Ascorbic acid enhances adipogenesis of 3T3-L1 murine preadipocyte through differential expression of collagens

Abstract

Background: Adipogenesis from preadipocytes into mature adipocyte is precisely coordinated by transcription factors such as CCAAT-enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor γ (PPARγ), cytokines, and hormones, which is accompanied by extracellular matrix remodeling. Besides anti-oxidant activity, ascorbic acid (ASC) is participating in collagen biosynthesis and increase production and processing of collagens. Moreover, several studies demonstrated that ASC enhanced differentiation from preadipocytes into mature adipocytes.

Methods: The adipogenic effect of ascorbic acid was evaluated in chemical induced 3T3-L1 by Oil Red O staining. This effect was elucidated by immunoblotting which detected the expression level of collagens and transcription factors in adipogenesis. The immunocytochemical determination of type I collagen was performed in 3T3-L1 adipocyte to show the change of extracellular matrix during adipogenesis.

Results: In this study, Oil Red O staining in 3T3-L1 preadipocytes was increased dose-dependently by addition of ASC. These ASC-treated adipocytes increased collagen processing of α1(I) and α1(V) and expressed α1(VI) and α2(VI) collagens differentially. ASC also stimulated expression of C/EBPα and PPARγ, which is preceded by collagen enhancement. In addition, inhibition of ASC activity by ethyl-3,4-dihydroxybenzoate showed reduction of lipid accumulation by removal of large lipid droplets, not by inhibition of lipid production. This observation went with loss of α1(I) deposition on adipocyte surface, increase of α1(V) and α2(VI) collagens and decrease of C/EBPs.

Conclusion: Our findings imply that various actions of ASC on adipogenesis through differential collagen expression may provide diverse applications of ASC to adipose tissue technology.

Figure 1

ORO staining in 3T3-L1 cells was increased by ASC. (A) Effective phase of ASC treatment was determined by ORO staining on Day 8 after chemical induction. Numbers on x-axis are the day when ASC was treated. -, non-treated; all, ASC treated in overall phase. Data are expressed as means ± SEM (bars). **p < 0.01 vs not-treated. (B) Pictures were taken on Day 8 after chemical induction (Differentiated) or not (Non-differentiated). Various concentrations (0-50 μg/ml) of ASC were added on overall phase. (C) Effect of various ASC concentration was determined by ORO staining on Day 8 after chemical induction (DA_-2,0,2 for early phase and DAa for overall phase) or not (NAa for overall phase).

Figure 2

Expression of collagens and transcription factors was modulated by ASC. Cell extract of cell layer on Day 0, 2, 4, 6, and 8 was immunoblotted with (A) anti-collagen antibodies or (B) anti-transcription factor antibodies. ASC was treated early phase (A_-2,0,2) or overall phase (Aa) or not-treated (control, C). Pro, procollagen; pN, N-procollgen; α1(I), mature α1 chain of type I collagen; α1(V), mature α1 chain of type V collagen.

Figure 3

Inhibition of ASC in late phase removed lipid droplets from adipocytes. Cells were treated with high concentration (50 μg/ml) of ASC on early phase (A_-2,0,2) or low concentration (1 μg/ml) of ASC on overall phase (Aa). EDHB (100 uM) was applied to ASC-treated cells on early, late, or overall phase. EDHB non-treated cells were regarded as control. Inhibitory effect was determined by ORO staining on Day 8 after chemical induction. After pictures (A) were taken, ORO stain was extracted and absorbance (B) was measured at 520 nm. Data are expressed as means ± SEM (bars). **p < 0.01 vs control of high ASC, ##p < 0.01 vs control of low ASC. (C) Microscopic determination of inhibitory effect. Arrow heads indicated large lipid droplets while arrows indicated the empty spots of large lipid droplet. (D) Immunoblot determination of inhibitory effect on collagens. Pro, procollagen; pN, N-procollgen; α1(I), mature α1 chain of type I collagen; α1(V), mature α1 chain of type V collagen. (E) Immunoblot determination of inhibitory effect on transcription factors. Expression level of a2(VI) (F) and CEBPa (30 kDa) (G) in high concentration of ASC (50 μg/ml, A_-2,0,2) was semi-quantified. After adjusting control as 100%, band densities are expressed as means ± SEM (bars). *p < 0.05 and **p < 0.01 vs control.

Figure 4

Inhibition of ASC on late phase caused alteration of type I collagen on adipocyte surface. Immunostaining analysis was performed on the cells cultured with chemical induction (B), chemical induction plus low concentration (1 μg/ml, Overall phase) of ASC (C and D), chemical induction plus high concentration (50 μg/ml, Early phase) of ASC (E – H) and without chemical induction (A). EDHB was added on early phase (F) and overall phase (D and H). Pictures were taken on Day 8 (A – D, G, and H) and Day 4 (E and F).