Ghrelin Promotes Proliferation and Inhibits Differentiation of 3T3-L1 and Human Primary Preadipocytes.

Hui Miao,Hongbo Yang,Huijuan Zhu,Linjie Wang,Fengying Gong,Hui Pan

doi:10.3389/fphys.2019.01296

Abstract

ObjectiveGhrelin is a 28-amino-acid peptide that regulates energy hemostasis, glucose and lipid metabolism. We aimed to explore the effects of ghrelin on the proliferation and differentiation of 3T3-L1 and human primary preadipocytes.Methods3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining, intracellular glycerol-3-phosphate dehydrogenase (G-3-PDH) assays and semiquantitative reverse transcription polymerase chain reaction were used to investigate the action of ghrelin.ResultsGhrelin (0.01–1000 ng/ml) significantly increased the numbers of 3T3-L1 cells, and the maximum stimulatory effect was observed with the 10 ng/ml ghrelin treatment for 24 h (p < 0.05). Ghrelin also promoted the proliferation of human primary preadipocytes from 24 h (p < 0.05) to 48 h (p < 0.05) at a concentration of 1000 ng/ml. Further investigation showed that IGF-1 levels were notably increased in ghrelin-treated 3T3-L1 and human preadipocytes, and IGF-1 antibody was capable to attenuate this stimulatory action of ghrelin (all p < 0.05). Additionally, ghrelin significantly suppressed the differentiation of 3T3-L1 and human primary preadipocytes; 10 ng/ml ghrelin notably downregulated G-3-PDH activities in 3T3-L1 cells on day 3 and in human cells from days 4 to 12 following differentiation (all p < 0.05), and the intracellular lipoprotein lipase mRNA levels were lower than that of the controls (p < 0.05). Further investigation showed that the mRNA levels of peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT/enhancer binding protein α (C/EBPs) were also suppressed in ghrelin-treated human differentiating adipocytes.ConclusionGhrelin promotes the proliferation of 3T3-L1 and human primary preadipocytes by increasing the expression of IGF-1. Ghrelin inhibits murine and human adipocyte differentiation by downregulating PPARγ2 and C/EBPα levels, consequently leading to decreased lipid accumulation and lipogenic enzymes expression.

Highlights

Obesity and its related disorders, such as type 2 diabetes, dyslipidemia, and cardiovascular disease has increased in prevalence in recent years and has become a vital health concern worldwide (Corica et al, 2015; Martin et al, 2015)
The results suggest that ghrelin-induced 3T3-L1 cell proliferation may not be mediated through the genes we investigated in the present study
As the enzyme glycerol-3phosphate dehydrogenase (G-3-PDH) is expressed in differentiating adipocytes, we further investigated the effect of ghrelin on G-3-PDH activity. 10 ng/ml ghrelin was added to 3T3L1 preadipocytes in a serum-free differential medium for 1, 3 and 6 days, and G-3-PDH activity was measured

Summary

Introduction

Obesity and its related disorders, such as type 2 diabetes, dyslipidemia, and cardiovascular disease has increased in prevalence in recent years and has become a vital health concern worldwide (Corica et al, 2015; Martin et al, 2015). In the process of differentiation, some transcription factors have been reported to activate adipogenesis. Two known factors are peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT/enhancer binding protein α (C/EBPs), which cooperate as a complex to promote preadipocyte differentiation and are required for the maintenance of adipocytes (Rosen et al, 2002; Imai et al, 2004). These transcription factors are associated with activating the expression of adipocytespecific enzymes, which are associated with lipogenesis and lipid retention. Lipoprotein lipase (LPL) reveals lipid storage in the early stages of differentiation, whereas fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and glycerol-3phosphate dehydrogenase (G-3-PDH) are involved in triglyceride metabolism in late stages (Ailhaud, 1996; Lenhard, 2011; Mracek et al, 2013; Kersten, 2014)

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Discussion

Conclusion