In previous studies, we demonstrated that down-regulation of lipoprotein lipase in L6 muscle cells increased insulin-stimulated glucose uptake. In the current study, we used RNA interference technology to silence the LPL gene in L6 cells and generate a LPL-knock-down (LPL-KD) cell line. ShRNA transfected cells showed a 88% reduction in the level of LPL expression. The metabolic response to insulin was compared in wild-type (WT) and LPL-KD cells. Insulin-stimulated glycogen synthesis and glucose oxidation were respectively, 2.4-fold and 2.6-fold greater in LPL-KD cells compared to WT cells. Oxidation of oleic acid was reduced by 50% in LPL-KD cells compared to WT cells even in the absence of insulin. The contribution of LPL in regulating fuel metabolism was confirmed by adding back purified LPL to the culture media of LPL-KD cells. The presence of 10 mu g/mL LPL resulted in LPL-KD cells reverting back to lower glycogen synthesis and glucose oxidation and increased fatty acid oxidation. Thus, LPL depletion appeared to mimic the action of insulin. These finding suggests an inverse correlation between muscle LPL levels and insulin-stimulated fuel homeostasis. (C) 2015 Elsevier Inc. All rights reserved.