Results

15d-PGJ₂ stimulates the differentiation of sebocytes with increasing intracellular levels of TG

We first examined the relationship between PG levels and lipogenesis during the differentiation of hamster sebocytes. As intracellular lipid droplets accumulate in sebocytes mostly consist of TG (^{Sato et al, 2001a}), we evaluated sebocytic differentiation by measuring intracellular levels of TG. As shown in Figure 1a, levels of TG (closed squares) were found to increase in cell culture time dependently. The level of PGF₂ (open squares) was somewhat higher than that of PGE₂ (open triangles) and 15d-PGJ₂ (open circles) on day 3 in culture, but was found to decrease time dependently for up to 12 d (to <10 pg per g DNA). The level of PGE₂ was considerably lower (<10 pg per g DNA). 15d-PGJ₂ levels were augmented for 6 d and this augmented level (20–30 pg per g DNA) was sustained for up to 12 d, indicating that 15d-PGJ₂ is predominant in differentiated hamster sebocytes. It was found that hamster sebocytes constitutively produce COX-2 with the same molecular weight as the human COX-2 protein and its production slightly decreased for up to 12 d (Figure 1b, lower panel). Furthermore, COX-2 was found to be expressed in sebaceous glands (see SG, Figure 1b, lower panel). There was no detectable COX-1 in either sebaceous glands or cultured sebocytes (see Figure 1b, upper panel).

Figure 1.

Levels of prostaglandins (PG) during sebocyte differentiation in vitro. Hamster sebocytes at the 2nd passage were cultured for 12 d, with replacement of fresh culture medium every 3 d. (A) The harvested culture medium at each indicated period was subjected to measurement of PGE₂ (open triangles), PGF₂ (open squares), and 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂) (open circles). Levels of intracellular triacylglycerol (TG) (closed squares) were time dependently augmented during cell culture. Data are shown as meanstandard deviation of triplicate dishes. (B) The harvested cells at each indicated period and sebaceous glands (SG) were subjected to western blot analysis for cyclooxygenase (COX)-1 and COX-2. St indicates human COX-1 (upper panel) and COX-2 (lower panel). Three independent experiments were reproducible, and typical data are shown.

Full figure and legend (44K)

To clarify whether 15d-PGJ₂ might induce the differentiation of sebocytes, we investigated the effect of exogenous 15d-PGJ₂ on the formation of lipid droplets in hamster sebocytes. When 15d-PGJ₂ (1–10 M) was added in the sebocyte culture, the formation of intracellular lipid droplets (Figure 2b vs a) and TG levels (Figure 2c) increased in a concentration-dependent manner. As shown in Table I, 15d-PGJ₂-induced augmentation of TG production was found to be associated with an increase in the activity of diacylglycerol acyltransferase (DGAT), a rate-limiting enzyme of TG synthesis (p<0.001).

Figure 2.

Exogenous 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂) augments the formation of lipid droplets by increasing intracellular levels of triacylglycerol (TG) in hamster sebocytes. Oil-red-O staining of cultured hamster sebocytes at the 2nd passage shows that the accumulation of lipid droplets is spontaneously observed in untreated cells (A) and augmented by addition of 15d-PGJ₂ (10 M) for 12 d (B). Intracellular levels of TG (C) also increase in a concentration-dependent manner using 15d-PGJ₂ (1–10 M). Four independent experiments were reproducible, and typical data are shown. Data are shown as meanstandard deviation of triplicate dishes. ^*, ^**, and ^*** indicate results that were significantly different from untreated control cells (Cont) at p levels of <0.05, <0.01, and <0.001, respectively. Magnifications in panels A and B: 200.

Full figure and legend (156K)

Table I - Increase of DGAT activity by 15d-PGJ2 and COX inhibitors in hamster sebocytes.

Full table

Effects of COX inhibitors on the production of PGs in hamster sebocytes

To clarify the possible involvement of 15d-PGJ₂ in increasing sebocyte lipogenesis, we investigated the effects of COX inhibitors on the production of TG and the formation of intracellular lipid droplets in cultured hamster sebocytes. As shown in Figure 3, levels of PGE₂ (panel A) and PGF₂ (panel B) were significantly diminished (p<0.001) by non-selective COX inhibitors, indomethacin (10 M) and diclofenac (10 M), and by a COX-2-specific inhibitor, NS-398 (20 M). The level of 15d-PGJ₂ in the medium was found to increase in sebocytes treated with indomethacin (p<0.01), diclofenac (p<0.01), and NS-398 (p<0.001) (Figure 3c). In contrast, there was no change in the level of PGD₂ between untreated and indomethacin (10 M)-treated sebocytes (0.490.06 vs 0.540.09 pg per g DNA, respectively). Neither COX-1 nor COX-2 production was altered in the COX inhibitor-treated cells (see Fig S1). Moreover, in primarily cultured hamster skin fibroblasts, these three inhibitors were found to inhibit the production of PGE₂, PGF₂, and 15d-PGJ₂ (data not shown).

Figure 3.

Regulation of prostaglandin (PG) production by cyclooxygenase (COX) inhibitors in hamster sebocytes. Hamster sebocytes at the 2nd passage were treated every 3 d with indomethacin (Ind) (10 M), diclofenac (Dic) (10 M), and NS-398 (NS) (20 M) for 12 d. Culture media were harvested at 12 d and subjected to measurement of PGE₂ (A), PGF₂ (B), and 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂) (C). Four independent experiments were reproducible, and typical data are shown. Data are shown as meanstandard deviation of triplicate dishes. ^** and ^*** indicate significant differences from untreated control cells (Cont) with p values of <0.01 and <0.001, respectively.

Full figure and legend (28K)

Augmentation of lipogenesis and development of sebaceous glands by COX inhibitors

Because of our finding that 15d-PGJ₂ production was increased in sebocytes even when COX inhibitors were present, we examined whether the inhibitors could augment lipogenesis in sebaceous glands. As shown in Figure 4, the production of TG was found to increase concentration dependently in the presence of indomethacin (1–10 M), diclofenac (0.1–10 M), and NS-398 (5–20 M). The formation of intracellular lipid droplets also increased (data not shown). There were no significant changes in the levels of free fatty acids and cholesterol compared with those of TG in the lipids (data not shown). The selective augmentation of TG production by indomethacin, diclofenac, and NS-398 was found to be associated with an increase in DGAT activity in hamster sebocytes (Table I). When auricles from 5-wk-old male golden hamsters were topically treated with 2% (wt/vol) indomethacin for 14 d, the development of sebaceous glands (arrow heads) with the deposition of sebum (asterisks) was observed (Figure 5b vs a).

Figure 4.

Effects of cyclooxygenase (COX) inhibitors on levels of triacylglycerol (TG) in hamster sebocytes. Intracellular levels of TG were concentration-dependently augmented in indomethacin (1–10 M)-, diclofenac (0.1–10 M)-, and NS-398 (5–20 M)-treated hamster sebocytes at the 2nd passage. Four independent experiments were reproducible and typical data are shown. Data are shown as meanstandard deviation of triplicate dishes. ^*, ^**, and ^*** indicate significant differences from untreated control cells (Cont) with p values of <0.05, <0.01, and <0.001, respectively.

Full figure and legend (20K)

Figure 5.

Development of sebaceous glands by topically administering indomethacin in vivo. Histochemical staining of hamster auricles shows that topical treatment of indomethacin for 2 wk (B) leads to the development of sebaceous glands (arrow heads), and augments the deposition of sebum (asterisks) compared with vehicle-treated auricles (A). Three independent experiments were reproducible, and typical data are shown. Scale bar, 300 m.

Full figure and legend (103K)

Involvement of a novel CYP-mediated pathway of 15d-PGJ₂ synthesis in sebocyte lipogenesis

Based on our finding that indomethacin, diclofenac, and NS-398 cause the selective augmentation of 15d-PGJ₂ production in hamster sebocytes, a COX-independent pathway may be involved in the increase of 15d-PGJ₂ production. As it has been reported that arachidonic acid is metabolized not only by COX but also by CYP (^{Laniado-Schwartzman et al, 1988;Capdevila et al, 2000}), we investigated the effect of a CYP inhibitor on the production of 15d-PGJ₂ and on the synthesis of intracellular TG in COX inhibitor-treated sebocytes. As shown in Figure 6a, a non-selective CYP inhibitor, SKF-525A (10 and 20 M), suppressed indomethacin and NS-398-induced production of 15d-PGJ₂. These COX inhibitor-augmented levels of intracellular TG were diminished by SKF-525A (Figure 6b). SKF-525A did not influence the basal levels of 15d-PGJ₂ and TG in hamster sebocytes (Figure 6). Similar results were obtained when another non-selective CYP inhibitor, methoxsalen, was used (data not shown). As it has been reported that 15d-PGJ₂ as well as PGJ₂ is an endogenous activator of peroxisome proliferation-activating receptor (PPAR) (^{Kliewer et al, 1995;Yu et al, 1995;Shibata et al, 2002;Scher and Pillinger, 2005}), and that the activation of PPAR1 in sebocytes causes an increase in lipogenesis (^{Rosenfield et al, 1999}), we examined the effect of SKF-525A on the synthesis of TG in hamster sebocytes treated with a ligand activator of PPAR, troglitazone. As shown in Table II, troglitazone-augmented TG synthesis was not influenced by SKF-525A (5–20 M).

Figure 6.

A cytochrome P-450 (CYP) inhibitor suppresses indomethacin- and NS-398-augmented production of 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂) and triacylglycerol (TG) in hamster sebocytes. Hamster sebocytes at the 2nd passage were treated with indomethacin (Ind) (10 M) and NS-398 (NS) (20 M) in the presence of SKF-525A (10 and 20 M) for 12 d. Culture media and cells harvested at 12 d were subjected to measurement of 15d-PGJ₂ (A) and intracellular TG (B). Three independent experiments were reproducible, and typical data are shown. Data are shown as meanstandard deviation of triplicate dishes. a, b, and c indicate significant differences from untreated control cells (Cont) with p values of <0.05, <0.01, and <0.001, respectively. ^*, ^**, and ^*** indicate significant differences from indomethacin (Indo) alone with p values of <0.05, <0.01, and <0.001, respectively. ### indicates significant differences from NS-398 (NS) alone with p values of <0.001.

Full figure and legend (39K)

Table II - No effect of CYP inhibitor on troglitazone-augmented TG synthesis in hamster sebocytes.

Full table

Discussion

The proliferation and differentiation of keratinocytes have been reported to be regulated by PGE₂, which may be associated with the development of the epidermis and epidermal repair under wound healing (^{Pentland and Needleman, 1986;Unemori et al, 1994;Sato et al, 1997}). Sebocytes are classified as epithelial cells, and cause terminal differentiation with the intracellular accumulation of lipids (^{Rosenfield et al, 1998;Deplewski and Rosenfield, 1999}), it seems that PG might be involved in the regulation of sebaceous development and functions. In this study, the basal level of 15d-PGJ₂ was found to increase, whereas that of PGF₂ decreased and that of PGE₂ did not change during spontaneous differentiation of hamster sebocytes in vitro. We demonstrated that the formation of intracellular lipid droplets was augmented by exogenously administering 15d-PGJ₂, and was associated with an increase in DGAT-mediated TG biosynthesis in hamster sebocytes.^{Neufang et al (2001)} reported that the development of sebaceous glands with sebum deposition is augmented in transgenic mice overexpressing COX-2 in the epidermis, in which the levels of PGF₂ and PGE₂ are elevated. But, they did not show data for 15d-PGJ₂ in their transgenic mice. As a COX-mediated arachidonic acid metabolite, PGH₂, is a common precursor for 15d-PGJ₂, PGF₂, and PGE₂ (^{Narumiya et al, 1999;Tiano et al, 2002;Helliwell et al, 2004;Scher and Pillinger, 2005}), it is assumed that levels of not only PGF₂ and PGE₂ but also 15d-PGJ₂ are augmented in the epidermis in the transgenic mice. Taken together, these findings suggest that the increase in levels of 15d-PGJ₂ rather than PGF₂ and PGE₂ could cause augmentation of lipogenesis in sebaceous glands.

Non-steroidal anti-inflammatory drugs such as indomethacin and diclofenac have been useful in elucidating the involvement of PG in pathological and physiological reactions in vivo and in vitro by their inhibition of COX activity (^{Kobayashi et al, 1985;Smith et al, 1998}). In this study, we demonstrated that hamster sebocytes express COX-2 constitutively, but not COX-1, and that indomethacin, diclofenac, and NS-398 inhibit the production of PGE₂ and PGF₂ in sebocytes, which is dependent on a COX-2 pathway. Unexpectedly, however, there was no change in the level of PGD₂ in indomethacin-treated sebocytes. Furthermore, the treatment of hamster sebocytes with indomethacin, diclofenac, and NS-398 caused an increase in 15d-PGJ₂ production, suggesting that the production of PGD₂ and 15d-PGJ₂ in hamster sebocytes is regulated by the COX-independent pathway. In accord with the augmentation of 15d-PGJ₂ production, the formation of intracellular lipid droplets and of DGAT-mediated biosynthesis of TG was found to be increased in these COX inhibitor-treated sebocytes. Topical administration of indomethacin to auricles of male golden hamsters resulted in the development of sebaceous glands and the enhancement of sebum accumulation in vivo. These findings strongly suggested that hamster sebocytes possess at least two mechanisms for the biosynthesis of PG: (i) a general COX-2-dependent pathway for prostanoid synthesis (^{Narumiya et al, 1999;Tiano et al, 2002}) and (ii) a novel pathway for 15d-PGJ₂ synthesis, which may be stimulated by inhibiting COX-2 activity (Figure 7).

Figure 7.

A possible mechanism for sebocyte lipogenesis through a cytochrome P-450-15-deoxy-^12,14-PGJ₂-peroxisome proliferation activating receptor 1 (CYP-15d-PGJ₂-PPAR1) pathway. Cyclooxygenase (COX)-2-dependent and -independent pathways of 15d-PGJ₂ production are present in hamster sebocytes. Although the basal production of PGs is mediated by a COX-2-dependent pathway, the inhibition of COX-2 activity by non-steroidal anti-inflammatory drugs (NSAIDs) specifically leads to an increase in 15d-PGJ₂ production though a CYP-mediated pathway. The basal and the CYP-mediated augmentation of 15d-PGJ₂ production result in an increase of diacylglycerol acyltransferase (DGAT)-mediated production of TG through PPAR1 activation.

Full figure and legend (21K)

Regarding COX-independent PG production, it has been reported that CYP participates in arachidonic acid metabolism (^{Laniado-Schwartzman et al, 1988;Capdevila et al, 2000}).^{Keeney et al (1998)} reported that CYP2B12 is specifically expressed in rat sebaceous glands.^{Thiboutot et al (2003)} reported that CYP11A and CYP17 are expressed in sebaceous glands and ducts of human facial skin. In this study, our finding that SKF-525A suppressed indomethacin and NS-398-induced production of 15d-PGJ₂ and TG suggests that there is a novel CYP-mediated pathway for the synthesis of 15d-PGJ₂ in hamster sebaceous glands, which may be activated by inhibiting COX-2 activity (Figure 7). There was no change in the basal level of 15d-PGJ₂ in sebocytes treated with CYP inhibitor alone. Taken together with data for hamster sebocytes that constitutively produce COX-2 in vivo and in vitro, these results suggest the possibility that the basal level of 15d-PGJ₂ may be produced through a COX-2-dependent pathway (Figure 7). Further experiments will be needed to identify the subtype of CYP in hamster sebocytes, and to clarify the mechanism for the production of 15d-PGJ₂ through COX-2- and CYP-dependent pathways during the synthesis of sebum in sebaceous glands.

15d-PGJ₂ is an endogenous activator of PPAR for lipogenesis in adipocytes (^{Kliewer et al, 1995;Yu et al, 1995;Shibata et al, 2002;Scher and Pillinger, 2005}).^{Rosenfield et al (1999)} reported the involvement of PPAR1 in the augmentation of lipogenesis in rat sebocytes. We also demonstrated that hamster sebocytes constitutively express PPAR and 1 in vitro, whose activation by ligand activators for PPAR and augments the formation of lipid droplets and the production of TG (^{Akimoto et al, 2005}). Therefore, it is likely that the elevated levels of 15d-PGJ₂ cause the activation of PPAR1, which in turn augments the synthesis of TG in hamster sebocytes. Moreover, we found that PPAR1-mediated augmentation of DGAT-dependent production of TG is not influenced by SKF-525A. This suggests that the CYP-mediated augmentation of 15d-PGJ₂ production may occur upstream of the PPAR1 pathway in sebocyte lipogenesis (Figure 7).

In conclusion, we suggest that 15d-PGJ₂ is a crucial stimulator of sebocyte lipogenesis by augmenting DGAT-mediated synthesis of TG or by decreasing the activity of TG lipase (^{Dolinsky et al, 2004}). In addition to the COX-2 pathway, we suggest a CYP-mediated sebocyte-specific pathway for 15d-PGJ₂ synthesis, the activity of which may be evoked by selectively inhibiting COX-2 (Figure 7). Finally, these findings should provide an increased understanding of the functions of sebaceous glands in normal and diseased skin, and could contribute to the development of drugs and clinical strategies for skin diseases such as acne vulgaris.

Materials and Methods

Cell culture and treatments

Hamster sebocytes were established from sebaceous glands of auricles of 5-wk-old male golden hamsters as described previously (^{Ito et al, 1998;Sato et al, 2001a}). Sebocytes (2.35 10⁴ cells per cm²) in Dulbecco's modified Eagle's medium/Ham's F12 medium (1:1) (DMEM/F12) (Invitrogen, Carlsbad, California) supplemented with 6% (vol/vol) heat-denatured fetal bovine serum (Asahi Techno Glass, Tokyo, Japan), 2% (vol/vol) human serum (ICN Biochemicals, Costa Mesa, California), 0.68 mM L-glutamine (Invitrogen), and recombinant human EGF (10 ng per mL) (Progen Biotechnik GmbH, Heidelberg, Germany) were plated in 35, 60, or 100 mm diameter culture dishes (Becton Dickinson, Tokyo, Japan) for 24 h to achieve complete cell adhesion. Next, the cells were treated every 3 d with non-selective COX inhibitors, indomethacin and diclofenac (Sigma Chemical, St. Louis, Missouri); a COX-2-specific inhibitor; NS-398 (Calbiochem-Novabiochem, San Diego, California); and ligand activators of PPAR, 15d-PGJ₂ (Cayman Chemical, Ann Arbor, Michigan) and troglitazone (kindly provided by Sankyo, Tokyo, Japan), in the presence or absence of non-selective CYP inhibitors, SKF-525A or methoxsalen (Sigma), in DMEM/F12 supplemented with heat-denatured fetal bovine serum, human serum, and L-glutamine for up to 14 d. In this series of experiments, hamster sebocytes were used at up to the 3rd passage level.

Oil-red-O staining

Cultured hamster sebocytes were washed once with Ca²⁺- and Mg²⁺-free phosphate-buffered saline and fixed with 4% (wt/vol) paraformaldehyde (Wako Pure Chemicals, Osaka, Japan) diluted with Ca²⁺- and Mg²⁺-free phosphate-buffered saline for 1 h at room temperature. The cells were washed with distilled H₂O and then stained with 0.3% (wt/vol) oil-red O (Sigma) in isopropanol:distilled H₂O (3:2, vol/vol) at 37°C for 15 min. The stained cells were washed with distilled H₂O, and then viewed with a light microscope equipped with a digital camera (Olympus Optical, Tokyo, Japan).

Histochemical staining

Auricles of 5-wk-old male golden hamsters were topically treated everyday with 50 L of 2% (wt/vol) indomethacin in 95% (vol/vol) ethanol and 5% (wt/vol) glycerol, or with the same volume of vehicle for 14 d. After the treatment, the tissues were fixed with 4% (wt/vol) paraformaldehyde, embedded in paraffin, and then their sections were subjected to Mayer's hematoxylin–eosin staining (Wako Pure Chemicals). After terminating the staining, tissue secretions were washed with ethanol and xylene, and then viewed with a light microscope equipped with a digital camera. The animals had free access to food and water according to the Guidelines of Experimental Animal Care issued by the Prime Minister's Office of Japan. The experimental protocol was approved by the Committee of Animal Care and Use of Tokyo University of Pharmacy and Life Science.

Quantitative analysis of intracellular lipids

The lipid composition of extracts of intracellular lipids was analyzed by an automatic TLC, Iatroscan (Iatron Laboratories, Tokyo, Japan) using authentic tripalmitin, palmitic acid, cholesterol, cholesterol palmitate (Doosan Serdary Research Laboratories, Englewood Cliffs, New Jersey), and palmityl palmitate (wax ester) (Nu-Chek-Prep, Elysian, Minnesota) as described previously (^{Sato et al, 2001a}). The amounts of lipid components were also calculated using an internal control concomitantly performed using authentic cholesterol acetate (2 g) (Doosan Serdary Research Laboratories). Otherwise, the sonicated-cell lysates were used to measure the level of intracellular TG using Liquitech TG-II (Roche Diagnostics, Tokyo, Japan) according to the manufacturer's instructions. The amount of TG was calculated using an authentic trioleinate-standard solution (60 g per mL). The intracellular DNA content was measured using authentic salmon sperm DNA (6.25–100 g per mL) and 3,5-diaminobenzoic acid dihydrochloride (Sigma) as described previously (^{Sato et al, 2001a}).

DGAT activity

Hamster sebocytes were homogenized in 20 mM Tris-HCl (pH 7.6), 0.25 M sucrose, 2 mM MgCl₂, and 50 mM KCl, and then centrifuged at 12,000 g for 15 min at 4°C. The harvested supernatants were further centrifuged at 105,000 g for 60 min at 4°C. The precipitates were resuspended in 20 mM Tris-HCl (pH 7.6), 0.15 M KCl, and 1 mM EDTA, and then centrifuged under the same conditions. The resultant precipitates were resolved in distilled H₂O and then subjected to the measurement of DGAT activity (^{Coleman and Bell, 1976}). Briefly, aliquots (3 g) of the samples were incubated with 40 nM 1,2-dioleoyl glycerol (Cayman Chemical), and 5 nM [¹⁴C]palmitoyl-CoA (Amersham Biosciences, Tokyo, Japan) in 175 mM Tris-HCl (pH 7.6), 8 mM MgCl₂, and 1% (wt/vol) fatty-acid-free bovine serum albumin for 10 min at 37°C. The reaction was terminated by adding 2-propanol:heptane:distilled H₂O (40:10:1; vol/vol/vol) solution. After adding heptane:distilled H₂O (2:1; vol/vol) solution, the radioactivity of synthesized ¹⁴C-labeled TG in the extracted heptane layer was measured by a liquid-scintillation counter. We note that the main component of the heptane extraction on silica-gel 60 F₂₅₄-coated TLC (Merck, Darmstadt, Germany) by iodic visualization had the same relative flow as that of authentic TG (2 mg per mL) (data not shown). In addition, we confirmed that the radioactivity of synthesized ¹⁴C-labeled TG decreased in the presence of 1 mM EDTA or 0.1% (wt/vol) Tween 20 (data not shown) as described previously (^{Coleman and Bell, 1976}), suggesting that this assay is reliable for the measurement of DGAT activity in vitro.

Enzyme immunoassay for PGE₂, PGF₂, PGD₂, and 15d-PGJ₂

Amounts of PGE₂, PGF₂, PGD₂, and 15d-PGJ₂ in harvested culture media were measured using enzyme immunoassay kits for PGE₂, PGF₂, and PGD₂ (Cayman Chemical), and for 15d-PGJ₂ (Assay Designs, Ann Arbor, Michigan) according to the manufacturers' instructions.

Western blot analysis for COX

Cells and sebaceous glands were homogenized in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (wt/vol) NP-40, 0.1% (wt/vol) SDS, 1 mM EDTA, 10 g per mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride, and then subjected to sonication as described previously (^{Noguchi et al, 2003}). After centrifugation at 8000 g for 15 min at 4°C, proteins (80 g) in the resulting supernatant were subjected to SDS-polyacrylamide gel electrophoresis using 10% (wt/vol) acrylamide gel, and then electrotransferred onto a nitrocellulose membrane as described previously (^{Sato et al, 2001b}). The membranes were reacted with rabbit anti-(human COX-1) or anti-(human COX-2) antibody (Cayman Chemical), which was then complexed with horseradish peroxidase-conjugated goat anti-(rabbit IgG)IgG (Sigma). Immunoreactive COXs were visualized with enhanced chemiluminescence–Western blotting detection reagents (Amersham Biosciences) according to the manufacturer's instructions.

Statistical analysis

Data are presented as the meanstandard deviation, and were analyzed by a one-way analysis of variance and by the Fisher test for multiple comparisons. A value of p<0.05 was considered to indicate a statistically significant difference.

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Acknowledgments

This work was supported in part by grants from The Kao Foundation for Arts and Sciences, and from Matching Fund Subsidy for Private School of Japan. We wish to thank Dr. K. Tanzawa of Sankyo Co, Tokyo, Japan for generously providing troglitazone.