Insulin Receptor Kinase Activity Research Articles

A High-Throughput Radiometric Kinase Assay.

Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small-molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases.

The beneficial effects of tetracosanol on insulin-resistance by insulin receptor kinase sensibilisation

In this study, we investigated whether tetracosanol, an aliphatic alcohol isolated from Saccharum sinense, enhances insulin receptor kinase activity to exhibit an insulin synergistic effect in vitro and in vivo. The insulin plus tetracosanol enhanced insulin receptor kinase showed that AKT activity was down-regulated by S961 in differentiated L6 myotubes. Meanwhile, insulin plus tetracosanol restored the ability of glucose transporter translocation and glucose uptake in differentiated myotubes with S961-induced insulin resistance in vitro. The modification of carbon chain lengths and the hydroxyl group of tetracosanol showed that it served as a critical chemical structure for the glucostasis effect in vivo. This study provides new evidence to show that tetracosanol can improve glycaemic control via insulin receptor kinase activity induced and leads to the enhancement of glucose transporter translocation to improve glucose uptake. The hydroxyl group of tetracosanol plays a critical role for insulin receptor kinase activity.

Insulin/Insulin-like growth factors in cancer: new roles for the aryl hydrocarbon receptor, tumor resistance mechanisms, and new blocking strategies.

The insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor (IR) are receptor tyrosine kinases that are expressed in cancer cells. The results of different studies indicate that tumor proliferation and survival is dependent on the IGF1R and IR, and that their inhibition leads to reductions in proliferation and increases in cell death. Molecular targeting therapies that have been used in solid tumors include anti-IGF1R antibodies, anti-IGF1/IGF2 antibodies, and small molecule inhibitors that suppress IGF1R and IR kinase activity. New advances in the molecular basis of anti-IGF1R blocking antibodies reveal they are biased agonists and promote the binding of IGF1 to integrin β3 receptors in some cancer cells. Our recent reports indicate that pharmacological aryl hydrocarbon receptor (AHR) ligands inhibit breast cancer cell responses to IGFs, suggesting that targeting AHR may have benefit in cancers whose proliferation and survival are dependent on insulin/IGF signaling. Novel aspects of IGF1R/IR in cancer, such as biased agonism, integrin β3 signaling, AHR, and new therapeutic targeting strategies will be discussed.

A novel insulin receptor-binding protein from Momordica charantia enhances glucose uptake and glucose clearance in vitro and in vivo through triggering insulin receptor signaling pathway.

Diabetes, a common metabolic disorder, is characterized by hyperglycemia. Insulin is the principal mediator of glucose homeostasis. In a previous study, we identified a trypsin inhibitor, named Momordica charantia insulin receptor (IR)-binding protein (mcIRBP) in this study, that might interact with IR. The physical and functional interactions between mcIRBP and IR were clearly analyzed in the present study. Photo-cross-linking coupled with mass spectrometry showed that three regions (17-21, 34-40, and 59-66 residues) located on mcIRBP physically interacted with leucine-rich repeat domain and cysteine-rich region of IR. IR-binding assay showed that the binding behavior of mcIRBP and insulin displayed a cooperative manner. After binding to IR, mcIRBP activated the kinase activity of IR by (5.87 ± 0.45)-fold, increased the amount of phospho-IR protein by (1.31 ± 0.03)-fold, affected phosphoinositide-3-kinase/Akt pathways, and consequently stimulated the uptake of glucose in 3T3-L1 cells by (1.36 ± 0.12)-fold. Intraperitoneal injection of 2.5 nmol/kg mcIRBP significantly decreased the blood glucose levels by 20.9 ± 3.2% and 10.8 ± 3.6% in normal and diabetic mice, respectively. Microarray analysis showed that mcIRBP affected genes involved in insulin signaling transduction pathway in mice. In conclusion, our findings suggest that mcIRBP is a novel IRBP that binds to sites different from the insulin-binding sites on IR and stimulates both the glucose uptake in cells and the glucose clearance in mice.

Human biliverdin reductase‐based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor

Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. (290)KYCCSRK, which increased IRK V(max) without changing K(m), stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C(292)→A mutant did not stimulate glucose uptake; K(296)→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988-1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C(1056), C(1138), or C(1234), were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.

Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation

Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro.

Momordica charantia and Its Novel Polypeptide Regulate Glucose Homeostasis in Mice via Binding to Insulin Receptor

Momordica charantia (MC) has been used as an alternative therapy for diabetes mellitus. This study analyzed and elucidated therapeutic targets contributing to the hypoglycemic effect of aqueous extract of MC seeds (MCSE) by transcriptomic analysis. Protein ingredients aimed at the hypoglycemic target were further identified by proteomic, docking, and receptor-binding assays. The data showed that MSCE (1 g/kg) significantly lowered the blood glucose level in normal and diabetic mice. Moreover, MCSE primarily regulated the insulin signaling pathway in muscles and adipose tissues, suggesting that MCSE might target insulin receptor (IR), stimulate the IR-downstream pathway, and subsequently display hypoglycemic activity in mice. It was further revealed that inhibitor against trypsin (TI) of MC directly docked into IR and activated the kinase activity of IR in a dose-dependent manner. In conclusion, the findings suggested that MCSE regulated glucose metabolism mainly via the insulin signaling pathway. Moreover, TI was newly identified as a novel IR-binding protein of MC that triggered the insulin signaling pathway via binding to IR.

Effects of Fat and Cinnamon Feeding on Adiponectin after Rat Injection with Dexamethasone

Chronically excessive numbers of glucocorticoids are known to induce insulin resistance, which is possibly involved in impairing the insulin signaling in liver, muscle, and adipose tissues. Excessive levels of cortisol are characterized by the symptoms of abdominal obesity, hypertension, glucose intolerance or diabetes and dyslipidemia ,all of these features are shared by the condition of insulin resistance .Consumption of high levels of dietary fat is thought to be a major factor in the promotion of obesity and insulin resistance [7]. Fatty acid composition in the diet is another mechanism implicated in the development of insulin resistance [6]. Several environmental factors, including high-fat diet, are reported to activate the functioning of the hypothalamus-pituitary-adrenal axis [HPA]. Frequently evoked HPA-axis secretes excessive amount of cortisol [21] and elevated cortisol level is implicated in the development of entire spectrum of the metabolic syndrome, including insulin resistance, visceral obesity and dyslipidemia as well as the kinds of [21]. Cinnamon contains biologically active substances that have demonstrated insulin-mimetic properties. In vitro [3] and in vivo [1, 20] studies have shown that cinnamon enhances glucose uptake by activating insulin receptor kinase activity, autophosphorylation of the insulin receptor, and glycogen synthase activity. Other recent studies have demonstrated the ability of cinnamon to reduce lipid levels in fructose-fed rats, potentially via inhibiting hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity [2]. Adiponectin is the most abundant plasma protein synthesized for the most part in adipose tissue, and it is an insulin-sensitive hormone,playing a central role in glucose and lipid metabolism [7] Furthermore, adiponectin levels are inversely associated with visceral adiposity [15]. Moreover, adiponectin has been reported to exhibit anti-atherosclerotic and anti-inflammatory effects [17,16].

Influence of PTPN1 polymorphism among people with type 2 diabetes: A Thanjavur-based study

Type 2 diabetes is a multifactorial and polygenic disease that involves insulin resistance and impaired glucose induced insulin release. Besides environmental factors, the influence of genetic changes also drives the susceptibility of the disease. One such attractive candidate gene influencing the glucose metabolism is the gene encoding for protein tyrosine phosphatase 1B (PTPN1), located on the chromosome 20. The gene product is negatively regulating the insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase activation segment. This study examined the association between one of the allelic polymorphisms (−1023C/A) in PTPN1 gene with type 2 diabetes in the local population of Thanjavur, India. Genotypic analysis was performed on 96 diabetic and 96 non-diabetic patients using polymerase chain reaction–based restriction fragment length polymorphism. The frequency of allele A in the homozygous form of −1023C>A polymorphism was found to be lesser (19%) when compared with the control population. In the body mass index (BMI)-based classification, the homozygous A allele was absent in case participants with BMIs lower than 30 but it was present in four of the controls with variant allele. Chi square were used to determine out the significant association of the polymorphism with the disease. There was no statistical significance obtained for the studied population; the result may be validated by analyzing larger sample size in the future.

Cocoa polyphenols suppress adipogenesis in vitro and obesity in vivo by targeting insulin receptor

To investigate the inhibitory effect of cocoa polyphenol extract (CPE) on adipogenesis and obesity along with its mechanism of action. 3T3-L1 preadipocytes were cultured with isobutylmethylxanthine, dexamethasone and insulin (MDI), and male C57BL/6N mice (N=44) were fed a high-fat diet (HFD) for 5 weeks with or without CPE. CPE at 100 or 200 μg ml(-1) inhibited MDI-induced lipid accumulation without diminishing cell viability. In particular, CPE reduced the protein expression levels of PPARγ and CEBPα, and blocked mitotic clonal expansion (MCE) of preadipocytes by reducing proliferating signaling pathways. This in turn attenuates lipid accumulation during the differentiation of 3T3-L1 preadipocytes. CPE effectively suppressed MDI-induced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and their downstream signals. We then examined whether CPE regulates insulin receptor (IR), a common upstream regulator of ERK and Akt. We found that although CPE does not affect the protein expression level of IR, it significantly inhibits the activity of IR kinase via direct binding. Collectively, the results suggested that CPE, a direct inhibitor of IR kinase activity, inhibits cellular differentiation and lipid accumulation in 3T3-L1 preadipocytes. Consistently, CPE attenuated HFD-induced body weight gain and fat accumulation in obese mice fed with a HFD. We also found that HFD-induced increased fasting glucose levels remained unaffected by CPE. This study demonstrates that CPE inhibits IR kinase activity and its proliferative downstream signaling markers, such as ERK and Akt, in 3T3-L1 preadipocytes, and also prevents the development of obesity in mice fed with a HFD.

Piceatannol, Natural Polyphenolic Stilbene, Inhibits Adipogenesis via Modulation of Mitotic Clonal Expansion and Insulin Receptor-dependent Insulin Signaling in Early Phase of Differentiation

Piceatannol, a natural stilbene, is an analog and a metabolite of resveratrol. Despite a well documented health benefit of resveratrol in intervention of the development of obesity, the role of piceatannol in the development of adipose tissue and related diseases is unknown. Here, we sought to determine the function of piceatannol in adipogenesis and elucidate the underlying mechanism. We show that piceatannol inhibits adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner at noncytotoxic concentrations. This anti-adipogenic property of piceatannol was largely limited to the early event of adipogenesis. In the early phase of adipogenesis, piceatannol-treated preadipocytes displayed a delayed cell cycle entry into G(2)/M phase at 24 h after initiation of adipogenesis. Furthermore, the piceatannol-suppressed mitotic clonal expansion was accompanied by reduced activation of the insulin-signaling pathway. Piceatannol dose-dependently inhibited differentiation mixture-induced phosphorylation of insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/Akt pathway in the early phase of adipogenesis. Moreover, we showed that piceatannol is an inhibitor of IR kinase activity and phosphatidylinositol 3-kinase (PI3K). Our kinetics study of IR further identified a K(m) value for ATP of 57.8 μm and a K(i) value for piceatannol of 28.9 μm. We also showed that piceatannol directly binds to IR and inhibits IR kinase activity in a mixed noncompetitive manner to ATP, through which piceatannol appears to inhibit adipogenesis. Taken together, our study reveals an anti-adipogenic function of piceatannol and highlights IR and its downstream insulin signaling as novel targets for piceatannol in the early phase of adipogenesis.

Enhancement of doxorubicin cytotoxicity of human cancer cells by tyrosine kinase inhibition of insulin receptor and type I IGF receptor

The type I insulin-like growth factor receptor (IGF1R) contributes to cancer cell biology. Disruption of IGF1R signaling alone or in combination with cytotoxic agents has emerged as a new therapeutic strategy. Our laboratory has shown that sequential treatment with doxorubicin (DOX) and anti-IGF1R antibodies significantly enhanced the response to chemotherapy. In this study, we examined whether inhibition of the tyrosine kinase activity of this receptor family would also enhance chemotherapy response. Cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP) inhibited IGF1R and insulin receptor (InsR) kinase activity and downstream activation of ERK1/2 and Akt in MCF-7 and LCC6 cancer cells. PQIP inhibited both monolayer growth and anchorage-independent growth in a dose-dependent manner. PQIP did not induce apoptosis, but rather, PQIP treatment was associated with an increase in autophagy. We examined whether sequential or combination therapy of PQIP with DOX could enhance growth inhibition. PQIP treatment together with DOX or DOX followed by PQIP significantly inhibited anchorage-independent growth in MCF-7 and LCC6 cells compared to single agent alone. In contrast, pre-treatment with PQIP followed by DOX did not enhance the cytotoxicity of DOX in vitro. Furthermore, OSI-906, a PQIP derivative, inhibited IGF-I signaling in LCC6 xenograft tumors in vivo. When given once a week, simultaneous administration of OSI-906 and DOX significantly enhanced the anti-tumor effect of DOX. In summary, these results suggest that timing and duration of the IGF1R/InsR tyrosine kinase inhibitors with chemotherapeutic agents should be evaluated in clinical trials. Long-term disruption of IGF1R/InsR may not be necessary when combined with cytotoxic chemotherapy.

Structural Basis for the Interaction between the Growth Factor-binding Protein GRB10 and the E3 Ubiquitin Ligase NEDD4

In addition to inhibiting insulin receptor and IGF1R kinase activity by directly binding to the receptors, GRB10 can also negatively regulate insulin and IGF1 signaling by mediating insulin receptor and IGF1R degradation through ubiquitination. It has been shown that GRB10 can interact with the C2 domain of the E3 ubiquitin ligase NEDD4 through its Src homology 2 (SH2) domain. Therefore, GRB10 might act as a connector, bringing NEDD4 close to IGF1R to facilitate the ubiquitination of IGF1R by NEDD4. This is the first case in which it has been found that an SH2 domain could colocalize a ubiquitin ligase and its substrate. Here we report the crystal structure of the NEDD4 C2-GRB10 SH2 complex at 2.0 Å. The structure shows that there are three interaction interfaces between NEDD4 C2 and GRB10 SH2. The main interface centers on an antiparallel β-sheet composed of the F β-strand of GRB10 SH2 and the C β-strand of NEDD4 C2. NEDD4 C2 binds at nonclassical sites on the SH2 domain surface, far from the classical phosphotyrosine-binding pocket. Hence, this interaction is phosphotyrosine-independent, and GRB10 SH2 can bind the C2 domain of NEDD4 and the kinase domain of IGF1R simultaneously. Based on these results, a model of how NEDD4 interacts with IGF1R through GRB10 has been proposed. This report provides further evidence that SH2 domains can participate in important signaling interactions beyond the classical recognition of phosphotyrosine.

Protein tyrosine phosphatase 1B gene polymorphisms and essential hypertension: A case-control study in Chinese population

Protein tyrosine phosphatase (PTP)- 1B, encoded by the PTPN1 gene, negatively regulates insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase activation segment. Several rare single nucleotide polymorphisms (SNP) have been linked to diseases accompanying insulin resistance such as Type 2 diabetes, obesity, and dyslipidemia in different populations. To investigate whether PTP-1B SNP are associated with hypertension and hypertension-related metabolic traits in Chinese subjects. A total of 239 Chinese patients with hypertension and 141 non-hypertensive subjects were screened. The genotypes of PTP-1B gene polymorphisms were determined by PCR-restriction fragment length polymorphism methods. The case-control study showed associations between the frequencies of T allelic g54281 T>A and A allele of I5/37C>A and hypertension (p<0.0001, p=0.0371, respectively). In addition, significant associations were observed between the IVS6+G82A polymorphism and waist circumference, total cholesterol, and LDL-cholesterol levels in hypertensive patients (p=0.0005 approximately 0.0260). And g54281 T allele was associated with higher plasma triglyceride (p=0.0390) and LDL-cholesterol concentration (p=0.0141), while g58585 T>C was associated with body mass index (BMI) (p=0.0308), waist circumference (p=0.0216), and homeostasis model assessment of insulin resistance (p=0.047). Multivariate logistic regression analysis showed that T allele carriers of g54281T>A and A allele carriers of I5/37C>A had higher risks of hypertension independent of age, gender, BMI, glucose levels, and lipids profiles [odds ratio (OR): 1.79, 95% confidence interval (CI): 1.09- 2.96, p=0.02; OR: 1.66, 95% CI: 1.13-2.44, p=0.01, respectively]. PTP-1B polymorphisms contribute to pathogenesis of hypertension in Chinese subjects and PTP-1B SNP may be involved in the development of several features including dyslipidemia and obesity in hypertensive subjects.

Modulation of Insulin Receptor Substrate-1 and Some Inflammatory Variables in Hyperinsulinemic Rats Treated with Cinnamon Extract

Cinnamon Extract (CE) has shown to be generally safe when ingested and to have many pharmacological properties. Problem statement: Study the effects of daily intake of CE on the modulation of hepatic, cardiac Insulin Receptor Substrate-1 (IRS-1) and their relations to some inflammatory variables in hyperinsulinemic rats. Approach: The influence of CE administered orally was studied in hyperinsulinemic rats. Eighteen male Wistar rats were divided into 3 groups of 6 rats each. Group 1; control animals received starch as control diet, while Groups 2; rats were fed a "high-fructose diet" (60%). Group 3; fructose-fed rats received orally CE (0.5 mL/rat/day) from the 16th day of fructose feeding in experimental period. The animals were maintained in their respective groups for 30 days. At the end of the experimental period, Serum levels of glucose, insulin, lipid profile, Total Antioxidant Capacity (TAC), Malondialdehyde (MDA), sialic acid and soluble Fas (sFas) were assayed. Hepatic and cardiac IRS-1 levels were also evaluated. Results: Fed high fructose diet to rats induced significant elevations in serum levels of glucose, insulin, triacylglycerol, HDL-c, sialic acid, sFas and MDA, while the level of serum TAC was significantly reduced as compared to controls. Also significant reduction in the levels of hepatic and cardiac IRS-1 were recorded as compared to controls. Oral administration of cinnamon extract to fructose-fed rats alleviated the effects of fructose and these rats showed a normal level of the parameters studied. The percentage changes of IRS-1 level in fructose-fed rats before and after treatment with CE were 38.51 for liver and 31.92% for cardiac muscle. This increase in IRS-1 level after treatment is still lowered than control level with the percentage change -11.82 and -9.93% for liver and cardiac muscle respectively. There was a significant positive correlation between IRS-1 and TAC level whereas there was negative correlation between IRS-1 and MDA, sialic acid and sFas. Conclusion: This study reports interest findings that cinnamon extract enhances glucose uptake by activating insulin receptor kinase activity in rats fed high fructose diet and it has additional roles as antioxidant and anti-inflammatory agents. Thus, clinical application of cinnamon could be considered as a potential therapeutic option in humans in the treatment of insulin resistance states. So inclusion of cinnamon in the daily diet could be recommended.

A Type I Insulin-Like Growth Factor Receptor Kinase Inhibitor (PQIP) Enhances the Cytotoxicity of Doxorubicin in Human Cancer Cell Lines.

Abstract The type-I insulin like growth factor (IGF-IR) is either activated and/or overexpressed in a wide range of tumor types and contributes to tumorigenicity, proliferation, survival, metastasis and drug resistance. Disruption of IGF-IR signaling alone or in combination with other cytotoxic agents has emerged as an important strategy in cancer therapy. Our laboratory has shown that sequentially combining doxorubicin with anti-IGF-IR antibodies significantly enhances the response of chemotherapy, while the opposite sequence causes cells resistant to chemotherapy. Here, we show that a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP) inhibited IGF-IR and insulin receptor (IR) kinase activity and proliferation in MCF-7 and MDA435/LCC6 cell lines. Moreover, PQIP treatment blocked both IGF-I and insulin stimulated activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase in both cell lines. At doses that inhibited monolayer cell growth, the compound also inhibited cell motility, blocked IGF-I stimulated S-phase progression and induced autophage; at higher doses, it also caused PARP cleavage. Combining PQIP with doxorubicin significantly enhanced cytotoxicity but did not further enhance doxorubicin-induced PARP cleavage in monolayer cell growth. Furthermore, our sequencing studies showed that combining PQIP with doxorubicin simultaneously or doxorubicin followed by PQIP significantly inhibited the anchorage-independent growth in both MCF-7 and MDA435/LCC6 cells. In contrast, pre-treatment with PQIP followed by doxorubicin did not enhance the cytotoxicity of doxorubicin in anchorage-independent growth, which is similar as anti-IGF-IR antibodies in combination with doxorubicin. In summary, these results suggest that the IGF-IR tyrosine kinase inhibitor PQIP can be used alone or in combination with chemotherapy to enhance cytotoxicity of human tumor cell lines, and the timing of IGF-IR inhibition may affect responses to doxorubicin. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6110.

Visfatin regulates insulin secretion, insulin receptor signalling and mRNA expression of diabetes-related genes in mouse pancreatic β-cells

The role of the adipocyte-derived factor visfatin in metabolism remains controversial, although some pancreatic beta-cell-specific effects have been reported. This study investigated the effects of visfatin upon insulin secretion, insulin receptor activation and mRNA expression of key diabetes-related genes in clonal mouse pancreatic beta-cells. beta-TC6 cells were cultured in RPMI 1640 and were subsequently treated with recombinant visfatin. One-hour static insulin secretion was measured by ELISA. Phospho-specific ELISA and western blotting were used to detect insulin receptor activation. Real-time SYBR Green PCR array technology was used to measure the expression of 84 diabetes-related genes in both treatment and control cells. Incubation with visfatin caused significant changes in the mRNA expression of several key diabetes-related genes, including marked up-regulation of insulin (9-fold increase), hepatocyte nuclear factor (HNF)1beta (32-fold increase), HNF4alpha (16-fold increase) and nuclear factor kappaB (40-fold increase). Significant down-regulation was seen in angiotensin-converting enzyme (-3.73-fold) and UCP2 (-1.3-fold). Visfatin also caused a significant 46% increase in insulin secretion compared to control (P<0.003) at low glucose, and this increase was blocked by co-incubation with the specific nicotinamide phosphoribosyltransferase inhibitor FK866. Both visfatin and nicotinamide mononucleotide induced activation of both insulin receptor and extracellular signal-regulated kinase (ERK)1/2, with visfatin-induced insulin receptor/ERK1/2 activation being inhibited by FK866. We conclude that visfatin can significantly regulate insulin secretion, insulin receptor phosphorylation and intracellular signalling and the expression of a number of beta-cell function-associated genes in mouse beta-cells.

P-110: Measurement of insulin receptor kinase activity in muscle and erythrocytes of a family with mutations of the insulin receptor gene

P-110: Measurement of insulin receptor kinase activity in muscle and erythrocytes of a family with mutations of the insulin receptor gene

Phospholipid transfer protein reduces phosphorylation of tau in human neuronal cells

Tau function is regulated by phosphorylation, and abnormal tau phosphorylation in neurons is one of the key processes associated with development of Alzheimer's disease and other tauopathies. In this study we provide evidence that phospholipid transfer protein (PLTP), one of the main lipid transfer proteins in the brain, significantly reduces levels of phosphorylated tau and increases levels of the inactive form of glycogen synthase kinase-3beta (GSK3 beta) in HCN2 cells. Furthermore, inhibition of phosphatidylinositol-3 kinase (PI3K) reversed the PLTP-induced increase in levels of GSK3 beta phosphorylated at serine 9 (pGSK3 beta(Ser9)) and partially reversed the PLTP-induced reduction in tau phosphorylation. We provide evidence that the PLTP-induced changes are not due to activation of Disabled-1 (Dab1), insofar as PLTP reduced levels of total and phosphorylated Dab1 in HCN2 cells. We have also shown that inhibition of tyrosine kinase activity of insulin receptor (IR) and/or insulin-like growth factor 1 (IGF1) receptor (IGFR) reverses the PLTP-induced increase in levels of phosphorylated Akt (pAkt(Thr308) and pAkt(Ser473)), suggesting that PLTP-mediated activation of the PI3K/Akt pathway is dependent on IR/IGFR receptor tyrosine kinase activity. Our study suggests that PLTP may be an important modulator of signal transduction pathways in human neurons.

Cytoskeletal components enhance the autophosphorylation of retinal insulin receptor

Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, and we recently reported that deletion of IR from rod photoreceptors resulted in stress-induced photoreceptor degeneration. Retinal insulin receptor has a high basal level autophosphorylation compared to liver and the reasons for higher autophosphorylation are not known. In the current study we report a novel finding that cytoplasmic actin associates with and activates the retinal IR in vivo. Similar to insulin, actin also induced autophosphorylation at tyrosines 1158, 1162 and 1163 in the catalytic loop of IR. Our studies also suggest that globular actin activates the retinal IR more effectively than does filamentous actin. Retinal IR kinase activity has been shown to decrease in hyperglycemia and we found a decreased binding of actin to the IR under hyperglycemia. This is the first study which demonstrates that cytoplasmic actin regulates autophosphorylation of the retinal IR.