Insulin activates glycerol-3-phosphate acyltransferase (de novo phosphatidic acid synthesis) through a phospholipid-derived mediator. Apparent involvement of Gi alpha and activation of a phospholipase C.
We studied the mechanism whereby insulin activates de novo phosphatidic acid synthesis in BC3H-1 myocytes. Insulin rapidly activated glycerol-3-phosphate acyltransferase (G3PAT) in intact and cell-free preparations of myocytes in a dose-related manner. The apparent Km of the enzyme was decreased by treatment with insulin, whereas the Vmax was unaffected. No activation was found by ACTH, insulin-like growth factor-I, angiotensin II, or phenylephrine, but epidermal growth factor, which, like insulin, is known to activate de novo phosphatidic acid synthesis in intact myocytes, also stimulated G3PAT activity. In homogenates or membrane fractions, the effect of insulin on G3PAT was fully mimicked by nonspecific or phosphatidylinositol (PI)-specific phospholipase C (PLC). An antiserum raised against PI-glycan-PLC completely blocked the effect of insulin on G3PAT. Although the above findings suggested involvement of a PLC in insulin-induced activation of G3PAT, neither diacylglycerol nor protein kinase C activation appeared to be involved. On the other hand, insulin stimulated the release of a cytosolic factor, which activated membrane-associated G3PAT. This cytosolic factor had a molecular weight of less than 5K as determined by Sephadex G-25 chromatography. NaF, a phosphatase inhibitor, blocked the activation of G3PAT by insulin, suggesting involvement of a phosphatase. Insulin-induced activation of G3PAT was also blocked by pretreatment of intact myocytes with pertussis toxin and by prior addition, to homogenates, of an antiserum that recognizes the C-terminal decapeptide of Gi alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Vila
M C
MC
Research Service, James A. Haley Veterans Hospital, University of South Florida College of Medicine, Tampa 33612.
Milligan
G
G
Standaert
M L
ML
Farese
R V
RV
D000818:Animals; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D010712:Phosphatidic Acids / Q000096:biosynthesis*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D015398:Signal Transduction; D012969:Sodium Fluoride / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007295:Inositol Phosphates; D007328:Insulin; D010712:Phosphatidic Acids; D011134:Polysaccharides; D010566:Virulence Factors, Bordetella; C028039:inositol phosphate glycan; D012969:Sodium Fluoride; D005992:Glycerol-3-Phosphate O-Acyltransferase; D037342:Pertussis Toxin; D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase