Borrelidin Isolated from Streptomyces sp. Inhibited Adipocyte Differentiation in 3T3-L1 Cells via Several Factors Including GATA-Binding Protein 3

Abstract

An inhibitor of 3T3-L1 adipocyte differentiation was isolated from Streptomyces sp. TK08330 and identified by spectroscopy as the 18-membered macrolide borrelidin. Treatment with 1.0 μM borrelidin suppressed intracellular lipid accumulation by 80% and inhibited the expression of adipocyte-specific genes. Borrelidin suppressed the mRNA expression of two master regulators of adipocyte differentiation, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein (C/EBPα). Studies on well-known upstream regulators of PPARγ revealed that borrelidin down-regulated C/EBPδ mRNA expression but did not affect expression of C/EBPβ. Borrelidin increased mRNA expression of negative regulators of differentiation such as GATA-binding protein (GATA) 3, Krüppel-like factor (KLF) 3 and KLF7, as well as positive regulators, KLF4, KLF6 and KLF15, at early stages of differentiation. To elucidate a primary mediator of borrelidin differentiation inhibitory activity, small interfering RNA (siRNA) transfection experiments were performed. The mRNA expression of PPARγ, which was down-regulated by borrelidin, was not changed by KLF3 and KLF7 siRNA treatment. In contrast, expression of PPARγ in GATA-3 siRNA-treated cells was not significantly different from that of control siRNA-treated cells. Borrelidin significantly inhibited lipid accumulation in control siRNA-treated cells, and treatment with GATA-3 siRNA slightly reduced the inhibitory effect of borrelidin. These results indicate that borrelidin inhibited adipocyte differentiation partially via GATA-3.

Adipocytes accumulate fat and play important roles in energy storage.¹⁾ However, excessive accumulation of fat is a risk factor for various diseases such as osteoarthritis, diabetes mellitus type 2 and cardiovascular diseases.²⁾ New effective methods for controlling obesity are necessary and the inhibition of adipocyte differentiation is a potential target for therapeutic development. Pre-adipocyte 3T3-L1 cells are often used for adipocyte differentiation studies since they are easily differentiated to adipocytes using a standard differentiation cocktail containing dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX) and insulin.³⁾

Previous studies have identified several regulators of adipocyte differentiation (cf. Fig. 8 in Results and Discussion). In the 3T3-L1 differentiation system, CCAAT/enhancer binding protein beta (C/EBPβ) and C/EBPδ are primarily expressed and these factors regulate peroxisome proliferator-activated receptor gamma (PPARγ) and C/EBPα, which are master regulators of adipocyte differentiation.^4,5) PPARγ and C/EBPα are expressed 2 d after differentiation induction, reaching maximum expression after 4 d of induction.⁶⁾ PPARγ is necessary for adipocyte differentiation from pre-adipocytes,^7,8) although the mechanisms regulating PPARγ expression remain to be defined.

Recently, members of the Krüppel-like factor (KLF) and GATA-binding protein (GATA) families were identified as regulators of the early stages of 3T3-L1 adipocyte differentiation. The KLF family has a characteristic zinc finger at the C terminus with a reported role in cell proliferation, growth, and differentiation in mammals.^9,10) Among the family members, KLF2, 3, and 7, negatively regulate differentiation and are highly expressed in pre-adipocytes with reduced expression levels after induction.^11–13) In contrast, expression of the positive regulators KLF4, 5, 6, and 15 are increased after induction.^14–17) GATA family members share highly conserved zinc-finger DNA-binding domains and bind specifically to a consensus sequence (A/T)GATA(A/G).¹⁸⁾ Six GATA family members are found in eukaryotes; GATA-2 and GATA-3 are predominantly expressed in mammalian white adipose tissue. GATA-2 and GATA-3 proteins are usually detected in cultured 3T3-L1 cells; their levels decrease after induction and become undetectable after 2 d. These proteins suppress adipogenesis by directly binding to the GATA-binding site on the PPARγ-promoter.^19,20) Thus, forced expression of GATA-2 and GATA-3 inhibit adipocyte differentiation. GATA-2 and GATA-3 appear to act as gatekeepers to block expression of PPARγ and the role of GATA-3 was clarified using GATA-3 knockout ES cells,¹⁹⁾ which support studying GATA-2 and GATA-3 as suitable targets to inhibit adipocyte differentiation. Recently, berberine and salvianolic acid B were reported to up-regulate GATA-2 and GATA-3 expression during inhibition of 3T3-L1 adipocyte differentiation,^21,22) although the role of GATA-2 or GATA-3 was not defined in these reports.

In the course of screening for inhibitors of lipid accumulation from microorganisms, we identified the 18-membraned macrolide borrelidin as an active compound. Studies of the action mechanism indicated that borrelidin affected some negative and positive regulators of differentiation. Small interfering RNA (siRNA) transfection experiments demonstrated that borrelidin inhibited early stages of 3T3-L1 differentiation partially via GATA-3. Here, we describe the isolation and the identification of borrelidin and its mechanism of action as an inhibitor of adipocyte differentiation.

MATERIALS AND METHODS

General Experimental Procedures

NMR spectra were measured using a JMN-ECA-600 NMR spectrometer (Jeol, Tokyo, Japan), using tetramethylsilane as an internal standard in chloroform-d (CDCl₃). HPLC chromatograms and UV-Vis spectra were recorded using a GL-7410 HPLC pump and a GL-7452 PDA detector (GL Science, Tokyo, Japan). A Cosmosil 5C18-AR-II (Nacalai Tesque, Kyoto, Japan) column with an acetonitrile (MeCN)–H₂O solvent system was used for chromatography. LC-Electrospray ionization (ESI)-MS data were measured using a 1200 series HPLC system and an API-3200 triple quadrupole mass spectrometer (Applied Biosystems, Tokyo, Japan). For HPLC fractionation, a L-6200 HPLC pump and a L-4200 UV detector (Hitachi, Ibaraki, Japan) were used. Optical density (OD) was measured using the SH-1000 Lab microplate reader (Corona, Ibaraki, Japan). Quantitative real-time polymerase chain reaction (qPCR) was performed using Chromo4 (Bio-Rad Laboratories Inc., Tokyo, Japan). All other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Culture and Differentiation of 3T3-L1

3T3-L1 cells (Human Science, Tokyo, Japan) were cultured at 37°C in a humidified atmosphere of 95% air–5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 mg/mL streptomycin until confluent. Differentiation was stimulated 2 d after reaching confluence (day 0) with induction medium containing 0.5 mM IBMX, 0.25 µM DEX and 1.7 µM insulin. After 3 d of differentiation (day 3), induction medium was changed to maintenance medium containing 1.7 µM insulin and incubated for 2 additional days (day 5), followed by 2 d of incubation in fresh maintenance medium (day 7). The test samples were dissolved in dimethyl sulfoxide (DMSO) and 0.3% DMSO used as a control, which did not affect cell viability or differentiation. The cultures were treated with test samples for the entire culture period (days 0–7).

Oil Red O Staining

At the end of the incubation (day 7), intracellular lipid droplets were stained with Oil Red O (Sigma-Aldrich, Tokyo, Japan), as mentioned below. After removal of the culture medium, the cells were washed with phosphate-buffered saline (PBS) three times and fixed with 10% formalin at room temperature for 15 min. The cells were washed again with PBS and stained with freshly diluted Oil Red O solution (3 parts of 0.3% Oil Red O in isopropyl alcohol and 2 parts of water) for 20 min. After removing the staining solution, the cells were washed with PBS three times. To quantitate the amount of intracellular lipids, stained Oil Red O was eluted with 4% NP-40 in isopropyl alcohol and absorbance at 520 nm was measured using a microplate reader. The amount of intracellular lipids was calculated as the percentage ratio of the OD absorbance of the tested cells to the control cells treated with DMSO alone.

Borrelidin

We selected the active strain TK08330 for screening of inhibitors of lipid accumulation during 3T3-L1 differentiation. Phylogenetic analysis of the strain was performed using the nearly complete 16S ribosomal DNA (rDNA) gene (1490 bp: AB973399) sequence and compared with other sequences in the GenBank database by BLAST searching. More than one hundred sequences exhibited over 99% similarity from various Streptomyces species, thus the strain TK08330 was classified as a Streptomyces sp. Cultivation was carried out in starch-casein medium (1.0% starch, 0.03% casein, 0.2% sodium chloride (NaCl), 0.2% potassium hydrogen phosphate (K₂HPO₄), 0.005% magnesium sulfate (MgSO₄), 0.002% calcium carbonate (CaCO₃), 0.001% iron(II) sulfate heptahydrate (FeSO₄), pH 7.2, 10 L) for 7 d at 30°C with aeration and agitation. The cultured broth was filtered and the filtrate was extracted with ethyl acetate (EtOAc). The EtOAc extract (66.6 mg) was purified by reversed-phase HPLC (Cosmosil 5C18-AR II; 10×250 mm) using a 60% MeCN isocratic solvent system to obtain an active compound (6.5 mg).

The physicochemical properties of the active compound were as follows; white amorphous, ¹H-NMR (600 MHz, CDCl₃) δ: 0.73 (1H, m), 0.80 (3H, d, J=6.3 Hz), 0.84 (3H, d, J=6.9 Hz), 0.85 (3H, d, J=6.2 Hz), 1.05 (3H, d, J=6.9 Hz), 0.89–1.44 (6H, m), 1.51–1.70 (3H, m), 1.75–2.09 (6H, m), 2.32 (1H, m), 2.44 (1H, dd, J=17.2, 10.3 Hz), 2.48–2.75 (4H, m), 3.87 (1H, br d, J=10.3 Hz), 4.11 (1H, d, J=9.7 Hz), 4.97 (1H, dt, J=11.0, 3.4 Hz), 6.21 (1H, m), 6.39 (1H, dd, J=14.4, 11.7 Hz), 6.83 (1H, d, J=11.7 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.9, 17.0, 18.2, 20.1, 25.2, 26.2, 27.1, 29.6, 31.2, 35.2, 35.6, 35.9, 37.4, 39.3, 43.0, 45.7, 47.8, 48.4, 69.8, 73.1, 76.5, 115.9, 118.3, 126.9, 138.6, 144.0, 172.2, 179.4. UV λ_max: 257 nm. ESI-MS m/z: 488 [M−H]⁻. [α]_D²⁶ −50 (c=0.11, ethanol). The structure was identified as borrelidin by two dimensional (2D)-NMR analysis, the spectroscopic data noted above and comparative literature values.^23–25) The purity of the compound was 95% by HPLC analysis coupled with evaporative light scattering detection.

Cytotoxicity of Borrelidin on 3T3-L1 Cells

Inhibition of lipid accumulation was evaluated as described above. Borrelidin was added at days 0, 3 and 5 to final concentrations of 0.063, 0.13, 0.25, 0.5 and 1.0 µM and intracellular lipids were measured using Oil Red O staining at day 7. The cytotoxicity of borrelidin was measured using an XTT assay (Sigma-Aldrich, Tokyo, Japan), according to the manufacturer’s instructions at day 7. The results were quantified by measuring absorbance at 450 nm and cell viability calculated as a percentage relative to the DMSO control.

Effects of Borrelidin on Gene Expression

Total cellular RNA was isolated from 3T3-L1 cells treated with 1.0 µM borrelidin using RiboZol (ARMESCO, OH, U.S.A.) at days 1, 3 and day 5 or 0.5, 6.0 and 12.0 h from induction. cDNA was synthesized using the ReverTraAce kit (Toyobo, Osaka, Japan). The qKOD real-time PCR kit (Toyobo) was used for qPCR using 1.0 µg aliquots of cDNA to analyze mRNA expression of β-actin, fatty acid binding protein 4 (FABP4), lipoprotein lipase (LPL), 11β-hydroxysteroid dehydrogenase type 1 (HSD11β1), glucose transporter 4 (GLUT4), PPARγ, C/EBPα, C/EBPβ, C/EBPδ, GATA-2, GATA-3, KLF2, KLF3, KLF4, KLF5, KLF6, KLF7 and KLF15. The specific primers used for qPCR analyses are listed in the supplemental data (Supplementary 1). The mRNA levels of each gene were normalized to β-actin in each sample, and expressed as the ratio to non-induced cells (pre-adipocytes, day 0) calculated by the ΔΔCt method.

Effects of Borrelidin on PPARγ Expression

We prepared total cellular protein from 3T3-L1 cells at days 0 and 7 after induction with and without borrelidin treatment (1.0 µM). Total protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (ThermoScientific, Rockford, IL, U.S.A.). Electrophoresis was performed using e-PAGEL (ATTO CORPORATION, Osaka, Japan) loaded with the same amount of total protein samples. Proteins were transferred to a polyvinylidene difluoride membrane (Amersham Hybond-P; GE Healthcare, Buckinghamshire, U.K.). The membrane was incubated with dilute solutions of primary antibodies against β-actin and PPARγ (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) with BlockAce (DS Pharmabiomedical, Osaka, Japan). Anti-mouse IgG (H+L)-HRP (MEDICAL & BIOLOGICAL LABORATORIES Co., Ltd., Osaka, Japan) was used as a secondary antibody. The Amersham ECL Prime Western blotting Detection Reagent (GE Healthcare) was used for chemiluminescence and the image recorded with ImageQuant LAS 4000 mini (GE Healthcare Japan, Tokyo, Japan).

siRNA Transfection Experiment

3T3-L1 cells were cultured in penicillin- and streptomycin-free DMEM supplemented with 10% FBS for one day before addition of siRNA. A solution of siRNA [control siRNA or a mixture of KLF3 and KLF7 siRNAs or GATA-3 siRNA] (TaKaRa Bio Inc., Shiga, Japan) and Lipofectamine 2000 (Life Technologies) was diluted in Opti-MEM (Life Technologies, Tokyo, Japan) and was added to the cells based on the Lipofectamine 2000 protocol provided by the manufacturer. After 3 d, differentiation was induced in medium containing IBMX, DEX and insulin with and without 1.0 µM borrelidin. After incubation for a further 3 d, total RNA samples were prepared for qPCR to assess PPARγ expression. The lipid accumulation in the cells was also measured at day 7.

Statistical Analysis

Statistical analyses were performed using SPSS 12.0 J for Windows (SPSS Inc., Chicago, IL, U.S.A.). Values are expressed as mean±standard deviation (S.D.). In all analyses, p<0.05 indicates statistical significance.

RESULTS AND DISCUSSION

Screening assays for inhibitors of lipid accumulation in 3T3-L1 cells identified borrelidin from Streptomyces sp. TK08330 (Fig. 1A). When 3T3-L1 cells were treated with several concentrations of borrelidin for 7 d, lipid accumulation was inhibited dose-dependently and 80% inhibition was observed at 1.0 µM (Fig. 1B, bar). Borrelidin had little effect on cytotoxicity, with >90% cell viability at the highest concentration of borrelidin tested (Fig. 1B, line). Thus, the inhibitory activity of borrelidin on lipid accumulation was not due to cytotoxic effects. The IC₅₀ value of borreldin (0.25 µM) was lower than those of known lipid accumulation inhibitors, berberine (approx. 2.0 µM) and salvianolic acid B (over 100 µM),^21,22) and borrelidin is probably more potent than these inhibitors. Berberine and salvianolic acid B exhibited anti-obesity effect in vivo using KK-Ay mice and high-fat diet-induced obesity mice, respectively.^26,22)

Fig. 1. Structure and Effects of Borrelidin on Lipid Accumulation and Cell Viability

(A) Structure of borrelidin. (B) Lipid accumulation and viability of borrelidin-treated 3T3-L1 cells compared to control cells and non-induced cells (ni). Oil Red O staining and an XTT assay were used to measure intracellular lipid accumulation and cell viability at day 7, respectively, n=3±S.D.; * p<0.05, ** p<0.01 and *** p<0.001 from control.

The accumulation of intracellular lipids has been used as a marker of adipocyte differentiation.²⁷⁾ Effects of borrelidin on differentiation were evaluated by measuring the mRNA expression of adipocyte-specific genes, FABP4, LPL, HSD11β1 and GLUT4 (Fig. 2 white bars). The levels of these four genes in control cells increased time-dependently for 1–5 d after induction, but were suppressed completely in the borrelidin-treated cells (Fig. 2 black bars). In addition, these adipocyte specific genes are highly expressed in mature adipocytes^28–31) and reduced expression indicated that borrelidin interrupted differentiation.

Fig. 2. Effects of Borrelidin on the Expression of Adipocyte-Specific Genes

(A) FABP4, (B) LPL, (C) HSD11β1 and (D) GLUT4. Cells were treated with 1.0 µM borrelidin at 1, 3, and 5 d after induction, and gene expression was evaluated by qPCR and normalized to β-actin. The results are expressed as ratios to pre-adipocytes (day 0 control), n=3±S.D.; * p<0.05 and ** p<0.01 from control.

The mechanism of borrelidin action was examined by assessing the expression of several regulators that contribute to the differentiation of 3T3-L1 cells. We initially measured the expression of the master regulators of differentiation, PPARγ and C/EBPα (Fig. 3). High levels of PPARγ and C/EBPα expression in the control cells were observed at 3 and 5 d after induction, and subsequently significantly suppressed by borrelidin (Figs. 3A, B), although these expressions at day 1 were significantly up-regulated by borrelidin. This will be discussed below. Furthermore, PPARγ protein levels in borrelidin-treated cells were decreased at 7 d after induction as compared with control cells (Fig. 3C, lane 3). These results confirm that borrelidin inhibited adipocyte differentiation.

Fig. 3. Effects of Borrelidin on the Expression of Master Regulators

Cells were treated with 1.0 µM borrelidin at 1, 3 and 5 d after induction, and expression levels of (A) PPARγ and (B) C/EBPα were determined by qPCR and normalized to β-actin. The results are expressed as ratios to pre-adipocytes (day 0 control), n=3±S.D.; ** p<0.01 from control. (C) Western blot analysis for PPARγ from total protein isolated from pre-adipocytes and cells after 7 d of induction.

The upstream regulators, C/EBPβ and C/EBPδ, control expression of these master regulators in the early stages of adipocyte differentiation.⁵⁾ Thus, we examined the effect of borrelidin on expression of C/EBPβ and C/EBPδ at 0.5, 6.0 and 12.0 h after induction (Fig. 4). Expression of C/EBPβ and C/EBPδ increased gradually after induction in control cells. Although borrelidin did not affect expression of C/EBPβ (Fig. 4A), it decreased expression of C/EBPδ by ca. 50% of control cells at 12.0 h (Fig. 4B). However, even when expression of C/EBPβ or C/EBPδ was completely suppressed, adipocyte differentiation was only slightly inhibited.⁵⁾ These results suggest that the effects of borrelidin involve other regulators.

Fig. 4. Effects of Borrelidin on the Expression of C/EBP Genes

Cells were treated with borrelidin (1.0 µM) for 0.5, 6.0 and 12.0 h after induction. The mRNA expression of (A) C/EBPβ and (B) C/EBPδ was determined by qPCR and normalized to β-actin. The results are expressed as ratios to pre-adipocytes (0 h control). n=3±S.D.; * p<0.05 from control.

Preliminary experiments examined several regulators, such as Tob2, GATA family, and KLF family (data not shown) and we found that mRNA expression of GATA and KLF families were affected by borrelidin. Negative regulators, including GATA-2, GATA-3, KLF2, KLF3 and KLF7, tended to decrease during differentiation after induction in control cells. The effects of borrelidin on mRNA levels of GATA-2 and GATA-3 are shown in Fig. 5. Expression of GATA-2 was not changed by borrelidin (Fig. 5A) but that of the GATA-3 was significantly increased at 6.0 and 12.0 h (Fig. 5B). The results for the KLF family members are displayed in Fig. 6, whereby the mRNA expression of the negative regulators, KLF2, KLF3, and KLF7, are shown in Figs. 6A, B and C, respectively. The KLF2 mRNA level was not changed by borrelidin. The mRNA levels of KLF3 and KLF7 in the control cells were decreased after induction; however, they were increased significantly by borrelidin at 6.0  and 12.0 h. The remaining KLF family members in Fig. 6 are positive regulators. The mRNA levels of KLF4 and KLF15 were increased significantly by borrelidin at 6.0 and 12.0 h (Figs. 6D, G). That of KLF6 was also increased at 12.0 h (Fig. 6F) but that of KLF5 was not affected by borrelidin (Fig. 6E). As mentioned previously, the up-regulation of PPARγ and C/EBPα expressions at day 1 by borrelidin were observed, and this might be caused by up-regulation of these positive regulators at quite early stage. Since the up-regulation of negative (GATA-3, KLF3, KLF7) and positive (KLF4, KLF6 and KLF15) regulators by borrelidin occurred simultaneously at 6.0 and 12.0 h, the positive regulators seemed to affect more immediately than the negative regulators. After all, because the differentiation was eventually inhibited, the negative regulators prevailed against the positive ones in borrelidin-treated cells.

Fig. 5. Effects of Borrelidin on the Expression of GATA Genes

Cells were treated with 1.0 µM borrelidin for 0.5, 6.0 and 12.0 h after induction. The mRNA expression of (A) GATA-2 and (B) GATA-3 was determined by qPCR and normalized to β-actin. The results are expressed as ratios to pre-adipocytes (0 h control). n=3±S.D.; * p<0.05 from control and ** p<0.01 from control.

Fig. 6. Effects of Borrelidin on the Expression of KLF Family Genes

Cells were treated with 1.0 µM borrelidin for 0.5, 6.0 and 12.0 h after induction. The mRNA expression of negative regulators, (A) KLF2, (B) KLF3 and (C) KLF7, and positive regulators, (D) KLF4, (E) KLF5, (F) KLF6 and (G) KLF15, was determined by qPCR and normalized to β-actin. The results are expressed as ratios to preadipocytes (0 h control). n=3±S.D.; * p<0.05 and ** p<0.01 from control.

To identify the primary negative regulator for the inhibitory activity of borrelidin, siRNA transfection experiments were performed using GATA-3, KLF3 and KLF7 siRNAs. The mRNA levels of KLF3 and KLF7 were almost suppressed by the mixture of KLF3 and KLF7 siRNAs compared with control siRNA-treated cells, whereas that of GATA-3 was suppressed about 60% by GATA-3 siRNA (Supplementary 2). The mRNA level of a master regulator PPARγ was used as a marker of the differentiation. Control siRNA had no effect on PPARγ expression and were classified into group b, as shown in Fig. 7A. Since GATA-3 is a negative regulator, the expression of PPARγ in GATA-3 siRNA-treated cells (Fig. 7A, GATA-3 siRNA +, Borrelidin −) was increased significantly (group c). KLF3 and KLF7 were also reported to be negative regulators; however, the expression of PPARγ in KLF3 and KLF7 siRNA-treated cells (Fig. 7A, KLF3, 7 siRNA +, Borrelidin −) was not increased (group b). Nevertheless, KLF3 and KLF7 mRNA levels were almost suppressed by siRNA. Therefore, GATA-3 seemed more potent than KLF3 and KLF7 as a gatekeeper of adipocyte differentiation in 3T3-L1 cells. The effects of siRNAs on the expression of PPARγ in borrelidin-treated cells were also examined. Borrelidin almost completely suppressed PPARγ expression (group a) in control siRNA treated cells (Fig. 7A, Control siRNA +, Borrelidin +), and KLF3 and KLF7 siRNAs could not change the situation (Fig. 7A, KLF3, 7 siRNA +, Borrelidin +, group a). In contrast, the effect of borreridin on the expression of PPARγ was canceled (group b) by GATA-3 siRNA treatment (Fig. 7A, GATA-3 siRNA +, Borrelidin +). Additionally, the lipid accumulation was also measured at day 7. Borrelidin significantly inhibited lipid accumulation (group a) in control siRNA-treated cells (Fig. 7B, Control siRNA +, Borrelidin +). Treatment of GATA-3 siRNA slightly but certainly reduced (group b) the inhibitory effect of borrelidin (Fig. 7B, GATA-3 siRNA +, Borrelidin +). These results indicated that borrelidin inhibited adipocyte differentiation partially via GATA-3.

Fig. 7. Effects of KLFs and GATA-3 siRNA Transfection on PPARγ Expression and Lipid Accumulation in Borrelidin-Treated Cells

(A) Effects of siRNAs transfection on PPARγ expression. After transfection with siRNAs (control siRNA, GATA-3, KLF3 and KLF7 siRNA), the cells were differentiated according standard methods with and without borrelidin. After incubation for 3 d, the mRNA levels of PPARγ were determined by quantitative real-time PCR and normalized to that of β-actin. (B) Effects of GATA-3 siRNA transfection on lipid accumulation. After transfection of the siRNA and differentiation of the cells, intracellular lipids were measured by Oli red O staining at day 7. The results are expressed as ratios to cells treated with control siRNA. n=3±S.D.. The data were subjected to one-way ANOVA followed by Tukey’s test (p<0.05), resulting in three significantly different groups designated a, b and c.

Borrelidin is first isolated from Streptomyces rochei in 1949 by Berger et al. as an antibiotic possessing anti-borrelia activity,³²⁾ which is a unique 18-membered macrolide with a cyano substituent group. Pharmacological studies have revealed that borrelidin exhibits anti-malarial, anti-angiogenesis and anti-fungal activities,^33–35) and borrelidin is also known as a strong inhibitor of threonyl-tRNA synthetase.^36,37) Although a variety of biological activity has been reported, there are no reports related to anti-obesity effect of borrelidin.

In this study, we found for the first time that borrelidin inhibits adipocyte differentiation. We summarized in Fig. 8 the effects of borrelidin on modulation of regulators in 3T3-L1 cells. Borrelidin affected expressions of diverse important regulators, such as members of GATA and KLF families, at the early stages of adipocyte differentiation. To reveal the action mechanism of borrelidin, we investigated the expressions of PPARγ and its negative regulators KLF3, KLF7 and GATA-3 in the borrelidin-treated cells. GATA-3 siRNA transfection experiments clearly demonstrated that PPARγ expression was down-regulated by borrelidin mainly via GATA-3. GATA-3 is the most influential of the positive (KLF4, KLF6, KLF15) and negative (KLF3, KLF7) regulators up-regulated by borrelidin. This would be useful in determining the relationship between these regulators. However, lipid accumulation inhibitory activity of borrelidin was only slightly reduced by GATA-3 siRNA, therefore, the other regulator except PPARγ may play an important role in the action mechanism of borrelidin. Further work is required to elucidate the details of the action mechanism. The results of this study suggest that borrelidin would be a useful seed compound for anti-obesity drug development.

Fig. 8. A Schematic Illustration of Adipocyte Differentiation and Effects of Borrelidin for Early Regulators in 3T3-L1 Cells

Acknowledgments

We thank Dr. Ryo Hatano (Department of Pharmacy, Ritsumeikan University), Dr. Emi Yoshigai (Department of Biomedical Sciences, Ritsumeikan University) and Prof. Kenji Suzuki (Department of Pharmacy, Ritsumeikan University) for advice regarding the Western blotting, quantitative real-time PCR and siRNA transfection experiments, respectively.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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