Arachidonic Acid Added during the Differentiation Phase of 3T3-L1 Cells Exerts Anti-Adipogenic Effect by Reducing the Effects of Pro-Adipogenic Prostaglandins

Abstract

A linoleic acid (LA) metabolite arachidonic acid (AA) added to 3T3-L1 cells is reported to suppress adipogenesis. The purpose of the present study aimed to clarify the effects of AA added during the differentiation phase, including adipogenesis, the types of prostaglandins (PG)s produced, and the crosstalk between AA and the PGs produced. Adipogenesis was inhibited by AA added, while LA did not. When AA was added, increased PGE2 and PGF2α production, unchanged Δ12-PGJ2 production, and reduced PGI2 production were observed. Since the decreased PGI2 production was reflected in decreased CCAAT/enhancer-binding protein-β (C/EBPβ) and C/EBPδ expression, we expected that the coexistence of PGI2 with AA would suppress the anti-adipogenic effects of AA. However, the coexistence of PGI2 with AA did not attenuate the anti-adipogenic effects of AA. In addition, the results were similar when Δ12-PGJ2 coexisted with AA. Taken together, these results indicated that the metabolism of ingested LA to AA is necessary to inhibit adipogenesis and that exposure of AA to adipocytes during only the differentiation phase is sufficient. As further mechanisms for suppressing adipogenesis, AA was found not only to increase PGE2 and PGF2α and decrease PGI2 production but also to abrogate the pro-adipogenic effects of PGI2 and Δ12-PGJ2.

Eicosapentaenoic Acid Induces the Inhibition of Adipogenesis by Reducing the Effect of PPARγ Activator and Mediating PKA Activation and Increased COX-2 Expression in 3T3-L1 Cells at the Differentiation Stage

“…We added PA, SA, and OA, which are abundant and the typical fatty acids in red meat, ALA, which is abundant and the typical fatty acid in vegetable fats, and EPA, a metabolite of ALA, which is abundant and the typical fatty acid in fish meat, to 3T3-L1 cells at the differentiation stage and examined their effect on MDI-evoked adipogenesis during the maturation stage as described in Figure 1 A. AA was used as a positive control because we and other groups reported that AA added during the differentiation stage suppresses adipogenesis evoked by MDI after the maturation stage [ 24 ]. Figure 1 B shows that EPA reduced the intracellular TAG accumulation as an indicator of the level of adipogenesis.…”

“…Pre-adipogenic mouse 3T3-L1 cells (JCRB9014; JCRB Cell Bank, Osaka, Japan) were cultured following previously established protocols [ 24 , 25 ]. In summary, 3T3-L1 cells were seeded in 35- or 60-mm dishes containing growth medium (GM) at a density of 1 × 10 5 or 2 × 10 5 , respectively, in the growth stage.…”

“…To quantify intracellular triacylglycerol (TAG) and proteins, the same methods as previously described [ 24 , 25 ] were employed. Cultured mature adipocytes were collected and suspended in phosphate-buffered saline (PBS) without Ca 2+ and Mg 2+ (PBS [-]) containing 0.05% trypsin and 0.53 mM EDTA.…”

“…The gene expression levels were quantitatively analyzed following previously established methods [ 24 , 25 ]. In summary, reverse transcription (RT) was performed using M-MLV reverse transcriptase (ribonuclease H activity caused by point mutation), using total RNA (1 g) that was extracted from the cells using acid guanidium thiocyanate/phenol/chloroform after 24 and 48 h after the differentiation stage and on days 6 of the maturation stage.…”

Adipose tissue reconstruction facilitated with low immunogenicity decellularized adipose tissue scaffolds

“…As proliferation and the growth of the preadipocytes slow down, phenotypically relevant proteins begin to be expressed in the adipocyte, which might explain the promotion of secretion of leptin by DAT and D + DAT, favoring the growth of lipid droplets and enhancing the maturation of adipocytes after 7 and 14 d of culture. In figures 5 and 6, we can visualize that not all cells showed the typical imprinted ring shape, which is because 3T3-L1 cells accumulate triglycerides when entering a resting state [30], but not all of cells are able to enter the state. The percentage of cells that can enter the resting state is about 10%-50% [31].…”