and adipogenesis [8].
https://doi.org/10.1016/j.bbrc.2019.11.037 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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FFAR4 is also involved in the control of inflammation as a receptor of u3 fatty acids in macrophages and adipocytes [9]. 6-octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified as a peroxisome proliferator-activated receptor g (PPARg) agonist in the methanol extract of Marrubium vulgare L [10]. Thiazolidinediones, synthetic PPARg agonists such as pioglitazone and rosiglitazone, that are used in the treatment of diabetes, are known as FFAR1 agonists [11]. These results prompted us to examine whether 6-ODA and 9-ODA transduce signals through FFAR1 or FFAR4. First, we examined the activation of ERK by 6-ODA and 9ODA in HEK 293 cells that express FFAR1 [12] or FFAR4 in a doxycycline-dependent manner. Secondly, to verify the biological function of 6-ODA and 9-ODA, we examined the production of insulin from mouse insulinoma MIN6 cells induced by 6-ODA and 9ODA. Glucose stimulated a biphasic pattern of insulin secretion: the first phase occurs rapidly but continues only a few minutes and then a second phase that was sustained for a longer time is followed. Since the second phase of insulin secretion is regulated by actin polymerization that facilitates the recruitment and fusion of the insulin-containing granules to the membrane by stimulation [13]. Lastly, we examined actin polymerization by 6-ODA or 9-ODA in MIN6 cells. 2. Materials and methods 2.1. Chemicals 6-octadecynoic acid (6-ODA) was synthesized as described previously [10]. 9-octadecynoic acid (9-ODA) was purchased from Alfa Aesar and a-linolenic acid (a-Lnn) from Cayman chemical. GW1100, a selective antagonist of FFAR1, was purchased from Focus Biomolecules and digitonin from Tokyo Chemical Industry. Other chemicals were from Wako or Nakalai tesque. 2.2. Cell line T-REx FFAR1 (previously called T-REx GPR40) cells that express FFAR1 in a doxycycline (Dox)-dependent manner were constructed using Flp-In™ T-REx™-293 cells as described previously [12]. T-REx FFAR4 (T-REx GPR120) cells that express FFAR4 in a doxycycline (Dox)-dependent manner were constructed in the same way as TREx FFAR1 cells. These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Wako) containing 10% heatinactivated fetal-bovine-serum (FBS, Biosera), 100 mg/ml Hygromycin B (Wako) and 10 mg/ml Blasticidin S hydrochloride (Wako). Both cells were incubated at 37 C in a humidified 5% CO2 incubator. Expression of FFAR1 and FFAR4 were induced by 1 mg/ml Dox (Wako). Mouse insulinoma MIN6 cells were provided by Professor Junichi Miyazaki, Osaka University. MIN6 cells were maintained in DMEM supplemented with 15% heat-inactivated FBS, and 55 mM 2mercaptoethanol (Wako). Each medium contains 100 U/ml penicillin and 100 mg/ml streptomycin (Nakalai tesque). 2.3. Reagents Each free fatty acid was dissolved at 100 mM in 0.1 M NaOH. The solutions were heated for 30 min at 70 C and diluted at 5 mM in a solution of 10% fatty acid free-albumin, from bovine serum (BSA, Wako). These solutions were heated for 10 min at 55 C, and then sterilized by filtration using a 0.45 mM filter. The sterilized solutions were diluted with Krebs solution [14] containing glucose at different concentrations. Rosiglitazone and phorbol 12-myristate 13-acetate (PMA) were
dissolved in DMSO at 100 and 1 mM, respectively. 2.4. Preparation of cell lysates T-REx FFAR1 or T-REx FFAR4 cells were seeded in 12-well plates (1.0 105 cells per well) and incubated for 24 h. Then 1 mg/ml of Dox was added and the cells were incubated for another 24 h. The medium was replaced by serum-free DMEM containing Dox at 1 mg/ml and cells were cultured for 24 h and then each fatty acid, rosiglitazone or PMA was added. After 0e60 min, cells were washed with phosphate-buffered saline (PBS) and lysed in PBS containing 0.5% digitonin, 1 mM Na3VO4 and 10 mM NaF on ice for 15 min. The lysates were centrifuged at 15,000 rpm, 10 min, 4 C, and the supernatants were analyzed by western blotting. 2.5. Western blotting Each cell lysate in 1x SDS PAGE sample buffer (Nacalai) was heated 70 C for 10 min, and each lysate containing 10 mg proteins was deparated in 8% polyacrylamide gels and blotted onto PVDF membranes (Merck Millipore). Anti-Erk-1,2 (1:2000, 137F5, Cell Signaling), anti-phospho-Erk-1/2 (1:1000, D13.14.4E, Cell Signaling), and anti-GAPDH (1:3000, FL-335, Santa Cruz) were used as first-antibodies. Horseradish peroxidase-conjugated anti-rabbit IgG antibodies (1:3000, NA937, GE Healthcare) was used as a second antibody. ECL™ Western Blotting Detection System (GE Healthcare) and ImageQuant LAS 500 (GE Healthcare) were used for detection. 2.6. Insulin secretion assay MIN6 cells were incubated in 96-well plates (2.0 104 cells per well) for 48 h in maintenance medium. After washing twice with Krebs solution, MIN6 cells were subsequently incubated in Krebs solution for 1 h. The solution was replaced by Krebs solution with or without each fatty acid and cells were incubated at 37 C. Insulin concentrations in the supernatants were determined by the Insulin High Range Assay Kit (Cisbio). 2.7. Fluorescent microscopy of F-actin and insulin MIN6 cells on coverslips in 12-well plate (2.0 105 cells per well) in maintenance medium were incubated for 48 h. MIN6 cells were treated with or without fatty acid as described above and incubated for 30 min. The cells were fixed in 10% formaldehyde neutral buffer solution (Wako) for 30 min at 37 C, and permeabilized in 0.2% Triton X-100 (Nakalai tesque) in PBS for 5 min at room temperature. The fixed coverslips were treated by a blocking solution (5% fatty acid free-BSA in PBS) for 1 h. The coverslips were placed on solution of anti-insulin antibody (1:50, H-86, Santa cruz) and incubated for 1 h, and insulin and F-actin was detected by Alexa Fluor 488 conjugated Goat anti-Rabbit IgG (1:100, A-11034, Invitrogen) and Alexa Fluor™ 568 Phalloidin (2U, A-11034, Invitrogen), respectively, using a FLUOVIEW FV10 laser scanning microscope (Olympus). SlowFade™ Antifade kit (Life Technologies) was used to prevent fading. 2.8. Statistical analysis Statistical significance was tested by Dunnett’s test (multiplesample comparison) using R (ver. 1.1.383, RStudio). Differences were considered statistically significant if the p -value was less than 0.05.
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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3. Results 3.1. 6-ODA and 9-ODA activated ERK through FFAR1 but not through FFAR4 expressed in HEK293 cells T-REx FFAR1 [12] and T-REx FFAR4 cells expressed FFAR1 and FFAR4, respectively, in a doxycycline (Dox)-dependent manner in HEK293 cells,. Firstly, we examined ERK activation by 6-ODA or 9ODA using T-REx FFAR1 cells with induction of FFAR1 by Dox. Fig. 1A shows the ERK activation by these fatty acids or rosiglitazone. PMA was used as a positive control that activates ERK in an ~ E). As reported by FFAR1 or FFAR4 -independent manner (Fig. 1 A Smith et al. [15], a PPARg synthetic agonist rosiglitazone activated ERK after 10 min after treatment and activation slowly declined but was sustained at 60 min. ERK by 6-ODA and 9-ODA showed a similar activation pattern (Fig. 1A). Secondly, dose-dependent activation of ERK for 10 min by 6-ODA or 9-ODA was examined in T-REx FFAR1 or T-REx FFAR4 cells. ERK was phosphorylated only by stimulation with 6-ODA or 9-ODA but not by the vehicle control in T-REx-FFAR1 cells when expression of FFAR1 was induced by Dox (Fig. 1B). The activated ERK band was not detected by stimulation of 6-ODA or 9-ODA without Dox-induced FFAR1 expression (Fig. 1C), suggesting that activation of ERK by 6-ODA and 9-ODA was dependent on FFAR1 expressed in T-REx FFAR1 cells. However, after expression of FFAR4 was induced in T-REx FFAR4 cells, an FFAR4
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agonist linoleic acid (Lin) could stimulate phosphorylation of ERK but not that by 6-ODA or 9-ODA (Fig. 1D and E), suggesting that neither 6-ODA nor 9-ODA was an agonist for FFAR4. Lin activated ERK both in T-REx-FFAR1 and T-REx FFAR4 cells in the presence of Dox, while a-linolenic acid (a-Lnn) activated ERK in T-REx FFAR1 cells but weakly in T-REx FFAR4 cells (Fig. 1 B ~ E). Unlike Lin, 6-ODA and 9-ODA activated ERK in an FFAR1-dependent manner but not in an FFAR4-dependent manner in HEK293 cells that express FFAR1 or FFAR4 (Fig. 1B ~ E). 3.2. Dose- and FFAR1-dependent production of insulin in MIN6 cells by 6-ODA or 9-ODA Secretion of insulin from MIN 6 cells in the presence of 2 mM or 25 mM glucose induced by 6-ODA or 9-ODA at 60 min was measured by homogeneous time-resolved fluorescence (HTRF) insulin assay with an insulin high range assay kit. 6-ODA and 9-ODA induced secretion of insulin dose dependently at the dose of 20 or 60 mM, where production of insulin in the presence of 25 mM glucose was higher than that in the presence of 2 mM glucose, suggesting that glucose-dependent secretion of insulin by MIN6 cells was enhanced in this dose range of 6-ODA or 9-ODA (Fig. 2). The production of insulin by 6-ODA and 9-ODA was inhibited by the FFAR1 antagonist GW1100, suggesting that the production of insulin by 6-ODA and 9-ODA was FFAR1-dependent (Fig. 2).
Fig. 1. Phosphorylation of ERK by fatty acids. ERK phosphorylation in T-REx FFAR1 cells (A, B, C) or T-REx FFAR4 cells (D, E) with (A, B, D) or without (C, E) Dox treatment with fatty acids was analyzed by Western Blotting. (A) ERK phosphorylation in T-REx FFAR1cells. Rosiglitazone and PMA (Phorbol 12-myristate 13-acetate) were used as positive controls. (BeE) Cells were treated with fatty acids for 10 min. PMA was used as a positive control.
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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Fig. 2. Effect of fatty acids on insulin secretion from MIN6 cells. Values are means ± SD, n ¼ 3. Insulin secretion in the presence of 0 mM glucose (white), 2 mM glucose (gray), and 25 mM glucose (Black). *, p < 0.05; **, p < 0.01 vs vehicle control. ♯, p < 0.05; ♯♯, p < 0.01 vs without 1 mM GW1100.
3.3. Production of insulin in MIN6 cells by 6-ODA or 9-ODA The time of insulin secretion by 6-ODA or 9-ODA from MIN 6 cells in the presence of 2 mM or 25 mM glucose was analyzed. At the dose of 60 mM, 6-ODA and 9-ODA induced insulin secretion from MIN6 cells in a pattern similar to that of a well-known FFAR1 agonist, a-Lnn at 60 mM (Fig. 3). Two phases of insulin secretion by multiple stimuli are known. The first phase of insulin secretion occurs within 10 min, followed by a second phase of secretion. FFAR1 ligands of fatty acid, such as
oleic acid is known to stimulate second phase of insulin secretion from pancreatic b-cells [16]. Stimulation of insulin secretion by 6ODA or 9-ODA was observed significantly 30 min after addition of each fatty acid to the medium of MIN6 cells, suggesting that 6-ODA and 9-ODA increased insulin secretion in the second phase (Fig. 3). The constant difference in value of secreted insulin with and without 6-ODA, 9-ODA or a-Lnn after 120 min suggested that stimulation of insulin secretion by these fatty acids was supposed to cease around this time point. 3.4. F-actin remodeling supported production of insulin in the second-phase by 6-ODA or 9-ODA stimulation Although clear insulin granules could not be observed (Fig. 4) without using the total internal reflection fluorescence microscopy (TIRFM) system [17] or high-resolution microscopy, F-actin remodeling in the second phase of insulin secretion after stimulation by 6-ODA or 9-ODA was observed as an increase of F-actin beneath plasma membrane and this increase was inhibited by GW1100, suggesting that this phenomenon was dependent on FFAR1 (Fig. 4). 4. Discussion
Fig. 3. Insulin secretion from MIN6 cells stimulated by various fatty acids in the presence of 25 mM glucose. Vehicle control (open circle), or 60 mM of 6-ODA (Black circle), 9-ODA (open triangle) and a-Lnn (Black triangle). Values are means ± SD, n ¼ 3. *, p < 0.05; **, p < 0.01 vs vehicle control.
The biological functions of fatty acids with triple bond(s) remain to be determined. For example, the C10 acetylenic acid, masutakic acid A was cytotoxic against Kato III cells [18], and 6-nonadecynoic acid had strong anti-fungal activity against the human fungal pathogens [19]. 6-ODA, was known only for its anti-fungal activity [19]. Previously, we found that 6-ODA and its derivative 9-ODA acted as a PPARg agonist [10] like thiazolidinediones, such as rosiglitazone and pioglitazone, that are utilized as anti-diabetic medicine. The finding that the synthetic PPARg agonist, rosiglitazone, that is not a fatty acid, could also act as an FFAR1 fatty acid receptor agonist [15,20] prompted us to examine the biological function of 6-ODA or 9-ODA through FFAR1.
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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Fig. 4. Fluorescent image of F-actin and insulin in MIN6 cells. A) 25 mM glucose. (B) 60 mM fatty acids in 25 mM glucose. (C) 60 mM fatty acids þ 1 mM GW1100 in 25 mM glucose. Scale bar ¼ 20 mm.
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Fatty acid receptor FFAR1, also known as GPR40 or FFA1, was deorphanized as a medium and long-fatty acid receptor [1,3,20] expressed in brain and pancreas [1]. FFAR1 is known to transduce signals through Gaq subunit of G-protein since intracellular calcium is increased by oleic acid, Lin, g-linolenic acid (g-Lnn), arachidonic acid and docosahexaenoic acid (DHA) in insulinoma MIN6 cells and stimulated insulin secretion [1]. FFA-dependent ERK activation was induced in human FFAR1 expressing Chinese hamster ovary cells by Lin, oleic acid and DHA [3]. FFAR1-deficient mice had essentially normal glucose tolerance and glucose-responding insulin secretion, but intralipid-responding insulin secretion was reduced by ~50% [21]. FFAR4, also known as GPR120, with no homology to FFAR1, is a fatty acid receptor; stimulated saturated FFAs with a chain length for C14 to C18, and by unsaturated FFAs with a chain length of C16 to C22 [7]. Stimulation of FFAR4 by long-chain fatty acids also resulted in elevation of intracellular calcium and activation of ERK cascade which suggested interaction with Gaq family of G proteins [7]. Oh et al. reported that FFAR4-mediated expression of serum response element-luciferase reporter was induced not only by FFAR4 agonist GW9508 (that is usually known as an FFAR1 agonist) but also by DHA and eicosapentaenoic acid (EPA), but not by saturated fatty acid palmitate [22]. In RAW264.7 cells, DHA and EPA, but not saturated fatty acid palmitate activated ERK via FFAR4 [22]. In this study, 6-ODA and 9-ODA, both fatty acids with a triple bond, could activate ERK through FFAR1 (Fig. 1B and C) but not through FFAR4 (Fig. 1D and E). As compared with Lin which could activate ERK through both FFAR1 (Fig. 1B and C) and FFAR4 (Fig. 1D and E), 6-ODA and 9-ODA activates ERK selectively through FFAR1 but not FFAR4 (Fig. 1B ~ E). Since FFAR4 expressed in HEK293 cells did not respond to g-Lnn in the SRE-luc reporter system [22], while g-Lnn could increase intracellular calcium in colon epithelial cell lines, HCT116 and HT29 that express FFAR4 endogenously [23], our results that a-Lnn weakly activated ERK in T-REx-FFAR4 cells expressing FFAR4 (Fig. 1D and E), but not 6-ODA and 9-ODA, might be a phenomenon specific to this cell type. Thiazolidinedione “glitazone” drugs, synthetic agonists for PPARg, are also known to function as FFAR1 ligands. Smith et al. described that activation of ERK by rosiglitazone was dependent on FFAR1 since an FFAR1 antagonist GW1100 inhibited ERK phosphorylation by rosiglitazone [15]. Previously, we showed that acetylenic fatty acids 6-ODA and 9-ODA function as a PPARg agonist [10]. The present study revealed that 6-ODA and 9-ODA also function as a ligand for fatty acid receptor FFAR1 since 6-ODA and 9-ODA activated ERK only when expression of FFAR1 was induced by Dox in T-Rex-FFAR1 cells (Fig. 1B and C). Smith et al. also reported that the patterns of ERK activation, whether sustainable or transient, was dependent on each thiazilidinedione. This study revealed that 6-ODA and 9-ODA could phosphorylate ERK through FFAR1 10 min after stimulation and phosphorylated ERK decayed but still could clearly be observed 60 min after stimulation by these fatty acids (Fig. 1A). The mechanism for prolonged sustainable activation of ERK by 6-ODA and 9-ODA through FFAR1 should be revealed. Although the position of a triple bond was important for antifungal activity of 6-ODA compared to 9-ODA against some fungi [19], 6-ODA and -ODA were almost equally effective for induction of FFAR1-dependent insulin secretion for MIN6 cells (Fig. 3). The biphasic pattern of glucose stimulated insulin secretion (GSIS) consists of the first phase that occurs rapidly and continues for only a few minutes and the second phase [13]. 6-ODA and 9-ODA induced not the first phase but the second phase of insulin secretion (Fig. 3). Seino et al. proposed a model that the first phase of insulin secretion occurs from glanules near plasma membrane and the second phase of insulin secretion occurs from granules
Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037
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associated with the cortical actin network [13]. F-actin remodeling that mobilizes insulin granules inside of the F-actin barrier is believed to be important for the second phase of insulin secretion. Actin remodeling associated with the second phase of insulin secretion after stimulation of 6-ODA or 9-ODA to FFAR1 should be important, since this remodeling and secretion of insulin was inhibited by the FFAR1 antagonist GW1100 (Figs. 2 and 4). ERK activation through GPCRs is known to be regulated by two pathways [24], G protein-dependent and G-protein-independent, arrestin-dependent pathways. The pattern of spatiotemporal activation of ERK by the two pathways may be important for determining the functions of the ligands of GPCRs [25]. In this study 6ODA and 9-ODA stimulated ERK activation and insulin secretion through one of the GPCRs, FFAR1. Further studies are needed to reveal the function of 6-ODA and 9-ODA through FFAR1.
[9]
[10]
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Acknowledgment [13]
This work was supported by the JST-JICA’s Science and Technology Research Partnership for Sustainable Development (SATREPS) of Japan (JPMJSA1506) and by KAKENHI (25660294). We thank Professor Jun-ichi Miyazaki, Osaka University, for providing MIN6 cells.
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Please cite this article as: K. Nishino et al., Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.037