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The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.

Pro-opiomelanocortin (POMC) neurons function as key regulators of metabolism and physiology by releasing prohormone-derived neuropeptides with distinct biological activities. However, our understanding of early events in prohormone maturation in the ER remains incomplete. Highlighting the significance of this gap in knowledge, a single POMC cysteine-to-phenylalanine mutation at position 28 (POMC-C28F) is defective for ER processing and causes early onset obesity in a dominant-negative manner in humans through an unclear mechanism. Here, we report a pathologically important role of Sel1L-Hrd1, the protein complex of ER-associated degradation (ERAD), within POMC neurons. Mice with POMC neuron-specific Sel1L deficiency developed age-associated obesity due, at least in part, to the ER retention of POMC that led to hyperphagia. The Sel1L-Hrd1 complex targets a fraction of nascent POMC molecules for ubiquitination and proteasomal degradation, preventing accumulation of misfolded and aggregated POMC, thereby ensuring that another fraction of POMC can undergo normal posttranslational processing and trafficking for secretion. Moreover, we found that the disease-associated POMC-C28F mutant evades ERAD and becomes aggregated due to the presence of a highly reactive unpaired cysteine thiol at position 50. Thus, this study not only identifies ERAD as an important mechanism regulating POMC maturation within the ER, but also provides insights into the pathogenesis of monogenic obesity associated with defective prohormone folding., Introduction Hypothalamic pro-opiomelanocortin (POMC) neurons critically mediate leptin signaling and regulate feeding behavior and systemic metabolic homeostasis via the secretion of several bioactive neuropeptides derived from the POMC protein precursor [...]

The thyrotropin- (TRH) releasing hormone precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating N-terminal 15 kDa/9.5 kDa or C-terminal 16.5/10 kDa intermediate forms that are processed further to yield five copies of TRH-Gly and seven non-TRH peptides. Glucocorticoids (Gcc) have been shown to enhance TRH gene expression in three different cell systems in vitro, an effect that occurs, at least in part, through transcriptional activation. Although this implies that an increase of TRH prohormone biosynthesis would take place, this had not been demonstrated as yet. We report here that the synthetic glucocorticoid dexamethasone (Dex) substantially elevated the de novo biosynthesis of the intact 26-kDa TRH prohormone and its intermediate products of processing in cultured anterior pituitary cells, an observation that is consistent with an overall upregulation of both the biosynthesis and degradation of the TRH precursor. We reasoned that Gcc may act not only at the transcriptional, but also at the translational/posttranslational level. To address this question we chose a different cell system, AtT20 cells transfected with a cDNA encoding preproTRH. Since TRH gene expression in these cells is driven by the CMV-IE promoter and not by an endogenous "physiological" promoter, these cells provide an ideal model to study selectively the effects of Gcc on the translation and posttranslational processing of proTRH without interference from a direct transcriptional activation of the TRH gene. Dex caused a significant 75.7% increase in newly synthesized 26-kDa TRH prohormone, suggesting that the glucocorticoid raised the translation rate. We then demonstrated that Dex treatment accelerated TRH precursor processing. Of interest, processing of the N- vs the C-terminal intermediate was influenced differentially by the glucocorticoid. Although the N-terminal intermediate product of processing accumulated, the C-terminal intermediate was degraded more rapidly. Consistent with these observations was the finding that the intracellular accumulation of the N-terminally derived peptide preproTRH 25-50 was enhanced, but levels of the C-terminally derived peptide preproTRH208-255 were reduced. Accumulation of TRH itself, whose five copies are N- and C-terminally derived, was also enhanced. We conclude that Gcc induce changes in the biosynthesis and processing of proTRH by increasing the translation rate and by differentially influencing the processing of N- vs C-terminal intermediates of the precursor molecule. These effects of Gcc at the translational and posttranslational levels result in an increase in TRH production accompanied by differential effects on the accumulation of N- and C-terminal non-TRH peptides.

The hypothalamic-pituitary-thyroid (HPT) axis is a major contributor in maintaining energy expenditure and body weight, and the adipocyte hormone leptin regulates this axis by increasing TRH levels in the fed state. Leptin stimulates TRH directly in the hypothalamic paraventricular nucleus (PVN; direct pathway) and indirectly by regulating proopiomelnocortin neurons in the hypothalamic arcuate nucleus (ARC; indirect pathway). Whereas the indirect pathway is fully functional in lean animals, it is inactive during diet-induced obesity (DIO) because of the establishment of leptin resistance. Despite this, the HPT axis activity in obese humans and rodents remains within the normal levels or slightly higher. Therefore, in this study, we aimed to determine the mechanism(s) by which the HPT axis is still active despite leptin resistance. With a combination of using the Sprague-Dawley rat physiological model and the Zuker rat that bears a mutation in the leptin receptor, we were able to demonstrate that under DIO conditions the HPT axis is regulated at the central level, but only through the direct pathway of leptin action on TRH neurons. Deiodinase enzymes, which are present in many tissues and responsible for converting thyroid hormones, were not statistically different between lean and DIO animals. These data suggest that the increase in T4/3 seen in obese animals is due mostly to central leptin action. We also found that [T.sub.3] feedback inhibition on the prepro-TRH gene is controlled partially by leptin-induced pSTAT3 signaling via the TRH promoter. This interactive relationship between [T.sub.3] and pSTAT3 signaling appears essential to maintain the HPT axis at normal levels in conditions such as obesity. doi: 10.1152/ajpendo.00448.2010.

The [alpha]-melanocyte-stimulating hormone ([alpha]-MSH), derived from proopiomelanocortin (POMC), is generated by a posttranslational processing mechanism involving the prohormone convertases (PCs) PC1/3 and PC2. In the brain, [alpha]-MSH is produced in the arcuate nucleus (ARC) of the hypothalamus and in the nucleus of the solitary tract (NTS) of the medulla. This peptide is key in controlling energy balance, as judged by changes observed at transcriptional level. However, little information is available regarding the biosynthesis of the precursor POMC and the production of its processed peptides during feeding, fasting, and fasting plus leptin in the ARC compared with the NTS in conjunction with the PC activity. In this study we found that, in the ARC, pomc mRNA, POMC-derived peptides, and PC1/3 all decreased during fasting, and administration of leptin reversed these effects. In contrast, in the NTS, where there is a large amount of a 28.1-kDa peptide similar in size to POMC, the 28.1-kDa peptide and other POMC-derived peptides, including [alpha]-MSH, were further accumulated in fasting conditions, whereas pomc mRNA decreased. These changes were not reversed by leptin. We also observed that, during fasting, PC2 levels decreased in the NTS. These data suggest that, in the NTS, fasting induced changes in POMC biosynthesis, and processing is independent of leptin. These observations indicate that changes in energy status affect POMC in the brain in a tissue-specific manner. This represents a novel aspect in the regulation of energy balance and may have implications in the pathophysiology of obesity. processing; adrenocorticotropin hormone; [alpha]-melanocyte-stimulating hormone doi:10.1152/ajpendo.00466.2006

The prohormone convertases (PCs), PC1/3 and PC2, are involved in the tissuespecific endoproteolytic posttranslational processing of many hormonal precursors within the secretory pathway. One important prohormone, pro-thyrotropin-releasing hormone (TRH), is expressed in both hypophysiotropic (where it regulates the secretion of thyroid-stimulating hormone) and nonhypophysiotropic regions of the brain. Pro-TRH is processed at specific sites in the secretory pathway, primarily by PC1/3 followed by PC2. We hypothesized that thyroid hormone status in specific nuclei of the brain would alter pro-TRH processing by inducing changes in PC1/3 and PC2 expression. Therefore, we examined pro-TRH, PC1/3, and PC2 coexpression and coregulation in the paraventricular nucleus (PVN), lateral hypothalamus (LH), and ventromedial nucleus (VMN) of hypothyroid and euthyroid rats. Our results show that 6-n-propyl-2-thiouracil (PTU) treatment producing hypothyroidism induced a significant increase in the expression of PC1/3, PC2, and pro-TRH in the PVN and LH, but not VMN. When confocal studies were performed, an increase in colocalization of PC1/3 or PC2 in pro-TRH was observed only in PVN, a response that was especially prominent in the ventral and medial areas of the PVN. PTU did not regulate colocalization in the VMH or LH. Regulation of colocalization of processing enzyme and prohormone expression is a novel mechanism to alter hormonal biosynthesis. posttranslational processing; hyperthyroidism; hypothyroidism; prohormone convertase; hypothalamus; pro-thyrotropin-releasing hormone; peptide

The central melanocortin system is critical in the regulation of appetite and body weight, and leptin exerts its anorexigenic actions partly by increasing hypothalamic proopiomelanocortin (POMC) expression. The POMC-derived peptide [alpha]-melanocyte-stimulating hormone ([alpha]MSH) is a melanocortin 4 receptor agonist, and its potency in reducing energy intake is strongly increased by N-acetylation. The reason for the higher biological activity of N-acetylated [alpha]MSH (Act-[alpha]MSH) compared with that of N-desacetylated [alpha]MSH (Des-[alpha]MSH) is unclear, and regulation of acetylation by leptin has not been investigated. We show here that total hypothalamic [alpha]MSH levels are decreased in leptin-deficient ob/ob mice and increased in leptin-treated ob/ob and C57BL/6J mice. The increase in total [alpha]MSH occurred as soon as 3 h after leptin injection and was entirely due to an increase in Act-[alpha]MSH. Consistent with this observation, leptin rapidly induced the enzymatic activity of a N-acetyltransferase in the hypothalamus of mice. In 293T cells expressing the melanocortin 4 receptor, Act-[alpha]MSH is far more potent than Des-[alpha]MSH in stimulating cAMP accumulation, an effect caused by a dramatically increased stability of Act-[alpha]MSH. Moreover, Des-[alpha]MSH is rapidly degraded in the hypothalamus after intracerebroventricular injection in rats and was less potent in inhibiting energy intake. The results suggest that leptin activates a N-acetyltransferase in POMC neurons, leading to increased hypothalamic levels of Act-[alpha]MSH. Due to its increased stability, this posttranslational modification of [alpha]MSH may play a critical role in leptin action via the central melanocortin pathway.

The last decade had witnessed a tremendous progress in our understanding of the causes of metabolic diseases including obesity. Among the contributing factors regulating energy balance are nutrient sensors such as sirtuins. Sirtuin1 (Sirt1), a NAD + - dependent deacetylase is affected by diet, environmental stress, and also plays a critical role in metabolic health by deacetylating proteins in many tissues, including liver, muscle, adipose tissue, heart, endothelium, and in the complexity of the hypothalamus. Because of its dependence on NAD+, Sirt1 also functions as a nutrient/redox sensor, and new novel data show a function of this enzyme in the maturation of hypothalamic peptide hormones controlling energy balance either through regulation of specific nuclear transcription factors or by regulating specific pro-hormone convertases (PCs) involved in the post-translational processing of pro-hormones. The post-translational processing mechanism of pro-hormones is critical in the pathogenesis of obesity as recently shown that metabolic and physiological triggers affect the biosynthesis and processing of many peptides hormones. Specific regulation of pro-hormone processing is likely another key step where final amounts of bioactive peptides can be tightly regulated. Different factors stimulate or inhibit pro-hormones biosynthesis in concert with an increase in the PCs involved in the maturation of bioactive hormones. Adding more complexity to the system, the new studies describe here suggest that Sirt1 could also regulate the fate of peptide hormone biosynthesis. The present review summarizes the recent progress in hypothalamic SIRT1 research with a particular emphasis on the tissue-specific control of neuropeptide hormone maturation. The series of studies done in mouse and rat models strongly advocate for the first time that a deacetylating enzyme could be a regulator in the maturation of peptide hormones and their processing enzymes. These discoveries are the culmination of the first in-depth understanding of the metabolic role of Sirt1 in the brain. It suggests that Sirt1 behaves differently in the brain than in organs such as the liver and pancreas, where the enzyme has been more commonly studied. [ABSTRACT FROM AUTHOR]

Hypothalamic proopiomelanocortin (POMC) neurons constitute a critical anorexigenic node in the central nervous system (CNS) for maintaining energy balance. These neurons directly affect energy expenditure and feeding behavior by releasing bioactive neuropeptides but are also subject to signals directly related to nutritional state such as the adipokine leptin. To further investigate the interaction of diet and leptin on hypothalamic POMC peptide levels, we exposed 8- to 10-wk-old male POMC-Discosoma red fluorescent protein (DsRed) transgenic reporter mice to either 24-48 h (acute) or 2 wk (chronic) food restriction, high-fat diet (HFD), or leptin treatment. Using semiquantitative immunofluorescence and radioimmunoassays, we discovered that acute fasting and chronic food restriction decreased the levels of adrenocorticotropic hormone (ACTH), α-melanocytestimulating hormone (α-MSH), and β-endorphin in the hypothalamus, together with decreased DsRed fluorescence, compared with control ad libitum-fed mice. Furthermore, acute but not chronic HFD or leptin administration selectively increased α-MSH levels in POMC fibers and increased DsRed fluorescence in POMC cell bodies. HFD and leptin treatments comparably increased circulating leptin levels at both time points, suggesting that transcription of Pomc and synthesis of POMC peptide products are not modified in direct relation to the concentration of plasma leptin. Our findings indicate that negative energy balance persistently downregulated POMC peptide levels, and this phenomenon may be partially explained by decreased leptin levels, since these changes were blocked in fasted mice treated with leptin. In contrast, sustained elevation of plasma leptin by HFD or hormone supplementation did not significantly alter POMC peptide levels, indicating that enhanced leptin signaling does not chronically increase Pomc transcription and peptide synthesis. [ABSTRACT FROM AUTHOR]

Protein posttranslational processing is a cellular mechanism fundamental to the generation of bioactive peptides, including the anorectic α-melanocyte-stimulating hormone (α-MSH) and thyrotropin-releasing hormone (TRH) peptides produced in the hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei, respectively. Neuropeptide Y (NPY) promotes positive energy balance in part by suppressing α-MSH and TRH. The mechanism by which NPY regulates α-MSH output, however, is not well understood. Our results reveal that NPY inhibited the posttranslational processing of α-MSH's inactive precursor proopiomelanocortin (POMC) by decreasing the prohormone convertase-2 (PC2). We also found that early growth response protein-1 (Egr-1) and NPY-Y1 receptors mediated the NPY-induced decrease in PC2. NPY given intra-PVN also decreased PC2 in PVN samples, suggesting a reduction in PC2-mediated pro-TRH processing. In addition, NPY attenuated the α-MSH-induced increase in TRH production by two mechanisms. First, NPY decreased α-MSH-induced CREB phosphorylation, which normally enhances TRH transcription. Second, NPY decreased the amount of α-MSH in the PVN. Collectively, these results underscore the significance of the interaction between NPY and α-MSH in the central regulation of energy balance and indicate that posttranslational processing is a mechanism that plays a specific role in this interaction. [ABSTRACT FROM AUTHOR]

The adipose tissue-derived hormone leptin regulates energy balance through catabolic effects on central circuits, including proopiomelanocortin (POMC) neurons. Leptin activation of POMC neurons increases thermogenesis and locomotor activity. Protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of leptin signaling. POMC neuron-specific deletion of PTP1B in mice results in reduced high-fat diet-induced body weight and adiposity gain due to increased energy expenditure and greater leptin sensitivity. Mice lacking the leptin gene (ob/ob mice) are hypothermic and cold intolerant, whereas leptin delivery to ob/ob mice induces thermogenesis via increased sympathetic activity to brown adipose tissue (BAT). Here, we examined whether POMC PTP1B mediates the thermoregulatory response of CNS leptin signaling by evaluating food intake, body weight, core temperature (TC), and spontaneous physical activity (SPA) in response to either exogenous leptin or 4-day cold exposure (4°C) in male POMC-Ptp1b-deficient mice compared with wild-type controls. POMC-Ptp1b-/- mice were hypersensitive to leptin-induced food intake and body weight suppression compared with wild types, yet they displayed similar leptin-induced increases in TC. Interestingly, POMC-Ptp1b-/- mice had increased BAT weight and elevated plasma triiodothyronine (T3) levels in response to a 4-day cold challenge, as well as reduced SPA 24 h after cold exposure, relative to controls. These data show that PTP1B in POMC neurons plays a role in short-term cold-induced reduction of SPA and may influence cold-induced thermogenesis via enhanced activation of the thyroid axis. [ABSTRACT FROM AUTHOR]

Background: Dyskinesias associated with involuntary movements and painful muscle contractions are a common and severe complication of standard levodopa (L-DOPA, L-3,4-dihydroxyphenylalanine) therapy for Parkinson's disease. Pathologic neuroplasticity leading to hyper-responsive dopamine receptor signaling in the sensorimotor striatum is thought to underlie this currently untreatable condition. Methodology/Principal Findings: Quantitative real-time polymerase chain reaction (PCR) was employed to evaluate the molecular changes associated with L-DOPA-induced dyskinesias in Parkinson's disease. With this technique, we determined that thyrotropin releasing hormone (TRH) was greatly increased in the dopamine-depleted striatum of hemi-parkinsonian rats that developed abnormal movements in response to L-DOPA therapy, relative to the levels measured in the contralateral non-dopamine-depleted striatum, and in the striatum of non-dyskinetic control rats. ProTRH immunostaining suggested that TRH peptide levels were almost absent in the dopamine-depleted striatum of control rats that did not develop dyskinesias, but in the dyskinetic rats, proTRH immunostaining was dramatically up-regulated in the striatum, particularly in the sensorimotor striatum. This up-regulation of TRH peptide affected striatal medium spiny neurons of both the direct and indirect pathways, as well as neurons in striosomes. Conclusions/Significance: TRH is not known to be a key striatal neuromodulator, but intrastriatal injection of TRH in experimental animals can induce abnormal movements, apparently through increasing dopamine release. Our finding of a dramatic and selective up-regulation of TRH expression in the sensorimotor striatum of dyskinetic rat models suggests a TRH-mediated regulatory mechanism that may underlie the pathologic neuroplasticity driving dopamine hyper-responsivity in Parkinson's disease. [ABSTRACT FROM AUTHOR]

Abstract: The hypothalamic-pituitary-thyroid (HPT) axis plays a critical role in mediating changes in metabolism and thermogenesis. Thus, the central regulation of the thyroid axis by Thyrotropin Releasing Hormone (TRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) is of key importance for the normal function of the axis under different physiological conditions including cold stress and changes in nutritional status. Before the TRH peptide becomes biologically active, a series of tightly regulated processes occur including the proper folding of the prohormone for targeting to the secretory pathway, its post-translational processing, and targeting of the processed peptides to the secretory granules near the plasma membrane of the cell ready for secretion. Multiple inputs coming from the periphery or from neurons present in different areas of the brain including the hypothalamus are responsible for the activation or inhibition of the TRH neuron and in turn affect the output of TRH and the set point of the axis. [Copyright &y& Elsevier]

ProThyrotropin-releasing hormone (proTRH) is a prohormone widely distributed in many areas of the brain. After biosynthesis, proTRH is subjected to post-translational processing to generate TRH and seven non-TRH peptides. Among these non-TRH sequences, we found previously that preproTRH178–199 could regulate the secretion of prolactin in suckled rats by their pups. Dopamine (DA), the main regulator of prolactin secretion, is produced in dopaminergic tyrosine hydroxylase (TH)-positive neurons in the hypothalamic arcuate nucleus (ARC). In this study we investigated whether prolactin release during the estrous sexual cycle is regulated by prepro TRH178–199 through its effecton DA neurons of the ARC. We observed that biotinylated prepro TRH178–199 bound to neurons in the ARC; this was higher during proestrus than during diestrus. Binding of preproTRH178–199 to DA neurons was seen only during proestrus in the ARC. Using primary neuronal hypothalamic cultures we found that preproTRH178–199peptide decreased TH levels in a dose-responsive manner, whereas intra-ARC administration of preproTRH178–199 induced a 20-fold increase in plasma prolactin levels. Together, these results suggest a potential role for preproTRH178–199 in regulating dopaminergic neurons involved in the inhibition of pituitary prolactin release. [ABSTRACT FROM AUTHOR]

The central melanocortin system is critical in the regulation of appetite and body weight and leptin exerts its anorexigenic actions partly by increasing hypothalamic proopiomelanocortin (POMC) expression. The POMC-derived peptide α-melanocyte- stimulating hormone (αMSH) is a melanocortin 4 receptor agonist and its potency in reducing energy intake is strongly increased by N-acetylation. The reason for the higher biological activity of N-acetylated αMSH (Act-αMSH) compared with that of N-desacetylated αMSH (Des-αMSH) is unclear, and regulation of acetylation by leptin has not been investigated. We show here that total hypothalamic αMSH levels are decreased in leptin-deficient ob/ob mice and increased in leptin-treated oh/oh and CS7BL/6J mice. The increase in total αMSH occurred as soon as 3 h after leptin injection and was entirely due to an increase in Act-αMSH. Consistent with this observation, leptin rapidly induced the enzymatic activity of a N-acetyltransferase in the hypothalamus of mice. In 293T cells expressing the melanocortin 4 receptor, Act-αMSH is far more potent than Des-αMSH in stimulating cAMP accumulation, an effect caused by a dramatically increased stability of Act-αMSH. Moreover, Des-αMSH is rapidly degraded in the hypothalamus after intracerebroventricular injection in rats and was less potent in inhibiting energy intake. The results suggest that leptin activates a N-acetyltransferase in POMC neurons, leading to increased hypothalamic levels of Act-αMSH. Due to its increased stability, this posttranslational modification of αMSH may play a critical role in leptin action via the central melanocortin pathway. [ABSTRACT FROM AUTHOR]

Rat erythrocytes, infected with Plasmodium berghei, were disrupted by freezing and thawing or parasites were released by mechanical breaking of the agglutinated erythrocytes. The released soluble antigens, inducing blast transformation in non-adherent immune rat spleen cells, were fractionated by a sequence of salting out, ion exchange chromatography and gel filtration. At the final step a purification factor of 83.7-fold was achieved. This fraction contained three components discernible by SDS-PAGE of molecular weights 56, 66 and 72 kD respectively. The free parasites, also induced blast transformation in immune rat spleen cells. After disruption by freeze-thawing the majority of the active antigens was found in the sediment after centrifugation at 17,300 g. [ABSTRACT FROM AUTHOR]

Rat erythrocytes infected with Plasmodium berghei were disrupted by gentle passage of Coneanavalin A (ConA) agglutinated cells through a 100 mesh stainless steel grid. The free parasites were separated from cell debris, unbroken infected cells, and from uninfected rat erythrocytes on a Percoll gradient. The free parasites remained morphologically intact, metabolically active, and infective to mice. The parasites were observed by light and electron microscopy. The incorporation ofH-isoleucine andH-hypoxanthine was compared in intact and infected cells, and the infectivity was measured by the injection of parasites into susceptible mice. It seems that the combination of the two techniques used, produces a high yield of intact free parasites suitable for physiological or immunological studies. [ABSTRACT FROM AUTHOR]

Sirt1 is an evolutionarily conserved NAD+ dependent deacetylase involved in a wide range of processes including cellular differentiation, apoptosis, as well as metabolism, and aging. In this study, we investigated the role of hypothalamic Sirt1 in energy balance. Pharmacological inhibition or siRNA mediated knock down of hypothalamic Sirt1 showed to decrease food intake and body weight gain. Central administration of a specific melanocortin antagonist, SHU9119, reversed the anorectic effect of hypothalamic Sirt1 inhibition, suggesting that Sirt1 regulates food intake through the central melanocortin signaling. We also showed that fasting increases hypothalamic Sirt1 expression and decreases FoxO1 (Forkhead transcription factor) acetylation suggesting that Sirt1 regulates the central melanocortin system in a FoxO1 dependent manner. In addition, hypothalamic Sirt1 showed to regulate S6K signaling such that inhibition of the fasting induced Sirt1 activity results in up-regulation of the S6K pathway. Thus, this is the first study providing a novel role for the hypothalamic Sirt1 in the regulation of food intake and body weight. Given the role of Sirt1 in several peripheral tissues and hypothalamus, potential therapies centered on Sirt1 regulation might provide promising therapies in the treatment of metabolic diseases including obesity. [ABSTRACT FROM AUTHOR]

Abstract: Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for peptidylglycine α-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM+/− mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM+/− mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency. [Copyright &y& Elsevier]

Prothyrotropin-releasing hormone (pro-TRH) is initially cleaved by the prohormone convertase-1/3 (PC1/3) in the trans-Golgi network generating N- and C-terminal intermediate forms that are then packed into secretory vesicles. However, it is not known whether these peptides are differentially sorted within the secretory pathway. This is of key importance because the processing products of several prohormones fulfill different biological functions. Using AtT20 cells stably transfected with prepro-TRH cDNA, we found that two specific N- and C-terminal peptides were located in different vesicles. Furthermore, the C-terminal pro-TRH-derived peptides were more efficiently released in response to KC1 and norepinephrine, a natural secretagogue of TRH. Similar sorting and secretion of N- and C-terminal peptides occurs in vivo. When we blocked the initial proteolytic processing by a mutagenic approach, the differential sorting and secretion of these peptides were prevented. In summary, our data show that pro-TRH-derived peptides are differentially sorted within the secretory pathway and that the initial cleavage in the trans-Golgi network is key to this process. This could be a common mechanism used by neuroendocrine cells to regulate independently the secretion of different bioactive peptides derived from the same gene product. [ABSTRACT FROM AUTHOR]