Adipose tissue is an important endocrine organ involved in the control of whole body energy homeostasis and insulin sensitivity. Considering the increased incidence of obesity and obesity-related disorders, including diabetes, it is important to understand thoroughly the process of adipocyte differe...
Adipose tissue is an important endocrine organ involved in the control of whole body energy homeostasis and insulin sensitivity. Considering the increased incidence of obesity and obesity-related disorders, including diabetes, it is important to understand thoroughly the process of adipocyte differentiation and its control. Therefore, we performed a differential proteome mapping strategy using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to identify intracellular proteins that are differentially expressed during adipose conversion of 3T3-L1 pre-adipocytes in response to an adipogenic cocktail. In the current study, we identified 46 differentially expressed proteins, 6 of which have not been addressed previously in 3T3-L1 cell differentiation. Notably, we found that phosphoribosyl pyrophosphate synthetase (PRPS), a regulator of cell proliferation, was preferentially expressed in pre-adipocytes than in fully differentiated adipocytes. In conclusion, our results provide valuable information for further understanding of the adipogenic process.
Adipose tissue is an important endocrine organ involved in the control of whole body energy homeostasis and insulin sensitivity. Considering the increased incidence of obesity and obesity-related disorders, including diabetes, it is important to understand thoroughly the process of adipocyte differentiation and its control. Therefore, we performed a differential proteome mapping strategy using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to identify intracellular proteins that are differentially expressed during adipose conversion of 3T3-L1 pre-adipocytes in response to an adipogenic cocktail. In the current study, we identified 46 differentially expressed proteins, 6 of which have not been addressed previously in 3T3-L1 cell differentiation. Notably, we found that phosphoribosyl pyrophosphate synthetase (PRPS), a regulator of cell proliferation, was preferentially expressed in pre-adipocytes than in fully differentiated adipocytes. In conclusion, our results provide valuable information for further understanding of the adipogenic process.
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제안 방법
5% [w/v] Oil Red O in isopropanol and two parts of water) for 30 min at room temperature followed by washing with PBS twice, 70% ethanol once, and water twice. Cells were kept in water and photographed using an Olympus (Tokyo, Japan) microscope equipped with digital image acquisition (Figure not shown). Fat droplets in adipocytes were stained red.
The relative OD and relative volume were also calculated to correct for differences in gel staining. Each spot intensity volume was processed by background subtraction and total spot volume normalization; the resulting spot volume percentage was used tor comparison.
대상 데이터
3T3-L1 mouse embryo fibroblasts were purchased from American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). DMEM and FBS were purchased from Gibco BRL (Invitrogen, Carlsbad, CA, U.
). Dexamethasone (DEX), 3-isobutyl-l -methylxanthine (IBMX), and insulin were purchased from Sigma Chemical (St. Louis, MO, U.S.A.).
데이터처리
All experimental results were compeed by one-way analysis of varieice (ANOVA) using the Statistical Package of Social Science (SPSS, ver. 11.0) program, and the data were expressed as means±SE. Group means were considered to be significantly different at p<0.
이론/모형
Protein analysis was performed using an Ettan MALDI-TOF (Amersham Biosciences). The peptides were evaporated with a N2 laser at 337 nm using a delayed extraction mode.
Because of the abundance of hydrophobic proteins and interference of lipid in the mature adipocytes, a modified rehydration buffer (8 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF, 65 mM DTT, and 2% IPG buffer) was used to improve sample solubility, avoid protein precipitation, and achieve efficient extraction of total protein from cells. Total protein content was determined by the Bradford assay [3].
성능/효과
In conclusion, adipogenesis is a complex process that involves thousands of proteins at different stages of differentiation. Proteome analysis techniques allow the changes in steady-state levels of numerous proteins in a biological sample to be determined simultaneously.
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