Selenoprotein T: An Essential Oxidoreductase Serving as a Guardian of Endoplasmic Reticulum Homeostasis.

Significance: Selenoproteins incorporate the essential nutrient selenium into their polypeptide chain. Seven members of this family reside in the endoplasmic reticulum (ER), the exact function of most of which is poorly understood. Especially, how ER-resident selenoproteins control the ER redox and ionic environment is largely unknown. Since alteration of ER function is observed in many diseases, the elucidation of the role of selenoproteins could enhance our understanding of the mechanisms involved in ER homeostasis. Recent Advances: Among selenoproteins, selenoprotein T (SELENOT) is remarkable as the most evolutionarily conserved and the only ER-resident selenoprotein whose gene knockout in mouse is lethal. Recent data indicate that SELENOT contributes to ER homeostasis: reduced expression of SELENOT in transgenic cell and animal models promotes accumulation of reactive oxygen and nitrogen species, depletion of calcium stores, activation of the unfolded protein response and impaired hormone secretion. Critical Issues: SELENOT is anchored to the ER membrane and associated with the oligosaccharyltransferase complex, suggesting that it regulates the early steps of N-glycosylation. Furthermore, it exerts a selenosulfide oxidoreductase activity carried by its thioredoxin-like domain. However, the physiological role of the redox activity of SELENOT is not fully understood. Likewise, the nature of its redox partners needs to be further characterized. Future Directions: Given the impact of ER stress in pathologies such as neurodegenerative, cardiovascular, metabolic and immune diseases, understanding the role of SELENOT and developing derived therapeutic tools such as selenopeptides to improve ER proteostasis and prevent ER stress could contribute to a better management of these diseases.

Effect of food deprivation on the hypothalamic gene expression of the secretogranin II-derived peptide EM66 in rat.

EM66 is a peptide derived from the chromogranin, secretogranin II (SG-II). Recent findings in mice indicate that EM66 is a novel anorexigenic neuropeptide that regulates hypothalamic feeding behavior, at least in part, by activating the POMC neurons of the arcuate nucleus. The present study aimed to investigate the mechanism of action of EM66 in the control of feeding behavior and, more specifically, its potential interactions with the NPY and POMC systems in rat. We analyzed by Q-PCR the gene expression of the EM66 precursor, SG-II, in hypothalamic extracts following 2, 3, or 4 days of food deprivation and compared it with the expression levels of the two major neuropeptidergic systems, that is, POMC and NPY, modulating feeding behavior. Our results show that fasting for 2 and 3 days has no effect on SG-II mRNA levels. However, 4 days of food deprivation induced a significant alteration in the expression levels of the three genes studied, with a significant increase in SG-II and NPY mRNAs, and conversely, a significant decrease in POMC mRNA. These data indicate that the EM66 gene expression is modulated by a negative energy status and suggest interactions between EM66 and NPY to regulate food intake through the POMC system.

Impact of aflatoxin B1 on hypothalamic neuropeptides regulating feeding behavior.

The presence of mycotoxins in food is a major problem of public health as they produce immunosuppressive, hepatotoxic and neurotoxic effects. Mycotoxins also induce mutagenic and carcinogenic effects after long exposure. Among mycotoxins that contaminate food are aflatoxins (AF) such as AFB1, which is the most powerful natural carcinogen. The AF poisoning results in symptoms of depression, anorexia, diarrhea, jaundice or anemia that can lead to death, but very few studies have explored the impact of AF on neuroendocrine regulations. To better understand the neurotoxic effects of AF related to anorexia, we explored in rat the impact of AFB1 on the major hypothalamic neuropeptides regulating feeding behavior, either orexigenic (NPY, Orexin, AgRP, MCH) or anorexigenic (α-MSH, CART, TRH). We also studied the effect of AFB1 on a novel neuropeptide, the secretogranin II (SgII)-derived peptide EM66, which has recently been linked to the control of food intake. For this, adult male rats were orally treated twice a week for 5 weeks with a low dose (150 µg/kg) or a high dose (300 µg/kg) of AFB1 dissolved in corn oil. Repeated exposure to AFB1 resulted in reduced body weight gain, which was highly significant for the high dose of AF. Immunocytochemical and quantitative PCR experiments revealed a dose-related decrease in the expression of all the hypothalamic neuropeptides studied in response to AFB1. Such orexigenic and anorexigenic alterations may underlie appetite disorders as they are correlated to a dose-dependent decrease in body weight gain of treated rats as compared to controls. We also found a decrease in the number of EM66-containing neurons in the arcuate nucleus of AFB1-treated animals, which was associated with a lower expression of its precursor SgII. These findings show for the first time that repeated consumption of AFB1 disrupts the hypothalamic regulation of neuropeptides involved in feeding behavior, which may contribute to the lower body weight gain associated to AF exposure.

Hypothalamic Neuropeptide 26RFa Acts as an Incretin to Regulate Glucose Homeostasis.

26RFa is a hypothalamic neuropeptide that promotes food intake. 26RFa is upregulated in obese animal models, and its orexigenic activity is accentuated in rodents fed a high-fat diet, suggesting that this neuropeptide might play a role in the development and maintenance of the obese status. As obesity is frequently associated with type 2 diabetes, we investigated whether 26RFa may be involved in the regulation of glucose homeostasis. In the current study, we show a moderate positive correlation between plasma 26RFa levels and plasma insulin in patients with diabetes. Plasma 26RFa concentration also increases in response to an oral glucose tolerance test. In addition, we found that 26RFa and its receptor GPR103 are present in human pancreatic ß-cells as well as in the gut. In mice, 26RFa attenuates the hyperglycemia induced by a glucose load, potentiates insulin sensitivity, and increases plasma insulin concentrations. Consistent with these data, 26RFa stimulates insulin production by MIN6 insulinoma cells. Finally, we show, using in vivo and in vitro approaches, that a glucose load induces a massive secretion of 26RFa by the small intestine. Altogether, the present data indicate that 26RFa acts as an incretin to regulate glucose homeostasis.

Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain.

Leptin secreted by adipocytes acts on the brain to reduce food intake by regulating neuronal activity in the mediobasal hypothalamus (MBH). Obesity is associated with resistance to high circulating leptin levels. Here, we demonstrate that peripherally administered leptin activates its receptor (LepR) in median eminence tanycytes followed by MBH neurons, a process requiring tanycytic ERK signaling and the passage of leptin through the cerebrospinal fluid. In mice lacking the signal-transducing LepRb isoform or with diet-induced obesity, leptin taken up by tanycytes accumulates in the median eminence and fails to reach the MBH. Triggering ERK signaling in tanycytes with EGF reestablishes leptin transport, elicits MBH neuron activation and energy expenditure in obese animals, and accelerates the restoration of leptin sensitivity upon the return to a normal-fat diet. ERK-dependent leptin transport by tanycytes could thus play a critical role in the pathophysiology of leptin resistance, and holds therapeutic potential for treating obesity.

The orexigenic activity of the hypothalamic neuropeptide 26RFa is mediated by the neuropeptide Y and proopiomelanocortin neurons of the arcuate nucleus.

26RFa is a hypothalamic RFamide neuropeptide that was identified as the endogenous ligand of the orphan G protein-coupled receptor, GPR103, and that stimulates appetite in mice. Up until now, the mechanism of action of 26RFa in the hypothalamic control of food intake remains unknown. The high density of GPR103 in the arcuate nucleus (Arc) prompted us to investigate, in the present study, the effects of 26RFa on the rat neuropeptide Y (NPY)/proopiomelanocortin (POMC) system. Intracerebroventricular injection of 26RFa stimulated NPY expression and release in the basal hypothalamus, whereas it decreased POMC expression and alpha-MSH release, and these effects were associated with an increase in food intake. A double in situ hybridization procedure indicated that the 26RFa receptor is present in NPY neurons of the Arc, but not in POMC neurons. Central administration of NPY Y1 and Y5 receptor antagonists abolished the inhibitory effects of 26RFa on POMC expression and alpha-MSH release, and reversed 26RFa-induced food consumption. Finally, 26RFa antagonized the effects of leptin on NPY expression and release, POMC expression and alpha-MSH release, and food intake. Altogether, the present data demonstrate for the first time that 26RFa exerts its orexigenic activity by stimulating the release of NPY in the Arc, which in turn inhibits POMC neurons by activating the Y1 and Y5 receptors. It is also suggested that the balance 26RFa/leptin is an important parameter in the maintenance of energy homeostasis.

Immunohistochemical distribution of the secretogranin II-derived peptide EM66 in the rat hypothalamus: a comparative study with jerboa.

EM66 is a 66-amino acid peptide derived from secretogranin II, a member of granin acidic secretory protein family, by proteolytic processing. EM66 has been previously characterized in the jerboa (Jaculus orientalis) hypothalamus and its potential implication in the neuroendocrine regulation of feeding behaviour has been demonstrated. In the present study, an immunohistochemical analysis of the localization of EM66 within hypothalamic structures of rat was performed and compared to the distribution of EM66 in the jerboa hypothalamus. In the rat hypothalamus, as in the jerboa, EM66 immunostaining was detected in the parvocellular paraventricular, preoptic and arcuate nuclei, as well as the lateral hypothalamus which displayed an important density of EM66-producing neurones. However, unlike jerboa, the suprachiasmatic and supraoptic nuclei of the rat hypothalamus were devoid of cellular EM66-immunolabeling. Thus, the novel peptide EM66 may exert common neuroendocrine activities in rat and jerboa, e.g. control of food intake, and species-specific roles in jerboa such as the regulation of biological rhythms and hydromineral homeostasis. These results suggest the existence of differences between jerboas and rats in neuroendocrine regulatory mechanisms involving EM66.

Structure and functions of the novel hypothalamic RFamide neuropeptides R-RFa and 26RFa in vertebrates.

A number of RFamide peptides have been characterized in invertebrate species and these peptides have been found to exert a broad spectrum of biological activities. In contrast, in vertebrates, our knowledge on RFamide peptides is far more limited and only a few members of the RFamide peptide family have been identified in various vertebrate classes during the last years. The present review focuses on two novel RFamide peptides, Rana RFamide (R-RFa) and 26RFa, that have been recently isolated from the amphibian brain. R-RFa shares the C-terminal LPLRFamide motif with other RFamide peptides previously identified in mammals, birds and fish. The distribution of R-RFa in the frog brain exhibits strong similarities with those of other LPLRFamide peptides, notably in the periventricular region of the hypothalamus. There is also evidence that the physiological functions of R-RFa and other LPLRFamide peptides have been conserved from fish to mammals; in particular, all these peptides appear to be involved in the control of pituitary hormone secretion. 26RFa does not exhibit any significant structural identity with other RFamide peptides and this peptide is the only member of the family that possesses an FRFamide motif at its C-terminus. The strong conservation of the primary structure of 26RFa from amphibians to mammals suggests that this RFamide peptide is involved in important biological functions in vertebrates. As for several other RFamide peptides, 26RFa-containing neurons are present in the hypothalamus, notably in two nuclei involved in the control of feeding behavior. Indeed, 26RFa is a potent stimulator of appetite in mammals. Concurrently, recent data suggest that 26RFa exerts various neuroendocrine regulatory activities at the pituitary and adrenal level.

Identification of 26RFa from frog brain: a novel hypothalamic neuropeptide with orexigenic activity in mammals.

In the present study, we report the identification, in the frog brain, of a novel neuropeptide, termed 26RFa, that belongs to the RFamide peptide family. The cDNAs encoding the precursors for 26RFa have been characterized in human and rats. In rats, prepro-26RFa mRNA is expressed exclusively in two hypothalamic nuclei involved in the control of feeding behavior. Intracerebroventricular injection of 26RFa in mice induced a dose-dependent increase in food consumption. Taken together, these data indicate that 26RFa is a novel neuropeptide that may have important biological functions in vertebrates.

Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity.

A neuropeptide was isolated from a frog brain extract by HPLC purification and characterized by mass spectrometry. This 26-aa neuropeptide, which belongs to the RFamide peptide family, was designated 26RFa, and its primary structure was established as VGTALGSLAEELNGYNRKKGGFSFRF-NH2. Research in databases revealed the presence of sequences homologous to frog 26RFa in the human genome and in rat ESTs. On the basis of this sequence information, the cDNAs encoding the human and rat 26RFa precursors were cloned. The two preproteins show a similar organization, with the 26RFa sequence located in the C-terminal region of the precursor. Human preprotein (prepro)-26RFa encodes an additional putative RFamide peptide that is not found in the rat precursor. The primary structures of human, rat, and frog 26RFa exhibit approximately 80% identity, and the C-terminal octapeptide has been fully conserved from amphibians to mammals. In situ hybridization histochemistry revealed that, in the rat brain, the 26RFa gene is exclusively expressed in the ventromedial hypothalamic nucleus and in the lateral hypothalamic area. 26RFa induced a dose-dependent stimulation in cAMP production by rat pituitary cells in vitro and markedly increased food intake in mice. The conservation of the primary structure of 26RFa during vertebrate evolution, the discrete localization of the mRNA encoding its precursor in hypothalamic nuclei involved in the control of feeding behavior, and the observation that 26RFa possesses orexigenic properties indicate that this neuropeptide may play important biological functions.

PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells.

To determine the possible role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the development of the sympathoadrenal cell lineage, we have examined the effects of this neurotrophic peptide, in comparison to nerve growth factor (NGF), on the morphology, electrophysiological properties, expression of neuronal and neuroendocrine marker genes, and activity of transcription factors during differentiation of sympathoadrenal-derived cells, using the rat pheochromocytoma PC12 cell model. Both PACAP and NGF elicited rapid neurite outgrowth, which was accompanied by induction of cell excitability and the development of both sodium and calcium currents. Concurrently, PACAP and NGF increased the expression of a marker of synaptic vesicles. By contrast, PACAP, but not NGF, regulated the expression of different constituents of neuroendocrine large dense core vesicles in PC12 cells. Furthermore, PACAP and NGF differentially regulated the expression of mammalian achaete-scute homologue and paired homeobox 2b genes, transcription factors instrumental for sympathoadrenal development. To compare downstream effectors activated by PACAP and NGF, we studied the effects of these factors on the binding activity of consensus 12-O-tetradecanoylphorbol-13-acetate- and cAMP-responsive elements to nuclear extracts of differentiating PC12 cells. We found that both PACAP and NGF markedly increase the binding activity of these cis-regulatory sequences and that PACAP preferentially recruits activator protein-1-like transcription factors to these elements. Taken together, these results show that PACAP and NGF exert common as well as different effects on neuronal and neuroendocrine traits in differentiating PC12 cells, strongly suggesting that these two trophic factors could play complementary roles in the development of the sympathoadrenal cell lineage.