Insulin receptors (IRs) and insulin signaling proteins are widely distributed throughout the central nervous system (CNS). To study the physiological role of insulin signaling in the brain, we created mice with a neuron-specific disruption of the IR gene (NIRKO mice). Inactivation of the IR had no impact on brain development or neuronal survival. However, female NIRKO mice showed increased food intake, and both male and female mice developed diet-sensitive obesity with increases in body fat and plasma leptin levels, mild insulin resistance, elevated plasma insulin levels, and hypertriglyceridemia. NIRKO mice also exhibited impaired spermatogenesis and ovarian follicle maturation because of hypothalamic dysregulation of luteinizing hormone. Thus, IR signaling in the CNS plays an important role in regulation of energy disposal, fuel metabolism, and reproduction.

Cross-talk between insulin and the adrenergic system is important in the regulation of energy homeostasis. In cultured, differentiated mouse brown adipocytes, beta3-adrenergic stimulation induced a 4.5-fold increase in uncoupling protein-1 (UCP-1) expression, which was diminished by 25% in the presence of insulin. Beta3-adrenergic stimulation also activated mitogen-activated protein (MAP) kinase by 3.5-fold and caused a decrease in basal phosphoinositide (PI) 3-kinase activity detected in p110gamma- and Gbeta-subunit-immunoprecipitates in a time-dependent manner, whereas insulin stimulated p110alpha- and phosphotyrosine-associated PI 3-kinase activity. Inhibition of MAP kinase or PI 3-kinase potentiated the beta3-adrenergic effect on UCP-1 expression, both alone and in the presence of insulin. Thus, insulin inhibits beta3-adrenergic stimulation of UCP-1, and both MAP kinase and PI 3-kinase are negative regulatory elements in the beta3-adrenergic control of UCP-1 expression. Cross-talk between the adrenergic and insulin signaling systems and impaired regulation of UCP-1 might contribute to the development of a reduced energy balance, resulting in obesity and insulin resistance.

We have recently reported that skeletal muscle of the ob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes. Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus. GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway. Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis. Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa. Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs. GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/ob mice by Northern blot analysis. Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA. Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold. Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate. Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation. Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.

The failure of insulin to stimulate muscle glucose uptake and suppress hepatic glucose production represents two of the fundamental pathophysiologic lesions in type 2 diabetes mellitus (DM). Defining insulin action at the molecular level, therefore, provides the critical background against which to elucidate the mechanisms of insulin resistance that underlie type 2 DM, obesity and many other disorders. Over the past two decades substantial progress has been made in identifying many of the molecular mechanisms involved in insulin signaling. Much of this progress has been due to the use of homologous recombinant gene targeting. The present review examines the various insights that have been provided by studies of knockout mice strains. Taken together, the results present the possibility of a unifying hypothesis for type 2 DM, in which insulin resistance in the beta-cell synergizes with insulin resistance in the periphery to produce the two classic defects of this disease: relative hypoinsulinemia and peripheral insulin resistance.

Friedreich's ataxia (FA) is an autosomal recessive disease that has been attributed to a GAA triplet repeat expansion in the first intron of the X25/frataxin gene. Impaired glucose tolerance is present in up to 39% of FA patients, and clinically apparent diabetes is seen in approximately 18% of the affected individuals. Subjects carrying the X25/frataxin GAA repeat in a heterozygous state do not develop FA and, therefore, represent an ideal model to study the underlying metabolic defects that contribute to the diabetes associated with this disorder. In the present study, we have compared 11 first-degree relatives of FA patients (i.e., parents or heterozygous siblings of FA patients) with matched normal control subjects to study the parameters of glucose metabolism. An oral glucose tolerance test revealed diabetes in one of the heterozygous subjects who was excluded from further analyses. Using an octreotide-based quantification of insulin sensitivity, 8 of the remaining 10 study subjects showed pronounced insulin resistance, reflecting a significant difference from the control group (P = 0.001). In conclusion, a heterozygous expansion of the X25/frataxin GAA repeat in healthy individuals is associated with insulin resistance and might be considered a genetic co-factor in the pathogenesis of mitochondrial subtypes of diabetes.

Insulin and insulin-like growth factor I signals are mediated via phosphorylation of a family of insulin receptor substrate (IRS) proteins, which may serve both complementary and overlapping functions in the cell. To study the metabolic effects of these proteins in more detail, we established brown adipocyte cell lines from wild type and various IRS knockout (KO) animals and characterized insulin action in these cells in vitro. Preadipocytes derived from both wild type and IRS-2 KO mice could be fully differentiated into mature brown adipocytes. In differentiated IRS-2 KO adipocytes, insulin-induced glucose uptake was decreased by 50% compared with their wild type counterparts. This was the result of a decrease in insulin-stimulated Glut4 translocation to the plasma membrane. This decrease in insulin-induced glucose uptake could be partially reconstituted in these cells by retrovirus-mediated re-expression of IRS-2, but not overexpression of IRS-1. Insulin signaling studies revealed a total loss of IRS-2-associated phosphatidylinositol (PI) 3-kinase activity and a reduction in phosphotyrosine-associated PI 3-kinase by 30% (p

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.

The development of type 2 diabetes is linked to insulin resistance coupled with a failure of pancreatic B-cells to compensate by adequate insulin secretion. Here, we review studies obtained from genetically engineered mice that have helped dissect the pathophysiology of this disease. Transgenic/knockout models with monogenic impairment in insulin action and insulin secretion have highlighted potential molecular mechanisms for insulin resistance and suggested a mechanism for the development of MODY in humans. Polygenic models have strengthened the idea that minor defects in insulin secretion and insulin action, when combined, can lead to diabetes, pointing out the importance of interactions of different genetic loci in the production of diabetes. Tissue-specific knockouts of the insulin receptor have challenged current concepts on the regulation of glucose homeostasis and have highlighted the importance of insulin action in pancreatic B-cells and brain. The impact of the genetic background on insulin action, insulin secretion and the incidence of diabetes is also evident in these models. These findings highlight potential new therapeutic targets in the treatment of type 2 diabetes.

While most receptor tyrosine kinases signal by recruiting SH2 proteins directly to phosphorylation sites on their plasma membrane receptor, the insulin receptor phosphorylates intermediary IRS proteins that are distributed between the cytoplasm and a state of loose association with intracellular membranes. To determine the importance of this distribution to IRS-1-mediated signaling, we constructed a prenylated, constitutively membrane-bound IRS-1 by adding the COOH-terminal 9 amino acids from p21(ras), including the CAAX motif, to IRS-1 (IRS-CAAX) and analyzed its function in 32D cells expressing the insulin receptor. IRS-CAAX migrated more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than did IRS-1 and demonstrated increased levels of serine/threonine phosphorylation. Insulin-stimulated tyrosyl phosphorylation of IRS-CAAX was slightly decreased, while IRS-CAAX-mediated phosphatidylinositol 3'-kinase (PI3'-kinase) binding and activation were decreased by approximately 75% compared to those for wild-type IRS-1. Similarly, expression of IRS-CAAX desensitized insulin-stimulated [(3)H]thymidine incorporation into DNA by about an order of magnitude compared to IRS-1. By contrast, IRS-CAAX-expressing cells demonstrated increased signaling by mitogen-activated protein kinase, Akt, and p70(S6) kinase in response to insulin. Hence, tight association with the membrane increased IRS-1 serine phosphorylation and reduced coupling between the insulin receptor, PI3'-kinase, and proliferative signaling while enhancing other signaling pathways. Thus, the correct distribution of IRS-1 between the cytoplasm and membrane compartments is critical to the normal balance in the network of insulin signaling.