Stimulation of glycogen synthesis by insulin in human erythroleukemia cells requires the synthesis of glycosyl-phosphatidylinositol.
Although the insulin-dependent hydrolysis of glycosyl-phosphatidylinositol (GPI) may play an important role in insulin action, an absolute requirement for this glycolipid has not been demonstrated. Human K562 cells were mutated to produce a cell line (IA) incapable of the earliest step in PI glycosylation, the formation of PI-GlcNAc. Another cell line (IVD) was deficient in the deacetylation of PI-GlcNAc to form PI-GlcN and subsequent mannosylated species. Each line was transfected with wild-type human insulin receptors. Similar insulin-stimulated receptor autophosphorylation was observed in all three lines, along with a nearly identical increase in the association of phosphorylated insulin receptor substrate 1 with endogenous PI 3-kinase. Both normal and GPI-defective lines also displayed a similar 2- to 3-fold increase in phosphorylation of the Shc protein and its association with growth factor receptor-bound protein 2 in response to insulin. In contrast to these results, striking differences were noted in insulin-stimulated glycogen synthesis. In normal cells, glycogen synthesis was significantly increased by insulin, whereas no insulin stimulation was observed in GPI-deficient IA cells, and only a trace of stimulation was detected in IVD cells. These results indicate that tyrosine phosphorylation produced by insulin is not dependent on GPI synthesis, and this effect is not sufficient to elicit at least some of the metabolic effects of the hormone. In contrast, GPI synthesis is required for the stimulation of glycogen synthesis by insulin in these cells. These findings support the existence of divergent pathways in the action of insulin.
Lazar
D F
DF
Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Ann Arbor, MI 48105.
Knez
J J
JJ
Medof
M E
ME
Cuatrecasas
P
P
Saltiel
A R
AR
D002460:Cell Line; D005434:Flow Cytometry; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000096:biosynthesis*; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D016296:Mutagenesis; D010766:Phosphorylation; D019000:Phosphotyrosine; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D017261:Glycosylphosphatidylinositols; D007328:Insulin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D006003:Glycogen; D011972:Receptor, Insulin; D005947:Glucose