Tumor necrosis factor alpha has a crucial role in increased reactive oxygen species production in platelets of mice injected with lipopolysaccharide.

Increased reactive oxygen species (ROS) production leads to tissue damage observed in sepsis and lipopolysaccharide (LPS)-exposed animals. LPS stimulates cytokines releasing, including tumor necrosis factor alpha (TNF-α), that is important to ROS production. Platelets, considered inflammatory cells, generate ROS when exposed to LPS in vivo, but not when they are incubated in vitro with this compound. Therefore, we investigated the role of TNF-α on the increased intraplatelet ROS levels after LPS treatment. Mice were injected with LPS (1 mg/kg) or TNF-α (10 ng/kg), and blood was collected to prepare the washed platelets. Animals were treated with infliximab (anti-TNF-α antibody), R-7050 (non-selective TNF-α receptor antagonist) or apocynin (NADPH oxidase inhibitor). At 48 h after LPS or TNF-α injection, the ROS levels in ADP (25 µM)-activated platelets were evaluated by flow cytometry. Our data showed that injection of mice with LPS increased by 4-fold the ROS production (p < 0.05), which was significantly reduced by the treatments with infliximab, R-7050 or apocynin. Injection of mice with TNF-α markedly elevated the ROS formation in platelets (p < 0.05) that was reduced by infliximab, R-7050 or apocynin treatments. In separate experiments, platelets from saline-injected mice were incubated with TNF-α (30 to 3000 pg/mL) in absence or presence of infliximab, R-7050, apocynin or GKT137831 (NOX1/NOX4 inhibitor) before ROS measurements. TNF-α in vitro markedly increased the ROS levels, an effect significantly reduced by all treatments. Therefore, platelets are involved in the oxidative stress induced by LPS through TNF-α action, and NADPH oxidase takes part in this effect.

Implication of Rho-kinase and soluble guanylyl cyclase enzymes in prostate smooth muscle dysfunction in middle-aged rats.

AIMS: Aging is highly associated with benign prostate hyperplasia (BPH). We investigated here the alterations of the contractile and relaxant machinery in prostates of middle-aged rats, focusing on the Rho-kinase, nitric oxide (NO)-soluble guanylyl cyclase (sGC), α1- and ß-adrenoceptor pathways. METHODS: Male Wistar young (3.5-month old) and middle-aged rats (10-month old) were used. Quantitative image analysis of prostates and functional assays evaluating the prostate contractions and relaxations were employed. Measurement of [3 H]-noradrenaline efflux, western blotting for α1 and ß1 sGC subunits, and cyclic nucleotide levels were carried out. RESULTS: Prostates of middle-aged rats showed significant increases in lumen and smooth muscle cells, but no alterations in the relative prostate weight were observed. In vivo, noradrenaline (10-7 -10-4 g/kg) produced greater prostatic contractions in middle-aged compared with control rats. Likewise, the in vitro contractions to phenylephrine (1 nM-100 µM) and α,ß-methylene ATP (1-10 µM) were greater in middle-aged rats. Electrical-field stimulation (EFS, 1-32 Hz) promoted higher [3 H]-noradrenaline efflux and prostate contractions in middle-aged rats. Reduced expressions of α1 and ß1 sGC subunits and diminished NO-mediated prostate relaxations in middle-age were observed. Isoproterenol-induced relaxations and cAMP levels were reduced in prostates of middle-aged rats. The Rho-kinase inhibitor fasudil (50 mg/kg, 2 weeks) normalized the prostate hypercontractility in middle-age rats. CONCLUSIONS: Prostate hypercontractility in middle-aging is associated with increased release of noradrenaline and Rho-kinase pathway, as well as with impairments of NO-sGC and ß-adrenoceptor pathways. Middle-aged rats are suitable to explore the enhanced prostatic tone in the absence of prostate overgrowth. Neurourol. Urodynam. 36:589-596, 2017. © 2016 Wiley Periodicals, Inc.

The soluble guanylyl cyclase activator BAY 60-2770 inhibits murine allergic airways inflammation and human eosinophil chemotaxis.

OBJECTIVES: Activators of soluble guanylyl cyclase (sGC) act preferentially in conditions of enzyme oxidation or haem group removal. This study was designed to investigate the effects of the sGC activator BAY 60-2770 in murine airways inflammation and human eosinophil chemotaxis. METHODS: C57Bl/6 mice treated or not with BAY 60-2770 (1 mg/kg/day, 14 days) were intranasally challenged with ovalbumin (OVA). At 48 h, bronchoalveolar lavage fluid (BALF) was performed, and circulating blood, bone marrow and lungs were obtained. Human eosinophils purified from peripheral blood were used to evaluate the cell chemotaxis. RESULTS: OVA-challenge promoted marked increases in eosinophil number in BAL, lung tissue, circulating blood and bone marrow, all of which were significantly reduced by BAY 60-2770. The IL-4 and IL-5 levels in BALF were significantly reduced by BAY 60-2770. Increased protein expression of iNOS, along with decreases of expression of sGC (α1 and ß1 subunits) and cGMP levels were detected in lung tissue of OVA-challenged mice. BAY 60-2770 fully restored to baseline the iNOS and sGC subunit expressions, and cGMP levels. In human isolated eosinophils, BAY 60-2770 (1-5 µM) had no effects on the cGMP levels and eotaxin-induced chemotaxis; however, prior incubation with ODQ (10 µM) markedly elevated the BAY 60-2770-induced cyclic GMP production, further inhibiting the eosinophil chemotaxis. CONCLUSIONS: BAY 60-2770 reduces airway eosinophilic inflammation and rescue the sGC levels. In human eosinophils under oxidized conditions, BAY 60-2770 elevates the cGMP levels causing cell chemotaxis inhibition. BAY 60-2770 may reveal a novel therapeutic target for asthma treatment.

Increased arterial stiffness in resistant hypertension is associated with inflammatory biomarkers.

BACKGROUND: Increased levels of inflammatory biomarkers such as interleukin-6 (IL-6), 10 (IL-10), 1ß (IL-1ß), tumor necrosis factor-α (TNF-α) high-sensitivity C-reactive protein (hs-CRP) are associated with arterial stiffness in hypertension. Indeed, resistant hypertension (RHTN) leads to unfavorable prognosis attributed to poor blood pressure (BP) control and target organ damage. This study evaluated the potential impact of inflammatory biomarkers on arterial stiffness in RHTN. METHODS: In this cross-sectional study, 32 RHTN, 20 mild hypertensive (HTN) and 20 normotensive (NT) patients were subjected to office BP and arterial stiffness measurements assessed by pulse wave velocity (PWV). Inflammatory biomarkers were measured in plasma samples. RESULTS: PWV was increased in RHTN compared with HTN and NT (p < 0.05). TNF-α levels were significantly higher in RHTN and HTN than NT patients. No differences in IL-6 levels were observed. RHTN patients had a higher frequency of subjects with increased levels of IL-10 and IL-1ß compared with HTN and NT patients. Finally, IL-1ß was independently associated with PWV (p < 0.001; R(2) = 0.5; ß = 0.077). CONCLUSION: RHTN subjects have higher levels of inflammatory cytokines (TNF-α, IL-1ß and IL-10) as well as increased arterial stiffness, and detectable IL-1ß levels are associated arterial stiffness. These findings suggest that inflammation plays a possible role in the pathophysiology of RHTN.

Long-term disruption of maternal glucose homeostasis induced by prenatal glucocorticoid treatment correlates with miR-29 upregulation.

Excess of glucocorticoids (GCs) during pregnancy is strongly associated with the programming of glucose intolerance in the offspring. However, the impact of high GC levels on maternal metabolism is not clearly documented. This study aimed to test the hypothesis that mothers exposed to elevated levels of GCs might also display long-term disturbances in glucose homeostasis. Dexamethasone (DEX) was administered noninvasively to the mothers via drinking water between the 14th and the 19th days of pregnancy. Mothers were subjected to glucose and insulin tolerance tests at 1, 2, 3, 6, and 12 mo postweaning. Pregnant rats not treated with DEX and age-matched virgin rats were used as controls. Pancreatic islets were isolated at the 20th day of pregnancy and 12 mo postweaning in order to evaluate glucose-stimulated insulin secretion. The expression of the miR-29 family was also studied due to its responsiveness to GCs and its well-documented role in the regulation of pancreatic ß-cell function. Rats treated with DEX during pregnancy presented long-term glucose intolerance and impaired insulin secretion. These changes correlated with 1) increased expression of miR-29 and its regulator p53, 2) reduced expression of syntaxin-1a, a direct target of miR-29, and 3) altered expression of genes related to cellular senescence. Our data demonstrate that the use of DEX during pregnancy results in deleterious outcomes to the maternal metabolism, hallmarked by reduced insulin secretion and glucose intolerance. This maternal metabolic programming might be a consequence of time-sustained upregulation of miR-29s in maternal pancreatic islets.

Selective regulation of hepatic lipid metabolism by the AMP-activated protein kinase pathway in late-pregnant rats.

The liver plays an essential role in maternal metabolic adaptation during late pregnancy. With regard to lipid metabolism, increased secretion of very low-density lipoprotein (VLDL) is characteristic of late pregnancy. Despite this well-described metabolic plasticity, the molecular changes underlying the hepatic adaptation to pregnancy remain unclear. As AMPK is a key intracellular energy sensor, we investigated whether this protein assumes a causal role in the hepatic adaptation to pregnancy. Pregnant Wistar rats were treated with vehicle or AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) for 5 days starting at gestational day 14. At the end of treatment, the rats were subjected to an intraperitoneal pyruvate tolerance test and in situ liver perfusion with pyruvate. The livers were processed for Western blot analysis, quantitative PCR, thin-layer chromatography, enzymatic activity, and glycogen content measurements. Blood biochemical profiles were also assessed. We found that AMPK and ACC phosphorylation were reduced in the livers of pregnant rats in parallel with a reduced level of hepatic gluconeogenesis of pyruvate. This effect was accompanied by both a reduction in the levels of hepatic triglycerides (TG) and an increase in circulating levels of TG. Treatment with AICAR restored hepatic levels of TG to those observed in nonpregnant rats. Additionally, AMPK activation reduced the upregulation of genes related to VLDL synthesis and secretion observed in the livers of pregnant rats. We conclude that the increased secretion of hepatic TG in late pregnancy is concurrent with a transcriptional profile that favors VLDL production. This transcriptional profile results from the reduction in hepatic AMPK activity.

Insulin relaxes bladder via PI3K/AKT/eNOS pathway activation in mucosa: unfolded protein response-dependent insulin resistance as a cause of obesity-associated overactive bladder.

We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration-response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1-100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice.

Chaperone insufficiency links TLR4 protein signaling to endoplasmic reticulum stress.

Inflammation plays an important pathogenic role in a number of metabolic diseases such as obesity, type 2 diabetes, and atherosclerosis. The activation of inflammation in these diseases depends at least in part on the combined actions of TLR4 signaling and endoplasmic reticulum stress, which by acting in concert can boost the inflammatory response. Defining the mechanisms involved in this phenomenon may unveil potential targets for the treatment of metabolic/inflammatory diseases. Here we used LPS to induce endoplasmic reticulum stress in the human monocyte cell-line, THP-1. The unfolded protein response, produced after LPS, was dependent on CD14 activity but not on RNA-dependent protein kinase and could be inhibited by an exogenous chemical chaperone. The induction of the endoplasmic reticulum resident chaperones, GRP94 and GRP78, by LPS was of a much lower magnitude than the effect of LPS on TLR4 and MD-2 expression. In face of this apparent insufficiency of chaperone expression, we induced the expression of GRP94 and GRP78 by glucose deprivation. This approach completely reverted endoplasmic reticulum stress. The inhibition of either GRP94 or GRP78 with siRNA was sufficient to rescue the protective effect of glucose deprivation on LPS-induced endoplasmic reticulum stress. Thus, insufficient LPS-induced chaperone expression links TLR4 signaling to endoplasmic reticulum stress.

Airway exposure to staphylococcal enterotoxin A potentiates allergen-induced bone marrow eosinophilia and trafficking to peripheral blood and airways.

Bone marrow (BM) eosinopoiesis is a common feature during allergen exposure in atopic individuals. Airway exposure to staphylococcal superantigens aggravates allergic airway disease and increases the output of BM eosinophils. However, the exact mechanisms regulating eosinophil mobilization and trafficking to the peripheral circulation and airways remain to be elucidated. Therefore, this study aimed to investigate the mechanisms determining the BM eosinopoiesis in allergic mice under exposure to staphylococcal enterotoxin A (SEA). Ovalbumin (OVA)-sensitized male BALB/C mice were intranasally exposed to SEA (1 µg), and at 4, 12, 24, and 48 h later animals were challenged with OVA (10 µg, twice a day). Measurement of IL-5, eotaxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, flow cytometry for CCR3(+), VLA4(+), and CCR3(+)VLA4(+), as well as adhesion assays to VCAM-1 were performed in BM. Prior airway exposure to SEA time dependently increased the BM eosinophil number in OVA-challenged mice. Eosinophils gradually disappear from peripheral blood, being recruited over time to the airways, where they achieve a maximal infiltration at 24 h. SEA exposure increased the levels of IL-5 and eotaxin (but not GM-CSF) in BM of OVA-challenged mice. Marked increases in CCR3(+) and CCR3(+)VLA4(+) expressions in BM eosinophils of OVA-challenged mice were observed, an effect largely reduced by prior exposure to SEA. Adhesion of BM eosinophils to VCAM-1 was increased in OVA-challenged mice, but prior SEA exposure abrogated this enhanced cell adhesion. Accumulation of BM eosinophils by airway SEA exposure takes place through IL-5- and CCR3-dependent mechanisms, along with downregulation of CCR3/VL4 and impaired cell adhesion to VCAM-1.

Melatonin acts through MT1/MT2 receptors to activate hypothalamic Akt and suppress hepatic gluconeogenesis in rats.

Melatonin can contribute to glucose homeostasis either by decreasing gluconeogenesis or by counteracting insulin resistance in distinct models of obesity. However, the precise mechanism through which melatonin controls glucose homeostasis is not completely understood. Male Wistar rats were administered an intracerebroventricular (icv) injection of melatonin and one of following: an icv injection of a phosphatidylinositol 3-kinase (PI3K) inhibitor, an icv injection of a melatonin receptor (MT) antagonist, or an intraperitoneal (ip) injection of a muscarinic receptor antagonist. Anesthetized rats were subjected to pyruvate tolerance test to estimate in vivo glucose clearance after pyruvate load and in situ liver perfusion to assess hepatic gluconeogenesis. The hypothalamus was removed to determine Akt phosphorylation. Melatonin injections in the central nervous system suppressed hepatic gluconeogenesis and increased hypothalamic Akt phosphorylation. These effects of melatonin were suppressed either by icv injections of PI3K inhibitors and MT antagonists and by ip injection of a muscarinic receptor antagonist. We conclude that melatonin activates hypothalamus-liver communication that may contribute to circadian adjustments of gluconeogenesis. These data further suggest a physiopathological relationship between the circadian disruptions in metabolism and reduced levels of melatonin found in type 2 diabetes patients.

UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression.

Endocrine pancreas from pregnant rats undergoes several adaptations that comprise increase in ß-cell number, mass and insulin secretion, and reduction of apoptosis. Lactogens are the main hormones that account for these changes. Maternal pancreas, however, returns to a nonpregnant state just after the delivery. The precise mechanism by which this reversal occurs is not settled but, in spite of high lactogen levels, a transient increase in apoptosis was already reported as early as the 3rd day of lactation (L3). Our results revealed that maternal islets displayed a transient increase in DNA fragmentation at L3, in parallel with decreased RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation (pAKT), a known prosurvival kinase. Wortmannin completely abolished the prosurvival action of prolactin (PRL) in cultured islets. Decreased pAKT in L3-islets correlated with increased Tribble 3 (TRB3) expression, a pseudokinase inhibitor of AKT. PERK and eIF2α phosphorylation transiently increased in islets from rats at the first day after delivery, followed by an increase in immunoglobulin heavy chain-binding protein (BiP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islets from L3 rats. Chromatin immunoprecipitation (ChIP) and Re-ChIP experiments further confirmed increased binding of the heterodimer ATF4/CHOP to the TRB3 promoter in L3 islets. Treatment with PBA, a chemical chaperone that inhibits UPR, restored pAKT levels and inhibited the increase in apoptosis found in L3. Moreover, PBA reduced CHOP and TRB3 levels in ß-cell from L3 rats. Altogether, our study collects compelling evidence that UPR underlies the physiological and transient increase in ß-cell apoptosis after delivery. The UPR is likely to counteract prosurvival actions of PRL by reducing pAKT through ATF4/CHOP-induced TRB3 expression.

MKP-1 mediates glucocorticoid-induced ERK1/2 dephosphorylation and reduction in pancreatic ß-cell proliferation in islets from early lactating mothers.

Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phospho-ERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression.

Long-term methylglyoxal intake aggravates murine Th2-mediated airway eosinophil infiltration.

Asthma outcomes is aggravated in obese patients. Excess of methylglyoxal (MGO) in obese/diabetic patients has been associated with diverse detrimental effects on cell function. This study aimed to evaluate the effects of long-term oral intake of MGO on ovalbumin-induced eosinophil inflammation. Male C57/Bl6 mice received 0.5% MGO in the drinking water for 12 weeks. Mice were sensitized and challenged with ovalbumin (OVA), and at 48 h thereafter, bronchoalveolar lavage (BAL) fluid and lungs were collected for cell counting, morphological analysis, and ELISA, mRNA expressions and DHE assays. In MGO-treated mice, OVA challenge significantly increased the peribronchiolar infiltrations of inflammatory cells and eosinophils compared with control group. Higher levels of IL-4, IL-5, and eotaxin in BAL fluid were also detected in MGO compared with control group. In addition, lung tissue of MGO-treated mice displayed significant increases in mRNA expressions of NF-κB and iNOS whereas COX-2 expression remained unchanged. The high TNF-α mRNA expression observed in lungs of OVA-challenged control mice was not further increased by MGO treatment. In MGO group, OVA-challenge increased significantly the NOX-2 and NOX-4 mRNA expressions, without affecting the NOX-1 expression. Levels of reactive-oxygen species (ROS) were significantly higher in lungs of MGO-treated mice, and no further increase by OVA-challenge was observed. In conclusion, 12-week intake of MGO exacerbates Th2-mediated airway eosinophil infiltration by activation of NF-kB/iNOS-dependent signaling pathway and positive regulation of NOX-2 and NOX-4 in the lung tissues. Scavengers of MGO could be an option to prevent obesity-related asthma.

Fructose Consumption by Adult Rats Exposed to Dexamethasone In Utero Changes the Phenotype of Intestinal Epithelial Cells and Exacerbates Intestinal Gluconeogenesis.

Fructose consumption by rodents modulates both hepatic and intestinal lipid metabolism and gluconeogenesis. We have previously demonstrated that in utero exposure to dexamethasone (DEX) interacts with fructose consumption during adult life to exacerbate hepatic steatosis in rats. The aim of this study was to clarify if adult rats born to DEX-treated mothers would display differences in intestinal gluconeogenesis after excessive fructose intake. To address this issue, female Wistar rats were treated with DEX during pregnancy and control (CTL) mothers were kept untreated. Adult offspring born to CTL and DEX-treated mothers were assigned to receive either tap water (Control-Standard Chow (CTL-SC) and Dexamethasone-Standard Chow (DEX-SC)) or 10% fructose in the drinking water (CTL-fructose and DEX-fructose). Fructose consumption lasted for 80 days. All rats were subjected to a 40 h fasting before sample collection. We found that DEX-fructose rats have increased glucose and reduced lactate in the portal blood. Jejunum samples of DEX-fructose rats have enhanced phosphoenolpyruvate carboxykinase (PEPCK) expression and activity, higher facilitated glucose transporter member 2 (GLUT2) and facilitated glucose transporter member 5 (GLUT5) content, and increased villous height, crypt depth, and proliferating cell nuclear antigen (PCNA) staining. The current data reveal that rats born to DEX-treated mothers that consume fructose during adult life have increased intestinal gluconeogenesis while recapitulating metabolic and morphological features of the neonatal jejunum phenotype.

In Utero Dexamethasone Exposure Exacerbates Hepatic Steatosis in Rats That Consume Fructose During Adulthood.

Distinct environmental insults might interact with fructose consumption and contribute to the development of metabolic disorders. To address whether in utero glucocorticoid exposure and fructose intake modulate metabolic responses, adult female Wistar rats were exposed to dexamethasone (DEX) during pregnancy, and the offspring were administered fructose at a later time. Briefly, dams received DEX during the third period of pregnancy, while control dams remained untreated. Offspring born to control and DEX-treated mothers were defined as CTL-off and DEX-off, respectively, while untreated animals were designated CTL-off-CTL and DEX-off-CTL. CLT-off and DEX-off treated with 10% fructose in the drinking water for 8 weeks are referred to as CTL-off-FRU and DEX-off-FRU. We found that fructose promoted glucose intolerance and whole-body gluconeogenesis in both CTL-off-FRU and DEX-off-FRU animals. On the other hand, hepatic lipid accumulation was significantly stimulated in DEX-off-FRU rats when compared to the CTL-off-FRU group. The DEX-off-FRU group also displayed impaired very-low-density lipoprotein (VLDL) production and reduced hepatic expression of apoB, mttp, and sec22b. DEX-off-FRU has lower hepatic levels of autophagy markers. Taken together, our results support the unprecedented notion that in utero glucocorticoid exposure exacerbates hepatic steatosis caused by fructose consumption later in life.

Signalling pathways involved in p47phox -dependent reactive oxygen species in platelets of endotoxemic rats.

Thrombocytopenia during sepsis is associated with a less favourable clinical outcome. Overproduction of reactive oxygen species (ROS) by different cell types contributes to sepsis. Platelets generate ROS, but the upstream pathways of NADPH oxidase activation are not completely understood. Here, we designed experiments in washed platelets from lipopolysaccharide (LPS)-treated rats to investigate the p47phox activation and ROS generation, and its modulation by c-Src family kinase (c-Src), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC) and protein kinase G (PKG). Rats were injected intraperitoneally with LPS (1 mg/kg), and at 48 hours thereafter, arterial blood was collected and washed platelets were obtained. Washed platelets were pre-incubated with different inhibitors and subsequently activated or not with ADP. Flow cytometry, Western blotting and ELISA were performed. We found that LPS significantly increased the p47phox phosphorylation and ROS generation compared with the control group (P < 0.05). The enhanced ROS production in the LPS group was unaffected by the non-selective SFKs inhibitor PP2, the PI3K inhibitor wortmannin or the Akt inhibitor PPI-1. The cyclic GMP levels were 115% higher in activated platelets of LPS compared with the saline group (P < 0.05). Moreover, in the LPS group, the sGC inhibitor ODQ, the PKG inhibitor Rp-8-Br and the PKC inhibitor GF109203X abrogated the increased p47phox phosphorylation and reduced the ROS levels. In conclusion, selective inhibitors of cGMP-PKG and PKC-p47phox pathways that regulate ROS generation by LPS in platelets may help control the redox balance in sepsis improving the survival of patients.

Activation of soluble guanylyl cyclase with inhibition of multidrug resistance protein inhibitor-4 (MRP4) as a new antiplatelet therapy.

The intracellular levels of cyclic GMP are controlled by its rate of formation through nitric oxide-mediated stimulation of soluble guanylate cyclase (sGC) and its degradation by phosphodiesterases. Multidrug resistance protein 4 (MRP4) expressed in human platelets pumps cyclic nucleotides out of cells. In search for new antiplatelet strategies, we tested the hypothesis that sGC activation concomitant with MRP4 inhibition confers higher antiplatelet efficacy compared with monotherapy alone. This study was undertaken to investigate the pharmacological association of the sGC activator BAY 60-2770 with the MRP4 inhibitor MK571 on human washed platelets. Collagen- and thrombin-induced platelet aggregation and ATP-release reaction assays were performed. BAY 60-2770 (0.001-10 µM) produced significant inhibitions of agonist-induced platelet aggregation accompanied by reduced ATP-release. Pre-incubation with 10 µM MK571 alone had no significant effect on platelet aggregation and ATP release, but it produced a left displacement by about of 10-100-fold in the concentration-response curves to BAY 60-2770. Pre-incubation with MK571increased and decreased, respectively, the intracellular and extracellular levels of cGMP to BAY 60-2770, whereas the cAMP levels remained unchanged. The increased VASP-serine 239 phosphorylation in BAY 60-2770-treated platelets was enhanced by MK571. In Fluo-4-loaded platelets, BAY 60-2770 reduced the intracellular Ca2+ levels, an effect significantly potentiated by MK571. Flow cytometry assays showed that BAY 60-2770 reduces the αIIbß3 integrin activation, which was further reduced by MK571 association. Blocking the MRP4-mediated efflux of cGMP may be a potential mechanism to enhance the antiplatelet efficacy of sGC activators.

Signal transducer and activator of transcription 3-regulated sarcoendoplasmic reticulum Ca2+-ATPase 2 expression by prolactin and glucocorticoids is involved in the adaptation of insulin secretory response during the peripartum period.

During pregnancy, the maternal endocrine pancreas undergoes, as a consequence of placental lactogens and prolactin (PRL) action, functional changes that are characterized by increased glucose-induced insulin secretion. After delivery, the maternal endocrine pancreas rapidly returns to non-pregnant state, which is mainly attributed to the increased serum levels of glucocorticoids (GCs). Although GCs are known to decrease insulin secretion and counteract PRL action, the mechanisms for these effects are poorly understood. We have previously demonstrated that signal transducer and activator of transcription 3 (STAT3) is increased in islets treated with PRL. In the present study, we show that STAT3 expression and serine phosphorylation are increased in pancreatic islets at the end of pregnancy (P19). STAT3 serine phosphorylation rapidly returned to basal levels 3 days after delivery (L3). The expression of the sarcoendoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2), a crucial protein involved in the regulation of calcium handling in beta-cells, was also increased in P19, returning to basal levels at L3. PRL increased SERCA2 and STAT3 expressions and STAT3 serine phosphorylation in RINm5F cells. The upregulation of SERCA2 by PRL was abolished after STAT3 knockdown. Moreover, PRL-induced STAT3 serine phosphorylation and SERCA2 expression were inhibited by dexamethasone (DEX). Insulin secretion from islets of P19 rats pre-incubated with thapsigargin and L3 rats showed a dramatic suppression of first phase of insulin release. The present results indicate that PRL regulates SERCA2 expression by a STAT3-dependent mechanism. PRL effect is counteracted by DEX and might contribute to the adaptation of maternal endocrine pancreas during the peripartum period.

High-fat diet-induced obesity impairs insulin signaling in lungs of allergen-challenged mice: Improvement by resveratrol.

Insulin resistance plays an important role in obesity-associated asthma exacerbations. Using a murine model of allergic airway inflammation, we evaluated the insulin signaling transmission in lungs of obese compared with lean mice. We further evaluated the effects of the polyphenol resveratrol in the pulmonary insulin signaling. In lean mice, insulin stimulation significantly increased phosphorylations of AKT, insulin receptor substrate 1 (IRS-1) and insulin receptor ß (IRß) in lung tissue and isolated bronchi (p < 0.05), which were impaired in obese group. Instead, obese mice displayed increased tyrosine nitrations of AKT, IRß and IRS-1 (p < 0.05). Two-week therapy of obese mice with resveratrol (100 mg/kg/day) restored insulin-stimulated AKT, IRS-1 and IRß phosphorylations, and simultaneously blunted the tyrosine nitration of these proteins. Additionally, the c-Jun N-terminal kinase (JNK) and inhibitor of NF-κB Kinase (IκK) phosphorylations were significantly increased in obese group, an effect normalized by resveratrol. In separate experiments, the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (20 mg/kg/day, three weeks) mimicked the protective effects exerted by resveratrol in lungs of obese mice. Lungs of obese mice display nitrosative-associated impairment of insulin signaling, which is reversed by resveratrol. Polyphenols may be putative drugs to attenuate asthma exacerbations in obese individuals.