Nyggf4 On Insulin Sensitivity Of Fat Cells And Mechanism Analysis

Childhood obesity is a significant health problem that has reached epidemic proportions around the world and is associated with several metabolic and cardiovascular complications. Insulin resistance is a common feature of childhood obesity and is considered to be an important link between adiposity and the associated risk of type 2 diabetes. The prevalence of insulin resistance in the paediatric population is increasing, particularly among obese children and adolescents. Hence, exploring the pathogenesis of insulin resistance is necessary for peadiatric researchers.In an earlier study, we isolated and characterized NYGGF4, a novel gene whose expression was higher in adipose tissue from obese patients than in that from normal controls. This gene was primarily expressed in insulin-sensitive tissues, including skeletal muscle, adipose tissue and heart. We also found that the expression of NYGGF4 mRNA could be regulated by a variety of factors that are related to insulin sensitivity (such as TNFα,IL-6, et al). Our findings suggested that NYGGF4 might be a new candidate gene related to obesity-associated insulin resistance. So, in this study, we aimed to examine the effects of NYGGF4 on the insulin sensetivity of mature adipocytes and determine the molecular events underlying these effects.Part I: Effects of NYGGF4 overexpression on the insulin sensitivity and secretion founction of adipocytes and effects on the insulin signal pathwayAim: To investigate the effects of NYGGF4 on the insulin sensitivity and secretion founction of 3T3-L1 adipocytes and further to explore its association with insulin signal pathway. Methods: 3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1Myc/His B) or an NYGGF4 expression vector were differentiated into mature adipocytes. Glucose uptake was determined by measuring 2-deoxy-D- [3H] glucose uptake into the adipocytes. Immunoblotting was performed to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4). Immunoblotting also was used to measure the phosphorylation and total protein contents of insulin signaling proteins such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt, ERK1/2, p38, and JNK. The supernatant concentration of TNF-α, IL-6, adiponectin and resistin were measured by enzyme - linked immunosorbent assay. Results: 1) Tehre were no significant differences in cuture supernatant levels of TNF-α, IL-6, adiponectin and resistin between 3T3-L1 transfected with NYGGF4 and the control cells. 2) NYGGF4 overexpression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. 3) NYGGF4 overexpression impaired insulin stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, and JNK.Conclusion: Overexpression of NYGGF4 induced insulin resistance in 3T3-L1 adipocytes via attenuated phosphorylation of IRS-1 and Akt and had no effect on the secretory function of adipocytes.Part II: Effects of NYGGF4 overexpression on mitochondrial functionAim: To investigate the effects of NYGGF4 overexpression on mitochondrial function of 3T3-L1 adipocytes.Methods: 3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1Myc/His B) or an NYGGF4 expression vector were differentiated into mature adipocytes. The mitochondrial morphology was examined by transmission electron microscope. The mitochondrial DNA (mtDNA) copy number was evaluated by real-time quantitative PCR. Cellular ATP production was determined using luciferase-based luminescence assay. The mitochondrial membrane potential (ΔΨm) was detected by JC-1 with flow cytometry and reactive oxygen species (ROS) by DCFDA(2',7'-Dichlorofluorescein diacetate,DCFDA)with confocal laser microscopy and flow cytometry. The mRNA levels of uncoupling protein 2, 4 (UCP2, UCP4), Peroxisome proliferator-actived receptorγ(PPARγ) coactivator-1 (PGC-1), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (mtTFA), mitofusin gene 1 (Mfn1), mitofusin gene 2 (Mfn2), dynamin-related protein 1 (Drp1) and Fis1 were measured by real time quantitive RT-PCR.Results: 1) The mitochondria in NYGGF4 overexpressing cells were smaller in size with condensed abnormal morphology. Signs of mitochondrial damage ranged from swelling and reduced density to virtually hollow mitochondria with broken double membranes. 2) The mitochondrial DNA (mtDNA) copy number in NYGGF4 overexpressing adipocytes was significantly decreased. 3) Total cellular ATP was dramatically decreased in NYGGF4 overexpressing adipocytes. 4) NYGGF4 dramatically increased the ROS levels in adipocytes and slightly decreased the mitochondrial membrane potential. 5) NYGGF4 significantly increased UCP2 and UCP4 mRNA levels in adipocytes. 6) NYGGF4 did not alter the mRNA expression of NRF1 and mtTFA but upregulated the mRNA levels of PGC-1αand PGC-1β. 7) NYGGF4 overexpression resulted in a significant increase in the Mfn1 mRNA and mild decrease in Fis1 mRNA, while it did not affect Mfn2 and Drp1 mRNA expression. Conclusion: The overexpression of NYGGF4 may cause mitochondrial dysfunction in adipocytes, which might be responsible for the development of NYGGF4 induced insulin resistance.Part III: Effects of gene silencing of NYGGF4 on insulin sensitivity and mitochondrial function in adipocytesAim: To investigate the effects of gene silencing of NYGGF4 on insulin sensitivity and mitochondrial function in adipocytes.Methods: 3T3-L1 preadipocyte transfected with pGPU6/GFP/Neo-shRNA -NYGGF4 expression vector were differentiated into mature adipocytes. After pretreatment with insulin or insulin combination with phosphatidylinositol-3 kinase inhibitor (LY294002), glucose uptake was determined by measuring 2-deoxy-D-[3H] glucose uptake into the adipocytes.The methods were adopted to detect mitochondrial function as described in Part II.Results: 1) Suppression of NYGGF4 expression by RNAi caused significant increase in basal glucose uptake of 3T3-L1 adipocytes. 2) Gene silencing of NYGGF4 dramatically enhanced insulin-stimulated glucose uptake and the effect was attenuated by pretreatment with LY294002. 3) NYGGF4 gene silencing had no effect on the morphology and number of mitochondrion in adipocytes. 4) Suppression of NYGGF4 expression substantially reduced celluar ATP production. 5) NYGGF4 gene silencing significantly increased the ROS levels in adipocytes and slightly increased the mitochondrial membrane potential. 6) Gene silencing of NYGGF4 upregulated mRNA level in Fis1 and had no effect on other detected genes.Conclusion: 1) Gene silencing of NYGGF4 enhanced insulin sensitivity of adipocytes through activating phosphatidylinositol-3 kinase pathways. 2) Gene silencing of NYGGF4 resulted in a certain degree of damage in mitochondrial function, which suggested that NYGGF4 might play some role in maintain normal function of mitochondrion.