Effect Of Adipocyte Coculture, IL-6and Resistin On Expression Of ABCA1in HepG2Cells And Its Mechanisms

BackgroundObesity especially visceral obesity is a significant component of metabolic syndrome, and it in humans is always associated with low level of high-density lipoprotein cholesterol (HDL-C). As an important active endocrine and paracrine organ, adipose tissue with obesity releases a large number of dysfunctional cytokines (adipokines) such as Interleukin-6(IL-6), resistin, Tumor necrosis factor-alpha (TNF-alpha) etc. Combined with the fact, that in the intron of Sterol-regulatory element binding protein (SREBP), a microRNA called miR-33could regulate the expression of ATP-binding cassette transporter Al (ABCA1) gene and HDL metabolism. We speculate that the relevant adipokines of obesity might affect SREBP-miR33, resulting in ABCA1and HDL-C decrease.ObjectiveThis study establishes one model that co-culture of hepatic cell and mature fat cell, to investigate the effect of mature fat cell、IL-6and resistin treatment on the expression of ABCA1, APOA1and SREBP-miR33of HepG2cells.MethodCulturing3T3-L1cell and promoting it differentiation to fully mature cell (8days). Human adipose-derived mesenchymal stem cells (ADSCs), derived from subcutaneous adipose tissue of patients undergoing abdominal surgery, were differentiated into mature adipocytes. Then co-culture HepG2cells with3T3-L1and mature mice adipocyte for 48hours; and co-culture HepG2cells with ADSCs and mature human adipocyte for48hours. While the HepG2cultured alone is signed as a normal control group. Extractting protein and RNA of those groups of HepG2cells, taking the following experiment:1. Detect ABCAl protein expression of those groups of liver cells by western blot method.2. Determine miR-33a, miR-33b, SREBP-1c, SREBP-2, ABCA1and APOA1mRNA expression of the three groups of liver cells by Real-time polymerase chain reaction (Real-time PCR).Then HepG2cells were treated with lOng/ml IL-6and25,50,100ng/ml resistin, respectively. The untreated cells were used as control group. After24hours, the total RNA and protein were extracted. The relative gene expression of miR-33a, miR-33b, SREBP-1c, SREBP-2, ABCA1and APOA1were measured by real-time PCR. The relative protein expression of ABCAl was measured by Western Blot.Results1. Mice or human adipocytes coculture significantly reduced the ABCAl protein and mRNA in HepG2cells (P<0.05);2. Compared with the control group, the ABCA1protein and mRNA in HepG2cells of IL-6group were significantly down-regulated (P<0.05); however, the gene expressions of APOAl were unchanged (P>0.05);3. Compared with the control group,50and100ng/ml Resistin significantly reduced the ABCA1protein in HepG2cells (P<0.05), whereas25ng/ml Resistin didn’t affect the ABCA1protein expression (P>0.05); but the three kinds of concentration Resistin didn’t change ABCA1and APOA1mRNA obviously (P>0.05);4. That mice or human adipocytes coculture reduced the SREBP2/miR-33a or SREBP-lc/miR-33b expression in HepG2cells (P<0.05);5. That IL-6or Resistin didn’t change SREBP2/miR-33a or SREBP- lc/miR-33b expression in HepG2cells (P>0.05).ConclusionsThat mice or human adipocytes coculture which were constructed by Transwell chambers reduced the ABCA1expression in HepG2cells may partly explain the phenomenon of obesity always associated with low HDL-C; IL-6reduced the ABCA1expression and Resistin increased ABCA1protein degradation in HepG2cells, suggesting that the low HDL-C level was due in part to the secretion abnormal adipokines by adipocytes.