Ling Ling Hwang

Sex Differences in high-fat Diet-induced Obesity, Metabolic alterations and Learning, and Synaptic Plasticity Deficits in Mice

Obesity is a potential risk factor for cognitive deficits in the elder humans. Using a high‐fat diet (HFD)–induced obese mouse model, we investigated the impacts of HFD on obesity, metabolic and stress hormones, learning performance, and hippocampal synaptic plasticity. Both male and female C57BL/6J mice fed with HFD (3 weeks to 9–12 months) gained significantly more weights than the sex‐specific control groups. Compared with the obese female mice, the obese males had similar energy intake but developed more weight gains. The obese male mice developed hyperglycemia, hyperinsulinemia, hypercholesterolemia, and hyperleptinemia, but not hypertriglyceridemia. The obese females had less hyperinsulinemia and hypercholesterolemia than the obese males, and no hyperglycemia and hypertriglyceridemia. In the contextual fear conditioning and step‐down passive avoidance tasks, the obese male, but not female, mice showed poorer learning performance than their normal counterparts. These learning deficits were not due to sensorimotor impairment as verified by the open‐field and hot‐plate tests. Although, basal synaptic transmission characteristics (input–output transfer and paired‐pulse facilitation (PPF) ratio) were not significantly different between normal and HFD groups, the magnitudes of synaptic plasticity (long‐term potentiation (LTP) and long‐term depression (LTD)) were lower at the Schaffer collateral‐CA1 synapses of the hippocampal slices isolated from the obese male, but not female, mice, as compared with their sex‐specific controls. Our results suggest that male mice are more vulnerable than the females to the impacts of HFD on weight gains, metabolic alterations and deficits of learning, and hippocampal synaptic plasticity.

Orexins depolarize rostral ventrolateral medulla neurons and increase arterial pressure and heart rate in rats mainly via orexin 2 receptors

An injection of orexin A or B into the cisterna magna or the rostral ventrolateral medulla (RVLM), where bulbospinal vasomotor neurons are located, elevated arterial pressure (AP) and heart rate (HR). We examined how orexins affected RVLM neurons to regulate cardiovascular functions by using in vitro recordings of neuronal activity of the RVLM and in vivo measurement of cardiovascular functions in rats. Orexin A and B concentration-dependently depolarized RVLM neurons. At 100 nM, both peptides excited 42% of RVLM neurons. Tetrodotoxin failed to block orexin-induced depolarization. In the presence of N-(2-methyl-6-benzoxazolyl)-N′-1, 5-naphthyridin-4-yl urea (SB-334867), an orexin 1 receptor (OX1R) antagonist, orexin A depolarized 42% of RVLM neurons with a smaller, but not significantly different, amplitude (4.9 ± 0.8 versus 7.2 ± 1.1 mV). In the presence of (2S)-1- (3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-3,3-dimethyl-2-[(4-pyridinylmethyl)amino]-1-butanone hydrochloride (TCS OX2 29), an orexin 2 receptor (OX2R) antagonist, orexin A depolarized 25% of RVLM neurons with a significantly smaller amplitude (1.7 ± 0.5 mV). Coapplication of both antagonists completely eliminated orexin A-induced depolarization. An OX2R agonist, [Ala11,d-Leu15]-orexin B, concentration-dependently depolarized RVLM neurons. Regarding neuronal phenotypes, orexins depolarized 88% of adrenergic, 43% of nonadrenergic, and 36 to 41% of rhythmically firing RVLM neurons. Intracisternal TCS OX2 29 (3 and 10 nmol) suppressed intracisternal orexin A-induced increases of AP and HR, whereas intracisternal SB-334867 (3 and 10 nmol) had no effect on the orexin A-induced increase of HR but suppressed the orexin A-induced pressor response at 10 nmol. We concluded that orexins directly excite RVLM neurons, which include bulbospinal vasomotor neurons, and regulate cardiovascular function mainly via the OX2R, with a smaller contribution from the OX1R.

Blockade of central orexin 2 receptors reduces arterial pressure in spontaneously hypertensive rats

•  What is the central question of this study? Central orexinergic activity is involved in tonic and phasic control of cardiovascular homeostasis. A potential role for elevated central orexinergic activity in the maintenance of hypertension in spontaneously hypertensive rats (SHRs) has not previously been explored. •  What is the main finding and what is its importance? We show that central or intra‐rostral ventrolateral medulla blockade of orexin 2 receptors produces a significant reduction of arterial pressure in SHRs, but not Wistar–Kyoto rats. This study demonstrates a previously unrecognized role of orexin 2 receptors in maintaining hypertension in SHRs.

Orexins contribute to restraint stress-induced cocaine relapse by endocannabinoid-mediated disinhibition of dopaminergic neurons

Orexins are associated with drug relapse in rodents. Here, we show that acute restraint stress in mice activates lateral hypothalamic (LH) orexin neurons, increases levels of orexin A and 2-arachidonoylglycerol (2-AG) in the ventral tegmental area (VTA), and reinstates extinguished cocaine-conditioned place preference (CPP). This stress-induced reinstatement of cocaine CPP depends on type 1 orexin receptors (OX1Rs), type 1 cannabinoid receptors (CB1Rs) and diacylglycerol lipase (DAGL) in the VTA. In dopaminergic neurons of VTA slices, orexin A presynaptically inhibits GABAergic transmission. This effect is prevented by internal GDP-β-S or inhibiting OX1Rs, CB1Rs, phospholipase C or DAGL, and potentiated by inhibiting 2-AG degradation. These results suggest that restraint stress activates LH orexin neurons, releasing orexins into the VTA to activate postsynaptic OX1Rs of dopaminergic neurons and generate 2-AG through a Gq-protein-phospholipase C-DAGL cascade. 2-AG retrogradely inhibits GABA release through presynaptic CB1Rs, leading to VTA dopaminergic disinhibition and reinstatement of cocaine CPP.

Central pressor effects of CART peptides in anesthetized rats.

Interrelationships between energy homeostasis and regulation of cardiovascular functions have been suggested by previous observations [Am. J. Physiol. 278 (2000) R692; Regul. Pept. 104 (2002) 75; Am. J. Physiol. 277 (1999) R1780]. Cocaine- and amphetamine-regulated transcript (CART) was first discovered in the striatum of rats treated with cocaine or amphetamine. The CART peptides were later found in the hypothalamus and functioned as anorectic peptides. We observed that intracisternally (I.C.) administered CART peptide fragments (CART 61-102 and CART 55-102) dose-dependently (1-4 nmol) increased heart rate and blood pressure in urethane-anesthetized adult male Sprague-Dawley rats. Intrathecal (levels T2-T3) and intravenous administrations of these peptides, however, showed little or no effects on the heart rate and blood pressure in the rat. Furthermore, an increase of c-Fos-like immunoreactivity in the rat rostral ventrolateral medulla (RVLM) following an I.C. CART 61-102 was observed. The results suggest that central pressor effects of anorectic CART peptides may involve in activation of the medullary sympathetic systems in the rat. Our observations support the hypothesis that energy homeostasis and cardiovascular regulations are closely related and regulated.

Spontaneously hypertensive rats have more orexin neurons in the hypothalamus and enhanced orexinergic input and orexin 2 receptor‐associated nitric oxide signalling in the rostral ventrolateral medulla

What is the central question of this study? Our previous study demonstrates that elevated orexin 2 receptor (OX2R) activity within the rostral ventrolateral medulla (RVLM) contributes to hypertension in spontaneously hypertensive rats (SHRs), and a lower OX2R protein level was detected in their RVLM. The present study aims to explore the mechanisms underlying elevated orexinergic activity in the RVLM of SHRs, compared with their normotensive counterparts, Wistar‐Kyoto rats. What is the main finding and its importance? Increased orexinergic input into the RVLM and enhanced OX2R responsiveness in the RVLM, which was mainly mediated by augmented OX2R–neuronal nitric oxide synthase signalling, may underlie the elevated OX2R activity within the RVLM of SHRs.

Mediating Effects of Aryl-Hydrocarbon Receptor and RhoA in Altering Brain Vascular Integrity: The Therapeutic Potential of Statins

We have demonstrated previously that focal adhesion kinase (FAK)/RhoA alteration by the aryl-hydrocarbon receptor (AhR) agonist 3-methylcholanthrene (3MC) is involved in the antimigratory effects of 3MC in human umbilical vascular endothelial cells. Here, we identified that signaling properties and molecular mechanisms of RhoA/β-catenin were both implicated in alterations to blood-brain barrier integrity. The mechanisms of action were the down-regulation of integrin, the extracellular matrix, and adherens junction stability. PTEN phosphorylation by 3MC-mediated AhR/RhoA activation increased the proteasomal degradation of β-catenin through PKCδ/pGSK3β-mediated β-catenin phosphorylation; the crucial roles of AhR/RhoA in this process were verified by using gain- or loss-of-function experiments. The decrease in β-catenin led to decreased expression of fibronectin and α5β1 integrin. Additionally, protein interactions among FAK, VE-cadherin, vinculin, and β-actin were simultaneously decreased, resulting in adherens junction instability. Novel functional TCF/LEF1 binding sites in the promoter regions of fibronectin and α5/β1 integrin were identified by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results indicate that the binding activities of β-catenin decreased in mouse cerebrovascular endothelial cells treated with 3MC. In addition, simvastatin and pravastatin treatment reversed 3MC-mediated alterations in mouse cerebrovascular endothelial cells by RhoA inactivation, and the in vitro findings were substantiated by an in vivo blood-brain barrier assay. Thus, endothelial barrier dysfunction due to 3MC occurs through AhR/RhoA-mediated β-catenin down-regulation, which is reversed by simvastatin treatment in vivo.

BPR0C261 is a novel orally active antitumor agent with antimitotic and anti-angiogenic activities

BPR0C261 is a synthetic small molecule compound cytotoxic against human cancer cells and active prolonging the lifespan of leukemia mice. In the present study, we further investigated the mechanisms of its anticancer action and found that BPR0C261 inhibited microtubule polymerization through interacting with the colchicine binding sites on tubulins, disrupted microtubule arrangement and caused cell cycle arrest at G2/M phase in cancer cells. BPR0C261 also inhibited the clonogenic growths of cancer cells and showed cytotoxicity against human cervical cancer cells of multidrug‐resistant phenotype. In addition, BPR0C261 concentration‐dependently inhibited the proliferation and migration of HUVECs and disrupted the endothelial capillary‐like tube formations in HUVEC and rat aorta ring cultures. Given orally, BPR0C261 inhibited angiogenesis in s.c. implanted Matrigel plugs in mice. Notably, its IC50 values against the endothelial cell growths were approximately 10‐fold lower than those against the cancer cells. It was found orally absorbable in mice and showed a good oral bioavailability (43%) in dogs. BPR0C261 permeated through the human intestinal Caco‐2 cell monolayer, suggesting oral availability in humans. Orally absorbed BPR0C261 distributed readily into the s.c. xenografted tumors in nude mice in which the tumor tissue levels of BPR0C261 were found oral dose‐dependent. BPR0C261 showed in vivo activities against human colorectal, gastric, and nasopharyngeal tumors in nude mice. Most interestingly, the combination of BPR0C261 plus cisplatin synergistically prolonged the lifespans of mice inoculated with murine leukemia cells. Thus, BPR0C261 is a novel orally active tubulin‐binding antitumor agent with antimitotic, apoptosis‐inducing, and vasculature disrupting activities. (Cancer Sci 2011; 102: 182–191)

Intravenous paclitaxel against metastasis of human gastric tumors of diffuse type

PurposeGastric cancer is one of the leading cancerous diseases worldwide. It is diagnosed often at the advanced stage for which chemotherapy is the main treatment option. The prognosis remains poor for metastatic, especially the diffuse type, gastric cancers. We investigated the efficacy of intravenously administered paclitaxel treating metastases of locally disseminated gastric tumors of diffuse type.MethodsTransfection of green fluorescent proteins (GFP)-expressing plasmid into human gastric cancer MKN45 cells of diffuse type was performed, and MKN45-GFP cells constitutively expressing GFP were isolated. The MKN45-GFP cells were orthotopically inoculated into the mouse peritoneal cavity, and tumor growth and organ metastases were monitored. Liver metastases were harvested, re-inoculated, monitored for liver metastases again, and harvested for further inoculation. This in vivo selection procedure was repeated to isolate a subline with high metastatic abilities demonstrated by in vitro invasion abilities using Transwell® system. By visualizing the GFP-expressing tumors, the effects of intravenously administered paclitaxel against the growing peritoneally disseminated and metastasized tumors in nude mice without laparotomy were measured.ResultsAn in vivo selected gastric cancer cell line MKN45-GFP-ip4 with high metastatic ability was established. Its invasion ability was inhibited by paclitaxel treatments in vitro. The growths of metastatic and intraperitoneally disseminated MKN45-GFP-ip4 tumors were significantly suppressed by intravenous paclitaxel treatments in nude mice.ConclusionsWe found that intravenous paclitaxel is active against the metastases of human gastric cancer of peritoneal diffuse type, which warrants further investigations on optimizing the perioperative regimens with intravenous paclitaxel therapy for gastric cancer in patients.

Cardiovascular pressor effects of orexins in the dorsomedial hypothalamus

Abstract Orexins are important regulators of cardiovascular functions in various physiological and pathological conditions. The dorsomedial hypothalamus (DMH), an essential mediator of cardiovascular responses to stress, contains dense orexinergic innervations and receptors. We examined whether orexins can regulate cardiovascular functions through their actions in the DMH in anesthetized rats. An intra‐DMH injection of orexin A (30 pmol) produced elevation of arterial pressure and heart rate. Orexin A‐sensitive sites were located within or immediately adjacent to the DMH and larger responses were induced at the compact part of the dorsomedial hypothalamic nucleus. Orexin A‐induced responses were attenuated by intra‐DMH pretreatment with an orexin receptor 1 (OX1R) antagonist, SB‐334867 (15 nmol) (17.7 ± 2.8 vs. 5.2 ± 1.0 mmHg; 54.6 ± 10.0 vs. 22.8 ± 7.4 beats/min). Intra‐DMH applied [Ala11,D‐Leu15]‐orexin B (300 pmol), an orexin receptor 2 (OX2R) agonist, elicited cardiovascular responses mimicking the responses of orexin A, except for a smaller pressor response (7.4 ± 1.7 vs. 16.4 ± 1.8 mmHg). In a series of experiment, effects of orexin B (100 pmol) and then orexin A (30 pmol), were examined at a same site. Two patterns of responses were observed in 12 intra‐DMH sites: (1) both orexin A and B (9 sites), and (2) only orexin A (3 sites) induced cardiovascular responses, respectively suggesting OX1 R/OX2R‐mediated and OX1R‐predominant mechanisms. In conclusion, orexins regulated cardiovascular functions through OX1R/OX2R‐ or OX1R‐mediated mechanisms at different locations in the DMH.