Studies On Regulation And Regulation Mechanism Of Nutrition And Hormone On Adipogenesis In Primary Rat Adipocytes

Adipose tissue is not only a specialized passive storage organ of energy in original and classical view, but also an active player in energy metabolism of whole body. Adipose tissue exerts a profound influence on metabolism and health of mammalians through producing and secreting dozens of factors that act either in an auto/paracrine or an endocrine fashion. In the past several decades, obesity and obesity-related diseases have become extremely common, with prevalence in rates skyrocketing among certain groups and communities, as traditional dietary approaches to combat obesity have largely failed. It is an important research goal in domains of animal breeding and feeding to decrease fat accumulation of animal body. Adipogenesis includes increase both in amount of and in volume of adipocytes, which involves proliferation of preadipocytes, differentiation of preadipocytes into mature fat cells, and triglyceride synthesis. Adipogenesis is influenced with many factors, such as nutrients and hormones. Carbohydrate is the main energy source and lipogenic substrate for animals, and it and hormones regulate adipogenesis together through several signalling pathways. Thus, it is highly important to elucidate the effects of nutritions and hormones on adipogenesis and molecular mechanism underlying these roles in control of fat accumulation of an animal body.In this paper, through isolating and culturing rat preadipocytes, we investigate regulations of glucose, insulin, glucocorticoid (dexamethasone), tumor necrosis factor-α (TNF-α) and nutrition status on preadipocyte proliferation and differentiation, and lipogenisis and transcriptional expressions of lipogenic-related genes in mature adipocytes in vitro, to explore mechanisms of nutritional and hormonal regulation of lipogenesis. The research results were summarized as following:1. Insulin markedly stimulated preadipocyte proliferation in dose- and time-dependent manner, and preadipocyte differentiation in dose-dependent manner.2. Dexamethasone significantly inhibited rat preadipocyte proliferation and this inhibition had a dose-effect relation within concentration of 150 nmol/L. High concentration of dexamethasone (above 100 nmol/L) markedly enhanced preadipocyte differentiation and the stimulation increased upon lasting time.3. The preadipocyte proliferation enhanced upon glucose concentration within 15 mmol/L glucose. The preadipocyte differentiation increased upon increasing glucose concentration within 15 mmol/L concentration, but proliferation and differentiation of preadipocytes were decreased by higher concentration of glucose (25 mmol/L) due to "glucose toxicity".4. Within 20 mmol/L of concentration, glucose stimulated fatty acid biosynthesis and lipogenisis through inducing mRNA expressions of lipogenic genes FAS, ACC1 and transcription factors ChREBP and SREBP-lc in primary cultured mature adipocytes.5. The mRNA levels of lipogenic genes FAS and ACC1 were increased by insulin treatmemts and this stimulation of insulin was dose-dependent within concentration of 300 nmol/L in primary cultured mature adipocytes under culture condition of low glucose (5 mmol/L). Insulin markedly increased expression levels of FAS and ACC1 mRNA in high glucose of media (15 mmol/L glucose). Higher concentration of insulin, however, especially in high glucose of media, reduced the stimulation of insulin on the transcription expressions of lipogenic enzyme genes. mRNA level of SREBP-lc was not altered by insulin treatments in low and high glucose media. The effect of insulin on ChREBP mRNA level was in accordance with FAS and ACC1 mRNA. The results suggested the insulin-Stimulated FAS and ACC1 gene expressions did not require increased SREBP-lc mRNA level, but ChREBP might be essential to thansactivation in primary cultured mature adipocytes.6. After insulin resistance of primary adipocytes was induced with 300 nmol/L dexamethasone, lipogenisis and mRNA levels of FAS, ACC1, ChREBP, SREBP-lc genes significantly were reduced, compared to untreated adipocytes. Insulin treatments could partly reduce inhibition of insulin resistance on lipogenisis and transcription expressions of FAS and ACC1 genes and had maximum in 200 nmol/L concentration. Though SREBP-lc mRNA level was not influenced, ChREBP mRNA level altered in coincidence with FAS and ACC1 by insulin in dexamethasone-reduced insulin resistant adipocytes, which supported the previous conclusion that insulin-Stimulated FAS and ACC1 gene expressions require ChREBP and do not require increased SREBP-lc mRNA level in primary cultured adipocytes.7. Experiment mode of serum-deprivation/re-serum culture was employed to study mechanism of nutrition status regulation of expression of lipogenic enzyme genes in vitro. The results showed that ChREBP mRNA level was dependent on nutrition status in primary adipocytes: reduced decreasingly upon lasting time of serum-deprivation and increased according to lasting time of re-serum culture, which coincided with lipogenesis and mRNA levels of ACC1 and FAS. Thus it could be concluded that nutrition status influenced transcription expressions of ACC 1 and FAS genes through regulation of ChREBP expression in adipocytes.8. mRNA levels of lipid metabolic-related genes were determined in primary adipocytes at different stages of differentiation to study their expression sequence. The results showed that none mRNA of examined genes including SREBP-lc, ChREBP, SCD, ACC1, FAS and HSL mRNAs expressed in preadipocyte. FAS mRNA level was highest in early stage ofdifferentiation and then decreased, but had no significant difference between middle and terminal stage of differentiation. SCD and ChREBP mRNAs did not express in early stage of differentiation, whereas did in middle and terminal stage of differentiation, and SCD mRNA level was significantly and ChREBP mRNA trended higher in middle stage than in terminal stage of differentiation. ACC1 mRNA did not express in early and middle stage of differentiation, though had high expression level in terminal stage of differentiation. HSL mRNA began to express in early stage of differentiation, and significantly increased in middle and terminal stage of differentiation. SREBP-lc mRNA expressed in differentiating adipocytes, and had the highest expression level in early stage of differentiation, and reduced dependent differentiating progress. These data suggested that SREBP-lc expression was responsible for regulation of adipocyte differentiation, whereas it was not as important to transcription regulation of lipogenic enzyme genes in mature adipocyte as in liver. ChREBP expression did not related to early differentiation of adipocytes, but might regulate transcription expression of lipogenic enzyme genes in terminal adipocytes.9. TNF-a is a pleiotropic cytokine that can be secreted in adipose tissue. To study the effects of TNF-a on adipogenesis stimulated by insulin in adipose tissue in vitro, rat preadipocytes were isolated and cultured in media added with TNF-a, insulin and TNF-a+insulin, respectively. The results showed that TNF-a effectively inhibited preadipocyte proliferation stimulated by insulin and decreased lipogenesis through inhibiting stimulation of insulin on transcriptional expressions of lipogenic genes such as ACC1, FAS in adipocytes, suggesting that TNF-a is an effective inhibitor of adipogenesis in adipose tissue. In addition, mRNA expression of ChREBP was significantly inhibited by TNF-a.