Methods and compositions for the differentiation of human preadipocytes into adipocytes

Abstract

The present invention provides methods and compositions for the consistent and quantitative differentiation of human preadipocytes isolated from adipose tissue into adipocytes bearing biochemical, genetic, and physiological characteristics similar to that observed in isolated primary adipocytes. The methods of the invention comprise incubating isolated human preadipocytes, plated at least about 25,000 cells/cm 2 , in a medium containing, glucose, a cyclic AMP inducer such as isobutylmethylxanthine or forskolin, a glucocorticoid or glucocorticoid analogue, insulin or an insulin analogue and a PPARγ agonist or a RXR agonist. The compositions of the invention include media for the differentiation of human preadipocytes, human adipocytes differentiated by the methods of the invention and transfected adipocytes. The present invention also provides methods for determining the ability of a compound to affect the differentiation of human preadipocytes to adipocytes, for determining the ability of a compound to act as a PPARγ antagonist. a glucocorticoid, a glucocoticoid analogue, or an insulin analogue, for transfecting cultured human adipocytes, and as a means to identify novel polypeptides secreted from human adipocytes into the conditioned medium. The methods and compositions have use in the drug discovery of compounds having relevance to the disease states of diabetes, obesity, and cardiovascular disease and in the studies of these diseases.

1 . A method for determining the ability of a compound to affect the differentiation of preadipocytes to adipocytes, the method comprising: 
 a) plating isolated human preadipocytes at a density of about 25,000 to 40,000 cells/cm 2  in a preadipocyte medium comprising a defined cell culture medium having or supplemented with 1.0-4.5 g/liter glucose;    b) differentiating said human preadipocytes with a differentiation medium comprising a defined cell culture medium; wherein said medium comprises 1.0-4.5 g/liter glucose, a cyclic AMP inducer, a glucocorticoid or a glucocorticoid analogue, insulin or an insulin analogue, a Peroxisome Proliferator Activated Receptor gamma agonist or a retinoic acid X receptor agonist effective, and said compound in an appropriate vehicle or vehicle alone to stimulate differentiation of said human preadipocytes;    c) determining the number or percentage of differentiated cells using said differentiation medium containing said compound from step (b);    d) determining the number of percentage of differentiated cells in the cells using said differentiation medium containing said vehicle alone from step (b); and    e) comparing the number or percentage of differentiated cells from steps (c) and (d).    
     
     
         2 - 6 . (canceled)  
     
     
         7 . The method of  claim 1 , wherein said differentiation medium further comprises 3-10% fetal bovine serum.  
     
     
         8 . The method of  claim 1 , wherein said differentiation medium further comprises 1-100 μM pantothenate and 1-100 μM biotin.  
     
     
         9 . The method of  claim 1 , wherein said differentiation medium further comprises a buffer having a pH of about 7.0 to 7.6.  
     
     
         10 . The method of  claim 1 , wherein said differentiation medium is Dulbeccos Modified Eagle/Hams' F-10 Nutrient Broth (1:1 vol/vol).  
     
     
         11 . The method of  claim 1 , wherein said Peroxisome Proliferator Activated Receptor gamma agonist is thiazolidinedione.  
     
     
         12 . The method of  claim 11 , wherein said thiazolidinedione is BRL 49653.  
     
     
         13 . The method of  claim 12 , wherein the concentration of said BRL 49653 is about 0.5-1.0 μM.  
     
     
         14 . The method of  claim 11  wherein said thiazolidinedione is troglitazone.  
     
     
         15 . The method of  claim 14 , wherein the concentration of said troglitazone is about 0.5-1.0 μM.  
     
     
         16 . The method of  claim 1 , wherein said glucocorticoid is dexamethasone, hydrocortisone or cortisol.  
     
     
         17 . The method of  claim 1 , wherein the concentration of said glucocorticoid is about 16 nM to 1.0 μM.  
     
     
         18 . The method of  claim 1 , wherein said cyclic AMP inducer is isobutylmethylxanthine or forskolin.  
     
     
         19 . The method of  claim 18 , wherein said isobutylmethylxanthine is present in said differentiation medium at a final concentration of about 0.2 to 0.5 mM.  
     
     
         20 . The method of  claim 1 , wherein the concentration of insulin is about 100 nM to 1.0 μM.  
     
     
         21 . The method of  claim 1 , wherein exogenous DNA has been introduced into said preadipocyte.