Protein Tyrosine Phosphatase-1B Negatively Regulates Insulin Signaling in L6 Myocytes and Fao Hepatoma Cells.

Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein tyrosine kinases and protein tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique and analyzed the effect of overexpression of a wild-type protein tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70%, and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as MAP kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver.


INTRODUCTION
After insulin binds to its own receptor, the insulin receptor is phosphorylated on its tyrosine residues, and tyrosine kinase activity is activated. The activated insulin receptor binds to insulin receptor substrates (IRSs) via the YMXM motif, and IRSs are also phosphorylated on tyrosine residues (1). The tyrosine phosphorylated IRSs activate their downstream signaling molecules, such as phosphatidylinositol (PI) 3kinase and p21 ras . Since tyrosine phosphorylation is essential for insulin signaling, the balance of activities between protein tyrosine kinases and protein tyrosine phosphatases (PTPase) appears to be very important for insulin's effects.
Several lines of evidence demonstrate that PTPase activity is increased in insulin resistant states such as obesity and type 2 diabetes mellitus. It has been reported that obese human subjects have increased PTPase activity in skeletal muscle and adipose tissue (2,3), and a 10% body weight reduction causes a decrease in overall adipose tissue PTPase activity with enhanced insulin sensitivity (4). Furthermore, insulin infusion in vivo produces a rapid 25% suppression of soluble-PTPase activity in muscles of insulin-sensitive subjects, but this response is severely impaired in subjects who are insulin resistant (5). Moreover, increased PTPase activity is also observed in the liver (6) and skeletal muscle (7) of diabetic rats. Taken together, abnormalities of PTPase activity are thought to be important to understand the molecular mechanism of insulin resistance.
Previous studies showed that the tandem-domain transmembrane enzymes, leukocyte antigen related (LAR) and leukocyte common antigen-related phosphatase (LRP)/RPTP-α, and the intracellular, single domain enzymes, protein tyrosine phosphatase-1B (PTP1B) and SHP2 are candidate PTPases for the regulation of the insulin signaling pathway. In particular, PTP1B directly interacts with the activated insulin receptor (8), and exhibits the highest specific activity towards IRS-1 (9). We have also reported that exposing Rat 1 fibroblasts expressing human insulin receptors by guest on March 24, 2020 http://www.jbc.org/ Downloaded from to a high glucose condition impairs insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 due to the increased PTP1B expression and activity (10).
Furthermore, overexpression of PTP1B by the electroporation method reduces the level of GLUT4 on the cell surface in primary cultured rat adipose cells (11). Moreover, it is reported that mice lacking the PTP1B gene show increased insulin sensitivity and resistance to high-fat diet induced obesity, which is supported by enhanced insulininduced tyrosine phosphorylation of the insulin receptor and IRS-1 in muscle and liver (12). Thus, PTP1B appears to play an important role in the regulation of insulin signaling.
In the present study, we employed the adenovirus mediating gene transfer technique, and analyzed the effect of PTP1B overexpression on insulin signaling in model cells of insulin target tissues, such as L6 myocytes and Fao cells. In both cells, PTP1B overexpression markedly inhibited insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1, and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of PI 3-kinase and Akt phosphorylation as well as MAP kinase phosphorylation. Moreover, glycogen synthesis was also inhibited under both basal and insulin-stimulated conditions. The present findings demonstrate that PTP1B negatively regulates insulin signaling in muscle and liver cells. Glycogen synthesis was measured as described previously (18)

DISCUSSION
We previously reported that exposing Rat 1 fibroblasts expressing human insulin receptors to high glucose conditions impaired the insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 due to the increased expression and activity of PTP-1B (10). More direct evidence has shown that overexpression of PTP1B reduced the level of GLUT4 on the cell surface in primary cultured rat adipose cells (11). Recently, it was reported that the PTP1B knockout mice showed increased insulin sensitivity and resistance to high-fat diet induced obesity with enhanced insulininduced tyrosine phosphorylation of the insulin receptor and IRS-1 in muscle and liver (12). However, in those mice, insulin-induced tyrosine phosphorylation of the insulin receptor and IRS-1 in adipose tissue was not affected. Another study also showed that increased insulin sensitivity in PTP1B-deficient mice was tissue specific, as insulinstimulated glucose uptake was elevated in skeletal muscle, whereas adipose tissue was unaffected (19). Furthermore, it has been reported that overexpression of PTP1B decreased insulin-induced tyrosine phosphorylation of the insulin receptor and IRS-1 by about 50%, but did not affect the glucose transport in 3T3-L1 adipocytes (20).
Therefore, the role of PTP1B for insulin effects appears to be tissue specific.
In the current studies, we overexpressed PTP1B wild-type in L6 myocytes and     After 56 h, the cells were serum starved for 16 h, then the medium was changed to DME with 1% BSA. The cells were incubated with 100 ng/ml of insulin and