The Study On The Relationship Between Ghrelin And Molecular Mechanism Of Obesity

Simple obesity is the consequence of a chronic positive energy balance. Childhoodobesity is a global epidemic and rising trends in overweight and obesity is apparent in bothdeveloped and developing countries. Simple obesity in particular constitutes a growinghealth problem throughout the world. It is widely accepted that childhood obesity is animportant risk factor for the appearance of obesity in adulthood. And the great increase inobesity portends future increases in incidence of heart diseases; diabetes, stroke, andpossibly cancers. There has been a growing concern about obesity worldwide.On cellular level, obesity is caused by increasing amount and volume of adipocytesinduced by unceasing differentiation of preadipocytes. Abnormal differentiation,proliferation and apoptosis of preadipocytes will affect glucolipid metabolism, induceobesity and insulin resistance, and then influence the development of type 2 diabetes. It issignificant to study the effects of drugs on proliferation, differentiation and apoptosis ofpreadipocytes for prevention and cure of those metabolic diseases which have a closerelationship with obesity and insulin resistance. Therefore, many scholars have recentlyfocused on the regulation of the adipocyte proliferation, differentiation and apoptosis, andits relationship with obesity.Ghrelin is a stomach-derived hormone. It was originally identified as an endogenousligand of growth hormone secretagogue receptor (GHSR). In addition to its predictable effect on growthhormone (GH) secretion, ghrelin has an important role in the short-termregulation of appetite and the long-term regulation of energy balance and glucosehomeostasis. It is reported that ghrelin could induce adipogenesis not only through centralmechanisms but also though its direct effects on adipocytes. Therefore in recent years,much-attention has been paid to the research on ghrelin.In this study, 3T3-L1 preadipocytes were cultured in vitro. The effects of ghrelin onthe proliferation, differentiation and apoptosis of 3T3-L1 preadipocytes and the possiblemechanisms were investigated in order to analyze the relationship between ghrelin and thepathogenesis of obesity from cellular level.Partâ… Growth Hormone Seeretagogue Receptor-la Gene Expressionduring the Period of 3T3-L1 Preadipoeyte DifferentiationObjective To investigate growth hormone secretagogue receptor-la (GHSR-la) geneexpression during the period of 3T3-L1 adipocyte differentiation and toexplore the relationship between the GHSR-la gene expression andadipocytes differentiation.Methods 3T3-L1 preadipocytes were cultured in vitro and induced to differentiate intomatured adipocytes by lmg/L Insulin+0.5mM IBMX+1.0μM DEX. Themorphological changes of 3T3-L1 adipocytes were observed by microscope,the differentiation rate was assayed by Oil-Red O staining, and triglyceride(TG) mass was detected by chemical colorimetry methods during the periodof 3T3-L1 adipocyte differentiation. Total RNA was extracted fromadipocytes at various time, and the levels of GHSR-1a gene mRNAexpression were measured by RT-PCR. Results 3T3-L1 preadipocytes were fibroblastic and had no obvious fat droplet incytoplasm. However, there were some fat drops in 3T3-L1 cells and thesecells became bigger and rounder on the forth day of differentiation. With timegone, more and more fat drops accumulated in these cells. Compared withpreadipocytes and control group, the concentration of TG mass began toincrease on the forth day of differentiation and increase more significantly onthe eighth day (P＜0.01). The GHSR-1a gene mRNA began to express low onthe first day of differentiation. From the fourth day to the eighth day, theGHSR-1a gene mRNA expression was obviously up-regulated (P＜0.05).There were significant differences between any two detected time points inthe levels of GHSR-1a gene mRNA expression (P＜0.05), except those pointsbetween 0 day to 1 day, and 1 day to 4 day (P＞0.05).Conclusion Insulin+IBMX+DEX can induce 3T3-L1 adipocytes differentiate, andstimulate the TG mass accumulate. GHSR-1a gene mRNA expression isup-regulated during 3T3-L1 adipocyte differentiation. Up-regulation in thelevels of GHSR-la gene mRNA expression during 3T3-L1 adipocytedifferentiation may be involved in the adipogenesis of adipocytes.Partâ…¡Effects of Ghrelin on the Proliferation of 3T3-L1 Preadipoeytes andIts Possible MechanismsObjective To investigate the effects of ghrelin on the proliferation of 3T3-L1preadipocyte and analyze the possible mechanisms. Methods 3T3-L1 preadipocytes were cultured in vitro. The proliferation potentials of3T3-L1 preadipocytes that were treated with different concentrations ofghrelin were evaluated by MTT method and the levels of c-myc andthymidine kinase gene mRNA expression were detected by RT-PCR.Results Ghrelin at concentrations of 10^-7mol/L to 10^-15mol/L significantly stimulatedpreadipocyte proliferation (P＜0.05), with the most pronounced effectobserved at 10^-11mol/L (P＜0.01). Treatment of 3T3-LI preadipocytes with10^-9mol/L and 10^-11mol/L ghrelin significantly increased the levels of c-mycand thymidine kinase gene mRNA expression (P＜0.01).Conclusions Ghrelin can promote the proliferation of 3T3-L1 preadipocytes and enhancethe expression of c-myc, activate thymidine kinase which may play animportant role in the proliferation of 3T3-L1 preadipocytes.Partâ…¢Effects of Ghrelin on the Differentiation of 3T3-L1 Preadipocytesand Its Possible MechanismsObjective To observe the morphological changes, the differentiation rate, TG mass andthe sequential expression of transcription factors in 3T3-L1 adipocytesdifferentiation induced by ghrelin.Methods 3T3-L1 preadipocytes were cultured in vitro and induced to differentiate intomatured adipocytes by 1mg/L Insulin+0.5mM IBMX+1.0μM DEX and10^-11mol/L ghrelin respectively. The morphological changes of 3T3-L1adipocytes were observed by microscope, the differentiation rate was assayedby Oil-Red O staining, TG mass was detected by chemical colorimetry methods, and the levels of peroxisome proliferation activated receptorγ(PPART) and CAAT/enhancer binding protein (C/EBPα) gene mRNAexpression were detected by RT-PCR during the period of 3T3-L1preadipocyte differentiation.Results 3T3-L1 preadipocytes were fibroblastic and had no obvious fat droplet incytoplasm. However, there were some fat drops in 3T3-L1 cells on the forthday induced by ghrelin. And there were a lot of fat drops in 3T3-L1 cells andthese cells became bigger and rounder on the sixth day induced by ghrelin.With time gone, more and more fat drops accumulated in these cells. The rateof differentiation induced by ghrelin was 70%, compared with that byInsulin+IBMX+DEX was 90%. Compared with preadipocytes and controlgroup, the concentration of TG mass began to increase on the forth day ofdifferentiation and increase more significantly on the eighth day (P＜0.01).Compared with the control group, 10^-11mol/L ghrelin significantly increasedthe levels of PPARy and C/EBPαgene mRNA expression during thedifferentiation (P＜0.05). There were significant differences between 4 day to8 day in the levels of PPARγand C/EBPαgene mRNA expression during thedifferentiation induced by ghrelin or Insulin+IBMX+DEX (P＜0.01).Conclusions Ghrelin can induce 3T3-L1 preadipocytes differentiate. During the period ofadipocyte differentiation induced by ghrelin, PPARγand C/EBPαwereinduced to express and keep high levels until the preadipocytes werecompletely differentiated into adipocytes. It suggests that the sequentialexpression of these transcription factors induced by ghrelin could explain apart of the mechanism for ghrelin to induce the preadipocytes differentiation. Partâ…£Effects of Ghrelin on the Apoptosis of 3T3-L1 Preadipocytes and ItsPossible MechanismObjective To explore the possible mechanisms of ghrelin inhibiting apoptosis of 3T3-L1preadipocytes, and analyze the relationship between ghrelin and thepathogenesis of obesity from cellular level.Methods 3T3-L1 preadipocytes were cultured in vitro. The apoptotic rates ofpreadipocytes that were treated with different concentrations of ghrelin weredetected by flow cytometry (FCM) with Annexinâ…¤-FITC and propidiumiodide (PI) double labeling technique. Cell cycle was analyzed by using FCMwith PI labeling technique. The levels of p-ERK1/2 and p-Akt proteinexpression were determined by Western blot.Results The apoptotic rates of 3T3-L1 preadipocytes treated by 10^-11mol/L,10^-13mol/L ghrelin were (42.3±4.6) % and (45.5±5.3) % respectively.Compared with the control group, the apoptotic rates in the above groupsdecreased significantly (P＜0.05), with the most pronounced effect observed at10^-11mol/L. Cell cycle analysis showed that, with the treatment of 10^-11mol/Lghrelin, the cell number in sub-G₀ peak (apoptotic peak) decreasedsignificantly (P＜0.05), while the cell number in G₀/G₁, S and G₂/M had nosignificant changes (P＞0.05). The levels of p-ERK1/2 and p-Akt proteinexpression were significantly enhanced by treatment with ghrelin in 3T3-L1preadipocytes and terminally differentiated adipocytes (P＜0.01).Conclusions Ghrelin can efficiently inhibit the apoptosis of 3T3-L1 preadipocytes withenhancing the expression of p-ERK1/2 and p-Akt.