Inhibition of Adipogenesis Assays
Introduction
The lipid accumulation assays provide the tools to study the compounds that inhibit cultured human adipocyte differentiation or lipogenesis. Such compounds may useful in the treatment of obesity. Our assays measure the ability of a compound to block lipid accumulation in cells treated with PPAR γ agonists and glucocorticoid. Assays measure accumulation of lipid after two weeks in culture or upregulation of the adipocyte fatty acid binding protein (aP2)
Gene Expression Analysis aP2
Adipose differentiation can be determined by measuring the expression of the adipose specific marker gene aP2. aP2 is a fatty acid binding protein specifically expressed in adipocytes. Its expression is tightly controlled by the transcription factor Peroxisome Proliferator Activated Receptor gamma (PPAR Gamma) which plays a critical role in adipocyte differentiation. Compounds that block lipid accumulation in cells could have use as obesity therapies. The expression level of aP2 can therefore be used for identifying potential anti-obesity drugs.
aP2 expression is induced within 24 hours after treatment with PPAR gamma agonists. Cells will be dosed with test compounds and a PPAR gamma agonist or controls for 72 hours to obtain significant induction of aP2 mRNA. The quantity of aP2 mRNA will be measured by Quantigene. technology. Values are compared to expression levels of a control gene (PPIB). Assay done in triplicate in a 96 well format. Positive control: PPAR Gamma agonist (10 µM); Negative control: PPAR Gamma agonist (10 µM)+ TNFa (5 ng/ml).
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Fig. 1 shows relative luminescence values (RLU) for aP2 and
PPIB under various treatment conditions after 72 hrs.
PC1= PPAR gamma agonist 1 (1 µM)
NC1=PPAR gamma agonist 1 (1 µM)+ TNF. (5 ng/ml)
PC2 = PPAR gamma agonist 1 (10 µM)
NC2=PPAR gamma agonist 1 (10 µM)+ TNF. (5 ng/ml)
DMSO=vehicle
Lipid Accumulation
Our inhibition of lipid accumulation assays examine the ability of compounds to inhibit lipid accumulation in cells that are in the process of differentiating from preadipocytes to adipocytes. Cultured preadipocytes will be incubated in the presence of differentiation medium and test compound. Under these culture conditions and in the presence of an effective test compound there should be a substantial decrease in the amount of lipid accumulation when compared to controls. Researchers have a choice between acute and chronic treatments with compound
The assay involves complete lysis of the cells. Triglyceride is converted to glycerol and free fatty acids and the glycerol concentration is measured.
Positive control: PPAR Gamma agonist (10µM) or Dexamethasone (1µM)
Negative control: PPAR Gamma agonist (10µM) or Dexamethasone (1µM) + TNFa (5ng/ml)
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Fig. 2 shows accumulation of triglycerides in 2 week old adipocytes.
Cells were exposed to a PPAR gamma agonist (PC), a PPAR gamma agonist
plus TNF alpha (NC), vehicle (VC) or an unknown test compound for 3 days.
At the end of three days treatments were removed. The cells were fed and allowed
to accumulate lipid for a period of 14 days. At the end of 14 days cells were lysed and glycerol measured with the Zen-Bio glycerol reagent.
Ordering Information
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