Leland Ellis

Differential regulation of multiple hepatic protein tyrosine phosphatases in alloxan diabetic rats.

The involvement of tyrosine phosphorylation in insulin action led us to hypothesize that increased activity of protein tyrosine phosphatases (PTPases) might contribute to insulin resistance in alloxan diabetes in the rat. Hepatic PTPase activity was measured using two artificial substrates phosphorylated on tyrosine: reduced, carboxyamidomethylated, and maleylated lysozyme (P-Tyr-RCML) and myelin basic protein (P-Tyr-MBP), as well as an autophosphorylated 48-kD insulin receptor tyrosine kinase domain (P-Tyr-IRKD). Rats that were made alloxan diabetic exhibited a significant increase in hepatic membrane (detergent-soluble) PTPase activity measured with P-Tyr-MBP, without a change in activity measured with P-Tyr-RCML or the P-Tyr-IRKD. The PTPase active with P-Tyr-MBP behaved as a high molecular weight peak during gel filtration chromatography. Characterization of this enzyme indicated it shared properties with CD45, the prototype for a class of transmembrane, receptor-like PTPases. Our results indicate that alloxan diabetes in the rat is associated with an increase in the activity of a large, membrane-associated PTPase which accounts for only a small proportion of insulin receptor tyrosine dephosphorylation. Nonetheless, increased activity of this PTPase may oppose tyrosine kinase-mediated insulin signal transmission, thus contributing to insulin resistance.

A New Transgenic Mouse Model of Chronic Hyperglycemia

Expression under the control of the mouse transferrin promoter of a transgene encoding a soluble secreted derivative of the ectodomain of the human insulin receptor in transgenic mice results in the accumulation of this high-affinity insulin-binding protein in the plasma. Alterations of glucose homeostasis are observed including postabsorptive hyperglycemia concomitant with increased hepatic glucose production and hyperinsulinemia. Thus, this is the first transgenic animal model of chronic hyperglycemia with alterations in glucose homeostasis that are produced without a targeted alteration of pancreatic function. These mice provide a new experimental model to follow the progression and long-term consequences of chronic hyperglycemia.

An assessment of human insulin receptor phosphorylation and exogenous kinase activity following deletion of 69 residues from the carboxyl-terminus of the receptor β-subunit

A mutant human insulin receptor with a carboxyl-terminal deletion of 69 amino acids (proreceptor residues 1287-1355) is expressed as a stable protein in transiently transfected COS cells. We find that in intact cells this mutant is phosphorylated in an insulin-dependent manner on core tyrosines 1158, 1163 and 1163. As expected, the carboxyl-terminal beta-subunit phosphorylation sites (serines 1305/6, tyrosines 1328/34 and threonine 1348) are absent from this mutant. However, the two major insulin-stimulated serine phosphopeptides remain. In intact cells, insulin stimulates exogenous substrate phosphorylation by the truncated receptor only approximately 1.9-fold (cf. approximately 9-fold for the wild-type receptor in these cells), a consequence of a approximately 4.8-fold elevation in basal insulin-independent kinase activity.

Insulin receptor tyrosine kinase domain auto-dephosphorylation

We have observed dephosphorylation of the soluble, 48 kDa insulin receptor tyrosine kinase domain following its tyrosine autophosphorylation. Dephosphorylation was associated with generation of inorganic phosphate, thereby making catalysis by reversal of the kinase reaction unlikely. The kinase domain preparations could not be shown to contain detectable, contaminating protein tyrosine phosphatase activity. In addition, dephosphorylation was insensitive to protein phosphatase inhibitors. However, it was blocked by the kinase inhibitor staurosporine. These results are consistent with insulin receptor kinase domain auto-dephosphorylation via catalysis involving the kinase itself. These findings raise the possibility of a novel mechanism for termination of the insulin receptor signal.