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.

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 0.05) in the KO cells. The phosphorylation and activity of Akt, a major downstream effector of PI 3-kinase, as well as Akt-dependent phosphorylation of glycogen synthase kinase-3 and p70S6 kinase were not affected by the lack of IRS-2; however, there was a decrease in insulin stimulation of Akt associated with the plasma membrane. These results provide evidence for a critical role of IRS-2 as a mediator of insulin-stimulated Glut4 translocation and glucose uptake in adipocytes. This occurs without effects in differentiation, total activation of Akt and its downstream effectors, but may be caused by alterations in compartmentalization of these downstream signals.

The signaling pathway by which insulin stimulates insulin secretion and increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in isolated mouse pancreatic beta-cells and clonal beta-cells was investigated. Application of insulin to single beta-cells resulted in increases in [Ca(2+)](i) that were of lower magnitude, slower onset, and longer lifetime than that observed with stimulation with tolbutamide. Furthermore, the increases in [Ca(2+)](i) originated from interior regions of the cell rather than from the plasma membrane as with depolarizing stimuli. The insulin-induced [Ca(2+)](i) changes and insulin secretion at single beta-cells were abolished by treatment with 100 nm wortmannin or 1 micrometer thapsigargin; however, they were unaffected by 10 micrometer U73122, 20 micrometer nifedipine, or removal of Ca(2+) from the medium. Insulin-stimulated insulin secretion was also abolished by treatment with 2 micrometer bisindolylmaleimide I, but [Ca(2+)](i) changes were unaffected. In an insulin receptor substrate-1 gene disrupted beta-cell tumor line, insulin did not evoke either [Ca(2+)](i) changes or insulin secretion. The data suggest that autocrine-activated increases in [Ca(2+)](i) are due to release of intracellular Ca(2+) stores, especially the endoplasmic reticulum, mediated by insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Autocrine activation of insulin secretion is mediated by the increase in [Ca(2+)](i) and activation of protein kinase C.

The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70-80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration of egr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation.

To investigate the efficacy and mechanism of action of vanadium salts as oral hypoglycemic agents, 16 type 2 diabetic patients were studied before and after 6 weeks of vanadyl sulfate (VOSO4) treatment at three doses. Glucose metabolism during a euglycemic insulin clamp did not increase at 75 mg/d, but improved in 3 of 5 subjects receiving 150 mg VOSO4 and 4 of 8 subjects receiving 300 mg VOSO4. Basal hepatic glucose production (HGP) and suppression of HGP by insulin were unchanged at all doses. Fasting glucose and hemoglobin A1c (HbA1c) decreased significantly in the 150- and 300-mg VOSO4 groups. At the highest dose, total cholesterol decreased, associated with a decrease in high-density lipoprotein (HDL). There was no change in systolic, diastolic, or mean arterial blood pressure on 24-hour ambulatory monitors at any dose. There was no apparent correlation between the clinical response and peak serum level of vanadium. The 150- and 300-mg vanadyl doses caused some gastrointestinal intolerance but did not increase tissue oxidative stress as assessed by thiobarbituric acid-reactive substances (TBARS). In muscle obtained during clamp studies prior to vanadium therapy, insulin stimulated the tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1 (IRS-1), and Shc proteins by 2- to 3-fold, while phosphatidylinositol 3-kinase (PI 3-kinase) activity associated with IRS-1 increased 4.7-fold during insulin stimulation (P = .02). Following vanadium, there was a consistent trend for increased basal levels of insulin receptor, Shc, and IRS-1 protein tyrosine phosphorylation and IRS-1-associated PI 3-kinase, but no further increase with insulin. There was no discernible correlation between tyrosine phosphorylation patterns and glucose disposal responses to vanadyl. While glycogen synthase fractional activity increased 1.5-fold following insulin infusion, there was no change in basal or insulin-stimulated activity after vanadyl. There was no increase in the protein phosphatase activity of muscle homogenates to exogenous substrate after vanadyl. Vanadyl sulfate appears safe at these doses for 6 weeks, but at the tolerated doses, it does not dramatically improve insulin sensitivity or glycemic control. Vanadyl modifies proteins in human skeletal muscle involved in early insulin signaling, including basal insulin receptor and substrate tyrosine phosphorylation and activation of PI 3-kinase, and is not additive or synergistic with insulin at these steps. Vanadyl sulfate does not modify the action of insulin to stimulate glycogen synthesis. Since glucose utilization is improved in some patients, vanadyl must also act at other steps of insulin action.

Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.

Leptin is a 16-kDa hormone secreted by adipocytes and plays an important role in control of feeding behavior and energy expenditure. In obesity, circulating levels of leptin and insulin are high because of the presence of increased body fat mass and insulin resistance. Recent reports have suggested that leptin can act through some of the components of the insulin signaling cascade, such as insulin receptor substrates (IRS-1 and IRS-2), phosphatidylinositol 3-kinase (PI 3-kinase), and mitogen-activated protein kinase, and can modify insulin-induced changes in gene expression in vitro and in vivo. Well differentiated hepatoma cells (Fao) possess both the long and short forms of the leptin receptor and respond to leptin with a stimulation of c-fos gene expression. In Fao cells, leptin alone had no effects on the insulin signaling pathway, but leptin pretreatment transiently enhanced insulin-induced tyrosine phosphorylation and PI 3-kinase binding to IRS-1, while producing an inhibition of tyrosine phosphorylation and PI 3-kinase binding to IRS-2. Leptin alone also induced serine phosphorylation of Akt and glycogen synthase kinase 3 but to a lesser extent than insulin, and the combination of these hormones was not additive. These results suggest complex interactions between the leptin and insulin signaling pathways that can potentially lead to differential modification of the metabolic and mitotic effects of insulin exerted through IRS-1 and IRS-2 and the downstream kinases that they activate.

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.