Wen Yu, Wang Hong-wei, Wu Jing, Lu Hui-ling, Hu Xiu-fen, Cianflone Katherine
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Chin Med J (Engl). 2006 Oct 20;119(20):1701-8.
The levels of long-term elevated serum or intracellular free fatty acid (FFA) induce insulin resistance associated with central obesity. The insulin-mimetic protein visfatin is preferentially produced by visceral adipose tissues and has been implicated in obesity and insulin resistance. To identify that FFA is capable of inducing insulin resistance and to clarify the role of FFA on visfatin, we examined the effect of monounsaturated FFA oleate (C18:1) and saturated FFA palmitate (C16:0) on glucose transport and visfatin gene expression in cultured 3T3-L1 adipocytes or preadipocytes.
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 or preadipocytes and incubated overnight. Glucose transport was assessed as (3)H-2-deoxy-glucose uptake. Total RNA was extracted and subjected to RT-PCR for the measurement of visfatin mRNA levels. Statistical comparisons between control group and other groups were performed with the two-tailed paired t test, and one-way ANOVA was used to compare the mean values among the groups.
Insulin increased specific membrane glucose transport in 3T3-L1 preadipocytes. Upregulation was evident from 15 minutes to 1 hour exposure to insulin. However, after 6-hour exposure to insulin, there was a downregulation in the response to insulin. Dose response studies demonstrated that 2-deoxy glucose transport was increased by 336% at 50 nmol/L insulin (P < 0.01), and reached a maximal effect at 100 nmol/L insulin (P < 0.01). 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 preadipocytes and adipocytes. In 3T3-L1 preadipocytes, insulin resistance could be achieved at 0.125 mmol/L oleate or palmitate (P < 0.05, respectively), and the inhibition was dose dependent. In adipocytes, the inhibition was noted at 0.5 mmol/L oleate or 1.0 mmol/L palmitate. Visfatin mRNA expression increased during differentiation more than 1.5-fold. Bovine serum albumin (BSA) did not influence visfatin mRNA expression compared with the control group. Dose-response studies demonstrated that addition of 0.125 mmol/L oleate and palmitate to 3T3-L1 adipocytes decreased visfatin mRNA expression significantly (78%, 77%, respectively, relative to untreated control, P < 0.05), and further to 65% (relative to untreated control, P < 0.05) and 55% (relative to untreated control, P < 0.01) at 1.0 mmol/L FFA. Furthermore, the suppression on preadipocytes was similar to that of adipocytes, which reached a maximal reduction of 44% (oleate, P < 0.05) and 47% (palmitate, P < 0.05) at 1.0 mmol/L FFA.
Oleic acid and palmitic acid may induce insulin resistance in 3T3-L1 adipocytes and preadipocytes. Downregulation of visfatin mRNA may contribute to impair insulin sensitivity caused by oleate and palmitate.
长期升高的血清或细胞内游离脂肪酸（FFA）水平会导致与中心性肥胖相关的胰岛素抵抗。胰岛素模拟蛋白内脂素优先由内脏脂肪组织产生，并与肥胖和胰岛素抵抗有关。为了确定FFA能够诱导胰岛素抵抗，并阐明FFA对内脂素的作用，我们研究了单不饱和FFA油酸（C18:1）和饱和FFA棕榈酸（C16:0）对培养的3T3-L1脂肪细胞或前脂肪细胞中葡萄糖转运和内脂素基因表达的影响。
将不含FFA的DMEM/F12、0.125 mmol/L、0.5 mmol/L和1.0 mmol/L的油酸或棕榈酸添加到培养的3T3-L1脂肪细胞或前脂肪细胞中，并孵育过夜。通过（3）H-2-脱氧葡萄糖摄取评估葡萄糖转运。提取总RNA并进行RT-PCR以测量内脂素mRNA水平。对照组与其他组之间的统计比较采用双侧配对t检验，单向方差分析用于比较各组的平均值。
胰岛素增加了3T3-L1前脂肪细胞中特异性膜葡萄糖转运。从暴露于胰岛素15分钟到1小时，上调明显。然而，在暴露于胰岛素6小时后，对胰岛素的反应出现下调。剂量反应研究表明，在50 nmol/L胰岛素时，2-脱氧葡萄糖转运增加了336%（P < 0.01），并在100 nmol/L胰岛素时达到最大效应（P < 0.01）。油酸和棕榈酸处理不影响基础葡萄糖转运（无胰岛素刺激），而在前脂肪细胞和脂肪细胞中，过夜的油酸和棕榈酸处理后，胰岛素刺激的葡萄糖转运受到抑制。在3T3-L1前脂肪细胞中，0.125 mmol/L的油酸或棕榈酸即可导致胰岛素抵抗（分别为P < 0.05），且抑制呈剂量依赖性。在脂肪细胞中，0.5 mmol/L的油酸或1.0 mmol/L的棕榈酸时出现抑制。内脂素mRNA表达在分化过程中增加超过1.5倍。与对照组相比，牛血清白蛋白（BSA）不影响内脂素mRNA表达。剂量反应研究表明，向3T3-L1脂肪细胞中添加0.125 mmol/L油酸和棕榈酸可显著降低内脂素mRNA表达（分别相对于未处理对照组为78%、77%，P < 0.05），在1.0 mmol/L FFA时进一步降至65%（相对于未处理对照组，P < 0.05）和55%（相对于未处理对照组，P < 0.01）。此外，对前脂肪细胞的抑制与脂肪细胞相似，在1.0 mmol/L FFA时最大降低分别为44%（油酸，P < 0.05）和47%（棕榈酸，P < 0.05）。
油酸和棕榈酸可能在3T3-L1脂肪细胞和前脂肪细胞中诱导胰岛素抵抗。内脂素mRNA的下调可能导致油酸和棕榈酸引起的胰岛素敏感性受损。
