Date of Award


Degree Type


Degree Name

Doctor of Philosophy


The primary objective of this study was to determine the regulating factor(s) responsible for the coordinate changes in lipid metabolism observed as L{dollar}\sb{lcub}6{rcub}{dollar} myoblasts switch from predominantly triacylglycerol (TAG)-synthesizing cells to primarily phospholipid-synthesizing cells during fusion into myotubes (ie. skeletal myogenesis). Myoblasts, but not myotubes, showed a dramatic accumulation of TAG in fatty acid-supplemented growth medium. Both fatty acid oxidation and phospholipid synthesis increased significantly during skeletal myogenesis. Pulse-chase studies showed products of TAG degradation to be used for phospholipid synthesis (prior to oxidation), which resulted in a 4-fold increase in phospholipid content during skeletal myogenesis. A 2-fold increase in calcium content and a transient activation of calcium uptake observed during skeletal myogenesis indicate that phospholipid and calcium metabolism may be interrelated.;In vitro studies of catabolism (TAG-lipase) and synthesis (diacylglycerol acyltransferase, DAGAT) showed that TAG catabolism increased, and TAG synthesis decreased, during skeletal myogenesis. The dominant TAG-lipase was shown to be the lysosomal acid lipase based on its pH optimum, cosedimentation with acid phosphatase, and its marked inhibition by the lysosomotropic agents chloroquine (CQ) and chlorpromazine (CZ). In pulse-chase studies of L{dollar}\sb6{dollar} myoblasts, CQ or CZ prevented the shift of TAG products into phospholipid, implicating the above lipase in the catabolism of endogenously-synthesized TAG. The decrease in DAGAT activity was not due to decreased enzyme or DAG. A cell-permeant cAMP derivative (bt{dollar}\sb2{dollar}-cAMP) and the calcium ionophore (A{dollar}\sb{lcub}23/87{rcub}{dollar}) increased TAG synthesis in differentiating L{dollar}\sb6{dollar} myoblasts without affecting TAG catabolism or phospholipid synthesis. The tumor-promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA), a known activator of protein kinase C (PKC), antagonized these effects. Bt{dollar}\sb2{dollar}-cAMP and A{dollar}\sb{lcub}23/87{rcub}{dollar} decreased, but TPA increased, fatty acid oxidation in both L{dollar}\sb6{dollar} myoblasts and myotubes. These observations suggest that TAG synthesis and fatty acid oxidation may be under hormonal regulation.;Significant increases in phospholipid synthesis, calcium uptake and PIP turnover were observed during calcium-regulated myoblast fusion. A transient activation of PKC, possibly initiated by PIP{dollar}\sb2{dollar} breakdown, was postulated and explored (using TPA and A{dollar}\sb{lcub}23/87{rcub}{dollar}) in relation to the transient increase in phospholipid synthesis observed during skeletal myogenesis. TPA, a known activator of PC synthesis, enhanced {dollar}\sp{lcub}32{rcub}{dollar}P incorporation into PC dramatically, but this observation was likely due to a TPA-activated increase in P{dollar}\sb1{dollar} uptake and PC hydrolysis. TPA did not increase (methyl-{dollar}\sp3{dollar}H) choline incorporation into PC. A{dollar}\sb{lcub}23/87{rcub}{dollar} did not affect PC metabolism but did enhance PIP{dollar}\sb2{dollar} turnover in fusing myoblasts only (ie. not myotubes). Neither PKA, PKC nor calcium appear to be implicated in the activation of phospholipid synthesis during skeletal myogenesis. PIP{dollar}\sb2{dollar} breakdown may play a fundamental role during myoblast fusion. Studies with an L{dollar}\sb6{dollar} myoblast mutant (D1) show that TPA-activated PC hydrolysis plays no role in myoblast fusion. A reported drop in cAMP levels leading to diminished PKA-activated TAG synthesis, in combination with marked increases in phospholipid synthesis and fatty acid oxidation, likely manifest in the previously-described switch in lipid metabolism observed during skeletal myogenesis.



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