Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog

Abstract

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 nonradioactive 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-Dglucose 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.

Effect of Troglitazone on Glut4 Gene Expression in 3t3-L1 Adipocyte Differentiation

“…The cells treated by adipogenic induction media with and without Troglitazone were large and had oil droplets were arranged in loose cluster. In contrast, the cells un-treated by adipogenic induction media were small, with compact clusters of oil droplets 13 .…”

EFFECT OF TROGLITAZONE ON MORPHOLOGICAL CHANGES AND PPAR-γ GENE EXPRESSION IN 3t3-L1 ADIPOCYTE CELLS

“…However, identification of agents or molecules that modulate this transcription process also provides insight into the signal transduction pathways involved. These agents and molecules then play important roles in modulating adipocyte differentiation by permitting the morphological changes and adipocyte specific gene expression that accompany differentiation (Vishwanath et al 2013;Morrison and McGee 2015).…”

A colorimetric bioassay for quantitation of both basal and insulin-induced glucose consumption in 3T3-L1 adipose cells

“…Cells seeded on 24 or 48 well plates were grown in complete medium up to 100% confluence. At day 2 post confluence, cells were changed to differentiation medium (complete medium supplemented with 10 μg/ml insulin, 0.1 μM dexamethasone, 0.5 mM IBMX and 2 μM rosiglitazone) by 48 h, and then returned to complete medium for 7-8 days of differentiation, with media changes every other day [13,14,15].…”

“…Glucose consumption in cells and tissues is a major physiological process, and its alteration is related to diabetes and other diseases [1,2,3]. Traditional assays to evaluate glucose uptake have been based on the use of radiolabeled glucose analogues such as [U- 14 C] glucose, [ 14 C] 2-deoxi-D-glucose and [ 3 H]-2-deoxiglucose among others [4,5]. Although radioactive methods are highly sensitive, their main disadvantages are the cost of equipment and reagents, and the need for safety protocols and appropriate physical spaces to minimize health and environmental risks.…”