The broad nature of insulin resistant glucose metabolism in skeletal muscle of patients with type 2 diabetes suggests a defect in the proximal part of the insulin signaling network. We sought to identify the pathways compromised in insulin resistance and to test the effect of moderate exercise on whole-body and cellular insulin action. We conducted euglycemic clamps and muscle biopsies on type 2 diabetic patients, obese nondiabetics and lean controls, with and without a single bout of exercise. Insulin stimulation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, as measured by phosphorylation of the insulin receptor and IRS-1 and by IRS protein association with p85 and with PI 3-kinase, was dramatically reduced in obese nondiabetics and virtually absent in type 2 diabetic patients. Insulin stimulation of the MAP kinase pathway was normal in obese and diabetic subjects. Insulin stimulation of glucose-disposal correlated with association of p85 with IRS-1. Exercise 24 hours before the euglycemic clamp increased phosphorylation of insulin receptor and IRS-1 in obese and diabetic subjects but did not increase glucose uptake or PI 3-kinase association with IRS-1 upon insulin stimulation. Thus, insulin resistance differentially affects the PI 3-kinase and MAP kinase signaling pathways, and insulin-stimulated IRS-1-association with PI 3-kinase defines a key step in insulin resistance.

Type 2 diabetes is characterized by abnormalities of insulin action in muscle, adipose tissue, and liver and by altered beta-cell function. To analyze the role of the insulin signaling pathway in these processes, we have generated mice with combined heterozygous null mutations in insulin receptor (ir), insulin receptor substrate (irs-1), and/or irs-2. Diabetes developed in 40% of ir/irs-1/irs-2(+/-), 20% of ir/irs-1(+/-), 17% of ir/irs-2(+/-), and 5% of ir(+/-) mice. Although combined heterozygosity for ir/irs-1(+/-) and ir/irs-2(+/-) results in a similar number of diabetic mice, there are significant differences in the underlying metabolic abnormalities. ir/irs-1(+/-) mice develop severe insulin resistance in skeletal muscle and liver, with compensatory beta-cell hyperplasia. In contrast, ir/irs-2(+/-) mice develop severe insulin resistance in liver, mild insulin resistance in skeletal muscle, and modest beta-cell hyperplasia. Triple heterozygotes develop severe insulin resistance in skeletal muscle and liver and marked beta-cell hyperplasia. These data indicate tissue-specific differences in the roles of IRSs to mediate insulin action, with irs-1 playing a prominent role in skeletal muscle and irs-2 in liver. They also provide a practical demonstration of the polygenic and genetically heterogeneous interactions underlying the inheritance of type 2 diabetes.

In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an in vitro reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca(2+)-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5'-[gamma-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted in vitro to a limited extent by using isolated membranes. This association appears to involve protein-protein interactions among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.

Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP.Rad was also reconverted to GTP.Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

Insulin receptor substrate-1 (IRS-1) is pivotal in mediating the actions of insulin and growth factors in most tissues of the body, but its role in insulin-producing beta islet cells is unclear. Freshly isolated islets from IRS-1 knockout mice and SV40-transformed IRS-1-deficient beta-cell lines exhibit marked insulin secretory defects in response to glucose and arginine. Furthermore, insulin expression is reduced by about 2-fold in the IRS-1-null islets and beta-cell lines, and this defect can be partially restored by transfecting the cells with IRS-1. These data provide evidence for an important role of IRS-1 in islet function and provide a novel functional link between the insulin signaling and insulin secretion pathways. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) by receptor tyrosine kinases for growth factors is crucial for neuronal cell survival and proliferation. This class of kinases is comprised of heterodimers, each consisting of one regulatory and one catalytic subunit. Multiple isoforms of regulatory subunits exist, including p85alpha and its alternative splice products p50alpha and AS53/p55alpha, and p85beta and p55(PIK), which are derived from different genes. The regional distribution of these PI 3-kinase regulatory isoforms was mapped in the adult murine brain by in situ hybridization histochemistry. All isoforms were demonstrated in abundance in choroid plexus and anterior pituitary. In neuronal compartments, however, PI 3-kinase isoforms were distributed in a regionally specific manner. In general, the mRNAs for p85alpha, p50alpha, AS53, and p85beta were widespread, with the highest level in the olfactory system, in neuronal groups of the forebrain and hypothalamus, in the hippocampus, cortex, inferior and superior colliculus, pituitary, and cerebellum. However, each isoform had specific variations. Lower expression levels of these isoforms were found in the thalamus, diencephalon, mesencephalon, and brainstem. In contrast, abundant mRNA expression of p55(PIK) was limited to cerebellum and anterior pituitary, with moderate levels of p55(PIK) in the olfactory bulb and hippocampus and low levels elsewhere. The distribution pattern of PI 3-kinase isoforms in the brain indicates pluripotent signaling properties for PI 3-kinase isoforms p85alpha, p50alpha, AS53/p55alpha, and p85beta for a variety of receptor tyrosine kinases, whereas the restricted expression of p55(PIK) implies a regionally specific role for this isoform in neuronal signaling. The unique integrated expression profiles of PI 3-kinase isoforms in distinct neuronal compartments denote complex intracellular signaling pathways for each neuronal region to ensure specificity of receptor tyrosine kinase signal transduction.

Organ weight was compared in adult mice with deletion of one (IRS-1-/+) or both (IRS-1-/-) copies of the insulin receptor substrate-1 (IRS-1) gene and IRS-1+/+ littermates. IRS-1-/+ mice showed modest reductions in weight of most organs in proportion to a decrease in body weight. IRS-1-/- mice showed major reductions in weight of heart, liver, and spleen that were directly proportional to a decrease in body weight. In IRS-1-/- mice, kidney and particularly small intestine and brain exhibited proportionately smaller weight reductions, and gastrocnemius muscle showed a proportionately greater weight reduction than the decrease in body weight. Growth deficits in IRS-1-/- mice could reflect impaired actions of multiple hormones or cytokines that activate IRS-1. To assess the requirement for IRS-1 in insulin-like growth factor I (IGF-I)-dependent postnatal growth, IRS-1-/+ mice were cross-bred with mice that widely overexpress a human IGF-I transgene (IGF+) to generate IGF+ and wild-type mice on an IRS-1+/+, IRS-1-/+, and IRS-1-/- background. IGF-I overexpression increased body weight and weight of brain, small intestine, kidney, spleen, heart, and gastrocnemius muscle in IRS-1+/+ mice. IGF-I overexpression could not completely reverse the body growth retardation in IRS-1-/- mice. Absolute or partial IRS-1 deficiency impaired IGF-I-induced body overgrowth more in females than in males. In males and females, IGF-I stimulated similar overgrowth of brain regardless of IRS-1 status, and intestine and spleen showed dose dependence on IRS-1 for IGF-I-induced growth. IGF-I-induced growth of gastrocnemius muscle had an absolute requirement for IRS-1. IGF-I-induced growth of kidney and heart was impaired by IRS-1 deficiency only in females. In vivo, therefore, most organs do not require IRS-1 for IGF-I-induced growth and can use alternate signaling molecules to mediate IGF-I action. Other organs, such as gastrocnemius muscle, require IRS-1 for IGF-I-induced growth in vivo.

Activity of the sympathetic nervous system is an important factor involved in the pathogenesis of insulin resistance and associated metabolic and vascular abnormalities. In this study, we investigate the molecular basis of cross-talk between beta(3)-adrenergic and insulin signaling systems in mouse brown adipocytes immortalized by SV40 T infection. Insulin-induced tyrosine phosphorylation of the insulin receptor, insulin receptor substrate 1 (IRS-1), and IRS-2 was reduced by prestimulation of beta(3)-adrenergic receptors (CL316243). Similarly, insulin-induced IRS-1-associated and phosphotyrosine-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity, but not IRS-2-associated PI 3-kinase activity, was reduced by beta(3)-adrenergic prestimulation. Furthermore, insulin-stimulated activation of Akt, but not mitogen-activated protein kinase, was diminished. Insulin-induced glucose uptake was completely inhibited by beta(3)-adrenergic prestimulation. These effects appear to be protein kinase A-dependent. Furthermore inhibition of protein kinase C restored the beta(3)-receptor-mediated reductions in insulin-induced IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. Together, these findings indicate cross-talk between adrenergic and insulin signaling pathways. This interaction is protein kinase A-dependent and, at least in part, protein kinase C-dependent, and could play an important role in the pathogenesis of insulin resistance associated with sympathetic overactivity and regulation of brown fat metabolism.

KNOCKOUT MICE: The generation of knockout mice has largely improved our understanding of the function of a variety of gene products. Gene inactivation experiments in mice have yielded numerous animal models for human diseases, thereby expanding our understanding of the underlying pathophysiological mechanisms. The use of conventional knockout experiments is limited if the phenotyp of gene disruption results in embryonic letality. CONDITIONAL MUTAGENESIS: Conditional mutagenesis aims to overcome this limitation by regional and temporal control of gene inactivation in mice. CRE-LOXP SYSTEM: The bacteriophage-enzyme Cre recognizes loxP-sites in the genome and excises loxP-flanked DNA-regions. Using this system loxP-sites can be introduced into intron regions of a target gene and mice can be created carrying this functional, but loxP-marked gene. When crossed with transgenic mice expressing the Cre-recombinase under control of a tissue-specific and/or inducible promoter the gene will be inactivated in vivo in a timely and regionally controlled fashion.