Abstract
ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a approximately 15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase C and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.
Highlights
ACSL1 has been shown to be localized to the endoplasmic reticulum and mitochondria-associated membranes, whereas in adipocytes, ACSL1 was found associated with the plasma membrane, the lipid droplet surface [13], and glucose transporter 4-containing vesicles [14, 15]
Recent studies have postulated a cooperative role of FATP1 and ACSL1 in the movement of LCFAs across the plasma membrane via a process termed vectoral acylation [16], in which the CoA- and ATP-dependent esterification of internalized fatty acid provides the thermodynamic force necessary for net lipid influx
Our laboratory has recently undertaken a gene silencing approach to the evaluation of proteins implicated in adipocyte fatty acid influx and efflux, and prior studies have focused on FATP1, FATP4, and CD36 [6]
Summary
To evaluate the effect of ACSL1 silencing on total lenced adipocytes exhibited a slight increase in the expression cellular acyl-CoA synthetase activity, adipocyte cell extracts of FATP1, whereas the levels of another acyl-CoA synthetase, were assayed for fatty acid esterification using a variety of Acyl-CoA synthetase activity ؎ S.E. shScr shACSL1-3 incubated with 100 nM insulin for 30 min, the ACSL1-3 kd adipocytes displayed a small but statistically significant increase in the triacylglycerol (TAG) (ϳ19%, p ϭ 0.047) and diacylglycerol pools (ϳ34%, p ϭ 0.052) when compared with the control cells.
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