Overexpression Of Insulin Receptor Research Articles

Down-Regulation of INSR Restores Th17/Treg Immune Balance and Alleviates Airway Hyperviscosity in Asthmatic Mice via Inactivation of STAT3 Pathway.

Allergic asthma (AA) is a prevalent chronic airway inflammation disease. In this study, this study aims to investigate the biological functions and potential regulatory mechanisms of the insulin receptor (INSR) in the progression of AA. BALB/c mice (n = 48) were randomly divided into the following groups: control group, AA group, AA+Lentivirus (Lv)-vector short hairpin RNA (shRNA) group, AA+Lv-vector group, AA+Lv-INSR shRNA group, and AA+Lv-INSR group. The pulmonary index was calculated. mRNA and protein expression levels of INSR, signal transducer and activator of transcription 3 (STAT3), Janus kinase 2 (JAK2), phosphorylated-STAT3 (p-STAT3), phosphorylated-JAK2 (p-JAK2), alpha-smooth muscle actin (α-SMA), febrile neutropenia (FN), mucin 5AC (MUC5AC), and mucin 5B (MUC5B) were examined using reverse-transcription quantitative PCR (RT-qPCR) and western blot assays. Positive expressions of INSR, retinoic acid-related orphan receptor gamma-t (RORγt), and forkhead box protein P3 (Foxp3) were quantified by immunohistochemistry. Fluorescence intensities of α-SMA and FN were detected by immunofluorescence. Pathological morphology was observed through hematoxylin-eosin (H&E) staining, Masson staining, and Periodic Acid-Schiff (PAS) staining. Contents of immunoglobulin E (IgE), interleukin-6 (IL-6), eotaxin, interleukin-4 (IL-4), interleukin-13 (IL-13), interferon-γ (IFN-γ), interleukin-17 (IL-17), and interleukin-10 (IL-10) were quantified using enzyme-linked immunosorbent assay (ELISA). The percentage of T helper 17 (Th17) and regulatory T (Treg) cells was determined through flow cytometry. Compared to the control group, expression levels of INSR, p-STAT3, p-JAK2, α-SMA, FN, MUC5AC, MUC5B, RORγt, and Foxp3, as well as IgE, IL-6, eotaxin, IL-4, IL-13, and IL-17 contents, pulmonary index, glycogen-positive area (%), and Th17 cell percentage significantly increased (p < 0.05). Additionally, pulmonary histopathological deterioration and collagen deposition were aggravated, while Treg cell percentage and IFN-γ and IL-10 contents remarkably decreased (p < 0.05). The overexpression of INSR further exacerbated the progression of allergic asthma, but the down-regulation of INSR reversed the trends of the above indicators. The down-regulation of INSR alleviates airway hyperviscosity, inflammatory infiltration, and airway remodeling, restoring Th17/Treg immune balance in AA mice by inactivating the STAT3 pathway.

1482-PUB: Insulin Receptors in Vascular Smooth Muscle Cells Regulate Plaque Stability by Its actions on Glycolysis and Oxidative Phosphorylation

Vascular smooth muscle cells (VSMC) have prominent roles in the pathology of atherosclerotic plaque, where it forms intimal hyperplasia, maintains extracellular matrix (ECM) and interacts with lipoproteins to accelerate inflammation and formation of foam cells. Diabetes and insulin resistance exacerbate all of these histopathologies of atherosclerotic plaques. Further, diabetes increase the formation of unstable plaques that feature less VSMC and ECM, but more inflammatory cells and necrotic lesions, which, on rupture, cause thrombosis and acute myocardial infarction. We reported that insulin receptors (IR) are responsible for increasing arterial restenosis since mouse with IR deletion in the VSMC showed less proliferation, more apoptosis, and less arterial hyperplasia after intimal injury. Surprisingly, in mice with specific deletion of IR in VSMC (SMIRKO/ApoE-/-) , severity of atherosclerosis was increased and the plaques exhibited unstable pathologies of less VSMC and ECM, but greater levels of macrophages and necrosis than ApoE-/- mice. Analysis of glucose glycolysis (extracellular acidification rate, ECAR) and oxidative phosphorylation (oxygen consumption rate, OCR) , using Seahorse assay, showed dramatically decreased metabolic rates in VSMC from SMIRKO/ApoE-/- compared to ApoE-/- mice. Overexpression of IR in VSMCs increased ECAR and OCR, which were further stimulate by insulin, only in VSMC with IR. [J1] Insulin increased expressions of glycolytic enzymes including hexokinase 2 and phopshofructose kinase in VSMCs overexpressing IR. These findings have identified the importance of IR on the regulation of glycolysis and mitochondrial metabolism in VSMC, which could increase the stability of the atherosclerotic plaques by increased VSMCs proliferation and ECM, but reduced their apoptosis and productions of inflammatory cytokines. [J1]Hard to understand⋯maybe combine with the next phrase? Disclosure Q.Li: None. J.Fu: None. K.Park: None. Q.Li: None. G.L.King: Advisory Panel; Medtronic, Research Support; Janssen Pharmaceuticals, Inc.

Improvement of Lipotoxicity-Induced Islet β Cellular Insulin Secretion Disorder by Osteocalcin.

Background Osteocalcin (OCN) has been proved to be closely related with the development of type 2 diabetes mellitus (T2DM). We aimed to study if OCN could improve the disorder of islet cell caused by lipotoxicity. Methods Alizarin red staining was used to investigate the mineralization. Western blotting and ELISA methods were used to measure protein expression. Immunofluorescence staining was used to investigate the protein nuclear transfer. Results High glucose and high fat inhibited the differentiation of osteoblast precursors. Overexpression of insulin receptor (InsROE) significantly promoted the Runx2 and OCN expression. The increase of insulin, Gprc6a, and Glut2 by osteoblast culture medium overexpressing insulin receptor was reversed by osteocalcin neutralizing antibody. Undercarboxylated osteocalcin (ucOC) suppressed the lipotoxic islet β-cell damage caused by palmitic acid. The FOXO1 from intranuclear to extranuclear was also significantly increased after ucOC treatment compared with the group PA. Knockdown of Gprc6a or suppression of PI3K/AKT signal pathway could reverse the upregulation of GPRC6A/PI3K/AKT/FoxO1/Pdx1 caused by ucOC. Conclusion OCN could activate the FOXO1 signaling pathway to regulate GLUT2 expression and improve the insulin secretion disorder caused by lipotoxicity.

Insulin Receptor in Pancreatic Cancer-Crown Witness in Cross Examination.

Simple SummaryPancreatic cancer (PDAC) is a highly malignant disease with low survival rates. Due to its close proximity to the insulin producing pancreatic islets, PDAC should be exposed to comparatively higher concentrations of the growth promoting hormone insulin. We wanted to know if PDAC might take advantage of this circumstance. Therefore we cross examined the insulin receptor’s (IR) role in PDAC and precursor lesions and put it into context with the expression of the insulin-like growth factor 1 receptor (IGF1R). Our study of 160 PDAC patient samples showed that IR overexpression is already present at the precursor level. IR overexpression in PDAC was associated with adverse clinical features. The IGF1R was found to play a different role than formerly assigned. We hypothesize that the close proximity to the pancreatic islets is exploited by PDAC up to the point of the islets’ ultimate destruction by local cancer growth.Background: The proximity of pancreatic cancer (PDAC) to the physiological source of the growth promoting hormone insulin might be exploited by this highly malignant cancer entity. We investigated if (I) PDACs express the insulin receptor (IR) in cancer cells and cancer vasculature, (II) if IR correlates with clinicopathological patient characteristics, including survival, and hence is involved in PDAC biology, (III) if IR is already expressed in precursor lesions, if (IV) the IGF1 receptor (IGF1R) is associated with clinicopathological patient characteristics and survival and (V) is linked to IR expression. Methods: 160 PDAC samples were examined for IR and IGF1R expression by immunohistochemistry. A modified HistoScore was correlated with clinicopathological characteristics and survival. Results: IR overexpression was already observed in pancreatic intraepithelial neoplasia. Furthermore, it was more frequently observed in advanced disease and associated with distant metastasis, UICC stage, lymphatic invasion and an increased lymph node ratio, but without impacting survival in the end. IGF1R expression was not associated with clinicopathological parameters or survival, in contrast to former paradigms. Conclusions: We hypothesize that the close proximity to the pancreatic islets might be advantageous for cancer growth at first, but it experiences self-limitation due to surgical removal or local destruction following accelerated cancer growth.

SUN-131 The Roles of Two Insulin Receptor Isoforms in Triple Negative Breast Cancer Growth

Systemic hyperinsulinemia is believed to be an important factor in the progression of a number of cancers, including breast cancer by activating the insulin receptor (IR) signaling cascade in the tumor cells. The IR is expressed in two isoforms, IR-A and IR-B. IR-B is the full-length isoform, while IR-A is lacking 12 amino acids in the α-subunit due to exon 11 alternative splicing. IR-A is predominantly expressed in cancer tissues, while IR-B is mostly expressed in metabolic tissues. The IR and closely related insulin-like growth factor 1 receptor (IGF-1R) are expressed in different ratios in cancer cells. Compared with estrogen receptor positive breast cancers, triple negative breast cancers (TNBC) frequently have higher ratios of IR to IGF-1R. Hyperinsulinemia is associated with increased prevalence of TNBC in pre-menopausal women. Although new targeted therapies are emerging, among breast cancer subtypes TNBC continues to carry the worst prognosis and therefore developing a greater understanding of the links between IR signaling and TNBC progression is critical. The aim of this study is to understand the role of IR-A and IR-B on proliferation, metastasis and metabolism in breast cancer cells. We stably overexpressed human IR-A (IR-A OE) and IR-B (IR-B OE) in TNBC MDA-MB-231 (231) and murine c-myc/vegf overexpressing Mvt1 cells with lentiviral transduction using pLVX-IRES-puro HIV-1-based expression vectors with cDNA encoding the human IR-A,IR-B and control cDNA sequences. Native murine IR was silenced using lentiviral transduction of shRNA in the Mvt1 cells. Overexpression of IR was confirmed at a protein level by western blot, and RNA isoform expression was confirmed using real time PCR. Cell proliferation assays were performed in DMEM/10% FBS and revealed that MDA-MB-231 cells with IR-A OE cells had 15% higher proliferation rates than 231 IR-B OE cells. We then examined the IR signaling pathways by western blot in DMEM/10% FBS. No differences in phosphorylated or total ERK1/2 were observed between control, 231 IR-A OE and 231 IR-B OE cells. 231 IR-A OE cells were found to have 15-fold greater Akt phosphorylation (Ser473) than 231 control cells (p=0.0008) and 4 fold higher pAkt(Ser473) compared with 231 IR-B OE cells (p=0.0016). Further, we found that 231 IR-A OE cells had approximately 2 fold greater expression of c-myc protein compared with both 231 control (p=0.047) and 231 IR-B OE cells (p=0.026). No differences in c-myc expression were observed between 231 IRB OE and 231 control cells. In our previous studies we found that insulin stimulates c-myc expression and silencing the IR reduces c-myc expression in cancer cells. Our current studies show that IR-A, rather than IR-B is the insulin receptor isoform that regulates c-myc expression in human TNBC. Reference: (1) Belfiore et al., Endocr Relat Cancer. 2011; 18(4):R125-R147. (2) Ferguson et al., Breast Cancer Res. 2012; 14(1): R8.

The IGF-II-Insulin Receptor Isoform-A Autocrine Signal in Cancer: Actionable Perspectives.

Insulin receptor overexpression is a common event in human cancer. Its overexpression is associated with a relative increase in the expression of its isoform A (IRA), a shorter variant lacking 11 aa in the extracellular domain, conferring high affinity for the binding of IGF-II along with added intracellular signaling specificity for this ligand. Since IGF-II is secreted by the vast majority of malignant solid cancers, where it establishes autocrine stimuli, the co-expression of IGF-II and IRA in cancer provides specific advantages such as apoptosis escape, growth, and proliferation to those cancers bearing such a co-expression pattern. However, little is known about the exact role of this autocrine ligand–receptor system in sustaining cancer malignant features such as angiogenesis, invasion, and metastasis. The recent finding that the overexpression of angiogenic receptor kinase EphB4 along with VEGF-A is tightly dependent on the IGF-II/IRA autocrine system independently of IGFIR provided new perspectives for all malignant IGF2omas (those aggressive solid cancers secreting IGF-II). The present review provides an updated view of the IGF system in cancer, focusing on the biology of the autocrine IGF-II/IRA ligand–receptor axis and supporting its underscored role as a malignant-switch checkpoint target.

1785-P: Mice with Muscle-Specific Insulin Receptor Overexpression Are Protected from Diet-Induced Obesity and Glucose Intolerance but Develop Post-Receptor Insulin Resistance

Skeletal muscle insulin resistance is a prominent feature of type 2 diabetes (T2D) that precedes and predicts the development of disease in high risk populations. In individuals with T2D or pre-T2D, this insulin resistance is due, at least in part, to a decrease in the number of functional insulin receptors (IR) that localize on the cell membrane. To restore the number of IR, we generated mice that specifically overexpress IR in skeletal muscle (IRMOE). On chow diet, IRMOE mice had similar body weight compared to control, but displayed reduced body fat and higher lean mass percentage. Basal phosphorylation of IR, IRS and Akt were significantly higher in muscles from IRMOE mice compared to control, resulting in their improved glucose tolerance. Surprisingly, however, after i.v. injection of insulin, stimulated Akt phosphorylation was significantly lower in IRMOE muscle compared to control, despite persistent higher phosphorylation in IR and IRS, indicating activation of negative regulators in the IR signaling pathway when IR is chronically activated. Consistent with this, euglycemic-hyperinsulinemic clamp studies revealed significantly lower glucose infusion rate in insulin stimulated IRMOE mice compared to control. Increased basal and decreased insulin stimulated Akt phosphorylation was also observed in muscles of IRMOE mice after challenge with HFD. However, when fed HFD, IRMOE mice displayed both improved glucose tolerance and insulin tolerance which was in part due to being resistant to diet-induced obesity, despite consuming more calories. Taken together, these data indicate that muscle specific IR overexpression can protect mice from diet-induced obesity and its effects on glucose metabolism, but that chronic overexpression of IR can lead to post-receptor desensitization indicating additional post-receptor regulators of insulin signaling, which could provide new targets for therapy of T2D. Disclosure G. Wang: None. W. Cai: None. T.M. Batista: None. S. Softic: None. C. Kahn: Advisory Panel; Self; MedImmune. Board Member; Self; Kaleido Biosciences. Consultant; Self; AntriaBio, Inc., Cobalt Therapeutics, Flagship Pioneering. Research Support; Self; Alnylam. Funding American Diabetes Association (9-17-CMF-016 to G.W.)

Tissue-Specific Upregulation of Drosophila Insulin Receptor (InR) Mitigates Poly(Q)-Mediated Neurotoxicity by Restoration of Cellular Transcription Machinery.

Polyglutamine [poly(Q)] disorders are a class of trinucleotide repeat expansion neurodegenerative disorders which are dominantly inherited and progressively acquired with age. This group of disorders entail the characteristic formation of protein aggregates leading to widespread loss of neurons in different regions of the brain. SCA3 and HD, the two most commonly occurring types of poly(Q) disorders were examined in the present study. With the aim of elucidating novel genetic modifiers of poly(Q) disorders, the Drosophila insulin receptor (InR) was identified as a potential suppressor of poly(Q)-induced neurotoxicity and degeneration. We demonstrate for the first time that targeted upregulation of InR could effectively mitigate poly(Q)-mediated neurodegeneration in fly models. A significant reduction in poly(Q)-mediated cellular stress and apoptosis was noted upon InR overexpression in poly(Q) background. We further reveal that targeted upregulation of InR causes a substantial reduction in poly(Q) aggregate formation with the residual inclusion bodies localised to the cytoplasm. We also demonstrate that InR achieves suppression of poly(Q) toxicity by replenishing the cellular pool of CREB binding protein and improving the histone acetylation status of the cell. This leads to restoration of the cellular transcriptional machinery which is otherwise severely compromised in poly(Q) disease conditions. Interestingly, there also appeared a possibility of autophagy-mediated rescue of poly(Q) phenotype due to upregulation of InR. Therefore, our study strongly suggests that modulation of the insulin signalling pathway could be an effective therapeutic intervention against poly(Q) disorders.

Insulin Modulates Hippo Signaling by Impairing YAP Function

Yes‐associated protein (YAP) is the main transcriptional coactivator of the Hippo pathway, which regulates organ size, tissue homeostasis, and regeneration. YAP integrates multiple inputs from different signaling cascades. Recent evidence implicates YAP also in the control of nutrient and energy status of the cell, but the mechanisms underlying this function are unclear. In this study, we show that insulin modulates YAP activity in classic insulin target cells. We found by subcellular fractionation and Western blotting that insulin increases YAP phosphorylation, with a significant decrease in the abundance of YAP in the nuclei of HepG2 liver cells. Further, this regulation is likely mediated through the insulin receptor (IR), as overexpression of IR increased YAP phosphorylation. Proximity ligation assay analysis revealed a marked reduction in the interaction of YAP with TEA domain (TEAD) transcription factors in the nuclei of insulin‐treated cells. Consistent with these findings, insulin significantly impaired YAP/TEAD‐mediated transcription as measured by a dual luciferase assay in both HepG2 and C2C12 muscle cells. Further, quantitative PCR of HepG2 cells treated with insulin revealed reduced transcription of YAP regulated genes. Insulin alters transcription of numerous genes relevant to energy homeostasis, and we observed that knockdown of YAP by siRNA alters expression of genes in gluconeogenesis. Insulin stimulates activation of the phosphoinositide 3‐kinase (PI3K) pathway. The inhibitory effect of insulin on YAP transcription was abrogated by small‐molecule inhibitors of PI3K. Collectively, our results identify insulin as a previously undescribed suppressor of YAP activity and provide insight into an important link between the insulin and Hippo pathways.Support or Funding InformationThis work was supported by the National Institutes of Health Intramural Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced insulin receptor, but not PI3K, signalling protects podocytes from ER stress

Disruption of the insulin-PI3K-Akt signalling pathway in kidney podocytes causes endoplasmic reticulum (ER) stress, leading to podocyte apoptosis and proteinuria in diabetic nephropathy. We hypothesised that by improving insulin sensitivity we could protect podocytes from ER stress. Here we use established activating transcription factor 6 (ATF6)- and ER stress element (ERSE)-luciferase assays alongside a novel high throughput imaging-based C/EBP homologous protein (CHOP) assay to examine three models of improved insulin sensitivity. We find that by improving insulin sensitivity at the level of the insulin receptor (IR), either by IR over-expression or by knocking down the negative regulator of IR activity, protein tyrosine-phosphatase 1B (PTP1B), podocytes are protected from ER stress caused by fatty acids or diabetic media containing high glucose, high insulin and inflammatory cytokines TNFα and IL-6. However, contrary to this, knockdown of the negative regulator of PI3K-Akt signalling, phosphatase and tensin homolog deleted from chromosome 10 (PTEN), sensitizes podocytes to ER stress and apoptosis, despite increasing Akt phosphorylation. This indicates that protection from ER stress is conferred through not just the PI3K-Akt pathway, and indeed we find that inhibiting the MEK/ERK signalling pathway rescues PTEN knockdown podocytes from ER stress.

Insulin receptor based lymphocyte trafficking in the progression of type 1 diabetes

The insulin receptor (IR) is a transmembrane receptor which recognizes and binds the hormone insulin. We describe two models that were devised to explore the role of IR over-expression on T-lymphocytes and their chemotactic motility in the progression of type 1 diabetes. FVB/NJ-CD3-3×FLAG-mIR/MFM mice were generated to selectively over-express 3×FLAG tagged murine IR in T-lymphocytes via an engineered CD3 enhancer and promoter construct. Insertion of the 3×FLAG-mIR transgene into FVB/NJ mice, a known non-autoimmune prone strain, lead to a minor population of detectable 3×FLAG-mIR tagged T-lymphocytes in peripheral blood and the presence of a few lymphocytes in the pancreas of the Tg+/- compared to age matched Tg-/- control mice. In order to induce stronger murine IR over-expression then what was observed with the CD3 enhancer promoter construct, a second system utilizing the strong CAG viral promoter was generated. This system induces cell specific IR over-expression upon Cre-Lox recombination to afford functional 3×FLAG tagged murine IR with an internal eGFP reporter. The pPNTlox2-3×FLAG-mIR plasmid was constructed and validated in HEK-Cre-RFP cells to ensure selective Cre recombinase based 3×FLAG-mIR expression, receptor ligand affinity towards insulin, and functional initiation of signal transduction upon insulin stimulation.

S961, a biosynthetic insulin receptor antagonist, downregulates insulin receptor expression & suppresses the growth of breast cancer cells.

Background & objectives:Insulin resistance associated with hyperinsulinaemia and overexpression of insulin receptors (IRs) have been intricately linked to the pathogenesis and treatment outcomes of the breast carcinoma. Studies have revealed that upregulated expression of IRs in breast cancer pathogenesis regulates several aspects of the malignant phenotype, including cell proliferation and metastasis. This study was aimed to investigate the pivotal role of an IR antagonist S961 on IR signalling and other biological parameters in MCF-7, MDA-MB-231 and T47D cell lines.Methods:The effect of human insulin and S961 on growth, proliferation rate and clonogenic potential of breast cancer cells was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay and clonogenic assay. The mRNA expression of IR isoforms (IR-A and IR-B) was measured in the breast carcinoma cells using quantitative PCR.Results:The study revealed that breast cancer cells predominantly expressed IR-A isoform and showed extensive growth and proliferation owing to IR overexpression. It was found that S961 downregulated the IRs (IR-A and IR-B) with nanomolar dose and efficiently blocked expression of IRs even in the presence of insulin. IR mRNA expression levels were significantly downregulated in the continued presence of S961. S961 also inhibited cellular proliferation and colony formation in breast tumour cells.Interpretation & conclusions:IR antagonist, S961 showed distinct antagonism in vitro and appeared to be a powerful therapeutic modality that might provide insight into the pathogenesis of impaired IR signalling.

Bi-directional regulation of brown fat adipogenesis by the insulin receptor.

Insulin is a potent inducer of adipogenesis, and differentiation of adipocytes requires many components of the insulin signaling pathway, including the insulin receptor substrate IRS-1 and phosphatidylinositol 3-kinase (PI3K). Brown pre-adipocytes in culture exhibit low levels of insulin receptor (IR), and during differentiation there is both an increase in total IR levels and a shift in the alternatively spliced forms of IR from the A isoform (–exon 11) to the B isoform (+exon 11). Brown pre-adipocyte cell lines from insulin receptor-deficient mice exhibit dramatically impaired differentiation and an inability to regulate alternative splicing of the insulin receptor. Surprisingly, re-expression of either splice isoform of IR in the IR-deficient cells fails to rescue differentiation in these cells. Likewise, overexpression of IR in control IRlox cells also results in inhibition of differentiation and a failure to accumulate expression of the adipogenic markers peroxisome proliferator-activated receptor gamma, Glut4, and fatty acid synthase, although cells overexpressing IR retain the ability to activate PI3K and down-regulate mitogen-activated protein kinase (MAPK) phosphorylation. Thus, differentiation of brown adipocytes requires a timed and regulated expression of IR, and either the absence or overabundance of insulin receptors in these cells dramatically inhibits differentiation.

Overexpression of insulin receptor partially improves obese and diabetic phenotypes in db/db mice.

Type 2 diabetes mellitus (T2DM) is one of the major health concern among the world. Several treatment options for T2DM are in clinical use, including injecting insulin, promoting insulin secretion by insulin secretagogues, and improving insulin sensitivity by insulin sensitizers. However, increasing the amount of insulin receptor in insulin-target tissues has not been explored. In order to test the efficacy of insulin receptor overexpression for improving glucose control, we established a transgenic mouse line expressing human insulin receptor (INSR). We analyzed, growth, energy balance, and glucose control of INSR-overexpressing db/db mice (INSR; db/db), which we produced by mating INSR transgenic mice with db/db mice, a genetic model of obesity due to insufficient leptin signaling. Compared to db/db mice, INSR; db/db mice were rescued from hyperphagia and obesity, leading to improved blood glucose levels. Unexpectedly, however, INSR; db/db mice presented with stunted growth, accompanied by decreased plasma levels of free IGF1 and IGFBP-3, indicating the down-regulation of GH/IGF1 axis. These phenotypes were observed in INSR; db/db mice but not in INSR littermates. Meanwhile, bone defects observed in db/db male mice were not rescued. Moreover, improved blood glucose was not accompanied by improved insulin sensitivity. Therefore, overexpression of insulin receptor improves obese and diabetic phenotypes in db/db mice, with consequences on growth.

Oncogenic functions of IGF1R and INSR in prostate cancer include enhanced tumor growth, cell migration and angiogenesis.

We scrutinized the effect of insulin receptor (INSR) in addition to IGF1R in PCa using in vitro and in vivo models. In-vitro overexpression of IGF1R and INSRA, but not INSRB increased cell proliferation, colony formation, migration, invasion and resistance to apoptosis in prostate cancer cells (DU145, LNCaP, PC3). Opposite effects were induced by downregulation of IGF1R and total INSR, but not INSRB. In contrast to tumor cells, non-cancerous epithelial cells of the prostate (EP156T, RWPE-1) were inhibited on overexpression and stimulated by knockdown of receptors. In-vivo analyses using the chicken allantoic membrane assay confirmed the tumorigenic effects of IGF1R and INSR. Apart of promoting tumor growth, IGF1R and INSR overexpression also enhanced angiogenesis indicated by higher vessel density and increased number of desmin-immunoreactive pericytes. Our study underscores the oncogenic impact of IGF1R including significant effects on tumor growth, cell migration, sensitivity to apoptotic/chemotherapeutic agents and angiogenesis, and characterizes the INSR, in particular the isoform INSRA, as additional cancer-promoting receptor in prostate cancer. Both, the insulin-like growth factor receptor 1 and the insulin receptor exert oncogenic functions, thus proposing that both receptors need to be considered in therapeutic settings.

Metabolic syndrome and colorectal cancer: is hyperinsulinemia/insulin receptor-mediated angiogenesis a critical process?

Components of metabolic syndrome (MS) have been individually linked to colorectal cancer risk and prognosis; however, an understanding of the dominant mechanisms is lacking. Twenty-one patients (10 MS; 11 non-MS) with resectable colorectal cancer were prospectively enrolled. Patients were classified for MS by the World Health Organization criteria and tested for circulating vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), fasting insulin, and tumor expression of IGF-1 receptor (IGF-1R), insulin-receptor (IR) and receptor for advanced glycation end-products (RAGE). Circulating markers were re-tested 6 months after surgery. The MS group had significantly higher baseline and post-operative fasting insulin levels (p < 0.001 and 0.003). No differences were observed in circulating IL-6, VEGF, IGF-1 and free IGF-1. By immunohistochemistry (IHC), IGF-1R expression was significantly higher in tumor vs. normal tissues (p < 0.001) while IR expression showed no difference. Interestingly, 64% of tumors demonstrated high IR positivity in the vessels within or surrounding the tumor stroma, but not in the vessels away from the tumor. By reverse transcription polymerase chain reaction (RT-PCR), tumor IGF-1R over-expression (80%) was confirmed, but there was no difference between MS and non-MS patients. Tumor RAGE over-expression was found in 67% of patients and was equally distributed between the two groups. Hyperinsulinemia was the only significant factor distinguishing patients with colorectal cancer who have MS. The preferential over-expression of IR in the peri-tumoral microvessels suggests that hyperinsulinemia might contribute to colorectal cancer growth by enhancing angiogenesis.

Depletion of histone deacetylase 3 antagonizes PI3K-mediated overgrowth of Drosophila organs through the acetylation of histone H4 at lysine 16

Core histone modifications play an important role in chromatin remodeling and transcriptional regulation. Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. Herein, we demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster resulted in a reduction in body size. Further genetic studies showed that Hdac3 counteracted the organ overgrowth induced by overexpression of insulin receptor (InR), phosphoinositide 3-kinase (PI3K) or S6 kinase (S6K), and the growth regulation by Hdac3 was mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the overexpression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, resulted in changes in body size. Furthermore, we found that H4K16ac was modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracted the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, our studies indicated that Hdac3 served as an important regulator of the PI3K pathway and revealed a novel link between histone acetylation and growth control.

Abstract 18877: Novel Mouse Model for Testing NADPH Oxidase (Nox2) Role in Insulin Resistance Mediated Endothelial Dysfunction

Introduction: Insulin resistance (IR) plays a pivotal role in type 2 diabetes, obesity and hypercholesterolemia. Reactive oxygen species (ROS) play a key role in IR related endothelial dysfunction. We have reported that, superoxide (SO), an important ROS is produced at higher levels in IR and established that NADPH oxidase (Nox) is the primary source of SO in IR endothelium. Here we report our study to ascertain the role of Nox2 in IR. Methods: A unique murine model of endothelium specific IR, Endothelial Specific Mutated Insulin Receptor Over-expression (ESMIRO) and mice with universal deletion of Nox2 gene (Nox2 y/- ) were used here. Nox2 y/- mice was crossed with ESMIRO to generate double transgenics (ESMIRO/Nox2 y/- ). Endothelial function was analysed by measuring 1. aortic ring relaxation. 2. superoxide production. Expression of Nox and ROS related genes were examined by PCR and Western blotting. Results: Realtime PCR and Western blot of organs and pulmonary endothelial cells (PEC) confirmed the total loss of Nox2 in ESMIRO/Nox2 y/- . Growth rate and organ weights were unaltered in ESMIRO/Nox2 y/- . However, there was a trend towards reduced BP in double transgenics (119±1 mmHg in ESMIRO Vs 114±2 mmHg in ESMIRO/Nox2 y/- ; p=0.06). Metabolic characteristics including ITT and GTT showed no difference between two lines. Blood insulin levels before and after glucose injection were similar. Basal superoxide in PEC of ESMIRO/Nox2 y/- were 240±11% (p=0.02) reduced when compared to ESMIRO. Nitric oxide (NO) mediated vascular relaxation was improved in double transgenic animals (100±4% in response to 1000 nM Ach in ESMIRO/Nox2 y/- Vs 77±6% in ESMIRO). There was no change in endothelial independent relaxation (109±5% in response to 1000 nM SNP in ESMIRO/Nox2 y/- Vs 104±6% in ESMIRO). Realtime PCR showed that the important ROS scavenger superoxide dismutase 1 (SOD1) expression was increased above 300% in ESMIRO lungs compared to ESMIRO/Nox2 y/- . Other Nox and ROS related genes (Nox4, p22, p47, p67, SOD2, SOD3, eNOS &amp; catalase) expression were unchanged. Conclusions: These results show that Nox2 NADPH oxidase is a key source of the excess superoxide seen in a model of endothelium specific insulin resistance. Targeting Nox2 may be a useful therapeutic strategy in IR conditions.

Insulin resistance in breast cancer: relevance and clinical implications

Breast cancer risk is increased in women who have attributes of the insulin resistance syndrome, such as obesity (postmenopausal), central obesity (premenopausal and postmenopausal), high endogenous insulin levels, clinical diabetes and physical inactivity. There is a large body of evidence that obesity is associated with a 25 to 50% relative increase in risk of breast cancer recurrence or death, with adverse effects that appear to be independent of hormone receptor status. Obesity, particularly when it is central, is strongly associated with insulin resistance in healthy individuals and breast cancer patients. Several studies have shown that higher insulin and/or C-peptide levels (a marker of insulin secretion), both of which are linked to insulin resistance, are associated with an increased risk of recurrence and death in women with early stage breast cancer, even in the absence of diabetes. Risk is increased twofold to threefold in those with insulin levels in the highest (versus lowest) quartile. Data from our group suggest that these prognostic associations of insulin are most marked in the first 5 years post diagnosis. A role of insulin in breast cancer outcomes is biologically plausible given overexpression of insulin receptors (IR), most frequently the fetal form of the receptor (IR-A), by the majority of human breast cancers. IR-A often hybridizes with insulin-like growth factor 1 receptors to stimulate mitogenic signaling pathways; hybrid receptor activation has been associated with poor clinical outcomes. The current observational and preclinical evidence linking insulin to breast cancer is sufficiently compelling that neoadjuvant and adjuvant intervention studies have been initiated to evaluate clinical anti-cancer effects of metformin, an agent that lowers insulin levels and has other potential non-insulin-mediated anti-cancer effects (mainly through activation of adenosine monophosphate-activated protein kinase). Early results from window of opportunity neoadjuvant studies suggest short-term, single-agent metformin (2 to 3 weeks) lowers insulin levels, reduces proliferation and increases apoptosis. NCIC MA32, an ongoing randomized, multicenter, placebo-controlled, adjuvant trial involving 3,582 women with early stage breast cancer, will provide more definitive evidence regarding potential anti-cancer effects. Additional studies of metformin in the metastatic setting are underway and/or planned. Because other factors (such as inflammation, adipocytokines (leptin, adiponectin), higher estrogen levels) may also mediate prognostic effects of obesity and/or insulin resistance in breast cancer, additional research targeting these mediators as well as obesity per se is also needed.

Insulin receptor and cancer

The widespread epidemic of obesity and type 2 diabetes has raised concern for the impact of these disorders as risk factors for cancer and has renewed the interest for studies regarding the involvement of hyperinsulinemia and insulin receptor (IR) in cancer progression. Overexpression of IR in cancer cells may explain their increased sensitivity to hyperinsulinemia. Moreover, IR isoform A (IR-A) together with autocrine production of its ligand IGF2 is emerging as an important mechanism of normal and cancer stem cell expansion and is a feature of several malignancies. De novo activation of the IR-A/IGF2 autocrine loop also represents a mechanism of resistance to anticancer therapies. Increasing knowledge of the IR role in cancer has important implications for cancer prevention, which should include control of insulin resistance and hyperinsulinemia in the population and meticulous evaluation of new antidiabetic drugs for their metabolic:mitogenic ratio. We are now aware that several anticancer treatments may induce or worsen insulin resistance that may limit therapy efficacy. Future anticancer therapies need to target the IR-A pathway in order to inhibit the tumor promoting effect of IR without impairing the metabolic effect of insulin.