Chronic infusion of amyloid-β peptide and sustained attention altered α7 nicotinic receptor density in the rat brain

It is already known that progressive degeneration of cholinergic neurons in brain areas such as the hippocampus and the cortex leads to memory deficits, as observed in Alzheimer's disease. This work verified the effects of the infusion of amyloid-beta (A beta) peptide associated to an attentional rehearsal on the density of alpha 7 nicotinic cholinergic receptor (nAChR) in the brain of male Wistar rats. Animals received intracerebroventricular infusion of A beta or vehicle (control - C) and their attention was stimulated weekly (Stimulated A beta group: S-A beta and Stimulated Control group: SC) or not (Non-Stimulated A beta group: N-SA beta and Non-Stimulated Control group: N-SC), using an active avoidance apparatus. Conditioned avoidance responses (CAR) were registered. Chronic infusion of A beta caused a 37% reduction in CAR for N-SA beta. In S-A beta, this reduction was not observed. At the end, brains were extracted and autoradiography for alpha 7 nAChR was conducted using [I-125]-alpha-bungarotoxin. There was an increase in alpha 7 density in hippocampus, cortex and amygdala of SA beta animals, together with the memory preservation. In recent findings from our lab using mice infused with A beta and the alpha 7 antagonist methyllycaconitine, and stimulated weekly in the same apparatus, it was observed that memory maintenance was abolished. So, the increase in alpha 7 density in brain areas related to memory might be related to a participation of this receptor in the long-lasting change in synaptic plasticity, which is important to improve and maintain memory consolidation.

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) gamma Activators and Pan-PPAR Partial Agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) gamma to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPAR gamma ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPAR gamma LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPAR gamma LBD, stronger partial agonists with full length PPAR gamma and exhibit full blockade of PPAR gamma phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPAR gamma also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/beta-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPAR gamma modulators with useful clinical profiles among natural products.

Increased vascular contractility and oxidative stress in beta(2)-adrenoceptor knockout mice: the role of NADPH oxidase

Background/Aims: beta(2)-adrenoceptor (beta(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal beta(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional beta(2)-AR (beta 2KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from beta 2KO and wild-type mice (WT) were studied. beta 2KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between beta 2KO and WT mice. Basal NO availability was reduced in aortas from beta 2KO mice. Incubation of beta 2KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. beta 2KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in beta 2KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of beta 2KO mice. This study extends the knowledge of the relevance of the endogenous activity of beta(2)-AR to the maintenance of the vascular physiology. Copyright (C) 2012 S. Karger AG, Basel

GQ-16, a Novel Peroxisome Proliferator-activated Receptor gamma (PPAR gamma) Ligand, Promotes Insulin Sensitization without Weight Gain

The recent discovery that peroxisome proliferator-activated receptor gamma (PPAR gamma) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPAR gamma activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPAR gamma. The structure of GQ-16 bound to PPAR gamma demonstrates that the compound utilizes a binding mode distinct from other reported PPAR gamma ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the beta-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPAR gamma-based therapeutics stabilize the beta-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPAR gamma modulators that retain antidiabetic actions while minimizing untoward effects.

Impaired beta-adrenoceptor-induced relaxation in small mesenteric arteries from DOCA-salt hypertensive rats is due to reduced K-Ca channel activity

beta-Adrenoceptor (beta-AR)-mediated relaxation plays an important role in the regulation of vascular tone. beta-AR-mediated vascular relaxation is reduced in various disease states and aging. We hypothesized that beta-AR-mediated vasodilatation is impaired in DOCA-salt hypertension due to alterations in the cAMP pathway. beta-AR-mediated relaxation was determined in small mesenteric arteries from DOCA-salt hypertensive and control uninephrectomized (Uni) rats. To exclude nitric oxide (NO) and cyclooxygenase (COX) pathways, relaxation responses were determined in the presence of L-NNA and indomethacin, NO synthase inhibitor and COX inhibitors, respectively. Isoprenaline (ISO)-induced relaxation was reduced in arteries from DOCA-salt compared to Uni rats. Protein kinase A (PKA) inhibitors (H89 or Rp-cAMPS) or adenylyl cyclase inhibitor (SQ22536) did not abolish the difference in ISO-induced relaxation between the groups. Forskolin (adenylyl cyclase activator)-induced relaxation was similar between the groups. The inhibition of IKCa/SKCa channels (TRAM-34 plus UCL1684) or BKCa channels (iberiotoxin) reduced ISO-induced relaxation only in Uni rats and abolished the relaxation differences between the groups. The expression of SKCa channel was decreased in DOCA-salt arteries. The expression of BKCa channel a subunit was increased whereas the expression of BKCa channel p subunit was decreased in DOCA-salt arteries. The expression of receptor for activated C kinase 1 (RACK1), which is a binding protein for BKG, channel and negatively modulates its activity, was increased in DOCA-salt arteries. These results suggest that the impairment of beta-AR-mediated relaxation in DOCA-salt mesenteric arteries may be attributable to altered IKCa/SKCa and/or BKCa channels activities rather than cAMP/PKA pathway. Impaired beta-AR-stimulated BKCa channel activity may be due to the imbalance between its subunit expressions and RACK1 upregulation. (C) 2012 Elsevier Ltd. All rights reserved.

17 beta-Estradiol replacement in young, adult and middle-aged female ovariectomized rats promotes improvement of spatial reference memory and an antidepressant effect and alters monoamines and BDNF levels in memory- and depression-related brain areas

Clinical and experimental evidence suggest that estrogens have a major impact on cognition, presenting neurotrophic and neuroprotective actions in regions involved in such function. In opposite, some studies indicate that certain hormone therapy regimens may provoke detrimental effects over female cognitive and neurological function. Therefore, we decided to investigate how estrogen treatment would influence cognition and depression in different ages. For that matter, this study assessed the effects of chronic 17 beta-estradiol treatment over cognition and depressive-like behaviors of young (3 months old), adult (7 months old) and middle-aged (12 months old) reproductive female Wistar rats. These functions were also correlated with alterations in the serotonergic system, as well as hippocampal BDNF. 17 beta-Estradiol treatment did not influence animals' locomotor activity and exploratory behavior, but it was able to improve the performance of adult and middle-aged rats in the Morris water maze, the latter being more responsive to the treatment. Young and adult rats displayed decreased immobility time in the forced swimming test, suggesting an effect of 17 beta-estradiol also over such depressive-like behavior. This same test revealed increased swimming behavior, triggered by serotonergic pathway, in adult rats. Neurochemical evaluations indicated that 17 beta-estradiol treatment was able to increase serotonin turnover rate in the hippocampus of adult rats. Interestingly, estrogen treatment increased BDNF levels from animals of all ages. These findings support the notion that the beneficial effects of 17 beta-estradiol over spatial reference memory and depressive-like behavior are evident only when hormone therapy occurs at early ages and early stages of hormonal decline. (C) 2011 Elsevier B.V. All rights reserved.

Mode of Peroxisome Proliferator-Activated Receptor gamma Activation by Luteolin

The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a target for treatment of type II diabetes and other conditions. PPAR gamma full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPAR gamma but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPAR gamma agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPAR gamma target genes in 3T3-L1 cells (LPL, ORL1, and CEBP alpha) and PPAR gamma-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPAR gamma ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPAR gamma LBD conformer and occupies a space near the beta-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPAR gamma, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Omega-loop among H2', H3, and the beta-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPAR gamma-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPAR gamma modulators.

Effects of Continuous Erythropoietin Receptor Activator in Sepsis-Induced Acute Kidney Injury and Multi-Organ Dysfunction

Background: Despite advances in supportive care, sepsis-related mortality remains high, especially in patients with acute kidney injury (AKI). Erythropoietin can protect organs against ischemia and sepsis. This effect has been linked to activation of intracellular survival pathways, although the mechanism remains unclear. Continuous erythropoietin receptor activator (CERA) is an erythropoietin with a unique pharmacologic profile and long half-life. We hypothesized that pretreatment with CERA would be renoprotective in the cecal ligation and puncture (CLP) model of sepsis-induced AKI. Methods: Rats were randomized into three groups: control; CLP; and CLP+CERA (5 mu g/kg body weight, i.p. administered 24 h before CLP). At 24 hours after CLP, we measured creatinine clearance, biochemical variables, and hemodynamic parameters. In kidney tissue, we performed immunoblotting-to quantify expression of the Na-K-2Cl cotransporter (NKCC2), aquaporin 2 (AQP2), Toll-like receptor 4 (TLR4), erythropoietin receptor (EpoR), and nuclear factor kappa B (NF-kappa B)-and immunohistochemical staining for CD68 (macrophage infiltration). Plasma interleukin (IL)-2, IL-1 beta, IL-6, IL-10, interferon gamma, and tumor necrosis factor alpha were measured by multiplex detection. Results: Pretreatment with CERA preserved creatinine clearance and tubular function, as well as the expression of NKCC2 and AQP2. In addition, CERA maintained plasma lactate at normal levels, as well as preserving plasma levels of transaminases and lactate dehydrogenase. Renal expression of TLR4 and NF-kappa B was lower in CLP+CERA rats than in CLP rats (p<0.05 and p<0.01, respectively), as were CD68-positive cell counts (p<0.01), whereas renal EpoR expression was higher (p<0.05). Plasma levels of all measured cytokines were lower in CLP+CERA rats than in CLP rats. Conclusion: CERA protects against sepsis-induced AKI. This protective effect is, in part, attributable to suppression of the inflammatory response.

Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils

Neutrophil migration to inflamed sites is crucial for both the initiation of inflammation and resolution of infection, yet these cells are involved in perpetuation of different chronic inflammatory diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts through G protein coupled receptors (GPCRs) involved in signal transmission in both central and peripheral nervous systems. Its receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is a selective GRPR antagonist, recently found to have antiinflammatory properties in arthritis and sepsis models. Here we demonstrate that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is blocked by RC-3095. Macrophage depletion or neutralization of TNF abrogates GRP-induced neutrophil recruitment to the peritoneum. In vitro, GRP-induced neutrophil migration was dependent on PLC-beta 2, PI3K, ERK, p38 and independent of G alpha i protein, and neutrophil migration toward synovial fluid of arthritis patients was inhibited by treatment with RC-3095. We propose that GRPR is an alternative chemotactic receptor that may play a role in the pathogenesis of inflammatory disorders.

Effect of Cholecalciferol as Adjunctive Therapy With Insulin on Protective Immunologic Profile and Decline of Residual beta-Cell Function in New-Onset Type 1 Diabetes Mellitus

Objective: To evaluate the effect of vitamin D-3 on cytokine levels, regulatory T cells, and residual beta-cell function decline when cholecalciferol (vitamin D-3 administered therapeutically) is given as adjunctive therapy with insulin in new-onset type 1 diabetes mellitus (T1DM). Design and Setting: An 18-month (March 10, 2006, to October 28, 2010) randomized, double-blind, placebo-controlled trial was conducted at the Diabetes Center of Sao Paulo Federal University, Sao Paulo, Brazil. Participants: Thirty-eight patients with new-onset T1DM with fasting serum C-peptide levels greater than or equal to 0.6 ng/mL were randomly assigned to receive daily oral therapy of cholecalciferol, 2000 IU, or placebo. Main Outcome Measure: Levels of proinflammatory and anti-inflammatory cytokines, chemokines, regulatory T cells, hemoglobin A(1c), and C-peptide; body mass index; and insulin daily dose. Results: Mean (SD) chemokine ligand 2 (monocyte chemoattractant protein 1) levels were significantly higher (184.6 [101.1] vs 121.4 [55.8] pg/mL) at 12 months, as well as the increase in regulatory T-cell percentage (4.55%[1.5%] vs 3.34%[1.8%]) with cholecalciferol vs placebo. The cumulative incidence of progression to undetectable (<= 0.1 ng/mL) fasting C-peptide reached 18.7% in the cholecalciferol group and 62.5% in the placebo group; stimulated C-peptide reached 6.2% in the cholecalciferol group and 37.5% in the placebo group at 18 months. Body mass index, hemoglobin A(1c) level, and insulin requirements were similar between the 2 groups. Conclusions: Cholecalciferol used as adjunctive therapy with insulin is safe and associated with a protective immunologic effect and slow decline of residual beta-cell function in patients with new-onset T1DM. Cholecalciferol may be an interesting adjuvant in T1DM prevention trials.

Analysis of the mRNA expression of the S100 beta protein in adipocytes of patients with diabetes mellitus, type 2

Objective: This study aims to explore the possible relationship between the expression level of S100 beta protein mRNA with diabetes mellitus type 2 in adipocytes from patients with this disease in comparison with normoglycemic individuals. Materials and methods: Samples of adipose tissue of eight patients from the coronary section of the Institute Dante Pazzanese of Cardiology (IDPC), four in Group Diabetes and four of Normoglycemic group, were evaluated by RT-PCR real time. Results: An increase around 15 times values, between the threshold cycle (Delta Ct), of mRNA expression of S100 beta protein in adipocytes of the diabetes group was observed in comparison to the control group (p = 0.015). Conclusion: Our results indicate, for the first time, that there is coexistence of increased expression of the S100 beta and the type 2 diabetes mellitus gene. Arq Bras Endocrinol Metab. 2012;56(7):435-40

Structural Insights into Human Peroxisome Proliferator Activated Receptor Delta (PPAR-Delta) Selective Ligand Binding

Peroxisome proliferator activated receptors (PPARs delta, alpha and gamma) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPAR delta more than 300-fold more tightly than PPAR alpha or PPAR gamma but the structural basis of PPAR delta: GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPAR delta:GW0742 complex. Comparisons of the PPAR delta:GW0742 complex with published structures of PPARs in complex with alpha and gamma selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPAR delta selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPAR alpha and gamma ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPAR delta ligand design.

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

Abstract Background Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. Methods Twenty-four adult Wistar rats, 60 days old (+/- 250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [100 μg/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a.m. Results Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. Conclusions We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

MiRNA-208a and miRNA-208b are triggered in thyroid hormone-induced cardiac hypertrophy - role of type 1 Angiotensin II receptor (AT1R) on miRNA-208a/α-MHC modulation

Hyperthyroidism promotes cardiac hypertrophy and the Angiotensin type 1 receptor (AT1R) has been demonstrated to mediate part of this response. Recent studies have uncovered a potentially important role for the microRNAs (miRNAs) in the control of diverse aspects of cardiac function. Then, the objective of the present study was to investigate the action promoted by hyperthyroidism on β-MHC/miR-208b expression and on α-MHC/miR-208a expression, as well as the possible contribution of the AT1R in this event. The findings of this study confirmed that AT1R is a key mediator of the cardiac hypertrophy induced by hyperthyroidism. Additionally, we demonstrated that like β-MHC, miR-208b was down-regulated in the hyperthyroid group. Similarly, like the expression of its host gene, α-MHC, miR-208a expression was up-regulated in response to hyperthyroidism. Finally, our data suggest for the first time that AT1R mediates the hyperthyroidism-induced increase on cardiac miRNA-208a/α-MHC levels, while does not influence on the reduction of miRNA-208b/β-MHC levels.