| Part I Sequential Expression of Acylation Stimulating Protein Receptor in Preadipocytes DifferentiationObjectiveTo elucidate the sequential changes in acylation stimulating protein (ASP) receptor expression during differentiation of 3T3-L1 preadipocytes.Methods3T3-L1 preadipocytes were induced differentiated by cocktail hormones containing 10μg/mL insulin, 1μmol/L dexamethasone and 0.5 mmol/L isobutylmethylxanthine. The morphologic changes of 3T3-L1 cells were observed by microscope. Fat droplets were observed in the cytoplasm by Oil red O staining. Then immunofluorescence was used to observe the ASP receptor location on the surface of 3T3-L1 cells. RT-PCR and flow cytometry were used to detected mRNA and protein expression of ASP receptor.Results1. 3T3-L1 preadipocytes were fibroblastic, and no fat droplet was noted in cytoplasm. After fully differentiated, the adipocytes became bigger, rounder and larges of fat drops accumulated, which looked like the finger ring.2. ASP receptor (C5L2) mRNA expression was very low on 0d after confluence, and the expression was increased significantly on day 1 after inducing differentiation. C5L2 mRNA kept on increasing with a significant 2.08-fold, 2.44-fold and 3.56-fold stimulation seen on day 3, day 6 and day 9 of differentiation, respectively, as compared to undifferentiated cells (p<0.05 to p<0.01). While C5L2 protein was up-regulated at the early phase of preadipocytes differentiation. C5L2 protein expression was on middle level in preadipocytes (50.71%±14.58%, positive gated). Then it increased significantly by 21% (61.42%±18.33% vs 50.71%±14.58%,positive gated, p<0.05) at 6h after inducing differentiation and reached a maximal effect at 12h of 38% increase(69.90%±11.88% vs 50.71%±14.58%, positive gated, p<0.01). C5L2 protein then decreased significantly on the third day after induction to the level on day 0 and kept the middle level on day 6 and day 9. C5L2 located on the surface of 3T3-L1 cells and the fluorescence intensity is similar to that of by flow cytometry.ConclusionsThese results demonstrate that the ASP-C5L2 pathway plays a role in preadipocytes differentiation. The difference between the C5L2-mRNA and protein during preadipocytes differentiation remains to be further elucidated.Part II Study of the Roles of Acylation Stimulating Protein-Receptor C5L2 Pathway Insulin Resistance Induced by Fatty Acids in Adipocytes and PreadipocytesObjectiveTo evaluate potential ASP resistance in both adipocytes and preadipocytes under the conditions which produce insulin resistance by free fatty acids (FFA).Then to explore the mechanism of ASP resistance on both receptor level and post-receptor level.Methods3T3-L1 preadipocytes were induced differentiated and 0mmol/L (FFA-free DMEM/F12), 0.125 mmol/L, 0.5 mmol/L and 1.0 mmol/L oleate or palmitate was added to cultured 3T3-L1 adipocytes and preadipocytes overnight. Glucose transport was assessed as [3H] 2-deoxy glucose uptake to evaluate insulin resistance and ASP resistance. RT-PCR and flow cytometry were used to detected mRNA and cell surface expression of ASP receptor. Both non-FFA treated and FFA treated 3T3-L1 cells were cultured with 5.0μmol/L ASP for 4 hours. Then the cell proteins were extracted and the expressions of Gβ,Gαq/11,p-PKCαand p-PKCζwere measured by western blot.Results1. Insulin increased specific membrane glucose transport in 3T3-L1 preadipocytes. At the time of 15min-1h, stimulation was upregulation. However, after 6h exposure to insulin, there was a downregulation in the response. Dose response studies demonstrated that 2-DG transport increased by 336% at 50nmol/L insulin (P<0.01) ,reaching a maximal effect at 100nmol/L insulin with a significant 4.94-fold stimulation as compared to control (P<0.01). According to the results, the experiments below were treated with 100nmol/L insulin for 1 hour to evaluate insulin resistance.2. Oleate and palmitate treatment did not influence basal glucose transport (without insulin stimulation), whereas insulin-stimulated glucose transport was inhibited after overnight oleate and palmitate treatment in adipocytes and preadipocytes. In adipocytes, the inhibition was noted at 0.5 mmol/L oleate or 1.0 mmol/L palmitate. In preadipocytes, insulin resistance could be achieved at 0.125 mmol/L oleate or palmitate (P<0.05, respectively), and the inhibition was dose dependent.3. Both adipocytes and preadipocytes were responsive to ASP. ASP stimulated glucose transport by 198% in adipocytes and by 287% in preadipocytes( p<0.01 respectively, vs. PBS alone). 0.125 mmol/L FFA did not change ASP-stimulated glucose transport significantly, but high dose oleate or palmitate effectively reduced the ASP response with a significant reduction by 47% (p<0.01, oleate) and 34% (p<0.05, palmitate) at 1 mmol/L FFA in adipocytes. Similarly in preadipocytes, Glucose uptake rates were decreased by 43% (p<0.01, oleate) and 62% (p<0.01, palmitate) at 1 mmol/L FFA. Effects were comparable to those obtained with insulin.4. Oleate and palmitate suppressed C5L2 mRNA and protein expression in 3T3-L1 adipocytes but not preadipocytes. Significant reduction could be achieved at 0.5mmol/L oleate and reached a maximal reduction of 84% (0.18±0.04 vs 1.08±0.46, p<0.05) for C5L2 mRNA expression at 1.0 mmol/L oleate. However, the inhibition for C5L2 protein was noticeable at 0.125 mmol/L oleate (34.77%±8.02% vs 54.18%±7.11%, positive gated, p<0.01). Furthermore, both C5L2 mRNA and protein expression were dose-dependently suppressed by palmitate in 3T3-L1 adipocytes with a significant reduction first observed at concentrations as low as 0.125 mmol/L with a maximal 41%(0.57±0.28 vs 0.97±0.41, p<0.05) and 55%(24.24%±13.01% vs 54.18%±7.11%, positive gated, p<0.01) reduction respectively, at concentrations of 1.0 mmol/L.5. Following ASP treatment of adipocytes, there were significant increases in Gβprotein by 26 % , Gαq/11 protein by 32 % , phosphorylated PKCα( p-PKCα) by 71 % and phosphorylated PKCζ( p-PKCζ) by 49% (p<0.05 to p<0.001). Similar changes were detected in preadipocytes for Gβ, Gαq/11, p-PKCαand p-PKCζ(p<0.05 to p<0.01).6. After overnight incubation with oleate and palmitate (in adipocytes and preadipocytes), Gβ, Gαq/11, p-PKCαand p-PKCζwere downregulated both in the absence of ASP treatment and in the presence of ASP treatment in adipocytes. In adipocytes, at 1.0mmol/L, oleate inhibited the ASP-induced Gβ, Gαq/11, p-PKCαand p-PKCζexpression by 47%,44%,39%(p<0.05 to p<0.01)and 20%(p>0.05), respectively. Palmitate also effectively blocked the ASP-induced Gβ, Gαq/11, p-PKCαand p-PKCζexpression by 50%,43%,44% and 43% (p<0.05 to p<0.01) at 1.0mmol/L in adipocytes. In preadipocytes, oleate only inhibited ASP-induced p-PKCαand p-PKCζsignificantly by 39% and 19%, respectively (p<0.05). However, overnight exposure of 3T3-L1 preadipocytes to 1mmol/L palmitate leaded to 45%,50%,52% and 21%(p<0.05 to p<0.01)inhibition of ASP-induced Gβ, Gαq/11, p-PKCαand p-PKCζexpression, respectively.Conclusions1. Oleate and palmitate inhibited insulin-mediated stimulation and ASP-mediated stimulation of glucose transports both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance for the first time under the conditions of insulin resistance induced by FFA in a cellular model.2. Asp binding to its receptor (C5L2, 7-transmembrane G ptotein-coupled receptor) activation of Gβγ, Gαq/11, PKCαand PKCζproteins will lead to modulate preadipocyte differentiation and glucose metabolism related to ASP biological function.3. The mechanism of action involves both changes in expression of C5L2 as well as signaling parameters, and provides a basis for understanding and examining ASP resistance in human studies. Fatty acid-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance/ an obesity phenotype.Part III Study of the Roles of Acylation Stimulating Protein-Receptor C5L2 Pathway in Insulin Resistance Induced by Sex Hormones in Adipocytes and PreadipocytesObjectiveTo evaluate potential ASP resistance in both adipocytes and preadipocytes under the conditions which produce insulin resistance by sex hormones including 17β-estradiol, testosterone and progesterone. Then to explore the mechanism of ASP resistance on both receptor level and post-receptor level.Methods3T3-L1 preadipocytes were induced differentiated and 0mol/L (sex hormone-free DMEM/F12), 10-8 mol/L, 10-7 mol/L and 10-6 mol/L 17β-estradiol, testosterone or progesterone was added to cultured 3T3-L1 adipocytes and preadipocytes overnight. Glucose transport was assessed as [3H] 2-deoxy glucose uptake to evaluate insulin resistance and ASP resistance. RT-PCR and flow cytometry were used to detected mRNA and cell surface expression of ASP receptor. Both non-sex hormone treated and sex hormone treated 3T3-L1 cells were cultured with 5.0μmol/L ASP for 4 hours. Then the cell proteins were extracted and the expressions of Gβ,Gαq/11,p-PKCαand p-PKCζwere measured by western blot.Results1. 10-8 mol/L～10-6 mol/L 17β-estradiol, testosterone and progesterone treatment did not influence basal glucose transport (without insulin stimulation), whereas insulin-stimulated glucose transport was inhibited after overnight 17β-estradiol and testosterone treatment in adipocytes and preadipocytes at high dose. In adipocytes, the inhibition was noted at 10-6 mol/L 17β-estradiol and 10-7 mol/L testosterone. In contrast, no inhibition was induced by progesterone in adipocytes. In preadipocytes, insulin resistance could be achieved at 10-6 mol/L 17β-estradiol and testosterone with a significant reduction by 32% and 40%, respectively (P<0.05, respectively). Progesterone decreased insulin-stimulated glucose transport in dose-dependent manner in preadipocytes with a maximal inhibition by 38% (p<0.01) at 10-6 mol/L.2. 10-8 mol/L 17β-estradiol and testosterone increased ASP-stimulated glucose transport slightly in adipocytes(p>0.05, respectively), but high dose 17β-estradiol and testosterone effectively inhibited the ASP response with a significant inhibition by 46% (p<0.01) and 46% (p<0.05), respectively, at 10-6 mol/L in adipocytes. Whereas progesterone decreased ASP response at 10-8 mol/L with an effective decrease by 55% (p<0.01) and 52% (p<0.01) at 10-7 mol/L and 10-6 mol/L in adipocytes, respectively. Similarly in preadipocytes, high dose sex hormones inhibited ASP-stimulated glucose uptake significantly at 10-6 mol/L.3. High dose 17β-estradiol and testosterone suppressed C5L2 mRNA and protein expression in 3T3-L1 adipocytes but not preadipocytes. At 10-8 mol/L, 17β-estradiol increased C5L2 mRNA and protein expression slightly in both adipocytes and preadipocytes (p>0.05, respectively). At 10-6 mol/L, 17β-estradiol inhibited C5L2 mRNA and cell surface C5L2 expression by 40% (p<0.05) and 21% (p<0.05), respectively. Testosterone inhibited C5L2 mRNA and cell surface C5L2 expression by 60% (p<0.01) and 27%(p<0.01), respectively. At 10-6 mol/L, progesterone only downregulated C5L2 protein expression significantly by 22% (p<0.01) in adipocytes. However, both C5L2 mRNA and protein expression were suppressed by progesterone with a significant reduction by 66%(p<0.01) and 29%(p<0.05) in preadipocytes, respectively, at concentrations of 10-6 mol/L.4. After overnight incubation with sex hormones (in adipocytes and preadipocytes), Gβ, Gαq/11, p-PKCαand p-PKCζwere downregulated in the presence of ASP treatment to a certain degree. In adipocytes, at 10-6 mol/L, 17β-estradiol inhibited the ASP-induced Gβ, Gαq/11, p-PKCαand p-PKCζexpression by 23%,17%,15% and 20%15%(p>0.05, respectively). Whereas testosterone effectively blocked the ASP-stimulated Gαq/11, p-PKCαand p-PKCζexpression by 52%,50% and 57% (p<0.05 to p<0.01, respectively) at 10-6 mol/L in adipocytes but not Gβ. Similar to testosterone, after overnight exposure 3T3-L1 adipocytes to 10-6 mol/L progesterone, caused 63%,41%,49%(p<0.05 to p<0.01, respectively)and 32%(p>0.05)reduction of ASP-stimulated Gβ, Gαq/11, p-PKCαand p-PKCζexpression, respectively. In preadipocytes, high dose 17β-estradiol only inhibited ASP-stimulated Gαq/11 significantly by 24%(p<0.05) and testosterone did not influence the four proteins expression(p>0.05, respectively). However, overnight incubation 3T3-L1 preadipocytes with progesterone, the reduction for ASP-induced Gβ, Gαq/11 and p-PKCζexpression was observed at concentrations as low as 10-8 mol/L, and the four proteins were inhibited at 10-6 mol/L at the same time.Conclusions1. Sex hormones inhibited insulin-mediated stimulation and ASP-mediated stimulation of glucose transports both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance once more under the conditions of insulin resistance induced by 17β-estradiol, testosterone or progesterone in a cellular model.2. The ASP resistance mechanism of action involves both changes in expression of C5L2 as well as signaling parameters to some extent. Sex hormones-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and some abnormalities related with sex hormones such as disorders in late pregnancy, gestational diabetes mellitus and polycystic ovarian syndrome and so on.
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