Compartmentalization and insulin-induced translocations of insulin receptor substrates, phosphatidylinositol 3-kinase, and protein kinase B in rat liver.

Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60-85% and 10-20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulin-induced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr308 and Ser473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling.

Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK beta-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

Role of hyperinsulinemia on hepatic insulin receptor concentration and autophosphorylation in the presence of high growth hormone levels in transgenic mice overexpressing growth hormone gene.

Overexpression of bovine growth hormone (bGH) in transgenic (PEPCK-bGH) mice induces resistance to insulin, which is compensated by a major increase in insulin levels. In these animals, hepatic insulin receptors (InsRs) are downregulated while tyrosine kinase activity of wheat germ agglutinin (WGA)-purified InsRs towards exogenous substrates is unexpectedly increased. By normalizing insulinemia, we attempted to determine whether the alterations detected in the early steps of insulin signal transduction are due to exposure to chronically high GH levels or are secondary to hyperinsulinemia. Transgenic PEPCK-bGH animals were treated with a single intraperitoneal administration of streptozotocin (STZ) or were deprived of food for 48 h, to normalize insulin levels. Both fasting and STZ treatment were effective in reducing insulin blood levels to control values or below, while GH levels remained unchanged (STZ treatment) or increased (fasted animals). In the liver of untreated transgenic mice, the number of InsRs as determined by 125I-insulin binding was significantly diminished (65+/-5% and 60+/-6% of normal values in microsomes and solubilized membranes respectively;P<0.01 vs control mice). In treated transgenic mice, the number of InsRs increased to values similar to or slightly higher than those found in normal control mice (STZ-treated: 139+/-26% and 126+/-8%; fasted: 128+/-5% (P<0.05) and 102+/-1.5%, for microsomes and solubilized membranes respectively). Neither treatment altered InsR affinity. InsR concentration in liver as determined by immunoblotting using an antibody against the beta-subunit of the insulin receptor was found to be reduced in transgenic mice (69+/-3% of normal values,P<0.001) and was normalized after both STZ treatment (105+/-4%) and fasting (109+/-4%). Insulin-stimulated autophosphorylation activity of InsRs in transgenic mice was increased (154+/-13%,P<0.01 compared with the control group), essentially normalized by STZ treatment (96+/-14%), and reduced by fasting, to below the values measured in normal control mice (56+/-15%,P<0.05). The potential influence of basal serine/threonine (Ser/Thr) phosphorylation of the InsR beta-subunit on the regulation of the InsRs from transgenic mice was also investigated. The autophosphorylation activity of WGA-purified InsRs from all groups of mice studied was essentially unchanged after dephosphorylation with alkaline phosphatase or mild trypsinization. Consequently, our results suggest that the observed changes in InsR number and autophosphorylation activity in the liver of bGH transgenic mice are directly related to changes in insulin blood levels, and that Ser/Thr phosphorylation is apparently not involved in the regulation of the InsR autophosphorylation activity in this model of insulin resistance.

Overexpression of bovine growth hormone in transgenic mice is associated with changes in hepatic insulin receptors and in their kinase activity.

Transgenic mice expressing a hybrid gene produced by linking the promoter regulatory region of phosphoenolpyruvate carboxykinase (PEPCK) gene to the bovine growth hormone (bGH) gene, were used to investigate the effects of GH on insulin binding and insulin dependent tyrosine kinase activity of hepatic insulin receptors. Transgenic mice had normal levels of blood glucose, despite hyperinsulinemia, indicating that these animals were insulin resistant. The number of insulin receptors in the liver of transgenic mice was significantly decreased in both the particulate fraction (25%) and the solubilized membranes (40%) indicating that expressed (functional) and non-expressed (cryptic) receptors were affected. Scatchard analysis of competitive binding curves for insulin indicated that the affinity of the receptor did not differ between transgenic and normal mice. Insulin dependent tyrosine kinase activity in insulin receptors partially purified by wheat germ agglutin (WGA) agarose chromatography from solubilized liver membranes, was measured. The stimulatory action of insulin on phosphorylation of the synthetic substrate (a copolymer Glu-Tyr, 4:1) was increased 100% in transgenic, as compared to normal mice, using the same binding activity. Since transgenic mice are hyperinsulinemic, it is likely that the decreased insulin binding in this group reflects down regulation of the expressed and non-expressed insulin receptors, and the increased kinase activity represents a compensatory mechanism. We conclude that alterations in the insulin receptor number and in the tyrosine kinase activity develop in response to changes in insulin levels. Thus, insulin resistance detected in the liver of transgenic mice overexpressing GH may be due to post receptor defects.

Specific and functional insulin receptors in rat renal papilla.

To investigate a possible action of insulin on the rat kidney papilla, the binding of 125I-insulin to papilla microsomes was examined. This binding was specific to insulin in that it was displaced by increasing concentrations of unlabelled porcine insulin and to a lesser extent by porcine proinsulin and IGF-I, but not by IGF-II and bGH. Scatchard plot of the binding data was curvilinear consistent with either two classes of receptors with different affinities or a single class of receptors that showed negative cooperativity. A small fraction of 125I-insulin (maximum 2%) was degraded during incubation, but with a Km two order of magnitude higher than the constant of affinity for binding. Insulin stimulates the incorporation of phosphate to phosphatidylcholine in a dose-dependent manner, reaching a maximum with 10 nM insulin. This data showed both the presence of specific insulin receptors in the kidney papilla and an insulin action through the synthesis of phospholipids by insulin.

Down regulation of masked and unmasked insulin receptors in the liver of transgenic mice expressing bovine growth hormone gene.

The interaction of insulin with its receptor was studied in microsomes from livers of transgenic mice expressing the bovine growth hormone gene with mouse metallothionein-1 promoter (MT/bGH) and in their normal (non-transgenic) littermates. Specific binding of 125I-insulin was detected in hepatic microsomes from normal and transgenic mice with an apparent Kd of 8 and 200 nM, for high and low affinity sites, respectively. The transgenic MT/bGH mice had a marked hyperinsulinism without significant elevation of plasma glucose levels. Under identical conditions of preparation and incubation, microsomes from the transgenic male and female mice bound 39% and 34% less insulin than those from their litter mates. Scatchard's analysis indicates that this decrease in binding is due to a decrease in the number of receptor sites. In contrast to the marked decrease in insulin binding to unmasked receptors, the levels of masked (also called cryptic) insulin receptors were similar (or slightly increased) in transgenic mice microsomes as compared to those of their normal litter mates.