Insulin controls cell metabolism via metabolic signal transduction pathways and cell proliferation via mitogenic signal pathways. Metabolic signalling occurs through receptor-activated phosphorylation of insulin receptor substrate (IRS) proteins that subsequently activate phosphatidylinositide 3-kinase (PI3-kinase) to generate second messengers that produce increased phosphorylation and activation of protein kinase B ⁄ Akt (PKB). PKB appears to be central to downstream control of both glucose uptake and glycogen synthesis by insulin [1,2]. Although adipocytes are terminally differentiated cells that do not divide further, insulin has the potential for genomic control via a mitogenic signalling pathway. This may also be mediated by IRS; insulin activation of the G-protein Ras leads to phosphorylation and activation of mitogenactivated protein (MAP) kinases -extracellular signal-related kinase (ERK) 1 and 2 [3], and p38 [4,5] Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO 2 at 37°C 2 . Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1 ⁄ 2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulinstimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgeryinduced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.Abbreviations ERK, extracellular signal-related kinase; GLUT4, insulin-sensitive glucose transporter-4; IRS, insulin receptor substrate; MAP, mitogenactivated protein; PKB, protein kinase B; PI3-kinase, phosphatidylinositide 3-kinase.

Purpose: Deregulation of key cellular pathways is fundamental for the survival and expansion of neoplastic cells. In cancer, regulation of gene transcription can be mediated in a variety of ways. The purpose of this study was to assess the impact of gene dosage on gene expression patterns and the effect of other mechanisms on transcriptional levels, and to associate these genomic changes with clinicopathologic parameters.Experimental Design: We screened 97 invasive diploid breast tumors for DNA copy number alterations and changes in transcriptional levels using array comparative genomic hybridization and expression microarrays, respectively.Results: The integrative analysis identified an increase in the overall number of genetic alterations during tumor progression and 15 specific genomic regions with aberrant DNA copy numbers in at least 25% of the patient population, i.e., 1q22, 1q22-q23.1, 1q25.3, 1q32.1, 1q32.1-q32.2, 8q21.2-q21.3, 8q22.3, 8q24.3, and 16p11.2 were recurrently gained, whereas 11q25, 16q21, 16q23.3, and 17p12 were frequently lost (P < 0.01). An examination of the expression patterns of genes mapping within the detected genetic aberrations identified 47 unique genes and 1 Unigene cluster significantly correlated between the DNA and relative mRNA levels. In addition, more malignant tumors with normal gene dosage levels displayed a recurrent overexpression of UBE2C, S100A8, and CBX2, and downregulation of LOC389033, STC2, DNALI1, SCUBE2, NME5, SUSD3, SERPINA11, AZGP1, and PIP.Conclusions: Taken together, our findings suggest that the dysregulated genes identified here are critical for breast cancer initiation and progression, and could be used as novel therapeutic targets for drug development to complement classical clinicopathologic features. Clin Cancer Res; 16(15); 3860-74. ©2010 AACR.

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC 50 for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t1 ⁄ 2 of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.The incidence of type 2 diabetes is rapidly increasing in all parts of the world that allows a sedentary and affluent lifestyle, as the disease is closely associated with obesity. It has been estimated that over 200 million people will be afflicted with the disease by the end of this decade. The pathogenesis of diabetes is not understood in any great detail, but it is generally believed that insulin resistance in skeletal muscle and especially in adipose tissue is an early, if not primary, event. The peripheral insulin resistance is compensated for by increased concentrations of circulating insulin, masking development to diabetes. Eventually the pancreatic ␤-cells fail to compensate for the insulin resistance, and type 2 diabetes can be diagnosed. The insulin resistance of muscle and fat tissues is the result of impaired signal transduction from the cell surface receptor of insulin to metabolic effects, such as to increase glucose transport and inhibit lipolysis in adipocytes (1, 2). Epidemiological studies have failed to provide a strong link between the disease in general and genetic polymorphisms in the genes coding for the signaling proteins. The occupied insulin receptor autophosphorylates and then phosphorylates the insulin receptor substrate-1 (IRS1) 2 protein on tyrosine residues to provide docking sites for downstream activation of signal-transducing proteins (1).Recently, the tyrosine phosphorylation of IRS1 was shown to be the first signaling step to exhibit reduced sensitivity to insulin in adipocytes from patients with type 2 diabetes (3). The finding depended on the realization that adipocytes obtained from human beings become insulin-resistant from the surgical cell isolation procedures and that the resistance is reversed by overnight incubation of the cells (3). This insulin resistance is manifest downstream o...

Caveolae are plasma membrane invaginations with several functions, one of which appears to be to organize receptor mediated signalling. Here we report that in primary human subcutaneous adipocytes the insulin receptor was localized to caveolae by electron microscopy/immunogold detection and by isolating caveolae from plasma membranes. Part of insulin receptor substrate 1 (IRS1), the immediate downstream signal mediator, was colocalized with the insulin receptor in the plasma membrane and caveolae, as demonstrated by immunofluorescence microscopy, immunogold electron microscopy, and immunogold electron microscopy of transfected recombinant HA-IRS1. In contrast, rat epididymal adipocytes lacked IRS1 at the plasma membrane. Depletion of cholesterol from the cells using b-cyclodextrin blocked insulin stimulation of glucose uptake, insulin inhibition of perilipin phosphorylation in response to isoproterenol, and insulin stimulation of protein kinase B and Map-kinases extracellular signal-related kinase (ERK)1/2 phosphorylation. Insulin-stimulated phosphorylation of the insulin receptor and IRS1 was not affected, indicating that caveolae integrity is required downstream of IRS1. In conclusion we show that insulin receptor and IRS1 are both caveolar proteins and that caveolae are required for both metabolic and mitogenic control in human adipocytes. Our results establish caveolae as foci of insulin action and stress the importance of examining human cells in addition to animal cells and cell lines.Keywords: extracellular signal-related kinase; ultrastructure; protein kinase B; b-cyclodextrin; glucose transport.Insulin exerts control over cell metabolism by binding to its cell surface receptor, which has been characterized in great detail [1][2][3][4][5]. The occupied receptor is autophosphorylated on tyrosine residues and can thereby tyrosine phosphorylate other cellular proteins, to transduce the insulin signal into the signal network of the cell. Chief among these proteins are the insulin receptor substrate (IRS) family of proteins [1]. When tyrosine is phosphorylated they can transmit metabolic and mitogenic signals. The further downstream events involve the generation of second messengers and phosphorylation of protein kinase B/Akt (PKB). Eventually glucose transporter GLUT4 is translocated to the plasma membrane for glucose uptake and other target proteins (e.g. perilipin [6]) are phosphorylated/dephosphorylated. Insulin's ability for mitogenic signalling is transmitted via the Map-kinases extracellular signal-related kinase (ERK)1/2 for phosphorylation control of transcription factors. The precise mechanisms for insulin's cellular control are not yet known in detail, and especially not so in human cells and tissues.Caveolae are 25-150 nm invaginations of the plasma membrane and are found in most cell types. They are particularly abundant in rat adipocytes and increase dramatically in number when 3T3-L1 fibroblasts are differentiated into fat cells and become insulin responsive [7][8][9]. Cholesterol and sphingoli...

Brain tumors are the leading cause of cancer-related death in children but high-grade gliomas in children and adolescents have remained a relatively under-investigated disease despite this. A better understanding of the cellular and molecular pathogenesis of the diseases is required in order to improve the outcome for these children. In vitro-cultured primary tumor cells from patients are indispensable tools for this purpose by enabling functional analyses and development of new therapies. However, relevant well-characterized in vitro cultures from pediatric gliomas cultured under serum-free conditions have been lacking. We have therefore established patient-derived in vitro cultures and performed thorough characterization of the cells using large-scale analyses of DNA methylation, copy-number alterations and investigated their stability during prolonged time in culture. We show that the cells were stable during prolonged culture in serum-free stem cell media without apparent alterations in morphology or growth rate. The cells were proliferative, positive for stem cell markers, able to respond to differentiation cues and initiated tumors in zebrafish and mice suggesting that the cells are cancer stem cells or progenitor cells. The cells accurately mirrored the tumor they were derived from in terms of methylation pattern, copy number alterations and DNA mutations. These unique primary in vitro cultures can thus be used as a relevant and robust model system for functional studies on pediatric brain tumors.