Proopiomelanocortin mRNA Levels Research Articles

Seasonal changes in body mass, serum leptin levels and hypothalamic neuropeptide gene expression in male Eothenomys olitor

The present study examined seasonal changes in body mass and energy metabolism in the Chaotung vole (Eothenomys olitor) and the physiological mechanisms underpinning these changes. Seasonal changes in the following parameters were measured in male E. olitor, body mass, food intake, thermogenesis, enzyme activity, masses of tissues and organs, hormone concentrations and expression of hypothalamic arcuate nucleus energy balance genes including neuropeptide Y (NPY), agouti-related protein (AgRP), pro-opiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART). Body mass was constant over the year, but the masses of tissues and organs differed significantly between seasons. There were significant changes in body fat mass and serum leptin levels over the four seasons. E. olitor showed significant seasonal changes in food intake and thermogenesis, uncoupling protein 1 (UCP1) content, enzyme activity, and serum tri-iodothyronine (T3) and thyroxine (T4) levels. Moreover, mRNA expression in the hypothalamus showed significant seasonal changes. All of our results suggested that E. olitor had constant body mass over the year, which was inconsistent with the prediction of the ‘set-point’ hypothesis. However, body fat mass and serum leptin levels were significantly different among the four seasons, providing support for the ‘set-point’ hypothesis. The changes in leptin, NPY, AgRP, POMC, and CART mRNA levels may play a role in the regulation of energy intake in E. olitor. Furthermore, the role of leptin and hypothalamic neuropeptide gene in the regulation of energy metabolism and body mass may be different in animals that are acclimated to different seasons.

Persistent increases in rat hypothalamic POMC gene expression following chronic withdrawal from chronic “binge” pattern escalating-dose, but not steady-dose, cocaine

Recent research suggests an involvement of pro-opiomelanocortin (POMC) gene products (e.g., beta-endorphin) in modulating cocaine-induced reward and addiction-like behaviors in rodents. In this study, we investigated whether chronic “binge” cocaine and its withdrawal altered POMC gene expression in the brain of rats. Male Fischer rats were treated with two different chronic (14-day) “binge” pattern cocaine administration regimens (three injections at 1-h intervals, i.p.): steady-dose (45mg/kg/day) and escalating-dose (90mg/kg on the last day). Although there was no POMC mRNA alteration after chronic steady-dose cocaine, a significant decrease in POMC mRNA levels in the hypothalamus was found after chronic escalating-dose cocaine. In contrast, after acute (1-day) withdrawal from chronic “binge” escalating-dose regimen, but not steady-dose regimen, there were increased hypothalamic POMC mRNA levels that persisted into 14days of protracted withdrawal. To study the role of the endogenous opioid systems in the cocaine withdrawal effects, we administered a single naloxone injection (1mg/kg) that caused elevated POMC mRNA levels observed 24h later in cocaine naïve rats, but it did not lead to further increases in cocaine-withdrawn rats. Our results suggest that during withdrawal from chronic “binge” escalating-dose cocaine: (1) there was a persistent increase in hypothalamic POMC gene expression; and (2) hyposensitivity of the POMC gene expression to naloxone indicates altered opioidergic tone at or above the hypothalamic level.

Increased prepubertal body weight enhances leptin sensitivity in proopiomelanocortin and neuropeptide y neurons before puberty onset in female rats.

Pubertal onset may be advanced by obesity, with leptin potentially acting as a permissive factor. We hypothesized that having increased body weight (BW) prepubertally affects the ability of leptin to activate intracellular signaling pathways and modulate the expression of hypothalamic neuropeptides involved in reproduction and metabolism. Because being raised in small litters (SLs) tends to increase BW at weaning, female rats were raised in litters of 4 or large litters (LLs) of 12 pups. Leptin (3 μg/g BW) or vehicle (saline) was injected sc at postnatal day (PND) 21 and 30. Rats raised in SLs weighed more at both ages, but relative visceral and subcutaneous fat was increased only on PND21. Serum leptin levels were not different at PND21 or PND30. At PND21, key elements of intracellular leptin signaling (phosphorylated signal transducer and activator of transcription 3 and phosphorylated Akt [p-Akt]) were lower in SL than in LL rats. Leptin injection stimulated phosphorylated signal transducer and activator of transcription 3 in both groups, with a greater increase in LL, whereas p-Akt rose only in SL rats. At PND30, basal leptin signaling did not differ between LL and SL rats. Leptin activation of Akt was similar at 45 minutes, but at 2 hours p-AKT levels were higher in SL than in LL rats, as was the decrease in neuropeptide Y mRNA and increase in pro-opiomelanocortin mRNA levels. No change in the reproductive axis was found. Thus, being raised in SLs increases BW and visceral body fat content, fails to increase plasma leptin concentrations, and increases the leptin responsiveness of both neuropeptide Y and pro-opiomelanocortin cells in the prepubertal hypothalamus.

Inhibitory effects of trichostatin A on adrenocorticotropic hormone production and proliferation of corticotroph tumor AtT-20 cells.

Cushing's disease is primarily caused by adrenocorticotropic hormone (ACTH)-producing pituitary adenomas. Pituitary tumor-transforming gene 1 (PTTG1) expression, a hallmark of pituitary tumors, stimulates pituitary cell proliferation. Histone deacetylases (HDACs) play an important role in regulating gene transcription and HDAC inhibitors induce cellular differentiation and suppress tumor cell proliferation. HDAC inhibitors also repress PTTG1 mRNA levels. Trichostatin A (TSA) is a potent cell-permeable HDAC inhibitor that blocks cell cycle progression. In the present study, we determined the effect of TSA on ACTH production and cellular proliferation in mouse AtT-20 corticotroph tumor cells. TSA decreased proopiomelanocortin (POMC) mRNA levels in AtT-20 cells and reduced ACTH levels in the culture medium of these cells. The TSA-induced decreases in POMC mRNA levels were not modulated when TSA and dexamethasone were simultaneously administered. Drug treatment also decreased AtT-20 cell proliferation, induced apoptosis, and increased the percentage of cells in G0/G1 phase using flow cytometry. TSA decreased PTTG1 mRNA levels. Furthermore, PTTG1 knockdown inhibited cellular proliferation. Its knockdown also inhibited POMC mRNA and ACTH levels. TSA inhibits ACTH production and corticotroph tumor cell proliferation. TSA may inhibit cellular proliferation, and ACTH synthesis and secretion by decreasing PTTG1 expression.

Aphidicolin inhibits cell proliferation via the p53-GADD45β pathway in AtT-20 cells.

Cushing's disease is primarily caused by pituitary corticotroph adenomas, which autonomically secrete adrenocorticotropic hormone (ACTH). ACTH production may be associated with tumor cell proliferation; however, the effects of cell cycle progression on ACTH production and cell proliferation are little known in corticotroph tumor cells. A DNA polymerase inhibitor, aphidicolin, arrests cells at the entrance to the S phase and blocks the cell cycle; aphidicolin also induces apoptosis in tumor cells. In the present study, we determined ACTH production and cell proliferation of AtT-20 corticotroph tumor cells following treatment with aphidicolin. Aphidicolin decreased proopiomelanocortin mRNA levels in AtT-20 cells and the levels of ACTH in the culture medium of these cells. Aphidicolin also decreased cell proliferation and induced apoptosis in AtT-20 cells. Fluorescence-activated cell sorting analyses revealed that this agent increased the percentage of G0/G1 phase cells, and decreased S phase cells. Aphidicolin decreased the phosphorylation of cyclic adenosine monophosphate response element-binding protein and Akt. Aphidicolin increased the levels of tumor protein 27 (p27) and 53 (p53), while it decreased cyclin E levels. Aphidicolin also increased the mRNA levels of the stress response gene growth arrest and DNA damage-inducible 45β (GADD45β), a putative downstream target of p53. The p53 knockdown increased GADD45β mRNA levels. The GADD45β knockdown inhibited the decreases in cell proliferation. Thus, aphidicolin inhibits cell proliferation via the p53-GADD45β pathway in AtT-20 cells.

Fetal alcohol exposure alters proopiomelanocortin gene expression and hypothalamic-pituitary-adrenal axis function via increasing MeCP2 expression in the hypothalamus.

Proopiomelanocortin (POMC) is a precursor gene of the neuropeptide β-endorphin in the hypothalamus and is known to regulate various physiological functions including stress response. Several recent reports showed that fetal alcohol exposure programs the hypothalamus to produce lower levels of POMC gene transcripts and to elevate the hypothalamic-pituitary-adrenal (HPA) axis response to stressful stimuli. We investigated the role of methyl CpG binding protein (MeCP2) in the effects of prenatal ethanol on POMC gene expression and hypothalamic-pituitary-adrenal (HPA) axis function. Pregnant Sprague Dawley rats were fed between GD 7 and 21 with a liquid diet containing 6.7% alcohol, pair-fed with isocaloric liquid diet, or fed ad libitum with rat chow, and their male offsprings were used at 60 days after birth in this study. Fetal alcohol exposure reduced the level of POMC mRNA, but increased the level of DNA methylation of this gene in the arcuate nucleus (ARC) of the hypothalamus where the POMC neuronal cell bodies are located. Fetal alcohol exposed rats showed a significant increase in MeCP2 protein levels in POMC cells, MeCP2 gene transcript levels as well as increased MeCP2 protein binding on the POMC promoter in the arcuate nucleus. Lentiviral delivery of MeCP2 shRNA into the third ventricle efficiently reduced MeCP2 expression and prevented the effect of prenatal ethanol on POMC gene expression in the arcuate nucleus. MeCP2-shRNA treatment also normalized the prenatal ethanol-induced increase in corticotropin releasing hormone (CRH) gene expression in the hypothalamus and elevated plasma adrenocorticotrophic hormone (ACTH) and corticosterone hormone responses to lipopolysaccharide (LPS) challenge. These results suggest that fetal alcohol programming of POMC gene may involve recruitment of MeCP2 on to the methylated promoter of the POMC gene to suppress POMC transcript levels and contribute to HPA axis dysregulation.

Individual differences in gene expression of vasopressin, D2 receptor, POMC and orexin: Vulnerability to relapse to heroin-seeking in rats

Individual vulnerability to stress-induced relapse during abstinence from chronic heroin exposure is a key feature of opiate addiction, with limited studies on this topic. Arginine vasopressin (AVP) and its V1b receptor, components of the brain stress responsive systems, play a role in heroin-seeking behavior triggered by foot shock (FS) stress in rats. In this study, we tested whether individual differences in the FS-induced heroin-seeking were associated with alterations of AVP and V1b, as well as other stress responsive systems, including pro-opiomelanocortin (POMC), orexin, plasma ACTH and corticosterone, as well as dopamine D2 receptor (D2) and plasma prolactin. Sprague–Dawley rats were subjected to 3-hour intravenous heroin self-administration (SA) and then tested in extinction, and FS-induced and heroin priming-induced reinstatements. The rats that self-administered heroin were divided into high and low reinstatement responders induced by FS (H-RI; L-RI). Over SA sessions, both the H-RI and L-RI displayed similar active lever responding, heroin infusion and total heroin intake. Compared to the L-RI, however, the H-RI showed greater active lever responses during stress-induced reinstatement, with higher AVP mRNA levels in medial/basolateral amygdala and lower D2 mRNA levels in caudate putamen. However, heroin priming resulted in similar reinstatement in both groups and produced similarly low POMC and high orexin mRNA levels in hypothalamus. Our results indicate that: 1) enhanced amygdalar AVP and reduced striatal D2 expression may be related to individual vulnerability to stress-induced reinstatement of heroin- seeking; and 2) heroin abstinence-associated alterations of hypothalamic orexin and POMC expression may be involved in drug priming-induced heroin-seeking.

Marked cortisol production by intracrine ACTH in GIP-treated cultured adrenal cells in which the GIP receptor was exogenously introduced.

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing’s syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (–) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.

Inhibition of heat shock protein 90 decreases ACTH production and cell proliferation in AtT-20 cells.

Cushing's disease is primarily caused by adrenocorticotropic hormone (ACTH)-producing pituitary adenomas. If excision of the tumor from the pituitary, which is the primary treatment for Cushing's disease, is unsuccessful, further medical therapy is needed to treat the resultant hypercortisolism. Some of the drugs used to treat this condition have shown potential therapeutic benefits, but a more effective treatment should be explored for the treatment of Cushing's disease. In the present study, we determined the effect of heat shock protein 90 inhibitors on ACTH production and cell proliferation of AtT-20 corticotroph tumor cells. AtT-20 pituitary corticotroph tumor cells were cultured. The expression levels of mouse proopiomelanocortin (POMC) and pituitary tumor transforming gene 1 (PTTG1) mRNA were evaluated using quantitative real-time PCR. Cellular DNA content was analyzed with fluorescence-activated cell sorting (FACS) analysis. The protein levels were determined by Western blot analysis. Both 17-allylamino-17-demethoxygeldanamycin and CCT018159 decreased POMC mRNA levels in AtT-20 cells and ACTH levels in the culture medium of these cells, suggesting that both drugs suppress ACTH synthesis and secretion in corticotroph tumor cells. Both drugs also decreased cell proliferation and induced apoptosis. FACS analyses revealed that both agents increased the percentage of AtT-20 cells in the G2/M phase. These drugs decreased cell proliferation, presumably due to the induction of cell death and arrest of the cell cycle in AtT-20 cells. Tumor weight in mice xenografted with AtT-20 cells and treated with CCT018159 was lower than in AtT-20-xenografted control mice. CCT018159 also decreased plasma ACTH levels, and POMC and PTTG1 mRNA levels in the tumor cells. CCT018159 inhibits ACTH production and corticotroph tumor cell proliferation in vitro and in vivo.

Inhibitory effects of SOM230 on adrenocorticotropic hormone production and corticotroph tumor cell proliferation in vitro and in vivo

Adrenocorticotropic hormone (ACTH) production by pituitary corticotroph adenomas is the main cause of Cushing’s disease. A drug that targets pituitary ACTH-secreting adenomas would aid treatment of Cushing’s disease. Octreotide, a somatostatin receptor type 2 (SSTR2)-preferring somatostatin analogue, has no effect on ACTH secretion in patients with Cushing’s disease. The multiligand SOM230 (pasireotide) displays a much higher affinity for SSTR1 and SSTR5 than octreotide and suppresses ACTH secretion in cultures of human corticotroph tumors to a greater extent than octreotide. In the present in vitro and in vivo study, we determined the effect of SOM230 on ACTH production and cell proliferation of AtT-20 corticotroph tumor cells. SOM230 decreased proopiomelanocortin (POMC) mRNA levels in AtT-20 cells and ACTH levels in the culture medium of these cells, suggesting that SOM230 suppresses ACTH synthesis and secretion in corticotroph tumor cells. SOM230 also decreased cell proliferation and both cyclic adenosine monophosphate response element-binding protein and Akt phosphorylation in AtT-20 cells. SSTR5 knockdown inhibited the SOM230-induced decreases in cell proliferation. Fluorescence-activated cell sorting analyses revealed that SOM230 did not attenuate cell cycle progression. Tumor weight in mice xenografted with AtT-20 cells and treated with SOM230 was significantly lower than in AtT-20-xenografted control mice. SOM230 also significantly decreased plasma ACTH levels, and POMC and pituitary tumor transforming gene mRNA levels in the tumor cells. Thus, SOM230 inhibits ACTH production and corticotroph tumor cell proliferation in vitro and in vivo.

Betahistine ameliorates olanzapine-induced weight gain through modulation of histaminergic, NPY and AMPK pathways.

Olanzapine is widely used to treat schizophrenia and other disorders, but causes adverse obesity and other metabolic side-effects. Both animal and clinical studies have shown that co-treatment with betahistine (a histaminergic H1 receptor agonist and H3 receptor antagonist) is effective for ameliorating olanzapine-induced weight gain/obesity. To reveal the mechanisms underlying these effects, this study investigated the effects of co-treatment of olanzapine and betahistine (O+B) on expressions of histaminergic H1 receptor (H1R), AMP-activated protein kinase (AMPK), neuropeptide Y (NPY), and proopiomelanocortin (POMC) in the hypothalamus associated with reducing olanzapine-induced weight gain. Olanzapine significantly upregulated the mRNA and protein expressions of H1R, while O+B co-treatment significantly downregulated the H1R levels, compared to the olanzapine-only treatment group. The NPY mRNA expression was significantly enhanced by olanzapine, but it was significantly reversed by O+B co-treatment. The hypothalamic H1R expression was positively correlated with total food intake, and NPY expression. Olanzapine also increased AMPKα activation measured by the AMPKα phosphorylation (pAMPKα)/AMPKα ratio compared with controls, whereas O+B co-treatment decreased the pAMPKα/AMPKα ratio, compared with olanzapine only treatment. The pAMPKα/AMPKα ratio was positively correlated with total food intake and H1R expression. Although olanzapine administration decreased the POMC mRNA level, this level was not affected by O+B co-treatment. Therefore, these results suggested that co-treatment with betahistine may reverse olanzapine-induced body weight gain via the H1R-NPY and H1R-pAMPKα pathways.

Modulation of proopiomelanocortin gene expression by ethanol in mouse anterior pituitary corticotrope tumor cell AtT20

In humans, alcoholism is associated with a decrease in basal ACTH and cortisol levels, and blunted pituitary ACTH responses to administered corticotropin-releasing hormone (CRH) during active drinking and after long-term abstinence. Preclinical studies indicate that a persistent decrease in pituitary activation after chronic exposure to ethanol is due to a direct effect of ethanol on the corticotrope of the anterior pituitary. The present studies were undertaken to determine if ethanol has effects on proopiomelanocortin (POMC) gene transcription activity in mouse anterior pituitary corticotrope tumor cell AtT20. We measured the levels of the POMC primary nuclear RNA transcript (PT), processing intermediate, and mature mRNA in the nucleus and the levels of the POMC mRNA in the cytoplasm after treatment of AtT20 cells with 5–15mM concentrations of ethanol. After 15mM ethanol for 60 to 120min, the POMC PT levels were significantly decreased. This decreased POMC gene transcription activity was coupled with a significant reduction of the POMC cytoplasmic mRNA levels. After ethanol for 4h, however, both the decreases were no longer observed. After 8h, a decrease in the ACTH secretion in the medium was found. We further investigated if CRH or glutamate modulates the effects of ethanol on the POMC gene transcription activity. CRH at 10nM after 60min increased the POMC PT levels, and 15mM ethanol attenuated the effect of CRH on the nuclear transcription activity. Glutamate receptor proteins, including NMDA receptor subtype NR1 (but not NR2A or NR2B) and GluR2, were identified by Western immunoblot analysis in AtT20 cells, with similar sizes to those in mouse hypothalamus. The inhibitory effect of 60min ethanol at 5 to 15mM on the POMC PT levels was attenuated by 50μML-glutamate. Together, our data showed that: (1) ethanol treatment in intoxicate doses significantly inhibited POMC gene transcription activity in a dose- and time-dependent manner in AtT20 cells, and (2) the POMC gene transcription activity in response to CRH or glutamate was altered by ethanol. Our results suggest that ethanol has an inhibitory effect on the POMC gene transcription activity in the anterior pituitary corticotrope, which may contribute to the persistent decrease in pituitary activation after chronic ethanol exposure.

Maternal obesity impairs brain glucose metabolism and neural response to hyperglycemia in male rat offspring

Hypothalamic appetite regulators neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) are modulated by glucose. This study investigated how maternal obesity disturbs glucose regulation of NPY and POMC, and whether this deregulation is linked to abnormal hypothalamic glucose uptake-lactate conversion. As post-natal high-fat diet (HFD) can exaggerate the effects of maternal obesity, its additional impact was also investigated. Female Sprague Dawley rats were fed a HFD (20 kJ/g) to model maternal obesity. At weaning, male pups were fed chow or HFD. At 9 weeks, in vivo hypothalamic NPY and POMC mRNA responses to acute hyperglycemia were measured; while hypothalami were glucose challenged in vitro to assess glucose uptake-lactate release and related gene expression. Maternal obesity dampened in vivo hypothalamic NPY response to acute hyperglycemia, and lowered in vitro hypothalamic glucose uptake and lactate release. When challenged with 20 mM glucose, hypothalamic glucose transporter 1, monocarboxylate transporters, lactate dehydrogenase-b, NPY and POMC mRNA expression were down-regulated in offspring exposed to maternal obesity. Post-natal HFD consumption reduced in vitro lactate release and monocarboxylate transporter 2 mRNA, but increased POMC mRNA levels when challenged with 20 mM glucose. Overall, maternal obesity produced stronger effects than post-natal HFD consumption to impair hypothalamic glucose metabolism. However, they both disturbed NPY response to hyperglycemia, potentially leading to hyperphagia.

Perinatal exposure to low-dose bisphenol A affects the neuroendocrine stress response in rats

Bisphenol A (BPA) is an estrogen-mimicking endocrine disruptor. Early-life exposures to low doses of BPA exert long-lasting effects on animals' reproductive and brain physiology. However, little is known about the effects of BPA on the stress-response system. Given the interaction of sex and stress hormones, we examined the effect of a low perinatal BPA exposure on the function of the hypothalamic-pituitary-adrenal (HPA) axis at rest and upon application of acute stress. Throughout pregnancy and lactation rats received daily 40 μg BPA/kg body weight orally via cornflakes. We studied the effect of this low but chronic exposure to BPA in the male and female offspring at puberty. BPA exposure led to abnormal adrenal histology including reduced zona reticularis especially in male offspring, hyperplasia of zona fasciculata in both sexes, and increased adrenal weight in female offspring. BPA-treated females had increased basal corticosterone and reduced hypothalamic glucocorticoid receptors (GR) levels. Stressed BPA-exposed females exhibited anxiety-like behavioral coping, a less rigorous corticosterone response, and did not downregulate GR in the hypothalamus, compared with control females. BPA-exposed males exhibited a heightened corticosterone stress response compared with females; they also displayed increased pro-opiomelanocortin mRNA levels and retained the prestress levels of pituitary corticotropin-releasing hormone-receptor 1, compared with control males. We found that perinatal chronic exposure to a low dose of BPA perturbs the basal and stress-induced activity of the HPA axis in a sexually dimorphic manner at adolescence. Exposure to BPA might contribute to increased susceptibility to stress-related disorders in later life.

Blunted HPA axis response in lactating, vasopressin-deficient Brattleboro rats

Adaptation to stress is a basic phenomenon in mammalian life that is mandatorily associated with the activity of the hypothalamic-pituitary-adrenal (HPA) axis. An increased resting activity of the HPA axis can be measured during pregnancy and lactation, suggesting that these reproductive states lead to chronic load in females. In this study, we examined the consequences of the congenital lack of vasopressin on the activity of the HPA axis during lactation using vasopressin-deficient Brattleboro rats. Virgin and lactating, homozygous vasopressin-deficient rats were compared with control, heterozygous rats. In control dams compared with virgins, physiological changes similar to those observed in a chronic stress state (thymus involution, adrenal gland hyperplasia, elevation of proopiomelanocortin mRNA levels in the adenohypophysis, and resting plasma corticosterone levels) were observed. In vasopressin-deficient dams, adrenal gland hyperplasia and resting corticosterone level elevations were not observed. Corticotropin-releasing hormone (Crh) mRNA levels in the hypothalamic paraventricular nucleus were elevated in only the control dams, while oxytocin (OT) mRNA levels were higher in vasopressin-deficient virgins and lactation induced a further increase in both the genotypes. Suckling-induced ACTH and corticosterone level elevations were blunted in vasopressin-deficient dams. Anaphylactoid reaction (i.v. egg white) and insulin-induced hypoglycemia stimulated the HPA axis, which were blunted in lactating rats compared with the virgins and in vasopressin-deficient rats compared with the controls without interaction of the two factors. Vasopressin seems to contribute to the physiological changes observed during lactation mimicking a chronic stress state, but its role in acute HPA axis regulation during lactation seems to be similar to that observed in virgins. If vasopressin is congenitally absent, OT, but not the CRH, compensates for the missing vasopressin; however, the functional restitution remains incomplete.

Deficiency of alpha-calcitonin gene-related peptide induces inflammatory responses and lethality in sepsis

In the present study, we examined the role of alpha-calcitonin gene-related peptide (αCGRP) on expression of neuropeptides in the brain, inflammatory responses, and survival rate in septic shock condition. We examined expression of neuropeptides such as αCGRP, proopiomelanocortin (POMC), corticotrophin releasing hormone (CRH), and proenkephalin (ProENK) in the hippocampus and hypothalamus in C57BL/6 (WT) or αCGRP−/− (KO) mice subjected to sepsis. Cecal ligation and puncture (CLP) or lipopolysaccharide/D-galactosamine (LPS/D-GalN) treatment showed significant increases of hippocampal and hypothalamic αCGRP, POMC, CRH, and ProENK mRNA levels in WT mice, but not ProENK mRNA in the hypothalamus at 6h after on-set of sepsis. However, enhanced mRNA levels of POMC, CRH, and ProENK genes were not increased in the hippocampus and hypothalamus of CLP-subjected KO mice at 6h following sepsis. KO mice treated with LPS/D-GalN displayed a significant enhancement of plasma corticosterone, aspartate aminotransferase, and alanine aminotransferase levels compared to LPS/D-GalN treated WT mice at 12h after induction of sepsis. In addition, plasma levels of pro-inflammatory cytokines, such as IL-1β and TNF-α, were also further increased in KO mice compared to WT mice at 24h after CLP or LPS/D-GalN treatment. Interestingly, mRNA expressions of IL-6 and IL-10, anti-inflammatory cytokines, were synergistically enhanced in liver and lymph node of KO mice compared to WT mice at 6h after CLP. However, plasma level of IL-10 but not IL-6 was significantly decreased in KO mice compared to WT mice at 24h after CLP or LPS/D-GalN challenge. The survival rate of KO mice was significantly reduced compared to WT mice following mild (1 punch) and moderate (2 punch) CLP and LPS/D-GalN administration. Taken together, our findings suggest that the activation of αCGRP may induce other neuropeptides associated with immunomodulation at CNS level and modulate immune responses as enhancing anti-inflammatory cytokines and reducing pro-inflammatory cytokines during the sepsis.

Suppression of Hypothalamic–Pituitary–Adrenal Axis by Acute Heroin Challenge in Rats During Acute and Chronic Withdrawal from Chronic Heroin Administration

It is known that heroin dependence and withdrawal are associated with changes in the hypothalamic-pituitary-adrenal (HPA) axis. The objective of these studies in rats was to systematically investigate the level of HPA activity and response to a heroin challenge at two time points during heroin withdrawal, and to characterize the expression of associated stress-related genes 30min after each heroin challenge. Rats received chronic (10-day) intermittent escalating-dose heroin administration (3×2.5mg/kg/day on day 1; 3×20mg/kg/day by day 10). Hormonal and neurochemical assessments were performed in acute (12h after last heroin injection) and chronic (10days after the last injection) withdrawal. Both plasma ACTH and corticosterone levels were elevated during acute withdrawal, and heroin challenge at 20mg/kg (the last dose of chronic escalation) at this time point attenuated this HPA hyperactivity. During chronic withdrawal, HPA hormonal levels returned to baseline, but heroin challenge at 5mg/kg decreased ACTH levels. In contrast, this dose of heroin challenge stimulated the HPA axis in heroin naïve rats. In the anterior pituitary, pro-opiomelanocortin (POMC) mRNA levels were increased during acute withdrawal and retuned to control levels after chronic withdrawal. In the medial hypothalamus, however, the POMC mRNA levels were decreased during acute withdrawal, and increased after chronic withdrawal. Our results suggest a long-lasting change in HPA abnormal responsivity during chronic heroin withdrawal.

Melatonin receptor activation suppresses adrenocorticotropin production via BMP-4 action by pituitary AtT20 cells

The role of melatonin, a regulator of circadian rhythm, in adrenocorticotropin (ACTH) production by corticotrope cells has not been elucidated. In this study, we investigated the effect of melatonin on ACTH production in relation to the biological activity of bone morphogenetic protein (BMP)-4 using mouse corticotrope AtT20 cells that express melatonin type-1 (MT1R) but not type-2 (MT2R) receptors. We previously reported that BMP-4 inhibits corticotropin-releasing hormone (CRH)-induced ACTH production and proopiomelanocortin (POMC) transcription by inhibiting MAPK signaling. Both melatonin and an MT1R/MT2R agonist, ramelteon, suppressed CRH-induced ACTH production, POMC transcription and cAMP synthesis. The inhibitory effects of ramelteon on basal and CRH-induced POMC mRNA and ACTH levels were more potent than those of melatonin. Treatment with melatonin or ramelteon in combination with BMP-4 additively suppressed CRH-induced ACTH production. Of note, the level of MT1R expression was upregulated by BMP-4 stimulation. The suppressive effects of melatonin and ramelteon on POMC transcription and cAMP synthesis induced by CRH were not affected by an MT2R antagonist, luzindole. On the other hand, BMP-4-induced Smad1/5/8 phosphorylation and the expression of a BMP target gene, Id-1, were augmented in the presence of melatonin and ramelteon. Considering that the expression levels of BMP receptors, ALK-3/BMPRII, were increased by ramelteon, MT1R action may play an enhancing role in BMP-receptor signaling. Among the MT1R signaling pathways including AKT, ERK and JNK pathways, inhibition of AKT signaling functionally reversed the MT1R effects on both CRH-induced POMC transcription and BMP-4-induced Id-1 transcription. Collectively, MT1R signaling and BMP-4 actions were mutually augmented, leading to fine-tuning of ACTH production by corticotrope cells.

Gestational Choline Supplementation Normalized Fetal Alcohol-Induced Alterations in Histone Modifications, DNA Methylation, and Proopiomelanocortin (POMC) Gene Expression in β-Endorphin-Producing POMC Neurons of the Hypothalamus

Prenatal exposure to ethanol (EtOH) reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, because these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of EtOH on these neurons. Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal- and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunosorbent assay. Prenatal EtOH treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10), and increased the repressive marks (H3K9me2, G9a, Setdb1), DNA-methylating enzyme (Dnmt1), and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and β-EP and elevated corticosterone response to LPS. Gestational choline normalized the EtOH-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1, and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, β-EP production, and the corticosterone response to LPS. These data suggest that prenatal EtOH modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction in POMC gene expression. Gestational choline supplementation prevents the adverse effects of EtOH on these neurons.

Proopiomelanocortin (POMC) expression and conditioned place aversion during protracted withdrawal from chronic intermittent escalating-dose heroin in POMC-EGFP promoter transgenic mice

In heroin-dependent individuals, the drive to avoid or ameliorate the negative affective/emotional state associated with the discontinuation of heroin contributes to the chronic relapsing nature of the disease. Here, we investigate changes in proopiomelanocortin (POMC) expression at three time points across an extended period of heroin withdrawal in a clinically relevant rodent model of addiction using conditioned place aversion (CPA) in POMC-EGFP (POMC-enhanced green fluorescent protein) bacterial artificial chromosome (BAC) transgenic mice. Neurons expressing POMC-EGFP were found in the medial nucleus of the amygdala (MeA), basomedial amygdala (BMA) and dentate gyrus of hippocampus (DG), as well as the arcuate nucleus of hypothalamus (ARC). Heroin-treated mice displayed robust CPA after acute spontaneous withdrawal (12h), which persisted across the extended (14days) withdrawal period. After 12-h withdrawal, heroin-treated mice showed lower signal intensity of POMC-EGFP-positive cells in the ARC, higher levels of POMC mRNA in the amygdala but lower levels in the hippocampus than saline controls. After 7-d withdrawal, heroin-treated mice showed fewer POMC-EGFP-positive cells in the MeA and lower POMC mRNA in the amygdala than saline controls. After extended (14days) withdrawal, heroin-treated mice showed more POMC-EGFP-positive cells in BMA and DG, increased intensity of POMC-EGFP signal in DG, and higher POMC mRNA levels in the hippocampus compared to controls. Our results show dynamic changes in POMC in hypothalamic and extra-hypothalamic regions that may contribute to the negative affective/emotional state of heroin withdrawal shown by CPA from acute to extended periods of heroin withdrawal.