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Planta Med 2012; 78(5): 434-439
DOI: 10.1055/s-0031-1298155
Biological and Pharmacological Activity
Original Papers
© Georg Thieme Verlag KG Stuttgart · New York

Terpene Alcohols Inhibit De Novo Sphingolipid Biosynthesis

Kyong-Oh Shin1 , Myong-Yong Park1 , Cho-Hee Seo1 , Yong-Ill Lee2 , Hyun Sik Kim3 , Hwan-Soo Yoo1 , Jin Tae Hong1 , Jae-Kyung Jung1 , Yong-Moon Lee1
  • 1College of Pharmacy and MRC, Chungbuk National University, Chongju, Korea
  • 2Department of Chemistry, Changwon National University, Changwon, Korea
  • 3Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang, Korea
Further Information

Prof. Yong-Moon Lee

College of Pharmacy and MRC
Chungbuk National University

San 48, Kaesin-Dong, Hungduk-Ku

Chongju 361-763

Korea

Phone: +82 4 32 61 28 25

Fax: +82 4 32 68 27 32

Email: ymleefn@cbnu.ac.kr

Publication History

received October 13, 2011 revised Dec. 14, 2011

accepted Dec. 15, 2011

Publication Date:
24 January 2012 (online)

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Table of Contents #

Abstract

The terpene alcohols geranyllinalool, phytol (diterpene alcohol), and farnesol (sesquiterpene alcohol) were newly found to inhibit sphingolipid de novo biosynthesis in LLC-PK1 cells (pig kidney epithelial cells). A simple chromatographic bioassay was established for the screening of inhibitory compounds able to reduce the amount of sphinganine, an intermediate metabolite of sphingolipid biosynthesis. The screening strategy was based on the degree of suppression of fumonisin B1 (FB1)-induced sphinganine accumulation following co-treatment with selected terpene alcohols. L-cycloserine and ISP-1, specific serine palmitoyltransferase (SPT) inhibitors, were used as positive controls. Our results show that measuring reduced sphinganine levels after treatment with 2 µM FB1 in combination with the putative inhibitory compounds provides a useful screening bioassay for evaluating compounds causing sphingolipid depletion. Intracellular sphinganine concentrations were analyzed using the fluorescent peak areas of the o-phthalaldehyde (OPA) derivatives of sphinganine eluted with 87 % acetonitrile on a reversed-phase column. Geranyllinalool, phytol, and farnesol were identified as novel SPT inhibitors that reduce FB1-induced sphinganine accumulation and thus inhibit the first step of sphingolipid de novo synthesis.

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Key words

terpene alcohol - geranyllinalool - phytol - sphinganine - fumonisin B1 - ISP-1

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Introduction

Terpene alcohols are the primary constituents of the essential oils of many types of plants and flowers. Geranyllinalool is a fragrance ingredient used for cosmetics, cleaners, and detergents [1]. Phytol is an acyclic diterpene alcohol that is a precursor of vitamin E and effectively blocks retinol-induced teratogenicity [2]. Fumonisins are mycotoxins that are mainly produced by Fusarium verticillioides, fungal contaminants of corn and its related products. Fumonisins are strongly associated with specific farm animal diseases, such as leukoencephalomalacia in horses and pulmonary edema in swine, in addition to being suspected in the high incidence of human esophageal cancer in some regions of South Africa [3] and China [4].

Fumonisin (FB1) disrupts sphingolipid metabolism by inhibiting de novo biosynthesis. The toxin's effects are mainly due to the accumulation of endogenous sphinganine, the depletion of complex sphingolipids, and an increase in sphinganine degradation products [5]. An early increase in sphinganine levels by FB1 precedes cell growth arrest and cytotoxicity [6].

ISP-1 and L-cycloserine selectively inhibit the enzyme serine palmitoyltransferase (SPT) which catalyzes the first step of sphingolipid de novo biosynthesis [7]. Pretreatment with either L-cycloserine or ISP-1 prevents the increase in sphinganine levels as well as the growth arrest, DNA fragmentation, and cell death induced by FB1 [8], [9]. Therefore, FB1-induced outcomes may reflect early sphinganine accumulation in cultured cells.

The aim of this study was to identify inhibitors of sphingolipid de novo biosynthesis from medicinal plant sources using a simple HPLC screening method. This study began with the basic concept that blocking an early stage of the de novo synthetic route might reduce sphinganine accumulation [10]. Our approach is shown to have analytical applications as a result of the chromatographic advantages conferred by the separation of hydrophobic lipids, the strong fluorogenic properties of o-phthalaldehyde (OPA), and its high stability. It was successfully applied to determine the changes in sphingolipids levels triggered by various agonists [11], [12].

Depending on the cell type and size, the endogenous concentrations of sphingosine and sphinganine were generally below 10 pmol/mg protein [9]. Our method to determine sphinganine concentrations of 0.1 pmol/mg protein was further sensitized by inserting a preheating step to increase sphinganine solubility ahead of the OPA reaction [13]. Using this established chromatographic screening, we identified several plant compounds, i.e., geranyllinalool (GLO), phytol, and farnesol, which were able to reduce the sphinganine levels in a dose-dependent manner. This report describes terpene alcohols showing ISP-1-like activity in cultured cells.

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Materials and Methods

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Materials

FB1, ISP-1, and sphinganine were purchased from Biomol Research, Inc. The internal standard, C20-sphinganine, was purchased from Matreya, Inc. GLO (≥ 95 %), phytol (≥ 97 %), farnesol (≥ 97 %), dolichol (95 %), and dolichol phosphate (90 %) were obtained from Fluka. L-cycloserine (95 %) and bufalin (≥ 98.0 %) were obtained from Sigma. Serum and culture medium were obtained from Life Technologies, Inc. The HPLC-grade acetonitrile was purchased from Mallinckrodt Baker, Inc. The o-phthalaldehyde (OPA) was obtained from Nacalai Tesque. All of the organic solvents used in sphingolipid extraction were purchased from Merck.

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Cell culture and treatment of test compounds

Pig kidney epithelial (LLC-PK1) cells obtained from ATCC were seeded at 5 × 105 cells/well in 6-well dishes and cultured in DMEM/F12 medium supplemented with 1.2 g sodium bicarbonate/L, 100 units penicillin-streptomycin/mL, and 5 %(v/v) FBS at 37 °C in a humidified 5 % CO2 atmosphere. The medium was changed 12 h after seeding, and the cells were then incubated with DMEM/F12 containing 2 µM FB1 for 24 h to increase the intracellular free sphinganine content. Subsequently, 2 mL of medium was replaced with fresh medium containing both the test compounds and 2 µM FB1. The cells were incubated at 37 °C and harvested after 24 h using trypsin-EDTA (Life Technologies, Inc.).

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Extraction of cellular sphinganine

The harvested cells were rapidly precipitated by brief centrifugation at 10 000 rpm for 10 sec. After the supernatant had been removed, the pellets were washed twice with 1 mL of phosphate-buffered saline (PBS, pH 7.2), resuspended in 200 µL of methanol, and sonicated for 10 sec. For the protein assay, a 20-µL aliquot was transferred to the remaining suspension, while 200 µL of chloroform (CHCl3), 400 µL of 0.15 M methanolic KOH, and 40 pmol of C20-sphinganine in ethanol (as the internal standard) were added. The samples were mixed well and then incubated at 37 °C for 1 h followed by partitioning with 400 µL of CHCl3, 500 µL of alkaline water, and 100 µL of 2 N NH4OH. The CHCl3 phase was washed three times with alkaline water (150 µL of 2 N NH4OH in 700 mL of distilled water, pH 10.0). The lower organic phase was transferred to a fresh tube and dried under a nitrogen stream.

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HPLC measurement of sphinganine accumulation

The above-described samples were mixed well with 15 µL of the OPA reagent [50 mg OPA, 1 mL ethanol, 100 µL 2-mercaptoethanol, and 50 mL 3 % (w/v) boric acid solution adjusted to pH 10.5] and incubated at room temperature for 20 min. HPLC analyses were performed using a Hitachi Model L-6200 series pump and Rheodyne 7125 injector together with prepacked C18 reversed-phase columns (Cosmosil, 5C 18-AR; 4.6 mm i. d. × 150 mm or Mightysil 5C18; 4.6 mm i. d. × 150 mm). The isocratic eluent consisted of acetonitrile : deionized distilled water (87 : 13, v/v), and the flow rate of 1 mL/min was controlled accurately during the HPLC analysis. The OPA derivatives were detected selectively using a Jasco FP-920 fluorescence detector, with an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The resulting data and chromatographic profiles were evaluated using the Borwin® system manager software (JMBS).

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SPT activity assay

SPT activity was measured by following the improved HPLC protocol [14]. In brief, 190 µL of cell lysate (300 µg protein) was briefly mixed with 10 µL of 20× HPLC assay buffer [50 mM HEPES (pH 8) containing 1 mM EDTA and 0.1 % (w/v) sucrose monolaurate, 5 mM L-serine, 50 µM palmitoyl-CoA, and 20 µM pyridoxal 5′-phosphate]. The SPT enzyme reaction was started by placing the samples at 37 °C for 60 min. To terminate the reaction and to reduce 3-ketosphinganine into sphinganine, NaBH4 (5 mg/mL) was added and the samples left at room temperature for 5 min. Lipid extraction and product detection by HPLC were as described in the appendix protocol [14].

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Protein assay

The total protein concentration in the samples was measured in a 96-well plate by mixing an aliquot with the Bio-Rad® Bradford protein assay reagent. The samples were incubated at room temperature for 15 min, and the protein concentration was then measured using a Molecular Devices® ELISA reader at 570 nm. The results were then compared with the BSA standard calibration curve.

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Statistical analysis

These data were presented as mean ± SD. Statistical significance was assessed with one-way analysis of variance (ANOVA) by Newman-Keuls test, and in all cases the criterion for significance was p < 0.05.

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Results and Discussion

The mammalian diseases associated with fumonisins exposure are believed to be caused by a disruption of sphingolipid metabolism as both the initial and precipitating events since these compounds are potent inhibitors of sphinganine N-acyltransferase in a variety of cell types, as shown in [Fig. 1] [15].

Zoom Image

Fig. 1 FB1 causes the accumulation of sphinganine by inhibiting sphinganine N-acyltransferase in the sphingolipid de novo biosynthetic pathway. It also inhibits sphingosine N-acyltransferase, which results in an increase in sphingoid bases and the depletion of complex sphingolipids, including ceramides. Other inhibitors of the sphingolipid metabolic pathway are also shown.

The sphinganine that accumulated under various FB1 concentrations in cells exposed to the toxin for 48 h was chromatographically detected as a fluorescent peak of its OPA derivative. While sphinganine levels increased abruptly, sphingosine levels remained relatively unchanged, as previously described [16]. FB1 caused an initial steep increase in the sphinganine level up to 24 h, followed by a gradual diminishing thereafter. In the absence of FB1, the sphinganine levels remained extremely low. Under the screening conditions by the test compounds ([Fig. 2]) and in the presence of 2 µM FB1 as the test compound, the fragmented DNA content in cells was below 5 %, indicating the absence of cytotoxicity (data not shown) and thus the inability to assess the protective effects of GLO, phytol, and farnesol. However, high doses of FB1 (20 µM) significantly reduced cell viability to 64 ± 8 %, and while co-treatment with 50 µM GLO, phytol, and farnesol partially reduced the FB1-mediated sphinganine accumulation, both compounds failed to reduce FB1-induced cell cytotoxicity.

Zoom Image

Fig. 2 Chemical structures of the test compounds used in the inhibitory assay of sphingolipid biosynthesis. A fumonisin B1, B geranyllinalool, C phytol, D farnesol, E FTY720, F bufalin, G L-cycloserine, and H ISP-1.

ISP-1 and L-cycloserine are selective inhibitors of the SPT enzyme, which catalyzes the initial step of sphingolipid de novo biosynthesis. Both compounds are thought to reduce FB1 toxicity by blocking the increase in the intracellular sphinganine level and, hence, sphingolipids biosynthesis [8], [9].

Simultaneous incubation of the cells with 10, 100, and 1000 µM of L-cycloserine and 2 µM FB1 dose-dependently reduced sphinganine accumulation by 32 %, 62 %, and 86 % compared with the control, respectively ([Fig. 3]). Because de novo biosynthesis is a one-way route, as shown in [Fig. 1], a high sphinganine level after FB1 treatment was expected. These results suggest that our HPLC assay can be used to screen for selective inhibitors of FB1.

Zoom Image

Fig. 3 FB1 inhibits sphinganine N-acyltransferase activity and causes the intracellular accumulation of free sphinganine in a time-dependent manner. A LLC-PK1 cells treated with (a) or without (b) 2 µM FB1 were harvested, and the sphinganine level was measured after OPA fluorogenic derivatization in a HPLC system. The inhibitory effects were estimated by the increased sphinganine levels. B L-cycloserine dose-dependently inhibits sphinganine levels elevated in response to 2 µM FB1 following 24 h of coincubation of the cells with these two compounds. The results are expressed as the sphinganine content after 24 h as a percentage of the positive control ([1]: +FB1, 2 µM). The sphinganine content was normalized in pmol/5 × 105 cells (62 µg protein) using a Bradford protein assay. The data are shown as a mean ± SD (n = 3). ** P < 0.01 and *** p < 0.001 compared to FB1-treated group.

For the practical screening of FB1 inhibitors, bioactive lipids, including free fatty acids, eicosanoids, phospholipids, ceramides, and isoprenoids, were added to the cells, and the effects compared with the inhibitory effect of L-cycloserine. Among the tested compounds, strong inhibition of sphinganine accumulation was mainly achieved with the isoprenoids. These compounds, which include geranylgeraniol (GGO) and farnesol, bind to the small G-protein and subsequently inhibit the potential oncogenic activity of the Ras protein [17]. GLO is structurally similar to GGO and might therefore mimic GGO function, as previously described. FTY720, synthesized to obtain improved ISP-1 activity, is based on an extract from Isaria sinclarii, and its immunosuppressive function in CTLL-2 cells has been demonstrated [18]. Phytol suppresses prostaglandin synthesis in vitro [19] and weakly protects tumor promoters in mouse skin [20]. These isoprenoids were positively classified as potent inhibitors by comparing their effects with those of L-cycloserine. The results showed that treatment with these compounds (50 µM) dramatically reduced the accumulation of sphinganine 15.2- (GLO), 4.2- (phytol), and 3.8-fold (farnesol) compared with 100 µM L-cycloserine ([Fig. 4]). In addition, GLO and phytol reduced the level of FB1-induced sphinganine accumulation in a dose-dependent manner ([Fig. 5]). Thus, GLO, phytol, and farnesol were able to effectively prevent the early step of sphingolipid de novo biosynthesis. However, FTY720, dolichol and its phosphate derivative, an analog of GGO, had little effect on the decrease in FB1-induced sphinganine accumulation.

Zoom Image

Fig. 4 GLO, phytol, and farnesol strongly inhibit the increase in sphinganine levels induced by 2 µM of FB1 after a 24-h coincubation of the test compounds (L-cycloserine, 100 µM; FTY720, 20 µM; GLO, 50 µM; phytol, 50 µM; farnesol, 50 µM; dolichol, 50 µM; dolichol phosphate, 50 µM). Sphinganine content (%) is expressed as shown in [Fig. 3]. Each column and bar represents the mean ± SD (n = 3). ** P < 0.01 and *** p < 0.001 compared to the FB1-treated group.

Zoom Image

Fig. 5 GLO (A) and phytol (B) dose-dependently inhibit the increase in sphinganine levels induced by 2 µM of FB1 after a 24-h coincubation of the test compounds. Sphinganine content (%) is expressed as shown in [Fig. 3]. The data are shown as the mean ± SD (n = 3). ** P < 0.01 and *** p < 0.001 compared to the FB1-treated group.

The addition of 50 µM each of GLO, phytol, and farnesol to the cultures for 24 h inhibited SPT activity, as expected from the ability of these compounds to inhibit sphinganine accumulation by FB1. In particular, inhibition by GLO of SPT activity was similar to that achieved with 1 µM of ISP-1 ([Fig. 6 A]). After a 2-h incubation of cell lysates with the isoprenoid test compounds, SPT activities were strongly inhibited not only by GLO but also by phytol and farnesol, indicating that these three terpene alcohols are direct SPT inhibitors ([Fig. 6 B]). Although the mechanisms of SPT inhibition and its beneficial outcome are still under investigation, several pathways appear to be involved in the therapeutic activity of these compounds. For example, inhibition of sphingolipid de novo synthesis by ISP-1 was shown to abolish the interaction of the ATP-binding cassette transporter A1 (ABCA1) with the SPTLC1 subunit, which forms a heterodimer with the SPTLC2 or SPTLC3 subunits of the enzyme [21]. The subsequent increase in the expression of ABCA1 at the plasma membrane facilitates cholesterol efflux, with the removal of accumulated cholesterol from macrophages in the subintima of the vascular vessel wall; a process mediated by ABCA1. Indeed, the inhibition of sphingolipid de novo synthesis increases the levels of anti-atherogenic lipoproteins and decreases atherosclerosis in hyperlipidemic ApoE-deficient mice [22].

Zoom Image

Fig. 6 SPT activities are significantly inhibited by GLO, phytol, and farnesol. A LLC-PK1 cells were treated with GLO, phytol, and farnesol for 24 h. B The cell lysates (300 µg protein) were incubated for 2 h with GLO (50 µM), phytol (50 µM), farnesol (50 µM), and ISP-1 (1 µM as a positive control). Error bars indicate the standard deviation, n = 4. The data are shown as the mean ± SD (n = 3). ** P < 0.01 and *** p < 0.001 compared to the control.

In conclusion, a simple chromatographic method for screening inhibitors of FB1 in cultured cells was established. The inhibitory potential of the tested compounds is expressed as a reduction (%) in sphinganine elevation by FB1. Using this method, three isoprenoids, GGO, phytol, and farnesol, were found to be SPT inhibitors.

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Acknowledgements

This work was supported by the NRF grant funded by the Korean government (MRC, 2010–0029483) and by a KBSI grant (D32120) to HS Kim.

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Conflict of Interest

None of the authors have any potential conflicts of interest to disclose.

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References

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Prof. Yong-Moon Lee

College of Pharmacy and MRC
Chungbuk National University

San 48, Kaesin-Dong, Hungduk-Ku

Chongju 361-763

Korea

Phone: +82 4 32 61 28 25

Fax: +82 4 32 68 27 32

Email: ymleefn@cbnu.ac.kr

#

References

  • 1 Lapczynski A, Bhatia S P, Letizia C S, Api A M. Fragrance material review on geranyllinalool.  Food Chem Toxicol. 2008;  46 S176-S178
    Search in Google Scholar
  • 2 Arnhold T, Elmazar M M A, Nau H. Prevention of vitamin A teratogenesis by phytol or phytanic acid results from reduced metabolism of retinol to the teratogenic metabolite, all-trans-retinoic acid.  Toxicol Sci. 2002;  66 274-282
    Search in Google Scholar
  • 3 Rheeder J P, Marasas W F O, Thiel P G, Sydenham E W, Shephard G S, van Schalkwyk D J. Fusarium moniliforme and fumonisins in corn in related to human esophageal cancer in Transkei.  Phytophathology. 1992;  82 353-357
    Search in Google Scholar
  • 4 Chu F S, Li G Y. Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collected from the People's Republic of China in regions with high incidences of esophageal cancer.  Appl Environ Microbiol. 1994;  60 845-852
    Search in Google Scholar
  • 5 Merrill Jr. A H, Wang E, Gilchrist D G, Riley R T. Fumonisins and other inhibitors of de novo sphingolipid biosynthesis.  Adv Lipid Res. 1993;  26 215-234
    Search in Google Scholar
  • 6 Smith E R, Merrill Jr. A H. Regulation of the “burst” of free sphingosine and sphinganine, and their 1-phosphate and N-acyl-derivatives, that occurs upon changing the medium of cells in culture: differential roles of de novo sphingolipid biosynthesis and turnover in J774A.1 macrophages.  J Biol Chem. 1995;  270 18749-18758
    Search in Google Scholar
  • 7 Yoo H S, Norred W P, Riley R T. A rapid method for quantitating free sphingoid bases and complex sphingolipids in microgram amounts of cells following exposure to fumonisin B1.  Toxicol In Vitro. 1996;  10 77-84
    Search in Google Scholar
  • 8 Miyake Y, Kozutumi Y, Nakamura S, Fujita T, Kawasaki T. Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin.  Biochem Biophys Res Commun. 1994;  211 396-403
    Search in Google Scholar
  • 9 Yu C H, Lee Y M, Yun Y P, Yoo H S. Differential effects of fumonisin B1 on cell death in cultured cells: the significance of the elevated sphinganine.  Arch Pharm Res. 2001;  24 136-143
    Search in Google Scholar
  • 10 Riley R T, Norred W P, Wang E, Merrill A H. Alteration in sphingolipid metabolism: bioassays for fumonisin- and ISP-1-like activity in tissues, cells and other matrices.  Nat Toxins. 1999;  7 407-414
    Search in Google Scholar
  • 11 Enongene E, Sharma R P, Bhandari N, Miller J D, Meredith F I, Voss K A, Riley R T. Persistence and reversibility of the elevation in free sphingoid bases induced by fumonisin inhibition of ceramide synthase.  Toxicol Sci. 2002;  67 173-181
    Search in Google Scholar
  • 12 Johnson V J, He Q, Kim S H, Kanti A, Sharma R P. Increased susceptibility of renal epithelial cells to TNFa-induced apoptosis following treatment with fumonisin B1.  Chem Biol Interact. 2003;  145 297-309
    Search in Google Scholar
  • 13 Yoon H T, Yoo H S, Shin B K, Lee W J, Kim H M, Hong S P, Moon D C, Lee Y M. Improved fluorescent determination method of cellular sphingoid bases in high-performance liquid chromatography.  Arch Pharm Res. 1999;  22 294-299
    Search in Google Scholar
  • 14 Rutti M F, Richard S, Penno A, von Eckardstein A, Hornemann T. An improved method to determine serine palmitoyltransferase activity.  J Lipid Res. 2009;  50 1237-1244
    Search in Google Scholar
  • 15 Wang E, Norred W P, Bacon C W, Riley R T, Merrill Jr. A H. Inhibition of sphingolipid biosynthesis by fumonisins: implications for diseases associated with Fusarium moniliforme.  J Biol Chem. 1991;  266 14486-14490
    Search in Google Scholar
  • 16 Rother J, van Echten G, Schwarzmann G, Sandhoff K. Biosynthesis of sphingolipids : dihydroceramide and not sphinganine is desaturated by cultured cells.  Biochem Biophys Res Commun. 1992;  189 14-20
    Search in Google Scholar
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Prof. Yong-Moon Lee

College of Pharmacy and MRC
Chungbuk National University

San 48, Kaesin-Dong, Hungduk-Ku

Chongju 361-763

Korea

Phone: +82 4 32 61 28 25

Fax: +82 4 32 68 27 32

Email: ymleefn@cbnu.ac.kr

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Fig. 1 FB1 causes the accumulation of sphinganine by inhibiting sphinganine N-acyltransferase in the sphingolipid de novo biosynthetic pathway. It also inhibits sphingosine N-acyltransferase, which results in an increase in sphingoid bases and the depletion of complex sphingolipids, including ceramides. Other inhibitors of the sphingolipid metabolic pathway are also shown.

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Fig. 2 Chemical structures of the test compounds used in the inhibitory assay of sphingolipid biosynthesis. A fumonisin B1, B geranyllinalool, C phytol, D farnesol, E FTY720, F bufalin, G L-cycloserine, and H ISP-1.

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Fig. 3 FB1 inhibits sphinganine N-acyltransferase activity and causes the intracellular accumulation of free sphinganine in a time-dependent manner. A LLC-PK1 cells treated with (a) or without (b) 2 µM FB1 were harvested, and the sphinganine level was measured after OPA fluorogenic derivatization in a HPLC system. The inhibitory effects were estimated by the increased sphinganine levels. B L-cycloserine dose-dependently inhibits sphinganine levels elevated in response to 2 µM FB1 following 24 h of coincubation of the cells with these two compounds. The results are expressed as the sphinganine content after 24 h as a percentage of the positive control ([1]: +FB1, 2 µM). The sphinganine content was normalized in pmol/5 × 105 cells (62 µg protein) using a Bradford protein assay. The data are shown as a mean ± SD (n = 3). ** P < 0.01 and *** p < 0.001 compared to FB1-treated group.

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Fig. 4 GLO, phytol, and farnesol strongly inhibit the increase in sphinganine levels induced by 2 µM of FB1 after a 24-h coincubation of the test compounds (L-cycloserine, 100 µM; FTY720, 20 µM; GLO, 50 µM; phytol, 50 µM; farnesol, 50 µM; dolichol, 50 µM; dolichol phosphate, 50 µM). Sphinganine content (%) is expressed as shown in [Fig. 3]. Each column and bar represents the mean ± SD (n = 3). ** P < 0.01 and *** p < 0.001 compared to the FB1-treated group.

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Fig. 5 GLO (A) and phytol (B) dose-dependently inhibit the increase in sphinganine levels induced by 2 µM of FB1 after a 24-h coincubation of the test compounds. Sphinganine content (%) is expressed as shown in [Fig. 3]. The data are shown as the mean ± SD (n = 3). ** P < 0.01 and *** p < 0.001 compared to the FB1-treated group.

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Fig. 6 SPT activities are significantly inhibited by GLO, phytol, and farnesol. A LLC-PK1 cells were treated with GLO, phytol, and farnesol for 24 h. B The cell lysates (300 µg protein) were incubated for 2 h with GLO (50 µM), phytol (50 µM), farnesol (50 µM), and ISP-1 (1 µM as a positive control). Error bars indicate the standard deviation, n = 4. The data are shown as the mean ± SD (n = 3). ** P < 0.01 and *** p < 0.001 compared to the control.