Atomic force microscopy, biochemical analysis of 3T3-L1 cells differentiated in the absence and presence of insulin.
BACKGROUND
There are ample evidences to demonstrate that differentiation of preadipocytes is associated with deposition of fat in cells. Still, it is unclear whether the differentiation process also alters membrane topology as well as cholesterol levels and whether insulin contributes to it.


METHODS
Membrane scanning of differentiated cells, along with freshly plated and 11 day preadipocytes, was performed using Atomic Force Microscopy (AFM) to gain qualitative information about cell surface properties as well as roughness. Moreover, glucose uptake, lipid analysis, expression profiling of transcription factors and signaling molecules involved in the process of differentiation was also performed.


RESULTS
We report (i) differentiation in the presence of 500 microM isobutylmethylxanthine (IBMX), 0.25 microM dexamethasone (DEX) with or without 0.1 microM (0.57 microg/ml) insulin directly alters membrane topology. (ii) At nano-levels, addition of insulin maintains plasma membrane roughness during differentiation in comparison with IBMX and DEX only. (iii) At macro levels, decreased fat accumulation in preadipocytes exposed to insulin during the initial stages of differentiation is a result of reduced expression and nuclear localization of sterol regulatory element binding protein (SREBP)-1.


CONCLUSIONS
This study reports a significant reduction of membrane cholesterol and total cholesterol (p<0.01) in cells differentiated in the presence of insulin.
Pandey
Vimal
V
National Centre for Cell Science, Ganeshkhind, Pune-411007, India.
Vijayakumar
Malepillil Vavachan
MV
Kaul-Ghanekar
Ruchika
R
Mamgain
Hitesh
H
Paknikar
Kishore
K
Bhat
Manoj Kumar
MK
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D001692:Biological Transport; D002454:Cell Differentiation; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D020869:Gene Expression Profiling; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology / Q000502:physiology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D018625:Microscopy, Atomic Force; D015398:Signal Transduction; D051780:Sterol Regulatory Element Binding Protein 1 / Q000378:metabolism; D014280:Triglycerides / Q000378:metabolism
D000943:Antigens, Differentiation; D005230:Fatty Acids, Nonesterified; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D051780:Sterol Regulatory Element Binding Protein 1; D014280:Triglycerides; D002784:Cholesterol; D005947:Glucose