本研究的目的為利用3T3-L1前脂肪細胞篩選出具抗脂質生成活性之分離大豆蛋白(ISP)水解物，並探討其影響3T3-L1前脂肪細胞脂質生成活性的可能訊息傳遞途徑。實驗中測定甘油-3-磷酸去氫?(GPDH)活性作為評估3T3-L1前脂肪細胞分化的指標，GPDH活性愈低表示分化的程度愈低，即抗脂質生成活性愈高。ISP以Neutrase水解4小時所得水解物(NH4h)以及以Flavourzyme水解2小時所得水解物(FH2h)，於前脂肪細胞分化期間，均較其他水解物有更高的抗脂質生成活性。因此進一步探討NH4h及FH2h添加量與添加方式對細胞GPDH活性的影響，添加方式包括stageＩ(day 0及分化全期每隔兩天均添加水解物)與stageⅡ(僅分化初期day 0添加水解物)。其中GPDH活性隨添加劑量的增加而有下降的趨勢，但兩種添加方式在添加濃度400-1600ppm間，於相同濃度下均無顯著差異，可知分化前期添加水解物即可達到抗脂質生成的效果。當細胞分化前期添加400ppm NH4h和FH2h時，其GPDH活性顯著由485.3U/每毫克蛋白質分別下降至267.03與353.46 U/每毫克蛋白質(p<0.05)。為了進一步提升水解物抗脂質生成活性之專一性，確定具最佳抗脂質生成活性胜?之範圍，本研究利用1-30 kDa之間的分子量限值(Molecular weight cut-off, MWCO)濾膜對NH4h和FH2h進行區分，結果指出利用1kDa MWCO 濾膜分別區分所得的NH4h 1kDa濃縮物及FH2h 1kDa濾液，可再提升其抗脂質生成活性達1.72-2.02倍，且隨著添加劑量的增加，GPDH活性有下降的趨勢。最後評估大豆胜?造成3T3-L1前脂肪細胞具抗脂質生成活性的可能機制；結果顯示於前脂肪細胞分化前期，分別添加NH4h 1kDa濃縮物及FH2h 1kD濾液之凍乾水解物100ppm，會使過氧小體增生活化受體(PPAR)γ與CCAAT/增強子結合蛋白(C/EBP)α的蛋白質表現量隨培養時間增加而顯著的下降(p<0.05)，因此3T3-L1前脂肪細胞分化期間，大豆胜?可能藉由此途徑展現其抗脂質生成的活性。 The aim of this research was to screen the isolated soy protein (ISP) hydrolysates with anti-adipogenic activity and to evaluate the possible signaling pathway involved in their effects on the lipogenesis in 3T3-L1 preadipocytes. Glycerol-3-phosphate dehydrogenase (GPDH) activity was measured and acted as a marker for the cell differentiation. The lower the GPDH activity represents the higher the anti-adipogenic activity. Both neutrase-ISP hydrolysate at hydrolysis time of 4h (NH4h) and flavourzyme-ISP hydrolysate at hydrolysis time of 2h (FH2h) showed much higher anti-adipogenic activity than other hydrolysates in the cells. So probe into NH4h and FH2h dosages and add the impact on cell GPDH activation of the way further. The cells were individually incubated with NH4h and FH2h using two treatments denoted as stageＩ and stageⅡ. The former added each hydrolysate at day 0 and every 2-day until the end of day 9 during cell differentiation. The later added the hydrolysate only at early phase (day 0). Both treatments showed GPDH activity decreased as the concentration of NH4h and FH2h increased. There was no significant difference (p>0.05) in GPDH activity between treatments for addition of each hydrolysate at the same concentration ranged from 400-1600 ppm. Apparently the major effect of anti-adipogenic activity was contributed by adding the hydrolysate at early phase of cell differentiation. As 400 ppm of NH4h and FH2h were added individually under stageⅡ, the GPDH activities were significantly (p<0.05) decreased from 485.3 to 267.03 and 353.46 U/mg protein, respectively. In order to enhance the specific anti-adipogenic activity, confirm that molecular weight for soy peptides, each hydrolysate was further fractionated by 1-30kDa molecular weight cut-off (MWCO) membranes. Both NH4h and FH2h could enhance their anti-adipogenic activity 1.72-2.02 fold under stageⅡ using 1kDa MWCO membrane to obtain the fractions of NH4h 1kDa concentrate and FH2h 1kDa permeate. The protein expression of peroxisome proliferators-activated receptor (PPAR)γ and CCAAT/enhancer binding protein (C/EBP)α significantly decreased as incubation time increased during cell differentiation treated by each of the fractions under stageⅡ. These were the possible pathway for the hydrolysates to reveal anti-adipogenic activity during 3T3-L1 preadipocyte differentiation.
