Regulation of the iron regulatory proteins by reactive nitrogen and oxygen species.

Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are RNA binding proteins that posttranscriptionally regulate the expression of mRNAs coding for proteins involved in the maintenance of iron and energy homeostasis. The RNA binding activities of the IRPs are regulated by changes in cellular iron. Thus, the IRPs are considered iron sensors and the principle regulators of cellular iron homeostasis. The mechanisms governing iron regulation of the IRPs are well described. Recently, however, much attention has focused on the regulation of IRPs by reactive nitrogen and oxygen species (RNS, ROS). Here we focus on summarizing the iron-regulated RNA binding activities of the IRPs, as well as the recent findings of IRP regulation by RNS and ROS. The recent observations that changes in oxygen tension regulate both IRP1 and IRP2 RNA binding activities will be addressed in light of ROS regulation of the IRPs.

Sequence-independent assembly of spermatid mRNAs into messenger ribonucleoprotein particles.

During mammalian spermatogenesis, meiosis is followed by a brief period of high transcriptional activity. At this time a large amount of mRNA is stored as messenger ribonucleoprotein (mRNP) particles. All subsequent processes of sperm maturation occur in the complete absence of transcription, primarily using proteins which are newly synthesized from these stored mRNAs. By expressing transgene mRNAs in the early haploid spermatids of mice, we have investigated the sequence requirements for determining whether specific mRNAs in these cells will be stored as mRNP particles or be assembled into polysomes. The results suggest that mRNAs which are transcribed in spermatids are assembled into mRNP particles by a mechanism that acts independently of mRNA sequence. Our findings reveal a fundamental similarity between the mechanisms of translational control used in spermatogenesis and oogenesis.

Hypoxia post-translationally activates iron-regulatory protein 2.

Iron-regulatory proteins 1 and 2 (IRP1 and IRP2) are RNA-binding proteins that post-transcriptionally regulate the expression of mRNAs that code for proteins involved in the maintenance of iron and energy homeostasis. Here we show that hypoxia differentially regulates the RNA binding activities of IRP1 and IRP2 in human 293 and in mouse Hepa-1 cells. In contrast to IRP1, where hypoxic exposure decreases IRP1 RNA binding activity, hypoxia increases IRP2 RNA binding activity. The hypoxic increase in IRP2 RNA binding activity results from increased IRP2 protein levels. Cobalt, which mimics hypoxia by activation of hypoxia-inducible factor 1 (HIF-1), also increases IRP2 protein levels; however, cobalt-induced IRP2 lacks RNA binding activity. Addition of a reductant to cobalt-treated extracts restored IRP2 RNA binding activity. Hypoxic activation of IRP2 is not because of an increase in transcriptional activation by HIF-1, because IRP2 accumulates in Hepa-1 cells lacking a functional HIF-1beta subunit, nor is it because of an increase in IRP2 mRNA stability. Rather, our data indicate that hypoxia increases IRP2 levels by a post-translational mechanism involving protein stability. Differential regulation of IRP1 and IRP2 during hypoxia may regulate specific IRP target mRNAs whose expression is required for hypoxic adaptation. Furthermore, these data imply mechanistic parallels between the hypoxia-induced post-transcriptional regulation of IRP2 and HIF-1alpha.

Interactions among chronic cold, corticosterone and puberty on energy intake and deposition.

We have shown that chronic cold stress strongly interacts with corticosterone (B) to determine subsequent regulation of the hypothalamo-pituitary-adrenal (HPA) axis responses to novel stress. These studies, using the same 2 sets of rats, show that chronic cold also interacts with B and testosterone on signals of energy balance. The two groups of rats differed in weight by 20% and in age by 2 weeks (44-59 days of age). Adrenalectomized rats, replaced with varying doses of B, were exposed to cold or served as controls. Food intake and body weight during the experiments and hormones, metabolites and fat depots were measured on day 5. B, but not cold, affected food intake in the younger rats; by contrast, cold, but not B, affected food intake in the older rats. Testosterone was higher in older control rats and was markedly depressed by cold; younger rats had lower testosterone that was minimally affected by cold. Weight gain decreased in all rats at room temperature with increasing B, whereas they all lost weight in cold independently of B. Cold stimulated and B inhibited interscapular brown adipose tissue DNA content (reflecting sympathetic stimulation of thermogenesis). B stimulated insulin, whereas cold inhibited leptin and insulin; B also increased white adipose tissue weight gain in controls and inhibited its loss in cold. Leptin was unrelated to white adipose tissue depots in older control rats but was strongly related to these stores in younger rats and in all rats in cold. We conclude that: 1. By decreasing signals that act centrally to inhibit food intake (insulin, leptin and testosterone) cold allows B to stimulate food intake; 2. B inhibits weight gain although it causes accrual of fat; 3. Cold, probably through sympathetic stimulation of white adipose tissue, causes fat loss which is modulated by the inhibitory effect of B on sympathetic outflow; and, 4. The slope of the relationship between fat depot size and leptin becomes flatter in cold, possibly because of increased sympathetic outflow to these depots.

Regulation of iron regulatory protein 1 during hypoxia and hypoxia/reoxygenation.

Given the important relationship between O2 and iron (Fenton chemistry) a study was undertaken to characterize the effects of hypoxia, as well as subsequent reoxygenation, on the iron-regulatory proteins 1 and 2 (IRP1 and IRP2) in a rat hepatoma cell line. IRP1 and IRP2 are cytosolic RNA-binding proteins that bind RNA stem-loops located in the 5'- or 3'-untranslated regions of specific mRNAs encoding proteins that are involved in iron homeostasis. In cells exposed to hypoxia, IRP1 RNA binding was decreased approximately 2. 8-fold after a 6-h exposure to 3% O2. Hypoxic inactivation of IRP1 was abolished when cells were pretreated with the iron chelator desferrioxamine, indicating a role for iron in inactivation. IRP1 inactivation was reversible since re-exposure of hypoxically-treated cells to 21% O2 increased RNA binding activity approximately 7-fold after 21 h with an increase in activity seen as early as 1-h post-reoxygenation. IRP1 protein levels were unaffected during hypoxia as well as during reoxygenation. Whereas the protein synthesis inhibitor cycloheximide did not block IRP1 inactivation during hypoxia, it completely blocked IRP1 reactivation during subsequent reoxygenation. Reactivation of IRP1 during reoxygenation was also partially blocked by the phosphatase inhibitor okadaic acid. Finally, reactivated IRP1 was found to be resistant to inactivation by exogenous iron known to down-regulate its activity during normoxia. These data demonstrate that IRP1 RNA binding activity is post-translationally regulated during hypoxia and hypoxia/reoxygenation. Regulation of IRP1 by changing oxygen tension may provide a novel mechanism for post-transcriptionally regulating gene expression under these stresses.

Elevated corticosterone is not required for the rapid induction of neuropeptide Y gene expression by an overnight fast.

Fasting stimulates corticosterone (B) secretion and the expression and secretion of hypothalamic neuropeptide Y in rats. These studies tested the hypothesis that the rapid and marked fasting-induced increases in plasma B are responsible for stimulation of neuropeptide Y (NPY) gene expression. Plasma leptin and insulin were measured because they are also signals known to affect NPY messenger RNA (mRNA). Intact or adrenalectomized rats given a low fixed level of corticosterone (B replaced) were fasted for 48 h. NPY mRNA in the mediobasal hypothalamus, measured by nuclease protection assay, was elevated similarly above ad lib-fed controls in both intact and B replaced groups at 15 and 48 h after the onset of fasting. NPY immunoreactivity in the mediobasal hypothalamus increased between 3 and 48 h after onset of the fast in intact but not in B replaced groups. The fasting-induced decreases in leptin observed in intact rats at 48 h did not occur in B replaced rats. Fasting-induced decreases in insulin occurred in B replaced rats but not in intact rats. We conclude that: 1) elevated B is not required for fasting-induced increases in hypothalamic NPY gene expression; and 2) decreases in neither leptin nor insulin alone signal the changes that occur in NPY mRNA in fasted rats.

T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex.

After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase. At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized. Several specific protein-protein contacts within the complex are described in this paper. We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini.

The neural network that regulates energy balance is responsive to glucocorticoids and insulin and also regulates HPA axis responsivity at a site proximal to CRF neurons.

The structure of a large neural system that responds to and regulates energy balance and that encompasses that PVN and activity of the HPA axis has begun to emerge from these experiments (Fig. 6). Several large loops have been delineated within this context of the maintenance of energy balance. Corticosteroids stimulate both feeding and insulin secretion. The actions of corticosteroids in the periphery are catabolic, causing mobilization of energy stores; their actions in the central nervous system are stimulatory to energy acquisition (food intake). By contrast, the action of insulin in the periphery is anabolic, causing energy storage; its action in the central nervous system is inhibitory to energy acquisition (food intake). At the level of the CNS, insulin inhibits and corticosteroids stimulate expression of NPY mRNA in the arcuate nuclei, and these actions may explain, in part, the reciprocal actions of the hormones on energy acquisition. Thus over the long term, stimulation of insulin secretion by corticosteroids tends to supply an automatic brake on the effects of corticosteroids on feeding. The neural system that controls energy balance and responds to the reciprocal signals of corticosterone and insulin also regulates responsivity to restraint stress in the HPA axis. The low-amplitude ACTH responses to restraint, corticosteroid feedback, and prior stress-induced facilitation that are observed under conditions of relative fasting in the PM can be produced in the AM by a 14-h, overnight fast. By contrast, NPY injected ivt stimulates identical ACTH responses in the AM in fed rats and in rats fasted overnight, suggesting that NPY acts to stimulate CRF secretion at a site closer to the PVN than the stress of restraint, which is filtered through the neural energy balance system. In the periphery, corticosteroids and insulin also have reciprocal effects on energy storage; effects that are opposite those exerted in the CNS on energy acquisition. Thus, together, the two hormones may be construed as a bihormonal system that regulates overall energy balance. Although under normal conditions this system is well designed to accomplish energy balance, and provides a mechanism by which total energy stores may be increased appropriately (e.g., prior to hibernation or migration), it seems probable that under conditions of chronic stress, this regulatory system may be maladaptive. Chronic stress and glucocorticoid treatment cause increases in mean daily concentrations of both corticosteroids and insulin. Increases in the absolute levels of both hormones, with the normal ratio between them maintained, results in remodeling of body energy stores-away from muscle stores and toward fat stores, particularly abdominal fat stores. It seems quite likely that some conditions of abdominal obesity in man may be explained, at least in part, by increased activity in the HPA axis. Because abdominal obesity is associated with cardiovascular diseases, these responses, when they persist, are clearly maladaptive. Exploration of the role and control of the HPA axis in and by the larger neural network that regulates energy balance has to date been instructive. Clearly this work has just begun and is primarily still at the level of phenomenology. However, once the phenomenology is understood, mechanistic work can be performed that will flesh out our understanding of this very large and physiologically essential system.

Aldosterone and dexamethasone both stimulate energy acquisition whereas only the glucocorticoid alters energy storage.

Corticosteroids stimulate and insulin inhibits energy acquisition (food intake); conversely, corticosteroids inhibit and insulin stimulates energy storage (body weight gain). Thus, together these hormones mediate long-term energy balance. This study tested whether the stimulatory action of corticosteroids on food intake was mediated by association with high affinity mineralocorticoid receptors (MRs) or lower affinity glucocorticoid receptors (GRs). Young male rats were adrenalectomized (ADX) and given vehicle (control) or streptozotocin (diabetic); subgroups of rats were infused with vehicle, aldosterone (Aldo, an MR agonist in vivo), dexamethasone (Dex, a GR agonist in vivo), or Aldo&Dex for the 5 days after ADX. Sham-ADX rats were included. Food intake, body weight gain, and epididymal white adipose and interscapular brown adipose tissue stores were weighed. ADX decreased food intake by approximately 24%, and food intake was not increased by diabetes as it was in sham-ADX rats. In control ADX rats, Dex, but not Aldo, stimulated insulin, and food intake was not significantly affected by either hormone; together, Aldo and Dex restored insulin and food intake to sham-ADX rats. Food intake in diabetic ADX rats was significantly increased by each treatment (ADX < Aldo < Dex < Aldo&Dex = sham). Aldo increased body weight through an increase in fluid volume (estimated by decreased plasma protein concentration); however, fat stores were not different from ADX. Dex reduced body weight in control rats but maintained fat stores; in diabetic rats, body weight and fat stores were less than or similar to ADX. We conclude that: 1) corticosteroids, acting through association with both MRs and GRs, stimulate food intake; 2) insulin counteracts the GR-mediated stimulation of food intake in control rats; and 3) Dex and insulin, which is stimulated by Dex, selectively maintain or increase body fat stores, probably at the expense of protein stores.

Neuropeptide Y (NPY) may integrate responses of hypothalamic feeding systems and the hypothalamo-pituitary-adrenal axis.

Neuropeptide Y (NPY) is a powerful stimulus to food intake in the rat. Exogenous NPY given into the third ventricle or into the paraventricular nucleus (PVN) of the hypothalamus stimulates both food consumption as well as the hypothalamus-pituitary-adrenal (HPA) axis. Presumably NPY activates the adrenocortical system through direct stimulation of CRF containing cells in the PVN. Food intake is also a major regulator of adrenocortical activation. Rhythms in HPA axis activity follow rhythms in food consumption, and rats that have been food deprived overnight have inhibited HPA axis responses to restraint stress and corticosteroid feedback the following morning. To investigate the interaction of NPY with both feeding and HPA axis activation three sets of experiments were performed: Animals fed ad lib were injected icv with NPY (2.5 micrograms) and allowed access to food or not post injection; animals were fasted overnight prior to NPY injection; finally, dose response experiments were performed to examine the relative sensitivities of feeding and HPA axis activation to exogenous NPY. Ad lib fed animals allowed access to food after NPY injection had slightly greater ACTH responses to NPY while glucocorticoid and insulin responses were not significantly different from ad lib fed animals not allowed access to food post injection. Animals allowed to eat post injection had significantly decreased food consumption the night following injection, however, total 24 h food consumption was not different between these animals and those given food 8 h post NPY injection. In overnight fasted animals NPY injections produced ACTH responses of equal magnitude to those in ad lib fed animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of activity in the hypothalamo-pituitary-adrenal axis is integral to a larger hypothalamic system that determines caloric flow.

We have previously reported that there are diurnal rhythms in the magnitude of ACTH responses to stressors and in the sensitivity of stress-induced ACTH responses to facilitation induced by prior stress and to corticosterone (B) feedback induced by exogenous B. In all cases ACTH was more responsive in the morning than in the evening in nocturnally feeding rats. We have also shown in adrenalectomized rats that an overnight fast reduces ACTH responses to restraint in the morning compared with rats fed ad libitum, and we have shown that calorie-containing gavage during the fast increases the amplitude of ACTH responses to restraint in fasted rats. Therefore, this diurnal rhythm is not associated with B feedback and is associated with calories. In these studies we asked whether young, male intact rats that were deprived of food overnight had: 1) hypothalamo-pituitary-adrenal (HPA) axis responses during the fasting period; 2) altered basal activity in the HPA axis; 3) altered responsivity of ACTH to restraint; and 4) altered sensitivity of restraint-induced ACTH responses to facilitation or B feedback. Our results show that food deprivation: 1) induces marked ACTH and B responses during the fast that mirrors the pattern of food intake in fed rats, with an approximately 3-h lag; 2) results in essentially no change in basal ACTH in the morning; 3) reduces ACTH responsivity to stress in the morning; and 4) reduces ACTH responsivity to prior stress-induced facilitation and exogenous B-induced feedback. We conclude that: 1) the HPA axis serves as a default pathway to feeding when food is not available; 2) the diurnal rhythms in restraint-induced ACTH secretion are determined by food intake; and 3) the HPA axis is integral to a larger hypothalamic system that mediates energy flow.

The diurnal rhythm in adrenocorticotropin responses to restraint in adrenalectomized rats is determined by caloric intake.

There is a diurnal rhythm in ACTH responses to stressors that peaks, in nocturnally feeding rats, at the time of lights on, in the morning (AM). To determine whether this rhythm is subordinate to the rhythm in food intake, we tested the effects of removing food during the night or the day on ACTH responses in the AM or evening (PM) to the stimulus of restraint in 5-day-adrenalectomized rats. An overnight fast reduced the ACTH response to restraint with tail blood sampling in the AM to the low magnitude observed in the PM in rats fed ad libitum; by contrast, a fast of equivalent duration imposed during the day had no effect on the ACTH response to the stressor in the PM. Short term fasts did not alter the normal AM-PM rhythm in basal ACTH levels. The fasts did, however, significantly decrease the pituitary ACTH concentration at both times of day, suggesting that lack of food had stimulated ACTH secretion during the preceding 14 h. Providing calories by either gavage or manipulation of food presentation increased ACTH responses to restraint in fasted adrenalectomized rats in both the AM and PM. Although four of four experiments showed that provision of calories to fasted rats resulted in increased ACTH responses to the stimulus of restraint, none of the manipulations of caloric intake fully restored ACTH responses in fasted rats to the high amplitude observed in ad libitum fed rats in the AM. We conclude that 1) unlike the circadian rhythm in basal activity in the hypothalamic-pituitary-adrenalocortical (HPA) system, the diurnal rhythm in ACTH responsiveness to stimuli is tightly coupled to the endogenous rhythm in energy intake; and 2) caloric deprivation per se appears to activate the HPA system at some time during the 14- to 17-h fast, but does not produce the normal facilitation in the AM response to acute restraint that is induced by chronic or prior stimulation of the HPA axis.

Feast and famine: critical role of glucocorticoids with insulin in daily energy flow.

The hypothesis proposed in this review is that normal diurnal rhythms in the hypothalamic-pituitary-adrenal (HPA) axis are highly regulated by activity in medial hypothalamic nuclei to effect an interaction between corticosteroids and insulin such that optimal metabolism results in response to changes in the fed or fasted state of the animal. There are marked diurnal rhythms in function of the HPA axis under both basal and stress conditions. The HPA axis controls corticosteroid output from the adrenal and, in turn, forward elements of this axis are inhibited by feedback from circulating plasma corticosteroid levels. Basal activity in the HPA axis of mammals fed ad lib peaks about 2 h before the peak of the diurnal feeding rhythm, and is controlled by input from the suprachiasmatic nuclei. The rhythm in stress responsiveness is lowest at the time of the basal peak and highest at the time of the basal trough in the HPA axis activity. There are also diurnal rhythms in corticosteroid feedback sensitivity of basal and stress-induced ACTH secretion which peak at the time of the basal trough. These rhythms are all overridden when feeding, and thus insulin secretion, is disrupted. Corticosteroids interact with insulin on food intake and body composition, and corticosteroids also increase insulin secretion. Corticosteroids stimulate feeding at low doses but inhibit it at high doses; however, it is the high levels of insulin, induced by high levels of corticosteroids, that may inhibit feeding. The effects of corticosteroids on liver, fat, and muscle cell metabolism, with emphasis on their interactions with insulin, are briefly reviewed. Corticosteroids both synergize with and antagonize the effects of insulin. The effects of stress hormones, and their interactions with insulin on lipid and protein metabolism, followed by some of the metabolic effects of injury stress, with or without nutritional support, are evaluated. In the presence of elevated insulin stimulated by glucocorticoids and nutrition, stress causes less severe catabolic effects. In the central nervous system, regulation of function in the HPA axis is clearly affected by the activity of medial hypothalamic nuclei that also alter feeding, metabolism, and obesity in rats. Lesions of the arcuate (ARC) and ventromedial (VMN) paraventricular (PVN) nuclei result in obesity and hyperactivity in the HPA axis. Moreover, adrenalectomy inhibits or prevents development of the lesion-induced obesity. There are interactions among these nuclei; one mode of communication is via inputs of neuropeptide Y (NPY) cells in the ARC to the VMN, dorsomedial nuclei, and PVN.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid plastic response following early retinal lesions in rats.

Following an early retinal lesion, aberrant uncrossed projections from the opposite, undamaged, retina form in the target visual nuclei. The present study has examined the development of such aberrant projections by making retinal lesions in newborn rat pups, and then examining the nature of the uncrossed retinocollicular projection at different ages following the lesion. Intravitreal injections of horseradish peroxidase were made into the intact eye, and the uncrossed projection was subsequently revealed histochemically. A mature aberrant projection forms as early as postnatal day 9. On postnatal days 5 and 2, aberrant projections are discernable amongst the exuberant uncrossed terminals of normal developing rats, although the former have not matured to form the dense terminal fields characteristic of older projections. Aberrant projections were also detectable as early as 12 h following the lesion, revealed as a relative increase in the density of uncrossed label. These results indicate that lesion-induced plastic responses by intact retinal arbors are initiated shortly after the insult, and they caution the use of retinal lesions in studies of normal retinotopic connectivity during development.