Document Detail


Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog.
MedLine Citation:
PMID:  23292944     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive (3)H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive (3)H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive (3)H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.
Authors:
Divya Vishwanath; Harini Srinivasan; Manjunath S Patil; Sowmya Seetarama; Sachin Kumar Agrawal; M N Dixit; Kakali Dhar
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Publication Detail:
Type:  Journal Article     Date:  2013-01-06
Journal Detail:
Title:  Journal of cell communication and signaling     Volume:  7     ISSN:  1873-9601     ISO Abbreviation:  J Cell Commun Signal     Publication Date:  2013 Jun 
Date Detail:
Created Date:  2013-05-22     Completed Date:  2013-05-23     Revised Date:  2013-06-03    
Medline Journal Info:
Nlm Unique ID:  101308338     Medline TA:  J Cell Commun Signal     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  129-40     Citation Subset:  -    
Affiliation:
Bioanalytical Laboratory for Large Molecules, Clinigene International Limited, Third floor, Tower 1, Electronics City, Bangalore, 560 100, India, Divya.Vishwanath@clinigeneintl.com.
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