Role Of NR4A1and Natl In Pathogenesis Of Type2Diabetes

Part I:Distribution of NR4A1in peripheral blood mononuclear cells from patients with Type2DiabetesObjective To investigate the expression levels of nuclear receptor subfamily4group A member1(NR4A1) in peripheral blood mononuclear cells (PBMCs), and further explore its correlation with inflammatory cytokine productions and free fatty acids (FFAs).Methods34healthy subjects and30patients with new diagnosed Type2Diabetes (T2D), were recruited from Wuhan University Zhongnan Hospital. Real-time PCR and ELISA were applied in this study on the role of NR4A1in the relationship of T2D and inflammation. We analyzed NR4A1expression levels in PBMCs from T2D patients and healthy controls, as well as fasting glucose (FBG), fasting insulin (Fins), free fatty acids(FFAs), total cholesterol(TC), triglyceride(TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), C-reactive protein (CRP) and homeostasis model assessment (HOMA-IR). Tumor necrosis factor a (TNF-a), interleukin (IL-6) were detected through ELISA. The association of NR4A1and parameters above were disclosed.Results The basal expression levels of NR4A1in human PBMCs, TNF-a, and IL-6were higher in T2D patients compared to controls, as well as FFAs, TG, LDL-C, CRP and HOMA-IR. Furthermore, NR4A1expression positively correlated with HOMA-IR, FFAs, TNF-a, IL-6, Fins and FBGConclusions NR4A1expression in PBMCs was higher in patients with T2D than controls, positively correlated with HOMA-IR, FFAs, TNF-a, IL-6, FIN and FBG. Finally, PA stimulation increased NR4A1expression and secretion of inflammatory cytokines in cultured PBMCs. Thus, increased NR4A1levels are correlated with chronic low-grade inflammatory state and disorder of lipid metabolism in T2D patients. Part ?:Impact of fat or glucose on NR4A1expression in peripheral blood mononuclear cells from healthy populationObjective To investigate the impact of high level of fat or glucose stimulation on NR4A1expression in PBMCs from healthy population.Methods PBMCs from healthy subjects were collected and cultured with33.3mM glucose or250?M palmitic acid (PA) for2hours. Levels of TNF-? and IL-6in supernatant were measured through ELISA. NR4A1expression was also detected using real-time PCR and western blot.Results In cultured PBMCs from healthy people, NR4A1mRNA increased in250?M PA induced PBMCs compared to controls (15.92±1.75vs.1.00±0.09, p<0.01), as well as NR4A1protein level (2.18±0.12vs.0.87±0.18, p<0.01). However,33.3mM glucose stimulation had no obvious effect on NR4A1expression at mRNA level (1.23±0.17vs.1.00±0.07, p>0.05) or protein level (0.57±0.12vs.0.51±0.09, p>0.05).250?M PA stimulation also induced secretion of TNF-? (543.31±40.08pg/ml vs.26.09±9.35pg/ml, p<0.01) and IL-6(276.82±24.88pg/ml vs.7.80±3.45pg/ml, p<0.01).33.3mM glucose did not affect supernatant level of TNF-? (32.599±37.84pg/ml vs.31.48±4.71pg/ml, p>0.05) and IL-6(6.82±25.91pg/ml vs.4.93±1.53pg/ml, p>0.05).Conclusions Stimulation of high-level fat on human PBMCs induced NR4A1expression, as well as TNF-?, IL-6. However Short-time stimulation of high-level glucose did not change expression of NR4A1, TNF-? or IL-6? Part ?:Impact of Natl on PI3K/Akt pathway of3T3-L1differentiated adipocytesObjective The results of Genome-wide association study (GWAS) revealed that human N-acetyltransferase2(NAT2) associated tightly with insulin resistance. Human NAT2amounts to mouse Natl. The aim of this study is to disclose the impact of Natl on PI3K/Akt pathway of3T3-L1differentiated adipocytes. Methods Transient transfection was performed on3T3-L1preadipocytes after complete differentiation (containing IBMX, dexamethasone, insulin) to upregulate or downregulate Natl expression.48hours later, the differentiated cells were stimulated with or without insulin for1hour. We collected the cells lysate to detect the activity of PI3K/Akt signaling pathway by western-blot. The expression levels of Natl mRNA with or without differentiation were measured by real-time PCR.Results Upregualtion or downregulation did not affect the expression of insulin signaling pathway members, total Akt, p-Akt, total IRS1and p-IRS1, in differentiated mouse3T3-L1adipocytes.Conclusions No obvious difference was found in the phosphorylation of insulin signaling members upon Natl silence or overexpression Part IV:Regulation of Natl on adipogenesis of3T3-L1pre-adipocytesObjective To investigate the role of N-acetyltransferase1(Natl) in adipogenesis of3T3-L1pre-adipocytes.Methods Transduction of pLV-GFP-sh?pLV-GFP?pWPI-GFP-Nat1?pWPI-GFP were performed to3T3-L1preadipocytes. Thus the expression of Natl was downregulated and upregulated. After that, the transduced cells were treated differentiation medium (containing IBMX, dexamethasone, insulin).14-day post differentiation, we measured the differentiation percentage by oil red O staining and flow cytometry.Results Compared to controls, pLV-GFP-sh transduced3T3-L1preadipocytes differentiated at lower percentage and pWPI-GFP-Natl transduced3T3-L1preadipocytes differentiated at higher percentage.Conclusions Silencing Natl decreases adipogenesis and overexpression of Natl induces adipogenesis.