Replication, neogenesis, and apoptosis play a main role in neonatal endocrine pancreas remodeling. IGFs are major contributors to β-cell growth and function and are highly sensitive to nutritional status. We previously showed that maternal malnutrition caused an increase in β-cell mass in fetuses related to the stimulation of β-cell proliferation due to increased pancreatic IGF-1. At 4 days of life, the β-cell mass was decreased in undernourished neonates and persisted until adult age. To clarify whether undernutrition disrupts islet remodeling, we quantified β-cell mass, neogenesis, replication, and apoptosis on days 4, 14, and 23. To determine the impact of food restriction on IGF ontogeny and the consequences for β-cell growth, we measured IGF-1/-2 protein content in pancreas and liver and pancreatic IGF-1 receptor (IGF-1R)-signaling pathway at the same days. Our results indicate that undernutrition alters the timing and intensity of neonatal β-cell ontogeny. However, although malnutrition causes β-cell deficiency in neonates, an active process of β-cell neogenesis and a lower incidence of β-cell apoptosis maintain the regenerative capacity of the endocrine pancreas. Interestingly, our data provide evidence that local production of IGFs seems to be instrumental in these processes. In particular, increased pancreatic IGF-2 in undernourished rats may contribute to the partial suppression of the developmental wave of β-cell apoptosis probably through the inhibition of glycogen synthase kinase-3. In addition, decreased pancreatic levels of IGFBP-1/-2/-3 in undernourished neonates could enhance IGF availability for interacting with IGF-1R/IR.
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Laura de Miguel-Santos, Elisa Fernández-Millán, María Ángeles Martín, Fernando Escrivá, and Carmen Álvarez
Yukinori Kazeto, Rie Goto-Kazeto, Peter Thomas, and John M Trant
Membrane-bound progestin receptors (mPR) were recently cloned and characterized as a new class of steroid receptors that transduce cell-signals through alteration of MAP kinase- and cAMP-dependent pathways. To further develop our understanding of this new class of steroid receptors, we characterized the cDNAs and genes of theα, β and γ forms of the channel catfish mPRs (IpmPR). The predicted α and β proteins have 49% sequence identity, whereas they only have 30% and 27% identities, respectively, with the γ form. Furthermore, IpmPRα and IpmPRβ genes have similar structures featuring intronless coding regions, while IpmPRγ gene is composed of 8 exons and 7 introns. The 5′-flanking region of each IpmPR gene differs, but each contains putative transcriptional regulatory elements of factors known to influence reproductive physiology and endocrine disruption, for example, responsive elements for cAMP and steroids and the recognition sites for steroidogenic factor-1 and for the aryl hydrocarbon receptor. The IpmPRα gene was detected in all the tissues tested with relatively greater expression in brain, pituitary, muscle and testis. The expression of IpmPRβ was much lower than that of IpmPRα and the transcript was predominantly observed in brain, pituitary, ovary and testis. In contrast, the IpmPRγ transcript was mainly detected in gill, ventral aorta, intestine, and trunk kidney. In conclusion, all the structural features of the IpmPRs strongly suggest that the closely related α and β forms are distantly related to the γ form. Additionally, regulatory features of the 5′-flanking regions and the differences in tissue-specific expression of each IpmPR gene suggest that they are involved in different endocrine functions in catfish.
HM Sheppard, S Matsuda, JC Harries, KB Kindle, and DM Heery
Steroid receptors activate transcription in yeast cells via interactions with endogenous coactivators and/or basal factors. We examined the effects of mutations in the ligand binding domain on the transcriptional activity of ERalpha in yeast. Our results show that mutations in Helix 3 (K366A) and Helix 12 (M547A, L548A) disrupt transcriptional activity of ERalpha in yeast, as previously observed in mammalian cells. However, replacement of a conserved tyrosine residue in Helix 12 with alanine or aspartate (Y541A and Y541D), which renders ERalpha constitutively active in mammalian cells, had only a weak stimulatory effect on ligand-independent reporter activation by ERalpha in yeast. Two-hybrid interaction experiments revealed that a Y541A mutant expressed in yeast was capable of ligand-independent binding to a mammalian coactivator, suggesting that there is a subtle difference in how this mutant interacts with mammalian and yeast cofactors. We also show that the ligand-dependent activities of ERalpha and progesterone receptor (PR) in yeast cells were strongly enhanced by the human p160 protein steroid receptor coactivator (SRC1), but not by CREB-Binding Protein (CBP) or the p300/CBP associated factor (P/CAF). Although the SRC1 activation domains AD1 and AD2 are functional in yeast, deletion of these sequences only partially impaired SRC1 coactivator function in this organism; this is in contrast to similar experiments in mammalian cells. Thus SRC1 sequences involved in recruitment of CBP/p300 and Co-Activator-Associated Arginine Methyltransferase (CARM-1) in mammalian cells are not essential for its function in yeast, suggesting that SRC1 operates via distinct mechanisms in yeast and mammalian cells.
Amy L Filby, Karen L Thorpe, and Charles R Tyler
Complex interrelationships in the signalling of oestrogenic effects mean that environmental oestrogens present in the aquatic environment have the potential to disrupt physiological function in fish in a more complex manner than portrayed in the present literature. Taking a broader approach to investigate the possible effect pathways and the likely consequences of environmental oestrogen exposure in fish, the effects of 17β-oestradiol (E2) were studied on the expression of a suite of genes which interact to mediate growth, development and thyroid and interrenal function (growth hormone GH (gh), GH receptor (ghr ), insulin-like growth factor (IGF-I) (igf1), IGF-I receptor (igf1r ), thyroid hormone receptors-α (thra) and -β (thrb) and glucocorticoid receptor (gr )) together with the expression analyses of sex-steroid receptors and ten other genes centrally involved in sexual development and reproduction in fathead minnow (fhm; Pimephales promelas). Exposure of adult fhm to 35 ng E2/l for 14 days induced classic oestrogen biomarker responses (hepatic oestrogen receptor 1 and plasma vitellogenin), and impacted on the reproductive axis, feminising ‘male’ steroidogenic enzyme expression profiles and suppressing genes involved in testis differentiation. However, E2 also triggered a cascade of responses for gh, ghr, igf1, igf1r, thra, thrb and gr in the pituitary, brain, liver, gonad and gill, with potential consequences for the functioning of many physiological processes, not just reproduction. Molecular responses to E2 were complex, with most genes showing differential responses between tissues and sexes. For example, igf1 expression increased in brain but decreased in gill on exposure to E2, and responded in an opposite way in males compared with females in liver, gonad and pituitary. These findings demonstrate the importance of developing a deeper understanding of the endocrine interactions for unravelling the mechanisms of environmental oestrogen action and predicting the likely health consequences.
R. M. Haigh and C. T. Jones
Glucocorticoids are known to have marked effects on blood pressure regulation, predominantly through altering cardiovascular sensitivity to noradrenaline. However, the molecular mechanisms underlying this action remain unclear. As part of our studies into these we have measured α1-adrenergic receptor binding using the ligand [3H]prazosin in plasma membrane fractions of aortas prepared from control, adrenalectomized and dexamethasone-treated adrenalectomized rats. In controls there were 50±8 (s.e.m.; n=6) fmol α1-adrenergic receptors/mg membrane protein (Bmax) with a dissociation constant (K d) of 0·52±0·10 nm (n=6). Adrenalectomy 8 days before tissue preparation caused a 40% decrease in Bmax and a 60% decrease in K d. Dexamethasone replacement after adrenalectomy returned these values close to those of controls. Noradrenaline competed for the [3H]prazosin-binding sites. Computer analysis by a non-linear curve-fitting program (LIGAND) showed that noradrenaline binding was to a heterogeneous population of high- and low-affinity receptors with K d values of 1·87±0·73 μm and 0·48±0·12 mm (n=5) respectively. Guanosine thiotriphosphate (GTP[S]) caused the conversion of high-affinity to low-affinity binding, consistent with the model of the high-affinity sites being coupled to a G protein. After adrenalectomy, noradrenaline binding was to a homogeneous population of low-affinity receptors; hence, the effect of GTP[S] was no longer apparent, suggesting that under these conditions the α1-adrenergic receptors were unable to couple to a G protein. The two-site model of binding and GTP[S] effect was returned by dexamethasone treatment. These data provide evidence that glucocorticoids not only modulate the number of α1-adrenergic receptors on vascular smooth muscle, but also cause disruptions in receptor—G protein coupling. This may be an important mechanism by which glucocorticoids exert their effect on cardiovascular sensitivity.
Dominique H Eghlidi, Selva L Luna, Donald I Brown, Vasilios T Garyfallou, Steven G Kohama, and Henryk F Urbanski
In mammals, the suprachiasmatic nucleus (SCN) is the location of a master circadian pacemaker. It receives photic signals from the environment via the retinal hypothalamic tract, which play a key role in synchronizing the body’s endogenously generated circadian rhythms with the 24-h rhythm of the environment. Therefore, it is plausible that age-related changes within the SCN contribute to the etiology of perturbed activity–rest cycles that become prevalent in humans during aging. To test this hypothesis, we used gene arrays and quantitative RT-PCR to profile age-related gene expression changes within the SCN of male rhesus macaques – a pragmatic translational animal model of human aging, which similarly displays an age-related attenuation of daytime activity levels. As expected, the SCN showed high expression of arginine vasopressin, vasoactive intestinal polypeptide, calbindin and nuclear receptor subfamily 1, group D, member 1 (NR1D1) (also known as reverse strand of ERBA (REV-ERBα), both at the mRNA and protein level. However, no obvious difference was detected between the SCNs of young (7–12 years) and old animals (21–26 years), in terms of the expression of core clock genes or genes associated with SCN signaling and neurotransmission. These data demonstrate the resilience of the primate SCN to normal aging, at least at the transcriptional level and, at least in males, suggest that age-related disruption of activity–rest cycles in humans may instead stem from changes within other components of the circadian system, such as desynchronization of subordinate oscillators in other parts of the body.
Colin R Jefcoate and Jinwoo Lee
Cholesterol is an important regulator of cell signaling, both through direct impacts on cell membranes and through oxy-metabolites that activate specific receptors (steroids, hydroxy-cholesterols, bile acids). Cholesterol moves slowly through and between cell membranes with the assistance of specific binding proteins and transfer processes. The prototype cholesterol regulator is the Steroidogenesis Acute Regulatory (STAR), which moves cholesterol into mitochondria, where steroid synthesis is initiated by cytochrome P450 11A1 in multiple endocrine cell types. CYP27A1 generates hydroxyl cholesterol metabolites that activate LXR nuclear receptors to control cholesterol homeostatic and transport mechanisms. LXR regulation of cholesterol transport and storage as cholesterol ester droplets is shared by both steroid-producing cells and macrophage. This cholesterol signaling which is crucial to brain neuron regulation by astrocytes and microglial macrophage, is mediated by ApoE and is sensitive to disruption by β-amyloid plaques. sm-FISH delivers appreciable insights into signaling in single cells, by resolving single RNA molecules as mRNA and by quantifying pre-mRNA at gene loci. sm-FISH has been applied to problems in physiology, embryo development and cancer biology, where single cell features have critical impacts. sm-FISH identifies novel features of STAR transcription in adrenal and testis cells, including asymmetric expression at individual gene loci, delayed splicing and 1:1 association of mRNA with mitochondria. This may represent a functional unit for the translation-dependent cholesterol transfer directed by STAR, which integrates into mitochondrial fusion dynamics. Similar cholesterol dynamics repeat with different players in the cycling of cholesterol between astrocytes and neurons in the brain, which may be abnormal in neurodegenerative diseases.
Belen Brie, Ana Ornstein, Maria Cecilia Ramirez, Isabel Lacau-Mengido, and Damasia Becu-Villalobos
Many sex differences in liver gene expression originate in the brain, depend on GH secretion and may underlie sex disparities in hepatic disease. Because epigenetic mechanisms may contribute, we studied promoter methylation and microRNA abundance in the liver, associated with expression of sexual dimorphic genes in mice with selective disruption of the dopamine D2 receptor in neurons (neuroDrd2KO), which decreases hypothalamic Ghrh, pituitary GH, and serum IGFI and in neonatally androgenized female mice which have increased pituitary GH content and serum IGFI. We evaluated mRNA levels of the female predominant genes prolactin receptor (Prlr), alcohol dehydrogenase 1 (Adh1), Cyp2a4, and hepatocyte nuclear transcription factor 6 (Hnf6) and the male predominant gene, Cyp7b1. Female predominant genes had higher mRNA levels compared to males, but lower methylation was only detected in the Prlr and Cyp2a4 female promoters. In neuroDrd2KO mice, sexual dimorphism was lost for all genes; the upregulation (feminization) of Prlr and Cyp2a4 in males correlated with decreased methylation of their promoters, and the downregulation (masculinization) of Hnf-6 mRNA in females correlated inversely with its promoter methylation. Neonatal androgenization of females evoked a loss of sexual dimorphism only for the female predominant Hnf6 and Adh1 genes, but no differences in promoter methylation were found. Finally, mmu-miR-155-5p, predicted to target Cyp7b1 expression, was lower in males in association with higher Cyp7b1 mRNA levels compared to females and was not modified in neuroDrd2KO or TP mice. Our results suggest specific regulation of gene sexually dimorphic expression in the liver by methylation or miRNAs.
M A Mirando, J P Harney, Y Zhou, T F Ogle, T L Ott, R J Moffatt, and F W Bazer
This study determined whether twice-daily intrauterine injections of ovine conceptus secretory proteins (oCSP) containing type-I trophoblast interferon (25 μg/uterine horn) from day 11 to day 15 post-oestrus (oestrus = day 0) could alter the binding capacities of endometrial receptors for oxytocin, progesterone and oestrogen in cyclic ewes when compared with control ewes receiving serum protein (SP) injections. Injections of oCSP on days 11–15 post-oestrus decreased concentrations of oestrogen receptors (P<0·06), oestrogen receptor mRNA (P<0·05) and progesterone receptors (P<0·08) in endometrium on day 16 when compared with SP-infused control ewes, which were undergoing corpus luteum regression on days 14–16. Injection of oCSP also decreased the number (P<0·10) and affinity (P<0·06) of oxytocin receptors. Inositol phosphate formation induced in the endometrium on day 16 by 100 nm oxytocin in vitro was highly correlated with the concentration (r≥0·93, P<0·001) and K d (r= –0·91, P<0·01) of oxytocin receptors in SP-infused ewes, but was not as highly correlated with concentration (r≤0·83, P<0·06) and K d (r≤ –0·40, P>0·40) of oxytocin receptors in oCSP-infused ewes. This indicates that oCSP disrupted the relationship between oxytocin receptor binding and oxytocin-induced activation of its second messenger system. These results indicate that antiluteolytic type-I trophoblast interferon may prevent oxytocininduced luteolytic pulsatile secretion of prostaglandin F2α during maternal recognition of pregnancy in sheep, by reducing the synthesis and affinity of endometrial oxytocin receptors. Inhibition of other components of the oxytocin receptor—phospholipase C system by ovine trophoblast interferon may also be involved in reducing endometrial responsiveness to oxytocin. Ovine trophoblast interferon may inhibit the synthesis of endometrial oestrogen receptors to inhibit responsiveness to oxytocin during early pregnancy in ewes.
Zhor Bouizar, Bruno Ragazzon, Lucie Viou, Mariuccia Hortane, Jerôme Bertherat, and Marthe Rizk-Rabin
Various types of protein kinase A (PKA) alterations have been observed in adrenocortical tumours and Carney complex (CNC). PKA is a heterotetramer of two regulatory and two catalytic subunits. The R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. A decrease in R2B protein levels has been observed in adrenal adenoma, whereas tumours from patients with CNC display a decrease in R1A protein levels. Dysregulation of the balance between R1A and R2B may thus be involved in adrenal tumourigenesis. We investigated the impact of the differences in the balance of PKA subunits on cell growth using specific cAMP analogues. We assessed the effects of 8-chloroadenosine-cAMP (8Cl-cAMP), a site-selective activator of PKA R2B, in H295R adrenocortical cells. 8Cl-cAMP stimulated PKA activity, decreased R1A levels and increased R2B levels. It had no cytotoxic effects, initially stimulating DNA synthesis and then inducing apoptosis by disrupting G2/M progression. We observed an initial accumulation of cells in the S phase, translocation of cyclin A to the nucleus, CDK2 activation, sustained DNA synthesis and proliferating cell nuclear antigen accumulation. Cell cycle arrest in the G2 phase was parallel with the accumulation of cyclin B and the inactivation of CDC2 kinase. The 8CPT-cAMP, which activates the R2B subunit, had similar effects. R2B silencing reduced the apoptosis induced by tumour necrosis factor α and transforming growth factor β. Thus, R2B is a key regulator of proliferation/differentiation in H295R cell line along with the complex balance between the PKA subunits. Activation of PKA R2B and dysregulation of the R1A/R2B balance regulate cell cycle progression and apoptosis in adrenocortical cells by modulating cyclin production and cyclin-dependent kinase activities.