PO3-16: Altered mouse adipose tissue IGF-1 expression influences glucose control

Posters / Growth Hormone & IGF Research 22S1 (2014) S25–S52

PO3-16 Altered mouse adipose tissue IGF-1 expression influences glucose control J. Trost, J. Alcaniz Rolli, G. Brockmann. Breeding Biology and Molecular Genetics, Humboldt-Universität zu Berlin / Albrecht Daniel Thaer-Institut for Agricultural and Horticultural Sciences, Berlin, Germany Introduction: Insulin like growth factor 1 (IGF1) is a hormone with a variety of effects on growth and differentiation but is also involved in blood glucose control. IGF-1 is mainly produced in the liver but has also been shown to be expressed in and excreted from white adipose tissue (WAT) in mice. Since WAT plays a role in energy metabolism and glucose homeostasis we assumed, that AT derived IGF-1 contributes to the overall control of blood glucose. Methods: To test this hypothesis, we designed an experiment with (AT-Igf-1KO mice). AT-Igf-1KO mice and wild type littermates were tested for glucose and insulin tolerance, hepatic and adipose tissue igf-1 expression were measured and total serum IGF-1 levels were determined. Results: We could show that igf-1 expression levels of AT-Igf-1KO mice were significantly lower in visceral and gonadal WAT and significantly higher in the liver compared to wild type littermates with overall unaltered IGF-1 serum level. The higher hepatic Igf-1 expression likely compensates for the reduced WAT expression to maintain the required serum level. Conclusions: Despite unaltered insulin and IGF-1 serum levels the AT-Igf-1KO mice showed a delayed glucose clearance when fed a high fat died which confirmed the crucial role of adipose tissue derived Igf-1 in murine glucose control. PO3-17 Hypermethylation and hypomethylation of IGF-II DMRO (Insulin like growth factors-II Differentially Methylated Region) correlated to increased or decreased expression of IGF-II in breast cancer cells and paired breast tissues of African American and Caucasian breast cancer patients V.K. Radhakrishnan, M.A. De León, D. DeLeon. CHDMM, LLUSM, Loma Linda, United States IGF-II is highly expressed in breast cancer (BC). We previously showed that IGF-II levels are higher in BC tissues from African American (AA) patients as compared to Caucasian (CA). The present study was designed to determine if differential methylation of the IGF-II gene was responsible for the higher levels detected in BC tissues and cell lines from AA women. Methylation analysis was performed using Combined Bisulfite Restriction Analysis (COBRA) and Methylation Sensitive McrBC. Genomic DNA (gDNA) was isolated and treated with sodium bisulphate and MSRE HapII and MDRE McrBC restriction digestion enzymes. The bisulphated gDNA fractions were subsequently amplified using methyl specific IGF-II DMR and CpG primers to quantify the methylation patterns and CpG regulatory regions. Finally, IGF-II mRNA and proIGF-II protein expression were assessed by qRT-PCR and Western blot respectively. Our data demonstrated that there are differentially methylated regions (DMRs) and CpG islands in the IGF-II gene of normal and malignant paired breast cancer tissues. These methylated regions of the IGF-II gene are crucial for differential expression of the proIGF-II m-RNA and proIGF-II protein expression. Comparison of IGF-II methylation pattern to mRNA and protein data revealed that one of the IGF-II DMRO contained a methylated region that correlated to the differential expression of proIGF-II in both AA and CA breast cancer tissues and cell lines. Hypermethylation and hypomethylation of the IGF-II DMRO leads to differential expression of proIGF-II. Our data provides a

S35

potential epigenetic mechanism for the higher levels of proIGF-II protein expression seen in AA as compared to CA breast cancer patients and cell lines. We propose that differentially methylated IGF-II DMRO region can be used as an epigenetic biomarker for a more aggressive breast cancer disease. PO3-18 Lipopolysaccharide (LPS) treatment increased the protein levels of GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein) followed by inhibition of insulinmediated glucose transporter (GLUT) 4 translocation to the plasma membrane and glucose uptake in adipocytes B.-C. Yu1,2, Y. Ando1, Y. Obata3, A. Takenaka3, K. Chida1, F. Hakuno1, S.-I. Takahashi1. 1Departments of Animal Sciences and Applied Biological Chemistry, The University of Tokyo, Tokyo, Japan, 2 Research Fellow of Japanese Society for Promotion of Science, Tokyo, Japan, 3Meiji University, Kanagawa, Japan Introduction: Insulin receptor substrates (IRSs) play central roles in insulin/ IGF signaling and mediate their variety of bioactivities. We had identified GKAP42 as an IRS-1-associated protein by yeast two-hybrid screening. We reported that tumor necrosis factor alpha (TNFa) treatment decreased GKAP42 protein level, followed by a decrease in GKAP42 binding to IRS-1 and this decrease caused impairment of insulin-dependent tyrosine phosphorylation of IRS-1 and glucose uptake in 3T3-L1 adipocytes. It is well known that LPS administration to animals induced secretion of cytokines, resulting in insulin resistance in vivo. This study was undertaken to elucidate the roles of GKAP42 in LPS-induced insulin resistance in both rat adipose tissue and 3T3-L1 adipocytes. Methods and results: After one-week intraperitoneal administration of LPS (2.5 mg/kg daily) to male Wistar rats (6 weeks old), in liver TNFa expression was not changed but IL-1b expression markedly increased. Under this condition, GKAP42 protein levels increased in adipose tissue. In 3T3-L1 adipocytes treated with LPS (100 ng/mL) for 24 hours, we observed an increase in GKAP42 protein levels and inhibition of insulin-dependent glucose uptake. To test the effects of increases in GKAP42 on GLUT4 translocation to the plasma membrane, we performed myc-GLUT4-GFP translocation assay using 3T3-L1 adipocytes transfected with both GKAP42 and myc-GLUT4-GFP expression vectors. Overexpression of GKAP42 inhibited GLUT4 translocation to the plasma membrane with insulin stimulation, whereas enhanced it under the basal condition. Conclusion: In our models, LPS increased GKAP42 protein levels leading to impairment of insulin-dependent glucose uptake through disruption of GLUT4 translocation to plasma membrane in adipocytes. Taken together with previous results, our data suggest that GKAP42 possesses multiple functions such as maintenance of IRS-1-mediated insulin signals at low expression levels and inhibition of GLUT4 translocation to the plasma membrane at high expression levels. PO3-19 IGF-1 receptor C terminal tail phosphorylation on Y1250 in cancer cells S.P. O Shea, I. Fernández, G.M. Kelly, R. O’ Connor. Biochemistry, University College Cork, Cork, Ireland The Insulin-like Growth Factor Receptor (IGF-1R) has an essential function in normal cell survival and growth, and it’s deregulation is associated with cancer progression. Previously we showed that IGF-1R activity and signalling in cancer cells is regulated by its C terminal tail, and in particular by a motif that encompasses Tyrosines 1250 and 1251 flanked by Serines 1248 and 1252 (1248-SFYYS-1252 ). This motif is important for IGF-1R kinase