Overexpression of Protein-tyrosine Phosphatase-1B in Adipocytes Inhibits Insulin-stimulated Phosphoinositide 3-Kinase Activity without Altering Glucose Transport or Akt/Protein Kinase B Activation

Carol L Venable,Ernst U Frevert,Young-Bum Kim,Britta M Fischer,Shubhangi Kamatkar,Benjamin G Neel,Barbara B Kahn

doi:10.1074/jbc.m908392199

Abstract

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirus-mediated gene delivery. PTP1B expression was increased approximately 3-5-fold above endogenous levels at 16 h, approximately 14-fold at 40 h, and approximately 20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h post-transduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50-60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced approximately 32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nm) insulin-stimulated glucose transport or on the EC(50) for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires </=45% of maximal tyrosyl phosphorylation of IR or IRS-1 and <50% of maximal activation of PI3K, 2) a novel PI3K-independent pathway may play a role in insulin-induced glucose transport in adipocytes, and 3) overexpression of PTP1B alone in adipocytes does not impair glucose transport.

Highlights

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway
Quantifying these results reveals an ϳ7-fold greater sensitivity of the anti-PTP1B antiserum for human, compared with mouse PTP1B; signals derived from endogenous mouse PTP1B must be multiplied by seven to compare with signals derived from the exogenous human PTP1B protein
The expression and activity of PTP1B, as well as certain other protein-tyrosine phosphatases (PTPs), has been reported to be elevated in adipose tissue and skeletal muscle of obese humans and rodents (16 –19, 22). These findings led to the hypothesis that increased activity of specific PTPs may play a role in the pathogenesis of insulin resistance

Summary

Introduction

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. Cells expressing high levels of PTP1B showed a 50 – 60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Insulin binds to its transmembrane receptor, causing receptor trans-phosphorylation on tyrosyl residues and activating receptor tyrosine kinase activity toward a variety of substrates including IRS-1– 4,1 shc, and Gab1 [2, 4, 5] These tyrosyl-phosphorylated proteins provide high affinity binding sites for the Src homology 2 domains of several signal relay molecules, such as the regulatory subunit of phosphoinositide 3-kinase (PI3K), which leads to kinase activation and stimulation of several downstream mediators, including the serine/threonine kinase Akt/protein kinase B [6, 7]. Whether Akt, atypical protein kinase C isoforms, or other, as yet unknown, downstream mediators are necessary for the effect of insulin-induced PI3K activation on GLUT4 translocation remains controversial [12,13,14,15]

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Results

Conclusion