A randomised controlled trial comparing a dietary antiplatelet, the water-soluble tomato extract Fruitflow, with 75 mg aspirin in healthy subjects.

BACKGROUND/OBJECTIVES: Increasing numbers of food ingredients are gaining acknowledgement, via regulated health claims, of benefits to human health. One such is a water-soluble tomato extract, Fruitflow (FF), a dietary antiplatelet. We examined relative platelet responses to FF and to 75 mg aspirin (ASA) in healthy subjects. SUBJECTS/METHODS: A total of 47 healthy subjects completed a double-blinded randomised controlled trial following a crossover design. Acute and 7-day treatments with 75 mg ASA were compared with control with and without concomitant FF, over a 5-h timecourse. Platelet aggregation response agonist, platelet thromboxane A2 release, plasma clotting times and time to form a primary haemostatic clot (PFA-100 closure time, TTC) were measured. RESULTS: Administration of all treatments lowered platelet function and thromboxane A2 generation, and extended the TTC, relative to baseline (P<0.001) and to control (P<0.001). Plasma clotting times were not affected. A single 75 mg dose of ASA showed approximately equal efficacy to a dose of FF, whereas daily 75 mg ASA was approximately three times as effective after 7 days (P=0.002). Platelet responses were heterogenous with distinct weak and strong responder groups. Weak ASA responders retained a functional platelet response to collagen agonist and were responsive to FF. Concomitant FF and ASA did not lead to significant additive effects. CONCLUSIONS: The suppression of platelet function observed after consuming FF is approximately one-third that of daily 75 mg ASA. The reversible action of FF renders it less likely to overextend the time to form a primary haemostatic clot than ASA, an important safety consideration for primary prevention.

Kiwifruit decreases blood pressure and whole-blood platelet aggregation in male smokers.

Lifestyle modifications to reduce risk factors for cardiovascular diseases such as blood pressure (BP) and smoking have been emphasized. Fruits and vegetables may modify such risk factors. The major aim of this randomized, controlled trial was to investigate the effects of (1) kiwifruits and (2) an antioxidant-rich diet compared with (3) a control group on BP and platelet aggregation (that is, whole-blood platelet aggregation) after 8 weeks in male smokers (age 44-74 years, n=102). The kiwifruit group received 3 kiwifruits per day, whereas the antioxidant-rich diet group received a comprehensive combination of antioxidant-rich foods. In the kiwifruit group, reductions of 10 mm Hg in systolic BP and 9 mm Hg in diastolic BP were observed (P=0.019 and P=0.016 (change from baseline in the kiwifruit group compared with change from baseline in the control group)). In the antioxidant-rich diet group, a reduction of 10 mm Hg in systolic BP was observed among hypertensives (P=0.045). Additionally, a 15% reduction in platelet aggregation and an 11% reduction in angiotensin-converting enzyme activity was observed in the kiwifruit group (P=0.009 and P=0.034). No effects on these parameters were observed in the antioxidant-rich diet group. This study suggest that intake of kiwifruit may have beneficial effects on BP and platelet aggregation in male smokers.

IFPA Meeting 2011 workshop report I: Placenta: Predicting future health; roles of lipids in the growth and development of feto-placental unit; placental nutrient sensing; placental research to solve clinical problems--a translational approach.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2011 there were twelve themed workshops, four of which are summarized in this report. These workshops related to both basic science and clinical research into placental growth and nutrient sensing and were divided into 1) placenta: predicting future health; 2) roles of lipids in the growth and development of feto-placental unit; 3) placental nutrient sensing; 4) placental research to solve clinical problems: a translational approach.

Docosahexaenoic acid stimulates tube formation in first trimester trophoblast cells, HTR8/SVneo.

Angiogenesis is a key factor in the placentation process and vascular remodeling that involves several growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin-like protein 4 (ANGPTL4). PPARs are involved in the placentation process but not much information is available on whether their ligands such as fatty acids have any effects on these processes. We therefore investigated the effect of fatty acids (arachidonic acid, 20:4 n-6(ARA), eicosapentaenoic acid, 20:5 n-3(EPA), docosahexaenoic acid, 22:6 n-3 (DHA) and oleic acid, 18:1 n-9 (OA)) on tube formation (as a measure of angiogenesis) on matrigel in the first trimester trophoblast cells, HTR8/SVneo. In addition we also investigated the effects of fatty acids on expression of genes involved in angiogenesis (VEGF and ANGPTL4) and lipid metabolism in these cells. Gene expression was determined after incubating these cells with different fatty acids for 24 h using real-time qRT-PCR, whereas VEGF and ANGPTL4 proteins were measured by respective ELISA kits. Of all the fatty acids tested, DHA increased tube formation to the greatest extent. DHA-induced increase in tube length was 583%, 247% and 70% over control, OA and EPA, respectively (p < 0.05). In addition, DHA stimulated cell proliferation by 150% of these cells. Of all fatty acids investigated, only DHA stimulated VEGF mRNA expression and protein secretion compared with control. Unlike DHA, other fatty acids (OA, EPA, ARA) stimulated ANGPTL4 mRNA expression and protein secretion in these cells. An inhibitor of VEGF decreased DHA stimulated tube formation in these cells. Altogether these data indicate that DHA may potently influence the placentation process by stimulating tube formation and this effect may be mediated in part via VEGF in first trimester trophoblast cells.

Expression of liver X receptors in pregnancies complicated by preeclampsia.

Preeclampsia is a pregnancy-specific disorder associated with hyperlipidemia. Liver X receptor (LXR) alpha and LXRbeta are key regulators of lipid homeostasis. In the current study, we investigated expression of LXRalpha, LXRbeta and their target genes in human term placenta, decidua and subcutaneous adipose tissue from pregnancies complicated by preeclampsia. Furthermore, we analyzed the protein levels of LXRalpha and LXRbeta in placenta. We also analyzed lipid concentrations in term placental tissue. Gene expression of LXRalpha, LXRbeta and fatty acid transporter CD36 was significantly decreased in placental tissues, while increased expression was observed for LXRalpha in adipose tissue, from pregnancies complicated by preeclampsia. The placental protein level of LXRbeta was reduced, and there was a positive correlation between placental LXRbeta mRNA expression and placental free fatty acids in preeclampsia. Our results suggest a possible role for LXRbeta as a transcriptional regulator in preeclampsia.

Long-chain polyunsaturated fatty acids stimulate cellular fatty acid uptake in human placental choriocarcinoma (BeWo) cells.

Supplementation of long-chain polyunsaturated fatty acids (LCPUFAs) is advocated during pregnancy in some countries although very little information is available on their effects on placental ability to take up these fatty acids for fetal supply to which the fetal growth and development are critically dependent. To identify the roles of LCPUFAs on placental fatty acid transport function, we examined the effects of LCPUFAs on the uptake of fatty acids and expression of fatty acid transport/metabolic genes using placental trophoblast cells (BeWo). Following 24 h incubation of these cells with 100 microM of LCPUFAs (arachidonic acid, 20:4n-6, eicosapentaenoic acid, 20:5n-3, or docosahexaenoic acid, 22:6n-3), the cellular uptake of [(14)C] fatty acids was increased by 20-50%, and accumulated fatty acids were preferentially incorporated into phospholipid fractions. Oleic acid (OA, 18:1n-9), on the other hand, could not stimulate fatty acid uptake. LCPUFAs and OA increased the gene expression of ADRP whilst decreased the expression of ACSL3, ACSL4, ACSL6, LPIN1, and FABP3 in these cells. However, LCPUFAs but not OA increased expression of ACSL1 and ACSL5. Since acyl-CoA synthetases are involved in cellular uptake of fatty acids via activation for their channelling to lipid metabolism and/or for storage, the increased expression of ACSL1 and ACLS5 by LCPUFAs may be responsible for the increased fatty acid uptake. These findings demonstrate that LCPUFA may function as an important regulator of general fatty acid uptake in trophoblast cells and may thus have impact on fetal growth and development.

Food intake of selenium and sulphur amino acids in tuberculosis patients and healthy adults in Malawi.

Wasting in tuberculosis (TB) patients is associated with the severity of lung disease and low serum level of selenium. Low serum levels of selenium may be due to low food intake or altered absorption/metabolism in the body. We therefore wanted to measure and compare the intake of selenium and sulphur amino acids in smear-positive TB patients and appropriately matched adults. We found a low intake of selenium but no significant difference in intake of selenium and sulphur amino acids between the groups in this study.

Long-chain polyunsaturated fatty acid transport across human placental choriocarcinoma (BeWo) cells.

Long-chain polyunsaturated fatty acids (LCPUFAs) such as docosahexaenoic acid (DHA) and arachidonic acid (AA) are essential for proper development of fetal brain and retina. These LCPUFAs are selectively enriched in the fetal circulation compared with the maternal circulation. In the current study we investigated the transfer of LCPUFAs and a non-essential fatty acid (oleic acid, OA) in a transwell monolayer system of placental choriocarcinoma (BeWo) cells. We show that incubation with OA results in increased triglyceride accumulation and lipid droplet formation compared with that of DHA. The relative amount of transfer of DHA across the cell monolayer was approximately 4-fold greater compared with that of OA when these fatty acids were added individually at 100 muM. This reflects the different fates of these two fatty acids in their metabolism and subsequent transport across the placental trophoblasts to the fetus. When using a mixture of fatty acids mimicking the composition of plasma non-esterified fatty acids during the last trimester of pregnancy, the transfer of OA and the LCPUFAs (DHA and AA) into the basolateral reservoir was not significantly different, whereas the transfer of palmitic acid (PA) was approximately 3.5-fold higher than OA transfer. However, since the concentration of OA compared to LCPUFAs was 10-fold higher in the donor chamber, the relative transport of the LCPUFAs was higher compared with that of OA. In addition, we show that inhibiting esterification of fatty acids into acyl-CoA can modulate, in part, the degree of transport through the cells. In conclusion, the transwell model system closely mimics the mechanisms of differential fatty acid transport as observed in vivo. LCPUFAs were transported through the cells more efficiently than shorter fatty acids such as OA.

Role of the M3 muscarinic acetylcholine receptor in beta-cell function and glucose homeostasis.

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

Liver X receptors mediate inhibition of hCG secretion in a human placental trophoblast cell line.

Liver X receptors (LXR) alpha and beta are important regulators of lipid homeostasis in liver, adipose and other tissues. However, no such information is available for the human placenta. We determined expression of both LXR alpha and beta in placental trophoblast cell lines, BeWo and JAR. Exposure of BeWo cells to a synthetic LXR agonist, T0901317, resulted in an increase in the amount of mRNA of LXR target genes, sterol regulatory element-binding protein-1 and fatty acid synthase. T0901317 also increased the synthesis of lipids. Moreover, T0901317 resulted in a reduced secretion of hCG during differentiation of these cells. Our data for the first time demonstrate a new role for LXRs in the human placenta.

M1-M5 muscarinic receptor knockout mice as novel tools to study the physiological roles of the muscarinic cholinergic system.

A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M(1)-M(5)) has proven a difficult task, primarily due to the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues and organs express multiple mAChRs. To circumvent these difficulties, we used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M(1)-M(5) mAChR genes. The different mAChR mutant mice and the corresponding wild-type control animals were subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. The M(1)-M(5) mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.

Expression of NK2 homologous transcripts during zebrafish development.

Expression of NK2 homologous transcripts during Zebrafish development was investigated by in situ hybridization technique. NK2 mRNA expression was first detected in a cluster of cells at the embryonic shield stage, and subsequently in the heart field of the 5-8-somite embryo. By the 15-17 somite stages, NK2 expressing cells were revealed in the midline of the developing embryo, and by 48 hpf within the heart tube of the larva. By 72 hpf, NK2 transcripts appeared in the jaw bones, pharyngeal arches, the cranium with minimal expression in striated muscle.

Asymmetric exchange is associated with P element induced male recombination in Drosophila melanogaster.

Spontaneous meiotic recombination events do not normally occur in the male germ line of Drosophila melanogaster. However, such events are induced in males when a P transposable element or a source of P element encoded transposase protein is present in its genome. This report concerns a molecular analysis of the meiotic exchanges that were induced in the male Drosophila by P elements within a genetically marked region of the third chromosome. The marked region also harbors a single P-element called P(lArB). Fifty-six percent of the P(lArB) region crossovers indicated some alterations in the P element 5' fragment. Such alterations appear to be related to asymmetric or unequal genetic exchanges. Finally, P(lArB) excision was found to be independent of P(lArB) region crossover events.

Cholinergic dilation of cerebral blood vessels is abolished in M(5) muscarinic acetylcholine receptor knockout mice.

The M(5) muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M(1)-M(5)) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M(5) receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M(5) receptor-deficient mice (M5R(-/-) mice). M5R(-/-) mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M(5) receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M(5) receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M(5) receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R(-/-) mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5R(-/-) mice. Our findings provide direct evidence that M(5) muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimer's disease and focal cerebral ischemia, cerebrovascular M(5) receptors may represent an attractive therapeutic target.

Hyperactivity and intact hippocampus-dependent learning in mice lacking the M1 muscarinic acetylcholine receptor.

Members of the muscarinic acetylcholine receptor family are thought to play key roles in the regulation of a large number of important functions of the CNS. However, the precise roles of the individual muscarinic receptor subtypes in modulating these processes are not well understood at present, primarily because of the lack of ligands with sufficient receptor subtype selectivity. To investigate the behavioral significance of the M(1) muscarinic receptor (M(1)R), which is abundantly expressed in the forebrain, we subjected M(1) receptor-deficient mice (M(1)R(-/-) mice) to a battery of behavioral tests. M(1)R(-/-) mice showed no significant impairments in neurological reflexes, motor coordination, pain sensitivity, and prepulse inhibition. Strikingly, however, M(1)R(-/-) mice consistently exhibited a pronounced increase in locomotor activity in various tests, including open field, elevated plus maze, and light/dark transition tests. Moreover, M(1)R(-/-) mice showed reduced immobilization in the Porsolt forced swim test and reduced levels of freezing after inescapable footshocks, suggesting that M(1)R(-/-) mice are hyperactive under stressful conditions as well. An increased number of social contacts was observed in a social interaction test. Surprisingly, M(1)R(-/-) mice displayed no significant cognitive impairments in the Morris water maze and in contextual fear conditioning. M(1)R(-/-) mice showed slight performance deficits in auditory-cued fear conditioning and in an eight-arm radial maze, most likely because of the hyperactivity phenotype displayed by the M(1)R(-/-) mice. Our results indicate that M(1) muscarinic receptors play an important role in the regulation of locomotor activity but appear to be less critical for cognitive processes, as generally assumed.

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice.

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.

Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean.

Members of the muscarinic acetylcholine receptor family (M1-M5) have central roles in the regulation of many fundamental physiological functions. Identifying the specific receptor subtype(s) that mediate the diverse muscarinic actions of acetylcholine is of considerable therapeutic interest, but has proved difficult primarily because of a lack of subtype-selective ligands. Here we show that mice deficient in the M3 muscarinic receptor (M3R-/- mice) display a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low levels of serum leptin and insulin. Paradoxically, hypothalamic messenger RNA levels of melanin-concentrating hormone (MCH), which are normally upregulated in fasted animals leading to an increase in food intake, are significantly reduced in M3R-/- mice. Intra-cerebroventricular injection studies show that an agouti-related peptide analogue lacked orexigenic (appetite-stimulating) activity in M3R-/- mice. However, M3R-/- mice remained responsive to the orexigenic effects of MCH. Our data indicate that there may be a cholinergic pathway that involves M3-receptor-mediated facilitation of food intake at a site downstream of the hypothalamic leptin/melanocortin system and upstream of the MCH system.

In vivo regulation of mu-opioid receptor density and gene expression in CXBK and outbred Swiss Webster mice.

Chronic in vivo treatment with the opioid agonist etorphine downregulates mu-opioid receptor density, produces tolerance, and regulates gene expression in the mouse. After cessation of treatment, there is an increase in mu-opioid receptor mRNA level associated with the recovery of mu-opioid receptors. However, the effect of etorphine on the regulation of mRNA during treatment is currently not known. In this study, etorphine-induced changes in mu-opioid receptor density, mRNA, and opioid analgesic potency were determined in two mouse strains that differ in basal mu-opioid receptor density in brain. CXBK mice (mu-opioid receptor deficient) and outbred Swiss Webster mice were implanted s.c. with placebo pellets (controls) or etorphine minipumps (250 microg/kg/day) for 1-7 days and mu-opioid receptor density or mRNA levels in whole brain were assessed or mice were tested for etorphine analgesia following 7 days of treatment. In control CXBK mice, mu-receptor density was approximately 40% less than that for the Swiss Webster, although mRNA abundance was similar in both strains. Etorphine's potency was 4-fold greater in control Swiss Webster compared to CXBK mice. Etorphine treatment decreased ( approximately 25-40%) mu-receptor density similarly in both strains throughout treatment. The magnitude of analgesic tolerance to etorphine was 8-fold in both mouse strains. Etorphine produced a biphasic effect on receptor mRNA in both strains with levels decreased (25%) by 3 days and increased (30-40%) at 7 days. mRNA levels remained elevated (55%) 16 h following the end of the 7 day etorphine treatment. Taken together, these data suggest that in vivo etorphine treatment that produces mu-opioid receptor downregulation and tolerance, can regulate mu-opioid receptor mRNA abundance. Receptor downregulation may initially induce decreases in mRNA levels since downregulation preceded a decrease in gene expression. Prolonged (>3 days) receptor downregulation may be responsible for increasing message levels and may be important in recovery of receptors following treatment. In addition, the magnitude of changes in receptor density, mRNA, and tolerance were similar in both CXBK and Swiss Webster mice, indicating that the mechanisms required for receptor regulation and its functional consequences are independent of basal mu-opioid receptor density.

Opioid receptor upregulation in mu-opioid receptor deficient CXBK and outbred Swiss Webster mice.

Chronic in vivo treatment with opioid antagonists increases opioid receptor density and the potency of opioid agonists without altering receptor mRNA levels. To determine if basal receptor density affects opioid receptor upregulation, we examined the effect of chronic naltrexone treatment on mu-opioid receptor density and mRNA in two mice strains that differ in mu-opioid receptor density. CXBK mice (mu-opioid receptor deficient) and outbred Swiss Webster mice were implanted s.c. with a placebo or 15 mg naltrexone pellet for 8 days, the pellets removed and 24 hr later opioid receptor density (mu, delta) and receptor mRNA level (mu) determined in whole brain; or morphine dose-response studies conducted. In placebo-treated CXBK mice, mu-opioid receptor density was approximately 40% less than in Swiss Webster mice, although mu-opioid receptor mRNA abundance was similar in both strains. In placebo-treated CXBK mice, morphine potency was approximately 6-fold less than Swiss Webster mice. Naltrexone treatment increased morphine potency (1.7-fold) and mu- (approximately 90%) and delta- (approximately 20-40%) opioid receptor density in CXBK and Swiss Webster mouse brain similarly. Mu-opioid receptor mRNA was unchanged by naltrexone treatment in either strain. There was no difference in the basal or naltrexone-treated whole brain G(i alpha2) protein levels in CXBK or Swiss Webster mouse. These data indicate that a deficiency in mu-opioid receptors does not alter the regulation of opioid receptors by opioid antagonists in vivo, and suggest that adaptive responses to chronic opioid antagonist treatment are independent of opioid receptor density.