IR Expression Research Articles

The tyrphostin NT157 suppresses insulin receptor substrates and augments therapeutic response of prostate cancer.

Insulin-like growth factor (IGF) signaling is associated with castrate-resistant prostate cancer (CRPC) progression. Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and antiapoptotic signaling from IGF1 receptor (IGF1R), insulin receptor, and other oncoproteins. This study demonstrates that IRS1/2 expression is increased in prostate cancer, and persists in CRPC. Furthermore, this study assesses the anticancer activity of NT157, a small molecule tyrphostin targeting IRS proteins, using androgen-responsive (LNCaP) and -independent (PC3) prostate cancer cells in vitro and in vivo. NT157 treatment resulted in dose-dependent inhibition of IGF1R activation, suppression of IRS protein expression, inhibition of IGF1-induced AKT activation, but increased ERK activation in NT157-treated cells in vitro. These effects were correlated with decreased proliferation and increasing apoptosis of LNCaP cells and increasing G2-M arrest in PC3 cells. NT157 also suppressed androgen-responsive growth, delayed CRPC progression of LNCaP xenografts, and suppressed PC3 tumor growth alone and in combination with docetaxel. This study reports the first preclinical proof-of-principle data that this novel small molecule tyrosine kinase inhibitor suppresses IRS1/2 expression, delays CRPC progression, and suppresses growth of CRPC tumors in vitro and in vivo. Demonstration that IRS expression can be increased in response to a variety of stressors that may lead to resistance or reduced effect of the therapies indicate that NT157-mediated IRS1/2 downregulation is a novel therapeutic approach for management of advanced prostate cancer.

11β-Hydroxysteroid dehydrogenase type 1 shRNA ameliorates glucocorticoid-induced insulin resistance and lipolysis in mouse abdominal adipose tissue.

Long-term glucocorticoid exposure increases the risk for developing type 2 diabetes. Prereceptor activation of glucocorticoid availability in target tissue by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) coupled with hexose-6-phosphate dehydrogenase (H6PDH) is an important mediator of the metabolic syndrome. We explored whether the tissue-specific modulation of 11β-HSD1 and H6PDH in adipose tissue mediates glucocorticoid-induced insulin resistance and lipolysis and analyzed the effects of 11β-HSD1 inhibition on the key lipid metabolism genes and insulin-signaling cascade. We observed that corticosterone (CORT) treatment increased expression of 11β-HSD1 and H6PDH and induced lipase HSL and ATGL with suppression of p-Thr(172) AMPK in adipose tissue of C57BL/6J mice. In contrast, CORT induced adipose insulin resistance, as reflected by a marked decrease in IR and IRS-1 gene expression with a reduction in p-Thr(308) Akt/PKB. Furthermore, 11β-HSD1 shRNA attenuated CORT-induced 11β-HSD1 and lipase expression and improved insulin sensitivity with a concomitant stimulation of pThr(308) Akt/PKB and p-Thr(172) AMPK within adipose tissue. Addition of CORT to 3T3-L1 adipocytes enhanced 11β-HSD1 and H6PDH and impaired p-Thr(308) Akt/PKB, leading to lipolysis. Knockdown of 11β-HSD1 by shRNA attenuated CORT-induced lipolysis and reversed CORT-mediated inhibition of pThr(172) AMPK, which was accompanied by a parallel improvement of insulin signaling response in these cells. These findings suggest that elevated adipose 11β-HSD1 expression may contribute to glucocorticoid-induced insulin resistance and adipolysis.

The β-amyloid precursor protein analog P165 improves impaired insulin signal transduction in type 2 diabetic rats.

This study was performed to understand whether P165 improves learning and memory by restoring insulin action using a diabetes mellitus (DM) rat model. A total of 34 male Sprague-Dawley rats were randomly divided into four groups: control group (n = 8), DM group (n = 8), DM group treated with a low dose of P165 (n = 9), and DM group treated with a high dose of P165 (n = 9). After 8 weeks of treatment, the animals were killed and the expression of insulin signaling-related proteins was examined in the hippocampus by Western blot and immunohistochemical staining. Administration of P165 in diabetic rats did not induce a significant effect on the fasting blood glucose level. The expression of IR, IRS-1, AKT, p-CREB, and Bcl-2 proteins was significantly enhanced in the hippocampus in diabetic rats. Treatment of diabetic rats with P165 at both low and high doses significantly attenuated the expression levels of these proteins. Moreover, immunohistochemistry staining showed that IR, IRS-1, AKT, p-CREB, and Bcl-2 were abundantly expressed in the CA1 region of the hippocampus. The number of cells positively stained for the above proteins was significantly higher in diabetic tissues compared to control tissues, whereas P165 treatments induced a significant reduction in the expression of these proteins. The expression of IR, IRS-1, AKT, p-CREB, and Bcl-2 was enhanced in DM rats, and administration of P165 normalized the expression of these molecules, suggesting that P165 can improve impaired insulin signal transduction.

Diabetes-associated changes in the fetal insulin/insulin-like growth factor system are organ specific in rats.

Diabetes in pregnancy affects fetal growth and development. The insulin/insulin-like growth factors (IGF) system comprising insulin, IGF, their receptors, and binding proteins, has been implicated in fetal growth regulation. This study tested the hypothesis that maternal diabetes alters the fetal insulin/IGF system in a tissue-specific manner. Wistar rats were rendered diabetic by neonatal administration of streptozotocin and mated with control rats. At day 21 of gestation, the weights of fetuses, placentas, and fetal organs (heart, lung, liver, stomach, intestine, and pancreas) were determined. Maternal and fetal plasma concentrations of insulin, IGF1, and IGF2 were measured by ELISA, and expression of IGF1, IGF2, IGF1R, IGF2R, IR, IGFBP1, BP2, and BP3 in placenta and fetal organs by qPCR. The well-known increase in fetal growth in this model of mild diabetes is accompanied by elevated insulin and IGF1 levels and alterations of the insulin/IGF system in the fetus and the placenta. These alterations were organ and gene specific. The insulin/IGF system was generally upregulated, especially in the fetal heart, while it was downregulated in fetal lung. In our model of mild diabetes, the effect of maternal diabetes on fetal weight and fetal insulin/IGF system expression is organ specific with highly sensitive organs such as lung and heart, and organs that were less affected, such as stomach.

A polyphenol rescues lipid induced insulin resistance in skeletal muscle cells and adipocytes

Skeletal muscle and adipose tissues are known to be two important insulin target sites. Therefore, lipid induced insulin resistance in these tissues greatly contributes in the development of type 2 diabetes (T2D). Ferulic acid (FRL) purified from the leaves of Hibiscus mutabilis, showed impressive effects in preventing saturated fatty acid (SFA) induced defects in skeletal muscle cells. Impairment of insulin signaling molecules by SFA was significantly waived by FRL. SFA markedly reduced insulin receptor β (IRβ) in skeletal muscle cells, this was affected due to the defects in high mobility group A1 (HMGA1) protein obtruded by phospho-PKCε and that adversely affects IRβ mRNA expression. FRL blocked PKCε activation and thereby permitted HMGA1 to activate IRβ promoter which improved IR expression deficiency. In high fat diet (HFD) fed diabetic rats, FRL reduced blood glucose level and enhanced lipid uptake activity of adipocytes isolated from adipose tissue. Importantly, FRL suppressed fetuin-A (FetA) gene expression, that reduced circulatory FetA level and since FetA is involved in adipose tissue inflammation, a significant attenuation of proinflammatory cytokines occurred. Collectively, FRL exhibited certain unique features for preventing lipid induced insulin resistance and therefore promises a better therapeutic choice for T2D.

Growth hormone abolishes beneficial effects of calorie restriction in long-lived Ames dwarf mice

Disruption of the growth hormone (GH) axis promotes longevity and delays aging. In contrast, GH over-expression may lead to accelerated aging and shorter life. Calorie restriction (CR) improves insulin sensitivity and may extend lifespan. Long-lived Ames dwarf (df/df) mice have additional extension of longevity when subjected to 30% CR. The aim of the study was to assess effects of CR or GH replacement therapy separately and as a combined (CR+GH) treatment in GH-deficient df/df and normal mice, on selected metabolic parameters (e.g., insulin, glucose, cholesterol), insulin signaling components (e.g., insulin receptor [IR] β-subunit, phosphorylated form of IR [IR pY1158], protein kinase C ζ/λ [p-PKCζ/λ] and mTOR [p-mTOR]), transcription factor p-CREB, and components of the mitogen-activated protein kinase (MAPK) signaling (p-ERK1/2, p-p38), responsible for cell proliferation, differentiation and survival. CR decreased plasma levels of insulin, glucose, cholesterol and leptin, and increased hepatic IR β-subunit and IR pY1158 levels as well as IR, IRS-1 and GLUT-2 gene expression compared to ad libitum feeding, showing a significant beneficial diet intervention effect. Moreover, hepatic protein levels of p-PKCζ/λ, p-mTOR and p-p38 decreased, and p-CREB increased in CR mice. On the contrary, GH increased levels of glucose, cholesterol and leptin in plasma, and p-mTOR or p-p38 in livers, and decreased plasma adiponectin and hepatic IR β-subunit compared to saline treatment. There were no GH effects on adiponectin in N mice. Moreover, GH replacement therapy did not affect IR, IRS-1 and GLUT-2 gene expression. GH treatment abolishes the beneficial effects of CR; it may suggest an important role of GH–IGF1 axis in mediating the CR action. Suppressed somatotrophic signaling seems to predominate over GH replacement therapy in the context of the examined parameters and signaling pathways.

Localization of kisspeptin neurons in the hypothalamus of peripubertal female lambs; possible connection with gonadotrophin releasing hormone and neuropeptide Y neurons

Hypothalamic peptide kisspeptin (kp) plays a major role in the initiation of pubertal events in mammals through the activation of gonadotrophin-releasing hormone (GnRH) neurons. The present study was designed to analyse the morpho-functional role of kisspeptin cells in the multimodal effects on GnRH secretion in female prepubertal lambs. The localization of kp immunoreactive (ir) expression was compared with that of GnRH release and neuropeptide Y (NPY) synthesis. The experiment was conducted on eight 32-week-old Merino lambs. The distribution of the peptides was estimated using immunohistochemistry and anti-kp, anti-GnRH and anti-NPY specific antibodies. The hypothalamic area containing numerous kp-ir perikarya and a dense network of kp-ir fibres was localized in the caudal part of the arcuate nucleus. Single axons and single perikarya were observed throughout the hypothalamus extending from the preoptic area to the mammillary bodies. Distinct bundles of kp-ir nerve terminals were observed in the lateral zone of the median eminence (ME) and dispersed in smaller quantities in its medial zone. Double immunostaining indicated that a third of the kp-ir neurons were in close apposition with NPY-ir afferents, and that the distribution of kp-ir nerve terminals in the ME matched that of GnRH-ir nerve terminals. To conclude, the presence of very large numbers of kp-ir neurons in the peripubertal period could be attributed to their stimulating action on the GnRH neuronal system during this period. This influence could act directly in the ME, where both peptides are released. In addition, kp-ir neurons could mediate the effect of NPY on GnRH secretion.

Effect of surgical stress on insulin signal in intestinal epithelial cells

Objective To discuss the surgical effects on insulin signaling pathway in intestinal epithelial cells.Methods The obstructive jaundice models of rats were established,then peripheral blood samples were harvested before and at 120 min,4 h and 24 h after surgery,respectively.The insulin resistance (IR) index was calculated.The expression of insulin receptor in intestinal epithelial cells was detected by immunohistochemistry.The expression of the insulin receptor substrate (IRS-1,-2) and protein kinase B (PKB) was tested by Western blotting.Results The IR index in operation group was 3.40 ±0.12,4.29 ±0.11 and 5.34 ±0.18 at 120 min,4 h and 24 h after surgery,respectively.As compared with the control group,the IR index at 24 h was increased significantly in operation group (P ＜ 0.05).The number of strong insulin receptor expression was 3/10,2/10,2/10 at 120 min,4 h and 24 h after surgery respectively,and the expression of insulin receptor at 24 h was reduced obviously (P ＜ 0.05).The expression of IRS-1 was decreased (0.42 ± 0.04,P ＜ 0.05) after operation,but there was no significant change in the IRS-2 expression.The relative amount of PKB was also reduced in operation group (0.52 ±0.10,P ＜ 0.05).Conclusion The occurrence of IR in intestinal epithelial cells is related to the reduced expression of insulin receptor and IRS after surgical stress.The low expression of PKB is an important factor of IR signaling pathway in intestinal epithelial cells. Key words: Surgical stress; Insulin resistance; Intestinal epithelium; Insulin receptor

Protein quality and the protein to carbohydrate ratio within a high fat diet influences energy balance and the gut microbiota in C57BL/6J mice.

Macronutrient quality and composition are important determinants of energy balance and the gut microbiota. Here, we investigated how changes to protein quality (casein versus whey protein isolate; WPI) and the protein to carbohydrate (P/C) ratio within a high fat diet (HFD) impacts on these parameters. Mice were fed a low fat diet (10% kJ) or a high fat diet (HFD; 45% kJ) for 21 weeks with either casein (20% kJ, HFD) or WPI at 20%, 30% or 40% kJ. In comparison to casein, WPI at a similar energy content normalised energy intake, increased lean mass and caused a trend towards a reduction in fat mass (P = 0.08), but the protein challenge did not alter oxygen consumption or locomotor activity. WPI reduced HFD-induced plasma leptin and liver triacylglycerol, and partially attenuated the reduction in adipose FASN mRNA in HFD-fed mice. High throughput sequence-based analysis of faecal microbial populations revealed microbiota in the HFD-20% WPI group clustering closely with HFD controls, although WPI specifically increased Lactobacillaceae/Lactobacillus and decreased Clostridiaceae/Clostridium in HFD-fed mice. There was no effect of increasing the P/C ratio on energy intake, but the highest ratio reduced HFD-induced weight gain, fat mass and plasma triacylglycerol, non-esterified fatty acids, glucose and leptin levels, while it increased lean mass and oxygen consumption. Similar effects were observed on adipose mRNA expression, where the highest ratio reduced HFD-associated expression of UCP-2, TNFα and CD68 and increased the diet-associated expression of β3-AR, LPL, IR, IRS-1 and GLUT4. The P/C ratio also impacted on gut microbiota, with populations in the 30/40% WPI groups clustering together and away from the 20% WPI group. Taken together, our data show that increasing the P/C ratio has a dramatic effect on energy balance and the composition of gut microbiota, which is distinct from that caused by changes to protein quality.

SS Doctors & the State of Exception

The Auschwitz camp doctors, the five of whom most notoriously remembered, Dr. Herta Oberhauser, Professor Carl Clauberg, Dr. Herta Oberhauser, Dr. Josef Mengele, and Dr. Franz Lucas, formed an imagined community complete with the power to determine life and death. The (ir)rationality of the camp based doctors stood in juxtaposition against the state’s expression of (ir)rationality. The primary focus of the doctors was found in the realm of striving towards scientific and medical success of novel experimentation, while the state’s cardinal concern (in the early regime years) rested upon the furtherance of joint economic and militaristic visions and then, in the last stages of the regime, ensuring basic state security. This imagined community operated within the paradigm which propounded, on the whole, racism and anti-Semitism (which) manifest themselves, not across national boundaries, but within them.

ERK Phosphorylation Is Predictive of Resistance to IGF-1R Inhibition in Small Cell Lung Cancer

New therapies are critically needed to improve the outcome for patients with small cell lung cancer (SCLC). Insulin-like growth factor 1 receptor (IGF-1R) inhibition is a potential treatment strategy for SCLC: the IGF-1R pathway is commonly upregulated in SCLC and has been associated with inhibition of apoptosis and stimulation of proliferation through downstream signaling pathways, including phosphatidylinositol-3-kinase-Akt and mitogen-activated protein kinase. To evaluate potential determinants of response to IGF-1R inhibition, we assessed the relative sensitivity of 19 SCLC cell lines to OSI-906, a small molecule inhibitor of IGF-1R, and the closely related insulin receptor. Approximately one third of these cell lines were sensitive to OSI-906, with an IC50 < 1 μmol/L. Cell line expression of IGF-1R, IR, IGF-1, IGF-2, IGFBP3, and IGFBP6 did not correlate with sensitivity to OSI-906. Interestingly, OSI-906 sensitive lines expressed significantly lower levels of baseline phospho-ERK relative to resistant lines (P = 0.006). OSI-906 treatment resulted in dose-dependent inhibition of phospho-IGF-1R and phospho-Akt in both sensitive and resistant cell lines, but induced apoptosis and cell-cycle arrest only in sensitive lines. We tested the in vivo efficacy of OSI-906 using an NCI-H187 xenograft model and two SCLC patient xenografts in mice. OSI-906 treatment resulted in 50% tumor growth inhibition in NCI-H187 and 30% inhibition in the primary patient xenograft models compared with mock-treated animals. Taken together our data support IGF-1R inhibition as a viable treatment strategy for a defined subset of SCLC and suggest that low pretreatment levels of phospho-ERK may be indicative of sensitivity to this therapeutic approach.

Abstract LB-28: The IGF/insulin signaling axis TMPRSS2:ERG and prostate cancer survival.

Abstract Background: One half of prostate cancers harbor the TMPRSS2:ERG fusion, a somatic event that may reflect a distinct disease molecular subtype. TMPRSS2 is androgen regulated and ERG is a transcription factor that is part of the ETS family. Based on experimental evidence, we hypothesized that TMPRSS2:ERG tumors would be more sensitive to the insulin like growth factor (IGF)/insulin signaling axis in prostate cancer survival. Methods: We studied participants in the prospective Physicians' Health Study (PHS) and the Health Professionals Follow-Up Study (HPFS) diagnosed with prostate cancer from 1982 - 2005. TMPRSS2:ERG was assessed in tumor tissue microarrays by immunohistochemical evaluation of ERG expression (n=1,236). Pre-diagnostic plasma concentrations of IGF1 (n=307), IGFBP3 (n=307), c-peptide (n=293), and adiponectin (n=120), and tissue expression of the IGF1 receptor (IGF1R; n=713), insulin receptor (IR; n=682), and adionectin receptor (AdipoR2; n=840) were available among men with measured ERG expression. Men have been followed for biochemical recurrence and cancer mortality through 2012. We used Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals (CI) of associations between circulating biomarkers, TMPRSS2:ERG and prostate cancer outcomes. Results: Mean follow-up for lethal prostate cancer was 12.8 years and for recurrence 11.1 years. The number of lethal and recurrence events among men with measured blood markers were: IGF1/IGFBP3 25 and 61, c-peptide 20 and 56, and adiponectin 9 and 17, respectively. Among ERG negative tumors, comparing high vs. low concentrations, the multivariable HR for recurrence was 0.4 (95% CI 0.2-1.1) for IGF1, 1.1 (95% CI 0.5-2.8) for IGFBP3, 1.6 (95% CI 0.7-3.6) for c-peptide, and 1.4 (95% CI 0.3-5.6) for adiponectin. The corresponding HRs among ERG positive tumors were 1.8 (95% CI 0.8-4.0; p interaction 0.04) for IGF1, 0.8 (95% CI 0.4-1.8; p interaction 0.61) for IGFBP3, 0.6 (95% CI 0.3-1.3; p interaction 0.16) for c-peptide, and 0.8 (95% CI 0.2-3.6; p interaction 0.53) for adiponectin. Among ERG negative tumors, comparing high vs. low concentrations of the blood markers, the multivariable HR for lethal disease was 0.7 (95% CI 0.2-3.2) for IGF1, 1.4 (95% CI 0.3-5.9) for IGFBP3, 3.64 (95% CI 0.6-20.9) for c-peptide, and 1.1 (95% CI 0.2-7.8) for adiponectin. The corresponding HRs among ERG positive tumors were 2.3 (95% CI 0.5-11.1; p interaction 0.53) for IGF1, 0.6 (95% CI 0.1-2.3; p interaction 0.86) for IGFBP3, 0.7 (95% CI 0.2-2.9; p interaction 0.37) for c-peptide, and 0.4 (95% CI 0.03-5.4; p interaction 0.44) for adiponectin. The expression of IR, IGF1R, and AdipoR2 were significantly (all p-values &amp;lt;0.01) higher in ERG positive vs. negative tumors. Conclusions: Although the number of events were relatively small, our results support the hypothesis that TMPRSS2:ERG fusion may modify the association between the IGF/insulin signaling axis and prostate cancer prognosis. Citation Format: Thomas U. Ahearn, Andreas Pettersson, Edward C. Stack, Jing Ma, Travis Gerke, Allison Meisner, Michael N. Pollak, Stephen Finn, Meir J. Stampfer, Massimo Loda, Edward L. Giovannucci, Lorelei A. Mucci. The IGF/insulin signaling axis TMPRSS2:ERG and prostate cancer survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-28. doi:10.1158/1538-7445.AM2013-LB-28

Xanthoceraside attenuates learning and memory deficits via improving insulin signaling in STZ-induced AD rats

Xanthoceraside, a triterpenoid saponin extracted from the fruit husks of Xanthoceras sorbifolia Bunge, has been shown to reverse the cognitive deficits observed in several Alzheimer's disease (AD) animal models. Increasing evidence suggests the involvement of the insulin signaling pathway in neurodegenerative disorders such as AD. Thus, we used an AD animal model of cognitive impairment induced by the intracerebroventricular (ICV) injection of streptozotocin (STZ) to test the effects of xanthoceraside on behavioral impairments and insulin signaling mechanisms. In our present study, memory impairment was assessed using the Morris water maze test. The expression of IR, IGF-1R and Raf-1/ERK/CREB was tested by western blotting. The STZ group showed memory deficits in the Morris water maze test and significant decreases in IR and IGF-1R protein levels in the hippocampus. Xanthoceraside treatment significantly rescued memory deficits, as well as IR and IGF-1R protein expression levels. STZ inhibited the Ras/ERK signaling cascade and decreased the phosphorylation of CREB; these effects were also attenuated by xanthoceraside treatment. These results suggest the potential use of xanthoceraside for the treatment of neurodegenerative disorders in which brain insulin signaling may be involved.

The effects of TNF‐alpha and Ceramide on Insulin Receptor signaling and its localization to Caveolae in C2C12 Myocytes

TNF‐α and ceramide are associated with the etiology of insulin resistance. There is evidence for the involvement of caveolae membrane rafts in the modulation of insulin signaling and glucose uptake. Thus, we propose to elucidate the signaling mechanisms by which TNF‐α and ceramide induce insulin resistance in C2C12 myocytes and the role of caveolins in the organization of this pathway within the membrane. We hypothesized that exposure of C2C12 myocytes to TNF‐α and ceramide will result in altered insulin signaling, decreased glucose uptake, and translocation of the insulin signaling cascade away from caveolae. C2C12 myocytes were treated with TNF‐α, ceramide, and insulin, individually and in combination. The treated cells were subjected to Western blot analysis for insulin receptor‐β (IR‐β), phosphorylated IR (p‐IR), Protein Kinase B (PKB or Akt), phosphorylated Akt (p‐Akt), Cav‐3, Glut‐4, and actin (as loading control). The total protein expression of IR, Cav‐3, and Glut‐4 was unaltered with treatment. However, there was a significant increase in the total expression of Akt in cells treated with TNF‐α. In addition, there was a significantly higher activation of Akt and IR in cells treated with TNF‐α after 48 hours exposure. This data suggests that the effect of TNF‐α on insulin signaling in a result of a cross‐talk mechanism. This data will further the identification of novel signaling mechanisms leading to insulin resistance as a prerequisite to improved diagnosis and therapeutic treatments for diabetes.

Saturated fat-rich diet increases fetal lipids and modulates LPL and leptin receptor expression in rat placentas

Metabolic alterations in obese and overweight mothers impact the placenta and the fetus, leading to anomalies in fetal growth and lipid accretion. The primary aim of the study was to examine the effect of a saturated fat-rich diet (FD) on growth, lipid accretion, and lipases, leptin and leptin receptor (ObR) expression in the placenta and fetal liver. We also aimed to find a role for fetal leptin in the modulation of placental and fetal liver lipase and ObR expression. Six-week-old rats were fed with a standard rat chow (control) or a 25% FD for 7 weeks until mating and during pregnancy. Also, in a group of control rats, fetuses were injected with leptin on days 19, 20, and 21 of pregnancy. On day 21, we assessed lipidemia, insulinemia, and leptinemia in mothers and fetuses. In the placenta and fetal liver, lipid concentration was assessed by thin layer chromatography (TLC) and the gene expression of lipoprotein lipase (LPL), endothelial lipase, insulin receptor (Insr), leptin, and ObR by RT-PCR. The FD induced hypertriglyceridemia and hyperleptinemia (P<0.01) in mothers and fetuses, an increase in maternal (P<0.05) and fetal weight (P<0.01), overaccumulation of lipids in fetal liver (P<0.01), and enhanced leptin expression in the placenta and fetal liver (P<0.05). Placental expression of IR and LPL was increased (P<0.05), and ObR decreased (P<0.05) in the FD group. Fetal administration of leptin induced the placental and fetal liver downregulation of ObR (P<0.05) and upregulation of LPL expression (P<0.05). The FD led to increased fetal lipid levels, which may result from high maternal lipid availability and fetal leptin effects.

Inhibitory Receptor Expression Depends More Dominantly on Differentiation and Activation than “Exhaustion” of Human CD8 T Cells

Under conditions of chronic antigen stimulation, such as persistent viral infection and cancer, CD8 T cells may diminish effector function, which has been termed “exhaustion.” Expression of inhibitory Receptors (iRs) is often regarded as a hallmark of “exhaustion.” Here we studied the expression of eight different iRs by CD8 T cells of healthy humans, including CTLA-4, PD1, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1. We show that many iRs are expressed upon activation, and with progressive differentiation to effector cells, even in absence of long-term (“chronic”) antigenic stimulation. In particular, we evaluated the direct relationship between iR expression and functionality in CD8 T cells by using anti-CD3 and anti-CD28 stimulation to stimulate all cells and differentiation subsets. We observed a striking up-regulation of certain iRs following the cytokine production wave, in agreement with the notion that iRs function as a negative feedback mechanism. Intriguingly, we found no major impairment of cytokine production in cells positive for a broad array of iRs, as previously shown for PD1 in healthy donors. Rather, the expression of the various iRs strongly correlated with T cell differentiation or activation states, or both. Furthermore, we analyzed CD8 T cells from lymph nodes (LNs) of melanoma patients. Interestingly, we found altered iR expression and lower cytokine production by T cells from metastatic LNs, but also from non-metastatic LNs, likely due to mechanisms which are not related to exhaustion. Together, our data shows that expression of iRs per se does not mark dysfunctional cells, but is rather tightly linked to activation and differentiation. This study highlights the importance of considering the status of activation and differentiation for the study and the clinical monitoring of CD8 T cells.

Preliminary investigation of the expression and functions of insulin receptor isoforms in endometrial carcinoma

To investigate the expression of insulin receptor isoforms (IR, including IR-A and IR-B) in endometrial carcinoma (EC), and explore the role of IR-A in the growth of endometrial carcinoma cells. The expression of IR isoforms were detected by reverse transcription (RT)-PCR and real-time PCR in 4 different endometrial cancer cell lines (HEC-1-A, Ishikawa, RL95-2 and KLE), with human breast cancer cell line MCF-7 cells and hepatocellular carcinoma cell line Hep-G2 as positive control and in the endometrial cancer tissue specimens of 42 cases, admitted in Peking University People's Hospital from November 2007 to July 2009, with normal endometrial tissues from 15 cases of ovarian neoplasms at the same period as controls. The relationships among the expression of IR, IR-A isoforms and the clinicopathological parameters of EC tissues were analyzed by Spearman rank correlation analysis. An eukaryotic IR-A expression plasmid was constructed and transfected into RL95-2 cells [RL95-2(IR-A)] for overexpression of IR-A in RL95-2 cells, in which the expression of IR-A originally was low. Non radioactive cell proliferation assay-MTS was used to determine the proliferation curves in the four EC cells listed above and in RL95-2 or RL95-2 (IR-A). (1) There were two isoforms of IR-A and IR-B co-expressed were detected in EC cells and EC tissues. Among four kinds of EC cell lines, the expression level of IR mRNA in RL95-2 cells was the highest, followed by Ishikawa, KLE and HEC-1-A cells, in which the relative IR mRNA expression levels were (26.54 ± 1.82)×10(-4), (15.44 ± 3.29)×10(-4), (10.14 ± 0.10)×10(-4) and (2.63 ± 0.23)×10(-4), respectively (P < 0.01). The expression level of IR-A mRNA was the highest in Ishikawa cells, followed by KLE, RL95-2 and HEC-1-A cells, with the relative expression levels were (12.07 ± 3.31)×10(-4), (4.68 ± 0.63)×10(-4), (3.03 ± 0.22)×10(-4) and(1.46 ± 0.03)×10(-4), respectively (P < 0.01). The relative expression level of IR and IR-A mRNA were 0.017 ± 0.013 and 0.011 ± 0.010 in the EC tissues, respectively, compared with 0.015 ± 0.014 and 0.010 ± 0.012 in the controls, in which there were no significant differences in the expression level of IR or IR-A mRNA between EC tissues and the control (P = 0.662, P = 0.780). The expression of IR and IR-A in EC tissues had no significant relevance with International Federation of Gynecology and Obstetrics (FIGO) stage, cell differentiation, depth of myometrial invasion, invasion of lymph-vascular space, lymph nodes metastasis, the expression status of estrogen receptor, human progesterone receptor, and PTEN gene (all P > 0.05). The expression of IR-A mRNA in EC patients with type 2 diabetes mellitus (DM) was significantly higher than that in patients without type 2 DM (P = 0.031), while there were no statistical correlation between the expression of IR mRNA and type 2 DM (P = 0.438). (2) The proliferation rates of the four kinds of EC cells was positively related with the IR-A expression ratio, with the most growth potential in Ishikawa, followed by HEC-1-A, KLE and RL95-2 cells. The overexpression of IR-A in RL95-2 (IR-A) cells showed a significant proliferation-promoting effect than that in control RL95-2 cells (P < 0.01). There are two isoforms of IR-A and IR-B co-expressed in EC. The overexpression of IR-A may promote the proliferation of EC cells.

A Mechanism for Circadian Control of Pacemaker Neuron Excitability

Although the intracellular molecular clocks that regulate circadian (~24 h) behavioral rhythms are well understood, it remains unclear how molecular clock information is transduced into rhythmic neuronal activity that in turn drives behavioral rhythms. To identify potential clock outputs, the authors generated expression profiles from a homogeneous population of purified pacemaker neurons (LN(v)s) from wild-type and clock mutant Drosophila. They identified a group of genes with enriched expression in LN(v)s and a second group of genes rhythmically expressed in LN(v)s in a clock-dependent manner. Only 10 genes fell into both groups: 4 core clock genes, including period (per) and timeless (tim), and 6 genes previously unstudied in circadian rhythms. The authors focused on one of these 6 genes, Ir, which encodes an inward rectifier K(+) channel likely to regulate resting membrane potential, whose expression peaks around dusk. Reducing Ir expression in LN(v)s increased larval light avoidance and lengthened the period of adult locomotor rhythms, consistent with increased LN(v) excitability. In contrast, increased Ir expression made many adult flies arrhythmic and dampened PER protein oscillations. The authors propose that rhythmic Ir expression contributes to daily rhythms in LN(v) neuronal activity, which in turn feed back to regulate molecular clock oscillations.

Excess aldosterone-induced changes in insulin signaling molecules and glucose oxidation in gastrocnemius muscle of adult male rat

Emerging evidences demonstrate that excess aldosterone and insulin interact at target tissues. It has been shown that increased levels of aldosterone contribute to the development of insulin resistance and thus act as a risk factor for the development of type-2 diabetes mellitus. However, the molecular mechanisms involved in this scenario are yet to be identified. This study was designed to assess the dose-dependent effects of aldosterone on insulin signal transduction and glucose oxidation in the skeletal muscle (gastrocnemius) of adult male rat. Healthy adult male albino rats of Wistar strain (Rattus norvegicus) weighing 180-200 g were used in this study. Rats were divided into four groups. Group I: control (treated with 1 % ethanol only), group II: aldosterone treated (10 μg /kg body weight, twice daily for 15 days), group III: aldosterone treated (20 μg /kg body weight, twice daily for 15 days), and group IV: aldosterone treated (40 μg/kg body weight, twice daily for 15 days). Excess aldosterone caused glucose intolerance in a dose-dependent manner. Serum insulin and aldosterone were significantly increased, whereas serum testosterone was decreased. Aldosterone treatment impaired the rate of glucose uptake, oxidation, and insulin signal transduction in the gastrocnemius muscle through defective expression of IR, IRS-1, Akt, AS160, and GLUT4 genes. Phosphorylation of IRS-1, β-arrestin-2, and Akt was also reduced in a dose-dependent manner. Excess aldosterone results in glucose intolerance as a result of impaired insulin signal transduction leading to decreased glucose uptake and oxidation in skeletal muscle. In addition to this, it is inferred that excess aldosterone may act as one of the causative factors for the onset of insulin resistance and thus increased incidence of type-2 diabetes.

Abstract 1901: EGFR and K-Ras mutations and resistance of lung cancers to IGF-1R TKI

Abstract Background: Most patients with non-small cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). We investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and the molecular determinants of resistance to IGF-1R TKIs. Methods: Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in a NSCLC tissue microarray. We analyzed the antitumor effects of an IGF-1R TKI (PQIP or OSI-906), either alone or in combination with a small-molecular inhibitor (PD98059 or U0126) or with siRNA targeting K-Ras or MAPK/extracellular signal-regulated kinase kinase (MEK), in vitro and in vivo in NSCLC cells with variable histologic features and EGFR or K-Ras mutations. Results: pIGF-1R/IR expression in NSCLC specimens was associated with a history of tobacco smoking, squamous cell carcinoma histology, mutant (mut) K-Ras, and wild-type (wt) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF-1R TKIs exhibited significant antitumor activity in NSCLC cells with wt EGFR and wt K-Ras but not in those with mutations in these genes. Introduction of mut K-Ras attenuated the effects of IGF-1R TKIs on NSCLC cells expressing wt K-Ras. Conversely, inactivation of MEK restored sensitivity to IGF-TKIs in cells carrying mut K-Ras. Conclusions: The mutation status of both EGFR and K-Ras could be predictive markers of response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF-1R TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1901. doi:1538-7445.AM2012-1901