Mechanism of acylation stimulating protein resistance induced by fatty acid in 3T3-L1 adipocytes and preadipocytes / 中国病理生理杂志

ABSTRACT

AIM: To evaluate the potential acylation stimulating protein (ASP) resistance in both adipocytes and preadipocytes under the conditions by which insulin resistance is produced by the stimulation of free fatty acids (FFA), and to explore the mechanism of ASP resistance on post-receptor level. METHODS: 3T3-L1 preadipocytes were induced to differentiate. Then the cells were treated with oleate or palmitate at concentration of 0 mmol/L (FFA-free DMEM/F12), 0.125 mmol/L, 0.5 mmol/L or 1.0 mmol/L overnight. Glucose transport was assessed by [~3H] 2-deoxyglucose uptake to evaluate insulin resistance and ASP resistance. Both non-FFA treated and FFA treated 3T3-L1 cells were cultured with ASP at concentration of 5.0 μmol/L for 4 h, then the cell proteins were extracted, and the expressions of guanine nucleotide binding protein beta (Gβ), guanine nucleotide-binding protein alpha-q/11(Gαq/11), phosphorylated-protein kinase Cα (p-PKCα) and phosphorylated-protein kinase Cζ (p-PKCζ) were measured by Western blotting. RESULTS: Both adipocytes and preadipocytes were responsive to ASP. ASP stimulation increased glucose transport by 198% in adipocytes and by 287% in preadipocytes (P<0.01 vs PBS). FFA at concentration of 0.125 mmol/L did not change ASP-stimulated glucose transport significantly, but high dose of oleate or palmitate effectively reduced the ASP response with a significant reduction by 47% (P<0.05 for oleate) and 34% (P<0.05 for palmitate) at 1 mmol/L FFA in adipocytes. Similarly in preadipocytes, glucose uptake rates were decreased by 43% (P<0.05 for oleate) and 62% (P<0.01 for palmitate) at 1 mmol/L FFA. Effects were comparable to those obtained with insulin. After overnight incubation with oleate or palmitate in adipocytes and preadipocytes, Gβ, Gαq/11, p-PKCα and p-PKCζ were downregulated both in the absence of ASP treatment and in the presence of ASP treatment in adipocytes. At concentration of 1.0 mmol/L, oleate inhibited the expressions of ASP-induced Gβ, Gαq/11, p-PKCα and p-PKCζ in adipocytes by 47%, 44%, 39% (P<0.05, P<0.01) and 20% (P>0.05), respectively. Palmitate also effectively blocked the expressions of ASP (at concentration of 1.0 mmol/L)-induced Gβ, Gαq/11, p-PKCα and p-PKCζ by 50%, 43%, 44% and 43% (P<0.05, P<0.01) in adipocytes. In preadipocytes, oleate only inhibited ASP-induced p-PKCα and p-PKCζ significantly by 39% and 19%, respectively (P<0.05). However, overnight exposure of 3T3-L1 preadipocytes to 1 mmol/L palmitate leaded to 45%, 50%, 52% and 21% (P<0.05, P<0.01) inhibition of ASP-induced expressions of Gβ, Gαq/11, p-PKCα and p-PKCζ, respectively. CONCLUSION: Oleate and palmitate inhibit ASP-mediated stimulation of glucose transport both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance under the condition of insulin resistance induced by FFA in a cellular model. The mechanism of action involves both changes in expression of C5L2 as well as signaling parameters. Fatty acid-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and obesity phenotype.