Ching-Han Yu

Role of Reactive Oxygen Species-Related Enzymes in Neuropeptide Y and Proopiomelanocortin-Mediated Appetite Control: A Study Using Atypical Protein Kinase C Knockdown

AIMS Studies have reported that redox signaling in the hypothalamus participates in nutrient sensing. The current study aimed to determine if the activation of reactive oxygen species-related enzymes (ROS-RE) in the hypothalamus participates in regulating neuropeptide Y (NPY)-mediated eating. Moreover, possible roles of proopiomelanocortin (POMC) and atypical protein kinase C (aPKC) were also investigated. Rats were treated daily with phenylpropanolamine (PPA) for 4 days. Changes in the expression levels of ROS-RE, POMC, NPY, and aPKC were assessed and compared. RESULTS Results showed that ROS-RE, POMC, and aPKC increased, with a maximal response on Day 2 (anorectic effect) and with a restoration to the normal level on Day 4 (tolerant effect). By contrast, NPY expression decreased, and the expression pattern of NPY proved opposite those of ROS-RE and POMC. Central inhibition of ROS production by ICV infusion of ROS scavenger attenuated PPA anorexia, revealing a crucial role of ROS in regulating eating. Cerebral aPKC knockdown by ICV infusion of antisense aPKC modulated the expression of ROS-RE, POMC, and NPY. CONCLUSION Results suggest that ROS-RE/POMC- and NPY-containing neurons function reciprocally in regulating both the anorectic and tolerant effects of PPA, while aPKC is upstream of these regulators. INNOVATION These results may further the understanding of ROS-RE and aPKC in the control of PPA anorexia.

Involvement of neuropeptide Y Y1 receptor in the regulation of amphetamine-mediated appetite suppression.

Recently, we reported that an initial decrease followed by recovery of food intake was observed during four days of amphetamine (AMPH) treatment and suggested that these changes in response were mediated by changes in neuropeptide Y (NPY) and proopiomelanocortin (POMC). Here we investigated if Y1 receptor (Y1R) and/or Y5 receptor (Y5R) might be involved in this regulation. Rats were treated daily with AMPH for four days. Changes in the expression levels of Y1R, Y5R, melanocortin receptor 3 (MC3R), and NPY were assessed and compared. Results showed that Y1R and MC3R increased, with a maximal increase of about 210% on Day 2 but with a restoration to the normal level on Day 4. In contrast, NPY decreased with a biggest reduction of about 45% on Day 2 and the pattern of expression during AMPH treatment was opposite to those of Y1R and MC3R, while the expression of Y5R was not changed. Central inhibitions of NPY formation or Y1R activity modulated the anorectic response of AMPH and the reciprocal regulation of NPY and MC3R, revealing a crucial role of Y1R in this action. It is suggested that Y1R participates in the reciprocal regulation of NPY- and MC3R-containing neurons in the hypothalamus during the anorectic effect of AMPH. These results may further the understanding of Y1R in the control of eating.

Involvement of hypothalamic PI3K–STAT3 signalling in regulating appetite suppression mediated by amphetamine

Appetite suppression induced by amphetamine has been attributed to its inhibition of neuropeptide Y (NPY) neurons and activation of pro‐opiomelanocortin (POMC) neurons in the hypothalamus. This study examined whether STAT3 was involved in these actions of amphetamine.

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 4days. 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 1h 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.

Both neuropeptide Y knockdown and Y1 receptor inhibition modulate CART-mediated appetite control

Amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibition of neuropeptide Y (NPY)-containing neurons in the hypothalamus. This study examined whether hypothalamic cocaine- and amphetamine-regulated transcript (CART)-containing neurons and NPY Y1 receptor (Y1R) were involved in the action of AMPH. Rats were treated daily with AMPH for four days, and changes in feeding behavior and expression levels of NPY, CART, and POMC were assessed and compared. The results showed that both feeding behavior and NPY expression decreased during AMPH treatment, with the biggest reduction occurring on Day 2. By contrast, the expression of CART and melanocortin 3 receptor (MC3R), a member of the POMC neurotransmission, increased with the maximum response on Day 2, directly opposite to the NPY expression results. The intracerebroventricular infusion of NPY antisense or Y1R inhibitor both modulated AMPH-induced anorexia and the expression levels of MC3R and CART. The results suggest that in the hypothalamus both POMC- and CART-containing neurons participate in regulating NPY-mediated appetite control during AMPH treatment. These results may advance the knowledge of molecular mechanism of anorectic drugs.

Involvement of oxidative stress in the regulation of NPY/CART-mediated appetite control in amphetamine-treated rats.

Amphetamine (AMPH) treatment can suppress appetite and increase oxidative stress in the brain. AMPH-induced appetite suppression is associated with the regulation of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. The present study explored whether antioxidants, including glutathione S-transferase (GST) and glutathione peroxidase (GP), were involved in this NPY/CART-mediated appetite control. Rats were treated daily with AMPH for four days. Changes in food intake and expression levels of hypothalamic NPY, CART, GST, and GP were examined and compared. Results showed that, in AMPH-treated rats, (1) food intake and NPY expression decreased, while CART, GST, and GP expression increased; (2) NPY knockdown in the brain enhanced the decrease in NPY and the increases in CART, GST, and GP expression; and (3) central inhibition of reactive oxygen species production decreased GST and GP and modulated AMPH anorexia and the expression levels of NPY and CART. The present results suggest that oxidative stress in the brain participates in regulating NPY/CART-mediated appetite control in AMPH-treated rats. These results may advance the knowledge regarding the molecular mechanism of AMPH-evoked or NPY/CART-mediated appetite suppression.

Central dopamine action modulates neuropeptide‐controlled appetite via the hypothalamic PI3K/NF‐κB‐dependent mechanism

Hypothalamic neuropeptides, including neuropeptide Y (NPY) and proopiomelanocortin (POMC), have been found to control the appetite‐suppressing effect of amphetamine (AMPH). In this study, we have examined whether dopamine receptor (DAR), phosphatidylinositol 3‐kinase (PI3K) and nuclear factor‐kappaB (NF‐κB) are involved in AMPHs action. We administered AMPH to rats once a day for 4 days and assessed and compared changes in hypothalamic NPY, melanocortin receptor 4 (MC4R), PI3K, pAkt and NF‐κB expression. We found that the inhibition of DAR increased NPY, but decreased MC4R, PI3K and NF‐κB expression, compared with AMPH‐treated rats. Moreover, MC4R, PI3K, pAkt and NF‐κB increased with the maximum response on Day 2, which was consistent with the response of feeding behavior, but was opposite to the expression of NPY. Furthermore, we found that the intracerebroventricular infusion of the PI3K inhibitor or NF‐κB antisense could attenuate AMPH‐induced anorexia, and partially reverse the expression of NPY, MC4R, PI3K, Akt and NF‐κB back toward a normal level. We, therefore, suggest that DAR–PI3K–NF‐κB signaling in the hypothalamus plays functional roles in the modulation of NPY and POMC neurotransmissions and in the control of AMPH‐evoked appetite suppression.

Role of oxidative stress in disrupting the function of negative glucocorticoid response element in daily amphetamine-treated rats.

Amphetamine (AMPH)-induced appetite suppression is associated with changes in hypothalamic reactive oxygen species (ROS), antioxidants, neuropeptides, and plasma glucocorticoid. This study explored whether ROS and glucocorticoid response element (GRE), which is the promoter site of corticotropin-releasing hormone (CRH) gene, participated in neuropeptides-mediated appetite control. Rats were treated daily with AMPH for four days, and changes in food intake, plasma glucocorticoid and expression levels of hypothalamic neuropeptide Y (NPY), proopiomelanocortin (POMC), superoxide dismutase (SOD), CRH, and glucocorticoid receptor (GR) were examined and compared. Results showed that food intake decreased and NPY gene down-regulated, while POMC, SOD, and CRH gene up-regulated during AMPH treatment. GR and GRE-DNA bindings were disrupted on Day 1 and Day 2 when glucocorticoid levels were still high. Pretreatment with GR inhibitor or ROS scavenger modulated mRNA levels in NPY, POMC, SOD and CRH in AMPH-treated rats. We suggest that disruptions of negative GRE (nGRE) on Day 1 and Day 2 are associated with an increase in oxidative stress during the regulation of NPY/POMC-mediated appetite control in AMPH-treated rats. These results advance the understanding of molecular mechanism in regulating AMPH-mediated appetite suppression.

Growth Modulation of Diabetic Factors and Antidiabetic Drugs on Prostate Cancer Cell Lines

Risk factors for prostate cancer (PCa) include age, hormones, race, family history and diet. Recently, epidemiologic evidence has indicated that history of diabetes mellitus (DM) is inversely associated with risk of PCa. However, epidemiological investigations have yielded inconsistent results. Hence, the exact mechanism of DM-induced reduction in the incidence of PCa has yet to be fully elucidated. The aim of this study was to investigate the effects of DM factors, including glucose, insulin and insulin-like growth factor-1 (IGF-1), on the proliferation of PCa cell lines in vitro. Cell proliferation and expression of hormone receptors was examined in MTT assay and Western blot analysis, respectively. The results showed that DM factors did not affect the viability of androgen receptor (AR)-expressing PCa cell lines. However, cell proliferation increased after treatment with DM factors in androgen-independent PCa cell lines. On PCa tissue arrays, intensities of total AR and nuclear IGF-1R were higher in malignant tissues than in normal prostate glands. In terms of hormonal receptors, androgen-dependent LNCaP cells treated with insulin and IGF-1 in a low-serum medium showed decreased expression of insulin receptor beta (IRβ) and elevated expression of IGF-1 receptor beta (IGF-1Rβ). Moreover, expression of AR was upregulated after insulin and IGF-1 treatment in LNCaP cells, but not in the other PCa cell lines. Most of the studied antidiabetic drugs promoted the viability of PCa cells. However, metformin decreased the viability of AR-expressing PCa cells. These results suggest that diabetic factors modify the expression of AR, IR and IGF-1R to increase cancer cell proliferation. Moreover, the growth suppressing effects of metformin on PCa may be via the regulation of the AR signaling pathway.

Participation of ghrelin signalling in the reciprocal regulation of hypothalamic NPY/POMC-mediated appetite control in amphetamine-treated rats.

Hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) have been documented to participate in amphetamine (AMPH)-induced appetite suppression. This study investigated whether ghrelin signalling is associated with changes in NPY/POMC-mediated appetite control. Rats were given AMPH daily for four days, and changes in food intake, body weight, plasma ghrelin, hypothalamic NPY, melanocortin 3 receptor (MC3R), ghrelin O-acyltransferase (GOAT), acyl ghrelin (AG) and ghrelin receptor (GHSR1a) were examined and compared. Food intake, body weight and NPY expression decreased, while MC3R expression increased and expressed reciprocally to NPY expression during AMPH treatment. Plasma ghrelin and hypothalamic AG/GOAT/GHSR1a expression decreased on Day 1 and Day 2, which was associated with the positive energy metabolism, and returned to normal levels on Day 3 and Day 4, which was associated with the negative energy metabolism; this expression pattern was similar to that of NPY. Infusion with a GHSR1a antagonist or an NPY antisense into the brain enhanced the decrease in NPY and AG/GOAT/GHSR1a expression and the increase in MC3R expression compared to the AMPH-treated group. Peripheral ghrelin and the central ghrelin system participated in the regulation in AMPH-induced appetite control. These results shed light on the involvement of ghrelin signalling in reciprocal regulation of NPY/POMC-mediated appetite control and may prove useful for the development of anti-obesity drugs.