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Adipogenesis Assays

Introduction

The lipid accumulation assays provide the tools to study the compounds that stimulate cultured human adipocyte differentiation or lipogenesis. Such compounds may be PPARγ agonists or a combination of thiazolidinediones and glucocorticoids that are potentially useful in the treatment of diabetes. Our assays measure potential PPAR γ agonists or glucocorticoid analogs. Assays include 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 increase PPAR Gamma activity generally increase insulin sensitivity and thus improve the diabetes condition. The expression level of aP2 can therefore be used for identifying potential anti-diabetic drugs.

aP2 expression is induced within 24 hours after treatment with PPAR Gamma agonists. Cells will be dosed with test compounds 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: 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 ┬ÁM) 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 lipid accumulation assays examine the ability of compounds to stimulate lipid accumulation in the process of differentiating preadipocytes to adipocytes. Cultured preadipocytes will be incubated in the presence of appropriate initiation medium, and test compound. Under these culture conditions and in the presence of the test compound there should be a substantial increase in the amount of lipid accumulation when compared to vehicles. Researchers have a choice between acute and chronic treatments with compound. Researchers must also choose between testing potential PPAR Gamma agonists, or glucocorticoid analogues.

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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Prices for contract services vary depending on number of sample numbers, special conditions, compound solubility, etc. Minimum charges will apply. Please contact us for detailed information and a price quote on your project.

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Omental Adipocytes

Zen-Bio announces availability of Omental Adipocytes.

Cells pictured above are cultured in indomethacin free media.

Call Zen-Bio at 866-234-7673 or click here for more information.