Pre-B-cell transcription factor 1 and steroidogenic factor 1 synergistically regulate adrenocortical growth and steroidogenesis

variety of transcription factors including Wilms tumor gene (Wt-1), steroidogenic factor 1 (Sf-1), dosage-sensitive sex reversal, adrenal hypoplasia congenita on the X-chromosome, Gene 1 (Dax-1), and pre-B-cell transcription factor 1 (Pbx1) have been defined as necessary for regular adrenocortical development. However, the role of Pbx1 for adrenal growth and function in the adult organism together with the molecular relationship between Pbx1 and these other transcription factors have not been characterized. We demonstrate that Pbx haploinsufficiency (Pbx1(+/-)) in mice is accompanied by a significant lower adrenal weight in adult animals compared with wild-type controls. Accordingly, baseline proliferating cell nuclear antigen levels are lower in Pbx1(+/-) mice, and unilateral adrenalectomy results in impaired contralateral compensatory adrenal growth, indicating a lower proliferative potential in the context of Pbx1 haploinsufficiency. In accordance with the key role of IGFs in adrenocortical proliferation and development, real-time RT-PCR demonstrates significant lower expression levels of the IGF-I receptor, and up-regulation of IGF binding protein-2. Functionally, Pbx1(+/-) mice display a blunted corticosterone response after ACTH stimulation coincident with lower adrenal expression of the ACTH receptor (melanocortin 2 receptor, Mc2-r). Mechanistically, in vitro studies reveal that Pbx1 and Sf-1 synergistically stimulates Mc2-r promoter activity. Moreover, Sf-1 directly activates the Pbx1 promoter activity in vitro and in vivo. Taken together, these studies provide evidence for a role of Pbx1 in the maintenance of a functional adrenal cortex mediated by synergistic actions of Pbx1 and Sf-1 in the transcriptional regulation of the critical effector of adrenocortical differentiation, the ACTH receptor.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcR-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Riluzole elevates GLT-1 activity and levels in striatal astrocytes

Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and (3)H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1muM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100muM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1mM) did not regulate GLT-1. Finally, CDP-choline (10muM-1mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.

A gene variation (rs12691) in the CCAT/enhancer building α modulates glucose metabolism in metabolic syndrome

Background and aims CCAAT/enhancer-binding protein alpha (CEBPA) is a transcription factor involved in adipogenesis and energy homeostasis. Caloric restriction reduces CEBPA protein expression in patients with metabolic syndrome (MetS). A previous report linked rs12691 SNP in CEBPA to altered concentration of fasting triglycerides. Our objective was to assess the effects of rs12691 in glucose metabolism in Metabolic Syndrome (MetS) patients. Methods and results Glucose metabolism was assessed by static (glucose, insulin, adiponectin, leptin and resistin plasma concentrations) and dynamic (disposition index, insulin sensitivity index, HOMA-IR and acute insulin response to glucose) indices, performed at baseline and after 12 weeks of 4 dietary interventions (high saturated fatty acid (SFA), high monounsaturated fatty acid (MUFA), low-fat and low-fat-high-n3 polyunsaturated fatty acid (PUFA)) in 486 subjects with MetS. Carriers of the minor A allele of rs12691 had altered disposition index (p = 0.0003), lower acute insulin response (p = 0.005) and a lower insulin sensitivity index (p = 0.025) indicating a lower insulin sensitivity and a lower insulin secretion, at baseline and at the end of the diets. Furthermore, A allele carriers displayed lower HDL concentration. Conclusion The presence of the A allele of rs12691 influences glucose metabolism of MetS patients. Clinical Trials Registry number NCT00429195.

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.

Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

Intake of total and subgroups of fat minimally affect the associations between selected single nucleotide polymorphisms in the PPARγ pathway and changes in anthropometry among European adults from cohorts of the DiOGenes study

BACKGROUND: Although the peroxisome proliferator-activated receptor γ (PPARγ) pathway is central in adipogenesis, it remains unknown whether it influences change in body weight (BW) and whether dietary fat has a modifying effect on the association. OBJECTIVES: We examined whether 27 single nucleotide polymorphisms (SNPs) within 4 genes in the PPARγ pathway are associated with the OR of being a BW gainer or with annual changes in anthropometry and whether intake of total fat, monounsaturated fat, polyunsaturated fat, or saturated fat has a modifying effect on these associations. METHODS: A case-noncase study included 11,048 men and women from cohorts in the European Diet, Obesity and Genes study; 5552 were cases, defined as individuals with the greatest BW gain during follow-up, and 6548 were randomly selected, including 5496 noncases. We selected 4 genes [CCAAT/enhancer binding protein β (CEBPB), phosphoenolpyruvate carboxykinase 2, PPARγ gene (PPARG), and sterol regulatory element binding transcription factor 1] according to evidence about biologic plausibility for interactions with dietary fat in weight regulation. Diet was assessed at baseline, and anthropometry was followed for 7 y. RESULTS: The ORs for being a BW gainer for the 27 genetic variants ranged from 0.87 (95% CI: 0.79, 1.03) to 1.12 (95% CI: 0.96, 1.22) per additional minor allele. Uncorrected, CEBPB rs4253449 had a significant interaction with the intake of total fat and subgroups of fat. The OR for being a BW gainer for each additional rs4253449 minor allele per 100 kcal higher total fat intake was 1.07 (95% CI: 1.02, 1.12; P = 0.008), and similar associations were found for subgroups of fat. CONCLUSIONS: Among European men and women, the influence of dietary fat on associations between SNPs in the PPARγ pathway and anthropometry is likely to be absent or marginal. The observed interaction between rs4253449 and dietary fat needs confirmation.

Supranutritional selenium induces alterations in molecular targets related to energy metabolism in skeletal muscle and visceral adipose tissue of pigs

While selenium (Se) is an essential micronutrient for humans, epidemiological studies have raised concern that supranutritional Se intake may increase the risk to develop Type 2 diabetes mellitus (T2DM). We aimed to determine the impact of Se at a dose and source frequently ingested by humans on markers of insulin sensitivity and signalling. Male pigs were fed either a Se-adequate (0.17 mg Se/kg) or a Se-supranutritional (0.50 mg Se/kg; high-Se) diet. After 16 weeks of intervention, fasting plasma insulin and cholesterol levels were non-significantly increased in the high-Se pigs, whereas fasting glucose concentrations did not differ between the two groups. In skeletal muscle of high-Se pigs, glutathione peroxidase activity was increased, gene expression of forkhead box O1 transcription factor and peroxisomal proliferator-activated receptor- coactivator 1 were increased and gene expression of the glycolytic enzyme pyruvate kinase was decreased. In visceral adipose tissue of high-Se pigs, mRNA levels of sterol regulatory element-binding transcription factor 1 were increased, and the phosphorylation of Akt, AMP-activated kinase and mitogen-activated protein kinases was affected. In conclusion, dietary Se oversupply may affect expression and activity of proteins involved in energy metabolism in major insulin target tissues, though this is probably not sufficient to induce diabetes.

Ethylene and 1-methylcyclopropene differentially regulate gene expression during onion sprout suppression

Onion (Allium cepa) is regarded as a nonclimacteric vegetable. In onions, however, ethylene can suppress sprouting while the ethylene-binding inhibitor 1-methylcyclopropene (1-MCP) can also suppress sprout growth; yet, it is unknown how ethylene and 1-MCP elicit the same response. In this study, onions were treated with 10 mu L L(-1) ethylene or 1 mu L L(-1) 1-MCP individually or in combination for 24 h at 20 degrees C before or after curing (6 weeks) at 20 degrees C or 28 degrees C and then stored at 1 degrees C. Following curing, a subset of these same onions was stored separately under continuous air or ethylene (10 mu L L(-1)) at 1 degrees C. Onions treated with ethylene and 1-MCP in combination after curing for 24 h had reduced sprout growth as compared with the control 25 weeks after harvest. Sprout growth following storage beyond 25 weeks was only reduced through continuous ethylene treatment. This observation was supported by a higher proportion of down-regulated genes characterized as being involved in photosynthesis, measured using a newly developed onion microarray. Physiological and biochemical data suggested that ethylene was being perceived in the presence of 1-MCP, since sprout growth was reduced in onions treated with 1-MCP and ethylene applied in combination but not when applied individually. A cluster of probes representing transcripts up-regulated by 1-MCP alone but down-regulated by ethylene alone or in the presence of 1-MCP support this suggestion. Ethylene and 1-MCP both down-regulated a probe tentatively annotated as an ethylene receptor as well as ethylene-insensitive 3, suggesting that both treatments down-regulate the perception and signaling events of ethylene.

Relationship between milk consumption and prostate cancer: a short review

Epidemiological evidence based on both case–control and prospective cohort studies points to an overall positive relationship between consumption of milk/dairy products and the risk of developing prostate cancer. There are inconsistencies in the data, but taken together, the increased relative risk does not seem to be high. A number of mechanisms have been proposed to account for the relationship, with most attention being focused on the involvement of calcium/vitamin D, insulin-like growth factor-1 and oestrogens, although it is unlikely that a single factor in milk is implicated. In any event, any added risk of prostate cancer from increased milk consumption has to be set alongside other evidence, which shows that increased milk consumption can provide substantially reduced risk of coronary heart disease, stroke and colorectal cancer, particularly because cardiovascular disease accounts for vastly more deaths than prostate cancer (although the latter is of course restricted to men).

A general synthesis of macrocyclic pi-electron-acceptor systems

Cyclocondensations of aromatic diamines with 1,1'-bis(2,4-dinitrophenyl)-4,4'-bipyridinium salts afford doubly or quadruply charged, macrocyclic, N,N'-diarylbipyridinium cations. These are tolerant of a wide range of acids, bases, and nucleophiles, although they appear to undergo reversible, one-electron reduction by tertiary amines. Single-crystal X-ray analysis demonstrates the presence of a macrocycle conformation in which the 4,4'-bipyridinium and 4,4'-biphenylenedisulfonyl residues are suitably spaced and aligned for complexation with pi-donor arenes, and NMR studies in solution indeed confirm binding to 1,5-bis[hydroxy(ethoxy)ethoxy]naphthalene.

Attention through self-synchronisation in the spiking neuron stochastic diffusion network

The paper discusses ensemble behaviour in the Spiking Neuron Stochastic Diffusion Network, SNSDN, a novel network exploring biologically plausible information processing based on higher order temporal coding. SNSDN was proposed as an alternative solution to the binding problem [1]. SNSDN operation resembles Stochastic Diffusin on Search, SDS, a non-deterministic search algorithm able to rapidly locate the best instantiation of a target pattern within a noisy search space ([3], [5]). In SNSDN, relevant information is encoded in the length of interspike intervals. Although every neuron operates in its own time, ‘attention’ to a pattern in the search space results in self-synchronised activity of a large population of neurons. When multiple patterns are present in the search space, ‘switching of at- tention’ results in a change of the synchronous activity. The qualitative effect of attention on the synchronicity of spiking behaviour in both time and frequency domain will be discussed.

Anti-proliferative effects of physiological concentrations of enterolactone in models of prostate tumourigenesis

SCOPE: There is evidence that a mammalian lignan, enterolactone (ENL), decreases the proliferation rate of prostate cancer cells, although previous studies have used concentrations difficult to achieve through dietary modification. We have therefore investigated the anti-proliferative effects of ENL in an in vitro model of prostate tumourigenesis at concentrations reported to occur in a range of male populations. METHODS AND RESULTS: The effects of 0.1 and 1 μM ENL on three markers of viability and proliferation (metabolic activity, growth kinetics, and cell cycle progression) were assessed in the RWPE-1, WPE1-NA22, WPE1-NB14, WPE1-NB11, WPE1-NB26, LNCaP, and PC-3 cell lines over 72 h. Based on these data, we quantified the expression levels of 12 genes involved in the control of DNA replication initiation using TaqMan real-time PCR in the WPE1-NA22, WPE1-NB14, WPE1-NB11, and WPE1-NB26 cell lines. ENL significantly inhibited the abnormal proliferation of the WPE1-NB14 and WPE1-NB11 cell lines and appears to be a consequence of decreased expression of abnormal chromatin licensing and DNA replication factor 1. CONCLUSION: In contrast to previous studies, concentrations of ENL that are reported after dietary intervention restrict the proliferation of early-stage tumourigenic prostate cell lines by inhibiting the abnormal formation of complexes that initiate DNA replication.

A functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA and secreted protein level (75 and 94%, respectively) and elicited a 77% reduction in CYP17A1 mRNA level and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 mRNA level (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ~2-fold. The CYP17 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling.

Association of low adiponectin levels with the metabolic syndrome--the Chennai Urban Rural Epidemiology Study (CURES-4)

The aim of the study was to assess the relation of adiponectin levels with the metabolic syndrome in Asian Indians, a high-risk group for diabetes and premature coronary artery disease. The study was conducted on 100 (50 men and 50 women) type 2 diabetic subjects and 100 age and sex matched subjects with normal glucose tolerance selected from the Chennai Urban Rural Epidemiology Study, an ongoing population study in Chennai in southern India. Metabolic syndrome was defined using modified Adult Treatment Panel III (ATPIII) guidelines. Adiponectin values were significantly lower in diabetic subjects (men: 5.2 vs 8.3 microg/mL, P=.00l; women: 7.6 vs 11.1 microg/mL, P<.00l) and those with the metabolic syndrome (men: 5.0 vs 6.8 microg/mL, P=.01; women: 6.5 vs 9.9 microg/mL, P=.001) compared with those without. Linear regression analysis revealed adiponectin to be associated with body mass index (P<.05), waist circumference (P<.01), fasting plasma glucose (P=.001), glycated hemoglobin (P<.001), triglycerides (P<.00l), high-density lipoprotein (HDL) cholesterol (P<.001), cholesterol/HDL ratio (P<.00l), and insulin resistance measured by homeostasis assessment model (P<.00l). Factor analysis identified 2 factors: factor 1, negatively loaded with adiponectin and HDL cholesterol and positively loaded with triglycerides, waist circumference, and insulin resistance measured by homeostasis assessment model; and factor 2, with a positive loading of waist circumference and systolic and diastolic blood pressure. Logistic regression analysis revealed adiponectin to be negatively associated with metabolic syndrome (odds ratio [OR], 0.365; P<.001) even after adjusting for age (OR, 0.344; P<.00l), sex (OR, 0.293; P<.001), and body mass index (OR, 0.292; P<.00l). Lower adiponectin levels are associated with the metabolic syndrome per se and several of its components, particularly, diabetes, insulin resistance, and dyslipidemia in this urban south Indian population.

Interaction between vitamin D receptor gene polymorphisms and 25-hydroxyvitamin D concentrations on metabolic and cardiovascular disease outcomes

AIM: 25-hydroxyvitamin D (25OHD) concentrations have been shown to be associated with major clinical outcomes, with a suggestion that individual risk may vary according to common genetic differences in the vitamin D receptor (VDR) gene. Hence, we tested for the interactions between two previously studied VDR polymorphisms and 25OHD on metabolic and cardiovascular disease-related outcomes in a large population-based study. METHODS: Interactions between two previously studied VDR polymorphisms (rs7968585 and rs2239179) and 25OHD concentrations on metabolic and cardiovascular disease-related outcomes such as obesity- (body mass index, waist circumference, waist-hip ratio (WHR)), cardiovascular- (systolic and diastolic blood pressure), lipid- (high- and low-density lipoprotein, triglycerides, total cholesterol), inflammatory- (C-reactive protein, fibrinogen, insulin growth factor-1, tissue plasminogen activator) and diabetes- (glycated haemoglobin) related markers were examined in the 1958 British Birth cohort (n up to 5160). Interactions between each SNP and 25OHD concentrations were assessed using linear regression and the likelihood ratio test. RESULTS: After Bonferroni correction, none of the interactions reached statistical significance except for the interaction between the VDR SNP rs2239179 and 25OHD concentrations on waist-hip ratio (WHR) (P=0.03). For every 1nmol/L higher 25OHD concentrations, the association with WHR was stronger among those with two major alleles (-4.0%, P=6.26e-24) compared to those with either one or no major alleles (-2.3%, P≤8.201e-07, for both) of the VDR SNP rs2239179. CONCLUSION: We found no evidence for VDR polymorphisms acting as major modifiers of the association between 25OHD concentrations and cardio-metabolic risk. Interaction between VDR SNP rs2239179 and 25OHD on WHR warrants further confirmation.