Ali Aus T, Penny Clem B, Paiker Janice E, Psaras George, Ikram Faisel, Crowther Nigel J
Department of Chemical Pathology, National Health Laboratory Service, University of Witwatersrand Medical School, Parktown 2193, South Africa.
Anal Biochem. 2006 Jul 15;354(2):247-54. doi: 10.1016/j.ab.2006.04.028. Epub 2006 May 11.
Alkaline phosphatase (ALP) is expressed in 3T3-L1 preadipocytes, and its activity increases during adipogenesis. The purpose of this study was to determine whether ALP activity could be used as a measure of intracellular lipid accumulation in human preadipocytes and 3T3-L1 cells and which of the factors that induce adipogenesis are responsible for stimulating ALP activity. Adipogenesis was initiated in 3T3-L1 cells by incubation with differentiation medium containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The effect of leaving out each of the differentiation medium components was studied. Adipogenesis was also assessed in human preadipocytes and 3T3-L1 cells in the presence of the ALP inhibitor histidine. ALP activity was measured using an automated colorimetric assay and intracellular lipid accumulation was measured using the lipid-specific dye oil red O. Removal of insulin or dexamethasone from the differentiation medium had little effect on either ALP activity or lipid accumulation in 3T3-L1 cells, while removal of IBMX blocked both. Histidine inhibited ALP activity and adipogenesis in human preadipocytes and 3T3-L1 cells. Pearson univariate correlation analysis demonstrated strong correlations between ALP activity and lipid accumulation in human preadipocytes (r=0.78, n=69) and in 3T3-L1 cells (r=0.92, n=27). These data suggest that ALP and fat storage are tightly linked during preadipocyte maturation and that the measurement of ALP activity may be a novel technique for the quantification of intracellular lipid accumulation that is more sensitive and rapid than currently used methods.
碱性磷酸酶（ALP）在3T3-L1前脂肪细胞中表达，并且其活性在脂肪生成过程中增加。本研究的目的是确定ALP活性是否可作为衡量人前脂肪细胞和3T3-L1细胞内脂质积累的指标，以及诱导脂肪生成的哪些因素负责刺激ALP活性。通过用含有胰岛素、地塞米松和3-异丁基-1-甲基黄嘌呤的分化培养基孵育，在3T3-L1细胞中启动脂肪生成。研究了省略每种分化培养基成分的效果。还在存在ALP抑制剂组氨酸的情况下，对人前脂肪细胞和3T3-L1细胞中的脂肪生成进行了评估。使用自动比色法测量ALP活性，使用脂质特异性染料油红O测量细胞内脂质积累。从分化培养基中去除胰岛素或地塞米松对3T3-L1细胞中的ALP活性或脂质积累影响很小，而去除异丁基甲基黄嘌呤则两者均被阻断。组氨酸抑制人前脂肪细胞和3T3-L1细胞中的ALP活性和脂肪生成。Pearson单变量相关分析表明，人前脂肪细胞（r=0.78，n=69）和3T3-L1细胞（r=0.92，n=27）中ALP活性与脂质积累之间存在强相关性。这些数据表明，在前脂肪细胞成熟过程中，ALP与脂肪储存紧密相关，并且ALP活性的测量可能是一种用于定量细胞内脂质积累的新技术，比目前使用的方法更灵敏、更快速。
