Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

1. The literature has been reviewed with respect to the dietary intake and subsequent metabolism of polyunsaturated fatty acids (PUFA), of both the n-6 and n-3 series, in teleost fish. Particular emphasis has been made to the physiological roles of PUFA with respect to cell membrane function and eicosanoid production. 2. Atlantic salmon post-smolts were fed practical-type diets, based on fish meal, in three separate dietary experiments of 10-16 weeks duration. The first trial compared dietary lipid supplied either as fish oil (FO) or as sunflower oil (SO) with the diets having an n-3/n-6 PUFA ratio of 9.4 and 0.2 respectively. The second trial used diets formulated with blends of FO, SO, grape seed oil and safflower oil to provide linoleic acid at 10, 25 and 45% of total dietary fatty acids. The third trial was similar to the first but with an additional diet in which the lipid component was supplied by linseed oil (LO). All diets satisfied the nutritional requirements of salmonid fish for n-3 PUFA. There were no statistically significant differences in final weights between dietary treatments in the third trial. However, in the second trial fish fed the intermediate level of linoleic acid (25%) attained a significantly higher final weight compared to both other treatments while fish fed the highest level of linoleic acid (45%) had significantly lower final weights compared to both other treatments. In the first trial the effect of diet on growth (weight gain) could not be ascertained as the initial weights of the fish were significantly different. 3. A number of fish fed SO developed severe cardiac lesions which caused thinning of the ventricular wall and heart muscle necrosis. In addition the fish fed diets containing SO were susceptible to a transportation-induced shock syndrome that resulted in 30% mortality. 4. Incorporation of linoleic acid (18:2n-6) into membrane phospholipids increased in response to dietary intake with fish fed SO having increased levels of 18:2n-6 (up to 15-fold), 20:2n-6 (up to 12-fold), 20:3n-6 (up to 25-fold) and arachidonic acid (AA; 20:4n-6) (up to 3-fold), and decreased levels of eicosapentaenoic acid (EPA; 20:5n-3) (up to 3-fold). The ratio of n-3/n-6 PUFA was decreased (up to 4-fold) and the20:4n-6/20:5n-3 ratio increased (up to 9-fold) in membrane phospholipids from fish fed SO compared to those fed fish oil. While the tissue phospholipids from fish fed La had increased levels of 18:2n-6, 20:2n-6 and 20:3n-6, the levels of AA, 22:4n-6 and 22:5n-6 were similar to or significantly reduced compared to fish fed FO. Membrane phospholipids from fish fed LO also had increased 18:3n-3 and 20:4n-3 compared to both other treatments while in some tissues and phospholipid classes EPA was increased compared to fish fed FO. 5. These dietary induced changes in phospholipid eicosanoid precursor ratio were reflected in altered eicosanoid production. In gill cells, stimulated with the calcium ionophore A23187, 12-hydroxy-8, 10, 14, 17-eicosapentaenoic acid (12-HEPE) was the major 12-lipoxygenase product in fish fed Fa. In stimulated gill cells from fish fed SO and LO, 12-HEPE, 12-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12-HETE), 14- hydroxy-4, 7, 10, 13, 16, 19-docosahexaenoic acid (14-HDHE) and thromboxane B2 (TXB2) were all decreased compared to fish fed FO. However, the ratio of 12- HETE/12-HEPE was significantly elevated in stimulated gill cells from SO-fed fish compared to both other treatments. In stimulated blood leucocytes leukotriene B4 (LTB4)' 12-HETE and TXB2 were significantly increased while LTB5 and 12-HEPE were significantly decreased in fish fed SO compared to those fed FO. Blood leucocytes from fish fed LO produced less TXB2 compared to fish fed SO and prostaglandin E2 was reduced compared to both other treatments. In isolated cardiac myocytes stimulated with A23187, TXB2 production was increased in SO fed fish compared to those fed FO. 6. The activity of cardiac sarcoplasmic reticulum Ca2+-Mg2+ATPase was not affected by dietary treatment. 7. An established cell line derived from chum salmon heart (CHH-1) was utilised to study PUFA metabolism. The CHH-1 cells exhibited considerable A6 desaturase activity but showed no preference towards n-3 over n-6 PUFA. CHH-1 cells did exhibit significant A5 desaturase activity which showed a preference towards n-3 PUFA. No A4 desaturation activity was observed. Elongation of C20 PUFA was especially active in CHH-1 cells with C22 PUFA being specifically incorporated into phosphatidylethanolamine (PE) and phosphatidylserine (PS). CHH-1 cells supplemented with 20:3n-6 showed reduced growth rate, cell death and unusual pycnotic appearance, compared to those supplemented with other PUFA. 8. The lipid compositions of hearts and livers from wild and farmed parr and presmolts were analysed and compared. The fatty acid compositions of triacylglycerols (TAG) and phospholipids from both farmed parr and pre-smolts contained greater amounts of monoenoic fatty acids compared to their wild counterparts. TAG, phosphatidylcholine (PC) and PE from heart and liver of wild fish contained more 18:2n-6 and AA compared to farmed fish. Linolenic acid, EPA and 22:Sn-3 were increased in hearts and livers of wild fish compared to farmed. Docosahexaenoic acid (DHA; 22:6n-3) levels were higher in heart and liver of farmed fish, particularly in heart PC, PS and TAG. The n-3/n-6 PUFA ratio was generally lower in wild compared to farmed fish, largely due to higher n-6 PUFA, in particular AA, in wild fish. 9. The results are discussed with respect to the competitive interactions between PUFA of the n-6 and n-3 series which determine the fatty acid compositions of membrane phospholipids in salmon. The ratio of n-3/n-6 PUFA in membrane phospholipids, and in particular the ratio of AAIEPA, appears important in terms of membrane physiology and biochemistry, eicosanoid production and the development of cardiac histopathological lesions.

No description available.

No description available.

Three experiments were conducted to determine how dietary lipid sources influence lipid and polyunsaturated fatty acid (PUFA) metabolism when fed to young, growing chickens. In the first experiment, commercial meat-type chickens were fed one of four dietary lipids: 1) linseed oil (LO); 2) menhaden oil (MO); 3) soybean oil (SBO); or 4) chicken fat (CF). Chickens fed the polyunsaturated lipids, LO, MO, and SBO all had similar very low density lipoprotein + low density lipoprotein (VLDL + LDL) triacylglycerol concentrations which were lower than those for chickens fed CF. Tissue lipids from chickens fed LO contained more 20:5n3 compared with those fed SBO or CF. The amounts of 20:5n3 in tissues from chickens fed LO approached those found in tissues from chickens fed MO. Tissue lipids from LO and MO treatments exhibited decreased 20:4n6 concentrations compared with SBO or CF treatments. The data indicate that dietary n-3 lipid sources influence the fatty acid compositions of tissues and can be effectively used to enrich edible chicken tissues. The second experiment examined the effects of varying combinations of CF and MO on plasma triacylglycerols in broiler chickens. As the amount of dietary n-3 fatty acids and the polyunsaturate:saturate ratio increased, the concentration of triacylglycerols in plasma and the plasma VLDL + LDL fraction decreased. On the other hand, plasma triacylglycerol levels increased as the dietary n-6 fatty acids increased. The dietary n-3 fatty acids in the MO treatment led to higher levels of PUFA in the tissues evaluated. In the third experiment, female chickens from two genetic lines, high body weight (HW) and low body weight (LW), were fed SBO (rich in n-6 polyunsaturates) or MO (rich in n-3 polyunsaturates). The amounts of triacylglycerols in the plasma VLDL + LDL fractions were elevated in the LW chickens compared with the HW groups. Amounts of 18:1 isomers and total monounsaturates were highest in the livers and hearts of HW chickens. Feeding MO enriched the plasma, liver and heart tissues with n-3 polyunsaturates in both genetic lines. Plasma triacylglycerol concentrations were decreased in chickens fed MO at 56, but not at 84 days of age. The data suggest differences in lipid metabolism between the HW and LW lines which were not greatly affected by dietary n-6 or n-3 polyunsaturated fatty acids. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / Each biological cell is surrounded by a membrane that consists of many different kinds of lipids. The lipids are mainly composed of phospholipids, which form a fluid bilayer that serves as the platform for the function of membrane bound proteins regulating cellular activity. In the research described in this thesis we employed solid state 2H NMR, complemented by DSC (differential scanning calorimetry) and MD (molecular dynamics) simulations, to study the effect of PUFA (polyunsaturated fatty acids) and TFA (trans fatty acids) on molecular organization in protein-free model membranes of controlled composition. These two classes of unsaturated fatty acid incorporate into membrane lipids and have, respectively, a beneficial and harmful impact on health. The aim is to gain insight into the molecular origin of this behavior. DHA (docosahexaenoic acid), which with 6 "natural" cis double bonds is the most highly unsaturated PUFA found in fish oils, and EA (elaidic acid), which with only a single "unnatural" trans double bond is the simplest manmade TFA often found in commercially produced food, were the focus. 2H NMR spectra for [2H31]-N-palmitoylsphingomyelin ([2H31]16:0SM) in SM/16:0-22:6PE (1-palmitoyl-2-docosahexaenoylphosphatidylethanolamine)/cholesterol (1:1:1 mol) mixed membranes were recorded. This system served as our PUFA-containing model. The spectra are consistent with lateral separation into nano-sized (< 20 nm) domains that are SM-rich/cholesterol-rich (raft), characterized by higher chain order, and DHA-rich/cholesterol-poor (non-raft), characterized by lower chain order. The aversion cholesterol has for DHA, as opposed to the affinity cholesterol has for predominantly saturated SM, excludes the sterol from DHA-containing PE-rich domains and DHA from SM-rich/cholesterol-rich domains. It is the formation of highly disordered membrane domains that we hypothesize is responsible, in part, for the diverse health benefits associated with dietary consumption of DHA. 2H NMR spectra for 1-elaidoyl-2-[2H35]stearoylphosphatidylcholine (t18:1-[2H35]18:0PC) and 1-oleoyl-2-[2H35]stearoylphosphatidylcholine (c18:1-[2H35]18:0PC) were recorded to compare membranes with respect to a trans vs. cis ("natural") double bond. The spectra indicate that while a trans double bond produces a smaller deviation from linear conformation than a cis double bond, membrane order is decreased by a comparable amount because the energy barrier to rotation about the C-C single bonds either side of a <italic>trans</italic> or <italic>cis</italic> double bond is reduced. Although EA adopts a conformation somewhat resembling a saturated fatty acid, the TFA is almost as disordered as its <italic>cis</italic> counterpart oleic acid (OA). We speculate that EA could be mistaken for a saturated fatty acid and infiltrate lipid rafts to disrupt the high order therein that is necessary for the function of signaling proteins.

Indiana University-Purdue University Indianapolis (IUPUI) / This dissertation studies the effects of fatty acids on prostate cancer. Prostate cancer is one of the most common malignant diseases in males in the U.S. Because of the slow progression of this disease, early intervention methods, especially, dietary fatty acid interventions are considered very important to control the disease in early stages. This study describes how the depletion of the enzyme for endogenous fatty acid synthesis, fatty acid synthase, influences the expression of enzymes that metabolize dietary fatty acids and show how dietary fatty acids affect prostate cancer protein expression and function. Fatty acid synthase is an oncoprotein overexpressed in prostate cancer and its expression is suppressed with omega-3 fatty acid treatment. This study finds that the depletion of fatty acid synthase by siRNA knockdown induces suppression of cyclooxygenase-2 and fatty acid desaturase-1. Our results also show that fish oil (omega-3 fatty acid), but not oleic acid (omega-9 fatty acid), suppresses prostate cancer cell viability. Assessment of fatty acid synthesis activity indicates that oleic acid is a more potent inhibitor than fish oil of de novo fatty acid biosynthesis. In addition, the inhibition of its activity occurs over several days while its effects on cell viability occur within 24 hours. To better understand this relationship, label free LC-MS/MS based mass spectrometry was carried out to determine global proteomic and phosphoproteomic profiles of the prostate cell line PC3, with longitudinal treatment with fish oil or oleic acid. With short-term fish oil treatment, sequestosome-1was elevated. Prolonged treatment induced downregulation of microseminoprotein, a proinflammation factor, as well as proteins in the glycolysis pathway. In the phosphoproteomics study, we confidently identified 828 phosphopeptides from 361 phosphoproteins. Quantitative comparison between fish oil or oleic acid treated groups and the untreated group suggests that the fish oil induces changes in phosphorylation of proteins involved in the pathways associated with cell viability and metabolic processes, with fish oil inducing significant decreases in the levels of phospho-PDHA1Ser232 and phospho-PDHA1Ser300 and they were accompanied by an increase in PDH activity, suggesting a role for n-3 polyunsaturated fatty acids in controlling the balance between lipid and glucose oxidation.

The time course of this increase in muscle n-3 PUFA composition and anabolic protein expression is currently unknown. In Chapter 2 of this thesis ten healthy male participants consumed 5g.d-1 of n-3 PUFA-enriched fish oil for 4 weeks. Muscle biopsies samples were collected in the fasted, rested state 2 weeks prior, immediately before (Week 0), at Week 1, Week 2 and Week 4 after initiation of fish oil supplementation for assessment of changes in lipid composition and expression of anabolic signalling proteins over time. Muscle lipid profile, (% total n-3 PUFA/total fatty acids) increased from W0 to W2 (3.8 ± 0.2 to 5.1 ± 0.3 %) and continued to rise at W4 (6.7 ± 0.4 %). Total protein content of FAK increased from W0 to W4 (3.9 ± 1.5 fold) whereas total mTOR was increased from W0 at W1 (2.4 ± 0.6 fold) with no further significant increases at W2 and W4. For the first time this study demonstrates that oral fish oil consumption results in an increase of n-3 PUFA levels in human skeletal muscle that is associated with increases in the expression of anabolic signalling proteins. Our understanding of the anabolic signalling process that underpins muscle protein synthesis has been advanced by the application of the WB technique. However, the semi-quantitative nature and poor dynamic range associated with the WB technique may lead to incongruence regarding the molecular response of skeletal muscle to anabolic stimulation. Chapter 3 of this thesis developed and applied a quantitative in vitro [γ-32P] ATP kinase assay (KA) alongside a traditional WB methodology to assess p70S6K1 signalling responses in human skeletal muscle to RE and protein feeding. Following validation in tissue culture with rapamycin and optimization of the assay in human skeletal muscle, this methodology was tested in a physiologically relevant context. In this regard, six males performed unilateral resistance exercise (RE) followed by the consumption of 20 g of protein. Skeletal muscle biopsies were obtained at pre-RE, at 1 h and 3 h post-RE. In response to RE and protein consumption, p70S6K1 activity was significantly increased from pre-RE at 1 h and 3 h post-RE (8.84 ± 0.78 to 17.18 ± 2.62 and 15.62 ± 3.12 µU/mg). However, phosphorylated p70S6K1thr389 was not significantly elevated. To assess if a combined stimulus of RE and feeding can influence AMPK activity we directly measured AMPK activity. AMPK activity was suppressed from pre-RE at 3 h post-RE (24.15 ± 1.6 to 15.64 ± 1.07 mU/mg), whereas phosphorylated ACCser79 was unchanged. These data therefore highlight the utility of the KA to study skeletal muscle plasticity. Previous studies have shown that ingestion of n-3 PUFA potentiates the phosphorylation of mTORC1 and associated kinases in response to nutrition. However, no study has identified whether n-3 PUFA supplementation potentiates anabolic kinase activity when RE is performed prior to nutrient provision. In Chapter 4 of this thesis, twenty healthy males consumed 5g.d-1 of either fish oil (FO) or coconut oil (CO) capsules for 8 weeks. Muscle biopsy samples were collected in the fasted, rested state before and after 8 weeks of supplementation for assessment of changes in lipid composition. Following 8 weeks of supplementation muscle samples also were obtained at rest (Rest), post RE in both the exercise leg (Post-RE) and the rested leg (Pre-FED) and also at 3 h post RE and protein feeding from both the exercise leg (3 h post-REF) and rested leg (3 h post-FED). There was a 2-fold increase in muscle (5.53 ± 0.3 to 11.16 ± 0.45 % of total fatty acids) n-3 PUFA composition after supplementation in the FO group but no change in the CO group. Following supplementation there was an increase in p70S6K1 activity at 3 h post-REF from Rest in the CO group (5.6 ± 1.4 to 12.2 ± 2.1 µU/mg) but no change in the FO group. In the CO group, AMPKα2 was significantly increased at Post-RE from Rest (3.7 ± 0.7 to 9.9 ± 2.0 mU/mg). These data show that 8 weeks of n-3 PUFA enriched fish oil supplementation suppresses the activity of p70S6K1 in response to RE and protein feeding.

Thessis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase-3 activation or the apoptotic index. EPA and ARA (20 jiM, present before and after SI) significantly reduced caspase-3 activation, PARP-cleavage and the apoptotic index during reperfusion. This was associated with increased ERK- and decreased p38 phosphorylation. Vanadate (a tyrosine phosphatase inhibitor), but not okadaic acid (a serine-threonine phosphatase inhibitor), significantly reduced ARAinduced inhibition of p38 phosphorylation, suggesting involvement of tyrosine phosphatases during Sl/R. MKP-1, a dual-specificity phosphatase, was targeted and a significant induction of MKP-1 by ARA and EPA was observed. An in vitro dephosphorylation assay confirmed that this phosphatase might be responsible for the inhibition of p38 activation. It was also demonstrated that the protective actions of ARA are PI3-K dependent. The results suggest that p38 has a pro-apoptotic role while JNK phosphorylation is protective and that these kinases act via caspase-3 to prevent or promote cell survival in response to SI/R-induced injury. It was demonstrated for the first time that EPA and ARA protect neonatal cardiac myocytes from ischaemia/reperfusion-induced apoptosis through induction of a dual-specific phosphatase, MKP-1, causing dephosphorylation of the proapoptotic kinase, p38. These beneficial effects of ARA and EPA were also reflected by improvement in functional recovery during ischaemia/reperfusion of the isolated perfused rat heart model. / AFRIKAANSE OPSOMMING: Dit word algemeen aanvaar dat lang-ketting poli-onversadigde vetsure teen kardiovaskulere siektes beskerm, maar hul meganisme van aksie sowel as hul invloed op intrasellulere seinoordragpaaie is egter onbekend. Die doelwitte van hierdie studie is dus tweevoudig: (i) om die belang van mitogeen-geaktiveerde proteien kinases (MAPKs) en protein kinase B (PKB/Akt) in isgemie/herperfusie-geinduseerde apoptose vas te stel en (ii) om te bepaal of lang-ketting poli-onversadigde vetsure die hart, deur manipulering van hierdie kinases, beskerm. Rot neonatale ventrikulere miosiete, blootgestel aan gesimuleerde isgemie en herperfusie (Sl/H), is gebruik om die aktivering van ekstrasellulere seingereguleerde kinase (ERK), p38, c-Jun NH2-terminale protein kinase (JNK) asook PKB/Akt tydens apoptose, te karakteriseer. Die effek van ‘n omega-3 vetsuur (eikosapentaenoSsuur - EPA) en ‘n omega-6 vetsuur (aragidoonsuur - ARA) op die respons van bogenoemde kinases in neonatale kardiomiosiete tydens Sl/H, is ondersoek. Blootstelling van miosiete aan SI (energie-uitputting gemduseer deur kaliumsianied en 2-deoksi-D-glukose) het ‘n afname in die vermoe van die sel om te oorleef, soos gemeet deur die MTT (3-[4,5-dimetieltiazol-2-yl]-2,5- difeniel tetrazolium bromied) bepaling, tot gevolg gehad. ‘n Toename in apoptose (kaspase-3 aktivering en poli(ADP-ribose) polimerase (PARP) kliewing) is ook waargeneem. Morfologiese bewyse van apoptose (Hoechst 33342 kleuring) was egter eers tydens herperfusie sigbaar. SI is gekenmerk deur vinnige aktivering van p38 en PKB/Akt Ser473, terwyl ERK en JNK fosforilering slegs tydens herperfusie waargeneem is. Vooraf-behandeling met SB203580, ‘n p38 inhibitor, het ‘n beduidende toename in sellewensvatbaarheid asook ‘n afname in die apoptotiese indeks tydens Sl/H teweeggebring, terwyl SP600125, ‘n spesifieke JNK inhibitor, apoptose bevorder het. PD98059, ‘n ERK inhibitor, het geen invloed op apoptose tydens Sl/H gehad nie. Wortmannin, ‘n PI3-kinase inhibitor, het Thr308 (nie Ser473) fosforilering onderdruk, gepaargaande met ‘n toename in PARP kliewing, maar dit het geen invloed op kaspase-3 aktivering of die apoptotiese indeks gehad nie. EPA en ARA (20 (iM, teenwoordig voor en na SI) het kaspase-3 aktivering en PARP kliewing asook die apoptotiese indeks tydens herperfusie beduidend verminder. Beide vetsure het ook ‘n beduidende toename in ERK en afname in p38 fosforilering veroorsaak. Vanadaat (‘n serien-threonien fosfatase inhibitor), maar nie “okadaic” suur (‘n tirosien fosfatase inhibitor), kon die ARA-gemduseerde inhibisie van p38 ophef nie. Induksie van MKP-1, ‘n tweeledige-spesifieke fosfatase, is beduidend deur ARA en EPA tydens herperfusie verhoog. 'n In vitro defosforileringbepaling het bevestig dat hierdie fosfatase wel betrokke by die inhibisie van p38 kan wees. Daarbenewens is gevind dat die beskermende aksie van ARA PI3-K afhanklik is. Hierdie resultate wys dat fosforilering van p38 pro-apoptoties is, terwyl JNK beskermend is en dat hierdie kinases via kaspase-3 seldood of oorlewing tydens SI/H-geinduseerde beskadiging bemiddel. In hierdie model is daar vir die eerste keer getoon dat EPA en ARA neonatale kardiale miosiete teen isgemie/herperfusie-geinduseerde apoptose beskerm deur induksie van MKP- 1, wat defosforilering van die pro-apoptotiese kinase, p38 teweegbring. Hierdie voordelige effekte van EPA en ARA is ook sigbaar in die funksionele herstel tydens isgemie/herperfusie van die geisoleerde rothart model.