Lysosomal triacylglycerol lipase activity in L6 myoblasts and its changes on differentiation.
L6 myoblasts, before fusion, accumulate large stores of neutral lipid when cultured in medium supplemented with fatty acid. Upon fusion to terminally differentiated myotubes, a noticeable decrease in these neutral-lipid stores was observed. Triacylglycerol lipase activity was examined in L6 myoblasts at various stages of cell differentiation to assess a possible role for this enzyme in the above phenomenon. In this first study to demonstrate lipolytic activity in cultured muscle cells, the activity was found to be totally dependent on the presence of a detergent, either Cutscum or Triton X-100, during homogenization. The inhibition by many thiol-specific reagents [N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5'-dithiobis-(2-nitrobenzoic acid)] suggest that a thiol group is at or near the active site. The observed acidic pH optimum (5.5-6.0), the acute inhibition by chlorpromazine (a lysosomal lipase inhibitor) and the distribution of lipolytic activity upon cell fractionation (which co-sediments with acid phosphatase, a lysosomal marker enzyme) suggest that the lipase may be of lysosomal origin. Under the optimal conditions described, the triacylglycerol lipase activity of L6 myoblasts was determined to be 2.9 +/- 0.4 nmol of oleic acid released/min per mg of DNA. This activity increased 3-fold, to 9.0 +/- 1.6 nmol/min per mg, in the myotube phase. This increase in lipolytic activity may be responsible for the observed decrease in neutral-lipid stores of differentiating myoblasts.
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D008049:Lipase / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology*; D009132:Muscles / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013997:Time Factors
D003902:Detergents; D008049:Lipase; D000135:Acid Phosphatase