title: Decreased in situ insulin receptor dephosphorylation in hyperglycemia-induced insulin resistance in rat adipocytes.
abstract: The regulation of insulin receptor (IR) tyrosine (tyr) phosphorylation is a key step in the control of insulin signaling. Augmented IR tyr dephosphorylation by protein tyrosine phosphatases (PTPs) may contribute to insulin resistance. To investigate this possibility in hyperglycemia-induced insulin resistance, primary cultured rat adipocytes were rendered insulin-resistant by chronic exposure (18 h) to 15 mmo/l glucose combined with 10(-7) mol/l insulin. Insulin-resistant adipocytes showed a decrease in insulin sensitivity and a maximum response of 2-deoxyglucose uptake, which was associated with a decrease in maximum insulin-stimulated IR tyr phosphorylation in situ. To assess tyr dephosphorylation, IRs of insulin-stimulated permeabilized adipocytes were labeled with [gamma-32P]ATP and chased for 2 min with unlabeled ATP in the presence of EDTA. In a nonradioactive protocol, insulin-stimulated adipocytes were permeabilized and exposed to EDTA and erbstatin for 2 min, and IRs were immunoblotted with anti-phosphotyrosine (pY) antibodies. Both methods showed a similar diminished extent of IR tyr dephosphorylation in resistant cells. Immunoblotting of four candidate IR-PTPs demonstrated no change in PTP1B or the SH2 domain containing phosphatase-2 (SHP-2), whereas a significant decrease in leukocyte antigen-related phosphatase (LAR) (51 +/- 3% of control) and an increase in PTP-alpha (165 +/- 16%) were found. Activity of immunoprecipitated PTPs toward a triple tyr phosphorylated IR peptide revealed a correlation with protein content for PTP1B, SHP-2, and LAR but a decrease in apparent specific activity of PTP-alpha. The data indicate that decreased IR tyr phosphorylation in hyperglycemia-induced insulin resistance is not due to enhanced dephosphorylation. The diminished IR tyr dephosphorylation observed in this model is associated with decreased LAR protein content and activity.
authors.affiliation: Banting and Best Diabetes Centre, University of Toronto, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Tang
authors.forename: H
authors.initials: H
authors.lastname: Le-Tien
authors.forename: B J
authors.initials: BJ
authors.lastname: Goldstein
authors.forename: P
authors.initials: P
authors.lastname: Shin
authors.forename: R
authors.initials: R
authors.lastname: Lai
authors.forename: I G
authors.initials: IG
authors.lastname: Fantus
mesh_terms: D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D003847:Deoxyglucose / Q000493:pharmacokinetics; D006943:Hyperglycemia / Q000378:metabolism / Q000503:physiopathology*; D015151:Immunoblotting; D007333:Insulin Resistance / Q000502:physiology*; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D019000:Phosphotyrosine; D014443:Tyrosine; D003847:Deoxyglucose; D011972:Receptor, Insulin; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases