Receptor Beta-subunit Research Articles

Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization.

Crohn's Disease (CD) afflicts over half a million Americans with an annual economic impact exceeding $10 billion. Granulocyte macrophage colony-stimulating factor (GM-CSF) can increase patient immune responses against intestinal microbes that promote CD and has been effective for some patients in clinical trials. We have made important progress toward developing GM-CSF variants that could be more effective CD therapeutics by virtue of being less prone to neutralization by the endogenous GM-CSF autoantibodies that are highly expressed in CD patients. Yeast display engineering revealed mutations that increase GM-CSF variant binding affinity by up to ∼3-fold toward both GM-CSF receptor alpha and beta subunits in surface plasmon resonance experiments. Increased binding affinity did not reduce GM-CSF half-maximum effective concentration (EC50) values in conventional in vitro human leukocyte proliferation assays. Affinity-enhancing mutations did, however, promote a 'refacing effect' that imparted all five evaluated GM-CSF variants with increased in vitro bioactivity in the presence of GM-CSF-neutralizing polyclonal antisera. The most improved variant, H15L/R23L, was 6-fold more active than wild-type GM-CSF. Incorporation of additional known affinity-increasing mutations could augment the refacing effect and concomitant bioactivity improvements described here.

Synergistic antitumor activity of the CD122-biased immunostimulatory cytokine NKTR-214 when combined with anti-PD-1 in murine tumor models

Meeting abstracts NKTR-214 is an immunotherapeutic drug that exerts its biological activity by binding and activating the interleukin-2 (IL-2) receptor beta subunit (IL-2Rb), thereby causing expansion of memory effector T cells in the tumor. NKTR-214 consists of 4-6 releasable polyethylene glycol (

Abstract 5032: Synergy between an engineered cytokine, NKTR-214, and CTLA-4 blockade in murine colon and breast tumor models

Abstract Background: Immunotherapy offers the potential for durable responses in a growing list of cancer indications. NKTR-214 is comprised of the cytokine IL2 conjugated o multiple PEG units. Upon in vivo administration some of these PEG molecules are released to leave activated IL2-conjugates that bind selectively at the IL2 receptor beta subunit (IL2Rβ). The active IL-2-conjugates favor proliferation of tumor-killing CD8+ memory T cells (CD8T) over immunosuppressive regulatory T cells (Treg) in the tumor microenvironment (Charych, JCO 2013, 31:15, Suppl. 1).We have also shown that NKTR-214 results in sustained exposure of drug in the tumor and robust immune system activation that translates between rodents and monkeys at safe doses, (AACR; Mol Cancer Ther 2013;12 (11 Suppl): Abstract B296). The receptor selectivity and improved pharmacokinetic profile leads to substantially improved single-agent efficacy and safety over the original IL-2 cytokine. Combinations of different checkpoint blockade antibodies have shown great promise clinically and in preclinical tumor models, highlighting the importance of targeting multiple immune activation pathways. Here we examine the combination of NKTR-214 with anti-CTLA4 antibody in murine breast and colon tumor models to explore the interaction of two mechanisms of action: checkpoint inhibition and direct CD8T cell activation via the IL-2 receptor beta. Methods: Female BALB/c mice bearing established tumors implanted with CT-26 (Murine Colon Carcinoma) or EMT6 (Murine Mammary Carcinoma) cells were treated with single agent NKTR-214, murine anti-CTLA-4 antibody or the two agents in combination. NKTR-214 was administered at 0.8 mg/kg i.v. and anti-CTLA-4 at 100 μg/mouse i.p. Results: In both the EMT6 breast and the CT-26 colon models, the combination of NKTR-214 with anti-CTLA-4 yielded significantly better tumor growth inhibition compared to either agent dosed alone. In the EMT6 model 10/12 animals were tumor free by day 20 in the combination arm and stayed tumor free until the end of study (60 days). Similarly in the CT-26 model, 8/12 animals were tumor free by day 25 in the combination arm and stayed tumor free until the end of study (day 60). Single agent anti CTLA-4 or NKTR-214 did not show significant tumor inhibition in either model. No toxicities were observed when NKTR-214 was combined with anti CTLA-4 antibody. Conclusions: NKTR-214 is a highly differentiated immunotherapy with a new mechanism of action that shows efficacy both as a single agent (as previously shown in a mouse melanoma model) and in combination. Combining NKTR-214 mediated T cell activation with CTLA-4 blockade is highly synergistic in murine models of cancer and holds the promise for durable responses in patients. Citation Format: Steve Lee, Murali Addepalli, Ute Hoch, Rhoneil Pena, Payal Shirsat, Yolanda Kirksey, Stephen K. Doberstein, Deborah Charych, Seema Kantak. Synergy between an engineered cytokine, NKTR-214, and CTLA-4 blockade in murine colon and breast tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5032. doi:10.1158/1538-7445.AM2014-5032

Novel de novo mutations of the interleukin-10 receptor gene lead to infantile onset inflammatory bowel disease

Background and aims: Defects in the interleukin 10 (IL-10) signalling pathway have been shown to cause very early onset inflammatory bowel disease (IBD). We report a patient with severe infantile-onset IBD with a compound heterozygous IL-10 receptor alpha subunit (IL-10RA) mutation, one of which was paternally-inherited and the other occurring de novo. Methods: Deep sequencing of IL-10, IL-10RA and IL-10 receptor beta subunit (IL-10RB) were performed. Peripheral blood mononuclear cell (PBMC) surface expression of IL-10RA was analysed by flow cytometry. IL-10 signalling pathway was examined by measuring phosphorylated STAT3 in PBMC cultured in the presence of IL-6 or IL-10. Result: We identified a missense mutation in exon 4 of IL-10RA (c.583T>C) in one allele and a nonsense mutation in exon 7 of IL-10RA (c.1368G>T) in the other allele. Neither mutation has been reported previously. The patient has functional IL-10RA deficiency despite normal IL-10RA expression. Conclusion: This represents the first case report of a de novo mutation of IL-10RA that is associated with very early onset severe IBD. Therefore, IL-10 pathway defect should be considered in patients with infantile-onset IBD even if the parents are non-consanguineous.

Expression of Caveolin 1 is enhanced by DNA demethylation during adipocyte differentiation. status of insulin signaling.

Caveolin 1 (Cav-1) is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms must have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised.

Expression and significance of glycine receptor beta subunit in the model of chronic pancreatitis for pain study

Objective To study the expression of glycine receptor beta subunit in the model of chronic pancreatitis,and evaluate the relationship between glycine receptor beta subunit and pain in chronic pancreatitis.Methods Thirty male Wistar rats were randomly divided into three groups:normal group,phosphate buffer (PBS) perfusion group,and trinitrobenzene sulfonic acid perfusion group.Histopathological and pain examinations were done to confirm the establishment of chronic pancreatitis model.Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was applied to detect the expression of glycine receptor beta subunit.Immunohistochemistry was used to localize the expression site of glycine receptor beta subunit.Results Pain examination in this model showed dynamic,decrease at first,increase at last.Compared with another groups,the trinitrobenzene sulfonic acid perfusion group is lower in the aspect about the pain during the fourth week (comparing the three groups:x2 =4.279＜x0.05 (8),P ＞ 0.05 ; comparing with each other:P ＜0.05),but higher than others in gene expression (2.5 times,P ＜0.05),and strongly expression was observed in cytoplasm.Conclusion Enhanced expression of glycine receptor beta subunit,the inhibitory neurotransmitter,is involved in the pain of chronic pancreatitis.For self-protection,it reduces the degree of pain below normal lever. Key words: Chronic pancreatitis; Glycine receptor beta subunit; Pain

IL3-Receptor Signaling Is Dispensable For The Generation and Maintenance Of Jak2V617F-Induced Myeloproliferative Neoplasm (MPN

Myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and primary myelofibrosis are clonal diseases driven by acquired somatic mutations in hematopoietic stem cells (HSCs), the most common of which is JAK2V617F. JAK2V617F leads to cytokine hypersensitivity and activation of JAK-STAT signaling in the presence of an erythropoietin (EPO), thrombopoietin (TPO) or interleukin-3 (IL3) cytokine receptor scaffold (Nature 2005; 434:1144-8, Leukemia 2008; 22:1828-40). Physiological Jak2V617F expression in long-term hematopoietic stem cells (LT-HSCs; lineagelowcKit+Sca1+CD150+CD48-) is necessary and sufficient to generate MPN, and these LT-HSCs contain the sole reservoir of Jak2V617F MPN-initiating stem cells (Cancer Cell 2010; 17:584-96; Blood 2012; 120:166-72). Although Jak2V617F-mediated EPO hypersensitivity drives erythroid expansion and in vitro EPO-independent colony formation, the EPO receptor is not expressed on Jak2V617F HSC populations (Blood 2012; 120:166-72). This suggests that hypersensitivity to IL3 and/or TPO signaling is responsible for driving LT-HSC proliferation and survival in vivo.To determine the respective contributions of IL3 and TPO signaling to Jak2V617F-induced MPN, pStat5 was measured in HSC and progenitor populations from E2ACre+Jak2V617F+/- knockin mice (hereafter Jak2VF) after stimulation with rmIL3 or rmTPO. Committed myeloid progenitors showed robust pStat5 stimulation with rmIL3, but only low level stimulation with rmTPO (TPO 460.25 ± 25.02 MFI vs. IL3 598.25 ± 69.05 MFI, p<0.05, Vehicle 362.25 ± 76.66 MFI). In contrast, LT-HSCs showed strong induction of pStat5 signaling with rmTPO stimulation but less so with rmIL3 stimulation (TPO 571 ± 47.36 MFI vs. IL3 487.5 ± 53.69 MFI, p=0.05, Vehicle 249.5 ± 47.63 MFI). Concordant with these results, TPO signaling appears essential for the generation of Jak2V617F-induced MPN (ASH 2012 Abstract 427). To determine the contribution of IL3 receptor signaling in Jak2V617F-induced MPN, we crossed Jak2VF knockin mice with mice lacking the common beta subunit of the IL3 receptor that is responsible for signal transduction of IL3, IL5 and GM-CSF (Jak2VFIL3Rb-/-). Jak2VFIL3Rb-/- mice developed MPN with similar latency and mortality to Jak2VF controls. There were no differences in peripheral blood white cell count (16.51 ± 1.5×109/L vs. 14.89 ± 2.4×109/L, p=0.39, n=3), haematocrit (67.97 ± 7.85×109/L vs. 61.97 ± 5.73×109/L, p=0.35, n=3) or extramedullary erythropoiesis (spleen weight, 665 ± 107mg vs. 541 ± 106mg, p=0.22, n=3) between Jak2VFIL3Rb-/- and Jak2VF mice respectively. In competitive bone marrow transplantation assays, all recipients of Jak2VFIL3Rb-/- or Jak2VF bone marrow developed MPN with similar diagnostic parameters such as elevated white cell count (14.29 ± 3.41×109/L vs. 18.38 ± 5.78×109/L, p=0.11, n=8), hematocrit (63.4 ± 16.5% vs. 50.5 ± 13%, p=0.16, n=8) and splenomegaly (529 ± 86mg vs. 465 ± 119mg, p=0.23, n=8) in Jak2VFIL3Rb-/- and Jak2VF mice respectively. Jak2VFIL3Rb-/- bone marrow cells initially showed reduced short-term (4 weeks) engraftment and white cell count in recipients compared to the Jak2V617F group (p<0.0005). However after 16 weeks post-transplant there was no difference in chimerism between recipients of Jak2VFIL3Rb-/- or JakVF cells. IL3 was unable to stimulate pStat5 in Jak2VFIL3Rb-/- LT-HSCs or progenitors, but was preserved in Jak2VF LT-HSCs and progenitors.These data show that IL3Rb signaling is dispensable for Jak2V617F-induced MPN and LT-HSC function, however may regulate short-term myeloid progenitor cell expansion. TPO signaling appears preferentially important for Jak2VF LT-HSC pStat5 induction, whereas IL3 is more important for pStat5 induction in progenitor cells. These findings will help to inform strategies aimed at targeting long term Jak2V617F-initiating HSC populations. Disclosures:No relevant conflicts of interest to declare.

Bilaterally cleft lip and bilateral thumb polydactyly with triphalangeal component in a patient with two De novo deletions of HSA 4q32 and 4q34 involving PDGFC, GRIA2, and FBXO8 genes

We report on a newborn boy with a bilateral cleft of the primary palate, duplicated triphalangeal thumbs, and a patent foramen ovale. During childhood he had moderate developmental delay. Brain MRI at 4 years was normal. The concurrence of non-syndromic clefts of the lip/palate (CL/P) and duplicated thumbs with triphalangeal component has, to our knowledge, not been reported so far. In our case, array-CGH analysis documented two de novo deletions (∼1.2 Mb and ∼400 Kb) of the long arm of chromosome 4, containing four genes: platelet-derived growth factor C (PDGFC), glycine receptor beta subunit (GLRB), glutamate receptor ionotropic AMPA2 (GRIA2), and F-box protein 8 gene (FBXO8). PDGFC codes for a mesenchymal cell growth factor already known to be associated with clefts of the lip. Pdgfc(-/-) mice have skeletal anomalies, and facial schisis resembling human cleft/lip palate. GRIA2 codes for a ligand-activated cation channel that mediates the fast component of postsynaptic excitatory currents in neurons, and may be linked to cognitive dysfunction. FBXO8, a gene of unknown function, is a member of the F-box gene family, among which FBXW4, within the minimal duplicated region associated with human split-hand/foot malformation type 3 (SHFM type 3). The presence of overlapping deletions in patients who do not share the same phenotype of our case suggests incomplete penetrance, and a possible effect of modifier genetic factors.

Association of IFNAR2 and IL10RB genes in chronic hepatitis B virus infection

In this study, we investigated the effects of two functional polymorphisms, type I interferon receptor 2 gene (IFNAR2)-F8S and interleukin-10 receptor subunit beta gene (IL10RB)-K47E, on chronic hepatitis B virus (HBV) infection. We included 227 Thai patients with chronic HBV infection [100 with hepatocellular carcinoma (HCC) and 127 non-HCC], 170 individuals with self-limited HBV infection and 150 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze these two single nucleotide polymorphisms (SNPs). In this study, the C allele of IFNAR2-F8S was found to be significantly increased in chronic HBV patients when compared with healthy controls [odds ratio, OR (95% confidence interval, CI)= 3.31 (2.11-5.21), P = 6.214 × 10(-9) and corrected P-value, P(c)= 1.864 × 10(-8)]. The effect of this allele was similar to that of an autosomal dominant gene in the presence of CC and CT genotype, when compared to TT with an OR of 4.02 (P = 4.631 × 10(-9) and P(c)= 1.389 × 10(-8)). Furthermore, AA genotype of IL10RB-K47E was found to be significantly decreased in chronic HBV patients compared with individuals with self-limited HBV infection (P = 0.006, P(c)= 0.018 and OR = 0.45). For haplotype analysis, we found CA and CG haplotypes were associated with susceptibility to chronic HBV (P = 0.014, OR = 6.84 and P = 0.002, OR = 3.75, respectively) when compared with healthy individuals. This study suggests that IFNAR2-F8S polymorphisms might be involved in the susceptibility to chronic HBV infection. Moreover, AA genotype of IL10RB-K47E may provide a protective effect in this disease. However, an association study using a larger sample size should be performed to confirm these findings.

Subetta treatment increases adiponectin secretion by mature human adipocytes in vitro.

Purpose. To investigate the mechanism of action in peripheral tissues of novel complex drug containing release-active dilutions of antibodies to the beta subunit of the insulin receptor and antibodies to endothelial nitric oxide synthase (Subetta), which has shown efficacy in animal models of diabetes. Methods. Human mature adipocytes were incubated either with Subetta, with one of negative controls (placebo or vehicle), with one of nonspecific controls (release-active dilutions of antibodies to cannabinoid receptor type I or release-active dilutions of rabbit nonimmune serum), or with dimethyl sulfoxide (DMSO) at 37°C in a humidified incubator at 5% CO2 for three days. Rosiglitazone was used as reference drug. Secretion of adiponectin was measured by quantitative enzyme-linked immunosorbent assay (ELISA). Results. Only Subetta significantly stimulates adiponectin production by mature human adipocytes. Nonspecific controls did not significantly affect adiponectin secretion, resulting in adiponectin levels comparable to background values of the negative controls and DMSO. Conclusion. Increasing adiponectin production in absence of insulin by Subetta probably via modulating effect on the beta subunit of the insulin receptor might serve as one of the mechanisms of the antidiabetic effect of this drug. These in vitro results give first insight on possible mechanism of action of Subetta and serve as a background for further studies.

Myelin Basic Protein-primed T Helper 2 Cells Suppress Microglial Activation via AlphaVBeta3 Integrin: Implications for Multiple Sclerosis

Multiple sclerosis (MS) is the most common autoimmune demyelinating disease in human and T helper type 2 (Th2) cells have been shown to be beneficial for this disease. However, mechanisms by which Th2 cells ameliorate disease in MS are poorly understood. Microglial activation plays an important role in the pathogenesis of MS and other neurodegenerative disorders. Here, we delineate that Th2 cells are capable of suppressing microglial activation via cell-to-cell contact. After polarization of MBP-primed Th1 cells to Th2 by gemfibrozil and other drugs, we observed that MBP-primed Th2 cells dose dependently inhibited the production of interleukin-1β (IL-1β) and nitric oxide (NO) in LPS-stimulated microglia via cell-to-cell contact. Similarly, Th2 cells also suppressed the microglial inflammatory response in the presence of different pathological stimuli of Alzheimer’s disease (AD), Parkinson’s disease (PD), and HIV associated dementia (HAD). Interestingly, Th2 cells expressed higher levels of alphaV (αV) and beta3 (β3) integrins as compared to Th1 cells, and functional blocking antibodies against αV and β3 integrins impaired the ability of Th2 cells to suppress microglial activation. Furthermore, we demonstrate that microglia expressed the beta subunit of PDGF receptor (PDGFRβ) and that neutralization of PDGFRβ abrogated the ability of Th2 cells to suppress microglial inflammation. Activation of microglial cAMP response element-binding (CREB) by Th2 cells, suppression of CREB activation by neutralization of either αV and β3 integrins on Th2 cells or PDGFRβ on microglia, abrogation of anti-inflammatory activity of Th2 cells by siRNA knockdown of microglial CREB, highlights the importance of αVβ3 and PDGFRβ in guiding the anti-inflammatory activity of Th2 cells via activation of CREB, which may be responsible for beneficial effect of Th2 cells in MS and other related disorders.

Traditional Medicine in Management of Type 2 Diabetes Mellitus

The incidence of type 2 diabetes mellitus has now reached epidemic proportions. Although the disease manifests in the form of hyperglycemia, the cause could be varied ranging from disturbance in insulin secretion, insulin action, insulin resistance, glucose production and glucose uptake, interplay between different hormones, and various kind of stress. Due to such varied etiology, the management of diabetic condition poses a great medical challenge. No single agent has so far been unequivocally accepted as the antidiabetic drug. Alternative systems of medicine based on traditional wisdom have thrived through ages and are still practiced by a large population for the management of diabetes. A large number of plants have proved their efficacy in management of diabetes especially hyperglycemia. In many cases, scientific studies have validated the antidiabetic nature of plant-based medicines, and the bioactive principle has been isolated and characterized. It is important that more research is done to understand the mechanism(s) involved in the antidiabetic action of large number of plant-based medicines used as traditional therapy for the management of diabetic condition. The present special volume has brought together some interesting papers reporting the findings of the use of traditional medicines for the treatment of diabetes mellitus. S. I. Rizvi and N. Mishra provide a good review of the anti-diabetic potential and the bioactive compounds present in Ficus religiosa, Pterocarpus marsupium, Gymnema sylvestre, Allium sativum, Eugenia jambolana, Momordica charantia, and Trigonella foenum-graecum. All these plants are widely used in the Indian subcontinent for the management of diabetic condition. O. Eleazu et al. explore the chemical composition of cocoyam and unripe plantain flours and their potential in the dietary prevention of diabetic complications. D. Bailbe and colleagues evaluate the effects of Subetta (containing release-active dilutions of antibodies to beta-subunit of insulin receptor and antibodies to endothelial nitric oxide synthase) in Goto Kakizaki diabetic rats and demonstrate that 28-day administration improves glucose control to an extent similar to that of Rosiglitazone. H. Y. Jin et al. have tested the efficacy of DA-9801, a mixture of extracts from Dioscorea japonica and Dioscorea nipponica in the treatment of diabetic peripheral neuropathy in experimental diabetes. They have also presented a comparison of the effect of DA-9801 with lipoic acid. Statins are very widely used during dyslipidemia. Since type 2 diabetes is frequently associated with dyslipidemia, it is important to investigate the effect of statins on glucose homeostasis in diabetes. Wang et al. report that simvastatin may cause hyperglycemia and have an adverse effect on glucose homeostasis in diabetic rats. Syed Ibrahim Rizvi Elena Matteucci Pinar Atukeren

Hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells

The flavanone hesperetin is known to decrease basal glucose uptake, although the inhibitory mechanism is largely unknown. Here, we used MDA-MB-231 breast cancer cells to investigate the molecular pathways affected by hesperetin. The results indicate that the suppression of glucose uptake is caused by the down-regulation of glucose transporter 1 (GLUT1). Hesperetin was also found to inhibit insulin-induced glucose uptake through impaired cell membrane translocation of glucose transporter 4 (GLUT4). In addition, the phosphorylation of the insulin receptor-beta subunit (IR-beta) and Akt was suppressed. Hesperetin also decreased cellular proliferation, which is likely due to the inhibition of glucose uptake. Cancer cells are highly dependent on glucose and hesperetin may, therefore, have potential application as an anticancer agent.

Aromatase cytochrome P450 enzyme expression in prolactinomas and its relationship to tumor behavior

The aim of the study was to evaluate the presence of aromatase cytochrome P450 enzyme (P450AROM) expression in normal pituitary tissues and tumor tissues of patients with prolactinoma and to examine the impact of the P450AROM expression on clinical outcome. Twenty-six consecutive human pituitary tissue samples were obtained from autopsies performed at the Institute of Forensic Medicine. Sixty-four patients who had an adenomectomy between 2000 and 2009 after prolactinoma diagnosis with histologically confirmed pituitary tumor tissues were retrospectively included in this study. The slices from the pituitary tissues were subjected to immunohistochemical staining for evaluation of P450AROM and estrogen receptor beta (ER beta) subunit. Immunohistochemistry results were compared according to age, gender, remission rate, resistance and invasion status of the patients. Higher than normal P450AROM expression was found in the pituitary tissues of the patients with prolactinoma (p < 0.001). P450AROM intensity had no relation to resistance or remission in patients with prolactinoma (p = 0.44, p = 0.45, respectively). The subgroup analysis showed that compared to males without invasive adenoma, males with invasive adenoma had higher P450AROM expression (p = 0.048). ER beta was found to have an impact on resistance (p = 0.049). This study shows that P450AROM expression is present in the pituitary tissues of patients with prolactinoma and that this presence could be important in development and tumor behavior of prolactinomas.

Chemerin inhibits IGF-1-induced progesterone and estradiol secretion in human granulosa cells

Chemerin is a novel adipokine involved in the regulation of adipocyte development, inflammation and metabolic functions. To date, no role of this adipokine in reproductive functions has been described. In the present study, we identified chemerin and its receptor, CMKLR1 (chemokine-like receptor 1), in primary human granulosa cells (hGCs) and in a human ovarian granulosa-like tumour cell line (KGN). We also investigated the effects of recombinant human chemerin (rhChem) on steroid production and on various signalling pathways. By RT-PCR immunoblotting and immunohistochemistry, we showed that chemerin and CMKLR1 are expressed in hGCs and KGN cells. By ELISA, we also found chemerin in human follicular fluid and we observed that in 8 of 10 women the chemerin level was at least 2-fold higher in follicular fluid than in plasma. rhChem (10 or 100 ng/ml) significantly decreased insulin-like growth factor-1 (IGF-1) (10(-8) M)-induced secretion of progesterone and estradiol (as determined by radioimmunoassay) but did not affect basal-or FSH (10(-8) M)-induced steroid secretion in hGCs and KGN cells. In parallel, it also decreased IGF-1-induced p450 aromatase protein levels without affecting the protein levels of other factors involved in steroidogenesis (steroidogenic acute regulatory protein, 3-beta-hydroxysteroid dehydrogenase and p450 side-chain cleavage enzyme) in hGCs cells. All these changes were associated with a decrease in the IGF-1-induced tyrosine phosphorylation of IGF-1 receptor beta subunit and phosphorylation of mitogen-activated protein kinase extracellular signal-regulated kinases 1/2 (MAPK ERK1/2) and Akt. In hGCs and KGN cells, rhChem also decreased IGF-1-induced thymidine incorporation. Finally, we showed that rhChem rapidly activates MAPK ERK1/2, MAPK P38 and Akt phosphorylation and more slowly AMP-activated protein kinase phosphorylation under basal conditions (no IGF-1 or FSH) in primary hGC cells. Taken together, chemerin and its receptor (CMKLR1) are present and active in hGCs. Chemerin reduces IGF-1-induced steroidogenesis and cell proliferation through a decrease in the activation of IGF-1R signalling pathways in primary hGCs.

New Insights into the Role of ENPP1 in Insulin Resistance.

New Insights into the Role of ENPP1 in Insulin Resistance Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a transmembrane glycoprotein highly expressed in adipocytes and also found in other tissues involved in glucose and lipid metabolism, including liver, skeletal muscle and beta cell of the pancreas. Early studies demonstrated that ENPP1 interacts with the insulin receptor at the level of the alpha subunit. This interaction results in decreased insulin-mediated activation of the tyrosine phosphorylation in the beta-subunit of the insulin receptor, and downstream insulin signaling activation.

Plasma Membrane Topology of the Insulin Receptor: Insights from Computational Modeling

The insulin receptor in the plasma membrane is composed of two alpha-subunits, each linked to a beta-subunit and to each other by disulfide bonds. It has been proposed that both alpha-subunits are extracellular and anchored to the plasma membrane via a single transmembrane (TM) helix in the N-terminal region of each beta-subunit. However, we find that newer sequence analysis algorithms predict that the alpha-subunits of human insulin receptor (P06213) are largely intracellular and each contains a single TM helix in the N-terminal region. The MEMSAT-SVM method predicts (after correcting for a 19 residue N-terminal signal peptide) that the alpha-subunit contains a TM helix between residues 64 and 79, with a 63 amino acid ectodomain and a large 676 residue cytoplasmic domain. The existence of a large intracellular domain is consistent with the presence of 10 caveolin-binding (CB) motifs (OxOxxxxO, OxxxxOxxO, where O is the aromatic amino acid F, Y, or W), which exist within most caveolin-associated proteins where caveolin is accessible only intracellularly. Our computational analysis of the insulin receptor beta-subunit sequence confirms a single TM helix between residues 194 - 218 (UniProtKB places the TM helix within residues 194-213), with 1 CB motif proximal to the TM helix (W175 - Y182). Similar topologies were obtained with insulin receptor sequences from a variety of species. The insulin receptor co-isolates with lipids and the purified insulin receptor readily forms lipid complexes. Thus, caveolin binding motifs may direct the insulin receptor tetramer to caveolin-rich lipid rafts. The topology of the insulin receptor and CB motifs is relevant to understanding the microdynamics of insulin receptor movement between caveolar and non-caveolar regions of the plasma membrane as well as insulin-mediated events such as endocytosis, membrane hyperpolarization, and regulation of Na+/K+/H+ ions.

Insulin Receptor Expression and Activity in the Brains of Nondiabetic Sporadic Alzheimer’s Disease Cases

We investigated the contents of the insulin receptor-beta subunit (IRβ) and [Tyr1162/1163]-phosphorylated IRβ as surrogate indices of total IR content and IR activation in postmortem hippocampal formation brain specimens from nondiabetic sporadic Alzheimer's disease (AD) cases. We found no significant changes in the brain contents of total IRβ or [Tyr1162/1163]-phosphorylated IRβ, suggesting normal IR content and activation in the brains of nondiabetic sporadic AD cases. Moreover, total IRβ and [Tyr1162/1163]-phosphorylated IRβ levels in the hippocampal formation are not correlated with the severity of amyloid or tau-neuropathology. Exploring the regulation of glycogen synthase kinase 3 (GSK3) α/β, key IR-signaling components, we observed significantly lower levels of total GSK3 α/β in brain specimens from nondiabetic AD cases, suggesting that impaired IR signaling mechanisms might contribute to the onset and/or progression of AD dementia. Outcomes from our study support the development of insulin-sensitizing therapeutic strategies to stimulate downstream IR signaling in nondiabetic AD cases.

Intermediate-affinity interleukin-2 receptor expression predicts CD56bright natural killer cell expansion after daclizumab treatment in the CHOICE study of patients with multiple sclerosis

Objective: The objective of this study was to evaluate whether interleukin-2 (IL-2) receptor expression on CD56bright natural killer (NK) cells predicted CD56bright NK cell expansion and therapeutic response to daclizumab (DAC) in multiple sclerosis (MS). Methods: DAC exposure, CD56bright NK cell counts, IL-2 receptor alpha (CD25) and beta (CD122) subunits, and new or enlarged lesions on brain MRI were measured in 64 subjects in a pharmacokinetic/pharmacodynamic substudy of the phase 2 CHOICE trial at multiple time points. Peripheral blood mononuclear cell (PBMC) samples were obtained from healthy subjects to assess the relationship among DAC treatment, intermediate affinity IL-2 signaling, and CD56bright NK cell expansion. Results: Increased CD56bright NK cell counts in DAC/interferon beta (IFNβ)-treated subjects were observed by day 14, the first post-dosing time point (mean [SD] ln{CD56bright NK cell count}: DAC high/IFNβ, 2.01 [1.25]; DAC low/IFNβ, 2.29 [1.06]; placebo/IFNβ, 1.01 [1.03]; adjusted p = 0.003), and persisted throughout the treatment period. Higher DAC dose predicted a faster rate of CD56bright NK cell expansion (p < 0.001), but individual subjects’ increases in CD56bright NK cells from baseline levels were only weakly correlated with DAC exposure (r2 = 0.167). Higher expression of the intermediate-affinity IL-2 receptor subunit (CD122) on CD56bright NK cells at baseline predicted fewer new gadolinium-enhanced (Gd+) lesions during the treatment period (1.77 vs. 0.62 adjusted mean new Gd+ lesions during weeks 8–24, lowest vs. highest quartile of percentage CD122+ CD56bright NK cells; p = 0.033) and a greater increase in CD56bright NK cell counts at the end of DAC dosing (p = 0.029). Conclusion: CD56bright NK cell expansion after DAC treatment appears to reflect individual differences in the capacity for intermediate-affinity IL-2 signaling and could provide a basis for predicting clinical response to DAC in MS.

Thyroid disruption by Di-n-butyl phthalate (DBP) and mono-n-butyl phthalate (MBP) in Xenopus laevis.

BackgroundDi-n-butyl phthalate (DBP), a chemical widely used in many consumer products, is estrogenic and capable of producing seriously reproductive and developmental effects in laboratory animals. However, recent in vitro studies have shown that DBP and mono-n-butyl phthalate (MBP), the major metabolite of DBP, possessed thyroid hormone receptor (TR) antagonist activity. It is therefore important to consider DBP and MBP that may interfere with thyroid hormone system. Methodology/Principal Findings Nieuwkoop and Faber stage 51 Xenopus laevis were exposed to DBP and MBP (2, 10 or 15 mg/L) separately for 21 days. The two test chemicals decelerated spontaneous metamorphosis in X. laevis at concentrations of 10 and 15 mg/L. Moreover, MBP seemed to possess stronger activity. The effects of DBP and MBP on inducing changes of expression of selected thyroid hormone response genes: thyroid hormone receptor-beta (TRβ), retinoid X receptor gamma (RXRγ), alpha and beta subunits of thyroid-stimulating hormone (TSHα and TSHβ) were detected by qPCR at all concentrations of the compounds. Using mammalian two-hybrid assay in vitro, we found that DBP and MBP enhanced the interactions between co-repressor SMRT (silencing mediator for retinoid and thyroid hormone receptors) and TR in a dose-dependent manner, and MBP displayed more markedly. In addition, MBP at low concentrations (2 and 10 mg/L) caused aberrant methylation of TRβ in head tissue.ConclusionsThe current findings highlight potential disruption of thyroid signalling by DBP and MBP and provide data for human risk assessment.