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The Nutrient and Energy Sensor Sirt1 Regulates the Hypothalamic-Pituitary-Adrenal (HPA) Axis by Altering the Production of the Prohormone Convertase 2 (PC2) Essential in the Maturation of Corticotropin-releasing Hormone (CRH) from Its Prohormone in Male Rats*

  • Anika M. Toorie
    Affiliations
    From the Division of Endocrinology, Department of Medicine, The Warren Alpert Medical School of Brown University/Rhode Island Hospital, Providence, Rhode Island 02903,

    the Graduate Program in Pathobiology and
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  • Nicole E. Cyr
    Affiliations
    From the Division of Endocrinology, Department of Medicine, The Warren Alpert Medical School of Brown University/Rhode Island Hospital, Providence, Rhode Island 02903,

    the Biology Department and Neuroscience Program, Stonehill College, Easton, Massachusetts 02357
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  • Jennifer S. Steger
    Affiliations
    From the Division of Endocrinology, Department of Medicine, The Warren Alpert Medical School of Brown University/Rhode Island Hospital, Providence, Rhode Island 02903,
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  • Ross Beckman
    Affiliations
    From the Division of Endocrinology, Department of Medicine, The Warren Alpert Medical School of Brown University/Rhode Island Hospital, Providence, Rhode Island 02903,
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  • George Farah
    Affiliations
    the Biology Department and Neuroscience Program, Stonehill College, Easton, Massachusetts 02357
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  • Eduardo A. Nillni
    Correspondence
    To whom correspondence should be addressed: Brown University. 40 Pine Grove Ave, Sharon, MA 02067. Tel.: 781-784-8525 or 617-755-3621;
    Affiliations
    From the Division of Endocrinology, Department of Medicine, The Warren Alpert Medical School of Brown University/Rhode Island Hospital, Providence, Rhode Island 02903,

    Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02903, and
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  • Author Footnotes
    * This work was supported by National Institutes of Health GrantwR01 DK085916 and DK085916S from NIDDK (to E.A.N.) and the Dr. George A. Bray Research Scholars Award (to N. E. C.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Open AccessPublished:January 11, 2016DOI:https://doi.org/10.1074/jbc.M115.675264
      Understanding the role of hypothalamic neuropeptides and hormones in energy balance is paramount in the search for approaches to mitigate the obese state. Increased hypothalamic-pituitary-adrenal axis activity leads to increased levels of glucocorticoids (GC) that are known to regulate body weight. The axis initiates the production and release of corticotropin-releasing hormone (CRH) from the paraventricular nucleus (PVN) of the hypothalamus. Levels of active CRH peptide are dependent on the processing of its precursor pro-CRH by the action of two members of the family of prohormone convertases 1 and 2 (PC1 and PC2). Here, we propose that the nutrient sensor sirtuin 1 (Sirt1) regulates the production of CRH post-translationally by affecting PC2. Data suggest that Sirt1 may alter the preproPC2 gene directly or via deacetylation of the transcription factor Forkhead box protein O1 (FoxO1). Data also suggest that Sirt1 may alter PC2 via a post-translational mechanism. Our results show that Sirt1 levels in the PVN increase in rats fed a high fat diet for 12 weeks. Furthermore, elevated Sirt1 increased PC2 levels, which in turn increased the production of active CRH and GC. Collectively, this study provides the first evidence supporting the hypothesis that PVN Sirt1 activates the hypothalamic-pituitary-adrenal axis and basal GC levels by enhancing the production of CRH through an increase in the biosynthesis of PC2, which is essential in the maturation of CRH from its prohormone, pro-CRH.

      Introduction

      Since its discovery in 1981 (
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      Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and β-endorphin.
      ), corticotropin-releasing hormone (CRH)
      The abbreviations used are: CRH, corticotropin-releasing hormone; PVN, paraventricular nucleus; GC, glucocorticoid; HPA, hypothalamic-pituitary-adrenal; PC, prohormone convertase; qrtPCR, quantitative real time PCR; RIA, radioimmunoassay; DIO, diet-induced obese; T3, triiodothyronine; T4, thyroxine; AVP, arginine vasopressin; CPE, carboxypeptidase E; GC, glucocorticoid; HFD, high fat diet; ME, median eminence; POMC, pro-opiomelanocortin; ARC, arcuate nucleus of the hypothalamus; α-MSH, α-melanocyte-stimulating hormone; TRH, thyrotropin; releasing hormone.
      has been demonstrated to be involved in mediating various physiological processes, including those involved in organismal homeostasis. Renowned for its critical role in mediating the stress response (
      • Kovács K.J.
      CRH: the link between hormonal-, metabolic- and behavioral responses to stress.
      ), CRH functions to regulate metabolic, immunologic, and homeostatic changes both basally and under various pathologic conditions (
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      ). CRH heterogeneously expresses in the periphery and the brain with high expression in the hypothalamic paraventricular nucleus (PVN) (
      • Ziegler C.G.
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      ). Arginine vasopressin (AVP) is also produced in the PVN and acts to synergize CRH actions. It is the CRH that is produced in the medial parvocellular division of the PVN that functions as the central regulator of the HPA axis (
      • Aguilera G.
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      ).
      Levels of bioactive CRH are dependent on the post-translational processing of its precursor pro-CRH. Prohormone post-translational processing is the mechanism by which all peptide hormones become biologically active (
      • Nillni E.A.
      Regulation of prohormone convertases in hypothalamic neurons: implications for prothyrotropin-releasing hormone and proopiomelanocortin.
      ,
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      The proprotein convertases.
      ). In rodents and humans, aberrations in prohormone processing results in deleterious health consequences, including metabolic dysfunctions (
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      Deletion of the Nhlh2 transcription factor decreases the levels of the anorexigenic peptides α-melanocyte-stimulating hormone and thyrotropin-releasing hormone and implicates prohormone convertases I and II in obesity.
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      ). CRH is initially produced as a large inactive precursor, preproCRH, that is made of 196 amino acids (
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      • Rivier C.
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      Primary structure of corticotropin-releasing factor from ovine hypothalamus.
      ,
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      Characterization of rat hypothalamic corticotropin-releasing factor.
      ). After cleavage of the signal sequence, the prohormone (pro-CRH) enters the lumen of the rough endoplasmic reticulum and is routed to the trans-Golgi network where it undergoes enzymatic post-translational modifications to generate several intermediate forms as well as the bioactive CRH(1–41) peptide that is produced from the C-terminal region of the precursor polypeptide (
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      ). Specifically, pro-CRH is cleaved by prohormone convertase 2 (PC2) or PC1 at Arg-152–Arg-153, thereby releasing a 43-residue CRH peptide (
      • Perone M.J.
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      ,
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      ). The PC2 protease is synthesized as a zymogen that undergoes its own post-translational processing autocatalytic maturation within the secretory pathway. CRH is further processed into its bioactive form via the removal of the C-terminal lysine residue by the actions of carboxypeptidase E (CPE), and it is subsequently amidated at the exposed carboxyl group of a glycine residue by peptidylglycine hydroxylase (also referred to as peptidylglycine α-amidating monooxygenase) (Fig. 1) (
      • Eipper B.A.
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      ).
      Figure thumbnail gr1
      FIGURE 1.DIO rodents display increased PVN Sirt1 and basal GC. Rats were fed a diet high in fat for 12 weeks to generate DIO (gray bars) rodents, whereas lean controls were maintained on standard chow (black bars). A, lean and DIO rats were fasted for 24 h and sacrificed, and the PVN was collected and subjected to Western blot analysis for Sirt1 protein levels (n = 6/group). **, p < 0.01. B and C, lean and DIO rats were sacrificed either in the morning or mid-afternoon and trunk blood was collected and serum-extracted, and GC (ng/ml) was measured by RIA. B, 9 a.m. basal GC (n = 4 lean and 5 DIO). C, 2 p.m. basal GC (n = 7 lean and 10 DIO). D and E, lean rats were icv-infused twice with 5 μg of resveratrol or vehicle control at 9 a.m. and 4 p.m. on day 1. Rats were sacrificed at 9 a.m. the following day; serum was isolated from trunk blood and measured for GC. D, serum GC levels (n = 3/group). E, PVN was assessed for CRH mRNA (n = 4/group). F, pro-CRH processing cascade is modeled here and has been described previously. G–J, lean and DIO rats were icv-infused with 5 μg of Ex-527 or vehicle control at the beginning of the fasting period and again 8 h later. Rats were fasted for 24 h total and sacrificed, and the PVN was collected and processed for protein or mRNA analysis. G–L, PC2 protein and PC1 protein (n = 6/group) and CRH mRNA from the PVN of lean and DIO rats with or without central Sirt1 inhibition. G, PC2 in DIO PVN. H, PC1 in DIO PVN. I, CRH mRNA in DIO PVN (n = 3 vehicle and 4 Ex-527). J, PC2 in lean PVN. K, PC1 in lean PVN. L, CRH mRNA in lean PVN. (n = 4/group). Data are mean ± S.E. *, p value < 0.05.
      Under both basal and stimulated conditions, CRH produced in the PVN is released from nerve terminals that anteriorly juxtapose the median eminence where it is traversed into the hypothalamo-hypophyseal portal system (
      • Rho J.H.
      • Swanson L.W.
      Neuroendocrine CRF motoneurons: intrahypothalamic axon terminals shown with a new retrograde-Lucifer-immuno method.
      ). Upon binding to its cognate receptor, corticotropin-releasing hormone receptor 1 (CRHR1) (
      • Lovejoy D.A.
      • Chang B.S.
      • Lovejoy N.R.
      • del Castillo J.
      Molecular evolution of GPCRs: CRH/CRH receptors.
      ), which is expressed by corticotropic cells of the adenohypophysis, CRH stimulates the synthesis and secretion of adrenocorticotropic hormone (ACTH) as well as other bioactive molecules such as β-endorphin (
      • Solomon S.
      POMC-derived peptides and their biological action.
      ). ACTH engages the melanocortin 2 receptor expressed by cells of the adrenal cortex and stimulates the production and secretion of steroid hormones such as GC (
      • Veo K.
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      • Liang L.
      • Moser E.
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      Observations on the ligand selectivity of the melanocortin 2 receptor.
      ).
      Both ACTH and glucocorticoids (GCs) function to regulate the HPA axis activity via long and short negative feedback loops that signal at the level of the hypothalamus, extra-hypothalamic brain sites, and the adenohypophysis. CRH functions to promote negative energy balance by suppressing appetite and enhancing thermogenesis (
      • Richard D.
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      The corticotropin-releasing hormone system in the regulation of energy balance in obesity.
      ,
      • Mastorakos G.
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      The hypothalamic-pituitary-adrenal axis in the neuroendocrine regulation of food intake and obesity: the role of corticotropin-releasing hormone.
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      Long-term infusion of brain-derived neurotrophic factor reduces food intake and body weight via a corticotrophin-releasing hormone pathway in the paraventricular nucleus of the hypothalamus.
      ), and the GC functions to promote positive energy balance in part by affecting glucose metabolism, lipid homeostasis, and increasing appetite drive (
      • Tataranni P.A.
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      • Snitker S.
      • Young J.B.
      • Flatt J.P.
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      Effects of glucocorticoids on energy metabolism and food intake in humans.
      ). Although a consensus on the exact role of adrenal activity in relation to energy dysfunction has yet to be reached, increased and sustained basal GC is implicated in the development of visceral obesity, insulin resistance, and metabolic disease (
      • Laryea G.
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      ,
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      ).
      Silent mating type information regulation 2 homolog 1 (Sirt1) is a NAD+-dependent class III deacetylase that regulates gene expression and protein activity via the deacetylation of its effector targets, which include histones and transcription factors. Sirt1's dependence on NAD+ supports its role as an energy sensor (
      • Cantó C.
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      AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity.
      ), and several studies have demonstrated that Sirt1 is nutrient-sensitive and plays a role in energy balance both in the periphery and in the brain (
      • Cakir I.
      • Perello M.
      • Lansari O.
      • Messier N.J.
      • Vaslet C.A.
      • Nillni E.A.
      Hypothalamic Sirt1 regulates food intake in a rodent model system.
      • Dietrich M.O.
      • Antunes C.
      • Geliang G.
      • Liu Z.W.
      • Borok E.
      • Nie Y.
      • Xu A.W.
      • Souza D.O.
      • Gao Q.
      • Diano S.
      • Gao X.B.
      • Horvath T.L.
      Agrp neurons mediate Sirt1's action on the melanocortin system and energy balance: roles for Sirt1 in neuronal firing and synaptic plasticity.
      ,
      • Ramadori G.
      • Lee C.E.
      • Bookout A.L.
      • Lee S.
      • Williams K.W.
      • Anderson J.
      • Elmquist J.K.
      • Coppari R.
      Brain SIRT1: anatomical distribution and regulation by energy availability.
      • Kanfi Y.
      • Peshti V.
      • Gozlan Y.M.
      • Rathaus M.
      • Gil R.
      • Cohen H.Y.
      Regulation of SIRT1 protein levels by nutrient availability.
      ). Studies from our laboratory and others had demonstrated that reducing central Sirt1 level or activity resulted in decreased food intake and body weight gain in lean rodents (
      • Cakir I.
      • Perello M.
      • Lansari O.
      • Messier N.J.
      • Vaslet C.A.
      • Nillni E.A.
      Hypothalamic Sirt1 regulates food intake in a rodent model system.
      ,
      • Dietrich M.O.
      • Antunes C.
      • Geliang G.
      • Liu Z.W.
      • Borok E.
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      • Xu A.W.
      • Souza D.O.
      • Gao Q.
      • Diano S.
      • Gao X.B.
      • Horvath T.L.
      Agrp neurons mediate Sirt1's action on the melanocortin system and energy balance: roles for Sirt1 in neuronal firing and synaptic plasticity.
      ,
      • Cyr N.E.
      • Steger J.S.
      • Toorie A.M.
      • Yang J.Z.
      • Stuart R.
      • Nillni E.A.
      Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.
      ), whereas pharmacological activation of ARC Sirt1 resulted in increased food intake and body weight gain (
      • Cakir I.
      • Perello M.
      • Lansari O.
      • Messier N.J.
      • Vaslet C.A.
      • Nillni E.A.
      Hypothalamic Sirt1 regulates food intake in a rodent model system.
      ).
      Moreover, inhibition of central Sirt1 activity conferred an anorectic response specifically via enhanced melanocortin signaling and melanocortinergic tone, respectively, (
      • Cakir I.
      • Perello M.
      • Lansari O.
      • Messier N.J.
      • Vaslet C.A.
      • Nillni E.A.
      Hypothalamic Sirt1 regulates food intake in a rodent model system.
      ,
      • Dietrich M.O.
      • Antunes C.
      • Geliang G.
      • Liu Z.W.
      • Borok E.
      • Nie Y.
      • Xu A.W.
      • Souza D.O.
      • Gao Q.
      • Diano S.
      • Gao X.B.
      • Horvath T.L.
      Agrp neurons mediate Sirt1's action on the melanocortin system and energy balance: roles for Sirt1 in neuronal firing and synaptic plasticity.
      ). In addition, we demonstrated that Sirt1 protein is increased in the ARC of diet-induced-obesity (DIO) rodents. In that same study, central inhibition of Sirt1 activity enhanced melanocortin signaling, increased thyroid activity and energy expenditure, and was associated with reduced overnight body weight gain independent of alterations in food consumption (
      • Cyr N.E.
      • Steger J.S.
      • Toorie A.M.
      • Yang J.Z.
      • Stuart R.
      • Nillni E.A.
      Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.
      ). In another study, pan-neuronal knock-out (limited to neurons of the central nervous system) of functional Sirt1 resulted in enhanced peripheral and central insulin sensitivity (
      • Lu M.
      • Sarruf D.A.
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      • Osborn O.
      • Sanchez-Alavez M.
      • Talukdar S.
      • Chen A.
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      Neuronal Sirt1 deficiency increases insulin sensitivity in both brain and peripheral tissues.
      ). Collectively, these findings suggest that reducing hypothalamic Sirt1 activity can promote negative energy balance and protect against the development of type 2 diabetes and obesity.
      In this study, we investigated the role of PVN Sirt1 on CRH production from its precursor under different nutritional conditions and the resulting impact on energy balance. We utilized a combination of in vitro experiments as well as an in vivo model wherein we pharmacologically manipulated central and PVN Sirt1 activity in lean rats and the Sprague-Dawley model of adult-onset DIO.

      Discussion

      This study shows the first evidence supporting the hypothesis that PVN Sirt1 activates the HPA axis and basal GC levels by enhancing the production of CRH through an increase in the biosynthesis of PC2, which is essential in the maturation of CRH from its prohormone, pro-CRH. Additionally, in the DIO state PVN Sirt1 increases basal (not stress-induced) circulating GC in a manner independent of preproCRH transcriptional changes. Instead, Sirt1's effects on adrenal activity are mediated via a post-translational processing mechanism in concert with an increase in PC2. Increased CRH release from the PVN increased pituitary ACTH synthesis and release, and in turn it increased circulating basal GC concentrations (FIGURE 1., FIGURE 10.). Using the combination of two in vitro systems (N43 and AtT20 cells), where Sirt1 was either cDNA overexpressed or inhibited by shRNA, we validated the proposed hypothesis that increased Sirt1 activity positively regulates PC2. Our in vivo studies support the assertion that Sirt1 significantly regulates PVN PC2. We also show that Sirt1 increased CRH production and release (Figs. 3, E–H, and 4, A–H). Although these results demonstrate that Sirt1 regulates PC1 and PC2 expression in a positive manner, we did not investigate the independent contributions of each convertase on the Sirt1-mediated processing of pro-CRH. However, it is worthy of noting that although AtT20 cells have been used to characterize the processing of pro-CRH by PC1, these cells are adrenocorticotropic in nature and can be influenced by CRH signaling (
      • Castro M.G.
      • Brooke J.
      • Bullman A.
      • Hannah M.
      • Glynn B.P.
      • Lowry P.J.
      Biosynthesis of corticotrophin-releasing hormone (CRH) in mouse corticotrophic tumour cells expressing the human pro-CRH gene: intracellular storage and regulated secretion.
      ). Thus, Sirt1's effect on PC1 may be a secondary consequence to changes in secreted CRH in the in vitro system employed.
      Our in vivo data demonstrate that stimulating Sirt1 activity in the PVN of lean (Fig. 5) and DIO (Fig. 6) rodents results in elevated serum basal ACTH and GC levels. We were unable to assess PVN and ME for CRH content; however, we assessed the anterior pituitaries for changes in POMC, CPE, and PC1 as their expressions are induced in corticotroph cells upon CRH-mediated stimulation of the CRHR1. We observed an increase in POMC, PC1, and CPE expressed in the anterior pituitary of rodents with PVN Sirt1 stimulation. Together, these findings suggest that increasing PVN Sirt1 activity results in an increase in the amount of CRH targeted to the anterior pituitary, thereby enhancing ACTH signaling and basal GC concentration. Furthermore, our results demonstrate that inhibiting Sirt1 specifically in the PVN of obese rodents caused a reduction in PVN pro-PC2 and active forms of PC2, but not PC1, protein (Fig. 8A). This decrease in PC2 was associated with a decrease in CRH in the ME (Fig. 8E), as well as reduced circulating GC (Fig. 8F), effects that were not observed in lean individuals (Fig. 7). In vitro data support these finding as increasing Sirt1 activity enhanced PC2 levels and CRH production, whereas diminished Sirt1 activity decreased PC2 levels and CRH production.
      In terms of the possible mechanism regulating PC2, we found that the increase of the transcription factor FoxO1 in N43 cells up-regulated PC2 protein but not PC1. In further support, we showed that Sirt1 inhibition reduced the levels of deacetylated FoxO1 along with PC2 levels suggesting an important role for FoxO1 in regulating PC2. However, because FoxO1 also functions as a positive transcriptional regulator of Sirt1 (Fig. 9H), we were unable to reconcile whether these effects were a direct effect or a secondary effect of altered Sirt1 levels. Thus, we cannot exclude the possibility that FoxO1 acts upstream of Sirt1 to alter PC2. Future studies will investigate the extent to which each of these mechanisms regulates PC2 and CRH levels. Overall, our data suggest that Sirt1 in the DIO PVN specifically increases PC2, which increases CRH to ultimately elevate circulating levels of basal GC. Our preliminary results from PC2 promoter activity studies suggested that Sirt1 up-regulated PC2 (data not shown), which may suggest a direct action of Sirt1 on PC2 in addition to FoxO1 either acting on Sirt1 or directly on PC2. However, gene expression may not fully reflect altered protein or peptide content. For example, hypothalamic Sirt1 protein is increased during calorie deprivation, yet the Sirt1 transcript is unaltered (
      • Cakir I.
      • Perello M.
      • Lansari O.
      • Messier N.J.
      • Vaslet C.A.
      • Nillni E.A.
      Hypothalamic Sirt1 regulates food intake in a rodent model system.
      ,
      • Ramadori G.
      • Lee C.E.
      • Bookout A.L.
      • Lee S.
      • Williams K.W.
      • Anderson J.
      • Elmquist J.K.
      • Coppari R.
      Brain SIRT1: anatomical distribution and regulation by energy availability.
      ). In addition, preproPC2 mRNA, but not protein, is reduced under conditions of nutrient depletion (
      • Sanchez V.C.
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      • Bjorbaek C.
      • Nillni E.A.
      Regulation of hypothalamic prohormone convertases 1 and 2 and effects on processing of prothyrotropin-releasing hormone.
      ). Results of this study reveal that Sirt1 stimulation induced the expression of PC1 and PC2 at the protein level, which complements the promoter activity data but not at the same ratio. This is supported by the well known fact that changes in promoter activity do not necessarily correlate directly to protein biosynthesis, a mechanism that is mostly controlled by a cell biology mechanism (folding, post-translational modifications, intracellular traffic, etc.). In addition to evidence that Sirt1 alters PC1 and PC2 promotor activity, we also observed that augmented Sirt1 increases endogenous levels of both pro-PC1 and pro-PC2 in N43-5 cells. The Sirt1-induced changes in PC2 using AtT20 cells further suggest that Sirt1 may affect PC2 at a post-translational level by an unknown mechanism. Additionally, central inhibition of Sirt1 caused a decrease in both pro-PC2 and the active form of PC2 in the DIO PVN, but the magnitude of decrease in active PC2 was greater (Fig. 8N). In addition, the maturation of PC2 to its active form can be regulated by many factors, including levels and activity of the “chaperone”-like protein 7B2 and pH conditions in the secretory pathway. Future studies will explore how Sirt1 affects PC2 maturation.
      Because we observed a significant decrease in plasma GC levels with Sirt1 inhibition in the PVN, we also assessed whether reduced adrenal activity would confer changes in energy balance. However, potentially due to the acute experimental paradigms used, we did not observe alterations in overnight food intake or energy expenditure (Fig. 8, K and L), yet these animals exhibited a net loss in body weight (Fig. 8M). We were unable to reconcile the cause of body weight loss in this study; however, future studies will investigate the mechanism mediating this effect. The aforementioned observation is in contrast to the reported effects of acute central Sirt1 manipulation, long term ARC Sirt1 knockdown, and chronic cell type-specific Sirt1 ablation models, wherein Sirt1 activity altered energy balance and body weight by affecting food intake, energy expenditure, sympathetic tone, and body fat composition (
      • Cakir I.
      • Perello M.
      • Lansari O.
      • Messier N.J.
      • Vaslet C.A.
      • Nillni E.A.
      Hypothalamic Sirt1 regulates food intake in a rodent model system.
      ,
      • Dietrich M.O.
      • Antunes C.
      • Geliang G.
      • Liu Z.W.
      • Borok E.
      • Nie Y.
      • Xu A.W.
      • Souza D.O.
      • Gao Q.
      • Diano S.
      • Gao X.B.
      • Horvath T.L.
      Agrp neurons mediate Sirt1's action on the melanocortin system and energy balance: roles for Sirt1 in neuronal firing and synaptic plasticity.
      ,
      • Cyr N.E.
      • Steger J.S.
      • Toorie A.M.
      • Yang J.Z.
      • Stuart R.
      • Nillni E.A.
      Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.
      ,
      • Lu M.
      • Sarruf D.A.
      • Li P.
      • Osborn O.
      • Sanchez-Alavez M.
      • Talukdar S.
      • Chen A.
      • Bandyopadhyay G.
      • Xu J.
      • Morinaga H.
      • Dines K.
      • Watkins S.
      • Kaiyala K.
      • Schwartz M.W.
      • Olefsky J.M.
      Neuronal Sirt1 deficiency increases insulin sensitivity in both brain and peripheral tissues.
      ,
      • Ramadori G.
      • Fujikawa T.
      • Anderson J.
      • Berglund E.D.
      • Frazao R.
      • Michán S.
      • Vianna C.R.
      • Sinclair D.A.
      • Elias C.F.
      • Coppari R.
      SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance.
      • Ramadori G.
      • Fujikawa T.
      • Fukuda M.
      • Anderson J.
      • Morgan D.A.
      • Mostoslavsky R.
      • Stuart R.C.
      • Perello M.
      • Vianna C.R.
      • Nillni E.A.
      • Rahmouni K.
      • Coppari R.
      SIRT1 deacetylase in POMC neurons is required for homeostatic defenses against diet-induced obesity.
      ,
      • Sasaki T.
      • Kikuchi O.
      • Shimpuku M.
      • Susanti V.Y.
      • Yokota-Hashimoto H.
      • Taguchi R.
      • Shibusawa N.
      • Sato T.
      • Tang L.
      • Amano K.
      • Kitazumi T.
      • Kuroko M.
      • Fujita Y.
      • Maruyama J.
      • Lee Y.S.
      • et al.
      Hypothalamic SIRT1 prevents age-associated weight gain by improving leptin sensitivity in mice.
      • Sasaki T.
      • Kim H.J.
      • Kobayashi M.
      • Kitamura Y.I.
      • Yokota-Hashimoto H.
      • Shiuchi T.
      • Minokoshi Y.
      • Kitamura T.
      Induction of hypothalamic Sirt1 leads to cessation of feeding via agouti-related peptide.
      ). Moreover, several metabolic parameters associated with energy homeostasis were also affected in the aforementioned studies, including glucose homeostasis, insulin and leptin signaling, and adipose tissue dynamics. As glucocorticoids play a critical role in glucose and insulin homeostasis, we assessed whether PVN Sirt1's effect on adrenal activity also influenced these parameters.
      The results indicate that inhibition of PVN Sirt1 activity did not significantly alter sated glucose levels; however, there was a trend for decreased glucose in DIO rodents with PVN Sirt1 inhibition in DIO (Fig. 8I) individuals. However, it resulted in a significant increase in sated serum insulin in obese rats (Fig. 8J) in the face of reduced basal GC (Fig. 8F), an observation that was not evident in lean rats (Fig. 7J). Although not assessed in this study, we speculate that a rise in sated insulin concentration may serve to diminish elevated circulating glucose levels with time and may precede enhanced insulin sensitivity. Indeed, Lu et al. (
      • Lu M.
      • Sarruf D.A.
      • Li P.
      • Osborn O.
      • Sanchez-Alavez M.
      • Talukdar S.
      • Chen A.
      • Bandyopadhyay G.
      • Xu J.
      • Morinaga H.
      • Dines K.
      • Watkins S.
      • Kaiyala K.
      • Schwartz M.W.
      • Olefsky J.M.
      Neuronal Sirt1 deficiency increases insulin sensitivity in both brain and peripheral tissues.
      ) demonstrated that a loss of functional Sirt1 in neurons of the central nervous system enhanced both brain and peripheral insulin sensitivity and reversed the hyperglycemia associated with obesity. However, Knight et al. (
      • Knight C.M.
      • Gutierrez-Juarez R.
      • Lam T.K.
      • Arrieta-Cruz I.
      • Huang L.
      • Schwartz G.
      • Barzilai N.
      • Rossetti L.
      Mediobasal hypothalamic SIRT1 is essential for resveratrol's effects on insulin action in rats.
      ) demonstrated that an acute increase in the activity of Sirt1 in the medial ARC of lean rats was sufficient to improve glucose homeostasis and increase insulin sensitivity. Collectively, these findings support a role of brain Sirt1 in regulating insulin and glucose homeostasis and point to region- and cell-specific influences in this regulation. Future studies are aimed at chronically reducing PVN Sirt1 activity to ascertain whether this will indeed alter glucose and insulin dynamics and/or have a substantial effect on energy balance.
      In vivo data from other laboratories strongly support a role of PC2 in modulating CRH production. Under basal conditions, CRH neurons primarily express PC2, which is the likely PC responsible for the enzymatic processing of pro-CRH into CRH (
      • Dong W.
      • Seidel B.
      • Marcinkiewicz M.
      • Chrétien M.
      • Seidah N.G.
      • Day R.
      Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids.
      ). However, CRH expressing neurons do express minute levels of PC1, and its expression is up-regulated under conditions of acute stress and glucocorticoid insufficiency (
      • Ge J.F.
      • Peng L.
      • Cheng J.Q.
      • Pan C.X.
      • Tang J.
      • Chen F.H.
      • Li J.
      Antidepressant-like effect of resveratrol: involvement of antioxidant effect and peripheral regulation on HPA axis.
      ). In addition, arginine vasopressin (AVP) cells of the PVN also express PC1, and both PC2 and PC1 equally processes pro-AVP (
      • Ge J.F.
      • Peng L.
      • Cheng J.Q.
      • Pan C.X.
      • Tang J.
      • Chen F.H.
      • Li J.
      Antidepressant-like effect of resveratrol: involvement of antioxidant effect and peripheral regulation on HPA axis.
      ). We did not investigate the involvement of AVP in the context of Sirt1 regulation of the HPA axis in this study. However, it is possible that Sirt1's positive regulation of the PCs enhances basal adrenal activity by affecting both CRH and AVP production, although the latter remains unknown.
      The observation that no changes in PVN PC1 protein in rodents with central Sirt1 inhibition (Fig. 1, H and K) and in contrast to our in vitro results may be explained by the following. As aforementioned, DIO rats with central Sirt1 inhibition display increased α-MSH that results in enhanced activation of TRH neurons (
      • Cyr N.E.
      • Steger J.S.
      • Toorie A.M.
      • Yang J.Z.
      • Stuart R.
      • Nillni E.A.
      Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.
      ). Furthermore, PC1 is enhanced upon stimulation of the TRH neuron (
      • Sanchez V.C.
      • Goldstein J.
      • Stuart R.C.
      • Hovanesian V.
      • Huo L.
      • Munzberg H.
      • Friedman T.C.
      • Bjorbaek C.
      • Nillni E.A.
      Regulation of hypothalamic prohormone convertases 1 and 2 and effects on processing of prothyrotropin-releasing hormone.
      ). As we failed to examine cell-specific fluctuations in PC1 and PC2 upon Sirt1 manipulation, and instead analyzed PVN micropunches for PC content, it is possible that a down-regulation in PC1 (presumably in CRH neurons) due to central Sirt1 inhibition may be offset by α-MSH's induction of PVN PC1 (presumably in TRH neurons). Alternatively, glucocorticoid receptor resistance has also been reported in obese individuals (
      • de Guia R.M.
      • Rose A.J.
      • Herzig S.
      Glucocorticoid hormones and energy homeostasis.
      ,
      • Jessop D.S.
      • Dallman M.F.
      • Fleming D.
      • Lightman S.L.
      Resistance to glucocorticoid feedback in obesity.
      ), and a study revealed that adrenalectomy increased the expression of PC1 in hypophysiotropic CRH neurons, an effect that was blunted via administration of exogenous GCs (
      • Dong W.
      • Seidel B.
      • Marcinkiewicz M.
      • Chrétien M.
      • Seidah N.G.
      • Day R.
      Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids.
      ). We have shown previously that Sirt1 in the ARC regulates, at the central level, the thyroid axis via its actions on several components of the central melanocortin system (
      • Cakir I.
      • Perello M.
      • Lansari O.
      • Messier N.J.
      • Vaslet C.A.
      • Nillni E.A.
      Hypothalamic Sirt1 regulates food intake in a rodent model system.
      ,
      • Cyr N.E.
      • Steger J.S.
      • Toorie A.M.
      • Yang J.Z.
      • Stuart R.
      • Nillni E.A.
      Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.
      ). Our prior studies revealed that central Sirt1 inhibition increased ARC α-MSH production, an effect that was mediated via increased ARC CPE protein. This resulted in an increase in hypophysiotropic thyroid activity in obese individuals; however, we failed to investigate its effect on the HPA axis in those prior studies.
      α-MSH is also targeted to CRH neurons of the PVN and augments energy balance in part by activation of CRH neurons, which is mediated by transcription of the CRH gene (
      • Dhillo W.S.
      • Small C.J.
      • Seal L.J.
      • Kim M.S.
      • Stanley S.A.
      • Murphy K.G.
      • Ghatei M.A.
      • Bloom S.R.
      The hypothalamic melanocortin system stimulates the hypothalamo-pituitary-adrenal axis in vitro and in vivo in male rats.
      ,
      • Fekete C.
      • Légrádi G.
      • Mihály E.
      • Tatro J.B.
      • Rand W.M.
      • Lechan R.M.
      α-Melanocyte stimulating hormone prevents fasting-induced suppression of corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus.
      ). Thus, in this study, we indirectly assessed whether α-MSH was mediating the effects of central Sirt1 on the hypophysiotropic adrenal axis by assessing CRH mRNA in PVN. There was no difference in CRH mRNA levels from the PVN of individuals centrally infused to inhibit Sirt1 activity when compared with their vehicle-infused controls, suggesting that α-MSH's induction of CRH transcription did not occur. α-MSH's effect on energy balance, particularly feeding, is in part facilitated through CRH signaling (
      • Ramadori G.
      • Fujikawa T.
      • Anderson J.
      • Berglund E.D.
      • Frazao R.
      • Michán S.
      • Vianna C.R.
      • Sinclair D.A.
      • Elias C.F.
      • Coppari R.
      SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance.
      ). As we observed no effect of central Sirt1 inhibition on feeding in DIO rodents (
      • Cyr N.E.
      • Steger J.S.
      • Toorie A.M.
      • Yang J.Z.
      • Stuart R.
      • Nillni E.A.
      Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.
      ), it is likely that α-MSH was preferentially targeted to TRH neurons as evidenced by enhanced thyroid activity and energy expenditure (
      • Cyr N.E.
      • Steger J.S.
      • Toorie A.M.
      • Yang J.Z.
      • Stuart R.
      • Nillni E.A.
      Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.
      ), a proposition that is under current investigation. In addition to its regulation of the hypophysiotropic thyroid axis, a recent study has revealed a role of central Sirt1 in regulating the hypophysiotropic gonadal axis. Sirt1 was shown to mediate Leydig and Sertoli cell maturity by regulating steroidogenic gene expression and, in particular, at the central level by increasing the expression of gonadotropin-releasing hormone, thereby resulting in increased circulating luteinizing hormone and intratesticular testosterone levels (
      • Kolthur-Seetharam U.
      • Teerds K.
      • de Rooij D.G.
      • Wendling O.
      • McBurney M.
      • Sassone-Corsi P.
      • Davidson I.
      The histone deacetylase SIRT1 controls male fertility in mice through regulation of hypothalamic-pituitary gonadotropin signaling.
      ).
      Another study (
      • Ge J.F.
      • Peng L.
      • Cheng J.Q.
      • Pan C.X.
      • Tang J.
      • Chen F.H.
      • Li J.
      Antidepressant-like effect of resveratrol: involvement of antioxidant effect and peripheral regulation on HPA axis.
      ) revealed that a peripheral administration of resveratrol resulted in reduced GC in a chronic unpredictable mild stress model that is characterized by hyperactivity of the HPA axis. Although it is possible that resveratrol could act through Sirt1 to mediate the aforementioned effects, the investigators did not examine Sirt1's involvement in this process. Sirt1's involvement in down-regulating HPA activity at first would seem to contradict the observations presented in this study; however, the down-regulatory effects of resveratrol on hypophysiotropic adrenal activity was attributed by the authors of that study to resveratrol action in the periphery rather than in the brain (
      • Ge J.F.
      • Peng L.
      • Cheng J.Q.
      • Pan C.X.
      • Tang J.
      • Chen F.H.
      • Li J.
      Antidepressant-like effect of resveratrol: involvement of antioxidant effect and peripheral regulation on HPA axis.
      ). Sirt1, like many other metabolic sensors (i.e. AMP-activated kinase and mechanistic target of rapamycin), has opposing roles in the brain versus its functions in the periphery. Generally, Sirt1 action in the periphery functions to promote a negative energy balance and to protect against the development of metabolic dysfunctions and obesity. In the hypothalamus, Sirt1's role remains opaque, yet a number of studies have demonstrated central Sirt1 to promote a positive energy balance. Future studies will employ the use of genetic models to ascertain the specific role of Sirt1 expressed in CRH neurons of the PVN in regard to energy balance and its regulation of the HPA axis. Collectively, the findings demonstrate that Sirt1 regulates the CRH peptide by modulating the processing enzyme PC2 and FoxO1 adding a novel regulatory link between PVN Sirt1 and HPA axis activity.

      Author Contributions

      E. A. N., A. M. T., and N. E. C. designed the study and wrote the paper. A. M. T. and N. E. C. designed, performed, and analyzed the experiments shown in FIGURE 1., FIGURE 9.. A. M. T. and N. E. C. designed and R. B. performed and analyzed the experiments in Fig. 2. A. M. T. and E. A. N. designed, performed, and analyzed the experiments in FIGURE 3., FIGURE 4., FIGURE 5., FIGURE 6., FIGURE 7., FIGURE 8.. J. S. S. and G. F. provided technical assistance and contributed to the preparation of figures. All authors reviewed the results and approved the final version of the manuscript. The final revision was done and approved by E. A. N.

      Acknowledgments

      We thank Ronald Stuart, Claudia Arevalo, Lindsay Steele, Katherine Barcay, and Ashleigh Burton for technical assistance.

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