Osmotic loading of neutralizing antibodies demonstrates a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway.
Protein-tyrosine phosphatases (PTPases) have been postulated to balance the steady-state phosphorylation and the activation state of the insulin receptor and its substrate proteins. To explore whether PTP1B, a widely expressed, non-receptor-type PTPase, regulates insulin signaling, we used osmotic shock to load rat KRC-7 hepatoma cells with affinity-purified neutralizing antibodies that immunoprecipitate and inactivate the enzymatic activity of recombinant rat PTP1B in vitro. In cells loaded with PTP1B antibody, insulin-stimulated DNA synthesis and phosphatidylinositol 3'-kinase activity were increased by 42% and 38%, respectively, compared with control cells loaded with preimmune IgG (p < 0.005). In order to characterize the potential site(s) of action of PTP1B in insulin signaling, we also determined that insulin-stimulated receptor autophosphorylation and insulin receptor substrate 1 tyrosine phosphorylation were increased 2.2- and 2.0-fold, respectively, and that insulin-stimulated receptor kinase activity toward an exogenous peptide substrate was increased by 57% in the PTP1B antibody-loaded cells. Osmotic loading did not alter the cellular content of PTP1B protein, suggesting that the antibody acts in the cell by sterically blocking catalytic interactions between PTP1B and its physiological substrates. These studies demonstrate that PTP1B has a role in the negative regulation of insulin signaling and acts, at least in part, directly at the level of the insulin receptor. These results also show that insulin signaling can be enhanced by the inhibition of specific PTPases, a maneuver that has potential clinical relevance in the treatment of insulin resistance and Type II diabetes mellitus.
Ahmad
F
F
Dorrance H. Hamilton Research Laboratories, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Li
P M
PM
Meyerovitch
J
J
Goldstein
B J
BJ
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000494:pharmacology*; D000918:Antibody Specificity; D002460:Cell Line; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000276:immunology* / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism
D000906:Antibodies; D007074:Immunoglobulin G; D007328:Insulin; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases