Roles of protein kinase Cα isozyme in the regulation of oxidative stress and neuropeptide Y gene expression in phenylpropanolamine-mediated appetite suppression

Hypothalamic neuropeptide Y (NPY) is an appetite stimulant in the brain. Although regulation of NPY expression has been reported to contribute to the appetite-suppressing effect of phenylpropanolamine (PPA), it is still unknown if protein kinase C (PKC) is involved in this effect. Rats were daily treated with PPA for 4 days. Changes in food intake, hypothalamic NPY, PKC, and proopiomelanocortin (POMC) mRNA levels were assessed and compared. Results showed that the NPY gene was down-regulated following PPA treatment, which was parallel with the decrease of feeding. Moreover, several isotypes of PKC mRNA level (α, βI, βII, γ, δ, η, λ, ε, and ζ) were changed. Among these, α, δ, and λ PKC were up-regulated along with POMC gene expression which coincided with down-regulation of the NPY gene. To further determine if PKCα was involved, infusions of antisense oligonucleotide into the cerebroventricle were performed at 1 h before daily PPA treatment in free-moving rats. Results showed that PKCα knock-down could modify both anorexia induced by PPA and the NPY mRNA levels. Moreover, PKCα knock-down could also modify superoxide dismutase (SOD) gene expression. It is suggested that PKCα participates in the regulation of PPA-mediated appetite suppression via the modulation of NPY and SOD gene expression.     

Immunohistochemical and genomic evidence for the involvement of hypothalamic neuropeptide Y (NPY) in phenylpropranolamine-mediated appetite suppression

Phenylpropanolamine (PPA) is an appetite suppressant. The mechanism for the anorectic effect of PPA has been attributed to its action on the site of hypothalamic paraventriculum. Neuropeptide Y (NPY) is an appetite stimulant that is widely distributed in the site of hypothalamus. It is not clear whether hypothalamic NPY is involved in the anorectic action of PPA. This study was aimed to investigate the mechanism underlying the involvement of NPY gene in the anorectic action of PPA. Results revealed that PPA treatment in rats could decrease both NPY content and mRNA level in the hypothalamus. In addition, the expression of NPY immunoreactivity following PPA treatment was decreased in areas of hypothalamic arcuate nucleus, paraventricular nucleus and periventricular area using immunohistochemical staining, suggesting an involvement of NPYergic pathway in the action of PPA anorexia. Our results provided immunohistochemical and genomic evidence to suggest that PPA might reduce feeding by altering NPY gene expression.     

Intracerebral administration of protein kinase A or cAMP response element-binding protein antisense oligonucleotide can modulate amphetamine-mediated appetite suppression in free-moving rats

Although amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibitory action on neuropeptide Y (NPY), an appetite neurotransmitter abundant in the brain, molecular mechanisms underlying this effect are not well known. This study examined the possible role of protein kinase A (PKA) and cAMP response element-binding protein (CREB) signaling in this anorectic effect, and the results showed that both PKA and CREB mRNA levels in hypothalamus were increased following AMPH treatment, which was relevant to a reduction of NPY mRNA level. To determine whether PKA or CREB was involved in the anorectic response, intracerebroventricular infusions of antisense oligonucleotide (or missense control) were performed 60 min before daily AMPH treatment in conscious rats, and results showed that either PKA or CREB knockdown could block AMPH-induced anorexia as well as restore NPY mRNA level, indicating the respective involvement of PKA and CREB signaling in the regulation of NPY gene expression. It is suggested that hypothalamic PKA and CREB signaling may involve the central regulation of AMPH-mediated feeding suppression via the modulation of NPY gene expression.     

Inhibiting neuropeptide Y Y1 receptor modulates melanocortin receptor- and NF-κB-mediated feeding behavior in phenylpropanolamine-treated rats

Neuropeptide Y (NPY) and nuclear factor-kappa B (NF-κB) are involved in regulating anorexia elicited by phenylpropanolamine (PPA), a sympathomimetic drug. This study explored whether NPY Y1 receptor (Y1R) is involved in this process, and a potential role for the proopiomelanocortin system was identified. Rats were given PPA once a day for 4 days. Changes in the hypothalamic expression of the NPY, Y1R, NF-κB, and melanocortin receptor 4 (MC4R) levels were assessed and compared. The results indicated that food intake and NPY expression decreased, with the largest reductions observed on Day 2 (approximately 50% and 45%, respectively), whereas NF-κB, MC4R, and Y1R increased, achieving maximums on Day 2 (160%, 200%, and 280%, respectively). To determine the role of Y1R, rats were pretreated with Y1R antisense or a Y1R antagonist via intracerebroventricular injection 1 h before the daily PPA dose. Y1R knockdown and inhibition reduced PPA anorexia and partially restored the normal expression of NPY, MC4R, and NF-κB. The data suggest that hypothalamic Y1R participates in the appetite-suppression from PPA by regulating MC4R and NF-κB. The results of this study increase our understanding of the molecular mechanisms in PPA-induced anorexia.     

Role of hypothalamic neuropeptide Y (NPY) in the change of feeding behavior induced by repeated treatment of amphetamine

Neuropeptide Y (NPY), an orexigenic peptide, is involved in the control of food intake. Repeated administration of amphetamine (AMPH), an anorectic agent, results in an anorectic effect on day 1 and a tolerant anorectic effect on the followings. In an attempt to know the role of hypothalamic NPY in these effects of AMPH, contents of hypothalamic NPY were determined by radioimmunoassay at first. In AMPH-treated groups, the contents of hypothalamic NPY decreased rapidly on day 1 but restored gradually to the normal level on the following days as observed in repeated AMPH. An involvement of hypothalamic NPY in the feeding change of repeated AMPH can thus be considered. Moreover, daily injection of NPY antisense oligonucleotide into brain (10 microg/10 microl/day, i.c.v.) to inhibit the gene expression of hypothalamic NPY were performed at 1 hour before daily 2 mg/kg AMPH. The reversion of food intake from the anorectic level to the normal level (tolerant anorexia) was abolished by this antisense pretreatment. It is suggested that hypothalamic NPY may play a role in the change of feeding behavior induced by repeated AMPH administration.     

Stimulation of Muscarinic Receptors Induces Expression of Individual fos and jun Genes Through Different Transduction Pathways

Abstract: The transduction pathways coupling muscarinic receptors to induction of fos and jun genes were investigated in neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced expression of c- fos , fosB , c- jun , junB , and junD . This effect was abolished by pretreatment with atropine, indicating an involvement of muscarinic receptors. These genes were also induced by activation of protein kinase C with phorbol ester or by elevating the intracellular Ca²⁺ concentration with a Ca²⁺ ionophore. The Ca²⁺ effect was inhibited by KN-62, suggesting an induction through Ca²⁺/calmodulin-dependent kinase II. Inhibition of protein kinase C with GF109203X suppressed the carbachol-stimulated increase in mRNA levels of c- fos , fosB , and junB by ∼70% but had only minor effects on the expression of c- jun and junD . On the other hand, preincubation with KN-62 attenuated the carbachol-induced increase in c- jun and junD expression by 70% but had no effect on c- fos , fosB , and junB mRNA levels. Simultaneous inhibition of both protein kinase C and Ca²⁺/calmodulin-dependent kinase II completely abolished the carbachol-stimulated expression of c- jun and junD , but c- fos , fosB , and junB were still expressed to a certain extent under this condition. Comparison of the inhibitory effects of GF109203X and Gö 6976 suggests the involvement of classical protein kinase C isozymes in muscarinic receptor-stimulated expression of fos and jun genes. These results demonstrate that the muscarinic receptor-induced expression of individual fos and jun genes is regulated via different pathways, primarily protein kinase C or Ca²⁺/calmodulin-dependent kinase II.     

Stimulation of the Cyclic GMP Pathway by NO Induces Expression of the Immediate Early Genes c-fos and junB in PC12 Cells

Abstract: Stimulation of several second messenger pathways induces the expression of immediate early genes such as c- fos , c- jun , junB , and junD , but little is known about their induction via the stimulation of the cyclic GMP pathway. Here we looked at the expression of early genes in pheochromocytoma PC12 cells after activation of cytosolic guanylate cyclase by sodium nitroprusside. This compound spontaneously releases NO, a molecule known to be involved in cell communication. We found that expression of c- fos and junB but not of c- jun or junD is increased upon activation of cyclic GMP pathway. c- fos mRNA expression was the most activated (fourfold at 30 min), whereas junB response was more modest (2.2-fold activation at 60 min). Nuclear extracts of stimulated cells show increased binding capacity to the AP1 binding site consistent with the dose-response curve. The activating effect of nitroprusside could be reproduced by dipyridamole, a selective cyclic GMP phosphodiesterase inhibitor and by 8- p -chlorophenylthio-cyclic GMP, a permeant selective cyclic GMP-dependent protein kinase activator, and abolished by KT5823, an inhibitor of that kinase. The results show that NO promotes early gene activation and AP1 binding enhancement through the stimulation of the cyclic GMP pathway.     

Involvement of hypothalamic neuropeptide Y in regulating the amphetamine-induced appetite suppression in streptozotocin diabetic rats

BACKGROUND AND AIM: Amphetamine (AMPH) is a well-known anorectic agent. In normal rats, AMPH-induced anorexia has been attributed to its inhibitory action on hypothalamic neuropeptide Y (NPY), an appetite stimulant in the brain. In diabetic rats, however, if this anorectic response of AMPH might still be observed was uncertain. METHODS: Rats (including normal, diabetic and insulin-treated diabetic rats) were given daily with saline or AMPH for 6 days. Changes in food intake, plasma glucose level (PGL) and NPY content of these rats were measured and compared. RESULTS: The AMPH-induced anorectic response was altered in diabetic rats. Although the anorectic effects of AMPH on the first day of dosing were similar between diabetic and control rats, diabetic rats developed tolerance to this anorexia more rapidly than control rats. This alteration was independent of PGL since PGL levels were not changed following AMPH treatment and PGL normalization induced by phlorizin could not restore the level of AMPH anorexia. On the other hand, this alteration was dependent on the action of NPY because NPY contents were decreased following AMPH treatment and the replacement of insulin in diabetic rats could restore both NPY content and AMPH anorexia. CONCLUSION: These results suggested that the elevated hypothalamic NPY content in diabetic rats was involved in modifying the anorectic response of AMPH.