Relationship Between Skeletal Muscle LPL Activity and the Ability of Endothelial Cells to Bind LPL

Abstract

Lipoprotein lipase (LPL) in skeletal muscle has been a focus of many exercise studies. The interesting feature of this protein is that while it is synthesized in muscle fibers, it is functional when bound to the vascular endothelium lumen. However because of prior technical limitations, almost nothing is known about the endothelial regulation of LPL protein in skeletal muscle. Thus, we have developed methodologies to quantify the binding of radiolabeled LPL to rat tissues in vivo and to isolated perfused muscle. PURPOSE To determine if the endothelial binding of exogenous 125I-LPL is associated with the amount of endogenous LPL activity residing in the muscle tissue. We tested the hypothesis that the ability of muscles to bind 125I-LPL would be correlated with the amount of endogenous LPL activity on the endothelium. METHODS Tyrosine residues of the LPL protein were iodinated. Dimeric LPL was purified by heparin-sepharose chromatography. The 125I-LPL was either injected intravenously into the jugular vein or perfused into the femoral artery of an isolated rat hindlimb. Tissues were obtained after thoroughly flushing the limb with perfusate to remove any tracer from the blood. In some of the experiments, heparin was perfused prior to taking tissue samples in order to determine how much of the 125I-LPL was binding to the heparin-sensitive sites that anchor LPL. RESULTS The dimeric 125I-LPL rapidly bound to the skeletal muscle vasculature (>95% complete in 10 minutes). A subset of experiments indicated that the tracer was very likely binding to vascular sites also anchoring the functional pool of endogenous LPL because heparin perfusion released 98–100% of the 125I-LPL from skeletal muscles into the venous outflow. There was a 34% coefficient of variation (CV) for LPL binding when comparing different rats. When comparing contra-lateral muscles within each rat, the CV for LPL binding was only 8%. Predominantly oxidative muscles bound 200–300% more 125I-LPL than glycolytic muscles (e.g. the endothelium of the red gastrocnemius bound 220±15% more LPL than observed in the white gastrocnemius; P = 0.0001). Most interestingly, there was a striking relationship between endogenous heparin-releasable LPL activity in skeletal muscle and the ability of 125I-LPL to bind to the endothelium (R2=0.833, P=0.0006). CONCLUSION These studies indicate that the binding of exogenous LPL to the vascular endothelium accounts for 83% of the variance in rat skeletal muscle LPL activity. Thus the results are consistent with the hypothesis that the endothelium may be a primary determinant of the amount of skeletal muscle LPL activity. Supported in part by NIH Grant HL07482