Hypolipidemic effect of dietary water-soluble protein extract from chicken: impact on genes regulating hepatic lipid and bile acid metabolism.

BACKGROUND: Amount and type of dietary protein have been shown to influence blood lipids. The present study aimed to evaluate the effects of a water-soluble fraction of chicken protein (CP) on plasma and hepatic lipid metabolism in normolipidemic rats. METHODS: Male Wistar rats were fed either a control diet with 20 % w/w casein as the protein source, or an experimental diet where casein was replaced with CP at 6, 14, or 20 % w/w for 4 weeks. RESULTS: Rats fed CP had markedly reduced levels of triacylglycerols (TAG) and cholesterol in both plasma and liver, accompanied by stimulated hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase 2 activity in the 20 % CP group compared to the control group. In addition, reduced activities and gene expression of hepatic enzymes involved in lipogenesis were observed. The gene expression of sterol regulatory element-binding transcription factor 1 was reduced in the 20 % CP-fed rats, whereas gene expression of peroxisome proliferator-activated receptor alpha was increased. Moreover, 6, 14, and 20 % CP-fed rats had significantly increased free carnitine and acylcarnitine plasma levels compared to control rats. The plasma methionine/glycine and lysine/arginine ratios were reduced in 20 % CP-treated rats. The mRNA level of ATP-binding cassette 4 was increased in the 20 % CP group, accompanied by the increased level of plasma bile acids. CONCLUSIONS: The present data suggest that the hypotriglyceridemic property of a water-soluble fraction of CP is primarily due to effects on TAG synthesis and mitochondrial fatty acid oxidation. The cholesterol-lowering effect by CP may be linked to increased bile acid formation.

Krill oil versus fish oil in modulation of inflammation and lipid metabolism in mice transgenic for TNF-α.

PURPOSE: Biological effects of marine oils, fish oil (FO) and krill oil (KO), are mostly attributed to the high content of n-3 polyunsaturated fatty acids (n-3 PUFAs), predominantly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The study was aimed to investigate the influence of FO and KO on lipid homeostasis and inflammation in an animal model of persistent low-grade exposure to human tumor necrosis factor α (hTNF-α) and to evaluate whether these effects depend on the structural forms of EPA and DHA [triacylglycerols (TAG) vs. phospholipids]. METHODS: Male C57BL/6 hTNF-α mice were fed for 6 weeks a high-fat control diet (24.50 % total fats, w/w) or high-fat diets containing either FO or KO at similar doses of n-3 PUFAs (EPA: 5.23 vs. 5.39 wt%, DHA: 2.82 vs. 2.36 wt% of total fatty acids). RESULTS: We found that KO, containing bioactive n-3 PUFAs in the form of phospholipids, was capable of modulating lipid metabolism by lowering plasma levels of TAG and cholesterol and stimulating the mitochondrial and peroxisomal fatty acid ß-oxidation, as well as improving the overall carnitine turnover. Though the administration of FO was not as effective as KO in the lowering of plasma TAG, FO significantly improved the levels of all cholesterol classes in plasma. Except from the increase in the levels of IL-17 in FO-fed mice and a trend to decrease in MCP-1 levels in KO-fed animals, the levels of pro-inflammatory cytokines were not substantially different between treatment groups. CONCLUSION: Our findings demonstrate that FO and KO are comparable dietary sources of n-3 PUFAs. However, when quantitatively similar doses of n-3 PUFAs are administered, KO seems to have a greater potential to promote lipid catabolism. The effect of dietary oils on the levels of inflammatory markers in hTNF-α transgenic mice fed a high-fat diet needs further investigations.

Normalization strategy is critical for the outcome of miRNA expression analyses in the rat heart.

Since normalization strategies plays a pivotal role for obtaining reliable results when performing quantitative PCR (qPCR) analyses, this study investigated several miRNA normalization candidates in regards to their efficiency as normalization standards in the ischemic reperfused ex vivo rat heart, with special reference to regulation of the miRNAs miR-1 and miR-101b. The possibility of including primers for several miRNAs in one reverse transcription (RT) reaction was also investigated. Langendorff perfused rat hearts were subjected to 30 min regional ischemia and 0, 1, 5, 15, or 120 min reperfusion. Total RNA was isolated and reverse transcribed for miRNA qPCR analysis. Normalization candidates were evaluated by the NormFinder and geNorm algorithms and the following stability expression rank order was obtained: sno202 < U6B < U87 < snoRNA < 4.5S RNA A < Y1 < 4.5S RNA B < GAPDH. Applying U6B as a normalizer it was found that miR-1 and miR-101b was downregulated in the ischemic reperfused myocardium. Furthermore, up to three primers could be included in one RT reaction by replacing RNase-free water with two supplemental sets of primers in the TaqMan MicroRNA assay protocol. This study demonstrates the importance of validating normalization standards when performing miRNA expression analyses by qPCR, and that miR-1 and miR-101b may play an important role during early reperfusion of the ischemic rat heart.

Long-term effects of environmentally relevant doses of 2,2',4,4',5,5' hexachlorobiphenyl (PCB153) on neurobehavioural development, health and spontaneous behaviour in maternally exposed mice.

BACKGROUND: Polychlorinated biphenyls (PCBs) are widespread in the environment, human food and breast milk. Seafood is known to contain nutrients beneficial for the normal development and function of the brain, but also contaminants such as PCBs which are neurotoxic. Exposure to non-coplanar PCBs during brain development can disrupt spontaneous behaviour in mice and lead to hyperactive behaviour. Humans are chronically exposed to the highest relative levels of organochlorines in early childhood during brain development, though usually at doses which do not give clinical symptoms of toxicity. This study aimed to elucidate the developmental and behavioural effects of 2,2',4,4',5,5' hexachlorobiphenyl (PCB153) in mice, mimicking human exposure during gestation and lactation. METHODS: Environmentally relevant doses of PCB153 were added to the experimental diets. Feed concentrations were approximately 0.5, 6.5, and 1500 µg PCB153/kg feed, representing a realistic and a worst case scenario of frequent consumption of contaminated fish. The study also investigated the effects of maternal nutrition, i.e. a standard rodent diet versus a high inclusion of salmon. Mice pups were examined for physical- and reflex development, sensorimotor function and spontaneous behaviour from five days after birth until weaning. A selection of pups were followed until 16 weeks of age and tested for open field behaviour and the acoustic startle response (ASR) with prepulse inhibition (PPI). Blood thyroid hormones and liver enzymes, blood lipids and PCB153 content in fat were examined at 16 weeks. Statistical analyses modelled the three way interactions of diet, PCB exposure and litter size on behaviour, using generalized linear models (GLM) and linear mixed effect models (LME). The litter was used as a random variable. Non-parametric tests were used for pair wise comparisons of biochemical analyses. RESULTS: Litter size consistently influenced pup development and behaviour. Few lasting PCB153 related changes were observed, but results indicated effects on synchronization of physical development. Perinatal PCB153 exposure appeared to reduce habituation and cause aggression in males, though not statistically significant. CONCLUSIONS: Litter size and maternal diet influenced physical development and function more than PCB153 in perinatally exposed mouse pups and supports the developmental importance of maternal care and the social environment.

Cerebral gene expression in response to single or combined gestational exposure to methylmercury and selenium through the maternal diet.

Controversy remains regarding the safety of consuming certain types of seafood, particularly during pregnancy. While seafood is rich in vital nutrients, it may also be an important source of environmental contaminants such as methylmercury (MeHg). Selenium (Se) is one essential element present in seafood, hypothesised to ameliorate MeHg toxicity. The aim of the present study was to ascertain the impact of Se on MeHg-induced cerebral gene expression in a mammalian model. Microarray analysis was performed on brain tissue from 15-day-old mice that had been exposed to MeHg throughout development via the maternal diet. The results from the microarray analysis were validated using qPCR. The exposure groups included: MeHg alone (2.6 mg kg(-1)), Se alone (1.3 mg kg(-1)), and MeHg + Se. MeHg was presented in a cysteinate form, and Se as Se-methionine, one of the elemental species occurring naturally in seafood. Eight genes responded to Se exposure alone, five were specific to MeHg, and 63 were regulated under the concurrent exposure of MeHg and Se. Significantly enriched functional classes relating to the immune system and cell adhesion were identified, highlighting potential ameliorating mechanisms of Se on MeHg toxicity. Key developmental genes, such as Wnt3 and Sparcl1, were also identified as putative ameliorative targets. This study, utilising environmentally realistic forms of toxicants, delivered through the natural route of exposure, in association with the power of transcriptomics, highlights significant novel information regarding putative pathways of selenium and MeHg interaction in the mammalian brain.

Cerebral gene expression and neurobehavioural development after perinatal exposure to an environmentally relevant polybrominated diphenylether (BDE47).

Nutrients in seafood are known to be beneficial for brain development. Effects of maternal exposure to 2,2',4,4' tetrabromo diphenylether (BDE47) was investigated, alongside the potential ameliorating impact of seafood nutrients, through assessment of neurobehaviour and gene expression in brain and liver. Developing mice were exposed during gestation and lactation via dams dosed through casein- or salmon-based feed, spiked with BDE47. Two concentrations were used: a low level (6 µg/kg feed) representing an environmentally realistic concentration and a high level (1,900 µg/kg feed) representing a BDE47 intake much higher than expected from frequents consumption of contaminated seafood. Experimental groups were similar with respect to reproductive success, growth and physical development. Minor, transient changes in neurobehavioural metrics were observed in groups given the highest dose of BDE47. No significant differences in behaviour or development were seen on postnatal day 18 among maternally exposed offspring. Cerebral gene expression investigated by microarray analyses and validated by RT-qPCR showed low fold changes for all genes, despite dose-dependent accumulation of BDE47 in brain tissue. The gene for glutamate ammonia ligase was upregulated compared to control in the casein-based high BDE47diet, suggesting potential impacts on downstream synaptic transmission. The study supported a previously observed regulation of Igfbp2 in brain with BDE47 exposure. Genes for hepatic metabolic enzymes were not influenced by BDE47. Potential neurotoxic effects and neurobehavioural aberrations after perinatal exposure to high levels of BDE47 were not readily observed in mice pups with the present experimental exposure regimes and methods of analysis.

Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies.

BACKGROUND: Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium. RESULTS: The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions. CONCLUSIONS: The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

PREPARE: guidelines for planning animal research and testing.

There is widespread concern about the quality, reproducibility and translatability of studies involving research animals. Although there are a number of reporting guidelines available, there is very little overarching guidance on how to plan animal experiments, despite the fact that this is the logical place to start ensuring quality. In this paper we present the PREPARE guidelines: Planning Research and Experimental Procedures on Animals: Recommendations for Excellence. PREPARE covers the three broad areas which determine the quality of the preparation for animal studies: formulation, dialogue between scientists and the animal facility, and quality control of the various components in the study. Some topics overlap and the PREPARE checklist should be adapted to suit specific needs, for example in field research. Advice on use of the checklist is available on the Norecopa website, with links to guidelines for animal research and testing, at https://norecopa.no/PREPARE .

Dual serotonergic regulation of ventricular contractile force through 5-HT2A and 5-HT4 receptors induced in the acute failing heart.

Cardiac responsiveness to neurohumoral stimulation is altered in congestive heart failure (CHF). In chronic CHF, the left ventricle has become sensitive to serotonin because of appearance of Gs-coupled 5-HT4 receptors. Whether this also occurs in acute CHF is unknown. Serotonin responsiveness may develop gradually or represent an early response to the insult. Furthermore, serotonin receptor expression could vary with progression of the disease. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation with nonligated sham-operated rats as control. Contractility was measured in left ventricular papillary muscles and mRNA quantified by real-time reverse-transcription PCR. Myosin light chain-2 phosphorylation was determined by charged gel electrophoresis and Western blotting. Ca2+ transients in CHF were measured in field stimulated fluo-4-loaded cardiomyocytes. A novel 5-HT2A receptor-mediated inotropic response was detected in acute failing ventricle, accompanied by increased 5-HT2A mRNA levels. Functionally, this receptor dominated over 5-HT4 receptors that were also induced. The 5-HT2A receptor-mediated inotropic response displayed a triphasic pattern, shaped by temporally different activation of Ca2+-calmodulin-dependent myosin light chain kinase, Rho-associated kinase and inhibitory protein kinase C, and was accompanied by increased myosin light chain-2 phosphorylation. Ca2+ transients were slightly decreased by 5-HT2A stimulation. The acute failing rat ventricle is, thus, dually regulated by serotonin through Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT4 receptors.

Appearance of a ventricular 5-HT4 receptor-mediated inotropic response to serotonin in heart failure.

BACKGROUND: Current pharmacological treatment of congestive heart failure (CHF) addresses changes in neurohumoral stimulation or cardiac responsiveness to such stimulation. Yet, undiscovered neurohumoral changes, adaptive or maladaptive, may occur in CHF and suggest novel pharmacological treatment. Serotonin [5-hydroxytryptamine (5-HT)] enhances contractility and causes arrhythmias through 5-HT(4) receptors in human atrium and ventricle but not through rat ventricular 5-HT(4) receptors. OBJECTIVE: We investigated whether CHF could induce ventricular responsiveness to serotonin. METHODS: Postinfarction CHF was induced in male Wistar rats by coronary artery ligation. Contractility was measured in left ventricular papillary muscles 6 weeks after infarction. Messenger RNA was quantified by RT-PCR and cAMP by RIA. RESULTS: Serotonin caused positive inotropic (-logEC(50)=7.5) and lusitropic effects in CHF but not Sham papillary muscles. The inotropic effect of 10 muM serotonin in CHF (31.3+/-2.2%) was of similar size as the effect of 10 muM isoproterenol (34.0+/-1.7%). The effects of serotonin were antagonised by GR113808 (0.5-5 nM), consistent with mediation through 5-HT(4) receptors. This was further supported by positive inotropic effects of the 5-HT(4)-selective partial agonist RS67506. Carbachol blunted the serotonin responses and serotonin increased ventricular and cardiomyocyte cAMP, consistent with coupling to G(s) and adenylyl cyclase. Quantitative RT-PCR revealed fourfold increased 5-HT(4(b)) mRNA expression in CHF vs. Sham ventricles. CONCLUSION: Functional ventricular 5-HT(4) receptors are induced by myocardial infarction and CHF of the rat heart. We propose that they are a model for ventricular 5-HT(4) receptors of human failing heart and may play a pathophysiological role in heart failure.

Macronutrient composition determines accumulation of persistent organic pollutants from dietary exposure in adipose tissue of mice.

Accumulation of persistent organic pollutants (POPs) has been linked to adipose tissue expansion. As different nutrients modulate adipose tissue development, we investigated the influence of dietary composition on POP accumulation, obesity development and related disorders. Lifespan was determined in mice fed fish-oil-based high fat diets during a long-term feeding trial and accumulation of POPs was measured after 3, 6 and 18months of feeding. Further, we performed dose-response experiments using four abundant POPs found in marine sources, PCB-153, PCB-138, PCB-118 and pp'-DDE as single congeners or as mixtures in combination with different diets: one low fat diet and two high fat diets with different protein:sucrose ratios. We measured accumulation of POPs in adipose tissue and liver and determined obesity development, glucose tolerance, insulin sensitivity and hepatic expression of genes involved in metabolism of xenobiotics. Compared with mice fed diets with a low protein:sucrose ratio, mice fed diets with a high protein:sucrose ratio had significantly lower total burden of POPs in adipose tissue, were protected from obesity development and exhibited enhanced hepatic expression of genes involved in metabolism and elimination of xenobiotics. Exposure to POPs, either as single compounds or mixtures, had no effect on obesity development, glucose tolerance or insulin sensitivity. In conclusion, this study demonstrates that the dietary composition of macronutrients profoundly modulates POP accumulation in adipose tissues adding an additional parameter to be included in future studies. Our results indicate that alterations in macronutrient composition might be an additional route for reducing total body burden of POPs.

Marine fatty acids aggravate hepatotoxicity of α-HBCD in juvenile female BALB/c mice.

Oily fish, a source of long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs), may contain persistent organic pollutants (POPs), including α-hexabromocyclododecane (α-HBCD). In experimental studies, marine LC n-3 PUFAs ameliorate fatty liver development while HBCD exposure was found to cause liver fatty acid (FA) changes. The present study investigated interactions of FAs and α-HBCD in juvenile female BALB/c mice using a factorial design. Mice (n = 48) were exposed for 28 days to a low (100 µg*kg body weight (BW)-1*day-1) or high dose (100 mg*kg BW-1*day-1) of α-HBCD in diets with or without LC n-3 PUFAs. High dose α-HBCD affected whole body lipid metabolism leading to changes in body weight and composition, and pathological changes in hepatic histology, which surprisingly were aggravated by dietary LC n-3 PUFAs. Hepatic FA profiling and gene expression analysis indicated that the dietary modulation of the hepatotoxic response to the high dose of α-HBCD was associated with differential effects on FA ß-oxidation. Our results suggest that in a juvenile mouse model, marine FAs accentuate hepatotoxic effects of high dose α-HBCD. This highlights that the background diet is a critical variable in the risk assessment of POPs and warrants further investigation of dietary mediated toxicity of food contaminants.

Prucalopride is a partial agonist through human and porcine atrial 5-HT4 receptors: comparison with recombinant human 5-HT4 splice variants.

Prucalopride is a gastrointestinal prokinetic drug that acts through 5-HT4 receptors, but its potential effects on cardiac atrial function are unknown. We investigated the effects of prucalopride on human right atrium, piglet left atrium, and piglet sinoatrial node. The effects of prucalopride on 5-HT4 receptor splice variants a, b, g and i, known to be expressed in human atrium, were studied for comparison. Prucalopride was an inotropic partial agonist, compared with 5-HT, on paced human atrial trabeculae (-logEC50M=7.4) and porcine left atria (-logEC50M=7.2), with intrinsic activity of 0.77 and 0.63 respectively. Prucalopride (1 microM) surmountably antagonized the positive inotropic effects of 5-HT on human (pK(P)=7.2) and porcine (pK(P)=7.1) atrium. Prucalopride was also a chronotropic partial agonist (-logEC50M=7.4, intrinsic activity=0.72 with respect to 5-HT) on spontaneously beating piglet atria. The cardiostimulant effects of prucalopride were prevented by GR113808 (1 microM), consistent with mediation through 5-HT4 receptors. Prucalopride bound to recombinant 5-HT4(a), 5-HT4(b), 5-HT4(g), and 5-HT4(i) receptors, labeled by [3H]GR113808, with pKi values of 7.6, 7.5, 7.4, and 7.8 respectively. Prucalopride stimulated adenylyl cyclase as a partial agonist on 5-HT4(a), 5-HT4(b), and 5-HT4(i) receptors with intrinsic activities of 0.82, 0.86, and 0.78 and -logEC50 values of 7.2, 7.3, and 7.2 respectively. At the 5-HT4(g) receptor prucalopride acted as a full agonist (-logEC50M=8.0) compared with 5-HT in the cell line tested, which was probably due to high receptor expression levels. We conclude that prucalopride is a cardiostimulatory partial agonist through human and porcine 5-HT4 receptors. Since prucalopride acts similarly through 5-HT4(a), 5-HT4(b), 5-HT4(g), and 5-HT4(i) receptors, any of these variants could be involved in the mediation of cardiostimulation.

Intake of farmed Atlantic salmon fed soybean oil increases hepatic levels of arachidonic acid-derived oxylipins and ceramides in mice.

Introduction of vegetable ingredients in fish feed has affected the fatty acid composition in farmed Atlantic salmon (Salmo salar L). Here we investigated how changes in fish feed affected the metabolism of mice fed diets containing fillets from such farmed salmon. We demonstrate that replacement of fish oil with rapeseed oil or soybean oil in fish feed had distinct spillover effects in mice fed western diets containing the salmon. A reduced ratio of n-3/n-6 polyunsaturated fatty acids in the fish feed, reflected in the salmon, and hence also in the mice diets, led to a selectively increased abundance of arachidonic acid in the phospholipid pool in the livers of the mice. This was accompanied by increased levels of hepatic ceramides and arachidonic acid-derived pro-inflammatory mediators and a reduced abundance of oxylipins derived from eicosapentaenoic acid and docosahexaenoic acid. These changes were associated with increased whole body insulin resistance and hepatic steatosis. Our data suggest that an increased ratio between n-6 and n-3-derived oxylipins may underlie the observed marked metabolic differences between mice fed the different types of farmed salmon. These findings underpin the need for carefully considering the type of oil used for feed production in relation to salmon farming.

Functional 5-HT receptors in human occipital artery.

5-HT receptors were studied in human occipital arteries, obtained from patients during neurosurgery. We detected mRNA for the following receptors (incidence): 5-HT(1B) (14/18), 5-HT(1D) (15/18), 5-HT(2A) (16/18), 5-HT(2B) (8/8), 5-HT(4(a)) (13/18), 5-HT(4(b)) (5/18), 5-HT(4(g)) (7/18), 5-HT(4(i)) (1/18), 5-HT(7(a/b)) (10/18) and 5-HT(7(d)) (12/18). 5-HT contracted and relaxed arterial rings at low (-logEC(50) M=7.0) and high (-logEC(50) M=4.2) concentrations, respectively. 5-HT-evoked contractions were antagonized partially by both 5-HT(1B)-selective SB224289 (200 nM) and 5-HT(2A)-selective ketanserin (1 microM) but not by 5-HT(1D)-selective BRL15572 (500 nM) or prazosin (1 microM). Sumatriptan caused contractions (-logEC(50) M=6.8, intrinsic activity with respect to 5-HT=0.3). Sumatriptan-evoked contractions were antagonized by SB224289 with high potency (pK(B)=9.4) but not by BRL15572. 5-HT-induced relaxations were resistant to blockade by 5-HT(1B)-selective SB224289 (1 microM), 5-HT(1D)-selective BRL15572, 5-HT(2B)-selective SB204741 (1 microM), 5-HT(4)-selective GR113808 (100 nM) and 5-HT(7)-selective SB269970 (1 microM), and a combination of SB204741 and SB269970, inconsistent with an involvement of 5-HT(1B), 5-HT(1D), 5-HT(2B), 5-HT(4) and 5-HT(7) receptors. Triton X-100 treatment of the arteries abolished acetylcholine-induced relaxations of rings precontracted by prostaglandin F(2alpha), but a reduction of the relaxant effects of 5-HT did not reach significance. Nitro-L-arginine (1 mM) reduced 5-HT-induced relaxations, suggesting a contribution of nitric oxide released from endothelial cells. Ketanserin (1 microM) prevented the relaxant effects of 5-HT. We conclude that 5-HT contracts human occipital artery through 5-HT(1B) receptors at low concentrations and through 5-HT(2A) receptors at high concentrations. Sumatriptan contracts mostly through 5-HT(1B) receptors. These results are consistent with the 5-HT(1B) and 5-HT(2A) mRNA data. 5-HT-induced relaxation is mediated, in part, through ketanserin-sensitive receptors, but 5-HT(1B), 5-HT(1D), 5-HT(2B), 5-HT(4) and 5-HT(7) receptors appear not to be involved.

Cloning, pharmacological characterisation and tissue distribution of a novel 5-HT4 receptor splice variant, 5-HT4(i).

5-HT4 receptor pre-mRNA is alternatively spliced in human (h) tissue to produce several splice variants, called 5-HT4(a) to 5-HT4(h) and 5-HT4(n). Polymerase chain reaction (PCR) with primers designed to amplify both 5-HT4(a) and 5-HT4(b) amplified three additional bands in different tissues, two representing different mRNA species both encoding 5-HT4(g) and one representing mRNA for a novel splice variant named 5-HT4(i), cloned from testis and pancreas respectively. Primary and nested PCR detected both 5-HT4(g) and 5-HT4(i) in multiple tissues. Whereas 5-HT4(i), was found in all cardiovascular tissues analysed, 5-HT4(g) was mainly present in atria. However, quantitative RT-PCR indicated 5-HT4(g) expression also in cardiac ventricle. The pharmacological profiles and ability to activate adenylyl cyclase (AC) were compared between four recombinant h5-HT4 splice variants (a, b, g and i) expressed transiently and stably in HEK293 cells. Displacement of [(3)H]GR113808 with ten ligands revealed identical pharmacological profiles (affinity rank order: GR125487, SB207710, GR113808>SB203186>serotonin, cisapride, tropisetron>renzapride, 5-MeOT>5-CT). In transiently transfected HEK293 cells cisapride was a partial agonist compared to serotonin at 5-HT4(b), 5-HT4(g) and 5-HT4(i) receptors. In membranes from HEK293 cells stably expressing 5-HT4(g) (3,000 fmol/mg protein) or 5-HT4(i) (500 fmol/mg protein), serotonin and 5-MeOT were full agonists while cisapride was full agonist at 5-HT4(g) and partial agonist at 5-HT4(i), probably due to different receptor expression levels. At both 5-HT4(g) and 5-HT4(i), the behaviour of 5-HT4 receptor antagonists was dependent on receptor level. At high receptor levels, tropisetron and SB207710 and to a variable extent SB203186 and GR113808 displayed some partial agonist activity, whereas GR125487 and SB207266 reduced the AC activity below basal, indicating both receptors to be constitutively active. We conclude that the novel 5-HT4(i) receptor splice variant is pharmacologically indistinguishable from other 5-HT4 splice variants and that the 5-HT4(i) C-terminal tail does not influence coupling to AC.

Functional serotonin 5-HT4 receptors in porcine and human ventricular myocardium with increased 5-HT4 mRNA in heart failure.

Serotonin (5-hydroxytryptamine, 5-HT) increases contractile force and elicits arrhythmias through 5-HT(4) receptors in porcine and human atrium, but its ventricular effects are unknown. We now report functional 5-HT(4) receptors in porcine and human ventricle. 5-HT(4) mRNA levels were determined in porcine and human ventricles and contractility studied in ventricular trabeculae. Cyclic AMP-dependent protein kinase (PKA) activity was measured in porcine ventricle. Porcine and human ventricles expressed 5-HT(4) receptor mRNA. Ventricular 5-HT(4(b)) mRNA was increased by four times in 20 failing human hearts compared with five donor hearts. 5-HT increased contractile force maximally by 16% (EC(50)=890 nM) and PKA activity by 20% of the effects of (-)-isoproterenol (200 microM) in ventricular trabeculae from new-born piglets in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine. In ventricular trabeculae from adult pigs (3-isobutyl-1-methylxanthine present) 5-HT increased force by 32% (EC(50)=60 nM) and PKA activity by 39% of (-)-isoproterenol. In right and left ventricular trabeculae from failing hearts, exposed to modified Krebs solution, 5-HT produced variable increases in contractile force in right ventricular trabeculae from 4 out of 6 hearts and in left ventricular trabeculae from 3 out of 3 hearts- range 1-39% of (-)-isoproterenol, average 8%. In 11 left ventricular trabeculae from the failing hearts of four beta-blocker-treated patients, pre-exposed to a relaxant solution with 0.5 mM Ca(2+) and 1.2 mM Mg(2+) followed by a switch to 2.5 mM Ca(2+) and 1 mM Mg(2+), 5-HT (1-100 microM, 3-isobutyl-1-methylxanthine present) consistently increased contractile force and hastened relaxation by 46% and 25% of (-)-isoproterenol respectively. 5-HT caused arrhythmias in three trabeculae from 3 out of 11 patients. In the absence of phosphodiesterase inhibitor, 5-HT increased force in two trabeculae, but not in another six trabeculae from 4 patients. All 5-HT responses were blocked by 5-HT(4) receptor antagonists. We conclude that phosphodiesterase inhibition uncovers functional ventricular 5-HT(4) receptors, coupled to a PKA pathway, through which 5-HT enhances contractility, hastens relaxation and can potentially cause arrhythmias.

A salmon protein hydrolysate exerts lipid-independent anti-atherosclerotic activity in ApoE-deficient mice.

Fish consumption is considered health beneficial as it decreases cardiovascular disease (CVD)-risk through effects on plasma lipids and inflammation. We investigated a salmon protein hydrolysate (SPH) that is hypothesized to influence lipid metabolism and to have anti-atherosclerotic and anti-inflammatory properties. 24 female apolipoprotein (apo) E(-/-) mice were divided into two groups and fed a high-fat diet with or without 5% (w/w) SPH for 12 weeks. The atherosclerotic plaque area in aortic sinus and arch, plasma lipid profile, fatty acid composition, hepatic enzyme activities and gene expression were determined. A significantly reduced atherosclerotic plaque area in the aortic arch and aortic sinus was found in the 12 apoE(-/)- mice fed 5% SPH for 12 weeks compared to the 12 casein-fed control mice. Immunohistochemical characterization of atherosclerotic lesions in aortic sinus displayed no differences in plaque composition between mice fed SPH compared to controls. However, reduced mRNA level of Icam1 in the aortic arch was found. The plasma content of arachidonic acid (C20:4n-6) and oleic acid (C18:1n-9) were increased and decreased, respectively. SPH-feeding decreased the plasma concentration of IL-1ß, IL-6, TNF-α and GM-CSF, whereas plasma cholesterol and triacylglycerols (TAG) were unchanged, accompanied by unchanged mitochondrial fatty acid oxidation and acyl-CoA:cholesterol acyltransferase (ACAT)-activity. These data show that a 5% (w/w) SPH diet reduces atherosclerosis in apoE(-/-) mice and attenuate risk factors related to atherosclerotic disorders by acting both at vascular and systemic levels, and not directly related to changes in plasma lipids or fatty acids.

Fish oil and the pan-PPAR agonist tetradecylthioacetic acid affect the amino acid and carnitine metabolism in rats.

Peroxisome proliferator-activated receptors (PPARs) are important in the regulation of lipid and glucose metabolism. Recent studies have shown that PPARα-activation by WY 14,643 regulates the metabolism of amino acids. We investigated the effect of PPAR activation on plasma amino acid levels using two PPARα activators with different ligand binding properties, tetradecylthioacetic acid (TTA) and fish oil, where the pan-PPAR agonist TTA is a more potent ligand than omega-3 polyunsaturated fatty acids. In addition, plasma L-carnitine esters were investigated to reflect cellular fatty acid catabolism. Male Wistar rats (Rattus norvegicus) were fed a high-fat (25% w/w) diet including TTA (0.375%, w/w), fish oil (10%, w/w) or a combination of both. The rats were fed for 50 weeks, and although TTA and fish oil had hypotriglyceridemic effects in these animals, only TTA lowered the body weight gain compared to high fat control animals. Distinct dietary effects of fish oil and TTA were observed on plasma amino acid composition. Administration of TTA led to increased plasma levels of the majority of amino acids, except arginine and lysine, which were reduced. Fish oil however, increased plasma levels of only a few amino acids, and the combination showed an intermediate or TTA-dominated effect. On the other hand, TTA and fish oil additively reduced plasma levels of the L-carnitine precursor γ-butyrobetaine, as well as the carnitine esters acetylcarnitine, propionylcarnitine, valeryl/isovalerylcarnitine, and octanoylcarnitine. These data suggest that while both fish oil and TTA affect lipid metabolism, strong PPARα activation is required to obtain effects on amino acid plasma levels. TTA and fish oil may influence amino acid metabolism through different metabolic mechanisms.