Primary culturing and effects of insulin glargine on proliferation, differentiation of human preadipocytes from subcutaneous and omental adipose tissue

Abstract

To compare the morphological and functional differences of human primary preadipocytes from different fat depots and explore the effects of insulin glargine on their proliferation and differentiation. Primary preadipocytes isolated from human subcutaneous and omental adipose tissue by collagenase I were passaged in vitro.Inverted phase contrast microscope was used to observe the morphological differences of two kinds of preadipocytes. Then two kinds of preadipocytes were cultured or induced to differentiation with different doses of insulin glargine. The methyl thiazolyl tetrazolium (MTT) assay was used to detect their proliferative differences.Reverse transcription-polymerase chain reaction (RT-PCR) was used to observe the effects of insulin on adipogenic gene expression. (1) Both preadipocytes could be successfully cultured from adipose tissue and amplified in vitro.Subcutaneous preadipocytes were more slender and proliferated more quickly while omental preadipocytes were polygonal and aged easily.(2) MTT results showed that insulin glargine could inhibit the proliferation of omental preadipocytes in a dose-dependent fashion. After 72 h incubation, compared with negative control, the absorbance (A) value of 1000 nmol/L insulin glargine group decreased greatly (0.144 ± 0.021 vs 0.267 ± 0.040, P < 0.01). But it had no effect on subcutaneous preadipocytes (0.305 ± 0.045 vs 0.350 ± 0.037, P > 0.05). (3) Insulin at 500 nmol/L was a suitable concentration for inducing differentiation.RT-PCR analysis showed that, for subcutaneous adipocytes, adipogenic genes such as peroxisome proliferator-activated receptor gamma (PPARγ) (F = 31.31, P < 0.01) and CCAAT enhancer binding protein α (C/EBPα) (F = 9.86, P < 0.05) had the highest mRNA expression while preadipocytes gene Pref-1 had the lowest expression at this concentration. But insulin dose had no obvious effect on PPARγ or C/EBPα mRNA (P > 0.05) for omental adipocytes. Insulin glargine could inhibit the proliferation of omental preadipocytes, and enhance the differentiation of subcutaneous and omental preadipocytes.