Tsutomu Nagaya

Endogenous 15-deoxy-Δ12,14-prostaglandin J2 synthesized by adipocytes during maturation phase contributes to upregulation of fat storage

15‐Deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) has been identified as a natural ligand for peroxisome proliferator‐activated receptor (PPAR) γ to promote adipogenesis. However, it remains elusive about the ability of PPARγ‐expressing adipocytes to produce PGJ2 series and the role in the life cycle of adipocytes. Here, we developed an enzyme‐linked immunosorbent assay specific for 15d‐PGJ2. The analysis using this method revealed the increase in the endogenous synthesis of immunoreactive 15d‐PGJ2 in cultured adipocytes during the maturation phase. Further studies using cyclooxygenase inhibitors clarified the contribution of endogeous 15d‐PGJ2 produced by mature adipocytes to upregulation of fat storage in an autocrine manner.

Regulation and role of arachidonate cascade during changes in life cycle of adipocytes

Although some eicosanoids serve as potent natural ligands to activate peroxisome proliferator-activated receptor (PPARγ), the ability of adipocytes to produce eicosanoids and regulate PPARγ remains unclear. Here, adipogenic 3T3-L1 cells were employed to determine the gene expression of isoforms of biosynthetic enzymes in the arachidonate cyclooxygenase (COX) pathway and the synthesis of prostaglandins (PGs). The expression of COX-2 was induced transiently in a biphasic manner upon the triggering of the differentiation and maturation phases while COX-1 was constitutive. The exclusive expression of lipocalin-type PGD synthase occurred and gradually increased during the maturation process along with the stable expression of PPARγ. Moreover, we confirmed the formation of PGD2 from arachidonic acid by the mature adipocytes, suggesting conversion into PGJ2 derivatives. Even though cytosolic and membrane-associated subtypes of PGE synthase were expressed at relatively constant levels, the ability of preadipocytes to produce PGE2 was greater than that of mature adipocytes in the cell response. The treatment of the mature adipocytes with exogenous PGD2, 15-deoxy-Δ12,14-PGJ2 and PGE2, in the presence of aspirin, enhanced the adipogenesis. These findings imply the specific roles of prostanoids produced by the mature adipocytes in the maintenance of terminal differentiation through an autocrine control mechanism.

Up-regulation of adipogenesis in adipocytes expressing stably cyclooxygenase-2 in the antisense direction.

Adipocytes and the precursor cells express two types of cyclooxygenase (COX) isoforms that are involved in the biosynthesis of different types of prostaglandins (PGs) exerting opposite effects on adipogenesis. To evaluate the role of the inducible COX-2 isoform in the control of the differentiation and maturation of adipocytes, we employed an antisense technology to suppress specifically the expression of COX-2 in adipocytes. Cultured 3T3-L1 preadipocytes were transfected stably with a mammalian expression vector having the full-length cDNA encoding mouse COX-2 oriented in the antisense direction. The cloned transfectants with antisense COX-2 exhibited stable expression of antisense RNA for COX-2, which was accompanied by the suppressed expression of mRNA and protein levels of sense COX-2. However, almost no alteration in the expression of COX-1 was detected. The transfectants with antisense COX-2 showed significant decreases in the delayed synthesis of PGE(2) involving the inducible COX-2 in response to cell stimuli. By contrast, the immediate synthesis of PGE(2) associated with the constitutive COX-1 was not influenced appreciably. The stable expression of antisense mRNA of COX-2 resulted in significant stimulation of fat storage during the maturation phase without affecting the cell proliferation associated with the clonal expansion phase. The gene expression studies revealed higher expression levels of adipocyte-specific markers in the transfectants with antisense COX-2, indicating the mechanism that stimulates adipogenesis program. The up-regulation of fat storage was appreciably prevented by anti-adipogenic prostanoids, such as PGE(2) and PGF(2alpha), during the maturation phase. These results suggest that COX-2 is more preferentially involved in the generation of endogenous anti-adipogenic prostanoids during the maturation phase of adipocytes.

Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms

Prostaglandins (PGs) are known to play a variety of roles in adipocytes and precursor cells, which have the arachidonate cyclooxygenase (COX) pathway to generate several series of PGs at different stages of life cycle of adipocytes. To gain a unique insight into the specific roles of the COX isoforms during the life cycle of adipocytes, 3T3-L1 preadipocytes were stably transfected with a mammalian expression vector harboring either cDNA coding for murine COX-1 or COX-2. The cloned stable transfectants with COX-1 or COX-2 exhibited higher expression levels of their corresponding mRNA and proteins, and greater production of PGE(2) upon stimulation with free arachidonic acid or A23187 than the parent cells and the transfectants with vector only. However, either type of transfectants brought about the marked reduction in the accumulation of triacylglycerols after the standard adipogenesis program. Unexpectedly, aspirin or other COX inhibitors at different phases of life cycle of adipocytes failed to reverse the reduced storage of fats. The transfectants with COX-2 were sensitive to exogenous 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and troglitazone as peroxisome proliferator-activated receptor gamma (PPARgamma) agonists during the maturation phase for restoring the adipogenesis. By contrast, the transfectants with COX-1 were much less sensitive, which was reflected by much lower gene expression levels of PPARgamma and the related adipocyte-specific markers. Taken together, the results suggest that the sustained overexpression of either COX-1 or COX-2 resulted in the interference of adipogenesis program through a PG-independent mechanism with a different mode of action of COX isoforms.

Development of enzyme-linked immunosorbent assay for Δ12-prostaglandin J2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase.

Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to J(2) series of PGs including 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2), which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ(12)-PGJ(2) has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Δ(12)-PGJ(2). According to the standard curve, the amount of Δ(12)-PGJ(2) can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ(2), while it only showed the cross-reaction of 28% with unstable PGJ(2). This immunological assay was applied to the determination of the endogenous formation of Δ(12)-PGJ(2) in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ(12)-PGJ(2) increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ(2). Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ(12)-PGJ(2) in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ(2) series at various doses on adipogenesis during the maturation phase. Although Δ(12)-PGJ(2) was slightly less potent than 15d-PGJ(2), each of these PGJ(2) series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Δ(12)-PGJ(2) contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ(2) during the maturation phase of cultured adipocytes.

Molecular Cloning and Functional Expression of Soybean Allene Oxide Synthases

A plant allene oxide synthase (AOS) reacting with 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (13-HPOT), a lipoxygenase product of α-linolenic acid, provides an allene oxide which functions as an intermediate for jasmonic acid (JA) synthesis, making AOS a key enzyme regulating the JA level in plants. Although AOSs in various plants have been investigated, there is only limited information about AOSs in soybean (Glycine max). In this study, we cloned and characterized two soybean AOSs, GmAOS1 and GmAOS2, sharing 95% homology in the predicted amino acid sequences. GmAOS1 and GmAOS2 were composed of 564 and 559 amino acids respectively, with predicted N-terminal chloroplast-targeting signal peptides. Both AOSs expressed in Escherichia coli were selective for 13S-hydroperoxides of α-linolenic and linoleic acids, suggesting the potential of GmAOS1 and GmAOS2 to contribute to JA synthesis. GmAOS1 and GmAOS2 were expressed in leaves, stems, and roots, suggesting broad distribution in a soybean plant.

Regulation of apoptosis through arachidonate cascade in mammalian cells.

The arachidonate cascade includes the cyclooxygenase (COX) pathway to form prostanoids and the lipoxygenase (LOX) pathway to generate several oxygenated fatty acids, collectively called eicosanoids. Eicosanoids are suggested to play a dual role in regulating cell survival and apoptosis in various types of cells through an unknown mechanism. We found apoptosis in cultured Madin-Darby canine kidney (MDCK) cells treated with 12-O-tetradecanoylphorbol β-acetate (TPA), a potent tumor promoter, and nordihydroguaiaretic acid (NDGA), a LOX inhibitor. The effect of TPA was synergistically stimulated along with NDGA. Aspirin, a COX inhibitor, was not effective. The target of NDGA might be different from the mechanism involving a LOX activity in some kinds of carcinoma cells because the increased expression of 12-LOX was not detected in MDCK cells treated with TPA. Caspase and poly(ADP-ribose) metabolites were found to be involved in the signal transduction pathway of the TPA- and NDGA-induced apoptosis in MDCK cells. Alternatively, hydrogen peroxide-induced apoptosis was not affected by NDGA. Thus, the TPA-induced response involved the mechanism independent of the oxidative stress. Obesity is a risk factor for severe diseases including noninsulin-dependent diabetes and atherosclerosis characterized by the changes of cell properties of adipocytes. We found that conjugated linolenic acid from bitter gourd was able to induce apoptosis in mouse preadipogenic 3T3-L1 cells. The findings provide the potential use of conjugated fatty acids to regulate obesity.

Development of enzyme-linked immunosorbent assay for 8-iso-prostaglandin F2α, a biomarker of oxidative stress in vivo, and its application to the quantification in aged rats

8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) is one of the isoprostanes that are mainly generated nonenzymatically in vivo from arachidonic acid through free radical-induced lipid peroxidation. To assess oxidative stress in vivo, we developed a quantitative enzyme-linked immunosorbent assay (ELISA) for 8-iso-PGF2alpha. A sensitive calibration curve allowed the quantification of the amounts from 0.23 pg to 98.4 pg with 4.7 pg of 50% displacement in one assay. The ELISA method was applied to the measurement of the plasma levels of 8-iso-PGF2alpha in young rats (4-8 weeks of age) and aged rats (106-123 weeks). The average level of esterified form in the plasma from aged rats was 30.6-fold higher than that in the plasma from young rats, reflecting the enhanced status of oxidative stress in aged animals. In addition, the aged rats exhibited higher levels of this F2-isoprostane esterified to lipids from liver and kidney, suggesting local oxidative injury in specific organs. These results indicate the utility and accuracy of our ELISA for 8-iso-PGF2alpha as a biomarker in vivo to assess systemic oxidative stress in animals or humans as well as oxidative injury at local sites.

Endogenous Prostaglandins E2 and F2α Serve as an Anti-Apoptotic Factor against Apoptosis Induced by Tumor Necrosis Factor-α in Mouse 3T3-L1 Preadipocytes

Adipocytes can function as endocrine cells secreting a variety of adipocytokines including tumor necrosis factor (TNF)-α. Treatment of cultured mouse 3T3-L1 preadipocytes with TNF-α induced apoptosis, as was evident from increases in nuclear condensation and caspase-3 activity, but differentiated adipocytes during the maturation phase showed resistance to apoptosis by TNF-α. Antioxidants effectively reduced TNF-α-induced apoptosis in preadipocytes, indicating the involvement of reactive oxygen species. Exposure of preadipocytes to calcium ionophore A23187 reduced TNF-α-induced apoptosis, which was accompanied by increased production of prostaglandins (PGs) E2 and PGF2α. TNF-αpreferentially promoted gene expression of cyclooxygenase (COX)-2 without affecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-α-induced apoptosis, which was reversed by exogenous PGE2 and PGF2α. These results indicate that endogenous PGE2 and PGF2α synthesized by preadipocytes through the induction of COX-2 can serve as anti-apoptotic factors against apoptosis by TNF-α.

Control of life cycle of mouse adipogenic 3T3-L1 cells by dietary lipids and metabolic factors

Adipocytes function not only as in the storage and mobilization of lipids but also as endocrine cells by secreting tumor necrosis factor-α (TNF-α), free fatty acids, and other cytokines. To study the effects of dietary lipids and metabolic factors on the control of the life cycle of adipocytes, we utilized mouse 3T3-L1 preadipocytes that could be induced to differentiate into adipocytes. To evaluate the role of endogenous prostaglandins (PGs) in the adipogenic changes, we examined the effect of specific inhibitors of cyclooxygenase (COX). SC-560, a specific COX-1 inhibitor, suppressed adipogenesis dose dependently, suggesting a role of constitutive COX-1 in the endogenous synthesis of PGs, including PGJ2 derivatives formed by mature adipocytes with the ability to promote adipogenesis. NS-398, a COX-2 inhibitor, had little influence on the maturation processes. Both COX inhibitors were effective in stimulating apoptosis of preadipocytes induced by TNF-α, indicating that both PGE2 and PGF2α produced by preadipocytes through the action of both COX isoforms serve as survival factors. However, the effect of both inhibitors was negligible for the proliferation of preadipocytes. Moreover, conjugated linolenic acid from bitter gourd at lower concentrations that was without effects by itself synergistically stimulated TNF-α-induced apoptosis. Therefore, dietary lipid factors are capable of controlling the life cycle of adipocytes together with metabolic factors.